Leathes7). On the other hand the depot fats of domesticated animals. typical terrestrial vertebrate fat depot. The liver fatty acids of man,

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1 FATTY ACID METABOLISM IN THE LIVER. II. THE RELATION OF THE FATTY ACIDS IN THE FOOD OF PLAICE TO THOSE IN THEIR LIVERS AND MYOTOMES'. BY V. H. MOTTRAM. (From the School of Physiology, Liverpool, and the Marine Biology Station, Port Erin, I.O.M.) THE outstanding feature of the fatty acids of marine vertebrates is their high iodine value. Thus clupanodonic acid from sardine oil, herring oil or whale oil has an iodine value of The liver oils of marine vertebrates have iodine values from (shark) to 181V3 (cod)3; the blubber oils as high values as 193 (seal) and the fish oils, 19.3 (sardine)3. The fatty acids from these oils have values correspondingly higher. In this characteristic these fatty acids resemble the fatty acids of terrestrial vertebrate tissues and are unlike the fatty acids of the typical terrestrial vertebrate fat depot. The liver fatty acids of man, the pig, the cat, the rabbit, the bullock, the cock and the guinea-pig have iodine values of about 130 (Hartley4, Leathes5 and others7). The same is true of the kidney, heart and skeletal muscle (Hartley4, Leathes7). On the other hand the depot fats of domesticated animals have iodine values from 35 (mutton tallow)3 to 70 (lard)3. The fatty acids therefrom have values about 6 /o higher. In wild animals the value is higher, especially in those that feed onfish. To explain this sudden step between dep6t fatty acids and tissue fatty acids, Leath es8 has suggested that the rdle of the liver in fatty I Part of the expense of this research was defrayed by a grant from the Royal Society. 2 Lewkowitscb. Chemical technology and analysis of oils, fats and waxes, i. p I Op. cit. p This Journal, xxxvi. p ; and xxxviii. p Lancet, Feb. 27th, This Journal, xxxvm. p ; and XL. p Proc. Physiol. Soc. p. ii (This Journal, xxu.) 8 Lancet, Feb. 27th, 1909.

2 364 V. H. MOTTRAM. acid metabolism is that of desaturating the mobilised fat on its way to the tissues. Depot fat, which is derived from the food without much change, must be altered and transmuted before it becomes fit material for tissue metabolism. This theory, supported as it is by much experimental data, is now on its way to general acceptation. Is it possible to extend this theory to include marine vertebrates? The question is at once raised whether marine vertebrates owe their peculiar fatty acids to their food stuffs or whether fatty acid metabolism is of a high grade of activity and the ability to desaturate fats and fatty acids is particularly great in marine vertebrates' livers. This question was placed before Professor Herdman, and there being excellent material for research in the Marine Biology Station of Port Erin, of which he is director, the following experiments were instituted. Material and Method. Plaice, about the five year standard in size, were used for analysis. These fish had been in the spawning pond, soine one, some two and some three years. Since the preceding autumn they bad been fed on parboiled Morecambe rnussels exclusively. On May 20th and 21st, a number were Ai1led by decapitation and their livers and a certain group of myotomes dissected out. The myotomes taken were, in every case, those on the blind side of the fish between the lateral line and the dorsal fin-in brief, the dorsal fillet. To eliminate, as far as possible, individual variations, the fish were dealt with in groups of three. Their livers, after removal of obvious connective tissue, were analysed together. The myotomes were minced, thoroughly mixed, and aliquot portions analysed. Further, a sample of the mussels was taken, parboiled, minced, mixed thoroughly and analysed. The method of fatty acid estimation was that described in Because the material after the preliminary dissolution in caustic potash had to be transported to Liverpool, slight modifications of the method were made. The heating was done in an atmosphere of nitrogen and not in vacuo and, after the material had gone into solution, nitrogen was passed through the pressure bottles till all was cold. The vents were then securely closed. The alkaline solutions of tissues were thus kept in the presence of nitrogen till they were analysed in Liverpool, and the method has shown itself to be very satisfactory. That such a precaution is necessary is seen from the following experiment Four samples of bullock's liver were dissolved in 40 0/0 caustic potash as usual, two estimated at once and two stored in well-stoppered bottles in the dark for five weeks. Results:-A. Control analysed at once. Amount used Fatty acid 0/0 fatty acid Iodine value 14 25g g [above 129] * This Journal, XL. p

3 FATTY ACID METABOLISM. 365 B. Analysed after standing five weeks in dark. Amount used Fatty acid 0/0 fatty acid Iodine value g g It is seen that much of the fatty acid (present as soap) is oxidised and lost to estimation, and that the iodine value of the remaining fatty acid is considerably lowered. At the conclusion of the fatty acid estimation, the fatty acid was dissolved in carbon tetrachloride and its iodine value obtained by Wijs' method. The results obtained are as follow: Exp. 1. Three plaice, one about seven years old, the others five years, were beheaded and their livers and dorsal fillets cut out. The former united and weighed into a pressure bottle; the latter minced, mixed thoroughly and four portions weighed into pressure bottles. 40 0/0 caustic potash added. Nitrogen passed through pressure bottles for halfan-hour. Then heated for 1I hours at 1000 C. While cooling nitrogen passed, and at the end bottles sealed. Fatty acid estimations made at Liverpool. Tissue Amount used Fatty acid 0/l fresh tissue Iodine value Livers g g Myotomes [above 180] [above 151] Mean The first and second iodine value estimations of myotome fatty acid have been discarded because, owing to a leakage in the vacuum drying oven, the two samples of fatty acid concerned were oxidised and partially converted into a brown varnish insoluble in carbon tetrachloride. It is obviously important when working with such small yields of such highly oxidisable fatty acid to use as large a number of parallel estimations as possible. The figures show a much lower iodine value for the fatty acids of liver than for those of the myotomes, the significance of which will be discussed later. Exp. 2. Three plaice, all about the five year standard. Treated as in Exp. 1. Tissue Amount used Fatty acid 0/0 fresh tissue Iodine value Livers g g Myotomes Mean This is the most concordant series of estimations of myotome fatty; acid, and the iodine values show but small deviations from the mean.

4 366 V. H. MOTTRAM. Probably this experiment, as regards estimation, is the high water mark of the efficiency of the method and apparatus employed. Again the iodine value of the liver fatty acids is well below that of the myotomes. Exp. 3. Three plaice, all about the five year standard, but larger than those in Exp. 2. Treated as in Exp. 1. Tissue Amount used Fatty acid 0/0 fresh tissue lodine value Liver g g Myotomes [above 184] A429 [above 164] Mean In two cases of myotome fatty acid estimation oxidation had occurred: a brown insoluble varnish had been formed. The iodine values of the soluble portions (calculated on the total mass of fatty acid oxidised and otherwise) are only relevant in so far as they show that, had oxidation not occurred, they would be well above 160 in one case and 180 in the other. The evidence obtained is as before: the iodine value of myotome fatty acid is well above that of liver fatty acid. Resuming the results of the analyses of the livers and the myotomes of the nine plaice examined, we see (a) that the iodine value of myotome fatty acid is in every case higher than that of liver fatty acid; (b) that there is a rough parallelism between the two sets of figures'; and (c) that there is an antiparallelism between the percentages of fatty acid in the livers and the fatty acid iodine value2. These three results are in close agreement with Leathes' theory of desaturation of fatty acid by means of the liver. It remains to relate the results with the analysis of the food stuff. The food of the plaice, since the time they first entered the spawning pond, had been chiefly Morecambe mussels. In the months of July, August and September pieces of mackerel and herring were also given. But for the seven months preceding the experiment the food was exclusively mussels and a sample of these was chosen for analysis. Exp. 4. Thirty-five average mussels were immersed one at a time in boiling water till the shells gaped, the animals were removed from the shells, and after the moisture was removed from their surface, they were minced and thoroughly mixed. Five aliquot portions of the mince were weighed out and analysed as in Exp. 1 for fatty acid. When the fish is immature, liver and tissue fatty acids have a higher iodine value than when the fish is more mature. In Exp. 1, where the liver iodine value is low, one of the fish was sexually mature and about to spawn /0, /0, /0 correspond with 149, 171 and 193 respectively.

5 FATTY ACID METABOLISM. 367 Amount used Fatty acid 0/0 fresh tissue Iodine value g g '027 [above 184] Mean 1.01 [5] 197 The result of these analyses was not expected. It was thought that, following the paradigm of fatty acid metabolism in mammalia, the iodine value of the fatty acids of the food would be well below that of the liver fatty acids and still further below that of the tissue fatty acids. Instead we find it intermediate between that of the liver fatty acids and that of the myotome fatty acids. High iodine value fatty acids are not an invention of the vertebrate and observations on plaice do not agree with those made on domnesticated animals. Collecting the results we obtain the following table. Iodine values of fatty acids. I, Of myotomes II, Of livers Plaice 1, 2 and , 5 and , 8 and Mean 208 approx. 171 III, Of mussels 197 In every case the iodine value of the fatty acids of the food of these plaice is intermediate between that of the liver fatty acids and that of the myotome fatty acids. Indeed when the mean of the means is taken it falls much nearer the latter than the former. Consequently these results yield no evidence for a desaturating action of the plaice liver on the fatty acids supplied to it. Desaturation may take place thereit must take place somewhere-but if it does it is masked by other reactions. Nor can any consideration of probable errors of estimation throw discredit on the results. From the amounts of tissue used, 50 to 80 mgrms of fatty acid were obtained in myotomes; 130 to 170 mgrms in mussels; and 620 to 1820 mgrms in livers. These were all handled in apparatus of the same size. Consequently the fatty acids that would suffer most by oxidation are those (i) of the highest iodine values, and (ii) those present in the least quantity. Those of the myotomes come under both headings, and if any fatty acids have suffered oxidation in estimation it is these. [There is evidence-see the protocols above- 1 Brown varnish, insoluble in carbon tetrachloride, formed. 2 Small loss in initial stage of fatty acid extraction-no correction made.

6 368 V. H. MOTTRAM. that this is so.] Intermediate come those of the food and last, those of the liver. If, then, these errors could be corrected, the correction would spread the results still further, and in the same direction. A consideration of the possible and probable errors simply confirms the deductions from the above results. It must be admitted, then, that as the experiments stand, they give no evidence for a desaturating action of the liver of plaice such as is probable in the mammalia. But although these experiments give no support to Leathes' theory, they must by no means be taken as refuting it. At the most they mean that any such desaturation is masked. And a hundred and one other suggestions leap to mind to explain these anomalous results. The possible seasonal changes of mussel fatty acid, the seasonal changes in the fish (though but one was sexually mature), and the manufacture by the plaice liver of fat from the shell-fish glycogen (always abundant) are a few. Until these suggestions can be realised or refuted, the research must remain an isolated group of facts, rather of the nature of a preliminary research. SUMMARY. (i) The iodine value of the fatty acids of the mussel is high-circa 197-and falls between that of the plaice liver fatty acids and that of the plaice myotome fatty acids-circa 171 and 208 respectively. (ii) Therefore fatty acids of high iodine value are not characteristic of the vertebrates: they appear in animal economy before the appearance of a true liver. Their manufacture is not exclusively the function of the liver. (iii) Nevertheless the above experiments on the feeding of plaice on mussels cannot be taken as a refutation of Leathes' theory of the desaturating'influence of the liver in fatty acid metabolism. Evidence for that theory may easily be masked by other hepatic transformations. (iv) The experiments fiurnish another example of the general rule that the fatty acids of the tissues, where material in the highest state of elaboration is found, have a higher iodine value than those of the food. Desaturation has presumably taken place-otherwise there must have been a specific consumption of the less oxidisable fatty acids, an assumption that is somewhat improbable. I should like to thank Mr Chadwick, Curator of the Marine Biology Station at Port Erin, very heartily for his kindness and help during my work there.

7 FATTY ACID METABOLISM. 369 ADDENDUM. While the above was in the press the following analyses of normal plaice caught in the Port Erin neighbourhood were completed. They are included for comparison with those of the plaice held for so long in captivity in the spawning pond. Flat fish are not migratory so that both sets of fish are presumably of the same stock. Exp. 5. Three plaice, all of the five year standard, treated as in Exp. 1 with the slight exception that the alkaline solution was neutralised with hydrochloric acid before transport to Liverpool. Atmosphere in pressure bottles carbon dioxide. Tissue Amount used Fatty acid %/s fresh tissue Iodine value Livers g g Myotomes 'Mean 0X52 198X5 Exp. 6. Three plaice, all of the five year standard, treated as in Exp. 5. Tissue Amount used Fatty acid 0/0 fresh tissue Iodine value Livers g g Myotomes These results show that the plaice of the spawning pond have myotome fatty acids only a trifle more unsaturated than those of normal plaice. The liver fatty acids have low iodine values in the normal plaice, accompanied, as is usual, by high percentage values. We have here a fiurther confirmation of the anti-parallelism of iodine values of fatty acids and their percentage in the liver. On the whole, then, the unexpected results of the first part of the research are not due to any abnormality of the fish examined, and they may be accepted, with the reservations stated above (p. 368), as typical of the relations of fatty acids of the food, of the livers and of the myotomes of plaice.