A special chapter by Causeret (Volume II, Chapter 5 ) presents in a comprehensive way the mineral composition of fish and other aquatic food animals. The most complete compilation of analytical data in this
6. ORGANIC CONSTITUENTS OF FISH 175 respect is the monograph by Vinogradov (1935-37), originally in Russian but later translated and available through the Sears Foundation for Marine Research (Vinogradov, 1953). In this chapter only some organic compounds into which these basic elements enter will be discussed.
A . PHOSPHORUS
Phosphorus is essential to cell metabolism including that of fish.
It has more functions than any other single mineral. Most of the phos
phorus is concentrated to the nucleus. It combines to form phospho-proteins, which initiate muscle action. Phospholipids are essential in lipid metabolism. The blood of fish is rich in organic acids, soluble phos
phorus compounds, but flows as inorganic phosphoric acid in the blood stream. The two B-vitamins, Bx and B i2, are in fact complex phosphoric acid.
There are three closely related phospholipids, these being esters of phosphatidic acids and nitrogen-containing alcohols (choline, ethanola-mine, and serine). The one derived from choline is the well-known lecithin, nowadays isolated from a great number of fish.
Glycerophosphatides have been identified in several fishes and are characterized by the presence of appreciable and frequently high pro
portions of C2o and C22 highly unsaturated acids (chiefly arachidonic and clupanodonic) (Lovern and Olley, 1953a, b ) .
The fact that phosphorus occurs in fish in the form of high-energy phosphates, phosphoproteins, nucleic acids, etc., limits the value of total phosphorus determinations. In summary, organic phosphorus com
pounds play a key role in various basic metabolic processes. This best becomes evident through a listing compiled by Tarr (1950) (Table X I I I ) .
Adenosinepolyphosphates are key substances to an understanding of the basic changes taking place in fish flesh held at different tempera
tures, particularly when freezing is involved (see this volume Chapter 9, and Volume III, Chapter 1 1 ) . Saito and Arai (1957) have reported noteworthy differences in carp held at 1 6 ° C , 0 ° C , and — 8 ° C , and in liquid air. At -\-16°C, ATP and ADP are rapidly broken down into adenine and adenosine; at 6 ° C , these changes are slow, and the main degradation product is AMP. When frozen at — 8 ° C , ATP and ADP decrease measurably and the major breakdown product is inosine mono
phosphate ( I M P ) . In liquid air, almost no changes take place.
TABLE XIII ACID-SOLUBLE PHOSPHORUS COMPOUNDS OF THE FISH MUSCLE AS COMPARED TO THOSE OF THE (Micromoles per 100 grams) WHITE RAT<* Tomcod (muscle Rat Rat stored Blue (Le Page, (present Starry Whiting Whiting 2 days sea Ling Compound 1946) work) flounder (1) (2) Tomcod at 0°C.) perch6 cod Total phosphorus 5,500 7,170 12,400 8,970 15,300 11,300 5,790 8,250 5,670 Inorganic phosphorus 800 2,920 9,150 5,620 14,100 9,150 5,500 4,580 4,250 Phosphocreatine 1,600 1,470 1,680 845 45 0 0 553 535 ATP plus ADP 800 405 123 371 240 197 27 372 52 Fructose-l,6-diphosphate 25 29 13 128 27 35 20 45 4.7 Phosphoglyceric acids 400 618 88 352 119 103 52 85 35 Glucose-l-phosphate 100 87 39 56 42 58 49 29 120 Glucose-6-phosphate 400 204 47 97 9 38 39 136 58 Fructose-6-phosphate 10 44 6.8 82 23 8 17 25 6.5 Pentose-phosphate 50 20 7.6 183 0 14 22 149 161 Myoadenylic acid 75 14 51 214 158 240 0 90 80 Coenzyme 25 16 3.8 11 5.7 5 2.4 5 8.5 Ρ recovered in two fractions (%) 93-96 91 96 93 96 96 87 97 97 <* Reference: Tarr (1950). & Helioperca incisor.
6. ORGANIC CONSTITUENTS OF FISH 177
B . SULFUR
Sulfur-containing amino acids are the chief carriers of this mineral in fish. The sulfate content is low, and other sulfurous compounds rare.
The sulfur of protein is centered in the following amino acids: cystine, cysteine, and methionine. Tsuchiya (1944) reported on these amino acids and devoted a special study to methionine. Beveridge (1946) established that lingcod, halibut, lemon sole, and spring salmon are ex
cellent sources of methionine. Outside of this amino acid and cystine, only minor quantities of other sulfur-bearing compounds exist in fish flesh.
Other organic forms of sulfur found in the fish body are glutathione, taurocholic acids, etc.
Figures indicating total content of sulfur obviously have less signifi
cance. To what degree it occurs in the above-mentioned derivatives is far more pertinent.
C. MISCELLANEOUS
Copper and iron enter into the constitution of a complex of iron-copper nucleoproteins (Saito, 1954) from which they are liberated by pepsic or trypsic digestion (Saha and Guha, 1940). The complex in
cludes 3 0 - 4 0 % ionizable iron (Saha, 1941a, b ) which is rapidly ab
sorbed by the intestinal membrane (Saha and Banerjee, 1943). The com
plex exerts a high hematopoietic power in a rat anemiated by a milk diet. The elimination of copper clearly reduced the antianemic reaction, and the association of iron and copper to doses corresponding to those of the complex also has a hemotopoietic power smaller than that of the triple association (Saha, 1941b). Red muscle contains more organically-bound iron than ordinary white muscle (Namiki, 1934).
Another important organic compound with built-in minerals is Vita
min Bi2, which contains cobalt.
As to organic iodine compounds, mention should be made of the fact that thyroxine is intimately related to the motility of fish. Sedentary types contain less iodine and thyroxine. Anadromous fishes store iodine for increased thyroxine production, ensuing from the exerting activities of spawning migration, taking them into iodine-poor fresh water (Fon
taine and Leloup (1950a, b ) .
It is generally concluded that several shellfish with a high arsenic or fluorine content, far above permissible levels for human safety, can be explained only on the basis of assuming their being bound into complex organic molecules, from which they are only gradually being released.
VI. Vitamins
A. V I T A M I N A
There is a general tendency among aquatic animals preferentially to accumulate xanthophylls rather than carotenes, and fish are no excep
tion. They are largely devoid of carotenes. Only occasionally do caro
tenes appear in the liver, and in the ovaries only ß-carotene is found.
Euler (1933) reported minor quantities of carotene in salmon oil, but Bailey et al. (1952) could find none.
The xanthophyll distribution in fish is unique in that only three pig
ments have been found, although a large number of species have been examined in this respect. No basic differences have been established between marine and fresh-water fish (Goodwin, 1951). Hirao et al.
(1957) list certain differences in this respect. These problems are, how
ever, discussed more in detail in this volume, Chapter 13.
The pigments of most fishes are localized in the skin, and in only a few, such as salmon, is the muscle tissue pigmented. The color of salmon flesh is due mainly to astaxanthin (Bailey, 1937; Kanemitsu and Aoe, 1958), a pigment found in most Crustacea. Steven (1948) found astaxan
thin to be present in the flesh of brown trout and of the char of Scottish lochs. None was obtained from trout with white muscle. Baalsrud
(1956) reported on a cod which had red-colored flesh due to the pres
ence of astaxanthin (esterified). Templeman and Sandeman (1958) examined a redfish, the fillets of which were colored salmon-pink.
Bligh and Dyer (1959) reported that in such an atypical cod with reddish flesh, zeaxanthin constituted one-third of the pigment, the re
mainder being astaxanthin. In similar haddock with slightly pink flesh, only zeaxanthin was found.
Fishes do not synthesize astaxanthin, but derive this carotenoid from their diet (Goodwin, 1951). Sources in the diet of such fish are numer
ous, and euphausiids, copepods, and shrimps all provide this compound.
See further Templeman and Sandeman (1958) for references in this respect.
There exist in fish different forms of vitamin A with a varying degree of physiological efficiency. An exception is the subvitamin A, showing only minor or even doubtful vitamin activity. Creae'h (1955) gives the characteristics for these variants as shown in the tabulation.
According to Schantz (1948), the active p o w e r of vitamin A2 in crys
t a l l i z e d form is e q u a l to 4 0 % of t h a t of a x e r o p h t h o l . In the C r u s t a c e a
6 . ORGANIC CONSTITUENTS OF FISH 179
Form
Maximal absorption
(Ä units) Origin
Vitamin Ax (axerophthol) 3.280 Almost all species Neovitamin Ax 3.280 Dogfish, cod, halibut Vitamin A2 3.500 Fresh-water and anadromous
fish
Subvitamin A 2.900 Shark
Retinene — Roe and eggs
and also in fish, Grangaud et al. (1950-51) have brought into evidence the presence of the above-mentioned carotenoid pigment astaxanthin, which carries an oxidized nucleus and is endowed with antixerophthal-mic properties.
In the liver of whales one finds significant quantities of kitol, a sub
stance which is transformed into vitamin A through heating at 170°C.
In the intestines of whales, kitol is partially transformed into axeroph
thol. Its degree of vitamin activity varies with species. The rat does not utilize it as a precursor of vitamin A. In other species it supposedly plays the role of a provitamin (Tawara and Fukazawa, 1950; Swain, 1949b).
The genesis of vitamin A in fish poses many unsolved problems.
Larger fish eat smaller ones and through them get pre-existing vitamin A which they can store. Small-sized fish get their nourishment from plankton, as is the case with large marine mammals. In the spring, phytoplankton dominates. It contains ß-carotene capable of being trans
formed into axerophthol, as shown by Neilands (1947) with cod, as well as by Morton and Creed (1939) with fresh-water fishes. The Zooplank
ton, preponderant toward the end of summer, is composed of small
C r u s t a c e a , eggs, and l a r v a e . Certain species have a considerable content of vitamin A. (Meganyctiphanes norvegica: 260 I.U./g.) but more often, the content is low (copepods: 0.3 I.U./g.) or absent. But, Zooplankton is rich in astaxanthin.
On the basis of these relationships, three hypotheses seem plausible:
( a ) astaxanthin is a precursor of vitamin A, as suggested by Gran
gaud (1950-51);
( b ) vitamin A is preformed by Zooplankton and constitutes a source for the building of axerophthol for the fish, as suggested by Goodwin
(1952);
( c ) copepod C r u s t a c e a contain a noncarotenoid precursor of vitamin A, as maintained by Lane (1950).
The fat-soluble vitamin A follows the pathways of body lipids in its anatomical localization. It is found in the fats of the viscera, muscles, membranes, but above all in the liver oils. Table X I V shows the dis
tribution of vitamin A in the pilchard (Black and Schwartz, 1950).
TABLE XIV
DISTRIBUTION OF O I L AND VITAMIN A IN THE PILCHARD** (S. AFRICA) Vitamin A Oil in organ in the Weight of the Oil Oil in the in % of whole Ex organ in % of in the organ in % total
fish amined weight of the organ of total oil vitamin A Date (%) organs whole fish ( % ) in fish in fish
10-16-45 3.7 Head 16.9 6.7 30.3 1.7
Body 69.2 3.3 61.7 5.2
Liver 2.5 4.0 2.7 69.0
Other viscera 11.3 1.7 5.2 24.3
5-26-47 14.2 Head 18.0 10.1 12.8 5.8
Body 68.1 14.0 66.9 19.9
Liver 1.1 5.1 0.4 49.3
Other viscera 12.8 22.1 12.8 25.7
a Source: Black and Schwartz (1950).
The hepatic reserve of vitamin A can be considerable in certain species and, as a rule, aquatic animals have much larger stores than mammals (except polar mammals) and birds. In large-size livers, as in those of sharks, the distribution of vitamin A is uneven (Bucher et al., 1948). In Squalus suckleyi, differences are found ranging from 13,300 international units per gram of oil in the exterior parts of the hepatic lobes to 16,900 units in the parts close to the gall bladder (Butler, 1948).
These variations explain the difficulty encountered in processing repre
sentative samples for establishing the average vitamin A content of the liver. Part of the vitamin A present in shark livers may be bound to proteins (Swain, 1944; Kini, 1945; Kini and Chidambaram, 1947).
As is the case with the fat content of liver, the amount of vitamin A depends on a number of factors:
( a ) Size and weight: Large-sized specimens show more of a trend to store than do smaller-sized ones. This is the case with both bony and cartilaginous fishes (MacPherson, 1933). Liver oil from Eopsetta jordani contained approximately 5,000 USP per gram in specimens measuring 33-40 cm. but 23,500 units in those with a length of 49 cm. An exception to this rule is found in liver oil of Genypterus blacodes, the vitamin
con-6. ORGANIC CONSTITUENTS OF FISH 181 tent of which remained stable regardless of size. The oil content varies in the viscera in much the same way as for liver oils.
( b ) Sex: The differences between sexes are sometimes very appre
ciable and in other cases absent. According to Ripley and Bolomey (1946), liver oil of the soup-fin shark, contains three times more vitamin A in males than in females. Archer (1949) finds no difference in the vitamin content of male and female South African hake, when the individual specimens are of the same size. In many species the liver of the female is richer than that of the male. This is the case for Squalus acanthias (Templeman, 1944), Squalus suckleyi (Pugsley, 1939), grey mullet, Australian salmon, and barracuda (Jowett and Davies, 1938).
( c ) The sexual stage: This factor is often predominant. Sanford et al. (1949) inform about three cases in which livers of sexually immature Apristurus brunneus did not contain vitamin A in spite of its abundance in this species. Ripley and Bolomey (1946), studying soup-fin shark, show that females carrying well-developed embryos furnish a liver oil which is considerably richer in vitamin A (190,000-260,000 I.U./g.) than the females in a less-advanced stage of pregnancy (65,000 I.U./g.). This fact is less obvious in Squalus acanthias (Templeman, 1944).
(d) Seasonal variations: Besides the intrinsic influences presented above, the vitamin content of liver oils depends on environmental con
ditions, especially water temperature, availability and quality of nutri
ents. Taken all together, these factors cause a great variation in the vitamin A content of livers as to seasons and fishing places. Gradually, as cod fishing advances, i.e., from May to August for the Pacific, the yield of liver oil steadily increases, while the vitamin A content de
creases. In July and in the beginning of August, the quantity of vitamin per liver unit stays almost stable. The influence of feeding conditions was fully proved by Lovern (1935): in the Atlantic halibut the impres
sive increase in the vitamin A content of the livers starting in the month of May corresponds to a growing abundance of diatoms, particularly rich in carotene. A second maximum appears in September at the peak of the season for the plankton crustaceans. This, too, explains variations in regard to fishing localities. According to Archer (1949), livers of the female South African hake from northern latitudes have a lower vitamin A content (1.87 million units per kilogram) than livers from southern animals ( 5 million units per kilogram). Oil from the shark Laemargus macrocephalus contains up to 8,100 I.U./g. in specimens from the coast of the Faeroe Islands and only 1,900 I.U. for those from the east coast
of Greenland at 69° north (Aure et al, 1951). Pugsley (1938) finds differences from one to three times (40-120 S.P.U./g.) in the A vitamin values of body oil of herring caught the same day (October 15, 1936) at different points off the coast of British Columbia.
Fish liver oils were widely utilized as a source of vitamin A up to recent years, when a strong competition started from synthetically made vitamin A. Leading sources so far have been livers of cod, halibut, and tuna. For the benefit of further development research, the list of refer
ences shown in the tabulation below has been compiled for liver oils.
General Butler (1946, 1948)
Creac'h (1955) Grangaud (1950)
Inhoffen and Pommer (1954) Cod (Norway) Braekkan and Lambertsen (1952) Cod (Bering Sea) Sanford et al (1950b)
Flounder (Bering Sea) Sanford et al (1950b)
Grayfish Sanford et al (1950a)
Sharks Pradhan and Magar (1956)
Brazilian fishes Carvalho Rios (1954) Indian fresh-water fishes Balasundaram et al (1956a) Indian marine fishes Balasundaram et al (1956b) Marine mammals Schmidt-Hebbel (1950) Marine sea Hons Sanford (1949)
A detailed discussion on the occurrence of vitamin A in other body organs of fish is found in the special chapter on vitamin C (Chapter 13 by Higashi). Only mention may be made here of the fact that roe and milt, so rich in B-complex vitamins, are very poor in vitamin A.
The flesh of lean fish is undisputably poor in vitamin A and may even be completely devoid of this vitamin. This holds true for ling, coalfish, and whiting (Bailey, 1950; Causeret, 1950). A hundred grams of cod fillet contain hardly more than 50 I.U. (Lunde, 1939), and the tables of FAO, 1954, estimate this quantity as negligible and indicate it as zero. The flesh of fat fish is considerably richer; the above-mentioned tables give the average content as 100 I.U. per 100 g. of edible flesh.
In general, meat of fatty fishes has a higher vitamin A content than cattle meat. Table X V lists some analytical data in this respect [see also Bailey (1931) as regards salmon].
Canning and smoking do not involve noticeable losses in vitamin A (Lovern, 1943). Changes in processing are further analyzed in Volume II, Chapter 6.
6. ORGANIC CONSTITUENTS OF FISH 183 TABLE X V