Palmitate labeled, oxidation

T.R. de Grade, S. Wang, J.E. Holden, R.J. Mickles, M. Taylor, C.K. Stone, Synthesis and preliminary evaluation of [ F]-labelled 4-thia palmitate a pet tracer of myocardial fatty acid oxidation, Nucl. Med. Biol. 27 (2000) 221-231. 

Oxidation of Initiated Habilitate Pakrritate uniformly labeled with tritium (3H) to a specific activity of 2.48 X 10 counts per minute (cpm) per micromole of palmitate is added to a mitochondrial preparation that oxidizes it to acetyl-CoA. The acetyl-CoA is isolated and hydrolyzed to acetate. The specific activity of the isolated acetate is 1.00 X 107 cpm//xmol. Is this result consistent with the /1-oxidation pathway Explain. What is the final fate of the removed tritium ... 

Despite the lower rate in the presence of inhibitor, radioactively labelled palmitate was added to a CHCla-inhibited system. Table IV shows that butyrate was formed at a faster rate than formate, acetate, or propionate. The fact that butyrate was now one of the major end products of palmitate dissimilation indicates that secondary reactions involving acetate and/or propionate were probably serving to remove hydrogen produced during dissimilation since methanogenesis was inhibited in these experiments. This was partially verified by the findings that radioactively labelled acetate was converted to formate and butyrate at faster rates in inhibited than in uninhibited sludge. It is also possible that formation of butyrate indicates some alternative to -oxidation as a dissimilatory reaction. Acetate itself was formed from C02 in the presence or absence... 

Leyton et al. (1987) used young rats (23-26 d old) and recovered CO2 from breath in a metabolic chamber at various time-points up to 24 h after oral dosing of 5-6 nCi of tracer in 0.2 mL olive oil. They found that 65% of the a-linolenate dose was completely oxidized within 24 h, an amount two to three times greater than for palmitate or stearate (Table 1). The same amount of tracer was given regardless of the amount typically found in the diet or in body lipids. This inequivalent dilution of the tracer potentially confounded the interpretation of the data. However, oxidation of oleate was closest to that of a-linolenate, yet there is 10- to 20-fold more oleate in tissue lipids. In agreement with Leyton et al. (1987), we have also found that "C-labeled a-linolenate and oleate are similarly p-oxidized in healthy women (McCloy et al. 2000). Thus, the effects of differential tracer dilution do not necessarily affect the rank order of p-oxidation of these long-chain fatty acids. 

As palmitate enters the tricarboxylic acid cycle via acetyl-CoA, their results with labeled palmitate suggest that the inhibited step is the oxidation of palmitate and pyruvate after their pathways have joined. 

Radioactive acetyl CoA can be generated by direct synthesis from C-acetate or from (3 oxidation of radioactive fatty acids, such as uniformly labeled palmitate. Examination of the reactions of the citric acid cycle reveals that neither of the two carbons that enter citrate horn acetate is removed as carbon dioxide during the first pass through the cycle. Labeled carbon from C-methyl-labeled acetate appears in C-2 and C-3 of oxaloacetate, because succinate is symmetrical, with either methylene carbon in that molecule labeling C-2 or C-3 of oxaloacetate. The conversion of oxaloacetate to phosphoenolpyruvate yields PEP labeled at C-2 or C-3 as well. Formation of glyceraldehyde 3-phosphate and its isomer dihydroxyacetone phosphate gives molecules, both labeled at carbons 2 and... 

In vitro testing may involve the oxidation of C-labelled or H-labelled pal-mitate and/or myristate by lymphocytes or fibroblasts [22] or the oxidation of C-labelled palmitate by fibroblasts with subsequent measurement of acylcarnitines in the supernatant [23]. Both approaches are for specialized labs only. 

C-labeled di-, tri- and tetraenoic fatty acids are oxidized by isolated mitochondria at nearly the same rate as palmitic acid (Stoffel et al. 1965). 

The technique of the intravenous administration of a fat has made possible a study of the rates of oxidation, by the intact animal, of various naturally occurring fats, and in particular, the rates at which the individual carbons of a given fatty acid are converted to CO. When fasted rats are injected intravenously with emulsions containing C Mabeled palmitic acids, significant amounts of the C appear in the expired CO2 as early as 30 minutes after injection, and 70% is converted to C 02 during 24 hours. On the other hand, with the shorter chain triglyceride, trilaurin-l-C, 71% of the administered C is converted to C 02 in 4.5 hours by normal or fasted rats. In that time, a labeled carbon of palmitic acids produces only about 50% of the administered C . Triglycerides of fatty acids with fewer than 14 carbon atoms are not deposited as... 

In the intact rat, a difference in the rate of oxidation of the 2 carbons of 2-carbon fragments, for example, between the 1st and 6th carbons of palmitic acid, was not observed. The oxidation experiments with the variously labeled tripalmitins were conducted on fasted rats, and in the light of present knowledge, considerable amounts of acetoacetate and other ketone bodies must have been formed from the fatty adds. The over-all processes involved in the metabolism of the 2-carbon fragment and of acetoacetate may be such as to lead to no difference in the in vivo rate of oxidation of the two carbons. 

ALA was long considered to be important only as a precursor of longer-chain compounds however, it is now recognized that ALA is also metabolized by other pathways which may be quantitatively more important than the formation of DHA. Studies in humans and animals have revealed that a major catabolic route of metabolism of ALA is P-oxidation. In rats, -60% of an oral dose of labelled ALA was expired as CO within 24 h, compared with less than 20% for palmitic acid, stearic acid, and AA (Leyton et al., 1987). Pan and Storlien (1993) reported that the higher the proportion of PUFA in the diet, the higher the rate of oxidation of labelled ALA in rats. In humans, 16-20% of ALA was expired as CO within 12 h (Vermunt et al, 2000). 

Localization of radioactivity following a dose of [1,6- C]lipoate was greatest in liver, intestinal contents, and muscle. Retention of the label was approximately twice as high after per os administration than i.p. injection at earlier (4-hour) and later (24-hour) periods. The uptake of lipoate by liver mitochondria and subsequent 3-oxidation to yield CO2 was found to be similar to shorter-chain fatty acids, e.g. octanoate, rather than longer-chain ones, e.g. palmitate. Lipoate and octanoate were relatively insensitive to additions of d7-carnitine to supply the 7-substrate for the long-chain acyl transferase upon which mitochondrial translocation of palmitate is dependent. Moreover, (+)-decanoylcarnitine inhibited the latter but had no effect on the short-chain compounds. 

Other common carboxylic acids—e.g., propionic, butyric, myristic, and palmitic acids—were also found to be incorporated into cytochalasin D (6) (Vederas et aL, 1975). Feeding of sodium [1- C]propionate produced an enhancement at C(4) in the NMR spectrum, indicating the production of phenylalanine labeled at C(l) via the shikimate pathway. However, this pathway seems to be of secondary importance. The other even-numbered saturated acids underwent j8-oxidation to yield C(l)-labeled acetate, which was incorporated in the usual manner. It is concluded that neither saturated nor unsaturated fatty acids are biogenetic intermediates of the cytochalasins. 

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