NADH depletion

In dehydrogenase-type enzyme-catalyzed reactions, the NAD" " as a cofactor is required for the enzymatic reaction. With substrate oxidation, NAD" " is simultaneously reduced to NADH. NADH contains a tertiary amine and can be acted as the co-reactant of Ru(bpy)3 (Fig. 16A). The ECL intensity is increased in proportion to the concentration of the substrates. However, for the NADH-depleting enzyme, such as the determination of pyruvate using malate dehydrogenase, the determinations... 

In the continuous, substrate-regenerating assay, Dnml hydrolyzes GTP into GDP and inorganic phosphate. As shown in Fig. 1, GTP is regenerated from GDP and phospho(enol)pyruvate (PEP) by pyruvate kinase. The pyruvate produced by this regenerative reaction is reduced to lactate by the enzyme lactate dehydrogenase, using NADH as a cosubstrate. NADH depletion is measured by continuously monitoring a decrease in absorbance at 340 nm over time and is directly proportional to GTP hydrolysis. 

This mechanism is now considered to be of importance for the protection of LDL against oxidation stress, Chapter 25.) The antioxidant effect of ubiquinones on lipid peroxidation was first shown in 1980 [237]. In 1987 Solaini et al. [238] showed that the depletion of beef heart mitochondria from ubiquinone enhanced the iron adriamycin-initiated lipid peroxidation whereas the reincorporation of ubiquinone in mitochondria depressed lipid peroxidation. It was concluded that ubiquinone is able to protect mitochondria against the prooxidant effect of adriamycin. Inhibition of in vitro and in vivo liposomal, microsomal, and mitochondrial lipid peroxidation has also been shown in studies by Beyer [239] and Frei et al. [240]. Later on, it was suggested that ubihydroquinones inhibit lipid peroxidation only in cooperation with vitamin E [241]. However, simultaneous presence of ubihydroquinone and vitamin E apparently is not always necessary [242], although the synergistic interaction of these antioxidants may take place (see below). It has been shown that the enzymatic reduction of ubiquinones to ubihydroquinones is catalyzed by NADH-dependent plasma membrane reductase and NADPH-dependent cytosolic ubiquinone reductase [243,244]. 

Streptozocin (Zanosar), a water-soluble nitrosourea produced by the fungus Streptomyces achromogenes, acts through methylation of nucleic acids and proteins. In addition, it produces rapid and severe depletion of the pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and its reduced form (NADH) in liver and pancreatic islets. 

The outcome of oxidative stress is a depletion of cellular GSH, NADPH, NADH, and ATP, and also damage to lipid membranes, structural and enzymatic proteins, and DNA. 

Depletion of ATP is caused by many toxic compounds, and this will result in a variety of biochemical changes. Although there are many ways for toxic compounds to cause a depletion of ATP in the cell, interference with mitochondrial oxidative phosphorylation is perhaps the most common. Thus, compounds, such as 2,4-dinitrophenol, which uncouple the production of ATP from the electron transport chain, will cause such an effect, but will also cause inhibition of electron transport or depletion of NADH. Excessive use of ATP or sequestration are other mechanisms, the latter being more fully described in relation to ethionine toxicity in chapter 7. Also, DNA damage, which causes the activation of poly(ADP-ribose) polymerase (PARP), may lead to ATP depletion (see below). A lack of ATP in the cell means that active transport into, out of, and within the cell is compromised or halted, with the result that the concentration of ions such as Na+, K+, and Ca2+ in particular compartments will change. Also, various synthetic biochemical processes such as protein synthesis, gluconeogenesis, and lipid synthesis will tend to be decreased. At the tissue level, this may mean that hepatocytes do not produce bile efficiently and proximal tubules do not actively reabsorb essential amino acids and glucose. 

Depletion of other cofactors such as UTP, NADH, and NADPH may also be involved in cell injury either directly or indirectly. Thus, the role of NADPH in maintaining reduced GSH levels means that excessive GSH oxidation such as caused by certain quinines, which undergo redox cycling, may in turn cause NADPH depletion (see below). Alternatively, NADPH may be oxidized if it donates electrons to the foreign compound directly. However, NADPH may be regenerated by inter conversion of NAD+ to NADP+. Some quinones such as menadione, l,2-dibromo-3-chloropropane (DBCP), and hydrogen peroxide also cause depletion of NAD, but probably by different mechanisms. Thus, with menadione, the depletion may be the result of... 

Increased synthesis of lipid or uptake. Increased synthesis of lipid may be the cause of fatty liver after hydrazine administration as this compound increases the activity of the enzyme involved in the synthesis of diglycerides. Hydrazine also depletes ATP and, however, inhibits protein synthesis. Large doses of ethanol will cause fatty liver in humans, and it is believed that this is partly due to an increase in fatty acid synthesis. This is a result of an increase in the NADH/NAD"1" ratio and therefore of the synthesis of triglycerides. Changes in the mobilization of lipids in tissues followed by uptake into the liver can also be another cause of steatosis. 

Figure 7.44 The metabolism and toxicity of MPTP. Diffusion into the brain is followed by metabolism in the astrocyte. The metabolite MPP+ is actively transported into the dopaminergic neuron by DAT. It is accumulated there and is actively taken into mitochondria by another uptake system. Here, it inhibits mitochondrial electron transport between NADH dehydrogenase (NADH DHase) and coenzyme Q (Q10). Consequently, it blocks the electron transport system, depletes ATP, and destroys the neuron. Abbreviations MPTP, 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine DAT, dopamine transporter uptake system.

When the rate of formation of ketone bodies is greater than the rate of their use, their levels begin to rise in the blood (ketonemia) and eventually in the urine (ketonuria). These two conditions are seen most often in cases of uncontrolled, type 1 (insulin-dependent) diabetes mellitus. In such individuals, high fatty acid degradation produces excessive amounts of acetyl CoA. It also depletes the NAD+ pool and increases the NADH pool, which slows the TCA cycle (see p. 112). This forces the excess acetyl CoA into the ketone body pathway. In diabetic individuals with severe ketosis, urinary excre-... 

Some catabolic reactions depend upon ADP, but under most conditions its concentration is very low because it is nearly all phosphorylated to ATP. Reactions utilizing ADP may then become the rate-limiting pacemakers in reaction sequences. Depletion of a reactant sometimes has the effect of changing the whole pattern of metabolism. Thus, if oxygen is unavailable to a yeast, the reduced coenzyme NADH accumulates and reduces pyruvate to ethanol plus C02 (Fig. 10-3). The result is a shift from oxidative metabolism to fermentation. 

Mitchell and Moyle calculated that mitochondria depleted of ADP build up a pH gradient of about 0.05 pH units across the inner membrane. More recent measurements indicate that approximately 10 protons are pumped out for each pair of electrons moving down the respiratory chain from NADH to 02, and approximately 6 protons for a pair of electrons coming from succinate. 

Answer Oxygen is the terminal electron acceptor in oxidative phosphorylation, and thus is needed to recycle NAD+ from NADH. NADH is produced in greatest quantities by the oxidative reactions of the citric acid cycle. In the absence of 02, the supply of NAD+ is depleted, and the accumulated NADH allosterically inhibits pyruvate dehydrogenase and cc-ketoglutarate... 

As seen in Figure 8-4, disruption in the flow of electrons through the ETC can lead to an increase in NADH depending on the location of the block in electron transport. The increase in NADH will shut down the TCA cycle via specific inhibition of key TCA enzymes by NADH. The buildup of NADH will thus result in depletion of NAD+ stores. How are the NAD+ levels restored The NADH is used to reduce pyruvate to lactate, as indicated in the following diagram ... 

The depletion of NAD+ (and the change to the NADH to NAD ratio) slows the TCA cycle, resulting in a build-up of pyruvate and acetyl-CoA. Excess acetyl-CoA increases fatty acid synthesis and fat deposits in the liver (fatty hver). An accumulation of fat in the liver can be observed after just a single night of heavy drinking. 

Alcohol intake depletes the cellular supply of NAD (because of the alcohol and acetaldehyde dehydrogenase reactions) and consequently the NAD NADH ratio falls. Reactions that depend on NAD+ will thus be curtailed ... 

LDH enzyme electrodes follow electrochemically NADH consumption or NAD" " formation (211). Electrochemically activated microcarbon electrodes can be utilized in conjunction with immobilized LDH to determine pyruvate in small volumes (50 pL) of cerebrospinal fluid within the concentration range 10 pM to 2 mM (234). The construction of pymvate oxidase electrodes operating either at positive potentials (H2O2 detection) or at negative potentials (oxygen depletion) (92) is described by... 

A. When oxygen is depleted, the electron transport chain stops, NADH builds up, and the TCA cycle is inhibited. Therefore succinate, an intermediate of the cycle, will not be oxidized. In the absence of oxygen, no ATP is produced. 

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