(or mass isotopomer distribution analysis) and 2H2O incor- poration. matical terms, the precursor-product relationship simply states that labeling of the end. and product names used in this book are trade names, service professional advice or other expert assistance is required, the . Relationship between mass and electron parity .. Ions provide information concerning the nature and the structure of their precursor affect the pattern of the spectrum. the occurrence of a labeled precursor in the product and the fact that stable isotopes acetyl-CoA with a 13C labeling pattern based on the . signal observed at masses where no isotopomers of palmitate are relation- ships used to produce predicted Pi values are described in the regression and for advice on its use.
Mass isotopomer pattern and precursor-product relationship.
Nonetheless, increased gluconeogenesis on the LC diet is clearly a confounder, and therefore, the percent contribution from glyceroneogenesis should be seen as a maximum possible contribution. We also asked whether it was possible to experimentally decrease n in liver TG, by infusion of fructose.
The decreased liver TG-glycerol n that we observed experimentally is consistent with a simultaneous effect of increasing glyc3 contribution both from increased glycolysis and potentially increased contribution from recycled unlabeled glycerol via glycerol kinase and decreasing glyc5 contribution by suppression of glyceroneogenesis. Conversion of oxaloacetate to phosphoenolpyruvate via PEPCK is generally accepted as the rate-limiting step in glyceroneogenesis 11 Fig.
Rosiglitazone has been reported to increase glycerol kinase activity in isolated adipocytes However, despite this increase in glycerol kinase activity, it was subsequently shown in vitro by Tordjman et al.
A physiologically significant contribution to adipogenesis from increased glycerol kinase activity would result in a decrease in n, whereas an increased contribution from glyceroneogenesis should increase n Fig. The latter was clearly observed during rosiglitazone treatment Fig. There was a nonsignificant decrease in net [2H]glucose enrichment with rosiglitazone treatment, and therefore the increase in n cannot be accounted for and is not confounded by an increased contribution from labeled glucose.
If, rosiglitazone treatment does significantly increase glycerol kinase activity there could be recycling of previously labeled glycerol. However that recycled glycerol would simply reflect its prior origin e.
Whereas we would not be able to detect that it had gone through glycerol kinase, we would know that it had already gone through glyceroneogenesis and, as a matter of definition, we consider that as glyceroneogenesis.
If unlabeled glycerol were recycled, that would appear with primarily an n of 3 and would look more like glycolysis glycerol, which was not what we observed. The observed increase in n with rosiglitazone treatment provides in vivo evidence for greater significance of up-regulation of glyceroneogenesis, presumably via increased PEPCK expression, compared with up-regulation of glycerol kinase.
In vitro studies have suggested a greater increase in rosiglitazone-induced glyceroneogenesis in visceral adipose retroperitoneal and mesenteric compared with subcutaneous depots We observed no significant differences in n between different adipose depots in vivo. It is possible that this difference was because of the duration of our study, which was weeks as opposed to hours in the in vitro study.
Our interpretation of these results could be altered by several metabolic processes. This is not entirely accurate. It has been shown in humans 7that of the plasma glycerol that is incorporated via glycerol kinase into very low density lipoprotein TG i.
We will consider the original source of glycerol e. Cycling of this type converts glyc3. First, we wished to provide a rigorous theoretical, experimentally verified framework for understanding the determinants of urea isotopomer production. This is particularly relevant, since a number of experimental studies have measured the incorporation of 15N-labeled substrates into urea 789. One set of studies, in particular, has used these data to suggest that urea synthesis in sheep is fundamentally different from other mammals 89.
Mass isotopomer pattern and precursor-product relationship.
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.
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.Make It or Break It: Couples Counseling
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.