Studies on APO-beta-cartoenals and related compounds

Abstract

The existence of an aesthetically fascinating group of organisms, the fruiting myxobacteria, was first recognised by Thaxter (309). He studied carefully the life cycles of six species of this group and found them to be quite unique among the Schizomycetes. Thaxter placed these organisms in a new order, which he termed Myxobacteriaceae, in view of their striking similarity to Mycetozoa. In the succeeding years he collected several more of these forms (310, 311, 312). His reports were soon followed by Dahn’s (123) rearrangement of the known species; in fact, Dahn was the first to attempt a detailed classification of myxobacteria (124). Organisms closely related to the above forms but lacking the capacity to form fruiting structures were encountered frequently in decomposing cellulose. At that time, they were placed among the true bacteria (194, 319), spirochaetes (114), and actinomycetes (33). Stanier (293, 294, 295) recognised their relationship to fruiting myxobacteria. Since Dahn’s original definition of the order Myxobacterales did not include the non fruiting forms, Stanier (295) redefined the order to permit their inclusion. Dahn’s system of classification, revised by Stanier, is currently in vogue (Bergey’s Manual, 7th Ed., 1957) (36). The order includes five families: Archangiaceae, Sorangiaceae, Polyangiaceae, Myxococcaceae, and Cytophagaceae. The last family consists of only one genus-Cytophaga, comprised of organisms that form neither resting cells nor fruiting bodies. The genus Sporocytophaga, which consists of organisms forming resting cells but not fruiting bodies, is placed in the family Myxococcaceae. For convenience, the lower myxobacteria, i.e. those belonging to the genera Cytophaga and Sporocytophaga, will henceforth be referred to as “cytophagas” and the fruiting myxobacteria (higher myxobacteria) as “myxobacteria” (300). Available information on the occurrence of cytophagas and myxobacteria in various parts of the world points to their ubiquity in nature (11, 37, 38, 88, 120, 121, 125, 133, 141, 155-160, 185, 198, 199, 215-217, 244-246, 267, 279, 280, 296, 309-312, 320, 321, 348). Cellulolytic and chitinolytic cytophagas are common in soil as well as marine environments. Some facultatively anaerobic cytophagas have been reported from fresh water and sea water. Similarly, myxobacteria are widely distributed in nature-in soil, composts, decaying plant debris, animal dung, and on the bark of living trees. While Stanier’s review (295) on cytophagas covers most aspects of these organisms, Dworkin (65) has recently discussed the information accumulated on myxobacteria over the years after Thaxter’s discovery of these organisms. Various aspects of these organisms have also been discussed in a recent symposium (12, 53, 200, 247, 272, 277, 332). It is considered worthwhile to briefly describe the morphology, life cycle, morphogenesis and other particulars of these organisms in the following pages.

Life Cycle, Morphology and Morphogenesis Briefly stated, the life cycle of myxobacteria consists of a vegetative or swarm stage, followed by a fruiting stage. The latter is absent in cytophagas. In the vegetative stage, myxobacteria have elongated cells which multiply by fission and creep over the substratum by their gliding movement. Although various hypotheses have been put forth (95, 124)…

(The second half, starting “In the light of information available thus far…”, is treated below.)

In the light of information available thus far, it has been suggested that terminal oxidation of carotenoids may be one of the important routes of biosynthesis of vitamin A from its carotenoid precursors in animals. The formation of apocarotenoids has been envisaged as active intermediates in this scheme. In order to explore the possibility of the involvement of these carotenoids as intermediates in the biosynthesis of vitamin A, in vivo and in vitro studies with such compounds have been undertaken. Studies on the metabolism of apo carotenals show that they are biologically active and are slowly degraded to vitamin A. The relative biopotencies of 12 , 10 and 8 apo carotenals are 44.2%, 75.9% and 51.59%, respectively, as compared to all trans carotene. Time distribution studies indicate that part of 8 apo carotenal escapes cleavage, is absorbed from the intestine and stored in the liver as such, whereas 12 and 10 apo carotenals are completely degraded in the intestine. Apo carotenals are oxidised in vivo to their corresponding apo carotenoic acids, which could be detected in the intestine. Unlike retinal and 3 dehydroretinal (Glover, 1948a; John et al., 1966), apo carotenals, when administered either orally or intraperitoneally, are stored as such without prior reduction to their corresponding alcohols and esterification. This is probably due to the inability of enzyme systems present in both intestine and liver to reduce them to their corresponding alcohols. Metabolic studies on apo carotenals suggest that they are cleaved at the 15,15 double bond although the extent of vitamin A formation is low. Unlike apo carotenals, epoxides of 12 and 8 apo carotenals are biologically inactive and are not transformed to vitamin A in the animal system, suggesting that compounds which cannot be transformed to vitamin A are biologically inactive. It also supports the previous suggestion of Krishna Mallia et al. (1970) that the double bond in the ionone ring is a prerequisite for vitamin A activity. Like 8 apo carotenal, its 5,6 epoxy derivative is absorbed from the intestine and stored in the liver. It is also oxidised to its corresponding acid in the intestine. Epoxides of 8 apo carotenyl acetate are hydrolysed to their corresponding alcohols when administered either orally or intraperitoneally. The inability to detect epoxides of vitamin A upon feeding epoxides of 12 and 8 apo carotenals as well as 8 apo carotenyl acetate suggests that they are not attacked at the 15,15 double bond. These studies therefore suggest that modification of the ionone ring of apo carotenals by epoxidation affects their cleavage at the 15,15 double bond. Metabolic studies on 5 hydroxy 8 apo carotenal indicate that it is esterified at the free hydroxyl group and, like 8 apo carotenal, is oxidised to its corresponding acid in the intestine. The 3 hydroxy 8 apo carotenal is not absorbed from the intestine, suggesting that modification of the ionone ring by introduction of a hydroxyl group affects its absorption. Neither 3 hydroxy retinal nor 3 hydroxy vitamin A could be detected after administration of 3 hydroxy 8 apo carotenal, suggesting that, like epoxides, this compound is also not cleaved at the 15,15 double bond. Apo carotenoic acids are biologically active. Their biopotencies (12 , 10 , 8 apo carotenoic acids) administered orally in oil are 88.40%, 95.25% and 92.55%, respectively, compared to all trans carotene. Although the extent of vitamin A formation from these acids is low, their high biological activities may arise partially from retinoic acid like activity. In vivo studies suggest that they may be cleaved at the 15,15 double bond but at a slower rate than apo carotenals. Carotene 15,15 dioxygenase has been isolated and partially purified from the intestinal mucosa of guinea pig and rabbit. The enzyme from both sources shows remarkable similarities in time course, substrate affinity, activation by GSH and Fe² , inhibition by SDS, sulfhydryl binding and iron chelating agents, and substrate specificity. The only notable difference is that the guinea pig enzyme has a slightly higher pH optimum (8.5) than the rabbit enzyme (7.8). The enzyme is nonspecific and cleaves several carotenoids at the 15,15 double bond. Conversion of one ionone ring to an ionone ring reduces enzyme activity. Epoxidation or hydroxylation of the ionone ring reduces activity further. Introduction of two hydroxyl groups, one in each ring, renders the molecule completely inactive. Apo carotenals and apo carotenols are also cleaved, but at slower rates. Among these, the 10 derivatives are cleaved most readily. Hydroxylation or epoxidation of the ionone ring of apo carotenals makes them inert. Enzymatic cleavage of apo carotenoic acids could not be demonstrated, although nutritional studies show biological activity; this may be due to a rate of cleavage too slow for detection. In vivo studies from this laboratory on mono ring substituted carotenoids had earlier suggested eccentric cleavage. However, detection of ring substituted retinal derivatives now clearly demonstrates that these compounds can be cleaved at the 15,15 double bond in vitro. Whether the same mechanism operates in vivo remains unresolved, as the ring substituted fragments may be metabolised rapidly and escape detection.