Formation Of Pyruvic Acid Research Articles

Bimetallic Fe/Co-MOF dispersed in a PVA/chitosan multi-matrix hydrogel as a flexible sensor for the detection of lactic acid in sweat samples.

Anovel bimetallic Fe/Co-metal-organic framework (MOF) hydrogel-based wearable sweat sensorwas developed. Morphological and structural analysis of the hydrogel shows uniformly sized spines and spindle-shaped particles of the Fe/Co-MOF, and it has a high surface area (132.306 m2g-1) and porosity (0.059 cm3g-1) as confirmed by Brunauer-Emmett-Teller (BET) studies. The integration of the bimetallic MOF into a polyvinyl alcohol/chitosan (PVA/CS)-mixed matrix resulted in a multiple network hydrogel. The optimisation study investigated the effects of different pH of the PBS electrolyte, scan rates, and accumulation time in voltammetry. The electrochemical methods such as cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS) provided information on the redox behaviour, electrochemical stability, and catalytic activity of the hydrogel. The sensor demonstrates a wide linear detection range from 0.05µM to 100mM, a superior sensitivity of 0.02mAmM-1cm-2, and a lower limit of detection of 0.01µM . Active sites distributed over the hydrogel surface, specifically Fe2+ and Co2+ within the MOF structure, catalyse the oxidation of L-lactic acid, resulting in electron transfer and the formation of pyruvic acid. Notably, the fabricated sensor exhibits high selectivity, effectively discriminating against interfering species such as uric acid, ascorbic acid, glucose, urea, dopamine, NaCl, and CaCl2. Real-time analysis conducted in a simulated sweat sample via the standard addition method resulted in good recovery percentages of a minimum of 98%. The work presented here is a versatile and simple platform for point-of-care testing, especially for athletes and military personnel.

Papaia fermentada: estratégias para monitorizar e melhorar a fermentação das amostras - estudo preliminar

Introduction: Fermented papaya is known as a nutraceutical, with a unique composition and several health benefits. Its fermentation, being a central process of metabolism, converts the sugars in the fruit into alcohols or acids, with the intermediate formation of pyruvic acid. However, there is a lack of control and uniformity in terms of processes. Objectives: Carry out fermentation cycles of papaya in order to determine which conditions contribute to improving the pyruvic acid content and verify if there is any change of it after freezing preservation. Methodology: The papaya was washed with distilled water and cut into small cubes. About a quarter of each fruit was divided into two sterile flasks, adding ultrapure water, and placing it at 30°C. The flasks were manually shaken every 24 hours, an aliquot was taken for pH measurement. At the end of each fermentation cycle, the sample was subjected to centrifugation followed by gravity filtration and storage at -20°C. At the time of analysis, the aliquots were thawed, and the pH was measured again. The sodium pyruvate content (sodium salt of pyruvic acid) was quantified through a method adapted from another study and validated by this research team. The various fermentation cycles were carried out in duplicate, with changes in the fermentation conditions. Results: The highest content of sodium pyruvate was obtained at the end of the fermentation cycle where the whole fruit (pulp, peel and seeds) was used with sucrose supplementation (1 g.L-1). This indicates that the peel and seeds of papaya, due to their phytochemical composition, have constituents that improve the fermentation process while reducing food waste. The lowest content of sodium pyruvate was obtained on the second day of the fermentation cycle where only fresh papaya pulp and seeds were used, confirming that the presence of the peel is fundamental, probably due to the existence of autochthonous flora in the fruit that contributes to the fermentation process. Furthermore, it was also possible to verify that the pH does not change with the freezing process, indicating that there was no alteration of the pyruvic acid content during the preservation period. Conclusion: It was found that the fermentation conditions that promote a higher content of pyruvic acid is using the whole fruit and sucrose, at 30°C, for 7 days. However, other temperatures, as well as other fermentation conditions, should still be tested in order to understand if the fermentation process can be improved.

Analysis of the Formation of Characteristic Aroma Compounds by Amino Acid Metabolic Pathways during Fermentation with Saccharomyces cerevisiae.

Amino acid metabolic pathways can have profound impacts on the activities of key enzymes in the biosynthesis of specific aroma compounds during yeast fermentation. Aroma compounds, pyruvic acid and glucose were monitored in relation to the key enzymes of leucine aminotransferase (LTR), phenylalanine aminotransferase (PAL), pyruvate kinase (PK) and acetyl-CoA in the amino acid metabolic pathways during the fermentation of simulated juice systems with added amino acids in order to explore the formation of characteristic aroma compounds. The addition of L-phenylalanine or L-leucine to the simulated juice systems significantly improved the activities of PK, PAL and LTR, and the content of acetyl-CoA, and significantly increased the concentrations of phenylethyl alcohol, octanoic acid, isoamyl acetate, phenylethyl acetate, ethyl hexanoate and ethyl caprylate during fermentation. Correlation analysis showed that there was a significant positive correlation between PAL, LTR, PK and acetyl-CoA and pyruvic acid formation. Path analysis revealed that the addition of amino acids affected the metabolism of pyruvate to alcohols, acids and esters to some extent.

Interstellar Formation of Biorelevant Pyruvic Acid (CH3COCOOH)

<h2>Summary</h2> Pyruvic acid represents a key molecule in prebiotic chemistry to form metabolites and amino acids. Without liquid water on the early Earth, endogenous formation of pyruvic acid is unlikely, and an exogenous delivery constitutes an appealing alternative. However, despite the detection of more than 200 molecules in space, pyruvic acid is elusive. Here, we describe its formation by barrierless recombination of hydroxycarbonyl (HOCO⋅) and acetyl (CH₃CO⋅) radicals in ices of acetaldehyde (CH₃CHO) and carbon dioxide (CO₂) modeling interstellar conditions driven by cosmic rays. Exploiting isotopically labeled ices and photoionization reflectron time-of-flight mass spectrometry, the reaction products were selectively photoionized in the temperature-programmed desorption phase and isomers discriminated based on their ionization energies. This reveals a key reaction pathway for pyruvic acid synthesis through non-equilibrium reactions in interstellar cold molecular clouds and star-forming regions, thus offering a unique entry point to abiotic organic synthesis in deep space.

Disorders of carbohydrate metabolism in critical conditions

Objective. To characterize the features of the occurrence and treatment of disorders of carbohydrate metabolism in critical conditions.&#x0D; Materials and methods. The search of publications was conducted using the engines PubMed and Google Scholar and analyzed the available domestic literature sources on the topics: carbohydrate metabolism disorders and critical conditions.&#x0D; Results and discussion. In critical conditions, the following disorders of carbohydrate metabolism develop: diabetic and non-diabetic ketoacidosis, hyperosmolar syndrome, hypoglycemia and lactic acidosis. Carbohydrate disorders in critical conditions develop against the background of the underlying disease, masked by it and worsen the patient’s condition. To diagnose these conditions, a nitroprusside ketonuria test is often used, which does not reflect the actual level of ketonuria (β-oxybutyric acid) and responds to their level in a delayed manner, so most ketosis is diagnosed late – in a decompensated state. Therefore, it is necessary to be guided first of all by a condition of the patient and clinical signs of existence of ketoacidosis. Xylitol (Xylat) solutions are used to treat these conditions. It is believed that xylitol has the greatest antiketogenic effect among all known substances. In particular, Xylat reduces the amount of free fatty acids; increasing of the formation of pyruvic acid, which promotes the oxidation of acetyl-coenzyme A in the Krebs cycle; enhances glycogen production in the liver; stimulates insulin secretion. Do not wait for the appearance of “4+” acetone in the urine analysis. If the patient has nausea, vomiting, dizziness, drowsiness, dry skin and dry mouth, deep noisy breathing and frequent urination on the background of hyperglycemia (&gt;13.9 mmol/l) – should use xylitol-containing drug (Xylat) at a dose of 6-10 ml/kg intravenously.&#x0D; Clinical case. Patient M., 67 years old, was admitted to the trauma department with a hip fracture. He was treated conservatively (skeletal traction) for 3 weeks. Sudden deterioration of state of health is registered: periodically non-contact, loses consciousness. Blood pressure is not determined, heart rate cannot be calculated due to high frequency. On ECG – ventricular tachycardia. Bolus administration of amiodarone had no effect. Carefully analyzed history (diabetes does not get sick, but 3 weeks did not eat enough, almost starved). The level of glycemia is 5.2 mmol/l, ketonemia is 8.4 mmol/l (N=1.7 mmol/l). Diagnosed with non-diabetic ketoacidosis. Xylat infusion (7.5 ml/kg) was performed and amiodarone was reintroduced. As a result, sinus rhythm was restored, the patient regained consciousness.&#x0D; Conclusions. With the development of critical conditions, patients often have disorders of carbohydrate metabolism, which are often stopped by the introduction of xylitol (Xylat).

Interstellar Formation of Biorelevant Pyruvic Acid (CH &lt;sub&gt;3&lt;/sub&gt;COCOOH)

Pyruvic acid represents a key molecular building block in prebiotic chemistry to form metabolites and amino acids. As conditions on early Earth were not favorable for an endogenous formation of pyruvic acid, an exogenous delivery by comets and meteorites constitutes an appealing alternative. However, despite the detection of more than 200 molecules in interstellar and circumstellar environments, pyruvic acid has yet remained elusive. Here we report on the formation of pyruvic acid by barrierless recombination of hydroxycarbonyl (HOCO) and acetyl (CH3CO) radicals in interstellar ices modelling interstellar conditions of acetaldehyde (CH3CHO) and carbon dioxide (CO2) driven by galactic cosmic rays. Exploiting isotopically labelled ices and photoionization reflectron time-of-flight mass spectrometry, the subliming neutral reaction products were selectively photoionized in the temperature programmed desorption phase and isomers discriminated based on their distinct ionization energies. These results reveal a key reaction pathway for the synthesis of pyruvic acid through non-equilibrium reactions in interstellar ices in cold molecular clouds like TMC-1 and star forming regions such as the Orion Molecular Cloud complex (OMC-1), thus offering a unique entry point to abiotic organic synthesis in deep space.

Dynamics of and relationship between fat accumulation and enzyme activities of precocious walnut

Two precocious walnut varieties, Lvling and Lvzao were used as materials in this study, the kernel fat contents and 3 related enzyme activities in different development periods after flowe-ring 50 days were analyzed. The key enzymes affecting walnut fat in different periods were illumina-ted. The results showed that the kernel fat accumulation trends of the two varieties were basically the same. The kernel began to solidify 50 days after flowering, and the increase in kernel fat content was rapid 60-90 days after flowering, slowed down 90-120 days after flowering, and stopped 120-130 days after flowering. The Logistic model was used to fit the fat accumulation (P<0.01). Fat content rapid accumulation period was 57.8-85.8 days after flowering for Lvling, and 67.4-92.1 days after flowering for Lvzao. The activities of acetyl-CoA carboxylase (ACCase), 6-phosphate dextrose dehydrogenase (G6PDH) and pyruvate kinase (PK) increased from 50 to 100 days after flowering, and then the activities of enzymes began to decrease. The kernel fat content was positively correlated with the activity of ACCase. The kernel fat accumulation rate was positively correlated with PK enzyme activity. The correlation between fat content and enzyme activity was different at different development stages. The 50-100 days after flowering was the exuberant period of walnut kernel fat synthesis, and at this time the fat content could be improved by strengthening the field cultivation management measures. At the early stage of the walnut fat synthesis, G6PDH was the major enzyme to affect fat content, and PK activity influenced the formation of pyruvic acid, so as to indirectly affect the synthesis of fat. ACCase activity affected the final fat content and ACCase played an important regulating role in every period of fat synthesis. It was speculated that ACCase might be a key enzyme to affect the fat synthesis of walnut kernel.

Cofactor-embedded nanoporous activated carbon matrices for the immobilization of intracellular enzymes and degradation of endocrine disruptor.

The mixed intracellular enzyme (MICE) from Citrobacter freundii, capable of degrading o-phenylene diamine (OPD), was extracted and characterized. Cofactors such as zinc and copper ions enhanced the MICE activity. The functionalized nanoporous-activated carbon (FNAC) matrix, zinc-impregnated FNAC matrix (Zn2+ -FNAC), copper-impregnated FNAC matrix (Cu2+ -FNAC), and zinc- and copper-impregnated FNAC matrix (Zn2+ -Cu2+ -FNAC) were prepared and characterized to immobilize MICE. The parameters such as time (0-240 Min), pH (1-10), temperature (20-50ºC), amount of MICE (1-5 mg), particle size of carbon (100-600 μm), and mass of carbon (0.5-2.5 g) were optimized for immobilization of MICE on different FNAC matrices. The carrier matrices in the free and MICE immobilized form were characterized using SEM, FT-IR, XPS, XRD, thermogravimetric analysis (TGA), and DSC analyses. The kinetic and adsorption models for the immobilization of MICE on FNAC matrices were studied. The parameters such as time, pH, temperature, concentration of OPD, and agitation speed were optimized for the degradation of OPD using FNAC-MICE and MICE-immobilized metal-impregnated FNAC matrices. The maximum amount of pyruvic acid formed was found to be 133 μg/mg of OPD using Zn2+ -Cu2+ -FNAC-MICE matrix. The kinetic models were studied for the formation of pyruvic acid on OPD degradation and confirmed using FT-IR spectroscopy.

Statistical optimization of the enzymatic breakdown of 2-Nitrophloroglucinol using thermo tolerant mixed Intracellular enzymes from Serratia marcescens

Serratia marcescens, capable of degrading 2-Nitrophloroglucinol (NPG), an endocrine disruptor, was isolated from the soil contaminated with tannery wastewater. The mixed intracellular enzymes (MICE) from S. marcescens were extracted and characterized. The activity of MICE was found to be the maximum at pH, 7 and at temperature, 40 ºC. The optimum conditions for degradation of NPG by thermo tolerant MICE were determined using Plackett–Burman design and response surface methodology. The maximum formation of pyruvic acid, the degradation end product was obtained at time, 3 h; pH, 7; temperature, 40 ºC; concentration of MICE, 4 mg and agitation speed, 75 rpm. The amount of pyruvic acid formed upon degradation (94.65%) of NPG from its initial value of 75 µg was 31.25 µg. The degradation of NPG by thermo tolerant MICE was found to be enhanced by the addition of metal ions such as Zn2+, Cu2+, Ca2+ and V3+ ions with complete conversion of NPG into pyruvic acid. The degradation of NPG by MICE was evaluated through UV–visible, fluorescence and Fourier Transform-Infra Red spectroscopy. The NPG degradation by MICE was confirmed using high pressure liquid chromatography, nuclear magnetic resonance and GC–MS spectroscopy.

Factors affecting the activation and inhibition of intracellular enzymes for degradation of 1,2 diamino benzene: kinetics and thermodynamic studies.

Citrobacter freundii, the bacterium isolated from marine sediments was capable of degrading 1,2 diamino benzene (DAB), an endocrine disruptor. The mixed intracellular enzymes from C. freundii were extracted and purified. The mixed intracellular enzymes were used for the degradation of DAB and degree of degradation was evaluated in terms of pyruvic acid, the end product, formed. The variables such as effect of pH, temperature and metal ions on the degradation of DAB using mixed intracellular enzymes (MICE) were investigated. The maximum amount of pyruvic acid formed was found to be 569 ± 5 µg with 96% degradation efficiency at pH 7; temperature 25 °C; zinc nitrate 0.1 mM; and copper sulphate ions 0.15 mM. The stability of MICE at different temperatures and the interaction of MICE with metal ions were confirmed using FT-IR spectroscopy. The formation of pyruvic acid from degradation of DAB followed pseudo-second-order rate kinetics and it was a spontaneous, exothermic process. The activation energy of degradation of DAB by MICE was found to be 82.55 kJ/mol.

Liquid-phase oxidation of propylene glycol using heavy-metal-free Pd/C under pressurized oxygen

The liquid-phase oxidation of propylene glycol using Pd/C undoped with any heavy metal such as tellurium in the presence of aqueous NaOH under pressurized oxygen resulted in a significant production of lactic acid and a minimal amount of hydroxyl acetone, although, under these conditions, sodium lactate as sodium analogue of lactic acid has been known to easily convert to sodium pyruvate. The advantageous effect of doping Pd/C with a heavy metal, which has been reported for various catalytic reactions in an aqueous phase, was not observed in the present oxidation. Previous papers have reported that oxidation using heavy-metal-free Pd/C and Pd/C doped with a heavy metal at atmospheric pressure and a pH 8 produced pyruvic acid via the formation of hydroxyl acetone and lactic acid, respectively, and that Pd/C doped with a heavy metal rather than Pd/C was suitable for the formation of pyruvic acid from propylene glycol. However the results of the present study show that the use of pressurized oxygen conditions as used in the present study can result in the exclusion of doping with a heavy metal in the preparation of the catalyst, which is amenable to green chemistry.

Proteome-based physiological analysis of the metabolically engineered succinic acid producer Mannheimia succiniciproducens LPK7

Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry, the proteome of a metabolically engineered succinic acid-overproducing bacterium, Mannheimia succiniciproducens LPK7, was examined and compared with that of its wild type strain, MBEL55E, to elucidate the physiological and metabolic changes responsible for succinic acid overproduction and cell growth. Comparative proteomic studies clearly showed that the expression levels of enzymes involved in the ATP formation and consumption (AtpD, Ppa, SerS, ProS, Pnp, PotD, MalK, RbsB, and TbpA), pyruvate metabolism (AceF and Lpd), glycolysis (GapA, Pgk, Fba, and TpiA), and amino acid biosynthesis (Asd, DapA, DapD, Gdh, ArgD, and ArgG) varied significantly in the LPK7 strain compared with those in the MBEL55E strain. Based on the comparative proteome profiling, the formation of pyruvic acid, a newly formed byproduct in the engineered LPK7 strain, could be reduced by adding into the culture medium pantothenate and L: -cysteine, which serve as precursors of CoA biosynthesis.

Improved production of ethanol by deleting FPS1 and over-expressing GLT1 in Saccharomyces cerevisiae

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant KAM-3, the FPS1 gene, which encodes a channel protein responsible for glycerol export, was deleted. The mutant KAM-11 had the GLT1 gene (encoding glutamate synthase) placed under the PGK1 promoter while having the FPS1 deletion. Growth rate and biomass concentration remained virtually unchanged with the mutant KAM-11, compared to that of the parent. Over-expression of GLT1 by the PGK1 promoter along with FPS1 deletion resulted in a 14% higher ethanol production and a 30% lower glycerol formation compared to the parental strain under anaerobic fermentation conditions. Furthermore, acetate and pyruvic acid formation was also reduced in order for cells to maintain redox balance.

The role of β-hydroxyamino acids in the Maillard reaction—transamination route to Amadori products

Analyses of degradation products of β-hydroxyamino acids—l-serine and l-threonine have indicated that the formation of 2-ketoacids and 2-amino alcohols such as pyruvic acid and ethanolamine constitutes a major degradation pathway. Simple mechanisms of decarboxylations and dehydrations can explain their formation in these systems as confirmed by labeling studies. Interestingly, the amino carbonyl interaction between the resulting pyruvic acid and the amino ethanol can lead to the formation of an Amadori product identical in structure to that formed from alanine and glycolaldehyde, indicating the existence of another route to this important Maillard intermediate. In this study, using variously [13C]-labeled pyruvic acids, amino ethanol and alanine, detailed investigation of this interaction was carried out. The initial imine formed during this reaction undergoes an isomerization through a 1,3-prototropic shift followed by enolization and formation of an Amadori product. Aternatively, the isomerized imine can undergo hydrolysis to produce an amino acid and an α-hydroxyaldehyde. The results of these labeling studies have also confirmed the formation of pyruvic acid in the reaction mixture of alanine/glycolaldehyde and the presence of alanine and glycolaldehyde in pyruvic acid/ethanolamine mixture, consistent with the proposed mechanism. It can be concluded therefore that in addition to sugar/amino acid mixtures, 2-ketoacid and α-amino alcohol interaction can also lead to the formation of Amadori product and initiation of Maillard reaction through transamination process.

Formation of sugar-specific reactive intermediates from (13)C-labeled L-serines.

Analysis of the pyrolysis products of [1-(13)C], [2-(13)C], and [3-(13)C]-labeled L-serines has indicated the presence of three initial degradation pathways. Decarboxylation followed by deamination produces aminoethanol and acetaldehyde, respectively; a retro-aldol pathway generates formaldehyde and glycine. Dehydration of L-serine can lead to the formation of pyruvic acid, which eventually can be converted into the amino acid alanine. Formation of alanine and glycine was confirmed due to the detection of 2, 5-diketo-3,6-dimethylpiperazine and cycloglycylalanine. Most of the advanced decomposition products of L-serine can be rationalized on the basis of these initial degradation products. Label incorporation studies have elucidated the origin of carbonyl precursors of methyl- and 2,3-dimethylpyrazines formed in the thermal decomposition mixture of L-serine. Three mechanistic pathways were identified for the formation of carbonyl precursors of methyl- and 2, 3-dimethylpyrazines. The major pathway (70%) for the formation of the precursor of methylpyrazine involved aldol addition of formaldehyde to glycolaldehyde to form glyceraldehyde. On the other hand, the major pathway (60%) for the formation of the precursor of 2,3-dimethylpyrazine involved an aldol condensation of acetaldehyde with glycolaldehyde to form 2,3-butanedione.

Oxidative dehydrogenation of lactic acid to pyruvic acid over iron phosphate catalyst

Vapor-phase air oxidation of lactic acid has been carried out using an iron phosphate catalyst with a P Fe atomic ratio of 1.2. It was found that lactic acid is selectively converted to form pyruvic acid by oxidative dehydrogenation. The one-pass yield reached 50 mol-%. The effects of the reaction variables on the formation of pyruvic acid were studied.

Formation of Pyruvic Acid by Oxidative Dehydrogenation of Lactic Acid

Abstract Lactic acid is converted to pyruvic acid with a high selectivity by a vapor-phase air oxidation over iron phosphate catalysts with a P/Fe atomic ratio of 1.2. The partially reduced iron phosphates show better catalyst performances than the fresh, fully reduced, or reoxidized iron phosphates.

Streamlining Onion Pungency Analyses

Breeding onions for desired pungency requires reliable methods for discriminating between individuals in a segregating population. Organoleptic evaluation has been used traditionally in onion breeding programs; however, it lacks precision and repeatability, especially when evaluating highly pungent onions. Schwimmer and Weston’s method (1961) has been proven to be effective in quantifying onion pungency (Lancaster and Boland, 1990; Whitaker, 1976). Pungency and other flavors are formed from the hydrolysis of S-alk(en)yl cysteine sulfoxide precursor molecules by alliinase following cell disruption. This cleavage produces S-alk(en) sulfenic acid, pyruvic acid, and ammonia. Schwimmer and Weston’s method quantifies pyruvic acid formation from the enzymatic cleavage of the flavor precursor molecules. Pyruvic acid reacts with 2,4-dinitrophenyl hydrazine to produce a colored derivative, which can be measured spectrophotometrically. Although pyruvic acid is a relatively stable compound in this reaction, it is also a common metabolic product in the plant that can upwardly bias enzymatic hydrolysis measurements. As a result, pyruvic acid background levels are determined by thermal deactivation of the enzyme system before analysis. To determine the amount of pyruvic acid formed enzymatitally, the background concentration is subtracted from total pyruvic acid. Schwimmer and Weston’s method, however, is time-consuming and cumbersome, which makes routine and systematic evaluation of breeding material and experimental treatments costly. We propose a routine method for evaluating onion pungency that reduces time and costs. Processing a single bulb in our laboratory using Schwimmer and Weston’s method took ≈74 min-58 min to extract and purify the onion juice for total and background measurements, and 16 min to quantify pyruvic acid. To shorten the time required to extract and purify the juice, we designed and devel-

High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate β-lyase activity: Application to S-1,2-dichlorovinyl- l-cysteine and S-2-benzothiazolyl- l-cysteine

An HPLC—fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate β-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic β-lyase, notably S-1,2-dichorovinyl- l-cysteine (DCVC) and S-2-benzothiazolyl- l-cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the β-elimination activity of β-lyase during the β-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the β-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of β-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl- l-cysteine conjugates.

High performance liquid chromatography assay for alanine amino transferase

An improved method of alanine-amino transferase determination is proposed. The reaction is carried out with alanine and 2-oxoglutarate as substrates and analysis is by HPLC on a reversed-phase chromatographic system using a C18 column and tetrabutylammonium phosphate in phosphate buffer (pH 7.0)-acetonitrile as mobile phase. The enzyme activity was determined by directly following the formation of pyruvic acid without employing any secondary reaction, which is necessary in the spectrophotometric method. The detection limit of pyruvic acid is 10 pmole μl−1 and the standard deviation for the enzymatic activity of standard solutions is 5.4%. Furthermore under the chromatographic conditions selected it is possible to detect the presence of some intermediate species.