The osmotic adjustment in the euryhaline teleosts, the flounder, Pleuronectes flesus L. and the three-spined stickleback, Gasterosteus aculeatus L.

Comp. Biochem. Physiol., 1965, Vol. 15, pp. 283 to 292. Pergamon Press Ltd. Printed in Great Britam

THE OSMOTIC ADJUSTMENT IN THE EURYHALINE TELEOSTS, THE FLOUNDER, P L E U R O N E C T E S F L E S U S L. AND THE THREE-SPINED STICKLEBACK, G A S T E R O S T E U S A C U L E A T U S L. R. L A N G E and K. F U G E L L I Institute of Zoophysiology, University of Oslo, Blindern, Norway (Received 10 February 1965)

A b s t r a c t - - 1 . The euryhaline teleosts, the flounder, Pleuronectesflesus, and the three-spined stickleback, Gasterosteus aculeatus, evidently possess the system

of intracellular osmotic regulation. 2. This system implies that cells, which have been exposed to a variation of the osmolarity in the surrounding fluids, possess the ability of volume regulation with a concomitant adjustment of the number of intracellular osmotically active particles. 3. The active role of the free ninhydrin-positive substances, as well as of trimethylamine oxide, in the intracellular osmotic regulations of muscle cells in these fishes, are demonstrated. INTRODUCTION THE freezing point of serum of euryhaline teleosts is consistently lower when the fish lives in sea water than when it lives in fresh water (Black, 1957). Such findings imply that the cells of these fishes might be exposed to variations in the osmolarity of their surrounding fluids. On the assumption that the body fluids and the cells of fishes are isosmotic, a striking similarity exists between the osmotic demands to. the cells of the homoiosmotic euryhaline teleosts and the isosmotic euryhaline invertebrates. In the euryhaline invertebrates which adjust osmotically with volume regulation, the osmotic regulations may be ascribed to the interaction of two different regulatory systems (Florkin, 1961-62; Lange, 1964). The extraceUular system regulates the volume and composition of the animal's extracellular fluids by means of specialized excretory organs. With few exceptions, the study of osmotic regulations in animals has been devoted to the elucidation of this system (see, e.g. Ports & Parry, 1964). The intracellular system is responsible for the proper osmotic regulations of the animal's cells--the isosmotic intracellular regulation (Florkin, 1961-62). It has been suggested that the intracellular osmotic regulation might involve three subsequent steps, the osmotic, the intermediate and the regulatory step (Lange,. 1964; and Fig. 1). 283

284

R. LANGEAND K. FUGELLI

D u r i n g the regulatory step the volumes of the cells are adjusted to their original sizes, and, simultaneously, the cells adjust their n u m b e r of osmotically active particles, which in the invertebrates so far studied have been s h o w n to consist largely of free ninhydrin-positive substances (Florkin, 1962). T h e extracellular fluids of the euryhaline invertebrates have almost the same total concentration of inorganic ions as that of the s u r r o u n d i n g sea water. T h e composition of the osmotically active particles of the cells are definitely different f r o m those of these fluids. F o r these reasons the intracellular system of osmotic regulations seems to be the d o m i n a t i n g o s m o r e g u l a t o r y system in the isosmotic invertebrates. T h e main o s m o r e g u l a t o r y role is taken over by the extracellular system in the h o m o i o s m o t i c teleosts, since the excretory organs of these animals are capable of maintaining the animal's osmolarity at an almost constant level and nearly independent of the s u r r o u n d i n g water. However, the fact that variations of the osmolarity of these fishes m a y occur, o p e n e d the possibility to demonstrate w h e t h e r or not the intracellular system of osmotic regulation is present also in vertebrates. T h e p u r p o s e of the present paper is thus to elucidate this question by experiments on the euryhaline teleosts, the flounder, Pleuronectes flesus, and the stickleback, Gasterosteus aculeatus, respectively. MATERIALS AND METHODS Newly caught flounders, Pleuronectes flesus, with a length of 26-30 cm, were obtained from the BiologicalStati on, University of Oslo, Droebak ,in October. On arrival at this laboratory they were separated into two groups of fishes. One group was placed in a refrigerated sea-water aquarium (Temp. = 5°C, freezing point = -1.88°C). The second group was acclimatized for 2 days in an aquarium containing 50% sea water, then subsequently transferred to the freshwater aquarium (Temp. = 5°C, f.p. = -0'01°C). The flounders were kept in the sea-water and freshwater aquaria, respectively, for at least 10 days before they were used for experimental purposes. Sticklebacks (Gasterosteus aculeatus) were caught in brackish water at Sandvika in May, which is the time of migration to fresh water at this location of the Oslofjord. The sticklebacks were, on arrival at the laboratory, separated into two groups and placed in the sea-water and the freshwater aquaria respectively. The sticklebacks were kept for at least 3 weeks in these aquaria before they were used for experimental purposes. Neither the flounders nor the sticklebacks were fed during their period of captivity. In order to avoid experimental disturbances, which might have been introduced by a different period of starvation, the fishes used in the experiments were alternatively taken from seawater and freshwater aquaria. All fishes were in good condition throughout their period of captivity. Physical methods. Freezing points of the water in the aquaria, and of sera obtained by heart punctures of the fishes, were determined according to the technique described by Ramsay (1949). All determinations were made in duplicate. The water content of the muscle tissue was determined on approximately 400 mg samples of tissue. The samples were taken from the epaxial musculature of the flounders, then carefully dried between two pieces of filter paper and quickly transferred to glassstoppered vials. The water content was determined from the difference in weight before and after drying at 105°C for 24 hr. Five samples of the musculature of each flounder were used, and the average of these gave the water content of that muscle. Due to the size of the sticklebacks all the axial musclature from each fish had to be used either for the water

THE OSMOTIC ADJUSTMENT IN EURYHALnNE TELEOSTS

285

determinations or for the chemical analyses. In the flounders, however, all the different analyses were done on each separate fish, with the exceptions of the analyses of the ionic content and the specific-weight determinations. The specific weight of the muscle tissue of the flounderswas calculated from the tissue

wet weight and its buoyancy, which was estimated by weighing the tissue immersed in liquid paraffin with known density. Chemical analyses. The total free ninhydrin-positive substances and trimethylamine oxide were determined in protein-free extracts of muscle tissues obtained by the following procedures. Aqueous homogenates (10% w/v) of muscle tissue were made by homogenization for 10 rain in a Potter-Elvehjem glass homogenizer. Protein-free extracts of the homogenates and of plasma were obtained according to the method of Kalman & Lombrozo (1961), and the total free ninhydrin-positive substances was determined by the ninhydrin procedure of Moore & Stein (1948). At least three different volumes (amounts) of each protein-free extract were analysed, and each of these in triplicate. Qualitatively analyses of protein-free extracts of the flounder muscle ("Thin-layer chromatography", butanol:acetic-acid:water) revealed five dominant spots, which tentatively are determined to be: isoleucin-leucin, valine, alanine, unknown and lysine. Since the free ninhydrin-positive substances, however, exert their effect as osmoregulating particles primarily through their total quantity, the analyses have subsequently been restricted to determination of their total concentration. Aqueous homogenates (15% w/v) of muscle tissue were made by grinding the tissue in a mortar with quartz sand. Tungstic acid extracts of the homogenates and blood plasma were prepared according to the method of Cohen & Krupp (1958). The trimethylamine oxide concentration of muscle tissue and of blood plasma were determined from triplicate analyses of the protein-free extracts according to the method described by Conway (1962) with the modifications of Cohen & Krupp (1958). The sodium and the potassium concentrations of muscle tissue and of blood plasma were determined by means of an Eel flame photometer. The muscle tissues analysed were identical to the dried samples of the water content determinations described above. The samples had, prior to the analyses of the ions, been digested by concentrated nitric acid and subsequently diluted to contain no more than 5 and 10 ppm of sodium and potassium, respectively. The standard solutions of these ions used contained the same proportion of sodium, potassium and nitric acid as the samples to be analysed. The chloride concentrations were determined according to the method of Schales & Schales (1941). It was titrated directly in blood plasma and in the aqueous tissue homogenates.

RESULTS AND DISCUSSION

The apparent tissue water It is known that the water content of tissues of euryhaline fishes is higher when the fishes live in fresh water than when they live in sea water (Gordon, 1959; Parry, 1961; and Table 1). T h e difference observed has been interpreted as a change in the water content of the fish cells (Gordon, 1959), respectively as a change of the extracellular space (Houston, 1959; Parry, 1961). However, the possibility exists that the differences observed might be ascribed to the known change of the animals' osmolarity, similar to that of the comparable observations on the isosmotic sea urchin, Strongylocentrotus droebachiensis, for reasons shortly reviewed below (Lange, 1964). I9

286

g . L A N G E AND

K.

FUGELLI

T h e water content of animal tissues is generally measured as g water/100 g wet tissue. T h e result obtained is therefore dependent on the specific weight of the tissue. Since the specific weight is determined by the constituents of the tissue itself, it is clear that a variation in the concentration of any of the tissue components gives rise to an apparent change of the water content of the tissue. T A B L E 1 - - S O M E EFFECTS OF TRANSFERRING THE FLOUNDER,

Pleuronectes flesus,

AND THE

STICKLEBACK, Gasterosteus aculeatus, FROM SEA WATER TO FRESH WATER

Fish

Ext. milieu

Serum mOsM* _+S.D.

Flounder

Sea water Fresh water

Stickleback

Sea water Fresh water

Muscle g H20/lO0 g wet w t _+S.D.

Sp. wt _+S.D.

364 + 7(8)t 304+ 13(5)

80'57 ± 0"15(8) 81'24_+0'15(5)

1'0574 + 4 x 10-3(5) 1.0565-+4x 10 3(2)

340 + 17(6) 290 +_11(4)

80'25 _+0'20(8) 82"01 -+0-10(7)

* Calculated from the freezing point of serum. t Number of fishes analysed in parentheses. As mentioned in the Introduction, the osmotic adjustment of the cell volumes is accompanied by a concomitant regulation of the n u m b e r of osmotically active particles in the cells (Fig. 1). T h e concentration of these particles will for this reason display a correlation to the animal's osmolarity. Since the specific weight of OSMOLARITY OF EXTRACELLULAR

OSMOLARITY OF EXTRACELLULAR

FLUID

FLUID

100%

f

© VOLUME = 1

75"/. (H20) n

VOLUME--I

, ~

VOLUME =1 * A

?

(H20)n* x

VOLUME-- 1+ A

VOLUME=I

FIG. 1. The suggested steps of the intracellular osmotic regulation (according to Lange, 1964). 1. The osmotic step. Isosmotic conditions between the extra- and intracellular fluids are primarily re-established by dilution of the osmotically active particles of the cell. II. The intermediate step. Time consumed by suggested unknown metabolic processes. III. The regulatory step. Regulation of the cell volume to its original size and the simultaneous adjustment of the number of osmotically active particles of the cell.

THE OSMOTIC ADJUSTMENT IN EURYHALINE TELEOSTS

287

the tissue is dependent on the concentration of the osmotically active particles of the tissue too, the result is that the water content of a tissue apparently varies with the animal's osmolarity even when the animal's volume regulation is complete. Quantitatively the difference observed is primarily determined by the change of the animal's osmolarity, and secondarily by the nature and mean molecular weight of the osmotically active particles. Thus, when a tissue content, e.g. of water, is measured in per cent of wet tissue, the value observed should preferentially be given in relation to the animal's osmolarity. Furthermore, a correct interpretation of the significance of an observed difference in, for example, tissue water has to be evaluated on the basis of the quantitative relationships observed. In the light of the above arguments, the data of Table 1 suggest that the euryhaline teleosts, the flounder, Pleuronectesflesus, and the stickleback, Gasterosteus aculeatus, adjust osmotically with volume regulation. If volume regulation had been completely absent, the differences between the tissue water of the sea-water and the freshwater fishes would have been larger, as shown in the following example. The mean serum osmolarity of sea-water flounders was found to be 364 mOsM, which after transfer and adaptation of flounders to fresh water, decreased to 304 mOsM (Table 1). On the assumption that blood, other tissue fluids and cells are isosmotic, the observed decrease in serum osmolarity leads to an osmotic movement of water into the cells. If the cells were incapable of volume regulation, the increment of cell water due to osmosis would have persisted. This increment can be calculated on the basis of the data of Table 1. The volume of water/ml "sea water muscle": W = 1057'4 mg/ml x 0"8057 mg H20/mg wet tissue, W -- 852"3 mg H~O/ml tissue, V-- 852-3/~1 H~O/ml tissue (sp. wt. of water = 1"0). The transfer of flounders to fresh water will lead to an increase of the tissue-water according to the following calculations: 852.3/~1 x 364 mOsM 304 mOsM h = 1020"5/~1. Hence, the increase in tissue-water/ml of "sea water tissue" is: H = 1020.5/,1-852.3/,1 H = 168"2/~1. h--

According to the calculations each ml of muscle tissue would, after transfer of the flounders to fresh water, swell to about 1.2 ml in complete absence of volume regulation. The original weight of 1 ml muscle-l.0574 g (Table 1), would correspondingly increase to 1.2256g (1.0574g+0.1682g); and the specific weight decrease to 1.0490. In this event the apparent water content of tissue of freshwater adapted flounders is 83.27 per cent. This value yields an increase in the apparent water content more than four times that observed (Table 1). Evidently therefore,

288

R . L A N G E AND K . F U G E L L I

the flounder, Pleuronectesflesus, possesses the property of volume regulation. The data of Table 1 are furthermore compatible with a complete volume regulation if the minimum mean molecular weight of the particles used in the osmotic regulation of the tissue is about 150 (calculated on the basis of the data of Table 1). The data therefore suggest that the fraction of the small molecular organic compounds of the total amount of osmotically active particles in the muscle tissue is large. For reasons equivalent to those above, the data on the stickleback, Gasterosteus aeuleatus (Table 1), are interpreted as those on the flounder.

Intracellular solutes In Table 2 the concentrations of sodium, potassium and chloride in the muscle tissue and the blood plasma of sea-water and fresh-water adapted flounders, are given. The concentrations found are comparable to those reported for other fishes (Gordon, 1959). In the light of the present problem, however, interesting information can be obtained from the pattern of these ions. It can be seen (Table 2) that the difference in osrnolarity between sea-water and freshwater adapted flounders is adequately accounted for in the serum by the difference in the concentration of sodium chloride. In the muscle tissue, however, the concentrations of the ions studied were almost unaffected by the change of the TABLE 2--THE FLOUNDER,

CONCENTRATIONS OF NA, K AND CL IN MUSCLE TISSUE AND PLASMA OF THE

Pleuronectes flesus,

LIVING IN SEA WATER AND FRESH WATER RESPECTIVELY

Sea-water fishes Serum mOsM* + S.D.

Fresh-water fishes

364 + 7(8)t

304 _+11(5)

Na K C1

193'7 + 2"4(7) 5.4 ± 0.2(7) 166-1± 11.6(4)

156"8 +_8"2(4) 5.1 + 1.3(3) 113.7+_22.0(3)

Muscle mM/kg tissue water + S.D. Na K C1

14-7 +_1-4(4) 157.7+_10.4(4) 41.5 +_3.4(3)

10"2 + 1-9(3) 156-7_+13.2(3) 30.0 + 6.1(3)

Plasma mM/1 + S.D.

* Calculated from the freezing point of serum. t Number of fishes analysed in parentheses. osmolarity. This finding is in accordance with the above suggestion that the tonicity of the cells is maintained to a large extent by means of organic molecules. The suggestion thus implies that the cellular response to a variation of the osmolarity of the surrounding fluids involves a variation of the intracellular concentration of organic molecules. The data of Table 2 may also give an idea of the relative extracellular space of the muscle tissue of the flounders. Thus, on the assumption that the sodium ions

THE OSMOTIC ADJUSTMENT IN EURYHALINE TELEOSTS

289

are evenly distributed in the blood plasma and the extracellular fluids of the muscle tissue, the observed distribution of sodium indicates that the relative volume of the extracellular fluids in flounders which had lived in sea water, as well as in the fresh-water adapted specimens, is less than 5 per cent. This value is in agreement with the finding of Gordon (1959), who reported the relative extracellular space of the muscle tissue of Salmo trutta to be 5-7 per cent. Since the extracellular volume of muscle tissue of fishes apparently is small in relation to the intracellular volume, the concentrations of different compounds of the muscle tissue are subsequently taken as expressions of the intracellular concentrations. The possible roles of NPS* and TMAO~ in the osmotic adjustment of fishes have been the subject for several previous investigations (Shewan, 1951 ; Velankar & Govindan, 1958; Cowey et al., 1962), but their roles were doubted because the amount of the amino acids measured seemed too low in relation to the total osmolarity of the fish (Cowey et al., 1962), and since inconsistencies in the expected correlation between the T M A O content of the fish and the osmolarity of the surrounding water were found (Shewan, 1951; Parry, 1961). Evidently, however, the osmolarity of a fish is not immediately determined by the osmolarity of the surrounding water (Gordon, 1959; and Table 3). For this and other reasons it seemed interesting to reinvestigate the possible role of NPS and T M A O in the osmotic adjustments of the flounder and the stickleback. In Table 3, data on the concentrations of NPS and T M A O in the muscle tissue and blood plasma of the flounder and the stickleback, respectively, are given in relation to the osmolarity of the serum of the fishes. The concentrations of NPS as well as T M A O found, fall in the range of those previously reported (Shewan, 1951; Parry, 1961). In conformation with the previous studies, the present data demonstrate that NPS as well as T M A O are preferentially distributed to the muscle tissue of the fishes. In contrast to the previous investigations, however, the tissue concentrations of NPS as well as T M A O are definitely higher in the fishes which had lived in sea water than in freshwater adapted fishes (Table 3). On an average, the serum osmolarities of the freshwater adapted flounders and sticklebacks were respectively 62 and 50 mOsM lower than the serum osmolarities of the sea-water fishes (Table 3). The question is therefore to what extent the corresponding differences in the cells of these fishes can be accounted for by the changes in the concentrations of NPS and TMAO. It can be seen that on an average, the differences are 43 mM/kg tissue water in the flounders and 21 mM/kg tissue water in the sticklebacks, or, NPS and T M A O account for about 70 and 40 per cent of the total difference in the flounder and the stickleback, respectively. In this comparison molarities of the cells have been related to osmolarities of the sera. Since NPS, at least partly, are dissociated, the above figures express minimum values of the quantitative role of NPS and T M A O in the establishment of the osmotic pressure of the cells. The possibility has, however, to be considered that other organic compounds participate in the osmotic equilibration between the cells * NPS = total, free ninhydrin positive substances. t TMAO=trimethylamine oxide.

290

R. LANGEAND K. FUGELLI

and the surrounding fluids of these fishes. Since the difference between the osmolarity of the extra- and intracellular fluids in all probability is further reduced by a higher total intracellular concentration of inorganic ions in the sea-water fishes than in the fresh-water fishes, it appears unlikely that these other organic compounds are more important than N P S and T M A O in this respect. On the assumption that the cells are isosmotic to the surrounding fluids, a linear correlation between the total activity of intracellular solutes and the osmolarity of the blood be will observed. I n Fig. 2 the sum of the tissue concentrations of N P S and T M A O in the flounders are related to the osmolarity, and, as can be TABLE 3--THE

DECREASE OF

NPS*

AND OF

SERUM O S M O L A R I T Y I N THE FLOUNDER,

aculeatu$, RESPECTIVELY,

Fish

Flounder

Ext. milieu

Sea water

I N MUSCLE TISSUE AND PLASMA, AND OF AND THE STICKLEBACK,

Gasterosteus

AFTER A D A P T A T I O N OF THE FISHES TO FRESH WATER

Serum mOsM~: + S.D.

Muscle mMoles/kg tissue water +S.D. NPS

TMAO

Plasma mM NPS

TMAO

379 360 362 364

70'6 84-1 73'6 56'8

30'6 21 "4 27'9 41-2

5'0 5"3 2'7 3"0

---0'7

366 +_8

71"3 + 11'4

30'3 _+8-2

4.0

0"7

304 293 316 304___11

39'4 44"2 49'0 44'2+_4"8

14"3 9-4 19'5 14.4+5"0

3"3 3-2 3"0 3-2

0"4 --0"4

Sea water

340 _+17

83"3+_8-9

20"0 + 1-7

7'2

2"5

1"88°C Fresh water -0-01°C

(6) § 290 +_11 (4)

(13) 66'6 _+11"9 (13)

(11) 10"5+ 3"9 (12)

(10) 5"3 (12)

(4) --

- 1"88°C

Fresh water -0'01°C Stickleback

TMAOt

Pleuronectes flesus,

-

* N P S = total, free ninhydrin positive substances measured as taurine equivalents. t TMAO = trimethylamine oxide. + Calculated from the freezing point of serum. § Number of fishes analysed in the parentheses. seen, the sum fits well to such a linearity, contrary to the separate concemrations. T h e individual variations observed in the relative composition of N P S and T M A O in the flounder muscle, which suggest a differentiated cellular response to osmotic demands, might be one of the reasons for the failure of the previous attempts to demonstrate an osmoregulatory role of these substances in fish tissues. In summary then, the data presented above strongly indicate that the teleosts, the flounder, Pleuronectesflesus, and the stickleback, Gasterosteus aculeatus, possess the system of intracellular osmotic regulation. Evidently, the cells of these fishes

THE O S M O T I C A D J U S T M E N T I N E U R Y H A L I N E TELEOSTS

291

are able to regulate their volume (Table 2). Furthermore, since the data of T a b l e 3 (on the differences in osmolarity and concentrations of N P S and T M A O ) fit reasonably well with the prediction made on the basis of T a b l e 2 (on the differences of apparent water content and specific weights), it seems warranted to conclude

;

110

, TMAO

~---~

NPS

,,T 9O

70

v- L ~s0F

lO

T T ,,

*

I

300

';'

.

.

.

.

320

.

.

.

340

L---

,

360

380

SERUM 0SMOLARtTY, m 0 s M

FIG. 2. T h e concentrations of N P S and T M A O

of the muscle tissue in relation

to serum osmolarity of the flounder, Pleuronectes flesus. NPS = Total, free ninhydrin positive substances, determined as taurine equivalents. TMAO = Trimethylamine oxide. Serum osmolarity calculated from the freezing point. Each column represents data obtained on one single fish. that the volume regulation of the muscle cells of these fishes is complete, i.e. that the cell volumes are of equal sizes in the sea-water and in the freshwater adapted fishes, respectively. REFERENCES BLACKV. S. (1957) Excretion and osmoregulation. In The Physiology of Fishes (Edited by BROWN M. E.), Vol. I. Academic Press, New York. COHEN J. J., KRUPP M. A. & CHIDSEYC. A. (1958) Renal conservation of trimethylamine oxide by the spiny dogfish, Squalus acanthias. Amer. J. Physiol. 194, 229-235. CONWAYE. J. (1962) Microdiffusion Analysis and Volumetric Error, 5th ed. Crosby Lockwood & Son Ltd., London. COWEY C. B., DAISLEY K. W. & PARRY G. (1962) Study of amino acids, free or as components of protein, and of some B-vitamins in the tissues of the atlantic salmon (Salmo salar) during spawning migration. Comp. Biochem. Physiol. 7, 29-38. FLORK1N M. (1961-62) Regulation anisosmotique extracellulaire regulation isosmotique intracellulaire et euryhalinite. Ann. Soc. Zool. Belg. 92, 183-186. FLORKIN M. (1962) La regulation isosmotique intracellulaire chez les Invertebres matins euryhalins. Bull. Acad. Belg. CI. Sci. 48, 687-694. GORDON M. S. (1959) Ionic regulation in the brown trout (Salmo trutta L.). J. Exp. Biol. 36, 227-252. HOUSTON A. H. (1959) Osmoregulatory adaptation of steelhead trout (Salmo gairdnerii Richardson) to sea water. Canad. J. Zool. 37, 729-748.

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R. LANGEAND K. FUGELLI

KALMAN S. M. & LOMBROZO M. E. (1961) T h e effect of estradiol on the free amino acids of the rat uterus, ft. Pharmacol. Exp. Ther. 131, 265-269. LANGE R. (1964) The osmotic adjustment in the echinoderm, Strongylocentrotus droebachiensis. Comp. Biochem. Physiol. 13, 205-216. MOORE S. & STEIN W. H. (1948) Photometric ninhydrin method for use in the chromatography of amino acids..7. Biol. Chem. 176, 367-388. PARRY G. (1961) Osmotic and ionic changes in blood and muscle of migrating salmonids. ft. Exp. Biol. 38, 411427. POTTS W. T. & PARRYG. (1964) Osmotic and Ionic Regulation in Anbnals. Pergamon Press, Oxford. RAMSAY J. A. (1949) A new method of freezing-point determination for small quantities. .7. Exp. Biol. 26, 57-64. SCHALES O. & SCHALES S. (1941) A simple and accurate method for determination of chloride in biological fluids, ft. Biol. Chem. 140, 879-884. SHEWAN J. M. (1951) The chemistry and metabolism of the nitrogenous extractives in fishes. In The Biochemistry of Fishes. Biochem. Soc. Symposia (Cambridge), No. 6, pp. 28-48. VALENKAR N. K. & GOVINDAN T. K. (1958) A preliminary study of the distribution of non-protein nitrogen in some marine fishes and invertebrates. Indian Acad. Sci. Proc. Sect. B. 47, 202-209.