Distribution Of Lactate Dehydrogenase Isoenzymes Research Articles

Misconceptions regarding basic thermodynamics and enzyme kinetics have led to erroneous conclusions regarding the metabolic importance of lactate dehydrogenase isoenzyme expression.

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate involving the coenzyme NAD+ . Part of the foundation for the proposed shuttling of lactate from astrocytes to neurons during brain activation is the differential distribution of LDH isoenzymes between the two cell types. In this short review, we outline the basic kinetic properties of the LDH isoenzymes expressed in neurons and astrocytes, and argue that the distribution of LDH isoenzymes does not in any way govern directional flow of lactate between the two cellular compartments. The two main points are as follows. First, in line with the general concept of chemical catalysis, enzymes do not influence the thermodynamic equilibrium of a chemical reaction but merely the speed at which equilibrium is obtained. Thus, differential distribution of LDH isoenzymes with different kinetic parameters does not predict which cells are producing and which are consuming lactate. Second, the thermodynamic equilibrium of the reaction is toward the reduced substrate (i.e., lactate), which is reflected in the concentrations measured in brain tissue, suggesting that the reaction is at near-equilibrium at steady state. To conclude, the cellular distribution of LDH isoenzymes is of little if any consequence in determining any directional flow of lactate between neurons and astrocytes. © 2017 Wiley Periodicals, Inc.

An atypical distribution of lactate dehydrogenase isoenzymes in the hooded seal (Cystophora cristata) brain may reflect a biochemical adaptation to diving.

The brains of some diving mammals can withstand periods of severe hypoxia without signs of deleterious effects. This may in part be due to an enhanced cerebral capacity for anaerobic energy production. Here, we have tested this hypothesis by comparing various parameters of the lactate dehydrogenase (LDH) in the brain of the hooded seal (Cystophora cristata) with those in the brains of the ferret (Mustela putorius furo) and mouse (Mus musculus). We found that mRNA and protein expression of lactate dehydrogenase a (LDHA) and lactate dehydrogenase b (LDHB), and also the LDH activity were significantly higher in the ferret brain than in brains of the hooded seal and the mouse (p < 0.0001). No conspicuous differences in the LDHA and LDHB sequences were observed. There was also no difference in the buffering capacities of the brains. Thus, an enhanced capacity for anaerobic energy production likely does not explain the higher hypoxia tolerance of the seal brain. However, the brain of the hooded seal had higher relative levels of LDHB isoenzymes (LDH1 and LDH2) compared to the non-diving mammals. Moreover, immunofluorescence studies showed more pronounced co-localization of LDHB and glial fibrillary acidic protein in the cortex of the hooded seal. Since LDHB isoenzymes primarily catalyze the conversion of lactate to pyruvate, this finding suggests that the contribution of astrocytes to the brain aerobic metabolism is higher in the hooded seal than in non-diving species. The cerebral tolerance of the hooded seal to hypoxia may therefore partly rely on different LDH isoenzymes distribution.

Cerebrospinal fluid lactate dehydrogenase isoenzymes in children with bacterial and aseptic meningitis

Differentiation of bacterial from aseptic meningitis may be difficult. Our aim was to determine the pattern of distribution of lactate dehydrogenase (LDH) isoenzymes in the cerebrospinal fluid (CSF) of patients with bacterial and aseptic meningitis. One hundred and fifty-seven patients with suspected meningitis were enrolled in the study. They were divided into 3 groups according to the culture- or bacterial antigen assay-proven diagnosis and CSF findings: bacterial meningitis (n = 31), aseptic meningitis (n = 65), and non-meningitis (n = 61). Total LDH level and percentages of LDH isoenzymes in the CSF were measured in each patient. Each group showed a distinct LDH isoenzyme distribution pattern, with a statistically significant difference among the groups in the percentages of the various isoenzymes. Compared with the non-meningitis group, total LDH activity in the CSF was high in the aseptic meningitis group (49.82+/-35.59 U/L, P < 0.001) and exaggerated in the bacterial meningitis group (944.53+/-112.3 U/L, P < 0.001). Low LDH-2 levels were unique to bacterial meningitis (P < 0.01), whereas high LDH-3 levels were characteristic of aseptic meningitis (P < 0.05). Both groups had low levels of LDH-1 and high levels of LDH-4 and LDH-5. In conclusion, the LDH isoenzyme pattern may be of clinical diagnostic value in meningitis, particularly when culture results are pending.

Lactate dehydrogenase isoenzyme distribution and patterns in chicken organs

Unlike most mammals, chicken lactate dehydrogenase isoenzymes cannot be separated using the ‘Titan-Gel’ electrophoresis. However, using isoelectric focusing at a pH range of 3·0 to 9·0, a good and clear separation of all five isoenzymes was achieved. Generally, three characteristic groups were seen: (a) those having a cathodic domination (breast muscle and serum) with mainly lactate dehydrogenase–5 (b) those having an anodic domination (heart, muscle, liver, pancreas, kidney, erythrocytes) of mainly lactate dehydrogenase – 1 and 2 and (c) those with a more uniform distribution (spleen, lung, and brain).The total lactate dehydrogenase activity was the highest in the breast muscle, followed by the heart muscle, liver and serum with the lowest activities in the lung and pancreas.

Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle.

To evaluate the potential role of mitochondrial lactate dehydrogenase (LDH) in tissue lactate clearance and oxidation in vivo, isolated rat liver, cardiac, and skeletal muscle mitochondria were incubated with lactate, pyruvate, glutamate, and succinate. As well, alpha-cyano-4-hydroxycinnamate (CINN), a known monocarboxylate transport inhibitor, and oxamate, a known LDH inhibitor were used. Mitochondria readily oxidized pyruvate and lactate, with similar state 3 and 4 respiratory rates, respiratory control (state 3/state 4), and ADP/O ratios. With lactate or pyruvate as substrates, alpha-cyano-4-hydroxycinnamate blocked the respiratory response to added ADP, but the block was bypassed by addition of glutamate (complex I-linked) and succinate (complex II-linked) substrates. Oxamate increased pyruvate (approximately 10-40%), but blocked lactate oxidation. Gel electrophoresis and electron microscopy indicated LDH isoenzyme distribution patterns to display tissue specificity, but the LDH isoenzyme patterns in isolated mitochondria were distinct from those in surrounding cell compartments. In heart, LDH-1 (H4) was concentrated in mitochondria whereas LDH-5 (M4) was present in both mitochondria and surrounding cytosol and organelles. LDH-5 predominated in liver but was more abundant in mitochondria than elsewhere. Because lactate exceeds cytosolic pyruvate concentration by an order of magnitude, we conclude that lactate is the predominant monocarboxylate oxidized by mitochondria in vivo. Mammalian liver and striated muscle mitochondria can oxidize exogenous lactate because of an internal LDH pool that facilitates lactate oxidation.

Lactate dehydrogenase quantification and isoenzyme distribution in physiological response to stress in red deer ( Cervus elaphus)

As a contribution to the description of the physiological response to stress in red deer ( Cervus elaphus), the potential use of the intracellular glycolytic enzyme, lactate dehydrogenase ( LDH) and its isoenzyme LDH-5, as a marker of muscle damage was assessed. The distribution of LDH isoenzymes within red deer tissues was similar to that shown in other ruminants: tissue characterisation showed isoenzyme LDH-5 to be particularly associated with skeletal muscle. High plasma concentrations of creatine kinase, a muscle-specific enzyme, were associated With both high total LDH activity and the percentage of LDH-5 activity in deer undergoing a potentially stressful procedure, transportation followed by simulated abattoir lairage, Which is further evidence of the specificity of the isoenzyme for skeletal muscle damage. The activity of LDH was not correlated with the plasma concentration of cortisol, a Widely used physiological measure of psychological stress. This may have been due to different time courses for release and the fact that, in the present study, the immediate pre-collection procedures may have induced a short-term stress response.

Selective distribution of lactate dehydrogenase isoenzymes in neurons and astrocytes of human brain.

In vertebrates, the interconversion of lactate and pyruvate is catalyzed by the enzyme lactate dehydrogenase. Two distinct subunits combine to form the five tetrameric isoenzymes of lactate dehydrogenase. The LDH-5 subunit (muscle type) has higher maximal velocity (Vmax) and is present in glycolytic tissues, favoring the formation of lactate from pyruvate. The LDH-1 subunit (heart type) is inhibited by pyruvate and therefore preferentially drives the reaction toward the production of pyruvate. There is mounting evidence indicating that during activation the brain resorts to the transient glycolytic processing of glucose. Indeed, transient lactate formation during physiological stimulation has been shown by 1H-magnetic resonance spectroscopy. However, since whole-brain arteriovenous studies under basal conditions indicate a virtually complete oxidation of glucose, the vast proportion of the lactate transiently formed during activation is likely to be oxidized. These in vivo data suggest that lactate may be formed in certain cells and oxidized in others. We therefore set out to determine whether the two isoforms of lactate dehydrogenase are localized to selective cell types in the human brain. We report here the production and characterization of two rat antisera, specific for the LDH-5 and LDH-1 subunits of lactate dehydrogenase, respectively. Immunohistochemical, immunodot, and western-blot analyses show that these antisera specifically recognize their homologous antigens. Immunohistochemistry on 10 control cases demonstrated a differential cellular distribution between both subunits in the hippocampus and occipital cortex: neurons are exclusively stained with the anti-LDH1 subunit while astrocytes are stained by both antibodies. These observations support the notion of a regulated lactate flux between astrocytes and neurons.

Distribution of lactate dehydrogenase isoenzymes in coelomic fluid and fetal adnexae

The objective of this study was to evaluate the distribution of lactate dehydrogenase isoenzymes in the human gestational sac. Total lactate dehydrogenase and its isoenzymes were measured in matched coelomic fluid and samples of villous and decidual tissues collected at the time of pregnancy termination in a group of 16 healthy women who were between 7 and 12 weeks of gestation. In addition intact secondary yolk sacs ( n=2) and fragments of fetal liver ( n=3) were collected. For comparison, samples of placental tissue, amniotic membrane and chorion obtained at term, were also investigated. In early pregnancy, H-type lactate dehydrogenase isoenzymes predominate in placental villous tissue and coelomic fluid, whereas M-type lactate dehydrogenase isoenzymes predominate in extracts of decidua, secondary yolk sac and fetal liver. These results highlight the important contribution of placental bioproducts to the constitution of the coelomic fluid. We observed also that placental patterns of lactate dehydrogenase isoenzymes change between the first and the third trimester of gestation, from the H-type to the M-type lactate dehydrogenase isoenzymes. We suggest that the lactate dehydrogenase pattern might be an indicator of proliferative or differentiative potential of the trophoblastic cells throughout pregnancy.

Changes in expression and distribution of lactate dehydrogenase isoenzymes in the developing chick retina

We have investigated the expression and distribution of lactate dehydrogenase (LDH) isoenzymes in the developing and adult chicken retina. Non-denaturing polyacrylamide gel electrophoresis was used to follow the appearance and expression of the five main LDH isoenzymes in tissue homogenates. Immunohistochemistry was used to define the distribution of the aerobic heart type LDH and the anaerobic muscle type LDH in paraffin sections of embryonic and adult chick retina. The electrophoretic results show that the expression of the anaerobic isoenzyme increases in the retina as development proceeds. Immunoreactivity against the anaerobic isoenzyme localizes to the inner plexiform layer and other regions of the inner chick retina. The aerobic isoenzyme immunoreactivity is localized to the inner segments of the photoreceptor cells as well as the ganglion cell layer. Both antibodies bind weakly in other layers of the retina with the notable exception that the anaerobic protein does not localize in the photoreceptor cell inner segments. These results provide further evidence for the anaerobic nature of the adult inner retina. As retinal development proceeds, the expression of the anaerobic isoenzyme in the retina increases.

Effect of reticulocytosis on lactate dehydrogenase isoenzyme distribution in serum: in vivo and in vitro studies

We investigated the effect of reticulocytosis on the lactate dehydrogenase (LD; EC 1.1.1.27) isoenzyme LD1/LD2 ratio in patients with and without evidence of hemolytic disease. Analysis of sera from patients with reticulocytosis and in vivo hemolysis showed a mean LD1/LD2 ratio of 0.92 compared with a ratio of 0.69 in patients with in vivo hemolysis and normal reticulocyte counts. Determination of LD isoenzymes in erythrocyte lysate revealed significantly increased LD1/LD2 ratios for patients with marked reticulocytosis compared with those for patients with normal-to-minimal increases in reticulocytes. Finally, separation of mature erythrocytes and reticulocytes by flow cytometry revealed marked differences in the LD1/LD2 isoenzyme distribution between these two cell types. The ability of hemolysis to cause a "flipped" LD1/LD2 ratio is dependent on the proportion of the hemolyzed cells that are reticulocytes.

Distribution of lactate dehydrogenase isoenzymes in potato (Solanum tuberosum) and other plants

Distribution of lactate dehydrogenase isoenzymes in potato (<i>Solanum tuberosum</i>) and other plants

Cytochemical changes in lactate dehydrogenase isoenzymes in human brain tumours.

The lactate dehydrogenase (LDH) isoenzyme patterns in benign and malignant brain tumours were determined by means of electrophoresis of the cell extracts and selective cytochemical stain of the smears. The LDH isoenzyme distribution of the cell extracts showed a pronounced cathodal shift in the malignant gliomas and metastatic carcinomas. Normal brain tissues and histologically benign gliomas, however, showed an anodal pattern with a dominance of the H-type LDH. Schwannomas and meningiomas had a midzone isoenzyme pattern with a dominant LDH3 fraction. Pituitary adenomas usually showed the LDH pattern similar to that of the normal cerebrum. The LDH M fraction could be cytochemically verified using an inhibitory effect by 2.6 M urea in staining. Astrocytomas grades 3-4 and metastatic carcinomas were characterized by loss or marked reduction of stainability by urea treatment, while astrocytomas grades 1-2 and oligodendrogliomas were resistant to urea inhibition.

LDH isoenzyme distribution in human Tγ lymphocytes

The lactate dehydrogenase (LDH) isoenzyme distribution was measured in T γ + lymphocytes from normal individuals. T γ + lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The B:A subunit ratio was clearly lower in the T γ + lymphocytes. Phenotyping of the T γ + lymphocytes showed a vast majority of OKT11 +, OKM1 +, OKT3 − lymphocytes. In one experiment, T γ + lymphocytes were enriched by OKT3 depletion of T lymphocytes. The low B:A ratio was also found in these cells indicating that the LDH pattern is not the consequence of an in vitro activation by immune complexes. As the T γ + lymphocytes were considerably enriched in cells having the characteristics of NK cells according to their phenotyping, morphology and NK cell activity, we may assume that NK cells have a low B:A ratio.

Adaptive changes in uterine distribution of lactate dehydrogenase isoenzymes during protein malnutrition and steroid maintained pregnancy in rats.

The electrophoretic studies revealed that in non-pregnant and pregnant rats fed with protein-free diet (PFD), a significant increase in percentage of LDH1 and LDH2 (H-isozymes) was observed with a concomitant decrease in concentration of LDH4 and LDH5 (M-isozymes). The changes in LDH3 were not significant. Further, during protein deficiency heart-type activity (H-subunits) was increased markedly with a parallel decline in muscle-type activity; thus resulting in an increase in H/M ratio or analogue ratio (A.R.). However, the decrease in A.R. (below 2.00), observed during normal and steroid maintained pregnancy was due to higher muscle-type than heart-type activity. These observations suggest that protein malnutrition in rats favours a metabolic shift from anaerobic state to aerobic predominance in the uterus. The physiological significance of these adaptations has been discussed.

Abnormal lactic dehydrogenase patterns in carcinoma of stomach and in peritumoral tissues.

Segments of gastric mucosa from 32 surgical and biopsy specimens containing gastric carcinoma have been studied to determine the total content of lactate dehydrogenase (LDH). The LDH isoenzyme distribution patterns in tumoral tissues have been compared to that of peritumoral tissue and to the gastric mucosa of a healthy control group. LDH isoenzyme activity in the samples from tumoral tissue was significantly increased. The adjacent peritumoral tissue had a slight increment in the polypeptide M as compared to the distant and normal tissue. This suggests that LDH isoenzyme measurement may prove a valuable addition to histologic study, in the recognition of the tumoral distribution in stomach carcinoma.

Distribution of lactate dehydrogenase isoenzymes in normal and inflamed bovine udders and milk

The patterns of distribution of lactate dehydrogenase (LDH) isoenzymes were determined in normal and inflamed bovine udder tissues, in normal and mastitic milk-leucocytes and serum. LDH1 was the most common isoenzyme found in all the types of tissues examined, normal tissues contained the lowest proportions of LDH5 whereas the inflamed tissues and leucocytes from mastitic milk showed a higher proportion of LDH4 and LDH5. It seems that the origin of the elevated LDH in mastitic milk is the leucocytes and the parenchyma cells of the udder. The significance of the shift in the molecular forms of LDH isoenzymes is discussed in relation to possible alternations in energy metabolism in the inflamed bovine mammary gland.

Changes in lactate dehydrogenase isoenzyme patterns in patients with tumours of the central nervous system.?

The total activities of lactate dehydrogenase (LD) and the LD isoenzyme distribution were determined on homogenates from 61 benign and malignant intracranial tumours, and in serum and cerebrospinal fluid from 45 and 28 of the tumour patients respectively. The LD activity and the LD isoenzyme distribution in serum were not changed in patients with intracranial tumours. The LD activity in the cerebrospinal fluid was raised in about 50% of the tumour patients, but without any significant differences between patients with benign and malignant tumours. Except for very low activities in acoustic schwannomas and in pituitary adenomas, the tumour tissue did not differ significantly from normal brain in total LD activity. The LD isoenzyme distribution showed a pronounced cathodal shift in the grade 3-4 astrocytomas and the metastatic carcinomas, but an anodal shift in the grade 1-2 astrocytomas and the oligodendrogliomas. The meningiomas and the ocoustic schwannomas showed a midzone isoenzyme pattern with a dominant LD3 fraction. Knowledge of the LD isoenyme distribution can thus sometimes be a complement to the histological examination in the classification of brain tumours.

Lactate dehydrogenase (LD), hydroxybutyrate dehydrogenase (HBD), and LD-isoenzymes in brain tissue.

The total activities of lactate dehydrogenase (LD) and hydroxybutyrate dehydrogenase (HBD), and the LD isoenzyme distribution were determined in tissue samples taken in vivo from different parts of the rabbit brain, and in cortical grey, subcortical white, and cerebellar tissue from man. The total LD and HBD activities were found to be higher in the brain stem than in the cerebral hemisphere, basal ganglia, and cerebellum of the rabbit brain. The isoenzyme distribution in the brain stem and the cerebellum showed an anodal shift, i.e., significantly higher activities of LD1 compared with the cerebral hemisphere and the basal ganglia. In man the total LD activity was found to be lower in white than in grey matter, although the isoenzyme patterns did not differ. The implications of these findings in relation to the LD isoenzyme pattern in serum after traumatic brain injury are discussed.

Total lactate dehydrogenase and its isoenzymes in serum in the presence of penicillamine and other sulfhydryl compounds.

Toward delineation of changes in total lactate dehydrogenase (LDH) and in the distribution of LDH isoenzymes as assessed by polyacrylamide disc electrophoresis, we inbucated human and rat sera with various agents, notably sulfhydryl compounds. Although artefacts were apparent when these agents were used without preliminary adjustment of pH, we saw little alteration in total unitage when one or two volumes of serum was mixed with one volume of any of several thiols, especially penicillamine, at an initial concentration of 0.4 mol/liter and pH 7.0-7.5. Under these conditions, penicillamine caused a loss in LDH-5 after incubation for 1 h at 25 degrees C together with small decreases in mobility of the other four isoenzymes toward the anode. A zymosan region appeared below the albumin and tracking dye area. With longer periods of incubation of rat serum with penicillamine or alpha-mercaptosuccinate, a novel band in the zymogram was noted just above the LDH-4 peak. The observations are discussed in terms of allosteric effectors.

Regional distribution of lactate dehydrogenase isoenzymes in adult rat brain.

The highest lactate dehydrogenase (LDH) activity was found in thalamus, statistically significantly less in cerebral and cerebellar cortex and the lowest in pons. LDH1 and LDH4+5 represented 58% and 23% of the total activity in cerebral cortex, 54% and 20% in thalamus, 42% and 4% in cerebellar cortex and 55% and 7% in pons, respectively.