Stability of solutions of human granulocyte lactate dehydrogenase stored at different temperatures

Granulocyte suspensions from eight healthy individuals were lysed ultrasonically in 0.15 M NaCl or serum. The leukolysates were tested for lactate dehydrogenase after being stored at 22 C, 5 C, -20 C and -196 C for one, two, three, four and seven days. The LDH content of granulocytes was 291 IU/10(10) cells (mean) and the enzyme had a characteristic isoenzyme pattern rich in LDH-5 with stepwise increases in each fraction from LDH-1 to LDH-5. The enzyme was unstable in saline solutions. This was marked at -20 C and related to the duration of storage. All the isoenzyme fractions showed significant inactivation. A mean 10% loss of activity occurred at -196 C. This loss was independent of the duration of storage and related to the process of freezing and thawing. Moderate inactivation occurred at room temperature while refrigerated samples were stable for four days. Serum protected the enzyme from denaturation and samples stored at -20, -196 and 22 C were stable for the seven days of the experiment.     

Coagulation parameters of thawed fresh-frozen plasma during storage at different temperatures

Once thawed, fresh-frozen plasma (FFP) should be used, according to guidelines, within 24 h. In hospital practice, this may be associated with wastage. This study has been performed to investigate the coagulation levels of thawed quarantine FFP as used in the Netherlands. Five units of quarantine FFP, obtained by plasmapheresis, were thawed and by sterile docking divided into satellite bags (SB). SB 2-4 were stored at room temperature (RT) for, respectively, 1, 3 and 6 h and SB 5-9 at 4 degrees C for 6, 12 and 24 h and 1 and 2 weeks. At each time point, activated partial thromboplastin time (APTT), prothrombin time (PT), fibrinogen, factor V (FV), factor VIII (FVIII) and ADAMTS13 activity were measured. During storage at RT for up to 6 h, no major differences were found in the levels of FV, PT, fibrinogen and ADAMTS13 activity. FVIII activity showed a decrease of 16% and the APTT was prolonged by 6%. During storage at 4 degrees C for 2 weeks, FV and FVIII were reduced by 35 and 45%, respectively. The APTT and PT were prolonged by 17 and 15%, respectively. Fibrinogen was decreased by 8%. No change in ADAMTS13 activity was found. FFP stored at RT for 6 h or at 4 degrees C for 2 weeks can provide sufficient support for adequate haemostasis except for patients with a known deficiency for FVIII and can be used for plasmapheresis in patients with thrombotic thrombocytopenic purpura (TTP).     

The effect of storage at different temperatures on cholinesterase activity in human serum

The effect of storage on the catalytic concentration of cholinesterase and on the reference values of cholinesterase in human serum were studied. When serum was stored at room temperature (20 degrees C), at 4 degrees C and at - 20 degrees C (one year) there was no change in catalytic activity. Even after nine times freezing and thawing (over nine weeks) the catalytic activity was unaffected. The average reference value was significantly higher for males than for females (3.11 +/- 0.57 vs 2.50 +/- 0.43 kU/l).     

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.     

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.     

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.     

Stability of human blood serum aminoacids after storage at different pH and temperature conditions

The stability of the aminoacids in human blood serum ultrafiltrates and in an aminoacid standard solution was investigated under different pH and storage conditions. At close to neutral pH values the aminoacid concentrations remained constant for at least 12 months at -50 degrees C (n = 6), except for lysine. With acid but particularly with alkaline conditions a time- and temperature-dependent decrease was observed for glutamine and asparagine with concomitant increases of glutamate and aspartate. Similar, but less prominent alterations were noted for the concentrations of methionine, glycine, tyrosine, histidine, arginine and ornithine. Almost independent of pH, there was an effect of temperature; after 24 h at 55 degrees C a significant increase of several per cent in a number of serum aminoacid concentrations was observed, presumably due to the hydrolysis of small proteins and peptides. For the purpose of aminoacid analysis it is recommended that samples be stored deproteinized, deep-frozen and at neutral pH.     

抗凝血不同贮存温度对血凝试验的影响

目的 探讨不同的抗凝血体外贮存条件对凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)的影响.方法 将同一标本分别置于室温(18～25℃)、4℃、-20℃,在不同的时间测不同贮存温度下的PT、APTT,再与初始测定值进行统计学分析.结果 标本贮存在-20℃,对PT、APTT测定72 h无明显影响;而贮存在室温和4℃经过16～24 h,就会使结果产生不同程度的差异.结论 血凝试验前的标本预处理是保证结果可信度的关键.     

Some observations on serum lactate dehydrogenase (pyruvate leads to lactate) assays optimised at 37 degrees

One hundred and four sera were assayed for serum lactate dehydrogenase (l-lactate : NAD oxidoreductase, EC 1.1.1.27.) activity (pyruvate→ lactate) by three methods: the Henry assay used at 37°, and the McQueen and Eskalab assays which are claimed to be optimised at 37°. The McQueen assay gave significantly less lactate dehydrogenase activity, due to substrate inhibition, than the other two methods which appeared to be equivalent. An excess proportion of M-type lactate dehydrogenase subunits in samples was slightly overestimated by both the Eskalab and McQueen assays. It was concluded that an assay using pyruvate concentrations between 0.6 mM (Henry assay) and 1.5 Mm (Eskalab assay) would be more satisfactory.     

Hereditary deficiency of lactate dehydrogenase M-subunit

A family with a complete deficiency of lactate dehydrogenase M-subunit was investigated. The propositus was an 18-year-old male who complained of exertional pigmenturia and easy fatigue. Marked discrepancy was observed in the ratio between creatine kinase and lactate dehydrogenase (CK/LDH). Electrophoretic analysis of serum LDH isoenzymes of the propositus demonstrated only one activity band of LDH H₄. A complete lack of the LDH M-subunit was similarly demonstrated in erythrocytes, leukocytes and in the intermediate vastus muscle. LDH levels in the muscle specimen were markedly decreased in the patient, whereas CK and aspartate aminotransferase were almost the same as in a control subject. LDH isoenzymes of erythrocytes were analyzed in 5 siblings and in the parents. This demonstrated a complete lack of LDH M-subunit in 3 siblings. The ratio between H-subunit and M-subunit (H/M) in erythrocyte LDH suggested a partial absence of the M-subunit in two siblings and in the parents.An abortive increase of blood lactate and a marked increase in blood pyruvate were observed immediately after ischémie work of the forearm, accompanied by an increase in serum creatine kinase and myoglobinuria. The present case represents a newly described form of genetically determined myopathy.