Inhibition of lactate dehydrogenase isoenzymes by sodium perchlorate evaluated

We evaluated a method of measuring lactate dehydrogenase isoenzyme 1 (LD-1) selectively (Clin Chem 1987;33:991-2), in which all other LD isoenzymes were inhibited by adding sodium perchlorate to the reaction medium to a final concentration of 0.825 mol/L. In this study we used the different isoenzymes purified from human autopsy tissue and found that the originally published amount of inhibitor (a) increased the original LD-1 activity and (b) did not eliminate all enzyme activity of LD-2 and LD-3. Interference by LD-2 was further demonstrated. Thus we conclude that this method cannot be used for the selective determination of LD-1 because its results do not accurately reflect the original LD-1 activity.     

The relation of clinical catastrophes, endogenous oxalate production, and urolithiasis

A dose-related toxicity syndrome of renal, cerebral, and liver dysfunction; metabolic acidosis; and deposition of calcium oxalate crystals in tissues is reported in association with various apparently unrelated treatments for a wide range of diseases. The parenteral nutrient xylitol, the hyperosmolar agent glycerol, the polysorbate emulsifiers (e.g., in vitamin E preparations), the anesthetic methoxyflurane, and possibly the experimental hypoglycemic agent dichloroacetate all produce a toxicity syndrome very similar to that of ethylene glycol poisoning. In long-term, high-dose oral toxicity studies with rodents, these or similar agents also produce calcium oxalate bladder stones and bladder tumors. Studies with both unlabeled and labeled agents in humans and animals and in vitro experiments with purified enzymes, tissue homogenates, and isolated hepatocytes have provided both strong circumstantial and direct evidence for the existence of minor pathways of carbohydrate metabolism and of oxidative dealkylation and dehalogenation reactions in drug biotransformations that link these agents to endogenous oxalate production. Because urinary oxalate is now considered to be a critical factor in stone formation and because it is increasingly accepted that 80-90% of urinary oxalate is produced endogenously, it is now possible to formulate pathways that link oxalate production with dietary macronutrients. Therapeutic modifications of diet, in vivo hormonal milieu, and intracellular metabolic controls in relation to endogenous oxalate production may provide new forms of treatment for urolithiasis.     

Sensitivity of the direct oxalate oxidase assay of urinary oxalate improved

The direct colorimetric method for urinary oxalate has been modified to improve its sensitivity. Oxalate is precipitated overnight with calcium chloride and ethanol, the precipitate is redissolved, and the oxalate is measured by use of oxalate oxidase (EC 1.2.3.4), methylbenzothiazolinone hydrazone, and dimethylaniline. The color developed is more intense, analytical recovery averages 102%, and the overall imprecision is less than 5%. To assess the accuracy of the method, we used a gas-chromatography comparison method and control sera. Interference from ascorbate is negligible. The modified method retains its simplicity and is less expensive.     

Use of pyruvate oxidase to overcome pyruvate inhibition during the lactate to pyruvate reaction for assaying lactate dehydrogenase in serum

Automated assays of lactate dehydrogenase (LD) in serum are based on measuring the rate of NADH produced in a reverse LD reaction using lactate and NAD. The observed nonlinearity of LD reaction used in earlier assays performed in phosphate buffers has generally been attributed to the formation of a ternary complex of NAD, pyruvate, and phosphate. This is not satisfactory to explain the course of assay reaction carried out in organic buffers. Investigation of the possible causes of nonlinearity during the course of the reverse LD reaction during LD assays performed in Tris or other organic buffers indicated that inhibition of LD activity by pyruvate may be chiefly responsible for the observed effects, especially in serum exhibiting abnormally high LD enzyme activity. Most of the LD activity in serum was inhibited by 5 mMoles/L pyruvate. By contrast, the LD isoenzyme activities were inhibited partially at 0.5 mMole/L pyruvate, LD1 being the most and LD4 the least susceptible. In assays of serum samples with abnormally high LD and PYR concentration using LD reagent containing Tris buffer, pH 9.3, the inclusion of a bacterial pyruvate oxidase (PO) enabled the removal of pyruvate accumulating in situ, making it possible to assay LD activity in the absence of inhibitory concentration of pyruvate. The inclusion of 10 U/L of PO in our routine LD reagent was sufficient to overcome pyruvate inhibition, thus permitting the assay of serum exhibiting high LD activity, hence the extension of the upper limits of linearity of LD assay without compromising assay performance.     

Reversible loss of lactate dehydrogenase isoenzymes and lactate dehydrogenase activity in patient's serum

An abnormal lactate dehydrogenase (LD; EC 1.1.1.27) electrophoretogram (only one band, at the application site) and a low LD activity (7 U/L) was seen for a patient's serum during storage at 22 and 4 degrees C. Both reverted to normal when the serum was incubated at 37 degrees C.     

不同检测系统LDH测定结果的可比性研究

目的探讨同一临床实验室不同检测系统间乳酸脱氢酶(LDH)测定结果是否只有可比性,为血清酶测定的标准化提供实验数据。方法参考NCCLS的EP9-A文件,以日立7170生化分析仪、罗氏原装试剂、c.f.a.s校准品和质控品组成的检测系统(已通过ISO/IEC17025实验室认可)为比较方法,检测系统1-4为实验方法,用患者新鲜血清对LDH进行检测,计算试验方法(Y)和比较方法(X)之间的相对偏差(SE%),以CLIA’88规定的室间质量评价允许误差范围的1/2为标准,判断不同检测系统的可比性。结果LDH测定结果各系统的误差临床均可以接受。结论各检测系统测定LDH结果具有可比性。当同一实验室同一检验项目存在2个以上的检测系统时,应进行方法比对和偏差评估,以保证检验结果的可比性。     

强生Vitros 5.1 FS生化分析仪上乳酸脱氢酶的性能验证

目的探讨强生Vitros 5.1 FS生化分析仪上乳酸脱氢酶(LDH)的性能验证。方法根据美国临床和实验室标准化协会的检测仪器评价标准,并参照美国强生公司提供的验证方案对LDH的精密度、正确度、线性范围、最大稀释度、生物参考区间进行验证。结果 LDH批内及批间精密度试验均≤3.30%;正确度试验≤4.00%;线性试验决定系数为0.997 2;LDH用生理盐水稀释最大稀释度为8倍;生物参考区间试验证实《VITROS方法学手册》提供的参考区间313~618U/L可以引用。结论强生Vitros 5.1 FS生化分析仪检测的LDH性能验证基本符合质量目标要求和厂商说明要求,满足临床检验的需求。     

Alanine glyoxylate aminotransferase and the urinary excretion of oxalate and glycollate in hyperoxaluria type I and the Zellweger syndrome

We have studied the urinary excretion of oxalate and glycollate in patients with the Zellweger syndrome and hyperoxaluria type I and have measured the activity of alanine glyoxylate aminotransferase (a peroxisomal enzyme in man) in the patients. In agreement with earlier reports we found that alanine glyoxylate aminotransferase was strongly deficient in liver from a hyperoxaluria type I patient, thus explaining the increased urinary excretion of oxalate and glycollate in these patients. In livers from Zellweger patients, however, in which morphologically distinguishable peroxisomes are absent, the enzyme was not deficient, which is in accordance with our finding that the urinary excretion of oxalate and glycollate was normal in these patients.     

Hammerhead ribozymes: biochemical and chemical considerations

Ribozymes can be used to cleave specific mRNAs so as to prevent their translation. This presents an alternative to the use of antisense oligonucleotides for drug target validation or therapeutic purposes. The present review covers only one class of ribozymes, the hammerheads, which are small enough for chemical synthesis. Following an account of their structure and ability to catalyze the cleavage of target RNA is a discussion of some problems associated with their use as exogenous therapeutics. Chemical modifications that are used to address these issues are described.     

Time-related changes in the diagnostic utility of total lactate dehydrogenase, lactate dehydrogenase isoenzyme-1, and two lactate dehydrogenase isoenzyme-1 ratios in serum after myocardial infarction

Using receiver-operating characteristic (ROC) curve and likelihood ratio analysis, we examined the diagnostic utility of total lactate dehydrogenase (LD; EC 1.1.1.27) activity (I). LD isoenzyme-1 activity (II), and the LD-1 percentage of total LD activity (III), LD-1 LD-2 (IV), and LD-1/LD-4 (V) in 347 persons admitted to the Cardiac Care Unit (of whom 173 were subsequently proven to have had myocardial infarction). Blood was sampled from these subjects at about 6-h intervals for up to 96 h from the onset of chest pain. Defining an "effective" test as one having an area under the ROC curve of greater than or equal to 0.9, we determined the ranked utility (greatest to least) of these tests as V = IV greater than III greater than II greater than I. Tests III, IV, and V had by this criterion, diagnostic effectiveness equivalent to measurements of creatine kinase-2 in serum but in samples obtained at later time intervals. The decision thresholds for both high (constant) test sensitivity and specificity varied with time, to differing extents, over the entire 96-h period, a finding with important diagnostic implications. We document positive and negative likelihood ratio values for each of these tests throughout the entire period of study.     

Optimal conditions for assaying human lactate dehydrogenase by the lactate-to-pyruvate reaction: Arrhenium relationships for lactate dehydrogenase isoenzymes 1 and 5.

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.     

Use of purified lyophilized human lactate dehydrogenase isoenzymes in a study of the measurement of lactate dehydrogenase activity

We examined the stability of human lactate dehydrogenase (EC 1.1.1.27; LD) isoenzymes 1, 2, and 3--purified to specific activities of about 200 kU/g--when lyophilized in a buffered stabilized matrix of bovine albumin. Each isoenzyme was prepared at two activity concentrations and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. LD-1 activity decayed with zero-order kinetics, LD-2 and LD-3 with first-order kinetics. The extrapolated half-lives of these preparations at -20 degrees C varied between 80 and 530 years. Stability of reconstituted samples stored at 4 degrees C was excellent for LD-1 but poor for LD-2 and LD-3. We suggest that preparations of human LD-1 be further investigated as a possible reference material.     

A critical assessment of the roles of circulating hydrogen ion and lactate in the production of exercise-induced asthma.

To evaluate the roles of circulating hydrogen ion and lactate in the production of exercise-induced asthma, two experiments were performed. In the first, we exercised six asthmatic subjects to exhaustion on a bicycle ergometer while recording arterial pH at periodic intervals. Multiple aspects of pulmonary mechanics were measured before and after the work load. After recovery, the identical procedures were repeated, but sufficient quantities of sodium bicarbonate were infused to keep the pH at the pre-exercise level. In both experiments, statistically identical attacks of asthma were induced. To study the effect of lactate, five subjects were exercised on several occasions in order to determine the lowest level of work, and hence arterial lactate, that was reproducibly associated with an acute asthma attack. When this was known, sufficient quantities of sodium lactate were infused into the resting subjects so as to equal or exceed the amount produced with exercise. Pulmonary mechanics were not altered with this intervention. These findings demonstrate that lactic acidemia is not the cause of exercise-induced asthma.