It is probable that some of the differences noted by Green and his colleagues between the behavior of the "cyclophorase " system and data earlier reported by Lehninger may depend upon somewhat different conditions of study. For instance, Grafflin and Green have employed much greater concentrations of the particles in the reaction media, and this factor, together with the presence of added cycle intermediates as " sparker," causes the Krebs cycle pathway to be accentuated, with a correspondingly lower yield of acetoacetate even in the liver system. Secondly, Grafflin and Green found the oxidase to be very sensitive to malonate, whereas Lehninger had reported no significant inhibition by 0.01 M malonate. However, unpublished experiments by Lehninger have shown that inhibition by malonate is not only dependent upon the proportion of fatty acid entering the Krebs cycle (much higher under the conditions of Grafflin and Green) but also involves an aging phenomenon. Freshly prepared mitochondria from liver show little inhibition by malonate; if aged or treated with H2O, the sensitivity to malonate increases markedly.

Fatty acid oxidation has been observed in particulate systems not only from liver, but also from kidney (19) and heart muscle (40). In the latter tissues, essentially all the oxidation proceeds via the cycle, with little accumulation of acetoacetate. Acetoacetate is readily oxidized by extrahepatic tissue preparations, but is probably not an intermediate in fatty acid oxidation. In addition, Geyer, Matthews, and Stare (18) have found with C14-labeled fatty acids and glycerides that slices of lung, spleen, brain, and skeletal muscle also oxidize fatty acids with formation of radioactive CO2, lung and spleen having very intense activity. In recent work, Weinhouse (62) has been able, using essentially similar techniques, to demonstrate fatty acid oxidation by a variety of tumors'. It would appear, however, that fatty acid oxidation by isolated mitochondria from tumors has not yet been successfully studied.

Substrate specificity. A wide variety of fatty acids and derivatives has been tested for oxidizability by either washed saline preparations or isolated mitochondria. Among the saturated straight chain fatty acids, all of those tested, from butyrate to stearate inclusive, were found to be oxidized by isolated mitochondria (28). Although the "cyclophorase " of Green and colleagues was inactive against acids having more than 13 carbon atoms, it is probable that this failure was due to the technical difficulty of preparing suitable emulsions of the fatty acids at pH 7.4. Although rates of oxidation are difficult to compare since it is necessary to use varying concentrations of substrate and the reaction pathways may differ with chain length, it appears that the rate of oxidation increases with chain length up to C8-C10, and then remains constant to Ge. In the presence of a constant concentration of G dicarboxylic acid, the longer the carbon chain of even-carbon acids, the greater the proportion of G units which are oxidized via the cycle, rather than to acetoacetate, a phenomenon apparently due to the variation in relative preponderance of two types of G units (28). Under the same conditions odd-carbon acids are also oxidized at about the same rate, although the shorter ones have a greater tendency to be oxidized via the cycle than their even-carbon neighbors.

Oleic, linoleic, linolenic, and vaccenic acids are all rapidly oxidized and all require " priming." Autoxidation of these acids was negligible under conditions of the tests. It is remarkable that elaidic acid, the unnatural trans isomer of oleic acid, is as rapidly oxidized as oleic acid (28). In fact, the fatty acid oxidase system is within limits rather omnivorous. Grafflin and Green (19) have found that a variety of unsaturated acids (both cis and trans forms of crotonic acid, a, β-unsaturated acids, etc.), β-hydroxyacids, of which both D and L isomers were attacked, and j8-keto acids were readily oxidized. On the other hand, certain a- or β-substituted acids were not attacked. The findings allow no clear-cut postulations regarding the mechanism of oxidation but they are at least consistent with classical views of the mechanism.

The oxidase system requires a free carboxyl group; long-chain fatty alcohols, aldehydes, amides, and N-acylglycine derivatives are not oxidized. Fatty acid esters and phospholipides are oxidized, although there is no indication that they are oxidized as such, since the enzyme preparations are rich in esterase activity. At least four dicarboxylic acids, adipic, glutaric, pimelic, and azelaic acids, have found to be inert in the system (19, 33).

So far no success has attended efforts to isolate or identify intermediates between the fatty acid stage and the C2-unit stage in preparations from animal tissues. However, some important clues from work on C. kluyveri extracts, discussed below, may possibly provide a rationale for reconstruction of the oxidation in extracts of mitochondria.

Activation of fatty acid oxidation. Leloir and Munoz (44), in their early studies on fatty acid oxidation, observed that the simultaneous oxidation of fumarate considerably stimulated the oxidation of fatty acids by their preparations. This stimulation of fatty acid oxidation by the concurrent oxidation of fumarate or other Krebs cycle intermediates has since been observed in several other laboratories, and has been subjected to considerable study and discussion. The effect has been variously called the " priming " or " sparking " of fatty acid oxidation.

On the basis of recent work with mitochondria from sucrose homogenates of liver tissue, isolated by the method of Hogeboom et al. (22), two types of enzyme preparations may be distinguished, varying as to the requirement for a " priming " simultaneous oxidation of another substrate to initiate fatty acid oxidation. Freshly prepared mitochondria from isotonic or hypertonic sucrose homogenates, protected from exposure to temperatures higher than 0° C, and from structural changes induced by high salt concentrations or markedly hypotonic media, carry out the oxidation of fatty acids at rapid rates without the addition of priming co-substrates, as Lardy (32) and other investigators have noted. This type of preparation also exhibits only a slight inhibition of fatty acid oxidation upon the addition of malonate (32,33). However, if such mitochondrial preparations are subjected to prolonged wash procedures at temperatures slightly above 0° C, or are incubated in the absence of substrate, or are briefly exposed to distilled water, then significant oxidation of fatty acid occurs only when the test system is supplemented with catalytic amounts of other substrates. The malonate inhibition of fatty acid oxidation is much greater in these preparations which require " priming." Since the mitochondria are undoubtedly the subcellular components responsible for the fatty acid oxidation activity in the older type of washed particle preparations studied by Lehninger (35), it would appear that the requirement for " priming " shown by these preparations was also due to the slight degradation of the mitochondrial content brought about by the earlier procedures. This same reasoning may apply to the " cyclophorase " preparations of Green, which are essentially washed residues of liver or kidney homogenates.