Nicotinamide adenine dinucleotide assay

The kinetic assay of LDH is based on the conversion of lactic acid to pyruvic acid, in the presence of nicotinamide adenine dinucleotide (NAD), and is closely monitored at intervals of 30 seconds or 1 minute by measuring the increase in absorbance at 340 nm. In this particular instance lactic acid available in an excess to ensure that the increase in pyruvic acid is linear with time, i.e., directly proportional to time. The reaction involved may be expressed as follows ... 

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). 

An enzyme assay measures the conversion of substrate to product, under conditions of cofactors, pH and temperature at which the enzyme is optimally active. High substrate concentrations are used so that the initial reaction rate is proportional to the enzyme concentration. Either the rate of appearance of product or the rate of disappearance of substrate is measured, often by following the change in absorbance using a spectrophotometer. Reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), which absorb light at 340 nm, are often used to monitor the progress of an enzyme reaction. 

Conditions for cytosolic incubations depend on the aim of the assay e.g. to cover specific enzymatic activity present in the cytosol. For this purpose it is essential to fortify the incubation medium with the specific cofactor for the reaction-if needed (Ekins 1999). (J> -Nicotinamide adenine dinucleotide (NAD) is needed for alcohol and aldehyde dehydrogenases, flavin adenine dinucleotide (FAD) for polyamine oxidase, P-nicotinamide adenine dinucleotide phosphate (NADPH) for Dihydropyrimidine dehydrogenase. Phase II reactions depend on PAPS (sulfotransferases,... 

Spectrophotometric assay (BIO, L7, S29) is accomplished by a similar incubation, without hydrazine, where excess added triosephosphate isomerase converts triose phosphate formed to DAP, in turn removed as glycerophosphate by reduced nicotinamide-adenine dinucleotide (NADH2, formerly termed DPNH) and added glycerophosphate dehydrogenase. 

Fluorescence detection is also inherently more selective than absorbance detection, since both the excitation and emission wavelengths may be chosen to suit a particular reaction product. For example, assays employing dehydrogenase enzymes may monitor NAD+ or nicotinamide adenine dinucleotide phosphate (NADP+) absorbance at 340 nm with reasonable sensitivity and selectivity. However, if excited at 340 nm, the nicotinamide coenzymes fluoresce at 460 nm. Not only do the fluorescence measurements inherently have lower detection limits, but they also provide selectivity against potential interferents that may also absorb at 340 nm but do not emit at 460 nm. 

The amount of inulin present is determined firom the reduction of nicotinamide-adenine dinucleotide, reduced form (NADH) measured as a decrease in absorbance at 340 nm. The method is calibrated with either inulin or fructose endogenous fructose in each sample is measured by incubation with an inactivated inulinase reagent. Urine samples require predilution (typically 1 in 40) before analysis. A typical between-run imprecision of less than 2% for plasma and less than 4% for urine can be obtained with an automated assay. 

Gatto et al m characterized the mechanism of L-pipecolic acid formation by cyclodeaminase RapL from L-lysine within rapamycin biosynthesis, which is a hybrid NRP—polyketide antibiotic (Figure 25(a)). RapL was characterized by biochemical assays to require cofactor nicotinamide adenine dinucleotide (NAD+) and an oxidative cyclodeamination reaction mechanism corresponding to ornithine cyclodeamination was proposed based on ESI-FTMS analysis of RapL reaction products (Figure 25(b)). 

In vitro experiments can sometimes provide valuable insight into what is happening in vivo that is limiting oral bioavailability. The typical experiments, often employed in tandem, to understand bioavailablilty are determinations of compound solubility, membrane permeability, and stability in subcellular fractions. The membrane permeability assays that are most often employed are either a measurement of permeability through an artificial membrane (Parallel artificial membrane permeability assay, PAMPA, is the most common technique) or a cell monolayer (Caco-2, a human colon carcinoma-derived cell line, is the most common cell monolayer). The subcellular fractions most often employed are plasma (for ester-containing compounds) and liver microsomes with the addition of either reduced nicotinamide adenine dinucleotide phosphate (NADPH) or uridine diphosphoglucuronic acid (UDPGA) as cofactor. 

Since NADPH absorbs light at 340 nm and NADP+ does not (Figure 8-3), this reaction can be followed by measurement of the change in absorbance at this wavelength with time. A similar assay (with lactate and NAD+) can be used for serum lactate dehydrogenase. The difference in absorbance is measured so readily that a number of assays make use of it directly or indirectly. In the latter case, the reaction to be measured is linked by one or more steps to a reaction in which a nicotinamide adenine dinucleotide is oxidized or reduced. Thus, in the measurement of aminotransferases (Chapter 17), one of the products of the primary reaction serves as a substrate for a secondary reaction in which NADH is required. For example, in the assay of aspartate aminotransferase,... 

In nicotinamide adenine dinucleotide-linked assays, endogenous metabolites and enzymes in the serum may cause oxidation or reduction of the coenzyme, thereby introducing errors. For example, in the AST assay, the NADH of the indicator reaction may be oxidized by the presence of pyruvate and of LDH in the serum ... 

Byers, S., Anderson, D., Brobst, D., and Cowan, F. (2000). Automated assay for nicotinamide adenine dinucleotide (NAD ). J Appl Toxicol 20 Suppl 1, S19-22. 

The MT T assay is another colorimetric method to assess cell viability. NAD(P)H (nicotinamide adenine dinucleotide(P)H)-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes are capable of reducing the tetrazolium dye MTT 3-(4,5-diwiethylfhiazol-2-yl)-2,5-diphenylletrazolium bromide to its insoluble purple-coloured formazan (Figure 7.71). In living cells, the originally yellow MTT is converted by cellular reductase to purple formazan (Mosmann, 1983). A solubilisation solution (usually either dimethyl sulfoxide or sodium dodecyl sulfate dissolved in diluted hydrochloric acid) is added to dissolve the insoluble... 

McNeil, C. J., Spoors, J. A., Cocco, D., Cooper, J. M., Bannister, J. V., Thermostable Reduced Nicotinamide Adenine Dinucleotide Oxidase Application to Amperometric Enzyme Assay , Anal. Chem. 61 (1989) 25-29. 

There are no recognized enzyme standards or reference materials. The accepted basis for measurement is the rate of reduction of the substrate, commonly nicotinamide adenine dinucleotide (NAD) for many reactions. The reduced form, NADH, absorbs at 340 nm, and the rate of change of this absorbance is measured in an enzyme-activity assay. The absorptivity a in Beer s law) is known for NADH from this and the rate of change of absorbance per unit time, the activity of the enzyme can be calculated in micromoles of substrate converted per minute. This is referred to as an International Unit (lU), expressing the activity as lU/liter. Therefore, an accurate, absolute absorbance scale must be established in each case in order to make a valid assay. 

ADH is widely used for serum ethanol assay. ADH kinetics is sophisticated due to the reversibility of reaction and the inhibition by both acetaldehyde and NADH as products. To simplify ADH kinetics, some special approaches are employed to make ADH reaction apparently irreversible on single substrate (alcohol). Thus, reaction pH is optimized to 9.2 to scavenge hydrogen ion semicabarzide at final 75 mmol/L is used to remove acetaldehyde as completely as possible final nicotinamide adenine dinucleotide (NA1>) is 3.0 mmol/L final ADH is about 50 U/L (Liao, et al., 2007a). By assigning the maximal absorbance at 340 nm for reduced nicotinamide adenine dinucleotide (NADH) by the equilibrium method to Ame and that by kinetic analysis of reaction curve to Amk, kinetic analysis of ADH reaction curve should predict Amk consistent with Am but requires some special efforts. 

An automated kinetic assay for the determination of D-glucose in blood has employed D-glucose dehydrogenase [EC 1.1.1.47] in the presence of muta-rotase and nicotinamide adenine dinucleotide as hydrogen acceptor. The technique is highly specific and can be applied to blood and other body fluids. 

Matsumura H and Miyachi S (1980) Cycling assay for nicotinamide adenine dinucleotides. Methods in Enzymo-logy 69 465—470. 

Bernofsky C, Swan M (1973) An improved cycling assay for nicotinamide adenine dinucleotide. Anal Biochem 53 452-458... 

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