Enzymatic determination of the branched-chain α-keto acids

ANALYTICAL BIOCHEMISTRY 162,536-539

(1987)

Enzymatic Determination

of the Branched-Chain

cr-Keto Acids’

G. W. GOODWIN, M. J. KUNTZ, R. PAXTON,* AND R. A. HARRIS Department of Biochemistry, Indiana University School of Medicine. 635 Barnhill Dr., Indianapolis, Indiana 462 ‘23 Received August 2 I, I986 A spectrophotometric endpoint assay for determination of branched-chain cu-keto acids is described. The assay depends on measurement of the NADH produced after addition of branched-chain cY-ketoacid dehydrogenase. Interference by pyruvate and cy-ketobutyrate was eliminated by pretreating the sample with pyruvate dehydrogenase. The method yielded a peripheral venous plasma value of 59 + 5 JIM (mean + SE) for the branchedchain a-keto acids of five overnight fasted healthy humans. Q 1987 Academic press Inc. KEY WORDS: branched-chain ol-keto acids; branched-chain a-keto acid dehydrogenase; blood; plasma; branched-chain amino acids; leucine.

drogenase (5) is of comparable utility to the method described in this article. Thus, the two enzymatic assays have the advantage of relative speed and simplicity; sample preparation requires only deproteinization, and the assays require only a uv spectrometer. However, these two methods suffer from the disadvantage that the sum of the three BCKAs is obtained. This parameter, however, is useful because the three BCKAs are metabolized similarly and are elevated in the same pathological conditions. It should be added that branched-chain cY-keto acid dehydrogenase, or BCKDH, is not presently commercially available. However, pyruvate dehydrogenase, or PDH, is relatively inexpensive, and supply houses could easily furnish BCKDH as a side product of the PDH preparation if a demand for the enzyme is indicated. In addition, while leucine dehy’ This work was supported by N.I.H. Research Grant drogenase is commercially available (Sigma AM19259 and a Fellowship Grant from the American Chemical Co., St. Louis, MO), it is prohibiHeart Association, Indiana Affiliate, Inc. tively expensive (about 5 dollars per assay 2 Present address: Department of Biological Sciences, under the conditions described by Livesey Box 4149, Texas Tech University, Lubbock, TX 79409and Lund (5)). 4149. 3 Abbreviations used: BCRA, branched-chain cu-keto BCKDH (EC 1.2.4.4 + no EC No. for diacid; BCKDH, branched-chain cr-keto acid dehydrogehydrolipoamide acyltransferase + 1.8.4.3) nase; PDH, pyruvate dehydrogenase; Mes, Cmorphoand PDH (EC 1.2.4.1 + 2.3.1.12 + 1.8.4.3) lineethanesulfonic acid; Mops, 4-morpholinepropanecatalyze the following irreversible reaction: sulfonic acid.

Interest in an assay for branched-chain (Yketo acids, or BCKAS,~ stems from the fact that normal metabolism of the branchedchain amino acids (leucine, valine, and isoleucine) involves transamination and interorgan transport of the corresponding a-keto acids (a-ketoisocaproate, cu-ketoisovalerate, and cu-keto-&methylvalerate). In addition, elevated levels of blood BCKAs are characteristic of an inborn error of metabolism called maple syrup urine disease. This interest was initially satisfied by gas-liquid chromatography ( 1,2) and, later, by high-performance liquid chromatography (3). These techniques yield values for the individual BCKAs and require sample derivatization under a nitrogen atmosphere (4). An enzymatic assay for the sum of the three BCKAs using bacterial leucine dehy-

0003-2697187 $3.00 Copyright Q 1987 by Academic F’Iw, Inc. All rigim of reproduction in any form IFSB-V~~

536

SPECTROPHOTOMETRIC

DETERMINATION

OF BRANCHED-CHAIN

a-KETOACIDS

537

Rat blood was drawn into a heparinized syringe by cardiac puncture under ether anesRiCOC+ CoA + NAD+ + thesia. Plasma was prepared by centrifugation at 1OOOgfor 10 min. Ice-cold 15% (w/v) perchloric acid (0.2 ml) R:CoA + CO 2 + NADH. containing 0.4 &i/ml ‘HZ0 was vigorously mixed with 0.4 ml of sample and maintained For PDH, the cu-keto acid substrates include on ice for 15 min. 3H20 may be omitted if pyruvate and cu-ketobutyrate, but not the sample dilution is determined by convenBCKAs. The a-keto acid substrates for tional (i.e., volumetric or gravimetric) BCKDH include the BCKAs, pyruvate, LYketobutyrate, and a-ketomethionine or y- methods. After centrifugation for 3 min in an Eppendorf centrifuge, 0.3 to 0.4 ml of methylthio-cr-ketobutyrate (6). Pyruvate supernatant was withdrawn with care to levels in blood or plasma are normally much avoid insoluble material. After addition of 50 higher than the BCKAs. Although pyruvate ~1 of universal indicator (Fisher Scientific) is a relatively poor substrate for BCKDH (lu, the supernatant was brought to a neutral = 0.8 mM, I’,,,, = 20% of that for cy-ketoisoendpoint with 3 M KOH containing 0.25 M valerate (7)) it will interfere with a spectro4-morpholineethanesulfonic acid (Mes) and photometric assay for the BCKAs utilizing 0.25 M 4-morpholinepropanesulfonic acid BCKDH (shown in this study). Interference (Mops). Potassium perchlorate was sediin the assay by pyruvate and a-ketobutyrate, mented for 30 s in an Eppendorf centrifuge however, can be eliminated readily by preinimmediately before adding the sample (0.3 cubation of the sample with PDH, although possible interference by cY-ketomethionine is ml unless otherwise indicated) to the cuvette. Determination of BCkTAs. Assays were not eliminated by this procedure. conducted in 1.5-ml glass cuvettes (path length 1 cm) in a final assay volume of 1 ml MATERIALS AND METHODS containing 30 mM Kpi, 2 mM MgC12, 0.4 Sources of materials. 3H20 was from mM thiamine pyrophosphate, 0.4 mM CoA, Amersham Corp. (Arlington Heights, IL). 1 mM dithiothreitol, 2 mM NAD+, 0.1% (v/v) PDH, sp act 2.7 U/mg, was from Sigma Triton X-100, pH 7.5, 1.25 U/ml porcine Chemical Co. (St. Louis, MO). Sources of all heart dihydrolipoamide reductase (EC other materials and preparation and storage 1.8.4.3) and 16 mu/ml PDH. Reactions of rabbit liver BCKDH, sp act 9.0 U/mg, were conducted at 37°C. Upon completion have been previously described (89). of the PDH reaction (approximately 30 min BCKDH activity is lost at a rate of a few as indicated by a stable absorbance), a basepercent per month in storage. Male Wistar line absorbance was established on the chart rats (500-600 g) were from Harlan Industries recorder set at a full scale of 0.1, period of 10 (Indianapolis, IN). Absorbance changes were s, and the BCKDH reaction was initiated by determined with a Cat-y 2 19 recording spec- addition of 5 ~1 of 8.5 U/ml BCKDH. Betrophotometer with automatic sample cause of the small absorbance changes and changer. Radioactivity was measured with a the inhomogeneity of most cuvettes, care Beckman LS 1801 liquid scintillation must be taken not to disturb the position of counter. the cuvette after a baseline absorbance is esSample preparation. All samples were pro- tablished, though positioning of the turette cessed immediately, the assays were com- with this particular instrument was suffipleted within a few hours, and all operations ciently consistent so that the automatic samwere at 0-4°C unless otherwise indicated. ple changer could be used (i.e., five assays Human blood was withdrawn from an ante- could be performed at one time). Generally, cubital arm vein into a heparinized syringe. reactions go to completion within 15 min.

538

GOODWIN

The absorbance change at 340 nm, as determined from the chart paper, is corrected for a reagent blank in which water replaces sample. The blank amounted to 0.0034 absorbance unit for the BCKDH preparation used in this study. However, this must be determined for each preparation. Total sample dilution was determined by relating the radioactivity of 0.1 ml of the contents of the cuvette to the same volume of the ‘H20spiked perchloric acid solution, counted in 4 ml of Scinti-verse II (Fisher Scientific).

ET AL.

e-10

MIN4

FIG. 2. Conditions were as described in Fig. 1, using the same plasma sample, except the PDH preincubation was omitted.

RESULTS AND DISCUSSION

Typical tracings illustrating both the PDH and BCKDH reactions are shown in Fig. 1. In the basic assay, PDH is initially included in the cuvette to save time. A second addition of BCKDH did not result in any further change in absorbance. In addition, 98% of a subsequent addition of a-ketoisovalerate was recovered (allowing for a drop in absorbance by 2% resulting from volume expansion by 20 pl), indicating that both the PDH and BCKDH reactions had gone to completion (Fig. 1). Absorbance changes were completely inhibited by 1 mM arsenite in the cu-

3 NYOL a-KETOIS0”ALERAT.Z I

PDH

i

I i

+

0.0; 100 10 MINI

1. Rat plasma was processed for the basic assayas described under Materials and Methods, except PDH was initially omitted. PDH refers to the addition of 5 ~1 of 3.2 U/ml PDH. BCKDH refers to the addition of 5 ~1 of 8.5 U/ml BCKDH. The pyruvate concentration in the original sample, as total PDH substrate, was 125 PM, and the BCKA concentration was 24 pM. Breaks in the scale are 10 min. FIG.

vette, confirming that they resulted solely from lipoamide-dependent reactions. In the absence of preincubation of the sample with PDH, the endpoint was indeterminate (Fig. 2), presumably from slow utilization of pyruvate by BCKDH, indicating the necessity of the PDH preincubation. Within- and between-day variations in the assay were determined by freezing 0.4-ml aliquots of whole human blood in liquid Nz and assaying five aliquots on each of five consecutive days after storage at -70°C. The average intraassay coefficient of variation was 6.3%, while the interassay coefficient of variation between the five means was 4.5% for a sample that was 35 PM in the BCKAs. The BCKAs are therefore stable under these storage conditions. The assay was linear with amount of sample added to the cuvette (r = 0,997, Fig. 3). Sensitivity, taken as 10 times the background (+0.0005 absorbance unit) is approximately 1 nmol. Under the conditions of standard assay and extractions, the sensitivity in the original sample is therefore approximately 5 PM.

Recovery of the individual BCKAs was determined in human plasma and blood by addition of 30 nmol of the BCKA to the original sample. Percentage recoveries were calculated after correction for the endogenous values (Tabie 1). The explanation for shghtly lower recovery of cr-ketoisocaproate in these

SPECTROPHOTOMETRIC

0

0.1

0.2

ML EXTRACT

DETERMINATION

0.3

0.4

ASSAYED

FIG. 3. Rat plasma was processed for the basic assayas described under Materials and Methods. The indicated volumes of plasma extract were assayed for change in absorbance over the blank upon addition of BCKDH. Each point is the mean f SE for three determinations. The BCKA concentration in the original sample was 26 PM. The final absolute absorbance with the largest amount of extract assayed was 0.85.

experiments is not known, although it has been previously reported (5). The following compounds were without effect on the assay of 8 nmol of cr-ketoisovalerate: 40 nmol of pyruvate, cw-ketobutyrate, a-ketoglutarate, acetoacetate, oxaloacetate, glyoxalate, /3-hydroxybutyrate, lactate, and 1 mg/ml bilirubin. The presence of crketomethionine, however, resulted in an indeterminate endpoint (as in Fig. 2). arKetomethionine levels appear to be fairly small compared to the sum of the 3CKAs in normal human serum ( 10). However, it should be kept in mind that certain patholog-

TABLE 1 PERCENTAGERECOVERIES~FTHEINDIVIDUAL BCKASINHUMANBLOODANDFTASMA

% Recovery BCKA cY-Ketoisovalerate ol-Ketoisocaproate cr-Keto-fl-methylvalerate

Plasma

Whole blood

91+4* 85 +4

91 + 2 85 + 1

90 f 5

97 +6

* Values are the means + SE for four or five samples obtained from different people.

OF BRANCHED-CHAIN

a-KETOACIDS

539

ical conditions (i.e., hypermethioninemia) may cause a spurious determination of the BCKAs by this method. Determination of sample dilution during deproteinization and neutralization of the deproteinized extract by means of ‘HZ0 was compared to a gravimetric procedure using whole blood. The average difference in fold dilution between the two method was 0.26 f 0.26% (mean f. SE for 26 samples). The use of ‘Hz0 should be widely applicable so long as comparison is made to standard procedures to rule out possible artifacts from differential quenching. It should be emphasized that 3H20 was used for convenience, though other methods for determining sample dilution are equally applicable to this assay. The BCKA assay was applied to peripheral venous plasma from five healthy humans who had fasted overnight (four males and one female, age range 26 to 46) and yielded a value of 59 + 5 PM (mean +- SE for the mean of duplicate determinations) which is comparable to values reported by other investigators (2,3,9).

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