Normal values at 37 degrees for serum lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase and glycerate dehydrogenase

The distribution of serum lactate dehydrogenase and α-hydroxybutyrate dehydrogenase activity in a sample population of 280 volunteers is presented. Enzyme activity was measured at 37° using optimal concentrations of substrate, coenzyme and phosphate buffer. Significant increases in lactate dehydrogenase activity using the substrates pyruvate, 2-oxobutyrate and hydroxypyruvate were found with increasing age. No significant differences between males and females were found with pyruvate as substrate, but were found when 2-oxobutyrate and hydroxypyruvate were used.     

Use of pyruvate oxidase to overcome pyruvate inhibition during the lactate to pyruvate reaction for assaying lactate dehydrogenase in serum

Automated assays of lactate dehydrogenase (LD) in serum are based on measuring the rate of NADH produced in a reverse LD reaction using lactate and NAD. The observed nonlinearity of LD reaction used in earlier assays performed in phosphate buffers has generally been attributed to the formation of a ternary complex of NAD, pyruvate, and phosphate. This is not satisfactory to explain the course of assay reaction carried out in organic buffers. Investigation of the possible causes of nonlinearity during the course of the reverse LD reaction during LD assays performed in Tris or other organic buffers indicated that inhibition of LD activity by pyruvate may be chiefly responsible for the observed effects, especially in serum exhibiting abnormally high LD enzyme activity. Most of the LD activity in serum was inhibited by 5 mMoles/L pyruvate. By contrast, the LD isoenzyme activities were inhibited partially at 0.5 mMole/L pyruvate, LD1 being the most and LD4 the least susceptible. In assays of serum samples with abnormally high LD and PYR concentration using LD reagent containing Tris buffer, pH 9.3, the inclusion of a bacterial pyruvate oxidase (PO) enabled the removal of pyruvate accumulating in situ, making it possible to assay LD activity in the absence of inhibitory concentration of pyruvate. The inclusion of 10 U/L of PO in our routine LD reagent was sufficient to overcome pyruvate inhibition, thus permitting the assay of serum exhibiting high LD activity, hence the extension of the upper limits of linearity of LD assay without compromising assay performance.     

Reaction-rate assay of serum sorbitol dehydrogenase activity of 37 degrees C.

The sorbitol dehydrogenase (EC 1.1.1.14) activity of human serum and liver was investigated at 37 degrees C. Various buffers were examined but tris(hydroxymethyl)aminomethane HCI in a final concentration of 90 mmol/liter (pH 6.6, at 37 degrees C) was the one with which activity was the greatest. The enzyme is inhibited by its substrate, D-fructose, and activity was greatest with a substrate concentration (in the final assay mixture) of 500 mmol/liter and an NADH concentration of 247 mumol/liter. The specimen volume was 100 mul. The enzyme from liver and serum was shown to have similar Km values and activation energies, and we conclude that the liver enzyme was unchanged on passing into the extrahepatic space. Normal values for this assay are 0--2.6 U/liter (2.5 and 97.5 percentiles).     

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C.

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate were determined for isoenzymes 1 and 5 at 25, 30, and 37 degrees C. Three of the nine different buffers examined--imidazole, triethanolamine, and N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid--are satisfactory. Beta-NADH, pyruvate, and hydrogen ion concentrations were chosen to measure both isoenzymes with maximal-equal-sustainable efficiency at the lowest substrate concentrations. Approximately 95% of each isoenzyme is measured, for activities up to threefold the upper normal limit, if the measurements are made immediately after the reaction is initiated. The Arrhenius relationship for each isoenzyme is unique. Interconversion of results from one temperature to another is practical only with reservations. Results at 37 degrees C are not as reliable as those at 25 degrees C.     

Optimal conditions and comparison of lactate dehydrogenase catalysis of the lactate-to-pyruvate and pyruvate-to-lactate reactions in human serum at 25, 30, and 37 degrees C.

We report optimal conditions for assaying highly purified human lactate dehydrogenase isoenzymes with the lactate-to-pyruvate and pyruvate-to-lactate reactions, as they apply to human serum. Interconversion of results between reactions is not practicable. Measurements of lactate dehydrogenase in either reaction direction at 25, 30, or 37 degrees C can be equally reliable if the volume fraction and the resulting deltaA/min is small. However, for interinstrument and interlaboratory comparisons, results from the lactate-to-pyruvate reaction are more reliable.     

Immunoglobulin A (lambda chains) conjugated with lactate dehydrogenase in serum

An atypical pattern for lactate dehydrogenase (EC 1.1.1.27) isoenzymes in a patient with sclerosis of the bladder neck was ascribable to complexing between lactate dehydrogenase and IgA. This complex formation was also replicable "in vitro." We determined that the IgA bound to lactate dehydrogenase was of the lambda type, a very unusual occurrence.     

Overexpression of monocarboxylate transporter and lactate dehydrogenase alters insulin secretory responses to pyruvate and lactate in beta cells

Previous investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic beta cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 beta cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1-overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.     

离心时间对血浆乳酸脱氢酶活性的影响

乳酸脱氢酶(lactate dehydrogenase,LDH)广泛存在于各种组织中,以肝、心肌、肾脏、骨骼肌、胰腺、肺最多,组织中酶的活力为血清中的1 000倍.因LDH分布广泛,指标特异性差,心肌梗死、肝炎、肝硬化、肾脏疾病、恶性肿瘤、某些贫血患者均增高.在心肌梗死患者中LDH 8～18h开始超过参考上限,24～72h达高峰值,6～10d恢复正常.所以该酶与肌酸激酶相比增高出现较慢,阳性率也较低,但维持时间长,故仍作为诊断心肌梗死的一个有用指标.     

Photodensitometry of mouse serum lactate dehydrogenase isoenzymes separated on polyacrylamide gel

A photodensitometric method for the quantitative estimation of mouse serum lactate dehydrogenase isoenzymes after electrophoresis on polyacrylamide is described. Polyacrylamide provided better resolution of individual isoenzyme fractions than could be obtained using cellulose acetate support media. Photodensitometry allowed for easier and more accurate quantitation.     

The in-vitro degradation at 37 degrees C of vancomycin in serum, CAPD fluid and phosphate-buffered saline

Solutions of vancomycin in phosphate-buffered saline, peritoneal dialysis effluent fluid and human serum were incubated at 37 degrees C for ten days and sampled at daily intervals. The samples were assayed for vancomycin content by a microbiological assay, HPLC and polarisation fluoroimmunoassay (Abbott TDX). The results obtained by HPLC and microbiological assay agreed well and indicated approximately 50% loss over ten days in serum and buffered saline and over 70% loss in dialysate. TDX results indicated losses of only 20% and 40%, respectively. Degradation products were prepared from vancomycin by acid hydrolysis and purified by HPLC. These purified products were shown to cross-react in the TDX assay. It is suggested that the TDX assay becomes non-specific in the presence of vancomycin breakdown products and thus overestimates true vancomycin concentrations.