[Effects of ascorbic acid on relaxation of ex vivo Bufo gastrocnemius during sustained isometric contraction].

OBJECTIVE: To investigate the effect of ascorbic acid (VC) on relaxation of ex vivo Bufo gastrocnemius during sustained isometric contraction. METHODS: Dynamic tension of the muscle was recorded under constant voltage stimulation within 7.0 min at 2 s intervals. The rest tension and relaxation rate of the muscle was obtained by weighted fitting to the relaxation process of tension <90% of its peak with a mono-exponential model to characterize the muscular relaxation. RESULTS: VC at 2.0 mmol/L alone or in combination with the inhibitors of the antixoidation enzymes (surperoxide dismutase, glutathione peroxidase and catalase) resulted in negligible alterations in the muscular relaxation kinetics. VC combined with the inhibitor of surperoxide dismutase resulted in significantly lowered relaxation rate while increased rest tension, but VC with the inhibitor of either catalase or glutathione peroxidase showed negligible action. VC combined with the inhibitors of all the 3 enzymes also caused significant effect on the muscular relaxation kinetics, which was similar the effect of VC with superoxide dismutase inhibitor. CONCLUSION: VC at high concentration may result in oxidative toxicity to the biological system rich in transitional metal ion complexes but with low antioxidation capacity by causing superoxide-mediated oxidative damages.

[Rest tension-based characterization of isometric contractility of Bufo gastrocnemius ex vivo].

OBJECTIVE: To characterize the isometric contractility of Bufo gastrocnemius ex vivo in light of the rest tension. METHODS: Bufo gastrocnemius treated with SOD inhibitor and ascorbate was stimulated electrically (12 V DC, 2 ms duration with a 2 s interval) to record the tension within 10 min. Weighted fitting to the relaxation curve of the tension below 90% of the peak tension with a mono-exponential model yielded the rest tension and relaxation rate. RESULTS: The control gastrocnemius showed monotonic decrease of the rest tension, but treatment with SOD inhibitor and ascorbate resulted in a decrease of the rest tension followed by a fast increase within a 1.0 min contraction. The increase of the rest tension at 7.0 min of contraction of the treated muscle was significantly greater than that of the control muscle. The control muscle showed a monotonic decrease of the relaxation rate in 10 min, whereas treatment with SOD inhibitor and ascorbate produced increased relaxation rate followed by monotonic decrease till a plateau was reached. In the course of the 10 min recording, the relaxation rate of the treated muscle was lower than that of the control after the same duration of contraction. CONCLUSION: Rest tension is a characteristic index to represent the skeletal muscle contractility.

Correlation of serum arylesterase activity on phenylacetate estimated by the integrated method to common classical biochemical indexes of liver damage.

The correlation of serum arylesterase (PON1) activity on phenylacetate determined by an integrated method to classical biochemical indexes of liver damage was investigated for the use of PON1 activity to evaluate liver damage. PON1 reaction curve as absorbance at 270 nm for 0.20 mmol/L phenylacetate hydrolysis was analyzed by the integrated method to determine maximal PON1 reaction rate. Classical biochemical indexes of liver damage were determined routinely. The 95% confidence threshold of PON1 activity in sera from healthy individuals was 2.12 mkat/L [(4.73+/-1.31) mkat/L, n=105]. PON1 activity in clinical sera was closely correlated to serum albumin, total protein and the ratio of albumin to globulins, but was weakly correlated to both direct and total bilirubin in serum. There were no correlations of PON1 activity to gamma-glutamyltransferase, alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. Among 127 clinical sera with PON1 activity>2.12 mkat/L, there were 92% healthy individuals examined by albumin, 90% healthy individuals examined by total protein, 88% healthy individuals examined by total bilirubin, 86% healthy individuals examined by direct bilirubin and 64% healthy individuals examined by the ratio of albumin to globulins, respectively. In each group of healthy individuals judged by classical biochemical indexes of close correlation to PON1 activity, percentage of healthy individuals examined by PON1 activity was always >80%. These results suggested PON1 activity on phenylacetate estimated by the integrated method was also suitable for the evaluation of liver damage.

The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables.

The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation ln(S(0)/S)+(S(0)-S)/K(m)=(V(m)/K(m))xt was investigated and compared to that by fitting to (S(0)-S)/t=V(m)-K(m)x[ln(S(0)/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K(m) from 5 to 100 micromol/l was estimated with Eq. (1) while K(m) from 5 to 50 micromol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K(m) with Eq. (2), but did not affect K(m) estimated with Eq. (1). Both equations gave better estimation of V(m). The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K(m) consistent with the Lineweaver-Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K(m) by Eq. (1) linearly increased, while V(m) were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver-Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors.

Kinetic substrate quantification by fitting the enzyme reaction curve to the integrated Michaelis-Menten equation.

The reliability of kinetic substrate quantification by nonlinear fitting of the enzyme reaction curve to the integrated Michaelis-Menten equation was investigated by both simulation and preliminary experimentation. For simulation, product absorptivity epsilon was 3.00 mmol(-1) L cm(-1) and K(m) was 0.10 mmol L(-1), and uniform absorbance error sigma was randomly inserted into the error-free reaction curve of product absorbance A(i) versus reaction time t(i) calculated according to the integrated Michaelis-Menten equation. The experimental reaction curve of arylesterase acting on phenyl acetate was monitored by phenol absorbance at 270 nm. Maximal product absorbance A(m) was predicted by nonlinear fitting of the reaction curve to Eq. (1) with K(m) as constant. There were unique A(m) for best fitting of both the simulated and experimental reaction curves. Neither the error in reaction origin nor the variation of enzyme activity changed the background-corrected value of A(m). But the range of data under analysis, the background absorbance, and absorbance error sigma had an effect. By simulation, A(m) from 0.150 to 3.600 was predicted with reliability and linear response to substrate concentration when there was 80% consumption of substrate at sigma of 0.001. Restriction of absorbance to 0.700 enabled A(m) up to 1.800 to be predicted at sigma of 0.001. Detection limit reached A(m) of 0.090 at sigma of 0.001. By experimentation, the reproducibility was 4.6% at substrate concentration twice the K(m), and A(m) linearly responded to phenyl acetate with consistent absorptivity for phenol, and upper limit about twice the maximum of experimental absorbance. These results supported the reliability of this new kinetic method for enzymatic analysis with enhanced upper limit and precision.