Liver, acetoacetic acid formation

During prolonged starvation or when carbohydrate metabolism is severely impaired, as in uncontrolled diabetes mel-iitus (see Chapter 25), the formation of acetyl-CoA exceeds the supply of oxaioacetate. The abundance of acetyl-CoA results from excessive mobilization of fatty acids from adipose tissue and excessive degradation of the fatty acids by p-oxidation in the liver. The resulting acetyl-CoA excess is diverted to an alternative pathway in the mitochondria and forms acetoacetic acid, P-hydroxybutyric acid, and acetone—three compounds known collectively as ketone bodies (Figure 26-9). The presence of ketone bodies is a frequent finding in severe, uncontrolled diabetes melUtus. 

A variety of thiokinases probably exist, but only a few of them have been identified. Acetic acid and butyryl thiokinase have been purified from a variety of sources, including yeast, liver, and muscle. These two enzymes differ in their specificity for the substrate. Acetic thiokinase catalyzes only the oxidation of propionic, acetic, and acrylic acids, but butyryl thiokinase activates fatty acids of chain lengths ranging from 4-to 12-carbon units. A third thiokinase was also discovered. It acts on fatty acid chains with 5- to 22-carbon units and is found in the microsomes. This intracellular distribution is in striking contrast with the cellular location of all other enzymes involved in fatty acid oxidation, which are all in mitochondria. The palmityl enzyme, which is active in the presence of ATP and CoA, becomes inactive when incubated in the absence of CoA therefore, it has been proposed that the active form of the enzyme involves the formation of an enzyme-CoA complex. The heart, the skeletal muscle, and the kidney also contain a thiokinase that specifically activates acetoacetic acid. Acetoacetic acid thiokinase is absent in liver this observation is significant in the pathogenesis of ketosis. 

Dihydroxyphenylpyruvate was not oxidized to homogentisate, thus it cannot be an intermediate in the oxidation of p-hydroxyphenyl-pyruvate. The results suggest that the hydroxylation shift of the side chain and decarboxylation of the p-hydroxyphenylpyruvate are simultaneous processes. Additional evidence that 2,5-dihydroxyphenylpyruvate is not the intermediate was obtained by experiments in which the relative rates of oxidation of this compound and of p-hydroxyphenylpyruvate were compared in homogenates of rat liver, where the reaction proceeded to formation of acetoacetic acid. Oxidation of p-hydroxyphenylpyruvate proceeded much more rapidly. Other analogs of p-hydroxyphenylpyruvate were found to be inactive as substrates. 

In 1945 Lipmann found that a novel coenz3mae—coenzyme A— is required for the enzymic acetylation of sulfanilamide in pigeon liver preparations. Soon afterwards Nachmannsohn and Berman (see also ) found that a coenzyme is also required for the synthe of acetyl choline from choline and acetate in brain tissue, and this was found to be identical with the coenzyme of the acetylation of sulfanilamide, i Subsequently, three other reactions of acetate were found to involve coenzyme A the formation of acetoacetic acid from acetate, the s3mthesis of citrate from oxalacetate and acetate, - and the exchange reaction between acetyl phosphate and inorganic phosphate in bacterial extracts. " Thus, coenzyme A was shown to be a general coenzyme of acetylations, and... 

Oxidation of Fatty Acids.— The molecule of an unsaturated fatty acid such as oleic, is most likely to undergo oxidative attiack at the point of unsaturation the saturated acids, stearic and palmitic, are degraded by terminal oxidation. Fat oxidation occurs chiefly, if not entirely, in the liver, and under normal conditions the process is complete, and ends in COg and HjO. However, in diabetes and other conditions of carbohydrate inadequacy, fat oxidation in the liver is unable to proceed beyond acetoacetic acid, which suggests that this compound is an intermediate in fat metabolism. The natural fatty acids almost without exception contain an even total number of carbon atoms, and to explain the process of acetoacetic formation, Knoop proposed, in 1904, his theory of /5-oxidation of the fatty acids, according to which, the point of oxidative attack is the carbon atom in the /5-position, or next but one to the terminal carboxyl group. By this means the fatty acids are degraded two carbon atoms at a time. 

Multiple Alternate Oxidation.—Fatty acids with a carboxyl group at each end of the hydrocarbon chain have been found in the urine after fat ingestion, which shows that jS-oxidation is not the only path traversed in fat metabolism, and that terminal or oxidation also can occur. Furthermore, Jowett and Quastel (1935) have found that although liver tissue can form acetoacetic acid from a variety of acids containing an even number of carbon atoms, the rate of formation is less with butyric acid than with higher acids in the same series, which indicates that butyric acid is not an essential intermediate. They have proposed a theory of multiple alternate oxidation, according to which the fatty acid is oxidised at different points on alternate carbon atoms before the linkage is broken. 

The gene mutation inhibits hydrolytic cleavage of fumarylacetoacetate into fumarate and acetoacetate. Consequently, the toxic precursors maleylacetoacetate and fumarylacetoacetate accumulate in the liver and kidneys. They possess a reactive double bond and can therefore react with macromolecules to assume the properties of alkylating substances. In addition, intracellular glutathione deficiency develops due to the stable complex formation with glutathione, favouring lipid peroxidations. Enhanced formation of 5-aminolaevulinic acid can also be observed during occasional attacks of acute intermittent porphyria (G. Mitchel et al., 1990). 

Ketone body formation, which occurs within the matrix of liver mitochondria, begins with the condensation of two acetyl-CoAs to form acetoacetyl-CoA. Then acetoacetyl-CoA condenses with another acetyl-CoA to form /3-hydroxy-/3-methylglutaryl-CoA (HMG-CoA). In the next reaction, HMG-CoA is cleaved to form acetoacetate and acetyl-CoA. Acetoacetate is then reduced to form /3-hydroxybutyrate. Acetone is formed by the spontaneous decarboxylation of acetoacetate when the latter molecule s concentration is high. (This condition, referred to as ketosis, occurs in uncontrolled diabetes, a metabolic disease discussed in Special Interest Box 16.3, and during starvation. In both of these conditions there is a heavy reliance on fat stores and /3-oxidation of fatty acids to supply energy.)... 

In muscle, most of the fatty acids undergoing beta oxidation are completely oxidized to C02 and water. In liver, however, there is another major fate for fatty acids this is the formation of ketone bodies, namely acetoacetate and b-hydroxybutyrate. The fatty acids must be transported into the mitochondrion for normal beta oxidation. This may be a limiting factor for beta oxidation in many tissues and ketone-body formation in the liver. The extramitochondrial fatty-acyl portion of fatty-acyl CoA can be transferred across the outer mitochondrial membrane to carnitine by carnitine palmitoyltransferase I (CPTI). This enzyme is located on the inner side of the outer mitochondrial membrane. The acylcarnitine is now located in mitochondrial intermembrane space. The fatty-acid portion of acylcarnitine is then transported across the inner mitochondrial membrane to coenzyme A to form fatty-acyl CoA in the mitochondrial matrix. This translocation is catalyzed by carnitine palmitoyltransferase II (CPTII Fig. 14.1), located on the inner side of the inner membrane. This later translocation is also facilitated by camitine-acylcamitine translocase, located in the inner mitochondrial membrane. The CPTI is inhibited by malonyl CoA, an intermediate of fatty-acid synthesis (see Chapter 15). This inhibition occurs in all tissues that oxidize fatty acids. The level of malonyl CoA varies among tissues and with various nutritional and hormonal conditions. The sensitivity of CPTI to malonyl CoA also varies among tissues and with nutritional and hormonal conditions, even within a given tissue. Thus, fatty-acid oxidation may be controlled by the activity and relative inhibition of CPTI. 

Two acetyl CoAs can combine to form acetoacetyl CoA by the reverse of b-ketothiolase. The acetoacetyl CoA then combines with another acetyl CoA to make hydroxymethyl glutaryl CoA (HMG CoA) by the enzyme hydroxymethyl glutaryl CoA synthase. The HMG CoA in the mitochondrion can be cleaved by HMG CoA lyase in the mitochondrion to form acetoacetate and acetyl CoA. In this conversion, the formation of acetoacetyl CoA from two acetyl CoAs releases a free CoA and formation of HMG CoA from acetyl CoA and acetoacetyl CoA also releases a free coenzyme A. Thus, the release of free coenzyme A allows beta oxidation to continue with the production of acetoacetate. During diabetes and starvation, almost 90% of carbon from a fatty acid such as oleate can be accounted for in the form of ketone bodies during experiments with perfused livers. At this time, it would be worth noting that this process occurs in the mitochondrion later it will be seen that HMG CoA in the cytosol is a major precursor for cholesterol synthesis. 

Spectral analysis of the incubation mixture that catalyzes fatty acid synthesis in vitro has demonstrated the presence of a typical j8-ketothiolester bond that probably appears during the formation of protein-bound acetoacetate. As soon as NADPH is added to the system, the spectral characteristics of the j8-ketothiolester disappear. This change in the spectrum suggests that the protein-bound keto acid is reduced to the ]8-hy-droxy acid. Reductases catalyzing such a reaction have been found in yeast by Lynen s group and in bird livers by Walker s group. 

Fig. 3 Diagram to show pathways of ketogenesis in the liver. The formation of ketone bodies (acetoacetate and 3-hydroxybutyrate) in the liver, by partial oxidation of free fatty acids.

Acetoacetate Metabolism. An active deacylase in liver is responsible for the formation of free acetoacetate from its CoA derivative. The j8-hydroxybutyric dehydrogenase mentioned above and a decarboxylase are capable of converting acetoacetate into the other ketone bodies, /3-hydroxybutyrate, and acetone. liver does not contain a mechanism for activating acetoacetate. Heart muscle has been found to contain a specific thiophorase that forms acetoacetyl CoA at the expense of suc-cinyl CoA. Acetoacetate is thus used by peripheral tissues by activation through transfer, then reaction with either the enzymes of fatty acid synthesis or jS-ketothiolase and the enzymes that use acetyl CoA. 

More precise information on the pathway of leucine catabolism was obtained from studies on the formation of ketone bodies in liver slices incubated with and C Mabeled leucine and isovaleric acid. In these experiments it was found that leucine-3-C yielded acetoacetate in which the label was virtually all contained in the methyl and methylene carbons, and to approximately the same extent in each of these. Only a trace of radioactivity was found in the carboxyl carbon. On incubation with leucine-4-Ci the label occurred solely in the carbonyl group. This suggested that the isopropyl group of the amino acid had been directly converted to acetone. The over-all conclusion was that the isopropyl group forms acetone, and carbons 2 and 3 of the amino acid yield a 2-car-bon fragment which can condense to acetoacetate. The acetoacetate formed from leucine-4-C was not symmetrically labeled, the isotope being present only in the carbonyl carbon. 

Advances in the knowledge of the metabolic reactions of acetoacetate and the further studies of Coon on leucine metabolism solved the remaining uncertainties in the interpretation of the previous experimental data. This author demonstrated that the 3 carbons of the isopropyl group of leucine are incorporated as a unit into the 2-, 3-, and 4-positions of aceto-acetic acid. The carboxyl carbon probably arises by a CO2 fixation reaction, which had not been recognized previously. According to this scheme the complete breakdown of a mole of leucine by liver leads to the formation of approximately 1.5 moles of ketone bodies. 

Before leaving the subject of Lipmann s laboratory I should mention the results of two other studies that were key to an understanding of the mechanism of fatty acid metabolism. These studies were carried out in collaboration with Michael Douderoff who spent a few weeks in Lipmann s laboratory while I was there. To determine whether acetoacetate synthesis involved the condensation of two molecules of acetyl CoA or one equivalent each of acetyl-CoA and acetate, Douderoff and I studied the formation of acetoacetate in a coupled enzyme system composed of phosphotransacetylase and acetoacetate synthetase from pigeon liver. We found that the acetoacetate produced from 1-P C] acetyl-P and unlabeled acetate was equally labeled in the carboxyl and carboxyl carbons, whereas acetoacetate was produced from unlabeled acetyl-P and P C] acetate contained no... 

The catabolism of glycine by way of serine and pyruvic acid is probably of little quantitative significance. This is indicated by the low degree of acetoacetate formation from glycine and serine in liver slices in contrast to the formation of acetoacetate from lactate SO). Furthermore, it is probable that serine serves as a source of glycine in the organism rather than the reverse. This is discussed under serine biosynthesis in Chapter 15. 

Endogenous production of HMB occurs in muscle and liver (Figure 12.1) and possibly other tissues. The first step in HMB formation is the transamination of leucine to KIC, which occurs in both the cytosol and mitochondria of muscle cells. In the mitochondria, KIC is irreversibly oxidized to isovaleryl-CoA by the enzyme branched-chain a-keto acid dehydrogenase. Isovaleryl-CoA then undergoes further metabolic steps within the mitochondria (Figure 12.1), yielding P-hydroxy- -methylglutaryl-CoA (HMG-CoA). Further metabolism by the enzyme HMG-CoA lyase results in the end products acetoacetate and acetyl-CoA. Approximately 90% of KIC is oxidized to isovaleryl CoA in liver mitochondria and ultimately to acetoacetate and acetyl-CoA. 

The formation of acetoacetate in washed liver homogenates is consistent with the view that from 40% to 50% of the terminal 2 carbons of a fatty acid becomes identical in reactivity with the 2-carbon fragments derived from the rest of the molecule. Table VI gives the theoretical and observed values for R and Rt for 40% and 50% conversion of (CH3CO—)... 

As applied to the washed liver homo nate system where tim formation of acetoacetate from fatty acids is quantitative (Crandall e< al. >) ... 

The 3-C + 1-C condensation offers a pathway for the incorporation of (bi)carbonate-C or formate-C into a 2-carbon fragment, provided acetoacetate can be split into two 2-carbon fragments. Recall here that liver has only a limited ability to split the ketone body. Acetoacetate formed by a 3-C + 1-C condensation could possibly give rise to a carboxyl-labeled 2-carbon fragment in extrahepatic tissues, which are known to utilize acetoacetate more rapidly than does liver. The isotope from (bi)carbonate-C could then, via such labeled 2-carbon fragments, give rise to fatty acids labeled in odd carbons, as has been observed with rats. The incorporation of CO2 into fatty acids cannot occur through known reactions of the tricarboxylic acid cycle. 

Ketogenic reaction in normal rat liver was studied with various substrates in vitro. As shown in Fig.7, when palmitoyl carnitine or palmitate was used as the substrate, the ketogenic reaction was stimulated, although to a small extent, by pantethine as well as palmitoyl-S-pantetheine. Palmitoyl-S-pantetheine was somewhat more effective than pantethine, but palmitoyl-S-pantetheine by itself did not serve as the substrate of the reaction, unless CoA and carnitine were added to the reaction mixture. When octanoic acid was used as the substrate, the formation of acetoacetate was not affected by these two compounds at all. 

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