Cytoplasm NADPH

How is the cytoplasmic [NADPH]/[NADP+] ratio maintained at a value higher than that of [NADH]/ [NAD+] Part of the answer is from operation of the pentose phosphate pathway (Section E,3). The reactions of Eq. 17-12, if they attained equilibrium, would give a ratio of cytosolic [NADPH]/[NADP+] > 2000 at 0.05 atm C02. Compare this with the ratio 1 /634 for [NADH/[NAD+] deduced from the observation on the reactions of Eq. 17-42. 

Domanska-Janik K, Wideman J. Regulation of thiols in the brain. 2. Effect of hypoxia on the activities of cytoplasmic NADPH-producing enzymes in different parts of the rat brain. Resuscitation 1974 3 37-41. 

The increase in the cytoplasmic NADPH may be due to lower utilization (Veech et al., 1969), as it is well established that fatty acid synthesis is early inhibited in this nutritional state, presumably because of the lack of ATP (Rous et al., 1967). 

In addition to moving acetyl-CoA from the mitochondria to the cytoplasm, this cycle also converts an NADH to an NADPH. If we assume that the amount of ATP that we could get from NADH and NADPH oxidation is the same, making NADPH from NADH and NADP+ doesn t cost any energy. So we can conclude that the cost of just moving the acetyl-CoA out of the mitochondria is 2 ATPs per acetyl-CoA. 

The second major breakthrough in understanding the defect in CGD neutrophils came through the development of assays in which the NADPH oxidase can be activated in a cell-free system in vitro ( 5.3.2.3). In these systems, activation of the oxidase can be achieved by the addition of cytoplasm to plasma membranes in the presence of NADPH and arachidonic acid (or SDS or related substances). Interestingly, the oxidase cannot be activated in these cell-free systems using extracts from CGD neutrophils however, cytosol and plasma membranes from normal and CGD neutrophils may be mixed, and in most cases activity is restored if the correct mixing pattern is used. For example, as may be predicted, in X-linked CGD it is the membranes that are defective (because the cytochrome b is deficient), whereas in autosomal recessive CGD the cytosol is defective in the cell-free system. 

Cholesterol is required for membrane synthesis, steroid synthesis, and in the liver, bile acid synthesis. Most cells derive their cholesterol from LDL or HDL, but some cholesterol may be synthesized de novo. Most de novo synthesis occurs in the liver, vfhere cholesterol is synthesized from acetyl CoA in the cytoplasm. The citrate shutde carries mitochondrial acetyl CoA into the cytoplasm, and NADPH is provided by the HMP shunt and malic enzyme. Important points are noted in Figure 1-15-9,... 

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

The pentose phosphate pathway (PPP, also known as the hexose monophosphate pathway) is an oxidative metabolic pathway located in the cytoplasm, which, like glycolysis, starts from glucose 6-phosphate. It supplies two important precursors for anabolic pathways NADPH+H+, which is required for the biosynthesis of fatty acids and isopren-oids, for example (see p. 168), and ribose 5-phosphate, a precursor in nucleotide biosynthesis (see p. 188). 

In the vertebrates, biosynthesis of fatty acids is catalyzed by fatty add synthase, a multifunctional enzyme. Located in the cytoplasm, the enzyme requires acetyl CoA as a starter molecule. In a cyclic reaction, the acetyl residue is elongated by one C2 unit at a time for seven cycles. NADPH+H is used as a reducing agent in the process. The end product of the reaction is the saturated Cie acid, palmitic acid. 

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