Henri Brunengraber

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.

Fructose as a carbohydrate source yields stable pH and redox parameters in microcarrier cell culture

Abstract The pH and cytosolic NADH/NAD+ redox potential in microcarrier cultures of Madin-Darby canine kidney cells remain within physiological range when fructose is substituted for glucose in medium formulation. This difference is accounted for by the low rate of lactic acid production in cultures utilizing fructose as a primary carbohydrate source.

Zonation of labeling of lipogenic acetyl-CoA across the liver: Implications for studies of lipogenesis by mass isotopomer analysis

Measurement of fractional lipogenesis by condensation polymerization methods assumes constant enrichment of lipogenic acetyl-CoA in all hepatocytes. mass isotopomer distribution analysis (MIDA) and isotopomer spectral analysis (ISA) represent such methods and are based on the combinatorial analyses of mass isotopomer distributions (MIDs) of fatty acids and sterols. We previously showed that the concentration and enrichment of [13C]acetate decrease markedly across the dog liver because of the simultaneous uptake and production of acetate. To test for zonation of the enrichment of lipogenic acetyl-CoA, conscious dogs, prefitted with transhepatic catheters, were infused with glucose and [1,2-13C2]acetate in a branch of the portal vein. Analyses of MIDs of fatty acids and sterols isolated from liver, bile, and plasma very low density lipoprotein by a variant of ISA designed to detect gradients in precursor enrichment revealed marked zonation of enrichment of lipogenic acetyl-CoA. As control experiments where no zonation of acetyl-CoA enrichment would be expected, isolated rat livers were perfused with 10 mm [1,2-13C2]acetate. The ISA analyses of MIDs of fatty acids and sterols from liver and bile still revealed a zonation of acetyl-CoA enrichment. We conclude that zonation of hepatic acetyl-CoA enrichment occurs under a variety of animal models and physiological conditions. Failure to consider gradients of precursor enrichment can lead to underestimations of fractional lipogenesis calculated from the mass isotopomer distributions. The degree of such underestimation was modeled in vitro, and the data are reported in the companion paper (Bederman, I. R., Kasumov, T., Reszko, A. E., David, F., Brunengraber, H., and Kelleher, J. K. (2004) J. Biol. Chem. 279, 43217-43226).

Fatty Acid, 3-β-Hydroxysterol, and Ketone Synthesis in the Perfused Rat Liver

The effects of oleate and hydroxycitrate on the rate of long-chain fatty acid and 3-beta-hydroxysterol synthesis were measured in perfused rat livers. Metabolite measurements show that in livers from fed animals inhibition of fatty acid synthesis by oleate or hydroxycitrate is associated with an increase in the tissue content of glucose 6-phosphate and fructose 6-phosphate, and a diminution in glycolytic intermediates from fructose diphosphate to phosphoenolpyruvate. Oleate also causes an increase in the tissue content of long-chain fatty acyl-CoA and citrate. The increase in long-chain fatty acyl-CoA is larger in livers from starved as compared to fed rats, while the increase in citrate is larger in livers from fed as compared to starved rats. However, the increase in the citrate content of livers from fed rats occurs in a range where it causes no further activation of acetyl-CoA carboxylase in vitro. Ketogenesis by livers from fed rats perfused without free fatty acids is strongly inhibited by hydroxycitrate. However, ketogenesis is not inhibited by hydroxycitrate when livers from starved rats are perfused with oleate, and ketogenesis is increased somewhat by hydroxycitrate when livers from fed rats are perfused with oleate. These results are interpreted in terms of an extramitochondrial pathway of ketogenesis which operates in carbohydrate-fed animals. The intramitochondrial pathway predominates in starved animals, or when the concentration of fatty acids is high, or both. Other interpretations, which cannot be ruled out at present, are also considered.

Metabolism of S-3-hydroxybutyrate in the perfused rat liver☆

The metabolism of millimolar concentrations of S-3-hydroxybutyrate (the unnatural enantiomer) has been studied in perfused livers from fed and starved rats. Protocols were designed to test whether S-3-hydroxybutyrate is metabolized in the cytosol or in the mitochondria via a racemase, a dehydrogenase, or a ligase. Our data show that only a minor fraction of S-3-hydroxybutyrate metabolism could occur via L-3-hydroxyacid dehydrogenase. Most of the metabolism of S-3-hydroxybutyrate proceeds via mitochondrial activation. In rat liver, S-3-hydroxybutyrate is converted to physiological ketone bodies (i.e., R-3-hydroxybutyrate, acetoacetate, acetone), lipids, and CO2. Carbons from S-3-hydroxybutyrate are transferred from the mitochondria to the cytosol mostly via citrate and the citrate cleavage pathway.

Contributions of Cytosolic and Mitochondrial Acetyl-CoA Syntheses to the Activation of Lipogenic Acetate in Rat Liver

Acetate derived from ethanol oxidation is activated by cytosolic and mitochondrial acetyl-CoA synthetases before contributing to the extra-mitochondrial processes of fatty acid and 3-beta-hydroxysterol synthesis. Mitochondrially-generated acetyl-CoA is transferred to the cytosol via citrate and ATP-citrate lyase; this transfer is blocked by (-)-hydroxycitrate. Rats were injected IV with 3.3 mmol/kg of [2-3H,2-14C] acetate and IP with either 0.5 mmol/kg hydroxycitrate or saline. After one hour, the rats were killed and the incorporation of label was measured in liver fatty acids and 3-beta-hydroxysterols. The 3H/14C ratio was increased by 12 and 13% in the fatty acids and 3-beta-hydroxysterols of the hydroxycitrate-treated group. The lower ratio in the fatty acids and 3-beta-hydroxysterols derived from mitochondrially-generated acetyl-CoA is ascribed to a loss of 3H in the citrate synthase reaction. The data showed that (1) fatty acids and 3-beta-hydroxysterols syntheses use the same pool of cytosolic acetyl-CoA; and (2) in the absence of an isotope effect in the citrate synthase reaction, mitochondrially-generated acetyl-CoA contributes about 36% to lipogenesis from acetate.

In vitro modeling of fatty acid synthesis under conditions simulating the zonation of lipogenic [13C]acetyl-CoA enrichment in the liver.

In the companion report (Bederman, I. R., Reszko, A. E., Kasumov, T., David, F., Wasserman, D. H., Kelleher, J. K., and Brunengraber, H. (2004) J. Biol. Chem. 279, 43207-43216), we demonstrated that, when the hepatic pool of lipogenic acetyl-CoA is labeled from [13C]acetate, the enrichment of this pool decreases across the liver lobule. In addition, estimates of fractional synthesis calculated by isotopomer spectral analysis (ISA), a nonlinear regression method, did not agree with a simpler algebraic two-isotopomer method. To evaluate differences between these methods, we simulated in vitro the synthesis of fatty acids under known gradients of precursor enrichment, and known values of fractional synthesis. First, we synthesized pentadecanoate from [U-13C3]propionyl-CoA and four gradients of [U-13C3]malonyl-CoA enrichment. Second, we pooled the fractions of each gradient. Third, we diluted each pool with pentadecanoate prepared from unlabeled malonyl-CoA to simulate the dilution of the newly synthesized compound by pre-existing fatty acids. This yielded a series of samples of pentadecanoate with known values of (i) lower and upper limits for the precursor enrichment, (ii) the shape of the gradient, and (iii) the fractional synthesis. At each step, the mass isotopomer distributions of the samples were analyzed by ISA and the two-isotopomer method to determine whether each method could correctly (i) detect gradients of precursor enrichment, (ii) estimate the gradient limits, and (iii) estimate the fractional synthesis. The two-isotopomer method did not identify gradients of precursor enrichment and underestimated fractional synthesis by up to 2-fold in the presence of gradients. ISA uses all mass isotopomers, correctly identified imposed gradients of precursor enrichment, and estimated the expected values of fractional synthesis within the constraints of the data.

Lipogenesis from ketone bodies in perfused livers from streptozocin-induced diabetic rats

Production of ketone bodies and their contribution to lipogenesis were measured in isolated livers from normal and streptozocin-induced diabetic (STZ-D) rats perfused with tracer amounts of 3H2O and (R)-3-hydroxy[3-14C]butyrate. Diabetes decreased by 80–95% the total rates of fatty acid and 3-β-hydroxysterol synthesis in perfused livers and livers of live rats. The activity of cytosolic acetoacetyl-CoA synthetase was slightly (17%) decreased in livers from STZ-D rats. The incorporation of ketone bodies into fatty acids and sterols was markedly inhibited in perfused livers from STZ-D rats despite the stimulation of ketogenesis by diabetes and the presence of oleate. Treatment of the rats with insulin before liver perfusion led to a normalization of the rates of ketogenesis and fatty acid synthesis. The rates of sterol synthesis were only partially normalized by insulin treatment. We conclude that in STZ-D, ketosis does not stimulate hepatic lipogenesis via cytosolic activation of acetoacetate.

Uptake of artificial model remnant lipoprotein emulsions by the perfused rat liver

In comparison with their precursor lipoproteins, the remnants of the triacylglycerol-rich lipoproteins are reduced in contents of triacylglycerols and apolipoproteins AI and AIV, whereas the contents of cholesterol (free and esterified) and apolipoprotein E are increased. In this study, lipid emulsion models of remnant lipoproteins were used to explore which of these factors are necessary for physiological rates of remnant uptake by the perfused rat liver. Uptake rates of lipid emulsion models of remnant lipoproteins in the presence of apolipoprotein E were similar to in vivo uptake rates.

[56] Use of the perfused liver for the study of lipogenesis

Publisher Summary It is common practice to perfuse organs with whole blood, diluted blood, or Krebs-Ringer bicarbonate buffer containing albumin and washed erythrocytes. In this type of perfusion, the total red cell mass usually exceeds the weight of the liver, and one is dealing with a model in which one must consider the metabolism of the erythrocytes and of the liver. The high glycolytic capacity of red cells imposes a redox buffer on the liver. Aged red cells that glycolyze at a slower rate than fresh cells are prone to hemolyze. It is neither desirable nor necessary to use red cells when one wishes to use the perfused liver as a model to study the regulation of fatty acid synthesis. The albumin is dialyzed before use as a 10% solution against 30 volumes of Krebs-Ringer bicarbonate buffer at 5°C with 6 changes of buffer for 48 hours. Dialysis removes various impurities from the albumin such as citrate that is used to prevent clotting of the blood from which the albumin is prepared.