Urinary Phenylacetylglutamine Research Articles

Urinary phenylacetylglutamine as a possible biomarker for central nervous system disorders in forensic autopsy cases

Phenylacetylglutamine (PAG) is a metabolite that is excreted in human urine. Phenylalanine is metabolized to phenylacetic acid, which is then amide-bonded to glutamine to form PAG. We are currently studying PAG as a urinary biomarker in forensic autopsy cases. Materials and MethodsUrine samples were collected from 188 forensic autopsy cases and the urinary PAG concentration was analyzed quantitatively using GC-MS. Urinary creatinine (Cr) concentration was also analyzed by GC-MS. For statistical analysis, the JMP Pro 15.0.0 software program was used. The relationship between urine PAG/Cr (the ratio of each concentration), sex, age, postmortem interval (PMI), survival duration, and cause of death was statistically analyzed. Results and DiscussionThe median (range) of PAG/Cr was 0.12 (0.002-3.26). The PAG/Cr ratio showed no significant relationship to sex or survival duration. Regarding the cause of death, traumatic brain injuries had a significantly higher ratio than intoxication (p=0.023). Cerebrovascular disease, such as cerebral hemorrhage and subarachnoid hemorrhage, did not differ significantly from any cause of death group. However, when traumatic brain injuries and cerebrovascular accidents are combined as one cause of death group, the PAG/Cr value of CNS damages was significantly higher than that of intoxication (p=0.062). ConclusionUrinary PAG/Cr might be a biomarker not only for traumatic brain injuries but also for antemortem CNS damages.

Urine Phenylacetylglutamine Determination in Patients with Hyperphenylalaninemia

Phenylketonuria (PKU), an autosomal-recessive inborn error of phenylalanine (Phe) metabolism is the most prevalent disorder of amino acid metabolism. Currently, clinical follow-up relies on frequent monitoring of Phe levels in blood. We hypothesize that the urine level of phenylacetylglutamine (PAG), a phenyl-group marker, could be used as a non-invasive biomarker. In this cross-sectional study, a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS) method was used for urinary PAG quantification in 35 participants with hyperphenylalaninemia (HPA) and 33 age- and sex-matched healthy controls. We have found that (a) PKU patients present higher urine PAG levels than healthy control subjects, and that (b) there is a significant correlation between urine PAG and circulating Phe levels in patients with HPA. In addition, we show a significant strong correlation between Phe levels from venous blood samples and from capillary finger-prick dried blood spot (DBS) samples collected at the same time in patients with HPA. Further research in order to assess the potential role of urine PAG as a non-invasive biomarker in PKU is warranted.

Safety, tolerability, and pharmacokinetics of l-ornithine phenylacetate in patients with acute liver injury/failure and hyperammonemia.

OPA was well-tolerated in patients with ALI/ALF, and no safety signals were identified. Target [PA] was achieved at infusion rates of 20 g every 24 hours, leading to ammonia excretion in urine as PAGN in proportion to renal function. Randomized, controlled studies of high-dose OPA are needed to determine its use as an ammonia-scavenging agent in patients with ALF. (Hepatology 2018;67:1003-1013).

Urinary phenylacetylglutamine (U-PAGN) concentration as biomarker for adherence in patients with urea cycle disorders (UCD) treated with glycerol phenylbutyrate

Urinary phenylacetylglutamine (U-PAGN) concentrations in spot urine samples were analyzed as a dosing biomarker during glycerol phenylbutyrate (GPB) dosing in 68 healthy adults and 66 adult and pediatric patients with urea cycle disorders who participated in GPB clinical trials. Age- and body surface area (BSA)-specific 25th percentile cutoff points for spot U-PAGN concentrations (<~9000μg/mL for <2years old patients, <7000μg/mL for >2years with BSA ≤1.3m2, and <~5000μg/mL for >2years of age with BSA >1.3m2) were determined as an approach to identify patients for whom increased dosing and/or adherence to prescribed dosing should be assessed.

Population Pharmacokinetic Modeling and Dosing Simulations of Nitrogen‐Scavenging Compounds: Disposition of Glycerol Phenylbutyrate and Sodium Phenylbutyrate in Adult and Pediatric Patients with Urea Cycle Disorders

Sodium phenylbutyrate and glycerol phenylbutyrate mediate waste nitrogen excretion in the form of urinary phenylacetylglutamine (PAGN) in patients with urea cycle disorders (UCDs); rare genetic disorders characterized by impaired urea synthesis and hyperammonemia. Sodium phenylbutyrate is approved for UCD treatment; the development of glycerol phenylbutyrate afforded the opportunity to characterize the pharmacokinetics (PK) of both compounds. A population PK model was developed using data from four Phase II/III trials that collectively enrolled patients ages 2 months to 72 years. Dose simulations were performed with particular attention to phenylacetic acid (PAA), which has been associated with adverse events in non-UCD populations. The final model described metabolite levels in plasma and urine for both drugs and was characterized by (a) partial presystemic metabolism of phenylbutyric acid (PBA) to PAA and/or PAGN, (b) slower PBA absorption and greater presystemic conversion with glycerol phenylbutyrate, (c) similar systemic disposition with saturable conversion of PAA to PAGN for both drugs, and (d) body surface area (BSA) as a significant covariate accounting for age-related PK differences. Dose simulations demonstrated similar PAA exposure following mole-equivalent PBA dosing of both drugs and greater PAA exposure in younger patients based on BSA.

Ammonia Control in Children Ages 2 Months through 5 Years with Urea Cycle Disorders: Comparison of Sodium Phenylbutyrate and Glycerol Phenylbutyrate

To examine ammonia levels, pharmacokinetics, and safety of glycerol phenylbutyrate (GPB; also referred to as HPN-100) and sodium phenylbutyrate (NaPBA) in young children with urea cycle disorders (UCDs). This open label switch-over study enrolled patients ages 29 days to under 6 years taking NaPBA. Patients underwent 24-hour blood and urine sampling on NaPBA and again on a phenylbutyric acid-equimolar dose of GPB and completed questionnaires regarding signs and symptoms associated with NaPBA and/or their UCD. Fifteen patients (8 argininosuccinate lyase deficiency, 3 argininosuccinic acid synthetase deficiency, 3 ornithine transcarbamylase deficiency, 1 arginase deficiency) ages 2 months through 5 years enrolled in and completed the study. Daily ammonia exposure (24-hour area under the curve) was lower on GPB and met predefined noninferiority criteria (ratio of means 0.79; 95% CI 0.593-1.055; P=.03 Wilcoxon; 0.07 t test). Six patients experienced mild adverse events on GPB; there were no serious adverse events or significant laboratory changes. Liver tests and argininosuccinic acid levels among patients with argininosuccinate lyase deficiency were unchanged or improved on GPB. Eleven of 15 patients reported 35 symptoms on day 1; 23 of these 35 symptoms improved or resolved on GPB. Mean systemic exposure to phenylbutyric acid, phenylacetic acid, and phenylacetylglutamine (PAGN) were similar and phenylacetic acid exposure tended to be higher in the youngest children on both drugs. Urinary PAGN concentration was greater on morning voids and varied less over 24 hours on GPB versus NaPBA. GPB results in more evenly distributed urinary output of PAGN over 24 hours were associated with fewer symptoms and offers ammonia control comparable with that observed with NaPBA in young children with UCDs.

Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders

We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). Study designThese analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6–17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. ResultsPatients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r=0.730, p<0.001) or as total 24-hour excretion (r=0.791 p<0.001), followed by plasma phenylacetylglutamine AUC24-hour, plasma phenylacetic acid AUC24-hour and phenylbutyric acid AUC24-hour. Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. ConclusionThe findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.

Phase 2 comparison of a novel ammonia scavenging agent with sodium phenylbutyrate in patients with urea cycle disorders: Safety, pharmacokinetics and ammonia control

Glycerol phenylbutyrate (glyceryl tri (4-phenylbutyrate)) (GPB) is being studied as an alternative to sodium phenylbutyrate (NaPBA) for the treatment of urea cycle disorders (UCDs). This phase 2 study explored the hypothesis that GPB offers similar safety and ammonia control as NaPBA, which is currently approved as adjunctive therapy in the chronic management of UCDs, and examined correlates of 24-h blood ammonia. Methods An open-label, fixed sequence switch-over study was conducted in adult UCD patients taking maintenance NaPBA. Blood ammonia and blood and urine metabolites were compared after 7 days (steady state) of TID dosing on either drug, both dosed to deliver the same amount of phenylbutyric acid (PBA). Results Ten subjects completed the study. Adverse events were comparable for the two drugs; 2 subjects experienced hyperammonemic events on NaPBA while none occurred on GPB. Ammonia values on GPB were ∼30% lower than on NaPBA (time-normalized AUC = 26.2 vs. 38.4 μmol/L; Cmax = 56.3 vs. 79.1 μmol/L; not statistically significant), and GPB achieved non-inferiority to NaPBA with respect to ammonia (time-normalized AUC) by post hoc analysis. Systemic exposure (AUC 0–24) to PBA on GPB was 27% lower than on NaPBA (540 vs. 739 μg h/mL), whereas exposure to phenylacetic acid (PAA) (575 vs. 596 μg h/mL) and phenylacetylglutamine (PAGN) (1098 vs. 1133 μg h/mL) were similar. Urinary PAGN excretion accounted for ∼54% of PBA administered for both NaPBA and GPB; other metabolites accounted for <1%. Intact GPB was generally undetectable in blood and urine. Blood ammonia correlated strongly and inversely with urinary PAGN ( r = −0.82; p < 0.0001) but weakly or not at all with blood metabolite levels. Conclusions Safety and ammonia control with GPB appear at least equal to NaPBA. Urinary PAGN, which is stoichiometrically related to nitrogen scavenging, may be a useful biomarker for both dose selection and adjustment for optimal control of venous ammonia.

A metabonomic approach identifies human urinary phenylacetylglutamine as a novel marker of interstitial cystitis

An ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) based metabonomic approach was applied to identify a candidate metabolite with not known to be associated with interstitial cystitis (IC). IC is a chronic clinical syndrome associated with urinary frequency and urgency and/or pelvic pain. The ability to non-invasively diagnose the early stage of IC would be important for improving the patient's quality of life. The current standard IC diagnosis is cystoscopy, which is invasive and painful. Urine samples from the following were taken and analyzed: 10 IC patients, 10 bacterial cystitis (BC) patients, and 10 healthy volunteers (HVs) to identify an IC marker; and subsequently analyzed 5 IC patients and 5 HVs for marker validation. The urinary marker of IC was identified as phenylacetylglutamine (PAGN) using NMR and MS/MS analysis. In addition, quantitative methods were developed to determining the urinary PAGN levels using UPLC-UV. The urinary level of PAGN measured relative to creatinine (Cr) was significantly elevated in IC patients (mean 0.47 mg/mg Cr) compared with BC patients (mean 0.25 mg/mg Cr) and HVs (mean 0.11 mg/mg Cr). Interestingly, urinary PAGN/Cr ratios in patients with mild IC (grade I) and moderate IC (grade II) were higher than for patients with severe IC (grade III). Moreover, urinary PAGN/Cr ratios with mild and moderate IC patients (mean 0.30 mg/mg Cr) were higher than HVs (mean 0.059 mg/mg Cr), in the validation set. These findings establish urinary PAGN/Cr ratios as a novel urinary marker of IC, and may contribute to early diagnosis of IC patients.

Contribution of proteolytic and metabolic sources to hepatic glutamine by 2H NMR analysis of urinary phenylacetylglutamine 2H-enrichment from 2H 2O

Proteolytic and cataplerotic sources of hepatic glutamine were determined by 2H NMR analysis of urinary phenylacetylglutamine (PAGN) 2H-enrichments in eight healthy subjects after 2H 2O and phenylbutyric acid ingestion. Body water enrichment was 0.49±0.03%. PAGN was enriched to lower levels with significant differences between the various glutamine positions. PAGN position 2 enrichment=0.33±0.02%; 3 R=0.27±0.02%; 3 S=0.27±0.02% and position 4=0.17±0.01%. Position 3 R, S enrichments are conditional with the net conversion of citrate to glutamate and are therefore markers of cataplerosis. From the ratio of positions 3 R, S to body water enrichment, 55±3% of hepatic glutamine was derived from cataplerosis and 45±3% from proteolysis. In conclusion, enrichment of PAGN 3 R, S hydrogens relative to that of body water reflects the contribution of cataplerotic and proteolytic sources to hepatic glutamine.

L-ornithine and phenylacetate synergistically produce sustained reduction in ammonia and brain water in cirrhotic rats #

Treatment of hyperammonemia and hepatic encephalopathy in cirrhosis is an unmet clinical need. The aims of this study were to determine whether L-ornithine and phenylacetate/phenylbutyrate (administered as the pro-drug phenylbutyrate) (OP) combined are synergistic and produce sustained reduction in ammonia by L-ornithine acting as a substrate for glutamine synthesis, thereby detoxifying ammonia, and the phenylacetate excreting the ornithine-derived glutamine as phenylacetylglutamine in the urine. Sprague-Dawley rats were studied 4 weeks after bile duct ligation (BDL) or sham operation. Study 1: Three hours before termination, an internal carotid sampling catheter was inserted, and intraperitoneal saline (placebo), OP, phenylbutyrate, or L-ornithine were administered after randomization. BDL was associated with significantly higher arterial ammonia and brain water and lower brain myoinositol (P < 0.01, respectively), compared with sham-operated controls, which was significantly improved in the OP-treated animals; arterial ammonia (P < 0.001), brain water (P < 0.05), brain myoinositol (P < 0.001), and urinary phenylacetylglutamine (P < 0.01). Individually, L-ornithine or phenylbutyrate were similar to the BDL group. In study 2, BDL rats were randomized to saline or OP administered intraperitoneally for 6 hours or 3, 5, or 10 days and were sacrificed between 4.5 and 5 weeks. The results showed that the administration of OP was associated with sustained reduction in arterial ammonia (P < 0.01) and brain water (P < 0.01) and markedly increased arterial glutamine (P < 0.01) and urinary excretion of phenylacetylglutamine (P < 0.01) in each of the OP treated groups. The results of this study provide proof of the concept that L-ornithine and phenylbutyrate/phenylacetate act synergistically to produce sustained improvement in arterial ammonia, its brain metabolism, and brain water in cirrhotic rats.

Pharmacology Review: Sodium Phenylacetate and Sodium Benzoate in the Treatment of Neonatal Hyperammonemia

Ammonia is present in all body fluids and exists primarily as ammonium ion at physiologic pH. Hyperammonemia is defined as a blood ammonia concentration greater than about 100 mcmol/L in neonates or 50 mcmol/L in children and adults (precise cut-offs vary, depending on individual laboratory normative ranges). The concentration of ammonia is 10 times higher in tissue than in blood. A 5- to 10-fold increase in blood ammonia concentration usually is toxic to the nervous system. Hyperammonemia in the neonatal period, especially when due to inborn errors of metabolism, can progress rapidly and cause severe neurologic damage or early death. Hyperammonemia can be caused by inborn errors of metabolism as well as by a variety of acquired conditions (Tables 1 and 2). Urgent treatment is required because of the potential for irreversible neurologic sequelae that can, in many cases, be prevented by prompt diagnosis and institution of therapy. | Urea cycle defects | || | Amino acid transporter deficiencies | | Organic acidemias | | Fatty acid oxidation defects | | Pyruvate carboxylase deficiency | | Mitochondrial disorders | | Hyperinsulinism/hyperammonemia syndrome (glutamate dehydrogenase mutations) | | Delta1-pyrroline-5-carboxylate synthase deficiency | Table 1. Inborn Errors of Metabolism Associated With Hyperammonemia | Sampling artifact | || | Cardiovascular | | Perinatal asphyxia | | Liver failure | | Bacterial colonization (urease-positive organisms) | | Iatrogenic | Table 2. Causes of Acquired Hyperammonemia The combination of sodium phenylacetate (NAPA) and sodium benzoate (NABZ) in a 10%/10% solution is an intravenously administered United States Food and Drug Administration (FDA)-approved drug used as adjunctive therapy for …

Integration of [U-13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis.

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.

An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans.

Hepatic glucose synthesis from glycogen, glycerol, and the tricarboxylic acid (TCA) cycle was measured in five overnight-fasted subjects by (1)H, (2)H, and (13)C NMR analysis of blood glucose, urinary acetaminophen glucuronide, and urinary phenylacetylglutamine after administration of [1,6-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate. This combination of tracers allows three separate elements of hepatic glucose production (GP) to be probed simultaneously in a single study: 1) endogenous GP, 2) the contribution of glycogen, phosphoenolpyruvate (PEP), and glycerol to GP, and 3) flux through PEP carboxykinase, pyruvate recycling, and the TCA cycle. Isotope-dilution measurements of [1,6-(13)C(2)] glucose by (1)H and (13)C NMR indicated that GP in 16-h-fasted humans was 10.7 +/- 0.9 micromol.kg(-1).min(-1). (2)H NMR spectra of monoacetone glucose (derived from plasma glucose) provided the relative (2)H enrichment at glucose H-2, H-5, and H-6S, which, in turn, reflects the contribution of glycogen, PEP, and glycerol to total GP (5.5 +/- 0.7, 4.8 +/- 1.0, and 0.4 +/- 0.3 micromol.kg(-1).min(-1), respectively). Interestingly, (13)C NMR isotopomer analysis of phenylacetylglutamine and acetaminophen glucuronide reported different values for PEP carboxykinase flux (68.8 +/- 9.8 vs. 37.5 +/- 7.9 micromol.kg(-1).min(-1)), PEP recycling flux (59.1 +/- 9.8 vs. 27.8 +/- 6.8 micromol.kg(-1).min(-1)), and TCA cycle flux (10.9 +/- 1.4 vs. 5.4 +/- 1.4 micromol.kg(-1).min(-1)). These differences may reflect zonation of propionate metabolism in the liver.

Glutamate, a Window on Liver Intermediary Metabolism

In isotopic experiments, the labeling pattern of glutamate opens a window on hepatic metabolism, particularly the citric acid cycle, gluconeogenesis and fatty acid oxidation. This is because glutamate is in isotopic equilibrium with alpha-ketoglutarate, whose labeling pattern is influenced by the following: 1) the contributions of glucose and fatty acids to acetyl-CoA, 2) the relative contributions of pyruvate carboxylase and pyruvate dehydrogenase to the entry of pyruvate carbon into the citric acid cycle, and 3) the rate of gluconeogenesis in relation to citric acid cycle activity. In humans and primates, hepatic glutamate can be sampled noninvasively via urinary phenylacetylglutamine, which is formed in liver from phenylacetate (a side product of phenylalanine catabolism) and glutamine (which equilibrates with liver glutamate and alpha-ketoglutarate). The (14)C- or (13)C-labeling pattern of the glutamate moiety of phenylacetylglutamine can be measured by sequential degradations to (14)CO(2), gas chromatography-mass spectrometry or nuclear magnetic resonance (NMR). When phenylacetylglutamine is labeled from singly labeled [(14)C]- or [(13)C]substrates, relative metabolic rates can be computed from the labeling pattern using Landau's model. In diabetic patients infused with [3-(13)C]pyruvate, the noninvasive sampling of hepatic glutamate via phenylacetylglutamine allows one to test the degree of liver insulinization via the (pyruvate carboxylase)/(pyruvate dehydrogenase) activity ratio. This ratio regulates gluconeogenesis in part. Its measurement may allow the identification of patients who might benefit from the intraperitoneal administration of insulin, or from recently developed antidiabetic drugs.

13C NMR measurements of human gluconeogenic fluxes after ingestion of [U-13C]propionate, phenylacetate, and acetaminophen.

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 +/- 1.03 (SD) relative to citrate synthase. This was not significantly different from glucuronide and phenylacetylglutamine analyses (6.08 +/- 1.16 and 7. 14 +/- 1.94, respectively). Estimates of pyruvate recycling from glucose and glucuronide isotopomer distributions were almost identical (3.55 +/- 0.99 and 3.66 +/- 1.11, respectively), whereas phenylacetylglutamine reported a significantly higher estimate (5.74 +/- 2.13). As a consequence, net gluconeogenic flux reported by phenylacetylglutamine (1.41 +/- 0.28) was significantly less than that reported by glucose (2.37 +/- 0.64) and glucuronide (2.42 +/- 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.

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.

Validation of the Use of Urinary Phenylacetyl-Glutamine to Assess the Labeling Pattern of Liver Citric Acid Cycle Intermediates in Monkeys

Validation of the Use of Urinary Phenylacetyl-Glutamine to Assess the Labeling Pattern of Liver Citric Acid Cycle Intermediates in Monkeys

Metabolism of [2-14C]acetate and its use in assessing hepatic Krebs cycle activity and gluconeogenesis.

To examine the fate of the carbons of acetate and to evaluate the usefulness of labeled acetate in assessing intrahepatic metabolic processes during gluconeogenesis, [2-14C]acetate, [2-14C]ethanol, and [1-14C]ethanol were infused into normal subjects fasted 60 h and given phenyl acetate. Distributions of 14C in the carbons of blood glucose and glutamate from urinary phenylacetylglutamine were determined. With [2-14C]acetate and [2-14C]ethanol, carbon 1 of glucose had about twice as much 14C as carbon 3. Carbon 2 of glutamate had about twice as much 14C as carbon 1 and one-half to one-third as much as carbon 4. There was only a small amount in carbon 5. These distributions are incompatible with the metabolism of [2-14C]acetate being primarily in liver. Therefore, [2-14C]acetate cannot be used to study Krebs cycle metabolism in liver and in relationship to gluconeogenesis, as has been done. The distributions can be explained by: (a) fixation of 14CO2 from [2-14C]acetate in the formation of the 14C-labeled glucose and glutamate in liver and (b) the formation of 14C-labeled glutamate in a second site, proposed to be muscle. [1,3-14C]Acetone formation from the [2-14C]acetate does not contribute to the distributions, as evidenced by the absence of 14C in carbons 2-4 of glutamate after [1-14C]ethanol administration.

Noninvasive tracing of Krebs cycle metabolism in liver.

To quantify intrahepatic Krebs cycle metabolism, phenyl acetate, excreted in urine as a glutamine conjugate, was given to healthy subjects infused with [3-14C]lactate. They were studied after 60 h of fasting and when given glucose after an overnight fast. Distributions of 14C in glutamate from urinary phenylacetylglutamine and blood glucose were determined. Corrections to the distributions because of the fixation of 14CO2 formed from the [3-14C]lactate were determined by administering [14C]bicarbonate. Comparisons of distributions in glucose and glutamate support the assumption that the glutamate distributions reflect those in hepatic alpha-ketoglutarate. From the distributions in glutamate, the extent of exchange of labeled with unlabeled carbons and relative flow rates in the cycle in liver were estimated. Dilution of 14C by 12C in the cycle was found in the fasted but not the fed state. In the fasted state, pyruvate carboxylation was estimated to be at least twice the rate of Krebs cycle flux and the rate of pyruvate's decarboxylation less than 1/25 the rate of its carboxylation. In the fed state, the rate of decarboxylation was estimated to be between one-sixth and one-half the rate of carboxylation. The rate of conversion of oxalacetate to fumarate in both states appeared to be greater than 6 times the rate of Krebs cycle flux.