Optimum Reaction Conditions for Human Lactate Dehydrogenase Isoenzymes as They Affect Total Lactate Dehydrogenase Activity

1968 ◽  
Vol 14 (8) ◽  
pp. 740-753 ◽  
Author(s):  
Royal J Gay ◽  
Robert B McComb ◽  
George N Bowers
Keyword(s):  
Lactate Dehydrogenase ◽  
Comparative Study ◽  
Substrate Concentration ◽  
Spectrophotometric Assay ◽  
Serum Samples ◽  
Reaction Conditions ◽  
Lactate Dehydrogenase Activity ◽  
Ldh Isoenzymes ◽  
Optimum Reaction ◽  
Lactate Dehydrogenase Isoenzymes

Abstract Optimum reaction conditions at 30° ± 0.5° for two continuous spectrophotometric assay procedures, lactate to pyruvate (L → P) and pyruvate to lactate (P → L), were determined with respect to pH at 30° (pH30) substrate concentration, and coenzyme concentration for the human LDH isoenzymes. For the P → L procedure, broad pH30 optima were within the range of 7.20-7.40 for all the LDH isoenzymes. The coenzyme optima were identical for all of the isoenzymes tested at a reduced NAD concentration of 1.5 x 10-4 M. Pyruvate substrate optima ranged from 7.5 x 10-4 M for LDH1 to 1.7 x 10-3 M for LDH5 at pH30 7.30. For the L → P procedure, the pH30 optima were within the range of 8.30-8.88 for the LDH5 through LDH1 isoenzymes, respectively. Optimum activity was obtained at a NAD concentration of 6.0 x 10-3 M and remained constant at least to 1.8 x 10-2 M for each of the isoenzymes tested. L-Lactate substrate optima ranged from 4.0 x 10-2 M for LDH1 to at least 7.2 x 10-2 M for LDH5. From the isoenzyme studies, the degree of variation possibly involved in either method due to variations in isoenzyme distribution was calculated for total LDH samples. These calculations showed that both methods, P → L and L → P, were essentially equivalent. This equivalency was verified by a comparative study of the two methods on human serum samples.

Improved column method for separating lactate dehydrogenase isoenzymes 1 and 2.

1978 ◽  
Vol 24 (3) ◽  
pp. 480-482 ◽  
Author(s):  
D W Mercer
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Creatine Kinase ◽  
Heart Disease ◽  
Lactate Dehydrogenase ◽  
Sodium Chloride ◽  
Lactate Dehydrogenase Activity ◽  
Column Method ◽  
Creatine Kinase Mb ◽  
Lactate Dehydrogenase Isoenzymes

Abstract Lactate dehydrogenase (LD) isoenzymes 1 and 2 in human serum were separated on a column of diethylaminoethyl-Sephadex. Samples layered on mini-columns were eluted with buffered sodium chloride (100, 150, and 200 mmol/liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was evaluated by electrophoresis on polyacrylamide gel. Results for column-fractionated LD-1 and LD-2 were expressed in two ways: LD-1/LD-2 ratios and total LD-1 + LD-2 activities. The former is a more specific indicator of myocardial infarction than the latter. Sera from 10 patients with acute myocardial infarction (increased creatine kinease isoenzyme MB activity) exhibited ratios in the range of 0.92 to 1.56, ratios for 10 patients without heart disease (normal creatine kinase MB) ranged from 0.33 to 0.69.

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

1989 ◽  
Vol 35 (8) ◽  
pp. 1774-1776 ◽  
Author(s):  
D A Smith ◽  
G C Moses ◽  
A R Henderson
Keyword(s):  
Lactate Dehydrogenase ◽  
Half Life ◽  
Specific Activity ◽  
Lactate Dehydrogenase Activity ◽  
Bovine Albumin ◽  
First Order ◽  
First Order Kinetics ◽  
The Stability ◽  
Lactate Dehydrogenase Isoenzymes ◽  
Excellent Stability

Abstract 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.

Effect of Lactate Dehydrogenase Isoenzymes on the Coupled Enzymatic Assay for Alanine Aminotransferase Activity

1975 ◽  
Vol 21 (3) ◽  
pp. 330-333 ◽  
Author(s):  
Michael M Chang ◽  
Tai Wha Chung
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Alanine Aminotransferase ◽  
Rabbit Muscle ◽  
Aminotransferase Activity ◽  
Lactate Dehydrogenase Activity ◽  
Enzymatic Assays ◽  
Substrate Specificities ◽  
Lactate Dehydrogenase Isoenzymes ◽  
Alanine Aminotransferase Activity

Abstract We show an example of the importance of specifying the form of isoenzyme and source of indicator enzymes to be used in coupled enzymatic assays. When we compared H4 (pig heart) and M4 (rabbit muscle) isoenzymes of lactate dehydrogenase for their suitability as indicator enzymes in the assay for alanine aminotransferase activity, we found that about fourfold as much M4 as H4 was required in terms of lactate dehydrogenase activity to reflect accurately equivalent amounts of alanine aminotransferase activity. Moreover, the substrate specificities of the two isoenzymes differed quantitatively.

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

1986 ◽  
Vol 32 (5) ◽  
pp. 758-762 ◽  
Author(s):  
D A Smith ◽  
G C Moses ◽  
A R Henderson
Keyword(s):  
Lactate Dehydrogenase ◽  
Zero Order ◽  
Lactate Dehydrogenase Activity ◽  
First Order ◽  
First Order Kinetics ◽  
Activity Concentrations ◽  
Zero Order Kinetics ◽  
Specific Activities ◽  
The Stability ◽  
Lactate Dehydrogenase Isoenzymes

Abstract 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.

Interconverting measurements of human lactate dehydrogenase activities in three buffers.

1977 ◽  
Vol 23 (2) ◽  
pp. 200-204 ◽  
Author(s):  
S N Buhl ◽  
A H Richards ◽  
K Y Jackson ◽  
R Lubinski ◽  
R E Vanderlinde
Keyword(s):  
Lactate Dehydrogenase ◽  
Analytical Method ◽  
Serum Lactate ◽  
Serum Lactate Dehydrogenase ◽  
Normal Range ◽  
Serum Samples ◽  
Reaction Conditions ◽  
Clinical Laboratories ◽  
Wide Range ◽  
The Matrix

Abstract The lactate-to-pyruvate reaction for serum lactate dehydrogenase (LD) is most frequently assayed in one of three buffers, pyrophosphate (PPi), tris(hydroxymethyl)amino-methane (Tris), or 2-amino-2-methyl-1-propanol (AMP). We described interconverting results for serum samples and for highly purified LD isoenzymes I (dissolved in one of these matrixes) assayed in these buffers under optimized reaction conditions. The equation for converting results obtained for sera in Tris (x) to those in PPi(y) (both at 30 degrees C) is y = 0.74x+10 (n = 98). Since AMP is used extensively in Technicon procedures, we determined the LD activity of sera with an SMA 12/60, at 37 degrees C. The equation for convering these AMP results to results obtained in PPi at 30 degrees C is y = 0.45x-16 (n = 90). Very different equations were obtained with highly purified LD isoenzyme I maintained in two different matrixes and with both isoenzymes assayed in the same matrix. The matrix in which LD is dissolved and the proportion of various LD isoenzymes affect the magnitude of difference in observed LD activity under various conditions. Therefore, in clinical laboratories that use more than one analytical method or when conversion equations are used in the comparison of interlaboratory results, it is important to define the LD source, isoenzyme content, and the matrix, as well as the reaction conditions, and to use many samples with a wide range of activities when determining the conversion equations. For any changes in reagent source, substrate concentration, or alteration in procedure, a new normal range and new conversion equations should be determined.

Simultaneous Separation of Serum Creatine Kinase and Lactate Dehydrogenase Isoenzymes by Ion-Exchange Column Chromatography

1975 ◽  
Vol 21 (8) ◽  
pp. 1102-1106 ◽  
Author(s):  
Donald W Mercer
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Creatine Kinase ◽  
Lactate Dehydrogenase ◽  
Time Course ◽  
Serum Creatine Kinase ◽  
Lactate Dehydrogenase Activity ◽  
Ion Exchange Column ◽  
Simultaneous Separation ◽  
Lactate Dehydrogenase Isoenzymes

Abstract Lactate dehydrogenase isoenzymes were partially separated by use of a previously described column technique for creatine kinase [Clin. Chem. 20, 36 (1974)]. Extracts of lactate dehydrogenase-rich tissues were used to evaluate column resolution. Samples layered on mini-columns containing DEAE-Sephadex were eluted with Tris-buffered sodium chloride (100 and 200 mmol/ liter). Lactate dehydrogenase activity in column effluents was measured by the Wacker method, and their isoenzyme content was assessed by electrophoresis on polyacrylamide gel. Dehydrogenase isoenzymes 3, 4, and 5 were separated from isoenzymes 1 and 2, and the separation was tissue-specific and reproducible. The electrophoretic technique for isoenzymes 3, 4, and 5 gave values about 20% lower than did the column technique. Sera from 15 healthy laboratory technicians contained total lactate dehydrogenase, isoenzymes 1 and 2, and isoenzymes 3, 4, and 5 in the ranges 94 to 152, 34 to 64, and 38 to 75 U/liter, respectively. Activities of sera from 15 patients with acute myocardial infarction (total lactate dehydrogenase) ranged from 212 to 800 U/liter and lactate dehydrogenase isoenzymes 1 and 2 ranged from 138 to 628 U/liter. Lactate dehydrogenase and creatine kinase isoenzymes were rapidly and easily measured after being simultaneously separated. The procedure is specific and sensitive for following the post-infarct time course of changes in isoenzyme activities.

Lactate dehydrogenase isoenzyme pattern in sera of patients with malignant diseases.

1989 ◽  
Vol 35 (3) ◽  
pp. 396-399 ◽  
Author(s):  
E E Giannoulaki ◽  
D L Kalpaxis ◽  
C Tentas ◽  
P Fessas
Keyword(s):  
Lactate Dehydrogenase ◽  
Heat Stability ◽  
Surgical Removal ◽  
Anatomical Location ◽  
Malignant Diseases ◽  
Serum Samples ◽  
Ldh Isoenzymes ◽  
Ldh Activity ◽  
The Relationship ◽  
Isoenzyme Patterns

Abstract Total lactate dehydrogenase (LDH; EC 1.1.1.27) activity and the percentage distribution of LDH isoenzymes were determined in 127 patients with malignant diseases. A shift in the isoenzyme patterns was observed toward the M-type, with an increase in the percentage of LDH-4 and LDH-5 isoenzymes and a slight increase in total LDH activity of all patients. Serum samples from 68 of the patients contained an abnormal isoenzyme of LDH, "LDH-1 ex," that, on agarose gel electrophoresis at pH 8.6, migrated between albumin and LDH-1 isoenzyme. Chemotherapy, radiotherapy, or surgical removal of the tumor was accompanied by disappearance of this abnormal isoenzyme. The heat stability of LDH-1 ex isoenzyme appears to be similar to that of LDH-1 but greater than that of the other LDH isoenzymes. Statistical analysis of these data demonstrated a significant correlation between malignancy and the appearance of LDH-1 ex isoenzyme (P less than 0.001). In contrast, the relationship between LDH-1 ex isoenzyme and metastasis or anatomical location of the malignancy is not statistically important (P less than 0.1).

Functional and adaptive significance of differentially expressed lactate dehydrogenase isoenzymes in tissues of four obligatory air-breathing Channa species

Biologia ◽  
2009 ◽  
Vol 64 (1) ◽  
Author(s):  
Riaz Ahmad
Keyword(s):  
Lactate Dehydrogenase ◽  
Species Differences ◽  
Adaptive Significance ◽  
Heat Inactivation ◽  
Channa Punctata ◽  
Air Breathing ◽  
The Third ◽  
Ldh Isoenzymes ◽  
General Order ◽  
Lactate Dehydrogenase Isoenzymes

AbstractThis study investigates the differential expression of lactate dehydrogenase (LDH) isoenzymes in the genus Channa using PAGE. With the help of obligate air-breathing, all of the selected species can sustain water deprivation to varying degrees. In subunit composition and higher electrophoretic mobility of LDH-A4, the profiles of channid species were similar to other teleosts documented in the literature. However, inter- and intra-species differences, with particular reference to aerobic/anaerobic metabolic options, existed. Whereas glycolysis in Channa punctata appears to depend largely on aerobic LDH-B and partly on anaerobic LDH-A, metabolism in C. gachua, C. striata and C. marulius depends exclusively on the activity of anaerobic LDH-A. Expression of the third locus Ldh-C was recorded in the eyes of C. marulius, in addition to C. gachua. Heat inactivation experiments reveal species differences between LDH isoenzymes and a general order of the relative stabilities: LDH-C > DH-B > LDH-A. Metabolic and evolutionary implications of the findings have also been discussed.

scholarly journals Subnanogram Determination of Aniracetam in Pharmaceutical Preparations and Biofluids by Flow Injection Analysis with Chemiluminescence Detection Based on Its Enhancement of the Myoglobin-Luminol Reaction

2011 ◽  
Vol 94 (5) ◽  
pp. 1461-1466
Author(s):  
Shao Xiaodong ◽  
Li Ying ◽  
Li Fagen ◽  
Liu Yangqin ◽  
Song Zhenghua
Keyword(s):  
Flow Injection ◽  
Pharmaceutical Preparations ◽  
Serum Samples ◽  
Reaction Conditions ◽  
Whole Process ◽  
Optimum Reaction ◽  
Human Urine And Serum ◽  
Flow Injection Chemiluminescence ◽  
Urine And Serum

Abstract A novel flow injection chemiluminescence method with a myoglobin-luminol system is described for determining aniracetam. Myoglobin-bound aniracetam produced a complex that catalyzed the chemiluminescence reaction between luminol and myoglobin, leading to fast chemiluminescence. The chemiluminescence intensity in the presence of aniracetam was remarkably enhanced compared with that in the absence of aniracetam. Under the optimum reaction conditions the chemiluminescence increment produced was proportional to the concentration of aniracetam in the range of 0.1–1000.0 ng/mL (R2 = 0.9992), with a detection limit of 0.03 ng/mL (3δ). At a flow rate of 2.0 mL/min, the whole process, including sampling and washing, could be completed in 0.5 min, offering a sampling efficiency of 120/h; the RSD was less than 3.0% (n = 5). The method was satisfactory for determination of aniracetam in pharmaceutical preparations and human urine and serum samples. A possible mechanism of the reaction is also discussed.

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