Lactate, continued oxidation

The two-enzyme system was also used to convert L-lactate into D-lactate with a yield better than 97%. L-Lactate is oxidized by L-lactate dehydrogenase to give pymvate. The keto acid is reduced in an electrochemical system at the cathode to racemic lactate and NADH is oxidized to NAD at the anode. The continuous... 

Figure 16.12. Maintaining Redox Balance. The NADH produced by the glyceraldehyde 3-phosphate dehydrogenase reaction must be reoxidized to NAD+ for the glycolytic pathway to continue. In alcoholic fermentation, alcohol dehydrogenase oxidizes NADH and generates ethanol. In lactic acid fermentation (not shovm), lactate dehydrogenase oxidizes NADH while generating lactic acid.

Cells that lack mitochondria (e.g., erythrocytes) or contain mitochondria but under hypoxic conditions (e.g., vigorously and repeatedly contracting muscle) reduce pyruvate to lactate by lactate dehydrogenase (LDH), which uses NADH as a reductant. This reduction regenerates NAD+, which is required for continued oxidation of glyc-eraldehyde 3-phosphate. 

CK catalyzes the reversible phosphorylation of creatine in the presence of ATP and magnesium. When creatine phosphate is the substrate, the resulting creatine can be measured as the ninhydrin fluorescent compound, as in the continuous flow Auto Analyzer method. Kinetic methods based on coupled enzymatic reactions are also popular. Tanzer and Gilvarg (40) developed a kinetic method using the two exogenous enzymes pyruvate kinase and lactate dehydrogenase to measure the CK rate by following the oxidation of NADH. In this procedure the main reaction is run in a less favorable direction. 

Simultaneous and continuous measurements of extracellular pH, potassium K+, and lactate in an ischemic heart were carried out to study lactic acid production, intracellular acidification, and cellular K+ loss and their quantitative relationships [6, 7], The pH sensor was fabricated on a flexible kapton substrate and the pH sensitive iridium oxide layer was electrodeposited on a planar platinum electrode. Antimony-based pH electrodes have also been used for the measurement of myocardial pH in addition to their application in esophageal acid reflux detection. 

The ADP produced by the hydrolysis of ATP is continuously used up by added purified pyruvate kinase, which in the presence of phosphoenol pyruvate produces pyruvate and ATP (Fig. 3.8.6). Pyruvate is then utilized by added lactate dehydrogenase, which in the presence of NADH produces lactate and NAD+. Complex V activity is estimated from the rate of NADH oxidation at 340 nm (e 4870-M 1-cm 1 isosbestic point 380 nm), after subtracting the oligomycin-resistant activity. It should be kept in mind that oligomicyn sensitivity requires the preserved attachment of the Fr component of the enzyme to the membranous F0 component. The attachment is readily lost upon freeze-thaw cycles. Consequently, it is reasonable to measure the activity on fresh material only. 

After a period of intense muscular activity, the individual continues breathing heavily for some time, using much of the extra 02 for oxidative phosphorylation in the liver. The ATP produced is used for gluconeogenesis from lactate that has been carried in the blood from the muscles. The glucose thus formed returns to the muscles to replenish their glycogen, completing the Cori cycle (Fig. 23-18 see also Box 15-1). 

The product of this metabolic sequence, pyruvate, is a metabolite of caitral importance. Its fate depends upon the conditions within a cell and upon the type of cell. When oxygen is plentiful pyruvate is usually converted to acetyl-coenzyme A, but under anaerobic conditions it may be reduced by NADH + H+ to the alcohol lactic acid (Fig. 10-3, step h). This reduction exactly balances the previous oxidation step, that is, the oxidation of glycer-aldehyde 3-phosphate to 3-phospho-glycerate (steps a and b). With a balanced sequence of an oxidation reaction, followed by a reduction reaction, glucose can be converted to lactate in the absence of oxygen, a fermentation process. The lactic acid fermentation occurs not only in certain bacteria but also in our own muscles under conditions of extremely vigorous exercise. It also occurs continuously in some tissues, e.g., the transparent lens and cornea of the eye. 

This glucose may be released into the bloodstream and returned to the muscles. The overall process is known as the Con cycle. Lactic acid accumulates in muscle after vigorous exercise. It is exported to the liver slowly, but if mild exercise continues the lactate may be largely oxidized within muscle via the tricarboxylic acid cycle. Recent NMR studies have shown that lactic acid is formed rapidly during muscular contraction,... 

This metabolic scheme, which is called lactate fermentation, is shown in Fig. 11-7. The coreactant cycle between the two dehydrogenase enzymes, glyceraldehyde-3-phosphate dehydrogenase (Step 6) and lactate dehydrogenase, ensures that there is regeneration of NAD+ in this particular oxidation state so that glycolysis, lactate fermentation, and the production of ATP can continue. 

As in alcoholic fermentation, there is no net oxidation-reduction. The NADH formed in the oxidation of glyceraldehyde 3-phosphate is consumed in the reduction of pyruvate. The regeneration of NAD + in the reduction ofpyruvate to lactate or ethanol sustains the continued operation of glycolysis under anaerobic conditions. 

Lactate dehydrogenase is closely associated with the final step in fermentative metabolism conversion of pyruvate to lactate- Citrate synthase is closely associated with oxidative metabolism, because this enzyme catalyzes the introduction of acetyl groups into the Krebs cycle. Continued operation of the Krebs cycle is dependent on the continued transfer of electrons from reduced NAD and FAD to the respiratory chain, which catalyzes the reduction of Oi to HjO. Table 4.14 lists lactate dehydrogenasc/citrate synthase activity ratios for muscles that contain a large proportion of white or red muscle fibers. The data demonstrate that the ratio is relatively high in white cell muscles but low in red cell muscles. 

Two moles of lactate are transported to neurons, and metabolized oxidatively to supply ATP for the energy demands of the latter cell [80]. Continuous (4 days) intracerebroventricular lep-tin infusion enhances insulin-stimulated glucose metabolism and favors the expression of the uncoupling proteins UCPl, UCP2 and UCP3 [81]. 

Pyruvate formed in the ALT reaction is reduced to lactate by LD. The substrate, NADH, and the auxiliary enzymes, MD or LD, must be present in sufficient quantity so that the reaction rate is limited only by the amounts of AST and ALT, respectively As the reactions proceed, NADH is oxidized to NAD. The disappearance of NADH is followed by measuring the decrease in absorbance at 340 nm for several minutes, either continuously or at frequent intervals. The change in absorbance per minute (AA/min) is proportional to the micromoles of NADH oxidized and in turn to micromoles of substrate transformed per minute. A preliminary incubation period is necessary to ensure that NADH-dependent... 

As seen in the above, NAD works as the coenzyme in glucose metabolism, therefore, NADH has to be re-oxidized to NAD to support continued glucose oxidation. Anaerobic bacteria such as Clostridia re-oxidizes NADH by producing organic acids like lactate, acetate and butyrate as well as alcohols like ethanol, butanol and butanediol, from pyruvate. Many bacteria have multiple metabolic pathways, and change the pathway in accordance with metabolite availability and/or pH in the culture liquid. Therefore, in general, it is very... 

---