Mass isotopomer pattern and precursor product relationship test

mass isotopomer pattern and precursor product relationship test

product relationships in biological systems, as described more fully below. Lastly, because of their safety, several stable isotope tracers can be used of various test interventions to be compared on the basis of paired statistics, with precursor-product events from the distribution of mass isotopomer patterns in the . Cholesterol biosynthesis measurement by mass isotopomer distribution analysis . Data were tested for normality before using paired t-tests to compare FSR and . of the precursor-product relationship: definition of the precise precursor pool by the pattern of excess enrichments among mass isotopomers of the product. tein synthesis by MIDA is described and tested here: First, in vitro Mass isotopomer distribution analysis (MIDA) is a technique mass isotopomer2 pattern of the polymeric product is their relationship to other precursor pools or other.

Mass isotopomer pattern and precursor-product relationship.

Over periods as long as 48 h, protons that derive from water and NADPH are in equilibration with total body water, whereas those originating from acetyl-CoA are unlabeled, thereby limiting Dmax to 22 of the total 46 hydrogens in cholesterol 45.

Beyond 48 h, the acetyl-CoA pool may become labeled due to label recycling and thus contribute to the enrichment of cholesterol 45. Dmax has been calculated to be as high as 27 28 or 30 29 by different methods in rats exposed to deuterium in their drinking water over 1 week. Over the long term, such factors as dietary fat type 30 and ethanol ingestion 31 may also influence Dmax through changes in NADPH equilibrium 45.

Mass isotopomer pattern and precursor-product relationship.

Dmax cannot be determined directly in humans because of the slow turnover of the cholesterol pool, adverse effects of high doses of deuterated water, and the relative insensitivity of GC-MS.

Therefore, although the effects of changing metabolic conditions remain to be defined fully, Dmax is considered to be 22 for studies of cholesterol synthesis over 24 h. Theoretically if precursor enrichment is not constant over time or varies at different sites of synthesis, the labeling pattern of the product will be affected, as will estimates of p and FSR.

Reviews have assessed the impact of precursor pool nonhomogeneity in the broader context of MID analysis of intermediary metabolism 7 This may support the existence of multiple acetyl-CoA pools; however, precisely which of these pools would serve as the precursor of cholesterol is unknown. Higher mass isotopomers can become more highly enriched in cultured cells than in vivo, thereby allowing more rigorous analysis of the MID pattern of cholesterol.

The Kelleher et al. The potential impact of these uncertainties about precursor pools can be minimized by several modeling approaches as discussed by Hellerstein and Neese 712 and as was applied in the present study. Selecting an as near to linear relation between p and R as possible when calculating p from R can minimize the impact of multiple precursor pools.

The high correlation of estimates of p obtained from high and low mass ratios R3 and R1 in this study minimizes the practical impact of potential precursor pool nonhomogeneity on calculated cholesterol synthesis. The fundamental measurement of the FSR can be extended to an absolute synthesis rate by a variety of approaches.

We calculated the ASR for both techniques on the basis of the appearance of label in the rapidly exchanging cholesterol pool multiplied by the size of the pool.

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This approach, also commonly used to calculate plasma protein kinetics, is restricted to the initial monoexponential phase of label incorporation a condition satisfied in this study.

Alternatively, the rate constant of decay of higher mass isotopomers after termination of acetate infusion has been described as another approach to ASR calculation Calculation of the ASR is also dependent on how cholesterol pool size is estimated. The rapidly exchanging cholesterol pool is defined as half the M1 pool, as calculated from the equation of Goodman et al. One set of studies, in particular, has used these data to suggest that urea synthesis in sheep is fundamentally different from other mammals 89.

The second reason for carrying out these studies was to introduce a methodology that would permit the determination of the isotopic enrichment of the two nitrogenous precursor pools involved in urea synthesis. Such methodology will enable us to test Meijer's hypothesis that there is metabolic channeling between glutaminase and carbamoyl-phosphate synthetase I such that the amide nitrogen of glutamine has preferential access to carbamoyl-phosphate synthetase without mixing with the mitochondrial pool of ammonia We have employed the single-pass isolated perfused liver as our experimental model, since this avoids problems due to recycling of substrate such as incorporation of ammonia into glutamine in perivenous hepatocytes and subsequent use of this glutamine nitrogen for urea synthesis in periportal hepatocytes, which could occur either in incubated hepatocytes or in a recirculating perfusionwhich could confound the interpretation.

mass isotopomer pattern and precursor product relationship test

When 15NH3 is provided as substrate the urea formed may have a mass of 60, 61, or 62, depending on whether zero, one, or two 15N atoms are incorporated. This, in turn, depends on the enrichment of 15N in the two relevant nitrogen pools, the mitochondrial ammonia pool and the cytoplasmic aspartate pool. We present here a theoretical scheme that predicts the proportions of these three isotopomers of urea as a function of the 15N enrichment and an experimental means of determining the actual 15N enrichment of these pools.

We have also considered the synthesis of glutamine isotopomers. When synthesized in the presence of 15NH3 four separate glutamine mass and positional isotopomers are produced, i.

We present here a theoretical scheme that predicts the proportions of these four isotopomers of glutamine as a function of the 15N enrichment in the precursor pools, i. Perfusion flow rate, pH, pCO2, and pO2 in influent and effluent media were monitored throughout, and oxygen consumption was calculated.

After 15 min of perfusion, we changed to a medium containing 15NH4Cl final concentration, 0. Separate perfusate reservoirs, each containing ammonia of different 15N enrichment, were employed to facilitate changes in perfusion media. Samples were taken from the influent and effluent media for chemical and GC-MS analyses.

At the end of the perfusions livers were freeze-clamped with aluminum tongs precooled in liquid N2, the frozen livers were ground into a fine powder and extracted into perchloric acid, and the extracts were used for the analysis of adenine nucleotides by enzymatic techniques Urea and ammonia concentrations in the perfusion media were assayed by standard methods 13 Amino acid concentrations were determined by HPLC, utilizing precolumn derivatization with o-phthaldehyde A few perfusions were carried out to determine the rate of glutamine production due to proteolysis.

For these experiments the rats were pretreated with the glutamine synthetase inhibitor, methionine sulfoxamine, and this was included in the perfusate 6. The columns were washed with 3 ml of deionized water. Glutamate and aspartate were eluted with 3 ml of 1 N HCl. Arginine remained bound to this resin, whereas citrulline, urea, and other amino acids were eluted with 3 ml of water.

For GC-MS analysis, urea and amino acids were converted into t-butyldimethylsilyl derivatives. However, the t-butyldimethylsilyl derivative does not provide a measure of 15N enrichment in the amide nitrogen. Therefore, we utilized the N,N-bis-trifluoroacetyl derivative of glutamine Correction for possible overlapping ions in the MS was as described by Wolfe Statistical analyses between means were by the Student t test or the Newman-Keuls multiple comparison test, as appropriate.

Regression analysis was carried out using the Sigma Plot Program. Materials and Animals Chemicals were of analytical grade and obtained from Sigma or from Aldrich.

mass isotopomer pattern and precursor product relationship test

Then the fractional abundance of the urea isotopomers will be, Eq. One of the most important deductions from these equations is that even in a stable physiological situation the proportions of the different isotopomers need not be constant but will vary depending on the absolute 15N abundance of the pools.