Energy Metabolism in Rat Brain: Inhibition of Pyruvate Decarboxylation by 3-Hydroxybutyrate in Neonatal Mitochondria

Abstract: The effect of 3-hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1 -¹⁴C]pyruvate decarboxylation (1 mm final concentration) by brain synaptosomes derived from 8-day-old rats was inhibited by 10% in the presence of 2 mm -d ,l -3-hydroxybutyrate and by more than 20% in the presence of 20 mm -d ,l -3-hydroxybutyrate. The presence of 2 mm -l ,d -3-hydroxybutyrate did not affect the rate of [1-¹⁴T]pyruvate decarboxylation (1 mm final concentration) by brain mitochondria; however, at a concentration of 20 mm -d ,l -3-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-day-old (35% inhibition) and 21-day-old (24% inhibition) but not in those from adult rats. Although the presence of 100 mm -K⁺ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in the presence of 1 mm -K⁺, the presence of 20 mm -d ,l -3-hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mm -K⁺). The presence of 20 mm -d ,l -3-hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8-day-old rats. The concentrations of ATP, ADP, NAD⁺, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8-day-old rats incubated in the presence of 1 mm -pyruvate alone or 1 mm -pyruvate plus 20 mm -d ,l -3-hydroxybutyrate. Neither the ATP/ADP nor the NADH/NAD⁺ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3-hydroxybutyrate. The possible mechanisms and physiological significance of 3-hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain mitochondria are discussed.     

A spectrophotometric, enzymatic assay for D-3-hydroxybutyrate that is not dependent on hydrazine

Because of the potential carcinogenic properties of hydrazine and because of other health hazards associated with its use in the laboratory, an enzymatic assay has been developed for D-3-hydroxybutyrate that is not dependent on hydrazine to drive the reaction toward completion. The use of a high concentration of NAD+ and a buffer at pH 9.5 resulted in a favorable conversion of D-3-hydroxybutyrate to acetoacetate by D-3-hydroxybutyrate dehydrogenase even though the reaction favors D-3-hydroxybutyrate formation under physiological conditions. The assay was also completed faster than previous assays using hydrazine so that the amount of enzyme used for the assay could be reduced. The recovery of D-3-hydroxybutyrate added to liver samples was 98 +/- 1% (mean +/- SEM, n = 6). The assay was found to be suitable for the measurement of D-3-hydroxybutyrate in samples such as perchloric acid extracts of isolated hepatocytes even when the acetoacetate to D-3-hydroxybutyrate ratio was 4 to 1. This assay presents a reliable alternative to the use of hydrazine and may be used for the assay of D-3-hydroxybutyrate in a variety of physiological and experimental samples.     

β-Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine

Abstract— Rat brain cortex slices were incubated with 10 mm -glucose and trace amounts of [6-³H]glucose and [3-¹⁴C]β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and ³H:¹⁴C ratios calculated. Incorporation of [¹⁴C]β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of ¹⁴C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [³H-,¹⁴C]-glucose. (–)-Hydroxycitrate (5.0 m^M) reduced incorporation of [³H]glucose and [¹⁴C]β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm ) showed a decrease in ¹⁴C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm ) also produced a decreased ¹⁴C incorporation into ACh and its precursors. At 10 mm , acetoacetate reduced ³H and ¹⁴C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.     

Effects of diabetes and insulin on ketone bodies metabolism in heart

This work investigates the effect of alloxan-induced short-term diabetes (24 h) on D-3-hydroxybutyrate metabolism at physiological and non-physiological concentrations of the ketone body in the isolated non-working perfused rat heart. Also the effect of insulin (2 mU.ml⁻¹) on D-3-hydroxybutyrate metabolism was investigated in hearts from normal and diabetic rats. The rates of D-3-hydroxybutyrate utilization and oxidation and of acetoacetate production were proportional to D-3-hydroxybutyrate concentration. The utilization of D-3-hydroxybutyrate showed saturation kinetics in hearts from normal and diabetic rats, in the presence and absence of insulin. Acute short-term diabetes augmented D-3-hydroxybutyrate utilization and oxidation at 1.25 and 2.5 mM DL-3-HB, with no significant effect at higher concentrations, but increased acetoacetate production at all investigated concentrations. In hearts from normal rats, insulin enhanced D-3-hydroxybutyrate utilization and oxidation at 2.5, 5, and 10 mM DL-3-HB, but no effect was observed at the lowest (1.25 mM) and highest (16 mM) DL-3-HB concentrations. Insulin had no effect on D-3-hydroxybutyrate metabolism in hearts from diabetic rats. No significant effect of insulin on the rate of acetoacetate production in normal and diabetic states was observed.     

Competitive protein-binding radioassay of 24,25-dihydroxyvitamin D in sera from normal and anephric subjects

A competitive protein-binding radioassay for 24,25-dihydroxyvitamin D [24,25-(OH)₂D] in human serum has been developed. Whereas small amounts of [³H]24,25-(OH)₂D must be biosynthesized in order to trace the efficiency of the extraction and chromatographic procedures, tritiated 25-hydroxyvitamin D₃ ([³H]25-OHD₃) can be used as the assay tracer. Since 25-OHD₃ and 24,25-(OH)₂D₃ are equipotent in their competitive displacement of [³H]25-OHD₃ from rat serum, 25-OHD₃ can be used as the assay standard. Liquid-gel partition chromatography on small columns of Sephadex LH-20 can reliably isolate 24,25-(OH)₂D by batch elution. The purity of biosynthesized [³H]24,25-(OH)₂D₃ and the 24,25-(OH)₂D fraction isolated from serum was confirmed by high-pressure chromatography on 0.2 × 50 cm columns of 10-μm silica. Serum 24,25-(OH)₂D levels averaged 16% of the serum 25-OHD concentrations in normal subjects. Since chronic hemodialysis patients, without kidneys, had normal serum 24,25-(OH)₂D levels, significant extrarenal 25-hydroxycalciferol 24-hydroxylase activity occurs in these subjects. Since the present assay represents a reasonably simple extension of 25-OHD assay methodology, it should prove to be a useful technique in the analysis of clinical disorders of vitamin D metabolism.     

Estimation of the Number of Polyhydroxyalkanoate (PHA)-Degraders in Soil and Isolation of Degraders Based on the Method of Most Probable Number (MPN) Using PHA-Film

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 × 10⁵ and 2.15 × 10⁵ aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 × 10⁵ aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.     

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

(1) d(-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d- and l-lactate, n-butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD⁺ or NADH. With d-3-hydroxybutyrate as the variable reactant (K_{m app} = 0.26 mM) the inhibition constant of methylmalonate (K_is) was 0.09 mm. (3) The rate of utilization of d-3-hydroxybutyrate (78 μm) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μm methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n-octanoate in the absence of added ADP, methylmalonate (1–3 mm) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 mm) although the inhibition of total ketone formation from octanoate was more severe.     

Cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase from Acidovorax sp. strain SA1 and purification of the enzyme

The gene of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase (i3HBOH) was cloned and sequenced from a poly(3-hydroxybutyrate) (PHB)-degrading bacterium, Acidovorax sp. strain SA1. The i3HBOH gene has 876 nucleotides corresponding to the deduced sequence of 292 amino acids. In this amino acid sequence, the general lipase box sequence (G-X₁-S-X₂-G) was found, whose serine residue was determined to the active sites serine by site-directed mutagenesis. An i3HBOH was purified to electrophoretical homogeneity from SA1. The molecular mass of the purified enzyme was estimated to be 32 kDa by SDS-PAGE. The N-terminal amino acid sequence of the purified enzyme corresponded to the deduced N-terminal amino acid sequence in the cloned i3HBOH gene. This is the first cloning and sequencing of an intracellular D(-)-3-hydroxybutyrate oligomer hydrolase gene to date. Received: 19 October 2001 / Accepted: 7 December 2001     

Measurement of ketone bodies in subcellular fractions using a spectrophotometric iron-chelate assay

A sensitive spectrophotometric assay for 3-hydroxybutyrate determination in biological samples is described. Linearity between the amount of 3-hydroxybutyrate and ΔA₅₄₆ was obtained in the range of 0.3 to 4.0 nmol 3-hydroxybutyrate/assay. The same method is applicable for acetoacetate determination after its enzymatic reduction. The assay proved to be useful for the study of the subcellular distribution of ketone bodies in isolated liver cells. The assay procedure is adequate to measure the concentration of ketone bodies in 5-mg and 20μl samples from liver and blood, respectively.     

Biocatalytic production of (S)-4-bromo-3-hydroxybutyrate and structurally related chemicals and their applications

The enzymatic production of (S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with (S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum β-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of (S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of (S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds.     

Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-¹⁴C]d-(–)-hydroxybutyric acid, [1-¹⁴C]d-lactic acid, [1-¹⁴C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(–)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-¹⁴C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-¹⁴C]lactyl-CoA was used as a substrate at a rate that was only less than 10^–4 of the rate than with [3-¹⁴C]d-(–)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-¹⁴C]d-lactyl-CoA as substrate at a relatively high rate. Correspondence to: A. Steinbüchel     

Lipogenesis in the brain of suckling rats. Studies on the mechanism of mitochondrial–cytosolic carbon transfer

1. Studies on the incorporation of [3-¹⁴C]pyruvate and d-3-hydroxy[3-¹⁴C]butyrate into the brain lipid fraction by brain homogenates of the suckling (7-day-old) rat have been carried out. 2. Whereas approximately twice as much CO₂ was evolved from pyruvate compared with 3-hydroxybutyrate metabolism, similar amounts of the radioactivity of these two precursors were incorporated into the lipid fraction. Furthermore, in both cases the incorporation into lipid was almost tripled when glucose (10mm) or NADPH (2.5mm) was added to the incubation media. 3. If 5mm-(—)-hydroxycitrate, an ATP–citrate lyase inhibitor, was added to the incubation the incorporation of carbon from pyruvate was inhibited to 39% of the control and from 3-hydroxybutyrate to 73% of the control, whereas CO₂ production from both precursors was not affected. 4. The incorporation from pyruvate or 3-hydroxybutyrate into lipids was not affected by the presence of 10mm-glutamate in the medium (to encourage N-acetylaspartate production). However, incorporation from pyruvate was inhibited by 21% in the presence of 5mm-amino-oxyacetate (a transaminase inhibitor) and by 83% in the presence of both hydroxycitrate (5mm) and amino-oxyacetate. 5. Incorporation from 3-hydroxybutyrate into brain lipids was inhibited by 20% by amino-oxyacetate alone, but by 55% in the presence of both hydroxycitrate and amino-oxyacetate. 6. It is concluded that the mechanism of carbon transfer from pyruvate into lipids across the mitochondrial membrane in the suckling rat brain is mainly via citrate and N-acetylaspartate. 3-Hydroxybutyrate, in addition to using these routes, may also be incorporated via acetoacetate formation and transport to the cytosol.     

Validity of DNA analysis to determine cell numbers in tissue engineering scaffolds

The potential of a DNA content assay, PicoGreen, for use in 3D bioengineered constructs was examined. The assay was tested on ATDC5 cells in situ during culture in typical tissue engineering 3D constructs. Comparisons of cell standards from cell lines and primary cells to λDNA standards was also conducted. An effective working range of the assay within 3D constructs was shown up to 2.5 × 10⁵ cells ml⁻¹. From significant variation found in DNA content between cell lines and primary cells, it was concluded that the most accurate standard to use for the assay was from the cell type being examined.     

A novel radiochemical method for the microdetermination of total and individual ketone bodies in biological materials

A novel radiochemical method has been developed for ultramicrodetermination of acetone based on the principle that ¹²⁵I-labeled iodoform is produced by iodination of acetone with ¹²⁵ICl. [¹²⁵I]Iodoform is readily counted as a measure of acetone after separation from unreacted iodide ions. Quantitative conversion of 3-hydroxybutyrate to acetoacetate takes place when NAD-dependent oxidation of 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase is coupled with NADH-dependent reduction of pyruvate (or 2-oxoglutarate) by lactic dehydrogenase (or glutamic dehydrogenase). Acetoacetate thus formed produces acetone spontaneously when the acidified (deproteinized) reaction mixture is maintained at 50°C for 2 hr. Thus, total and individual ketone bodies in plasma are determined conveniently by combining the radiochemical determination of acetone with these conversion procedures.     

Assay of 3-hydroxybutyrate in the picomolar range

A radioisotopic procedure for the assay of 3-hydroxybutyrate is presented. It is based on the measurement of NADH, generated in the 3-hydroxybutyrate dehydrogenase reaction, through the conversion of 2-[U-14C]ketoglutarate to 14C-labeled L-glutamate in the presence of beef liver glutamate dehydrogenase. The assay is linear in the range of 2.5 to 20.0 pmole/sample and about 100-times more sensitive than previous methods. The procedure proved useful for the measurement of 3-hydroxybutyrate in liver samples not exceeding 25 micrograms wet weight.     

Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans

Summary Recombinant strains of Pseudomonas oleovorans, which harbour the poly(3-hydroxybutyrate)-biosynthetic genes of Alcaligenes eutrophus, accumulated poly(hydroxyalkanoates), composed of 3-hydroxybutyrate(3HB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyactanoate (3HO), up to 70% of the cell dry weight if the cells were cultivated with sodium octanoate as the carbon source. Physiological and chemical analysis revealed multiple evidence that this polymer is a blend of the homopolyester poly(3HB) and of the copolyester poly(3HHx-co-3HO) rather than a random or a block copolyester of 3HB, 3HHx and 3HO. The molar ratio between poly(3HHx-co-3HO) and poly(3HB) varied drastically during the process of fermentation. Whereas synthesis of poly(3HHx-co-3HO) started immediately after ammonium was exhausted in the medium, synthesis of poly(3HB) occurred only after a lag-phase. From freeze-dried cells poly(3HHx-co-3HO) was much more readily extracted with chloroform than was poly(3HB). The blend was fractionated into petrol-ether-insoluble poly(3HB) and petrol-ether-soluble poly(3HHx-co-3HO). The molecular weight values of these polyesters measured by gel permeation chromatography were 2.96 × 10⁶ and 0.35 × 10⁶ and were similar of those polymers accumulated by A. eutrophus or by wild-type P. oleovorans, respectively. Offprint requests to: A. Steinbüchel     

Preparation and characterization of monoclonal antibodies against adenylate kinase

Six hybridoma cell lines that can continuously secrete monoclonal antibodies against adenylate kinase (AK) have been produced. The characteristics including the subclass and molecular weight of monoclonal antibodies manufactured by these strains are also determined. Further studies show that the two monoclonal antibodies McAb3D3 and McAMD8 bind easily with AK absorbed on microtitration plates, with affinity constants of 8.4 × 108 M-1 and 9.6 × 108 M-1, while their interactions to AK in solution are much weaker, with affinity constants of 7.0 × 104 M-1 and 3.9×106M-1, respectively. Thus, McAb3D3 and McAMD8 react preferentially to the immobilized AKs. Since pro-teins are often partially denatured when absorbed on microtitration plates, it is suggested that both McAb3D3 and McAMD8 are directed against non-native AK.     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

Characterization of β-hydroxybutyrate transport in rat erythrocytes and thymocytes

A method was developed for study of β-hydroxybutyrate transport in erythrocytes and thymocytes. Critical to the method was a centrifugal separation of cells from medium which took advantage of β-hydroxybutyrate transport's temperature dependence and inhibition by phloretin and methylisobutylxanthine, all of which are demonstrated in this work. These properties suggested mediated transport, as did saturation kinetics and inhibition by several agents including pyruvate and α-cyanocinnamate. Most conclusive in this regard was a 2-fold preference for d- over l-β-hydroxybutyrate. Entry was not Na⁺ dependent. It was stimulated by substitution of SO₄^2? for most of the Cl^?. The equilibrium β-hydroxybutyrate space was much higher than the Cl^? space of thymocytes, suggesting that β-hydroxybutyrate entry is not associated with net inward negative current and is not coupled to outward Cl^? or inward K⁺ movement (assuming that K⁺ is at electrochemical equilibrium). Coupling to H⁺ entry or OH^? exit is compatible with the result. These findings are consistent with β-hydroxybutyrate entry by the carboxylate transport site which has been studied extensively with pyruvate and lactate as permeants. The Cl^?/HCO₃^? exchange carrier did not appear to contribute significantly to β-hydroxybutyrate transport.     

Quantitation of 1,3-butanediol and its acidic metabolites by gas chromatography-mass spectrometry

A number of problems present themselves during the gas chromatographic-mass spectrometric assay of R,S-1,3-butanediol as its bis-tert-butyldimethylsilyl ether. To circumvent these problems, three labeled internal standards were synthesized: (i) R,S-1,3-[3,4-13C2]-butanediol, (ii) R,S-1,3-[1,1,3-2H3]butanediol, and (iii) R,S-1,3-[1,1,3-2H3,3,4-13C2]butanediol. The availability of internal standards with different degrees of labeling allows (i) assaying of either unlabeled or 13C-labeled R,S-1,3-butanediol and (ii) analysis of 1,3-butanediol in either blood or urine samples. Reproducible standard curves were obtained using both electron impact and ammonia chemical ionization modes. The latter provides greater sensitivity and a lower limit of detection (5 microM). We have also designed an indirect assay of S-3-hydroxybutyrate, a catabolite of R,S-1,3-butanediol, which is difficult to analyze by conventional methods. This assay relies on the difference between (i) the concentration of R,S-3-hydroxybutyrate assayed by gas chromatography-mass spectrometry and (ii) the concentration of R-3-hydroxybutyrate assayed enzymatically.