: z ; JiC'wflfik RARE INBORN ERRORS OF METABOLISM IN CHILDREN WITH MENTAL RETARDATION \ ; I ($3 CHILDREN’S BUREAU PUBLICATION NUMBER 429-4965 \_\ RARE IN BORN ERRORS OF METABOLISM IN CHILDREN WITH MENTAL RETARDATION DONOUGH O’BRIEN, M.D., F.R.C.P. Prafumr of Pediatriu, Univmitj 0f Calomda School of Medicine ‘ U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE WELFARE ADMINISTRATION 0 Children's Bureau 0 1965 For sale by the Superintendent of Documents, US. Government Printing Oflice, Washington, D.C., 20402 - Price 70 cents FOREWORD One of the major obstacles to further ad- vances in understanding mental retardation is that of etiological diagnosis. It is important therefore that the many recent advances in associated metabolic errors be widely known in order that appropriate cases may be earlier diagnosed, better understood and more effec- tively treated. Phenylketonuria and galactosemia have been the subjects of earlier publications by this Bureau, and certain other conditions such as gargoylism, the lipoidoses, the aberrations of thyroxin metabolism and the hypoglycemic syn— dromes are not included here as they are already plentifully documented in standard texts. The descriptions in this booklet are for the most part confined to states with ill-defined physical signs in which the diagnosis depends on laboratory confirmation. The great majority of these syn- ii dromes relate to some disorder of amino acid metabolism. In each case, the clinical and laboratory findings are briefly stated, together with a sum- mary of current thought on the underlying bio- chemical disorder, the genetics, and the treat- ment. Diagnostic and screening tests within the compass of any good clinical laboratory are out- lined with individual syndromes and are also set out in greater detail in a special section. Key references only are supplied. This publication is intended to offer to pediatricians and other allied professions inter- ested in mental retardation up-to—date clinical information and diagnostic guidelines on this group of individually rare, but important syn- dromes on which little information is available in currenttextbooks. «Ms. W KATHERINE B. OETTINGER Chief, Children’: Bureau WELFARE ADMINISTRATION W504 MHOQ 1.26 CONTENTS 52,236. LIBRARY PART A. THE CLINICAL SYNDROMES Disturbances of Amino Acid Metabolism In the sulfur-containing amino acids- - 1 I n proline metabolism ______________ 28 C stathioninun'a: c stathionine Proline oxidase deficiency ________ 28 y y cleavage enzyme deficiency--- __ 1 Hyperprolinemia due to A’ pyITo-' Homocystinuria: cystathionine syn- line-5-carboxy1ic acid dehydrogen- thetase deficiency ______________ 3 ase deficiency ------------------ 31 Cystinuria ______________________ 6 Hydroxyprolinemia With mental re- Methionine malabsorption and men- tardatlon_ _ ._ _ _ _ _.- _ - - - _________ 32 tal retardation _________________ 7 Atax1a tlelangEZCthzh Wltll an ab- 33 . . . norma pept1 em eunne ------ I n thcleammo aczds of the urea syntheses 7 Joseph’s syndrome _______________ 34 cyc """"""""""""""" Osteopathy, peptiduria, and mental Two forms of argininosuccinic acid retardation ____________________ 35 Cleavage enzyme defiCiency ------ 7 In histidine mtabolism _____________ 36 Ornithine transcarbamylase defi- _ _ . , _ ciency _________________________ 11 H1st1d1ne a-deamlnase deficwncy: Congenital lysine intolerance With H hlsgdifl 6111.13" ' '.‘ ‘ ' :t—h— i. ‘ ' ". ' ‘. — ' ' 36 periodic ammonia intoxication-__ 12 ype 0 1,0 8.01. 6111.18” W1 ormimino- C' . . . . . . . . glutamlcac1dur1a followmg h1st1— 1trullmur1a. argmmosuccmic acld . . . . - dine loadmg: form1m1notrans- synthetase deficiency ----------- 15 ferase deficiency _______________ 38 Carbamyl phosphate syn thetase de— Imidazole aminoaciduria in cerebro- fielency_ ' "' ' ‘ ‘ ‘ ': """"""" 17 macular degeneration ----------- 40 In tryptophane metabolism —————————— 18 In tyrosine mtabolism ______________ 42 COélgenilital tryptophanurla mm 1 Transient p-hydroxyphenylpyruvic wa sm """"""""""" 8 acid oxidase deficiency ---------- 42 Hartnup dlsease- 1- “ ' ' - ' ‘ - j - - ": - - 20 Hyperphenylalaninemia ___________ 44 Indolylacroyl glycme excretlon In a Urinary 3_4 dihydroxyphenylala- family with mental retardation__ 22 nine (DOP A) excretion in chil- Hyperserotoninemia -------- .— - - -' - — 23 dren of short stature ___________ 44 Pyndoxme defi01ency , pyrldoxme Phenylketonuria With a-hydroxy- dependency and abnormalities of butyric aciduria: the oast-house kynureninase __________________ 25 syndrome --------------------- 45 iii In tyrosine metabolism—Continued Familial dysautonomia ___________ 48 Miscellaneous disorders _______________ 48 Idiopathic hyperglycinemia ________ 48 iv The oculo-cerebro-renal syndrome__ 51 Maple syrup urine disease: branched chain ketoaciduria _____________ 53 Ataxia, megaloblastic anemia form- iminoglutamic aciduria and mental retardation ____________________ 56 Sarcosinemia and sarcosinuria with mental retardation _____________ 57 “T” substance in the urine ________ 62 Green acyl dehydrogenase deficiency__ 63 The syndrome of hyperuricemia, choreoathetosis and mental re- tardation ______________________ 64 Idiopathic hypercalcemia __________ 65 Hereditary oroticaciduria, megalo— blastic anemia and mental retarda- tion __________________________ 57 Idiopathic hypervaljnemia _________ 58 The syndrome of spastic diplegia, microcephaly, mental retardation and amino aciduria ____________ 60 Glutamic acidemia and cerebral de- generation _____________________ 61 Mental retardation, cortical atrophy and increased glutamic acid in the C.S.F ________________________ 62 Other Rare Inborn Errors of Metabolism Associated with Mental Retardation Lactic and pyruvic acidosis _______ 67 Hyperlipidemia and mental retarda- tion __________________________ 68 Acid mucopolysacchariduria (hep- aritin sulfate) and mental retarda- tion __________________________ 69 PART B. DIAGNOSTIC TECHNICAL PROCEDURES Determination of plasma ammonia- 70 Pap er chromatography of amino ac ids _________________________ 71 Determination of serum calcium_- _ 77 Determination of n—formiminoglu- tamic acid in urine _____________ 79 Chromatographic identification of sulfated mucopolysaccharides in- cluding heparin ________________ 81 Determination of histidine in serum_ 81 Lactic and pyruvic acid determina- tion in serum __________________ 83 Determination of serum lipids _____ 86 Determination of plasma proline_- - 89 The tryptophan loading test and assay of kynurenine derivatives- _ 90 Determination of serum uric acid-_ 95 Paper chromatography of phenolic acids of urine, including 3—meth- oxy-4-hydroxy—mandelic acid (VMA) and homovanillic acid (HVA) ________________________ 97 The nitroprusside cyanide test for cystine and homocystine in urine- 100 The ferric chloride test ----------- 100 PART A. THE CLINICAL SYNDROMES Disturbances of Amino Acid Metabolism In the Jannr-conmining amino nciab Cystathioninuria: Cystathionine cleavage enzyme deficiency Clinical and laboratory findings Four cases only of this condition have been described. The first 1 was in a 64—year-old woman with an IQ of 42. She had been back- ward from birth but showed no additional physical signs beyond a mild bilateral talipes calcaneo valgus. Cystathionine was identified in the urine by paper chromatography and found to be excreted in amounts around 2.2 m mols/24 hrs. The second case2 was a 44-year-old man with obvious features of acromegaly. He also had small ears with deformities of the external canal, a bilateral primarily conductive deafness, and a modest enlargement of the thyroid. The liver edge was palpable 5 cm below the costal margin and there was some loss of sensation to light touch and pinprick over the fingertips. He was depressed, anxious, and tense, taking refuge in fantasies; his IQ, was 100 however, although his overall mental status was hard to assess because of deafness. Significant laboratory investigations, as in the first case, relate to the finding of cystathio- nine in the urine. Excretion varied between 4.3—5.8 m mols/24 hrs. Plasma and cerebro- spinal fluid values were 0.014 “mols and 0.021 amok/ml respectively; coincidental inulin clearances showed that cystathionine is cleared completely by the glomerulus. The third case3 occurred in an irritable, retarded, hyperactive, brown-haired, grey-eyed infant girl who was identified as a phenylketo- nuric at the age of 21/2 years. Serum phenyl- alanine was 15 mg/ 100 ml and urinary phenyl— pyruvic acid was sufliciently high to produce its characteristic odor. When the child failed to improve intellectually on a phenylalanine-low diet, further studies by column chromatography showed .1 to .9 m mols/24 hrs of cystathionine in the urine, depending upon the diet. Oral methionine loading produced an increase in cystathioninuria, with a delayed clearance of cystathionine in the urine. The fourth case, not retarded, had low platelets and calculi.11 Biochemistry The natural synthesis of cystathionine 4 is achieved by the transulfuration of the —SH group on homocysteine onto the a—,B unsaturated derivative of the aldimine derivative of pyri- doxal phosphate and serine as shown below. Pyridoxal phosphate is then dissociated to form cystathionine. Cystathionine is further broken down by a hydrolytic cleavage into cysteine and homoserine. In a first attempt to define the biochemical lesion more specifically, 5 g of DL methionine, the methylated derivative of homocysteine, was 1 THE METABOLISM OF CYSTATHIONINE figure 1. $“3 ‘3 SH Ho-c—c-CH3 c'H n I 2 0 CH2 l PYRUVIC ACID CHNH2 o I NH ll COOH 3 CH H o METHIONINE o 2 HO \ (mac-O ll HO-C-C=CH / I 2 H36 N clesH PYRI NH2 DOXAL (EH2 a—AMINOACRYLIC PHOSPHATE (:HNH2 ACID 9 CIOOH Ho-c—CH. NHTCHon HOMOCYSTEINE SERINE SERINE j DEHYDRASE o H S-CH—CH—CHNH coon Ho 0 H \\ I , 2 2 2 2 II I H20 HO-C-C-CH 9 Ho-c — C-CHZOH I 7 - _ = | x” HO c (3 CH2 N H. \‘CH x H,I‘\CH ’ x \ ' \ l ° \ CH50-® o CH-20-® \ CH—o-®—Z——' H3C N/ H c H c / a-IsUNSATURATED 3 H o 3 N DERIVATIVE H20 2 01:3;i'T’75E 9 DISSOCIATION H C H FROM PYRIDOXAL-(D émCHEO’G H3C\N/ PYRIDOXALO °\\ '1'?” CYSTATHIONINE CLEAVEAGE ‘3 fl. S"CHECHECHNH2C°°H ENZYME HO-C-E-CHZ L HO—C—Cl—CH2 NH N”2 CH20H 2 (IZH CYSTATHIONINE I 2 HO CI-INI-I2 2 I COOH HOMOSERINE given orally to the first case. As a result there was a 21/2-fold increase in the urinary excretion of cystathionine, suggesting a block in the break- down of this amino acid. Cystathionine cleav- age has been shown to require pyridoxal phos- phate 5 and cystathionine has been demonstrated in excess in the urine of a B6 deficient infant,6 as well as in the brain of B6 deficient rats.7 In the second patient the intramuscular injection of 90 mg of pyridoxal phosphate daily actually led to a perceptible fall in cystathionine excre- tion, which rose again when the dose was discon- tinued. A similar effect could be produced by 400 mg daily of B6 orally. B3 deficiency seems unlikely as there was no impairment of kynu- reninase‘activity as judged by the urinary ex- cretion of kynurenine derivatives following an oral dose of tryptophan. The evidence, such as it is, would, however, suggest an abnormality of the ooenzyme binding site on the cystathio- nine cleavage enzyme. It is not clear in what way excess cysta— thionine is of importance in developing cerebral metabolism, particularly as cystathionine de- ficiency in association with mental deficiency may also occur in homocystinuria. It is known, however, that cystathionine exists in relatively high concentrations in human brain 8 without being actually incorporated into protein. Diagnostic and screening tests Excess urinary cystathionine can be sus- pected from the one-dimensional paper chroma- tography screening test.9 Two-dimensional paper chromatography 1° or column chromatog- raphy is required for confirmation. Genetics The condition probably represents the homozygous condition of an autosomal recessive trait in that a moderate increase in urinary cys- tathionine was detected in one sibling of each case, as well as in the nephew of the first case and in two nephews and two nieces of the second. \Treatment There is now evidence that cystathionine excretion may be reduced by the intramuscular or oral administration of pyridoxine and that prolonged treatment in this way can improve intellectual performance.12 It would seem to be a desirable approach, especially, if the condi- tion were detected in an infant. References 1. Harris, H., Penrose, L. S., and Thomas, D. H. H.: Cystathioninuria. Ann. Human Gen. 23:442, 1958—59. 2. Frimpter, G. W., Haymovitz, A., and Horwith, M.: Cystathioninuria. New Eng. J. Med. 2681333, 1963. 3. Shaw, K. N. F., Koch, R., Donnell, G. N., Lieber- mann, E., Jacobs, E. E., Gutenstein, M., and Rags- dale, N.: Cystathioninuria in a phenylketonuric infant. American Pediatric Society, 1963, p. 84 (abstr.). 4. Selim, A. S. M., and Greenberg, D. M.: An enzyme that synthesizes cystathionine and deaminates L-serine. J. Biol. Chem. 234: 1474, 1959. 5. Binkley, F., Christensen, G. M., and Jensen, W.: Pyridoxine and transfer of sulfur. J. Biol. Chem. 194 z 109, 1952. 6. Scriver, C. R., Hutchison, J. H., and Ooursin, D. B.': Vitamin B3 deficiency in a human infant: Bio- chemical observations of amino acid and co- enzyme metabolism. Amer. J. Dis. Child. 102: 632 (abstr.). 7. Hope, D. B.: Cystathionine accumulation in the brains of pyridoxineadeficient rates. J. Neuro- chem. 11:327, 1964. 8. Tallan, H. H., Moore, 8., and Stein, W. H.: L—cysta- thionine in human brain. J. Biol. Chem. 230: 707, 1958. 9. See technical section page 71. 10. O’Brien, D. and Ibbott, F. A.: Laboratory manual pediatric micro- and ultamicrobiochemical tech- niques. Hoeber, New York, 3d ed. 1962, p. 25. 11. Mongeau, J. G., Frimpter, G. W., Hilgartner, M. W. and Worthen, H. G.: Cystathioninuria, throm- bocytopenic purpura and urinary lithiasis. Am. Fed. Soc. 1965. p. 47 (abstr.). 12. Berlow, S., and Efren, M. L.: Cystathioninuria. Mid-West Soc. Ped. Res. 1964 (abstr.). Homocystinuria: Cystathionine synthetasc deficiency Clinical and laboratory findings Some 70 cases of this condition are known and 6 have been reported in detail in the litera— 3 ture?‘4 This recently described inborn error of metabolism shows a variable clinical picture characterized by mental retardation, cataracts, lenticular dislocation, friable fair hair, a malar flush, thromboembolic disease, bone changes, and fatty infiltration of the liver.14 Mental retardation was present in most re- corded cases; in one instance it would appear to have been present since birth 3 whilst in an- other the child was thought to be normal until the age of 41/2 years when an acute encephalitic episode occurred following immunization against poliomyelitis.4 Occasional grand mal convulsions were noted and most of the cases showed some degree of spastic paraplegia. Dis— location of the lenses was seen in three of the cases 27 4, bilateral zonular cataracts in another; 3 however, in one child there were no eye signs. The fair hair may be fine or coarse and tends to break off easily, especially around the occiput. Again the majority of cases showed a very Figure 2. characteristic malar flush, and in one of these 2 erythema ab igne like patches were found else- where on the body. Extensive fatty change, but no necrosis or cirrhosis, was found on liver bi- opsy of one case who had an enlarged liver,2 but this finding appeared to be unique and was not confirmed in the two cases for whom autopsy in- formation is available. Genu valgum, pes cavus, and long fingers with peri-articular thickening of the interphalangeal joints are also reported. Pulmonary and other emboli were found in both of the cases who terminated fatally. This tendency to thromboembolic phenomena was the basis of a special investiga— tion 5 in which increased platelet adhesiveness was demonstrated in four children with homo- cystinuria; the effect, moreover, could be dupli- cated by the addition of homocystine in vitro to normal blood. . Conventional laboratory tests were unre- markable except that in one case leucine amino— THE METABOLISM OF HOMOCYSTINE CH20H l CH2 CH3 l ' C|H3 TO ONE H COOH CH(NH‘2) CARBON POOL ' ' ' S 5 CHon CH(NH2) COOH I i I I I CH2 CH2 + CHlNHQ] —«>- CH2 HOMOSER'NE | | I I CH2 CH2 COOH s + I I I CH(NH2) CH(NH2) SERINE CH2 SH I . AS ALDIMINE | l COOH COOH DERIVATIVE CH2 CH2 HOMOCYSTEINE WITH ' | METHIONINE PYRIDOXAL® CH(NH2) CH(NH2) (see Fig.1) ' I COOH COOH H2C _ s _ s _ CH2 CYSTATHIONINE CYSTEINE l I H2C| (le2 CH(NH2] CH(NH2) | I COOH COOH HOMOCYSTINE peptidase and isocitric dehydrogenase levels were slightly raised. Homocystine was detected in the urine first by paper chromatography and later by column chromatography. In the urine, base level excretion rates of homocystine varied between 26 and 300 ,umols/24 hrs (normal 0), methionine excretion was increased in two cases to between 25 to 80 mole/24 hrs 3 (normal 30 to ‘60 ,umols/24 hrs for an adult); this latter variability may relate to the total protein intake at the time of collection. In the plasma both homocysteine and homocystine have been found, about one-third being homocysteine, with levels around 0.1 limols/ml.1 Methionine in the plasma was strikingly increased in one case to 1.4 mols/ml. and in another to 0.1 pmols/ml. a level still above the normal range of .01 to .04 mols/ml. Biochemistry Homocysteine, which is in equilibrium with its reduced S—S form homocystine, is formed by the demethylation of methionine. The -SH group is then transulfurated6 to the a-B un- saturated derivative of the aldimine of pyro— doxal phosphate and serine. In the final step, as shown below and in figure 1, cystathionine syn- thetase catalyses cystathionine formation with the splitting off of pyridoxal phosphate. F ur- ther breakdown of cystathionine is affected 7 by the pyridoxal-dependent enzyme cystathionase, which breaks it down to ammonia, a-keto butyric acid, and cysteine. Oral loads with methionine 2’ a. 8 produce a sustained rise in plasma methionine with no ele- vation of plasma homocysteine and homocys- tine, and variable increases in urinary methio- nine, homocystine, homocysteine, and the mixed disulfide 1/; homocystine—% cystine. Despite some variability in the loading tests, the most probable explanation of the metabolic abnormality would seem to be offered by a defect in cystathionine synthetase. To test this possibility 3 a liver biopsy was taken from an 8-year-old girl with the disease, and methio- nine activating enzyme and cystathionine syn- thetase activity compared with five control cases. Methionine activation enzyme activity was slightly elevated, but no cystathione synthe- tase activity was detected in the child with 772-952 0 - 66 - 2 homocystinuria. Betaine and thetin-homocys- teine transmethylases were normal.9 The importance of cystathionine availabil- ity to cerebral metabolism is still not known, and it is particularly diflicult to attribute a causal role in mental retardation to a deficiency in cystathionine when cystathioninuria, a pre- sumed cystathionine cleavage enzyme defect, is also associated with mental deficiency. All that is known is that cystathionine is an important non-protein constituent of human brain 1°, 11 and that it was not present in a cerebral biopsy 12 or autopsies 17 of patients with homocystinuria. Diagnostic and screening tests Diagnoses can be made by finding homo- cystine on paper chromatography 13 or col- umn chromatography of the urine. Homocys- tine may also be measured as cystine polaro— graphically. All of these cases gave a positive nitroprusside—cyanide screening test.16 In this test 5 ml of urine, acidified by the addition of 0.5 ml of 1N HCl, is mixed with 2 m1 of fresh 5 per- cent NaCN and left for 30 minutes at room tem- perature. A deep red color develops on the addition of 1 ml of 5 percent nitroprusside so- lution in the presence of cystine or homocystine. Differentiation from cystinuria can be made from clinical appearances and by paper or col- umn chromatography of the urine. Genetics Family pedigrees investigated so far sug- gest that inheritance is as an autosomal reces- sive, although multiple genetic loci may be involved. In a single instance hepatic cystathio- nine synthetase levels in the parents were shown to be 40 percent of unrelated controls." Treatment An early theory was that the disease was caused by a deficiency of cystine.2 One child was therefore given cystine orally in a dose of 1 g three times a day. Initially the child ap- peared livelier and happier. but after 21/2 months she had a febrile convulsion and the amino acid was discontinued. Certainly, how- ever, dietary supplement with cysteine would be justified in any case diagnosed in early infancy. Later it was resumed for 31/2 months, but no con- 5 clusively beneficial efl'ect could be demonstrated. It is of interest that the paternal cousin of the last reported case 13 had homocystinuria, and a level of hepatic cystathionine synthetase be- tween that of the affected child and her parents, but was not retarded. It is suggested that the relatively high content of cystine in breast milk may have protected this case from cerebral dam- age in the early weeks of life. There is a little evidence that when L-serine intake is increased,12 or when methionine and serine intake are both increased,“1 homocystine excretion is decreased and a small amount of cystathionine excreted in the urine. Cystathi— onine therapy is rational but costly. Recently a low methionine, cystine supplemented diet has shown some success.“ References 1. Gerritsen, T., Vaughn, J. G., and Waisman, H. A.: The identification of homocystine in the urine. Biochem. Biophys. Res. Comm. 9: 493, 1962. 2. Carson, N. A. J ., Cusworth, 'D. 0., Dent, C. E., Field, 0. M. B., Neill, D. W., and Westall, R. G. : Homo- cystinuria: A new inborn error of metabolism as- sociated With mental deficiency. Arch. Dis. Child. 38: 425, 1963. 3. Gerritsen, T., and Waisman, H. A.: Homocystin- uria: An error in the metabolism of methionine. Pediatrics, 33:413, 1964. 4. Komrower, G. M., and Wilson, V. K.: Homocystin- uria. Proc. Roy. Soc. Med. 56:26, 1963. 5. McDonald, L., Bray, 0., Field, 0., Love, F., and Davies, B.: Homocystinuria, thrombosis, and the blood platelets. Lancet i: 745, 1964. 6. Selim, A. S. M., and Greenberg, D. M.: An enzyme that synthesizes cystathionine and deaminates Lserine. J. Biol. Chem. 231;: 1474, 1959. 7. Matsuo, Y., and Greenberg, D. M.: A crystalline enzyme that cleaves homoserine and cystathio- nine. J. Biol. Chem. 234: 515, 1960. 8. Gaull, G. E., Cusworth, D. C., Brenton, D., and Dent, C. E.: The biochemical defect in homo- cystinuria. J. Pediat. 65: 1049, 1964. 9. Mudd, S. H., Finkelstein, J. D., Irreverre, F., and Laster, L.: Homocystinuria: An enzymatic de- fect. Science 143: 1443, 1964. 10. Tallan, H. H., Moore, S., and Stein, W. H. : L-cysta- thionine in human brain. J. Biol. Chem. 230: 707 1958. 11. Hope, D. B.: Cystathionine accumulation in the brains of pyridoxinedeficient rats. J. Neuro- chem, 11: 327, 1964. 12. Gerritsen, T., and Waisman, H. A.: Homocystin- uria: Absence of cystathionine in the brain. Sci- ence 145 : 588, 1964. 13. See technical section page 71. 14. Schimke, R. N., McKusick, V. A., Huang, T. and Pollack, A. D.: Homocystinuria: studies of 20 families with 38 afiected members. J.A.M.A. 193: 711, 1965. 15. Brenton, D. P., Cusworth, D. C. and Gaull, G. 14].: Homocystinuria: metabolic studies on 3 patients. J. Pediat. 67 : 58, 1965. 16. See technical section page 100. 17. Finkelstein, J. D., Mudd, S. H., Irreverre, F., and Laster, L.: Homocystinuria due to cystathionine synthetase deficiency: The mode of inheritance. Science 146: 785, 1964. 18. Brenton, D. P., Cusworth, D. C. and Gaull, G. E.: Homocystinuria: biochemical studies of tissues including a comparison with cystathioninuria. Pediatrics 35: 50, 1965. Cystinuria CyStinuria reflects several amino acid dis- orders in which there is a defect in renal and enteric reabsorption of lysine, arginine, and ornithine, together with an abnormality of cys- tine-cysteine interrelationships.” The condi- tion appears to be clinically benign except for the occasional formation of renal calculi. Inheritance is commonly as a recessive with the typical amino aciduria only in the homozy— gote or as an incomplete recessive where the heterozygote excretes cystine and lysine in ex- cess. Not infrequently found in institutions for the retarded it is also associated with am- monia intoxication.7 There is also a report in a family2 showing the typical amino aci— duria, together with a form of osteogenesis im- perfecta. So far as is lmown, retardation is not associated with hereditary pancreatitis,3 a condition demonstrating a similar amino aciduria. Further investigation may establish an etiological relation between the amino aciduria in these cases and the cerebral defect. In the meantime, the metabolic disorder should be con- sidered in any comprehensive workup. The nitroprusside—cyanide test 4 is a suitable screen- ing procedure, and a definitive diagnosis can be obtained by paper 5' 6 or column chroma- tography and by polarog'raphy.8 ‘ References 1. Fox, M., Thier, S., Rosenberg, L., Kiser, W., and Segal, 8.: Evidence against a single renal transport defect in cystinuria. New Eng. J. Med. 270:556, 1964. 2. Berry, H. K.: Cystinuria in mentally retarded sib- lings with atypical osteogenesis imperfecta. A.M.A. J. Dis. Child. 97:196, 1959. 3. Gross, J. B., Gambill, E. E., and Ulrich, J. A.: Heredi— tary pancreatitis. Am. J. Med. 33:358, 1962. . See technical section page 100. . See technical section page 71. . Rosenberg, L. E., Durant, J. L. and Holland, J. N.: Intestinal absorption and renal extraction of cystine. New Eng. J. Med. 273 :1239, 1965. 7. Perheentupa, J. and Visakorpi, J. K.: Protein in- tolerance with deficient transport of basic amino acids. Lancet ii: 813, 1965. 8. O’Brien, D., and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro biochemical techniques. Hoeber, New York, 3d ed. 1964, p. 106. 0301*— Methionine malabsorption and mental retardation A single recent report 1 describes a syn— drome of convulsions, episodes of hyperventila— tion and intermittent diarrhea in a mentally retarded two and a quarter year old child with fair hair and blue eyes. A curious smell pervaded the infant Which was found to be due to a-hydroxybutyric acid as in the Cast-House Syndrome.2 On methio- nine loading, there was an increase in faecal a-hydroxybutyric acid and on restriction of methionine, the hydroxy acid not only decreased in the urine and faeces but the EEG reverted to normal. . Animal experiments have suggested that a—hydroxybutyric acid is formed from methio- nine in the large bowel by bacterial action. The biochemical defect would appear to be a specific transport defect across the 'bowel epithelium. There is no evidence, however, as to whether there is a similar defect in the renal tubule or at present any explanation of the role of this transport defect in engendering intellec- tual impairment. The syndrome would appear to be identified clinically. From the laboratory aspect, urinary a-hydroxybutyric acid can be identified chroma- tographically.1 References 1. Hooft, 0., Timmermans. J., Snoeck, J., Antener, I., Oyaert, W. and Van den Hende, Ch.: Methionine malabsorption in a mentally defective child. La/ncet ii : 20, 1964. 2. Smith, A. J. and Strang, L. B.: An inborn error of metabolism with the urinary excretion of a-hydroxy-butyric acid and phenylpyruvic acid. Arch. Dis. Child. 3332109, 1958. In tloe nmz'ne needy of the urea mint/yen} cycle Two forms of argininosuccinic acid cleav- age enzyme deficiency Clinical and laboratory findings Two types of the argininosuccinic aciduria have been described : The first of the reported cases 1 was born normally to unrelated parents and except for some backwardness of speech remained so until the age of 32 months, at Which time she had a series of brief generalized convulsions once or twice a day for a week. Nine months later she was readmitted to a hospital after a series of increasingly severe convulsive episodes with loss of consciousness and symmetrical general- ized clonic movements lasting up to 20 minutes. Physical examination was unremarkable save 7 for postictal equivocal Babinski responses and a left parasternal grade 3 systolic murmur. However, on recovery it was found that she was significantly retarded and that she had a marked ataxia of her upper limbs. This slowly improved but she remained retarded with an IQ. of 32. The EKG showed evidence of left ventricular hypertrophy and the EEG showed a dysrhythmia predominantly on the right, but without typical epileptic features. In the same family were two normal chil- dren, one who had died aged 41/2 months of bronchopneumonia and the second case1 a 6- year-old boy. The latter had been thought to be normal initially by an intelligent mother, but was finally accepted as being retarded at around the age of 21/2 years, with an IQ. of 50. Like his sister, he had a left parasternal mur- mur, but with an EKG suggesting right ven— tricular hypertrophy. He had no fits and no ataxia. The EEG showed definite evidence of epilepsy dominant in the right hemisphere. Both children had a rather similar facies; dry, light brown, friable hair that easily came out, a condition known as trichorrhexis nodosa 2 and in the first case the skin on the dorsum of the forearm was noticeably rough. In an extensive series of conventional labo- ratory investigations in both cases the only ab- normal finding was a modest elevation of serum alkaline phosphatase. On paper chromatog— raphy of the urine there was a distinct unknown spot in all specimens which was later identified as argininosuccinic acid in amounts of the order of 10 m mols/24 hrs. In the urine there was also some elevation of tyrosine and a variable reduction in the amounts of threonine, serine, glutamine, glycine, and histidine.4 The concen- tration of argininosuccinic acid was small but detectable in the plasma (0.1 ,umols/ ml) and the renal clearance very high. Glycine was also raised in the plasma to around 0.5 nmols/ml (normal 0.15—0.25 nmols/ml), and there was some elevation of tyrosine, with low values for Valine and the basic amino acids. The concen- tration of argininosuccinic acid in cerebrospinal fluid was about three times that in plasma.3 Ammonia levels were not estimated ‘but blood urea and daily urea excretion were normal, the 8 latter varying directly with the protein intake. Loading with 2 g/24 hrs of arginine did not increase the excretion of argininosuccinic acid, but the addition of 4 g/24 hrs of ornithine or citrulline led to an approximately 30 percent increase. The third case 5 was again born to noncon- sanguineous parents. He was apathetic and fed badly from birth until the seventh day of life when he became comatose and started to con- vulse, with vomiting and pedal edema. Some liver enlargement became apparent and there was intermittent vomiting until he was 4 weeks old. / This child became obviously considerably retarded. A low-protein diet reduced the de- gree of argininosuccinic aciduria considerably but produced skin lesions. A special diet low in arginine had a similar but much less pro- nounced effect. The whole program of dietary management was made more difficult by re- peated intercurrent infections, and there was no evidence of any clinical improvement. At 2 years of age, after repeated episodes of convulsions, the child could stand only with support, had no speech, and was severely re- tarded. On physical examination there was gross liver enlargement, and trichorrhexis nodosa. A wide series of laboratory investigations were unremarkable except for the finding of argininosuccinic acid in the urine, which again amounted to 5—10 m mols/24 hrs. Values for plasma and cerebrospinal fluid were the same as in the previous patients,4 and the high clearance of argininosuccinic acid was confirmed. It was shown that restricting protein intake would diminish urine argininosuccinic acid. Restric— tion of arginine intake had a like efl'ect. Am- monia levels in blood were not estimated. Two additional cases, both severely re- tarded, one of whom had ataxia, have been very briefly reported.6 In two other cases 7 one was a 13-year-old with an IQ. of 45 and conspicuous difficulties in muscular coordination. The other was an 11-year-old girl with an IQ. of 67. The two cases are of interest in that neither has de- fective hair. The last three cases to be re- ported 5 are of interest in that argininosuccinic acid was not elevated in the C.S.F. A second type of argininosuccinic aciduria has also been reported in association with moni- lethrix. This is a rare autosomal dominant tri- chopathy characterized by regularly alternative spindle-like swellings and structures along the hair. These findings may be associated with follicular hyperkeratosis, short brittle nails, cataracts, limitation of peripheral vision, dental lesions and mental retardation. There is Some evidence to suggest the epidermal isozyme of the arginino succinic acid cleavage enzyme may be defective. This in turn may affect the role of citrulline in the formation of keratin. Biochemistry Argininosuccinic acid is an intermediate in the urea synthesis cycle (figs. 3 and 4) . By anal- ogy with other biochemical abnormalities in this group it would be expected that the defect would be in argininosuccinase; such a defect has been shown in red blood cells though not in liver or other tissues.10 Certain features of the dis- ease are of special interest; the first is the char- acteristic observation that blood urea levels are normal, as were the total amounts of urea ex- creted. This suggests that either urea synthesis under normal load conditions is unimpaired by a partial enzymatic block or that an alternative pathway for urea synthesis, possibly via lysine and homocitrulline exists.“ 11 Ammonia levels in blood are now known to be high as in other disorders of this group.19 The second is the changes in the hair. Hair medulla and internal root sheath are the only natural proteins known to contain citrulline,12 and since both arginine and ornithine were low in the plasma 4 it might be that the same was true of citrulline to the ex- tent of interfering with normal hair growth. Finally, there is the finding in some cases of considerably higher levels of argininosuccinic acid in the brain than in the plasma. Since argininosuccinase is now known to be active in mammalian brain,13 the suggestion has been made that the particular isozyme in this tissue is absent but that it remains active in liver and kidney. This is, of course, possible but it seems equally possible that the transport of arginino- succinic acid from the cerebrospinal fluid to plasma does not match the remarkably effective glomerular filtration rise. Another suggestion has been made, namely that since the argininosuccinic acid cleavage re- action is reversible the block may be in the uti- lization of this substance in other pathways.“ Diagnostic and screening tests The finding of a typical peak for argini- nosuccinic acid on column chromatography 15 is the most satisfactory diagnostic test, although one- 1° or two- 1" dimensional paper chromatog- raphy can be equally satisfactory. There is a somewhat elaborate colorimetric test for argi- ninosuccinic acid using orthonitrobenzaldehyde and a modified Sakaguchi color reaction; 1‘ otherwise one-dimensional paper 16 chromatog- raphy is the only method that approximates to the simplicity required for screening. Genetics The transmission is autosomal in that both sexes are involved. In the families described, the disease has occurred in more than one sib- ling, except in two reports 8'19 and in a single generation. This suggests a recessive inherit- ance: some support comes from the finding of minimal argininosuccinic aciduria in the father and paternal uncle in the affected sibling 8 and in a wider range of relatives in another.19 Treatment No attempt has been made to treat an early case with a low-protein diet; however, by an— alogy with other syndromes in the group this might be expected to be successful. References 1. Allan, J. D., C‘usworth, D. 0., Dent, C. E., and Wilson, V. K.: A disease, probably hereditary, characterized by severe mental deficiency and a constant gross abnormality of amino acid metab- olism. Lancet 1‘: 182, 1958. 2. Crounse, R. G.: Trichorrhexis nodosa and amino acid metabolism. Arch. DermatoL 86: 391, 1962. 3. Westall, R. G.: Argininosucclnic aciduria: identi- fication and reactions of the abnormal metabolite in a newly described form of mental disease, with some preliminary metabolic studies. Biochem. J. 77: 135, 1960. p 4. Ousworth, D. 0., and Dent, C. 11).: Renal clearances of amino acids in normal adults and in patients 9 m Figure 5. THE KREBS-HENSELEIT CYCLE FOR UREA SYNTHESIS NH NH2 2 COOH Cl: C: /o I H o NH/"\ 2 TP CH— N— C—NH2 2 CH—NH NH2 CO+N*H§ A H ('5 2 UREA ' CIH *ACIH2 2 II H CH2 HN—CO-P—(OH) COOH CAEBAMYLPHOSPHATE CARBAMYL SHNm + ASPARTIC ACID COOH + CH2NH2 ARGININE NH COOH COOH CH2 cl NH —CH CHNH2 CH2 I CH CHNH2 (IZHENH CH2 2 , c'oorI COOH TH2 CIHQ C'OOH = CH ASPARHC ACID ORN'THHQE ? O I 2 V CH3NH coo C‘HNH I H CH2 I COOH I + CHNH2 CH2 CIH ARGININO 2 H o HNH l/ 2 SUCCINIC ACID C 2 COOH I COOH ASPARTIC ACID CITRULLINE (Corbomyl Ornithine) COOH I CH NH J ll 3 H Coo HC. C'=0 COOH COOH $“2 I CHOH FUMARIC ACID COOH CH2 4///4\ OXALOACETIC ACID CI020H H20 MALIC ACID NADH+H+ NAD+ 10. 11. 12. 13. 14. Figure 4. ARGININOSUCCINIC ACID CLEAVAGE ENZYME H (IZOOH l CHz—N—C—NHZ - ' a l l —>— —>—. CH2 CH2 ‘9 I I H2C C COOH H2C CH COOH CIH2 C|H2 \/ \N/ HCNH2 C0 , N H I . A.PIPERIDINE 2 COOH COOH CARBOXYLYIC ACID PIPECOUC ACID L-LYSINE aé.KETO-€-AMINO CAPROIC ACID COOH COOH COOH I I I CH2 H HCH H HCH CO /\ 2 I 2 I , H C CH CH CH2 CH2 2 2 _). I 2 —>— I ->- I —>— I I (lin _ ('IH2 CIH2 HC CHCOOH CH CH CH \ I 2 I 2 I 2 N CHO COOH COOH o A PIPERIDINE.2. aL-AMINO-ADIPIC. oC-KETO~ CARBOXYLIC ACID d'AM'NO-AD'P C ACID ADIPIC ACID I -SEM|ALDEHYDE COOH COOH COOH (:in (:20 H2H(::H CH2 + CH2 + CH2 COOH COOH COOH GLUTARIC ACID o(.KETO—GLUTARIC GLUTAMIC ACID 14 References 1. Colombo, J. P., Richterich, R., Donath, A., Spahr, A., and Rossi, E.: Congenital lysine intolerance with periodic ammonia intoxication. Lancet 1: 1014,1964. 2. Fox, M., Thier, S., Rosenberg, L., Kiser, W. and Segal, S.: Evidence against a single renal trans- port defect in cystinuria. New Eng. J. Med. 270: 556,1964. 8. Woody, N. 0.: Hyperlysinemia. Child. 1408: 543, 1964. 4. Ghadimi, H., Binnington, V. I., and Pecora, P.: Hyperlysinemia associated with mental retarda- tion. J. Pedlat, 6'5 : 1120, 1964 (abstr) 5. Rothstein, M. and Greenberg, D. M.: The metabo- ~ lism of DL-pipecoiic acid-2-G“ J. Biol. Chem. 235: 714, 1960. 6. Ryan, W. L., and Wells, 1. C.: Homocitrulline and homoarginine synthesis from lysine. Science, 141,: 1122, 1964. 7. Hunter, A., and Downs, O. E.: The inhibition of arginase by amino acids. J. Biol. Chem. 157: 427, 1945. 8. Cittadini, D., Pietropaolo, 0., de Cristofaro, D., and d’ijello-Caracciolo, M. : In vivo effect of L-lysine on rat liver arginase. Nature, 208:643, 1964. 9. Ghadimi, H., Kottmeier, P., Achs, R., Prabhu, R. and Jaffé, R.: Hereditary hyperlysinemia and lysine-induced crises. J. Pediat. 67:945, 1965 (abstr.) 10. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro- biochemical techniques. Hoeber. New York, 3d edit. 1962, p. 25. 11. See technical section page 71. A.M.A.J. Dis. Citrullinuria: Argininosuccinic acid syn- thetase deficiency Clinical and laboratory findings Only one case has been reported with this condition 1' 2 and that in a severely retarded boy. He developed normally for the first 9 months of life, after Which he began to have episodes of severe vomiting with alkalosis and coma. From then on he started to regress and at the age of 18 months following one such episode he ap- peared ill, dehydrated, and irritable, with a coarse tremor of his head and hands. Apart from hypotonia and a liver edge palpable 3 cm below the costal margin, there were no signifi- cant abnormal physical signs. The hemoglobin, white blood count and dif- ferential, and sedimentation rate, as well as serum levels for sodium, potassium, chloride, carbon dioxide, calcium, phosphorus, alkaline phosphatase, glucose, proteins, urea nitrogen, bilirubin, and SGOT were all normal. Urea and creatinine clearance was normal. There was a modest lowering of FBI to 3.8 pg/ 100 ml, and the cephalin flocculation test gave a 3+ result. The bone age was rather less than half the chronological age. The skull X—ray was normal, but the EEG showed a grossly abnormal pat- tern with low—voltage slow waves in the delta and theta range. The EKG was likewise ab- normal, with large, wide T waves and elevation of the ST segment in leads 3 and AVF. Amino acid analyses by column chromatog- raphy showed a marked rise in plasma citrul— line 1.4 to 2 pmols/ml over the normal of .01 to .03 umols/ml. Methionine levels were also high and those for arginine, cystine, isoleucine, leu— cine, tyrosine, and valine were low. In the urine there was a very large increment in citrulline, 2.7 to 14.3 m mols/24 hrs over the normal < 0.05 m mols/24 hrs. The excretion of alanine, aspar- tic acid, glycine, glutamic acid, and histidine was also elevated. Finally, citrulline was ele— vated in the cerebrospinal fluid, 0.3 pmols/ml. compared to the normal level of <0.005 meIS/ ml. In the postabsorptive state, blood ammonia levels rose remarkably to as high as 1000 pg- NH4(N) per 100 ml. Biochemistry Citrulline and aspartic acid condense in the presence of ATP, magnesium, and argininosuc- cinic acid synthetase to form argininosuccinic acid. Citrullinuria would therefore imply a deficiency in the condensing enzyme (figs. 3 and 6). Appropriately, a liver biopsy in this pa— tient showed a pronounced deficiency in argi- ninosuccinic acid synthetase with normal values for carbamyl phosphate synthetase, ornithine transcarbamylase, argininosuccinic acid cleav— age enzyme, and arginase. Plasma citrulline and urinary urea excretion increased in direct proportion to protein intake. Urea excretion was also within normal levels. These observa- tions suggest that the Krebs-Henseleit urea syn- 15 Figure 6. THE ACTION OF ARGININOSUCCINIC ACID SYNTHETASE rz C=O NH COOH | II I CH2—NH C—NH—CH I ARGININOSUCCINIC I I CH2 COOH ACID SYNTHETASE CH2—NH CH2 , l | l 1 CH2 + CIZHNH2 ¢ CH2 COOH ' I CH(NI-I,) CH2 $H2 COOH COOH CHNH2 | COOH CITRULLINE ASPARTIC ACID ARGININOSUCCINIC ACID thesis cycle is not essential to urea production and that some other pathway may become opera- tive, at least at high ammonia levels. It is pos- sible that a lysine, homocitrulline, homoargi- nine cycle may perform this function.3 Diagnostic and screwing tests High citrulline levels in urine and plasma must be confirmed by column chromatography.‘ The increased level in the urine is so striking, however, that it would certainly be noted on one-5 or two-6 dimensional paper chromatog- raphy. No very simple screening test is avail- able, and at the moment the simplest method large-scale survey is one-dimensional paper chromatography.” Genetics The genetics of this condition have not been clearly defined, but the mother and father were first cousins in the single reported case, suggest- ing a recessive inheritance. Treatment This child improved slightly on thyroid ad- ministration. A low-protein diet lowered plasma citrulline levels but neither program 16 was apparently able to avert severe mental re- tardation. From evidence in related condi- tions, however, it would seem that treatment of affected cases in the first months of life with a low-protein diet might be of significant pro- phylactic value. References 1. McMurray, W. 0., Mohyuddin, F., Rossiter, R. J., Rathbun, J. 0., Valentine, G. H., Koegler, S. J., and Zarfas, D. E.: Citrullinuria, a new aminoaciduria associated with mental retardation. Lancet 1:: 138, 1962. 2. McMurray, W. 0., Rathbun, J. 0., Mohyuddin, F., and Koegler,=S. J .: Citrullinuria. Pediatrics, 32: 347, 1963. 3. Ryan, W. L., and Wells, 1. 0.: Homocitrulline and homoarginine synthesis from lysine. Science, 144: 1122, 1964. 4. Spackman, D. H., Stein, W. H., and Moore, S.: Au- tomatic recording apparatus for use in the chroma- tography of amino acids. Analyt. Chem. 30: 1190, 1958. 5. See technical section page 71. 6. O’Brien, D., and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultra-micro biochemical tech- niques. Hoeber, New York. 3d ed., 1964, p. 25. Figure 7. THE ACTION OF CARBAMY L PHOSPHATE SYNTHETASE mg 0 O H II c02+ NH3 + 2ATP ——>— H2N— c— o —P—(OH2) Carbamyl phosphate synthetase deficiency Clinical and laboratory findings Only a single brief account presently exists on this condition.1 The patient was the sixth and only surviving. infant of unrelated parents. Four previous children had died from uncon- nected causes and the fifth had died at 5 months with symptoms similar to this case. At 10 days of age symptoms commenced with severe vomit- ing, hypotonia, lethargy, and dehydration, all of which responded to a protein-free intake and recurred with any return to conventional for- mulae. Blood ammonia was elevated (480 ,ug NH4 - N-/ 100 ml), as was the cerebrospinal fluid level (500 ,ug/ 100 ml). Blood urea nitrogen was only 4 mg/ 100 ml, and the total urea excretion only 51 mg/24 hrs. Plasma amino acids showed a significant elevation of glycine to 0.45—0.68 ,umols/ml (normal 0.15 to 0.25 mols/ml) . Fol- lowing oral administration of N15 glycine and N15 ammonium chloride, very little incorpora- tion of N15 into urea could be demonstrated in contrast to a sustained elevation of N15 am- monia. A liver biopsy showed normal levels for ornithine transcarbamylase and for arginino- succinic acid cleavage enzyme. There was a slightly low level for arginase, but carbamyl phosphate synthetase was only 25 percent of normal. Biochemistry As shown in figures 3 and 7, carbamyl phos- phate represents the first stage in the incorpora- CARBAMYL PHOSPHATE tion of ammonia into urea. There is an impair- ment of carbamyl aspatate formation which will in turn afl'ect citrulline formation and indirectly the whole operation of the urea cycle. Diagnostic and screening tests A preliminary group diagnosis may be made in any child who vomits in relation to pro- tein feeding by finding an excessive blood am- monia level,2 especially in the postabsorptive state. The definitive diagnosis must be carried out by the estimation of carbamyl phosphate synthetase in a liver biopsy.3 No simple routine screening test is available. Genetics The inheritance of this condition has not yet been defined. Treatment On a low-protein diet of 1 g/kg/24 hrs the child has been symptom-free and has developed normally. Precedents from other defects in this metabolic pathway suggest, however, that pro- longed ammonia intoxication leads to severe re- tardation of survivors in the absence of treat- ment. References 1. Freeman, J. M., Nicholson, J. F., Masland, W. 8., Rowland, L. P., and Carter, S. : Ammonia intoxica- tion due to a congenital defect in urea synthesis. J. Fed/tat. 65 : 1039, 1964 (abstr). 2. See technical section page 70. 3. Brown, G. W., and Cohen, P. P.: Comparative bio- chemistry of urea synthesis. J. Biol. Chem. 234: 1769,1959. 17 In tryptophane metaboliym Congenital tryptophanuria with dwarfism Clinical and laboratory findings There is a single well-documented reference to this condition.1 The patient was a 9-year-old girl who was referred to hospital because of mental and physical retardation. Her IQ, was 30 and her size that of a normally proportioned 5-year-old. Photosensitivity had been noticed since she was taken to the seaside at 6 months of age. At that time she developed telangiectasia on the conjunctivae and a reticular erythema on the face. Over the years the exposed skin sur- face of the face, neck, hands, and legs became dry, hardened, and hyperpigmented. Telangi- ectasia and hyperemia persisted on the conjunc- tivae. The only other abnormal physical find- ings were a wide-based, unsteady gait, intention tremor of the hands, and slow, high-pitched speech. The EEG showed a monotonous trace, with a lack of arousal pattern on stimulus; the chromosomal pattern was normal. An extensive series of. conventional labora- tory investigations, which included a liver biopsy, were normal except for a raised gamma globulin level of 2.7 g/ 100 ml. However, paper chromatography of the urine showed an ap— proximately threefold increase in tryptophane excretion, without any significant change in other amino acids. There was a modest increase in indole-3-acetic acid excretion, but no indican was detected. After an oral tryptophane load of 40 mg/kg, plasma levels in the patient were still approximately twice the fasting level after 4 hours, whereas the control caseshad returned to fasting levels. Measurement of kynurenine pathway and other tryptophane metabolites in . the urine showed low levels of kynurenine and NI-methylnicotinamide, a normal excretion of 5—hydroxy-indoleacetic acid, a marked incre- ment in indole—3-acetic acid excretion, and again no indican. Tryptophane loading of the consanguine- ous parents showed increased urinary excretion 18 of tryptophane in both cases, although kynurenine excretion was normal. Biochemistry This case presents many similarities with the Hartnup syndrome, notably the mental re- tardation, ataxia, and pellagra-like skin lesions. The degree of retardation appeared to be more severe, however, and the striking dwarfism has not lbeen seen in Hartnup disease. Also in con- trast with Hartnup disease from the laboratory point of view was the observation that the amino aciduria was confined to tryptophane and that there was no indicanuria. The amount of indole-3-acetic acid also appeared to be much less than in Hartnup disease. Finally, the fasting plasma tryptophane was elevated, whereas it is low in Hartnup disease. Despite the small number of control subjects and the well-recognized high variance of many of the methods used, the evidence is certainly in favor of a distinct metabolic block in the formation of kynurenine from tryptophane (fig. 8). The key observations were the sustained plasma tryptophane levels following an oral trypto- phane load, the increased urinary clearance of tryptophane, and the diminished urinary kynurenine excretion. In the conversion of tryptophane to kynurenine, formylkynurenine is first produced by the enzyme trytophane pyrrolase213, the subsequent hydrolysis of formylkynurenine to formic acid and kynurenine is by formylasef There is no evidence to date as to which of 'these two enzyme systems is affected in this syndrome, but it is of interest that tryptophane pyrrolase is apparently not found in the fetus,5 and is induced postnatally. As with the kynureninase defects, the impact of this enzyme block of cerebral development is not yet understood. Diagnostic and screening tests This condition may be distinguished from most other forms of mental deficiency because Figure 8. THE METABOLISM OF TRYPTOPHANE 5HYDROXVTRYPTAMINE [SEROTONIN) HO CHZCHZNH2 CH2COOH 5HYDROXYINDOLEACETIC INDOLE LACTIC no CHICHNHZ coon 9&1“; ann coon [bar—4 c coon ACID N INDOLEA ETIC ACID SHYDROXYTRYPTOPHANE on TRYPTOPHANE INDOLEPYRUVIC ACID coon N KYNURENIC A\CID <3 coon C-CH7CH—COOH _‘ __ :c|nz Nn-cocn3 an ann coon Nn2 ANTHRANILIC ACID N. ACETYL KYNURENINE KYNURENINE O-AMINOHIPPURIC ACID + NH 9" s. ANTHRANILIC ACID 9 I 2 C\ GLUCURONIDE C CHECH COOH CH .___>_ Nn2 /c coon N on on o 3HYDROXYKYNURENINE XANTHUREN'C u ACID C-NH coon co2 E NICOTINIC ACID of the clinical triad of retardation, ataxia, and conjunctival telangiectasia, with dryness and hyperpigmentation of the exposed skin. Diag- nostic confirmation and difl'erentiation from Hartnup disease can be achieved by a trypto- phane load test,6 following which the 4—hour serum tryptophane level,7 remains well above the fasting level, and there is both an increase in urinary tryptophane and indole—3-acetic acid,8 along with a decrease in other kynurenine NICOTINAMIDE 0 COOH N NH2—+ CH3 N ME 2 PYRIDONE 5. CARBOXAMIDE c'I-I3 NMETHVLNICOTINAMIDE pathway metabolites.6 The tryptophanuria is detectable in the older child by paper chromatographic screening methods} 9 but might be less easily distin- guished in the newborn because of the imma- turity of tryptophane pyrrolase. Genetics The parents of the single reported case were first cousins. Two other possible cases 19 were noted in the family in two boys who had died in the first months of life; in one of these cases the parents were likewise first cousins. The pattern is suggestive of an autosomal recessive inheritance. Treatment Nicotonic acid should be given for the skin lesions. There is no report as yet of any bene- ficial efl'ect of a low-protein or tryptophane-low diet. References 1. Tada, K., Ito, H., Wada, Y., and Arakawa, T.: 00n- genital tryptophanuria with dwarflsm. Tohoku J. Ewper. Med. 80: 11:8, 1963. 2. Hayaishi, 0., and Stanier, R. Y.: The bacterial oxi- dation of tryptophan. J. Bacteriol. 62: 691, 1951, 3. Tanaka, T., and Knox, W. E.: The nature and mechanism of the tryptophan pyrrolase (peroxi- dase-oxidase) reaction of pseudomonas and of rat liver. J. Biol. Chem. 231;: 1162, 1959. 4. Knox, W. E. and Mehler, A. H.: The conversion of tryptophan to kynurenine in liver. J. Biol. Chem. 187: 419, 431, 1950. 5. Nemeth, A. M., and Nachmias, V. T.: Ghanges in tryptophan peroxidase activity in developing liver. Science, 128: 1085, 1958. 6. O’Brien, D., and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro biochemical techniques. Hoeber, New York, 3d ed. 1964, page 301. 7. O’Brien, D. and Ibbott, F. A. : Laboratory manual of pediatric micro- and ultramicro biochemical tech- niques. Hoeber, New York, 3d ed. 1964, page 242. 8. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro biochemical tech- niques. Hoeber, New York, 3d ed. page 311. 1964. 9. See technical section page 90. Hartnup disease Clinical and laboratory findings Baron and others1 first described in 1956 a syndrome which they entitled, “Hereditary pellagra-like skin rash with temporary cerebel— lar ataxia, constant renal amino aciduria, and other bizarre biochemical features”. Since that time some 18 cases 2'11 have been described in the 20 literature in 10 separate kindreds, and much has been added to the understanding of this syn- drome which has come to be called “H” or “Hartnup” disease after the first aflected family described. The most uniformly present clinical finding is a pellagra-like rash on the ex- posed skin surfaces, which are characteristically dry, scaly, red, and hyperpigmented; in addi- tion about two—thirds of all cases show fluctuat- ing evidence of ataxia in the upper and lower limbs, nystagmus, intention tremors and pry- amidal tract involvement. There was a ten- dency for these symptoms to be aggravated by intercurrent respiratory or enteric infections on the one hand, and to gradually improve with age on the other. Mental retardation was noted in rather less than half the cases, although evanescent psychiatric disturbances were re— ported in many more. The characteristic biochemical finding was a marked and typical increase in urinary amino acids and indole derivatives. Plasma amino acid levels, however, were normal 1’ 13 in con- trast to the tryptophanuria reported by Tada 15. In the urine alanine, serine, asparagine, gluta- mine, valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophane, and histidine were pres— ent in greatly increased amounts. In contrast to the excretion of amino acids such as glycine and cystine, the basic amino acids, lysine, arginine and ornithine were relatively low 1. 4' 12' 13’ 14. In random urines there was an increase in indican and indoleacetic acid 1’ 1’. Biochemistry A series of observations have now estab- lished that this condition represents a selective rejection of amino acids by both the intestinal mucosa and the proximal renal tubular epi- thelium, with the indoluria a specific conse- quence of tryptophane malabsorption. The defect is thus analagous to cystinuria, where there is increased renal tubular and intestinal re- jection of cystine and the basic amino acids. The data in favor of this hypothesis may be summarized as follows: the evidence, it should be noted, is not presented in the same time sequence as it was obtained. 1. The pattern of enteric rejection of amino acids is similar to that by the renal tubule when the fecal amino acid pattern of a case was compared to the normal and to the urinary pattern.16 2. Intravenously administered DL-trypto- phan18 is metabolized normally with normal indoluria. 3. After the oral ingestion of L tryptophane there is a delayed and incomplete absorption from the bowel,14 with appreciable quantities reaching the colon. 4. On tryptophane loading there is an in- creased and sustained urinary excretion of indoleacetic and indolelactic acids, of indican, indoleacetylglutamine, indoleacetlyglucuronide, indolylacroyl glycine 14’ 17 and tryptamine.“ 22 5. The indoluria does not appear to be due to abnormal fecal fermentation and 2° can be abol- ished by neomycin.“ 18 Indoles formed in the bowel do not affect the rate of tryptophane ab— sorption,18 nor does neomycin administration. It was originally supposed that there might be a defect in tryptophane pyrrolase because of diminished kynurenine metabolites in the urine.14 Evidence on this point is conflicting, however. Recent data 19 suggest that indole can inhibit both tryptophane pyrrolase and kynu- renine formamidase, an action which would increase tryptophanuria and diminish urinary kynurenine metabolites. The relationship of these defects to the neurological findings has not been clearly defined, although the increased ab- sorption of tryptamine has been incriminated. Diagnostic and screening tests In all cases so far the diagnosis has been made either from clinical appearances or from routine checking for aminoaciduria in other members of an affected family. Screening of urine amino acids by one 23 or two-dimensional techniques 24 is satisfactory, although again more accurate figures are achieved by column chromatography.25 The diagnostic pattern of urinary indole excretion is best observed by paper chromatography after a tryptophane load-26, 27 772-952 0 - 66 - 4 Genetics Present evidence is that the disease repre- sents the homozygous state of an autosomal re- cessive inheritance. Treatment Oral nicotinic acid therapy 10 mg daily ap- pears to improve the skin condition and more erratically the ataxia. .It is of special interest that one report 13 claims that it occasions an in- creased enteric absorption of tryptophane. There is some evidence that acute exacerbations of ataxia may relate to increased indole absorp- tion, in which case both sterilization of the bowel 14 and alkalinization of the urine 21 would seem to be justified therapeutically. A dietary and therapeutic regimen along these lines which would control indoluria, would appear to be justified in any case diagnosed in early infancy. References 1. Baron, D. N., Dent, C. E., Harris, H., Hart, E. W., and Jepson, J. B.: Hereditary pellagra-like skin rash with temporary cerebellar ataxia, constant renal aminoaciduria, and other bizarre biochem- ical features. Lancet ii: 421, 1956. 2. Hickish, G. W.: Pellagra in an English child. Arch. Dis. Child. 30: 195, 1955. 3. Hersov, L. A.: A case of childhood pellagra with psychosis. J. Ment. Sci. 101:878, 1955. 4. Hersov, L. A., and Rodnight, R.: Hartnup disease in psychiatric practice: clinical and biochemical features of three cases. J. Neurosurg. c6 Psychiat. 23:40,1960. 5. Henderson, W.: A case of Hartnup disease. Arch. Dis. Child. 38: 114, 1958. 6. JOIJXil, J. H.: Olgophrenia phenylpyruriea en de Hartnupsiekte. Nederl. T. Geneesk. 101: 569, 1957. 7. Weyers, H., and Bickel, H.: Photodermatose mit aminoacidurie, indolaceturie, und cerebralen manifestationen (Hartnup Syndrom) Klin. Wohnschr. 36: 893, 1958. 8. Halvorsen, K., and Halvorsen, S.: Hartnup disease. Pediatrics 31 : 29, 1963. 9. Fois, A., and Lecchini, L.: Acute cerebellar ataxia associated with some features of the Hartnup syndrome. Helvetica, Paed. Acid. 19:42, 1964. 10. Srikantia, S. G., Venkatachalam, P. 8., and Reddy, V.: Clinical and biochemical features of a case of Hartnup disease. Brit. Med. J. i: 282, 1964. 11. Hooft, 0., de Laey, P., Timmermans, J. and Snoeck, 21 13. 14. 15. 16. 17. 18. 19. 20. 21. 22 31.: La Maladie de Hartnup. Acta paediat. belg, 16: 281, 1962. . Gusworth, D. 0., and Dent, C. 19.: Renal clearances of amino acids in normal adults and in patients with aminoaciduria. Biochem. J. 71;: 550, 1960. Evered, D. F.: Excretion of amino acids by the human. A quantitative study with ion-exchange chromatography. Biochem. J. 62:416, 1956. Milne, M. D., Crawford, M. A., Girao, C. B., and Loughridge, L. W.: The metabolic disorder in Hartnup disease. Quart. J. Med. 29:407, 1960. Tada, K., Ito, H., Wada, Y., and Arakawa, T.: Congenital tryptophanuria with dwarfism Tohoku J. Eaper. Med. 80: 118, 1963. Scriver, C. B.: “Hereditary aminoaciduria” in Progress in Medical Genetics. Grune and Strat- ton, New York. Vol. 2, Ch. 4, p. 141, 1962. Shaw, K. N. F., Redlich, D., Wright, S. W., and Jepson, J. B.: Dependence of urinary indole ex- cretion in Hartnup disease upon gut flora. Fed. Proc. 19: 194, 1960. de Laey, P., Hooft, C., Timmermans, J., and Snoeck, J .: Biochemical aspects of the Hartnup disease Part I. Results of intravenous and oral tryptophan loading tests in a case of Hartnup disease. Arm. Paediat. 202: 145, 1964. de Laey, P., Hooft, C., Timmermans, J., and Snoeck, J.: Biochemical aspects of the Hartnup disease Part 11. Some observations on rats about tryptophan metalbolism. Arm. Paediat. 202: 253, 1964. Asatoor, A. M., Oraske, J ., London, D. R., and Milne, M. D.: Indole production in Hartnup disease. Lancet 4;: 126, 1963. Milne, M. D., Crawford, M. A., Girao, C. B., and Loughridge, L.: The excretion of indolylacetic acid and related indoh'c acids in man and the rat. Clin. Sci. 19: 165, 1960. . Sjoerdsma, A., Oates, J. A., Zaltzman, P., and Udenfriend, 8.: Identification and assay of Figure 9. urinary tryptamine: application as an index of monoamine oxidase inhibition in man. J. Pharmacol. Em). Therap. 126: 217, 1959. 23. See technical section page 71. 24. O’Brien, D. and Ibbott, F. A.: Laboratory manual of paediatric micro and ultra-micro biochemical techniques, Hoeber, New York. 3d edition, 1962, p.25. 25. Spackman, D. H., Stein, W. M. and Moore, S: Au- tomatic recording apparatus for use in the cro- matography of amino acids. Analyt. Chem. 30: 1190, 1958. 26. Sprince, H., Parker, 0., Dawson, J. T., Jameson, D. and Dohan, F. 0.: Paper chromatography of urinary indoles extracted under alkaline and acid conditions. J. Chromatography 8: 457, 1962. 27. See technical section page 90. Indolylacroyl glycine excretion in a family with mental retardation Clinical and laboratory findings There is a brief report1 of a family in which indolylacroyl glycine was identified in the urine/of five mentally retarded siblings and their mother. The condition has also been ob- served in a second family.2 Oral supplementa- tion with 240 to 480 mg/kg/24 hrs of oral L- tryptophane or intravenous loads of L-trypto- phane up to 160 mg/ kg did not increase indoly- lacroyl glycine excreted, thus discounting a pro- posal that it is derived from tryptophane.3 In four of the five children the compound disap— peared from the urine following an oral neo- IN DOLYLACROYL GLYCINE 0 || CH=CH— c—NH—CH2—c— OH mycin therapy program and reappeared when this was discontinued. Indolylacroyl glycine would appear to be derived from the bowel, possibly as a result of a specific transmucosal transport defect such as exists in Hartnup syndrome.‘” 4 It is of interest that indolylacroyl glycinuria also occurs in this latter condition. Biochemistry The source and role in indolylacroyl glycine in human metabolism is not at present known. Diagnostic and screening tests Indolylacroyl glycine can be identified and approximately assayed by the paper chromato- graphic technique 1: 2 used by Mellman. A 24- hour volume of urine is collected and stored at 4° C. At the time of the assay, 3 g. of NaCl is added to 10 ml of the urine and the whole brought to pH 2 by drop by drop addition of 1N sulfuric acid. The acidified urine is then ex- tracted three times for 5 minutes with 30 ml of dry anaesthetic grade ethyl ether using a me— chanical shaker. The pooled ether extracts are evaporated down under vacuum to about 4 ml. This is placed in a smaller vial and evaporated down to approximately 200 ,ul; the Whole is then spotted onto 20 cm by 20 cm Whatman #1 paper and chromatographed for 16 hours in isopropanol: ammonia :water (20:1 :2) and for 3—5 hrs in 8 percent w/v NaCl in 1 percent glacial acetic acid in a. Shandon tank (see tech- nical section on two dimensional chromatog- raphy). After drying at 30°C in an air current, the papers are dipped in 25 percent con— centrated HCl v/v in acetone and the color developed over 30 minutes. Indolylacroyl glycine can be identified by the orange color at Rf 30 in the first dimension and Rf 24 in the second. The spot is cut out, out into small frag- ments and the color eluted with a mixture of 4 ml methanol and 2 ml concentrated HCl. After centrifugation, the colored supernatant is read in a spectrophotometer at 509 mg. The results have to expressed in optical density units in comparison with a series of normals as de- termined in individual calibrations. This is be- cause pure indolylacroyl glycine is not avail- able and no valid figures for the extinction co- efficient exist. Genetics The genetics of this condition are not yet established. Treatment No treatment is presently available. References 1. Mellman, W. J ., Barness, L. A., Tedesco, ’1“. A., and Besselman, D.: Indolylacroyl glycine excretion in a family with mental retardation. Olin. Chim. Acta. 8: 843, 1963. 2. Mellman, W. J. : Personal communication. 3. Milne, M. D., Crawford, M. A., Girao, C. B., and Loughridge, L: The metabolic disorder in Hart- nup disease. Quart. J. Med. 29: 407, 1960. 4. Jepson, J. B. and Spiro, M. J.: in Stanbury, J. B., Fredrickson, D. S. and Wyngaarden, J. B. “The Metabolic Basis of Inherited Disease”. McGraw-Hill, New York. 1960, p. 1338. Hyperserotoninemia Clinical and laboratory findings There is a single report of this condition 1 in a 49-year-old woman who had a long history of intermittent flushing, ataxia, and seizures. This patient was not retarded, but had there been an earlier onset of symptoms this would very possibly have occurred. The case is recorded here as another potential source of re- tardation in childhood. Since early childhood this patient had been subject to severe rage re- actions with recurrent episodes of flushing of the face, neck, and arms. At the age of 32 there was a rapid onset of hand tremors, slurred speech, and stiffness and ataxia of the lower limbs. Six years later there was a slow and im- perfect remission of these symptoms. During the same period there was an onset of episodes of flushing of the face, staring, and unrespon- siveness. On a number of occasions the patient exhibited an intermittent elevation of blood pressure, but no confirmatory evidence of 23 Figure 10. THE ABNORMALITY OF TRYPTOPHAN METABOLISM IN HYPERSEROTONINEMIA / CH2CHNH2COOH HO CH2 CHNH2COOH HO CH2CI-12NH2 | I —» I I \ N N \ N TRYPTOPHAN Ho/ \ 5-HYDROXYINDOLEACETIC ACID CHZCOOH N phaeochromocytoma could be found. An extensive series of routine laboratory and radiological investigations were unremark- able; in addition, however blood serotonin levels were detected which were greatly in ex- cess of normal 4 to 30 ,ug/ml, as against a normal range of 0.2 to 0.4 Mg/ml. The excretion of 5-hydroxy-3-indoleacetic acid was normal. It was not increased after an iv injection of 5-hydroxy-tryptophane (5HT), although it was increased after the i.v. administration of serotonin or 5-hydr0xy- indoleacetic acid (5HIAA) itself. Intra— muscular administration of reserpine 1.0mg/ 24 hrs for 4 days produced a marked accentua- tion of symptoms, but neither a rise in serum serotonin or in 5-hydroxy-indoleacetic acid excretion. Biochemistry The normal pathway for the metabolism of tryptophane to 5HIAA is shown in figure 10. It is clear in this case from the results of i.v. loads of 5HT, serotonin, and 5HIAA that the conversion of serotonin to 5HIAA was normal, as was the renal excretion of the latter end product. The failure of DL hydroxy-trypto- phane to lead to more than an abnormally small increase in urinary 5HIAA suggests a block in 24 5-HYDROXYTRYPTOPHAN POSSIBLE SITE OF BLOCK 5-HYDROXYTRYPTAMINE ISEROTONIN) the normal synthetic pathway of serotonin. The authors postulate a functional deficiency of brain serotonin in the presence of an elevated serum serotonin. As an explanation of the seizures they cite the autonomic mediation of serotonin 2 and the similarity of this patient’s symptoms to the diencephalic dysautonomic seizures described by Penfield.8 Diagnostic and screening tests Diagnosis of this condition rests on the de- tection of high plasma serotonin levels,4 a pre- carious and difficult technique. Genetics The inheritance of this condition is not known. Treatment No specific treatment was attempted in the single reported case. References 1. :Southren, A. L., Warner, R.R.P., Christoff, N. I., and Weiner, H. E.: An unusual neurologic syndrome associated with hyperserotonemia. New Eng. J. Med. 260: 1265, 1959. 2. Brodie, B. B., and Shore, P. A.: Concept for a role of serotonin and norepinephrine as chemical media- tors in the brain. Amt. New York Acad. Sci. 66‘: 631, 1957. 3. Penfield, W.: Diencephalic autonomic epilepsy. Arch. N aural. and Psychiat, '22: 358, 1929. 4. Contractor, S. F.: The estimation of 5-hydroxy-tryp- tamine in human blood. Biochem. Pharmacol. 13: 1351, 1964. Pyridoxine deficiency, pyridoxine depend- ency, and abnormalities of kynureninase Clinical and laboratory findings and biochemistry The conversion of 3-hydroxy kynurenine to 3-hydroxyanthranilic acid in the kynurenine pathway of tryptophane breakdown (fig. 11) does not in itself'appear to play any integral part in cerebral metabolism, as an isolated de- fect in this reaction may occur in otherwise normal persons.1 The catalysing enzyme ky- nureninase requires pyridoxal-5-phosphate as a coenzyme and the reaction is extremely sensi- tive to vitamin B6 deficiency. However, there are a number of other syndromes, sometimes referred to as pyridoxine dependency states, Where there is a defect in kynureninase in asso- ciation with convulsions and/ or mental retarda- tion. Pyridoxine deficiency Pyridoxine deficiency was first produced artificially in two retarded infants.2 One de- veloped a marked anemia and the other more characteristically developed severe convulsions. Both infants promptly responded to intrave- nous pyridoxine and were ultimately stabilized on 150 Mg per day by mouth. Shortly thereafter a group of infants given a proprietary liquid milk formula were noticed to become hyperirri- table between 6 weeks and 4 months of age?—7 Generalized seizures followed, however, which could be successfully treated by oral pyridoxine or by a formula containing normal ampunts of this vitamin.3 A similar story was given by a mother whose breast milk was shown to contain unusually small amounts of pyridoxine.8 These infants all had normal interictal EEGs, showed no familial incidence of convulsions, and de- veloped unexceptionally on a proper diet. Xan- thurenic aciduria was apparent on tryptophane loading; 8 the correction of this abnormality was noticeable in requiring greater amounts of pyridoxine for its correction than did the con- vulsions. Although pyridoxal phosphate is a coenzyme in a wide range of reactions,9 the key pyridoxal-dependent reaction in causing con- vulsions is thought to ‘be that of the formation of gamma-aminobutyric acid from glutamic acid 'by glutamic acid decarboxylase.10 Pyridoxine dependency Although not strictly an abnormality of kynurenine metabolism, the syndrome is in- cluded in this section because of similarities with both pyridoxine deficiency and the abnor- malities of kynureninase. Characteristically, it affects infants in the neonatal period with the development of hyperirritability, hyperacusis, and generalized convulsions}?! 11' 12 In some in- stances the seizures have certain additional characteristics such as blinking 7 and a startled expression. There is a definite familial bias in these cases; the interictal EEG is abnormal. The convulsive tendency is completely resolved by 2—10 mg of pyridoxine daily by mouth, but withdrawal even after long periods may lead to a resumption of seizures.” 13. These cases show no abnormality of kynureninase activity or in- deed of any other readily accessible pyridoxine dependent enzyme. In this syndrome the prompt response to pyridoxine makes it clear that there is no delay in the conversion of the coenzyme to pyridoxal phosphate. Again the key enzyme involved is thought to be glutamic acid decarboxylase. The availability of gamma amino butyric acid is im— portant in cerebral oxidative metabolism, and some indirect support for the involvement of this system is the demonstration that cerebral oxygen consumption is diminished in pyridox- ine-dependent seizures as it is in insulin hypo- glycemia seizures but not in those due to analeptics or electroshock.13 Scriver has in- voked Bonner’s work 14 on neurospora to postu- late that the abnormality is one of the coenzyme binding sites of the apoenzyme glutamic acid decarboxylase. 25 Figure 1 1. THE KYNURENINE PATHWAY OF TRYPTOPHANE METABOLISM N CH2CHNH2COOH TRYPTOPHANE COOH N KYNURENIC ACID\ i: COOH ‘ NH2 .‘___ N/ ALANINE / C— —CH-CH- COOH NH- COCH3 2 N-ACETYL- MCHNH COOH —>- NH ANTHRANILIC ACID H2 KYNURENINE KYNURENINE OH I O-AMINOHIPPURIC ACID 9 ”“2 C\ a. ANTHRANILIC ACID c CHECH coo“ C” GLUC RE U NIDE NH2 —_>. [c coo“ coon H o” N o co 3 HYDROXYKYNURENINE XANTHURENIC ACID n 2* c-NH2 NH2 0 0 ND 3HYDROXY C” . ANTHRANILIC ACID C00” CH3 NH2 —>-\ N-ME-2-PYRIDONE 5-CARBOXAMIDE NICOTINAMIDE NICOTINIC ACID N CHMESTHYI.NICOTINAMIDE KYNURENINE PATHWAY Kynureninase deficiency In the third section of this related group of syndromes are those associated with an abnormality of kynureninase. In the first category were two mentally defective young women 1" with no history of convulsions and no abnormal physical findings. There was a kynureninase defect as judged by hyper-Kan- 26 thurenic aciduria and an abnormally high hydroxykynurenine: hydroxyanthranilic a c i d ' ratio of 6.0 in the urine in both cases (normal < 2.0) . These abnormalities reverted to normal either after 100 mg of pyridoxine intravenously or 100 mg pyridoxine orally for several days. The prompt response to pyridoxine intrave- nously and the normal excretion of 4—pyridoxic acid would seem to rule out relative Be defi- ciencies due to a defect of pyridoxal kinase or excessive conversion of pyridoxine to 4-pyri- doxic acid, but does not exclude excessive renal loss of pyridoxal or pyridoxamine.9 Also in this section are some of the infants with the syndrome of infantile spasms“! 17’ 13 These patients develop massive myoclonic jerks between 2 and 12 months of age and usually become severely retarded. Changes in kynu- renine metabolism are the same as reported above and these may again be restored by doses of pyridoxine of the order of 1 mg/kg i.v. However, although the biochemical abnormal- ity may be corrected in this manner, only about one—third of the cases show any response clin- ically to oral pyridoxine in doses from 4—10 mg/kg/24 hrs. Both these two groups again probably represent defects in the coenzyme binding site of kynureninase; the nature of cerebral disorder remains undiscovered. Finally, there is a recent report 1" of an 11- year-‘old-girl who suffered from severe stom- atitis and reddening of the buttocks in infancy. She “failed to thrive,” was anemic, and showed a number of bony abnormalities, including de- fects of the lower dorsal and first lumbar verte- brae, the ribs, short and long bones, and delayed ossification of the right femoral epiphyses. Vitamin B supplements were given but she made slow progress and was considered to be retarded at the age of 8 years, with an IQ. of 78. On tryptophane loading she was shown to have a virtual absence of kynureninase activity with a massive xanthurenicaciduria hydroxy- kynureninuria and no hydroxyanthranilic acid in the prine. N. methyl nicotinamide and its pyridone derivative were not measured, but the child improved in growth and intellectual func- tion on 10 mg nicotinic acid daily. This defect might be interpreted'as one of the substrate binding sites on the apoenzyme kynureninase. Diagnostic and screening tests The diagnosis of pyridoxine dependency is made on the clinical history and as a result of the dramatic response of the convulsions or the EEG to pyridoxine administration. In these cases tryptop‘hane metabolism is normal. In the laboratory diagnosis of kynureninase defects the tryptophane loading test 15’ 2° has proved a cumbersome but effective test. It is based on the fact that when the conversion of 3—hydroxykynurenine to 3-hydroxyanthranilic acid is impaired there is a considerable increase in urinary kynurenine, hydroxykynurenine, and xanthurenic acid after a tryptophane load. Xanthurenic aciduria is easy to detect by the alkaline ferric salt method but it has proved a rather unreliable pointer in the syndrome of infantile spasms,“ 18 possibly because of its technical unreliability.21 Chromatographic as- say of the hydroxykynurenine: hydroxyanthra- nilic acid ratio 15' 18’ 2° is probably the most effective discriminant of this condition. Genetics There is a definite familial incidence in the syndrome of pyridoxine dependency, but there have been insufficient cases to determine the exact mode of inheritance. There is at present no indication of the inheritance of the abnor- malities of kynureninase when they associate with convulsions and/or mental retardation. Treatment Pyridoxine deficiency states will respond promptly to oral and parenteral pyridoxine ad— ministration and can be subsequently main— tained in good health on an oral intake of 0.5 mg/24 hrs. Pyridoxine dependency convul- sions respond with equal promptness, but up to 50 mg of pyridoxine a day by mouth may be required for their control. Hydroxykynuren- inuria is unresponsive to large doses of pyridox- ine, although clinical improvement in growth and intellectual function was noted with 10 mg nicotinic acid daily. On the other hand, about a third of the cases with myoclonic epilepsy will improve either with steroids or with 4—10 mg/ kg/24 hrs of pyridoxine orally. References 1. Knapp, A. : U‘ber eine neue, .hereditare, von vitamin- Be abhangige storung im tryptophan-stotfwwchel. Olin. Ohim. Acta. 5: 6, 1960. 2. Snyderman, S. E., Holt, L. E., Carretero, R., and 27 Jacobs, K.: Pyridoxine deficiency in the human infant. A.M.A. J. Olin. Nntr. 1:200, 1953. 3. Melony, C. J ., land Parmelee, A. H.: Convulsions in young infants as a result of pyridoxine (vitamin Bo) deficiency. J.A.M.A. 151,: 405, 1954. 4. Coursin, D. B.: Convulsive seizures in infants with pyridoxine—deficient diet. J.A.M.A. 154: 406, 1954. 5. Coursin, D. B.: Vitamin Bu deficiency in infants: a follow-up study. A.M.A.J. Dis. Ohild. 90:344, 1955. 6. Coursin, D. B. : Effects of vitamin B9 on the central nervous system activity in childhood. Amer. J. Olin. N air. 4: 354, 1956. 7. May 0. D.: Vitamin Be in human nutrition: a critique and an object lesson. Pediatrics 11,: 269, 1954. 8. Bessey, O. A., Adam, D. J. D., and Hansen, A. B.: Intake of vitamin B0 and infantile convulsions: a first approximation of requirements of pyridoxine in infants. Pediatrics 20: 33, 1957. 9. Coursin, D. B.: Bresent status of vitamin Ba me- tabolism. Amer. J. Olin. Nutr. 9:304, 1961 10. Scriver, C. R. : Vitamin Be dependency and infantile convulsions. Pediatrics 26: 62, 1960. 11. Hunt, A. D., Stokes, J., McCrory, W. W., and Stroud, H. H.: Pyridoxine dependency report of a case of intractable convulsions in an infant con— trolled by pyridoxine. Pediatrics 13: 140, 1954. 12. Garty, R., Yonis, Z., Braham, J., and Steinitz, K.: Pyridoxine—dependent convulsions in an infant. Arch. Dis. Child. 37: 21, 1962. 13. Sokolofl, L., Lassen, N. A., McKhann, G. M., Tower, D. B., and Albers, W.: Effects of pyridoxine withdrawal on cerebral circulation and metab- olism in a pyridoxine-dependent child. Nature 183: 751, 1959. 14. Bonner, D. M., ‘Suyama, Y., Demoss, J. A.: Genetic fine structure and enzyme formation. Fed. Proc. 19: 926, 1960. 15. O’Brien, D., and Jensen, 0. B.: Pyridoxine depend- ency in two mentally retarded subjects. Olin. Sci. 24:179, 1963. 16. 'Oochrane, W. A. : The syndrome of infantile spasms and progressive mental deterioration related to amino acid and pyridoxine metabolism. Proc. IX Intern. Cong. Peds. Montreal p. 115, 1959. 17. Bower, B. D.: The tryptophan load test in the syndrome of infantile spasms with oligophrenia. Proc. Roy. Soc. Med. 54: 540, 1961. 18. French, J. H., Grueter, R. B., Druckman, R., and O’Brien, D.: Pyridoxine and infantile myoclonic seizures. Neurology 15: 101, 1965. 19. Komrower, G., Wilson, V., Clamp, J .1 R. and Westall, R. R.: Hydroxykynureninuria. Arch. Dis. Ohild. 39:250. 1964. 20. O’Brien, D., and I‘bbott, F. A.: Laboratory Manual of Pediatric Micro- and Ultramicro Biochemical Techniques. Hoeber, New York, 3d ed. 1962, p. 301. 21. Satoh, K. and Price, J. M. : Fluorometric determina- tion of kynurenic acid and xanthurenic acid in human urine. J. Biol. Ohem. 230: 781, 1958. In proline metabolism Proline oxidasc deficiency Clinical and laboratory findings There have recently been published two ac- counts of kindreds 1'” in whom there was fa— milial nephritis, deafness, renal hypoplasia, photogenic epilepsy, abnormal electroencepha- lographic patterns, and prolinemia with excess proline, hydroxyproline, and glycine in the urine. The index case presented as a 21/2-year- old boy who was first admitted to a hospital 28 with a urinary tract infection and febrile con- vulsions. The right kidney was hypoplastic, as was the pelvis on the left. There was a marked impairment of hearing but even taking this into consideration there was intellectual and speech retardation. His subsequent course was characterized by frequent “staring spells” and generalized convulsions, some of which were photogenic. The child died at the age of 51/2 from an episode of acute renal failure. At autopsy there appeared to be a difl'use loss of cortical neurones, as well as pyelonephritis. The distinctive laboratory findings in the 9 - 99 ' O ZSG'ZLL 62 Figure 12. THE PROLINE OXIDASE 02 i“??? ;__> CH2CH-COOH \N/ F K NAD NADH + H H PROLINE O=CH l HOOC + GLYOXYLIC PYRUVIC ACID ACID 0- BREAKDOWN OF PROLINE A'P-S-C DEHYDROGENASE H20 CHTCH2 0=CH CH—COOH I NH2 GLUTAMIC ACID AI-PYRROHNE 7. SEMIALDEHYDE 5—CARBOXYLIC ACID 0 NAD ADH+H+ THECH2 HOOC CH—COOH {TRANSAMINATIONI / NH? GLUTAMIC ACID ASPARTIC ACID OXALOACETIC ACID KETOGLUTARIC ACID index case and three siblings related to the pro- linemia with levels of 0.5 to 1.7 ,umols/ml (normal 0.1 to 0.3 mols/ml). Column chro- matography of 24 hr urine also showed an in- crease of both proline, up to 7.2 mmols/ 1.7 3m2 and hydroxyproline up to 0.3 mmols/1.73m2. Biochemistry The actual pathway of proline breakdown is the exact reverse of the synthesis (fig. 12), al— though the enzyme systems are distinct. Pro- line is first broken down to A’-pyrroline—5- carboxylic acid by proline oxidase and then to glut-amic acid by A’-pyrroline-5-carboxylic acid dehydrogenase. In the index case A’-pyrroline- 5-carboxylic could not be detected in the urine by paper chromatography, which indicated a proline oxidase defect; the actual enzyme de- ficiency being subsequently proven.2 The mechanism by which hyperprolinemia might impair cerebral development is not clear (fig. 13). Scriver 3 has postulated a scheme for the interrelationship of proline, glutamic acid, and the arginine: ornithine: citrulline cycle in the brain. It is possible that excess proline may disturb the control mechanism of this sys— tem and thereby the utilization of glutamic acid ' as an energy source. Prolinemia, as well as prolinuria, hydroxy— prolinuria, and glycinuria were detected in three sisters of the index case. Studies of renal clearance data 4 show that the prolinuria was a sequel to the hyperprolinemia and that the hydroxyprolinuria and glycinuria were the re- Figure 13. GLUTAMINE sult of increased tubular rejection of these amino acids. Infusion studies 5 support the con- cept that there is a common pathway for the tubular transport of the two amino acids. The glycine transport path may either be an alter- native for the amino acids or have some point in common. Diagnostic and screening tests The diagnosis depends on the finding of a high plasma p r o l in e by colorimetric ‘3' 7 paper 8' 9 or column chromatography methods. The same chromatographic techniques can be used for the detection of excessive proline, hy- droxyproline, and glycine in the urine. Screen- ing in the newborn would be diflicult because of the high clearance of proline and hydroxypro- line in normal subjects at this age. Genetics An extensive investigation of this and other families suggest that the familial nephropathy, the deafness, and the photic epilepsy are in- herited as dominants. The prolinemia appears to have been inherited recessively. The index case was the only one to demonstrate a full com- plement of the dominantly and‘recessively in- herited traits and to be retarded; it remains to be shown Whether prolinemia alone is associated with retardation. Treatment N 0 treatment for hyperprolinemia was attempted. INTERRELATIONSHIPS IN PROLINE METABOLISM ORN'ITHINE l GLUTAMIC ACID —# A'PYIIOLINE -5-CAIIOXYI.IC ACID V—‘PROLINE YAMINOIUTYRIC ACID _’- 30 SUCCINIC ACID —-—_>- KREIS CYCLE References 1. Schafer, I. A., Scriver, O. R., and Efren, M. L.: Familial hyperprolinemi‘a, cerebral dysfunction, and renal anomalies occurring in a family with hereditary nephropathy and deafness. New Eng. J. Med. 267: 51, 1962. 2. Berlew, 8., and Efren, M. L. : A new cause of hyper- prolinemia associated with the excretion of A- pyrroline-5-carboxylic acid. Sec. Ped. Res. p. 43, 1964 (abstn) 3. Scriver, C. R.: “Hereditary aminoaciduria” in Prog- ress in Medical Genetics, Grune and Stratton, New York. vol 2, ch 4, p. 128. 4. Scriver, O. R., Schafer, I. A., and Efren, M. L.: New renal tubular amino acid transport system and a new hereditary disorder of amino acid metabolism. Nature 192: 672, 1961. 5. Scriver, C. R., Schafer, I. A., and Efren, M. L.: Evidence for a renal tubular amino acid transport system common to glycine, L-proline, and hy- droxy-L-proline. J. Olin. Invest. 40: 1080, 1961. 6. Summer, G. K. and Hawes, J. A.: Determination of free preline in serum. Proc. Soc. Ewper. Biol. and Med. 112: 402, 1963. 7. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicrobiochemical techniques. Hoeber, New York. 3d edition, 1962, page 228. . See technical section page 71. . O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro— and ultramicrobiochemical techniques. Hoeber, New York, 3d edition, 1962, page 25. 10. Efren, M. L.: Familial hyperprolinemia. New Eng. J. Med. 272: 1243, 1965. C000 Hyperprolinemia due to A’ pyrroline—s— carboxylic acid dehydrogenase deficiency Clinical and laboratory findings There is a single brief abstract 1 of a patient with convulsions and coma in association with marked hyperprolinemila. This patient could be distinguished from those with proline oxidase deficiency by the presence (fig. 14) of A’ pyrro- line-5-carboxylic acid in the urine, as shown by paper chrmnartography and high voltage chro- matography. This finding implies a defect of A’ pyrroline-5-carboxylic acid dehydrogenase, which is necessary for the further breakdown of proline degradation to glutanfic acid. Biochemistry The precise mechanism whereby a disturb- anCe of proline metabolism may afl’ect cerebral function is not clear. However, in the case of familial hyperprolinemia due to proline oxidase deficiency,2 it is possible that hyperprolinemia in cerebral tissue may interfere with the metab- olism of glutamic acid (fig. 13) . Diagnostic and screening tests Hyperprelinemia may be detected in plasma or urine in one-3 or two-dimensional4 paper chrexnatograms stained with a ninlhydrin : isatin mixture as well as by column chromatog- raphy.5 Proline in plasma may also be meas— ured colorimetrically alone“ or in combination with hydroxyproline.7 Genetics The inheritance of this condition has not yet been evaluated. Treatment The efl’ect of a low-preline or low-protein intake has not yet been described. References 1. Berlow, S., and Efren, M.: A new cause of hyper- prelinemia associated with the excretion of A1- pyrroline-5-carboxylic acid. See. Fed. Res, p. 43, 1964. (abstr.) 2. Schafer, I. A., Scriver, C. R. and Efren, M. L.: Fa-- milial hyperprolinemia, cerebral dysfunction and renal anomalies occurring in a family with heredi- tary nephropathy and deafness. New Eng. J. Med. 267: 51, 1962. 3. See technical section page 71. 4. O’Brien, D., and Ibbott, F. A.: Laboratory Manual of Pediatric Micro- and Ultramicro Techniques. Hoeber, New York, 3d ed., p. 25. 5. Spackman, D. H., Stein, W. H. and Moore, 8.: Auto- matic recording apparatus for use in the chroma- tography of amino acids. Anal. Chem. 30: 1190, 1958. 6. Summer, G. K. and Hawes, J. A.: Determination of free proline in serum. Proc. Soc. Ecper. Biol. and Med. 112:402, 1963. 31 Figure 14. THE METABOLISM OF HYDROXYPROLINE °2 HoCH-CH, .C II,C H-COOH NAD NADH + I-I+ H HYDROXYPROLINE AI-PYIROLINE-3- HYDROXY-S-CAIIOXYLIC ACID H20 _¥____,. no-CIH-CIII, E IIO-CIvI-Iiu2 CH ,CH-COOH oscu CH-COOH \N / NH: 7-HYDROXYGLU‘I’AMIC ACID SEMIALDEHYDE H20 NAD ADII + H+ CII3 uo-cufcn HO-CH-CH O=CH I l I c <—-—-> HOOC C-COOH nooc CH—COOH HOOC + // \ I (TRANSAMINAYION) o coon 0 NH, ASPARTIC OXAlOACETIC ACID ACID ouoxvuc PYRUVIC o.I-IVDIonv.r. YHYDROXYGLUTAMIC ACID ACID ACID K,ETOGLUTARIC ACID 7. Gould, B. S., and Shwachman, H. Studies in cystic fibrosis: determination of plasma proline follow- ing protein feeding as a diagnostic test for pan- creatic insufficiency. A.M.A.J. Dis. Child. 91: 584, 1956. Hydroxyprolincmia with mental retardation Clinical and laboratory findings The only case described 1’ 5 is that of a se- verely retarded girl with an IQ. of 26 who had a history of good health and who showed no abnormal physical signs, except for flat feet and mild concomitant Istrabismus. There were in- creased numbers of both red and White cells in her urine, but the intravenous pyelogram, plasma urea and serum proteins were nor- mal. An electroencephalogram, and audio- 32 gram, and a radiological bone survey were likewise unremarkable. On both paper and column chromatography increased amounts of L—hydroxyproline were detected in the plasma and urine; other amino acids were all normal. Free hydroxyproline excreted was 1.5 mols/min/1.73m2: a total hy- droxyproline following acid hydrolysis was 1.7 meIS/ min/ 1.7 3m2. Bound hydroxyproline was thus considered normal?» 3 The actual level of hydroxyproline in the plasma was 0.34 It mols/ml (normal < 0.001 pmols/ml) and the urinary clearance 0.35 ml/min (normal 0) . Biochemistry Hydroxyproline occurs only in collagen. The incorporation is not direct, however, but depends on the hydroxylation of proline in a collagen precursor. Ingested hydroxyproline is broken down as shown below in a manner analagous to proline,2 a process which must usually be very complete, as this amino acid is not normally detectable in the urine of other children and adults and is present in only trace quantities in the plasma. Hydroxyproline from endogenous collagen turnover may also be broken down in this manner, but is also cleared by the urine in a “bound” form as hydroxypro— line peptide. In this child the hydroxyprolinuria is un- likely to have been alimentary as a high plasma level of 0.28 ,u. mol/ml persisted after 8 days on a hydroxyproline low diet. In addition, the amount of bound hydroxyproline in the urine suggests a normal turnover of collagen. The condition would therefore appear to be due to a block in the pathway between hydroxyproline and glyoxylic acid and pyruvate. “By analogy with hyperprolinemia 2 the enzyme involved seems to be the hydroxyproline oxidase; and this has now been confirmed.5 Diagnostic and screening tests Excess hydroxyproline is readily detectable in plasma or urine in one- 3 or two—dimensional 4 paper chromatograms stained with a ninhy— drin: isatin mixture, as well as by column chro— matography.’5 Hydroxyproline may also be determined colorometrically in urine6 and in plasma as combined hydroxyproline and pro- line.7 References 1. Efron, M. L., Bixby, E. M., Palattao, L. G., and Pryles, C. V.: Hydroxyprolinemia associated with mental deficiency New Eng. J Med 267:1193, 1962. 2. Berlow, S., and Efron, M. L. : A new cause of hyper- prolinemia associated with the excretion of A- pyrroline-5—carboxylic acid. Soc. Ped. Res. p. 43, 1964. (abstr.) 3. See technical section page 71. 4. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro-and ultramicrobiochemical techniques. Hoeber, New York. 3d edit, 1962, page 25. 5. Efron, M. L., Bixby, E. M. and Pryles, O. V.: Hy- droxyprolinemia: a rare metabolic disease due to a deficiency of the enzyme hydroxyprollne oxi- dase. New Eng. J. Med. 272:1299, 1965. 6. Mitoma, 0., Smith, T. E., Davidson, J. D., Uden- friend, S., Da Costa, F. M., and Sjoerdsma, A.: Improvements in methods for measuring hydroxy- proline. J. Lab. 01m. Med. 53: 970, 1959. 7. Gould, B. S. and Shwvachman, H.: Studies in cystic fibrosis: determination of plasma proline follow- ing protein feeding as a diagnostic test for pan- creatic insufficiency. A.M.A. J. Dis. Child. 91: 584, 1956. Ataxia telangiectasia with an abnormal peptide in the urine Clinical and laboratory findings Fourteen years ago Louis—Bar 1" first de- scribed a syndrome in short—statured children which was characterized by skin and conjunc- tival telangiectasia and a slowly progressive cerebellar ataxia developing as the child began to walk. Characteristically, the expression was fixed and inattentive, with a tendency to drool and dysarthria. Deep tendon reflexes were sometimes diminished and at times the skin showed patchy atrophy,ls cafe au lait spots, and pigmented naevi.6 Bleeding sometimes oc- curred from the mucous membranes 6 and mental retardation was variably present.‘ Pelc and Vis in 1960, 2 and Paine and Efren 3 years later,3 described an abnormal amino compound in the urine in a total of four cases of the syndrome. On the Beckman/Spinco 150 cm. column amino acid analyzer system this substance gave a peak in the 440 my. trace be— tween cystathionine and methionine. The un— known peak was further separated out by high voltage electrophoresis and ascending paper chromatography, hydrolysed and shown to con- tain only proline and hydroxyproline.8 The presence of the peptide in the serum is not reported. Biochemistry Since hydroxyproline occurs only in col— lagen and elastin, it is probable that this urin- ary substance is a dipeptide resulting from the partial breakdown of the glycine-proline-hy- 33 droxyproline sequence in collagen. The inci- dence of this abnormal biochemical finding in the syndrome as a whole is not known, nor for the present is there any apparent relationship between the neurological findings and the peptiduria. Diagnostic and screening tests The diagnosis of ataxia is made clinically so that the finding of the abnormal peptide re- mains of speculative interest until further in- formation on its origin is available and until there is a better understanding of the role of imino acids in cerebral metabolism. Column chromatography is required to confirm the pres- ence of the peptide in the urine. Genetics The syndrome is thought to be inherited as an autosomal recessive; one atypical case3 is described in Which the inheritance is dominant, but this girl did not excrete the peptide in the urine. Treatment No treatment devised to correct the bio- chemical defect has been attempted. References 1. Louis-Bar: Sur un syndrome progressif comprenant des télangiectasies capillaires cutanées et con- jonctivales symétriques, a disposition des naevoide et des troubles cérébelleux. Confln. N enrol. 1;: 32, 1941. 2. Pele, S., and Vis, H.: Artaxie familiale avec telan- giectasies oculaires. Acta Neurol. Belg. 60: 905, 1960. 3. Paine, R. 8., and Efron, M. L.: Atypical variants of the “Ataxia Telangiectasia” syndrome. Develop. Med. Child. N enrol. 5: 14, 1963. 4. Boder, E., and Sedgwick, R. P.: “Ataxia-telang‘iec- tasia”: familial syndrome of progressive cerebel- lar ataxia, oculocutaneous telangiectasia, and fre- quent pulmonary infection. Pediatrics 21:526. 1958. 5. Beveridge, T.: A-taxia-telangiectasia. Med. J. Aust. i: 613, 1960. 6. Reye, C.: Ataxia-telangiectasia: report of a case. A.M.A. J. Dis. Child. 99: 238, 1960. 34 7. Spackm-an, D. EL, Stein, W. H. and Moore, 5.: Auto- matic recording apparatus for use in the chroma- tography of amino acids. Anal. Chem. 30: 1190, 1958. Joseph’s syndrome Three cases in a single family have been described 1 in whom there occurred a syndrome characterized by the early onset of generalized convulsions, raised cerebral spinal fluid protein, and iminoaciduria. The convulsions com- menced within the first 2 weeks of life and were severe and repeated; in tw0 instances the in- fants died in status epilepticus at 10 months and 2 years respectively. Cerebrospinal fluid pro- tein levels were considered to be above normal, being between 60 and 120mg/ 100 ml at 6 weeks of age. The eldest child died before any amino acid estimations could be carried out and in the two younger siblings the reports of aminoaci- duria cannot wholly be accepted as they were based on paper chromatography and unsup- ported by column chromatography. Plasma amino acids were normal, but the urine ap- peared to contain excessive amounts of proline in all cases and of hydroxyproline in the young- est infant. The latter observation may not be abnormal as the normal 6-week-old infant will show a significant hyperhyroxyprolinuria.2 The text also contains an unsubstantiated re- port of excess amounts of a proline peptide in the urine. The apparent normal serum proline values would seem to differentiate this family from the hyperprolinemic syndromesf’ 4 although it is not possible to be sure that serum proline levels were normal solely on the basis of two-dimen- sional paper chromatography. Peptiduria is not described, nor would it be anticipated in the proline oxidase deficiency and A1 pyrroline-5-carboxylic acid dehydroge- nase deficiency syndromes. In addition, these children do not appear to resemble either those with ataxia telangiectasia 5 or those with mas- sive new bone formation and high alkaline phosphatase,“ both of which groups show an iminodipeptiduria. The appropriate laboratory diagnostic techniques are the same as those described under the hyperprolinemic syndromes. References 1. Joseph, R., Ribiere, M., Job, J—C., and Girault, M.: Maladie familiale associant des convulsions a délburt trés précoce, une hypera'lbuminorac’hie et une hyperaminoacidurie. Arch. franc. de pediat. 15: 374, 1958. 2. OBrien, D., and Butterfield, L. J .: Further studies on renal tubular conservation of free amino acids in early infancy. Arch. Dis. Child. 38: 437, 1963. 3. Schafer, I. A., S‘criver, C. R., and Efron, M. L.: Familial hyperprolinemia, cerebral dysfunction, and renal anomalies occurring in a family with hereditary nephropathy and deafness. New Eng. J. Med. 367: 51, 1962. 4. Efren, M. L.: Familial hyperprolinemia. New Eng. J. Med. 272: 1243, 1965. 5. Paine, R. S. and Efron, M. L.: Atypical variants of the “ataxia telangiectasia” syndrome. De- velop. Med. Child. Near. 5: 14, 1963. 6. ‘Seakins, J. W. T.: Peptiduria in an unusual bone disorder. Arch. Dis. Child, 38: 215, 1963. Osteopathy, peptiduria and mental retardation Clinical and laboratory findings A small number of cases of this syndrome have been reported; but in only one 1 has there been a detailed evaluation of the underlying biochemical disorder. This child was thought to be normal during the early months of life; but at a year he was noticed to have thickening of the tissues on either side of his nose. At 18 months of age medical advice was sought for bony swellings over both cheeks. On examination there were no abnormal physical findings apart from the skeletal sys- tem. The child could stand with support but could not walk or talk. He was considered to be mentally retarded; but no formal IQ. is available and it is possible that the intellectual impairment was in significant measure due to the somatic problem. The head was large with a thickening of the bridge of the nose, the maxillae and cen- trally in the frontal region. On X-ray the skull was generally thickened and porotic. N o lamina dura was seen. The long bones were bowed and thickened and on X-ray the shafts were seen to be thickened and porotic with extensive sub- periosteal new bone formation. A wide range of conventional laboratory procedures were normal except for a substantial rise in serum alkaline phosphatase levels. Bone biopsy showed considerable disorganiza- tion of the normal structure. Biochemical disorder A rather elaborate series of biochemical procedures identified excessive amounts of gly- cylproline and prolylhydroxyproline in the urine. This occurrence of hydroxyproline pep- tide in association with bone disease is a strong indication of excessive collagen turnover. It is not clear however why only bone collagen appears to be affected. Again it is not possible to trace any obvious connection with mental retardation although it is clear that in other disorders of imino acid metabolism this relation- ship exists. Genetics The inheritance of this condition has not been defined. Diagnosis and screening Diagnosis depends on the typical clinical picture and on the presence of striking glycine and peptide spots in simple two dimensional paper chromatography—see technical section. The peptide spots which have similar R, values to proline can be confirmed by their disappear- ance after acid hydrolysis. Screening tech- niques are at present inappropriate as it is not known to what extent the peptiduria may be present in advance of bone changes. Reference 1. Seakins, J. W. T.: Peptiduria in an unusual bone disorder. Arch. Dis. Child. 38: 215, 1963. 35 In histidine metabolism Histidine a—dcaminasc deficiency: Histidincmia Clinical and laboratory findings Altogether eight cases of this disorder have so far been described}-7 The clinical features have been quite variable, and the condition may occur in an apparently normal subject.1 The commonest suggestive finding is some form of speech disorder which was a feature of five of the eight reported cases. Speech tends to be slow to develop and once acquired may be vari- ably intelligible. The defect seems to be pri- marily one of auditory memory leading to in- correct grammar and syntax, but slurred and inarticulate speech may appear as well. The majority of these children are small for their age and some have shown a tendency to recur- rent infections. While the speech disorder may lead to a semblance of mental retardation in fact only three of the children were significantly retarded. Usually, but not always, the urine from these patients gave a green colour on testing with ferric chloride solution or Phenistix strips, and some were provisionally considered to have phenylketonuria until serum phenylalanine levels were shown to be normal. It was readily established on paper chromatography that the urine contained a considerable excess of histi- dine, as well as slightly increased amounts of alanine and threonine. This amounted to be- tween 5 and 10 times the normal daily excretion of up to 1.5 m mols/1.73m2 in children and 0.8 m mols/1.73m2 in adults. Plasma fasting levels of histidine were also consistently raised to be- tween 0.45 to 1.0 Mmols/ml (normal <0.12 ,umols/ml). When other samples of urine were chromatographed and stained with diazoben- zene sulfonic acid (Pauly’s reagent), they were shown to contain not only histidine but imida- zolepyruvic, imidazolelactic, and imidazole- acetic acids. Imidazolepyruvic acid was shown 36 to be responsible for the positive ferric chloride test.‘ After oral histidine loading it was shown that there followed a sustained rise in plasma histidine levels?! 3' 4’ 6 As compared with con- trols8 there was also an increased urinary ex- cretion of histidine, imidazolepyruvic acid,‘ imidazolelactic, and imidazoleacetic acid. Urocanic acid, 3 imidazolepropionic acid and formiminoglutamic" acid were not excreted, however. Following the oral administration of 2-C14-L-histidine in one case, the urine con- tained labelled C“-imidazolepyruvic and C“- imidazolelactic acids in excess of normal; 5 there was no labelling of glutamic acid, formi- minoglutamic acid, ‘or hydantoin propionic acid. Biochemistry The first step in the normal breakdown of histidine is to urocanic acid catalyzed by his— tidine a-deaminase or histidase. The pathway is shown in figure 15. Excessive urinary ex— cretion of histidine and imidazole pyruvic, imi- dazolelactic, and imidazoloneacetic acids after histidine loading in the absence of any increase in urocanic acid, imidazoleproprionic acid, or FIGLU strongly suggested a defect in histidine a-deaminase. Following intravenous urocanic acid loading, 3’ 4 moreover, it was evident that the histidinemic patient could form imidazo- lone-propionic acid and FIGLU normally, thus emphasizing the site of the metabolic block. By way of final confirmation an absence of histidase activity in stratum corneum cells has been shown in the histidinemic patient." 1° Laboratory diagnostic screening tests Although the early cases of this syndrome were diagnosed on the basis of a positive ferric chloride test, this cannot be accepted as a uni- formly reliable screening test. The increases in both urinary and plasma histidine levels are so striking, however, that they can readily be 9 ' 99 - 0 296-2“. LE Figure 15. H H I I la 3Methy| Hisvidine 4————— HC=C-(i-(ll-COOH I H NH2 NH HlsvidyI-fl-Aianine N\/ HISTI / lCARNOSINE] / c l-me»His¢idyl.,6-Alonino H (ANSERINE) Hisoidine (lmidaxolealanine) z—n I 1411.: 1.;5—1 z' I Histamine \ lmidaxole acetaldehyde 1.4Movhylhistamine Hisvidine Trunsaminase H l HC=C-C-C-COOH l l | H o N NH \ \C/ H l I I HC=‘C-CH5HO HC=C-C.-COOH HC=C-(IL-CI-COOH H H O ’ N N NH H ”SC CH3N\ éN \c/NH \3’ I H H H 1,4Methyl Imidazole ”"idOIOIQV-Ice'ic aca'aldohyde acid +c-cn5coon ”i=l HSC-N\ éN Imidazolelactic acid Imidazoleaceiic acid riboside C . . H 1,4Me0hyl lmidazale acetic acud Uracanic acid (Imidaza|eacry|ic acid) Imidazolepropionic acid THE METABOLISM OF HISTIDINE Imidazolone prapionic acid t”! 5‘ T '1 777 HC=C-C-C—COOH o c—c-c-c-coon oC—c-c-c-COOH —->— I l I M ——> | M DASE N\ ,N“ We "kc/N” “Kc/N" H H II Hydantoin prapianic acid H H H H HOOC-Cl-Cl-Cl-COOH l M H“: TH Ozc—c-cl—cl-COOH CH NH H H “H T” CH Fovmiminoglu'amic acid ll (FlGlU) O Formylisoglutamine COOH | Tehahydroiolic acid CHNH2 I Formiminontrahydroiolic CH2 acid CH2COOH Glutamic acid detected by one-dimensional paper chroma— tography.11 Confirmation may come from the measurement of serum histidine as its enol borate complex, 4 by two-dimensional paper chromatography of urine,12 or column chroma- tography of serum or urine.13 The absence of urocanic acid in sweat 4 in histidinemia is like— wise a simple confirmation test. Genetics The inheritance of this syndrome is not yet established, although it would seem to be the homozygous state of an autosomal recessive trait. FIGLU excretion after a histidine load, skin histidase levels, and sweat urocanic acid content all might prove discriminants for the heterozygote state and thus permit a more spe- cific genetic evaluation of affected kindred. Treatment Various suggestions have been made to ex- plain a possible link between histidine metabo— lism and mental retardation. These include a diminished availability of the one carbon pool for purine and pyrimidine metabolism and the possibility that low serotonin levels 3 might also be of importance. At the moment it is diflicult to accept these suggestions when less than half the cases are mentally retarded, and the possi- bility remains that this is due to a different but linked genetic aberration. At all events there does not at the moment appear to be any ra- tional approach to therapy. References 1. Ghadimi, H., Partington, M. W., and Hunter, A.: A familial disturbance of histidine metabolism. New Eng. J. Med. 265: 221, 1961. 2. Ghadimi, H., Partington, M. W., and Hunter, A.: Inborn error of histidine metabolism. Pediat- rics 29: 714, 1962. 3. Auerbach, V. H., DiGeorge, A. M., Baldridge, R. G., Tourtellotte, C. D., and Brigham, M. P.: Histi- dinemia J. Pediot. 60: 487, 1962. 4. LaDu, B. N., Howell, R. R., Jacoby, G. A., Seeg- miller, J. E., Sober, E. K., Zannoni, V. G., Canby, J. P. and Ziegler, I. K.: Clinical and biochemical studies on two cases of histidinemia. Pediatrics 32: 216, 1963. 5. Snyder, S. H., Myron, P., Kies, M. W., and Berlow, 38 S.: Metabolism of 2-C“-labelled L-histidine in histidinemia. J. Olin. Endocrin. Metab. 23: 595, 1963. 6. Davies, H. E., and Robinson, M. J .: A case of histi- dinemia. Arch. Dis. Child. 38:80, 1963. 7. Shaw, K. N. F., Boder, E., Gutenstein, M. and Jacobs, E. E.: Histidinemia. J. Pediat. 63: 720, 1963 (abstr.) 8. Ames, B. N., and Mitchell, H. K.: The paper chro- matography of imidazoles. J. Amer. Chem. Soc. 71;: 252, 1952. 9. Tabor, H. and Wyngarden, L.: A method for the determination of formiminoglutamic acid in urine. J. Clin. Invest. 37: 824, 1958. 10. Zannoni, V. G., and LaDu, B. N. : Determination of histidine a-deaminase in human stratum corneum and its absence in histidinemia. Biochem. J. 88: 160, 1963. 11. See technical section page 71. 12. O‘Brien, D.. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicrobiochemical techniques. Hoeber, New York, 35th Ed. 1962, p. 25. 13. Spackman, D. H., Stein, W. H. and Moore, S.: Au- tomatic recording apparatus for use in the chro- matography of amino acids. Anal. Chem. 30: 1190, 1958. Hyperfolicacidemia with formiminogluta— micaciduria following histidine loading: F ormiminotransferase deficiency Clinical and laboratory findings A single case of a syndrome has been de- scribed which was characterized by a round face, obesity, physical and intellectual retarda- tion, hypersegmentation of polymorphonuclear nuclei, high serum folic acid levels, and exces— sive urinary formiminoglutamic acid (FIGLU) excretion following histidine loading.1 This infant girl was born at term after a normal delivery. The first months of life were uneventful, but at 3 months of age breast milk was insuflicient and she was given rice gruel supplements. At the fifth month of life she was noted to have developed a generalized edema with occasional grand mal convulsions. It is not clear whether or not this was a nutritional edema, but at nearly 9 months of age she was a short (10%ile), fat (90%ile) infant who was unable to sit, to support her head, or to show Figure 16. THE BREAKDOWN OF FORMIMINOGLUTAMIC ACID TETRA HYDRO, FORMIMINO TETRAHYDRO FOLIC ACID FOLIC ACID HOOC-CH-CHECHECOOH I NH I CH u NH FORMIMINOGLUTAMHZACID (HGLw any social recognition of her mother. The liver edge was palpable approximately 3 cm below the costal margin; otherwise there were no ab- normal physical signs. The physical features described in this case are in some ways reminiscent of pseudopseudo- hypoparathyroidism.2 However, this child showed no real dwarfism and did not have any metacarpal or metatarsal deformity, or any ec— topic calcinosis or exostoses. Urinalysis, skeletal X-rays, liver function tests, chromosome analysis, 17 ketosteroid and 17 hydroxycorticosteroid excretion, thyroid I131 uptake, and paper chromatography of urine and column chromatography of plasma for amino acids were all normal. Serum phosphorus levels averaged as high as 6.0 mg/100 ml in 10 estimations over a period of 1 year; but calcium levels were within normal limits except for one occasion where a level of 4.9 mg/100 ml was recorded. The only abnormality noted in the peripheral blood was a tendency to hyperseg- mentation of the polymorphonuclear leucocytes, a condition normally encountered in well-estab— , lished megaloblastic anemias. CHO COOH I CH NH H20 , NH3 ' 2 s": CH2 l COOH GLUTAMIC ACID In this case the appearance of the bone marrow and serum level of BB were normal. Using a microbiological assay3 serum folic acid levels were found to range from 10 ng/ml to 300 ng/ml; 12 of 17 levels taken over a 1-year period were over the upper limit of normal for the method of 100 ng/ml. After an oral histidine loading test 4’ 5 of 0.25 g/kg of body weight the 8-hour urinary excretions of FIGrLU6 were 67 and 13 amols respectively, compared to 6 and 2 ,umols in two controls. Biochemistry Histidine is normally metabolized through urocanic acid and imidazoloproprinonic acid to F IGLU (figs. 15, 16), or via histamine or imidazolepyruvic acid to imidazole acetic, im- idazolelactic, and 1.4: methyl imidazole acetic acids. Further breakdown of FIGLU is cat- alyzed by the enzyme formiminotransferase, by which FIGLU forms glutamic acid, and the one carbon formimino group is transferred to a 39 tetrahydrofolate. In this particular case the relatively high levels of folic acid suggest that the accumulation of formiminoglutamic acid is not due to nonavailability of tetrahydrofolate. Confirmation of this was obtained by demon- strating a normal level of folic acid reductase in a liver biopsy of this child. Once this possi- bility had been excluded the most likely cause of the distortion of histidine breakdown was an impairment of formiminotransferase activity.6 When this was measured on a liver biopsy it was shown to be about 14 percent of the control value. Laboratory and screening tests Obesity and retardation are common enough associations to be of little diagnostic value, but hypersegmentation of leucocytes in a peripheral blood smear may prove of value as a simple laboratory test. In other respects measurement of FIGLU after a histidine load- ing test is the identifying laboratory procedure.’ Genetics The inheritance of this disease is not yet clear. Two siblings of the patient were unaf- fected, although the father, a paternal aunt, and the paternal grandmother all showed hyper- segmentation of neutrophils. Treatment No treatment has been devised or attempted. References 1. Arakawa, T., Ohara, K., Kudo, Z., Tada, K., Haya- shi, '1‘., and Mizuno, T.: Hyperfolicacidemia with formiminoglutamicacidura following histidine loading. Tohoku, J. Earner. Med. 80: 370, 1963. 2. Papaioannou, A. 0., and Matsas, B. E.: Albright’s hereditary osteodystrophy (without hypercalce- mia). Pediatrics, 31: 599, 1963. 3. Herbert, V., Fisher, R., Koontz, B. J .: The assay and nature of folic acid activity in human serum. J. Clin. Irw. 1,0: 81, 1961. 4. Luhby, A. L., Oooperman, J. M., and Teller, D. N.: Histidine metabolic loading test to distinguish folic acid deficiency from vitamin B12 in megalo- blastic anemias. Proc. Soc. Ewp. Biol. and Med. 101: 350, 1959. 40 5. Luhby, A. L., Gooperman, J.M. and Teller, D.N.: Urinary excretion of formiminoglutamic acid. Am J. Olin. Nutr. 7: 397, 1959. 6. Tabor, H., and Wyngarden, L.: The enzymatic for- mation of formiminotetrahydrofolic acid, 5, 10- methenyltetrahydrofolic acid, and 10-formy1tetra- hydrofolic acid in the metabolism of formimino- glutamic acid. J. Biol. Chem. 234: 1830, 1959. 7. See technical section page 79. Imidazolc aminoaciduria in cerebromacu— lar degeneration In three kindreds amongst whom there were five cases of the late cerebromacular degenera- tion,1 a marked imidazole aminoaciduria was observed. The urine contained greatly in— creased amounts of carnosine and anserine, the dipeptides of B-alanine and histidine, and ,B-alanine and l-methylhistidine respectively (fig. 17). There was also some increased uri- nary histidine, l-methylhistidine. On hydroly- sis with 4N hydrochloric acid for 6 hours at 100° C the urine showed an expected rise in histidine, l-methylhistidine, and B-alanine with disap- pearance of the peaks for carnosine and anserine.2 In the families concerned, the inheritance of the aminoaciduria appeared to be as a domi- nant trait and the neurological manifestations as a recessive. The authors discuss the possi- bility that this could be a random association, but they point out that in view of the individual rarity of these traits it seems more probable that .they are expressions of the same gene. The imidazole aminoaciduria alone may thus repre- sent the heterozygous state, and the neurologi- cal syndrome plus the aminoaciduria the homo- zygous disease. Plasma levels of these imida— zole compounds were normal, suggesting that the increased urinary amounts are due to en- hanced renal tubular rejection. Carnosine and anserine, as well as the hydrolysate products ,B-alanine, histidine and l-methyl histidine, can be readily detected on one-dimensional paper chromatograms 3 and specifically identified on column chromatog- raphy.‘ Figure 17. THE STRUCTURES OF CARNOSINE AND ANSERINE COOH O C N H =C-CH2—‘CH—NH-C—'CH2—CH2—NH2 Z \c/ “ H CARNOSINE (HISTIDYL-fl-ALANINE) COOH O I ll HC= c—CH2—CH —NH — c — CH -CH2—NH2 l l 2 N N § / \ C CH3 H ANSERINE (I-METHYl-HISTIDYL-fl-ALANINE) References Carnosine excretion in juvenile amaurotic idiocy. Lancet ii: 756, 1964. 3. See technical section page 71. 4. Spackman, D. EL, Stein, W. H. and Moore, S.: Auto- aciduria in cerebromacular degeneration. Sci— matic recording apparatus for use in the chroma- ence 135: 789, 1962. tography of amino acids. Anal. Chem. 80: 1190, 2. Levenson, J., Lindahl-Kiessling, K. and Rayner, S.: 1958. 1. Bessman, S. P., and Baldwin, R.: Imidazole amino- ‘ 41 In tyrosine metaboliym Transient p—hydroxyphenylpyruvic acid oxidase deficiency Clinical and laboratory findings A typical case 1 is reported in a 4-month—old male infant of healthy nonconsanguineous par- ents. At the age of 4 months the child was first admitted to the hospital with a history of seizures for 1 month. Pregnancy and delivery had been normal, but he had been difficult to feed from an early age. At 2 months of age he had an episode of vomiting and diarrhea and 1 month later had his first generalized convulsion. On examination there was some increase in muscle tone in all four extremities, head lag, and a persistent Moro reflex. The abnormal laboratory findings included an abnormal EEG with spike and wave discharges, a persistent metabolic acidosis, and positive 2 :4 dinotro— phenyl-hydrazine and ferric chloride tests. In view of the latter tests, the child was diagnosed as a phenylketonuric, a decision encouraged by the fact that a first cousin was known to have the disease. The infant was placed on a low phenyla- lanine diet on which he showed an immediate and remarkable improvement, so that by 7 months of age his convulsions had ceased, neu- rological signs had disappeared, and he seemed of normal intelligence. Other laboratory tests prior to the insti- tution of the diet had shown, however, that the serum phenylalanine level was within nor- mal limits at 2.3 mg/100 ml. As a result the urine was chromatographed for keto acids and phenolic acids. These showed considerably in- creased amounts of p-hydroxyphenyl pyruvic (pHPPA) , p-hydroxyphenyl lactic (pHPLA), and p—hydroxyphenyl acetic acids (p.HPAA) in the urine, small amounts of o—hydroxyphenyl acetic acid; but no homogentisic acid, 2—5 dihy- droxyphenyl pyruvic or 3—4 dihydroxyphenly— alanine (DOPA). Tyrosinuria was also shown as part of a generalized aminoaciduria, but ty— 42 rosine levels were not measured before the ini- tiation of dietary treatment. Thereafter they ' were normal. Biochemistry The normal pathway for tyrosine metabo- _‘ ' lism is shown in figure 18, tyrosine for the most L part being transaminated to pHPPA and ,-, thence, after further oxidation and breaking of g the ring to fumarate and acetoacetate. In this _v infant the accumulation of pHPPA, pHPLA, ' and pHPAA without homogentisic acid sug- gested an impairment of pHPPA oxidase ac- ‘ tivity. The enzyme requires an unknown qui- ' none, copper ion, and vitamine C as cofactors. Permanent deficiency of this reaction has been reported only once in a 49—year-old man with myasthenia gravis.2 Impairment of pHPPA oxidation may of course occur in liver disease _ and in scurvy,a neither of which conditions was , ‘V in any way manifest in this infant. Premature infants receiving inadequate vitamin C supple- ' ments 4 may also show tyrosyluria. Full term ‘1 infants 5’ 6 on an apparently normal Vvitamin C intake may show the same abnormality in the ' first 2 months of life, by which time it ceases ; spontaneously. The infant described here is remarkable in showing clear cut neurological ' signs. ,' One of the full term infants, with a tran— sient deficiency of p.hydroxyphenlypyruvic :. acid oxidase, has recently been reported in some detail.7 The problem in this child was failure? 3 to gain at 2 months of age, despite a good ap— . ' petite. There were no neurological signs, al—‘-{ though it is of interest that a sibling of this I child was under treatment for phenylketonuria. ,. Paper chromatography of the DNPH deriva- ._. tive showed evidence for large amounts of ? p. hydroxyphenyl pyruvic acid in the urine. Column chromatography of the plasma showed 2. a very large increase in tyrosine, with elevated: values of proline, valine, leucine, isoleucine,. methionine, and phenylalanine. The authors, make the interesting speculation that the raised Figure 18. THE METABOLISM OF TYROSINE CH2CHOHCOOH HO AND HO P-HYDR‘OXYPHENYL LACTIC ACID CH 2COCOOH HO P- HYDROXYPHENYL PYRUVIC ACID CH2CHNH2COOH CHZCOOH P- HYDROXYPHENYL ACETIC ACID CH COOH HO 2 FUMARATE pHPPA ACETOACETATE OXIDASE OH HOMOGENTISIC ACID HO TYROSINE \\ \\\ CH2CHNH2COOH CH2CH2NH \\\ OH 0“ \ +- HO HO 2 5-DIHYDROXY 1 2,5-DIHYDROXYPHENYL “0 CH2CH coo” PHENYLALANINE ETHYLAMWE HO 3,4-DIHYDROXYPHENYL : ALANINE (DOPA) . phenylalanine level may in part reflect the in- ; hibition of phenylalanine hydroxylase by tyro- ' s1ne which has been reported in animals. If -’ this is so, it is conceivable that a degree of 'phenylketonuria, suflicient for brain damage, , might result indirectly from evanescent : hypertyrosinemia. Screening and diagnostic procedures The condition 1s likely to be first suggested by the finding of a positive ferric chloride test 2 1n association with a normal serum phenyla- .1an1ne level. 8 Further confirmation 1s provided by finding a high serum tyrosine level, 8 . pHPPA, pHPLA, and pHPAA on phenolic acid chromatography 9 in the absence of homo- ’ gent1s1c acid. Genetics The genetics of this very rare condition is unknown. Treatment This child recovered promptly when placed on a low phenylalanine, low tyrosine diet (Lofenalac ®). One of the questions this raises is whether subclinical brain damage may occur in other infants With tyrosyluria. References 1. Menkm, J. H., Jervis, G. A.: Developmental retarda- tion associated with an abnormality of tyrosine metabolism. Pediatrics 28: 399, 196]. 43 2. Medes, G.: A new error of tyrosine metabolism: tyrosinosis, the intermediary metabolism of tyro- sine and phenylalanine. Biochem. J. 26 2 917, 1932. 3. Morris, J. E., Harpur, E. R. and Goldbloom, A.: The metabolism of L—tyrosine in infantile scurvy. J. Olin. Imzest. 29: 325, 1950. 4. Levine, S. Z., Gordon H. H., and Marples, E-.: A de- fect in the metabolism of tyrosine and phenyla- lanine in premature infants. II Spontaneous oc- currence and eradication by vitamin C. J. Olin. vaest. 20: 209, 194.1.- 5. Berry H. K., Sutherland, B.: Tyrosinosis in an in- fant. Soc. Ped. Res. 1960, p. 78 (abstr.). 6. Bloxam, H. R., Day, M. G., Gibbs, N. K. and Woolf, L. I. : An inborn defect in the metabolism of tyro- sine in infants on a normal diet. Biochem. J. 77: 320, 1960. 7. Auerbach, V. H., DiGeorge, A. M., Brigham, M. P., and Dobbs, J. M.: Delayed maturation of tyrosine metabolism in a full-term sibling of a child with phenylketonuria. J. Pediatrics 62: 939, 1963. 8. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultra-micro biochemical tech- niques. Hoeber, New York. 3d edit. 1964. p. 242. 9. See technical section page 97. Hyperphenylalaninemia The extension of phenylketonuria screen- ing programs has revealed a number of new- born infants having phenylalanine levels in ex- cess of the normal range. Some, of course, are due to methodological variance but in a signifi- cant number the high values are sustained and may or may not be associated with abnormally elevated tyrosine levels}! 2 especially in the pre- mature. Aetiologically these may reflect the heterozygous state,3 maternal phenylketonu- ria,4 phenylalanine hydroxylase immaturity or transient p-hydroxyphenylpyruvic acid oxidase deficiency.5 Phenylalanine levels are usually in the 10—15 mg/ 100ml. range, but there have been levels reported up to 40 mg/100 ml. Hyperphenylalaninemia has been shown in animals to produce both a significant distortion of free amino acids in the brain and a marked defect in cerebroside content of brain; 6 thus it would seem logical to keep serum phenylalanine and tyrosine levels within normal limits in these cases. It is not necessary to use special low phenylalanine formulae, as many of these chil- 44 dren respond to a conventional low protein milk mixture. These cases illustrate the importance of reserving judgment on the diagnosis of phen- ylketonuria until the hyperphenylalaninemia can be shown to require a low phenylalanine formula for its abatement, to be accompanied by phenylketonuria and to persist with any return to conventional formulae. References 1. Auerbach, V. H., DiGeorge, A. M., Carpenter, G. G. and Dobbs, J. M.: Phenylalaninemia. Soc. Ped. Res. p. 108, 1965 (Abstr.). 2. Schneider, A. J. and Garrard, S. D.: Persistent hy- perphenylalaninemia. Am. Pediat. Soc. 75th Ann. Mtg. p. 54, 1965 (Abstr.). 3. Wang, H. L., Morton, N. E. and Waisman, H. A.: Increased reliability for the determination of the carrier state in phenylketonuria. Amer. J. Hum. Gen. 13: 255, 1961. 4. Mabry, 0. 0., Denniston, J. 0., Nelson, T. L. and Son, 0. D.: Maternal phenylketonuria. New Eng. J. Med. 269: 1404, 1963. 5. Auerbach, V. H., DiGeorge, A. M., Brigham, M. P. and Dobbs, J. M.: Delayed maturation of tyrosine metabolism in a-full-term sibling of a child with phenylketonuria. J. Pediat. 62: 938, 1963. 6. O’Brien, D., Ibbott, F. A. and Dabiere, 0.: The ef- fect of prolonged phenylalanine loading on the free amino acid and lipid content of the infant monkey brain. Develop. Med. and Child Neurol. 8: —, 1966. To be published. Urinary 3.4 dihydroxyphenylalanine (DOPA) excretion in children of short stature Two unrelated 5-year-old male children have been described,1 both of whom were short, maltreated, undernourished, and showed hir- sutism, anhydrosis, and hypercreatinuria. Their I.Q.s were 87 and 57 respectively and both used immature and often unintelligible speech. Toilet training was significantly delayed in one case. Both showed increased amounts of 3.4 di- hydroxyphenylalanine (DOPA) in the urine on column chromatography which was accentuated by oral -DOPA loading. It was tentatively asserted that there might be either a delayed Figure 19. THE METABOLISM OF DOPA CHICHNHZ COOH CH 2I’.ZOCOON "O _—_»H 3, 4- DIHVDIOXV 3, 4- DIHYDIOXY PHENVLALANINE (DOPAI pHENVLPVRUVIC conversion of DOPA to 3.4 dihydroxyphenyl- acetic acid 2 and homovanillic acids (fig. 19) or that some inhibitor might be present affect- ing the nonenzymatic conversion of DOPA to melanin. There was also an increase in urine taurine, 3 methylhistidine, anserine and came- sine which decreased substantially on a meat- free diet. Both children increased markedly in height and weight when they were given a liberal diet, and DOPA disappeared from the urine. It could not be convincingly demonstrated that the biochemical error was more than an evanescent feature of the malnutrition. DOPA excretion, however was not a feature of children with kwashiorkor.3 References 1. Copps, S. 0., Gerritsen, T., Smith, D. W. and Wais— man, H. A.: Urinary excretion of 3,4-dihydroxy- phenylalanine (DOPA) in two children of short stature with malnutrition. J. Pediatrics 62:208. 1963. 2. Shaw, K. N. F., McMillan, A. and Armstrong, M. D.: The metabolism of 3,4-dihydroxyphenylalanine. J. Biol. Chem. 226: 255, 1957. 3. Edozien, J. 0., Phillips, E. J. and Collis, W. R. F.: The free amino acids of plasma and urine in kwashiorkor. La/ncet i: 615, 1960. Phenylketonuria with a—hydroxybutyric aciduria: The Cast—house syndrome Clinical and laboratory findings Only one case of this syndrome has been de- scribed.1 This was a female infant of healthy 772-952 0 - 66 - ’7 CH2CHO HO CH2COOH CH3 CHZCOOH HO HO 3, ‘- DIHVDROXYPHENVL ACETALDEHVDE 3,4-DIHVDIOXYPHENVL HOMOVANILLIC ACETIC ACID ACID unrelated parentage who was born normally at term. By the fourth day of life she had begun to have extensor spasms, with flaccidity and complete unresponsiveness in the interim. During the 10 months until her death she re- mained hypotonic and with no apparent aware- ness to her surroundings. In the latter 5 months of life she had frequent episodes of unexplained fever, hyperpnoea, and generalized pitting edema. The infant had a small amount of un- pigmented scalp hair. At post mortem the kidneys were large with an apparent hypertrophy of the proximal con- voluted tubules. Apart from an unduly soft consistency, the brain was grossly normal but microscopicaly showed widespread demyelina- tion. The autopsy findings were otherwise un- remarkable. A striking feature was the curious smell in this child’s urine which was noted on the first day of life. The urine gave a persistent green color on the addition of ferric chloride. The presence of phenylpyruvic acid was confirmed by one—dimensional paper chromatography and by direct measurement and shown to amount to 134 mg/24 hrs (normal <10 mg/24 hrs). The curious urinary smell however was associated with an area of the chromatogram which con— tained no keto acids. An ethereal extract of the urine was further chromatographed in a variety of solvent systems which demonstrated that the characteristic smell was due to a-hydroxybu- tyric acid and its lactones (fig. 20); phenyl- acetic acid was also shown to be present. Two- dimensional paper chromatography 2 also dem- onstrated that the urine contained excessive amounts of phenylalanine, methionine, and tyrosine. After some months of storage the urine was re-examined and shown also to con- 45 tain greatly increased amounts of leucine and isoleucine, as well, as of phenylalanine, methi- onine, and tyrosine; alanine on the other hand was significantly diminished. Other com- pounds formed in the urine in excess were in- dolelactic acid, p.hydroxyphenylacetic acid, and p—hydroxyphenylacetic acid, but not o-hydroxy- phenylacetic acid. Biochemistry The precise biochemical aberration in this disease is not yet known and unfortunately tis- sues were not preserved for more elaborate an- alysis, nor have any further cases been recorded. The increased urinary content of the keto acids of phenylalanine, tyrosine, and alpha amino butyric acids and some of their derivatives (see fig. 20) suggests some generalized inability to metabolize keto acids. It is possible that the urine also contained increased amounts of the keto acids of leucine, isoleucine, and methionine, but this was never confirmed. The syndrome in some ways resembles maple syrup urine dis- ease in its apparent biochemical lesion. The aminoaciduria, however, was quite distinct. Diagnostic and screening tests The condition should initially attract at- tention because of the curious smell in the urine and the positive ferric chloride tests. Simple one-dimensional paper chromatography would also show the characteristically increased amounts of the leucines, methionine, tyrosine, and phenylalanine? Two-dimensional paper chromatography or column chromatography 4. 5 should confirm the diagnosis in conjunction with paper chroma- tography of the keto acids and their deriva- tives. Genetics The inheritance of this condition has not been established. Treatment No treatment has been described for this condition. References Smith, A. J., and Strang, L. B.: An inborn error of metabolism with the urinary excretion of a-hy- droxybutyric acid and phenylpyruvic acid. Arch. Dis. Child. 33: 109, 1958. . Jepson, J. B., Smith, A. J., and Strang, L. B.: An inborn error of metabolism with urinary excretion of hydroxyacids, ketoacids, and aminoacids. Law Get ii:1334, 1958. . See technical section page 71. . O’Brien, D., and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro-biochemical techniques. Hoeber, New York, 3d ed. 1962, p. 25. . Spackman, D. H., Stein, W. H. and Moore, 8.: Auto- matic recording apparatus for use in the chroma- tography of amino acids. Anal. Chem. 30:1190. 1958. t'“ N 03 .4; O! Figure 20. THE FORMATION OF a-HYDROXYBUTYRIC ACID COOH COOH COOH . TRANSAMINATION . I CIHNH2+ R-CO—COOH c=o+ R-CHNH5COOH , CHOH CH2 KETO ACID CH2 AMINO ACID CH2 I I I CH, CH, CH, .L-AMINO JcKETOBUTYRIC ACID aL-HYDROXY BUTYRIC ACID 46 BUTYRIC ACID A? Figure 21. PRESSOR AMINE METABOLISM CH:CHNH;COOH lodo'yvosines CH7CHNHICOOH Phenylalanine 3 METHOXV 4 HYDROXY PHENYLALANINE CH,O CH2CHNH7COOH HO - Dopaquinona TYROSINE 5:6 Dihydvoxyindolo Melanin A HydvoxyphenyloceOuldehyde CHZCHNHICOOH 0H \ [Hydroxyphonylcuefic "o BADihydvo-yphonyl | , N methylvyvomine HO a cum. Hordenune CH:CH:NH2 CH2CH2NNz DOPAMINE NORSVNEPHRINE O (”.10 C00" no —— SVNEPHRINE 0“ "0 Yyromvne ”0 P-HYDROXVMANDELIC ACID HO 3 4 DuhydIo-yphonyl DV'uv-( and I CHOH CH2NH1 (“01° G NOREPINEPHRINE HO —— o no ancoou cmcoon /// H/O// O '—“’ O '/// 3 4 0- hvd'o-vou'oldehvd- / CHOHCH NHCH one No / 7 3 HO HO ,' —_ / cuoncoo/ CHOnanH "00 EPINEPHRINE HOMOVANILLIC ACID J‘D‘hYd'Olvvher‘ylcuelu no t / 00” :N:Q / on / (”two ("KM-N” / NORMEYANEPHRINE C"°“C“2N”C"J ‘ 3 4 Dmvoloxv / CH0 *7 cm 0 MANDEUC ACID cnjo HO HO HO Homovorull." 3 Me'holy'ylamvne METANEPHRINE COnOMCOOH 3 MEYHOXOVH 4 HVDROXV PHENVl GlVCOl AlDEHYDE QC 3 MEYHOXV A HYDROXV MANDEUC ACID Familial dysautonomia Familial dysautonomia 1' 2 is a genetically- determined syndrome seen primarily in Jewish children. Symptoms are noticeable from birth onwards. Autonomic dysfunction is a prom— inent feature, as evidenced by lack of emotional lacrimation, hyperhydrosis, transient skin blotching, swallowing disturbances, labile blood pressure with postural hypotension, and erratic temperature control. The entire nervous sys- tem, however, is involved with findings such as insensitivity to pain, deep tendon areflexia, poor motor coordination, emotional instability, and not infrequently, mental retardation. Reported objective findings include pupillary meiosis in response to local mecholyl, abnormal response to intradermal histamine, the absence of fungi— form papillae on the tongue, and an increased pressor response to epinephrine. The prog- nosis for life is poor, death being caused by respiratory infection or “autonomic decompen— sation” with vascular collapse. Recently it has been shown 3 that there is a relative inhibition of the metabolism of dop- amine to vanylmandelic acid (VMA) as 0p- posed to homovanillic acid (HVA) in the urine. The mean figures were as follows: VMA Mg/g HVA [Lg/g HVA/ creatinine creatine VMA Normal ....... 3.1i 1.8 3.8i 1.5 1.4i0.55 Dysautonomic. 1.7:l:0.84 7.7:]:3.7 4.7;i:l.6 This change in ratio can be approximately referred by paper chromatography of the phe- nolic acids in the urine.4 The biochemical ab- normality, however, does not explain the mental deficiency, nor at the present does it offer any potential for therapy. Urine o-tyrosine is also increased. References 1. Riley, C. M., Day, R. L., Greeley, D. M., and-Lang- ford, W. S.: Central autonomic dysfunction with defective lacrimatiocn: report of ,5 cases. Pedi- atrics 3: 468, 1949. 2. Riley, C. M.: Familial dysautonomia. Pediatrics IX: 157, 1957. 3. Smith, A. A., Taylor, T., and Wortis, S. 3.: Ab- normal catecholamine metabolism in familial dysautonomia. New Eng. J. Med. 268:705, 1963. 4. See technical section page 97. Adm/noes in Miycellaaeoax a’imrdem Idiopathic hyperglycinemia Clinical and laboratory findings Nine cases have been reported so far of this rare inherited syndrome whose clinical features include episodes of vomiting, severe dehydra- tion with acidosis, marked ketosis, hypotonia, repeated infections, somatic and mental retar- dation, and hyperglycinemi-a with glycinuria. The first case 1' 2' 3 showed vomiting, hyper- 48 pnoea, and ketosis a few hours after birth. These symptoms persisted so that at 10 days a pyloromyotomy was carried out. The vomit- ing continued and at 2 months the infant had been readmitted with the additional symptoms of neutropenia, episodic thrombocytopenia with purpura, frequent infections, and glycinuria. At this time also there occurred myoclonic jerks and staring spells. Physical examination showed a chronically ill, small, wasted child considerably retarded in both somatic and in- tellectual development. There was some brown- ish discolouration of the teeth, the liver edge was palpable 2 cm below the costal margin, and the hands and fingers showed athetoid movements. The second case,4 also a male, developed episodes of severe dehydration with acidosis at 4 months of age. Thereafter there were re- peated admissions to hospital, for the most part precipitated by episodes of drowsiness, leth- argy, anorexia, dehydration, and acidosis. At 13 months of age this infant was greatly re- tarded somatically and mentally. The liver edge was again palpable but there were no other abnormal physical signs. A third infant was briefly described by Cochrane et al.‘5 who presented at 12 days of age with difficulty in feeding, vomiting, dehydra- tion, ketosis, and progressive listlessness. The child was resuscitated with intravenous fluids, but regressed as soon as milk feeds were reintro- duced; death occurred at 2 months as a result of intractable acidosis. Of the last cases, one in a newborn male in- fant,6 died at 4 days of age in marked respira- tory acidosis, the other at 11 days.10 Other features of this series of cases were generalize-d osteoporosis seen in one instance, neutropenia with a decrease in bone marrow myeloid elements in two, intermittent thrombo- cytopenia in three, and hypogammaglobuline- mia in one. Autopsy findings were briefly recorded on three patients, but showed no consistent pattern. Aside from the evidence of acidosis and the above noted changes in the WBC and plate- let counts, routine laboratory procedures were unremarkable. The definitive abnormalities of the syndrome were noted in the amino acid com- position of plasma and urine. The predomi- nance of glycine on paper chromatography of urine originally focused attention on this amino acid. In the plasma, however, even though the glycine level may be five times the normal (0.18 —-0.3 pmols/ ml) , significant increases have also been shown for serine, alanine, and glu— tamic acid,”6 as well as less consistently for threonine, proline, valine, leucine, iso—leucine, and tyrosine“,6 In the infant’s urine, glycine may be as much as 20 times the normal (6:2.5 amols/1.73m2/min); there is also a smaller but still significant increase in valine and leucine levels. Taurine and serine excre- tion may be low, however. Biochemistry The possible pathways of glycine metabo— lism are shown in figure 22. In the process of investigating which of these might be at fault, it was found in the first case that whole blood glutathione levels were normal and the infer- ence made was therefore that there was no im- pairment in the conversion of glycine into glutathione.1 Conjugation of glycine, its con- version to urinary oxalate, and incorporation of labelled glycine into protein likewise appeared to be normal.1 Creatinuria also appeared to be within normal limits in relation to the small muscle mass. In one case5 the finding of a marked increase in iron-containing pigment in the Kupfer and parenchymal cells of the liver, as well as in the macrophages and reticulo— endothelial cells of the bone marrow implied a bone defect in the incorporation of glycine into porphyrins. The absence of similar findings in another autopsy on an infant 6 suggests however that this may not be the case; although the second infant was some weeks younger than the first. Although the administration of serine in- creased plasma glycine, the urinary excretion of serine was either absolutely low 2' 6 or low in relation to the glycine load,‘1L so that possibly the hyperglycinemia is due to an impaired con- version to serine.12 The level of glycinuria, in- cidentally, appeared to Show a significant nega- tive correlation with the polymorphonuclear count. The disorder in glycine utilization, al- though the most strikingly reflected in serum and urine chromatograms, is not the only dis- turbance of amino acid metabolism. In the investigation of the first case it was clearly shown that the oral administration of leucine, to a lesser extent threonine, and isoleucine, as well as valine and methionine, which also led to vomiting and lethargy, could produce a prompt exacerbation of symptoms}! 3 After oral loading with any of the amino acids 2’ 3 individually, plasma levels are abnormally sus- 49 tained, but it should be noted that high serum leucine levels 5, 6 are not necessarily accom— panied by an increased urinary output. As presently understood, the syndrome is thought to result from an impairment of the conversion of glycine to serine, leading to an increase in the total body glycine pool and to hyperglycinemia and hyperglycinuria. The hyperglycinemia itself may then induce a sec— ondary distortion of amino acid homeostasis in the extracellular water, and a disturbance of the normal balance of competition in the transport of amino acids into cells. In particular, the metabolism of leucine, isoleucine, threonine, valine and methionine is in some Way altered, which in turn is a principal cause of the symp- toms. Screening and diagnostic laboratory tests Present evidence is that symptoms start early in the newborn period in the majority of cases. In one case, at any rate,6 the hypergly- Figure 22. OXALATE GLYOXYLICACID l TISSUE PROTEINS-<— CH2COOH | NH2 GLYCINE / GLYCINE CONJUGATES cinemia and hyperglycinuria were apparent on the first day of life. A one-dimensional screen- ing test for amino acids8 will easily reveal changes of the magnitude found and the pre- dominance of glycine in both aminoacidemia and amino aciduria can be shown on two-dimen- sional paper chromatography. A more precise definition of amino acid changes is not needed for diagnosis, but can be obtained by column chromatography. The condition is easy to distinguish both clinically and in the amino acid changes from the hyperglycinuria, glu— cosuria, glycylprolinuria syndrome,7 and from hyperprolinemia with glycinuria.11 A new variant of this condition" has re- cently been described in which there was severe retardation, seizures, failure of somatic growth, microcephaly and spastic paraplegia. Neutrope- nia, thrombocy‘topenia and acidosis were not features in this variant which is thought to be a specific glycine-oxidase deficiency on account of the hypo-oxaluria. Some of the earlier cases (e.g. Mabry and Karam South. Med. J. 56': 1444, 1963) may have been of this type. THE METABOLISM OF GLYCINE /NH' CH3 CREATININE CREATINE A NH2 ——>— HN=C HNCHZCOOH \GLYCOCYAMINE cuzon I H2N*CH-COOH SERINE GLUTAMYL-CYSTEINYl-GLYCINE (GLUTATHIONE) 50 Genetics Three of the reported cases are in male in- fants, in the case of the fourth 4 the sex is not stated. Reports also suggested that at least three male siblings died with a similar syn- drome in the first few days of life. Consanguin— ity of the parents is not apparent and in two instances 1'6 normal amino acid patterns were seen in the plasma and urine of both parents. The condition is certainly familial and present evidence suggests that it may be a sex- linked recessive. Treatment It was apparent in the first recorded case that the episodes of vomiting, ketosis, and lethargy could be effectively contained by intra- venous glucose and electrolyte solutions, and that the reintroduction of formula might ex- acerbate these symptoms. It was at first thought that the hyperglycinemia per se might be the main cause of symptoms. Sodium benzoate was therefore administered.1 The conjugation of glycine with benzoate to form hippurate led to a pronounced fall in plasma and urine gly- cine with no change in urinary serine. The symptoms, however, persisted. However, the limitation of the protein intake to 1.0 g/kg/24 hrs has produced a favorable response in one case 3 with improvement in the clinical manifes- tations except continuing failure to grow. It has recently been shown that the keto- genic amino acids have less of an effect when ingested with nonketogenic acids.3 A trial is at present underway of a diet containing 45 g. a day of pure amino acids as the only amino nitrogen supplements. To this has been added minimal daily requirements of the ketogenic amino acids. References 1. Childs, B., Nyhan, W. L., Borden, M., Bard, L., and Cooke, R. E. : Idiopathic hyperglycinemia and hy— perglycinuria: a new disorder of amino acid metabolism. I. Pediatrics 27: 522, 1961. 2. Nyhan, W. L., Borden, M., and Childs, B.: Idio- pathic hyperglycinemia : a new disorder of amino acid metabolism. II. Pediatrics 27:539, 1961. 3. Ohilds, B. and Nyhan, W. L.: Further observations of a patient with hyperglycinemia. Pediatrics 33: 403, 1964. 4. Nyhan, W. L., Chisolm, J. J ., and Edwards, R. 0.: Idiopathic hyperglycinuria. J. Pedidt. 62:540, 1963. 5. Cochrane, W., Scriver, C. R., Krause, V.: Hyper- glycinemia-hypergly‘cinuria syndrome in a new- born infant. Soc. Pediatric. Research p. 102, 1963. (abstr). 6. Visser, H. K. A., Veenlstra, H. W., and Pik, 0.: Hyperglycinemia and hyperglycinuria in a new- born infant. Arch. Dis. Child. 39: 397, 1964. 7. Scriver, C. R., Goldbloom, R. B., and Roy, 0. C.: Hypophospha‘temic rickets with renal hypergly- cinura, renal glucosuria, and glycyl-prolinuria. Pediatrics 34 : 357, 1964. 8. See technical section page 71. 9. Gerritsen, T., Kareggia, E., and Waisman, H. A.: A new type of idiopathic hyperglycinemia with hypo-oxaluria. Pediatrics 36: 882, 1965. 10. Sass-Kortsak, A., Choitz, H. 0., Balfe, J. W., Levi- son, H., Hanley, W. B., and Jackson, S. H. : Neo- natal hyperglycinemia. J. Pediat. 67 : 945, 1965 (abstr). 11. Schafer, I. A., Scriver, C. R. and Efron, M. L.: Familial hyperprolimemia, cerebral dysfunction and renal anomalies occurring in a family with hereditary nephropathy and deafness. New Eng. J. Med, 267: 51,1962. 12. Nyhan, W. L. and Childs, B.: Hyperglycinemia. V. The miscible pool and turnover rate of gly- cine and the formation of serine. J. Clin. Invest. 43: 2404, 1964. The oculo—cerebro—renal syndrome Clinical and laboratory findings In 1952 Lowe, Terrey, and MacLachlan 1 first described a syndrome in male infants char- acterized by buphthalmos, cataracts, mental and somatic retardation, hypotonia, and hypo- phosphatemic rickets, together with a variety of renal tubular defects. These children com- monly present shortly after birth because of the cataracts 0r hydrophthalmus. In the three earliest reported cases the latter condition was due to anomalies of the canal of Schlemm in two cases, and to synechiae between the iris and lens in the third. Corneal milkiness 1’ 2 is also common in addition to the lenticular opacities. The eye signs, together with prominent epican- thal folds and a tendency to scaphocephaly and 51 frontal prominence, give these children some- thing of a uniform faciesF’S'4 Other abnor- malities reported include a high arched palate, tapered fingers, hypotonia, hyporeflexia, and deafness.2 There is a variable incidence of rickets, with elevation of serum alkaline phos— phatase, low serum phosphorus, low to normal serum calcium values, along with a hyper- chloremic acidosis. The abnormal findings in the urine include intermittent cylinduria 3 and tubular protein- uria, 4' 5 as well as increased tubular rejection of phosphorus 3' 4 .and occasional glucosuria. Other observed tubular defects are a tendency to form an alkaline urine and an impairment of ammonium and hydrion excretion, which is ac- centuated by ammonium chloride loading. Perhaps the most striking urinary abnormality is the very pronounced aminoaciduria. This may reach levels as high as 40 mg/kg/24hrs (normal 2 mg/kg/24 hrs). Apart from a single report 6 of raised serum glutamine and gluta- mic acid levels, plasma amino acids appear to be normal and the increased urinary loss a wholly renal phenomenon. The amino acid— uria 3 appears to affect all the common amino acids, but especially ornithine aspartic, proline and hydroxyproline. (Schwartz, Hall and Gabuzda. A.M.A.J. Dis. Child. 104:484, 1962 labstr.) Valine, leucine, and isoleucine excretion appear to be normal. The original authors 1 considered that hy- perorganicaciduria was a notable part of the syndrome; subsequent reports, 3’ 6 however, have only shown a rather modest increase above normal. In one instance an attempt to define individual organic acid components by paper chromatography 8 did not show any increase. Unlike some of the specific aminoacidurias, this syndrome offers no real explanation of the mental retardation. The characteristic exemp- tion of valine, leucine, and isoleucine, the triad involved in maple syrup urine disease, from the amino aciduria suggests a rather complex transport phenomenon, at least at the renal tubule. Although the normal plasma levels are against related defects in other tissues, it is possible that the cerebral maldevelopment may be brought about by some homeostatic distor- tion of intraneuronal amino acids such as may 52 lead to cerebroside defects in the phenylke— tonuric brain. Attempts have been made to explain the renal tubular defects on anatomic grounds. Although swan neck lesions 7 and tubular mito- chondrial changes3 have been reported, these have not been uniform. Screening and diagnostic tests The clinical syndrome is striking enough on its own and the determination of amino- aciduria confirmatory rather than diagnostic. The latter feature could be readily observed on any one or two—dimensional screening chroma- togramsfiv 1° Genetics With one exception of a fatal case in a 2-month-old apparent female, 7 all reported cases have been males and there is abundant evi- dence of the familial occurence of the syndrome. Present evidence is that this is a sex-linked re- cessive inheritance transmitted by the female carrier where the homozygous female state is nonviable. Lenticular opacities have been observed 1" in supposed female carriers. De- tailed studies of renal function in female kindred are not available, but in one case the mother had albuminuria and cystinuria, as well as cataracts.2 Treatment Surgical treatment of the eye defects may be required, but otherwise there is no specific treatment. Vitamin D requirements for the rickets are variable and range from 1,000 to 20,- 000 I.U./24 hrs. At the same time approxi- mately 20—40 mEq of alkali should be given orally as sodium citrate or sodium bicarbonate. References 1. Lowe, C. U., Terrey, M., MacLachlan, E. A.: Or- ganicaciduria, decreased renal ammonia produc- tion, hydrophthalmos, and mental retardation. A.M.A. J. Dis. Child. 83: 164, 1952. 2. Terslev, E.: Two cases of aminoaciduria, ocular changes, and retarded mental and somatic devel- opment (Lowe’s syndrome). Acta Paediatrica 49: 635, 1960. 3. Schoen, E. J., and Young, G.: Lowe’s syndrome: abnormalities in renal tubular function in combi- nation with other congenital defects. Am. J. Med. 27: 781, 1959. 4. Richards, W., Donnell, G. N., Wilson, W. A., and Stowens, D. S.: The oculo-cerebro-renal syn- drome of Lowe. A.M.A. J. Dis. Child. 100: 707, 1960. (abstr). 5. Butler, B. A., and Flynn, F. V.: The proteinuria of renal tubular disorders. Lancet ii: 978, 1958 6. Bickel, H., and Thursby-Pelham, D. 0.: Hyper- aminoaciduria in Lignac-Fanconi disease, in galactosemia, and in an obscure syndrome. Arch. Dis. Child. 29: 224, 1954. 7. Scholten, H. G.: Ein meisje met het syndroom von Lowe. Maandsohrlft v. Kindergn. 28: 251, 1960. 8. Dedmon, R. E., Dent, G. E., Scriver, C. R., and Westall, R. G.: The urinary excretion of organic acids in man: A survey of a variety of metabolic disturbances by two-dimensional paper partition chromatography. Clin. Chim. Acta. 6: 291. 1961 9. See technical section page 71. 10. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro and ultra micro biochemical techniques. Hoeber, New York. 3d edit. 1962. page 25. 11. Dent, C. E., and Smellie, J. M. : Two children with the oculocerebro-renal syndrome of Lowe, Ter- rey, and MacLachlan. Proc. Roy. Soc. Med. 54: 335, 1961. Maple syrup urine disease: Branched chain ketoaciduria Clinical and laboratory findings More than 20 cases of this disease have been reported since it was first described just over 10 years ago.1 'Characteristically, symp- toms start in the neonatal period, sometimes in the first few days of life, with difficulty in feeding, lethargy, anorexia, episodic rigidity, diminished awareness, and occasionally convul- sions. At the same time as the symptoms de- velop the urine acquires a characteristic odor from which the name of this disease derives. The condition affects both sexes, has been widely reported over the world, and appears to be uniformly fatal in its common form if untreated. The results of conventional laboratory pro- cedures are unremarkable, but 3 years afterthe early description 2 it was shown that there were greatly elevated values of valine, leucine, and isoleucine in the plasma and urine. Methionine levels were originally thought to be increased in plasma and urine as well, but the peak on column chromatography has since been shown to be alloisoleucine.3 Not long thereafter, as a result of the positive ferric chloride and dini- trophenylhydrazine test, a series of papers 4-“ demonstrated that the keto acids, a-ketoisova- leric, a—ketoisocaproic, and a-keto-B-methylva- leric corresponding to valine, leucine, and isoleucine accumulated in spinal fluid and plasma and were also excreted in the urine. The plasma levels of proline, serine, and taurine are also elevated above normal.8 Biochemistry The finding of increased plasma and urine levels of valine, leucine, and isoleucine and of their corresponding keto acids and a-hydroxy acids 12 suggests that the basic enzymatic block is in the oxidative' decar‘boxyl-ation of the keto acids, as illustrated in figure 23. So far, how- ever, the keto derivative of alloisoleucine has not been identified. Leukocytes have been used for the in vivo investigation of the exact defect. C“-la‘belled leucine, isoleucine, and valine were added to a leucocyte suspension 7 and the amount of labelled 002 evolved and keto acid formed were determined. Each amino acid showed an accumulation of counts in the pre- cipitated DNPH: keto acid complex with virtu— ally no ‘COZ produced, as compared to the nor- mal. The experiment additionally suggested that a single decarboxylase facilitated the on- ward metabolism of all three amino acids, rather than that there was one specific decar- boxylase deficiency with accumulated metabo- lites competitively inhibiting the others. Oral loads of 5 g. of the individual amino acids involved3 led to increased levels of the loaded amino acid in the plasma, as well as to slightly increased levels of the other branched chain amino acids. In the case of valine, no symptoms were caused in an infant rendered asymptomatic by previous dietary therapy, nor did any odor appear in the urine. This should 53 Figure 23. THE KETOACIDS 'IN MAPLE-SYRUP-URINE DISEASE CH3 \CH-CH-z-CHNH-2COOH —————>— / CH3 LEUCINE CH3 CH-CHNHECOOH —-» CH3CH2 ISOLEUCINE CH3 \ HCOOH ———>- _ /CHN 2 CH3 VALINE not imply however, that the hypervalinemic component may not be harmful as evidenced by the single case of hypervalinemia.8 L-iso- leucine likewise produced no abnormal neuro— logical symptoms, but did lead to a rapid ap- pearance of the characteristic odor in the urine. L-leucine, on the other hand, did not lead to urine changes but within three hours neuro- logical changes could be detected with irri- tability, lethargy, unsteadiness of gait, and involuntary movements. Despite the striking symptomatic effects of L-leucine, the actual mechanism for the cerebral disorganization must remain obscure until some explanation can be given for the nonlethal variety of the dis- ease,9 where the biochemical changes are only apparent intermittently with episodes of infec- tion. Two pointers to the mechanism have been given. The keto derivatives of leucine and valine have been shown to inhibit the activity of L-glutamic decarboxylase 1° which might in- 54 CH CH 0 3 H \CH-CHz-C-COOH 3 k-KETOISOCAPROIC ACID CH3 0 H CH— C'COOH CH3CH2 fi-KETO-B-METHYLVALERIC ACID CH3 0 CH'C'COOH CH3 L-KETOISOVALERIC ACID terfere with the availability of energy within the brain as has been suggested in pyridoxine dependency. In addition, post mortem studies have shown a defect of myelination.11 Screening and diagnostic procedures The urine from these children gives posi- tive ferric chloride and dinitrophenylhydrazine reactions. Plasma amino acid levels for phen- ylalanine and histidine are normal however. Confirmation can be obtained by one- 13 or two- dimensional paper chromatography, or more satisfactorily by colunm chromatography.15 Genetics Present evidence is that the condition is inherited as an autosomal recessive. Treatment There is now good evidence,3' 6 that the ab- normal biochemical findings can be controlled DIET (g. /day unless stated otherwise) after \Westall16 First Second Third Fourth Fifth Sixth Seventh Eighth 28-day month month month month month month month period Amino acid mixture: 1-alanine . . . . . ........ 0.72 0.84 0.40 0.46 0.46 0.46 0.46 0.46 l-aspartic acid. ....... 1.60 1.87 1.00 1.16 1.16 1.16 1.16 1.16 l-glutamic acid. ...... 3.92 4.55 2.00 2.32 2.32 2.32 2.32 2.32 l-cystine .......... . . . . 0.72 0.84 0.90 1.04 1.04 1.25 1.25 1.25 1-histidine. . ...... . . . . 0.48 0.56 0.56 0.65 0.65 0.65 0.65 0.65 l-proline. ...... . ..... 1.40 1.63 0.70 0.81 0.81 0.81 0.81 0.81 l-phenylalanine ..... . . 0.96 1.12 0.43 0.50 0.50 0.89 0.89 0.89 1-tyrosine ............. 0.88 1.03 1.05 1.21 1.21 1.23 1.23 1.23 l-serine... ..... 0.92 1.07 0.96 1.11 1.11 1.11 1.11 1.11 l-threonine ..... . . . . . . 0.92 1.07 0.23 0.27 0.27 0.54 0.54 0.54 l-arginine ............ 0.80 0.93 0.66 0.76 0.76 0.77 0.77 0.77 l-lysine HC1.......1.12 1.30 0.45 0.52 0.52 0.53 0.53 0.53 1-tryptophan ..... .. .. 0.36 0.42 0.10 0.12 0.12 0.23 0.23 ' 0.23 l-methionine ..... . . . . 0.40 0.46 0.10 0.12 0.12 0.24 0.24 0.24 Glycine ............... 0.48 0.56 1.00 1.16 1.16 1.16 1.16 1.16 Total............. 15.68 18.23 10.54 12.21 12.21 13.35 13.35 13.35 Gelatin........... ........ ........ . 5.0 5.8 5.8 5.8 5.8 7.0 Liquid milk (ml.)....... 75-100 110—130 120 130 135 ...... ........ Skimmed milk (dry) .............. . .. . . ........... . . . . . . . .. ......... 13.4 13.4 15.0 Arachis oil (ml.) . . . . . . . . 20 23 30 36 36 40 40 40 Sucrose... . . . . .......... 40 46 60 72 72 75 75 75 Baker’s yeast ............ . . . . . . . . .............. 5 5 10 10 Mashedcarrot................ . . ....... . . . 2 2 3 3 4 Apple purée (tsp) ...... . ........ 2 2 3 3 4 $13332; fifth??? See below (both A and B mineral and vitamin mixtures used). Mineral mixture (A): Calcium lactate 4.5 g., calcium chloride (CaClz, 2H20) 0.3 g., dipotassium hydrogen phosphate 1.5 g., disodium hydrogen phosphate 1.0 g., magnesium sulphate 0.8 g., ferrous sulphate 20 mg., zinc chloride 2 mg., manganese sulphate 2 mg., potassium iodide 80 pg, potash alum 30 ug., cobalt sulphate 30 fig” sodium molybdate 30 pg., copper sulphate 2 mg. Mineral mixture (B): Anhydrous sodium carbonate 0.17 g., potassium carbonate 0.22 g., and calcium carbonate 0.31 g. Added to prevent coagulation of milk proteins. Vitamin mixture (A): Vitamin A 5,000 i.u., vitamin D 1,000 i.u., aneurine 1.0 mg., riboflavine 0.4 mg., pyri- doxine 0.5 mg., nicotinamide 5.0 mg., and ascorbic acid 25 mg. Vitamin mixture (B) 2 Folic acid 0.5 mg., choline chloride 75 mg., p-aminobenzoic acid 1.0 mg., inositol 1.0 mg., biotin 50 pg, vitamin B1210 pg. Calcium pantothenate 5.0 mg. was incorporated with the amino acid mixture as it caused precipitation if added to the vitamin mixture. 55 by a diet that is low in leucine, isoleucine, and valine. Neurological signs are also controlled. Treatment, which may be an urgent need 17 in the first few days of life may lead to normal somatic and intellectual development. Pre— vention and control of intercurrent infection appears to be particularly important. One of the problems of diet therapy is that branched chain amino acids cannot be readily removed from protein hydrolysates. Formulae that pro- vided amino acids individually are expensive to compound and appear to lack an unknown essential nutritional factor. Some combination of a specially prepared casein hydrolysate or of a gelatin base with pure amino acid supplements is presently the most satisfactory solution. The program devised by Westall16 seems to offer a successful compromise using a gelatin and milk base supplemented by pure amino acids, arachis oil, yeast, sucrose, vegetable purée, and a min- eral and Vitamin mixture. References 1. Menkes, J. H., Hurst, P. L., and Craig, J .M. : A new syndrome: progressive familial infantile cerebral dysfunction associated with an unusual urinary substance. Pediatrics 14: 462, 1954. 2. Westall, R. G., Dancis, J., and Miller, 8.: Maple sugar urine disease A.M.A. J. Dis. Child. 91,: 571, 1957. 3. Snyderman, S. E., Norton, P. M., Roitman, E., and Holt, L. E.: Maple syrup urine disease, with particular reference to dietotherapy. Pediatrics 34: 454, 1964. 4. Menkes, J. H.: Maple syrup disease: isolation and identification of organic acids in the urine. Pedi- atrics 23:348, 1959. 5. Mackenzie, D. Y., and Woolf, L. 1.: Maple syrup urine diseaseL—an inborn error of the metabolism of valirne, leucine, and isoleucine associated with gross mental deficiency. Brit. Med. J. i: 90, 1959. 6. Dancis, J ., Levitz, M. Miller, S., and Westall, R. G. : Maple syrup urine disease. Brit. Med. J. i: 91, 1959. 7.. Dancis, J., Hutzler, J., and Levitz, M.: The diag- nosis of maple syrup urine disease. Pediatrics 32: 234, 1964. 8. Wada, Y., Tada, K., Minagawa, A., Yoshida, T., Morikawa, T. and Okamura, T.: Idiopathic hy- pervalinemia. Tohoku, J. Ewper. Med. 81:46, 1963. 56 9. Morris, M. D., Lewis, B. D., Doolan, P. D., and Harper, H. A.: Clinical and biochemical obser- vations on an apparently nonfatal variant of branched chain ketoaciduria (maple syrup urine disease). Pediatrics 28: 918, 1961. 10. Tashian, R. E.: Inhibition of brain glutamic acid decarboxylase by phenylalanine, valine and leu- cirne derivatives: a. suggestion concerning the etiology of the neurological defect in phenylke- tonuria and branched chain ketonuria. M etabo- lism 10: 393, 1961. 11. Silberman, J., Dancis, J., and Feigin, I.: Neuro— pathological observations in maple syrup urine disease: ,branched-chain ketoaciduria. Arch, Neurol. 5:351, 1961. 12. Patrick, A. D.: Maple syrup urine disease. Arch. Dis. Child. 36: 269, 1961. 13..See technical section page 71. 14. O‘Brien, D.‘and Ibbott, F. A.: Laboratory manual ' of pediatric micro and ultramicro biochemical techniques. Hoeber, New York, 3d ed. 1962, page 25. 15. Spackman, D. H., Stein, W. H. and Moore, S.: Au- tomatic recording apparatus for use in the chro- matography of amino acids. Anal. Chem. 30: 1190, 1958. 16. Westall, R. G.: Dietary treatment of a child with maple syrup urine disease (branched chain keto— aciduria). Arch. Dis. Child. 38: 485, .1963. 17. Lonsdale, D., and Barber, D. H.: Maple-Syrup Urine Disease. New Eng. Jonrn. of Med. 271: 1338, 1964. Ataxia, megaloblastic anemia, formimino— glutamic aciduria and mental retardation There is a single report 1 of two cases some— what resembling the one with formimino trans— ferase deficiency (see page 38). Two sisters portrayed a syndrome characterized by megalo— blastic anemia, ataxia, mental retardation, and convulsions. There was a marked defect in folic acid absorption from the gastrointestinal tract, and during acute exacerbations there was an increase in formiminoglutamic acid before and after histidine loading. There was a satis- factory hematological response to intramuscular folic acid administration, but no details are given of any impact on the mental retardation. It is suggested that this may be a complex dis- order of folic acid transport, as no evidence of any derangement of enzymic metabolism was detected. Reference 1. Luhby, A. L., Cooperman, J. M., and Pesci-Bourel, A. : A new inborn error of metabolism: Folic acid responsive megaloblastic anemia, ataxia, mental retardation, and convulsions. Am. Pediat. Soc. 75th Ann. Mtg. p. 42, 1965 (Abstr.) Sarcosinemia and sarcosinuria with men— tal retardation There is a single brief report 1 of a 1-year- old child who died at 14 months. During his lifetime he was hypotonic and retarded both intellectually and physically. The urinary sar- cosine (methyl glycine) excretion was approxi— mately one thousand times the normal for that age of around 1 pmol/24 hrs. and the serum sar- cosine some 20 times the normal level of less than 0.01 pmols/ml. Loading experiments with dimethylglycine and with sarcosine sug- gested a defect in the action of sarcosine oxidase. A 6-year-old sister of this child appeared to have a similar defect. The inheritance of the condition, however, remains obscure, and there are as yet no ideas for treament. Dimethyl glycine Choline—>Betaine——>(CH3)2'N—CH2-COOH Dimethyl glycine Sarcosme oxidas e oxidase t HgN-CHz—COOHeCHg-NH—CHg-COOH Glycine Sarcosine (methyl glycine) Reference 1. Gerritsen, T., and Waisman, H. A. : Sarcosinemia and Sarcosinuria: A new familial error of metabolism. Am. Pediat. Soc. 75th Ann. Mtg. p. 28, 1965 (Abstr.) Hereditary oroticaciduria, megaloblastic anemia and mental retardation Clinical and laboratory findings Three infants 1’ 2' 3 have been reported in whom good progress is made in the first 3 months who then become pale and listless with repeated episodes of respiratory infection and diarrhea. There is a severe hypochromic ane- mia with normal serum iron levels and severe megaloblastic changes in the bone marrow. Two were retarded mentally and in all three there was delay in somatic growth. Crystals of orotic acid are found in the urine. The deposit appears after allowing the urine to stand for several hours and character- istically adheres to the glass container. The crystals are colorless fine needles which may exceed 1 g. a day to the extent of producing an obstructive uropathy. To confirm the nature of the crystals the typical absorbance peaks at 278 mu in 0.01N. HCl and at 284 my in 0.01N. KOH can be determined. There is a typical elution peak from Dowex I. Biochemistry The biochemical pathway defining the posi— tion of orotic acid in the synthesis of pyrim— idines has been established in bacteria and some animal tissues for a number of years (see fig. 24). On the assumption that similar pathways existed in man it was proposed in the first case 1 that there might be an absence of orotidylic pyrophosphorylase and decarboxylase activity. This was eventually demonstrated in the red cells of the first case and in his family and again in the second 2 and third.3 A similar enzymic defect has been demonstrated in tissue culture of skin from one of these patients as well as in leukemic patients treated with 6-azauridine. I nberitance Enzymes studies of red cells of patients and their families suggest that the inheritance of this condition is as an autosomal recessive. Treatment These children did not show any thera- 57 Figure 24. THE METABOLISM OF OROTIC ACID THE METABOLISM or ononc ACID 0 || <3 H2N_ C— P coo” C / \ CARIAMYL PHOSPHATE Tn, l”: mnvonocaouss TH CH2 —---> —> + c cn C CH // \ / \ // \ / \ coon o N coon o N coon l n H CH2 l CARBAMYL olnvonocnonc ACID cnnn2 ASPARTIC ACID C'oon ounvoapcnonc ASPARTIC Ac”, DEHYDROGENASE on on I oaonovuc I on oaonovuc c PYROPHOS /c I DECARBOXYLASE // PHORYLASE N/ \ c 4— N CH 4— C“ // \ | I N T i” //C\ /c\ //C\ c\ C cn o 74 coon o N/ coon n // \ Pf/ R-P R-P ononovuc ACID OROT'C ‘C'D URIDYLIC ACID \ URIDINE CYTIDYLIC ACID pen-tic response to the administration of iron, folic acid or vitamin B. 12. There was a partial response to cortisone. Cytidilic acid produced some reduction in urinary orotic acid but no change in red cell precursor morphology. On the basis that there was an underlying uridine deficiency, treatment with uridine 150 mg. five times a day evoked a substantial reduction in oroticaciduria and a restoration of a normal bone marrow and peripheral blood film appear- ance. At the same time there was a substantial and sustained general clinical improvement. References 1. Huguley, C. M., Bain, J. A., Rivers, S. L., and Scog. gins, R. B.: Refractory megaloblastic anemia as- 58 sociated with excretion of orotic acid. J. Hema- tology 11;: 615, 1959. 2. Becroft, D. M. 0., and Phillips, L. 1.: Hereditary oroticaciduria and megaloblastic anemia ; A second case, with response to uridine. B.M.J. i: 547, 1965. 3. Haggard, M. E., and Lockart, L. M.: Hereditary orotic aciduria; A disorder of pyrimidine metab- olism responsive to uridine therapy. J. Pediat. 67: 906, 1965. Idiopathic hypervalinemia Clinical and laboratory findings A recent description has been given1 of a female infant with a syndrome characterized by failure to thrive, vomiting, nystagmus, hy- pervalinemia, and hypervalinuria. This infant was born at term after a normal pregnancy. The parents were nonconsanguine- ous. There was difliculty with sucking and some vomiting in the early neonatal period. There were also episodes of unexplained fever. These symptoms increased in severity and as a result she was brought to the hospital at 2 months of age. At that time she was below normal in length and had only gained 270 g over her birth weight. There was a horizontal nystagmus and she appeared to be blind. There was decreased tone in the extremities, and the child was drowsy, immobile, and unresponsive. Conven— tional laboratory investigations were all nor- mal. In addition the chromosome patterns was shown to be normal, but at 4 months of age ab- anue 25. CH3 CH3 CH CHNH COOH +_’. / 2 CH3 CH3 VAUNE CH CH3 c\ o c \ ll H/ CH§c—scoA CH3 HOCH2 H o 2 co 2 CH3 CoA SH \ ll CH C-S CoA normally shaped waves and spikes appeared in the EEG. Serum amino nitrogen was normal (2.3 ,umols/ml) and urine amino acids were 10.8 mg/kg/24 hrs., a figure which is high for the normal but difficult to assess in a mal- nourished infant. The first indication, how- ever, of a specific amino acid disorder was pro— vided by paper chromatography of the urine, which showed a disproportionate increase of valine in the urine. Subsequent estimations of amino acids were made on plasma and urine, using a Beckman/ Spinco amino acid analyzer. The results are somewhat diffiicult to interpret as the number and age of the controls were not given, and the figures for the controls are expressed as a range, THE METABOLISM OF VALINE O \ ll CH C COOH / 4-KETOISOVALERIC ACID l O \ II CH C S-CoA ISOBUTYRYL CoA . CH o 2s .. C-C—S Co A / CH3 0 METHYLACRYLYLCoA / fi-HYDROXYISOBUTYRYL CoA CH3CH2CHO -——>— CH3CH2COOH ——>— T0 GLUCOSE AND GLYCOGEN PROPIONIC ACID 59 rather than as a mean and standard deviation. No results are shown for the basic amino acids and the histidines. Nevertheless, a presum- ably fasting serum valine level of 0.9 amok/ml (normal 0.15—0.3 amok/ml) and a urine valine of 0.354 mg/mg creatinine (normal .017—.033) are strikingly above normal. No attempt was made to detect any specific enzyme defect in the metabolism of valine. Biochemistry The metabolism of valine is illustrated in figure 24. The first step is a transamination to alpha ketoisovaleric acid and thence by a series of steps to propionic acid and to glucose, via succinyl coenzyme A. In this case the ferric chloride test was negative so that it might be supposed that the appropriate keto acid, alpha ketoisovalerate was not present. This would place the defect at the level of the valine-ketoisovaleric transa— minase catalysed step. The mechanism for the neurological findings remain obscure, but valine loading in monkeys will produce retardation, convulsions, and unusual behavior.2 It is pos- sible, again, that this is analagous to phenylke- tonuria and is a condition where a disturbance of intraneuronal amino acid homeostasis leads to secondary disturbances of control, possibly in cerebroside synthesis. Screening and diagnostic tests The high levels of valine in plasma and urine are sufficient to be readily detectable by one3 or two4 dimensional paper chromato— graphy. Column chromatography is required for quantitative studies.5 Genetics Abnormal amounts of valine were reported by paper chromatography in the urine of both mother and father. Both parents were healthy so that this is possibly an autosomal recessive. Treatment No attempt at treatment was made; but it is rational to suppose that early dietary re- striction of valine might be of great help. 60 References 1. Wada, Y., Tada, K., Minagawa, A., Yoshida, T.. Morikawa, T., and Okamura, ’1‘.: Idiopathic hyper- valinemia. Tohoku J. Empe'r. Med. 81: 46, 1963. 2. Waisman, H. A., Gerritsen, T., Boggs, D. E., Poli- dora, J. J. and Harlow, H. F. 2 Mental Retardation in Monkeys: II, Branched-chain amino-aciduria and ketoaciduria. A.M.A. J. Dis. Oh/ild. 104: 488, 1962. 3. See technical section page 71. 4. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro and ultramicro biochemical techniques. Hoeber. New York. 3d edit. 1962, page 25. 5. Spackman, D. H., Stein, W. H. and Moore, 8.: Auto- matic recording apparatus for use in the chroma- tography of amino acids. Anal. Chem. 30: 1190, 1958. The syndrome of spastic diplegia, micro- cephaly, mental retardation, and amino— aciduria A single pedigree has been described 1 of a sex—linked syndrome in male infants who showed a combination of mental retardation, spastic diplegia, epilepsy, and a generalized aminoaciduria. At 15 months the index case was below the third percentile for height and weight, he showed no awareness of his surround- ings, was slightly spastic and hyper-reflexic, and had early optic atrophy. Shortly there- after he developed massive myoclonic seizures and died. Postmortem showed microcephaly and cerebellar ‘hypoplasia with a diminished number of cortical neurones and underdevelop- ment of structures in the pons and medulla. The 24 hr. excretion of urine showed 7.3 mg/kg of amino nitrogen, a level that is a little over three times the normal value. This figure was less strikingly elevated, however, in rela- tion to total urine nitrogen and creatinine. The excess amino acids were primarily glycine, ala- nine, threonine, serine, glutamine, and glutamic acids. On the evidence available it is not possible to accept the aminoaciduria as an integral part of the syndrome or as in any way ofi'ering an explanation of the anatomical distortions. The case is mentioned, however, as a reminder that aminoaciduria may occur in any inactive child who is either undernourished or losing muscle tissue for other reasons. Reference 1. Paine, R. S.: Evaluation of familial biochemically- determined mental retardation in children with special reference to‘ aminoaciduria. New Eng. J. Med. 262: 658, 1960. Glutamic acidemia and cerebral degeneration Clinical and laboratory findings There is a single report by Menkes and others 1 of five male children in a family who showed a sex linked recessive trait comprising hair changes, failure to gain, seizures, mental retardation, cerebral degeneration, and high serum glutamic acid levels._ These infants all progressed normally in the immediate postnatal period but began to fail to gain weight between 2 and 8 weeks. This failure to gain seemed to be primarily a reflexion of the infant’s disinter- est in feeding. Physical examination was for the most part normal, except for a variety of morphological defects. Micrognathia was seen in two cases, talipes equinovarus in one and pre— mature closure of the lambda] and anterior sagittal sutures in another. The hair in all the affected children was sparse, coarse, and devoid of pigment. More elaborate examination of the hair in one case showed spiral twisting and beading of the filaments. The hair also showed trichorrexis nodosa or excessive brittleness, a condition also seen in argininosuccinicaciduria.2 Pili torti, the condition of spiral twisting, has also been reported primarily in girls with enamel hypoplasia,3 cataracts, and mental re- tardation, ’ 5 but without the early and fulmi- nating cerebral degeneration shown in these cases. Focal or generalized seizures began be- tween 3 and 15 months but were relatively straightforward to control with conventional drugs. Two otherwise unaffected children in the family also had seizures. The EEG was uniformly abnormal, but showed no unique pat- tern. After some initial progress in which the infants might achieve head control and be able to smile, central nervous system function de- teriorated rapidly with the child becoming a spastic paraplegic, and finally opisthotonic. All cases died between the ages of 7 months and 31/2 years. The brains of two cases at post mortem showed widespread patchy degenera- tive areas of the cerebrum and cerebellum. A slight increase in urinary amino nitro- gen was noticed in one, case, which may well have been nutritional, and in two others in whom it was looked for, serum glutamic acid levels were repeatedly but modestly elevated to amounts twice normal levels. On the analogy that the cerebral histology resembled that in organic mercury compound poisoning," the au- thors suggested that there might be a defect in an enzyme with an —SH or disulfide group in their active site. Glutamic acid deh'ydrogenase is such an enzyme, and might impair the con- version of cerebral glutamate to ketoglutarate. It is difficult to see, however, Why the glutamate could not be decarboxylated and be available for energy via succinic semialdehyde. References 1. Menkes, J. 1-1., Alter, M., Steigleder, G. K., Weakley, D. B., and Sung, J. H.: A sex-linked recessive dis- order with retardation of growth, peculiar hair, and focal cereme and cerebellar degeneration. Pediatrics 29: 764, 1962. 2. Allan, J. D., Cusworth, D. 0., Dent, C. E. and Wilson, V. K.: A disease, probably hereditary, character- ized by severe mental deficiency and a constant gross abnormality of amino acid metabolism. Lancet 1': 182, 1958. 3. Appel, B., and Messina, S. J ., Pili torti hereditaria. New England J. Med. 226: 912, 1942. 4. Scheer, M.: Monilethrix. Arch. Derm. Syph. ’7: 275, 1923. 5. Ullmo, A.: Un nouveau type d’agenesie et de dys- trophie pilaire familiale et hereditaire. Derma- tologica. 90: 75, 1944. 61 6. Hunter, D., and Russell, D. 8.: Focal cerebral and cerebellar atrophy in a human subject due to or— ganic mercury compounds. J. N enrol. Neurosurg. Psychwt. 17: 235, 1954. Mental retardation, cortical atrophy and increased glutamic acid in the C.S.F. There is to date only a single case report 1 concerning the above syndrome. The descrip— tion is of a male infant who was first noticed to “fail to thrive” at around 3 months of age. At 10 months of age he was overtly retarded men- tally as well as physically. His awareness was greatly subdued as was normal motor activity. Apart from poor growth, increased bone in the extremities and hyperreflexia, the physical find- ings were normal. Three male cousins and an uncle had died in infancy from an apparently similar syndrome, but none had been investi- gated. Pneumoencephalograms showed bilat- eral cortical atrophy with normal angiograms. Column chromatography of serum reput- edly showed some elevation of glutamic acid, proline and leucine, but of all these only proline seemed remarkable in relation to other pub- lished figures. There was in addition a modest aminoaciduria of 7.3 mg./kg./24 hrs. Amino acid analysis of the C.S.F. showed an increase in the glutamic acid to about four times the normal level of 12 umols/ 1. with some reduction in glutamine. In the absence of an assurance that the samples from controls and the patient were identically treated there is a possibility that the biochemical change is arti— fact, since glutamine will normally form glu- tamic acid in C.S.F. on standing at room temperature. Galactosuria was also reported. The authors remark on certain similarities with a syndrome reported by Menkes.2 References 1. Yoshida, T., Tada, K., Mizuno, T., Wada, Y., Aka- bane, J ., Ogasawara, J ., Minagawa, A., Morikawa, '1‘., and Okamura, T.: A sex-linked disorder with mental and physical retardation characterized by cerebrocortical atrophy and increase of glutamic acid in the cerebrospinal fluid. Tohoku. J. Med. Sci. 83:261, 1964. 2. Menkes, J. H., Alter, M., Steigleder, G. K., Weakley, D. R., and Sung, J. H.: A sex-linked recessive disorder with retardation of growth, peculiar hair and focal cerebral and cerebellar degeneration. Pediatrics, 29: 764, 1962. Other Rare Inborn Errors of Metabolism Associated With Mental Retardation “T” substance in the urine Six cases have been reported 1’ 2 in four un- related kindreds of children who excreted an abnormal ninhydrin positive substance in the urine. The abnormal urinary constituent was found in both male and female members of the family in smaller amounts, suggesting a hetero- 62 zygote state in an autosomal recessive inheri- tance. Two of the affected homozygotes were of normal intelligence, but the others were men- tally and usually somatically retarded. No specific physical findings were associated with these children, although one was found to have a horseshoe kidney. When the urine was chromatographed on Whatman #1 paper3 in a phenol: ammo- nia and butanol : butanone: dicyclohexylamine: water system and stained with ninhydrin, the excretion of identifiable amino acids appeared to be normal. However, there was an addi- tional spot which moved close to taurine. This was reddish-purple in color, the color being de- stroyed by acetic acid and restored by ammonia. When eluted with 50 percent acetone and evapo— rated, the pale pink residue gave a purple color with ammonia. The substance was only pres- ent after electrolyte desalting of the urine, and disappeared promptly when purine substances were excluded from the diet. The spot did not reappear when 100 mg/2él hrs of cafl'eine, ade- nine, quanine, xanthine, cytosine, thymine, or uridine were added to the diet, but did reappear on resumption of normal diet. The diet was supposedly free of exogenous dyes and no such spot was seen in the urine of many children on the same diet. At one time it was thought to be alloxan. A more recent hazard was that it might be a quinone or quinolone; it has not been accurately identified, however. Until this is done, there can be no further speculation on the possible fundamental etiology of the associated mental retardation. References 1. Coles, H. M. T., Priestman, A., and Wilkinson, J. H.: T—substance anomaly, an inborn error of purine metabolism. Lancet ii: 1220, 1960. 2. Coles, H. M. T.: T-substance anomaly with horse- shoe kidney. Proc. Roy. 800. M ed. 51,: 330, 1961. 3. Bowden, C. H.: An improved solvent combination for amino acid chromatography. Olin. Chim. Acta. 4: 539, 1959. Green acyl dehydrogenase deficiency This syndrome has been only briefly re- ported.1 Moreover, all the children died within the first 2 weeks of life so that it can hardly yet be accepted as an established cause of mental retardation. Nevertheless the severity of the neurological symptoms was such that mental retardation would seem inevitable in any less severe form. Three of four children of second cousin par- ents all died in the neonatal period of an illness characterized by convulsions, lethargy, hepa- tomegaly, leucopenia, and thrombocytopenia. The urine, the blood, and the breath had an unusual stale smell. Laboratory investigations revealed no changes in amino acid metabolism. There was a marked metabolic acidosis which, however, was associated with normal levels of acetoacetic acid, B—hydroxybutyric acid, and total nonesterified fatty acids. Gas chromatog- raphy of the methylated esters showed normal plasma levels of lauric (012) fatty acids and those with longer chains. Paper chromatog- raphy, however, demonstrated a marked increase in butyric acid and hexanoic acids. These findings suggest that there may have been a deficiency of green acyl dehydrogenase. Biochemistry In the breakdown of fatty acids a series of five separate enzymatic steps are involved in the general reaction shown below : R—CHg—CHg—COOH (ll 0 | —>R—C—CoA+CHa——C—COA In the second of the steps a series of en- zymes called acyl dehydrogenases govern the general reaction: 0 0 ("I—SCoA C—SCoA at # Hi: 'CH. LH 'CH. 'CH. Butyryl CoA Crotonyl CoA This group of enzymes has a certain sub- strate specificity depending on the chain length 63 of the fatty acid. Green acyl dehydrogenase is so named because of a copper-containing prosthetic group; it is primarily active on butyryl CoA, with about 50 percent of the aflin- ity for hexanoyl CoA. Such an enzyme defect would account for the biochemical findings. The block is presumably not complete, as hex— anoyl and hexadecanoyl dehydrogenases also have a perceptible, if limited, effect on the butyryl CoA. Laboratory tests This diagnosis might be considered on the basis of clinical signs, essentially the accompan- iment of early neonatal convulsions with a curi- ous odour. This would, however, have to be differentiated from maple syurp urine disease and phenylketonuria with a-hydroxybutyric aciduria. These cases have negative ferric chloride tests and in addition would show butyric and hexanoic acids on low temperature gas chromatography of serum. Genetics The inheritance is assumed, but not proven to be an autosomal recessive. Treatment No treatment was attempted but these chil- dren might be amenable to a regimen that would control the utilization of fatty acids; that is, a low fat high carbohydrate diet. Reference 1. Sidbury, J. B., Harlanel, W. R. and Wittles, B.: Description of an apparent inborn error of short- chain fatty acid metabolism. A.M.A. J. Dis Child. 101,: 531, 1962. (Abstr) The syndrome of hyperuricemia, choreo- athetosis, and mental retardation Clinical and laboratory studies A syndrome has recently been described 1’ 2 in three families which is characterized by men- 64 ta] retardation, cerebral palsy, choreoathetosis, severe lip and nail biting, and hyperuricemia. Both of the index cases were well developed physically when first seen at the ages of 4 and 8 years respectively. Both showed choreoathe- tosis and had a spastic quadriplegia. Exact evaluation of their intelligence was diflicult be- cause .of the severe motor handicap, but they were considered to be significantly retarded mentally. Both showed a compulsive tendency to destructive biting of their fingers and lips. In a review of the literature on hyper— uricemia the author notes 1 two other possible cases, both of whom had choreoathetosis and mental'retardation, and one of whom showed visual evidence of lip biting.3 One of the two brothers had joint symp- toms but neither had X-ray evidence of joint involvement. Uric acid crystals were plenti- fully present in the urine and in one case this had led to hematuria. Serum uric acid was ele- vated to between 9 to 16 mg/ 100 ml. Biochemistry The exact nature of the abnormality in uric acid metabolism is not understood, nor is it com- prehended in what manner hyperuricacidemia may affect intellectual development. Uric acid excretion in the urine averaged 45 mg/kg/24 hrs and was about four times the normal. Stud— ies with labeled uric acid and labeled glycine showed that the total body pool of uric acid was 43 mg/ kg, a level of the same order as that found in adults with non-tophaceous gout, and that the rate of conversion of isotope-labelled glycine into urinary uric acid was some 200 times that of the control children. Laboratory diagnosis The diagnosis might be considered in any retarded child with choreoathetosis, especially if there is a history of compulsive lip or finger biting. Confirmation comes from finding hy- peruricemia.4 Genetics Two paternal uncles and a paternal grand- father showed asymptomatic hyperuricemia in one family. There is, however, insufl‘icient evi- dence to determine the mode of inheritance. Treatment In the 4-year-old boy the administration of probenecid 15 mg/kg/24 hrs for 4'weeks and then 25 mg/kg/24 hrs plus alkali in the form of Polycitra® decreased the serum acid level to below 5 mg/ 100 ml. It is not yet known whether treatment in the early stages of this disease could have any effect on the neurological findings. References 1. Lesch, M., and Nyhan. W. L.: A familial disorder of uric acid metabolism and central nervous sys- tem function. Amer. J. Med. 36: 561, 1964. 2. Nyhan, W. L., Lesch, M., Sweetman, L., and Hoef- nagel, D.: Uric acid overproduction and central nervous system dysfunction. Amer. Fed. Soc. 1965, p. 48. 3. Riley, 1. D. : Gout and cerebral palsy in a 3-year—old boy. Arch. Dis. Child. 35: 293, 1960. 4. See technical section page 95. Idiopathic hypercalcemia Clinical and laboratory findings Laboratory diagnosis This condition has been divided into two varieties, a chronic, more severe form1 and a milder, transitory variety}, 3’ 4 The severer type is characterized by symptoms referable to the hypercalcemia, namely, failure to thrive, anorexia, vomiting, constipation, polyuria, and muscular hypotonia, but there may also be physical and mental retardation, a character- istic “elfin” facies, significant systolic cardiac murmurs sometimes due to aortic stenosis,20 hypertension, osteosclerosis, and microcephaly with craniosynostosis. In patients with the mild type, the findings are generally limited to those usually associated with hypercalcemia. The characteristic facial configuration noted in many, but not all, patients with the severe cases of the disease and occasionally found in those with the milder form, is a notable part of the syndrome. The facies is due to the presence of a combination of features 5 including a pinched snub nose with the nostrils pointing forward, underdevelopment of the bridge of the nose and mandible, prominent epicanthal folds, a con- comitant squint, a prominent loose upper lip, and open mouth which may be asymmetrical, a slack hanging lower lip, narrow temples, large ears, and a rounded, sometimes prominent fore- head. The head is frequently held extended, thus making the ears appear low. The cause of the characteristic facies is unknown, al- though some authors have suggested that it is due to abnormal growth of the basisphenoid. However, it would appear likely that not all of the facial characteristics are due to bony changes, inasmuch as some of the features ap- pear to result from alterations in the contours of the soft tissues as well. In the typical case of idiopathic hypercal- cemia, the clinical features first appear between the second and eighth months of age. The ini- tial symptoms are not specific and may be in- sidious in their onset with irritability, loss of appetite, listlessness, and failure to thrive being the first changes noted by the parents. As the name of the disease implies, the char- acteristic laboratory finding is an abnormal ele- vation of serum calcium content. This ranges between 12 and 18 mg. per 100 ml. In cases uncomplicated by renal failure due to diffuse calcium deposits throughout the kidney, the serum phosphorus and phosphatase levels are within normal limits. The urine is usually acid and may contain a moderate excess of al- bumin and leukocytes. The blood urea nitro— gen level may be elevated and serum electro- phoresis mayshow an elevation of the alpha-2 and gamma globulin fractions in the serum.‘ 6 In some patients the ratio of free to esteri- fied cholesterol has been elevated, the beta lipo- protein—bound cholesterol level being increased. The results of vitamin A absorption tests may be abnormal in that fasting levels may be high and rise to unusually high levels after a stand- ard loading dose. The electrophoretic mobility of serum beta lipoproteins may be altered also.7 Associated renal acidosis has been reported on several occasionsfv 3 Early in the disease 65 roentgenograms may reveal bands of increased density at the metaphyseal margins of the long bones and metacarpals, while in the later stages a variable degree of osteosclerosis of the base of the skull and orbit as well as of the vertebrae and long bones, is usually found. In addition retardation of epiphyseal development may occur. In those patients who have died, diffuse calcium deposits have been found in the kidneys and other internal organs. The kidneys may also show evidence of tubular destruction, hyalinization, and fibrosis of the glomeruli. The prognosis in idiopathic hypercalcemia is variable. In many patients the severe form has been associated with a progressively down- hill course, death resulting within the first 5 years; others have survived but were mentally retarded.9 The milder type generally tends to remit spontaneously, with complete recovery occurring after several months. However, re- cent reports in the literature indicate that the division into the mild and severe forms is some- what artificial, for some infants with the “benign” form have died of the disease while others have had hypertension or cardiac mur- murs.2 Conversely, mental retardation and the typical facies are not invariably associated with the severe form.8 There would appear, in fact, to be a spectrum of severity rather than any clear differentiation into specific varieties.10 Biocbemistry Several possibilities have been suggested as to the pathogenesis of the disease. These in- clude abnormal sensitivity to ingested calcium in the presence of primary renal disease,1 a response to the administration of excessive amounts of alkaline medication, an association of separate congenital defects, vitamin D toxic- ity due to unusual sensitivity to small ex- cesses of the vitamin, or an inborn error of metabolism based on some enzyme defect in lipid and calcium metabolism.” 12 Despite the ele- vated serum calcium concentration that occurs, hyperparathyroidism probably has no role, since the alkaline phosphatase is not elevated in patients with hypercalcemia, and with steroid therapy the concentration of phosphorus in the serum is normal and the serum calcium level falls. Likewise, there is no evidence that 66 dietary deficiency of linoleic and arachnidonic acids plays any part in the disease.13 One form of hypercalcemia19 occurs in the “blue diaper syndrome” in association with a disorder of tryptophan metabolism. There does not, how- ever, appear to be any other common ground between these two conditions. Some more recent observations suggest that the fundamental defect is a distortion of choles- terol metabolism whereby an excessive quantity of an abnormal antirachitic steroid is produced in the serum in association with one of the lipo- protein fractions. The relatively high calcium content of cow’s milk may be an important ac- centuating factor, since almost all infants with hypercalcemia have been fed artificial formulas of cow’s milk which have‘four to five times more calcium than human milk. With regard to vitamin D, on the other hand, the balance of available evidence at present} suggests that daily doses of below 400 LU. do not aggravate the condition, although hypersensitivity to the vitamin may explain its hitherto greater inci- dence in the United Kingdom, where intakes of vitamin D of 2,000 I.U. per day were not rare. In some patients, however, evidence of activity has continued even when they were deprived of all exogenous vitamin D. Diagnostic and screening tests There are not satisfactory screening tests for this condition. The diagnosis is made on the basis of certain physical characteristics together with the finding of a raised serum calcium.” Genetics There is no evidence that this is an in- herited condition. ‘ Treatment Management of these children has thus far been aimed primarily at reducing the concen- tration of calcium in the serum, and various forms of therapy have been tried with mixed success. These include low-calcium and vita- min D-restricted diets,14 the administration of adrenocortical steroids)?" 1“ and the oral use of calcium-binding agents such as ethylene-dia- mine tetra acetate (EDTA),16 sodium phytate, and sodium sulfate 17 to lower calcium intake. Therapy with EDTA and sodium phytate was soon discarded because of gastro-‘intestinal symptoms in the case of the former and uncon- vincing eflicacy in the case of the latter. In all instances vitamin D supplements should be withheld and the children given some form of a low-calcium diet; a special low-calcium for- mula is particularly convenient. Adrenocorti- cal steroids are of especial value in the therapy of severe cases and when low«calcium milk and cereals are not readily available.“ 16 The dose of the steroids must be individualized; the opti- mum is the smallest which will efl’ectively lower the concentration of serum calcium to within the normal range. References 1. Butler, N. R., and Schlesinger, B. : Generalized Re- tardation with Renal Impairment, Hypercal— caemia and Osteosclenosis of Skull, Proc. Roy. Soc. Med. 44: 296, 1951. 2. Lightwood, R. : Idiopathic hypercalcemia in infants with failure to thrive, Arch. Dis. Childhood 27 : 302, 1952. _ 3. Lowe, K. G., Henderson, J. L., Park, W. W. and McGreal, D. A.: Idiopathic hypercalcaemic syn- dromes of infancy, Lancet ii: 101, 1954. 4. O’Brien, D., Peppers, T. D. and Silver, H. K.: Idio- pathic hypercalcemia of infancy. J.A.M.A. 173: 1106, 1960. 5. Joseph, M. C. and Parrott, D.: Severe infantile hy- percalcaemia with special reference to the facies. Arch. Dis. Childhood 33: 385, 1958. 6. Payne, W. W.: Blood chemistry in idiopathic hy- percalcaemia. Arch. Dis. Childhood (abstract) 27: 302, 1952. 7. Salt, H. B. and Wolff, 0. H.: Applications of serum lipoprotein electrophoresis in pediatric practice. Arch. Dis. Childhood 32: 404, 1957. 8. Rhaney, K. and Mitchell, R. G.: Idiopathic hyper- calcaemia of infants. Lancet i: 1028, 1956. 9. Schlesinger, B. E., Butler, N. R. and Black, J. A.: Severe type of infantile hypercalcaemia. B.M.J. 1: 127, 1956. 10. Creery, R. D. G.: Idiopathic hypercalcaemia of in- fants. Lancet ii: 17, 1953. 11 Fellers, F. X. and Schwartz, R.: Etiology of severe form of idiopathic hypercalcemia of infancy : De- fect in vitamin D metabolism. New England J. Med. 259: 1050, 1958. 12. Gribetz, D. and Wolf, B. S.: Idiopathic hypercal- cemia. Am. J. Med. 26: 936, 1959. 13. James, A. T., Webb, J ., Stapleton, '1‘. and MacDon- ald, W. B.: Essential fatty acids and idiopathic hypercalcemia of infancy. Lancet i: 502, 1958. 14. Stapleton, T., MacDonald, W. B. and Lightwood, R.: Management of “idiopathic” hypercalcaemia in infancy. Lancet 1:932, 1956. 15. Forfar, J. 0., Balf, C. L., Maxwell, G. M. and Tompsett, S. L.: Idiopathic hypercalcaemia of infancy: Clinical and metabolic studies with special reference to the aetiological role of vita- min. D. Lancet i:981, 1956. 16. Morgan, H. G., Mitchell, R. G., Stowers, J. M. and Thomson, J.: Metabolic studies on two infants with idiopathic hypercalcaemia. Lancet i:925— 931, 1956. 17. Kowarski, A.: ment with sodium sulfate. 1958. , 18. See technical section page 77. 19. Drummond, K. N., Michael, A. F., Ulstrom, R. A. and Good, R. A.: The blue diaper syndrome: familial hypercalcemia with nephrocalcinosis and indicanuria. A new familial disease with definition of the metabolic abnormality. Amer. J. Med. 37: 928, 1964. 20. Black, J. A. and Bonham Carter, R. E.: Associa- tion between aortic stenosis and facies of severe infantile hypercalcemia. Lancet iiz745, 1963. Idiopathic hypercalcaemia treat- Pediatrics 22: 533, Lactic and pyruvic acidosis Clinical and laboratory findings Five cases 1’ 2. 4 have been described of men- tally retarded children, one of whom had Down’s syndrome where the characteristic bio- chemical abnormality was hyperlactic-acidemia and hyperpyruvic-acidemia. Clinically these children were obese, hypotonic and severely mentally retarded. In all instances there seemed to be a. special predilection for respiratory in- fections, but it is possible these were initially mistaken for episodes of severe metabolic acido- sis associated with marked hyperpnoea, irrita- bility, drowsiness, convulsions and not infre- quently coma. During the episodes of acidosis the plasma 002 was usually below 8 m mols/ 1 and the blood pH might be as low as 6.8. Venous blood lactate levels varied from 60 to 120 mg 100 ml (normal 10—20 mg/ 100 ml) and blood pyruvate from 2.8 to 6.5 mg/100 ml (normal 0.2—0.7 mg/ 100 ml). 67 Biochemical lesion In two of these children” the lactate: py- ruvate ratio was at the upper level of the normal range for convalescent children 2' 3 which sug- gested that lactic dehydrogenase activity was normal. Estimations of the rate of glycolysis of heparinized whole blood at 37° C were nor- mal, pyruvate and lactate levels were unaffected by a high fat and low carbohydrate diet and the relative rates of oxidation of 01 and Cs isotope labelled glucose were normal. All of this evi- dence suggests that the accumulation of pyru- vate and lactate was due to a partial block in onward metabolism rather than to any increased tempo in the glycolytic pathways. This was supported by finding sustained blood lactate levels, as well as an abnormal rise in level, fol— lowing lactate infusion. There was some evidence from raised a-ketoglutarate levels in plasma and urine of an impairment in the Krebs cycle. Histochemical examination of a muscle biopsy showed a nor- mal content of succinic dehydrogenase, malic dehydrogenase, DPN diaphorase, lactic dehy- drogenase, gylcerophosphate dehydrogenase, phosphorylase and isocitric dehydrogenase. However early dominance of the M isozyme of LDH may account for the metabolic defect.‘1 Genetics The inheritance of_ this condition is not known, though there were 2 cases in a family.4 Treatment Acute acidosis can be controlled by the ad— ministration of sodium bicarbonate intrave- nously. With continued oral administration of sodium bicarbonate it is possible to maintain a normal blood pH. The high lactate levels 3 were unaffected by high fat or high carbohy- drate diet or by thiamine, calcium pantothenate or penicillamine administration. References 1. Hartmann, A. F., W‘ohl‘tmaznn, H. J ., Purkerson, M. L. and Wesley, M. E.: Lactate metabolism, studies 68 of a child with a serious congenital deviation. J. Pediat. 61: 165, 1962. 2. Israels, s., H‘aworth, J. 0., Gourley, B., and Ford, J. D.: Chronic acidofis due to an error in lactate and pyruvate metabolism. Pediatrics 34:346, 1964. 3. Huckabee, W. E.: Abnormal resting blood lactate. II. Lactic acidosis. Amer. J. Med. 30: 840, 1961. 4. Erickson, R. J.: Familial infantile lactic acidosis. J. Pediat. 66: 1004, 1965. Hyperlipidemia and mental retardation Clinical and laboratory findings This clinical syndrome has been reported 1 in two brothers, both of whom showed somatic and intellectual retardation associated with a disturbance of serum lipids, Both children were below the 3 percentile levels for height and weight at 9 and 51/2 years of age respectively. There was a pronounced hepatomegaly in both cases which had been noticed since infancy; the spleen, however, was not enlarged and there were no other symptoms or signs of liver disease. Conventional liver function and other laboratory tests were within normal limits. A liver biopsy in the elder sibling at the age of 6 years showed vacuolization of many parenchy- mal cells. The characteristic laboratory find- ings were in the distribution of serum lipo- proteins, cholesterol, phospholipid and trigly- cerides. On paper electrophoresis there was some reduction in the beta lipoproteins and a very striking reduction in the alpha1 lipo- protein with a prominent abnormal band in the alpha.2 region. On starch gel electrophoresis of serum it could be shown that there was a marked elevation in tryglyceride levels in all protein fractions and of phospholipids espe- cially in the alpha2 area. Although there was an increase in free cholesterol, total cholesterol was within normal limits distinguishing this syndrome from primary hypercholesterolemia. In idiopathic hyperlipemia the serum is lipae- mic in the fasting state, and the increased lipo- proteins are primarily in the beta fraction. The methods in use when these children were first seen are probably adequate to validate the find- ings as a separate syndrome, modern lipid techniques would now permit a much more ex- haustive analysis of the abnormality. Diagnostic and screening tests No screening tests are appropriate in this instance. The diagnosis can be confirmed and difl’erentiated from idiopathic hyperlipidemia and primary hypercholesterolemia on the basis of the physical signs and the distribution of lipoproteins, cholesterol, phospholipids, and tryglycerides in the serum.2 Genetics Both children were the offspring of a marriage between second cousins. Other mem- bers of the family were not overly afl'ected al- , though there was some evidence to suggest a change in serum lipoprotein distribution in the father and three siblings. The evidence suggests an autosomal recessive mode of inheritance. Treatment Treatment with thyroid, choline, methi— onine, desiccated hog stomach, low fat and site- sterols was attempted in both cases without any evidence of a modification of the clinical course or the abnormal serum lipid pattern. References 1. Bigler, J. A.., Mais, R. F., Dowben, R M. and Hsia, D. Y-Y.: An inborn error of lipid metabolism. Pediatrics 23:644, 1959. 2. See technical section page 86. Acid mucopolysacchariduria (heparitin sulfate) and mental retardation The classical forms of Hurler’s syndrome have been well described in standard textbooks. Recent studies,1 however, have shown that four types may be designated on the basis of the ex- cretion pattern of urinary mucopolysaccharides. In addition, these osteochondro—dystrophies are known to share in part their biochemical dis- order with the Morquio-Ullrich group.2 The classical sex-linked and more severe autosomal recessive forms are dwarfed with a characteristic and marked skeletal deformity and typical bone changes radiologically. The liver and spleen are enlarged, there may be corneal opacities and cardiac abnormalities as well as loss of hearing and mental retarda- tion. These children excrete greatly increased amounts of chrondroitin sulfate B and heparitin sulfate in the urine. The proportion of chon- droitin sulfate is somewhat higher in the sex-linked form. In the so-called “adult” auto- somal recessive form intelligence appears to be normal and the skeletal changes modest. These patients do excrete excess amounts of chondroi- tin sulfate B in the urine but in such a form that it cannot be precipitated as the calcium salt in 10—20 percent ethanol. Recently a series of children have been re- ported 1’ 3 whose appearance is within normal limits. They are mentally retarded and show hepatomegaly. Corneal opacities, however, are mild and require a slit-lamp for their detection. The bone changes are correspondingly minimal. The distinguishing feature of their acid muco- polysacchariduria being that the increase is in heparitin sulfate 4 and not in chondroitin sulfate B. References 1. Terry, K. and Linker, A.: Distinction among four forms of Hurler’s syndrome. Proo. Soc. Emper. Biol. and M ed. 115: 394, 1964. 2. Zellweger, H., Ponseti, J. V., Pedrini, V., Stamler, F. S. and von Noorden, G. K.; Morquio-Ullrich's disease. J. Pediat. 592549, 1961. 3. Sanfilippo, S. J., Podosin, R., Langer, L. and Good, R. A.: Mental retardation associated with acid mucopolysacchariduria (Heparitin sulfate type). J. Pediat. 63: 837, 1964 (abstr). 4. See technical section page 81. 69 PART B. DIAGNOSTIC TECHNICAL PROCEDURES Determination of plasma ammonia The standard methods for the determina- tion of plasma ammonia have, until recently, been adaptations of the microdifiusion method of Conway}, 2’ 3 The technical and theoretical disadvantages of diffusion techniques are well known insofar as volatile amines, such as iso— amylamine, and n-butylamine diffuse at approx- imately the same rate as ammonia and will be included in the final quantitation. In addition, the diffusion time required is sufficient to allow the alkaline hydrolysis of protein to occur, pro- ducing additional ammonia."7 The ion ex- change resin methods which have recently been developed 8‘10 have the advantage that they do not estimate these substances and are technically more simple. The method described below is a modification of those previously published. The same precautions are applicable to all methods of determining plasma ammonia; blood should be collected into a heparin tube placed in a beaker of ice; the vein puncture should be performed without stasis or muscular activity on the part of the patient; the estima— tion should be commenced within 30 minutes of the shedding of the blood, or if this is not pos- sible, the plasma may be frozen for a maximum of 2 hours; glassware must be scrupulously cleaned in alkali detergent or approximately O-IN sodium hydroxide, and rinsed several times in deionized water; the laboratory atmos— phere must be free of ammonia gas. Reagents 1. Water. All water used in the estimation both for the preparation of reagents and for the 70 rinsing of glassware, should be good quality distilled water passed through a mixed bed resin immediately before use. 2. Ion Exchange Resin. A strongly acidic cation exchange resin (DOWEX 50W X8,_8 percent cross linked, 200 mesh, obtainable from J. T. Baker Chemical Co. or Bio-Rad Labora- tories) is washed six times with twice its volume of deionized water over a period of 2 days to remove contaminants. It is stored under water in a sealed container. 3. Bufler [7H 7.40. 420 ml of 0 -4 N sodium hydroxide (8g/500 m1) is added to 500 ml of 0-4 M potassium dihydrogen phosphate (27-22 g. KH2P04/500 ml). The pH is adjusted to 7-40 with O-4N NaOH using a pH meter. 4. Sodium Pbenute Reagent. 2-5 g. of phe- nol is dissolved in deionized water, 7-8 ml of 4N NaOH is added and made up to 100 ml with deionized water. This reagent should be pre- pared fresh daily. 5. Sodium Nitroprusside (1%). 0-1 g. so- dium nitroprusside is dissolved in and made up to 10 ml with deionized water. 6. Sodium Hypocblorite. 6 percent sodium hypochlorite (approximately 0-8N) is diluted 1 in 4 with deionized water to give an approxi- mately 0 -2N solution. 7. Sodium Hypocblorite/Nitroprusside Re- agent. Immediately before use, mix 50 ml of sodium hypochlorite solution and 10 ml of 1 percent sodium nitroprusside. (Reagents 4, 5, and 6 may be obtained in preweighed packages from Hyland Laboratories, Los Angeles, Calif.) 8. Stock Ammonia Standard (100 pug/ml). 382 mg. NH4CI is dissolved in and made up to 1 liter with freshly deionized water. 9. Working Ammonia Standards. Dilute 1, 2, and 5 ml of stock standard to 100 ml with deionized water to give standards of 100, 200, and 500 pg ammonia nitrogen/ 100 ml. Procedure 1. Equilibration of Resin. Approximately 10 g. of wet resin is placed on several circles of filter paper to remove excess moisture and then transferred to a large stoppered tube. The resin is washed with 25 ml of deionized water fol— lowed by five washes with pH 7-4 buffer. The pH of the resin should now be 7-4. The resin is now washed with several changes of deionized water to remove the buffer solution. 2. 1 g. portions of the prepared resin are placed in suflicient 10 ml stoppered tubes; four tubes for standards and blank and one tube for each determination. 3. To each tube 1 ml plasma, 1 ml of de- ionized water, or 1 ml of standard is added. Shake for 5 minutes and allow the resin to settle (the tubes may be centrifuged briefly). The supernatant fluid is aspirated to waste. Wash the resin thoroughly four times with 5—6 ml of deionized water, aspirating each wash to waste. 4. Add 2 ml O-lN NaOH. Mix for 3 minutes. 5. Add 1 ml of sodium phenate reagent fol- lowed by 1 ml of sodium hypochlorite/nitro- prusside reagent to the resin. Mix and place in a water bath at 37° C for 10 minutes. 6. Read in a spectrophotometer at 640 mp. The color is stable for at least 30 minutes follow- ing incubation. Test-Blank Calculation: Standard-Blank X concentration of standard = pg ammonia nitrogen/100 ml plasma References 1. Conway, E. J. and Byrne, A.: Absorption apparatus for the micro-determination of certain volatile substances; micro-determination of ammonia. Biochem. J. 27: 419, 1933. 2. Seligson, D. and Seligson, H.: A microdiffulion method for the determination of nitrogen liber- ated as ammonia. J. Lab. and Olin. Med. 38: 324, 1951. 3. Seligsom, D. and Hirahara, K.: The measurement of ammonia in whole blood, erythrocytes, and plasma. J. Lab. and Olin. Med. 49: 962, 1957. 4. Bessman, S. P. : Ammonia metabolism in animals in “Inorganic nitrogen metabolism”. W. B. Mc- Elroy and B. Glass, eds. Johns Hopkins Press, 1956. 5. Bessman, S. P.: Blood ammonia in “Advances in Clinical Chemistry”. Vol. 2. H. Sobotka and C. P. Stewart, eds. Academic Press, 1959. 6. Conway, E. J .: Microdiffusion analysis and volu- metric error. 0. Lockwood, London. 1947. 7. Rosender, V. M.: The measurement of ammonia in whole blood. J. Olin. Path. 12 z 128, 1959. 8. Dienst, S. G.: An ion exchange method for plasma ammonia concentration. J. Lab. and Olin. Med. 58: 149, 1961. 9. Hutchinson, J. H. and Labby, D. E: New method for micro-determination of blood ammonia by use of cation exchange resin. J. Lab. and Olin. Med. 60: 170, 1962. 10. Dienst, S. G. and Morris, B.: Plasma ammonia de- termination by ion exchange. J. Lab. and Olin. Med. 64: 495, 1964. Paper chromatography of amino acids Paper chromatography 1’ 2’ 3’ 4' 5 is best used solely as an indicator of changes in amino acid pattern rather than as a means of absolute quan- titation. To obtain the most meaningful chro- matograms, the filter paper must be neither excessively over- nor under-loaded, the results being confused or overly faint, respectively. Several methods have been recommended for deciding the quantity of sample to be applied. These are based on the alpha amino nitrogen, total nitrogen, or creatinine concentrations or on the volume of urine passed in a given time, say 2 seconds. A reliable way is to apply that volume of urine which contains 10 pg. alpha amino nitrogen, this being a suitable load for the 200-mm. square papers. 71 Because of the presence of amino acids in sweat, chromatograms can be spoiled by careless handling and therefore the papers should be picked up only by the extreme corners. Strong acid fumes can completely decolor- ize papers stained with the ninhydrin isatin reagent and hence the atmosphere should be fume-free whenever the chromatograms are exposed. Without very special arrangements for en— vironmental control, it has been found impos— sible to quantitate the spots with less than $15 percent coefficient of variation. The visual comparison of unknowns with standards proved at least as accurate as densitometry‘ or elution followed by spectrophotometry of the eluate. A satisfactory arrangement is to use a one— dimensional system for screening plasma and urine samples and a two-dimensional system for the identification of individual amino acids. The one-dimensional system used here is essen— tially the same as that of Scriver, et al. (Lancet ii : 230, 1964) except that it uses larger papers to get improved resolution. The technique of Efron, et al. (New Eng. J. Med. 270: 1378, 1964) has some advantage in that whole blood samples on filter paper discs are used, which can easily be mailed. Our own experience however has been that the chromatograms tend to streak and lose resolution. The following are two straight-forward systems for one- and two-dimensional paper chromatography : A. One-dimensional System (Figs. 26 and 27.) 1. 46 x 57 cm. sheets of Whatman 3 mm. paper are folded once on a line 5 cm. from one of the short edges and again in the opposite direction 2 cm. from the same edge. These folds enable the sheet to be hung in the solvent trough. After folding, the sheet is laid flat and a pencil line drawn 3 cm. down from the first fold, i.e. 8 cm. from the edge. Samples are applied approximately 2.5 cm. intervals along this line. 2. Blood samples are drawn into heparin— ized capillary tubes. These are centrifuged 72 and broken off at the juncture of red cells and plasma. Tubes can be stored at this stage after sealing with clay or the contents can be ex- pelled into 400 [1.1 capped plastic tubes for storage. 3. Using a Kirk pipette, plasma is drawn up either directly from the capillary tube or from the plastic tube. 10 p1 of plasma or serum or 15 ,ul of urine is applied to the paper over about 5 seconds. 16 samples can be run on one sheet in this manner. 4. The chromatograms are allowed to equil- ibrate for 2—3 hours in the tank with the solvent in the bottom tray. They are developed over- night by descending chromatography in freshly mixed n-butanol, glacial acetic acid, and water (12: 3: 5). The papers are run until the sol- vent front reachos the bottom edge. They are then dried for 1 hour in a stream of air. I 5. Divide the papers longitudinally with scissors into halves and dip in a mixture of 0.25% ninhydrin 0.01% isatin 1% lutidine, all diluted in acetone. Heat for 15 min. at 75° C in a vented hot air drying oven. B. Two-dimensional System (Fig. 28). Apparatus Chromatography tank with tray and frame for twelve, 20 x 20 cm. papers (Shandon Scien- tific Co. Ltd., London, S.W.7, England; dis— tributors in the United States are Consolidated Laboratories, Inc., Chicago Heights, 111.). Whatman No. 1 papers, 20 x 20 cm. with 6 mm. circular perforations in corners to fit frame. Electric desalter (Research Specialties Co., 200 S. Garrabo Blvd, Richmond, Calif., Model A—1930). Figure 26. ONE-DIMENSIONAL PAPER CHROMATOGRAM OF AMINO ACIDS IN PLASMA N-Bulanol; Syndromes Acetic :Waler Rf Rf ‘2 i 3‘ 5 scale values MAPLE SYRUP URINE DISEASE leucine,isoleucine,valine PHENYLKETONURIA phenylalanine IDIOPATHIC HYPERVALINEMIA valine TRANSIENT p.HYDROXYPHENYL PYRUVIC ACID OXIDASE DEF tyrosine CONG. TRYPTOPHANURIA tryplophane in excess after oral load \\§s PROLINE OXIDASE DEFIC. A'-PYRROLINE-S-CARBOXYLIC ACID DEHYDROGENASE DEFIC. proline HYDROXYPROLINEMIA WITH RETARDATION hydroxyproline HYPERGLYCINEMIA glycine""serine,alanine . glutamic" HOMOCYSTINURIA methionine CITRULLINURIA cilrulline GLUTAMIC ACIDEMIA and CEREBRAL DEGENERATION glutamic acid HISTIDINEMIA hislidine LYSINE INTOLERANCE Z lysineT“ arginine' -72 #24 Leucine, lsoleucine (63) Phenylalanine (61) Valine (55) Tyrosine (54) SALE. (52) Trylophane (51) ac Am. Butyric (4Q Proline, flAlanine (3 a) Alanine,a6Am. Adipic (33) Hormocifrullgrs (31) eon Gluian'llf (Aid (29) Hydroxyproline (27) flhiomne 26 ycme 25 Aspargic 24 Citrpllme 2 Senne,Homocyinne(2§ Glucosamine (21) Taurine,GIuIamine (l9 Asparagine,Homoargmine (la) PtosfiiEnIggnolprnine 16) u Riflgdjne 3 fié'llfiill.’ 1 Arginlne IMe.Hist. 1 L sine,Cyst I ionine (12) §rnithine (cl 1 ysteic Acid. Cysfine (ll) Anserine,Carnosine (6) ORIGIN 73 Figure 27. ONE-DIMENSIONAL PAPER CHROMATOGRAM OF AMINO ACIDS IN URINE 74 N-BuIanoI : Syndromes AceIic :WaIer 12 : 3 2 5 MAPLE SYRUP URINE DISEASE leucine, isoleucine,va|ine PHENYLKETONURIA phenylalanine OAST HOUSE SYNDROME p enylalanine, lepcine,isoleucine meIhIonine ,Iyrosine, vaIine IDIOPATHIC HYPERVALINEMIA valine CONGENITAL TRYPTOPHANURIA IrypIophaneT‘aerr oral loa HARTNUP DISEASE GALACTOSEMIA generalised aminoacidutia PROLINE OXIDASE DEFICIENCY proline, hydroxyproline, glycine HYDROIG DEF. pro ine, Chy roxyprol Ine JOSEPH' S SYNDROME hydroxyproline HYDROXYPROLINEMIA AND M.R. DIPLEGIA AND AMINOACIDURIA g|ycine alanine,serine,glutami quIamIc acid IDIOPATH HYPECRGINCIN'"e MIA glycine‘“, eucnne,valn CITR LnLINURIA ciIrUI HOMOnCYSTINURIA homocyinne ARGINII’tOSUCCINIC- . RIA. . argInInosucchc acud HISTIDINEMIA hiindi YSTngIQNURIA cysIaI Ionlne CYSTIN RIA cysIIne, ysine,arginine orniIhine DOPA and SHORT STATURE Iaurine,anserine,carnosine I-me—hiindine CEREBRO-MACULAR DEGENERATION O anserine, carnosine Rf scale —68 —- 64 i—bo “-56 “-44 —36 Rf values Leucine, Isoleucine (be) Phenylalanine (61) V l‘ 55 Tam II.) B.A.LB. (52) TrypIophane (51) 0(- Am. BuIyric (4 4) Proline,fl Alanine (38) Alanine,oé Am. Adipic (33) HomociIruIline (31) Threonine 30 GluIamic ACId (2 9) Hydroxyproline 27 Methionine 26)( ) Glycine 2 A_sparIic 2 CiIrulIine 2 Serine Homoc inne 2: Glucoslamine (21)( ) Taurine,GIuIamine (I Asparogine, Homoarginine (18) Argininosuccinic Acid Hiindine 3Me. HisI. (I4) Arginine 1Me.HisI.13) sine, CysIthionine (12) aniIhine (n) CysIeic Acid. Cyinne (II) Phos. EIhanoIamine( (13; ) 15 Anserine, Carnosine (6) ORIGIN Figure 28. TWO-DIMENSIONAL PAPER CHROMATOGRAPHY OF AMINO ACIDS FIRST DIMENSION SECOND DIMENSION BUTANOL/PYRIDINE/WATER PHENYL/WATER/AMMONIA 60/60/60 460/40/2.5 Leuc,iso|euc, OPOI'I. Al OW Q“ 0 0H0 Pro. Ala. 'ThOr l.Me.Hisf Tour. Homocitl 00 B, Al& 8. 3. Me Hist OGIY _Ser. Cysteic. Q GHQO em. 0 / “'5' GluNH / Homoarg ‘ flAlanine O p.NH As BUTANOL/PYRIDINE/WATER / \ Asp. Cystathion. Arg,$ucc. Phosph.Ef|'I. PHENOL/WATER/AMMONIA 75 Kirk transfer pipettes (Microchemical Specialties Co., 1825 Eastshore Highway, Berkeley, Calif.) . Heat gun (Cole-Farmer, 7320 N. Clark Street, Chicago 26, I11., Cat. No. 5700). Reagents (All reagent grade unless otherwise specified.) ‘ _ P h e n o l, C6H50H (Mallinckrodt Gilt Label), ammonium hydroxide (NH4OH) solu- tion 58 percent, n-butanol, CH3(CH2)2CH20H, pyridine, C5H5N, sodium cyanide, NaCN, nin- hydrin, isatin, CsH5N02, triethylamine (C2H5) 3N, hydrogen peroxide 30 percent, sili- cone high vacuum grease, amino acid standards, activated charcoal. Composition of amino acid standard solution Dissolve the quantities below in about 70 ml. water. Add a little H'Cl until all are com- pletely in solution and make up to 100 ml. with Con- cen- mg./ L-Amino acid M.W. tra- 100 tion ml. in m mol/l 1. Alanine .......... 89.09 6 53.5 2. Asparagine ....... 132.12 4 52.8 3. Aspartic acid ..... 133.10 4 53.2 4. Cystine ........... 240.29 8 192.2 5. Glutamic acid. . . . 147.13 4 58.8 6. Glutamine ........ 146.15 6 87.7 7. Glycine .......... 75.07 6 45.0 8. Histidine ......... 155.16 8 124.2 9. Hydroxyproline . . 131.13 8 104.9 10. Leucine .......... 131.17 2 26.2 11. Lysine ............ 149.19 4 59.7 12. Phenylalanine. . . . 165.18 4 66.0 13. Proline .......... 115.13 8 92.1 14. Homocitrulline . . . 189.0 6 1 1 3.4 15. Serine ............ 105.09 4 42.0 16. Taurine .......... 125.14 8 100.2 17. Threonine ........ 119.12 4 47.6 18. Tryptophan ...... 204.22 4 81.7 19. Tyrosine ......... 181.19 8 145 20. Valine ........... 117.15 2 23.4 water. Store bulk in deep freeze, keeping a few milliliters for current use in refrigerator. For standard chromatogram, apply 10,1.1. Purification of ninhydrin 100 gm. ninhydrin is dissolved in 500 ml. of hot 2N HCl. The solution is filtered and 10 gm. activated charcoal added. The solution is boiled for 10 min. and filtered while hot. The crystals are allowed to form slowly at room temperature and the whole is placed in the re- frigerator at 4° C. The crystals are filtered off, washed 3 times with 50 ml. portions of ice-cold 1N HCl, and dried over KOH in vacuo. NOTE: Ninhydrin obtained from Pierce Chemical 00., Rockford, 111., does not need purification and may ‘be used as supplied. Preparation of urine samples It is preferable to carry out chromatog- raphy on a portion of a 24-hour specimen of urinecollected in a bottle containing a pre- servative such as a‘ few drops of 50 percent chlorbutol in ethanol or a few drops of chloro- form. Desalt the urine according to the in- structions with the electric desal-ter. Provided the volumes are not inconvenient, it is best to apply a volume of urine containing 10 pg of alpha amino nitrogen. Preparation of serum ‘One volume of serum is pipetted into 10 volumes of 1.0 percent picric acid. The precipi- tate is spun down in the centrifuge and a meas- ured volume of the supernatant is placed on a 2 cm. diameter column packed to a depth of 3 cm. with Dowex X8, which has been pretreated with 10 ml of 1N HCl and washed to neutrality with distilled water. The amino acids are eluted from the column by 5 washes of 3 ml. 0-01 N HCl. The eluate is evaporated to the original volume of serum and the pH adjusted to 7.0. Approximately 20 pl of final eluate is applied to the paper. Application of solutions Mark the point of application in one corner of each paper with a pencilled cross, 1 inch from each edge. To 1 volume of urine at 0° C add 0.1 volume cold 30 percent H202. Stand 1 to 2 minutes at 0° and then apply to the paper. This treatment oxidizes the sulfur-containing amino acids, rendering them more stable in the presence of the organic solvents used for de- velopment. Using micro pipettes, apply 50 to 150 pl. prepared sample or 10 [1.1. standard solu— tion with a stream of hot air from a heat gun blowing onto the application site. Put on 1 to 2 1111- each time and allow to dry before applying the next portion. The circle of paper moist- ened should be not greater than 4 mm. diameter. Solvent Mixtures First Dimension 60 ml. n-butanol 60 ml. pyridine 60 ml. water. Mix thoroughly. Second Dimension 184 ml. phenol solution 16 ml. water 1 ml. 58% ammonium hydroxide. Mix. With this solvent it is an advantage to place in the tank at the sides of the tray 3 petri dishes, each with a filter paper soaked in approximately 2% NaCN. Run in each solvent overnight and dry dur- ing the day. It is important that the tank should be in a warm draft-free place and should be level. After running in each solvent, place racks of papers in the chromatography oven or in the fume hood with the extractor fan on until all solvent has completely evaporated. This may take 3 hours for phenol but is considerably less for more volatile solvents. Do not use heat. Location Location reagent 0.25% ninhydrin w/v 0.01% isatin 1% 2.6-Lutidine Make up with acetone. Shake thoroughly. Wearing rubber gloves, dip each paper in the dye solution once only and replace on the frame. Place in a fume chamber until dry (15 min. at 75° C). A map using this solvent sys- tem of biologically important amino acids is shown in figure 27. The color tends to darken with time. Notes 1. A considerable amount of time can be saved and the escape of objectionable odors minimized by the use of a special oven, e.g., Research Specialties Co. Model C—425. Phenol papers can be dried in this at room temperature with the fan on. 2. Handle papers by the corners only at all times. 3. When dyeing papers and during the pe- riod of color development, the atmosphere must be free of strong acid fumes. References 1. Smith, I.: Aminoacids, amines, and related com- pounds. Chromatographic and Electrophoretic Techniques, Vol. I. Interscience Publishers, Inc., New York, 1960. p. 82. 2. Kolor, M. G., and Roberts, H. R.: A new reagent for the detection of hydroxyproline on paper chro- matograms. Arch. Biochem wnd Bioph/ys. 70: 620, 1957. 3. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro—bioehemical techniques. Hoeber, New York, 3d edit, repr. 1964, p. %. 4. Scriver, C. R., Davies, El, and Cullen, A. M. : Applica- tion of a simple micromethod to the screening of plasma for a variety of aminoacidopathies. Lan- cet ii: 230, 1964. 5. Efron, M. L., Young, D., Maser, H. W. and. Mac- Cready, R. A.: A simple chromatographic screen- ing test for the detection of disorders of amino acid metabolism New Eng. J. Med. 270: 1378, 1964. Determination of serum calcium The most convenient, rapid and accurate assay for serum calcium is a fluorometric one. 77 This determination is dependent on the forma- tion of a yellow-green fluorescent complex of calcium ion and calcein (3, 6-dihydroxy-2, 4- bis—(N N’-di-(carboxy methyl) -amino methyl) fluoron, a product of the condensation of fluores— cein with imino—acetic acid and formalde- hyde?‘3 The reaction takes place only with calcium, barium, and strontium. Since the concentra- tions of the latter two ions in serum is insignifi- cant, no loss in specificity is experienced. No interference is caused by the other major ions of serum, sodium, potassium, chloride, phos- phate, magnesium, or by protein. The prob- lem of adsorption of calcium onto glassware is avoided by the use of polystrene tubes as cuvettes, and the accuracy of earlier methods is improved on by the reduction of the number of pipetting steps to two.‘ Reagents 1. Water: Good quality distilled water passed through a column of “Amberlite MB3” deionizing resin should be used. 2. Calcium Standard (10 mg per 100 ml. 5 mEq per liter): 25 mg of dry calcium carbonate (CaOOS) (analytical grade) is dissolved in 0.6 ml of 1N hydrochloric acid. Heat to boiling in a beaker until the excess HCl has been driven off. Cool to 25° C. Wash the contents of the beaker into a 100 ml volumetric flask and make up to the mark with deionized water. .3. Potassium Hydroxide, KOH, 0.8N: Weigh out 44.8 g. analytical grade potassium hydroxide and dissolve in and make up to 1,000 ml with deionized water. Store in a polyethy-i lene bottle. 4. Stock Calcein Solution (100 mg per 100 ml): 100 mg of calcein is dissolved in and made up to 100 ml with.0.8 N KOH. This reagent should be stored in a brown polyethy- lene bottle at 4° C. Under this condition, it is stable for approximately a month. 5. Working Calcein Solution (0.7 mg per 100 ml): Dilute 0.7 ml of stock calcein solution to 100 ml with 0.8 N KOH. Store in a brown poly- 78 ethylene bottle at room temperature. Stable for approximately 5 days. Equipment 1. Turner Model 110 or Model 111 Fluorom- eter, equipped with primary filters #110—816 (2A) and #110—813 (47B) and secondary filter #110—818 (2A—12) plus an appropriate neutral density filter; or, other suitable spectrofluorom- eter with an activating wavelength of 436 my. and a fluorescent wavelength of 510 mp.. 2. Cuvettes. 12 x 75 mm polystyrene dis? posable culture tubes, FALCON PLASTICS, N0. 2003. Procedure 1. Pipette 5 ml of working calcein solution into a polystyrene cuvette. 2. Add 20 [1.1 of serum. 3. Set up a series of standards in the same way using 10, 15, 20, and 25 pl of calcium stand- ard equivalent to 5, 7 - 5, 10, and 12-5 mg/ 100 ml in place of serum. 4. Mix each tube by inverting several times. 5. Incubate in a water bath at any specific temperature between 20° C and 30° C for suit- able period (10—30 minutes). The optimum conditions should be determined by running a series of standard curves at varying tempera- tures and incubation times to obtain linearity over the desired range. 6. Read in fluorometer and compare tests with standard curve. References 1. Wallach, D. F. H., Surgenor, D. M., Soderburg, J. and Delano, 19.: Preparation and Properties of 3,6-Dihydroxy-2,4—bis— (N,N’—di- (carboxy methyl) - amino methyl) fluoran: Utilization for the ultra- microdetermination of calcium. Anal. Chem. 31: 456,1959. 2. Wallach, D. F. H. and Steck, T. L.: Fluorescence techniques in the microdeterminaltion of metals in biological materials. Anal. Chem. 35:1035, 1963. 3. Kepner, B. L. and Hercules, D. M.: Fluorometric determination of calcium in blood serum. Anal. Chem. 35: 1238, 1963. 4. G: K. Turner Associates: “Manual of Fluorometric Clinical Procedures,” Palo Alto, Calif. Determination of n-formiminoglutamic acid in urine N-formiminoglutamic acid (FIGLU) in urine has been determined by several different assay systems. These include: the microbio- logical assay of glutamic acid formed from FIGLU by heating, using the organism Lacto- bcwilhos ambimm,1'3 an enzymatic method based on the formation of 5,10-methyl-tetra— hydrofolic acid by the enzymes FIGLU trans- ferase, and formino-tetrahydrofolate cyclode— aminase, and the measurement of the product spectrophotometrically; 4 a combined enzy- maticmicrobiological technique which over- comes the difiiculty of using unstable tetrahy- drofolic acid as a starting material by the use of folic acid, folic acid reductase, and a TPN generating system to produce the active form; 5 a modification of the two previous procedures which estimates both FIGLU and its precursor urocanic acid spectrophotometrically; 6 paper chromatography; 2 and high voltage electro- phoresis.7 While some of these methods fulfill the requirements of sensitivity and specificity, they suffer from the disadvantages of technical complexity and/ or a requirement for expensive equipment, and are not, perhaps, compatible with the routine clinical laboratory. The use of conventional voltage electrophoresis on cel- lulose acetate membranes provides a simple semiquantitative method, which is adequate for screening following histidine loading.8 Should the requirement for sophisticated equipment not be a consideration, the method of choice would be the ion-exchange chromatography procedure of Edozien and Mudie.9 Reagents l. Bufler Solution pH 5 -3. 50 ml pyridine and 20 m1 glacial acetic acid are added to 4 liters of distilled water, and the pH adjusted to 5-3 if necessary. 2. Ninhydrin Locution Reagent. Dissolve 0 - 2 g. Ninhydrin in 6 ml of ethanol and make up to 100 ml with diethyl ether. A. small quantity of a strong cation-exchange resin in the acidic form (DOWEX 50W X8) is added to the re- agent and allowed to settle out after shaking. This removes traces of ammonia. 3. FIGLU Standards. A series of standards is prepared by adding 1 mg-lO mg of FIGLU (obtainable from Koch-Light Laboratories, Ltd, Poyle, Colnbrook, Bucks, England) to 100 m1 aliquots of normal urine. The urines thus prepared are preserved by addition of a few thymol crystals and may be stored for several months at 4° C. 4. Ammonia. 0.88 specific gravity. Materials Cellulose acetate strips—These may be ob- tained from Gelman Instrument 00., Ann Ar- bor, Mich. (Sepraphore), or Consolidated Laboratories, Inc., Chicago Heights, Ill. (Ox- oid) and should be cut to 12 x 5 cm. strips. Power supply—A constant voltage power supply capable of delivering 200 v. The cur- rent obtained should be approximately 2—2.5 m. amp. per strip. Pipettes—10 Ml- Drummond “Microcap” (Drummond Scientific Co., Philadelphia 20, Pa.) disposable capillary pipettes are suitable, although any 10 [A]. micropipette may be used. Electrophoresis apparatus—Any horizontal type electrophoresis tank may be used. If dis- tance between the electrode chambers is too great to accommodate 12 cm strips, filter paper wicks may be used to complete the circuit. Procedure 1. The cellulose acetate is marked as shown, certain ball-point pencils are suitable since the ink they contain does not diffuse. 2. The strip is soaked in buffer by first a1- lowing it to float on the surface, and then, when the surface uniformly darkens, submerging it completely. 79 t l ' S—tandard x + I . l Patient’s I Urine X 5 cm. I I Patient’s X : Urine I Standard X .1. 3. Blot the strip lightly between filter paper strips to remove excess moisture. 4., Place the strip in position in the electro- phoresis apparatus, and apply 10 M1 of patient’s urine, and 10 ul of the standard urine, to the spots designated above. 5. Apply a constant voltage of 200 volts for approximately 30 minutes. The time re- quired varies with the prevailing conditions. 6. Switch 011' current, remove strips and dry in an oven at 80° C for 15 minutes. Curl- ing of the strips can be prevented by light tensioning. 7. Cut each strip longitudinally along the center line. 8. Place one half-strip in an ammonia at- mosphere for 30 minutes by suspending it from the lid of container in which is placed a small beaker of ammonia. This converts FIGLU to glutamic acid. 9. Remove the strip and dry briefly at 80° C to remove the ammonia. 10. Pass both half-strips through the nin- hydrin staining solution contained in a petri dish, and immediately place between sheets of filter paper and return to the oven for 10 min- utes. Pressure should be applied to the strips to prevent curling. This may be achieved by placing the filter paper between cardboard sheets and clipping them together, or by plac- ing a weight on top of them. 11. Color development is complete after 25—30 minutes. 80 Evaluation A normal urine run by the above procedure will usually exhibit three spots: at the cathode end a spot containing mainly histidine, at or near the point of application, a spot containing glycine, and towards the anode a faint spot of glutamic acid. FIGLU, if it is present in the urine, runs to the same position as glutamic acid, hence the need for comparison between the ammoniated and the non-ammoniated half- strips. A semiquantitative estimate of concen- tration can be made by visual comparison of the standard solutions. If the divergency is too great, the patient’s urine should be diluted with acidified normal urine, in such a way as to give interpretation between two standards on the re- peat. The method is sensitive to approxi- mately 2 mg. of FIGLU per 100 ml. urine. Histidine administration and collection of urine sample L-Histidine monohydrochloride, 0.5 g. / kilo to maximum of 15 g., is given by mouth to the patient in a fasting state and food is Withheld for 1 hour after the administration of the dose. Because histidine is only slightly soluble, it is administered by mixing it with water in a glass and then the residue in the glass is washed .down with water until all the histidine has been taken. Three hours after taking the dose the pa- tient voids and this is discarded. All urine passed over the next 5 hours (3 to 8 hours after histidine) is collected in a bottle to which has been added 1 ml. of concentrated hydrochloric acid and a few thymol crystals. The urine vol— ume is measured and an aliquot taken for analy- sis from the collection period of between 3 and 8 hours is used as the determination of hourly FIGLU excretion. Most patients show the peak excretion of FIGLU during this time. References 1. Henderson, L. M. and Snell, E. E.: A uniform medium for determination of amino acids with various microorganisms. J. Biol. Chem. 172: 15, 1948. 2. Silverman, M., Gardiner, R. C. and Bakerman, H. A. : The nature of the glutamic acid excreted in folic acid deficiency. J. Biol. Chem. 194: 815, 1952. 3. Broquist, H. P.: Evidence for the excretion of for- mimino-glutamic acid following folic acid antago- nist therapy in acute leukemia. J. Amer. Chem. Soc. 78: 6205, 1956. 4. Tabor, H. and Wyngarden, L. : A method for the de- termination of formiminoglutamic acid in urine. J. Clin. Invest. 37 : 824, 1958. 5. Silverman, M., Gardiner, R. C. and Condit, P. T.: A method for the detection of N—formiminoglu— tamic acid in urine. J. Nat. Cancer Inst. 20: 71, 1958. 6. Chanarin, I. and Bennett, M. 0.: A spectrophoto- metric method for estimating formiminoglutamic and urocanic acid. Brit. Med. J. i:27, 1962. 7. Knowles, J. P., Prankerd, T. A. J. and Westall, R. G. : Simplified method for detecting formiminoglu- tamic acid in urine as a test of folic—acid defi- ciency. Lancet ii: 347, 1960. 8. Kohn, J ., Mollin, D. L. and Rosenhach, L. M.: Con- ventional voltage electrophoresis for formimjno glutamic acid determination in folic acid defi- ciency. J. Olin. Path. 14: 345, 1961. 9. Edozien, J. C. and Modie J. A.: Determination of formiminoglutamic acid by ion-exchange chroma— tography. Lancet ii: 1149, 1964. Chromatographic identification of sulfated mucopolysaccharides including heparin Isolation of urimry mucopolysaccbarides Thirty ml of urine from a random morning sample is acidified with concentrated HCl added drop by drop to pH 6.0.0 The sample is then dialyzed for 24 hours at 4° against a constant flow of water; the pH is adjusted to 5.0 after which 1 ml of aqueous 5 percent cetyl trimethylammonium bromide is added and the whole allowed to stand for not less than 16 hours at 4° C. The precipitated acid muco- polysaccharides are centrifuged off at 4° C and washed twice with 5 ml of 95 percent ethanol saturated with NaCl. The precipitate is resus- pended and recentrifuged with each washing. Finally, the precipitate is redissolved in 2 ml of 0.05 NaOH. Chromatography A one-dimensional chromatographic 1. 2 sys- tem is used with Whatman #1 papers 16”—22” long and 41/2 wide. On a line 3 inches from one end of the papers 20pl aliquots of the sample as well as solutions of beef heparin sulfate and chondroitin sulfate containing 30 pg per 20 11.1 are applied. The atmosphere in a suitable sized descending chromatography jar is allowed to equilibrate overnight with a mixture of 0.04 M ammonium formate pH 4.3 and isopropanol 60:40, v/v. Papers containing the sample and standards are then equilibrated in the tank for 4 hours. Solvent is then added to the trough and the papers chromatographed for 12 to 16 hours at 26°:2° C. The chromatograms are dried in an air current at the same temperature. With paper that has been stored in the labora- tory, it may be necessary to increase the propor- tion of ammonium formate in the solvent. The dry papers are dipped in a solution of 200 mg Azure A. (National Aniline Division, Allied Chemical & Dye Corp.) in 50 ml distilled water to which is then added 400 ml A.R. ace- tone and 1800 ml A.R. methanol. Heparin (bright pink spot) and heparitin sulfate (red violet spot) have an Rf of around 0.5 in the system and the chondroitin sulfates (purple spot) about 0.85, migration may be slower in stored papers. References 1. Spotter, L. and Marx, W.: Paper chromatography of heparin and related sulfated mucopolysac- charities. Biochimioa et Biophysica Acta 38: 123, 1960. 2. Terry, K and Linker, A.: Distinction among four forms of Hurler’s syndrome. Proo. Soc. Ewper. Biol. Med. 115 z 394, 1964. Determination of histidine in serum Histidine in serum may be determined by a modification1 of the method of LaDu and Michael for the estimation of phenylalanine.2 The method depends on the fact that the aromatic amino acids, phenylalanine, tyrosine and tryptophan are converted to keto acids by snake venom L-amino acid oxidase at pH 6.5 81 and in the presence of arsenate and borate ions enol—borate complexes result which have characteristic absorption spectra in the ultra— violet. Histidine, however, is not oxidized under such conditions, but is so oxidized to- gether with the previously mentioned amino acids, at pH 7.8. This provides a means of de- termining histidine by the difference between the values obtained at pH 6.5 and pH 7 .8. Reagents 1. Sodium arsenate solution (2.0M). 62.4 g. sodium arsenate (NazHAsO4 ° 7H20) is dis— solved in and made up to 100 ml. with deionized water. From this stock two solutions are pre- pared. One is adjusted to pH 6.5 and the second to pH 7.8 by the addition of HCl. 2. Borate-arsenate reagent. Two solutions are prepared as follows: 61.8 g. (1.0M) boric acid is dissolved in 1 liter of 2.0M Sodium Arsenate (NazHAsO4-7H20) prepared by dis- solving 624 g. sodium arsenate in deionized water adjusting the pH to 6.5 and 7.8 and making up to 1 liter with deionized water. 3. Phosphate Buffer (0.2M). 7.6 g. of so- dium phosphate (Na3P04-12H20) is dis- solved in deionised water, the pH adjusted to 6.5, and the volume made up to 100 ml, 4. L-arnino acid oxidase. (Crotalus ada- manteus venom obtainable from Ross Allen Reptile Institute, Silver Springs, Fla.) A sus— pension of dried venom in deionized water is prepared such that 1 ml. contains 10 mg. The suspension is centrifuged and the clear super- natant fluid removed. This should be stored at 4° C. and remains active for several days. 5. Catalase. Crystalline beef liver catalase (Worthington Biochemical Corp., Freehold, NJ.) is diluted 1: 5 with 0.2M phosphate buffer. The enzyme solution is stored at 4° and __ stable for several weeks. 6. Perchloric Acid 7%. 7. Potassium Hydroxide SN. 28 g. KOH dissolved in and made up to 100 ml, with deion- ized water. 82 8. Histidine Standard (0.6 pM per 7711.). 125.8 mg. L-Histidine hydrochloride mono- hydrate is dissolved in deionized water, ad- justed to pH 7.8 and diluted to 1,000 ml. Procedure 1. 2 ml. of serum is mixed with 2 ml. of 7 percent perchloric acid. The tube is centri- fuged and 0.4 ml. of 5N potassium hydroxide added to precipitate the excess perchlorate. 2. Tubes are set up, and reagents added in order shown in table on page 83. 3. Read pH 6.5 assay, within 14—16 minutes after the addition of serum filtrate, at 292 ma. 4. Read pH 7 .8 assay, 30 minutes after the addition of serum filtrate at 292 mp. Calculation Since the interfering absorbancies of the complexes formed from phenylalanine, tyro- sine and tryptophan are greater at pH 6.5 than at pH 7 .8 it is necessary to apply a correction factor. This has been found to be 0.70.1 Thus the QB. value obtained in the pH 6.5 assay is multiplied by 0.70 and the result sub- tracted from the value obtained in the pH 7 .8 assay. Then, O.D. pH 7.8— (O.7>< 4.4= micromoles Histidine/ ml. serum. References 1. Baldridge, R. 0., and Greenberg, N. A Method for the determination of histidine in blood. J. Lab. & Olin. Med. 61: 700, 1963. 2. LaDu, B. N., and Michael, P. J. An Enzymatic spectrophotometric method for the determination of phenylalanine in blood. J. Lab. & Olin. Med. 55 :491, 1960. pH 6.5 assay pH 7.8 assay Blank Test Blank Test Deionized water .................................. 0.70 ml. 0.70 ml. 0.70 ml. 0.70 ml. pH 6.5 Arsenate solution reagent #1 ...................... 1.50 .................. pH 6.5 Arsenate-borate solution reagent #2 .................... 1.50 ............ pH 7.8 Arsenate solution reagent #1 ................................... 1.50 ...... pH 7.8 Arsenate-borate solution reagent #2 . . . . . . . . . . . . . . . . . . . . . . . . ......... 1.50 Dilute catalase reagent #5 ......................... 0.03 0.03 0.03 0.03 L-amino acid oxidase reagent #4 .................. 0.30 0.30 0.30 0.30 MIX CONTENTS OF TUBES BY TAPPING Serum filtrate ..................................... 0.50 0.50 0.50 0.50 OR Standard ................................................ 0.50 0.50 MIX CONTENTS OF TUBES BY TAPPING Lactic and pyruvic acid determination in serum A. Determination of Lactate in Blood In the determination of pyruvate use is made of the decrease in absorbancy at 340 m,» as DPN H is converted to DPN in the reaction: COO ' COO " I Lactic I C = 0 + : HOCH l + DPNH + H Dehydro- l + DPN CH3 genase CH 3 Pyruvate Lactate This same reaction can be utilized to esti— mated lactate if the equilibrium can be shifted to the side of pyruvate. This can be achieved by means of excess DPN, an alkaline medium, and the removal of the pyruvate formed by the addition of hydrazine.1 Reagents 1. Glycine-Hydrazz'ne Bufler (pH 9). 3.75g. (0.5M) glycine and 2.35 ml of an 85 percent solution of hydrazine hydrate are dissolved in and made up to 100 ml. with deionized water. This solution should be stored in the refriger- ator at 4° C. 2. Dipbaspbopyfl'dine Nucleotide (DPN) 83 (0.027 M.). 100 mg. DPN are dissolved in and made up to 5 ml with deionized water. Store in the refrigerator at 4° C. 3. Lactic Debydrogenase (L.D.H.) 2 mg. en- zyme protein per ml. The preparation Boeh- ringer Rabbit Skeletal Muscle LDH #15145 suspended in 2.2 M ammonium sulfate, obtain- able from the California Biochemical Corp., Los Angeles, is suitable. Store in the refrig- erator. 4. Tricbloroacetic Acid 10% in 0-5N: HCl. 10g. of trichloroacetic acid is dissolved in and made up to 100 ml with 0.5N.HCl. 5. Blank Solution 5% Triohloroacetic acid in 0.25N HCl dilute reagent (4) 1:2 with de- ionized water. 6. Standard Solution 2.0 and 4.0 mEq./L. L-Lactate. Lithium lactate obtained from Sigma Chemical Co., St. Louis, Mo. must be assayed for L—Lactate content. This can be most simply achieved by the use of Boehringer Lactate Test Kit TEB 15972 obtainable from the‘California Biochemical Corp., Los Angeles. Example. A batch of lithium lactate as- sayed at 78.38% L-lactate. Singe 1 mEq. of lactate= 89 mg. Since 4 mEq. of lactate= 366 mg. Since however product is 78.38% L-lactate 366 X 100 to obtain 366 mg, 78 38 mg must be weighed out. i.e. 470 mg. dissolved in 1000 ml will in this case give a 4 m. Eq/Liter solution of L- lactate. Note: All glassware used must be soaked in chromic acid solution, rinsed with water, soaked in detergent, and thoroughly rinsed in deionized water, and then dried before use. Procedure 1. Immediate precipitation of blood is es- sential. 1 ml of precipitating reagent (reagent (4)) is placed in a tightly stopper-ed tube and weighed. Place tube in an ice bath and add an approximately equal volume of freshly drawn blood and mix well. Reweigh tube: (a) Weight with blood—weight without blood=weight of blood added. (b) Weight of blood added W (o) Dilution = Volume of blood added + volume ppting reagent Volume blood added. 2. Centrifuge =volume of blood 3. The following is set up in duplicate. To 3 ml. bufler solution (reagent 1) add 0.1 ml blank solution for BLANK; 0.1 standard solutions for STANDARDS; or 0.1 test super- natant for TEST. 4. To all tubes add 30 pl. LDH. Mix. 5. Add 200 ”.1. DPN. Cover with parafilm and mix by gentle inversion. 6. Incubate in 25° C water bath for 45 minutes. 7. Read optical density at 340 my. in spectrophotometer. LNote: The optical density is stable for 40—50 minutes after incubation. Calculation OoD-Test_0°D-Blmk Average O.D. Stdl mEq/LXDllunon =mEq/Liter L—Lactate (O.D. STD.2 mm,L—O.D. BLANK)+(O.D. STD.4 mum—O.D. BLANK): Average O.D. STD-1 mum 6 84 Reference 1. Scholz, R., Schmitz, H., Bucher, T. and Lampen, J. O. Uber die Wirkung von Nystatin auf Back- erhefe. Biochem. Z. 3:31:71, 1959. B. Determination of Blood Pyruvate The determination of blood pyruvate has, until recently, been performed by its reaction w i t h 2, 4-dinitrophenylhydrazine.1 T h i s method however in addition to having technical difficulties is non-specific, in that hydrazones are also formed by other a-keto acids present in blood. The method described here is based on the reduction of pyruvate to lactate by the en- zyme lactic dehydrogenase. This reaction uti- lizes DPNH as the "hydrogen donor and has an equilibrium constant such that at pH 6.9 it proceeds virtually to completion, furthermore the decrease in the amount of DPNH is stoi— chiometrically related to the reduction of pyru- Vate (1 mole’DPNI-I per 1 mole of pyruvate). Since DPN H has an absorption peak at 340m); and DPN does not, the conversion can be meas- ured spectrophotometrically.2' 3 Reagents 1. 10% Trichloroacetic acid in 0.5N. HCl 100g Trichloroacetic acid dissolved in and made up to 1,000 ml with 0.5N Hydrochloric acid. 2. 5% Tricbloroacetic acid in 0.25N. HCl. Dilute reagent (1) 1 : 1 with deionized water. 3. Di-basic Potassium Phosphate Bufler. (pH 9), A 1.1M solution of di-basic potassium phosphate (KZHPO4) is prepared by dissolv- ing 19.14 g. in deionized water and diluting to 100 ml. 4. Reduced Dipbosopyridine Nucleotide (DPNH). 3 X 104M solution (2mg/ml pre- pared by dissolving 2 mg DPNH in 1 ml of freshly prepared 1 percent sodium bicarbonate (NaHCOs). (Preweighed vials containing 2 mg DPNH are obtainable from Sigma Chemi- cal Co., St. Louis, Mo.) 5. lactic Debydrogenase (0.75 mg enzyme protein/ml). 75 Ml of LDH type III (Sigma) containing 20 mg. enzyme protein/ml ammo- nium sulfate solution are diluted to 2 ml with deionized water. 6. Stock Standard (10 Eq./L pyrucate). 110 mg sodium pyruvate (CHaCOCOONa) are dis- solved in and made up to 100 ml with deionized water. 7. Working Standards 0.2 m Eq/ L pyruvate (2 m1. stock standard dilute to 100 ml with reagent (2) ) 0.1 m Eq/L pyruvate (1 ml. stock standard diluted to 100 ml with reagent (2)) NB. the concentration of DPNH (reagent (4)) may re- quire adjusting so that an optical density read- ing at time zero of approximately .800 is obtained. Procedure 1. Before drawing blood place a volume of (e.g. 1 ml) of 10 percent Tric‘hloroacetic acid (reagent (1)) in a tightly stoppered tube and. weigh. Place tube in an ice bath. Inject an approximately equal volume of blood immedi- ately upon drawing into the precipitating re- agent and mix ‘well. Reweigh the tube : then (a) (Wt. of tube with blood)—(Wt. of tube without blood)=(Wt. of blood added) (lb) (Wt. of blood added) (Density of blood) =vol. blood added (0) Dilution=(Vol. blood added) + (vol. ppt. reagent) (V01. blood added) 2. Centrifuge. 3.,The following procedure is carried out in duplicate: To a cuvet’oe is added 250 pl of reagent (2) for the BLANK; 250 [1.1 of standard for STAND- ARD; or 250 pl of clear supernatant fluid from protein precipitation for TEST. 85 4. 250 [1.1 of phosphate buffer (reagent (3)) is added to each cuvette and mixed. :5. 50 [1.1 of DPNH are added to the cuvettes and mixed, noting time. 6. The first reading of optical density is made at 340 mu exactly 2 minutes after the addi- tion of the DPNH to the cuvette containing the reference solution (deionized water) . 7. Exactly 3 minutes after the DPNH ad- dition, 10 [1.1 of LDH are added to each cuvette and mixed. 8. Each cuvette is read again exactly 2 min- utes after the addition of the enzyme. Notes: 1. Tests may be run at room temperature. 2. The time intervals used in steps 6, 7, and 8 have been found to be convient, the intervals, while variable, must however, be the same for each cuvette. 3. Cells with a 10 mm. light path and 0.7 ml. capacity are convenient (obtainable from Pyrocell Manfucturing Co.) Calculation (O.D. Test)—(O.D. Blank) (Average O.D.—Standardo_ 1 m. Eq/L =mEq. Pyruvate/L. )XDilution References 1. Friedemann, T. E., and Haugen, G. E. Pyruvic Acid: II Determination of Keto acids in Blood and Urine. J. Biol. Chem. 147: 415, 1943. 2. Sega], 8., Blair, A. E., and Wyngaarden, J. B. An Enzymatic Sepctrophotometric‘ method for the de- termination of pyruvic acid in Blood. J. Lab. Olin. Med. 1,8: 137, 1956. 3. Gloster, J. A. and Harris, P. Observations on an enzymic method for the estimation of pyruvate in blood. Olin. Uhim. Aeta. 7:206, 1962. (O.D. Stdo 1 m_ Eq/L. —0.D. Blank)+ (O.D. Stdo_ 2m. EQ. /L_0'D‘ Blank) 3 Determination of serum lipids A. Determination of Total Lipids The method of Sperry (1) provides a means of determining serum total lipids withou‘t the disadvantages inherent in many of the earlier described procedures. In addition the lipids are available for further analysis after determination by weighing. Reagents 1. Cbloroform-metbanol (2:1). Two vol- umes of chloroform are mixed with one volume of methanol. 2. Calcium chloride (20 mg per 100 ml). 86 =Average O.D. Standard.o_1 mm” 20 mg of calcium chloride are diluted in and made up to 100 ml with deionized water. 3. Reagent (2) is saturated with chloro- form-methanol (2: 1) by placing 23.7 ml of (2) in a separatory funnel together with 96.3 ml of reagent (1). The phases are allowed to separate and the lower phase discarded. Procedure 1. Extraction: 8.3 of methanol is placed in a 25 m1 volumetric flask, and 1 ml of serum is added from a pipette whose tip is placed just above the point where the neck of the flask meets the bulb. The contents of the flask are swirled vigorously during the addition, and the tip of the pipette is “touched off” against the neck after draining. An approximately equal quantity of chloroform is added, and the flask brought to a boil on a steam bath. When cool the contents of the flask are made up to the mark with chloroform, mixed and filtered rapidly through lipid-free filter paper. A 20 ml. aliquot is pipetted at once for purification. 2. Purification: The 20 ml aliquot of the serum filtrate is pipetted into a 25 ml stoppered cylinder, 4 ml of water is added, the stopper inserted, and the contents shaken vigorously for 1 minute. (2). The cylinder is allowed to stand until the two phases separate completely. The upper phase is removed as completely as pos- sible and discarded. 3 ml of reagent (3) is used to wash down the walls of the cylinder, Without mixing with the lower phase. The cyl- inder is rotated gently to mix the wash solution with the residue of the original upper phase. The wash solution is removed to waste and the washing procedure repeated twice more. 3. Evaporation, filtration and weigbing: 2 m1. of methanol is added to the contents of the cylinder and mixed. The solution is trans- ferred to a 50 ml Ehrlenmeyer flask. The cylinder and stopper are washed with chloro- form-methanol (2:1) and the washings added to the Ehrlenmeyer flask. The contents of the flask are evaporated to dryness under a stream of nitrogen on a water bath at 400 C. The residue is dissolved in” chloroform-methanol (2: 1) and filtered through lipid-free paper into preweighed 5 ml beakers. The solvent is evap- orated under nitrogen as before, and the beakers placed in a dessicator with “Dricrete,” and un- der nitrogen at negative pressure, for at least 18 hours. The beakers are weighed and the weight of lipid determined. Calculation Weight of lipidX 125=mg/ 100 ml total se- rum lipid. References 1. Sperry, W. M. in “Methods of Biochemical Analysis” Vol 11, ed. D. Glick Interscience Publishers Inc. New York, 1955. 2. Folch, J., Lees, M., and Sloan-Stanley, G. H. A simple method for preparation of total pure lipid extracts from brain. Fed. Proo. 13: 209, 1954. B. Determination of Plasma Phospholipid Reagents 1. 60% Perchloric acid, HClOl. 2. 4% Perchloric acid. Dilute 60% 1: 15. 3. Extraction solution. 15 parts petroleum ether (B.P. 100°—120° C.) added to 85 parts iso- butyl alcohol. 4. 5% Ammonium molybdate, (NH...) 2M004, in 4N H2804, sulfuric acid. 5. 43% SnCIZZHZO, stannous chloride, in concentrated hydrochloric acid, HCl. Each day dilute 1:400 (e.g. 10 ,ul in 4.0 ml.) with 18N H2804. 6. Standard. Stock. 1 mg. P per ml. 4.394 gm. KH2P04, potassium phosphate monobasic, dissolved in and diluted to 1 liter with water. Add a few drops of CHCla, chloroform. Inter- mediate. 100 Mg./ ml. 10 ml. stock standard plus 3.3 ml. 60 percent HClO4, diluted to 100 ml. with water. Working. 1 tug/m1. 1.0 ml. intermediate standard diluted to 100 ml. with 2 percent H0104. 7. Bloor’s Reagent. nol : 1 part ethyl ether. 3 parts absolute etha- Procedure 1. Into a glass-stoppered tube pipette 4.9 ml. Bloor’s Reagent. Wash in 0.1 ml. plasma. Stopper and mix well. Stand a few minutes. Centrifuge. 2. Into a glass-stoppered tube pipette 1 ml. supernatant fluid. Evaporate to dryness in a sand bath at 100° C. Add 0.1 ml. 60 percent H0104. Digest in the sand bath at 120° C. un- til clear, and then allow to cool. Add 2 ml. water followed by 0.5 ml. molybdate solution and 2.5 ml. extracting solution and continue as 87 for inorganic phosphorus. 1 ml. of water plus 1 ml. of 4 percent perchloric acid are placed in a clean glass stoppered tube for blank or 2 ml. working standard. All tubes are mixed on a .Vortex mixer for 20 secs. Centrifuge briefly. 3. Into a clean glass stoppered tube pipette 2 ml, of the supernatant fluid from each tube. Add 0.3 ml. absolute ethanol. Mix. ‘ 0.2 ml. stannous chloride-sulfuric acid reagent. Mix. 4. Read for 15 minutes at 625 my. in a spectrophotometer using a dry cuvette. Calculation (O.D. Sample—O.D. Blank) X 2 X 100 (O.D. Standard—O.D. Blank) 0.02 1000 (O.D. Sample—O.D. Blank) (O.D. Standard—O.D. Blank) X10=mg/100 ml. Phosoholipid (as lecithin) =lipid phosphorus X2 5 Reference O’Brien, D., and Ibbott, F. A.,- Laboratory Manual of Pediatric Micro and Ultramiero Techniques. Hoe- ber, New York, 3d ed. repr. 1964, p. 250. C. Determination of Plasma Cholesterol Reagents 1. Cholesterol, standard. 100 mg. cholesterol, m.p. 149° C, is dissolved in and made up to 1,000 ml. with 95 percent ethanol. 2. 2N Potaxsium hydroxide, KOH, aqueous. 3. Acetone, CHsCOCHa. 4. 95% Ethanol. 5. Ethanol-acetone mixture, 1: 1 v/v. 6. Phenolphthalein, 1 percent in 95 percent ethanol. 88 7. 10% Hydrochloric acid, HCl, aqueous. 8. Digitonin, CMHMOZB, 0.5 percent in 50 percent ethanol. 5 gm. digitonin is di5501ved with gentle heating in 1,000 ml. 50 percent ethanol/water. The solution is filtered after being allowed to stand overnight. 9. Ethyl ether, CzHaoCsz, anhydrous. 10. Ether-acetone mixture, 2: 1 v/v. 11. Acetic anhydride, (CHSCO) 20 HCl free. 12. Sulfuric acid, H2804, sp. gr. 1.84. 13. Glacial acetic acid, CHaCOOH. 14. Modified Liebermann—Burchard rea- gent (prepare immediately before use). Slowly add 2 ml. concentrated H280, to 30 ml. acetic anhydride. Add 15 ml. glacial acetic acid. Cool to room temperature. Procedure Total I. 1. Into 12 ml. heavy wall graduated centri- fuge tubes, pipette: . Test. 0.1 ml. serum or plasma... Blank. Nil. Standards. 100 mg./ 100 ml. standard: 1 ml. stock cholesterol solu- tion. 300 mg./ 100 ml. standard: 3 ml. stock cholesterol solu- tion. 2. Add to each tube 0.5 ml. 2N KOH, and heat in 65° C water bath for 15 min. 3. Tests and Blank. Add 5 ml. ethanol- acetone mixture. Standards. 100 mg./100 ml. standard: Add 2 ml. 95 percent eth- anol and 31111. acetone. 300 mg./100 ml. standard: Add 3 ml. acetone. 4. Stir with 3 mm. glass rods and return tubes to 65° C water bath for 30 min., leaving rods intubes. 5. Remove tubes from water bath. Add 1 drop 1 percent p‘henolphthralein solution to each tube. Acidify with 10 percent HvCl (approx. 0.5 ml.). Add 3 ml. 0.5 percent digitonin solution. *Stir, drain rods, and remove rods to numbered block. Stopper tubes with No. 0 rubber stoppers, and allow tubes to stand over- night. (Minimum precipitating time is 12 hours.) II. Remove stoppers and centrifuge tubes for 15 minutes at 2,000 rpm. Aspirate and discard supernatant. Add 5 m1. ether-acetone mixture and suspend precipitate with corresponding rod. Drain rod and return to block. Centri- fuge tubes for 5 minutes at 2,000 rpm. Wash precipitate with ether, using above technique. Centrifuge for 5 minutes at 2,000 rpm. Care- fully aspirate supernatant; stopper tubes loosely with No. 00 rubber stoppers wrapped in aluminum foil. Heat in 65° C oven for 20 minutes, then in 100° C oven for 15 minutes. Push stoppers in tightly and allow to cool to room temperature. III. Color Development 4 ml. Liebermann-Burchard reagent are added to the blank at time 0, and to successive samples at 1-minute intervals, each precipitate being finely ground with its rod, stirred well, and placed in a darkened water bath at 25° C. Samples are read in Hilger, filter No. 61 or other suitable absorptiometer, at 610 mm. 25 mins. after addition of reagent. Free 1. To 0.1 ml. serum or plasma in a 12 ml. heavy wall graduated centrifuge tube, add 3 ml. ethanol-acetone mixture. Heat for 10 minutes in 65° C water bath. 2. Centrifuge for 5 minutes at 2,000 rpm. 3. Decant supernatant into clean tube. 4. Repeat extraction, pooling supernatants. 5. Add 1 drop 10 percent HCl and 3 ml. 0.5 percent digitonin. 6. Follow procedure for Total from* (1.5). Calculation (O.D. Test—O.D. Blank) TAverage (O.D. Std.—O.D. Blank)><100 =mg./100 ml. Cholesterol Cholesterol Ester = Total Cholesterol - Free Cho- lesterol Note : ’rBy average is meant the mean of the 100 mg./ 100 ml. standard and the 300 mg./ 100 ml. divided by 3. Reference After Colman, D. M., and McPhee, A. F., Amer. J. 01m. Path. 26: 181, 1956. Determination of plasma proline The technique presents no special problem although if the test is carried out infrequently, it is as well to run the standard curve before using the blood samples as the reagents do not keep indefinitely. This is also true of the stand- ard solution. In other respects the procedure is straightforward. Reagents l. Nitroprusside-aldebyde reagent. Dissolve 2 gm of sodium nitroprusside, Na2(Fe(CN)5 NO) -2H20 (nitroferricyanide) in about 50 ml of cold distilled water. Add 20 ml of cold highest quality acetaldehyde, CH3 - CHO, to the cold solution. Make up to 100 ml with cold distilled water. Carefully adjust to pH 7.0, using solid sodium bicarbonate and an external indicator paper. When the reagent is not in use, keep in the refrigerator. Reproducible results have been obtained with reagent that is several weeks old, but the stability varies from lot to lot. It is preferable to make up the reagent fresh, and it should always be checked against known standards at the time determinations are made. 89 2. 4% sodium carbonate, NagCOS. .3. 0.8% Uranyl acetate, UOZ(CZH302)2‘ 2H20, solution. 4. 0.3% sodium chloride, NaCl. 5. Standard proline, C4H8NCOOH, solu- tion. 100 jug/m1 in 0.3 percent NaCl. Test To the fasting child give 1.5 gin/kg body weight of casein as a 15 percent solution that has been suitably flavored. Blood samples fast- ing and at 120 minutes are taken. Procedure 1. Preparation of standard curve Pipette out into 12 X 75 mm test tubes as follows: Standard 0. 3 % NaCl Concentration proline solu- solution, of standard, tion, ml. ml. ug/ml. 0.0 l .0 0 0.2 0.8 20 0. 5 0. 5 5 0 l .0 0.0 1 00 Add to each tube 0.25 ml nitroprusside- aldehyde reagent, followed by 0.5 ml water. Stand for 2 minutes and then add 0.25 ml 4 percent sodium carbonate solution. Between 2 and 4 minutes read in 1 cm micro cells in Spekker using No. 7 filter or in Beckman DU at 600 mp against water. The standard curve for proline is almost linear through the range given and is essentially linear at the lowest concentrations. 2. Determination on serum (or plasma) Into a 2 ml glass-stoppered test tube pipette 0.4 m1 uranium acetate solution. Wash in rapidly 0.2 ml serum. Mix thoroughly and centrifuge. Transfer 300 pl SNF to another tu‘be. Add 50 pl nitroprusside aldehyde reagent. Stand 2 minutes. Then add 50 ,ul sodium car- 90 bonate solution. Stand 2-4 minutes. Read as for standard curve. Calculation Concentration of Standard><(Reading—Blank) Standard—Blank X 2 = pg Proline/ml 01’ Plot 21 standard curve. Read unknowns from curve. Value from curve><2=ug/ml. Interpretation Fasting 1. Normal 24.8 ug/ml (13.8—32.5). 2. Pancreatic insufficiency 14.1 pig/ml (80—230). 120-minute level 1. Normal 76 ,ug/ml. 2. Pancreatic insufficiency < 30 lug/ml. References 1. Gould, B. S. and Shwachman, 11.: Studies in cystic fibrosis. Determination of plasma proline following protein feeding as a diagnostic test for pancreatic insufficiency. AMA J. Dis. Child. 91:584, 1959. 2. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro-biochemical techniques. Hoeber, New York 3d edit. repr. 1964. p. 228. The tryptophan loading test and assay of kynurenine derivatives Urine is collected for a timed period after a loading dose in the fasting state of 100 mg/kg of L—tryptop’han, given orally, suspended in milk with a Waring Blender. Further elabora- tion of tryptophan metabolites can be elegantly carried out by chromatographing the urine (Coppini, D., et a1, Olin. Uhem. 5:391, 1959), a procedure also described in this section. The Fluorimetric Assay of Estimation of Xanthurenic and Kynurenic Acids in Urine Current opinion is that the simple colori— metric method for xanthurenic acid using alka- line ferric ammonium sulfate is not reliable, probably on account of the presence of other ion chelating substances in urine. For this reason the more elaborate fluorimetric assay is given. Reagents l. Hydrochloric acid, HCl. 0.2N, 0.5N, 1.0N and 5N. 2. 0.5M Phosphate hufler (pH 7.4). 56.8 gm anhydrous NagHPO4, disodium hydrogen phos- phate, and 1326 gm of KH2P04, potassium phosphate monobasic, are dissolved in and made up to 1,000 ml with ion-free water. Adjust pH. 3. 0.005M Phosphate buffer (pH 7.4). Di- lute 0.5M ’bufier 1: 100. 4. Concentrated sulfuric acid, H2804, re- agent grade. 5. Saturated aqueous sodium hydroxide, NaOH, reagent grade. Working Standards 1. Xanthurenic acid, approximately 660 mg of pure xanthurenic acid, CBH4N(OH)2COOH, free of uncleaved 8-methyl ether, is dissolved in and made up to 1,000 ml with ion-free dis— tilled water. 2. Kynurenic acid, approximately 610 mg of kynurenic acid, C9H5N(OH)COOH, is dis-- solved in and made up to 1,000 ml with ion- free distilled water. Reference Standards 1. Xanthurenic acid. Dilute 1 ml of work- ing standard to 400 ml with 0.005M phosphate buffer (pH 7.4). Treat 1 ml of dilute standard as for aliquot of column eluate. 2. Kynurenic acid. Dilute working stand- , eter. ard as for xanthurenic acid. Treat 1 ml of dilute standard as for aliquot of column eluate. Apparatus ,1. Aminco Bowman spectrofluorometer set as follows: Xanthurenic acid, exciting 370 mu, fluorescence 530 my. Kynurenic acid, exciting 340 mu, fluorescence 435 mp“ 2. Or, Beckman DU with fluorimetric at- tachment and the following filters: Xanthur- enic acid—exciting No. 5860, fluorescence No. 3486 and No. 5031. Kynurenic acid—exciting No. 5860, fluorescence No. 3389 and No. 5113. 3. Or, Coleman Model 12—0 Photofluorom- Xanthurenic acid—exciting No. 5874, fluorescence No. 3385. Kynurenic acid—excit— ing No. 5874,, fluorescence No. 3389 and No. 4305. Ion Exchange Column 3 cm. Dowex 50W—X12(200—400 mesh) packed in a force feed chromatograph column. Procedure 1. To each of two glass-stoppered 250-ml volumetric flasks containing 100 ml of deionized water add 20 ml of urine. To one sample add 1.0 ml of xanthurenic acid and 1.0 ml of kyn- urenic acid working standard and then to both add 30 m1 of 1N HCl. Mix. (RECOVERY and TEST.) 2. Pass through column previously washed with 50 ml of 5N HCl. Wash with 50 ml 0.2N H01, 100 m1 of 0.5N HCl, and 20 ml of water. Elute column with 400 m1 of ion-free water. 3. Treat 1 to 3 ml of eluate as follows: Use same volume for RECOVERY and TEST. Xanthurenic acid. Make up to 5 ml with 0.005M phosphate buffer. Add 5 ml of satur- ated NaOH slowly, cooling in iced water. Kynurenic acid. Make up to 6 ml with 0.005M phosphate buffer. Add 4 ml of concentrated H2804 slowly, cooling in iced water. 4. Centrifuge tubes for 5 minutes at 2,000 rpm if solutions are not clear. 91 5. Read fluorescence against STANDARD, and BLANK of 0.005M phosphate buffer after 1 hour with fluorometer settings as under “Apparatus.” Calculation F=fluorescence reading (on % Transmittance scale) Concentration of Working Standard (lug/ml) Fran“ FBlank 1 . —— — = ml urine XFRecovery_FTestX20 Mg/ The Chromatographic Assay of Kynurenine Derivatives of Tryptophan in Urine The chromatographic procedure described below provides a rapid method of separating eight of the urinary metabolites of tryptophan as distinct fluorescent spots on paper. Several procedures are then described by which the spots may be eluted and quantitatively meas- ured. Abnormalities in this pathway are seen in diabetes, mongolism, Wilson’s disease, con- genital aregenerative anemia, scleroderma, and in phenylketonuria. Also an increase in xan- thurenic acid excretion is characteristic of vi- tamin B6 deficiency dependency, and kynuren- inase defects. There is evidence, however, that an increased ratio of OH—kynurenine: OH—an- thranilic acid in the urine is a more sensitive index of this state. These methods are linear Within the experimental range and are repro- ducible within the following 95 percent confi- dence limits (i 2x/(Coelf. Var.,e.t)2+ (Coefl'. Vans”, )2): xanthurenic acid, 13.7 percent; kynurenine, 18.3 percent; kynurenic acid, 10.8 percent; N-oc- acetylkynurine, 13.7 percent; hydroxykynuren- ine, 14.5 percent; hydroxyanthranilic acid, 23.1 percent; xanthurenic acid/8-methy1 ether, 20.5 per cent. Column chromatography of xanthu- renic acid, kynurenic acid and XAE gives very similar results. 92 Normal Values The figures given below are based on values obtained from 15 apparently healthy, well- nourished, mentally defective children after an L-tryptophan load of 100 mg/kg. The results are expressed as ,u. mols/kg/7 hours and as a percentage of the total output of the metabolites listed below plus methylnicotinamide. The figures in the last colum represent the mean of the sum of individual percentages and there- fore are not necessarily the same as those that can be derived from the means in p. mols/kg/7 hours. It mol/kg/7 hours Range Mean Kynurenine ............... 3.8—50. 5 1 5.6 OH-kynurenine ............ (0.5-. 9.2 3.0 Kynurenic acid ........... 3.4—20.6 7.1 Xanthurenic acid .......... 0.8- 5.6 1.9 OH-anthranilic acid ....... 1.2- 4.9 3.0 N-alpha acetyl kynurenine. . 1.6-22.5 9.0 The results are also expressed as percent- ages of the total metabolites listed above plus methylnicotinamide. Percent of kynu- renine athway meta olites Range Mean Kynurenine ................ 17.9-58.7 36.2 OH—kynurenine ............ < 1 .0—20.0 7.6 Kynurenic acid ..... g ....... 1 1.9—3 1.6 18.7 Xanthurenic acid ........... 1.1- 9.8 5.4 OH-anthranilic acid ........ 2.4-26.9 10.0 N-alpha acetyl kynurenine . . 4.7-43.2 21.3 Figure 29. CHROMATOGRAPHY OF TRYPTOPHAN DERIVATIVES 5)——>| I]: (4 O ORIGIN n-BUTANO'L-ACETIC ACID-WATER 3-Hydroxy.. anthranilic AcidOQ O Xanthurenic Acid 8 Methyl Ether C50 Xanthurenic Acid DL 3-Hydroxykynureniry L Kynurenine Anthranilic Acid Nd-Acetyl- kynurenine Kynurenic Acid DISTILLED WATER . OH-kynurenine The ratlo m based OI]. the above individuals=1.2 (Range 0—4.1) Chromatography Reagents 1. n-Butyl alcohol, CHacHchZCHgoH, reagent grade. 2. Glacial acetic acid, CHSCOOH, reagent grade. 3. Distilled water. 4. Standard solutions, all 500 pg. ml. except Where otherwise stated. a. L-kynurenine sulfate (Mann Research Laboratories) dissolved in water, con- centration 2.5 mg/ ml. b. 3-Hydroxyanthranilic acid (Mann) dissolved in small amount of 1N HCl and made up to volume with water. c. Kynurenic acid (Mann) , dissolved in 93 a small amount of 2N NH4OH and made up to volume with absolute ethanol. d. Anthranilic acid (Nutritional Bio- chemicals), dissolved in water con- taining two drops saturated NaOH and made up to volume with water. e. Xanthurenic acid. 1. Pure (Calif. Corp.). 2. Containing the 8—methyl ether (Sigma) both dissolved as in d. f. Na-acetylkynurenine dissolved in ab- solute ethanol, concentration 1 mg/ml. g. 3-Hydroxy—DL-kynurenine dissolved as in b.1 mg/ml. Apparatus 1. Whatman No. 1 Chromatography paper (57 X 46 cm). 2. Chromatocab (Model A. Research Equipment Corp., Oakland, Calif.) . 3. Chromatography drying oven (Model C—425, Research Specialties Co., Rich— mond, Calif) 4. Ultraviolet light source (46495 Quad plus. Standard Scientific Supply, New York, N.Y.). 5. Heat Gun (Model 12200, Cole-Farmer Instrument & Equipment Co., Chicago, Ill. 6. Kirk pipettes, 5, 10, 20, and 25 pl. Procedure Urine Collection. L—tryptophan is given in a dose of 100 mg/kg and the urine is collected for 7 or 24 hours. Urine samples are chromat- ographed as soon as possible after collection. They can be stored at — 12° C. if necessary. Chromatography. Two-dimensional de- scending chromatography is used for all sam— ples. Standards and urines are applied by micro pipette to a spot 8 cm. from each side at one corner. A stream of warm air from the heat gun is blown at the spot during application. 94 The spot should not be more than 7 mm. in diameter. 100 ,ul of, each urine is used for the initial runs and 10 11.1 of each of the standards is used. Eight papers are chromatographed per run; at least one of these must contain only standards. ’ The solvent for the first dimension (57 cm. length) is the organic layer of butanol-acetic acid-water (4:1 :5). The aqueous phase is used in the tray at the bottom of the chromatocab. The papers must equilibrate with the aqueous phase for 4 hours. The organic phase is added to the troughs and the papers are run for 16 hours. At the end of the run, excess solvent is aspirated from the troughs, the papers are clipped to the glass supporting rods and re- moved to the drying oven, and dried at 40° C. for half an hour. The second dimension (46 cm. length) is distilled water and is run at right angles to the butanol run for 4 hours. The papers are again dried at 40° C., taking about 45 minutes. The papers are then viewed under ultra- violet light (253.7 Hip) and the spots are cir- cled in pencil. This must be done quickly as at least one of the substances (kynurenic acid) deteriorates rapidly under UV light. The spots are then cut out and placed in 15 ml glass- stoppered tubes. Pieces of paper, approxi- mately the same area as the fluorescent spots, are cut from fluorescence-free portions of the filter paper sheet and used as paper blanks. Elute with the appropriate reagents overnight. Elution Reagents 1. Absolute ethanol (spectroscopic grade). 2. Distilled water. 3. 0.4N HCl, hydrochloric acid. 4. 1% p-dimetbylaminobenzaldebyde (CH3)2 NCsH4 CHO, in 50 percent acetic acid. 5. 0.5% sodium nitrate, NaNOz. 6. Sulfanilic acid, NH2C6H4SOsH-H20, 0.5 percent. in 2 percent HCl. 7. Pyridine, C5H5N. 8. 0.25% sodium nitrite, NaN02. 9. 10% ammonium sulfamate, NH4NH2803. 10. 0.25% N -1 mapbtb yletb ylenediamine di- byd’rocblo'ride’ CloH-[HNCHZCI—IzNI—Iz ° QHCL Apparatus 1. Glass—stoppered 15-m1 test tubes. 2. 15-ml graduated conical test tubes. 3. Water bath, constant at 15° C. (Model MR—3210, Blue M Electric Go, Blue Island, Ill.) 4. Refrigerated centrifuge. 5. Beckman DU with hydrogen and tung- sten lamps and appropriate cuvettes. Procedures 1. Kynurenic acid and xanthurenic acid 8- methyl ether. Elute with 5 m1 of absolute ethanol. Shake thoroughly. At the end of 16 hours, pack down the paper piece and centri- fuge at 1,500 rpm for 5 minutes. Read the alco— hol solutions including paper BLANK at 243 my. for kynurenic acid and 237 m for xanth— urenic acid 8-methyl ether. 2. Xanthurenic acid, 3-hydroxykynurenine, 3-hydr0xyanthrani1ic acid. These substances eluted for 16 hours with 3.8 ml of distilled water. The tubes are then placed in a water bath held constant at 15° C. 1 ml of equal parts of 0.5 percent NaNOz and 0.5 percent sul— fanilic acid, freshly mixed, is added to each tube and mixed. Then 0.2 ml of pyridine is added and mixed. The solution from xanthurenic acid is poured off into a 15—ml conical test tube and centrifuged at 15° C. for 5 minutes at 1,000 rpm. The solutions are read immediately in the Beckman at 510 mp. The tubes containing 3-hydroxykynurenine and 3-hydroxyanthra- nilic acid are centrifuged at 15° 0., placed in the 15° C. water bath for 60—80 minutes, and read at 450 my. All readings must be made as soon as possible to ensure a minimum departure of the temperature from 15° C. 3. Kynurenine and Na—acetylkynurenine. These are eluted for 16 hours with 3 ml of 0.4N HCl. 0.2 ml of 0.25 percent NaNOz is added, the tubes are mixed and allowed to stand for 3 minutes. 0.2 ml of 10 percent ammonium sulfamate is added, the tubes are mixed, and allowed to stand 2 minutes. Then 0.2 ml of 0.25 percent N-l-naphthylethylenediamine dihy- drochloride is added. After 3 hours, the solu- tion and paper are poured into a funnel fitted in a centrifuge tube with a rubber stopper. The tubes with funnel containing the papers are centrifuged for 5 minutes at low speed in order to collect all possible liquid. The solution, in- cluding the paper BLANK, are read at 550 mu. 4. Anthranilic acid. Elute with 5 ml of 1 percent p-dimethylaminobenzaldehyde in 50 percent acetic acid for 16 hours. The solutions are read at 450 my. Calculations All calculations are done as follows: 0.D. Test — 0.D. Blank m X ,ug. Standard = pg. Fluorescent Spot References 1. Satoh, K. and Price, J. M.: Fluorometric determi— nation of kynurenic acid and xanthurenic acid in human urine. J. Biol. Chem. 230: 781, 1958. 2. Coppini, D., Benassi, C. A. and Montorsi, M.: Quantitative determination of tryptophan metabo- lites (via kynurenine) in biologic fluid. C’li/‘n. Chem. 5 : 391, 1959. 3. Brown, R. R. and Price, J. M.: Quantitative studies on metabolites of trytophan in the urine of the dog, cat, rat, and man. J. Biol. Chem. 2192985, 1956. 4. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro-biochemical techniques. Hoeber, New York, 3d edit. repr. 1964, page 301. Determination of serum uric acid Uric acid has a strong absorption in the ultraviolet part of the Spectrum, with a maxi— mum between 290 and 295 mp. This absorption 95 is abolished by the action of uricase in convert- ing its substrate to allantoin. The fall in O.D. at the wavelength of maximum absorption, with a 10-mm. light path, is 0.072 per ,ug. uric acid per milliliter. The method is simple and re- liable, the only drawback being that the Deter- matubes® last only about 3 months under re- frigeration after issue from the makers. The colorimetric procedure is less specific than the direct spectrophotometric method and may have a tendency to give 10w results owing to the adsorption of uric acid on the protein precipitate although the ultramicro version given below does not seem to be affected in this way. There is also a difliculty with the stand- ard, which, if treated exactly as serum, normally an advantage in an ultramicro procedure, gives an anomalous calibration caused by the presence of the protein precipitants. This does not occur when protein is present together with the standard, or if water is sub- stituted for the tungstate and sulfuric acid, when in both cases a linear calibration results. The method can thus be modified in one of sev- eral ways with respect to the standard: a. Substitute water for the protein precipi— tants, b. Use as standard a reliable commercially available control serum, c. Adjust the protein precipitation as fol- lows: Into 400-pl. polyethylene test tubes pipette Blank Standard Test 5% Albumin ...... 20 pl 20 pl ......... Serum ............................... 20 pl Standard .................. 20 p1 ......... Water ............. 20 pl ........... 20 pl Continue with the protein precipitation as given in the ultramicro procedure. ‘ 96 Normal Values 2.0 to 5.5 mg/ 100 ml serum. Procedure 1. Plasma a. Add 2.9 ml of water to dried enzyme and buffer (Determatube®). b. Prepare a reference cell containing 0.1 m1 of serum in 2.9 ml of 0.9 per- cent NaCl and set this at zero optical density at 293 mp. 0. Add 0.1 ml serum to the enzyme solu- tion, mix, start a stopwatch, and rec- ord the O.D. with a minimum of delay, noting the time to the nearest second. Serial readings of O.D. should be made over the next five minutes by presetting the O.D. scale and recording the time at which the needle passes the zero mark (intervals of 0.01 O.D. are convenient). d. After optical density has stopped falling, a minimum of 20 minutes, record extinction value. e. Plot the reading and extrapolate the line to zero time to get the initial reading and calculate as follows: . AE . . AE—fall 1n O.D. 0”Tr—mg uric add/100 ml 2. Urine. Add 2.0 ml distilled water to the dried bufi'ered enzyme. Dilute the urine 1: 100 in water. Reference cell. Add 1 ml dilute urine to 2 ml water and set this at zero O.D. Add another 1 ml urine to the enzyme solution and proceed as for serum. — mg Uric Acid/ 100 ml . AE Calculation. W Ultramicro Procedure Reagents l. 10% Sodium carbonate, Na2003H20. 2. 3.2% Sodium tungstate, Na2W042H20. 3. 0.2N Sulfuric acid, H2804. 4. Phosphotungstic acid, H3PW12040- 14H20. To 80 ml water in a glass—jointed 250- ml distillation flask, add 10 gm molybdate—free sodium tungstate, Na2WO4 '2H20, and dissolve. Then add 8 ml 85 percent phosphoric acid, H3P04, and reflux gently for 2 hours. Cool and dilute to 100 ml. For use, dilute 1: 10 with water. 5. Standard uric acid solution. Dissolve 0.46 gm anhydrous disodium hydrogen phos- phate, NaQHPO4 in 60 ml warm water. Weigh out 40 mg uric acid, C5H4N403, and wash into a 100 m1 volumetric flash using all the phos- phate solution. When the uric acid has com- pletely dissolved, add 0.18 ml glacial acetic acid, CHscOOH, and make up to 100 ml with water. To dilute for use, pipette 20 pl of the stock solu- tion into a 400—,ul polyethylene tube and add 180 ,ul water. Prepare fresh. Procedure Into 400—,ul polyethylene test tubes pipette 20 [1.1 serum, or water for BLANK. (For dis- cussion of the STANDARD, see comment in introduction.) Add 80 ,ul 3.2 percent sodium tungstate and 80 pl 0.2N sulfuric acid and mix. After mixing centrifuge for 15 seconds in a micro centrifuge. Transfer 100 pl supernatant to a second tube and add 20 ,ul sodium car- bornate solution, mix, and then add 20 ,ul dilute phosphotungstic acid reagent. Mix and allow to stand for 30 minutes. Read in a Spinco 151 Spectrocolorimeter at 650 my, setting water at zero optical density or in any other suitable colorimeter. Calculation “ V0.1). Test—OD. Blank mX4=mg/1°0 m1 References 1. Kalckar, H. M.: Differential spectrophotometry of purine compounds by means of specific enzymes. J. Biol. Chem. 167: 429, 1947. 2. Praetorius, E. : An enzymatic method for the deter- mination of uric acid by ultraviolet spectropho- tometry. Scand. J. Lab. Olin. Invest. 1: 222, 1949. 3. Praetorius, E. and Poulsen, H.: Enzymatic deter- mination of uric acid. Scand. J. Lab. Olin. Invest. 5:273, 1953. 4. Caraway, W. T.: Determination of uric acid in serum by a carbonate method. Am. J. Olin. Path. 25:840, 1955. 5. O’Brien, D. and Ibbott, F. A.: Laboratory manual of pediatric micro- and ultramicro-techniques. Hoeber, New York, 3d edit. repr. 1964. page 325. Paper chromatography of phenolic acids of urine, including 3—methoxy—4—hy— droxy—mandelic acid (VMA) and homovanillic acid (HVA) The chromatographic procedure described by Armstrong, et al.1 provides a simple method of separating the many phenolic acids occurring in human urine as shown on page 98. Reagents 1. Ethyl acetate analytical grade 2. Sodium bicarbonate solution (10%). 100 g. NaH003 are dissolved in and made up to 1,000 ml with deionized water. 3. Hydrochloric acid concentrated 4. Sodium chloride 5. First Solvent System. Isopropyl alco- hol, ammonia, and water are mixed together in the ratio, 8 volumes isopropyl alcohol to 1 volume ammonia to 1 volume of water. 6. Second Solvent System. 2 volumes of benzene are added to 2 volumes of propionic acid and 1 volume of water. 7. p-Nitroaniline (0.2%). 0.2 g. of p- nitroaniline are dissolved in and made up to 97 coco-ac: 10. 20 isopropyl alcohol ammonia water 8:1:1 T 10 l 9 19,21 i 16 2 1 lst ‘ 15 18 12 l 17 l 5 l l 14 7 4 6 13 3 benzine, propionic acid water __ _, 2nd 2 : 2 : 1 . 3-methoxy-4-hydroxy—mandelic acid—purple . 5-hydroxyindolacetic acid—pink . vanillic acid (3-methoxy-4—hydroxy benzoic acid)— purple . homovanillic acid—blue . o-hydroxyphenyl acetic acid—purple . phydroxy benzoic acid—~pink . m-hydroxybenzoic acid—light purple . p—hydroxypheny‘lacetic acid—purple . m—tyrosine—reddish purple o-tyrosine—reddish purple 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. salicylic acid—orange salicyluric acid—orange ferulic acid—blue indole-3-acetic acid—yellow p-hydroxyhippuric acid m-hydroxyhippuric acid—pink caffeic acid—light yellow protocatechuic acid—light yellow p—hydroxymandelic acid—pink m—hydroxyphenylacetic acid—yellow p—hydroxyphenylpyruvic acid—~dark gray 100 ml with 1N hydrochloric acid. 8. Sodium Nitrute (5%). 5 gm. of sodium nitrate are dissolved in and made up to 100 ml with deionized water. 9. Sodium carbonate (10%). 10 gm. of sodium carbonate are dissolved in and made up to 100 ml with deionized water. Equipment A chromatography tank capable of accom- modating 20x20 cm sheets of paper. Shandon 98 Universal Tank available from Consolidated Laboratories, Inc., Chicago Heights, 111. Procedure A. Extraction 1. The volume of a 24-hour sample of urine is measured and the creatinine concentra- tion determined by the J afi'é reaction. An aliquot (100—200 ml) of urine is diluted such that it contains 0.5—0.8 mg of crea- tinine per ml of urine. 2. The diluted urine is chilled in an ice bath, acidified to pH 1—2 using small addi- tions of concentrated hydrochloric acid, and saturated with sodium chloride by the addition of approximately 26 g. per 100 ml. 3. The urine is then extracted with three successive portions of ethyl acetate, each being one-half of the volume of the diluted acidified urine. (The initial extraction frequently causes the formation of an emul- sion, this may be overcome by separating the aqueous and organic layers, as far as possible, and centrifuging the residual emulsion. No emulsion forms on the sec- ond and third emulsion.) 4. The three ethyl acetate extracts are com— bined and extracted vigorously and with prolonged shaking, with small volumes of 10 percent sodium bicarbonate. The vol- umes of sodium carbonate used are such that 1 ml of the first extract corresponds to 20 mg of creatinine in the original urine, the second and third extractions should be made with a volume of sodium carbonate one- half that of the first extraction. 5. The sodium bicarbonate extracts are combined, chilled in an ice bath, acidified with concentrated hydrochloric acid and saturated with sodium chloride. They are then extracted with the portions of ethyl acetate, which are pooled in a graduated container. The final volume of the ethyl acetate extract is adjusted so that it cor- responds to a definite amount of creatinine in the original urine. 6. The extracts may be stored at this stage if desired. Water is removed by the ad- dition of a small quantity of anhydrous sodium sulfate, and the extract contained in a screw cap vial, placed in the deep freeze. Chromatography 1. An amount of the ethyl acetate extract corresponding to 1 mg. of creatinine in the original urine is applied to the paper with a micro-pipette, and dried in a stream of air without heat. 2. The chromatographs are developed for 16 hours (overnight) in the first solvent system. It has been found advantageous to perform this run in a cold room at 4° C. The lid of chromatography tank should be weighed down to prevent the loss of am— monia. No prior equilibration of the papers is necessary. 3. The papers are dried in a stream of air, at room temperature until the odor of am- monia no longer persists. 4. The papers are returned to the chroma— tography tank and allowed to equilibrate for 1—2 hours with the aqueous phase (lower) of the second solvent system. The organic phase is then added and the chro- matograms allowed to developed for 3 hours. 5. The papers are dried overnight at room temperature. If this is inconvenient, they may be placed in an oven at 100° C for 3 minutes, the former procedure is, however, recommended. C. Visualization 1. The chromatograms are sprayed lightly but completely with 10 percent sodium car- bonate solution to reduce background color due to phenol contamination, and allowed to dry. 2. The diazotised p-nitroaniline is prepared as follows: 5 ml of 0.2 percent p-nitroani- line in IN HCl, 50 m1 of 1N HCl, and 1 ml of 5 percent sodium nitrite, are placed in three tubes in an ice bath, allowed to come to its temperature and then mixed. After 3 minutes, 10 ml of ice-cold 10 percent sodium carbonate is added. The mixture should be used immediately. Note: It is essential that great care be taken to prevent contamination of the chromat- ograms by phenol which may be present in the laboratory atmOSphere. This contam- 99 ination causes background coloration after spraying which may obscure some spots. The chromatograms may also be sprayed with diazotised sulphanilic acid, but it has been found that the p-nitroaniline reagent gives a better range of colors for the various compounds and is therefore more satisfactory. Reference 1. Armstrong, M. D., Shaw, K. N. F. and Wall, P. E.: The phenolic acids of human urine. J. Biol. Chem. 218: 293, 1956. The nitroprussidc cyanide test for cystine and homocystine in urine Reagents 1. Approximately 1.0 N HCl. Take 10 ml concentrated HCl, dilute to 100 ml with water. 2. 5% Sodium cyanide, NaCN. 3. 0.5% Sodium nitroprusside, Nag (Fe(CN)5NO) '2H20. Procedure Acidify 5 ml. of the test urine with 0.5 ml. of 1N HCl, add 2 ml. of fresh sodium cyanide solution, and stand at room temperature for 30 minutes. Finally add 1 ml. of sodium nitro- prusside solution when a definite purple color indicates the presence of excess amounts of cystine or homocystine. Faintly positive reac- tions may be obtained in older normal children, and more definite ones in small premature infants in the first trimester of life. The ferric chloride test When a 10 percent aqueous solution of ferric chloride is added drop by drop to 5 ml of urine, a light precipitate of ferric phosphate is 100 often formed. Further addition of ferric chloride may lead to the formation of a colored compound if certain drugs or abnormal metabo- lites are present in the urine. The test is best known in the diagnosis of phenylketonuria. \ Phenistix® papers are a useful commercial var- iant of this test which does not stain the diaper or involve making up solutions. / It is impor- tant, however, that these filter paper strips are thoroughly saturated with urine and not just moistened. The following is a list of substances in urine which may produce a color in the presence of ferric chloride. Metabolites phenylpyruvic acid—green p-hydroxyphenyl pyruvic acid—greenifad- ing rapidly aketobutyric acid (oasthouse syndrome)— purple going to brownish-red 3-hydroxyanthranilic acid—brown Urocanic acid (histidinemia)—green Homogentisic acid—very dark brown Xanthurenic acid (pyridoxine disorders) — dark green going to brown Branched chain keto—acids (maple-syrup urine) —gray—green Pyruvic acid—yellow-brown Melanin—eblack Aceto-acetic acid—red-brown, red Imidazole pyruvic acid (histidinemia)— blue-green Drugs Salicylates—purple Phenothiazincs—purple p-amino salicylic acid—red-brown U.S. GOVERNMENT PRINTING G'FICE: 1968 0—772-952 cueauumsea CHILDREN’S BUREAU PUBLICATION NUMBER 429—1965 US. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE WELFARE ADMINISTRATION . Children’s Bureau *