3-Hydroxybutyrate: acetoacetate ratio

The concentration and associated ratio of the ketone bodies, aceto-acetate and 3-hydroxybutyrate, may also be helpful [15, 18, 19]. Ketosis and keto-aciduria are observed in certain patients with a mitochondrial disorder. A non-physiological increase of ketone bodies postprandially may be another indicator of a mitochondrial defect (Saudubray et al). Increased 3-hydroxybutyrate/acetoacetate ratio may suggest a defect in the respiratory chain in liver tissue. 

Moreover, the significance of these assays is limited they provide no information on free coenzyme concentration nor on cell compart-mentation. It is preferable to estimate the concentrations of oxidized and reduced substrates (Hohorst et al., 1959, 1961 Borst, 1963 Krebs, 1967 Krebs et al., 1967 Williamson et al., 1967 Veech and Krebs, 1969 Krebs and Veech, 1969 for example, the lactate pyruvate ratio gives information on the free cytoplasmic NADHiNAD ratio. The 8-hydroxybutyrate acetoacetate ratio corresponds to the mitochondrial NADH NAD ratio. 

In some poorly controlled diabetic patients the high rate of fatty acid oxidation decreases the mitochondrial NADVNADH concentration ratio so that the 3-hydroxybutyrate/acetoacetate concentration ratio can rise to as high as 15 in the blood. Since a test for ketone bodies in the urine (using Clinistix or similar material) detects only acetoacetate this can result in a serious underestimate of the concentration of ketone bodies in the urine. 

Somewhat surprisingly, within the mitochondria the ratio [NAD+]/[NADH] is 100 times lower than in the cytoplasm. Even though mitochondria are the site of oxidation of NADH to NAD+, the intense catabolic activity occurring in the (3 oxidation pathway and the citric acid cycle ensure extremely rapid production of NADH. Furthermore, the reduction state of NAD is apparently buffered by the low potential of the (3-hydroxybutyrate-acetoacetate couple (Chapter 18, Section C,2). Mitochondrial pyridine nucleotides also appear to be at equilibrium with glutamate dehydrogenase.169... 

Long chain acyl-CoA esters inhibit the activity of isolated citrate synthase specifically (Wieland, 1968 Hsu and Powell, 1975 Caggiano and Powell, 1979) but this effect has not been demonstrated with intact mitochondria and its possible involvement in the control of acetyl-CoA utilization for citrate formation in vivo remains uncertain. Similarly an elevation of palmi-toyl-CoA generation at the outside of mitochondrial membrane in vitro increases the relative rates of ketogenesis and p-hydroxybutyrate to acetoacetate ratio, and these events can be rationalized in terms of the known inhibition of mitochondrial adenine nucleotide translocase by long chain acyl-CoA esters (Pande and Blanchaer, 1971 Shug et al., 1971) but whether this inhibition is exerted in intact cells is equivocal (Hansford,... 

With these improved techniques P-hydroxybutyrate, which penetrates mitochondria easily and is oxidized to acetoacetate using NAD+ as H acceptor, gave a P/O ratio of 3, the value equivalent to that from the reoxidation of NADH found by Lehninger. Succinate, which bypassed the NAD+/NADH step, gave a ratio of 2. When cytochrome c-Fe2+ was... 

Alcoholics can also develop ketoaddosis. In alcoholic ketoacidosis, 3-hydroxybutyrate is the major ketone body produced because there is usually a high NADH/NAD ratio in the hver. The urinary nitroprusside test detects only acetoacetate and may dramatically underestimate the extent of ketosis in an alcoholic. 3-Hydroxybutyrate levels (P-hydroxybutyrate) should always be measured in these patients. 

Individuals with either type of diabetes are unable to take up glucose efficiently from the blood recall that insulin triggers the movement of GLUT4 glucose transporters to the plasma membrane of muscle and adipose tissue (see Fig. 12-8). Another characteristic metabolic change in diabetes is excessive but incomplete oxidation of fatty acids in the liver. The acetyl-CoA produced by JS oxidation cannot be completely oxidized by the citric acid cycle, because the high [NADH]/[NAD+] ratio produced by JS oxidation inhibits the cycle (recall that three steps convert NAD+ to NADH). Accumulation of acetyl-CoA leads to overproduction of the ketone bodies acetoacetate and /3-hydroxybutyrate, which cannot be used by extrahepatic tissues as fast as they are made in the liver. In addition to /3-hydroxybutyrate and acetoacetate, the blood of diabetics also contains acetone, which results from the spontaneous decarboxylation of acetoacetate ... 

Synthesis of the ketone bodies HMG CoA is cleaved to produce acetoacetate and acetyl CoA, as shown in Figure 16.23, Acetoacetate can be reduced to form 3-hydroxybutyrate with NADH as the hydrogen donor. Acetoacetate can also spontaneously decarboxylate in the blood to form acetone—a volatile, biologically non-metabolized compound that can be released in the breath. [Note The equilibrium between acetoacetate and 3-hydroxybutyrate is determined by the NADVNADH ratio. Because this ratio is high during fatty acid oxidation, 3-hydroxy-butyrate synthesis is favored.]... 

The glucagon/insulin ratio can rise imder certain pathological conditions (i.e., insulin-dependent diabetes). A small percentage of diabetics develop ketoacidosis, a condition that results from the overproduction and underutilization of ketone bodies. Increased concentrations of P-hydroxybutyrate and acetoacetate, which are acids, can cause a drop in the pH of the blood. This acidification, known as acidosis, can impair the ability of the heart to contract and result in a loss of consciousness and coma, which, in rare cases, may be fatal. Diabetic ketoacidosis may manifest as abdominal pain, nausea, and vomiting. A subject may hyperventilate (breathe quickly and deeply) to correct acidosis, as described vmder Sodium, Potassium, and Water in Chapter 10. It is the responsibility of the clinician, when confronted with a subject whose breath smells of acetone or who is hyperventilating, to facilitate prompt treatment. 

The pathways of formation of ketone bodies are shown in Figure 18-9. The major pathway of production of acetoacetate is from j6-hydroxy-j8-methylglutaryl-CoA (HMG-CoA). Hydrolysis of acetoacetyl-CoA to acetoacetate by acetoacetyl-CoA hydrolase is of minor importance because the enzyme has a high for acetoacetyl-CoA. HMG-CoA is also produced in the cytosol, where it is essential for the synthesis of several isoprenoid compounds and cholesterol (Chapter 19). The reduction of acetoacetyl-CoA to /8-hydroxybutyrate depends on the mitochondrial [NAD+]/[NADH] ratio. 

D-3-Hydroxybutyrate is formed by the reduction of acetoacetate in the mitochondrial matrix by D-3-hydroxybutyrate dehydrogenase. The ratio of hydroxybutyrate to acetoacetate depends on the NADH/NAD ratio inside mitochondria. 

The redox potential is a measure of oxidative phosphorylation maintained by the hepatic mitochondria. Changes of the redox potential are reflected by alterations to the ratio of acetoacetate to 3-hydroxybutyrate (also called the ketone body ratio). This ratio is dependent on mitochondrial 3-hydroxybutyrate dehydrogenase activity, which catalyzes the interconversion of acetoacetate and 3-hydroxybuyrate (Laun et al. 2001 Fukao, Lopaschuk, and Mitchell 2004 Hidaka 2004 Matsumoto et al. 

Acetoacetate can directly enter the blood or it can be reduced by (3-hydroxybu-tyrate dehydrogenase to (3-hydroxybutyrate, which enters the blood (see Fig. 23.18). This dehydrogenase reaction is readily reversible and interconverts these two ketone bodies, which exist in an equilibrium ratio determined by the NADH/NAD ratio of the mitochondrial matrix. Under normal conditions, the ratio of (3-hydroxybutyrate to acetoacetate in the blood is approximately 1 1. 

Fatty acids undergo 3-oxidation, producing acetyl CoA, NADH and FADH2. The NADH and FADH2 are oxidised by the respiratory chain to form ATP which is used for gluconeogenesis (Chapter 34) and for urea synthesis (Chapter 44). The acetyl CoA forms the ketoacids acetoacetate and P-hydroxybutyrate, known as the ketone bodies . Acetone, formed in small amounts from acetoacetate, causes the fruity smell of the breath in ketotic patients or people on low carbohydrate diets (e.g. the Atkins diet ). NB When the ratio of NADH NAD is high, as in diabetic ketoacidosis (DKA), the equilibrium of the P-... 

Ethyl (R)-3-hydroxybutyrate was prepared from the bioreducrion of ethyl acetoacetate using Acetobacter sp. (CCTCC M209061) cells in l-butyl-3-methyl-imidazolium hexafluorophosphate (C4mim PF6), an ionic liquid with good biocompatibility. The reaction was performed with a 1 1 ratio of buffer to C4mim PF6 and a substrate concentration of 55 mmol/L in buffer at pH 5.5. The reaction provided 90.8% conversion and more than 99% ee and was scaled to a 450 mL reaction volume. Also demonstrated in the paper was the successful recycle of the catalyst up to 10 times as an immobilized alginate [60]. 

Fig. 10.1 Metabolites in the urine of an untreated patient with branched-chain keto aciduria (maple syrup urine disease). Extracted using ethyl acetate and separated as their trimethylsilyl-oxime derivatives on a 25 m SE-30 capillary column, using temperature programming from 80°C to 110°C at 0.5°C min and an injection split ratio 1 12 at a temperature of 250°C. The peaks marked R are due to solvent and reagents. Peak identifications are 1, lactic 2, 2-hydroxyisobutyric 3, 2-hydroxybutyric 4, pyruvic 5, 3-hydroxybutyric 6, 2-hydroxyisovaleric 7, 2-oxobutyric 8, 2-methyl-3-hydroxy-isovaleric 10, a and b, 2-oxoisovaleric 11, acetoacetic 12, 2-hydroxyisocaproic 13, 2-hydroxy-3-methyl- -valeric 14, 2-oxo-3-methyl-/i-valeric (14a L- 14b D-) 15, 2-oxoisocaproic acids. The internal standard was malonic acid. (Redrawn with modifications from Jellum etal., 1976)...

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