Pyruvate, lactate determination

Pyronin B, fluorometry, 640 Pyruvate, lactate determination, 625, 626 Pyruvic acid, hydrogen peroxide determination, 655... 

The now classical example is lactate dehydrogenase. Sil-verstein and Boyer were the first to determine the rates of exchange between cognate pairs of reactants ie., lactate and pyruvate as well as NAD and NADH). Convenient [NADH]/[NAD ] and [pyruvate]/[lactate] ratios were chosen such that when combined they satisfied the apparent equilibrium constant for the LDH reaction. These investigators first established that each exchange rate was directly proportional to the duration of exchange and likewise directly proportional to enzyme concentration. As an additional control, they also demonstrated the equality of the pyruvate lactate exchange... 

Esters constituted the largest family and included acetates (ethyl, propyl, isobutyl, butyl, isoamyl, and phenethyl), ethyl esters of fatty acids (propanoate, isobutanoate, butanoate, hexanoate, octanoate, 3-hydroxybutanoate, 3-hydroxyhexanoate and furcate), ethyl esters of organic acids (pyruvate, lactate, ethyl myristate, diethyl malate and, mono- and diethyl succinate) and various other esters, such as methyl butanoate, isobutyl lactate and phenylethyl octanoate. The acids quantified included isobutanoic, butanoic, hexanoic, octanoic, decanoic, lauiic and 3-methylbutanoic. The lactones included y-butyrolactone, pantolactone, y-decalactone and E- and Z-oak lactone and the terpenes included neral d-terpineol, P-dtronellol and Z-nerolidol. The aldehyde family comprised acetaldehyde, benzaldehyde, furfural, 5-methylfurfural and octanal, and the phenol family included eugenol, 4-ethylphenol and 4-ethylguaiacol. Finally, 1,1-diethoxyethane, acetoin, sotolon, 2, 3-butanedione, p-cymene and methionol were also determined. 

Angelis CT, Dunn MF, Muchmore DC, Wing RM (1977) Lewis acid complexes which show spectroscopic similarities to an alcohol dehydrogenase ternary complex. Biochemistry 16 2922-2931 Arnold LJ, Kaplan NO (1974) The structure of the abortive diphospho-pyridine nucleotide-pyruvate-lactate dehydrogenase ternary complex as determined by proton magnetic resonance analysis. J Biol Chem 249 652-655... 

Lactate and pyruvate can be determined simultaneously in the same whole blood sample. Pyruvate is determined using an electrode constructed by immobilizing pyruvate oxidase on a platinum electrode to detect the H2O2 formed [177]. The L-lactate could also be transformed enzymatically into pyruvate, and then all the pyruvate in the sample reduced electrochemically by a mercury cathode polarized at -1.75 V [178]. 

Pyruvate is determined by setting the pH to a more acidic value (pH 7.4) to favor production of lactate. The NADH is then consumed, and the fluorescence diminishes with the pyruvate concentration, which can be measured from 0.2 to 1 pM. The presence of the equilibrium means that the response of the optical sensor is non-linear for both lactate and pyruvate. 

The enantiomeric excess (ee) of the hydrogenated products was determined either by polarimetry, GLC equipped with a chiral column or H-NMR with a chiral shift reagent. Methyl lactate and methyl 3-hydroxybutanoate, obtained from 1 and 2, respectively, were analized polarimetry using a Perkin-Elmer 243B instrument. The reference values of [a]o(neat) were +8.4° for (R)-methyl pyruvate and -22.95° for methyl 3-hydroxybutcinoate. Before GLC analysis, i-butyl 5-hydroxyhexanoate, methyl 5-hydroxyhexanoate, and n-butyl 5-hydroxyhexanoate, obtained from 1, 5, and 6, respectively, were converted to the pentanoyl esters, methyl 3-hydroxybutanoate was converted to the acetyl ester, and methyl 4-methyl-3-hydroxybutanoate obtained from 2 was converted the ester of (+)-a-methyl-a-(trifluoromethyl)phenyl acetic acid (MTPA). 

The concept of using colloids stabilized with chiral ligands was first applied by Bonnemann to hydrogenate ethyl pyruvate to ethyl lactate with Pt colloids. The nanoparticles were stabilized by the addition of dihydrocinchonidine salt (DHCin, HX) and were used in the liquid phase or adsorbed onto activated charcoal and silica [129, 130]. The molar ratio of platinum to dihydrocinchonidine, which ranged from 0.5 to 3.5 during the synthesis, determines the particle size from 1.5 to 4 nm and contributes to a slight decrease in activity (TOF = l s ). In an acetic acid/MeOH mixture and under a hydrogen pressure up to 100 bar, the (R)-ethyl lactate was obtained with optical yields of 75-80% (Scheme 9.11). 

The measurement of stability constants of complexes of yttrium, lanthanide, and actinide ions with oxalate, citrate, edta, and 1,2-diaminocyclohexanetetra-acetate ligands has revealed that there is a slight increase in the stability of complexes of the /-electron elements, relative to the others. A series of citric acid (H cit) complexes of the lanthanides have been investigated by ion-exchange methods and the species [Ln(H2cit)]", [Ln(H2cit)2] , [Ln-(Hcit)], and [Ln(Hcit))2] were detected. Simple and mixed complexes of dl- and jeso-tartaric acid have been obtained with La " and Nd ions, and the stability constants of lactate, pyruvate, and x-alaninate complexes of Eu and Am " in water have been determined. 

Amplification of the sensitivity of substrate or co-en me recycling is especially efficient in thermometric analysis since all the reactions involved frequently contribute to increasing the overall temperature change. One case in point is the determination of lactate or pyruvate by substrate recycling using co-immobilized lactate oxidase and lactate dehydrogenase [160]. 

L-Iactate is oxidized by lactate oxidase to pyruvate, which is reduced back to lactate by LDH. The total enthalpy change for this system can be further increased by addition of catalase, which makes the overall enthalpy change as large as -225 kJ/mol, so signal increases greater than 1000-fold can be obtained as a result. Co-enzyme recycling was also used for the determinations of ATP/ADP [161] and NAD(H) [162],... 

Lactate, pyruvate, acetoacetate (ACAC) and 3-hydroxybutyrate ( ) are intermediary metabolites that normally occur in blood and play an essential role in energy production. Their accumulation in blood is a frequent cause of metabolic acidosis in children. The determination of these metabolites in biological fluids is useful in the early detection, diagnosis and treatment follow-up of abnormalities such as those of ... 

Lactate and can be measured in blood, plasma, urine and the cerebrospinal fluid (CSF). Pyruvate and ACAC are highly unstable, so deproteinisation is performed immediately after sampling. Lactate, pyruvate, and ACAC determination can be measured in the same sample after deproteinisation using perchloric acid [17]. 

Deproteinisation using acids is required to avoid glycolysis, but has to be performed as soon as possible after sampling (within 5 min). Simultaneous determination of lactate, pyruvate, ACAC and is possible using this sample. 

Artuch R, Vilaseca MA, Farre C, Ramon F (1995) Determination of lactate, pyruvate, beta-hydroxybutyrate and acetoacetate with a centrifugal analyser. Eur J Clin Chem Clin Biochem 33 529-533... 

RGURE 7 An oxidation-reduction reaction. Shown here is the oxidation of lactate to pyruvate. In this dehydrogenation, two electrons and two hydrogen ions (the equivalent of two hydrogen atoms) are removed from C-2 of lactate, an alcohol, to form pyruvate, a ketone. In cells the reaction is catalyzed by lactate dehydrogenase and the electrons are transferred to a cofactor called nicotinamide adenine dinucleotide. This reaction is fully reversible pyruvate can be reduced by electrons from the cofactor. In Chapter 13 we discuss the factors that determine the direction of a reaction. 

From these data Krebs and Veech concluded that the oxidation state of the NAD system is determined largely by the phosphorylation state ratio of the adenylate system.169 If the ATP level is high the equilibrium in Eq. 17-42a will be reached at a higher [NAD+]/[NADH] ratio and lactate may be oxidized to pyruvate to adjust the [lactate]/[pyruvate] ratio. 

The catalytically important His-195 is unusually reactive toward diethylpyro-carbonate. This enabled the pKa in both the apo and holoenzymes to be determined directly from the pH dependence of the rate of modification (the pKa — 6.7).58 There is evidence that lactate binds preferentially to the holoenzyme containing the un-ionized histidine, whereas pyruvate binds preferentially to the enzyme-NADH complex containing protonated histidine. 

Flavocytochrome b2 catalyses the oxidation of lactate to pyruvate at the expense of cytochrome C. After reduction of flavin (FMN) by the substrate, reducing equivalents are transferred to heme b2 and from there to cytochrome C602. The mechanism of this process has been studied603 at 5.0 °C by determining the D KIE in the FMN reduction using L-[2-2H]lactate and wild-type enzyme and also with the Y143F mutant prepared from transformed Escherichia coli604. Tritium IE in the conversion of [2-3-H]lactate to... 

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