Biomedical subjects

J K Kelleher

Publications and source records attributed to J K Kelleher.

At least 19 recordsLinked to original sources

PubMed2002

Isotopomer spectral analysis of intermediates of cholesterol synthesis in human subjects and hepatic cells.

Steroid intermediates of the cholesterol synthesis pathway are characterized by rapid turnover rates relative to cholesterol due to their small pool size. Because the small pools will label rapidly, these intermediates may provide valuable information about the incorporation of isotopes in de novo synthesis of cholesterol and related compounds. The labeling of cholesterol synthesis intermediates from [1-(13)C]acetate was investigated in human subjects and in liver cell models by means of isotopomer spectral analysis (ISA). In human subjects, infusing [1-(13)C]acetate into the duodenum for 12 h demonstrated that approximately 50% of the plasma lathosterol pool was derived from de novo synthesis during this interval. The lipogenic acetyl-CoA precursor pool enrichment reached a constant value within 3 h of the start of the infusion. In vitro studies indicated that liver cell models decrease de novo lathosterol synthesis when cholesterol synthesis is inhibited by statins or cholesterol-containing serum. We propose a new calculation to increase the accuracy and precision of cholesterol synthesis estimates in vivo combining the ISA of lathosterol and cholesterol.

Acetates↗

PubMed2001

Flux estimation using isotopic tracers: common ground for metabolic physiology and metabolic engineering.

Metabolic physiologists and metabolic engineers share the need to estimate flux. However, the physiologist often works with systems that do not maintain steady state for long. Many sites cannot be sampled, and calculating mass and isotopic balance for the entire system may not be feasible. To deal with these constraints, metabolic physiologists have developed specialized isotopic techniques that may be unfamiliar to metabolic engineers. A selection of these techniques is presented here, not because it is anticipated that they would be used by engineers exactly as in the physiologist's setting, but because they illustrate novel applications of tracer methodology. Creative engineers may find new adaptations of these tools in metabolic engineering and opportunities to increase redundancy. Physiologists, entering into a dialog with engineers, may see more clearly the potential of comprehensive models and revisit the impediments to a more complete analysis of human metabolic systems.

Animals↗

PubMed2001

Progestins block cholesterol synthesis to produce meiosis-activating sterols.

The resumption of meiosis is regulated by meiosis-preventing and meiosis-activating substances in testes and ovaries. Certain C29 precursors of cholesterol are present at elevated levels in gonadal tissue, but the mechanism by which these meiosis-activating sterols (MAS) accumulate has remained an unresolved question. Here we report that progestins alter cholesterol synthesis in HepG2 cells and rat testes to increase levels of major MAS (FF-MAS and T-MAS). These C29 sterols accumulated as a result of inhibition of Delta24-reduction and 4alpha-demethylation. Progesterone, pregnenolone, and 17alpha-OH-pregnenolone were potent inhibitors of Delta24-reduction in an in vitro cell assay and led to the accumulation of desmosterol, a Delta5,24 sterol precursor of cholesterol. A markedly different effect was observed for 17alpha-OH-progesterone, which caused the accumulation of sterols associated with inhibition of 4alpha-demethylation. The flux of 13C-acetate into lathosterol and cholesterol was decreased by progestins as measured by isotopomer spectral analysis, whereas newly synthesized MAS accumulated. The combined evidence that MAS concentrations can be regulated by physiological levels of progestins and their specific combination provides a plausible explanation for the elevated concentration of MAS in gonads and suggests a new role for progestins in fertility.

Animals↗

PubMed2001

Neutral sterols of rat epididymis. High concentrations of dehydrocholesterols in rat caput epididymidis.

Phospholipids and sterols are known to have multiple functions in reproductive tissue of mammals. High concentrations of the cholesterol precursor desmosterol have been described in testis, epididymis, and spermatozoa of various species. These findings and the recent discovery of some cholesterol precursors as meiosis-activating sterols suggest important functions of cholesterol precursors in fertility. Many sterol intermediates appear from the 19-step conversion of lanosterol, the first sterol synthesized in the cascade of cholesterol synthesis, to cholesterol. The biochemical basis of the genetically inherited Smith-Lemli-Opitz syndrome has been described as a defective conversion of 7-dehydrocholesterol to cholesterol. Since this discovery, interest has focused on this special cholesterol precursor. Here, we report high concentrations of 7- and 8-dehydrocholesterol in caput epididymidis and spermatozoa derived from caput epididymidis of Sprague-Dawley and Wistar rats, which comprised up to 30% of total sterols. In contrast to caput epididymidis, 7- and 8-dehydrocholesterol were barely detected in cauda epididymidis or testis. Desmosterol increased several times from caput to cauda epididymidis. This is the first report of the natural appearance of high concentrations of dehydrocholesterols in mammalian tissue, and it underlines the putative importance of cholesterol precursors in reproductive tissue.

Animals↗

PubMed2000

Effect of dietary protein quality and fatty acid composition on plasma lipoprotein concentrations and hepatic triglyceride fatty acid synthesis in obese cats undergoing rapid weight loss.

OBJECTIVE: To determine effects of dietary lipid and protein on plasma lipoprotein and free fatty acid concentrations and hepatic fatty acid synthesis during weight gain and rapid weight loss in cats. ANIMALS: 24 ovariohysterectomized cats. PROCEDURE: Cats were fed a high energy diet until they gained 30% of their ideal body weight and then randomly assigned to receive 1 of 4 weight reduction diets (6 cats/diet) at 25% of maintenance energy requirements. Diets contained a low or high quality protein source and a lipid source deficient or sufficient in long chain essential fatty acids. Plasma samples and liver biopsy specimens were obtained before and after weight gain and during and after weight loss for determination of free fatty acid, triglyceride, and lipoprotein concentrations. Synthesis of these substances was measured by use of isotope enrichment. RESULTS: Plasma total cholesterol concentration and concentration of lipoprotein fractions increased after weight gain, compared with baseline values. Weight loss resulted in a significant decrease in concentrations of all lipoprotein fractions except high density lipoprotein. High density lipoprotein concentration was significantly greater in cats fed diets containing an oil blend, compared with cats fed diets containing corn oil. Fatty acid synthesis after weight loss was below the detection limit of the measurement technique. CONCLUSIONS AND CLINICAL RELEVANCE: In cats undergoing rapid weight loss there is neither increased triglyceride synthesis nor decreased transport of very low density lipoproteins from the liver, suggesting that their involvement in the development of hepatic lipidosis may be minimal.

Animal Nutritional Physiological Phenomena↗

PubMed1999

Estimating gluconeogenesis with [U-13C]glucose: molecular condensation requires a molecular approach.

Recently three equations for estimating gluconeogenesis in vivo have been proposed, two by J. A. Tayek and J. Katz [Am. J. Physiol. 270 (Endocrinol. Metab. 33): E709-E717, 1996, and Am. J. Physiol. 272 (Endocrinol. Metab. 35): E476-E484, 1997] and one by B. R. Landau, J. Wahren, K. Ekberg, S. F. Previs, D. Yang, and H. Brunengraber [Am. J. Physiol. 274 (Endocrinol. Metab. 37): E954-E961, 1998]. Both groups estimate gluconeogenesis from cycling of [U-13C]glucose to lactate and back to glucose, detected by mass spectrometry. Landau's approach is based on analysis of labeled molecules, whereas Tayek and Katz's is based on labeling of carbon atoms by use of the concept of "molar enrichment," which weights each mass isotopomer by the number of labeled carbons. We derived an equation very similar to Landau's using binomial probability. Our analysis demonstrates that the molecular-based approach is correct. Additionally, equations appropriate for 14C studies are not appropriate for 13C studies, because the method used to detect 14C, decay of atoms, differs from 13C mass isotopomers detected as labeled molecules. We conclude that the molar enrichment carbon-based approach is not useful in the derivation of equations for the polymerization of molecules detected by mass spectrometry of molecules, and we confirm the findings of Landau et al.

Carbon Isotopes↗

PubMed1998

Effect of tamoxifen on cholesterol synthesis in HepG2 cells and cultured rat hepatocytes.

The objective of this study was to investigate the mechanisms by which tamoxifen modifies cholesterol metabolism in cellular models of liver metabolism, HepG2 cells and rat hepatocytes. The effect of tamoxifen on cholesterol and triglyceride-palmitate synthesis was measured using isotopomer spectral analysis (ISA) and gas chromatography-mass spectrometry (GC-MS) and compared with the effects of progesterone, estradiol, the antiestrogen ICI 182,780, and an oxysterol, 25-hydroxycholesterol (25OHC). Cholesterol synthesis in cells incubated in the presence of either [1-(13)C]acetate, [U-13C]glucose, or [4,5-(13)C]mevalonate for 48 hours was reduced in the presence of 10 micromol/L tamoxifen and 12.4 micromol/L 25OHC in both HepG2 cells and rat hepatocytes. The ISA methodology allowed a clear distinction between effects on synthesis and effects on precursor enrichment, and indicated that these compounds did not affect enrichment of the precursors of squalene. Progesterone was effective in both cell types at 30 micromol/L and only in HepG2 cells at 10 micromol/L. Estradiol and ICI 182,780 at 10 micromol/L did not inhibit cholesterol synthesis. None of the compounds altered the synthesis of triglyceride-palmitate in either cell type. Treatment of cells with tamoxifen produced accumulation of three sterol precursors of cholesterol, zymosterol, desmosterol, and delta8 cholesterol. This pattern of precursors indicates inhibition of delta24,25 reduction in addition to the previously described inhibition of delta8 isomerase. We conclude that tamoxifen is an effective inhibitor of the conversion of lanosterol to cholesterol in cellular models at concentrations comparable to those present in the plasma of tamoxifen-treated individuals. Our findings indicate that this mechanism may contribute to the effect of tamoxifen in reducing plasma cholesterol in humans.

Acetates↗

PubMed1998

Role of acetyl-L-carnitine in rat brain lipogenesis: implications for polyunsaturated fatty acid biosynthesis.

This study was undertaken to explore the metabolic fate of acetyl-L-carnitine in rat brain. To measure the flux of carbon atoms into anabolic processes occurring at regional levels, we have injected [1-(14)C]acetyl-L-carnitine into the lateral brain ventricle of conscious rats. After injection of [1-(14)C]acetyl-L-carnitine, the majority of radioactivity was recovered as 14CO2 expired (60% of that injected). The percentage of radioactivity recovered in brain was 1.95, 1.60, 1.30, and 0.93% at 1, 3, 6, and 22 h, respectively. Radioactivity distribution in various lipid components indicated that the fatty acid moiety of phospholipid contained the majority of radioactivity. The radioactive profile of these fatty acids showed that the acetyl moiety of acetyl-L-carnitine was incorporated into saturated (60%), monounsaturated (15%), and polyunsaturated (25%) fatty acids [mainly present in 20:4 (5.2%) and 22:6 (7.8%)]. Injection in the brain ventricle of radioactive glucose, the major source of acetyl-CoA in the CNS, revealed that glucose was a precursor of saturated (85%) and monounsaturated (15%) but not of polyunsaturated fatty acids. Thus, this study demonstrated distinct fates of glucose and acetyl-L-carnitine following intracerebroventricular injection. In summary, these data implicate acetyl-L-carnitine as an important member of a complex acetate trafficking system in brain lipid metabolism.

Acetylcarnitine↗

PubMed1997

Applications of mass isotopomer analysis to nutrition research.

Investigations into regulating metabolic pathways with stable isotopes have, over the past decade, undergone major development with the use of nuclear magnetic resonance and mass spectrometry in studying labeling patterns of newly synthesized biomolecules. In this review, we concentrate on investigations of mass isotopomer distribution (MID) measured by mass spectrometry. We review the applications of MID to analytical problems, in particular the possibility of amplifying the measurement of low isotopic enrichments by incorporating multiple molecules or atoms of a primary analyte into the molecule of a secondary analyte, the MID of which is assayed. We also review new information on the regulation of intermediary metabolism gathered from the analysis of MID patterns of synthesized compounds. Lastly, we review the applications of MID to the synthesis of polymeric molecules, with emphasis on the validity of these techniques. A number of these techniques are applicable to investigations of nutrient metabolism in health and disease.

Humans↗

PubMed1997

Quantitative analysis of acetoacetate metabolism in AS-30D hepatoma cells with 13C and 14C isotopic techniques.

Experimental hepatoma cells utilize acetoacetate as an oxidative energy source and as a precursor for lipid synthesis. The significance of ketone body metabolism in tumors lies in the study of tumor-host metabolism and the ketoneMic condition that is often present in cancer patients. The quantitative importance of acetoacetate and glucose was investigated in AS-30D cells with use of 13C and 14C isotopic methods. In addition, the effects of acetoacetate were compared with those of dichloroacetic acid (DCA), an activator of pyruvate dehydrogenase (PDH). The 14CO2 ratio method evaluated the entry of pyruvate into the tricarboxylic acid (TCA) cycle and revealed that acetoacetate diverted pyruvate from PDH to pyruvate carboxylation. In contrast, DCA increased the oxidation of glucose largely through PDH, indicating that PDH is not maximally active in the absence of DCA. Isotopomer spectral analysis of lipid synthesis demonstrated that, in the absence of acetoacetate, glucose supplied 65% of the acetyl-CoA used for de novo lipogenesis. When 5 mM acetoacetate was included in the incubation, glucose was displaced as a lipogenic precursor and acetoacetate supplied 85% of the acetyl-CoA for lipogenesis vs. only 2% for glucose. Thus AS-30D cells have a large capacity for acetoacetate utilization for de novo lipogenesis.

Acetoacetates↗

PubMed1997

Acetyl-L-carnitine flux to lipids in cells estimated using isotopomer spectral analysis.

Acetyl-L-carnitine is known as a reservoir of activated acetyl units and as a modulator of metabolic function. The objective of this study was to quantify the fate of the acetyl moiety of acetyl-L-carnitine in lipogenic pathways. Lipogenesis was studied in an adipocyte model, differentiated 3T3-L1 cells, and a hepatoma cell, HepG2 cells. Lipogenesis and ketogenesis were examined in rat hepatocytes. Both de novo synthesis and elongation of fatty acids were investigated using gas chromatography/mass spectrometry and [1,2-(13)C]acetyl-L-carnitine. Comparisons were performed with [13C]glucose and [13C]acetate. Isotopomer Spectral Analysis, a stable isotope method for differentiating between the enrichment of the precursor and the amount of synthesis was used to analyze the data. Acetyl-L-carnitine was generally less effective than acetate as a precursor for de novo lipogenesis. The effects of acetyl-L-carnitine were not identical to those of acetate plus carnitine as expected if acetyl-L-carnitine flux to acetyl CoA is controlled by carnitine acetyl transferase. Acetyl-L-carnitine (2 mM) contributed approximately 10% of the lipogenic acetyl-CoA used for synthesis and elongation as well as 6% of the ketogenic acetyl-CoA. No differences were found between the precursor enrichment for de novo lipogenesis and for elongation of saturated fatty acids. Flux of acetyl-L-carnitine to lipid was increased, not decreased, by the ATP citrate lyase inhibitor, -hydroxycitrate. In contrast, flux of glucose to lipid was dramatically decreased by this inhibitor. These results indicate that flux of acetyl-L-carnitine to lipid can bypass citrate and utilize cytosolic acetyl-CoA synthesis.

3T3 Cells↗

PubMed1996

Monte Carlo spreadsheet modeling of stable isotope biosynthesis.

Metabolic physiologists often introduce stable isotopes, atoms containing additional neutrons, into molecules during biosynthesis. This tags the newly synthesized material by altering its mass. Monte Carlo analysis is implemented on a popular spreadsheet to analyze this process. An example is provided where acetoacetate is synthesized by condensation of two acetate moieties. The precursor acetate is present as a mixture of natural, and 13C enriched, acetate. Monte Carlo spreadsheet modeling captures the complexity of the multi-species isotope biosynthesis by repetitively performing multiple simultaneous Boolean calculations. The effects of increasing the number of molecules synthesized on the goodness of fit of between model and an exact analytical solution is illustrated.

Acetoacetates↗

PubMed1995

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation.

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of 14C-glutamine to 14CO2 and of 14C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle alpha-ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label alpha-ketoglutarate carbon 5, [2-14C]glucose, [1-14C]acetate and [5-14C]glutamine. The analysis indicated that the probability of flux of TCA cycle alpha-ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of alpha-ketoglutarate through alpha-ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [14C]glutamine to 14CO2, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through alpha-ketoglutrate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of alpha-ketoglutarate was demonstrated by measuring incorporation of [5-14C]glutamine into 14C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.

Animals↗

PubMed1994

Acetoacetate metabolism in AS-30D hepatoma cells.

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetyl CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells. The major findings were: 1. Acetoacetate was utilized by AS-30D cells, with 14C-lipid and 14CO2 as major products of [3-14C] acetoacetate. 2. Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium. 3. Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate. 4. Succinyl CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria. 5. Addition of acetoacetate, but not beta-hydroxybutyrate decreased conversion of [1-14C] acetate to 14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer. 6. In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetoacetates↗

PubMed1994

Isotopomer spectral analysis of cholesterol synthesis: applications in human hepatoma cells.

Cholesterol synthesis from 13C-labeled precursors produces a discrete spectrum of mass isotopomers detectable using gas chromatography-mass spectrometry. The isotopomer spectral analysis (ISA) method matches the observed spectrum of cholesterol isotopomers with a mathematical model to obtain the best fit of model spectrum to data spectrum. The model was based on multinomial probability expressions that simulate cholesterol synthesis as a condensation of mevalonate fragments. As many as four unknown parameters, representing fluxes between compartments, were included in the model. Models were developed to assess cholesterol synthesis from 13C-enriched precursors including mevalonate, acetate, acetoacetate or octanoate. Models were tested in the human hepatoma cell line, Hep G2, which readily incorporated the 13C substrates into cholesterol. The ISA approach was used to estimate the fractional amount of the cholesterol precursors derived from the 13C substrate and the fraction of total cellular cholesterol synthesized in the presence of the 13C substrate. The study demonstrated the feasibility of the ISA approach for a condensation biosynthesis that is not a simple polymerization and for models with more than two unknown parameters.

Acyl Coenzyme A↗

PubMed1992

Model equations for condensation biosynthesis using stable isotopes and radioisotopes.

Important syntheses in living systems occur by condensation reactions of the type nA----1B (where n is the number of A molecules needed to synthesize 1 molecule of B). Quantitative relationships for estimating the rate of synthesis of B from radioactive and stable isotope tracers are compared. With radioisotope tracers, only a single quantity is detected, the amount of radioactivity in B. In contrast, isotopes of varying mass produce multiple mass isotopomers B that are detected using mass spectrometry. The analysis demonstrates that the rate of synthesis of B is identifiable from stable isotope data but not from radioisotope data. This results because the isotopomer distribution of B at any time after tracer addition is a function of only the multinomial distribution representing the synthesis of B from n molecules of A and two parameters representing the fractional fluxes of isotopically enriched molecules to the sampled compartment of B. The model considers the possibility that the sampled compartment of B may not reach isotopic steady state during the experiment. A graphical method for obtaining initial estimates of the two parameters is presented.

Acetates↗

PubMed1992

Isotopomer spectral analysis of triglyceride fatty acid synthesis in 3T3-L1 cells.

A new analysis of stable isotope data for biosynthesis reaction, isotopomer spectral analysis (ISA), is demonstrated. ISA is theoretically applicable for polymerization biosynthesis where data are collected using selected ion-monitoring gas chromatography-mass spectrometry. ISA utilizes the discrete spectrum of isotopomer abundances and the multinomial distribution to estimate two key parameters related to the biosynthesis. These parameters are 1) the dilution of the precursor immediately before biosynthesis and 2) the dilution of the newly synthesized product in the sampled compartment. Differentiated 3T3-L1 cells incorporated 2 mM [1,2-13C]acetate into triglyceride palmitate, yielding a spectrum of mass isotopomers of palmitate. The set of equations for the first nine isotopomers were solved for the two parameters using nonlinear regression. We found that precursor dilutions for acetate and glucose were constant over time, whereas the product dilution parameter increased with time, as expected for cells accumulating triglyceride palmitate. Mathematical procedures are presented for calculating 1) the predicted isotopomer fractional abundance values and 2) the correction for atoms other than the tracer atom in the mass ion.

3T3 Cells↗

PubMed1990

Submicromolar Ca2+ regulates phosphorylating respiration by normal rat liver and AS-30D hepatoma mitochondria by different mechanisms.

The stimulation of 2-oxoglutarate and NAD(+)-isocitrate dehydrogenase by Ca2+ in mitochondria from normal tissues has been proposed to mediate partially the activation of oxidative energy metabolism elicited by physiological elevations in cytosolic Ca2+. This mode of regulation may also occur in tumor cells in which several aspects of mitochondrial metabolism are known to be altered. This study provides a comparison of the stimulation by submicromolar concentrations of Ca2+ on the rates of ATP-generating (state 3) respiration under physiologically realistic conditions by mitochondria isolated from normal rat liver and from highly malignant rat AS-30D ascites hepatoma cells. The K0.5 for activation of glutamate-dependent state 3 respiration by Ca2+ in the presence of ATP at 37 degrees C was determined to be 0.70 +/- 0.05 (S.E.) microM for hepatoma mitochondria and 0.90 +/- 0.03 microM for rat liver mitochondria. This activation was also reflected by a Ca2(+)-induced shift in the oxidation-reduction state of hepatoma mitochondrial pyridine nucleotides to a more reduced level and Ca2+ stimulation of 14CO2 production from [1-14C]glutamate. Whereas the Ca2+ sensitivity of state 3 respiration by hepatoma mitochondria can be explained by the activation of 2-oxoglutarate and possibly NAD(+)-isocitrate dehydrogenases, the Ca2+ sensitivity of liver mitochondrial respiration appears to be predominantly mediated by activation of electron flow through ubiquinone and Complex III of the electron transport chain, as indicated by the specificity of the effects of Ca2+ on respiration with different oxidizable substrates. Although rat liver and hepatoma mitochondria employ different modes of Ca2(+)-activated ATP generation, these results support the hypothesis that changes in cytosolic Ca2+ play a significant role in the potentiation of energy production in tumor, as well as normal tissue.

Animals↗