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Isovaleric acidemia: Identification of isovalerate, isovalerylglycine, and 3-hydroxyisovalerate in urine of a patient previously reported as having butyric and hexanoic acidemia
February, 1973 T h e Journal of P E D I A T R I C S
243
Isovaleric acidemia: Identification 4 isovalerate, isovalerylglycine, and 3-bydroxyisovalerate in urine of a patient previously reported as baying butyric and bexanoic acidemia Urine from one of the original patients with butyric and hexanoic aidemia was examined for the possible excretion of glycine conjugates of butyryl-CoA and hexanoyl-CoA. Thin-layer chromatography of acylglycines revealed a distinct spot with an R[ value similar to that of isovalerylglycine. Gas liquid chromatography of a distillate of urine showed a peak with the same retention time as that of isovaleric acid and a second peak with a retention time longer than that of hexanoic acid. Furthermore, gas liquid chromatography of the trimethylsilyl derivative of an extract of urine showed peaks of isovalerylglycine and fi-hydroxyisovaleric acid. The structure of these compounds was established by mass spectrometry. These findings indicate that the case reported actually had isovaleric acidemia.
Toshiyuki Ando, William L. Nyhan, Claude Bachmann, Karsten Rasmussen, La ]olla, Cali[., Ronald Scott, and Elizabeth K. Smith, Seattle, Wash.
T w o i N B 0 R N errors of metabolism, isovaleric acidemia 1 and butyric and hexanoic From the Departments of Pediatrics, University of California, San Diego, the University of Washington, and Children's Orthopedic Hospital and Medical Center. Supported in part by United States Public Health Service Grants Nos. GM 17702 from the National Institute of General Medical Sciences, and HD 04608 and HD 04406 from the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., and by a grant from the Liehtenstein-Frank Sti#ung, Basel, Switzerland. Reprint address: William L. Nyhnn, M.D., Ph.D., Department ol Ped~trlcs, University of California, San Diego, P.O. Box 109, La ]olla, Calif. 92037.
acidemia, 2 have been reported in which the patient may present with a "cheesy" or "sweaty feet" body odor. The clinical features in the young infant 3 are very similar, namely, early onset of an overwhelming illness with acidosis, thrombocytopenia, coma, and death. Isovalerylglycine is excreted in large amounts in patients with isovaleric acidemia.4, 5 The formation and excretion of the compound appears to be a detoxification mechanism by which excessive amounts of isovaleryl-CoA may be eliminated from the body. The detection and identification of a Vol. 82, No. 2, pp. 243-248
2 44
Ando et al.
volatile compound such as isovaleric acid is difficult in small amounts of body fluid. On the other hand, detection of isovalerylglycine~ is a much more reliable approach to diagnosis, because the compound is stable, nonvolatile, and excreted in large quantities. The mechanism of formation of acylglycine is common to a considerable number of acylCoA derivatives. The reaction is catalyzed by glycine-N-acylase. We therefore undertook a systematic study of the urinary excretion of acylglycines in patients with a variety of organic acidemias caused by the accumulation of acyl-CoA's. A case originally reported as having butyric and hexanoic acidemia excreted isovalerylglycine, fi-hydroxyisovaleric acid, and isovaleric acid in the urine. The detection of isovalerylglycine provided the first evidence that the patient actually had isovaleric acidemia.
MATERIALS AND METHODS A specimen of urine from Patient A. B. was obtained from the Children's Orthopedic Hospital where it had been stored after the patient died. The urine smelled like a shortchain fatty acid, such as valeric or isovaleric acid. At -20 ~ C. the specimen was not frozen hard. This appeared to reflect the large amount of organic acid in the specimen. In our experience, isovaleric acid and isovalerylglycine are stable for long periods of time at -20 ~ C.
Thin-layer chromatography of acylglycines. The method has previously been reported2 Gas chromatography of short-chain fatty acids. The methods for vacuum distillation and gas chromatography have previously been reported. 7 Analyses were done on two different columns. In one, Porapak PS (50/80 mesh, Waters Associates, Inc., Framingham, Mass.) was used as the column packing. In the other, neopentolglycoladipate (5 per cent) --H.~PO, (2 per cent) was used as the liquid phase on Chromosorb G acid-washed dimethylchlorosilane (DMCS) (100/120 mesh). A Hewlett-Packard Model 402 gas chromatograph with a 6 foot x 5 mm. glass U-column was used for all gas chromatographic analy-
The ]ournal o[ Pediatrics February 1973
ses. The chromatograph was equipped with a hydrogen flame detector. The carrier gas was helium. The column temperature was 155 ~ C., and the helium flow rate was 50 ml. per minute. Gas liquid chromatography of trlmethylsilyl derivatives of extract of urine. The procedures are based on the methods described by Homing. s, 9 Organic acids were extracted three times each with ethylacetate and ether. The residues after the evaporation of solvent were subjected to derivatization to form trimethylsilylesters and trimethylsilylether (TMS) using Tri-Sil and N, O-Bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (Pierce Chemical Co., Rockford, Ill.). Authentic acylglycines were synthesized in this laboratory2 The columns were packed with OV-1 (3 per cent on Chromosorb W (HP) 80/100 mesh, Pierce Chemical Co.) or OV-25 (3 per cent on Chromosorb G acid wash-DMCS) as the liquid phase. Methylene unit values were determined by injecting standard alkanes together with the samples and by programming the column temperature from 90 ~ C. to 300 ~ C. with OV-1 or 60 ~ C. to 250 ~ C. with OV-25 at a rate of 3 ~ C. per minute. For the quantitation of TMS-isovalerylglycine, isothermal chromatography was performed at 170 ~ C. Gas chromatography-mass spectrometry. An LKB 9000 mass spectrometer with a gas chromatographic inlet was used. A Porapak Q column was prepared for the short-chain fatty acids, and 1 per cent OV-1 was used for the TMS derivatives.
RESULTS Thln-layer chromatography of acylglytines, The thin-layer chromatogram is shown in Fig. 1. In the extract of the patient's urine there were four distinct spots: the first at the origin, the second at the area of isovalerylglycine, the third at the area of hippurate, and the fourth at the second solvent front. No butyrylglycine was detected.
Gas chromatography of short-chain fatty acids. Distillate of the urine contained an extremely large peak with the retention time of isovaleric acid (Fig. 2). The amount of
Volume 82 Number 2
Isovaleric acidemia
24 5
A
Fig. 1. Thin-layer chromatography of acylglycines. The urine (2.0 ml.) was acidified with 0.4 ml. of 5N H2SO~ and extracted three times with 5 ml. of chloroform-n-butanol (5:1). The dried extract was redissolved in 100 #1 of the extraction fluid, and a 10 #1 aliquot was spotted on a silica gel H plate. The chromatograph was developed with benzene:isoamylalcohol:formic acid ( 70:25:5). Acid spots were located by spraying with alcoholic bromcresol purple. Sol. 1 and Sol. 2 identify the two solvent fronts that occasionally develop using this method because of separations of aqueous solvents in the mixture. The numbers indicate the following: I ~ hippuric acid, II ~ acetylglycine, III ~ isovalerylglycine, IV ~ extract of the patient's urine, V ~ hippuric acid, and VI valerylglycine. distillate employed was equivalent to onethirtieth milliliter of original urine. I n norreal individuals distillates equivalent to 1.0 ml. urine have very small peaks in these areas u n d e r the same analytic conditions. O n the P o r a p a k Q column a fairly large second peak a p p e a r e d in the area of hexanoic acid, b u t it m i g r a t e d a little slower t h a n hexanoate. Fig. 2 clearly indicates t h a t the first p e a k formed a single peak on this column with a d d e d isovalerate a n d 2-methylbutyrate, while the second peak was separated from a d d e d hexanoate. O n the neopentolglycoladip a t e column the second peak m i g r a t e d slightly a h e a d of valerate.
5
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20
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MINUTES Fig. 2. Gas chromatograms of short-chaln fatty acids. A represents 10 #1 of distillate of urine of the patient (300 #1 of distillate = 1 ml. of urine). B represents i0 /d of 0.25 mM. standard mixture. A + B represents 4 #1 of distillate from the patient mixed with 4 #1 of the standard mixture. The numbers designate the following: 1 ~ propionate, 2 7__ isobutyrate, 3 ~- butyrate, 4 = isovalerate + 2-methylbutyrate, 5 = 3-methylcrotonate, 6 = tiglate, 7 ~ isohexanoate, and 8 ~ hexanoate. The 6 foot glass U column was packed with Porapak-PS (50/80 mesh). The inlet pressure was 28 p.s.i. The oven temperature was 1550 C.
T h e first p e a k was identified as isovaleric acid by mass spectrometry. T h e structure of the second c o m p o u n d has not yet been deduced.
Gas liquid chromatography of trlmethylsilyl derivative of extract. T w o m a j o r peaks were found in each of the gas liquid chrom a t o g r a p h i c systems for T M S derivatives (Fig. 3). T h e first peak was identified as 2 TMS-/?-hydroxyisovalerate by cochromat o g r a p h y with synthesized standard. Analysis of the mass spectrum of the first peak was consistent with the structure of TMS-/?-hydroxyisovalerate. T h e second peak, similarly, h a d a retention time and mass spectrum
246
Ando et al.
The Journal o[ Pediatrics February 1973
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Fig. 3. Gas chromatographic pattern of TMS derivatives of urine extract. Five microliters of the derivatized urine was injected into the 3 per cent OV-1 column together with standard alkanes. Temperature was programmed from 90 ~ C. to 300 ~ C. at a rate of 3 ~ C. per minute. identical to that of authentic TMSisovalerylglycine. The mass spectra of T M S isovalerylglycine from the sample and from the standard are illustrated in Fig. 4. Methylation of an extract of urine was also carried out using diazomethane. Gas liquid chromatography mass spectrometry was carried out using a 3 per cent OV-25 column at 148 ~ C. A peak was identified with the retention time of isovalerylglycine methylester and no peak of the methylester of butyrylglycine. The mass spectrum of the peak found was identical to that of methylisovalerylglycine. 4 The molecular ion was 173, and there were prominent ions at in/e 131, 114, 85, and 57. DISCUSSION The formation and excretion of glycine conjugates of acyl-CoA derivatives appear to be general phenomena among patients with organic acidemias in which the CoA derivatives of organic acids accumulate. Examples reported include the excretion of isovalerylglycine in isovaleric acidemia, 4, ~ of 3-methylcrotonylglycine and tiglylglycine in a patient with a presumed defect in 3-methylcrotonylCoA carboxylase, 1~ and of propionylglycine and tiglylglycine in propionie aciclemia? 1, 12 Metabolites of isoleucine between tiglic acid and propionic acid have not been found in
patients with propionic acidemia. The present study was undertaken to see if patients with butyric and hexanoic acidemia excrete butyrylglycine or hexanoylglycine in the urine. The discovery of isovalerylglycine in the specimen by thin-layer chromatography altered the course of the investigation. It was important to document carefully the chemical nature of the abnormal compounds present in the urine. The most conelusive evidence was the documentation by gas chromatography-mass spectrometry that the patient excreted large amounts of isovalerylglycine, hydroxyisovaleric acid, and isovaleric acid in the urine. All three compounds have previously been reported to be excreted in the urine of patients with isovaleric acidemia?, 4, is The mass spectra of the T M S derivatives have not previously been reported. These data raise questions as to the existence of butyric/hexanoic acidemia as a nosologic entity. Our studies were earned out on the material of only one of the original patients. Unfortunately , no specimens remain from the other three in the original report, 2 who were siblings. Certainly, the patient we studied had isovaleric acidemia and not butyric/hexanoic acidemia. Analyses of the short-chain fatty acids in the original report were done in different
Volume 82
Isovaleric acidemia
2 47
Number 2
100
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Fig. 4. The mass spectra of TMS-isovalerylglycine from the sample and from authentic standard.
places and with different methods for the patient we studied and for the other three patients. It is of interest that two peaks were observed. One was described as having the same retention time as standard butyric acid and the other to have a shorter retention time than the hexanoic acid. However, when the standards and the biological extract were mixed, only two peaks were obtained. The similarity of our findings relevant to the retention times of the unkown and that of hexanoic acid are striking. Certainly, the presence of a single peak on cochromatography does not establish identity. The problem is to effect separations. Improvement in methodology now has permitted a clearer separation than was previously possible. The two peaks originally identified as butyrate and hexanoate are now readily recognized as isovalerate and a compound other than hexanoate. It is not possible to come to a firm conclusion about all four patients originally described. The documentation of isovaleric acidemia in one of them raises the possibility
that they all had the same cause for their unusual odor and early demise. These observations raise important questions regarding the description of new inborn errors of metabolism. The migration of organic compounds on paper, on thin layers, on liquid-solid columns, and on gas liquid columns has made qualitative organic analysis easy. These methods have permitted the exploration of biological problems despite the availability of only small samples and microquantities of material. At the same time they have led to an acceptance of migration rates in the characterization of organic compounds that is less than rigorous. Older organic chemists required a melting point and two derivatives for the identification of a compound; this is clearly impossible with most biological samples. Certainly, rigorous identification is not necessary for the diagnosis of the hundredth case of homocystinuria. It is in the initial description of a condition that rigorous proof of structure is important. Alternatives have been the use of a
248
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number of different chromatographic systems to characterize a compound, possibly with the synthesis of a derivative and further chromatography of the derivative in comparison with authentic sample. Today, physical methods of organic chemistry are an enormous help in the elucidation of structure. Ultraviolet and infra-red spectra are very useful, as may be nuclear magnetic resonance. The most powerful current tool is the mass spectrometer. Coupled with the gas chromatographic inlet, this instrument should vastly increase our abilities to recognize with assurance the identity of unusual compounds in biological fluids. These approaches to characterization were not available in 1967. T h e availability of these methods today establishes a requirement for greater certainty in the identification of intermediates in the initial description of new syndromes. The authors are grateful to Mrs. Janette Holm for her technical assistance. REFERENCES 1. Tanaka, K., Budd, M. A., Efron, M. L., and Isselbacher, K. J.: Isovaleric acidemla: A new genetic defect of leucine metabolism, Proc. Natl. Acad. Sci. U. S. A. 56: 236, I966. 2. Sidbury, J. B., Jr., Smith, E. K., and Harlan, W.: An inborn error of short-chain fatty acid metabolism, J. PEDIATR. 70: 8, t967. 3. Newman, C. G. H., WiIson, B. D. R., Callagham, P., and Young, L.: Neonatal death associated with isovaleric acidemia, Lancet 2: 439, 1967.
The Journal o[ Pediatrics February 1973
4. Tanaka, K., and Isselbacher, K. J.: The isolation and identification of N-isovalerylglycine from urine of patients with isovaleric acidemia, J. Biol. Chem. 242: 2966, 1967. 5. Ando, T., Klingberg, W. G., Ward, A. N., Rasmussen, K., and Nyhan, W. L.: Isovaleric acidemia presenting with altered metabolism of glycine, Pediatr. Res. 5: 478, 1971. 6. Ando, T., and Nyhan, W. L.: A simple screening method for detecting isovalerylglycine in urine of patients with isovalerie acidemia, Clin. Chem. 16: 420, 1970. 7. Ando, T., Rasmussen, K., Nyhan, W. L., Donnell, G. N., and Barnes, N. D.: Propionic acidemia in patients with ketotic hypergtycinemia, J. PI~DIATR.78: 827, 197I. 8. Homing, M. G.: In Szymanski, A., editor: Biomedical applications of gas chromatography, New York, 1968, Plenum Press, vol. 2, p. 55. 9. Horning, M. G.: Gas chromatographic study of derivatives of acids of the Krebs cycle and related compounds, Anal. Letters 1: 713, 1968. 10. Gompertz, D., Draffan, G. H., Watts, J. L., and Hull, D.: Biotin-responsive #-methylcrotonylglycinuria, Lancet 2: 22, 1971. 11. Rasmussen, K., Ando, T., Nyhan, W. L., Hull, D., Cottom, D., Wadlington, W., and Killroy, A. W.: Excretion of propionylglycine in propionic acidemia, Clin. Sci. 42: 665, 1972. 12. Rasmussen, K., Ando, T., Nyhan, W. L., Hull, D., Cottom, D., Kilroy, A. W., and Wadlington, W.: Excretion of tiglylglycine in propionic acidemia, J. PEDIATR. 81: 970, 1972. 13. Tanaka, K., Orr, J. C., and IsseIbacher, K. J.: Identification of /?-hydroxyisovalerlc acid in the urine of a patient with isovaleric acidemia, Biochim. Biophys. Acta 152: 638, 1968.
243
Isovaleric acidemia: Identification 4 isovalerate, isovalerylglycine, and 3-bydroxyisovalerate in urine of a patient previously reported as baying butyric and bexanoic acidemia Urine from one of the original patients with butyric and hexanoic aidemia was examined for the possible excretion of glycine conjugates of butyryl-CoA and hexanoyl-CoA. Thin-layer chromatography of acylglycines revealed a distinct spot with an R[ value similar to that of isovalerylglycine. Gas liquid chromatography of a distillate of urine showed a peak with the same retention time as that of isovaleric acid and a second peak with a retention time longer than that of hexanoic acid. Furthermore, gas liquid chromatography of the trimethylsilyl derivative of an extract of urine showed peaks of isovalerylglycine and fi-hydroxyisovaleric acid. The structure of these compounds was established by mass spectrometry. These findings indicate that the case reported actually had isovaleric acidemia.
Toshiyuki Ando, William L. Nyhan, Claude Bachmann, Karsten Rasmussen, La ]olla, Cali[., Ronald Scott, and Elizabeth K. Smith, Seattle, Wash.
T w o i N B 0 R N errors of metabolism, isovaleric acidemia 1 and butyric and hexanoic From the Departments of Pediatrics, University of California, San Diego, the University of Washington, and Children's Orthopedic Hospital and Medical Center. Supported in part by United States Public Health Service Grants Nos. GM 17702 from the National Institute of General Medical Sciences, and HD 04608 and HD 04406 from the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md., and by a grant from the Liehtenstein-Frank Sti#ung, Basel, Switzerland. Reprint address: William L. Nyhnn, M.D., Ph.D., Department ol Ped~trlcs, University of California, San Diego, P.O. Box 109, La ]olla, Calif. 92037.
acidemia, 2 have been reported in which the patient may present with a "cheesy" or "sweaty feet" body odor. The clinical features in the young infant 3 are very similar, namely, early onset of an overwhelming illness with acidosis, thrombocytopenia, coma, and death. Isovalerylglycine is excreted in large amounts in patients with isovaleric acidemia.4, 5 The formation and excretion of the compound appears to be a detoxification mechanism by which excessive amounts of isovaleryl-CoA may be eliminated from the body. The detection and identification of a Vol. 82, No. 2, pp. 243-248
2 44
Ando et al.
volatile compound such as isovaleric acid is difficult in small amounts of body fluid. On the other hand, detection of isovalerylglycine~ is a much more reliable approach to diagnosis, because the compound is stable, nonvolatile, and excreted in large quantities. The mechanism of formation of acylglycine is common to a considerable number of acylCoA derivatives. The reaction is catalyzed by glycine-N-acylase. We therefore undertook a systematic study of the urinary excretion of acylglycines in patients with a variety of organic acidemias caused by the accumulation of acyl-CoA's. A case originally reported as having butyric and hexanoic acidemia excreted isovalerylglycine, fi-hydroxyisovaleric acid, and isovaleric acid in the urine. The detection of isovalerylglycine provided the first evidence that the patient actually had isovaleric acidemia.
MATERIALS AND METHODS A specimen of urine from Patient A. B. was obtained from the Children's Orthopedic Hospital where it had been stored after the patient died. The urine smelled like a shortchain fatty acid, such as valeric or isovaleric acid. At -20 ~ C. the specimen was not frozen hard. This appeared to reflect the large amount of organic acid in the specimen. In our experience, isovaleric acid and isovalerylglycine are stable for long periods of time at -20 ~ C.
Thin-layer chromatography of acylglycines. The method has previously been reported2 Gas chromatography of short-chain fatty acids. The methods for vacuum distillation and gas chromatography have previously been reported. 7 Analyses were done on two different columns. In one, Porapak PS (50/80 mesh, Waters Associates, Inc., Framingham, Mass.) was used as the column packing. In the other, neopentolglycoladipate (5 per cent) --H.~PO, (2 per cent) was used as the liquid phase on Chromosorb G acid-washed dimethylchlorosilane (DMCS) (100/120 mesh). A Hewlett-Packard Model 402 gas chromatograph with a 6 foot x 5 mm. glass U-column was used for all gas chromatographic analy-
The ]ournal o[ Pediatrics February 1973
ses. The chromatograph was equipped with a hydrogen flame detector. The carrier gas was helium. The column temperature was 155 ~ C., and the helium flow rate was 50 ml. per minute. Gas liquid chromatography of trlmethylsilyl derivatives of extract of urine. The procedures are based on the methods described by Homing. s, 9 Organic acids were extracted three times each with ethylacetate and ether. The residues after the evaporation of solvent were subjected to derivatization to form trimethylsilylesters and trimethylsilylether (TMS) using Tri-Sil and N, O-Bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (Pierce Chemical Co., Rockford, Ill.). Authentic acylglycines were synthesized in this laboratory2 The columns were packed with OV-1 (3 per cent on Chromosorb W (HP) 80/100 mesh, Pierce Chemical Co.) or OV-25 (3 per cent on Chromosorb G acid wash-DMCS) as the liquid phase. Methylene unit values were determined by injecting standard alkanes together with the samples and by programming the column temperature from 90 ~ C. to 300 ~ C. with OV-1 or 60 ~ C. to 250 ~ C. with OV-25 at a rate of 3 ~ C. per minute. For the quantitation of TMS-isovalerylglycine, isothermal chromatography was performed at 170 ~ C. Gas chromatography-mass spectrometry. An LKB 9000 mass spectrometer with a gas chromatographic inlet was used. A Porapak Q column was prepared for the short-chain fatty acids, and 1 per cent OV-1 was used for the TMS derivatives.
RESULTS Thln-layer chromatography of acylglytines, The thin-layer chromatogram is shown in Fig. 1. In the extract of the patient's urine there were four distinct spots: the first at the origin, the second at the area of isovalerylglycine, the third at the area of hippurate, and the fourth at the second solvent front. No butyrylglycine was detected.
Gas chromatography of short-chain fatty acids. Distillate of the urine contained an extremely large peak with the retention time of isovaleric acid (Fig. 2). The amount of
Volume 82 Number 2
Isovaleric acidemia
24 5
A
Fig. 1. Thin-layer chromatography of acylglycines. The urine (2.0 ml.) was acidified with 0.4 ml. of 5N H2SO~ and extracted three times with 5 ml. of chloroform-n-butanol (5:1). The dried extract was redissolved in 100 #1 of the extraction fluid, and a 10 #1 aliquot was spotted on a silica gel H plate. The chromatograph was developed with benzene:isoamylalcohol:formic acid ( 70:25:5). Acid spots were located by spraying with alcoholic bromcresol purple. Sol. 1 and Sol. 2 identify the two solvent fronts that occasionally develop using this method because of separations of aqueous solvents in the mixture. The numbers indicate the following: I ~ hippuric acid, II ~ acetylglycine, III ~ isovalerylglycine, IV ~ extract of the patient's urine, V ~ hippuric acid, and VI valerylglycine. distillate employed was equivalent to onethirtieth milliliter of original urine. I n norreal individuals distillates equivalent to 1.0 ml. urine have very small peaks in these areas u n d e r the same analytic conditions. O n the P o r a p a k Q column a fairly large second peak a p p e a r e d in the area of hexanoic acid, b u t it m i g r a t e d a little slower t h a n hexanoate. Fig. 2 clearly indicates t h a t the first p e a k formed a single peak on this column with a d d e d isovalerate a n d 2-methylbutyrate, while the second peak was separated from a d d e d hexanoate. O n the neopentolglycoladip a t e column the second peak m i g r a t e d slightly a h e a d of valerate.
5
I0
15
20
25
.%0
35
MINUTES Fig. 2. Gas chromatograms of short-chaln fatty acids. A represents 10 #1 of distillate of urine of the patient (300 #1 of distillate = 1 ml. of urine). B represents i0 /d of 0.25 mM. standard mixture. A + B represents 4 #1 of distillate from the patient mixed with 4 #1 of the standard mixture. The numbers designate the following: 1 ~ propionate, 2 7__ isobutyrate, 3 ~- butyrate, 4 = isovalerate + 2-methylbutyrate, 5 = 3-methylcrotonate, 6 = tiglate, 7 ~ isohexanoate, and 8 ~ hexanoate. The 6 foot glass U column was packed with Porapak-PS (50/80 mesh). The inlet pressure was 28 p.s.i. The oven temperature was 1550 C.
T h e first p e a k was identified as isovaleric acid by mass spectrometry. T h e structure of the second c o m p o u n d has not yet been deduced.
Gas liquid chromatography of trlmethylsilyl derivative of extract. T w o m a j o r peaks were found in each of the gas liquid chrom a t o g r a p h i c systems for T M S derivatives (Fig. 3). T h e first peak was identified as 2 TMS-/?-hydroxyisovalerate by cochromat o g r a p h y with synthesized standard. Analysis of the mass spectrum of the first peak was consistent with the structure of TMS-/?-hydroxyisovalerate. T h e second peak, similarly, h a d a retention time and mass spectrum
246
Ando et al.
The Journal o[ Pediatrics February 1973
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Fig. 3. Gas chromatographic pattern of TMS derivatives of urine extract. Five microliters of the derivatized urine was injected into the 3 per cent OV-1 column together with standard alkanes. Temperature was programmed from 90 ~ C. to 300 ~ C. at a rate of 3 ~ C. per minute. identical to that of authentic TMSisovalerylglycine. The mass spectra of T M S isovalerylglycine from the sample and from the standard are illustrated in Fig. 4. Methylation of an extract of urine was also carried out using diazomethane. Gas liquid chromatography mass spectrometry was carried out using a 3 per cent OV-25 column at 148 ~ C. A peak was identified with the retention time of isovalerylglycine methylester and no peak of the methylester of butyrylglycine. The mass spectrum of the peak found was identical to that of methylisovalerylglycine. 4 The molecular ion was 173, and there were prominent ions at in/e 131, 114, 85, and 57. DISCUSSION The formation and excretion of glycine conjugates of acyl-CoA derivatives appear to be general phenomena among patients with organic acidemias in which the CoA derivatives of organic acids accumulate. Examples reported include the excretion of isovalerylglycine in isovaleric acidemia, 4, ~ of 3-methylcrotonylglycine and tiglylglycine in a patient with a presumed defect in 3-methylcrotonylCoA carboxylase, 1~ and of propionylglycine and tiglylglycine in propionie aciclemia? 1, 12 Metabolites of isoleucine between tiglic acid and propionic acid have not been found in
patients with propionic acidemia. The present study was undertaken to see if patients with butyric and hexanoic acidemia excrete butyrylglycine or hexanoylglycine in the urine. The discovery of isovalerylglycine in the specimen by thin-layer chromatography altered the course of the investigation. It was important to document carefully the chemical nature of the abnormal compounds present in the urine. The most conelusive evidence was the documentation by gas chromatography-mass spectrometry that the patient excreted large amounts of isovalerylglycine, hydroxyisovaleric acid, and isovaleric acid in the urine. All three compounds have previously been reported to be excreted in the urine of patients with isovaleric acidemia?, 4, is The mass spectra of the T M S derivatives have not previously been reported. These data raise questions as to the existence of butyric/hexanoic acidemia as a nosologic entity. Our studies were earned out on the material of only one of the original patients. Unfortunately , no specimens remain from the other three in the original report, 2 who were siblings. Certainly, the patient we studied had isovaleric acidemia and not butyric/hexanoic acidemia. Analyses of the short-chain fatty acids in the original report were done in different
Volume 82
Isovaleric acidemia
2 47
Number 2
100
75
Unknown 172
50 216
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-
Isovalerylglycine
,~o 260 2~o 2~o
m/e
Fig. 4. The mass spectra of TMS-isovalerylglycine from the sample and from authentic standard.
places and with different methods for the patient we studied and for the other three patients. It is of interest that two peaks were observed. One was described as having the same retention time as standard butyric acid and the other to have a shorter retention time than the hexanoic acid. However, when the standards and the biological extract were mixed, only two peaks were obtained. The similarity of our findings relevant to the retention times of the unkown and that of hexanoic acid are striking. Certainly, the presence of a single peak on cochromatography does not establish identity. The problem is to effect separations. Improvement in methodology now has permitted a clearer separation than was previously possible. The two peaks originally identified as butyrate and hexanoate are now readily recognized as isovalerate and a compound other than hexanoate. It is not possible to come to a firm conclusion about all four patients originally described. The documentation of isovaleric acidemia in one of them raises the possibility
that they all had the same cause for their unusual odor and early demise. These observations raise important questions regarding the description of new inborn errors of metabolism. The migration of organic compounds on paper, on thin layers, on liquid-solid columns, and on gas liquid columns has made qualitative organic analysis easy. These methods have permitted the exploration of biological problems despite the availability of only small samples and microquantities of material. At the same time they have led to an acceptance of migration rates in the characterization of organic compounds that is less than rigorous. Older organic chemists required a melting point and two derivatives for the identification of a compound; this is clearly impossible with most biological samples. Certainly, rigorous identification is not necessary for the diagnosis of the hundredth case of homocystinuria. It is in the initial description of a condition that rigorous proof of structure is important. Alternatives have been the use of a
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number of different chromatographic systems to characterize a compound, possibly with the synthesis of a derivative and further chromatography of the derivative in comparison with authentic sample. Today, physical methods of organic chemistry are an enormous help in the elucidation of structure. Ultraviolet and infra-red spectra are very useful, as may be nuclear magnetic resonance. The most powerful current tool is the mass spectrometer. Coupled with the gas chromatographic inlet, this instrument should vastly increase our abilities to recognize with assurance the identity of unusual compounds in biological fluids. These approaches to characterization were not available in 1967. T h e availability of these methods today establishes a requirement for greater certainty in the identification of intermediates in the initial description of new syndromes. The authors are grateful to Mrs. Janette Holm for her technical assistance. REFERENCES 1. Tanaka, K., Budd, M. A., Efron, M. L., and Isselbacher, K. J.: Isovaleric acidemla: A new genetic defect of leucine metabolism, Proc. Natl. Acad. Sci. U. S. A. 56: 236, I966. 2. Sidbury, J. B., Jr., Smith, E. K., and Harlan, W.: An inborn error of short-chain fatty acid metabolism, J. PEDIATR. 70: 8, t967. 3. Newman, C. G. H., WiIson, B. D. R., Callagham, P., and Young, L.: Neonatal death associated with isovaleric acidemia, Lancet 2: 439, 1967.
The Journal o[ Pediatrics February 1973
4. Tanaka, K., and Isselbacher, K. J.: The isolation and identification of N-isovalerylglycine from urine of patients with isovaleric acidemia, J. Biol. Chem. 242: 2966, 1967. 5. Ando, T., Klingberg, W. G., Ward, A. N., Rasmussen, K., and Nyhan, W. L.: Isovaleric acidemia presenting with altered metabolism of glycine, Pediatr. Res. 5: 478, 1971. 6. Ando, T., and Nyhan, W. L.: A simple screening method for detecting isovalerylglycine in urine of patients with isovalerie acidemia, Clin. Chem. 16: 420, 1970. 7. Ando, T., Rasmussen, K., Nyhan, W. L., Donnell, G. N., and Barnes, N. D.: Propionic acidemia in patients with ketotic hypergtycinemia, J. PI~DIATR.78: 827, 197I. 8. Homing, M. G.: In Szymanski, A., editor: Biomedical applications of gas chromatography, New York, 1968, Plenum Press, vol. 2, p. 55. 9. Horning, M. G.: Gas chromatographic study of derivatives of acids of the Krebs cycle and related compounds, Anal. Letters 1: 713, 1968. 10. Gompertz, D., Draffan, G. H., Watts, J. L., and Hull, D.: Biotin-responsive #-methylcrotonylglycinuria, Lancet 2: 22, 1971. 11. Rasmussen, K., Ando, T., Nyhan, W. L., Hull, D., Cottom, D., Wadlington, W., and Killroy, A. W.: Excretion of propionylglycine in propionic acidemia, Clin. Sci. 42: 665, 1972. 12. Rasmussen, K., Ando, T., Nyhan, W. L., Hull, D., Cottom, D., Kilroy, A. W., and Wadlington, W.: Excretion of tiglylglycine in propionic acidemia, J. PEDIATR. 81: 970, 1972. 13. Tanaka, K., Orr, J. C., and IsseIbacher, K. J.: Identification of /?-hydroxyisovalerlc acid in the urine of a patient with isovaleric acidemia, Biochim. Biophys. Acta 152: 638, 1968.