Lactate assay

Individual NMF species can be measured by High Performance Liquid Chromatography (PCA and UCA), colorimetric assays (FAA) or by enzymatic assays (lactate and glycerol). 

On the base of alcohol oxidase and flavocytochrome b, the enzymatic kits for selective assay of ethanol, methanol, formaldehyde and L-lactate were developed. 

Assay of plasma enzymes aids diagnosis and prognosis. For example, a myocardial infarction elevates serum levels of lactate dehydrogenase isozyme I,. 

Rosalki, S. B. Standardization of isoenzyme assays with special reference to lactate dehydrogenase isoenzyme electrophoresis. Clin. Biochem. (1974), 2> 29-40. 

The bioluminescent determinations of ethanol, sorbitol, L-lactate and oxaloacetate have been performed with coupled enzymatic systems involving the specific suitable enzymes (Figure 5). The ethanol, sorbitol and lactate assays involved the enzymatic oxidation of these substrates with the concomitant reduction of NAD+ in NADH, which is in turn reoxidized by the bioluminescence bacterial system. Thus, the assay of these compounds could be performed in a one-step procedure, in the presence of NAD+ in excess. Conversely, the oxaloacetate measurement involved the simultaneous consumption of NADH by malate dehydrogenase and bacterial oxidoreductase and was therefore conducted in two steps. 

These biosensors were tested for glucose and lactate measurements in sera, and for lactate measurements in whey solutions. Good agreements were obtained between the present method and reference methods. For glucose analysis in serum, the coefficient of variation for 53 repeated measurements performed over a 10 h period was 4.8% while for lactate analysis, 80 assays performed over a 15 h period gave a coefficient of variation of 6.7%. Thus,... 

With the specific suitable oxidases, lactate, choline and glucose could be assayed. Concentration measurements of these metabolites could be performed over at least two decades wit a detection limit of 10 pM for lactate and choline and 20 pM for glucose. 

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

Figure 8.2 The effect of pH on the enzyme lactate dehydrogenase (EC 1.1.1.27). The enzyme shows maximum activity at pH 7.4 (A). When stored in buffer solutions with differing pH values for 1 h before re-assaying at pH 7.4, it shows complete recovery of activity from pH values between 5 and 9 but permanent inactivation outside these limits (B).

L-Amino acid oxidase has been used to measure L-phenylalanine and involves the addition of a sodium arsenate-borate buffer, which promotes the conversion of the oxidation product, phenylpyruvic acid, to its enol form, which then forms a borate complex having an absorption maximum at 308 nm. Tyrosine and tryptophan react similarly but their enol-borate complexes have different absorption maxima at 330 and 350 nm respectively. Thus by taking absorbance readings at these wavelengths the specificity of the assay is improved. The assay for L-alanine may also be made almost completely specific by converting the L-pyruvate formed in the oxidation reaction to L-lactate by the addition of lactate dehydrogenase (EC 1.1.1.27) and monitoring the oxidation of NADH at 340 nm. 

Finally, the In Utero through Uactation Assay involves the use of pregnant rats to assess postnatal development of the neonate after in utero and lactational exposure. This assay has not yet been assigned to either Tier 1 or 2. 

Standjord, P. E., and Clayson, K. J., The control of inhibitory impurities in reduced nicotinamide adenine dinucleotide in lactate dehydrogenase assays. J. Lab. Clin. Med. 67, 144 (1966). 

The LDH+ALT reactor provided a linear response from 0.1 to 50 pmol/L lactate, thereby increasing lactate conversion by 117-183% relative to LDH alone. The intra- and inter-assay CV were both less than 5%, and recoveries ranged from 93 to 106%. Even though roughly 100% of the LDH and ALT added bound to the support under the immobilization conditions used, the activities of the immobilized enzymes were ca. 3% of those of the free enzymes, which is consistent with previous results obtained by the same [67] and other authors [69,70]. Jointly immobilized LDH and ALT preserved ca. 50% of their original activity after 60-90 days of intermittent use. On the other hand, immobilized luciferase was less markedly inhibited than that in the free solution by substances present in the biological samples assayed [71]. 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

This type of extraction is employed in the BP assay of Cyclizine Lactate Injection the injection is diluted with dilute H2SO4 and then neutral and acidic excipients are extracted with ether. The solution is basified and the cyclizine is extracted into ether leaving the lactate ion, which would not have extracted during the initial ether extraction step, behind in the aqueous layer. For convenience in measurement by UV spectrophotometry and in order to carry out volumetric dilution of the extract, cyclizine is then back extracted into dilute H2SO4 and subjected to further dilution. 

Special assays (urine and plasma) lactate, orotidine, thiosulphate, carnitine, succinylacetone, hydroxyproline, urate, orotate, sialic acid, MPS, guanidino-acetate, HVA, pyroglutamate, 5H1AA, pipecolate, pyruvate, 3-hydroxybutyrate, phytanate, VLCFA, homocysteine, 7-dehydrocholesterol, phenylalanine ERNDIM 8 148... 

The same deproteinised blood as used for lactate, pyruvate, , and ACAC assays described above is used for enzymatic methods employing spectrophotometric measurement (Fig. 1.4.3) [10, 17]. The enzymes involved are LDH for pyruvate, lactate oxidase for lactate and HBDH for ACAC and . 

Lactate may be quantified spectrophotometrically after reaction with FeCl2, or by an enzymatic assay (using lactate dehydrogenase which can quantify both D- and L-isomers) or by HPLC. 

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