A Noninvasive Urine Metabolome Panel as Potential Biomarkers for Diagnosis of T Cell-Mediated Renal Transplant Rejection.

Acute T cell-mediated rejection (TCMR) is a major complication after renal transplantation. TCMR diagnosis is very challenging and currently depends on invasive renal biopsy and nonspecific markers such as serum creatinine. A noninvasive metabolomics panel could allow early diagnosis and improved accuracy and specificity. We report, in this study, on urine metabolome changes in renal transplant recipients diagnosed with TCMR, with a view to future metabolomics-based diagnostics in transplant medicine. We performed urine metabolomic analyses in three study groups: (1) 7 kidney transplant recipients with acute TCMR, (2) 15 kidney transplant recipients without rejection but with impaired kidney function, and (3) 6 kidney transplant recipients with stable renal function, using 1H-nuclear magnetic resonance. Multivariate modeling of metabolites suggested a diagnostic panel where the diagnostic accuracy of each metabolite was calculated by receiver operating characteristic curve analysis. The impaired metabolic pathways associated with TCMR were identified by pathway analysis. In all, a panel of nine differential metabolites encompassing nicotinamide adenine dinucleotide, 1-methylnicotinamide, cholesterol sulfate, gamma-aminobutyric acid (GABA), nicotinic acid, nicotinamide adenine dinucleotide phosphate, proline, spermidine, and alpha-hydroxyhippuric acid were identified as novel potential metabolite biomarkers of TCMR. Proline, spermidine, and GABA had the highest area under the curve (>0.7) and were overrepresented in the TCMR group. Nicotinate and nicotinamide metabolism was the most important pathway in TCMR. These findings call for clinical validation in larger study samples and suggest that urinary metabolomics warrants future consideration as a noninvasive research tool for TCMR diagnostic innovation.

Exploratory metabolomics of nascent metabolic syndrome.

INTRODUCTION: Metabolic syndrome (MetS) is a disorder defined by having three of five features: increased waist circumference (WC), hypertriglyceridemia, decreased high-density lipoprotein-cholesterol, hypertension and an elevated blood glucose (BG). Metabolic Syndrome ( MetS) affects 35% of American adults and significantly increases risk for Atherosclerotic cardiovascular disease (ASCVD) and type-2 diabetes (T2DM). An understanding of the metabolome will help elucidate the pathogenesis of MetS and lead to better management. We hypothesize that the metabolites, gamma-aminobutyric acid (GABA), d-pyroglutamic acid (PGA) and N-acetyl-d-tryptophan (NAT) will be altered in nascent MetS patients without the confounding of ASCVD or T2DM. We also correlated these metabolites with biomarkers of inflammation. PATIENTS AND METHODS: This was an exploratory study of 30 patients with nascent MetS and 20 matched controls undertaken in 2018. Metabolites were evaluated from patient's frozen early morning urine samples and were correlated with biomarkers of inflammation and adipokines. They were assayed by the NIH Western Metabolomics Center using liquid chromatography/mass spectrometry and standardized to urinary creatinine. All patients had normal hepatic and renal function. RESULTS: GABA and PGA levels were significantly increased in MetS patients compared to controls: 2.8-fold and 2.9-fold median increases respectively with p < 0.0001 and p = 0.004, possibly deriving from glutamate. NAT was significantly decreased by 90% in MetS patients compared to controls, p < 0.001. GABA correlates significantly with cardio-metabolic (CM) features including WC, blood pressure systolic (BP-S) while NAT correlated inversely with WC, BP-S, blood glucose (BG) and triglycerides (TG). GABA correlated positively with chemerin, leptin, Fetuin A and endotoxin. NAT correlated inversely with WC, BP-S, BG, TG, high sensitivity C - reactive protein (hsCRP), toll-like receptor-4 (TLR-4), lipopolysaccharide binding protein (LBP), chemerin and retinol binding protein-4 (RBP-4). CONCLUSIONS: We make the novel observation of increased GABA and PGA with decreased NAT in patients with MetS. While GABA and PGA correlates positively with CM features and biomediators of inflammation, the metabolite NAT correlated inversely. Thus, GABA and PGA could contribute to the pro-inflammatory state of MetS while NAT could mitigate this pro-inflammatory response.

Stacking-cyclodextrin-microchip electrokinetic chromatographic determination of gabapentinoid drugs in pharmaceutical and biological matrices.

A facile, rapid, and highly sensitive microchip-based electrokinetic chromatographic method was developed for the simultaneous analysis of two gabapentinoid drugs, gabapentin (GPN) and pregabalin (PGN). Both drugs were first reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) via nucleophilic substitution reactions to yield highly fluorescent products with λex/em 470/540nm. Analyses of both fluorescently labeled compounds were achieved within 200s in a poly(methyl methacrylate) (PMMA) microchip with a 30mm separation channel. Optimum separation was achieved using a borate buffer (pH 9.0) solution containing methylcellulose and ß-cyclodextrin (ß-CD) as buffer additives. Methylcellulose acted as a dynamic coating to prevent adsorption of the studied compounds on the inner surfaces of the microchannels, while ß-CD acted as a pseudo-stationary phase to improve the separation efficiency between the labeled drugs with high resolution (Rs>7). The fluorescence intensities of the labeled drugs were measured using a light emitting diode-induced fluorescence detector at 540nm after excitation at 470nm. The sensitivity of the method was enhanced 14- and 17-fold for PGN and GPN, respectively by field-amplified stacking relative to traditional pinched injection so that it could quantify 10ngmL-1 for both analytes, with a detection limit lower than 3ngmL-1. The developed method was efficiently applied to analyze PGN and GPN in their pharmaceutical dosage forms and in biological fluids. The extraction recoveries of the studied drugs from plasma and urine samples were more than 89% with%RSD values lower than 6.2.

Urinary Concentrations of Topically Administered Pain Medications.

A common treatment for chronic pain is prescription of analgesics, but their long-term use entails risk of morbidity, addiction and misuse. One way to reduce the risk of abuse is prescribing of analgesics in a topical form. Physicians are urged to perform urine drug testing to ensure that patients are compliant with their medication regimens. However, there is little data on the efficiency of transdermal delivery for many analgesic drugs, and no data on expected urine drug levels. This study includes data from over 29,000 specimens tested for gabapentin, ketamine, cyclobenzaprine or amitriptyline used orally or topically. Gabapentin and amitriptyline concentrations were more likely to be below the limits of detection (25-40 ng/mL) in the urine of patients using them topically as compared with patients using them orally. Levels in gabapentin-positive topical specimens were much lower than in gabapentin-positive oral specimens (261 ng/mL vs >10,000 ng/mL). In contrast, ketamine and cyclobenzaprine were more readily detectable in the urine of topical users. Ketamine topical specimens were positive 12% more often than oral specimens, and mean topical specimen levels were 68-100% those of oral specimens. Cyclobenzaprine specimens were equally likely to be positive whether the dose was oral or topical, although mean levels after topical dosing were approximately 13-21% those after oral dosing. These findings are consistent with the reported percutaneous absorption efficiencies of gabapentin and ketamine, and are likely to be related to the absorption efficiencies of cyclobenzaprine and amitriptyline.

A novel mutation of ALDH5A1 gene associated with succinic semialdehyde dehydrogenase deficiency.

Succinic semialdehyde dehydrogenase deficiency is a rare autosomal recessive metabolic disorder affecting γ-aminobutyric acid degradation. We described a boy with a severe phenotype of succinic semialdehyde dehydrogenase deficiency and novel mutations of ALDH5A1 gene. He was referred because of developmental delay, focal seizures, and choreoathetosis at 6 months of age. The diagnosis of succinic semialdehyde dehydrogenase deficiency was confirmed by increased level of γ-hydroxybutyric acid in urine and novel compound heterozygous mutations in the ALDH5A1 gene. His seizures were successfully controlled. However, the patient showed a slowly progressive clinical course with severe neurologic deficits. A magnetic resonance imaging (MRI) revealed abnormal high intensities in the putamen and globus pallidi on T2-weighted images when he was 6 months old, and more diffuse abnormal signal intensities over bilateral hemispheres were noted when he was 3 years old.

Determination of Gabapentin in Human Plasma and Urine by Capillary Electrophoresis with Laser-Induced Fluorescence Detection.

A simple and reliable method based on capillary electrophoresis with laser-induced fluorescence detection was developed for the analysis of the antiepileptic drug Gabapentin in human plasma and urine. 4-Chloro-7-nitrobenzofurazan was used for precolumn derivatization of the drug. With an uncoated fused silica capillary (40.0 cm effective length, 50.2 cm total length and 75 µm internal diameter), optimal separation was achieved with 30 mM sodium dodecyl sulfate, 40 mM sodium borate (pH 10.25) and acetonitrile 10% (v/v) as running buffer. The applied voltage was 20 kV and the samples were injected by pressure (3.45 kPa × 3 s). The method was fully validated with regard to linear range, sensitivity, precision, limit of detection and limit of quantification in human plasma and urine samples. Linear ranges were 0.1-15 µg mL(-1) for plasma and urine. The intra- and interday precisions were ≤9.02 and 13.90%, respectively. The recoveries were 96.0-109.3% for plasma and 94.3-98.0% for urine. The method was successfully applied for the determination of Gabapentin in human plasma and urine.

Pregabalin abuse among opiate addicted patients.

PURPOSE: Pregabalin is a novel GABA-analogue approved for the treatment of partial onset seizures, neuropathic pain, and general anxiety disorder. Pregabalin has been classified as a Schedule V drug with a low risk of inflicting abuse or addiction. However, some publications have indicated that pregabalin may have a potential for abuse among patients with past or current opiate addiction. Thus, we hypothesized that pregabalin might be abused by patients who were undergoing an opiate replacement therapy and never had an indication for taking pregabalin on medical grounds. METHODS: Urine specimens from 124 patients with opiate dependency syndrome and from 111 patients with other addiction disorders (alcohol, benzodiazepines, cannabis, amphetamines) were screened for pregabalin by means of a mass spectrometer analysis. RESULTS: We found 12.1 % of all urine specimens from patients with opiate addiction to be positive for pregabalin. None of the patients concerned had a medical indication for using pregabalin. In the control group, 2.7 % of the patients were tested positively for pregabalin, due to their taking it regularly for chronic pain or general anxiety. CONCLUSIONS: Our data suggest that pregabalin is liable to be abused among individuals with opiate dependency syndrome Thus, vigilance and caution are called for when patients with a past or current opiate dependency are exposed to treatment with pregabalin.

Direct determination of pregabalin in human urine by nonaqueous CE-TOF-MS.

Determination of pregabalin in urine samples was carried out by nonaqueous CE with TOF-MS via ESI, with a mixture of 10 mM ammonium formate and 0.05% acetic acid in methanol. By using TOF-MS, accurate mass information was obtained, thus causing a great improvement in qualitative ability. In order to avoid ionic suppression, urine samples dilution 1:10 was used. This was the only treatment to urine samples before the injection. Despite this dilution, the detection limit was as low as 0.03 µg/mL for pregabalin. The method was validated with respect to accuracy, precision, and linearity, LOD, and LOQ. This method was applied to the analysis of urine samples from seven different cancer patients undergoing treatment with pregabalin. The developed method may find wide application for the routine determination of pregabalin in biological samples in order to establish a more efficient and safe dosage.

Detection times of pregabalin in urine after illicit use: when should a positive specimen be considered a new intake?

BACKGROUND: Pregabalin has an abuse potential and is occasionally used as a recreational drug. To draw the right conclusions regarding new intake of pregabalin in situations of repeated urinary drug testing, the knowledge of its disappearance rate in urine is essential. METHODS: One healthy male volunteer took a single oral dose of pregabalin at 2 occasions, first 75 mg and thereafter 150 mg. All urine was collected in 8-hour portions for 5 days and analyzed for pregabalin. A systematic search for literature describing concentrations of pregabalin in urine was performed, and the results from these studies were interpreted on the basis of the findings from the healthy volunteer. RESULTS: In the healthy volunteer, specimens remained positive for 56 hours after intake of 75 mg and for 64 hours after intake of 150 mg. Urinary elimination half-lives based upon creatinine-normalized concentrations were 5.7-5.9 hours. The systematic literature search revealed only 1 article describing urinary concentrations of pregabalin. In that study, including 4799 urinary samples, the median concentration was not higher than the initial concentration found in the healthy volunteer. By applying a urinary elimination half-life of 6 hours on that material, at least 50% would be expected to have negative urine specimens within 3 days and a total of 5 days would be needed to achieve negative urine specimens in the subject with the maximum urinary concentration measured. CONCLUSION: In subjects with normal renal function, it seems highly unlikely that a urine specimen should remain positive for pregabalin for more than 5-6 days after intake.

Development, validation and comparison of two microextraction techniques for the rapid and sensitive determination of pregabalin in urine and pharmaceutical formulations after ethyl chloroformate derivatization followed by gas chromatography-mass spectrometric analysis.

The present article reports first time the use of solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME) to extract pregabalin (PRG) from urine and pharmaceutical formulations followed by GC-MS analysis after ethyl chloroformate (ECF) derivatization. PRG is an antiepileptic and analgesic drug, which is a structural analogue of γ-amino-butyric acid (GABA). It is approved by Food and Drug Administration (FDA) for the treatment of central nervous system (CNS) disorders and neuropathic pain. Initially PRG was derivatized with ECF in the presence of pyridine at room temperature for 30s. Experimental parameters were investigated for derivatization, SPME and DLLME conditions. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.019 µg/ml and 0.063 µg/ml for SPME and 0.022 µg/ml and 0.075 µg/ml for DLLME respectively. The percentage recovery, in case of SPME was in the range of 83-98% while for DLLME it is in the range of 84-98%. The intra and inter-day precisions were found to be less than 6%. The developed methods after ECF derivatization were found to be simple, fast, efficient and inexpensive. DLLME has several advantages like lesser extraction time and cost effectiveness as compared to SPME. The developed methods may find wide application for the routine determination of PRG in biological as well as in quality control samples of pharmaceutical formulations.

Determination of γ-hydroxybutyrate (GHB), ß-hydroxybutyrate (BHB), pregabalin, 1,4-butane-diol (1,4BD) and γ-butyrolactone (GBL) in whole blood and urine samples by UPLC-MSMS.

The demand of high throughput methods for the determination of gamma-hydroxybutyrate (GHB) and its precursors gamma-butyrolactone (GBL) and 1,4-butane-diol (1,4BD) as well as for pregabalin is increasing. Here we present two analytical methods using ultra-high pressure liquid chromatography (UPLC) and tandem mass spectrometric (MS/MS) detection for the determination of GHB, beta-hydroxybutyrate (BHB), pregabalin, 1,4BD and GBL in whole blood and urine. Using the 96-well formate, the whole blood method is a simple high-throughput method suitable for screening of large sample amounts. With an easy sample preparation for urine including only dilution and filtration of the sample, the method is suitable for fast screening of urine samples. Both methods showed acceptable linearity, acceptable limits of detection, and limits of quantification. The within-day and between-day precisions of all analytes were lower than 10% RSD. The analytes were extracted from matrices with recoveries near 100%, and no major matrix effects were observed. Both methods have been used as routine screening analyses of whole blood and urine samples since January 2010.

Impact of concomitant antacid administration on gabapentin plasma exposure and oral bioavailability in healthy adult subjects.

The aim of this open-label, randomized, and 3-period crossover study was to evaluate the influences of concomitant antacid administration on the plasma disposition, intestinal absorption, and urinary excretion of gabapentin in humans. Gabapentin (200 mg) was orally administered alone, with 1 g magnesium oxide (MgO), or with 20 mg omeprazole to 13 healthy adult subjects. Oral bioavailability (BA) of gabapentin was estimated by 24-h urine collection. The C(max), T(max) and AUC(0-∞) of gabapentin + MgO were significantly lower than that of gabapentin alone (by 33%, 36% and 43%, respectively) and gabapentin + omeprazole (by 29%, 46% and 40%, respectively). In contrast, no significant differences were observed in the plasma disposition parameters of gabapentin between the treatments with and without omeprazole. The gabapentin BA in the MgO treatment was significantly lower, by 32% and 39%, compared to the gabapentin alone and with omeprazole treatment, respectively. There was no significant difference in the gabapentin BA between the gabapentin alone and with omeprazole treatment. Concomitant MgO and omeprazole did not affect the renal clearance of gabapentin. In conclusion, concomitant MgO decreased the gabapentin exposure through the reduction of intestinal absorption extent and rate. This reduction may be independent of the suppression of gastrointestinal acidification caused by antacids.

Urine drug testing of chronic pain patients. IV. prevalence of gabapentin and pregabalin.

Gabapentin and pregabalin are well established for the treatment of seizures and neuropathic pain. Both drugs are eliminated primarily unchanged by renal excretion. As part of an ongoing research program to improve and expand drug testing methods for compliance monitoring of pain patients, the prevalence and concentrations of gabapentin and pregabalin in urine specimens from chronic pain patients were determined by a validated liquid chromatography-tandem mass spectrometry assay. The study was approved by an Institutional Review Board. A total of 57,542 urine specimens from 231 pain clinics located in 19 states were analyzed over the period of November 24, 2009, through May 2010. The limit of quantitation (LOQ) and upper LOQ of the assays for both drugs were 2.5 and 1000 µg/mL, respectively. Gabapentin was identified in 7013 specimens (12.2% prevalence), and pregabalin was identified in 4799 patients (8.3% prevalence). Generally, gabapentin concentrations were more than twofold higher than pregabalin, consistent with their relative potencies. Interestingly, both drugs were found in specimens from 249 patients, likely representing switching of prescriptions by the prescriber.

A randomized targeted amino acid therapy with behaviourally at-risk adopted children.

BACKGROUND: Increasing numbers of children are at-risk for behavioural and emotional disorders, a phenomenon contributing to increased use of pharmacological interventions for paediatric clients. Adverse side effects and other risks associated with pharmacological approaches have helped fuel interest in nutritional interventions for behaviourally at-risk children. METHODS: The current randomized clinical trial evaluates the efficacy of a neurochemical intervention involving the glutamine and glutamate analogue L-theanine and 5-hydroxytryptophan, the precursor for serotonin, with children adopted from traumatic backgrounds. RESULTS: Results include significant increases in urinary levels of the biomarkers for serotonin and gamma-aminobutyric acid, coupled with significant decreases in parent reports of the children's behaviour problems. CONCLUSIONS: While further research is needed, these initial findings are encouraging and are consistent with a growing number of studies indicating the efficacy of nutritional approaches to help behaviourally at-risk children.

Novel ELISAs for screening of the biogenic amines GABA, glycine, beta-phenylethylamine, agmatine, and taurine using one derivatization procedure of whole urine samples.

The inhibitory neurotransmitters GABA, glycine and agmatine and neuromodulators beta-phenylethylamine (beta-PEA) and taurine are important biogenic amines of the sympathetic and parasympathetic nervous systems in the body. Abnormalities in the metabolism of these biomarkers have been implicated in a vast number of neurological diseases. Novel competitive immunoassays, using one unique whole urine derivatization procedure applicable for all five biomarkers, have been developed. The determination of these biomarkers was highly reproducible: the coefficient of variance of inter- and intra-assay variation is between 3.9% and 9.8% for all assays. The assays show a good linearity in urine samples within the range of 100-400 mg Cr/dL and specificity when urine samples are spiked with biogenic amines. The recoveries are between 76 and 154%. The correlation between HPLC and ELISA for glycine and taurine (n = 10) showed regression coefficients of 0.97 and 0.98, respectively. An in vivo study on the urinary clearance of beta-PEA, agmatine and taurine after oral intake by healthy individuals demonstrated the specificity and clinical significance of these new immunoassays. The immunoassays are useful for clinical and basic research where a fast and accurate assay for the screening of biogenic amines in urine is required, without preclearance of the sample.

Hollow fiber-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of gabapentin in biological samples.

Three-phase hollow fiber microextraction technique combined with high performance liquid chromatography-ultra violet (HPLC-UV) was applied for the extraction and determination of gabapentin in biological fluids. Gabapentin (GBP) was derivatized with 1-fluoro-2,4-dinitrobenzene, as a UV absorbent agent in borate buffer (pH 8.2) before extraction. The derivative product of GBP was extracted from the 8.5 mL of acidic solution (source phase) into an organic phase (dihexyl ether) impregnated in the pores of a hollow fiber and finally back-extracted into 24 microL of the basic solution (pH 9.1) located inside the lumen of the hollow fiber (receiving phase). The extraction took place due to pH gradient between the inside and outside of the hollow fiber membrane. In order to achieve maximum extraction efficiency, different parameters affecting the extraction conditions were optimized. Under the optimized conditions, preconcentration factor of 95 and detection limit (LOD) of 0.2 microg L(-1) were obtained. The calibration graph was linear within the range of 0.6-5000 microg L(-1). Finally, the feasibility of the proposed method was successfully confirmed by extraction and determination of GBP in human urine and plasma samples in the range of microgram per liter and suitable results were obtained (RSDs<6.3%).

Clinical pharmacokinetics of pregabalin in healthy volunteers.

Pregabalin has shown clinical efficacy for treatment of neuropathic pain syndromes, partial seizures, and anxiety disorders. Five studies in healthy volunteers are performed to investigate single- and multiple-dose pharmacokinetics of pregabalin. Pregabalin is rapidly absorbed following oral administration, with peak plasma concentrations occurring between 0.7 and 1.3 hours. Pregabalin oral bioavailability is approximately 90% and is independent of dose and frequency of administration. Food reduces the rate of pregabalin absorption, resulting in lower and delayed maximum plasma concentrations, yet the extent of drug absorption is unaffected, suggesting that pregabalin may be administered without regard to meals. Pregabalin elimination half-life is approximately 6 hours and steady state is achieved within 1 to 2 days of repeated administration. Corrected for oral bioavailability, pregabalin plasma clearance is essentially equivalent to renal clearance, indicating that pregabalin undergoes negligible nonrenal elimination. Pregabalin demonstrates desirable, predictable pharmacokinetic properties that suggest ease of use. Because pregabalin is eliminated renally, renal function affects its pharmacokinetics.

beta-Alanine and gamma-aminobutyric acid in chronic fatigue syndrome.

BACKGROUND: Due to the occurrence of sleep disturbances and fatigue in chronic fatigue syndrome (CFS), an investigation was performed to examine if there is an abnormal excretion of gamma-aminobutyric acid (GABA) and/or its structural analogue beta-alanine in the urine from CFS patients. Both GABA and beta-alanine are inhibitory neurotransmitters in the mammalian central nervous system. METHODS: The 24 h urine excretion of GABA and beta-alanine was determined by isotope dilution gas chromatography mass spectrometry in 33 CFS patients and 43 healthy controls. The degree of symptoms in both patients and controls was measured by grading of three typical CFS symptoms using a Visual Analogue Scale. RESULTS: Men had a significantly higher excretion of both beta-alanine and GABA than women. Comparing CFS patients with healthy controls showed no significant difference in excretion of neither beta-alanine nor GABA. No correlation was found between the excretion of beta-alanine or GABA and any of the three characteristic CFS symptoms measured. However, two female and two male CFS patients excreted considerably higher amounts of beta-alanine in their 24 h urine samples than control subjects. CONCLUSIONS: Increased excretion of beta-alanine was found in a subgroup of CFS patients, indicating that there may be a link between CFS and beta-alanine in some CFS patients.

Determination of gabapentin in human plasma and urine by high-performance liquid chromatography with UV-vis detection.

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV-vis detection has been developed and validated for the determination of gabapentin (GBP) in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction with C18-cartridge. After the clean up procedure, the samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10mM orthophosphoric acid (pH 2.5) with isocratic elution (35:65). Baclofen was used as an internal standard (I.S.). The method developed for GBP was linear over the concentration range of 0.05-5.0 microg/ml and 0.1-10.0 microg/ml for plasma and urine, respectively. The method is precise (relative standard deviation, R.S.D. <4.05%) and accurate (relative mean error, RME <0.15%); mean absolute recoveries were 72.21% for plasma and 72.73% for urine.