Self-assembled monolayers with biospecific affinity for lactate dehydrogenase for the electroenzymatic oxidation of lactate

Surface modified gold electrodes with high biospecific affinity for NAD(H)-dependent lactate dehydrogenase have been prepared by covalent attachment of several traizine dyes to stepwise functionalized mixed alkanethiol self-assembled monolayers. The biospecific affinity of such ligand-anchored monolayers to bind submonolayer amounts of enzyme was demonstrated from the course of the protein adsorption events monitored by surface plasmon resonance. Electroenzymatic activity measurements of lactate dehydrogenase modified surfaces for the reaction of lactate oxidation, carried out `ex situ¿ at different stages of protein layer growth, allowed the optimization of the preparative procedure to yield reproducible enzymatic electrodes with a low amount of unspecifically bound protein. A short adsorption time, as well as a high concentration of enzyme in the solution used for protein layer growth, led to lactate dehydrogenase-modified gold electrode surfaces with a high electroenzymatic activity arising mainly from biospecifically bound species. The lowest amount of unspecifically adsorbed protein was found for ligand-anchored monolayers prepared from mixed alkanethiol underlayers with an excess of positively charged groups. The lack of electroenzymatic activity shown by lactate dehydrogenase modified electrodes in the absence of soluble coenzyme (NAD+) indicates that none of the investigated ligand-anchored monolayers could provide an efficient electronic pathway from the metal to the active site of the enzyme. Therefore, the monolayers acted just as an anchoring system for lactate dehydrogenase

Stabilization of lactate dehydrogenase

Addition of substances such as dimethylsulfoxide, glycerol, and gelatin to aqueous solutions of lactate dehydrogenase maintains enzymes in stable and fully active form when stored at 2 - 6 C

Evidences of basal lactate production in the main white adipose tissue sites of rats. Effects of sex and a cafeteria diet.

Female and male adult Wistar rats were fed standard chow or a simplified cafeteria diet for one month. Then, the rats were killed and the white adipose tissue (WAT) in four sites: perigonadal, retroperitoneal, mesenteric and subcutaneous (inguinal) were sampled and frozen. The complete WAT weight in each site was measured. Gene expression analysis of key lipid and glucose metabolism enzymes were analyzed, as well as tissue and plasma lactate and the activity of lactate dehydrogenase. Lactate gradients between WAT and plasma were estimated. The influence of sex and diet (and indirectly WAT mass) on lactate levels and their relationships with lactate dehydrogenase activity and gene expressions were also measured. A main conclusion is the high production of lactate by WAT, practically irrespective of site, diet or sex. Lactate production is a direct correlate of lactate dehydrogenase activity in the tissue. Furthermore, lactate dehydrogenase activity is again directly correlated with the expression of the genes Ldha and Ldhb for this enzyme. In sum, the ability to produce lactate by WAT is not directly dependent of WAT metabolic state.We postulate that, in WAT, a main function of the lactate dehydrogenase path may be that of converting excess available glucose to 3C fragments, as a way to limit tissue self-utilization as substrate, to help control glycaemia and/or providing short chain substrates for use as energy source elsewhere. More information must be gathered before a conclusive role of WAT in the control of glycaemia, and the full existence of a renewed glucose-lactate-fatty acid cycle is definitely established

Serum and Cerebrospinal Fluid Lactate Dehydrogenase in Children with Febrile Convulsions

  Objective Tissue damage caused by febrile convulsion has not still been proved or refuted completely. Given the fact that lactate dehydrogenase as an intracellular enzyme can be increased due to tissue damage, we decided to evaluate serum and cerebrospinal fluid lactate dehydrogenase in children with febrile convulsion. Materials & Methods This is a cross-sectional study on 166 children aged 6-24 month, in three groups of simple febrile convulsion (n=56), complex febrile convulsion (n=27) with 3 different subgroups (recurrence in 24 hours, duration >15 minutes, and with focal components), and control (n=83). Patients’ serum and cerebrospinal fluid specimens were collected after meeting the inclusion criteria. Demographic information was documented and patients’ serum and cerebrospinal fluid lactate dehydrogenase and glucose were measured. Data were analyzed using SPSS software. Result The mean serum lactate dehydrogenase in simple febrile convulsion, complex febrile convulsion, and controls were 501.57± 143.70, 553.07±160.22, and 505.87±98.73 U/L, respectively. The mean cerebrospinal fluid lactate dehydrogenase in simple, complex febrile convulsion, and control groups were 22.58±11.92, 29.48±18.18, and 21.56±17.32 U/L, respectively. Only cerebrospinal fluid lactate dehydrogenase difference between complex febrile convulsion and control group (p=0.039) (In the duration >15 minutes subgroup and controls, p=0.028) was statisticallysignificant. There was a significantThere was a significant difference between sex and serum lactate dehydrogenase in the same subgroup of complex group (p=0.012). Conclusion Complex febrile convulsion may lead to increase of lactate dehydrogenase in cns of CNS cellular damag

A study on lactate dehydrogenase levels in hypertensive disorders of pregnancy and its correlation with feto-maternal outcome

Background: Hypertensive disorders of pregnancy are one of the most common medical disorders seen during pregnancy. Lactate dehydrogenase (LDH) is an intracellular enzyme. The objective of this study was to compare lactate dehydrogenase levels in women with hypertensive disorders of pregnancy and normal pregnant women, to correlate lactate dehydrogenase levels with complications of hypertensive disorders of pregnancy and role of lactate dehydrogenase as an early predictor of hypertensive disorders of pregnancy. Methods: A study was conducted in the department of obstetrics and gynecology at JJ group of hospitals, Mumbai, India for a duration of 18 months from January 2020 to June 2021. This study has a sample size of 500 antenatal patients. Necessary information such as their detailed clinical, and obstetric history, clinical examination, investigations was noted. LDH were measured at 12-16 weeks of pregnancy and at the time of delivery. Results: In our study, the incidence of hypertensive disorders of pregnancy was 10.2% There was no association between LDH levels at 12-16 weeks of gestation and development of hypertensive disorders of pregnancy. There was association between levels of lactate dehydrogenase levels at time of delivery and severity of hypertensive disorders in our study. Higher serum LDH levels were associated with increased incidence of maternal and fetal complications like abruption placenta, HELLP syndrome, IUGR, IUFD, prematurity and oligohydramnios in our study. Conclusions: Hypertensive disorders of pregnancy are one of the medical conditions affecting pregnancy. Lactate dehydrogenase levels at 12-16 weeks of gestation is not early predictor of hypertensive disorders of pregnancy. Serum lactate dehydrogenase levels at time of delivery helps in prediction of severity of disease, adverse outcomes and complications of hypertensive disorders of pregnancy. Hence lactate dehydrogenase acts as prognostic factor in hypertensive disorders of pregnancy

Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a Gram-negative opportunistic pathogen and the proposed causative agent of localized aggressive periodontitis. A. actinomycetemcomitans is found exclusively in the mammalian oral cavity in the space between the gums and the teeth known as the gingival crevice. Many bacterial species reside in this environment where competition for carbon is high. A. actinomycetemcomitans utilizes a unique carbon resource partitioning system whereby the presence of L-lactate inhibits uptake of glucose, thus allowing preferential catabolism of L-lactate. Although the mechanism for this process is not fully elucidated, we previously demonstrated that high levels of intracellular pyruvate are critical for L-lactate preference. As the first step in L-lactate catabolism is conversion of L-lactate to pyruvate by lactate dehydrogenase, we proposed a model in which the A. actinomycetemcomitans L-lactate dehydrogenase, unlike homologous enzymes, is not feedback inhibited by pyruvate. This lack of feedback inhibition allows intracellular pyruvate to rise to levels sufficient to inhibit glucose uptake in other bacteria. In the present study, the A. actinomycetemcomitans L-lactate dehydrogenase was purified and shown to convert L-lactate, but not D-lactate, to pyruvate with a Km of approximately 150 µM. Inhibition studies reveal that pyruvate is a poor inhibitor of L-lactate dehydrogenase activity, providing mechanistic insight into L-lactate preference in A. actinomycetemcomitans

High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii

Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii Delta dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii Delta dhaD resulted in the strain C. werkmanii Delta dhaD Delta ldhA.adhE::ChlFRT which provided the maximum theoretical yield of 1.00 +/- 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 +/- 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached

The use of the ethanol pathway in goldfish Carassius auratus (L.) following anoxia

Goldfish (Carassius auratus) were subjected, for a period of 6 weeks, to 2h progressive hypoxia followed by 6h anoxia in closed respirometers at 15 degree C. The concentrations of glucose, lactate and ethanol were determined in whole goldfish following exposure to both hypoxia and anoxia. Lactate accumulation (mmol/kg/h) was 0.35 during the 1st week but declined to 0.14 in the 6th week of exposure to anoxia. In contrast, ethanol excreted to the surrounding water, increased from 65% to 92% of the total production in the lst and 6th week, respectively. The switch from lactate accumulation to ethanol pathway utilization, with the resultant metabolic depression and anoxia resistance is discusse

Distribution, organization and expression of genes concerned with anaerobic lactate utilization in human intestinal bacteria

Lactate accumulation in the human gut is linked to a range of deleterious health impacts. However, lactate is consumed and converted to the beneficial short-chain fatty acids butyrate and propionate by indigenous lactate-utilizing bacteria. To better understand the underlying genetic basis for lactate utilization, transcriptomic analyses were performed for two prominent lactate-utilizing species from the human gut, Anaerobutyricum soehngenii and Coprococcus catus , during growth on lactate, hexose sugar or hexose plus lactate. In A. soehngenii L2-7 six genes of the lactate utilization (lct) cluster, including NAD-independent d-lactate dehydrogenase (d-iLDH), were co-ordinately upregulated during growth on equimolar d- and l-lactate (dl-lactate). Upregulated genes included an acyl-CoA dehydrogenase related to butyryl-CoA dehydrogenase, which may play a role in transferring reducing equivalents between reduction of crotonyl-CoA and oxidation of lactate. Genes upregulated in C. catus GD/7 included a six-gene cluster (lap) encoding propionyl CoA-transferase, a putative lactoyl-CoA epimerase, lactoyl-CoA dehydratase and lactate permease, and two unlinked acyl-CoA dehydrogenase genes that are candidates for acryloyl-CoA reductase. A d-iLDH homologue in C. catus is encoded by a separate, partial lct, gene cluster, but not upregulated on lactate. While C. catus converts three mols of dl-lactate via the acrylate pathway to two mols propionate and one mol acetate, some of the acetate can be re-used with additional lactate to produce butyrate. A key regulatory difference is that while glucose partially repressed lct cluster expression in A. soehngenii , there was no repression of lactate-utilization genes by fructose in the non-glucose utilizer C. catus . This suggests that these species could occupy different ecological niches for lactate utilization in the gut, which may be important factors to consider when developing lactate-utilizing bacteria as novel candidate probiotics

Total tissue lactate dehydrogenase activity in endometrial carcinoma

Lactate dehydrogenase (LDH) is essential for continuous glycolysis necessary for accelerated tumor growth. The aim of this study was to reconsider if assay of total tissue activity of this enzyme could be useful as marker for endometrial carcinoma (EC). Activity of LDH was measured spectrophotometrically in homogenate supernatants of uterine tissue samples of 40 patients (10 normal endometria, 27 normal myometria, and 33 EC), including 30 matched pairs. Data obtained were analyzed in relation to clinical and histopathologic findings and compared with our previously published results on the tissue levels of the same enzyme in ovarian cancer and on the proteolytic activity of dipeptidyl peptidase III (DPP III) in EC (suggested biochemical indicator of this malignancy). Significantly increased (1.8-3.0 times; P < 1 x 10(-4)) LDH activity was observed in EC samples if compared with normal uterine tissues. This rise was not related to the clinicopathologic findings, however. In contrast to previous results on LDH in ovarian carcinomas, a significant rise in LDH activity was found already in grade 1 EC. Using the cutoff value of 1.06 U/mg, diagnostic sensitivity of 82%, specificity of 100%, and accuracy of 91% for total tissue LDH assay have been calculated. A correlation of tissue's LDH and DPP III activities was found, and their combined assay for EC showed increased diagnostic sensitivity (94%) and accuracy (96%)