BIOCHEMICAL MEDICINE 1, 87-91 (1967)

Diagnosis of Branched-Chain Ketonuria (Maple Syrup

Urine Disease) by Gas Chromatography1

MELVIN GREER AND CLYDE M. WILLIAMS

Departnwn ft\ of hledicine and Radiolog?/, University of Flom’tin Collrgt7 of Meflici?L,,. Gainesville, Florida 52601

Branched-chain ketonuria (maple syrup urine disease) is a metabolic disease involving the degradation of the branched-chain amino acids, leucine, isoleucine, and valine, in which a deficiency in oxidation decar- boxylation of the respective a-keto acids leads to :;n accumulation of both the keto acids and amino acids in the blood and urine. The entity was first described in 1954 (1)) and by 1963 (2)) 18 cases had been reported in the literature. The clinical picture most commonly begins about the fourth day of life with poor feeding, vomiting, and occasionally, hypertonicity and convulsions. The diagnostic feature is the maple syrup odor of the urine. If the patient survives :ong enough, mental retardation and neurologic signs appear. A variant of the classical onset in infancy pattern has been described in older children who exhibit no impairment in psychomotor function. (3, 4) The diagnosis is confirmed in the laboratory by demonstrating an elevation of the branched-chain keto acids or amino acids in urine, or by demonstrating a metabolic block in the peripheral leukocyte. Limited experience indicates that a diet con- taining reduced amounts of the branched-chain amino acids will prevent the serious complications of the disease. Recently we have had an op- portunity to examine the urine of a patient with the disease and establish optimum condidtions for the diagnosis of the disease by gas chromatog- raphy.

MATERIALS AND METHODS

n-cu-keto-P-methyl valeric acid was prepared from nL-isoleucine (Mann Research Labs, Inc.) using D-amino acid oxidase (hog kidney, Mann Research Labs, Inc.) according to the method of Meister (5). L-a-keto+ methyl valeric acid was prepared from nL-isoleucine by the action of L- amino acid oxidase (Crotaleus adamanteus venom, Calbiochem) accord- ing to the procedure of Wellner and Meister (6). Neither of the methyl

‘This study was aided by grants from the National Institutes of Health (AM- 08391-56 and HDO-13~6-03).

87

88 GREER AND WILLIAMS

esters of the two isomers could be separated on any of the columns. The other acids were obtained from commercial suppliers listed in Table 1. Methyl esters of these acids were prepared by reaction with ethereal diazomethane for 15 minutes. All of these acids produced single sym- metrical peaks. It is important not to exceed this time since Simmonds, Pettit, and Zlatkis (7) have shown that prolonged (4 hours) treatment of a-keto glutaric acid with diazomethane produces methyl 2- (carbo- methoxyethyl) glycidate.

TABLE 1 RETENTION TIMES OF METHYL ESTERS OF ACIDS

EXCRETED IN BRANCHED-CHAIN KETOXURIA

Methyl esters SE-30” EGtti XE-608 QF-lg

Methyl benzoste Methyl a-keto isovaleratei Methyl a-keto isocaproate Methyl cu-keto-p-methyl valerate Methyl a-hydroxy isovalerat& Methyl a-hydroxy isocaproate Methyl cu-hydroxy-p-methyl valerate

1.w l.Od l.Of l.Oh 0.1s O.li 0.24 0.45 0.40 0.36 0.56 0.89 0.39 0.29 0.48 0.79 0.22 0.28 0.27 0.26 0.48 0.60 0.61 0.49 0.51 0.55 0.56 0.49

a Seven percent coated on NO-120 mesh Chromosorb W. Conditions: 68” column temp., argon inlet pressure 12 psi, outlet pressure atmospheric, flow rate 90 ml/minute.

b Approximate retention time of methyl benzoate 28 minutes. c Five percent coated on 100-120 mesh Chromosorb W. Conditions: 82” column

temp., argon inlet pressure 8 psi, outlet pressure atmospheric, flow rate 20 ml/minute. d Approximate retention time of methyl benzoate 48 minutes. e Three percent coated on 80-100 Chromoport xxx Conditions: 69” column temp.,

argon inlet pressure 8 psi, outlet pressure atmospheric, flow rate 40 ml/minute. f. Approximate retention time of methyl benzoate 22 minutes.

0 Four percent coated on 100-120 mesh Chromosorb G (H.P) Conditions: 79” column argon inlet pressure 23 psi, outlet pressure atmospheric, flow rate 60 ml/minute.

h Approximate retention time of methyl benzoate 23 minutes. j California Biochemical Research Foundation. k Sigma Chemical Co.

Their retention times were established on three polar and one nonpolar columns (Table 1). These aliphatic acids were then extracted from urine by the same techniques used for the extraction of phenolic acids from human urine (8). When recoveries were carried out, it was discovered that in the last step where the reaction mixture is evaporated completely to dryness under a stream of nitrogen, that the volatility of these esters was such that substantial amounts were lost. The procedure was then revised as follows: After the 15 minute methylation, excess diazomethane is evaporated under a stream of air until approximately 200400 ~1 of ethereal solution remaining methyl benzoate is then added as an internal

DIAGNOSIS OF BRAITCHED-CIIAIK 89

standard after establishing that benzoic acid is not present in normal urine. When this was done recoveries were satisfactory ranging from go-100%.

A sample of 6 cc of urine from a patient with branched-chain keton- uria was obtained through the courtesy of Dr. Selma Snyderman of New York University College of Medicine. One-half ml samples were ex- tracted, treated with diazomethane as described, and chromatographed

I METHYLa- HYDROXY ISOVAIERATE

METHYLa- KETOISOCAPROAIE

IO

TlME , MlNUlES 1

20 30

FIG. 1. Gas chromatograph of urine from a patient with branched chain ketonuria treated as described in text on a 4% QF-1 column at 79”. The other column condi- tions were as described in Table 1. The amount injected in this chromatogram is equivalent to the acids from 24 pl of urine.

on a 3% &F-l column. This chromatograph is shown, in Figure 1. It was found that four of the peaks were at the exact retention times of four of the standard acids. Each of these four peaks was trapped with a teilon tube, the end of which was immersed in 509 ~1 of cold ether. Each peak was then rechromatographed on an EGA column and a single symmetrical peak was found with the same retention time and the same width at half- height as the authentic standard. The retention times of methyl-a- hydroxy+-methyl valerate and methyl a-hydroxyisocaproate are very close on a QF-1 column. On this column a small peak was observed near

90 GREER AND WILLIAMS

this time, but when trapped and subjected to chromatography on another column, this peak proved not to be either of these compounds. A control sample from a new born infant was found to have no peaks corresponding to any of the six acids on a &F-l column.

With the aid of an internal standard and an injection of lesser amounts, the amounts excreted were measured quantitatively. Table 2 indicates the measured values which agree well with previous figures (2).

TABLE 2 URINARY EXCRETION OF PKETO AND CPHYDROXY ACIDS

IN A CASE OF BRANCHED-CHAIN KETONVRIA

Acid e/ml

cu-ketoisocaproic 1,960 a-keto-p-methyl valeric 950 wketoisovaleric 250 whydroxyisovaleric 710 a-hydroxyisocaproic 0 a-hydroxy-p-methyl valeric 0

DISCUSSION

At the present time the disease branched-chain ketonuria may be detected by the determination of plasma valine, leucine, and isoleucine by one-dimensional paper chromatography, by column chromatography, or by microbiological assay. Of these methods column chromatography is preferred for confirmation of a provisional diagnosis (2), but a shorter less elaborate procedure is desirable for following the effects of dietary regimens. For this purpose paper chromatography (9) and thin layer chromatography (10) of the 2, 4-dinitrophenylhydrazones of the keto acids have been recommended. However, the latter procedure is lengthy and does not detect the a-hydroxy acids. The use of gas chromatography for the determination of the keto and hydroxy acids has been discussed (11)) but no detailed account of the method has been presented. The method presented here can be carried out in approximately two hours and all of the relevant hydroxy or keto acids can bc separated by a combination of the columns.

SUMMARY

A relatively simple method is described for the quantitative deter- mination of the diagnostically significant aliphatic keto and hydroxy acids in the genetic disease branched-chain ketonuria. This method involves the extraction of the acids from a salt-saturated acidified sample

DIAGNOSIS OF DRAY CHED-CH MI\ 91

of urine, and the preparation of methyl esters with diazomethane, fol- lowed by gas chromatography with the aid of an internal standard.

REFERENCES

1. MENBES, J. H., HURST, P. L., AND CRAIG, J. M., Pediatrics 14, 462 (1954). 2. DANCIS, J., .4ND LEVITZ, M., in “The Metabolic Nature of Inherited Diseases”

(J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds)., 2nd ed., p. 353. McGraw-Hill, New York, 1966.

3. GIL, R., AND ROKKONES, T., Acta Paediat. 53, 356 (1964).

4. MORRIS, M. D., LEWIS, B. D., DOOLAN, P. D., AND HARPER, H. A., Pediatrics 28, 918 (1961).

5. MEISTER, A., J. Bill. Chem. 190,269 (1951). 6. WELLNER, D., AND MEISTER, A4., J. Bid. Chem. 235, 2013 (1960)

7. SIMMONDS, P. G., PETTIT, B. C., AND ZLATKIS, A., Anal. Chem. 39, 163 (1967). 8. WILLIAMS, C. M., .~NII SWEELEY, C. C., in “Biomedical Application of Gas Chro-

matography” (H. A. Szymanski, ed.), p. 225. Plenum Press, New York, 1964. 9. NEISH, W. J. P., irz “Methods of Biochemical Analysis” (D. Glick, ed.) 5, 154.

Intcrscience, New York, 1957. 10. DANCIS, J., HUTZLER, J., AND LEVITZ, M., Biochem. Biophys. Acta. 78, 85 (1963).

11. DANCIS, J., AND LEVITZ, M., in “The Metabolic Nature of Inherited Diseases” (J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson, eds.), 1st ed., p. 473 McGraw-Hill, New York, 1960.