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Clinical Chemistry 46: 848-853, 2000;
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(Clinical Chemistry. 2000;46:848-853.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

Branched-Chain Keto-Acids and Pyruvate in Blood: Measurement by HPLC with Fluorimetric Detection and Changes in Older Subjects

Karine Pailla1,4, Françoise Blonde-Cynober2,4,a, Christian Aussel1,4, Jean-Pascal De Bandt3,4 and Luc Cynober3,4

1 Biochem Laboratory, Emile Roux Hospital, AP-HP, 1 Avenue de Verdun, 94456 Limeil-Brévannes Cedex, France.

2 Biology Laboratory, Joffre-Dupuytren Hospital, AP-HP, 91211 Draveil Cedex, France.

3 Biochem Laboratory, Hôtel-Dieu Hospital, AP-HP, 1 Place du Parvis Notre-Dame, 75181 Paris Cedex 04, France.

4 Nutrition Laboratory, EA 2498, Paris V University, 4 Avenue de l’Observatoire, 75006 Paris, France.
a Address correspondence to this author at: Laboratoire de Biologie, Hôpital Joffre-Dupuytren, 1, Rue Louis Camatte, 91211 Draveil Cedex, France. Fax 33-1-69-83-6495; e-mail f.blonde-cynober{at}jfr.ap-hop-paris.fr


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Background: Measurement of keto-acids is important in various clinical situations. The aim of the present work was to develop a rapid HPLC method for the determination of keto-acids in human serum and to assess the concentrations of these acids in young adults and institutionalized elderly adults. This method was applied to the determination of blood keto-acid concentrations of young adults and institutionalized elderly people, divided into age groups

Methods: Four keto-acids ({alpha}-ketoisocaproate, {alpha}-ketoisovalerate, {alpha}-keto-ß-methylvalerate, and pyruvate) were derivatized with o-phenylenediamine to give fluorescent derivatives. After the sample preparation step (75 min to prepare 20 samples), the derivatives were separated chromatographically on a reversed-phase column using a binary gradient.

Results: The fluorometric detection of the four keto-acids was rapid, <12 min. The method is repeatable and reproducible: the CVs were <6% and <11%, respectively, for each of the keto-acids. We found no significant difference between males and females. Concentrations of the branched-chain keto-acids decreased after age 60 years, especially {alpha}-ketoisocaproate, which decreased ~40%.

Conclusions: The proposed method allows rapid and reliable measurement of keto-acids. The data demonstrate that changes in branched-chain keto-acids concentrations in serum occur with age.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
In recent years, increased attention has been paid to the metabolism and biological functions of the branched-chain keto-acids (BCKAs),1 i.e., {alpha}-ketoisocaproate (KIC), {alpha}-ketoisovalerate (KIV), and {alpha}-keto-ß-methylvalerate (KMV). BCKAs are glucogenic (KIV and KMV) or ketogenic (KIC and KMV) precursors (1) and can regulate protein turnover (2)(3)(4). Hence, the investigation of BCKA metabolism is of interest for pathological situations such as sepsis (5), burns (6), and in hepatic disorders (7). Another keto-acid, pyruvate (PYR), is at a metabolic crossroads and is notably involved in alanine biosynthesis. It is therefore also important to follow the changes in its concentration in hypercatabolic states (6)(8)(9). A higher risk of infectious diseases has been reported in elderly people, particularly those living in institutions, because of protein malnutrition and a decline in the immune system (10). In these clinical latter situations, the measurement of keto-acids in venous blood is therefore of relevance. To the best of our knowledge, no prior work on this topic has been reported.

Many methods based on gas chromatography, using derivatives such as quinoxalinols (11)(12) or oximes (13)(14), and HPLC (15)(16)(17)(18)(19)(20)(21)(22) currently are used for the determination of keto-acids. However, many of these techniques are time-consuming.

The first purpose of this study was to develop, for clinical use, a reliable HPLC technique with fluorometric detection based on quinoxalinol derivatives for the determination in serum of BCKAs and pyruvate, using o-phenylenediamine (OPD) as a fluorogenic agent. Its second aim was to assess the blood concentrations of keto-acids in adults and elderly institutionalized people.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
reagents
Sodium salts of keto-acids [KIC (CAS no. 4502-00-5), KIV (CAS no. 3715-29-5), KMV (CAS no. 66872-74-0), PYR (CAS no. 113-24-6), ketovalerate (KV; CAS no. 13022-83-8)], OPD (CAS no. 95-54-5), HPLC-grade ethyl acetate (CAS no. 141-78-6), and anhydrous sodium sulfate (CAS no. 7757-82-6) were purchased from Sigma.

The OPD solution was prepared by dissolving 133 mg of OPD in 100 mL of 3 mol/L hydrochloric acid.

Methanol was HPLC grade (J.T. Baker). Distilled water was used for the preparation of all aqueous solutions.

Each component of the mobile phase (methanol and water) was filtered through 0.45 µm filters (type HV; Millipore), and then degassed ultrasonically before use.

Serum albumin concentrations were determined using the bromcresol green method (reagent from Boehringer) adapted to a multiparametric analyzer (911 Hitachi; Boehringer).

chromatographic conditions
The chromatographic system consisted of a model 680 automated gradient controller, a syringe-loading sample injector containing a 20-µL loop, two model 510 pumps, and a model 470 scanning fluorescence detector (all from Waters), a Spherisorb ODS-2 RP-18 column (250 x 4.6 mm; 5 µm particles) from Hypersil, and a column oven (Touzart Matignon).

Retention times and peak areas were determined by a model 746 integrator (Waters) set with an attenuation of 128.0 and a chart speed of 0.5 cm/min. The serum keto-acid concentrations were calculated with the internal standard (IS) method.

calibrators
Because KV does not occur in human serum, KV was used as an IS. A stock solution of KV was prepared at the concentration of 3.62 mmol/L and stored at -20 °C.

Individual stock aqueous solutions of PYR (4.55 mmol/L), KIC (3.29 mmol/L), KIV (3.62 mmol/L), and KMV (3.29 mmol/L) were prepared and stored at -20 °C. The working calibration mixture was then prepared by mixing the four stock solutions in various proportions in water to obtain final concentrations of 164.4 µmol/L for KIC, 36.2 µmol/L for KIV, 32.8 µmol/L for KMV, and 45.5 µmol/L for PYR.

subjects
For the determination of blood concentrations, sera were obtained from fasting healthy adult volunteers (25 men, 26 women), 19–59 years of age (mean ± SD, 40.0 ± 9.9 years), and from elderly people in an institution (25 men, 25 women), 60–99 years of age (81.3 ± 10.3 years). This study was performed in accordance with the current revision of the Helsinki Declaration of 1975. No elderly patient had total parenteral or enteral nutrition, but some had oral supplementation. All subjects exhibited normal hepatic function.

pretreatment of samples
Samples were deproteinated by the addition 50 mg of sulfosalicylic acid to 1 mL of serum. After vortex-mixing for 15 s, the mixture was kept at 4 °C for 10 min and then centrifuged at 2500g for 10 min. The supernatant was removed and frozen at -20 °C until analyzed.

Three distinct serum pools, collected randomly among hospitalized patients, were prepared in the same way. These pools were used to determine, respectively, the repeatability, the reproducibility, and the recoveries of the technique.

sample preparation
The conversion of keto-acids to the corresponding quinoxalinol derivatives was adapted from the procedure of Koike and Koike (15), and was as follows.

Samples and calibrators were thawed at room temperature. The IS (20 µL) and OPD (3 mL) were added to 250 µL of serum or to 500 µL of the calibration mixture in glass tubes. The derivatization was performed by heating the mixtures at 80 °C for 20 min. The samples were then cooled in an ice-water bath. Sodium sulfate (0.5 g) was added to each tube, and the quinoxalinol derivatives were extracted by 3 mL of ethyl acetate. The tubes were vortex-mixed for 1 min and then centrifuged. The upper ethyl acetate phase was transferred to a tube containing 0.5 g of sodium sulfate. After mixing and separation, the organic phase was evaporated to dryness under a stream of nitrogen at 37 °C (N-Evap model 111 evaporator; Organomation). The residue was then dissolved in 500 µL of methanol and filtered before injection.

hplc analysis
Samples (20 µL) of diluted derivatives were injected into the column. Keto-acids were chromatographed at 50 °C by a gradient at a flow rate of 1.6 mL/min. The composition of the mobile phase was as follows: the starting mobile phase consisted of 200 mL/L methanol–800 mL/L water. The mobile phase composition was changed by a linear gradient to 500 mL/L methanol–500 mL/L water over the course of 2 min. This composition was maintained for 1 min. Thereafter, a linear gradient elution was started, with a final concentration of 800 mL/L methanol at 10 min. The original conditions were then reestablished by a reverse gradient to 200 mL/L methanol–800 mL/L water from the 10th to 11th minutes.

The quinoxalinol derivatives of BCKAs were detected by monitoring the fluorescence emission at 410 nm (with excitation at 350 nm).

statistics
All of the data presented are expressed as the mean ± SD. Statistical significance was determined using the nonparametric Mann–Whitney test. Differences with P values <0.05 were considered significant.


   Results and Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
identification of keto-acids
A representative chromatogram of the quinoxalinol derivatives of keto-acids in human serum is shown in Fig. 1 .



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  		Figure 1. HPLC profile of the quinoxalinol derivatives of the keto-acids in a serum from an adult in the 19–59 years age group.

Peaks: 1, PYR (retention time, 6.3 min); 2, KV (IS; retention time, 9.6 min); 3, KIV (retention time, 10.2 min); 4, KIC (retention time, 10.6 min); 5, KMV (retention time, 11.3 min).

The HPLC method used here affords chromatograms that are easy to interpret, with a stable baseline and good peak separation. This HPLC technique permits the measurement of four keto-acids in less than 12 min. This speed has not been obtained with other techniques using either OPD as fluorogenic agent (15)(18)(19) (16–26 min), or 1,2-diamino-4,5-methylenedioxybenzene (21) (20 min). One technique based on the detection of 1,2-diamino-4,5-dimethoxybenzene derivatives gives the four keto-acids in almost the same time, i.e., within 14 min (22). However, in this latter technique, the preparation of the samples, in particular the derivatization, is time-consuming (2.5 h).

method validation (analytical variables)
Linearity and limit of detection.
For injected samples (calibration solutions) in the range 1–3000 µmol/L (20–60 nmol in 20 µL of injected samples), the detector response was linear (r2 >0.996) for each keto-acid.

The limit of detection was determined as the concentration corresponding to a signal 3 SD above the mean for a calibrator free of analyte. This limit of detection for each keto-acid was 1 µmol/L, i.e., well below the physiological range (15)(18)(21)(22).

Precision.
We assessed the within-batch precision of the whole method by 30 treatments of a pool of serum and then 30 individual injections a day (repeatability). The between-batch precision was measured after preparing and analyzing a sample from a pool of serum 12 times in 12 different experiments (reproducibility). The results are presented in Table 1 .


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  		Table 1. Precision of the technique.1

The CV for the determination of the within-batch precision was <6% for all compounds investigated. The CV for the determination of the between-batch precision was <11% for all compounds investigated.

Recovery.
To estimate the influence of the deproteinization process and sample preparation on the measurement of keto-acids, the recoveries of keto-acids added to a pool of serum were determined. The recoveries (mean ± SD) of keto-acids are presented in Table 2 . Recoveries were 97–109% for PYR, 87–94% for KIV, 90–95% for KIC, and 86–91% for KMV. Every keto-acid was satisfactorily recovered in comparison with other techniques (18)(21) for all concentrations added (80–640 µmol/L).


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  		Table 2. Recoveries of keto-acids added to pooled serum.

blood concentrations of keto-acids
The serum concentrations of keto-acids in a group of females and males, measured using the method described here, are presented in Tables 3 and 4. We observed no significant differences between females and males in a given age group (Table 3 ). In contrast, we report for the first time a significant decrease in the concentrations of KIV, KIC, and KMV in institutionalized patients >=60 years of age (Table 4 ). We found an inverse correlation between age and the concentrations of KIC (P <0.0001), KIV (P <0.001), and KMV (P <0.0001). The adult population studied here was older than in other studies (17)(22), which could explain why the BCKA values recorded for healthy adults in our study were slightly lower than in other studies (Table 5 ). The decrease in the concentrations of KIV, KIC, and KMV with increasing age may be related to the decrease in the lean muscular mass observed during aging because most circulating BCKAs come from muscle release (2)(6). Furthermore, it has been shown that low serum albumin concentrations are associated with reduced muscle mass in elderly people (23), and the lower albumin concentrations observed in elderly people (24) could explain this decrease in the BCKA concentrations. This hypothesis is supported by the fact that BCKAs are extensively bound to albumin: less than one-half of plasma BCKAs are ultrafiltrable (25)(26). It is therefore useful to determine the reference values of keto-acids as a function of albumin concentrations. In our study, the albumin concentration of the institutionalized elderly people was 35.4 ± 6.0 g/L (reference values in our laboratory, 40 ± 5 g/L). Supporting the hypothesis expressed above, we found a significant correlation between the albumin and KIC concentrations [KIC = 0.595(albumin) - 2.538; r = 0.499; z = 3.379; P <0.001], KIV concentrations [KIV = 0.215(albumin) + 3.309; r = 0.441; z = 2.921; P <0.01], and KMV concentrations [KMV = 0.294(albumin) + 3.268; r = 0.368; z = 2.378; P <0.05]. Finally, PYR, which does not bind to albumin, was not decreased in elderly subjects compared with adults 19–59 years of age.


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  		Table 3. Concentrations of keto-acids as a function of sex.


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  		Table 4. Concentrations of keto-acids as a function of age.


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  		Table 5. Comparison of serum/plasma concentrations of keto-acids in adults obtained by the present HPLC method and other methods.

In conclusion, the technique described in this report, although it includes a derivatization step, does not require long and complicated preparation of the samples. Chromatographic separation of four keto-acids is performed at a high flow rate of 1.6 mL/min at 50 °C, affording good separation of the peaks, good recoveries, and shorter retention times than other techniques described previously. With this method, 20 sera can be prepared (~75 min) and analyzed (12 min per sample) in <6 h. This technique, linear up to 3 mmol/L, enables the determination of widely different quantities of keto-acids, as observed for patients with metabolic disorders or suffering from various diseases (e.g., urea-cycle enzyme defects, uremia, hepatic encephalopathy). This study also showed that elderly institutionalized people have lower concentrations of BCKAs than younger adults, but similar concentrations of pyruvate. In view of the binding between BCKAs and albumin, it seems important to take the serum albumin concentration into consideration for the interpretation of BCKA assays in the elderly. A complementary study with noninstitutionalized elderly people needs to be performed to evaluate the effect of age on BCKA concentrations.


   Footnotes
 
1 Nonstandard abbreviations: BCKA, branched-chain keto-acid; KIC, {alpha}-ketoisocaproate; KIV, {alpha}-ketoisovalerate; KMV, {alpha}-keto-ß-methylvalerate; PYR, pyruvate; KV, ketovalerate; OPD, o-phenylenediamine; and IS, internal standard.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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