Study of Metabolic Pathways in Vivo Using Stable Isotopes Hans-Christoph Curtius, Jurg A. Vollmin, and Kurt Baerlocher Department of Pediatrics, University of Zurich, Kinderspital, CH-8032 Zurich, Switzerland
Deuterated tyrosine and leucine have been used for the study of their respective metabolisms in man. These labeled compounds have been given orally to healthy subjects as well as to patients, and the metabolites and their isotope content analyzed in urine using a combination of gas chromatograph-mass spectrometer. This technique elucidates the metabolic pathways in vivo without risk to the subjects.
The administration of radioactive isotopes is not without risk to human subjects and is, therefore, only rarely suitable for in vivo metabolic studies. Usually in vitro tests on biopsy tissue or. more recently, on tissue cultures are performed. These in vitro procedures can, however, often not replace in vivo studies. Despite the early pioneering work of Schoenheimer and Rittenberg in 1935 (1) and later, hitherto only a few in uivo experiments with nonradioactive stable isotopes, such as I3C, I5N, or deuterium, have been performed, since their determination posed some analytical problems. The most suitable instrument for detection of stable isotopes is the mass spectrometer. Separation and purification for mass spectrometric analysis is, however, elaborate and often gives poor yields. Since the introduction of combined instruments for gas chromatography-mass spectrometry (GC/MS), this problem has been considerably simplified. Complex biological mixtures with components in the nanogram range can be separated by gas chromatography and their isotope contents can be analyzed directly by mass spectrometry. The separation and sensitivity of the method can be improved significantly by the use of glass capillary columns (2, 3). We have previously described the application of the stable isotope technique to the study of metabolic diseases with GC/MS (4-6). In these studies, the phenylalaninetyrosine-dopa metabolism was investigated in vivo by loading healthy subjects and phenylketonuric patients with deuterated DL-phenylalanine and deuterated L-tyrosine. Protein synthesis has been studied in children by mass spectrometry using 15N labeled compounds (7, 8). Based on these experiments, we have developed a procedure for measuring the nitrogen retention in growth hormone deficiency (9). In the steroid field, the determination of the production rate of estrogens has been reported by Pinkus et al. (10). ( 1 ) R. Schoenheimer and D. Rittenberg. J. Biol. Chem., 111, 163 (1935). ( 2 ) J . A . Vollmin. Chromatograph/a, 3, 238 (1970). (3) J . A . Vollmin. Clin. Chim. Acta. 34, 207 (1971). (41 H.-Ch. Curtius. J . A . Vollrnin. and K . Baerlocher. Clin. Chim. Acta, 37, 277 (1972). (5) H . - C h . Curtius. Angew. Chem.. 84, I X (1972). (6) H . - C h . Curtius. K . Baerlocher, and J. A . Vollmin. Clin. Chim. Acta, 42, 235 (1972). (7) J. F. Nicholson, Pediat. Res. 4, 389 (1970) (8) 0. H. Gaeblerand H. C. Choitz, Metab. Clin. E x p . , 1 4 , 819 (1965). ( 9 ) M. Zachmann. J. A. Vollmin. and A . Prader. Presented at the European Society of Pediatric Endocrinology, Louvain. Sept 7-10, 1972. (10) J . L. Pinkus. D. Charles, and S. C. Chattoray, J. Bioi. Chem., 246, 633 (1971).
In the present paper, the usefulness of the stable isotope technique for metabolic studies in vivo is demonstrated on two examples. Phenylalanine-tyrosine metabolism has previously been examined with deuterated compounds on a number of healthy subjects and patients with phenylketonuria, and some interesting new aspects have been found by applying this technique (4-6). In this report, three children with mental retardation due to a hitherto unknown metabolic abnormality associated with increased amounts of urinary benzoic and hippuric acids are presented. Deuterated L-tyrosine was given orally in a single loading dose to these patients to study the urinary excretion of metabolites and deuterium content. Several diseases are known to be caused by disorders of leucine, isoleucine, and valine metabolism, such as “ketotic hyperglycinemia,” “sweaty feet syndrome,” and “maple syrup urine diseases.” These diseases are caused by enzymatic defects in the metabolism of the amino acids or their products. The results of the urinary analysis in a patient with suspected “sweaty feet syndrome” after a deuterated leucine oral load are reported. Procedure, results, and interpretation of these studies, which are only preliminary, will be presented here to demonstrate the application of this elegant and rather new method for studying metabolic pathways “in uiuo.”
EXPERIMENTAL Collected urinary samples were stored a t -20°C. Materials. All chemicals were of the highest purity grade available and solvents were redistilled before use. M e t h y l a t i o n was carried out with diazomethane according to Vogel(11). Reference C o m p o u n d s were obtained from Fluka, Switzerland, and Sigma Chemical Company, S t . Louis, Mo. The internal standard was enanthic acid (100 mg in 100 ml of methanol), Deuterated Compounds. 3,5-Dideutero-~-tyrosine has been synthetized by Dr. G. Schollenhammer, Department of Biology, University of Konstanz, West Germany. DL-Deuteroleucine was prepared according to J u n k and Svec (12). T h e procedure was modified by lowering the pD value and by refluxing for twice t h e time originally indicated. With these modifications. a higher deuterium incorporation in $ position was reached. Methods. Oral Load u i t h Deuterated Compounds. Deuterated L-tyrosine (150 mg/kg body weight) or 200 mg/kg body weight of deuterated DL-leucine were suspended in 50 ml of 2% (w/v) methyl cellulose in water and given orally after a n overnight fast. Extraction of Urine. The extraction and analysis of phenylalanine a n d tyrosine metabolites by gas chromatography-mass spectrometry have been described elsewhere (f3).T h e analysis of the metabolites of valine, leucine, and isoleucine is carried out a s follows: 10 ml of a 24-hr urine collection were adjusted to p H 8.5-9.0 with about 0.3 ml of a saturated solution of N a H C 0 3 and evaporated t o dryness i n U Q C U O a t 50 “C. T h e residue was dissolved in 2 ml of 6N HC1, and the solution was transferred to a separating funnel with a n additional 1 ml of 6N HCI and saturated with a m monium sulfate. This solution had a p H of about 0 and was extracted four times with 10 ml of ethyl acetate. T h e combined ex(1 1) A . I . Vogel. “Practical Organic Chemistry.” Longmans. Green and Co.. New York, N . Y . . 1964, p 971. (12) G . A . Junk and H . J. Svec, J. Org. Chem.. 29, 944 (1964) (13) J. A . Vollmin, H . R. Bosshard, M . Muller. S. Rampini. and H . - C h . Curtius. 2. Klin. Chem. Klin. Biochem.. 9, 402 (1971). A N A L Y T I C A L C H E M I S T R Y , V O L . 45, NO. 7, J U N E 1973
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Table I. Urinary Excretion of Benzoic Acid and Hippuric Acid (mg/24 hr) in a Patient after Oral Load with Deuterated L-Tyrosine (150 mg/kg) Benzoic acid Hippuric acid Before 4.8 4.3 0-24 h r 93.7 834.0 24-48 h r 5.1 1960.0
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II
~
134
89
Table I I . Urinary Excretion of Lactic Acid, 3-Hydroxyisovaleric Acid, 3-Hydroxybutyric Acid, Methylsuccinic Acid, and Succinic Acid (mg/24 hr) in a Patient after Oral Load with Deuterated DL-Leucine (200 mg/kg) 3-HydroxyMethylLactic isovaleric 3-Hydroxy- succinic Succinic acid acid butyric acid acid acid Before 17 41 16 03 08 0-24hr 39 18 8 1 1 10 16 24-48 h r 59 34 34 09 36
n 3
li MlN
30
I
I
1
20
10
0
Figure 1. Gas chromatographic separation of aromatic acids as methyl ester/trimethylsilyl ether in a patient with high excretion of hippuric acid after load with deuterated L-tyrosine
IS = Internal Standard (1 0-undecenoic acid), 1 = p-hydroxybenzoic acid, 2 = rn-hydroxyphenylacetic acid, 3 = p-hydroxyphenylacetic acid. 4 = rn-hydroxyphenylhydracrylic acid, 5 = homovanillic acid. 6 = phydroxyphenyllactic acid, 7 = vanilmandelic acid. 8 = p-hydroxyphenylpyruvic acid. 9 = hippuric acid.
tracts were dried with sodium sulfate and the solvent was concentrated to about 2 ml in uacuo at 40 "C and further to about 50 pl in a stream of nitrogen. After adding 50 ji1 of methanol, the sample was treated dropwise with a solution of diazomethane in ether until the yellow color persisted, After 5 min, the solvent was evaporated carefully to about 100 pl in a stream of nitrogen. A solution of internal standard (50 p l ) was added and 1-2 p1 of the sample was injected into the gas chromatograph. Gas Chromatography of Short Chain Carboxylic Acids. A Perkin-Elmer Model F 30 gas chromatograph was used with 2-m X 1108
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Figure 2. Mass spectrum of hippuric acid methyl ester, peak no. 9 in Figure 1 3-mm glass columns. T h e stationary phase was SP 1000 (PerkinElmer, Ltd.) (5% on gaschrom P 80-100 mesh). The carrier gas flow was 40 ml/min of nitrogen and the temperatures were as follows: column 4 min a t 70 "C, progressed 3 deg/min to 220 "C; injector block 250 "C; detector block 250 "C. Gas Chromatography-Mass Spectrometty. A glass column 2.5 m X 0.3 m m coated with FFAP (Varian-Aerograph, Ltd.) (15% on Chromosorb W 80-100 mesh) was used in a combination GC/MS LKB 9000. The carrier gas flow was 30 ml/min of helium. The chromatographic curve was recorded by monitoring the total ion current with a ionization voltage of 20 e\'. The mass spectra were recorded with a ionization voltage of 70 eV. The temperatures were as follows: flash heater 230 "C, separator 250 "C, ion source 270 "C. The scan time of the mass spectrum was 3 sec for a decade.
RESULTS Example 1 . Healthy controls excreted the following deuterated phenolic acids after a load of deuterated L-tyrosine: p-hydroxyphenylacetic, p-hydroxyphenylpyruvic, p-hydroxyphenyllactic, homovanillic, and vanillmandelic acid. In addition 3-methoxy-4-hydroxy-phenylglycol (MHPG) and dopamine, which were determined by a special gas chromatographic procedure (14), were also found to be deuterated. Benzoic acid was never found in normal controls (15). In the patients excreting excessive amounts of benzoic acid and hippuric acid, the loading with deuterated L-tyrosine gave the results summarized in Table I. The excretion of both compounds was markedly increased. A typical gas chromatogram of the aromatic acids in urine of one of these children is shown in Figure 1. GC/MS combination revealed that the same metabolites were deuterated as in the normal controls, but, in addition, benzoic acid and hippuric acid were found markedly deuterated. The mass spectra for hippuric acid, see Figure 2, indicated that the two labeling deuterium atoms were still present. Example 2. A mentally retarded patient with a urinary odor similar to the so-called "sweaty feet syndrome" was examined by loading with deuterated DL-leucine. The mass spectrum of this compound is presented in Figure 3. From the mass spectrum, it is obvious t h a t three deuterium atoms are incorporated; and from the mass numbers m l e 75 and 89, it can be concluded that two hydrogen atoms have been exchanged in the 0 or y position. The chromatogram of urinary acids of this patient is shown in Figure 4, and the concentrations of some organic acids found in urine before and after loading with DL-leucine are summarized in Table 11. The only metabolite which increased markedly was 3-hydroxyisovaleric acid and only this compound was deuterated. It is of special interest (14) Unpublished results from our laboratory. (15) T. L. Perry, S. Hansen. S. Diamond. S. B. Melancon, and D. Lesk, Clin. Chim. Acta. 31, 181 ( 1 9 7 1 ) .
A
IS
I
HT. ( X )
lo0
-
44
30 82
86
50
74
I
30 MIN
15
0
Figure 4. Gas chromatographic separation of short chain carboxylic acids as methyl esters of a patient with suspected "sweaty feet syndrome" IS = Internal Standard (enanthic acid), 1 = lactic acid. 2 = 3-hydroxyisovaleric acid, 3 = 3-hydroxybutyric acid, 4 = rnethylsuccinic acid. 5 = succinic acid
75
2 100
rn/e
Figure 3. Mass spectrum of DL-leucine ( A ) undeuterated. (6)deuterated
that methylsuccinic acid did not increase and was not deuterated (see Discussion).
DISCUSSION The present and earlier results (4-6, 9, 10) show that stable isotopes are well suited for in uivo metabolic studies. Compounds labeled with 13C would be even more suitable than the deuterated compounds used in the present study, since their isotope effect is extremely small and there is less danger of isotopic exchange in the body. However, the natural abundance of 13C is much higher than that of deuterium which decreases the sensitivity of the method; in addition, deuterated compounds are usually simpler to synthesize. An oral load of a stable isotope is without risk since small concentrations are given. Apart from physiological safety, there are analytical advantages in using stable isotopes. The components can be separated by gas chromatography and their isotope content may be directly analyzed by mass spectrometry. The combination GC/MS is superior in its separating capacity to conventional methods for determining radioactive isotopes, where the
components are usually separated by paper, thin layer, or column chromatography and then measured off line with a liquid scintillation counter. Radio gas chromatography is another interesting method but because of problems in temperature programming and in sensitivity, it is often not applicable to biological materials. Generally amino acid metabolism in the body is catalyzed by L-specific enzymes. Optically active compounds are therefore required for such metabolic studies. These compounds are often very difficult to synthesize. Either racemization must be inhibited during the synthesis or a step of enantiomer separation must be included. The application of DL-compounds, however, often cannot be circumvented. Yaturally, one has to be careful with conclusions drawn from experiments with racemates. While deuterating leucine, isoleucine, and valine, it must be remembered that the deuterium in the N position to the carboxyl group is already lost during the transaminase reaction. Deuteration is therefore necessary in 13 or y positions. For this reason, we performed the synthesis with pyridoxal and deuterium oxide. This reaction is well suited for the synthesis of these compounds but leads to racemization. High concentrations of hippuric and benzoic acids were detected in three mentally retarded patients. After loading with deuterotyrosine, these acids showed greatly increased concentrations and were deuterated. To our knowledge, the formation of hippuric acid from tyrosine has not been observed previously in man. We assume that a dehydroxylating enzyme removes the 4-hydroxyl group and the side chains are broken down in several steps. However, recent studies in one of these patients showed that deuterated L-tyrosine was no longer converted to deuterated benzoic and hippuric acid when neomycin was given (200 mg/kg body weight p.0.) for partial sterilization of the intestine during 3 days before the load. It seems, therefore, that deuterated benzoic acid and hippuric acid were formed from tyrosine by the intestinal flora. No isotope labeling of benzoic and hippuric acid was found in normal subjects after loading with deuterotyrosine. It is possible that degradation of tyrosine to hippuric acid also takes place normally but is below our detection
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limit. In animal experiments with 1%-phenylalanine, a similar observation was made by Armstrong et al. (16). Abnormalities of leucine, isoleucine, and valine metabolism are difficult to examine “in viuo.” The extremely water soluble compounds (hydroxy, keto, and dicarbonic acids) are difficult to extract from urine, and a number of compounds are extracted a t the same time, which makes the interpretation of gas chromatograms without mass spectrometry difficult. Many reports have been published on this subject (17-20), and a preliminary purification with an ion-exchange resin is recommended (18). In our patient with suspected “sweaty feet syndrome,” we detected deuterated 3-hydroxyisovaleric acid. This compound is excreted in increased amounts in P-methylcrotonylglycinuria (P-hydroxyisovalericacidemia), a pre-
(16) M. D. Armstrong, Fu-Chuan-Chao, V. J. Parker, and P. E. Wall, Proc. SOC.Exp. Biol. Med., 90, 675 (1955). (17) E. C. Horning and M . G. Horning, J. Chromatogr, Sci., 9, 129 (1971). (18) M. G. Horning in “Biomedical Applications of Gas Chromatography,” Vol. 2. H. A. Szymanski, Ed., Plenum Press, New York, N.Y.. 1968. (19) E. Jellum, 0. Stokke, and L. Eldjan, Scand. J. Clin. Lab. Invest., 27, 273 (1971). (20) K. 6.Hammond and S. J. Goodman, Clin. Chem., 16, 212 (1970).
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viously described metabolic defect of leucine degradation. Several authors have suggested that methylsuccinic acid is formed from isovaleric acid by an w-oxidation. However, methylsuccinic acid did not increase in our patient during the loading with deuterated DL-leucine and was not deuterated. Therefore it seems to be formed from another percursor in our patient. Our examples illustrate that loading studies with deuterated compounds and subsequent measurement of urinary metabolites by gas chromatography-mass spectrometry is a significant method for studying metabolic pathways in vivo, especially in patients with metabolic defects.
ACKNOWLEDGMENT The authors are grateful to G. Schollenhammer for the synthesis of deuterated L-tyrosine and to U. Redweik, and the Misses L. Nowrusow, K. Schaltegger, and G. Schnabel for skillful technical assistance. Received for review November 30, 1973. Accepted February 1, 1973. Dedicated to Professor F. Leuthard on the occasion of his 70th anniversary. Supported by the Schweizerischer Nationalfonds zur Forderung der wissenschaftlichen Forschung (No. 3.586.71).
