Determination Of Lactate Dehydrogenase Activity Research Articles

Biophysical features of using a recombination sensor to detect lactate dehydrogenase: sensitivity mechanisms analysis

Background. Most pathologies of the human body (in particular, malignant neoplasms, myocardial hypoxia, liver diseases, etc.) are accompanied by a violation of the integrity of cells in target tissues and the release of intracellular macromolecules into the extracellular environment. Thus, an important diagnostic and prognostic indicator is the level of activity of certain enzymes in blood serum, which are normally intracellular. One of the most promising areas of modern medical electronics and biophysics is the development and optimization of enzyme screening methods in biological fluids. In this study, we aimed to investigate the biophysical characteristics of using a recombination sensor for determining LDH activity in biological fluids. Materials and Methods. Experiments were performed on preparations of standard human blood serum. The reference determination of lactate dehydrogenase activity was carried out photometrically based on the change (decrease) in the concentration of the reduced form of the NADH coenzyme. The passage of the lactate dehydrogenase reaction was experimentally recorded by measuring the photocurrent of a silicon structure with a buried barrier under light irradiation from the region of strong absorption (λ = 532 nm). Results. The biophysical features of the device were studied. The detection of lactate dehydrogenase becomes possible due to the transfer of a hydrogen ion from nicotinamide adenine dinucleotide (NADH) to pyruvate, as a result of which lactate and NAD+ are formed. The effect is explained by the local electrostatic influence on the parameters of the recombination centers in the near-surface bending zone near the silicon surface, which leads to a change in the surface recombination rate. Conclusions. Our approach can be considered as a promising way to develop a highly sensitive method for the detection of lactate dehydrogenase. It has been experi­mentally shown that effective detection is possible in two changes at the surface ben­ding of the deep barrier silicon substrate zone.

Fluorescence determination of lactate dehydrogenase activity based on silicon quantum dots

Silicon quantum dots (SiQDs) synthesized based on 3-aminopropyltrimethoxysilane (ATPMS) as silicon source were used to detect the activity of lactate dehydrogenase (LDH) through changes of fluorescence intensity of SiQDs. In this system, the fluorescence of SiQDs was first quenched by nicotinamide adenine dinucleotide (NADH), and then recovered with the addition of LDH, as NADH was consumed by catalytic reaction of LDH. A linear calibration chart of LDH is obtained in the range of 0.77–385 U/mL. The assay displays high selectivity towards LDH detection, and was successfully applied to the analysis of LDH in human serum samples. This assay has great prospects for the diagnosis and prognosis of various diseases, especially melanoma.

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized 13C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-13C]lactate production from hyperpolarized [1-13C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using 31P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm3) and a diameter ranging from 3 to 6 mm.

Combined space-organized system for animals oocytes maturation in vitro

Aim. The aim of the work was to create a space-organized system for oocytes maturation based on rabbits granulosa cells, hyaluronic acid (for structural organization of this system) and insulin, as an important growth factor. Methods. Cultivation of granulosa cells and oocytes maturation in vitro, biochemical determination of lactate dehydrogenase activity and cholesterol concentration, and determination of progesterone and estradiol concentrations by enzyme immunoassay were done. Results. Hyaluronic acid in concentration of 0.5 μg/ml increased proliferation of granulosa cells and progesterone concentration in the conditioned medium on 72 h of cultivation, and estradiol on 48 and 72 hours of cultivation was found. The degree of oocyte maturation to metaphase-2 was 85% by the influence of 10 μg/ml insulin. Same concentration of insulin increased granulosa cells proliferation, estradiol, progesterone and cholesterol concentration in the conditioning medium after 24 hours of cultivation. Conclusions. The optimal parameters of the combined spaceorganized culture system are 1 million cells/ml of granulose with the addition of 0.5 μg/ml hyaluronic acid and 10 μg/ml insulin, which creates the optimal conditions for the maturation of oocytes to metaphase-2 cell cycle.&#x0D; Keywords: oocytes, granulosa cells, hyaluronic acid, insulin, in vitro.

In-cell determination of Lactate Dehydrogenase Activity in a Luminal Breast Cancer Model ⁻ ex vivo Investigation of Excised Xenograft Tumor Slices Using dDNP Hyperpolarized [1-13C]pyruvate.

[1-13C]pyruvate, the most widely used compound in dissolution-dynamic nuclear polarization (dDNP) magnetic resonance (MR), enables the visualization of lactate dehydrogenase (LDH) activity. This activity had been demonstrated in a wide variety of cancer models, ranging from cultured cells, to xenograft models, to human tumors in situ. Here we quantified the LDH activity in precision cut tumor slices (PCTS) of breast cancer xenografts. The Michigan Cancer Foundation-7 (MCF7) cell-line was chosen as a model for the luminal breast cancer type which is hormone responsive and is highly prevalent. The LDH activity, which was manifested as [1-13C]lactate production in the tumor slices, ranged between 3.8 and 6.1 nmole/nmole adenosine tri-phosphate (ATP) in 1 min (average 4.6 ± 1.0) on three different experimental set-ups consisting of arrested vs. continuous perfusion and non-selective and selective RF pulsation schemes and combinations thereof. This rate was converted to an expected LDH activity in a mass ranging between 3.3 and 5.2 µmole/g in 1 min, using the ATP level of these tumors. This indicated the likely utility of this approach in clinical dDNP of the human breast and may be useful as guidance for treatment response assessment in a large number of tumor types and therapies ex vivo.

Alkaline phosphatase and lactate dehydrogenase enzyme activity in gingival crevicular fluid during orthodontic tooth movements

Background: Bone remodeling is a biologic process involving an acute inflammatory response in periodontal tissues during orthodontic tooth movement. The early response of periodontal tissues to mechanical stress involves metabolic changes that allow tooth movement. Aim of the Study: The aim of the present study was to evaluate the changes in alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme activity in gingival crevicular fluid (GCF) when forces are applied during initial alignment (Phase 1 [P1]) and retraction (Phase 2 [P2]). Settings and Design: The study was performed in a hospital-based population with an experimental design. Materials and Methods: A total of 17 patients (11–21 years) were included in the study, all requiring first premolar extractions undergoing fixed orthodontic treatment. Maxillary canine and mandibular second molar were used as test tooth (TT) and control tooth (CT) in each patient, respectively. On the 14th day after the start of each phase, 2 μL of GCF was collected from mesial side of both TT and CT for ALP and LDH activity determination using spectrophotometer. Statistical Analysis: Statistical analysis was performed with SPSS (version 13, SPSS Inc., Chicago, USA) package. The significance of differences in both phases at test or, at control site for LDH and ALP enzyme activity, was evaluated by performing Student's paired t-test. The significance of differences in between test and control sites in each phase separately was evaluated by performing Student's unpaired t-test. Results: There was a variation in enzyme activity during orthodontic tooth movement. At TT, there was statistically significant increase in ALP and LDH activity in P2 as compared to P1 with P = 0.005 and P Conclusion: This study concluded that ALP and LDH activity could be successfully estimated in the GCF, and ALP and LDH activity in GCF reflects the biologic activity in the periodontium during orthodontic tooth movements.

Alkaline phosphatase and lactate dehydrogenase enzyme activity in gingival crevicular fluid during orthodontic tooth movement

Background: Bone remodeling is a biologic process involving an acute inflammatory response in periodontal tissues during orthodontic tooth movement. The early response of periodontal tissues to mechanical stress involves metabolic changes that allow tooth movement. Aim of the Study: The aim of the present study was to evaluate the changes in alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) enzyme activity in gingival crevicular fluid (GCF) when forces are applied during initial alignment (P1) and retraction (P2). Settings and Design: The study was performed in a hospital-based population with an experimental design. Materials and Methods: Seventeen participants (11–21 years) included in the study, all requiring first premolar extractions were undergoing fixed orthodontic treatment. Maxillary canine and mandibular second molar were used as test tooth (TT) and control tooth (CT) in each patient, respectively. On 14th day after the start of each phase, 2 μL of GCF was collected from mesial side of both TT and CT for ALP and LDH activity determination using a spectrophotometer. Statistical Analysis: The results were subjected for Student's t-test. Results: There was a variation in enzyme activity during orthodontic tooth movement. At TT, there was a statistically significant increase in ALP and LDH activity in P2 as compared to P1 with P = 0.005 and P ≤ 0.001, respectively. At CT, there was no statistically significant difference in ALP and LDH activity in P1 and P2 with P = 0.054 and P = 0.061, respectively. Conclusion: This study concluded that ALP and LDH activity could be successfully estimated in the GCF, and ALP and LDH activity in GCF reflects the biologic activity in the periodontium during orthodontic tooth movements.

An Novel Enzyme Fluorescence Capillary Analysis for Determination of Lactate Dehydrogenase Activity in Micro volume Serums

An Novel Enzyme Fluorescence Capillary Analysis for Determination of Lactate Dehydrogenase Activity in Micro volume Serums

Atividades de creatina quinase e lactato desidrogenase na identificação de frangos com estresse e filés PSE (pale, soft, exudative)

Porcine Stress Syndrome (PSS) is a syndrome that occurs in pigs. PSS animals are sensitive to the anesthetic halothane, have higher activities of Creatine Kinase (CK) and Lactate Dehydrogenase (LDH) and are predisposed to the development of PSE (Pale, Soft, Exudative) meat, compromising the meat quality. In chickens, little is known about the relationship between sensitivity to and halothane changes in the activities of these enzymes with the occurrence of PSE meat. The aim of this study was to evaluate the CK and LDH activities in sensitive and non-sensitive halothane chickens and for stress susceptibility identification and PSE meat development. Birds were exposed to halothane at a 3.0 % concentration in pure O2 for 5 min. Afterwords, they were classified into groups: sensitive to halothane (HAL +) (stiffening of the lower limbs) and Non-sensitive (HAL-) (no hardening). After 3 h and 48 h to the test, blood was collected from the wing vein gill, serum extracted and stored at –22 °C until analysis of CK and LDH. No differences were observed (p ? 0.05) in CK activity for HAL+ and HAL- groups. However, differences (p ? 0.05) were observed the LDH activity at 3 h and 48 h, and 44.2 % and 66.1 % higher for the group HAL +, respectively. The determination of lactate dehydrogenase activity indicates to be an alternative to identify animals susceptible to stress thus prone to produce PSE meat.

The role of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase in the diagnosis of subclinical intramammary infections in dairy sheep and goats

The objective was to investigate the changes occurring in the activities of the enzymes lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in sheep and goat milk as a result of subclinical intramammary infections (IMI) and to evaluate the use of these enzymes for the diagnosis of subclinical IMI in dairy sheep and goats. A total of 206 samples of sheep milk and 162 samples of goat milk, obtained from equal udder halves, were used in the study. For each species they were divided into two groups: a no-infection group and a subclinical infection group. Activities of LDH, ALP and AST were significantly higher in the subclinical infection group than in the no-infection group (P<0.05) in both sheep (LDH: 350.42+/-11.25 v. 120.91+/-4.41; ALP: 2773.43+/-105.18 v. 2189+/-94.24; AST: 29.57+/-0.74 v. 17.32+/-0.46) and goats (LDH: 354.07+/-13.33 v. 103.79+/-3.75; ALP: 311.13+/-25.74 v. 137.24+/-19.62; AST: 27.59+/-6.42 v. 15.87+/-0.45). The activity of LDH was identified as indicator for subclinical IMI in both sheep and goats. The optimum cut-off values for LDH activity, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic (ROC) analysis, were at 197 U/l, 185 U/l and 197 U/l for sheep, goats and both species, respectively. DSn for sheep, goats and both species at these cut-off values was 92.8%, 98.2% and 94.0%, whereas DSp was 95.4%, 96.3% and 96.3%, respectively. It was concluded that the determination of LDH activity in milk serum is a sensitive and reliable method for the detection of subclinical IMI in dairy sheep and goats.

Characterization of Candida parapsilosis infection of an in vitro reconstituted human oral epithelium

Oral candidosis is a common problem in immunocompromised patients, and whilst Candida albicans is regarded as the principal cause of infection, other non-Candida albicans Candida (NCAC) species are increasingly being recognized as human pathogens. Relatively little is known about the virulence factors associated with NCAC species, and the aim of this study was to use a reconstituted human oral epithelium (RHOE) to examine epithelial infection withCandida parapsilosis. Strains originating from the oral and vaginal mucosa and from the urinary tract were all shown to colonize RHOE in a strain-dependent manner. Strain differences were found in the colonizing morphology and in the extent of invasion of the RHOE. Low invasion of RHOE was detected for strains after 12 h, whereas extensive tissue damage was evident after 24 h when assessed using histological examination and lactate dehydrogenase activity determination. Tissue damage was reduced in the presence of pepstatin A, although C. parapsilosis invasion of the tissue was not inhibited. Real-time polymerase chain reaction of secreted aspartyl proteinase (SAP) genes (SAPP1-3) showed that expression was strain dependent, with an increased expression generally occurring for Candida infecting RHOE compared with planktonic equivalents. In summary, C. parapsilosis was not highly invasive of RHOE but did induce significant tissue damage, which could relate to specific SAPgene expression.

Determination of lactate dehydrogenase activity and urea content in milk by dry chemistry.

Comparison of the new technique of dry chemistry (EKTACHEM 700-XR, Eastman Kodak Co., USA) with conventional wet chemistry (HITACHI 717, Boehringer Mannheim, Germany) for quantitations of lactate dehydrogenase activity and urea content in bovine milk resulted in correlation coefficients of more than 0.9 even when measuring fresh raw milk by dry chemistry.

Simple method for platelet counting

A method is proposed for counting the number of adhered platelets based on the determination of lactate dehydrogenase activity in bulk after lysis of adhered platelets. This method was compared with the widely used radioisotope labelling technique. It was concluded that the present lactate dehydrogenase method is effective in counting the adhered platelets, as no significant difference was found between the readings of two methods when commercial polymers and glass were used as samples.

Synthesis of a new tetrazolium salt giving a water-soluble formazan and its application in the determination of lactate dehydrogenase activity

A pyridine analog of 3-(4-,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide was synthesized. The new reagent, 2-phenyl-3-(4,5-dimethyl-2-thiazolyl)-5-(4-pyridyl)tetrazolium bromide, gave a water-soluble formazan dye on enzymatic reduction and was applied in the spectrophotometric determination of lactate dehydrogenase (0–84 mU ml −1) in blood serum. The sensitivity (ϵ = 1.83 × 10 4 l mol cm −1 at 539 nm) was about 70% of that obtainable with nitrotetrazolium blue.

The correlation between lactate dehydrogenase activity in urine and serum and experimental renal damage in the rat

Abstract An increase in urinary and serum lactate dehydrogenase (LDH) activity was observed in rats following kidney damage induced by intravenous injections of mercuric chloride. The increase in the enzyme activity positively correlates with the increase in dose given as well as with the magnitude of the kidney damage demonstrated by histomorphological and enzyme-histochemical methods. It is suggested that the determination of lactate dehydrogenase activity in urine can be used as a method for detection of renal damage in rats, in different toxicity studies.

On the inhibitory influences of the reaction products in the determination of lactate dehydrogenase activity, using lactate as a substrate

It has been found that in the determination of lactate dehydrogenase activity (substrate: lactate, coenzyme: NAD +) as proposed by Amador et al. 1 the linearity between Δ A and time hardly holds true. This appeared to be the result of an inhibition, a phenomenon which increases with time. The best method for calculating the enzyme activity with this forward reaction was by means of a polynomial curve-fitting procedure. As NADH and pyruvate, when added to the reaction mixture, showed an inhibitory effect of the same order as found, it is probable that the inhibition is the result of these reaction products.

PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA

Abstract Case reports of eight patients with the PNH defect are presented.The diagnostic criteria applied are discussed: Ham's and Crosby's tests, the sucrose haemolysis test, acetylcholine esterase activity of the red cells, and signs of intravascular haemolysis with emphasis on the determination of lactate dehydrogenase activity in blood and bone marrow.The diversity of the presentation of PNH is outlined. One patient presented with attacks of abdominal pain for two years before diagnosis, three patients had longstanding pancytopenia with few clues to the diagnosis, two patients presented as cases of aplastic anaemia with initially negative Ham's tests, and two patients had concomitant myelofibrosis. The relationship of PNH to other blood diseases is discussed, and a possible connection with myelofibrosis is suggested. It thus appears that the PNH erythrocyte defect is encountered not only in the clinically classical PNH syndrome, but also in a variety of pancytopenias which are difficult to classify, and finally in some better defined conditions such as aplastic anaemia and myelofibrosis. This could be interpreted in several ways: 1. PNH is a nosological entity with an extremely wide clinical spectrum; 2. PNH is not a nosological entity, but erythroid cell lines carrying the PNH defect may be set up under many conditions when haemopoiesis is afflicted by a primary haematological disorder; 3. PNH exists (a) as a primary disorder, the clinically classical PNH syndrome, and (b) as a secondary defect in a variety of other haematological disorders. It is not yet possible to determine which of these possibilities, if any, is the right one, although at the present time the second interpretation would appear to be the most likely one.Clinical features are mentioned, particularly hypercoagulability and the occurrence of thromboses. An interesting aspect is that the majority of patients had an abnormal EEG. The significance of this remains to be determined. The multiple mechanism of anaemia in PNH is emphasized, including marrow hypoplasia, haemolysis, iron deficiency, autoimmunity, and folic acid deficiency.The value of blood transfusions, iron, adrenocorticoid and anabolic steroids in the management of PNH is discussed.

EFFECT OF PHENAZINE-METHOSULPHATE ON THE HISTOCHEMICAL LOCALIZATION OF LACTATE DEHYDROGENASE.

THE histochemical reaction for the localization of lactate dehydrogenase (LDH) is a multi-step reaction. The reduction of tetrazolium salts in tissue sections cannot be accomplished directly by LDH but only by means of the so-called tissue NADH-diaphorase1. Unfortunately, tissue diaphorases are not uniformly distributed in all tissues, cells or sub-cellular structures. The absence of these enzymes in certain places is therefore a limiting factor for the determination of LDH activity in tissue sections. Thus, in order to localize LDH at sites where no diaphorases are present, the use of an electron carrier seemed necessary.