Sylvain Desrochers

Interference of 3-hydroxyisobutyrate with measurements of ketone body concentration and isotopic enrichment by gas chromatography-mass spectrometry

Concentrations and 13C2 molar percentage enrichments of blood R-3-hydroxybutyrate and acetoacetate are measured by selected ion monitoring gas chromatography-mass spectrometry. Samples are treated with NaB2H4 to reduce unlabeled and labeled acetoacetate to corresponding deuterium-labeled RS-3-hydroxybutyrate species. Only the gas chromatographic peak for the tert-butyldimethylsilyl derivative of 3-hydroxybutyrate needs to be monitored. The various compounds are quantitated using an internal standard of RS-3-hydroxy-[2,2,3,4,4,4-2H6]-butyrate. Concentrations of ketone bodies are obtained by monitoring the m/z 159 to 163 fragments of tert-butyldimethylsilyl derivatives of labeled and unlabeled 3-hydroxybutyrate species. High correlations were obtained between ketone body concentrations assayed (i) enzymatically with R-3-hydroxybutyrate dehydrogenase and (ii) by gas chromatography-mass spectrometry. The limit of detection is about 10 nmol of substrate in blood samples. The current practice of monitoring the m/z 275 to 281 fragments overestimates the concentration of endogenous R-3-hydroxybutyrate, due to co-elution of 3-hydroxyisobutyrate, a valine metabolite. The method presented is used to measure ketone body turnover in vivo in 24-h-fasted dogs.

R,S-1,3-butanediol acetoacetate esters, potential alternates to lipid emulsions for total parenteral nutrition

Abstract We present the preparation and characterization of totally and partially water-soluble forms of fat which could replace emulsions of long-chain triacylglycerols for total parenteral nutrition. R,S-1,3-butanediol acetoacetate monoesters and diester represent pH-neutral, sodium-free, diffusible precursors of ketone bodies. The latter are water-soluble forms of fat that are well used by peripheral tissues except in prolonged starvation and diabetic ketoacidosis. The esters are rapidly hydrolyzed by plasma and tissue esterases. R,S-1,3-butanediol liberated is oxidized in liver to R,S-β-hydroxybutyrate. Reducing equivalents generated during this oxidation are trapped in the conversion of acetoacetate to R-β-hydroxybutyrate. So both the carbon and the hydrogen of the esters are exported from the liver to peripheral tissues in the form of R- + S-β-hydroxybutyrate. Thus, contrary to what occurs after administration of ethanol or R,S-1,3-butanediol alone, administration of the R,S-1,3-butanediol acetoacetate esters does not lead to major shifts in the livers [NADH] [NAD + ] ratio. Such shifts are responsible for the toxic effects of ethanol on the liver. It is therefore likely that long-term administration of the R,S-1,3-butanediol acetoacetate esters will not lead to liver toxicity.

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.