Measurement Of Lactate Dehydrogenase Activity Research Articles

The effect of aflatoxins on the incorporation of RNA and protein precursors by isolated hepatocytes.

Hepatocytes prepared by a simplified enzymatic technique were active in the incorporation of RNA and protein precursors into acid-insoluble material. The incorporation of RNA precursors was very markedly inhibited by low levels of aflatoxin B1 and G1 but not by aflatoxins B2 and G2. The activity of mixed function oxidases (MFO), the drug-metabolizing system of the endoplasmic reticulum, could be suppressed in these cells by SKF525A or stimulated by NADPH. SKF525A caused a reduction in the inhibition by aflatoxin B1 of the incorporation of RNA precursor into macromolecules. This finding suggests that a metabolite of aflatoxin B1 is the actual inhibitor of RNA synthesis in the cells. Measurement of lactate dehydrogenase activity showed these cells to be leaky on incubation at 37 degrees C and thus not suitable for studies of protein secretion.

Lactate dehydrogenase (LDH) activity in the inner ear under acoustic overstimulation (author's transl)

This paper gives the results of LDH activity of various tissue in the inner ear of a guinea pig, the organ of Corti organ, stria vascularis and perilyirphatic fluid under acoustic overstimulation. 47 normal guinea pigs, with good preyer reflex, were used for these experiments. These guinea pigs were exposed to white noise of 115 dB for various duration of time from 10 minutes to 72 hours. After acoustic stimulation, inner ears of animals were exposed by operation and perfused with LDH incubation. After animals were decapitated, the inner ear were fixed with Karnovsky's solution and embedded with paraplast. With the LDH staining method, aboundant deposits of reduced tetrazolium dyes were shown to be in the organ of Corti and stria vascularis. After exposure to noise for 60 minutes, almost all the formosan disappeared in both the organ of Corti and the stria vascularis. However, after exposure to noise for 15 hours, LDH activity increased in these areas again. As the next step of the experiment, the LDH activity in the organ of Corti and the stria vascularis were biochemically measured by socalled microbiochemieal technique. After decapitation of guinea pigs, their inner ears were put into 4°C physical NaCl solution. Using the surface preparation method, the organ of Corti and the stria vascularis of each turn were separately obtained and homegenated in minute glass tubes. After centrifusion, quantitative measurement of LDH activity and protein content was performed on the supernatant fluid. In normal guinea pigs, the organ of Corti and the stria vascularis showed almost the same level of LDH activity. The stria vascularis in the second turn showed the highest LDH activity, then first turn, third and the lowest activity in the fourth turn. After exposure to the noise for 60 minutes, the LDH activity of the stria vascularis decreased markedly in all turns and indicated the lowest level through the run. After exposure to noise for 15 hours, however, its activity increased to the highest level. After 72 hours, the LDH activity decreased again and returned to the normal level. The behaviour of LDH activity in the organ of Corti after acoustic overstimulation seemed to the same as it in stria vascularis. In perilymph, the LDH activity showed its highest level ( 5 times higher than normal level) on 15 hours exposure to the noise and then decreased gradually to the normal level.

Kinetic Colorimetric Measurement of Serum Lactate Dehydrogenase Activity

Abstract A kinetic colorimetric assay procedure is described for measuring lactate dehydrogenase activity, in which the reduction of NAD is coupled to the reduction of a tetrazolium salt, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), with phenazine methosulfate serving as an intermediate electron carrier. The molar absorptivity of the formazan of INT was found to be 19.3 x 103 at 503 nm, which provides a threefold increase in sensitivity over the ultraviolet (340 nm) kinetic assay. The results correlate well with those obtained by 340-nm kinetic methods and the procedure does not require an ultraviolet spectrophotometer. The method is simple, rapid, and does not rely on secondary enzyme standards. It requires only two reagents, which are stable.

Rapid Single-Step Kinetic Colorimetric Assay for Lactate Dehydrogenase in Serum

Abstract We report an improved kinetic colorimetric system for measuring lactate dehydrogenase activity in serum. In the system a tetrazolium salt, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride, is used as the chromogenic indicator of dehydrogenase activity, with diaphorase serving as the electron transfer agent. All ingredients required for an assay are combined in a single dry reagent that is stable at room temperature. The method is 2.5 times as sensitive as the ultraviolet method of Wacker and has a dynamic range three times that of the ultraviolet method. Reducing substances in serum do not affect the results. Precision, range of linearity, and stability of reagent after reconstitution are excellent. Results for fresh sera correlated well with those obtained by the "A-Gent" ultraviolet method (Wacker method at 37°C) and with the SMA 12/60.

Estimation of the growth of the T 1 strain of Mycoplasma mycoides in tryptose broth by the measurement of lactate dehydrogenase. II. Application to vaccine production.

The measurement of lactate dehydrogenase activity has been used to evaluate the growth of the T(1) vaccine strain of Mycoplasma mycoides var. mycoides in broth during the production of vaccine. Estimations were made during incubation at 37 C and storage at 4 C. The results were compared with titers of the vaccine obtained by two conventional methods of assessing viability titers: the counting of colonies formed on solid medium and a 50% end point titer found after dilutions were made in broth. The method of measuring lactate dehydrogenase activity allowed the rapid enumeration of organisms during incubation of the culture, but estimations made on vaccine stored at 4 C did not compare well with the two conventional methods of assessing viability. The lactate dehydrogenase activity of the culture has enabled rapid monitoring of the production stages of this vaccine.

Studies on Rates of Abortive Ternary Complex Formation of Lactate Dehydrogenase Isozymes

Abstract Rates of formation and dissociation of an abortive ternary complex between lactate dehydrogenase isozymes from various mammalian tissues, NAD+, and pyruvate were studied utilizing stopped-flow spectrofluorometry. Formation of the complex was accompanied by a decrease in protein fluorescence at 340 mµ and a loss of lactate dehydrogenase activity. Rates of formation of an abortive lactate dehydrogenase-1 and lactate dehydrogenase-5 complex were compared at 25° and 40° over a range of pyruvate and enzyme concentrations, at two NAD+ concentrations, and with two coenzyme analogues. Dialysis against buffer produced an increase in protein fluorescence and recovery of lactate dehydrogenase activity that suggested dissociation of the abortive complex. At pyruvate concentrations of 0.3 mm the abortive ternary complex formed twice as fast with lactate dehydrogenase-1 as with lactate dehydrogenase-5 and with 40.0 mm pyruvate it formed 5 times faster. The rate of complex formation increased with both isozymes when pyruvate concentrations were raised from 0.3 mm to 40.0 mm and when the temperature was elevated from 25° to 40°. However, the rates of abortive complex formation were inversely related to enzyme concentration. Compared to lactate dehydrogenase turnover numbers, the rates of formation of the abortive ternary complex were slow. Under physiological conditions of temperature (40°) and pyruvate concentration (0.3 to 1.0 mm), half the maximum fluorescence quenching required approximately 50 sec for partially purified lactate dehydrogenase-1. Changes in coenzyme concentration from 0.10 mm to 0.75 mm failed to alter the rates of abortive complex formation. With the 3-acetylpyridine analogue of NAD+, rates of abortive complex formation for both lactate dehydrogenase-1 and lactate dehydrogenase-5 were similar, whereas with deamino-NAD+ the abortive complex formed faster with lactate dehydrogenase-1 than with lactate dehydrogenase-5. Dialysis of the abortive complex resulted in recovery of enzyme activity; the rate of recovery was faster with lactate dehydrogenase-5 than with lactate dehydrogenase-1. The stopped-flow spectrophotofluorometer permitted measurement of lactate dehydrogenase activity at enzyme concentrations approaching those in tissue. At lactate dehydrogenase-1 concentrations of 3.5 x 10-6 m or 3.5 x 10-7 m no substrate inhibition was detected with pyruvate concentrations up to 20.0 mm, whereas with enzyme levels at and below 1.8 x 10-7 m, substrate inhibition appeared at pyruvate concentrations greater than 1.0 mm.