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1.  Lactate Dehydrogenase 5 Expression in Non-Hodgkin Lymphoma Is Associated with the Induced Hypoxia Regulated Protein and Poor Prognosis 
PLoS ONE  2013;8(9):e74853.
Lactate dehydrogenase 5 (LDH-5) is one of the major isoenzymes catalyzing the biochemical process of pyruvate to lactate. The purpose of this study was to investigate the expression of serum LDH-5 and test whether this enzyme is regulated by tumor hypoxia and represents a prognostic marker in patients with Non-Hodgkin’s lymphoma (NHL). In this study, LDH-5 levels were detected using agarose gel electrophoresis in NHL patients (n = 266) and non-NHL controls including benign lymphadenectasis (n = 30) and healthy cohorts (n = 233). We also explored the expression of LDH-5 and hypoxia-inducible factor (HIF) 1α in NHL and benign controls by immunohistochemistry and immunofluorescence staining, respectively. Moreover, the role of LDH-5 in the progression of NHL was assessed by multivariate Cox analyses and Kaplan-Meier survival estimates. Serum concentrations of LDH-5 were significantly higher in NHL patients (9.3%) than in benign patients and healthy controls (7.5% and 7.2%, respectively, P<0.01). Application of LDH-5 detection increased the sensitivity of NHL detection, identifying 53.4% of NHL patients as positive, compared with the measurement of total LDH levels (36.5% sensitivity). LDH-5 concentrations increased with clinical stage, extra-nodal site involvement, and WHO performance status of patients with NHL. Exposure to a hypoxic environment induced the expression of LDH-5 and its overexpression correlated with HIF1α cytoplasmic accumulation in NHL cells. In multivariate analyses, LDH-5 was an independent marker for progression-free survival in patients with NHL (P<0.001). Overall, the expression of LDH-5 was elevated in NHL, showing an association with tumor hypoxia and unfavorable prognosis. Thus, LDH-5 emerges as a promising prognostic predictor for NHL patients.
doi:10.1371/journal.pone.0074853
PMCID: PMC3781153  PMID: 24086384
2.  On-target Inhibition of Tumor Fermentative Glycolysis as Visualized by Hyperpolarized Pyruvate1 
Neoplasia (New York, N.Y.)  2011;13(1):60-71.
Many cancer cells display the Warburg effect, that is, enhanced glycolysis followed by fermentation (conversion of pyruvate to lactate). Recently, the molecular basis for these effects has started to be elucidated, and the up-regulation of the lactate dehydrogenase A (LDH-A) isoform of lactate dehydrogenase is felt to be a major molecular mediator of this phenomenon. Moreover, LDH-A expression in tumor tissue and LDH-A levels in blood portend a bad prognosis, and LDH-A blockade can lead to tumor growth inhibition in tumor transplant models. We have extended existing data (some of which were published during the time when we were carrying out our studies) in two important ways: 1) inhibition of LDH-A in a glycolytic lung cancer cell line results in reactive oxygen species-mediated apoptosis and increased sensitivity to the chemotherapeutic drug paclitaxel and 2) inhibition of fermentative glycolysis can also be accomplished by activation of the pyruvate dehydrogenase complex by the drug dichloroacetate, now undergoing clinical trials, and that this phenomenon can be monitored in vivo in a noninvasive real-time manner through magnetic resonance spectroscopy using hyperpolarized pyruvate. Collectively, these data suggest that in vivo effects of drugs that redirect the fate of pyruvate, and hence are aimed at reversing the Warburg effect, could be monitored through the use of hyperpolarized magnetic resonance spectroscopy, a method that is scalable to human use.
PMCID: PMC3022429  PMID: 21245941
3.  The utility of lactate dehydrogenase isoenzyme pattern in the diagnostic evaluation of malignant and nonmalignant ascites. 
OBJECTIVE: Lactate dehydrogenase (LDH), a tetrameric protein composed of four monomers, is expressed as five isoenzymes. Serum LDH isoenzymes may be useful in differential diagnosis of ascites etiology since tissue damage releases isoenzymes contained therein, leading to a change in their pattern. MATERIALS AND METHODS: We determined ascitic fluid LDH level and LDH isoenzyme activities in patients with malignant and nonmalignant ascites in a total of 76 patients (43 males and 33 females). RESULTS: LDH level, LDH-4 activity and LDH-5 activity were found to be significantly higher, and LDH-1 activity was found to be lower in malignant ascites when compared with nonmalignant ascites. LDH-1 activity was detected to be significantly higher in the sterile cirrhotic ascites when compared with spontaneous bacterial peritonitis, malignant ascites, tuberculous ascites and congestive heart failure-related ascites. LDH-2 activity was found to be higher in spontaneous bacterial peritonitis when compared with the other groups. LDH-3 activity was detected to be higher in spontaneous bacterial peritonitis, malignant ascites and tuberculous ascites when compared with the sterile cirrhotic ascites. In the diagnosis of malignant ascites, the sensitivity and specificity were 96% and 76% for LDH level, 90% and 70% for LDH-1 activity, 94% and 62% for LDH-4 activity, and 100% and 56% for LDH-5 activity, respectively. CONCLUSION: Ascitic LDH and its isoenzyme pattern may be helpful for the differential diagnosis of the most common causes of ascites: cirrhosis, spontaneous bacterial peritonitis, congestive heart failure, tuberculosis and malignancy.
PMCID: PMC2568581  PMID: 15719876
4.  Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers 
PLoS ONE  2013;8(2):e57697.
Objective
Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.
Experimental design
Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.
Results
Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.
Conclusions
Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.
doi:10.1371/journal.pone.0057697
PMCID: PMC3578788  PMID: 23437403
5.  Testis-specific lactate dehydrogenase is expressed in somatic tissues of plateau pikas☆ 
FEBS Open Bio  2013;3:118-123.
LDH-C4 is a lactate dehydrogenase that catalyzes the interconversion of pyruvate with lactate. In mammals the, Ldh-c gene was originally thought to be expressed only in testis and spermatozoa. Plateau pika (Ochotona curzoniae), belonging to the genus Ochotona of the Ochotonidea family, is a hypoxia tolerant mammal living at 3000–5000 m above sea levelon the Qinghai-Tibet Plateau. We found that the expression pattern of six LDH isoenzymes in the somatic tissues of female and male plateau pikas to be the same as those in testis and sperm, suggesting that LDH-C4 was expressed in somatic tissues of plateau pika. Here we report the detection of LDHC in the somatic tissues of plateau pika using RT-PCR, Western blotting and immunohistochemistry. Our results indicate that Ldh-c mRNA is transcribed in the heart, liver, lung, kidney, brain, skeletal muscle and testis. In somatic tissues LDHC was translated in the cytoplasm, while in testis it was expressed in both cytoplasm and mitochondria. The third band from cathode to anode in LDH isoenzymes was identified as LDH-C4. The finding that Ldh-c is expressed in both somatic tissues and testis of plateau pika provides important implications for more in-depth research into the Ldh-c function in mammals.
Highlights
▸ Plateau pika (Ochotona curzoniae) express 6 isoforms of lactate dehydrogenase (LDH). ▸ LDH-C4 was thought to be expressed only in the testis and sperm of mammals. ▸ We detected LDH-C4 in somatic tissues as well as the testis and sperm of plateau pika.
doi:10.1016/j.fob.2013.01.011
PMCID: PMC3668505  PMID: 23772382
Testis-specific lactate dehydrogenase C; Plateau pika (Ochotona curzoniae); Hypoxia; Somatic tissues; Qinghai-Tibet Plateau; LDH-C4, testis-specific lactate dehydrogenase; NBT, nitrobenzene thiocyanate chloride; PMS, phenazine methosulfate; cDNA, complementary DNA; EST, expressed sequence tag; CRE, cAMP-response element; IPTG, isopropyl-β-d-thiogalactopyranoside.
6.  Tyrosine Phosphorylation of Lactate Dehydrogenase A Is Important for NADH/NAD+ Redox Homeostasis in Cancer Cells ▿  
Molecular and Cellular Biology  2011;31(24):4938-4950.
The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD+. LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD+. However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD+ redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.
doi:10.1128/MCB.06120-11
PMCID: PMC3233034  PMID: 21969607
7.  Importance of glycolysis and oxidative phosphorylation in advanced melanoma 
Molecular Cancer  2012;11:76.
Serum lactate dehydrogenase (LDH) is a prognostic factor for patients with stage IV melanoma. To gain insights into the biology underlying this prognostic factor, we analyzed total serum LDH, serum LDH isoenzymes, and serum lactate in up to 49 patients with metastatic melanoma. Our data demonstrate that high serum LDH is associated with a significant increase in LDH isoenzymes 3 and 4, and a decrease in LDH isoenzymes 1 and 2. Since LDH isoenzymes play a role in both glycolysis and oxidative phosphorylation (OXPHOS), we subsequently determined using tissue microarray (TMA) analysis that the levels of proteins associated with mitochondrial function, lactate metabolism, and regulators of glycolysis were all elevated in advanced melanomas compared with nevic melanocytes. To investigate whether in advanced melanoma, the glycolysis and OXPHOS pathways might be linked, we determined expression of the monocarboxylate transporters (MCT) 1 and 4. Analysis of a nevus-to-melanoma progression TMA revealed that MCT4, and to a lesser extend MCT1, were elevated with progression to advanced melanoma. Further analysis of human melanoma specimens using the Seahorse XF24 extracellular flux analyzer indicated that metastatic melanoma tumors derived a large fraction of energy from OXPHOS. Taken together, these findings suggest that in stage IV melanomas with normal serum LDH, glycolysis and OXPHOS may provide metabolic symbiosis within the same tumor, whereas in stage IV melanomas with high serum LDH glycolysis is the principle source of energy.
doi:10.1186/1476-4598-11-76
PMCID: PMC3537610  PMID: 23043612
Melanoma; Lactate dehydrogenase; Glycolysis; Mitochondria; Oxidative phosphorylation; Monocarboxylate transporters
8.  Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted 
British Journal of Cancer  2006;94(4):578-585.
Tumours ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis.
doi:10.1038/sj.bjc.6602962
PMCID: PMC2361175  PMID: 16465194
pentose phosphate pathway (PPP); transketolase (TKT); transketolase-like-1 (TKTL1); aerobic glycolysis; Warburg effect; pharmacodiagnostic marker
9.  Phosphotyrosine-containing lactate dehydrogenase is restricted to the nuclei of PC12 pheochromocytoma cells. 
Molecular and Cellular Biology  1990;10(2):770-776.
There are five lactate dehydrogenase (LDH) isoenzymes, composed of various combinations of two types of subunits. LDH-5, which contains only the LDH A subunit, is known to be present in both the cytoplasm and the nucleus, to act as a single-stranded DNA-binding protein possibly functioning in transcription and/or replication, and to undergo phosphorylation of tyrosine 238 in approximately 1% of the enzyme after cell transformation by certain tumor viruses. We have characterized LDH from wild-type PC12 pheochromocytoma cells and from a PC12 variant (MPT1) that exhibits altered lactate metabolism and altered expression of multiple genes. Wild-type and MPT1 cells contain different proportions of LDH isoenzymes, with LDH-5 being more predominant in wild-type cells than in the variant. A small fraction of LDH from PC12 cells contains phosphotyrosine. Approximately 99% of the total LDH activity is located in the cytoplasm, but all of the phosphotyrosine-containing LDH is located in the nucleus. Furthermore, essentially all of the nuclear LDH contains phosphotyrosine. These results suggest that tyrosine phosphorylation can affect its role in the nucleus.
Images
PMCID: PMC360877  PMID: 1689001
10.  The prognostic influence of serum neuron specific enolase in small cell lung cancer. 
British Journal of Cancer  1988;58(6):805-807.
An analysis of prognostic factors in small cell lung cancer has been made using presentation data from 86 of 101 consecutive patients referred to The Finsen Institute for chemotherapy. Prognosis was in univariate analysis significantly correlated with performance status (PS), disease extent, serum lactate dehydrogenase (LDH), neuron specific enolase (NSE), alpha-1-acid glycoprotein and plasma sodium. Multivariate analysis, taking stage of disease into account, resulted in selection of PS and NSE as the most influential of the investigated variables. LDH was excluded as an independent prognosticator, but there was a strong correlation between the influence of LDH and NSE (coefficient: -0.38) as well as between their serum concentrations (coefficient: 0.72). LDH and NSE apparently have similar prognostic influence, and NSE seems superior to LDH. A firm conclusion should, however, await our investigation of a large series of patients.
PMCID: PMC2246866  PMID: 2852029
11.  Lactobacillus plantarum ldhL gene: overexpression and deletion. 
Journal of Bacteriology  1994;176(3):596-601.
Lactobacillus plantarum is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate with stereospecific enzymes designated L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. A gene (designated ldhL) that encodes L-(+)-lactate dehydrogenase from L. plantarum DG301 was cloned by complementation in Escherichia coli. The nucleotide sequence of the ldhL gene predicted a protein of 320 amino acids closely related to that of Lactobacillus pentosus. A multicopy plasmid bearing the ldhL gene without modification of its expression signals was introduced in L. plantarum. L-LDH activity was increased up to 13-fold through this gene dosage effect. However, this change had hardly any effect on the production of L-(+)- and D-(-)-lactate. A stable chromosomal deletion in the ldhL gene was then constructed in L. plantarum by a two-step homologous recombination process. Inactivation of the gene resulted in the absence of L-LDH activity and in exclusive production of the D isomer of lactate. However, the global concentration of lactate in the culture supernatant remained unchanged.
PMCID: PMC205095  PMID: 8300514
12.  Warburg effect in chemosensitivity: Targeting lactate dehydrogenase-A re-sensitizes Taxol-resistant cancer cells to Taxol 
Molecular Cancer  2010;9:33.
Background
Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells.
Results
Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells.
Conclusion
LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.
doi:10.1186/1476-4598-9-33
PMCID: PMC2829492  PMID: 20144215
13.  Clinicopathological Significance and Prognostic Value of Lactate Dehydrogenase A Expression in Gastric Cancer Patients 
PLoS ONE  2014;9(3):e91068.
Introduction
LDH-A, the enzyme responsible for transforming pyruvate into lactate, has been demonstrated to be up-regulated in many types of cancer and to give rise to more aggressive behavior by regulating proliferation and anti-apoptosis. However, its expression in gastric cancer (GC) has not been characterized thoroughly. The purpose of this study was to clarify the expression and potential impact of LDH-A in GC.
Methods
We examined LDH-A expression by immunohistochemistry on GC tissue microarray (TMA) and using Western blot on fresh GC tissues and cell lines. Prognostic value and correlation with other clinicopathologic factors were evaluated. We transfected siRNA into GC cells against LDH-A. LDH-A was analyzed by Western blotting and real-time RT-PCR. Cell growth was evaluated in vitro and in vivo. Lactate and ATP production by cells were determined.
Results
There was significantly higher LDH-A expression in carcinoma than in non-neoplastic mucosa (NNM). There was a positive correlation of LDH-A expression with age, histological type and Lymph node metastases. Survival analysis demonstrated that high expression of LDH-A in GC was associated with lower overall survival (OS). When stratified by Lauren grade and histological classification, significance appeared in diffuse type and undifferentiated type GC. In multivariate analysis, the LDH-A expression in GC was an independent prognostic risk factor for OS (hazard ratio = 1.829, 95%CI 1.375–2.433,P<0.0001). Specific siRNA against LDH-A in GC cell line retarded cell growth both in vitro and in mouse models. LDH-A knockdown also reduced lactate and ATP production in GC cells.
Conclusions
Our study indicated the oncogenic role of LDH-A in GC. LDH-A expression is an independent prognostic risk factor in GC patients and up-regulated expression of LDH-A could be predictive of poor outcomes in diffuse type and undifferentiated type GC. Our results suggested that LDH-A might be a potential therapeutic target in gastric cancer.
doi:10.1371/journal.pone.0091068
PMCID: PMC3946663  PMID: 24608789
14.  Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme. 
The Lactobacillus helveticus L-(+)-lactate dehydrogenase (L-LDH) gene (ldhL) was isolated from a lambda library. The nucleotide sequence of the ldhL gene was determined and shown to have the capacity to encode a protein of 323 amino acids (35.3 kDa). The deduced sequence of the 35-kDa protein revealed a relatively high degree of identity with other lactobacillar L-LDHs. The highest identity (80.2%) was observed with the Lactobacillus casei L-LDH. The sizes and 5' end analyses of ldhL transcripts showed that the ldhL gene is a monocistronic transcriptional unit. The expression of ldhL, studied as a function of growth, revealed a high expression level at the logarithmic phase of growth. The ldhL gene is preceded by two putative -10 regions, but no corresponding -35 regions could be identified. By primer extension analysis, the ldhL transcripts were confirmed to be derived from the -10 region closest to the initiation codon. However, upstream of these regions additional putative -10/-35 regions could be found. The L-LDH was overexpressed in Escherichia coli and purified to homogeneity by two chromatographic steps. The purified L-LDH was shown to be a nonaliosteric enzyme, and amino acid residues involved in allosteric regulation were not conserved in L. helveticus L-LDH. However, a slight enhancement of enzyme activity was observed in the presence of fructose 1,6-diphosphate, particularly at neutral pH. A detailed enzymatic characterization of L-LDH was performed. The optimal reaction velocity was at pH 5.0, where the kinetic parameters K(m), and Kcat for pyruvate were 0.25 mM and 643 S-1, respectively.
PMCID: PMC168581  PMID: 9212432
15.  Aqueous humour lactic dehydrogenase isoenzymes in retinoblastoma. 
LDH activity was determined in aqueous humour samples from 11 eyes (of 10 children), four of which contained retinoblastoma. Simultaneous serum LDH levels were also determined in eight of the children. There was no correlation between serum and aqueous humour LDH activity. Total aqueous humour LDH activity ranged from 0 to 99 i.u/l. in the seven eyes with non-neoplastic conditions. It was 56, 124, 158, and 1832 i.u./l. respectively, in the four eyes with retinoblastoma. In all four eyes the ratio of isoenzymes LDH5:LDH1 was greater than 5. The total aqueous humour LDH levels in retinoblastoma was neither consistently elevated, nor related to the total serum LDH. There was a characteristic LDH isoenzyme fractionation pattern which, it is suggested, may be present before the total aqueous humour LDH becomes elevated.
PMCID: PMC1042613  PMID: 1138854
16.  TKTL1 is overexpressed in a large portion of non-small cell lung cancer specimens 
Diagnostic Pathology  2008;3:35.
In several tumors the transketolase activity, controlled inter alia by enzymes of the pentose phosphate pathway which is an alternative, energy generating reaction-cascade to glycolysis, has been correlated with proliferation. The increase of thiamine-dependant transketolase enzyme reactions is induced especially through upregulated transketolase-like enzyme 1 (TKTL1)-activity; that shows TKTL1 to be a causative enzyme for tumors enhanced, anaerobic glucose degradation. We investigated TKTL1-expression in 88 human, formalin-fixed non-small cell lung cancer tissues and 24 carcinomas of the breast by immunohistochemical stainings applying a 0 to 3 staining-score system (3 = strongest expression). For means of validation we additionally stained 40 NSCLC fixed and paraffin-embedded utilizing the HOPE-technique; showing comparable results to the formalin-fixed, paraffin-embedded specimens (not shown). Potential correlations with age, sex, TNM-classification parameters and tumor grading as well as tumor transcription factor 1 (TTF1) and surfactant protein A (SPA) expression were investigated. 40.9% of the analyzed lung tumors expressed TKTL1 weakly (Score 1), 38.6% moderately (score 2) and 17.1% strongly (score 3). 3 tumors were diagnosed TKTL1-negative (3.4%; score 0). All Breast cancer specimen stainings were positive and scored 1: 32%; scored 2: 36%; scored 3: 32%. Alveolar macrophages and Alveolar Epithelial Cells Type II were also found to be TKTL1-positive.
None of the listed clinical parameters could be found to show a significant correlation to TKTL1 signal appearance.
Although we describe the expression of TKTL1 in lung cancers, we need to state that up till now there is no scientific indication for any treatment regimens based upon these findings.
doi:10.1186/1746-1596-3-35
PMCID: PMC2526982  PMID: 18700018
17.  A novel polyclonal antibody-based sandwich ELISA for detection of Plasmodium vivax developed from two lactate dehydrogenase protein segments 
Background
Immunoassays for Plasmodium detection are, presently, most frequently based on monoclonal antibodies (MAbs); Polyclonal antibodies (PAbs), which are cheaper to develop and manufacture, are much less frequently used. In the present study we describe a sandwich ELISA assay which is capable of detecting P. vivax Lactate Dehydrogenase (LDH) in clinical blood samples, without cross reacting with those infected with P. falciparum.
Methods
Two recombinant proteins were produced from different regions of the P. vivax LDH gene. Two sandwich ELISA assay were then designed: One which uses mouse anti-LDH 1-43aa PAbs as primary antibodies (“Test 1”) and another which uses anti-LDH 35-305aa PAbs (“Test 2”) as the primary antibodies. Rabbit anti-LDH 1-43aa PAbs were used as capture antibodies in both ELISA assays. Blood samples taken from P. vivax and P. falciparum infected patients (confirmed by light microscopy) were analysed using both tests.
Results
“Test 2” performed better at detecting microscopy-positive blood samples when compared to “Test 1”, identifying 131 of 154 positive samples (85%); 85 positives (55%) were identified using “test 1”. “Test 1” produced one false positive sample (from the 20 malaria-free control) blood samples; “test 2” produced none. Kappa coefficient analysis of the results produced a value of 0.267 when microscope-positive blood smears were compared with “test 1”, but 0.734 when microscope-positive blood smears were compared with the results from “test 2”. Positive predictive value (PPV) and negative predictive value (NPV) were observed to be 98% and 22% respectively, for “Test 1”, and 99% and 45%, for “test 2”. No cross reactivity was detected with P. falciparum positive blood samples (n = 15) with either test assay.
Conclusion
Both tests detected P. vivax infected blood and showed no evidence of cross-reacting with P. falciparum. Further studies will need to be conducted to establish the full potential of this technique for malaria diagnostics. As well as representing a promising new cost-effective novel technique for P. vivax diagnosis and research, the method for developing this assay also highlights the potential for PAb-based strategies for diagnostics in general.
doi:10.1186/1471-2334-14-49
PMCID: PMC3913629  PMID: 24475751
Malaria diagnosis; Lactate dehydrogenase; Recombinant protein; ELISA
18.  The ldhA Gene, Encoding Fermentative l-Lactate Dehydrogenase of Corynebacterium glutamicum, Is under the Control of Positive Feedback Regulation Mediated by LldR▿  
Journal of Bacteriology  2009;191(13):4251-4258.
Corynebacterium glutamicum ldhA encodes l-lactate dehydrogenase, a key enzyme that couples l-lactate production to reoxidation of NADH formed during glycolysis. We previously showed that in the absence of sugar, SugR binds to the ldhA promoter region, thereby repressing ldhA expression. In this study we show that LldR is another protein that binds to the ldhA promoter region, thus regulating ldhA expression. LldR has hitherto been characterized as an l-lactate-responsive transcriptional repressor of l-lactate utilization genes. Transposon mutagenesis of a reporter strain carrying a chromosomal ldhA promoter-lacZ fusion (PldhA-lacZ) revealed that ldhA disruption drastically decreased expression of PldhA-lacZ. PldhA-lacZ expression in the ldhA mutant was restored by deletion of lldR, suggesting that LldR acts as a repressor of ldhA in the absence of l-lactate and the LldR-mediated repression is not relieved in the ldhA mutant due to its inability to produce l-lactate. lldR deletion did not affect PldhA-lacZ expression in the wild-type background during growth on either glucose, acetate, or l-lactate. However, it upregulated PldhA-lacZ expression in the sugR mutant background during growth on acetate. The binding sites of LldR and SugR are located around the −35 and −10 regions of the ldhA promoter, respectively. C. glutamicum ldhA expression is therefore primarily repressed by SugR in the absence of sugar. In the presence of sugar, SugR-mediated repression of ldhA is alleviated, and ldhA expression is additionally enhanced by LldR inactivation in response to l-lactate produced by LdhA.
doi:10.1128/JB.00303-09
PMCID: PMC2698506  PMID: 19429617
19.  Serial lumbar and ventricle cerebrospinal fluid lactate dehydrogenase activities in patients with leptomeningeal metastases from solid and haematological tumours. 
Lactate dehydrogenase (LDH) activities were measured in cerebrospinal fluid in 350 patients with various neurological diseases to establish the sensitivity and specificity of the CSF LDH as a marker for the diagnosis of leptomeningeal metastases. Slight elevations of CSF LDH were observed in nonmalignant diseases, while marked elevations were observed in a considerable number of patients with bacterial meningitis. A sensitivity of 79% and a specificity of 83% were calculated. In the 34 patients with leptomeningeal metastases from solid and haematological tumours, the LDH in lumbar and ventricular CSF were measured simultaneously. The lumbar CSF LDH concentration in patients with leptomeningeal metastases was about five times greater than that in the ventricular CSF. No relationship was found between the CSF LDH and histology of the primary tumour. A good correlation was demonstrated between the lumbar CSF LDH and the effected area of the neuraxis. Serial determinations of CSF LDH showed a relationship between level changes and responses to therapy or progression. The findings of this study indicate that measurement of LDH in CSF can be used as an adjunctive diagnostic test for leptomeningeal metastases and in monitoring the efficacy of treatment.
PMCID: PMC1031796  PMID: 3559613
20.  Prognostic and predictive role of lactate dehydrogenase 5 ( LDH5) expression in colorectal cancer patients treated with PTK787/ZK 222584 (Vatalanib) anti-angiogenic therapy 
Purpose
The CONFIRM randomized trials, investigating the role of the VEGF-receptor inhibitor PTK787/ZK 222584 (vatalanib) in colorectal cancer (FOLFOX 4 ± vatalanib), showed some benefit in patients with high serum LDH levels. Here we investigated the expression of LDH5 (encoded entirely by the LDHA gene, regulated by the hypoxia inducible factors) in cancer tissues from patients recruited in the CONFIRM trials and relationship to response.
Experimental Design
Paraffin embedded materials from 179 patients recruited in the CONFIRM trials were analyzed by immunohistochemistry for the expression of the LDH5 protein. Correlations with serum LDH, response and survival were assessed.
Results
A significant association of tumor burden and of poor Performance Status (PS) with serum LDH was noted. Poor PS and high tumor LDH5 expression predicted for poor response rates. High tissue LDH5 was related to poor progression free survival (PFS) only in the placebo group of patients, while the addition of Vatalanib seemed to improved response and PFS in this subgroup. High serum LDH levels were linked with significantly poorer overall survival, which however was not sustained in multivariate analysis.
Conclusions
Serum LDH and tissue LDH5 levels are complementary features that help to characterize the activity of lactate dehydrogenase in colorectal cancer and have a potent value in predicting response to chemotherapy. The addition of vatalanib diminished the impact of LDH expression on the prognosis of patients.
doi:10.1158/1078-0432.CCR-10-2918
PMCID: PMC3145151  PMID: 21632858
LDH; PTK/ZK; vatalanib; colorectal cancer; CONFIRM
21.  Cloning, nucleotide sequence, and transcriptional analysis of the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene. 
Recombinant plasmids containing the Pediococcus acidilactici L-(+)-lactate dehydrogenase gene (ldhL) were isolated by complementing for growth under anaerobiosis of an Escherichia coli lactate dehydrogenase-pyruvate formate lyase double mutant. The nucleotide sequence of the ldhL gene predicted a protein of 323 amino acids showing significant similarity with other bacterial L-(+)-lactate dehydrogenases and especially with that of Lactobacillus plantarum. The ldhL transcription start points in P. acidilactici were defined by primer extension, and the promoter sequence was identified as TCAAT-(17 bp)-TATAAT. This sequence is closely related to the consensus sequence of vegetative promoters from gram-positive bacteria as well as from E. coli. Northern analysis of P. acidilactici RNA showed a 1.1-kb ldhL transcript whose abundance is growth rate regulated. These data, together with the presence of a putative rho-independent transcriptional terminator, suggest that ldhL is expressed as a monocistronic transcript in P. acidilactici.
PMCID: PMC167282  PMID: 7887607
22.  CcpA-Independent Glucose Regulation of Lactate Dehydrogenase 1 in Staphylococcus aureus 
PLoS ONE  2013;8(1):e54293.
Lactate Dehydrogenase 1 (Ldh1) is a key enzyme involved in Staphylococcus aureus NO·-resistance. Full ldh1-induction requires the presence of glucose, and mutants lacking the Carbon-Catabolite Protein (CcpA) exhibit decreased ldh1 transcription and diminished Ldh1 activity. The redox-regulator Rex represses ldh1 directly by binding to Rex-sites within the ldh1 promoter (Pldh1). In the absence of Rex, neither glucose nor CcpA affect ldh1 expression implying that glucose/CcpA-mediated activation requires Rex activity. Rex-mediated repression of ldh1 depends on cellular redox status and is maximal when NADH levels are low. However, compared to WT cells, the ΔccpA mutant exhibited impaired redox balance with relatively high NADH levels, yet ldh1 was still poorly expressed. Furthermore, CcpA did not drastically alter Rex transcript levels, nor did glucose or CcpA affect the expression of other Rex-regulated genes indicating that the glucose/CcpA effect is specific for Pldh1. A putative catabolite response element (CRE) is located ∼30 bp upstream of the promoter-distal Rex-binding site in Pldh1. However, CcpA had no affinity for Pldh1 in vitro and a genomic mutation of CRE upstream of Pldh1 in S. aureus had no affect on Ldh1 expression in vivo. In contrast to WT, ΔccpA S. aureus preferentially consumes non-glycolytic carbon sources. However when grown in defined medium with glucose as the primary carbon source, ΔccpA mutants express high levels of Ldh1 compared to growth in media devoid of glucose. Thus, the actual consumption of glucose stimulates Ldh1 expression rather than direct CcpA interaction at Pldh1.
doi:10.1371/journal.pone.0054293
PMCID: PMC3544828  PMID: 23342123
23.  Serum lactate dehydrogenase isoenzyme activities in patients with asthma 
Thorax  1974;29(6):685-689.
Usher, D. J., Shepherd, R. J., and Deegan, T. (1974).Thorax, 29, 685-689. Serum lactate dehydrogenase isoenzyme activities in patients with asthma. Increases in the serum activities of several enzymes have been reported in patients with asthma. Liver damage, resulting from altered tensions of oxygen and carbon dioxide in the circulation, has been held to be responsible for the majority of this increase, although it has also been suggested that allergic reactions in the lungs might make some contribution.
This communication describes the application of a more specific enzyme assay, the serum lactic dehydrogenase (LDH) isoenzyme pattern, to asthma patients in an attempt to elucidate the source of the increase in enzyme activity. Raised activities of two isoenzymes, LDH-3 and LDH-5, comprised the bulk of the increase in total LDH activity; in contrast, the activities of LDH-1 and LDH-2 were virtually unaltered. Analysis of the distribution of isoenzyme activity in lung tissue homogenate, coupled with knowledge of that in liver, suggested that both tissues contributed towards the effects observed. It appeared probable that the increment in LDH-3 activity arose from lung involvement, whereas the major portion of the increment in LDH-5 activity was derived from the liver.
PMCID: PMC470224  PMID: 4450178
24.  Isolation and Expression of Lactate Dehydrogenase Genes from Rhizopus oryzae 
Rhizopus oryzae is used for industrial production of lactic acid, yet little is known about the genetics of this fungus. In this study I cloned two genes, ldhA and ldhB, which code for NAD+-dependent l-lactate dehydrogenases (LDH) (EC 1.1.1.27), from a lactic acid-producing strain of R. oryzae. These genes are similar to each other and exhibit more than 90% nucleotide sequence identity and they contain no introns. This is the first description of ldh genes in a fungus, and sequence comparisons revealed that these genes are distinct from previously isolated prokaryotic and eukaryotic ldh genes. Protein sequencing of the LDH isolated from R. oryzae during lactic acid production confirmed that ldhA codes for a 36-kDa protein that converts pyruvate to lactate. Production of LdhA was greatest when glucose was the carbon source, followed by xylose and trehalose; all of these sugars could be fermented to lactic acid. Transcripts from ldhB were not detected when R. oryzae was grown on any of these sugars but were present when R. oryzae was grown on glycerol, ethanol, and lactate. I hypothesize that ldhB encodes a second NAD+-dependent LDH that is capable of converting l-lactate to pyruvate and is produced by cultures grown on these nonfermentable substrates. Both ldhA and ldhB restored fermentative growth to Escherichia coli (ldhA pfl) mutants so that they grew anaerobically and produced lactic acid.
PMCID: PMC110528  PMID: 10831409
25.  Quantification of Green Fluorescent Protein in Cellular Supernatant by Capillary Electrophoresis with Laser-Induced Fluorescence Detection for Measurement of Cell Death 
Talanta  2010;81(3):948-953.
A common method for quantifying cell death is measuring the concentration of lactate dehydrogenase (LDH) released by cells as their membranes become unstable. In cells expressing green fluorescent protein (GFP), degradation of the cell membrane also results in the release of GFP into the surrounding supernatant. In this study, we used capillary electrophoresis with laser-induced fluorescence detection to measure the levels of GFP in supernatants of UBIGFP/BL6 primary macrophages that had been infected with S. typhimurium, treated with staurosporine, or exposed to H2O2, all known inducers of cell death. We also used a standard LDH assay to measure the release of LDH into supernatants. We observed the rate of cell death quantified by release of GFP and LDH into supernatant to be essentially identical, demonstrating that GFP release is at least as good an indicator of macrophage cell death as the established LDH release method.
doi:10.1016/j.talanta.2010.01.041
PMCID: PMC2842605  PMID: 20298877
capillary electrophoresis; laser-induced fluorescence; green fluorescent protein; cell death; lactate dehydrogenase assay

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