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False-positive postmortem EMIT drugs-of-abuse assay due to lactate dehydrogenase and lactate in urine.

Three cases of multiple false-positive drug tests are described. Postmortem urine specimens were screened using the enzyme-multiplied immunoassay technique. All patients had proteinuria and lactic aciduria. These false-positive reactions were due to the presence of lactate dehydrogenase (LDH), lactic acid, and protein. This finding was confirmed by creating a multiple false-positive sample with a solution of LDH and lactate in 5% bovine serum albumin at pH 6.

Adult↗

A search for the best buffer to use in assaying human lactate dehydrogenase with the lactate-to-pyruvate reaction.

Highly purified human lactate dehydrogenases I and V were assayed in 17 different buffers, at a variety of reaction pH's. Diethanolamine and 2-amino-2-methyl-1,3-propanediol provided the best measurements of the enzyme, assayed lactate-to-pyruvate. However, the commercial preparation of 2-amino-2-methyl-1,3-propanediol contained insoluble matter and was relatively expensive. All of the four buffers nowmost commonly used were found to present difficulties. Glycine and pyrophosphate were inhibotory tolactate dehydrogenase activity with increasing buffer concentration. 2-Amino-2-methyl-1-propanol had three major disadvantages: it is chemically unstable during reagent preparation; activity is dependent on buffer concentration; and the pH optima for isoenzymes I and V are vastly different. The pKa of tris(hydroxymethyl)aminomethane is 8.0 at 30 degrees C, whereas to measure total activity the reaction pH should be greater than 8.5; thus tris(hydroxymethyl)aminomethane has limited buffering capacity at the reaction pH.

Buffers↗

The effects of sprint (300 m) running on plasma lactate, uric acid, creatine kinase and lactate dehydrogenase in competitive hurdlers and untrained men.

BACKGROUND: High intensity exercise may induce muscle damage especially in individuals unaccustomed to regular physical efforts. Our aim was to compare the impact of 300 m sprint running on muscle enzymes release into blood in competitive hurdlers and untrained but physically fit adults. METHODS EXPERIMENTAL DESIGN: Comparative study. SETTING: General community. PARTICIPANTS: Nine competitive hurdlers (CH; 21.3 yrs+/-3.1) and six matched moderately active untrained men (UT). INTERVENTIONS: Subjects performed a single 300 m sprint running test. MEASURES: Plasma creatine kinase (CK) and lactate dehydrogenase (LD) activities, and concentrations of lactate (LA) and uric acid (UA) measured before the warm-up and 5 min, 2 and 20 hrs postrun. RESULTS: Mean runner performance achieved by CH was significantly better (35.53-s+/-0.64) and LA concentrations significantly higher (14.95 mM+/-0.59) than those recorded in UT (41.52-s+/-2.22; 10.13 mM+/-0.59). Pre- and postrun CK activities until the 2nd hour of recovery were found to be significantly higher (p<0.05) in CH than in UT, whereas LD activities were similar in both groups pretest, but significantly higher in UT immediately postrun. In CH, the highest CK activity (32 percent increase) was recorded immediately post-test, whereas in UT the peak CK (135 percent increase) was attained in the 20th hour of recovery. In both groups UA reached the peak level in the 2nd hour of recovery. CONCLUSIONS: More pronounced and delayed postrun increase in CK, as recorded in UT, may provide evidence of a larger exercise-induced muscle injury in the untrained adults. Higher postexercise UA levels as recorded in CH might account for a more marked involvement of adenylate kinase reaction in ATP resynthesis.

Adult↗

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate at 25, 30, and 37 degrees C.

Optimal reaction conditions for assaying human lactate dehydrogenase pyruvate-to-lactate were determined for isoenzymes 1 and 5 at 25, 30, and 37 degrees C. Three of the nine different buffers examined--imidazole, triethanolamine, and N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid--are satisfactory. Beta-NADH, pyruvate, and hydrogen ion concentrations were chosen to measure both isoenzymes with maximal-equal-sustainable efficiency at the lowest substrate concentrations. Approximately 95% of each isoenzyme is measured, for activities up to threefold the upper normal limit, if the measurements are made immediately after the reaction is initiated. The Arrhenius relationship for each isoenzyme is unique. Interconversion of results from one temperature to another is practical only with reservations. Results at 37 degrees C are not as reliable as those at 25 degrees C.

Buffers↗

Clearance of lactate dehydrogenase by SJL/J mice infected with lactate dehydrogenase-elevating virus.

The plasma level of lactate dehydrogenase (LDH) activity rises to about ten times the normal level by 4 days after infection of mice with lactate-dehydrogenase elevating virus (LDV). The levels of seven other enzymes are also increased, but to a lesser degree. SJL/J mice demonstrate a unique, genetically controlled 20-fold increase in the plasma level of LDH enzyme after LDV infection, as well as enhanced levels of the other plasma enzymes elevated by LDV infection. Comparison of virus infection in SJL/J and Swiss mice as well as in cultures of peritoneal exudate cells made from them indicated that the time course and extent of virus replication was similar for the two strains of mice. The rate of clearance of intravenously injected rabbit or mouse LDH was found to be impaired to a similar extent in LDV-infected SJL/J and Swiss mice. The effect of LDV infection on the levels of endogenous LDH released as a result of injection of carbon tetrachloride or tumor growth was also similar in the two strains of mice. These results suggest that LDV infection may specifically induce a greater influx of LDH into the plasma of SJL/J mice from an as-yet-unknown source than in other strains of mice.

Animals↗

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.

Coloring Agents↗

Possible effect of lactational period on the milk-to-plasma drug concentration ratio in lactating women: results of an in vitro evaluation.

The fat and protein composition of human milk changes dramatically in the first several weeks postpartum. In order to investigate the possible effect of this compositional change on the milk-to-plasma drug concentration ratio (M/P), the following experiment was performed. Milk samples were collected from five healthy lactating women on days 3, 5, 7, and 14 postpartum; blood samples were obtained on these days, as well as on day 1. Serum and skim milk unbound fractions (fp and fm, respectively) and the skim milk-to-whole milk drug concentration ratio (S/M) were determined in vitro in the above samples for diazepam, propranolol, and etretin, an aromatic retinoid. In addition, the composition of these milk and serum samples was also assessed. Using a previously proposed mathematical model for the distribution of drugs between milk and plasma, M/P was calculated from values for fm, fp, S/M, milk pH, and literature values for the pKa values of the compounds. The M/P was calculated for each subject on each day of sample collection. Total serum protein and alpha-1-acid glycoprotein (AAG) levels increased in the first two weeks postpartum. Mean diazepam fp values fell from 0.020 to 0.014 during this period, while propranolol fp values changed inversely with serum AAG levels. Milk whey and total proteins decreased as lactation progressed, but changes in fat levels were not statistically significant. Calculated propranolol M/P increased during the study period, predominately due to changes in milk pH and propranolol S/M.(ABSTRACT TRUNCATED AT 250 WORDS)

Acitretin↗

A sensitive equilibrium-based assay for D-lactate using D-lactate dehydrogenase: application to penicillin-binding protein/DD-carboxypeptidase activity assays.

An assay for D-lactate (D-Lac) is described where D-Lac in the presence of NAD+ is equilibrated to NADH and pyruvate (Pyr) by Leuconostoc mesenteroides D-lactate dehydrogenase (DLDH). This assay was standardized using known concentrations of D-Lac and a linearized form of the equilibrium expression. The assay has a lower limit of about 20 microM D-Lac in a 1 ml assay mixture (20 nmol D-Lac). As a demonstration of this assay method it is used to characterize the hydrolysis of the standard penicillin-binding protein/DD-carboxypeptidase substrate Ac2-L-Lys-D-Ala-D-Lac by penicillin-binding protein 5 from Escherichia coli. The approach adopted here of using an inherently nonlinear response, which however follows precisely determinable physical behavior, has the advantages of providing a wider dynamic range and increased relative precision over analogous linear response-based methods. This approach may be applicable to the development of other enzyme-based assay methods.

Amino Acid Sequence↗

Co-ordinate regulation of lactate metabolism genes in yeast: the role of the lactate permease gene JEN1.

In the yeast Saccharomyces cerevisiae, the first step in lactate metabolism is its transport across the plasma membrane, a proton symport process mediated by the product of the gene JEN1. Under aerobic conditions, the expression of JEN1 is regulated by the carbon source: the gene is repressed by glucose and induced by non-fermentable substrates. JEN1 expression is also controlled by oxygen availability, but is unaffected by the absence of haem biosynthesis. JEN1 is negatively regulated by the repressors Mig1p and Mig2p, and requires Cat8p for full derepression. In this report we demonstrate that, in addition to these regulators, the Hap2/3/4/5 complex interacts specifically with a CAAT-box element in the JEN1 promoter, and acts to derepress JEN1 expression. We also provide evidence for transcriptional stimulation of JEN1 by the protein kinase Snf1p. Data are presented which provide a better understanding of the molecular mechanisms implicated in the co-regulation of genes involved in the metabolism of lactate.

Aerobiosis↗

Affinity precipitation of lactate dehydrogenase with a triazine dye derivative: selective precipitation of rabbit muscle lactate dehydrogenase with a procion blue H-B analog.

A simple methoxylated derivative of the triazine dye, Procion blue H-B, selectively precipitates rabbit muscle lactate dehydrogenase from solution. Optimum protein precipitation occurred at an enzyme subunit:dye ratio of approximately 2:1 and was fully reversible upon addition of competitive ligands such as NADH. With a crude extract of rabbit muscle, affinity precipitation with the dye followed by dissolution with NADH yielded homogeneous lactate dehydrogenase in 97% overall yield.

Animals↗

H4-isozyme of lactate dehydrogenase in a solution of sodium chloride-I. The molecular weight of H4-lactate dehydrogenase in relation to sodium chloride concentration, enzyme concentration and temperature.

1. The behavior of H4-lactate dehydrogenase in a solution of sodium chloride was studied with respect to its molecular weight. The molecular weight decreased as the concentration of sodium chloride increased. 2. On a controlled-pore glass column equilibrated by 0.5 M soldium chloride, H4-lactate dehydrogenase was found to have mol. wt 77,500. 3. In a low salt column (0.1 M sodium chloride or less), on the other hand, the molecular weight was found to be about 135,000. 4. Salt concentration dependent association-dissociation system of the enzyme was also observed by the sedimentation equilibrium method.

Animals↗

Pituitary response to thyrotropin, corticotropin, and gonadotropin-releasing hormones in lactating cows treated with sometribove for a fourth consecutive lactation.

The effect of chronic treatment with recombinant methionyl bovine somatotropin (USAN, sometribove) on anterior pituitary secretions and its target organs was investigated in six control and six sometribove-treated British Friesian cows. Cows averaged 112 and 119 d postpartum in their fourth lactation of treatment and, except for one control, had active corpora lutea. During each lactation, treated cows received sometribove injections (500 mg) every 2 wk (injection cycle) starting 60 +/- 3 d postpartum. On Day 9 of one injection cycle, blood was sampled for 390 min, starting 30 min before an intravenous injection of thyrotropin (TRH, 0.33 microgram/kg), corticotropin (100 microgram), and gonadotropin (GnRH, 200 micrograms)-releasing hormones. Baseline somatotropin (bST) and adrenocorticotropin (ACTH) were higher in sometribove-treated cows vs. controls (3.27 vs. 1.03 ng/ml and 35.24 vs. 19.28 pg/ml, respectively). Baseline total thyroxine, free thyroxine, triiodothyronine, prolactin, follicle stimulating and luteinizing hormones, estradiol, and progesterone (P4) were similar across treatments. Circulating cortisol levels did not differ between control and sometribove cows, indicating a reduced adrenal ACTH responsiveness in the latter. Releasing factors induced similar changes across treatments in hormones studied with the following exceptions: a bST spike was seen in control cows only, cortisol response to ACTH was reduced in treated cows, and a significantly higher P4 concentration was detected in the plasma of sometribove-treated cows, suggesting increased ovarian responsiveness to GnRH-stimulated P4 output. The study demonstrated reduced bST response to TRH, consistent with physiologic feedback mechanisms, whereas the release profiles of the other pituitary hormones were unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗

Selective inhibitors of human lactate dehydrogenases and lactate dehydrogenase from the malarial parasite Plasmodium falciparum.

Derivatives of the sesquiterpene 8-deoxyhemigossylic acid (2, 3-dihydroxy-6-methyl-4-(1-methylethyl)-1-naphthoic acid) were synthesized that contained altered alkyl groups in the 4-position and contained alkyl or aralkyl groups in the 7-position. These substituted dihydroxynaphthoic acids are selective inhibitors of human lactate dehydrogenase-H (LDH-H) and LDH-M and of lactate dehydrogenase from the malarial parasite Plasmodium falciparum (pLDH). All inhibitors are competitive with the binding of NADH. Selectivity for LDH-H, LDH-M, or pLDH is strongly dependent upon the groups that are in the 4- and 7-positions of the dihydroxynaphthoic acid backbone. Dissociation constants as low as 50 nM were observed, with selectivity as high as 400-fold.

Animals↗

Organ specificity and lactate-dehydrogenase activity. Some properties of human spermatozoal lactate dehydrogenase.

1. The presence of a characteristic lactate-dehydrogenase isoenzyme (LD(x)) in human, mouse and dog testis and in human spermatozoa has been confirmed by electrophoresis on cellulose acetate and on polyacrylamide gel. 2. The human spermatozoal isoenzyme exhibits a much higher affinity for 2-oxobutyrate than any of the five isoenzymes found in other tissues. K(m) values of 0.05mm for pyruvate and 0.18mm for 2-oxobutyrate were obtained. 3. LD(x) differs from other lactate-dehydrogenase isoenzymes in that its properties cannot be correlated with its electrophoretic mobility. It resembles LD(1) in being strongly inhibited by 0.2mm-oxalate and relatively resistant to 2m-urea, and in being relatively stable to heat. 4. The surprisingly high activity of LD(x) with 2-oxobutyrate suggests that this substance or 2-hydroxybutyrate may play a part in spermatozoal metabolism.

Animals↗

Reliability and validity of a continuous incremental treadmill protocol for the determination of lactate threshold, fixed blood lactate concentrations, and VO2max.

Fifteen male runners were tested on two occasions to determine the reliability of a continuous incremental level running treadmill protocol (C), with 3-min stages, for lactate threshold (LT) and fixed blood lactate concentration (FBLC) (2.0 mM, 2.5 mM, 4.0 mM) assessment. Test-retest (T-RT) reliability coefficients for velocity at LT, 2.0 mM, 2.5 mM, and 4.0 mM were r = 0.89, 0.91, 0.95, and 0.95, respectively (velocity ranged from 215.3 m/min at LT to 273.6 m/min at 4.0 mM). Mean differences in T-RT velocity values ranged from 0.7 m/min (at LT) to 6.0 m/min (at 2.5 mM; 252.3 m/min vs 258.3 m/min; NS) and the standard errors of measurement were less than +/- 10.0 m/min. Similar results were observed for VO2, with T-RT r values ranging from r = 0.82 (at LT) to r = 0.88 (at 2.0 mM) (VO2 ranged from 47.2 ml/kg.min-1 at LT to 60.9 ml/kg.min-1 at 4.0 mM; VO2 peak = 65.6 ml/kg.min-1). Mean differences in T-RT VO2 values were less than 1.4 ml/kg.min-1 (NS) and the standard errors of measurement were less than +/- 2.95 ml/kg.min-1. Interinvestigator and intrainvestigator reliability coefficients were high and ranged from r = 0.91 to r = 0.99. Validity of C was assessed in an additional 16 male runners who completed C and a criterion discontinuous (D) protocol (using a series of 10-min stages) for LT and FBLC assessment. Results indicated that C and D resulted in similar VO2 and velocity values at LT and FBLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Prediction of lactate threshold (LT) and fixed blood lactate concentrations (FBLC) from 3200-m running performance in women.

The present study evaluated the accuracy of predicting velocity and oxygen consumption (VO2) at the LT lactate threshold and FBLC fixed blood lactate concentrations from a 3200-m time trial in women. Forty-four women (mean age = 31.1 yrs, mean ht = 164.9 cm, mean wt = 65.0 kg) completed a treadmill protocol for the determination of LT and FBLC and a 3200-m time trial. Velocity and VO2 values at LT, FBLC of 2.0 2.5, and 4.0 mM, and peak were determined. Mean VO2 and velocity ranged from 27.8 +/- 10.8 ml/kg.min-1 at LT to 42.5 ml/kg.min-1 at peak and from 129.8 +/- 44.0 m.min-1 at LT to 187.0 +/- 52.4 m.min-1 at peak, respectively. Results indicated that a 3200-m time trial (mean time = 20.6 +/- 6.6 min) was a good predictor of VO2 and velocity at LT, FBLC, and peak. Correlation coefficients (using a quadratic model) for velocity ranged from R = 0.96 to R = 0.98 with SEE ranging from 9.0 to 13.1 m.min-1. Correlation coefficients for VO2 ranged from R = 0.94 to R = 0.96 with SEE ranging from 2.8 to 3.6 ml/kg.min. The validity of these regression equations was examined in 13 women who completed a 12-month running program (VO2 LT, VO2 at FBLC of 2.0, 2.5 and 4.0 mM, and VO2 peak increased by 34.7, 19.9, 16.9, 11.9, and 5.4%, respectively, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Prediction of lactate threshold and fixed blood lactate concentrations from 3200-m time trial running performance in untrained females.

The present study examined the effectiveness of a 3200-m time trial run for predicting VO2 and running velocity at lactate threshold (LT), and fixed blood lactate concentrations (FBLC) of 2.0, 2.5, and 4.0 mM and peak in untrained women. Thirty-nine female subjects completed a VO2peak/LT test a 3200-m time trial run. Twenty-eight subjects were randomly assigned to a validation sample and the remaining subjects were used for cross-validation purposes. In the validation sample, VO2 measurements at LT, FBLC of 2.0, 2.5, 4.0 mM, and peak were 22.5, 29.2, 31.2, 36.5, and 38.5 ml/kg.min-1, respectively. Velocities at LT, FBLC of 2.0, 2.5, 4.0 mM, and peak were 107.1, 129.7, 136.6, 155.1, and 163.2 m/min, respectively. Regression analysis in the validation group revealed that the 3200-m time trial was an accurate predictor of velocities at LT, FBLC of 2.0, 2.5, 4.0 mM, and peak with correlations of r = 0.70, r = 0.84, r = 0.85, r = 0.87, and r = 0.95, respectively, and standard errors of estimate ranging from +/- 9.5 m/min (for velocity peak) to +/- 13.7 m/min (velocity LT). Vor VO2 prediction, correlations ranged from r = 0.61 (3200-m time vs VO2 LT) to r = 0.77 (3200-m time vs VO2 peak) with the standard errors of estimate ranging from +/- 4.18 (VO2 2.0 mM) to +/- 4.87 ml/kg.min-1 (VO2 4.0 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Studies in human lactation: milk composition and daily secretion rates of macronutrients in the first year of lactation.

Time-dependent changes in milk composition and secretion from pregnancy through greater than or equal to 6 mo of exclusive breast-feeding were studied in 13 multiparous women. Concentrations and secretion rates of lipid, lactose, protein, sodium, chloride, potassium, total calcium, ionized calcium, magnesium, glucose, citrate, inorganic phosphate, creatinine, and urea and pH were analyzed longitudinally from day 6 until weaning commenced. The composition of the antepartum secretion was related to the permeability of the junctional complexes between mammary cells. Significant increases in lactose, glucose, pH, and ionized calcium and significant decreases in protein, sodium, potassium, chloride, and calcium concentrations were observed between 1 and 6 mo. Significant differences among individuals, which persisted through lactation, were observed for the concentrations of sodium, potassium, chloride, and inorganic phosphate. The amount of lactose, ionized calcium, and magnesium transferred to the infant was also characteristic of each mother-infant pair. Our data imply that lactation performance is determined in the first month postpartum.

Breast Feeding↗