Cumulative responses of muscle protein synthesis are augmented with chronic resistance exercise training.

AIM: The purpose of this study was to determine the anabolic response of a single bout of high intensity resistance exercise (RE) following 5 weeks of RE training. METHODS: To complete these studies, Sprague-Dawley rats were assigned by body mass to RE, exercise control (EC), or sedentary cage control (CC) groups and studied over 36 h after 5 weeks of RE (squat-like) training. Cumulative (final 36 h) fractional rates of muscle protein synthesis (FSR) were determined by ²H2O, and acute (16 h post-RE) rates of muscle protein synthesis (RPS) were determined by flooding with l-[2,3,4,5,6-³H]phenylalanine. Regulators of peptide-chain initiation, 4E-BP1, eIF4E and the association of the two were determined by Western blotting and immunoprecipitation respectively. RESULTS: No differences were observed with acute measures of RPS obtained 16 h following the final exercise bout in the plantaris or soleus muscles (P > 0.05). Consistent with this observation, 4E-BP1 was similarly phosphorylated and bound to eIF4E among all groups. However, upon determination of the cumulative response, FSR was significantly increased in the plantaris of RE vs. EC and CC (0.929±0.094, 0.384±0.039, 0.300±0.022% h(-1) respectively; P<0.001), but not the soleus. CONCLUSION: With the advantage of determining cumulative FSR, the present study demonstrates that anabolic responses to RE are still evident after chronic RE training, primarily in muscle composed of fast-twitch fibres.

Sources of blood glycerol during fasting.

To determine the source(s) of blood and very low density lipoprotein (VLDL)-triglyceride glycerol during fasting, four men ingested (2)H(2)O from 14 to 20 h into a 60-h fast to achieve ~0.5% body water enrichment. At 60 h of fasting, glycerol flux was measured using [2-(14)C]glycerol. Blood was taken for measurement of (2)H enrichment at carbon 6 of glucose and at carbon 3 of free glycerol and VLDL-triglyceride glycerol. (2)H enrichment of the 2 hydrogens bound to carbon 3 of VLDL-triglyceride glycerol was 105 +/- 2% of the (2)H enrichment of the 2 hydrogens bound to carbon 6 of glucose, indicating isotopic equilibrium between hepatic glyceraldehyde 3-P and glycerol 3-P. The (2)H enrichment of the 2 hydrogens bound to carbon 3 of free glycerol was 17 +/- 3% of VLDL-triglyceride glycerol, indicating that a significant percentage of free glycerol in blood originated from the hydrolysis of circulating VLDL-triglyceride or a pool of glycerol with similar (2)H enrichment. Glycerol flux was 6.3 +/- 1.1 micromol. kg(-1). min(-1). Glycerol appearing from nonadipose tissue sources was then approximately 1.1 micromol. kg(-1). min(-1). Seven other subjects were fasted for 12, 42, and 60 h. A small percentage of glycerol in the circulation after 12 h of fasting was enriched with (2)H. The enrichment of the 2 hydrogens bound to carbon 3 of free glycerol in the longer periods of fasting was approximately 16% of the enrichment of the 2 hydrogens bound to carbon 6 of glucose. Therefore, as much as 15-20% of systemic glycerol turnover during fasting is not from lipolysis of adipose tissue triglyceride.

Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats.

Activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofurano-side (AICAR) increases glucose transport in skeletal muscle via an insulin-independent pathway. To examine the effects of AMPK activation on skeletal muscle glucose transport activity and whole-body carbohydrate and lipid metabolism in an insulin-resistant rat model, awake obese Zuckerfa/fa rats (n = 26) and their lean (n = 23) littermates were infused for 90 min with AICAR, insulin, or saline. The insulin infusion rate (4 mU.kg(-1).min(-1)) was selected to match the glucose requirements during AICAR (bolus, 100 mg/kg; constant, 10 mg.kg(-1).min(-1)) isoglycemic clamps in the lean rats. The effects of these identical AICAR and insulin infusion rates were then examined in the obese Zucker rats. AICAR infusion increased muscle AMPK activity more than fivefold (P < 0.01 vs. control and insulin) in both lean and obese rats. Plasma triglycerides, fatty acid concentrations, and glycerol turnover, as assessed by [2-13C]glycerol, were all decreased in both lean and obese rats infused with AICAR (P < 0.05 vs. basal), whereas insulin had no effect on these parameters in the obese rats. Endogenous glucose production rates, measured by [U-13C]glucose, were suppressed by >50% during AICAR and insulin infusions in both lean and obese rats (P < 0.05 vs. basal). In lean rats, rates of whole-body glucose disposal increased by more than two-fold (P < 0.05 vs. basal) during both AICAR and insulin infusion; [3H]2-deoxy-D-glucose transport activity increased to a similar extent, by >2.2-fold (both P < 0.05 vs. control), in both soleus and red gastrocnemius muscles of lean rats infused with either AICAR or insulin. In the obese Zucker rats, neither AICAR nor insulin stimulated whole-body glucose disposal or soleus muscle glucose transport activity. However, AICAR increased glucose transport activity by approximately 2.4-fold (P < 0.05 vs. control) in the red gastrocnemius from obese rats, whereas insulin had no effect. In summary, acute infusion of AICAR in an insulin-resistant rat model activates skeletal muscle AMPK and increases glucose transport activity in red gastrocnemius muscle while suppressing endogenous glucose production and lipolysis. Because type 2 diabetes is characterized by diminished rates of insulin-stimulated glucose uptake as well as increased basal rates of endogenous glucose production and lipolysis, these results suggest that AICAR-related compounds may represent a new class of antidiabetic agents.

Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo.

To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1(-/-)) or IRS-2 (IRS-2(-/-)) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1(-/-), and IRS-2(-/-)) with [3-(3)H]glucose and [(2)H(5)]glycerol and assessed rates of glucose and glycerol turnover under basal (0-90 min) and hyperinsulinemic-euglycemic clamp (90-210 min; 5 mm glucose, and 5 milliunits of insulin.kg(-)(1).min(-)(1)) conditions. Both IRS-1(-)(/-) and IRS-2(-)(/-) mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1(-)(/-) mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2(-)(/-) mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2(-)(/-) mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolism in vivo in mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.

13C/31P NMR assessment of mitochondrial energy coupling in skeletal muscle of awake fed and fasted rats. Relationship with uncoupling protein 3 expression.

To examine the relationship between mitochondrial energy coupling in skeletal muscle and change in uncoupling protein 3 (UCP3) expression during the transition from the fed to fasted state, we used a novel noninvasive (31)P/(13)C NMR spectroscopic approach to measure the degree of mitochondrial energy coupling in the hind limb muscles of awake rats before and after a 48-h fast. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocnemius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, following a 48-h fast. Tricarboxylic acid cycle flux measured using (13)C NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 25 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respectively. Unidirectional ATP synthesis flux measured using (31)P NMR was 79 +/- 15 and 57 +/- 9 nmol/g/s (p not significant) in the fed and 48-h fasted groups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different between the fed and fasted states. To test the hypothesis that UCP3 may be involved in the translocation of long chain free fatty acids (FFA) into the mitochondrial matrix under conditions of elevated FFA availability, [U-(13)C]palmitate/albumin was administered in a separate group of rats with (+) or without (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ratio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb muscles was the same between groups, indicating that UCP3 does not appear to function as a translocator for long chain FFA in skeletal muscle following a 48-h fast. In summary, these data demonstrate that despite a 2-3-fold increase in UCP3 mRNA and protein expression in skeletal muscle during the transition from the fed to fasted state, mitochondrial energy coupling does not change. Furthermore, UCP3 does not appear to have a major role in FFA translocation into the mitochondria. The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.

Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction.

The liver plays a central role in the control of glucose homeostasis and is subject to complex regulation by substrates, insulin, and other hormones. To investigate the effect of the loss of direct insulin action in liver, we have used the Cre-loxP system to inactivate the insulin receptor gene in hepatocytes. Liver-specific insulin receptor knockout (LIRKO) mice exhibit dramatic insulin resistance, severe glucose intolerance, and a failure of insulin to suppress hepatic glucose production and to regulate hepatic gene expression. These alterations are paralleled by marked hyperinsulinemia due to a combination of increased insulin secretion and decreased insulin clearance. With aging, the LIRKO liver exhibits morphological and functional changes, and the metabolic phenotype becomes less severe. Thus, insulin signaling in liver is critical in regulating glucose homeostasis and maintaining normal hepatic function.

Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle.

Obesity and insulin resistance in skeletal muscle are two major factors in the pathogenesis of type 2 diabetes. Mice with muscle-specific inactivation of the insulin receptor gene (MIRKO) are normoglycemic but have increased fat mass. To identify the potential mechanism for this important association, we examined insulin action in specific tissues of MIRKO and control mice under hyperinsulinemic-euglycemic conditions. We found that insulin-stimulated muscle glucose transport and glycogen synthesis were decreased by about 80% in MIRKO mice, whereas insulin-stimulated fat glucose transport was increased threefold in MIRKO mice. These data demonstrate that selective insulin resistance in muscle promotes redistribution of substrates to adipose tissue thereby contributing to increased adiposity and development of the prediabetic syndrome.

Assessment of mitochondrial energy coupling in vivo by 13C/31P NMR.

The recently cloned uncoupling protein homolog UCP3 is expressed primarily in muscle and therefore may play a significant role in the regulation of energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this report, we demonstrate the use of a noninvasive NMR technique to assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining (13)C NMR to measure rates of mitochondrial substrate oxidation with (31)P NMR to assess unidirectional ATP synthesis flux. These combined (31)P/(13)C NMR measurements were performed in control, 10-day triiodo-l-thyronine (T(3))-treated (model of increased UCP3 expression), and acute 2,4-dinitrophenol (DNP)-treated (protonophore and mitochondrial uncoupler) rats. UCP3 mRNA and protein levels increased 8.1-fold (+/- 1.1) and 2.8-fold (+/- 0.8), respectively, in the T(3)-treated vs. control rat gastrocnemius muscle. (13)C NMR measurements of tricarboxylic acid cycle flux as an index of mitochondrial substrate oxidation were 61 +/- 21, 148 +/- 25, and 310 +/- 48 nmol/g per min in the control, T(3), and DNP groups, respectively. (31)P NMR saturation transfer measurements of unidirectional ATP synthesis flux were 83 +/- 14, 84 +/- 14, and 73 +/- 7 nmol/g per s in the control, T(3), and DNP groups, respectively. Together, these flux measurements, when normalized to the control group, suggest that acute administration of DNP (mitochondrial uncoupler) and chronic administration of T(3) decrease energy coupling by approximately 80% and approximately 60%, respectively, and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach could prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.

A critical evaluation of mass isotopomer distribution analysis of gluconeogenesis in vivo.

There are conflicting reports concerning the reliability of mass isotopomer distribution analysis (MIDA) for estimating the contribution of gluconeogenesis to total glucose production (f) during [(13)C]glycerol infusion. We have evaluated substrate-induced effects on rate of appearance (R(a)) of glycerol and glucose and f during [2-(13)C]glycerol infusion in vivo. Five groups of mice were fasted for 30 h and then infused with [2-(13)C]glycerol at variable rates and variable (13)C enrichments (group I: 20 micromol. kg(-1). min(-1), 99% (13)C; group II: 60 micromol. kg(-1). min(-1), 60% (13)C; group III: 60 micromol. kg(-1). min(-1), 99% (13)C; group IV: 120 micromol. kg(-1). min(-1), 40% (13)C; or group V: 120 micromol. kg(-1). min(-1), 99% (13)C). The total glycerol R(a) increased from approximately 104 to approximately 157 and to approximately 210 micromol. kg(-1). min(-1) as the infusion of [2-(13)C]glycerol increased from 20 to 60 and to 120 micromol. kg(- 1). min(-1), respectively. As the amount of 99% enriched [2-(13)C]glycerol increased from 20 to 60 and to 120 micromol. kg(-1). min(-1) (groups I, III, and V, respectively), plasma glycerol enrichment increased from approximately 21 to approximately 42 and to approximately 57% and the calculated f increased from approximately 27 to approximately 56 and to approximately 87%, respectively. Similar plasma glycerol enrichments were observed in groups I, II, and IV (i. e., approximately 21-24%), yet f increased from approximately 27 to approximately 57 and to approximately 86% in groups II and IV, respectively. Estimates of absolute gluconeogenesis increased from approximately 14 to approximately 33 and approximately 86 micromol. kg(-1). min(-1) as the infusion of [2-(13)C]glycerol increased from 20 to 60 and 120 micromol. kg(-1). min(-1). Plausible estimates of f were obtained only under conditions that increased total glycerol R(a) approximately 2-fold (P < 0.001) and increased glucose R(a) approximately 1.5-fold (P < 0.01) above basal. We conclude that in 30-h fasted mice, 1) estimates of f by MIDA with low infusion rates of [2-(13)C]glycerol yield erroneous results and 2) reasonable estimates of f are obtained at glycerol infusion rates that perturb glycerol and glucose metabolism.

Limitations in estimating gluconeogenesis and Cori cycling from mass isotopomer distributions using [U-13C6]glucose.

Tayek and Katz proposed calculating gluconeogenesis's contributions to glucose production and Cori cycling from mass isotopomer distributions in blood glucose and lactate during [U-13C6]glucose infusion [Tayek, J. A., and J. Katz. Am. J. Physiol. 272 (Endocrinol. Metab. 35): E476-E484, 1997]. However, isotopic exchange was not adequately differentiated from dilution, nor was condensation of labeled with unlabeled triose phosphates properly equated. We introduce and apply corrected equations to data from subjects fasted for 12 and 60 h. Impossibly low contributions of gluconeogenesis to glucose production at 60 h are obtained (23-41%). Distributions in overnight-fasted normal subjects calculate to only approximately 18%. Cori cycling estimates are approximately 10-15% after overnight fasting and 20% after 60 h of fasting. There are several possible reasons for the underestimates. The contribution of gluconeogenesis is underestimated because glucose production from glycerol and amino acids not metabolized via pyruvate is ascribed to glycogenolysis. Labeled oxaloacetate and alpha-ketoglutarate can exchange during equilibrium with circulating unlabeled aspartate, glutamate, and glutamine. Also, the assumption that isotopomer distributions in arterial lactate and hepatic pyruvate are the same may not be fulfilled.

Limitations of the mass isotopomer distribution analysis of glucose to study gluconeogenesis. Heterogeneity of glucose labeling in incubated hepatocytes.

We previously reported (Previs, S. F., Fernandez, C. A., Yang, D., Soloviev, M. V., David, F., and Brunengraber, H. (1995) J. Biol. Chem. 270, 19806-19815) that glucose made in isolated livers from starved rats perfused with physiological concentrations of lactate, pyruvate, and either [2-13C]- or [U-13C3]glycerol had a mass isotopomer distribution incompatible with glucose being made from a homogeneously labeled pool of triose phosphates. Similar data were obtained in live rats infused with [U-13C3]glycerol. We ascribed the labeling heterogeneity to major decreases in glycerol concentration and enrichment across the liver. We concluded that [13C]glycerol is unsuitable for tracing the contribution of gluconeogenesis to total glucose production. We now report isotopic heterogeneity of gluconeogenesis in hepatocytes, even when all cells are in contact with identical concentrations and enrichments of gluconeogenic substrates. Total rat hepatocytes were incubated with concentrations of glycerol, lactate, and pyruvate that were kept constant by substrate infusions. To modulate competition between substrates, the (glycerol)/(lactate + pyruvate) infusion ratio ranged from 0.23 to 3. 60. Metabolic and isotopic steady states were achieved in all cases. The apparent contribution of gluconeogenesis to glucose production (f) was calculated from the mass isotopomer distribution of glucose. When all substrates were 13C-labeled, f was 97%, as expected in glycogen-deprived hepatocytes. As the infusion ratio ([13C]glycerol)/(lactate + pyruvate) increased, f increased from 73% to 94%. In contrast, as the infusion ratio (glycerol)/([13C]lactate + [13C]pyruvate) increased, f decreased from 93% to 76%. In all cases, f increased with the rate of supply of the substrate that was labeled. Variations in f show that the 13C labeling of triose phosphates was not equal in all hepatocytes, even when exposed to the same substrate concentrations and enrichments. We also showed that zonation of glycerol kinase activity is minor in rat liver. We conclude that zonation of other processes than glycerol phosphorylation contributes to the heterogeneity of triose phosphate labeling from glycerol in rat liver.

Methods for measuring gluconeogenesis in vivo.

Recent developments in stable isotope technology have led to the conception of new protocols for measuring the contribution of gluconeogenesis to glucose production in humans. Earlier techniques were subject to variable underestimations resulting from isotopic exchanges occurring during the transfer of carbon label from the tracer to glucose. This review concentrates on four novel techniques: (1) mass isotopomer distribution analysis of glucose labelled from [13C]glycerol or [13C]lactate; (2) mass isotopomer distribution analysis of glucose and lactate during infusion of [U-13C6]glucose; (3) 2H-enrichment of body water by ingestion of 2H2O, and measuring the 2H-labelling on C5 and C2 of glucose, and (4) difference between glucose turnover and rate of hepatic glycogenolysis measured by nuclear magnetic resonance spectroscopy. The advantages and limitations of the four protocols are discussed. The 2H2O technique is the most practical; it is not subject to artifacts resulting from isotopic exchanges, and is not affected by zonation of hepatic metabolism.

Stable isotope studies of phytanic acid alpha-oxidation: in vivo production of formic acid.

The aim of this study was to test whether formate is formed during alpha-oxidation of phytanic acid in humans. To a healthy volunteer, [1-13C]phytanic acid was given as an oral substrate in a dose of 15 mg/kg body weight, after which plasma, urine and breath air samples were collected during 35 h. The plasma concentrations of [1-13C]-phytanic acid, 2-hydroxy[1-13C]phytanic acid, pristanic acid and [13C]formate were analysed. The [1-13C]phytanic acid concentration increased within 5-7 h to 105 mumol/l, then decreased. Formation of 2-hydroxy[1-13C]phytanic acid increased during the first 11 h after which it decreased during the next 20 h. Pristanic acid increased slightly during the test. In breath air, 13CO2 enrichment was measured, showing a cumulative output of ca. 30% of the ingested dose after 35 h. In both urine and plasma, enrichment of [13C]formate, higher than that of 13CO2 was demonstrated. These findings show that formate is a decarboxylation product in the alpha-oxidation of phytanic acid in vivo.

Tracing gluconeogenesis with deuterated water: measurement of low deuterium enrichments on carbons 6 and 2 of glucose.

The contribution of gluconeogenesis to glucose production in vivo can be measured by enriching body water with 0.5% 2H2O and measuring the glucose labeling ratio C6/C2 (Landau et al., J. Clin. Invest. 95, 172-178, 1995). We present further refinements of the measurements of the 2H enrichments on C6 and C2 of glucose. The transfer of 2H from C6 of glucose to hexamethylenetetramine (HMT) and extraction in preparation for gas chromatography-mass spectrometry can be done in a single test tube, without distillation of the intermediate formaldehyde. In addition, extraction of small amounts of HMT is greatly improved by making a HMT-iodine adduct. For C2, glucose is reduced to sorbitol, and 2H on C2 is transferred enzymatically to [U-13C3]pyruvate, forming [U-13C3,2-2H]lactate. The latter is assayed by negative chemical ionization gas chromatography-mass spectrometry of the pentafluorobenzyl derivative. The natural enrichment of the [U-13C3]lactyl ion is only 0.4%, allowing measurements of 2H enrichment down to 0.1%. These techniques were used in dogs infused with 2H2O and in isolated rat livers perfused with buffer containing 1 to 5% 2H2O. Our data reveal a difference in the rate of labeling of C6 and C2 of glucose in vivo. Lastly, in cows infused with [6,6-2H2]glucose, we show that the turnover of glucose can be economically measured by assaying low tracer enrichment (down to 0.1%) via hexamethylenetetramine.

Glycerol production and utilization in humans: sites and quantitation.

Liver is assumed to be the major site of glycerol uptake and fatty acid reesterification. [U-13C]glycerol was infused into ten 60 h-fasted healthy subjects. Measured were 1) blood glycerol concentrations and 13C enrichments in brachial and pulmonary arteries and in hepatic, renal, superficial, and deep forearm veins; 2) glycerol appearance rates in systemic circulation; and 3) splanchnic bed and kidney glycerol uptakes with use of balance and tracer methodology. Glycerol concentrations were one-fifth in hepatic, one-half in renal, 40% more in superficial, and the same in deep vein and pulmonary artery as in brachial artery blood. Glycerol enrichments were one-fifth in hepatic, two-thirds to three-quarters in renal and superficial veins, and the same in pulmonary as in brachial artery blood. Splanchnic glycerol uptake was 29% and kidney glycerol uptake was 17% of glycerol's rate of appearance, 5.11 mumol.min-1.kg-1. Splanchnic fatty acid uptake was 25% of calculated fatty acid release. Glycerol contributed 15% to glucose production. Most of the [13C]glycerol uptake by splanchnic bed and kidneys was incorporated into glucose. Thus, in 60 h-fasted individuals, most glycerol uptake does not occur in liver, and the extent of fatty acid reesterification in liver is in doubt.

Contributions of liver and kidneys to glycerol production and utilization in the dog.

The classical concept holds that liver and kidneys are the main sinks of glycerol released by adipose tissue. However, rates of glycerol appearance (Ra) exceed the rate of glycerol delivery to liver and kidneys. We measured the hepatic and renal contributions to glycerol production and utilization in anesthetized dogs that were fasted either overnight or for 24 h after 3 days on a carbohydrate-free diet. Dogs were infused with [2H5]glycerol, and the concentration and 2H enrichment of glycerol were measured across liver and kidney. After a baseline period, either norepinephrine or glucose plus insulin was infused to alter the rate of glycerol production. Our study shows that the production of glycerol by liver and kidneys amounted to 4-9% and 4-7% of the Ra of glycerol, respectively. Uptake of glycerol by liver and kidneys amounted to 26-30 and 10-19% of the Ra of glycerol, respectively. Thus, contrary to the classical concept, the bulk of glycerol utilization occurs in nonhepatic, nonrenal tissues that have very low glycerol kinase activity per gram.

Assay of the deuterium enrichment of water via acetylene.

A technique is presented for measuring the 2H enrichment of water in biological samples when this enrichment is greater than 0.2%. The sample is reacted with calcium carbide to form acetylene gas, which is determined by gas chromatography electron impact ionization mass spectrometry. Ion-molecule reactions, resulting in proton abstraction, are minimized by lowering the electron ionization energy from the usual 70 eV to 45 eV. This technique is much more rapid and economical than the classical isotope ratio mass spectrometric assay of the enrichment of hydrogen gas derived from reduction of water.

Assay of the 13C and 2H mass isotopomer distribution of phosphoenolpyruvate by gas chromatography/mass spectrometry.

The 13C mass isotopomer distribution of liver phosphoenolpyruvate (PEP) yields important information on the regulation of gluconeogenesis and the citric acid cycle. A convenient technique is presented for measuring the mass isotopomer distribution of PEP in tissue extracts. The procedure involves reduction of extant pyruvate to lactate with NaBH4, enzymatic conversion of PEP to pyruvate, extraction of pyruvate hydroxamate and gas chromatographic/mass spectrometric determination of pyruvate hydroxamate di-tert-butyldimethylsilyl derivative. When PEP is labeled with 2H, the enzymatic conversion of PEP to pyruvate results in the loss of 2H. Therefore, to assay the enrichment of [2H]PEP, the tissue extract is chromatographed on an anion-exchange column. The fraction containing PEP is treated to form PEP tri(trimethylsilyl) derivative. The procedures were applied to liver PEP labeled using [U-13C3]lactate, [U-13C3]glycerol or 2H2O. The results show the compatibility between the mass isotopomer distributions of PEP and glucose in rat livers perfused with [U-13C3]lactate or [U-13C3]glycerol. There is a 78% isotopic equilibration of 2H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.

Noninvasive probing of citric acid cycle intermediates in primate liver with phenylacetylglutamine.

In human and primate liver, phenylacetate and glutamine form phenylacetylglutamine, which is excreted in urine. Probing noninvasively the labeling pattern of liver citric acid cycle intermediates with phenylacetylglutamine assumes that the labeling pattern of its glutamine moiety reflects that of liver alpha-ketoglutarate. To validate this probe, we infused monkeys with [U-13C3]lactate, [3-13C]lactate, [1, 2-13C2]acetate, [2-13C]acetate, [U-13C3]glycerol, or 2-[3-13C]ketoisocaproate and compared the labeling patterns of urinary phenylacetyl-glutamine with those of glutamate and glutamine in liver, plasma, muscle, and kidney and liver alpha-ketoglutarate. Only with [U-13C3]lactate or [3-13C]lactate does the labeling pattern of phenylacetylglutamine reflect patterns of liver alpha-ketoglutarate and glutamate. With [13C]acetate, muscle and kidney glutamate are more labeled than liver metabolites. This confirms that with [13C]acetate, the labeling pattern of liver metabolites is influenced by 13CO2 and [13C]glutamine made in peripheral tissues. Our data validate the use of phenylacetylglutamine labeled from [3-13C]lactate or [3-13C]pyruvate to probe noninvasively the pyruvate carboxylase-to-pyruvate dehydrogenase flux ratio in human subjects.