Lactate Dehydrogenase Subunit Research Articles

Quantification of relative expression of genes with homologous sequences using fluorescence-based single-strand conformation polymorphism analysis--application to lactate dehydrogenase and cyclooxygenase isozymes.

The combination of reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis has been proposed for the quantitative determination of ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase subunits M and H, and cyclooxygenase 1 and 2. We designed fluorescence labeled common PCR primers in the sequences highly homologous between two subunit and isozyme cDNAs, and performed reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis. PCR efficiency was almost the same for the different target sequences, so analysis of mixtures of known amounts of lactate dehydrogenase M and H revealed linear and precise proportions of lactate dehydrogenase M mRNA. It was shown that template concentrations and number of PCR cycles did not affect the determination of proportions of lactate dehydrogenase M to total lactate dehydrogenase. The procedure was applicable to a determination of cyclooxygenase-2 proportion; furthermore, the present procedure could be easily applied to investigation of expression levels of genes encoding mRNAs with homologous sequences.

Chronic electrical stimulation increases MCT1 and lactate uptake in red and white skeletal muscle.

We examined whether chronic stimulation of red and white rat muscles increased the concentrations of the monocarboxylate transporter MCT1. Red and white tibialis anterior (RTA and WTA, respectively) and extensor digitorum longus (EDL) muscles were chronically stimulated via the peroneal nerve for 7 days. Stimulated and contralateral control muscles were examined for MCT1 content, L-lactate uptake, lactate dehydrogenase (LDH) isoforms, and muscle fiber composition. MCT1 was 1.5 times greater in stimulated RTA, 3 times greater in stimulated WTA, and 1.9 times greater in stimulated EDL compared with respective control muscles (P < 0.05). L-Lactate uptake increased in all stimulated muscles (P < 0.05), and this was highly correlated with the increase in MCT1 (r = 0.96). The heart-type LDH (H-LDH) subunits also increased in all stimulated muscles (P < 0.05). The H-LDH subunits correlated highly with MCT1 in the muscles (r = 0.83). There was no change in muscle-type LDH subunits (P > 0.05). There were negligible alterations in muscle fiber composition in the stimulated muscles, suggesting that the increase in MCT1 was independent of changes in muscle fiber composition. These studies are the first to demonstrate that chronic muscle contraction increases MCT1 concentrations in both red and white skeletal muscles.

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.

Relative Ratios of mRNA Molecules Encoded by Genes with Homologous Sequences Using Fluorescence-Based Single-Strand Conformation Polymorphism Analysis

The combination of reverse transcribed (RT)-PCR and fluorescence-based single-strand conformation polymorphism (SSCP) analysis is proposed for the quantitative determination of the ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase (LDH) subunits A and B. We designed fluorescence labeled common PCR primers in the sequences highly homologous between LDH-A and LDH-B cDNAs and performed RT-PCR-SSCP analysis. When PCR efficiency was almost the same between the different target sequences, analysis of mixtures of known amounts of LDH-A and LDH-B revealed linear and precise proportions of LDH-A mRNA. Template concentrations and PCR cycles did not affect the determination of proportions of LDH-A to total LDH. The present procedure could be easily applied to investigation of expression levels of genes encoding mRNAs with homologous sequences.

Myocardial lactate dehydrogenase subunit ratio in cardiac allograft recipients

Allograft coronary artery disease (CAD) is a major long-term complication in heart transplanted patients. However, the metabolic basis of allograft CAD remains to be fully elucidated. We analyzed the lactate dehydrogenase heart (H) and muscle (M) isoenzyme pattern in endomyocardial biopsy specimens and the evolution of the H/M ratio to test whether changes in this ratio could be the earliest manifestation of allograft CAD. Twenty-four heart transplant recipients were followed up for 12 months. Endomyocardial biopsy was performed at 1, 2, 3, 6, and 12 months after transplantation. Lactate dehydrogenase 1 through 5 isoenzymes were separated by electrophoresis, and the H/M ratio was calculated. Two groups of patients were identified: group 1 (n = 20), patients without allograft CAD; and group 2 (n = 4), patients with poor outcome (three deaths, 1 case of low cardiac output) and angiographic and histologic evidence of allograft CAD. Both groups had similar H/M baseline values. The H/M ratio was higher (p = 0.01) in group 1 at 6 months (3.48 +/- 0.64 versus 2.17 +/- 0.43) and 12 months (3.76 +/- 0.92 versus 2.18 +/- 0.45) when compared with group 2. The H/M ratio increased from 2.78 +/- 0.89 at 1 month to 3.76 +/- 0.92 at 12 months (p = 0.02) in group 1 and decreased in group 2 (2.86 +/- 0.49 versus 2.18 +/- 0.45; not significant). Changes in H/M ratio reflect an anaerobic shift in the lactate dehydrogenase isoenzyme composition and can be taken as an early indicator of allograft CAD.

Phosphorylation of the Transcription Factor NFATp Inhibits Its DNA Binding Activity in Cyclosporin A-treated Human B and T Cells

Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is thought to be largely mediated through inactivation of the phosphatase calcineurin, which in turn inhibits translocation of an NFAT component to the nucleus. Here we report that CsA treatment of Raji B and Jurkat T cell lines yields a phosphorylated form of NFATp that is inhibited in DNA-binding and in its ability to form an NFAT complex with Fos and Jun. Immunoblot analyses and metabolic labeling with [32P]orthophosphate show that CsA alters NFATp migration on SDS-polyacrylamide gel electrophoresis by increasing its phosphorylation level without affecting subcellular distribution. Dephosphorylation by in vitro treatment with calcineurin or alkaline phosphatase restores NFATp DNA binding activity and its ability to reconstitute an NFAT complex with Fos and Jun proteins. These data point to a new mechanism for CsA-sensitive regulation of NFATp in which dephosphorylation is critical for DNA binding.

Isoenzyme patterns of glucose-6-phosphate dehydrogenase, malate dehydrogenase and lactate dehydrogenase in Babesia rodhaini and Babesia microti.

Glucose-6-phosphate dehydrogenase (G6PD), malate dehydrogenase (MDH), and lactate dehydrogenase (LDH) activities and their isoenzyme patterns in B. rodhaini (BR) and B. microti (BM), the two major causative species of murine babesiosis, were examined. G6PD and LDH activities were higher in BR than those in BM, whereas MDH activity was lower in BR than that in BM. No differences were observed between BR and BM in the mobility of isoenzyme bands of G6PD and MDH. On LDH isoenzyme pattern, at least 5 bands were detected in BR, while only one band in BM. Since each subunit of LDH is known to be coded by different gene, these results suggests that BR and BM are able to be differentiated genetically.

Breast cancer diagnosis by lactate dehydrogenase isozymes in nipple discharge.

The diagnosis of breast cancer based on nipple discharge, often the only clinical manifestation of early breast cancer, is currently unsatisfactory. Because M subunits of lactate dehydrogenase (LDH) have been noted to increase in cancer tissue, the author assessed the value of using LDH isozyme patterns in nipple discharge for the diagnosis of breast cancer. LDH isozyme levels in (1) nipple discharge of patients diagnosed as having breast cancer, intraductal papilloma, mastopathy, drug-induced nipple discharge, mastitis, or benign nipple discharge; (2) control samples of normal nipple discharge (milk) 6 days, 1-5 months, and 6 months to 2 years postpartum; (3) the serum of patients presenting with nipple discharge; and (4) normal and cancerous breast tissue extracts were measured using a Ciba-Corning LDH isozyme system (Ciba Corning Diagnostic Corp., Tokyo, Japan). LDH isozyme levels in the nipple discharge of patients with benign breast diseases displayed various patterns. Levels in the nipple discharge of patients with breast cancer, including noninvasive carcinoma, tended to increase in ascending order from LDH1 to LDH5. This pattern was similar to that in breast cancer tissue and was unrelated to the pattern in serum. LDH isozyme assay of nipple discharge may be a useful technique for providing a supporting diagnosis of breast cancer.

Population screening of lactate dehydrogenase deficiencies in Fukuoka Prefecture in Japan and molecular characterization of three independent mutations in the lactate dehydrogenase-B(H) gene

Screening for lactate dehydrogenase (LDH) subunit deficiencies was performed on 2880 blood samples from healthy individuals in the Fukuoka Prefecture in Japan by means of electrophoresis. The frequencies of heterozygotes with either LDH-A or LDH-B deficiency were found to be 0.104% at each locus. These estimated frequencies of either LDH-A or LDH-B deficiencies were slightly lower than, but not significantly different from, those found previously in Shizuoka Prefecture. The genetic mutations in individuals heterozygous for LDH-B deficiency were analyzed by the polymerase chain reaction and DNA conformation polymorphism. Abnormal migration patterns were observed in individuals heterozygous for LDH-B deficiency. Subsequent sequence determination of the mutant alleles revealed three novel mutations: an eight-base duplication in exon 3, a four-base duplication in exon 4, and a one-base deletion in exon 7 of the LDH-B gene. These three mutations result in frame-shift translation and premature termination. In addition, the mutations resulting in the duplication of eight or four nucleotides appear to cause a decrease in the levels of LDH-B mRNA.

Effects of orchiectomy on EDL muscle ultrastructure and energy-supplying enzymes in growing male guinea pigs.

To investigate the influence of androgens on muscle maturation during growth, guinea pigs were orchiectomized (CX) or sham operated (SO) at the end of weaning. Extensor digitorum longus (EDL) muscles were removed at weeks 4, 8, and 12 of postnatal development. According to their contractile and metabolic characteristics, four fiber types were distinguished, called I, IIa, IIbox, and IIbglyc. Muscle maturation consisted of a concomitant decrease in percentages of type IIa and IIbglyc fibers and increase in the percentage of IIbox fibers in both groups. At week 12, fiber distributions were not different between the two groups. Citrate synthase activity fell and phosphofructokinase and lactate dehydrogenase (LDH) activities rose from week 4 to week 12 and were the same in CX and SO. Muscular LDH subunits increased in SO and decreased in CX during this period. In conclusion, fiber type distribution and enzyme activities at puberty were not androgen dependent in guinea pig EDL muscle. Conversely, these hormones acted on LDH isozyme distribution through the enhancement of the most glycolytic LDH fractions.

A mutation affecting the lactate dehydrogenase locus Ldh-1 in the mouse. II. Mechanism of the LDH-A deficiency associated with hemolytic anemia.

A procarbazine hydrochloride-induced mutation at the Ldh-1 structural locus encoding the A subunit of lactate dehydrogenase (LDH) was used to study the molecular and metabolic basis of severe hemolytic anemia due to LDH-A deficiency in the mouse. The mutant allele designated Ldh-1a-m1Neu codes for an enzyme that as homotetramer differs from the wild-type enzyme by a marked instability, acidic shift of the pH profile, increased Km for pyruvate and altered inhibition by high concentrations of this substrate. Except for the latter, all these altered properties of the mutant protein contribute to the diminished LDH activity in heterozygous and homozygous mutant individuals. Impaired energy metabolism of erythrocytes indicated by a relatively low ATP concentration is suggested to result in cell death at the end of the reticulocyte stage leading to the expression of hemolytic anemia with extreme reticulocytosis and hyperbilirubinemia. Despite the severe anemia, affected homozygous mutants exhibit approximately normal body weight and do not show noticeable impairment of viability or fertility. To date no such condition is observed in man. This discrepancy is likely due to the fact that in human erythrocytes both LDH-A and LDH-B subunits are expressed such that homozygotes for a LDH-A or LDH-B deficiency would not result in a comparably extreme LDH activity deficiency.

Fast Twitch Fibres May Predict Anaerobic Performance in Both Females and Males

The aim of the present study was to compare males and females with similar training backgrounds regarding the relationship between anaerobic performance and muscle characteristics and to test whether any of the analysed expressions of muscle characteristics could predict some of the difference in anaerobic performance between sexes. Subjects performed 30 s all-out sprints on a bicycle ergometer (Wingate test) and needle muscle biopsies were taken at rest. Peak and mean power were respectively 44% and 48% higher in males than in females. Activity of total lactate dehydrogenase (LD) was 33% higher and of M subunit of LD 38% higher in males. Anaerobic performance was directly related to the proportion of type II fibres, the relative M subunit activity or the activity of PFK in both males and females and the higher M subunit activity in males could predict some of the sex difference in anaerobic performance. It is suggested that anaerobic performance is directly related to fast contractile or/and anaerobic metabolic properties of skeletal muscle with no sex difference in this relationship. The difference in anaerobic performance between the sexes may partly be related to the sex difference in anaerobic metabolic properties of skeletal muscle.

Mechanism of differential inhibition of lactate dehydrogenase isoenzymes in the BMC LD-1 assay

The mechanism of inhibition of lactate dehydrogenase (LD) isoenzymes by guanidinium thiocyanate (GSCN) used in the LD-1 assay developed by Boehringer Mannheim Corporation (BMC) was investigated. Michaelis-Menten inhibition kinetics for the individual isoenzymes revealed that GSCN competitively inhibited LD-1 in the presence of lactate and NAD+, but is a noncompetitive inhibitor of LD-5. LD-2 and LD-3 exhibited mixed inhibition kinetics. The inhibition constants were two- to threefold smaller for LD-5 than for LD-1. Time-dependent studies also showed that the isoenzymes underwent a different rate of inactivation by GSCN. LD-5, LD-3, and LD-2 were rapidly inactivated within 1 min under the BMC assay conditions, whereas LD-1 lost only about 20% of activity after 10 min. The presence of lactate further protects LD-1, but not other isoenzymes. Under this condition, LD-1 was not inactivated during the initial 6 min of reaction. Separate experiments demonstrated that both guanidinium and thiocyanate ions are responsible for the inactivation process that was found to be irreversible. We speculate that GSCN selectively denatures the M subunit of LD. The H subunit is less susceptible to denaturation and is further stabilized by lactate.

Echinoderm ( Holothuria atra) lactate dehydrogenase: Affinities with the putative ancestral chordate enzyme

Lactate dehydrogenase (LDH) was purified from the echinoderm Holothuria atra. The enzyme occurs as a single L-specific homotetramer with a subunit molecular weight of 39,000, and kinetic properties that differ from those of the A 4, B 4 and C 4 enzymes of lower vertebrates and lower chordates. Amino acid compositional analysis and immunochemical titration indicate considerable structural affinity (⋍70% sequence similarity) with the single LDH subunit of the ascidan Pyura stolonifera. This supports the proposition that echinoderms lie close to the stemline leading from invertebrates to lower chordates, and that the ascidian LDH subunit represents the ancestral subunit type of vertebrate LDH.

Myocardial Lactate Dehydrogenase and its Isoenzyme Activities in Transplanted Human Hearts

Lactate dehydrogenase and its heart (H) and muscle (M) subunit activities were studied in right ventricular endomyocardial biopsies from eight transplanted human hearts and compared with five chronically failing hearts and six normal human hearts from brain-dead liver/kidney donors. Of the 17 transplant biopsies (taken 5-95 weeks postoperatively), only two showed histologic signs of chronic rejection: They were excluded from the group analysis. A higher proportion of the M subunit of lactate dehydrogenase (M%) was found in the transplanted and the chronically failing hearts than in the normal hearts, presumably reflecting increased myocardial anaerobic glycolytic stress. In the early post-transplantation period, M% was higher in the transplanted than in the chronically failing hearts. Thereafter M% gradually fell, but had not reached normal levels 1-2 years after transplantation. During that time it was similar to the values in the chronic-failure hearts. In the two biopsies with chronic rejection, M% was nearly twice as high as in contemporaneous biopsies showing mild or no rejection. Monitoring of enzymatic adaptation from endomyocardial biopsies may be of clinical interest.

Early clinical stages of nonseminomatous testis cancer. Evaluation of the primary treatment and follow-up procedures of the SWENOTECA project.

During a 5-year period, 588 consecutive patients with nonseminomatous testicular germ cell cancer were included by 16 hospitals into the Swedish-Norwegian Testicular Cancer Project (SWENOTECA). A total of 370 (63%) had early clinical stages (CS1, CS1Mk+ and CS2A), and 345 (93%) of these patients underwent pathological staging (PS) by retroperitoneal lymph node dissection (RPLND). The overall clinical staging accuracy was 75%, with no significant difference between hospitals with low, medium or high patient accrual rate. Addition of bipedal lymphography did not improve the clinical staging accuracy compared to evaluation of the retroperitoneum by CT alone. Tumor serum markers before and close monitoring of the levels after orchiectomy gave valuable information regarding risk of retroperitoneal metastases. After a median follow-up period of 5 years 30 (13.8%) of 217 patients with PS1 disease relapsed, only 3 of them later than 18 months from the RPLND. Short orchiectomy to RPLND time interval, vascular invasion and absence of teratoma elements in the primary tumour were significant predictors of relapse in PS1 cases according to multivariate analysis. Unilateral RPLND was not associated with higher relapse rate than a bilateral procedure, but significantly reduced the risk of dry ejaculation after RPLND. None out of 122 PS2 patients who received adjuvant cisplatin-based chemotherapy after RPLND relapsed, despite the fact that 37 of them had only undergone a unilateral RPLND. Repeated CT examinations and most routine blood tests except serum alpha foeto protein (AFP), beta subunit of human chorionic gonadotropin (HCG) and lactate dehydrogenase (LD) may safely be omitted in the follow-up period for patients who have been pathologically staged with RPLND, provided that effective adjuvant chemotherapy has been given to the PS2 patients.

Cloning and expression of the gene encoding the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans

The fructose-1,6-diphosphate-dependent lactate dehydrogenase from Streptococcus mutans JH1000 was purified by a modification of published methods. The sequence of 27 amino-terminal amino acids was determined, which allowed us to construct a 17-base DNA probe that had 32-fold degeneracy. The probe was used to screen a genomic library in pBR322. Of 18 reactive clones, 1 was found that expressed lactate dehydrogenase (LDH) activity identical to that of S. mutans with regard to dependence on fructose-1,6-diphosphate, thermal inactivation profile, and inhibition by sodium oxamate. Extracts of this clone possessed a protein band that comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with purified LDH from JH1000. Compared with controls, the clone was shown to produce elevated amounts of L-(+)-lactic acid during growth in the presence of glucose, thereby indicating that the activity was expressed in vivo. This result was substantiated by demonstrating that the activity could complement a mutation in the fermentative D-(-)-LDH of Escherichia coli. Subcloning showed that the S. mutans LDH subunit is encoded by a 1.2-kilobase gene. Our ability to clone this gene is expected to have great practical significance in the construction of an effector strain for use in the replacement therapy of dental caries.

Rhabdomyosarcoma-associated locus and MYOD1 are syntenic but separate loci on the short arm of human chromosome 11.

The MYOD1 locus is preferentially expressed in skeletal muscle and at higher levels in its related neoplasm, rhabdomyosarcoma. We have combined physical mapping of the human locus with meiotic and physical mapping in the mouse, together with synteny homologies between the two species, to compare the physical relationship between MYOD1 and the genetically ascertained human rhabdomyosarcoma-associated locus. We have determined that the myogenic differentiation gene is tightly linked to the structural gene for the M (muscle) subunit of lactate dehydrogenase in band p15.4 on human chromosome 11 and close to the p and Ldh-1 loci in the homologous region of mouse chromosome 7. Because the rhabdomyosarcoma locus maps to 11p15.5, MYOD1 is very unlikely to be the primary site of alteration in these tumors. Further, these analyses identify two syntenic clusters of muscle-associated genes on the short arm of human chromosome 11, one in the region of rhabdomyosarcoma locus that includes IGF2 and TH and the second the tightly linked MYOD1 and LDHA loci, which have been evolutionarily conserved in homologous regions of both the mouse and the rat genomes.

Criteria for detection of heterozygous individuals with lactate dehydrogenase subunit deficiencies

Criteria for detection of heterozygous individuals with lactate dehydrogenase subunit deficiencies

Laboratory and clinical features of lactate dehydrogenase subunit deficiencies

Heterozygous subjects with lactate dehydrogenase (LD) subunit deficiencies do not always show a low serum LD activity. Thus, it is necessary to examine the serum and erythrocyte LD isozyme patterns when screening for these conditions. We assessed patients with heterozygotes for H and M subunit deficiency by immunoblotting with anti-H subunit antibody. We could not find any protein or variant proteins in the heterozygous subjects with M subunit deficiency. On the other hand, the existence of variant protein was confirmed in the heterozygous subjects with H subunit deficiency. However, in one patient with homozygous for H subunit deficiency, no H subunit protein was observed. It is suggested that possibly many types of LD subunit deficiency may exist. Only patients with homozygous for M subunit deficiency show common patterns of clinical findings. All patients with LD subunit deficiencies have the risk of misdiagnosis of other clinical conditions from laboratory data because LD release is reduced. Thus it is important to detect them as a latent hereditary symptom poor disorders recognizing in laboratory medicine.