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PubMedDate not supplied

Effects of polyethylene glycol molecular weight and concentration on lactate dehydrogenase activity in solution and after freeze-thawing.

Lactate dehydrogenase (LDH) is a tetrameric enzyme that has been used as a model for labile protein drugs. Polyethylene glycols (PEGs) have been proposed as excipients to stabilize labile proteins in solution and during the freezing portion of the lyophilization cycle. The aim of this study was to determine the effects of PEG molecular weight and concentration on the activity of LDH. In general, PEG protection increases with PEG molecular weight and concentration. PEGs slow the loss of activity in LDH solutions stored at 4 degrees C, but are not sufficiently effective to allow for a solution product. PEGs 8000, 10,000, and 20,000 show full freezing protection at less than 0.01%, while lower molecular weight PEGs need higher concentrations to produce protection upon freezing. Circular dichroism (CD) studies of LDH solutions before and after freezing with PEG 400 and PEG 8000 confirm the activity studies. The CD spectrum of LDH before freezing shows the classic alpha helix pattern. After unprotected LDH solution is frozen and thawed, the CD spectrum erodes. Low concentrations of PEG 8000 (1% or less) preserve the alpha helix profile after freezing of the samples. PEG 400 preserves the alpha helix CD profile in a stepwise fashion with increasing concentrations. The CD and activity data suggest that PEGs can protect alpha helix structures and activity of LDH through the freezing process.

Animals↗
PubMed2005

Total serum lactate dehydrogenase activity in acute Plasmodium falciparum malaria infection.

INTRODUCTION: Lactate dehydrogenase (LDH) activity was assayed in the sera of 76 adult male and 76 adult female patients within the age group of 18-40 years presenting with acute, uncomplicated Plasmodium falciparum malaria infection and a control group of 80 healthy adults within the same age group. METHODS: Patient selection and pre-qualification were done by simple random sampling of individuals presenting at the Bauchi Specialist Hospital Outpatient Department with a history of fever and malaise within a period of one to eight days, and who were confirmed to be infected with the P. falciparum malaria parasite by microscopical examination of Giemsa-stained thin blood slides. RESULTS: The mean serum LDH activity in male patients was found to be 789.4 +/- 35.0 IU. This activity is significantly higher than the control LDH activity of 247.10 +/- 19.0 IU (p-value is less than 0.05). The mean serum LDH activity among female patients was 634.0 +/- 35.0 IU, which is a relatively higher activity compared to the control LDH activity of 247.10 +/- 19.0 IU (p-value is less than 0.05). CONCLUSION: The combination of acute hepatocellular injury and red cell haemolysis induced by the invading merozoites may account for the increase in serum LDH activity during this infection. Therefore serum LDH activity is a potentially valuable enzymatic marker of acute, uncomplicated P. falciparum malaria infection, especially in the absence of other complicating diseases known to be associated with the above normal serum LDH activities.

Acute Disease↗
PubMed1991

Lactate dehydrogenase, creatine kinase, and their isoenzymes in pleural effusions.

Lactate dehydrogenase (LD; EC 1.1.1.27) and creatine kinase (CK; EC 2.7.3.2) are widely distributed cytoplasmic enzymes. LD has five and CK has three isoenzymes distributed in different proportions in various tissues. The amounts of LD and CK and the distribution of isoenzymes in different body fluids are not thoroughly characterized. We have measured the total LD and CK concentrations and their isoenzyme distribution in pleural aspirates and in serum from 22 patients with benign conditions and from 14 patients with malignant effusions. In malignant pleural fluid, the mean total LD was 662 U/L; in benign conditions, it was nearly 5840 U/L with large variations (91-43 400 U/L) according to clinical diagnosis, the highest values being reached in inflammatory lesions. The mean total CK concentration in pleural fluid was close to the serum value in both groups of patients, as was the pleural CK isoenzyme distribution. The LD isoenzyme distribution in pleural effusions differed from that in serum in both groups, with LD-4 and -5 being the main isoenzymes in their pleural fluid specimens (greater than 42% of total LD). The total LD concentration correlated somewhat (r = 0.57) with the total pleural protein content. In conclusion, the pleural LD isoenzyme distribution, both in benign and malignant conditions, differs from that in serum, having shifted towards more anaerobic and embryonic isoenzymes (LD-4 and -5). Moreover, the greater the concentration of pleural total LD, the greater the proportion of LD-4 and -5. These data suggest that visceral or parietal pleural cells are rich in LD isoenzymes 4 and 5.

Adolescent↗
PubMed1990

Clinical evaluation of an automated chemical inhibition assay for lactate dehydrogenase isoenzyme 1.

We evaluated an automated assay for lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes, supplied by Boehringer Mannheim Diagnostics (BMD) and based on selective chemical inhibition of non-LD-1 isoenzymes by guanidine thiocyanate. Results were compared with the Roche Isomune LD-1 method. The Hitachi 717 analyzer was used to measure enzyme activity for both procedures in 229 serum samples. One hundred specimens were also analyzed by the Helena rapid electrophoresis (REP) method. We determined the limit of linearity of the BMD method to be about 1200 U of LD-1 per liter. The analytical correlation of BMD (y) with Isomune (x) yielded y = 1.0x + 0.5 U/L, r = 0.997, Sy/x = 16.9 (range 20-1397 U/L). The regression equation for BMD vs REP was y = 1.1x + 7.2% (r = 0.800, Sy/x = 7.4, range 14-83%). Average values for within-run precision for low (38 U/L), medium (180 U/L), and high (865 U/L) controls were 4.1%, 1.0%, and 0.5%, respectively (16 trials of six each). The average values for run-to-run precision were 4.1%, 1.7%, and 1.1%, respectively, for these controls (n = 16). We used receiver-operating characteristic curves to determine optimum decision limits. Using an LD-1 cutoff of 40% of total LD, we obtained a clinical sensitivity of 97-100% and a specificity of 95% when blood was collected during the optimum interval, 24-48 h after the onset of chest pain. We conclude that the BMD LD-1 assay is equivalent to the immunochemical and electrophoretic assays for measuring the LD-1 isoenzyme.

Autoanalysis↗
PubMed1981

Induction of lactate dehydrogenase by dibutyryl cAMP in primary cultures of central nervous tissue is an oligodendroglial marker.

In vitro induction of lactate dehydrogenase (L-lactate:NAD+-oxidoreductase, EC 1.1.1.27, LDH) in response to catecholamines occurs in neural cell lines expressing glial properties. LDH is a tetrameric protein, consisting of two types of subunits: M-type or LDH-5 (predominant in skeletal muscle), and H-type or LDH-1 (predominant in heart muscle). These subunits are encoded by two nonallelic genes. Several studies have shown that LDH is induced by catecholamines via a cAMP-mediated process. We have previously shown that the mechanism of catecholamine induction for LDH in C6 glial cells involves an increase in the differential rates of synthesis of the two LDH subunits, suggesting that the regulation occurs at both genetic loci. Recently, a method has become available to prepare separate cultures of oligodendrocytes and astrocytes from newborn cerebral tissue. Both of these cultures markedly increase their intracellular cAMP concentration following a challenge by beta-adrenergic agonists. We have now investigated the regulation of LDH activity by dibutyryl cAMP (Bt2cAMP) in separate cultures of neurons, oligodendrocytes and astrocytes. The induction involving an increase in the rate of synthesis by Bt2cAMP is solely characteristic of oligodendrocytes. Moreover, both LDH genes contribute to this regulation.

Animals↗
PubMed1993

Investigation of class I anti-arrhythmic drug actions on guinea-pig cardiac mitochondrial lactate dehydrogenase activity.

1. The effects of the Class I anti-arrhythmic drugs quinidine, procainamide, lidocaine, phenytoin and tocainide on mitochondrial lactate dehydrogenase activity were compared in guinea-pig heart preparations. 2. All the tested drugs inhibited the enzyme activity in a concentration-dependent fashion, exhibiting varying profiles in their actions. Lidocaine exhibited inhibitory concentration 20% (IC20) and IC50 values of 0.52 +/- 0.02 mmol/L and 25.6 +/- 0.5 mmol/L, procainamide 6.0 +/- 0.2 mmol/L and 108 +/- 7.2 mmol/L, phenytoin 3.4 +/- 0.06 mumol/L and 0.34 +/- 0.02 mmol/L, quinidine 39.2 +/- 1.2 mumol/L and 9.8 +/- 0.8 mmol/L and tocainide 2.7 +/- 0.3 mmol/L and 44.6 +/- 2.5 mmol/L. 3. According to the IC50 values, this is the order of their inhibitory potencies: phenytoin > quinidine > lidocaine > tocainide > procainamide. This trend is in general agreement with the lipophilicity rank of the drugs. 4. It is concluded, therefore, that inhibition of mitochondrial lactate dehydrogenase is a property shared by most Class I anti-arrhythmic drugs which may depend on their lipophilicity and possibly their membrane stabilizing effects.

Animals↗
PubMed1993

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon.

Amino Acid Sequence↗
PubMed1989

Oxidant stress in Neisseria gonorrhoeae: adaptation and effects on L-(+)-lactate dehydrogenase activity.

Neisseria gonorrhoeae, an obligate human pathogen, is subjected to oxidant stress when attacked by O2 reduction products formed by neutrophils. In this study, exposure of gonococci to sublethal concentrations of superoxide and hydrogen peroxide (and related O-centered radicals) resulted in phenotypic resistance to oxidant stress. Adaptation required new protein formation but was not related to increases in superoxide dismutase or catalase. We have previously demonstrated that gonococci use phagocyte-derived L-(+)-lactate. Oxidant stress of greater magnitude than that required for adaptation led to a generalized increase in bacterial metabolism, particularly in L-(+)- and D-(-)-lactate utilization and lactate dehydrogenase activity. Increased lactate utilization required new protein synthesis. These results suggest the possibility that lactate metabolism is of importance to N. gonorrhoeae subjected to oxidant stress. Use of lct mutant organisms unable to use L-(+)-lactate should allow examination of this hypothesis.

Adaptation, Physiological↗
PubMed2006

Lactate dehydrogenase 5 expression in operable colorectal cancer: strong association with survival and activated vascular endothelial growth factor pathway--a report of the Tumour Angiogenesis Research Group.

PURPOSE: Lactate dehydrogenase 5 (LDH-5) regulates, under hypoxic conditions, the anaerobic transformation of pyruvate to lactate for energy acquisition. Several studies have shown that serum LDH may be an ominous prognostic marker in malignant tumors. The clinical significance of tissue LDH-5, however, remains largely unexplored. PATIENTS AND METHODS: We investigated the immunohistochemical expression of LDH-5 in a series of 128 stage II/III colorectal adenocarcinomas treated with surgery alone. In addition, markers of tumor hypoxia (hypoxia-inducible factor 1 alpha [HIF1alpha]), angiogenesis (vascular endothelial growth factor [VEGF] and phosporylated kinase domain receptor [pKDR]/flk-1 receptor) and the tumor vascular density (CD31 positive standard vascular density [sVD] and pKDR positive activated vascular density [aVD]) were assessed. RESULTS: The expression of LDH-5, together with that of HIF1alpha and pKDR, was both nuclear and cytoplasmic. Assessment, with minimal interobserver variability, was achieved using a previously described scoring system. LDH-5 was significantly associated with HIF1alpha (P = .01), aVD (P = .001) and, particularly, with pKDR expression in cancer cells (P = .0001). Tissue LDH-5 expression was linked with elevated serum LDH levels, but serum levels failed to reflect tissue expression in 71% of LDH-5 positive cases. In univariate analysis tissue LDH-5 was associated with poor survival (P = .0003, HR 15.1), whereas in multivariate analysis this isoenzyme was the strongest independent prognostic factor (P = .0009). VEGF, pKDR, aVD, sVD and vascular invasion were all significantly related to unfavorable prognosis. CONCLUSION: The immunohistochemical assessment of tissue LDH-5 and pKDR provides important prognostic information in operable colorectal cancer. The strong association between LDH-5 and pKDR expression would justify their use as surrogate markers to screen patients for tyrosine kinase inhibitor therapy.

Adenocarcinoma↗
PubMed1986

Cerebrospinal fluid lactate and lactate dehydrogenase activity in the rapid diagnosis of bacterial meningitis.

The value of cerebrospinal fluid (CSF) lactate and lactate dehydrogenase (LD) activity in the rapid diagnosis of meningitis was investigated in three groups of patients--a 'no meningitis', an aseptic meningitis and a bacterial meningitis group. The sensitivity achieved in the detection of bacterial meningitis by CSF lactate values of 2.85 mmol/l (93.8%) and 3.9 mmol/l (89.6%) was greater than that reached by conventional chemical investigations using a CSF protein value of 1 g/l (81.5%) or a CSF glucose value of 2.2 mmol/l (68.8%) as the indicator. The sensitivity of an absolute CSF LD value of 40 U/l (86.3%) in the detection of bacterial meningitis was slightly lower than that of a CSF protein value of 1 g/l (87%) and better than the sensitivity of either a CSF/serum LD ratio of 0.1:1.0 (83.9%) or a CSF glucose level of 2.2 mmol/l (76.3%). As with conventional CSF chemistry, both investigations may give normal values in the presence of bacterial meningitis.

Child↗
PubMed1990

Site-specific incorporation of 5-fluorotryptophan as a probe of the structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli: a 19F nuclear magnetic resonance study.

The structure and function of the membrane-bound D-lactate dehydrogenase of Escherichia coli have been investigated by fluorine-19 nuclear magnetic resonance spectroscopy of 5-fluorotryptophan-labeled enzyme in conjunction with oligonucleotide-directed, site-specific mutagenesis. 5-Fluorotryptophan has been substituted for nine phenylalanine, tyrosine, and leucine residues in the enzyme molecule without loss of activity. The 19F signals from these additional tryptophan residues have been used as markers for sensitivity to substrate, exposure to aqueous solvent, and proximity to a lipid-bound spin-label. The nuclear magnetic resonance data show that two mutational sites, at amino acid residues 340 and 361, are near the lipid environment used to stabilize the enzyme. There are a number of amino acid residues on the carboxyl side of this region that are strongly sensitive to the aqueous solvent. The environment of the wild-type tryptophan residue at position 469 changes as a result of two of the substitution mutations, suggesting some amino acid residue-residue interactions. Secondary structure prediction methods indicate a possible binding site for the flavin adenine dinucleotide cofactor in the carboxyl end of the enzyme molecule. These results suggest that the membrane-bound D-lactate dehydrogenase may have the two-domain structure of many cytoplasmic dehydrogenases but with the addition of a membrane-binding domain between the catalytic and cofactor-binding domains. This type of three-domain structure may be of general significance for understanding the structure of membrane-bound proteins which do not traverse the lipid bilayer of membranes.

Amino Acid Sequence↗
PubMed1999

Signalling between mitochondria and the nucleus regulates the expression of a new D-lactate dehydrogenase activity in yeast.

We have adapted a LacZ promoter trap screen developed by Burns et al. (1994) to search for genes whose expression is dependent on Rtg2p, a protein with an N-terminal hsp70/actin/sugar kinase ATP binding domain. Rtg2p acts upstream of the basic helix-loop-helix/leucine zipper transcription factors, Rtg1p and Rtg3p. All three proteins are known to be required for the expression of the CIT2 gene, which encodes a peroxisomal isoform of citrate synthase whose expression is also dependent on the functional state of mitochondria. Using this screen, we have identified a previously uncharacterized gene, YEL071w, predicted to encode a protein of 496 amino acids that shares 80% homology and 60% sequence identity with actin interacting protein 2, encoded by the AIP2 gene; both proteins also share sequence similarity to aD-lactate dehydrogenase encoded by the DLD1 gene. Expression of YEL071w is dependent on the functional state of mitochondria and on all three of the Rtg proteins, whereas AIP2 expression is independent of the Rtg proteins and the functional state of mitochondria. Like CIT2, the 5' flanking region of YEL071w contains two R box binding sites for the Rtg1p/Rtg3p heterodimeric transcription complex. Both R boxes are necessary for full YEL071w expression. We show that YEL071w and AIP2 encode proteins withD-lactate dehydrogenase activity, the former located in the cytoplasm and the latter in the mitochondrial matrix. Our data thus provide gene assignments for two previously unrecognized D-lactate dehydrogenase activities in yeast.

Amino Acid Sequence↗
PubMed1980

Development of glial cells in primary cultures: energy metabolism and lactate dehydrogenase isoenzymes.

Primary cultures of glial cells prepared from brains of newborn rats were grown for periods of 1-5 weeks. After a proliferative phase of between 2 and 3 weeks, the cultures were maintained in stationary phase, during which a significant increase of oxygen consumption and of the activities of lactate dehydrogenase, succinate dehydrogenase, and mitochondrial glycerolphosphate dehydrogenase could be observed. Furthermore, qualitative changes in the lactate dehydrogenase isoenzyme pattern were found with time, characterized by a shift toward an enhanced synthesis of H subunits. A similar development was found in comparing the LDH isoenzyme pattern in the brain of 15-day-old rat embryo with those of newborn and adult rat brains. It is suggested that some aspects of maturation of glial cells in culture are comparable to those occurring in whole brain in vivo, namely a shift towards an enhanced aerobic metabolism.

Animals↗
PubMed1983

Molecular features and immunological properties of lactate dehydrogenase C4 isozymes from mouse and rat testes.

With the use of a computer graphics system, spacefilling models of mouse and rat testis lactate dehydrogenase C4 (LDH-C4) isozymes were constructed from amino acid sequence and published x-ray diffraction data. Thirty-two residues that differ between the mouse and rat LDH-C4 sequences are also displayed on the monomeric and tetrameric lactate dehydrogenase molecules. Immunological properties of mouse and rat LDH-C4 isozymes are compared and related to these 32 differences. Possible relationships between the structure, especially the amino acid sequence, and the unique enzymatic properties of LDH-C4 isozymes are also discussed.

Amino Acid Sequence↗
PubMedDate not supplied

Photoaffinity labeling of lactate dehydrogenase by the bis-azido analog of NAD+:P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio)butyl] diphosphate.

Lactate dehydrogenase from pig heart is inactivated by the NAD+-analog P1-N6-(4-azidophenylethyl)adenosine-P2-[4-(3-azidopyridinio) butyl]diphosphate (6) upon irradiation with UV light of wavelengths in the range from 300 to 380 nm. The decrease in enzyme activity can be prevented by the addition of NAD+ and oxalate. The modified enzyme shows a reduced binding capacity for its coenzyme as compared to native lactate dehydrogenase. The amount of incorporated coenzyme is deduced from the ribose content of inactivated enzyme. Tryptic digestion of the modified protein and separation of the peptides by HPLC yields 5 ribose-containing fractions. One of them, fraction 66, is split by treatment with nucleotide pyrophosphatase into two subfractions, 63 and 58. Only subfraction 63 contains ribose. Whereas peptide 58 shows a UV absorption spectrum similar to that of 4-(3-aminopyridinio)-butyl phosphate (3). Amino acid analyses of the peptides indicate that the inactivator forms covalent bonds with different parts of the protein: Peptide 63 is characterized by a great portion of hydrophobic amino acids whereas peptide 58 shows a high degree of hydrophilicity.

Affinity Labels↗
PubMedDate not supplied

[Structure-functional modification of lactate dehydrogenase isoforms under the effect of ultraviolet radiation and active forms of oxygen].

The investigation results on the UV-induced changes of lactate dehydrogenase structural and functional properties in the presence of some chemical substances, which are able to interact with the oxygen active forms have been summarized. As well the kinetic characteristics of the photoinactivation processes for heart and muscle LDH types in free state and in the complex with the exogenous agents have been studied. Singlet molecular oxygen has been shown to play an important role in the process of UV-modification of different protein isoforms. The scheme of probable physical and chemical processes, leading to the inactivation of lactate dehydrogenase molecules, has been suggested.

Isoenzymes↗
PubMed2000

[Differentiation of malignant and non-malignant origin of ascites by determination of levels of cholesterol and lactate dehydrogenase in ascitic fluid is not absolute].

The authors Castaldo et al. (Clin. Chem., 1994, 30: 478-83) state, that the ascitic lactate dehydrogenase and ascitic cholesterol association correctly identified 100% of malignant ascites from ascites associated with cirrhosis and/or hepatocellular carcinoma, with help of stepwise multiple linear discriminant analysis. The free software Capsules--Ascites is via internet available (http:¿www.leeds.ac.uk/acb), which use the mathematical formula from this article. As we argue, Castaldo's state is not correct. Three independent multidimensional statistical methods--bivariate reference regions (program EVAL-KIT), cluster analysis (program BioAnalyst), geometrical distance classification (program GEODICLA) applicated on Castaldo's original data showed that lactate dehydrogenase and cholesterol have not satisfactory absolute discriminative power between malignant from nonmalignant ascites in general, but the probability to determinate the right diagnosis is about 91-93%. Research conducted in correctly selected probands should provide information which is valid not only for the selected sample but for the entire population, to achieve more generally valid conclusions, useful for practical decisions. In addition, in the compiled table show we sensitivity and specificity of different laboratory parameters publicated in 11 original articles in the nineties, all with diagnostic efficiency less 100%.

Ascites↗
PubMed1977

Lactate dehydrogenase isoenzyme in detecting carriers of Duchenne muscular dystrophy.

Thirty mothers of patients with Duchenne muscular dystrophy were studied with serum enzyme tests, including serum glutamic-oxaloacetic transminase, creatine kinase, and lactate dehydrogenase isoenzymes. In addition, females from the mothers' pedigrees were studied. Lactate dehydrogenase isoenzyme 5 determinations were as senitive an indicator of carrier status as creatine kinase and also identified several mothers who had normal dehydrogenase isoenzyme 5 determinations, as well as extensive pedigree testing, identified 28 to 30 mothers as probable heterozygotes. These data independently support the suggestion that cases of Duchenne muscular dystrophy as a result of spontaneous mutation are more uncommon than currently accepted.

Adult↗