Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phosphate in the yeast Saccharomyces cerevisiae.
In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611-7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate. (+info)
Effect of obesity on red cell mass results.
Measurement of red cell mass with isotope dilution remains an important diagnostic test in the evaluation of patients with suspected polycythemia vera (PCV). Results and reference ranges are typically expressed in units normalized for body weight (mL/kg). Obesity is common in polycythemic patients, and it is important to know how the various published normative ranges compare across a wide range of body weights. METHODS: We retrospectively reviewed 51 consecutive patients referred for red cell mass determination with 51Cr red blood cell dilution. Results were expressed in milliliters per kilogram (mL/kg) by using the actual patient weight and after adiposity adjustments using ideal body weight, body mass index (BMI) and combinations of height-weight, including body surface area. Results were classified as normal, elevated or PCV. RESULTS: There was a high prevalence of obesity in our population (28/51 [55%] with BMI > 27 kg/m2, BMI range 16.0-54.8 kg/m2). The method used to compensate for obesity had a dramatic effect on the derived red cell mass, the fraction of patients with elevated measurements and the fraction of patients meeting criteria for PCV. Concordance for categorization as normal, elevated or PCV by all methods was only 47.1%. CONCLUSION: Obesity is a common confounding factor in the interpretation of red cell mass measurements. Currently published reference ranges generate inconsistent results when extrapolated to obese patients. Further normative data on obese subjects are needed to determine which method (if any) is optimal. (+info)
Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis.
Vitamin C (L-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and L-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains approximately 25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes. (+info)
Effects of a high-fat diet and voluntary wheel running on gluconeogenesis and lipolysis in rats.
The purpose of the present study was to determine the effects of diet composition and exercise on glycerol and glucose appearance rate (Ra) and on nonglycerol gluconeogenesis (Gneo) in vivo. Male Wistar rats were fed a high-starch diet (St, 68% of energy as cornstarch, 12% corn oil) for a 2-wk baseline period and then were randomly assigned to one of four experimental groups: St (n = 7), high-fat (HF; 35% cornstarch, 45% corn oil; n = 8), St with free access to exercise wheels (StEx; n = 7), and HF with free access to exercise wheels (HFEx; n = 7). After 8 wk, glucose Ra when using [3-3H]glucose, glycerol Ra when using [2H5]glycerol (estimate of whole body lipolysis), and [3-13C]alanine incorporation into glucose (estimate of alanine Gneo) were determined. Body weight and fat pad mass were significantly (P < 0.05) decreased in exercise vs. sedentary animals only. The average amount of exercise was not significantly different between StEx (3,212 +/- 659 m/day) and HFEx (3,581 +/- 765 m/day). The ratio of glucose to alanine enrichment and absolute glycerol Ra (micromol/min) were higher (P < 0.05) in HF and HFEx compared with St and StEx rats. In separate experiments, the ratio of 3H in C-2 to C-6 of glucose from 3H2O (estimate of Gneo from pyruvate) was also higher (P < 0.05) in HF (n = 5) and HFEx (n = 5), compared with St (n = 5) and StEx (n = 5) rats. Voluntary wheel running did not significantly increase estimated alanine or pyruvate Gneo or absolute glycerol Ra. Voluntary wheel running increased (P < 0.05) glycerol Ra when normalized to fat pad mass. These data suggest that a high-fat diet can increase in vivo Gneo from precursors that pass through pyruvate. They also suggest that changes in the absolute rate of glycerol Ra may contribute to the high-fat diet-induced increase in Gneo. (+info)
Glucose effectiveness is the major determinant of intravenous glucose tolerance in the rat.
To determine the importance of insulin for glucose disposal during an intravenous glucose tolerance test in rats, experiments were performed in four cohorts of conscious unrestrained rats fasted overnight. In cohorts 1-3, a bolus of tracer ([3-3H]glucose, 50 microCi) was given alone, with glucose (0.3 g/kg) to induce an endogenous insulin response (approximately 1,100 pmol/l), or with exogenous insulin to give physiological (1,700 pmol/l) or supraphysiological (12,000 pmol/l) plasma levels. Raising plasma insulin within the physiological range had no effect (P > 0.05), but supraphysiological levels induced hypoglycemia (7.3 +/- 0.2 to 3.6 +/- 0.2 mmol/l) and increased [3H]glucose disappearance rate (P < 0.001). In cohort 4, a primed, continuous tracer infusion was started 120 min before saline or glucose bolus injection. [3H]glucose levels fell 15-20%, and the disappearance rate rose 36% (P < 0.05) after glucose injection. These results indicate that in fasted rats a tracer bolus injection protocol is not sufficiently sensitive to measure the physiological effect of insulin released in response to a bolus of glucose because this effect of insulin is small. Glucose itself is the predominant mediator of glucose disposal after a bolus of glucose in the fasted rat. (+info)
The use of infrared spectrophotometry for measuring body water spaces.
BACKGROUND: The conventional method of measuring total body water by the deuterium isotope dilution method uses gas isotope ratio mass spectrometry (IRMS), which is both expensive and time-consuming. We investigated an alternative method, using Fourier transform infrared spectrophotometry (FTIR), which uses less expensive instrumentation and requires little sample preparation. METHOD: Total body water measurements in human subjects were made by obtaining plasma, saliva, and urine samples before and after oral dosing with 1.5 mol of deuterium oxide. The enrichments of the body fluids were determined from the FTIR spectra in the range 1800-2800 cm-1, using a novel algorithm for estimation of instrumental response, and by IRMS for comparison. RESULTS: The CV (n = 5) for repeat determinations of deuterium oxide in biological fluids and calibrator solutions (400-1000 micromol/mol) was found to be in the range 0.1-0.9%. The use of the novel algorithm instead of the integration routines supplied with the instrument gave at least a threefold increase in precision, and there was no significant difference between the results obtained with FTIR and those obtained with IRMS. CONCLUSION: This improved infrared method for measuring deuterium enrichment in plasma and saliva requires no sample preparation, is rapid, and has potential value to the clinician. (+info)
Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei.
The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the transport activity independently of the glycolytic flux. Phloretin proved a competitive inhibitor. When the effect of the intracellular glucose concentration on the inhibition was taken into account, the flux control coefficient of the glucose transporter was between 0.3 and 0.5 at 5 mM glucose. Because the flux control coefficients of all steps in a metabolic pathway sum to 1, this result proves that glucose transport is not the rate-limiting step of trypanosome glycolysis. Under physiological conditions, transport shares the control with other steps. At glucose concentrations much lower than physiological, the glucose carrier assumed all control, in close agreement with model predictions. (+info)
Method for the determination of total homocysteine in plasma and urine by stable isotope dilution and electrospray tandem mass spectrometry.
BACKGROUND: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. METHODS: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 microL of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d(4) was 1.5 min in a 2.5-min analysis. RESULTS: Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S(y|x) =1.595 micromol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S(y|x) =1.146 micromol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/L). CONCLUSIONS: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation. (+info)