Development of HPLC method for estimation of glyoxylic acid after pre-column fluorescence derivatization approach based on thiazine derivative formation: A new application in healthy and cardiovascular patients' sera.

Glyoxylic acid (GA) is the intermediate metabolite in various mammalian metabolic pathways. GA showed high reactivity towards formation of advanced glycation end-products (AGEs); the main cause of pathogenesis and complications of many diseases. The presented study aimed to detect GA in healthy and cardiovascular patients' (CV) sera; however analysis of GA in biological fluid is a challenge and requires chemical derivatization. Hence, a new, highly sensitive, time saving and reproducible pre-column fluorescence derivatization procedure coupled with high performance liquid chromatography (HPLC) method was developed. The derivatization method was based on reaction of 2-aminobenzenthiol (2-ABT), a fluorogenic reagent, with GA in acidic medium to form highly fluorescent thiazine derivative (290 and 390 nm for excitation and emission wavelengths respectively). The fluorescent derivative was separated within 6 min on a reversed-phase ODS column using an isocratic elution with a mixture of methanol-water (70:30, v/v%). The proposed method parameters were optimized and the method was validated. A good linearity in the concentration range (0.05-5.0 µM) was obtained with detection limit (LOD) of 10 nM (200 fmol/injection), which is more sensitive than several previous methods. Moreover, the recovery results were within the range of 85.0-95.5 % and the intra- and inter-day precision results were ≤3.5%. It should be emphasized that this method is the first one for monitoring of GA in CV patients; to investigate its role for diagnosis and monitoring the severity and complications of this disease in clinical laboratory.

Development and Validation of a New Gas Chromatography-Tandem Mass Spectrometry Method for the Measurement of Enrichment of Glyoxylate Metabolism Analytes in Hyperoxaluria Patients Using a Stable Isotope Procedure.

Primary hyperoxalurias (PH) are inborn errors of glyoxylate metabolism characterized by an increase in endogenous oxalate production. Oxalate overproduction may cause calcium-oxalate crystal formation leading to kidney stones, nephrocalcinosis, and ultimately kidney failure. Twenty-four hour urine oxalate excretion is an inaccurate measure for endogenous oxalate production in PH patients and not applicable in those with kidney failure. Treatment efficacy cannot be assessed with this measure during clinical trials. We describe the development and validation of a gas chromatography-tandem mass spectrometry method to analyze the samples obtained following a stable isotope infusion protocol of 13C2-oxalate and 1-13C-glycolate in both healthy individuals and PH patients. Isotopic enrichments of plasma oxalate, glycolate, and glyoxylate were measured on a gas chromatography-triple quadrupole mass spectrometry system using ethylhydroxylamine and N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) for analyte derivatization. Method precision was good for oxalate and glycolate (coefficients of variation [CV] were <6.3% and <4.2% for inter- and intraday precision, respectively) and acceptable for glyoxylate (CV <18.3% and <6.7% for inter- and intraday precision, respectively). The enrichment curves were linear over the specified range. Sensitivity was sufficient to accurately analyze enrichments. This new method allowed calculation of kinetic features of these metabolites, thus enabling a detailed analysis of the various pathways involved in glyoxylate metabolism. The method will further enhance the investigation of the metabolic PH derangements, provides a tool to accurately assess the therapeutic efficacy of new promising therapeutic interventions for PH, and could serve as a clinical tool to improve personalized therapeutic strategies.

Exposure-induced changes of plasma metabolome and gene expression in patients with panic disorder.

BACKGROUND: Anxiety disorders including panic disorder (PD) are the most prevalent psychiatric diseases leading to high disability and burden in the general population. Acute panic attacks are distinctive for PD but also frequent in other anxiety disorders. The neurobiology or specific molecular changes leading to and present during panic attacks are insufficiently known so far. METHODS: In the present pilot study, we investigated dynamic metabolomic and gene expression changes in peripheral blood of patients with PD (n = 25) during two exposure-induced acute panic attacks. RESULTS: The results show that the metabolite glyoxylate was dynamically regulated in peripheral blood. Additionally, glyoxylate levels were associated with basal anxiety levels and showed gender-related differences at baseline. As glyoxylate is part of the degradation circuit of cholecystokinin, this suggests that this neuropeptide might be directly involved in exposure-induced panic attacks. Only gene expression changes of very small magnitude were observed in this experimental setting. CONCLUSIONS: From this first metabolome and gene expression study in exposure-induced acute panic attacks in PD we conclude that metabolites can potentially serve as dynamic markers for different anxiety states. However, these findings have to be replicated in cohorts with greater sample sizes.

A novel dual labeling approach enables converting fluorescence labeling reagents into fluorogenic ones via introduction of purification tags. Application to determination of glyoxylic acid in serum.

Pre-column derivatization with fluorescence labeling reagents involves many problems including crowded chromatograms, possibility of the introduction of analytical errors, and poor selectivity. Herein we report a novel purification tag/fluorophore dual labeling approach based on a multi-component reaction to solve this major problem. Glyoxylic acid was recently identified as an early biomarker for diabetes, thus it was selected as a model analyte for our new dual labeling approach. Using the multi-component Petasis reaction, we could introduce a fluorophore (1-pyreneboronic acid, 1-PyBA) and a purification tag (taurine) to our target analyte (glyoxylic acid) in one step reaction. Using taurine as the amine reactant in Petasis reaction leads to the formation of a reaction product with a terminal sulfonic acid group which can be selectively retained on an anion exchange sorbent allowing excess fluorescent 1-PyBA reagent and its fluorescent decomposition products to be washed away. Then, quantification of the formed analyte-fluorophore-purification tag adduct was carried out by a simple isocratic HPLC-fluorescence detection method. The newly developed technique allowed highly selective, very rapid and efficient determination of glyoxylic acid in human serum eliminating endogenous components and excess reagent interference. Glyoxylic acid was determined in serum at a final concentration down to 30nM (600 fmol/injection) with good recovery (87.0%), accuracy (- 2.2 to 9.2) and precision (%RSD ≤ 8.7).

Eu3+ functionalized Sc-MOFs: Turn-on fluorescent switch for ppb-level biomarker of plastic pollutant polystyrene in serum and urine and on-site detection by smartphone.

The harm of plastic pollutants for human and environment is being paid more and more attention. Polystyrene (PS) and styrene are toxic compounds used in large quantities in the production of fiberglass reinforced polyesters. In this work, a simple method was designed for independent detecting polystyrene and styrene biomarker (phenylglyoxylic acid, PGA) in serum and urine. We prepared Eu3+ functionalized Sc-based metal-organic frameworks as turn-on fluorescent switch for PGA. The distinct enhanced luminescence is observed from the Eu@MOFs with addition of PGA. The fabricated fluorescent switch has several appealing features including high sensitivity (LOD = 4.16 ppb), quick response time (less than 5s) and broad linear range (0.02mg/mL to 0.5mg/mL). Furthermore, Eu@MOFs exhibits excellent selectivity that it is not affected by congeneric biomarkers. More interestingly, a paper-based probe has been devised. The paper-based fluorescence probe would perform an obvious fluorescence change from navy to red with the variety of PGA content. The practicability of the on-site detection platform for quantitative analysis using a colour scanning APP in smartphone has been also demonstrated by coupled with our proposed paper based fluorescence probe. This work first provides a fast, accurate and sensitive method for independent monitoring PS biomarker PGA, and the paper-based probe exhibit a new idea for design portable and easy to operate sensing devices combine with smartphone.

Glyoxylate, a new marker metabolite of type 2 diabetes.

Type 2 diabetes (T2D) is characterized by a variety of metabolic impairments that are closely linked to nonenzymatic glycation reactions of proteins and peptides resulting in advanced glycation end-products (AGEs). Reactive aldehydes derived from sugars play an important role in the generation of AGEs. Using metabolite profiling to characterize human plasma from diabetic versus nondiabetic subjects we observed in a recent study that the reactive aldehyde glyoxylate was increased before high levels of plasma glucose, typical for a diabetic condition, could be measured. Following this observation, we explored the relevance of increased glyoxylate in diabetic subjects and in diabetic C57BLKS/J-Lepr (db/db (-/-)) mice in the pathophysiology of diabetes. A retrospective study using samples of long-term blood donors revealed that glyoxylate levels unlike glucose levels became significantly elevated up to 3 years prior to diabetes diagnosis (difference to control P = 0.034). Elevated glyoxylate levels impact on newly identified mechanisms linking hyperglycemia and AGE production with diabetes-associated complications such as diabetic nephropathy. Glyoxylate in its metabolic network may serve as an early marker in diabetes diagnosis with predictive qualities for associated complications and as potential to guide the development of new antidiabetic therapies.

Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

Interference of ethylene glycol with (L)-lactate measurement is assay-dependent.

BACKGROUND: Metabolites of ethylene glycol (EG) can cross-react in l-lactate assays, leading to falsely elevated serum lactate levels in case of EG intoxication. In this study, we evaluated the effects of EG and its metabolites on routinely used lactate measuring methods. METHODS: Serum aliquots were spiked with either l-lactate, EG or one of its metabolites (all 12.5 mmol/L): glyoxal, glycolate, glyoxylic acid or oxalate. An unspiked sample (l-lactate 2.6 mmol/L) served as a control. l-Lactate levels in these samples were measured in 31 national hospitals on 20 different analysers from nine manufacturers. RESULTS: The l-lactate concentrations in the control sample and in the samples spiked with l-lactate, EG, glyoxal and oxalate provided correct results by all routinely used methods. However, the glycolate and glyoxylic acid spiked samples resulted in falsely elevated l-lactate concentration with all blood gas methods and with the majority of general chemistry methods using l-lactate oxidase. CONCLUSION: The EG metabolites glycolate and glyoxylic acid were shown to falsely elevate l-lactate results with most of the currently used methods due to cross-reactivity with the oxidase enzyme. Falsely elevated l-lactate results can lead to misdiagnosis and inappropriate management of patients with EG intoxication.

Solvent ototoxicity in the rat and guinea pig.

There is clear evidence that aromatic solvents can disrupt the auditory system in humans and animals. As far as animal models are concerned, solvent-induced hearing loss seems to be species-dependent. Indeed, most published data have been obtained with the rat, which shows mid-frequency cochlear deficits, whereas the guinea pig does not show any permanent hearing loss after solvent exposure. In the current investigation, the effects of two solvents, toluene (600 ppm) and styrene (1000 ppm), were studied in both Long-Evans rats and pigmented guinea pigs exposed 6 h/day for 5 consecutive days. Cochlear function was tested by using distortion product otoacoustic emissions (DPOAE) measured prior to the solvent exposure, 20 min after the end of the exposure and successively at 2 and 4 weeks post-exposure. In addition to cochlear testing, solvent concentrations in blood and urinary metabolites were measured. A cochlear histological analysis was performed at the end of the experiment. No decrease in DPOAE amplitude was observed in the guinea pig, even immediately following the end of exposure. The rat model showed severe disruption of auditory function and cochlear pathology, whereas the guinea pig had no disruption of DPOAE or cochlear pathological alterations. Therefore, the vulnerability of the cochlear function was strictly dependent on the species. As expected, an important difference in the styrene concentration in blood was observed: the solvent concentrations were fourfold higher in the rat than in the guinea pig. Therefore, it is clear that a pharmacokinetic or an uptake difference might explain the difference in susceptibility observed between the two species. Moreover, the metabolism pathways of the solvents were different depending on the species. Attempts to explain differences of vulnerability between the rat and guinea pig are addressed in the present paper.

Quantitative determination of CGS 26214, a cholesterol lowering agent, in human plasma using negative electrospray ionization liquid chromatography-tandem mass spectrometry.

CGS 26214 is a synthetic cholesterol-lowering agent shown to be active in the rat, dog and monkey. The present work was conducted to develop a sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for quantitative determination of the compound in human plasma following clinical doses of 10-100 microg per day. A number of analytical challenges were encountered during the development of the assay. The compound was an ester and susceptible to hydrolysis under experimental conditions. A lower limit of quantitation of 50 pg/ml was needed due to the low clinical doses. Positive electrospray ionization of CGS 26214 yielded insufficient sensitivity needed for the studies. Consequently, LC-MS-MS conditions were optimized for the negative ion mode of detection. The sample preparation steps proved to be critical in order to reduce the possibility of microbore column (50 mm x 1.0 mm I.D.) obstruction, chromatographic deterioration, and matrix mediated electrospray ion suppression. The present method addressed the above issues. The method was accurate and reproducible and was successfully applied to generate plasma concentration-time profiles for human subjects after low oral doses of the compound.

Determination of the chiral isomers of CGS 26214, a synthetic thyromimetic agent, in human plasma using microbore chiral chromatography-tandem mass spectrometry.

CGS 26214 is a racemic compound having cholesterol-lowering activity in rats, dogs, and monkeys. This compound has two equipotent chiral components CGS 28934(-) and CGS 28935(+). An analytical challenge was to develop a sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the analysis of the chiral components in human plasma following clinical doses of 1 mg or less. Several issues had to be addressed in order to devise a LC/MS/MS assay for the above compounds. First, the compounds were esters and susceptible to hydrolysis under experimental conditions. Second, a lower limit of quantitation (LLOQ) of 0.4 ng/ml was needed. Third, positive electrospray ionization of CGS 26214 did not yield sufficient sensitivity needed for the studies in humans. Consequently, LC/MS/MS conditions were optimized for negative ion mode of detection. Fourth, sample preparation steps proved to be critical in order to reduce the possibility of microbore chiral-HPLC column (100 x 1.0 mm i.d.) obstruction, chromatographic deterioration, and matrix mediated electrospray ion suppression. Although the present method addressed the above challenges, its major drawback was limited sample throughput capability. Nonetheless, the method was successfully applied to generate plasma concentration-time profiles for human subjects after oral doses (0.9 mg) of the racemate as well as the optically pure isomers.

Liver metabolism during treatment with estradiol and natural progesterone.

Serum concentrations of sex hormone-binding globulin (SHBG), corticosteroid binding globulin (CBG), ceruloplasmin, lipoprotein A and liver enzymes were measured in 30 postmenopausal women treated with 2 mg micronized 17 beta-estradiol daily and micronized progesterone orally in doses of 50, 100 and 200 mg daily, as progestogen supplementation. The treatment lasted for 4 months. The serum levels of SHBG and CBG increased during treatment and a weak association between progesterone dosage and CBG was observed. Levels of lipoprotein A and liver enzymes did not change. It is concluded that micronized natural progesterone is an attractive means of progesterone supplementation in postmenopausal hormone replacement therapy without any liver-related side-effects.

Human liver L-alanine-glyoxylate aminotransferase: characteristics and activity in controls and hyperoxaluria type I patients using a simple spectrophotometric method.

We have studied the characteristics of human liver alanine-glyoxylate aminotransferase, which is deficient in hyperoxaluria type I, an inherited disorder of glyoxylate metabolism. The enzyme was optimally active at pH 8.0 showing apparent Km values for L-alanine and glyoxylate of 8.3 and 1.3 mmol/l, respectively. Activity was found to proceed linearly for up to 4 h. Measurements under these optimal conditions enabled the biochemical diagnosis of hyperoxaluria type I to be made via enzyme activity measurements in percutaneous needle biopsy specimens of liver tissue.

Ion chromatographic determination of plasma oxalate in healthy subjects, in patients with chronic renal failure and in cases of hyperoxaluric syndromes.

An ion chromatographic procedure for the determination of plasma oxalate is proposed, in which the ultrafiltered sample is injected into an ion-chromatographic system. Sample processing appears effective in avoiding spontaneous oxalogenesis. Sensitivity (down to 1.0 mumol/l) allows determinations in normal and pathological samples; recoveries from plasma ultrafiltration are 94.6 +/- 11.7%. Protein binding was investigated and precautions to improve recoveries from plasma ultrafiltration are proposed. The technique is simple to perform from healthy controls averaged 6.75 +/- 2.62 mumols/l (mean +/- S.D. n = 18); samples from patients with primary hyperoxaluria and chronic renal failure undergoing regular dialysis were also analysed and some of the data obtained are reported and discussed.

The effect of additives on red cell 2,3 diphosphoglycerate levels in CPDA preservatives.

Forty-two chemical substances, chosen because they might influence red cell metabolism, were screened for effect on red cell adenosine triphosphate and 2,3 diphosphoglycerate (2,3 DPG) levels during storage in CPD or CPDA-1 at 4 degrees C. Of these substances, six appeared on initial screening to increase 2,3 DPG levels during storage; on repeated examination, four compounds, i.e., oxalate, glyoxalate, ethyl oxaloacetate, and L-phenylalanyl-L-alanine, consistently increased 2,3 DPG levels during storage. It was shown that glyoxalate was converted rapidly to oxalate in blood, presumably through the lactate dehydrogenase reaction. Ethyl oxaloacetate is known to hydrolyze, giving rise to oxalate. Thus, the effect of both glyoxalate and ethyl oxaloacetate can be explained by the formation of oxalate, a compound already known to increase 2,3 DPG levels. The effect of L-phenylalanyl-L-alanine remains to be explained, but it may be hydrolyzed to L-alanine and L-phenylalanine, both of which are thought to have the capacity to increase red cell 2,3 DPG levels by inhibiting pyruvate kinase activity.

Accumulation of glycolic acid and glyoxylic acid in serum in cases of transient hyperglycinemia after transurethral surgery.

Experimental data are presented here proving the accumulation of glycine in serum after transurethral prostatectomy and increased production of glycine metabolites: serine, alanine, glyoxylic acid, and glycolic acid. The presence of the metabolites glyoxylic acid and glycolic acid was demonstrated by gas-liquid chromatography and mass spectrometry. Glycine, glyoxylic acid, and glycolic acid possess neurological activity, so we examined the pathophysiology of the transurethral prostatectomy syndrome in view of the transient accumulation of these compounds in serum.

[Keto acids as regulators of hemoglobin reversible oxygenation]. / Ketokisloty kak reguliatory obratimoi oksigenatsii gemoglobina.

Several ketocarbonic acids covalently attached to the protein moiety of hemoglobin were tested as reversible oxygenation regulators. Two monocarbonic acids (glycoxylic and piruvic) and bicarbonic--alpha-ketoglutaric-acid were used for remoglobin modification. Functional properties and isoelectrical characteristics of modified hemoglobin were recorded. The binding of these acids to hemoglobin was shown to affect the net charge of hemoglobin molecule and the nature of its interaction with allosteric effectors (O2; CI-; H+). Under the influence of glyoxylic acid, the gas-transporting characteristics of hemoglobin underwent most distinct changes approaching those of the human whole blood.

Preliminary results of transmission experiments of non-A, non-B hepatitis by mononuclear leucocytes from a chronic patient.

Mononuclear leucocytes (mainly lymphocytes), isolated by Ficoll-Paque gradient centrifugation of blood freshly drawn from a haemophilia A patient with chronic non-A, non-B hepatitis (NANB), caused NANB when infused in a susceptible chimpanzee. Plasma from the same patient also transmitted the disease, as witnessed by elevated levels of liver enzymes in serum, histopathological signs of viral hepatitis and submicroscopic cytoplasmic alterations in the hepatocytes, considered to be specific for NANB in chimpanzees. In contrast, neither lymphocytes nor plasma from a chimpanzee apparently fully recovered from two episodes of NANB had the same effect.