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Transcript
13C NMR measurements of human gluconeogenic fluxes after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen JOHN G. JONES,1 MICHAEL A. SOLOMON,2 A. DEAN SHERRY,1,3 F. M. H. JEFFREY,1 AND CRAIG R. MALLOY1,2 1Department of Radiology, University of Texas Southwestern Medical Center, Dallas 75235; 2Department of Internal Medicine, University of Texas Southwestern Medical Center and Department of Veterans Affairs Medical Center, Dallas 75216; and 3Department of Chemistry, University of Dallas, Richardson, Texas 75083 glucose; glucuronide; glutamine; isotopomers; nuclear magnetic resonance offer promise for aiding diagnosis of abnormal glucose metabolism in the clinical setting. Although radioactive precursors can be used to trace human hepatic metabolism (28, 30), they are unlikely to be widely adopted in the clinical environment because of possible risks to the patient and the impracticality of systematic radiation containment. More recently, similar metabolic models have been successfully adapted for use with stable isotope tracers (3, 15), and well-established chemical biopsy methods have been used to target carbon skeletons of hepatic UDP-glucose and glutamine (12, 15, 27–30, 40). TRACERS OF HEPATIC METABOLISM The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Most common tracers of hepatic metabolism, such as the gluconeogenic substrates lactate and alanine, may be metabolized to some extent by peripheral tissues and thus possibly confound analysis of liver metabolism. Ideally, any tracer should be infused directly into the hepatic portal vein and extracted quantitatively by the liver. Portal vein infusions are too invasive for human studies, however, and most hepatic tracers are not quantitatively extracted from portal circulation by the mammalian liver (34). Propionate may be an important exception in that it is quantitatively extracted into liver from portal circulation in a wide range of mammalian species, likely including humans (2, 8). It is generated in significant quantities by colonic fermentation of a-amylase-resistant starch and nonstarch polysaccharides (2, 8). Propionate has been administered by gastric infusion to human subjects at rates of 4 mmol/h over a 3-h period with no ill effects and no alteration of hepatic glucose production (26). Being a weak acid, propionate is likely to pass rapidly and efficiently into both splanchnic and portal vein circulation from the stomach after ingestion, in a manner similar to aspirin. These features suggest that oral administration of propionate may be an effective method for noninvasive analysis of hepatic gluconeogenesis in humans. Although deployment of more hepatophilic tracers has minimized labeling contributions from peripheral metabolism (24, 30, 41), values of gluconeogenic flux reported by these newer methods remain controversial. Katz et al. (23) suggested that net gluconeogenic flux values of about three times the tricarboxylic acid cycle rate are unsustainable, because the ATP demand for gluconeogenesis exceeds the total ATP output of the citric acid cycle via substrate level and oxidative phosphorylation. Others have proposed that sufficient ATP is available if a significant portion of hepatic acetyl-CoA originates from b-oxidation (24). Because measurements of gluconeogenesis in humans to date have been derived by using tracers that enter the citric acid cycle via pyruvate or acetyl-CoA, it is important to verify these observations by use of a tracer that enters the citric acid cycle via a different metabolic pathway. Herein, we demonstrate that such measurements are easily derived from a single 13C NMR spectrum of human plasma glucose, urinary glucuronide, and urinary phenylacetylglutamine after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. 0193-1849/98 $5.00 Copyright r 1998 the American Physiological Society E843 Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 Jones, John G., Michael A. Solomon, A. Dean Sherry, F. M. H. Jeffrey, and Craig R. Malloy. 13C NMR measurements of human gluconeogenic fluxes after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. Am. J. Physiol. 275 (Endocrinol. Metab. 38): E843–E852, 1998.— Anaplerotic, pyruvate recycling, and gluconeogenic fluxes were measured by 13C isotopomer analysis of plasma glucose, urinary phenylacetylglutamine, and urinary glucuronide in normal, 24-h-fasted individuals after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. Plasma glucose isotopomer analysis reported a total anaplerotic flux of 5.92 6 1.03 (SD) relative to citrate synthase. This was not significantly different from glucuronide and phenylacetylglutamine analyses (6.08 6 1.16 and 7.14 6 1.94, respectively). Estimates of pyruvate recycling from glucose and glucuronide isotopomer distributions were almost identical (3.55 6 0.99 and 3.66 6 1.11, respectively), whereas phenylacetylglutamine reported a significantly higher estimate (5.74 6 2.13). As a consequence, net gluconeogenic flux reported by phenylacetylglutamine (1.41 6 0.28) was significantly less than that reported by glucose (2.37 6 0.64) and glucuronide (2.42 6 0.76). This difference in fluxes detected by analysis of phenylacetylglutamine vs. hexose is likely due to compartmentation of hepatic metabolism of propionate. Net gluconeogenic flux estimates made by use of this stable isotope method are in good agreement with recent measurements in humans with [14C]propionate. E844 MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE METHODS Fig. 1. Time course for administration of [U-13C]propionate, phenylacetate, and acetaminophen and sampling of blood and urine. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 Experimental protocol. Six healthy subjects [5 male and 1 female, 23–39 yr old, and weighing 54–88 kg (76 6 12 kg)] were studied under a protocol approved by the institutional human studies committee. Subjects were fasted for 24 h before the start of the experiment and allowed access to water. Figure 1 shows a flowchart of drug ingestion and blood and urine sampling. From 7:00–8:00 AM on the day of the study, each subject ingested 66 mg/kg phenylacetic acid distributed in 12 gelatin capsules (Gallipot, St. Paul, MN). At 8:00 AM, each subject ingested 2 tablets (500 mg each) of acetaminophen, and the first blood sample was also drawn into heparinized tubes (25 or 30 ml). Over the next hour, five of the subjects ingested a total of 20 or 25 mg/kg sodium [U-13C]propionate distributed in 6 gelatin capsules, and the sixth subject received 17 mg/kg of the sodium [U-13C]propionate. After the 8:00 AM sample, blood was drawn at 20-min intervals over the next 2 h, with additional draws at 2.5 and 3 h (total blood drawn was 225 or 270 ml). Urine was also collected every hour over a 6-h period starting at 9:00 AM, with the exception of one individual whose urine was collected every 2 h. Analytic procedures. Blood samples were chilled and centrifuged at 4°C in heparinized tubes immediately after being drawn. The plasma was deproteinized by addition of 0.5 ml of ice-cold 70% perchloric acid, and the precipitated protein was removed by centrifugation. The supernatant was adjusted to pH 5.0 with potassium hydroxide, and the insoluble potassium perchlorate was removed by a second centrifugation. The glucose content of each sample was assayed enzymatically before oxidation to gluconate with glucose oxidase (18). The samples were deproteinized a second time, and the pH was adjusted to 10.0 with KOH before lyophilization. Samples were resuspended in 600 µl 2H2O containing a potassium glycolate standard in which the glycolate carbons had natural 13C abundance (assumed to be 1.11%). For each NMR sample, the amount of glycolate added was equal to the amount of glucose assayed in the parent plasma extract (typically 20–40 µmol). Our initial studies revealed that lyophilized urine samples were unsuitable for high-resolution 13C NMR analysis of the phenylacetylglutamine and glucuronide moieties, because 1) they were highly viscous, 2) they had a high salt content, and 3) the glucuronide was partially converted into a mixture of a,b-glucuronate and a,b-6,3-glucuronolactone in many of the samples. Hence, all urine was subsequently treated with urease and b-glucuronidase and then partially purified by ion-exchange chromatography before 13C NMR spectroscopy. Urine samples were adjusted to pH 7.0 and incubated overnight with 10,000 U of urease per sample. The pH was then readjusted to 7.0, and the urine was further incubated for 24 h at 37°C with 10,000 Fishman units of Escherichia coli b-glucuronidase. A trace of sodium azide was also added to inhibit possible infection from airborne microorganisms. The samples were deproteinized by perchloric acid and centrifuged, the supernatant pH was adjusted to 10.0, and the sample was left at room temperature for 30 min. (This last step hydrolyzed any glucuronolactone that might have formed from the nascent glucuronate during incubation.) The pH was then adjusted to 8.0, the sample was loaded onto a 5- to 7-ml volume anion-exchange column (Dowex-138-acetate), and the column was washed with 40 ml of water. Phenylacetylglutamine and a,b-,D-glucuronic acid were released by eluting with 15 ml of 10 M acetic acid. This fraction was lyophilized and resuspended in 700 µl 2H2O. The pH was then adjusted to 9.0 with NaOD, the samples were centrifuged at 13,000 rpm with an Eppendorf centrifuge, and the supernatants were pipetted into 5-mm NMR tubes for NMR analysis. NMR spectroscopy. Proton-decoupled 13C NMR spectra of blood extracts were obtained using a 5-mm probe on a 9.4 T Bruker Omega spectrometer operating at 100.61 MHz. Freeinduction decays were digitized into 32 K points and were routinely multiplied by a 0.5- to 1.0-Hz exponential before Fourier transformation. Proton-decoupled 13C NMR spectra of urine extracts were obtained with a Unity INOVA 14.5 T spectrometer operating at 150.9 MHz (Varian Instruments, Palo Alto, CA). Free-induction decays were digitized into 44 K points and were multiplied by a 0.5-Hz exponential before Fourier transformation. Spectra from both systems were analyzed using the curve-fitting routine supplied with the NUTS PC-based NMR spectral analysis program (Acorn NMR, Fremont, CA). Typical numbers of acquisitions were 12,000– 18,000 for each blood extract and 6,000–12,000 for each urine extract. Collection times per extract ranged from 4.5 to 13.5 h. Quantitation of 13C enrichments. 13C enrichments of carbons in the glutamine moiety of phenylacetylglutamine were quantitated directly from the 13C NMR spectrum of the isolated phenylacetylglutamine by use of the method of Dugelay et al. (11). Gluconate 13C enrichments were quantitated directly from the 13C NMR spectrum by comparing the total area of each gluconate resonance with the C-2 resonance of the glycolate standard. Correction factors to account for T1 MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE E845 Figure 4 shows the complete 13C NMR spectrum of gluconate from the 120-min blood sample. The nineline C-2 resonance indicates that all four possible 13C isotopomers are present in the C-1 to C-3 triose unit, the relative ratios of which provide quantitative information about metabolism of [U-13C]propionate to glucose (18). The key components of this metabolic network are anaplerotic inflow (y), oxaloacetate-pyruvate (OAA-PYR) recycling (pk), net gluconeogenic outflow (g), and citrate synthase flux. Anaplerotic inflow is the total influx of both new and recycled C-4 units into the oxaloacetate pool. OAA-PYR recycling Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 2. 13C NMR spectra of gluconate carbons 1 (GL-1) and 2 (GL-2) obtained by oxidation of glucose from a single set of plasma extracts obtained at the stated time intervals after ingestion of [U-13C]propionate. and nuclear Overhauser enhancement differences between the glycolate C-2 and gluconate carbons were derived from the time 0 blood extract, where all the gluconate carbons are at natural abundance enrichment. Metabolic flux calculations. Estimates of total anaplerotic inflow into oxaloacetate (y), recycling of oxaloacetate carbons via pyruvate (pk), and net gluconeogenic flux (g) (all relative to citrate synthase flux) were calculated from the 13C-13C spin-coupled multiplets of gluconate C-2, phenylacetylglutamine C-2, and glucuronate C-5b by use of methods described previously (18). RESULTS 13C NMR spectra from blood samples. Figure 2 shows NMR spectra of gluconate C-1 and C-2 from blood drawn from an individual at various times after oral ingestion of [U-13C]propionate. Initially, only singlets from natural abundance 13C were detected, but by 20 and 40 min [U-13C]propionate conversion into glucose was already evident from the appearance of 13C-13C spin-coupled multiplets in all six gluconate resonances. The intensity of the multiplet resonances increased gradually for 120 min and then remained relatively constant between 120 and 180 min. In all subjects, the nine-line C-2 gluconate multiplet was clearly formed at 120 min, and the ratios of multiplet components remained constant between 150 and 180 min. The time course of gluconate fractional enrichment from 0 to 180 min is shown in Fig. 3. Carbons 1, 2, 5, and 6 of gluconate showed identical enrichment kinetics, reaching a peak enrichment of ,4.5%1, whereas carbons 3 and 4 reached peak enrichments of ,2.5%. Between 120 and 180 min, enrichment of all six gluconate carbons remained relatively constant. 13C 1 Enrichment values include the natural abundance tion (assumed to be 1.11%). 13C contribu- Fig. 3. Time course of enrichment of plasma gluconate carbons 1–6 (GL-1–GL-6) from 0–180 min after ingestion of [U-13C]propionate. Each plot represents means 6 SD for 5 individuals given 20–25 mg/kg [U-13C]propionate and does not include the 6th individual, who received 17 mg/kg propionate. E846 MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE flux is the combined activities of all pathways that convert OAA to phosphoenolpyruvate (PEP) and back to OAA via pyruvate carboxylase. These include OAA=PEP=PYR=OAA (the pyruvate kinase pathway) and OAA=MAL=PYR=OAA (the malic enzyme pathway) in hepatocytes, and, through peripheral catabolism and hepatic resynthesis of glucose, the Cori and glucose-alanine cycles. Gluconeogenic flux, the difference between total anaplerotic inflow and total OAA-PYR recycling, represents net export of carbons from the metabolic network. Although carbons can leave the citric acid cycle through pathways other than gluconeogenesis (for example as aspartate and glutamate), it is generally assumed that gluconeogenesis accounts for the bulk of the total anaplerotic carbon outflow in liver (30). Given certain assumptions2, simple mathematical equations relate the glucose 13C isotopomers (as reported by the C-2 resonance) to relative fluxes through these pathways (18). Urine analysis. The time course of urinary phenylacetylglutamine enrichment was similar to that of blood glucose, with peak enrichments being achieved 2–3 h after ingestion of propionate (Fig. 5). However, the 13C fractional enrichment in C-1, C-2, and C-3 of phenylacetylglutamine was significantly lower than that of C-1, C-2, C-5, or C-6 of glucose or glucuronate (compare Figs. 3 and 5). This difference is thought to be due to dilution of the glutamine pool by inflow of unenriched glutamine from peripheral tissues such as skeletal muscle (24, 30). Figure 6 shows the 20- to 100-ppm region of a 13C NMR spectrum of urine 2 Key assumptions are 1) isotopic steady-state conditions, 2) negligible labeling of acetyl-CoA, and 3) complete randomization of oxaloacetate carbons via fumarate. Fig. 5. Time course of enrichment of carbons 1–4 from glutamine moiety of urinary phenylacetylglutamine 30–330 min after ingestion of [U-13C]propionate. Times denote midpoint of each hourly urine collection. Each plot represents means 6 SD for 5 individuals given 20–25 mg/kg [U-13C]propionate and does not include the 6th individual, who received 17 mg/kg propionate. j, Carbon 1; k, carbon 2; s, carbon 3; r, carbon 4. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 4. Complete 13C NMR spectrum of plasma gluconate from a 120-min blood sample and isotopomer analysis of 9-line multiplet of carbon 2 (inset). GL-1 to GL-6 refer to resonances of gluconate carbons 1–6. In inset, S, singlet resonance of carbon 2; D12, doublet from coupling of carbon 2 with neighboring carbon 1; D23, doublet arising from coupling of carbon 2 with neighboring carbon 3; Q, doublet of doublets, or quartet, arising from coupling of carbon 2 with both its neighbors. collected 2 h after the last propionate ingestion. The C-2, C-3, and C-4 resonances of the glutamine moiety of phenylacetylglutamine are prominent, as is the natural abundance signal of the phenylacetyl methylene carbon at 43 ppm. The phenylacetylglutamine C-2 and C-3 resonances appear as multiplets arising from 13C-13C spin-coupling, whereas the C-4 resonance is largely a singlet (flanked by a weak doublet) arising primarily from natural abundance levels of 13C. This indicates that few of the 13C-enriched gluconeogenic precursors that leave the cycle actually reenter the cycle pools via acetyl-CoA, thus validating one important simplification of the metabolic model (18). Several key glucuronate multiplets are also fully resolved in the 13C NMR urinary spectrum, including the C-5b resonance (see Fig. 6). The C-5b carbon of glucuronate, like that of gluconate C-2 and phenylacetylglutamine C-2, has different coupling constants from its nearest neighboring carbons (JC-5–C-6 5 59 Hz and JC-5–C-4 5 40 Hz) and hence appears also as nine resolved resonances. The relative areas of the gluconate C-2, glucuronate C-5b, and glutamine C-2 multiplets are compared in Table 1. The multiplet areas of glucuronate C-5b will be identical to those of gluconate C-2 (from blood) if the distribution of 13C isotopomers is identical in dihydroxyacetone phosphate (triose unit reflected by gluconate C-2) and glyceraldehyde-3phosphate (triose unit reflected by glucuronate C-5b). Because both species originate from PEP with no rearrangement of the carbon skeleton, their labeling patterns should be identical, with the assumption of negligible pentose cycle activity (28). However, the fractional enrichments of the two pools can differ as a result of 1) incomplete equilibration of the label by triose phosphate isomerase and 2) influx of unlabeled E847 MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE glycerol into the dihydroxyacetone phosphate pool. Both of these effects dilute the 13C enrichment of dihydroxyacetone phosphate relative to glyceraldehyde3-phosphate; hence, carbons 4, 5 and 6 of glucose-6phosphate will be more enriched than carbons 1, 2 and 3. Within both glucuronate and gluconate, carbons 4, 5 and 6 had a slight but systematic elevation of 13C enrichment compared with the corresponding carbons 1, 2 and 3, consistent with a small dilution from this source. Significant excesses in labeling of the bottom Table 1. Multiplet areas from the 13C NMR spectra of gluconate C-2, glucuronate C-5b and phenylacetylglutamine C-2 for six normal individuals following a 24-hour fast Subject Gluconate C-2 Glucuronate C-5b Phenylacetylglutamine C-2 ML S D12 D23 Q 0.289 6 0.024 0.419 6 0.002 0.056 6 0.001 0.237 6 0.023 S D56 D45 Q 0.262 6 0.026 0.445 6 0.004 0.060 6 0.008 0.234 6 0.023 S D23 D12 Q 0.396 6 0.032 0.374 6 0.011 0.060 6 0.008 0.161 6 0.016 GA* S D12 D23 Q 0.260 6 0.032 0.420 6 0.039 0.059 6 0.008 0.244 6 0.017 S D56 D45 Q 0.353 6 0.004 0.409 6 0.016 0.053 6 0.011 0.163 6 0.032 S D23 D12 Q 0.469 6 0.062 0.358 6 0.063 0.050 6 0.001 0.124 6 0.001 DE S D12 D23 Q 0.336 6 0.030 0.390 6 0.018 0.072 6 0.016 0.202 6 0.008 S D56 D45 Q 0.343 6 0.035 0.428 6 0.001 0.051 6 0.011 0.178 6 0.008 S D23 D12 Q 0.513 6 0.039 0.345 6 0.031 0.047 6 0.004 0.116 6 0.020 MW S D12 D23 Q 0.322 6 0.063 0.403 6 0.027 0.068 6 0.009 0.205 6 0.040 S D56 D45 Q 0.217 6 0.031 0.432 6 0.024 0.067 6 0.010 0.284 6 0.003 S D23 D12 Q 0.527 6 0.069 0.317 6 0.034 0.043 6 0.008 0.113 6 0.028 SH † S D12 D23 Q 0.338 6 0.041 0.429 6 0.034 0.058 6 0.005 0.175 6 0.015 S D56 D45 Q 0.251 0.465 0.086 0.198 S D23 D12 Q 0.389 0.476 0.041 0.094 JJ S D12 D23 Q 0.478 6 0.047 0.350 6 0.034 0.043 6 0.001 0.129 6 0.011 S D23 D12 Q 0.567 6 0.062 0.330 6 0.030 0.036 6 0.015 0.069 6 0.016 ND ND ND ND Values are means6SD of 120-, 150-, and 180-min blood samples for each individual (gluconate) or of 2- to 3-h and 3- to 4-h urine samples for each individual (glucuronate and phenylacetylglutamine). Multiplet nomenclature is as in Fig. 6. ND, not determined. *Glucuronate multiplets (means6SD) were obtained from 1- to 2-h and 2- to 3-h urine spectra. †Glucuronate and phenylacetylglutamine multiples obtained from a single urine collection (2–4 h). Gluconate multiplet means were obtained from 120- and 180-min blood collections, since no blood was collected at 150 min from this subject. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 Fig. 6. Region (20 to 100 ppm) from a 150.9MHz proton-decoupled 13C NMR spectrum of a purified urine extract. Extract was obtained from urine collected 2–3 h after ingestion of [U-13C]propionate. PAG-2, PAG-3, and PAG-4 are carbons 2, 3 and 4 of the glutamine moiety of phenylacetylglutamine; PAM is the methylene carbon of phenylacetyl moiety of phenylacetylglutamine; H-1b, H-1a, and H-5b are carbons 1b, 1a, and 5b of glucuronate; 3-OHP-2 is carbon 2 of 3-hydroxypropionate; 3-OHP-3 is carbon 3 of 3-hydroxypropionate; A-2 is carbon 2 of acetate. For the PAG-2 and PAG-3 insets: S, singlet resonance; D12, doublet from coupling of carbon 2 with neighboring carbon 1; D23, doublet arising from coupling of carbon 2 with neighboring carbon 3; Q, doublet of doublets, or quartet, arising from coupling of carbon 2 with both its neighbors. For the H-5b inset: S, singlet resonance; D56, doublet arising from coupling of carbon 5 with neighboring carbon 6; D45, doublet arising from the coupling of carbon 5 with neighboring carbon 4; Q, doublet of doublets, or quartet, arising from coupling of carbon 5 with both its neighbors. E848 MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE components of the parent hepatic glucose-6-phosphate molecule had similar 13C isotopomer distributions. DISCUSSION Analytic considerations. We have shown that complex 13C isotopomer distributions of phenylacetylglutamine, glucuronate, and glucose produced by hepatic metabolism of [U-13C]propionate can be analyzed in a straightforward manner by use of 13C NMR spectroscopy. The analyte pools, particularly the urinary products, provide sufficient material to quantitate isotopomer distributions by 13C NMR at 13C enrichment levels only 1–2% above natural abundance. 13C NMR proved to be a particularly valuable tool for direct isotopomer analysis of the glucuronide hexose skeleton after its enzymatic hydrolysis from the parent acetaminophen b-glucuronide. There are three advantages to the methods reported here compared with previously published methods. First, the determination of positional 13C glucose isotopomers appears to be much simpler by 13C NMR than by gas chromatography-mass spectrometry. Second, others have isolated acetaminophen-b-glucuronide from urine and converted the glycone moiety to glucose by chemical reduction of the carboxy glycone C-6 followed by enzymatic cleavage of the b-glucoside product (27, 28). Again, the chemical approach reported here appears simpler and provides complete recovery of labeled urinary glycones, an important consideration given the relatively low sensitivity of 13C NMR compared with MS methods. In situ hydrolysis of the b-glucuronide may also have advantages in the clinical setting. A wide range of medications and drugs are cleared by glucuronidation; hence, a patient has the potential to produce a complex cocktail of urinary b-glucuronides even in the absence of acetaminophen. For such an individual, collective hydrolysis in situ should be far easier than quantitative isolation, reduction, and hydrolysis of the complex glucuronide mixture. Finally, our simple urine purification procedure retains both phenylacetylglutamine and glucuronide, thereby allowing a 13C isotopomer analysis of both metabolites from a single 13C NMR spectrum. Table 2. Estimations of total anaplerosis, oxaloacetate-pyruvate recycling, and net anaplerotic outflow from 13C multiplets of gluconate, glucuronate, and phenylacetylglutamine from 24-h-fasted human subjects Gluconate C-2 Subject ML GA DE MW SH JJ Means 6 SD Glucuronate C-5b Phenylacetylglutamine C-2 y pk g y pk g y pk g 6.48 6.12 4.42 4.93 6.40 7.14 3.25 2.98 2.61 2.91 4.38 5.14 3.23 3.14 1.81 2.02 2.02 2.00 6.42 6.72 7.39 5.45 4.41 ND 3.45 4.64 4.90 2.21 3.10 ND 2.97 2.08 2.49 3.24 1.31 ND 5.23 6.16 6.34 6.37 10.60 8.16 3.55 4.68 4.87 4.74 9.32 7.25 1.68 1.48 1.47 1.63 1.28 0.91 5.92 6 1.03 3.55 6 0.99a 2.37 6 0.64c 6.08 6 1.16 3.66 6 1.11a 2.42 6 0.76c 7.14 6 1.94 5.74 6 2.13b 1.41 6 0.28d y, Estimations of total anaplerosis; pk, oxaloacetate-pyruvate recycling; g, net anaplerotic outflow. each other (P , 0.05); c and d are significantly different from each other (P , 0.01). a and b are significantly different from Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 one-half vs. the top one-half of plasma glucose after infusion of [14C]lactate have been reported in fed humans, but both halves were equally labeled after 60 h of fasting (30). Our observations fall between these two extremes, consistent with the intermediate 24-h fasting period used in our study. The 13C NMR spectra of urine samples from three subjects featured weak multiplet signals at 58 and 39 ppm, which on the basis of chemical shifts and coupling constants have been tentatively assigned to carbons 2 and 3 of 3-[U-13C]hydroxypropionate. The C-1 carboxy resonance of this metabolite was obscured by numerous multiply labeled and natural abundance carboxy resonances that occupy a crowded region of the 13C NMR spectrum. 3-[U-13C]hydroxypropionate is the terminal b-oxidation product of [U-13C]propionyl-CoA, and its formation has been associated with saturation of the propionyl-CoA carboxylase pathway (1). The fact that only the uniformly labeled isotopomer was observed indicates that the carbon skeleton of [U-13C]propionate was intact when it entered the hepatic [U-13C]propionylCoA pool. Metabolic analysis. For calculations of y, pk, and g, we selected blood spectra from 120 to 180 min, when the 13C enrichment was relatively constant. These were compared with the 2- to 3-h and 3- to 4-h urine samples, which sampled the hepatic metabolite pools at ,1.25– 2.25 h and 2.25–3.25 h, respectively (7). The gluconate C-2, glucuronate C-5b, and phenylacetylglutamine C-2 multiplet areas (Table 1) were substituted into equations previously presented (18) to give estimates of y, pk, and g (Table 2). For all three metabolites, total anaplerotic flux relative to citrate synthase was estimated at 6.0–7.0. However, the phenylacetylglutamine analysis reported a significantly higher OAA-PYR recycling flux than either glucose and glucuronate, and this resulted in a significantly lower estimate of net gluconeogenesis compared with the hexose analyses (1.4 vs. 2.4). These data parallel the results of Landau et al. (24), in which net gluconeogenic fluxes reported by glucose and phenylacetylglutamine after infusion of [14C]propionate were ,3.0 and ,1.8, respectively (24). With the exception of one individual, the multiplet ratios of glucuronate C-5b and gluconate C-2 in all subjects were similar, providing consistent values for y, pk, and g (see Table 2). This indicates that both triose MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE µmol · kg21 · min21 (3.6/2.4 3 11.2). In relation to these fluxes, metabolism of 1.5 g of sodium [U-13C]propionate over 1–2 h represents a consumption rate of 1.6–3.2 µmol · min21 · kg21, which is 6–12% of the total anaplerotic inflow. Metabolic flux estimates. Anaplerotic flux (y), determined here by 13C NMR analysis of glucose/glucuronate, is slightly lower (6.0 vs. 7.0) than that reported by Magnusson et al. (30), whereas pyruvate-oxaloacetate recycling (pk) flux was similar (,3.6 vs. 3.7). Although estimates of pk may be inflated by flux from the Cori cycle (23, 30), we believe that our study protocol minimized any contribution from extrahepatic pathways. The Cori cycle has been estimated to contribute maximally ,15% to the total hepatic glucose output in starved rats (23). Any lactate generated in extrahepatic tissues from 13C-enriched glucose and returned to the liver via the Cori cycle would supplement estimates of intrahepatic pyruvate-oxaloacetate recycling. With the assumption of steady-state conditions and no glycogenolysis, a 15% Cori cycle contribution would result in a 15% underestimation of g, and pk would be correspondingly increased (y being unchanged). The effects of this level of Cori cycling on pk are rather modest; 15% of g amounts to 0.36 units or ,10% of our pk estimate. However, it has been estimated that about one-third of the total hepatic glucose output is derived from glycogenolysis (6a) after a 24-h fast, so dilution of blood glucose with unlabeled glucose via glycogenolysis would further attenuate the 10% overestimate of pk to ,3%. Thus we conclude that the bulk of the observed pk flux activity detected in this study originates from hepatic metabolism. Our estimate of net gluconeogenic flux was somewhat lower than the value (,2.4 vs. 3.3) reported by Magnusson et al. (30). One explanation could be the difference between the fasting periods in the two studies (24 h here vs. 60 h in Magnusson et al.). It has been shown that 3-carbon gluconeogenic sources contribute 67% of the total hepatic glucose output after a 22-h fasting period but .90% after a 42-h fasting period (6a). We would expect that this increased demand for 3-carbon precursors would be fulfilled in part by an increased ratio of gluconeogenic to citrate synthase flux. However, the additional gluconeogenic precursors could also be provided by a systematic increase in flux through all the pathways without any change in flux ratios. Hence, the modest differences between our flux estimates and those of Magnusson et al. may simply be methodological in origin. Conversion of relative to absolute fluxes by indexing g to the gluconeogenic portion of hepatic glucose output by use of additional tracers (15, 25, 30) will better resolve this uncertainty and is the focus of our ongoing studies. Finally, because net anaplerotic efflux can also be directed into nongluconeogenic pathways, such as glutathione biosynthesis, g represents the upper limit of glucose output from the tricarboxylic acid cycle. Effects of 13C reincorporation via bicarbonate on the measurements. Measurements of gluconeogenesis by use of singly labeled substrates such as [3-14C]lactate Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 Ingestion of gram quantities of [U-13C]propionate. Because 13C is a nonradioactive isotope and bulk substitution of 12C by 13C is not known to have a significant kinetic isotopic effect, substantial quantities of 13Cenriched substrates can be safely given to humans (7, 13, 14, 35). However, such experiments can generate supraphysiological substrate levels that could alter basal metabolic activities. In our study, fasted individuals ingested an average of 1.5 g of propionate over 1 h. With the assumptions of quantitative capture of propionate by portal circulation and a portal vein blood flow of ,700 ml/min (10), one can estimate that the average portal propionate concentration is no higher than 0.36 mM over the 1-h period. Portal vein propionate concentrations averaging 0.03 mM have been reported in patients undergoing gall-bladder surgery (9), but these probably represent fasting levels, which are likely to be much less than postprandial concentrations. The only other measurements of human portal vein propionate concentrations have been obtained by autopsy of sudden death victims, for whom levels ranging from 0.02 to 0.19 mM were reported (8). Measurements of portal vein propionate levels in the pig, a good model for human digestion, indicated levels of 0.08 mM under fasting conditions and 0.25–0.40 mM after feeding (38). These postprandial levels span the estimated portal vein propionate levels generated during the experimental protocol. Nonetheless, the fact that urinary 3-[U13C]hydroxypropionate was detected in several subjects suggests that the hepatic propionyl-CoA carboxylase can become saturated (1), and physiological consequences of saturating propionyl-CoA carboxylase in normal fasted humans are not known. In rat hepatocytes, high levels of propionate can inhibit the activities of several enzymes by lowering the free CoA levels (4, 5, 33), including pyruvate carboxylase. Because pyruvate carboxylase is the predominant anaplerotic pathway in the liver, inhibition of this enzyme can result in decreased gluconeogenic flux (4, 6). However, in fasted humans, the provision of 1.2 g of propionate over 3 h did not alter hepatic glucose production in fasted volunteers (26), suggesting that hepatic gluconeogenesis was maintained under these conditions. Our study featured comparable amounts of ingested propionate (,1.5 g), but the shorter ingestion period of our protocol probably generated higher levels of propionate in portal circulation. Nonetheless, our estimated portal vein propionate levels of 0.36 mM are still far below the 5 to 10 mM concentrations reported to inhibit gluconeogenesis and citric acid cycle fluxes (4, 6, 32). The maximal contribution of propionate carbons to the total anaplerotic flux can also be estimated. From the study of Chandramouli et al. (6a), gluconeogenic flux in a 24-h-fasted individual is ,5.6 µmol · kg21 · min21 out of a total hepatic glucose output of 8.6 µmol · kg21 · min21. Setting g equal to 11.2 µmol · kg21 · min21 of triose equivalents and applying the relative flux measurements from our study give a citrate synthase flux value of 4.7 µmol · kg21 · min21 (1/2.4 3 11.2), a total anaplerotic inflow value of 27.8 µmol · kg21 · min21 (5.9/ 2.4 3 11.2), and a pyruvate recycling flux value of 16.6 E849 E850 MEASUREMENTS OF GLUCONEOGENESIS WITH [U-13C]PROPIONATE 3 Gluconate C-6 analysis was chosen over the C-1 carboxy terminus because of its higher signal-to-noise ratio and the possibility of faster relaxation of the C-1 doublet relative to the C-1 singlet. the perivenous region, including periportal contributions (in the form of glutamate). Why are these labeling differences revealed with propionate but not with lactate? A key difference in the hepatic metabolism of propionate vs. lactate is that the former is quantitatively extracted from the circulation, whereas the latter is not (34). Because mitochondrial density is highest in the periportal region of the liver (39), it is possible that a propionate tracer is preferentially consumed by the periportal cells compared with a lactate tracer. Therefore, downstream regions could receive little direct labeling from propionate while receiving significant contributions from secondary labeled products of periportal propionate metabolism, including glucose. Reutilization of hepatic glucose by the perivenous region is consistent with the zonal model of regulated hepatic glucose output proposed by Jungermann and colleagues (19–22), in which periportal glucose synthesis is appropriately attenuated by perivenal glucose reutilization. Utilization of secondary labeled products by the perivenous region could generate quite a different oxaloacetate isotopomer distribution than that produced from periportal metabolism of [U-13C]propionate if the two regions have different metabolic flux characteristics. In contrast, a lactate tracer is metabolized more uniformly across the lobule and therefore generates an averaged labeling distribution in glucose and glutamine. The fact that glucose and glutamate were labeled equivalently in perfused rat livers supplied with supraphysiological and saturating levels of [U-13C]propionate, lactate, and pyruvate (18) suggests that intralobular substrate gradients may be an important determinant of labeling heterogeneity among different metabolites in the liver. In conclusion, we have shown that a detailed analysis of the relative rates of anaplerosis, oxaloacetatepyruvate cycling, and gluconeogenesis can be obtained in a rather simple procedure by 13C NMR analysis of blood and urine after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen. Our results are in good agreement with other recently published studies that used 14C-labeled radioisotopes. Finally, the simple processing protocols for both blood and urine are well within the capability of a standard medical analytic laboratory and its personnel. We acknowledge the excellent technical assistance and support provided by the staff of the General Research Clinical Center at University of Texas Southwestern. This research was supported by National Institutes of Health Grants RR-02584, HL-34557, and M01-RR-00633 and a Clinical Investigator Award from the Department of Veterans Affairs. Address for reprint requests: J. G. Jones, Mary Nell and Ralph B. Rogers Magnetic Resonance Center, 5801 Forest Park Rd., Dallas, TX 75235–9085. Received 29 April 1998; accepted in final form 23 July 1998. REFERENCES 1. Ando, T., K. Rasmussen, W. L. Nyhan, and D. Hull. 3-[U13C]hydroxypropionate: significance of b-oxidation of propionate in patients with propionic acidemia and methylmalonic acidemia. Proc. Natl. Acad. Sci. USA 69: 2807–2811, 1972. Downloaded from http://ajpendo.physiology.org/ by 10.220.33.1 on May 3, 2017 rely on determination of the ratio of label in glucose C-1/C-3 or glutamate C-2/C-1. These measurements are exquisitely sensitive to the reincorporation of label into glucose C-3 and glutamate C-1 via carboxylation of labeled bicarbonate (24, 30). To obtain the necessary correction factors, an additional labeling experiment using labeled bicarbonate must be performed (24, 30). By comparison, the glucose and phenylacetylglutamine multiplets observed in the 13C NMR spectra of these metabolites have differing degrees of sensitivity to enrichment from [13C]bicarbonate. The glucose C-1 (and C-6) and phenylacetylglutamine C-3 multiplets are completely insensitive to contributions from [13C]bicarbonate, because they originate from carbons 2 and 3 of oxaloacetate (18). 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