Existence of three forms of H2M2 isoenzyme of lactate dehydrogenase

Lactate dehydrogenase (LDH) was purified from rat and bovine tissues by affinity chromatography on immobilized colchicine and used for the separation of isoenzymes by high-performance anion-exchange liquid chromatography (HPLC). This analysis showed the splitting of rat H2M2 into three peaks and of bovine H2M2 into two peaks. The heat stability, inactivation rate of urea and electrophoretic mobility of isoenzymes were examined and these analyses indicated differences in physicochemical properties for the respective peaks of rat and bovine H2M2. In hybridization experiments, the splitting of H2M2 into three peaks was achieved only with the combination of rat H4 and rat M4, while the other combinations of bovine H4 and bovine M4, of rat H4 and bovine M4 and of bovine H4 and rat M4 resulted in two H2M2 peaks. These results demonstrate that H2M2 of LDH in normal rat and bovine tissues is always split into two or three peaks by HPLC and that these H2M2 peaks have different physicochemical properties, suggesting the existence of three possible geometrical isomers of H2M2.     

Radioimmunoassay for individual lactate dehydrogenase isoenzymes 1 and 2

Radioimmunoassays (RIA) for the determination of the individual lactate dehydrogenase (LDH) isoenzymes, LDH-1 and LDH-2 have been developed. LDH-1 can be measured in the range of 5–100 ng and LDH-2 in the range of 5–80 ng, if there is no significant cross reactivity. Immunization of several rabbits with LDH-1 and LDH-2 isoenzymes reveals that some animals do not produce antisera to LDH-2 while those injected with LDH-1 generated antiserum in each case. The results of the binding studies suggest that a 50% binding that is recommended for RIA can be achieved with a titer value of 12000 dilution of the antisera. Cross reactivity studies indicate that LDH-1 cross reacts with the antisera to LDH-2 if its concentration is higher than 30 ng/ml of the RIA mixture while LDH-2 cross reacts with the antisera to LDH-1 only if its concentration exceeds 80 ng/ml.     

Signal amplification by substrate recycling on polyaniline/lactate oxidase/lactate dehydrogenase bienzyme electrodes

The bienzyme electrodes were fabricated by coimmobilization of lactate oxidase (LOD) and lactate dehydrogenase (LDH) onto electrochemically prepared polyaniline (PANI) films. These PANI/LOD/LDH bienzyme electrodes were shown to provide signal amplification by substrate recycling, making it possible to detect l-lactate at lower concentrations (0.1-1 mM). The PANI/LOD/LDH bienzyme electrodes were found to be stable for about 21 d at 4–10°C.     

紫草萘醌类化合物对乳酸脱氢酶和乙醇脱氢酶的共价修 饰作用

为研究紫草萘醌类化合物的细胞毒性作用机制,从新疆软紫草根中分离出四种紫草萘醌类化合物β,β-二甲基丙烯酰阿卡宁(1),乙酰阿卡宁(2),β-乙酰氧基异戊酰阿卡宁(3)和阿卡宁(4)。研究了四种天然紫草萘醌类化合物对乳酸脱氢酶和乙醇脱氢酶的共价修饰作用。酶活力测定结果表明,这四种紫草萘醌类化合物对两种脱氢酶都具有不同程度的抑制作用;酶分子中游离氨基和巯基修饰率的测定结果表明,紫草萘醌类化合物对两种酶的抑制作用主要是通过与酶分子中的巯基共价结合产生的。     

Thermochemistry of the reaction catalyzed by lactate dehydrogenase

The thermochemistry of the reduction of pyruvate to lactate, in the presence of nicotinamide adenine dinucleotide (reduced form); catalyzed by the enzyme lactate dehydrogenase, has been studied. After approximately 120 experiments, a best value for the enthalpy of reaction has been determined to be ?14.80±0.30 kcal mol^?1. This reveals that the driving force for the reaction is almost completely enthalpic in nature. In addition, using the current methodology, it is possible to determine lactate dehydrogenase activity as low as 0.15 international units (325 Wroblewski units) per sample.     

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate

(13)C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized (13)C label exchange between exogenously administered [1-(13)C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-(13)C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-(13)C,U-(2)H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized (13)C signal in a heteronuclear (1)H/(13)C spin-echo experiment.     

Measurement of240Pu/239Pu ratio by fission track method and inductively coupled plasma mass spectrometry

Fission track /FT/ method and inductively coupled plasma mass spectrometry /ICP-MS/, that is a new analytical technique for the analysis of trace element, were used for the measurement of²⁴⁰Pu/²³⁹Pu ratios in environmental samples. The results obtained by both methods are in agreement within the relative deviation of 9–13%. The precision in ICP-MS was found to be better than in it the FT-method. These methods are applicable to measure the Pu isotopes ratio at low concentration levels in environmental samples.     

Bioactivity screening,extraction, and separation of lactate dehydrogenase inhibitors from Polygala tenuifolia Willd. based on a hyphenated strategy

Stroke is the second leading cause of death worldwide. Lactate dehydrogenase inhibitors are currently widely used in the treatment of ischemic stroke, and natural products are considered promising sources of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Polygala tenuifolia . Furthermore, five lactate dehydrogenase inhibitors, sibiricose A5, 3,6′‐di‐O‐sinapoyl‐sucrose, glomeratose A, tenuifoliside B, and tenuifoliside C, were selected as target lactate dehydrogenase inhibitors. In addition, the five target compounds with purities of 96.45, 97.65, 96.38, 94.34, and 93.29% were extracted and isolated using a new hyphenated strategy of microwave‐assisted extraction coupled with countercurrent chromatography with a two‐phase solvent system of n‐hexane/n‐butanol/ethanol/water (5.321:1.00:1.664:6.647). The bioactivities of the isolated compounds were analyzed using PC12 cells and the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. The results also demonstrated that microwave‐assisted extraction coupled with countercurrent chromatography is an efficient method of isolating chemical constituents from medicinal herbs. Moreover, the research route consisting of activity screening, extraction, separation, and activity verification of the compounds has the advantages of being efficient, orientated, and objective.     

Automatic analysis of serum lactate dehydrogenase isoenzymes by high-performance ion-exchange chromatography

The repetitive analysis of serum lactate dehydrogenase (LDH) isoenzymes has been performed on a weak anion exchanger (TSKgel DEAE-5PW), which was developed by introducing diethylaminoethyl groups into TSKgel G5000PW (10 microns particle diameter)--a hydrophilic polymer-based material of large pore size--for high-performance gel chromatography. By use of this anion exchanger, a high-pH (greater than 8.0) solvent could be used and the albumin peak was completely separate from the LDH isoenzyme peaks. After 10 successive analyses with an autosampler, the coefficient of variation of the LDH isoenzyme elution times was less than or equal to 0.90%, and the coefficient of variation for peak areas was less than or equal to 3.85%. After 40 successive analyses, resolution between isoenzymes was generally greater than 1.25. This column can be used for more than 300 intermittent injections of human serum.     

Kinetic determination of lactate dehydrogenase in blood serum by multi-detection with a cyclic flow-injection system

The use of a cyclic flow-injection system for the determination of lactate dehydrogenase (LDH) is proposed. This configuration allows the repeated passage of the reacting plug through the detector resulting in multiple peak recordings. From the data obtained, which correspond to a typical kinetic curve, the required sensitivity can be selected by using procedures based on fixed-time measurements (peak maxima or minima) or reaction-rate measurements (signal increment between two successive maxima or minima). The methods were applied successfully to the determination of LDH in blood sera; the average recovery was 100.9%.     

Extraction and separation of lactate dehydrogenase inhibitors from Poria cocos (Schw.) Wolf based on a hyphenated technique and in vitro methods

Stroke is one of the most common diseases worldwide. Lactate dehydrogenase inhibitors are widely used in the treatment of ischemic stroke, with natural products considered a promising source of lactate dehydrogenase inhibitors. In this study, ultrafiltration liquid chromatography coupled with mass spectrometry was used for the screening and identification of lactate dehydrogenase inhibitors from Poria cocos . Five lactate dehydrogenase inhibitors were selected: dehydropachymic acid, pachymic acid, dehydrotrametenolic acid, trametenolic acid, and eburicoic acid. The inhibitors were extracted and isolated with purities of 96.75, 98.15, 97.25, 95.46, and 94.88%, respectively, by using a new “hyphenated” strategy of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography by a two‐phase solvent system of n‐hexane/ethyl acetate/ethanol/water at the volume ratio 0.965:1.000:0.936:0.826 v/v/v/v. The bioactivity of the isolated compounds was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay in PC12 cells. The results also showed that the hyphenated technique of microwave‐assisted extraction coupled with counter‐current chromatography and centrifugal partition chromatography was an efficient method for the continuous extraction and online isolation of chemical constituents from medicinal herbs. Furthermore, the research route based on the activity screening, extraction, separation, and activity verification of the compounds offered advantages of efficiency, orientation, and objectivity.     

Trace/matrix separation of uranium and thorium from molybdenum and tungsten matrix. A batch method for ultratrace bulk analysis

Summary A combined analytical procedure for spectro-photometric determination of uranium and thorium traces in high-purity molybdenum and tungsten matrices using the ion-exchanger cellulose Hyphan (TM of Riedel de Haën AG, FRG) for preconcentration is described. Following their separation (batch mode) uranium and thorium are determined as arsenazo-III complexes. The limits of detection (3) are 3 ng g^–1 U and 20 ng g^–1 Th. The analytical procedures elaborated improve the detection limits for U and Th in molybdenum and tungsten matrices by four and three orders of magnitude, respectively, compared to their determination by ICP-OES or DCP-OES without matrix separation. Accordingly, the procedures are routinely applicable in an industrial laboratory. The capability of this batch method can be validated by measurements of ion beam ratio with the glow discharge mass spectrometer; comparable results are obtained.     

Preparation and partial characterization of a soluble site-to-site directed enzyme complex composed of alcohol dehydrogenase and lactate dehydrogenase

A soluble, bifunctional enzyme complex has been prepared by crosslinking lactate dehydrogenase and alcohol dehydrogenase with glutaraldehyde. The crosslinking was performed on a solid phase while the active sites of alcohol dehydrogenase and lactate dehydrogenase were held adjacent to one another with the aid of a bis-NAD analog. Subsequently, the enzyme complex was released from the solid phase. The soluble enzyme complex was then purified by using NAD-Sepharose as an affinity adsorbent. Based on gel filtration experiments, the complex was estimated to consist of one of each dehydrogenase. By using a third enzyme, lipoamide dehydrogenase, which competes with lactate dehydrogenase for NADH produced by alcohol dehydrogenase, the effect of site-to-site orientation was studied. It was found that about 83% of the NADH produced by alcohol dehydrogenase was oxidized by site-to-site oriented lactate dehydrogenase compared to a figure of only about 61% obtained in an identical system of separate enzymes. This indicates that given two alternative routes, the preference for the one to lactate dehydrogenase over the one to lipoamide dehydrogenase is enhanced when lactate dehydrogenase and alcohol dehydrogenase are site-to-site oriented.     

Abortive complexes as a cause of substrate inhibition of rabbit muscle lactate dehydrogenase

Rabbit skeletal muscle lactate dehydrogenase follows the Michaelis—Menten kinetics and is adapted to an ordered, sequential type mechanism. The determination of the initial reaction rates allows am empirical rate equation, allowing the substrate and reaction product inhibition of the lactate dehydrogenase to be deduced.An overall reaction scheme, including all the possible complex formations which may be responsible for the inhibition, is formulated. The theoretical rate equation is determined from this scheme by the method of King and Altman and it is compared with the experimental results. The inhibition brought about by the reaction products and by excess substrate is justified by the existence of the E—pyruvate and E—NAD—NAD abortive complexes, which have not been postulated previously for rabbit muscle lactate dehydrogenase. The proposed rate equation gives a general interpretation of the kinetic behavior of rabbit skeletal muscle lactate dehydrogenase, some of its aspects still being under discussion.     

Determination of lactate and lactic dehydrogenase by biamperometric monitoring of hexacyanoferrate(II) in a flowing stream

Summary Open tubular carbon electrodes were used to monitor the biamperometric current of hexacyanoferrate(II) ion produced by the LDH catalyzed reaction between hexacyanoferrate(III) ion and lactate, and that produced from the diaphorase catalyzed reaction between hexacyanoferrate(III) ion and NADH, product from the LDH catalyzed reaction between lactate and NAD. Reagents and samples were mixed in a flow stream. Reaction takes place between the mixing point and the electrodes, and measurements are taken after a constant time interval. Lactate and LDH standards in serum control solutions were measured using the NAD dependent reaction.

---

Bestimmung von Lactat und Lactatdehydrogenase durch biamperometrische Messung von Hexacyanoferrat(II) im Durchfluß

Zusammenfassung Kohlenstoff-Röhrenelektroden wurden zur Messung des biamperometrischen Stromes des Hexacyanoferrat(II)-ions eingesetzt, das aus der LDH-katalysierten Reaktion zwischen Hexacyanoferrat(III) und Lactat bzw. der Diaphorase-katalysierten Reaktion zwischen Hexacyanoferrat(III) und NADH (Produkt der LDH-katalysierten Reaktion zwischen Lactat und NAD) stammte. Die Reagentien wurden mit den Proben im Durchfluß vermischt. Die Reaktion fand zwischen der Mischungsstelle und den Elektroden statt und die Messungen wurden nach einem Bestimmten Zeitintervall durchgeführt. Mit Hilfe der NAD-abhängigen Reaktion wurden Lactat- und LDH-Standards in Serumkontrollösungen bestimmt.