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 METHODS: Urine 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 DISCUSSION: The 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). CONCLUSION: Urinary PAG/Cr might be a biomarker not only for traumatic brain injuries but also for antemortem CNS damages.

In older women, a high-protein diet including animal-sourced foods did not impact serum levels and urinary excretion of trimethylamine-N-oxide.

Diets including red meat and other animal-sourced foods may increase proteolytic fermentation and microbial-generated trimethylamine (TMA) and, subsequently, trimethylamine-N-oxide (TMAO), a metabolite associated with increased risk of cardiovascular disease and dementia. It was hypothesized that compared to usual dietary intake, a maintenance-energy high-protein diet (HPD) would increase products of proteolytic fermentation, whereas adjunctive prebiotic, probiotic, and synbiotic supplementation may mitigate these effects. An exploratory aim was to determine the association of the relative abundance of the TMA-generating taxon, Emergencia timonensis, with serum and urinary TMAO. At 5 time points (usual dietary intake, HPD diet, HPD + prebiotic, HPD + probiotic, and HPD + synbiotic), urinary (24-hour) and serum metabolites and fecal microbiota profile of healthy older women (n = 20) were measured by liquid chromatography-tandem mass spectrometry and 16S rRNA gene amplicon sequencing analyses, respectively. The HPD induced increases in serum levels of l-carnitine, indoxyl sulfate, and phenylacetylglutamine but not TMAO or p-cresyl sulfate. Urinary excretion of l-carnitine, indoxyl sulfate, phenylacetylglutamine, and TMA increased with the HPD but not TMAO or p-cresyl sulfate. Most participants had undetectable levels of E.timonensis at baseline and only 50% during the HPD interventions, suggesting other taxa are responsible for the microbial generation of TMA in these individuals. An HPD diet with or without a prebiotic, probiotic, or synbiotic elicited an increase in products of proteolytic fermentation. The urinary l-carnitine response suggests that the additional dietary l-carnitine provided was primarily bioavailable, providing little substrate for microbial conversion to TMA and subsequent TMAO formation.

Longitudinal metabolic and gut bacterial profiling of pregnant women with previous bariatric surgery.

OBJECTIVE: Due to the global increase in obesity rates and success of bariatric surgery in weight reduction, an increasing number of women now present pregnant with a previous bariatric procedure. This study investigates the extent of bariatric-associated metabolic and gut microbial alterations during pregnancy and their impact on fetal development. DESIGN: A parallel metabonomic (molecular phenotyping based on proton nuclear magnetic resonance spectroscopy) and gut bacterial (16S ribosomal RNA gene amplicon sequencing) profiling approach was used to determine maternal longitudinal phenotypes associated with malabsorptive/mixed (n=25) or restrictive (n=16) procedures, compared with women with similar early pregnancy body mass index but without bariatric surgery (n=70). Metabolic profiles of offspring at birth were also analysed. RESULTS: Previous malabsorptive, but not restrictive, procedures induced significant changes in maternal metabolic pathways involving branched-chain and aromatic amino acids with decreased circulation of leucine, isoleucine and isobutyrate, increased excretion of microbial-associated metabolites of protein putrefaction (phenylacetlyglutamine, p-cresol sulfate, indoxyl sulfate and p-hydroxyphenylacetate), and a shift in the gut microbiota. The urinary concentration of phenylacetylglutamine was significantly elevated in malabsorptive patients relative to controls (p=0.001) and was also elevated in urine of neonates born from these mothers (p=0.021). Furthermore, the maternal metabolic changes induced by malabsorptive surgery were associated with reduced maternal insulin resistance and fetal/birth weight. CONCLUSION: Metabolism is altered in pregnant women with a previous malabsorptive bariatric surgery. These alterations may be beneficial for maternal outcomes, but the effect of elevated levels of phenolic and indolic compounds on fetal and infant health should be investigated further.

Quantification of urinary derivatives of Phenylbutyric and Benzoic acids by LC-MS/MS as treatment compliance biomarkers in Urea Cycle disorders.

PURPOSE: Salts of phenylacetic acid (PAA) and phenylbutyric acid (PBA) have been used for nitrogen elimination as a treatment for hyperammonaemia caused by urea cycle disorders (UCD). A new analytical method for PBA measurement in urine which helps to evaluate the drug adherence has been implemented. METHODS: Urine specimens from UCD patients receiving PBA were analysed by tandem mass spectrometry to measure urine phenylacetylglutamine (PAGln). Some clinical and biochemical data for each patient were collected. RESULTS: Our study included 87 samples from 40 UCD patients. The PAGln levels did not correlate with height, weight or age. However, the PAGln values showed correlation with PBA dose (r = 0.383, P = 0.015). Plasma glutamine and ammonia levels presented a positive correlation (r = 0.537, P < 0.001). The stability for PAGln in urine was determined at different storage temperatures. CONCLUSIONS: We have developed a simple method for the determination of PAGln in urine, which acts as useful biomarker of effective drug delivery. PAGln in urine is stable at room temperature at least for 15 days, and for several months when frozen at -20 °C. This procedure is useful for the optimization and monitorization of the drug dose allowing the use of spot urine samples.

Urinary Metabolomics Profiles Associated to Bovine Meat Ingestion in Humans.

SCOPE: The impact of meat consumption on human health is widely examined in nutritional epidemiological studies, especially due to the connection between the consumption of red and processed meat and the risk of colon cancer. Food questionnaires do not assess the exposure to different methods of meat cooking. This study aimed to identify biomarkers of the acute ingestion of bovine meat cooked with two different processes. METHODS AND RESULTS: Non-targeted UPLC-MS metabolite profiling was done on urine samples obtained from 24 healthy volunteers before and 8 h after the ingestion of a single meal composed of intrinsically 15 N labelled bovine meat, either cooked at 55 °C for 5 min or at 90 °C for 30 min. A discriminant analysis extension of independent components analysis was applied to the mass spectral data. After meat ingestion, the urinary excretion of 1-methylhistidine, phenylacetylglutamine, and short- and medium-chained acylcarnitines was observed. 15 N labelling was detected in these metabolites, thus confirming their origin from ingested meat. However, no difference was observed in urinary metabolomic profiles according to the meat cooking process used. CONCLUSION: Meat ingestion led to the excretion of several nitrogen-containing compounds, but although a metabolic signature was detected for meat ingestion, the impact of the cooking process was not detectable at the level of urinary metabolic signature in our experimental conditions.

Untargeted metabolomics approach (UPLC-Q-TOF-MS) explores the biomarkers of serum and urine in overweight/obese young men.

BACKGROUND AND OBJECTIVES: Obesity is linked to metabolic diseases characterized by insulin resistance, such as diabetes and cardiovascular disease. In this study, we investigated the metabolic disorders of uncomplicated obesity to identify early alterations in biological systems. METHODS AND STUDY DESIGN: Metabolic differences between overweight/obese (n=36) and normal-weight (n=35) young Chinese men without known metabolic disorders were assessed. Metabolic profiling of the serum and urine was performed using ultra-performance liquidchromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Partial least squares discriminant analysis (PLS-DA) was undertaken to reveal and classify the differences between the two groups. RESULTS: Compared to normal-weight men, obese men had higher levels of the serum metabolites phenylalanine, Phe-Phe, and L-tryptophan, whereas those of p-cresol sulfate and p-cresol were less in obesity. Urinary metabolites phenylacetamide, L-glutamine, phenylacetylglutamine, indoxyl sulfate, p-cresol, and p-cresol sulfate were greater in obese men. CONCLUSIONS: These findings indicate that disorders involving aromatic amino acids and the tricarboxylic acid cycle (TCA) have microbiomic involvement in the uncomplicated phase of obesity.

Genome-Wide Association Studies of Metabolites in Patients with CKD Identify Multiple Loci and Illuminate Tubular Transport Mechanisms.

Background The kidneys have a central role in the generation, turnover, transport, and excretion of metabolites, and these functions can be altered in CKD. Genetic studies of metabolite concentrations can identify proteins performing these functions.Methods We conducted genome-wide association studies and aggregate rare variant tests of the concentrations of 139 serum metabolites and 41 urine metabolites, as well as their pairwise ratios and fractional excretions in up to 1168 patients with CKD.Results After correction for multiple testing, genome-wide significant associations were detected for 25 serum metabolites, two urine metabolites, and 259 serum and 14 urinary metabolite ratios. These included associations already known from population-based studies. Additional findings included an association for the uremic toxin putrescine and variants upstream of an enzyme catalyzing the oxidative deamination of polyamines (AOC1, P-min=2.4×10-12), a relatively high carrier frequency (2%) for rare deleterious missense variants in ACADM that are collectively associated with serum ratios of medium-chain acylcarnitines (P-burden=6.6×10-16), and associations of a common variant in SLC7A9 with several ratios of lysine to neutral amino acids in urine, including the lysine/glutamine ratio (P=2.2×10-23). The associations of this SLC7A9 variant with ratios of lysine to specific neutral amino acids were much stronger than the association with lysine concentration alone. This finding is consistent with SLC7A9 functioning as an exchanger of urinary cationic amino acids against specific intracellular neutral amino acids at the apical membrane of proximal tubular cells.Conclusions Metabolomic indices of specific kidney functions in genetic studies may provide insight into human renal physiology.

[Metabonomic analysis of the urine from rat model with abnormal sapra syndrome].

OBJECTIVE: To investigate the correlation between the change in metabolic components of urine and the abnormal sapra syndrome by using a rat model of abnormal sapra syndrome.
 Methods: Multiple factors, such as dry environment, dry feed, and chronic electrical stimulation, were used to establish the abnormal sapra syndrome in Wistar rats by Uyghur medicine. The differences in metabolites were detected through the metabonomics method.
 Results: The urine of rats in abnormal sapra syndrome group showed significant high abundance metabolites as follows: Leucine, isoleucine, and glycoprotein. And that significant low abundance metabolites as follows: Glutamine, creatine, citric acid, and phenylalanine.
 Conclusion: The urine of rats with the abnormal sapra syndrome displays abnormal energy metabolism. It is likely that the dysfunctional metabolisms of three major nutrients might be the molecular basis for the abnormal sapra syndrome.

Quantitation of phenylbutyrate metabolites by UPLC-MS/MS demonstrates inverse correlation of phenylacetate:phenylacetylglutamine ratio with plasma glutamine levels.

Urea cycle disorders (UCDs) are genetic conditions characterized by nitrogen accumulation in the form of ammonia and caused by defects in the enzymes required to convert ammonia to urea for excretion. UCDs include a spectrum of enzyme deficiencies, namely n-acetylglutamate synthase deficiency (NAGS), carbamoyl phosphate synthetase I deficiency (CPS1), ornithine transcarbamylase deficiency (OTC), argininosuccinate lyase deficiency (ASL), citrullinemia type I (ASS1), and argininemia (ARG). Currently, sodium phenylbutyrate and glycerol phenylbutyrate are primary medications used to treat patients with UCDs, and long-term monitoring of these compounds is critical for preventing drug toxic levels. Therefore, a fast and simple ultra-performance liquid chromatography (UPLC-MS/MS) method was developed and validated for quantification of phenylbutyrate (PB), phenylacetate (PA), and phenylacetylglutamine (PAG) in plasma and urine. The separation of all three analytes was achieved in 2min, and the limits of detection were <0.04µg/ml. Intra-precision and inter-precision were <8.5% and 4% at two quality control concentrations, respectively. Average recoveries for all compounds ranged from 100% to 106%. With the developed assay, a strong correlation between PA and the PA/PAG ratio and an inverse correlation between PA/PAG ratio and plasma glutamine were observed in 35 patients with confirmed UCDs. Moreover, all individuals with a ratio ≥0.6 had plasma glutamine levels<1000µmol/l. Our data suggest that a PA/PAG ratio in the range of 0.6-1.5 will result in a plasma glutamine level<1000µmol/l without reaching toxic levels of PA.

Microbial metabolites are associated with a high adherence to a Mediterranean dietary pattern using a 1H-NMR-based untargeted metabolomics approach.

The study of biomarkers of dietary patterns including the Mediterranean diet (MedDiet) is scarce and could improve the assessment of these patterns. Moreover, it could provide a better understanding of health benefits of dietary patterns in nutritional epidemiology. We aimed to determine a robust and accurate biomarker associated with a high adherence to a MedDiet pattern that included dietary assessment and its biological effect. In this cross-sectional study, we included 56 and 63 individuals with high (H-MDA) and low (L-MDA) MedDiet adherence categories, respectively, all from the Prevención con Dieta Mediterránea trial. A 1H-NMR-based untargeted metabolomics approach was applied to urine samples. Multivariate statistical analyses were conducted to determine the metabolite differences between groups. A stepwise logistic regression and receiver operating characteristic curves were used to build and evaluate the prediction model for H-MDA. Thirty-four metabolites were identified as discriminant between H-MDA and L-MDA. The fingerprint associated with H-MDA included higher excretion of proline betaine and phenylacetylglutamine, among others, and decreased amounts of metabolites related to glucose metabolism. Three microbial metabolites - phenylacetylglutamine, p-cresol and 4-hydroxyphenylacetate - were included in the prediction model of H-MDA (95% specificity, 95% sensitivity and 97% area under the curve). The model composed of microbial metabolites was the biomarker that defined high adherence to a Mediterranean dietary pattern. The overall metabolite profiling identified reflects the metabolic modulation produced by H-MDA. The proposed biomarker may be a better tool for assessing and aiding nutritional epidemiology in future associations between H-MDA and the prevention or amelioration of chronic diseases.

Urinary glutamine/glutamate ratio as a potential biomarker of pediatric chronic intestinal pseudo-obstruction.

Chronic intestinal pseudo-obstruction (CIPO) is a rare intestinal motility disorder with significant morbidity and mortality in pediatric patients. The diagnosis of CIPO is difficult, because it is clinically based on the symptoms and signs of bowel obstruction which are similar to the clinical manifestations of other gastrointestinal diseases like short bowel syndrome (SBS). Therefore, it is desirable to identify and establish new laboratory diagnostic markers for CIPO that are reliable and easily accessible. In our study we have identified the ratio of the urinary glutamine and glutamic acid as a promising biomarker for distinguishing suspected CIPO cases and simple SBS cases. The area under ROC curve was 0.83, at cutoff value = 7.04 with sensitivity of 65% and specificity of 92%.

Morphological and molecular identification of ticks infesting Boa constrictor (Squamata, Boidae) in Manaus (Central Brazilian Amazon) / Identificação morfológica e molecular de carrapatos coletados de jiboias (Boa constrictor) (Squamata, Boidae) da zona urbana de Manaus

The Boa constrictor is one of the world's largest vertebrate carnivores and is often found in urban areas in the city of Manaus, Brazil. The morphological identification of ticks collected from 27 snakes indicated the occurrence of Amblyomma dissimile Koch 1844 on all individuals sampled. In contrast, Amblyomma rotundatum Koch was found on only two snakes. An analysis of the 16S rRNA molecular marker confirmed the morphological identification of these ectoparasites.

A jiboia (Boa constrictor), vertebrado carnívoro, tem sido encontrada em abundância na área urbana de Manaus. A identificação morfológica dos carrapatos coletados em 27 dessas serpentes verificou a ocorrência de Amblyomma dissimile Koch 1844, em todos os exemplares avaliados e a presença de Amblyomma rotundatum Koch 1844, em duas dessas serpentes. A análise do marcador 16S rRNA confirma a identificação morfológica das espécies A. rotundatum e A. dissimile e apresenta novas sequências destes organismos.

[Kinetics of nitrogenous metabolites in the kidney during chronic tetrachloromethane hepatitis].

The kinetics of ammonia, glutamine, and urea in the kidney has been studied in experiments on 203 white rats (females) at the end of chronic tetrachloromethane (CCl4) exposure (65 days) and within 14 days after cessation of CCl4. It was found that on the 65th day of CCl4 administration the arterial hyperammoniemia is formed, which lasts for 14 days after the abolition of the toxin. This is accompanied by an increased excretion of ammonia in the urine and an increase in its concentration in the blood of renal veins, which does not prevent its accumulation in renal tissue. In chronic CCl4-hepatitis model are the changes of glutamine concentration in arterial blood are developing by type of hypo- and hyperglutaminemia. CCl4 stimulates accumulation of glutamine by the kidneys at the end of exposure and at early stage of the recovery period. Toxin cessation activates processes which are stabilizing the normal concentration of glutamine in the kidney by changing glutamine incretion from kidney to renal blood flow. Long-lasting CCl4 exposure increases the concentration of urea in the arterial blood and its urinary excretion. Simultaneously urea reabsorption is activated in the kidneys, which contributes to an increase in its concentration in the blood of the renal veins.

Pressurized CEC coupled with QTOF-MS for urinary metabolomics.

Pressurized CEC (pCEC) coupled with ESI-QTOF-MS using a sheathless interface was applied for metabolomics to develop an alternative analytical method for metabolic profiling of complex biofluid samples such as urine. The hyphenated system was investigated with mixed standards and pooled urine samples to evaluate its precision, repeatability, linearity, sensitivity, and selectivity. The applied voltage, mobile phase, and gradient elution were optimized and applied for the analysis of urinary metabolites. Multivariate data analysis was subsequently performed and used to distinguish lung cancer patients from healthy controls successfully. High separation efficiency has been achieved in pCEC due to the EOF. For metabolite identification, the pCEC-MS separation mechnism was helpful for discriminating the fragment ions of glutamine conjugates from co-eluted metabolites. Three glutamine conjugates, including phenylacetylglutamine, acylglutamine C8:1, and acylglutamine C6:1 were identified among 16 differential urinary metabolites of lung cancer. Receiver-operating-characteristic analysis of acylglutamine C8:1 resulted in an area-under-curve value of 0.882. Overall, this work suggests that this pCEC-ESI-QTOF-MS method may provide a novel and useful platform for metabolomic studies due to its superior separation and identification.

Glycerol phenylbutyrate treatment in children with urea cycle disorders: pooled analysis of short and long-term ammonia control and outcomes.

OBJECTIVE: To evaluate glycerol phenylbutyrate (GPB) in the treatment of pediatric patients with urea cycle disorders (UCDs). STUDY DESIGN: UCD patients (n=26) ages 2months through 17years were treated with GPB and sodium phenylbutyrate (NaPBA) in two short-term, open-label crossover studies, which compared 24-hour ammonia exposure (AUC0-24) and glutamine levels during equivalent steady-state dosing of GPB and sodium phenylbutyrate (NaPBA). These 26 patients plus an additional 23 patients also received GPB in one of three 12-month, open label extension studies, which assessed long-term ammonia control, hyperammonemic (HA) crises, amino acid levels, and patient growth. RESULTS: Mean ammonia exposure on GPB was non-inferior to NaPBA in each of the individual crossover studies. In the pooled analyses, it was significantly lower on GPB vs. NaPBA (mean [SD] AUC0-24: 627 [302] vs. 872 [516] µmol/L; p=0.008) with significantly fewer abnormal values (15% on GPB vs. 35% on NaPBA; p=0.02). Mean ammonia levels remained within the normal range during 12months of GPB dosing and, when compared with the 12months preceding enrollment, a smaller percentage of patients (24.5% vs. 42.9%) experienced fewer (17 vs. 38) HA crises. Glutamine levels tended to be lower with GPB than with NaPBA during short-term dosing (mean [SD]: 660.8 [164.4] vs. 710.0 [158.7] µmol/L; p=0.114) and mean glutamine and branched chain amino acid levels, as well as other essential amino acids, remained within the normal range during 12months of GPB dosing. Mean height and weight Z-scores were within normal range at baseline and did not change significantly during 12months of GPB treatment. CONCLUSIONS: Dosing with GPB was associated with 24-hour ammonia exposure that was non-inferior to that during dosing with NaPBA in individual studies and significantly lower in the pooled analysis. Long-term GPB dosing was associated with normal levels of glutamine and essential amino acids, including branched chain amino acids, age-appropriate growth and fewer HA crises as compared with the 12month period preceding enrollment.

Randomized, double-blind, controlled study of glycerol phenylbutyrate in hepatic encephalopathy.

UNLABELLED: Glycerol phenylbutyrate (GPB) lowers ammonia by providing an alternate pathway to urea for waste nitrogen excretion in the form of phenylacetyl glutamine, which is excreted in urine. This randomized, double-blind, placebo-controlled phase II trial enrolled 178 patients with cirrhosis, including 59 already taking rifaximin, who had experienced two or more hepatic encephalopathy (HE) events in the previous 6 months. The primary endpoint was the proportion of patients with HE events. Other endpoints included the time to first event, total number of events, HE hospitalizations, symptomatic days, and safety. GPB, at 6 mL orally twice-daily, significantly reduced the proportion of patients who experienced an HE event (21% versus 36%; P=0.02), time to first event (hazard ratio [HR]=0.56; P<0.05), as well as total events (35 versus 57; P=0.04), and was associated with fewer HE hospitalizations (13 versus 25; P=0.06). Among patients not on rifaximin at enrollment, GPB reduced the proportion of patients with an HE event (10% versus 32%; P<0.01), time to first event (HR=0.29; P<0.01), and total events (7 versus 31; P<0.01). Plasma ammonia was significantly lower in patients on GPB and correlated with HE events when measured either at baseline or during the study. A similar proportion of patients in the GPB (79%) and placebo groups (76%) experienced adverse events. CONCLUSION: GPB reduced HE events as well as ammonia in patients with cirrhosis and HE and its safety profile was similar to placebo. The findings implicate ammonia in the pathogenesis of HE and suggest that GPB has therapeutic potential in this population. (Clinicaltrials.gov, NCT00999167).

Safety of ornithine phenylacetate in cirrhotic decompensated patients: an open-label, dose-escalating, single-cohort study.

AIMS: Confirm in patients with cirrhosis and gastrointestinal bleeding the safety of ornithine phenylacetate (OP) and assess the pharmacokinetic profile of OP and its effects on plasma ammonia. BACKGROUND: OP is a drug that has shown experimentally to decrease hyperammonemia and improve hepatic encephalopathy. OP is safe in healthy subjects and in stable patients with cirrhosis, but there are no data in decompensated cirrhosis. METHODS: We performed a study to assess safety and tolerance of OP in cirrhotic patients after an episode of upper gastrointestinal bleeding.Ten patients were included within 24 hours of an upper gastrointestinal bleeding. OP was administered as a continuous infusion up to a maximum of 10 g/24 h (0.42 g/h) for 5 days. The infusion was started at 33% of the target dose and increased at 12-hour intervals achieving target dose at 24 hours. Ammonia was also assessed in control group of 10 patients. RESULTS: No severe adverse events were observed. Mild adverse events were reported in 4 patients. Plasma ammonia (baseline: 80±43 µmol/L) showed a progressive drop between baseline and 36 hours (42±15 µmol/L), 72 hours (44±15 µmol/L), 96 hours (40±24 µmol/L), and 120 hours (33±14 µmol/L). Plasma ammonia at 24 hours was significantly higher in the control group. Plasma glutamine showed a significant decrease (-37% at day 5) and its excretion in urine as phenylacetylglutamine, a progressive rise (52±35 mmol at day 5). CONCLUSIONS: OP is a safe and well-tolerated drug in decompensated cirrhotics that may decrease plasma ammonia by inducing its appearance as phenylacetylglutamine in urine.

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.

OBJECTIVES: 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). STUDY DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

UNLABELLED: 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 DESIGN: These 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. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/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 AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The 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.