Stability of lactate dehydrogenase at different storage temperatures

The effect of storing human serum, cord blood serum or heparinized plasma at 25 degrees C, 4 degrees C & -20 degrees C on the activity and isoenzyme distribution of lactate dehydrogenase (LD) was studied. Cellulose acetate and agarose electrophoresis, as well as an immunochemical inhibition technique, were used for isoenzyme quantification. In contrast to previous reports, cryo-instability was found only in specimens stored at 4 degrees C. Serum specimens stored at 25 degrees C and -20 degrees C retained 74% and 87% of total activity after 45 days of storage. LD-1 was stable at all three temperatures, with a maximum loss of 10%. LD-2, LD-3, LD-4, & LD-5 were most labile at 4 degrees C. Specimens that are to be analyzed for total LD or LD isoenzymes should be stored frozen or, if necessary, at room temperature, but not in a refrigerator. Thus, separate storage of specimens for cardiac isoenzymes (LD & creatine kinase) is not necessary. This may eliminate a possible source of falsely elevated LD-1/LD-2 ratios, as well as reducing the labor factor and the corresponding cost of cardiac isoenzyme determinations.     

自体血贮存在不同温度时红细胞形态的变化

背景：输血指南指出：全血应在（4±2）℃贮存,血液一旦离开正确的贮存条件,即有发生细菌繁殖或丧失功能的危险,受血者输注后则会发生不同程度的输血反应或输注无效。 目地：利用显微镜观察自体血贮存在不同温度下红细胞形态的变化。 方法：40例急性等容血液稀释患者取血后贮存在ACD枸橼酸血袋,分别贮存在4 ℃和常温23 ℃,于放自体血后即刻、自体血贮存1,2,3,4,5,6 h分别取样涂片,利用显微镜观察红细胞形态变化,计算红细胞畸形率。随机选取6 h段常温组血样与有效期内的ACD库存血各6份进行pH、K＋、游离血红蛋白等对比及细菌培养。 结果与结论：4 ℃组和常温组在各时间点红细胞畸形率差异无显著性意义, 6 h段常温组血样pH、K＋、游离血红蛋白等均优于有效期内的ACD库存血,培养均无细菌生长。提示常温下自体血贮存6 h内回输给患者是可行的。     

Stability of succinylcholine solutions stored at room temperature studied by nuclear magnetic resonance spectroscopy

The effect of storage temperature on the stability of two succinylcholine chloride solutions (20 and 50 mg/ml) was evaluated. Molecular composition was analysed using nuclear magnetic resonance spectroscopy. At room temperature, the degradation rate constant was 1.2%/month for the 20 mg/ml solution and 2.1%/month for the 50 mg/ml solution. The corresponding monthly degradation rates for the two solutions were 0.18% and 0.30% when stored at 4 degrees C, and 5.4% and 8.1% when stored at 37 degrees C. If a 10% loss of potency is considered acceptable, then the 20 and 50 mg/ml succinylcholine solutions can be stored in emergency resuscitation carts at room temperature for 8.3 and 4.8 months, respectively.     

True Arrhenius relationships of human lactate dehydrogenase.

The pyruvate and NADH concentrations and the buffer pH which gave maximal activity with LDH isoenzymes derived from human heart and liver tissue were established for the temperatures 25 degrees C, 30 degrees C, 35 degrees C, 37 degrees C,, 40 degrees C, 45 degrees C, and 50 degrees C. The velocities of the LDH isoenzymes using these maximal assay conditions were used to obtain Arrhenius plots, i.e. log initial velocity against inverse absolute temperature. The Arrhenius plots were linear with both isoenzyme preparations up to 45 degrees C. Between 45 degrees C and 50 degrees C it appeared that this linear relationship no longer held, particularly with the liver tissue. When the activation energies were calculated both isoenzyme preparations exhibited several points of inflexion, in each case occuring at the same temperatures. These inflexions represent a change in the reaction kinetics, possibly a conformational change in the enzyme. The results also indicate that the LDH 1 and 2 isoenzymes are more efficient than LDH 4 and 5.     

Use of purified lyophilized human lactate dehydrogenase isoenzyme 5 in a study of measuring lactate dehydrogenase activity

We examined the stability of human lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5--purified to a specific activity of about 400 kU/g--when lyophilized in a buffered, stabilized matrix of bovine albumin. This isoenzyme was prepared with a final activity of about 500 U/L and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. This isoenzyme decayed with approximate first-order kinetics, with an estimated half-life at -20 degrees C of about 475 years. Stability of reconstituted samples stored at 20 or 4 degrees C was poor, suggesting that the reconstituted material should be used without delay; material stored at -20 degrees C showed excellent stability for 15 days. We propose that such preparations might be further investigated as standards for use in electrophoresis of lactate dehydrogenase isoenzymes.     

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.     

Serum lactate dehydrogenase activity ratios with different substrates.

1. The lactate dehydrogenase activity of 89 sera from patients suffering myocardial infarction and of 55 sera from patients with hepatocellular damage was assayed under optimal conditions using pyruvate, alpha-oxobutyrate, hydroxypyruvate and glyoxylate as substrates. Activity was also measured with lactate as substrate at different pH values. 2. The ratios of activities under these different assay conditions were calculated for both series of patients. Correct differentiations for single ratios ranged from virtually nil for hydroxypyruvate/alpha-oxobutyrate to is greater than 93 per cent for glyoxylate/hydroxypyruvate and glyoxylate/alpha-oxobutyrate. This was little improved by the use of multiple ratios involving up to seven separate assays. 3. The activity ratio of hydroxypyruvate to pyruvate which is consistently greater than unity was found to be inverted in a case of morphine poisoning.     

New insights into salivary lactate dehydrogenase of human subjects

Lactate dehydrogenase (LDH) is a ubiquitous enzyme that plays a significant role in the clinical diagnosis of pathologic processes. The purpose of the current study was to examine LDH activity and isoenzyme profile of whole saliva and to compare it with the LDH activity of salivary glands and plasma before and after exposure to cigarette smoke (CS). The range of LDH activity in whole saliva at rest was 360 to 430 U/L. The mean +/- SEM of LDH activity in parotid and submandibular/sublingual salivary secretions was 41.3 +/- 19.2 U/L and 77.5 +/- 30.4 U/L, respectively, which implied that 75% of the whole-saliva LDH originated from an extra-salivary gland source. The profile of salivary LDH isoenzymes was found to have an entirely different pattern from that found in plasma, similar to that found in oral epithelium, indicating that the major source of salivary LDH is probably the oral epithelium-shedding cells. Therefore, salivary LDH may be evaluated for possible oral mucosal pathologies in a manner similar to that used for evaluating other tissue pathologies--such as those in heart, muscle, or liver--that can be detected in plasma. Exposure of whole saliva to CS in vitro resulted in a 41% reduction in LDH activity. However, CS exposure had no effect on LDH activity in plasma. Whole saliva, in contrast to plasma, contains redox-active metal ions such as iron and copper that may enhance LDH loss of activity. Therefore we conclude that whole saliva in the presence of CS becomes a potent protein-modifying agent that can destroy some of its endogenous components.     

Spectrophotometric method for selective assay of the five isoenzymes of human lactate dehydrogenase, based on their different stabilities at alkaline pH

After preincubation of human lactate dehydrogenase (EC 1.1.1.27) isoenzymes 1 through 5 with lactate at pH 9.4, 9.8, 10.25, and 10.6 at 30 degrees C for 10 min, the reduction of NAD+ was measured at the same pH values and temperature during the interval 1-2 min after adding the coenzyme. Relative to the reference activities measured at pH 8.7 by the method of Buhl et al. (Clin Chem 23: 1289-1295, 1977), the respective activities of isoenzymes 1 through 5 thus measured were 111, 104, 96, 68, and 0% at pH 9.4; 123, 108, 88, 0, and 0% at pH 9.8; 140, 100, 0, 0, and 0% at pH 10.25; and 138, 0, 0, 0, and 0% at pH 10.6. These relations allow selective assay of the respective isoenzymes in samples containing mixtures of them. Optimal conditions for such selective assay at 37 degrees C are reported.     

Normal values at 37 degrees for serum lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase and glycerate dehydrogenase

The distribution of serum lactate dehydrogenase and α-hydroxybutyrate dehydrogenase activity in a sample population of 280 volunteers is presented. Enzyme activity was measured at 37° using optimal concentrations of substrate, coenzyme and phosphate buffer. Significant increases in lactate dehydrogenase activity using the substrates pyruvate, 2-oxobutyrate and hydroxypyruvate were found with increasing age. No significant differences between males and females were found with pyruvate as substrate, but were found when 2-oxobutyrate and hydroxypyruvate were used.     

Complexes of lactate dehydrogenase and immunoglobulin G in human serum

Three patients with electrophoretically abnormal serum lactate dehydrogenase (LDH) isoenzyme patterns due to the presence of LDH-IgG complexes are described. It was found that normal LDH isoenzymes had been complexed with an anomalous immun oglobulin. Concerning the relation of the anomaly with a clinical disorder no definite conclusions could be drawn, although there are some indications that a relation exists between the occurrence of the LDH-IgG complexes and an autoimmune disease.     

Effect of reaction initiator on human lactate dehydrogenase assay.

Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.     

Identification of an extra lactate dehydrogenase band in human erythrocytes

Threehundred and thirty sera from hospitalized patients were analyzed for phospholipase A (PLA) activity. About 30% of unselected patients showed values above the normal range (0 to 10 U/l). The ratio of pathological to normal results was even higher in intensive care patients (around 1:1) and in patients with severe infections (2:1). 'Hyperphospholipasemia' was not typical for any defined organ system. From an etiological viewpoint, infectious diseases were most often related to increased PLA, followed by myocardial infarction and insufficiency and by malignant diseases. It is suggested that phospholipase A is a marker enzyme for the phagocytic activity in inflammation and necrosis.