ANALYTICAL

BIOCHEMISTRY

190,

309-313

(1990)

Determination of a-Keto Acids Including Phenylpyruvic Acid in Human Plasma by High-Performance Liquid Chromatography with Chemiluminescence Detection Toshihiro Faculty

Received

Nakahara,

of Pharmaceutical

May

Junichi

Ishida,

Masatoshi

Sciences, Fukuoka

University,

Yamaguchi,l Nanakuma,

Johnan-ku,

Fukuoka

Nakamura 814-01,

Japan

7, 1990

A highly sensitive method for the determination of a-keto acids including phenylpyruvic acid in human plasma is investigated. The method employs high-performance liquid chromatography with chemiluminescence detection. The acids and a-ketocaproic acid (internal standard) in human plasma are isolated by anion-exchange chromatography on a Toyopak DEAE cartridge, and then converted into the corresponding chemiluminescent derivatives with 4,5-diaminophthalhydrazide dihydrochloride, a chemiluminescence derivatization reagent for a-keto acids. The derivatives are separated within 50 min on a reversed-phase column, TSKgel ODS-12OT, with isocratic elution, followed by chemiluminescence detection; the chemiluminescence is produced by the reaction of the derivatives with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1). The detection limits for the acids are in the range 9-92 pmol/ml in plasma (signal-to-noise ratio = 3). This sensitivity permits precise determination of several a-keto acids including phenylpyruvic acid, which cannot be determined by other HPLC methods, in 10 ~1 of normal human plasma. The chemiluminescent product from phenylpyruvic acid was characterized as 3-benzyl-?&dihydropyridazino[4,5-g]quin-

oxaline-2,6,9(

lZ+trione.

0 mio

Academic

pr-0.

I~C.

a-Keto acids are important intermediates in the biosyntheses of amino acids, carboxylic acids, and sugars. Recently, considerable attention has been focused on the branched-chain cu-keto acids such as cr-ketoisovaleric, cy-ketoisocaproic, and a-keto+methylvaleric acids. Both starvation (l-3) and diabetes mellitus (4) can result in significant alteration in concentration of the acids. Additionally, the acids and phenylpyruvic

1 To whom

and Masaru

correspondence

should

be addressed.

0003-2697190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

acid exhibit greatly increased levels in plasma/serum from patients with hereditary metabolic diseases such as maple syrup urine disease, and phenylketonuria (5 9). In particular, phenylketonuria is characterized by the abnormal levels of phenylpyruvic acid in plasma. Therefore, a reliable method for the quantification of these cY-keto acids in human plasma is useful for the investigation of metabolism and diagnosis and/or treatment of such diseases. Various high-performance liquid chromatographic (HPLC) (10-16) and gas chromatographic (GC) (1,17) methods have been reported for the determination of cY-keto acids in plasma/serum. Branched-chain a-keto acids in human plasma/serum are successfully determined by these methods. However, the sensitivities of the methods are not enough to determine phenylpyruvic acid in normal human plasma, which occurs at very low concentrations. Recently, highly sensitive fluorimetric HPLC (l&19) and GC-mass specrometric (MS) methods (20,21) have been developed for the quantification of cY-keto acids. However, these methods have not been applied successfully to the determination of phenylpyruvic acid. Some workers have specifically stated that phenylpyruvic acid is undetectable in normal human blood/plasma (22,23). On the other hand, chemiluminescence detection has been successfully used for the HPLC analysis in biological and clinical fields because of its high sensitivity (2426). We have reported that 4,5-diaminophthalhydrazide dihydrochloride (DPH),’ a chemiluminogenic reagent for cu-keto acids, reacts selectively with cr-keto acids in dilute hydrochloric acid to produce the corresponding

’ Abbreviations used: DPH, 4,5&aminophthalhydrazide dihydrochloride; KB, a-ketobutyric acid; HPP,p-hydroxyphenylpyruvic acid, KV, a-ketovaleric acid, KIV, or-ketoisovaleric acid; KIC, a-ketoisocaproic acid; KC, a-ketocaproic acid; KMV, a-keto-B-methylvaleric acid, PP, phenylpyruvic acid. 309

310

NAKAHARA

products, which can be separated by reversed-phase HPLC with isocratic elution (27). The products produce chemiluminescence by the reaction with hydrogen peroxide in the presence of potassium hexacyanoferrate(II1) in alkaline media (27). The aim of this study is to develop a HPLC method with chemiluminescence detection utilizing the above derivatization for the quantification of several a-keto acids including phenylpyruvic acid in normal human plasma. cu-Ketocaproic acid does not occur in human plasma, and thus was used as an internal standard (IS).

EXPERIMENTAL

Chemicals and solutions. All chemicals and solvents were of analytical reagent grade, unless stated otherwise. Distilled water, purified with a Milli Q II system, was used for all aqueous solutions. Hydrogen peroxide (30%, v/v) was purchased from Mitsubishi Gas Kagaku (Tokyo, Japan). Sodium salts of a-ketobutyric acid (KB), p-hydroxyphenylpyruvic acid (HPP), a-ketovaleric acid (KV), a-ketoisovaleric acid (KIV), a-ketoisocaproic acid (KIC), D,L-cr-keto-/3-methylvaleric acid (KMV), cr-ketocaproic acid (KC), and P-phenylpyruvic acid (PP) were purchased from Sigma (St. Louis, MO) and used as received. DPH was prepared as described previously (27,28). DPH solution (1.2 mM) was prepared in 0.6 M hydrochloric acid containing 0.6 M b-mercaptoethanol. This solution was used within 5 h. A Toyopak DEAE cartridge (anion-exchange, diethylaminoethyl resin, 0.15 ml; Tosoh, Tokyo, Japan) was washed successively with 0.4 ml of 2.0 M sodium hydroxide (twice), 0.6 ml of water (three times), 0.4 ml of concentrated hydrochloric acidacetonitrile (1:9, v/v) (twice), 0.6 ml of water (three times), and 0.4 ml of Tris-hydrochloric acid buffer (10 mM, pH 9.0) (twice). Human blood was obtained from fasting healthy volunteers in our laboratories. The blood (1.0 ml) was taken into a test tube containing 3.8 mg of citric acid trisodium salt dihydrate and centrifuged at 1OOOg at 4°C for 15 min. The resulting plasma was stored at -20°C until just before use. Apparatus and chemiluminescence detection system. Infrared (IR) spectra were measured with a JASC0 IR-810 spectrophotometer using KBr pellets. ‘H nuclear magnetic resonance (NMR) spectra were obtained with a Hitachi R-22 (90 MHz) using approximately 2% (w/v) solutions in dimethyl sulfoxide-$ with tetramethylsilane as an internal standard. Splitting patterns were designated as follows: s, singlet; m, multiplet; br, broad. Fast atom bombardment mass spectra (MS) were taken with a JEOL D-300 spectrometer. Uncorrected melting points were measured with a Gallen Kamp melting point apparatus.

ET

AL.

Chromatography was performed with a Hitachi L-6200 high-performance liquid chromatograph (Tokyo, Japan) equipped with a Rheodyne 7125 syringeloading sample injector valve (20-~1 loop). Chromatograms were recorded with a Hitachi D-2500 chromatointegrator. The DPH derivatives of cu-keto acids were separated on a reversed-phase column, TSKgel ODS-12OT (250 X 4.6 mm, i.d.; particle size, 5 pm; Tosoh, Japan), by isocratic elution with a mixture of acetonitrile and 50 mM phosphate buffer (pH 7.0) (3:22, v/v) as eluent. The flow rate of the mobile phase was 1.0 ml/min. The column temperature was ambient (18-25°C). The chemiluminescence system was previously reported in detail (27). Briefly, the eluate from the HPLC column was mixed with 50 mM hydrogen peroxide and 30 mM potassium hexacyanoferrate in 2.0 M sodium hydroxide delivered by two pumps (JASCO 880 PU, Tokyo, Japan). The flow rate of the hydrogen peroxide and potassium hexacyanoferrate(II1) solutions were 1.0 and 2.0 ml/min, respectively. The generated chemiluminescence was monitored by an ATT0 AC-2220 luminomonitor (Tokyo, Japan) equipped with a 60-~1 flow cell. Isolation of the reaction product of phenylpyruvic acid with DPH. DPH (1.6 mmol) and phenylpyruvic acid (0.8 mmol) were dissolved in 20 ml of a mixture of ethanol and 2.0 M hydrochloric acid containing 1.2 M @mercaptoethanol (l:l, v/v). The mixture was heated for 2 h in a boiling water bath. The precipitate, which was produced during the heating, was filtered off, washed with ethanol, and then dried in vacua to give compound I [pale green powder; mp 352°C dec; yield, ca. 130 mg (ca. 46%)]. Analytical data were as follows: IR v,“,“: (cm-‘) 3100 (lactam NH), 1672 (lactam C = 0); ‘H NMR (dimethyl sulfoxide-$; ppm) 4.19 (2H, s), 7.117.69 (5H, m), 7.86 (lH, s), 8.27 (lH, s), 11.46 (1H X 2, br), 12.78 (lH, br); MS, m/z 321 (M + 1, base peak). Anal. Calcd for (C,,H,,N,O,): C, 63.77; H, 3.97; N, 17.63. Found: C, 63.75; H, 3.78; N, 17.49. Procedure. A lo-p1 aliquot of plasma was mixed with 20 ~1 of 3.1 nmol/ml KC (IS) and 0.4 ml of 10 mM Trishydrochloric acid buffer (pH 9.0), and the mixture was poured into a Toyopak DEAE cartridge. The cartridge was washed successively with 0.4 ml of water (five times) and 0.4 ml of aqueous 40% acetonitrile (five times). The adsorbed acids were eluted with 0.2 ml of a mixture of acetonitrile and 1.0 M sodium chloride (4~7, v/v). To 100 ~1 of the eluate placed in a screw-capped tube (100 X 15 mm i.d.) was added 100 ~1 of the DPH solution, and the tube was tightly closed and heated at 100°C for 45 min. The final reaction mixture (20 ~1) was injected into the chromatograph. For the establishment of the calibration graph, 20 ~1 of the KC (IS) solution was replaced with an IS solution containing cy-keto acids (1 pmol to 4 nmol). The net

CHROMATOGRAPHIC

FIG.

1.

Reaction

DETERMINATION

of phenylpyruvic

acid with

OF

AND

DPH.

DISCUSSION

HPLC and chemiluminescence derivatization tions were almost the same described previously Chemiluminescent Phenylpyruvic

Product of Reaction Acid and DPH

condi(27).

between

In order to investigate the structure of the chemiluminescent products, phenylpyruvic acid was employed as a model compound. The reaction product from phenylpyruvic acid was identified as 3-benzyl-7,8dihydropyridazino[4,5 -g]quinoxaline - 2,6,9(1H) - trione (compound I in Fig. 1) by the analytical data described under Experimental. When the product dissolved in water was applied directly onto the present HPLC, a single peak having exactly the same retention time as that of peak 8 in Fig. 2 appeared in the chromatogram. The efficiency of conversion of phenylpyruvic acid to the DPH derivative was examined by comparing the peak height obtained under the reaction conditions with that given by the pure reaction product; the extent of conversion (mean f standard deviation (SD), n = 5) was 88.7 t 3.3%. Furthermore, the product dissolved in water generated visible chemiluminescence immediately by the addition of the hexacyanoferrate(II1) and hydrogen peroxide solutions. These results indicate that compound I is the main product in the determination of phenylpyruvic acid with DPH. The products from the other a-keto acids might be estimated to be the corresponding quinoxaline derivatives. Deproteinization

and Cleanup

ACIDS

IN

HUMAN

311

PLASMA

the cartridge were effectively eluted ml) of acetonitrile and 1.0 M sodium The recoveries of KIV, KIC, KMV, the cartridge were 82.3 + 3.6,84.6 ? * 1.8, and 69.3 i 3.1% (mean +- SD, respectively.

peak height ratios of the individual a-keto acids and KC were plotted against the concentrations of a-keto acids added. RESULTS

ol-KETO

with a mixture (0.2 chloride (4:7, v/v). KC, and PP from 4.0,81.0 f 3.6, 76.5 n = 5 in each case),

Chromatography Figure 2 shows a typical chromatogram obtained with a standard mixture of seven a-keto acids of biological importance and KC. The DPH derivatives of these (Yketo acids could be completely separated within 50 min. The peaks for pyruvic and a-ketoglutaric acids, however, overlapped with those for the reagent blank, and so these acids could not be determined by the method. A typical chromatogram obtained from a normal human plasma is shown in Fig. 3. The components of peaks 4,5,6, and 8 (Fig. 3) were identified as the DPH derivatives of KIV, KIC, KMV, and PP, respectively, on the basis of their retention times, and by cochromato-

6

of Plasma

Plasma had to be deproteinized, otherwise the HPLC column packing was considerably damaged. When plasma was deproteinized with perchloric acid (16,18), unknown broad and large peaks due to endogenous substances in plasma appeared at retention time of 20-32 min. An anion-exchange chromatography with a Toyopak DEAE cartridge was successfully performed for the deproteinization and cleanup of plasma. ar-Keto acids were retained effectively in the cartridge when the pH of the plasma sample was adjusted to around 9.0; 0.4 ml of 10 mM Tris-hydrochloric acid buffer (pH 9.0) was used to adjust the pH of the sample. The acids adsorbed on

I 0

I 10

I 20 Time

4 30

1 40

I 50

(mini

FIG. 2. Chromatogram of the DPH derivatives of cy-keto acids. A portion (0.1 ml) of a standard mixture of a-keto acids (KB, KV, KC, and PP, 0.25 nmol/ml; HPP, KIV, and KIC, 0.75 nmol/ml; KMV, 1.5 nmol/ml) was treated according to the derivatization procedure. Peaks: 1, KB; 2, HPP; 3, KV; 4, KIV; 5, KIC; 6, KMV, 7, KC; 8, PP; 9, reagent blank.

NAKAHARA 5

ET

AL.

methods. The sensitivity of the present method permits precise determination of PP, which occurs at an extremely low concentration in normal human plasma. The within-day precision of the method was established by repeated determinations (n = 5) using a normal human plasma containing 11.4 (KIV), 38.0 (KIC), 22.5 (KMV), and 0.43 (PP) nmol/ml, respectively. The relative standard deviations were 5.0% or below for all the acids.

a-Keto A&&

1I I

, 0

Plasma

CONCLUSION I

I

10

I

20

Time

30

4b

5'0

(inin)

FIG. 3.

Chromatogram obtained with a normal human plasma. A portion (10 ~1) of the plasma was treated according to the procedure. The concentrations of cy-keto acids in the plasma (nmol/ml): KIV, 11.4; KIC, 38.0; KMV, 22.5; PP, 0.43. For peaks 4-8, see Fig. 2; other peaks, endogenous substances and reagent blank. Detector sensitivity: O-40 min, 1; 40-50 min, 8.

This study has provided the first practical HPLC method with chemiluminescence detection for the quantification of a-keto acids including phenylpyruvic acid. The method is the most sensitive of the methods so far reported. In this study, 10 ~1 of normal human plasma was used for the precise determination of phenylpyruvic acid. However, the method can be scaled down, possibly to &&, for the determination of only

graphy with the standard compounds. No peaks were observed in the chromatogram at the retention times for KIV, KIC, KMV, and PP when the derivatization reaction was not performed. KB could not be determined by interferring peaks from plasma components. HPP and KV could not be traced, as previously reported (12-18).

Linearity,

in Human

The concentrations of ar-keto acids in plasma from healthy volunteers were determined by this method (Table 1). The mean values and their standard deviations for KIV, KIC, and KMV were in good agreement with those obtained by Hayashi et al. (12), Koike and Koike (14), Hara et al. (16), and Wang et al. (18) (Table 2). However, the mean values were higher than those listed by the other workers (Table 2). The amount of PP in normal human plasma, which was first determined by the present method, was ca. 3-A those of KIV, KIC, and KMV.

4

I

Concentrations

Detection Limits,

and Precision

Linear relationships were observed between the ratios of the peak heights of a-keto acids to that of KC and the amounts of cu-keto acids added to plasma in the range 0.1-400 nmol/ml. The correlation coefficients of the calibration curves were higher than 0.998 for all the compounds. The lower limits of detection for KIV, KIC, KMV, and PP were 18,25,92, and 9 pmol/ml plasma (7, 10, 36, and 3.4 fmol/20 ~1 injection volume), respectively, at a signal-to-noise ratio of 3. The sensitivity for branched-chain a-keto acids was comparable with those of the HPLC methods reported by Wang et al. (18) and Kieber and Mopper (19), and the GC-MS method described by Ford et al. (20), and was higher than those of the other conventional methods (10-16). On the other hand, the sensitivity for PP is the highest of all the

TABLE

1

Concentrations of a-Keto Acids in Plasma from Healthy Persons Concentration Age

Sex”

KIV

KIC

20 21 21 22 22 22 24 22 22 22 24 31 41 Mean SD

F F F F F F F M M M M M M

13.7 7.8 11.4 9.3 10.7 10.0 10.0 15.4 13.6 13.7 16.5 10.4 16.2 12.2 2.8

44.4 31.3 38.0 32.9 34.3 33.5 29.1 48.0 45.0 36.4 37.8 29.1 39.9 36.9 6.1

o M, male;

F, female.

(nmol/ml) KMV 24.4 20.1 22.5 26.6 29.0 19.8 18.7 32.3 31.4 21.5 30.8 11.2 25.3 24.1 6.0

PP 0.23 0.48 0.43 0.30 0.32 0.28 0.29 0.48 0.29 0.29 0.38 0.42 0.26 0.34 0.09

CHROMATOGRAPHIC

DETERMINATION

OF TABLE

Comparison of Plasma Concentrations Obtained by the Present HPLC

a-KETO

ACIDS

IN

HUMAN

2

of Branched-Chain Method

and

a-Keto Acids

Other

Methods Concentration

Method HPLC HPLC HPLC HPLC HPLC HPLC

n

10 11

12 33 10 100 12 9 8 5 20 20 10 10 13

12 14 16 18

GC GC GC GC-MS Present

Ref.

Male Female

15

Male Female

17 21” HPLC

313

PLASMA

(nmollml,

KIV 12 8.8 14.6 13.0 22.7 13.2 12.0 8 13.3 19.0 9.4 9.0 12.2

mean

i SD)

KIC

23 + 4.9 + 5.6 f 2.9 -t 6.2 + 4.3 + 2.2 +4 * 3.3 * 7.4 k 6.0 +- 4.6 + 2.8

29 21.9 38.8 35.5 38.0 40.8 33.0 30 37.4 24.5 24.3 20.9 36.9

KMV

+ 8 Z!Z 10.9 k 15.8 t 2.7 +- 10.8 +- 9.3 z!Y 9.9 +-12 t 11.5 +- 8.9 +- 9.3 t 10.1 t 6.1

18 17.6 26.2 22.5 26.6 24.3 19.9 22 23.3 11.3 16.2 14.5 24.1

f4 + f + +f f f4 f + in + +

6.8 8.5 5.4 8.7 7.0 5.9 9.5 3.0 9.5 6.7 6.0

’ Children.

branched-chain cY-keto acids in plasma. The sensitivity of the method permits precise determination of PP even in 10 ~1 of normal human plasma; PP in the plasma was first determined by the method. It has long been supposed that excess phenylpyruvic acid in children is toxic to the central nervous system. However, the detailed role of the acid has remained unknown. The method should be useful for clinical and biological investigations of PP and branched-chain cu-keto acids in some hereditary metabolic diseases.

10.

Walser, M., Swain, them. 164, 287-291.

11. Smeaton, T. C., Owens, matogr. 487, 434-439.

r2.

This work was partly supported by a Grant-in-Aid for Encouragement of Young Scientist (02771689) from the Ministry of Education, Science and Culture. The authors are grateful to Miss J. Honda and Mr. H. Hanazono for elemental analysis and mass spectral measurements. We are also indebted to Miss M. Sugishita for skillful assistance.

and Alexander,

H., and Naruse,

13. Hayashi, T., Tsuchiya, H., Todoriki, Anal. Biochem. 122, 173-179. 14. Koike,

K., and Koike,

M.

(1984)

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Anal.

17. Woolf, L. I., Hasinoff, 231,237-245.

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H. (1983)

K., and Ohkura,

D. T., and Mopper,

K. (1983)

Moilanen, M.,

A., (1982)

Biomed.

C. R. (1986)

22. Jervis, 441.

G. A., and Drejza,

Chim.

3. Schauder, P., Herhertz, L., and Langenheck, U. (1985) Metabolism 34,58-61. 4. Schauder, P., Schriider, K., Matthaei, D., Henning, H. V., and Langenheck, U. (1983) Metabolism 32,323-327. 5. Funai, T., and Ichiyama, A. (1986) J. Biochem. 99, 579-589. 6. Ohmori, S., Mizuno, S., Ikeda, M., and Yao, K. (1982) Chem. Pharm. Bull. 30,2099-2104. 7. Livesey, G., and Lund, P. (1982) Bioehem. J. 204, 265-272.

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T., Kai, M., 226, 25-31.

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24. Kawasaki, T., 328, 121-126.

Maeda,

E. J. (1966)

Kohashi, M.,

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M. M.

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S. W. (1973)

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J. Chro-

Anal. T.,

Mass

13, 435-

A. (1985)

(1986)

135-

Acta

N., and Kawasaki,

J., Yamaguchi, M., Nakahara, Anal. Chim. Acta 23 1, l-6.

28. Williams, 10,891-898.

Clin.

430,

281,

21. Mamer, 0. A., Laschic, N. S., and Striver, Mass Spectrom. 13,553-558.

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and Oh-

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ACKNOWLEDGMENTS

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and

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