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The connective tissues and collagens of cod during starvation
Comp. Biochem. Physiol., 1976, Vol. 55B, pp. 487 to 492. Peroamon Press. Printed in Great Britain
THE CONNECTIVE TISSUES AND COLLAGENS OF COD DURING STARVATION* R. M. LOVE, K. YAMAGUCHI,Y. C"REAC'HAND J. LAVI~TY Torry Research Station, Aberdeen, Scotland (Received 25 March 1976) Abstract--1. Starvation causes the myocommata and skin of cod to thicken. 2. Collagen prepared from the thickened tissue appears to have identical properties to normal collagen-molecular shape, intramolecular crosslinking, amino and imino acid composition and thermal denaturation temperature. An exception is the intermolecular crosslinking which appears to be greater in starving myocomma collagen (in skin there was again no difference). 3. The collagen of cod seems to have a higher turnover throughout life than mammalian collagen. 4. Apart from any effects of starvation, it is found that the force required to break a unit thickness of myocomma decreases in older fish, but during life the fish more than compensate for this by increasing the thickness.
INTRODUCTION After mobilising some of their reserve lipids and carbohydrates, starving fish draw upon the contractile proteins of their body muscle to maintain life: the subject has been reviewed by Love (1970). The cells of the musculature are seen to become thinner (Love et al., 1968) and in extreme cases even the myofilamerits become disorganised or disappear (Gas, 1972). Connective tissue, however, is usually regarded as being stable, and although collagen from mouse skin is mobilised to some extent during subsistence on a protein-free diet (Harkness et al., 1958; Sobel et al., 1967), most publications describe increases in the proportion of animal collagen during starvation. These "increases" are in fact relative and at least partly the result of other constituents being preferentiaUy utilised (e.g. Thompson & Farragut, 1965). On the other hand, a thickening of the skin of Clupea harengus during depletion (Hughes, 1963) cannot be explained in this way, and a report by Lav6ty & Love (1972), that the myocommata of cod actually become mechanically stronger as starvation progresses, raises the possibility that more collagen might in fact be present. The purpose of the present work is to investigate the properties of cod connective tissues during starvation and to study some of the pure collagen isolated therefrom. MATERIAL AND METHODS Cod (Gadus morhua L.) caught near Aberdeen were kept for various periods without food in an aquarium at 9°C. The procedures for obtaining the myocommata and of measuring the water content and breaking stress have been described earlier (Lav6ty & Love, 1972). Strips of myocommata or skin 1 cm wide were used, and their thickness was determined by weight. A value of 1.09 was obtained for the specific gravity of both myocommata and skin from both starved and fed cod, so this value was used in the calculations. * Crown Copyright Reserved. C.Ba'.(B)55/4~--B
487
Acid-soluble collagen was prepared by a modification of the method of Lewis & Piez (1964), descried by Yamaguchi et al. (1976), and all the other experimental techniques are as described in the latter publication.
RESULTS Thickness o f myocommata A simple time scale of weeks without food is not suitable for plotting against properties of starving fish tissues, since the energy reserves of newly-caught fish vary from fish to fish, and the lag period during which the lipids and carbohydrates are utilised with no change in the protein is correspondingly variable. Figure 1 therefore shows the thickness of the myocommata on the basis of muscle water content, which rises above a "nourished" range of 80.0-80.9 in North Sea cod (Love, 1960) concurrently with the start of depletion of contractile proteins. The symbols increase in size with increase in water content. The fish have also been arbitrarily divided into normal or appreciably starved (up to 83.9% water) and more severely starved (84% and above) groups. Regressions of myocomma thickness (T) on body length (L) which were calculated for both groups (T = 0 . 4 0 7 L - 0.33 for starved fish, T = 0 . 3 3 3 L - 7.14 for better nourished fish, with standard deviations about the lines of 8.43 and 6.98 respectively) are shown in Fig. 1. For both groups the regressions were significant (correlation coefficients for the 41 severely starved fish being +0.59 and for the 86 more normal fish being +0.52 (P < 0.01 for both sets). The slopes of the 2 lines do not differ significantly but the lines are significantly separate from each other (P < 0.01) so we can now say definitely that myocommata thicken during starvation. The thickening of myocommata as cod grow has been reported earlier by Love (1970) who measured histological stained sections of muscle containing myocommata with a micrometer eyepiece in a microscope. Figure 2 shows observations made on the skin of the same fish. In contrast to rnyocommata, there was
488
R, M. Lov~ et al. Muscle water % 0"61 ? 90.0 - 9 1 . 9 88"0-89-9
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Fig. 1. Thickness of myocommata dissected from the thickest part of the musculature of cod of different body lengths, and undergoing different degrees of starvation. Each point is the mean of 5 determinations and the size of the symbol used indicates the severity of starvation as measured by the muscle water content--the bigger the symbol, the more severely starved. no significant correlation between body length and skin thickness. However, the mean thickness of the skin of the severely starved fish (0.86me; mean fish length= 72cm) is significantly (P <0.01) greater than the mean thickness of normal or somewhat starved fish (0.67 m e ; mean fish length = 74 cm).
related with muscle water content (correlation coefficient +0.34, P < 0.01). This means that myocommata are thickened during starvation with a mechanically stronger collagen than was present when the fish were adequately nourished; such collagen contains more intermolecular crosslinks. The breaking stress of a unit thickness of skin, on the other hand, did not change during starvation. The correlation coefficient of muscle water content on the breaking stress of the myocommata at constant thickness was +0.32 (P < 0.01) after adjusting
Mechanical strength
The tissues illustrated in Figs. 1 and 2 were then pulled until they broke. It was found that the force required to break a unit thickness of myocomma cor1.4- i90'0-91.g • 88.0- 8g.g Muscle - i 86.0-87-9 • 84.0_85.9 water 1.2-= 82.0 _83~J % • 78.0-81.9
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Fig. 2. Thickness of skin of cod of different body lengths and undergoing different degrees of starvation. Other details as in legend to Fig. 1.
Connective tissues and collagens of cod during starvation
489
Table 1. Relative amounts of monomer (at) and dimer (fl) components of thermally denatured cod collagens as determined by sedimentation analysis Myocommata collagen ct Component fl Component % % Control Starved
73 78.5
27 21.5
Skin collagen ~t Component fl Component % % 71 66.5
29 33.5
examined in a Spinco model E analytical ultracentrifuge at 25°C at 59,780rev/min with an An-D rotor. The Schlieren patterns showed 2 peaks, corresponding with the monomeric chains (~t component) and dimers (fl component). No trimer (y component) was seen in this work. The relative amount of/~ component indicates the amount of thermally stable intramolecular crosslinking. The results obtained by cutting out and weighing paper tracings of the peaks are shown in Table 1. The effects of starvation are seen to be small and inconsistent. (e) Chemical analysis. The amino acid composition determined by a Technicon auto-analyser after acid hydrolysis is shown in Table 2. Properties of the acid-soluble collagen There appeared to be more hydroxyproline in the (a) Source. Myocommata and skin were dissected starved collagen than in the control, so as a check from a male cod 98.5 cm long starved for 4 months it was determined again, separately, by the method and killed 31 May 1972. The water content of the of Leach (1960). By this method the hydroxyproline muscle was 91.4% and the body wt without guts (Wt~o) was 7.6 + 0.02 in the controls and 8.2 + 0.17 5.96 kg. As control a similar fish was taken from the in starved myocommata, and 7.6 + 0.3 in the controls sea at the same time, measuring 96.5 cm with body and 8.1 + 0.26% in starved skin. Proline (also somewt without guts 8,96 kg. The value for muscle water what greater in starved skin, Table 2) and hydroxyproline are thought to stabilise the collagen molecule content was unfortunately lost, but was <81.5~. The tissues were comminuted and extracted twice (Gustavson, 1953) and have been shown to correlate with excess of 0.5M sodium acetate pH7.1 the with the thermal stability of the collagens of different extracts being discarded. The insoluble residue was species of fish (Takahashi & Yokoyama, 1954; Berg then extracted twice with 0.5 M acetic acid and this & Prockop, 1973; Yamaguchi et al., 1976). However, extract, which comprises about 95% of the total col- in the present work the thermal stability of the conlagen of cod (Mohr, 1971) was used, after purification, trol and starved collagens did not appear to be different. Since the hydroxyproline content had seemed to in the subsequent studies. (b) Intrinsic viscosity. The viscosity of solutions of be the only important difference between the collagens of starved and normal cod, the whole work undenatured acid-soluble collagen was determined at 7°C at 4 different concentrations (0.01-0.08%) using was repeated by one of us (Y.C.), using a greater an Ostwald viscometer, sodium citrate buffer number of experimental animals. Acid-soluble collagen was isolated separately from (I = 0.089, pH = 3.5) being the solvent. The intrinsic viscosity obtained by extrapolating the results to in- each of 4 control fish (caught 25.1.74) ranging from finite dilution was found to be identical for the col- 76 cm to 96 cm in body length (mean --- 83) and havlagens from starved and from nourished cod: 5.4 dl/g ing water contents ranging from 80.4 to 81.8~o (mean = 80.9~o). Skin and myocornmata were isolated for skin and 8.4 dl/g for myocommata. (c) Thermal denaturation temperature (To). A col- from 5 individuals starved for about 7 months, ranglagen solution (0.7 mg/ml in sodium citrate buffer) ing in length from 74 to 99 cm (mean = 83.2 cm) and with water contents ranging from 85.2 to 90.5% was maintained for 1 hr at 9°C and 5 determinations of the viscosity were then made. Similar solutions (mean = 88.4~o). The results with standard deviations, were maintained at 11, 13°C etc at 2 ° increments up and also the thermal denaturation temperatures, are to 21°C. The thermal denaturation temperature was given in Table 3. It is clear from Table 3 that the imino acid compotaken as that at which the viscosity had fallen to 50°/o sition of the collagens of starving and fed cod are of the initial value. From the graphs it was clear that the To of skin was identical for starved and nourished essentially the same, in line with the similarity of cod (15°C) and it is unlikely that the To values of denaturation temperatures. The higher hydroxypromyocommata (16.3 and 15.7°C, respectively) are signi- line values found in the starving fish of the previous experiment (Table 2) cannot be explained, but the difficantly different. (d) Sedimentation analysis. The collagen solutions ference must be assumed to be not significant since the denaturation temperatures of control and starving (concentrations 0.3-0.5%) were denatured by incubating in sodium citrate buffer at 30°C for 2 hr and then fish did not differ on that occasion either. for the effects of body length. The correlation coefficient of body length on the breaking stress of the myocommata (constant thickness) after adjusting for muscle water content was -0.26 (P < 0.01). This means that the collagen of the myocommata of older fish is weaker (less crosslinked) than that of younger fish, but in life the older fish make up for this by increasing the thickness, as already mentioned. Thus doubling the length of a cod within the range of fish studied results in an average 50%0 loss in myocomma strength (using the calculated regression: Breaking Stress = 371.5-2.23L)but an average 170Yo increase in thickness (from the lower regression line, Fig. 1).
490
R . M . LOVE et al. Table 2. Amino acid composition of collagens from single fed and starving cod killed May 1972 (residues/1000 residues) Myocommata collagen Control Starved Ala Arg Asp Glu Gly His Hyl HyPro IsLeu Leu Lys Meth Phe Pro Ser Thr Tyr Val Recovery by weight Total N g/100g
106.0 59.1 42.3 82.2 313.6 16.3 8.2 40.7 18.6 32.3 36.3 20.4 14.5 87.6 62.9 25.8 6.0 26.1 88.1~o 17.8
110.0 61.6 38.4 74.9 356.8 11.1 8.4 45.1 14.3 23.4 31.1 19.3 12.3 87.3 59.1 23.3 3.4 20.2 88.3~o 17.6
DISCUSSION The results confirm that extra collagen is laid down in the m y o c o m m a t a and skin of cod during starvat±on, but as far as can be seen the new collagen as added to and mixed with the existing structure does not differ in molecular shape (from intrinsic viscosity), amino acid composition, imino acid stabilisation (from the analytical figures and the denaturation temperature) or intramolecular crosslinking (from sedimentation analysis). The breaking strength of a
Skin collagen Control Starved 105.6 62.5 41.5 77.4 332.2 11.8 7.6 40.7 17.1 29.7 33.1 20.6 13.8 89.6 60.8 26.1 5.0 25.2 98.6~o 18.0
103.8 58.2 38.2 74.5 341.0 12.0 7.6 56.2 15.1 24.6 38.8 19.3 12.1 93.6 59.0 22.0 3.5 21.5 99.3~o 17.3
constant thickness of m y c o m m a tissue is greater, indicating rather more intermolecular crosslinking in the collagen, but skin collagens from starving and nourished fish do not differ. N o w if the connective tissues of cod thicken each winter during the depletion associated with spawning, a time must come when they are eroded in some way to bring them back to their original thickness. In this laboratory we have seen a group of what appeared to be such fish only once---a uniform haul of cod caught on the Faroe Bank in June 1966. The myo-
Table 3. Amino acid composition of collagens from fed and starving cod killed early 1974 (residues/1000 residues) Myocommata collagen Control Starved Ala Arg Asp Glu Gly His HyPro lieu Leu Lys Met Phe Pro Ser Thr Tyr Val HyPro by Leach's method (g %) Denaturation temperature (°C)
116.5 43.7 55.2 76.2 347.3 8.8 54.2 8.0 19.3 30.2 15.7 11.9 97.7 70.3 25.7 4.4 14.8
+__2.8 ± 7.3 ± 0.6 ___0.7 ___21.5 ± 0.6 ± 2.6 + 1.7 ± 4.1 ± 0.9 ± 1.6 ± 1.9 ± 1.3 ± 0.6 ± 1.0 ± 0.8 ± 1.3
111.0 + 5.4 61.0 + 17.4 54.9 ± 2.3 75.2 ± 2.3 332.8 ± 12.5 9.7 ± 2.8 51.7 ± 2.9 10.3 ± 1.8 23.8 ± 3.9 28.8 + 1.6 13.4 ± 6.2 12.8 4- 1.7 102.9 _ 9.4 67.7 ± 4.0 25.1 ± 0.9 4.8 ± 1.0 14.8 ± 0.7
Skin collagen Control Starved 106.6 ± 57.2 ± 53.0 + 74.1 ± 346.8 ± 9.1 ± 52.4 ± 8.8 ± 20.2 + 30.1 ± 16.3 ± 12.0 ± 102.8 ± 69.1 ± 24.5 ± 3.8 ± 15.2 +
2.2 8.0 1.1 2.6 23.2 0.7 4.2 2.5 4.1 0.9 2.7 1.6 7.2 1.3 0.2 0.5 0.6
108.6 ± 6.9 56.7 ± 11.1 50.8 _ 3.2 72.2 ± 2.4 332.5 _ 27.9 8.7 ___ 1.4 54.3 + 5.3 9.4 ___ 1.7 20.8 + 3.6 27.4 +__1.1 17.8 ± 4.3 12.4 ± 0.4 105.6 ± 6.5 67.2 ± 2.5 23.7 ± 1.1 3.7 +__1.3 15.2 ± 0.9
8.2 _ 0.8
8.2 _ 0.7
7.8 ± 0.1
7.4 + 0.6
13.6 + 0.6
13.6 ___0.2
12.2 + 0.3
12.7 + 0.1
Connective tissues and collagens of cod during starvation commata of these fish could not be dissected cleanly, being in an extremely soft condition, and since the muscle was still twitching the softness was not a postmortem effect. Solubility experiments showed that the fraction insoluble in both neutral sodium chloride and citrate buffer (pH 3.5) amounted to only 0.65 pg hydroxyproline/mg wet tissue compared with 4.3 pg in "normal" fish from Aberdeen Bank caught in August (Love & Lav6ty, unpublished). Our single experiment indicates that the intermolecular crosslinking can be greatly reduced under certain circumstances, and reminds us that experiments with fish collagen are likely to lead to quite different conclusions from experiments with mammalian collagen which make up the bulk of the published work on this substance. Professor V. Mohr (private communication) has also observed a seasonal variation in the crosslinking of fish collagen, and it is clear that we are not here dealing with a tissue which in mammals "serves its function for many decades with little or no turnover in the adult animal" (Piez, :1968). Collagen becomes less digestible by collagenase as cattle age (Goll et al., 1964) and has been shown to become very much less soluble in salt or dilute acid during aging in cattle (Carmichael & Lawrie, 1967a; Szeredy, 1970), rats (Nageotte & Guyon, 1934 quoted by Harkness et al., 1954; Eichhom & Butzow, 1966), pigs (Szeredy, 1970) and man (Bakerman, 1962). In 5 experiments comparing the solubility of young (ca. 45cm long) and old ( > 1 0 0 c m long) cod myocommata, J. Lav6ty (unpublished) found that in each case the NaC1 and citrate-insoluble fraction did in fact increase with age, but only fractionally as compared with the many-fold increases seen in mammals. The increase in the insoluble fraction of older fish seems inconsistent with the present finding of a decrease in mechanical strength with age. However, the increase in insolubility reflects an increase in acidstable crosslinks: there could have been a greater decrease in acid-labile crosslinks, which would reduce the overall mechanical strength but not have shown up in the solubility studies. Another pointer in Lav6ty's work is that the salt-soluble fraction, the least crosslinked material, increased slightly as the fish aged, revealing once again a very different situation from that found in mammals. The lack of obvious signs of physical aging in cod may well relate to the annual renewal of a considerable proportion of the collagen. McBride et al. (1960) noticed a seasonal variation in the collagen content of spring-spawning Pacific herring (Clupea pallasii), the highest quantity being present at the spawning time. However, a study by Hughes (1963)on spring-spawning Clupea harengus (Atlantic herring) showed a maximum amount of collagen occurring in the winter, i.e. at other than the spawning time, varying inversely with the amount of oil and so presumably reflecting the state of nutrition. Even with skins removed the musculature still showed a collagen maximum in December; this seems to be the only published report which hints at the phenomenon described in the present paper. Acknowledgements--The work described in this paper formed part of the programme of the Ministry of Agriculture, Fisheries and Food. We would like to thank Dr. A.
491
J. Bailey for reading the manuscript and making helpful suggestions. Dr Ian Mackie carried out the sedimentation analysis on our behalf. The statistical analysis of the results was done by Mr. Gordon Smith.
REFERENCES BAKERMANS. (1962) Quantitative extraction of acid soluble human skin collagen with age. Nature, Lond. 196, 375-376. BERG R. A. & PgocKoP D. J. (1973) The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem. biophys. Res. Commns 52, 115-120. CARMICHAELD. J. & LAWRm R. A. (1967) Bovine collagen-1. Changes in collagen solubility with animal age. J. Fd Technol. 2, 299-311. Eica-mo~ G. L. & BUTZOWJ. J. (1966) Physical chemical studies on the crosslinking of collagen with age. Int. Cong. Gerontol. Proc. 7th, 2, 5~. GAS N. (1972) Structural alterations in the white muscle of carp (Cyprinus carpio L.) during prolonged starvation. C.r. Acad. Sci. Paris 275, Ser. D, 1403-1406. GOLL D. E., HOEKSTRAW. G. & BRAYR. W. (1964) Ageassociated changes in bovine muscle connective tissue. 1. Rate of hydrolysis by collagenase. J. Fd Sci. 29, 608413. GUSTAVSOr~K. H. (1953) Hydrothermal stability and intermolecular organisation of collagens from mammalian and teleost skins. Hydroxyproline content and degree of interchain linking. Svensk Kern. Tidskr. 65, 70-76. HARKNKSSM. L. R., HARKNESSR. D. & JAr,ms D. W. (1958) The effect of a protein-free diet on the collagen content of mice. J. Physiol., Lond. 144, 307-313. HAgr,r~ESS R. D., MARKO A. M., MtnR H. M. & NEUBERGm~A. (1954) The metabolism of collagen and other proteins of the skin of rabbits. Biochem. J. 56, 558-569. HuGI-ms R. B. (1963) Chemical studies on the herring (Clupea harengus). VII.---Collagen and cohesiveness in heatprocessed herring, and observations on a seasonal variation in collagen content. J. Sci. Fd Agric. 14, 432-441. LAV~TV J. & LOVE R. M. (1972) The strengthening of cod connective tissue during starvation. Comp. Biochem. Physiol. 41A, 39-42. LEACH A. A. (1960) Notes on a modification of the Neuman and Logan method for the determination of the hydroxyproline. Biochem. J. 74, 70-71. LEwis M. S. & PIEz K. A. (1964) The characterisation of collagen from the skin of the dogfish shark, Squalus acanthias. J. biol. Chem. 239, 3336-3340. LoVE R. M. (1960) Water content of cod (Gadus callarias L.) muscle. Nature, Lond. 185, 692 only. LoVE R. M. (1970) The Chemical Biology of Fishes. Academic Press, London. LOVE R. M., ROBERTSON I. & SrRACnAN I. (1968) Studies on the North Sea cod--VI. Effects of starvation--4. Sodium and potassium. J. Sci. Fd Agric. 19, 415-422. McBRIDE J. R., MACLEOD R. A. & IDLER D. R. (1960) Seasonal variation in the collagen content of Pacific herring tissues. J. Fish. Res. Bd Can. 17, 913-918. Morro V. (1971) On the constitution and physical-chemical properties of the connective tissue of mammalian and fish skeletal muscle. Ph.D. Thesis, University of Aberdeen, 231 pp. Pn~z K. A. (1968) Cross-linking of collagen and elastin. Ann. Rev. Biochem. 37, 547-570. SOB~L H., HEwLE'rT M. J., Hostage: S. & SACKER I. M. (1967) Effect of starvation on hyaluronic acid and extractable protein in skin of mice. Am. J. Physiol. 212, 773-776.
492
R.M. LOVE et al.
SZ~EDV I. (1970) Hydroxyproline contents and solubility conditions in various connective tissues---II. Examination of solubility in Ringer solution. Fleischwirtschaft SO, 481-482. Tara~,xs~tl T. & YOKOVAMAW. (1954) Physico-chemical studies on the skin and leather of marine animals--XII, The content of hydroxyproline in the collagen of differ-
ent fish skins. Bull. Jap. Soc. scient. Fish. 20, 525-529. THOMPSONM. H. & FARRAGUTR. N. (1965) Amino acid composition of the alewife (Alosa pseudoharenous). Fish. Ind. Res. 3, 47-53. YAMAGUCHIK., LAVI~TYJ. & LOW R. M. (1976) The connective tissues of fish. VIII. Comparative studies on hake, cod and catfish collagens. J. Fd Technol. (In press).
THE CONNECTIVE TISSUES AND COLLAGENS OF COD DURING STARVATION* R. M. LOVE, K. YAMAGUCHI,Y. C"REAC'HAND J. LAVI~TY Torry Research Station, Aberdeen, Scotland (Received 25 March 1976) Abstract--1. Starvation causes the myocommata and skin of cod to thicken. 2. Collagen prepared from the thickened tissue appears to have identical properties to normal collagen-molecular shape, intramolecular crosslinking, amino and imino acid composition and thermal denaturation temperature. An exception is the intermolecular crosslinking which appears to be greater in starving myocomma collagen (in skin there was again no difference). 3. The collagen of cod seems to have a higher turnover throughout life than mammalian collagen. 4. Apart from any effects of starvation, it is found that the force required to break a unit thickness of myocomma decreases in older fish, but during life the fish more than compensate for this by increasing the thickness.
INTRODUCTION After mobilising some of their reserve lipids and carbohydrates, starving fish draw upon the contractile proteins of their body muscle to maintain life: the subject has been reviewed by Love (1970). The cells of the musculature are seen to become thinner (Love et al., 1968) and in extreme cases even the myofilamerits become disorganised or disappear (Gas, 1972). Connective tissue, however, is usually regarded as being stable, and although collagen from mouse skin is mobilised to some extent during subsistence on a protein-free diet (Harkness et al., 1958; Sobel et al., 1967), most publications describe increases in the proportion of animal collagen during starvation. These "increases" are in fact relative and at least partly the result of other constituents being preferentiaUy utilised (e.g. Thompson & Farragut, 1965). On the other hand, a thickening of the skin of Clupea harengus during depletion (Hughes, 1963) cannot be explained in this way, and a report by Lav6ty & Love (1972), that the myocommata of cod actually become mechanically stronger as starvation progresses, raises the possibility that more collagen might in fact be present. The purpose of the present work is to investigate the properties of cod connective tissues during starvation and to study some of the pure collagen isolated therefrom. MATERIAL AND METHODS Cod (Gadus morhua L.) caught near Aberdeen were kept for various periods without food in an aquarium at 9°C. The procedures for obtaining the myocommata and of measuring the water content and breaking stress have been described earlier (Lav6ty & Love, 1972). Strips of myocommata or skin 1 cm wide were used, and their thickness was determined by weight. A value of 1.09 was obtained for the specific gravity of both myocommata and skin from both starved and fed cod, so this value was used in the calculations. * Crown Copyright Reserved. C.Ba'.(B)55/4~--B
487
Acid-soluble collagen was prepared by a modification of the method of Lewis & Piez (1964), descried by Yamaguchi et al. (1976), and all the other experimental techniques are as described in the latter publication.
RESULTS Thickness o f myocommata A simple time scale of weeks without food is not suitable for plotting against properties of starving fish tissues, since the energy reserves of newly-caught fish vary from fish to fish, and the lag period during which the lipids and carbohydrates are utilised with no change in the protein is correspondingly variable. Figure 1 therefore shows the thickness of the myocommata on the basis of muscle water content, which rises above a "nourished" range of 80.0-80.9 in North Sea cod (Love, 1960) concurrently with the start of depletion of contractile proteins. The symbols increase in size with increase in water content. The fish have also been arbitrarily divided into normal or appreciably starved (up to 83.9% water) and more severely starved (84% and above) groups. Regressions of myocomma thickness (T) on body length (L) which were calculated for both groups (T = 0 . 4 0 7 L - 0.33 for starved fish, T = 0 . 3 3 3 L - 7.14 for better nourished fish, with standard deviations about the lines of 8.43 and 6.98 respectively) are shown in Fig. 1. For both groups the regressions were significant (correlation coefficients for the 41 severely starved fish being +0.59 and for the 86 more normal fish being +0.52 (P < 0.01 for both sets). The slopes of the 2 lines do not differ significantly but the lines are significantly separate from each other (P < 0.01) so we can now say definitely that myocommata thicken during starvation. The thickening of myocommata as cod grow has been reported earlier by Love (1970) who measured histological stained sections of muscle containing myocommata with a micrometer eyepiece in a microscope. Figure 2 shows observations made on the skin of the same fish. In contrast to rnyocommata, there was
488
R, M. Lov~ et al. Muscle water % 0"61 ? 90.0 - 9 1 . 9 88"0-89-9
= 84.0-85.9 • 82"0-83"9
86"0-87-9
• 78"0-81"9
•
•
•
0.4
• •
,
I,
i"
)
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•, "
•
:
-.
.
•
. •
•
.-
. = , , ~ . . . "
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.
•
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.
•
!.
•
,70 : - 80
60
m
,
,
,
i
90
,
100
,
110
120
Length (cms)
Fig. 1. Thickness of myocommata dissected from the thickest part of the musculature of cod of different body lengths, and undergoing different degrees of starvation. Each point is the mean of 5 determinations and the size of the symbol used indicates the severity of starvation as measured by the muscle water content--the bigger the symbol, the more severely starved. no significant correlation between body length and skin thickness. However, the mean thickness of the skin of the severely starved fish (0.86me; mean fish length= 72cm) is significantly (P <0.01) greater than the mean thickness of normal or somewhat starved fish (0.67 m e ; mean fish length = 74 cm).
related with muscle water content (correlation coefficient +0.34, P < 0.01). This means that myocommata are thickened during starvation with a mechanically stronger collagen than was present when the fish were adequately nourished; such collagen contains more intermolecular crosslinks. The breaking stress of a unit thickness of skin, on the other hand, did not change during starvation. The correlation coefficient of muscle water content on the breaking stress of the myocommata at constant thickness was +0.32 (P < 0.01) after adjusting
Mechanical strength
The tissues illustrated in Figs. 1 and 2 were then pulled until they broke. It was found that the force required to break a unit thickness of myocomma cor1.4- i90'0-91.g • 88.0- 8g.g Muscle - i 86.0-87-9 • 84.0_85.9 water 1.2-= 82.0 _83~J % • 78.0-81.9
ml
1.0
• I
• •
0"8 •
•
mm
•m
Hi"
ilk
•
pl
" '
•
E t~8
O4 m
02,
,o
go
i
60
I
70 Length(cms)
I
80
I
go
i
100
Fig. 2. Thickness of skin of cod of different body lengths and undergoing different degrees of starvation. Other details as in legend to Fig. 1.
Connective tissues and collagens of cod during starvation
489
Table 1. Relative amounts of monomer (at) and dimer (fl) components of thermally denatured cod collagens as determined by sedimentation analysis Myocommata collagen ct Component fl Component % % Control Starved
73 78.5
27 21.5
Skin collagen ~t Component fl Component % % 71 66.5
29 33.5
examined in a Spinco model E analytical ultracentrifuge at 25°C at 59,780rev/min with an An-D rotor. The Schlieren patterns showed 2 peaks, corresponding with the monomeric chains (~t component) and dimers (fl component). No trimer (y component) was seen in this work. The relative amount of/~ component indicates the amount of thermally stable intramolecular crosslinking. The results obtained by cutting out and weighing paper tracings of the peaks are shown in Table 1. The effects of starvation are seen to be small and inconsistent. (e) Chemical analysis. The amino acid composition determined by a Technicon auto-analyser after acid hydrolysis is shown in Table 2. Properties of the acid-soluble collagen There appeared to be more hydroxyproline in the (a) Source. Myocommata and skin were dissected starved collagen than in the control, so as a check from a male cod 98.5 cm long starved for 4 months it was determined again, separately, by the method and killed 31 May 1972. The water content of the of Leach (1960). By this method the hydroxyproline muscle was 91.4% and the body wt without guts (Wt~o) was 7.6 + 0.02 in the controls and 8.2 + 0.17 5.96 kg. As control a similar fish was taken from the in starved myocommata, and 7.6 + 0.3 in the controls sea at the same time, measuring 96.5 cm with body and 8.1 + 0.26% in starved skin. Proline (also somewt without guts 8,96 kg. The value for muscle water what greater in starved skin, Table 2) and hydroxyproline are thought to stabilise the collagen molecule content was unfortunately lost, but was <81.5~. The tissues were comminuted and extracted twice (Gustavson, 1953) and have been shown to correlate with excess of 0.5M sodium acetate pH7.1 the with the thermal stability of the collagens of different extracts being discarded. The insoluble residue was species of fish (Takahashi & Yokoyama, 1954; Berg then extracted twice with 0.5 M acetic acid and this & Prockop, 1973; Yamaguchi et al., 1976). However, extract, which comprises about 95% of the total col- in the present work the thermal stability of the conlagen of cod (Mohr, 1971) was used, after purification, trol and starved collagens did not appear to be different. Since the hydroxyproline content had seemed to in the subsequent studies. (b) Intrinsic viscosity. The viscosity of solutions of be the only important difference between the collagens of starved and normal cod, the whole work undenatured acid-soluble collagen was determined at 7°C at 4 different concentrations (0.01-0.08%) using was repeated by one of us (Y.C.), using a greater an Ostwald viscometer, sodium citrate buffer number of experimental animals. Acid-soluble collagen was isolated separately from (I = 0.089, pH = 3.5) being the solvent. The intrinsic viscosity obtained by extrapolating the results to in- each of 4 control fish (caught 25.1.74) ranging from finite dilution was found to be identical for the col- 76 cm to 96 cm in body length (mean --- 83) and havlagens from starved and from nourished cod: 5.4 dl/g ing water contents ranging from 80.4 to 81.8~o (mean = 80.9~o). Skin and myocornmata were isolated for skin and 8.4 dl/g for myocommata. (c) Thermal denaturation temperature (To). A col- from 5 individuals starved for about 7 months, ranglagen solution (0.7 mg/ml in sodium citrate buffer) ing in length from 74 to 99 cm (mean = 83.2 cm) and with water contents ranging from 85.2 to 90.5% was maintained for 1 hr at 9°C and 5 determinations of the viscosity were then made. Similar solutions (mean = 88.4~o). The results with standard deviations, were maintained at 11, 13°C etc at 2 ° increments up and also the thermal denaturation temperatures, are to 21°C. The thermal denaturation temperature was given in Table 3. It is clear from Table 3 that the imino acid compotaken as that at which the viscosity had fallen to 50°/o sition of the collagens of starving and fed cod are of the initial value. From the graphs it was clear that the To of skin was identical for starved and nourished essentially the same, in line with the similarity of cod (15°C) and it is unlikely that the To values of denaturation temperatures. The higher hydroxypromyocommata (16.3 and 15.7°C, respectively) are signi- line values found in the starving fish of the previous experiment (Table 2) cannot be explained, but the difficantly different. (d) Sedimentation analysis. The collagen solutions ference must be assumed to be not significant since the denaturation temperatures of control and starving (concentrations 0.3-0.5%) were denatured by incubating in sodium citrate buffer at 30°C for 2 hr and then fish did not differ on that occasion either. for the effects of body length. The correlation coefficient of body length on the breaking stress of the myocommata (constant thickness) after adjusting for muscle water content was -0.26 (P < 0.01). This means that the collagen of the myocommata of older fish is weaker (less crosslinked) than that of younger fish, but in life the older fish make up for this by increasing the thickness, as already mentioned. Thus doubling the length of a cod within the range of fish studied results in an average 50%0 loss in myocomma strength (using the calculated regression: Breaking Stress = 371.5-2.23L)but an average 170Yo increase in thickness (from the lower regression line, Fig. 1).
490
R . M . LOVE et al. Table 2. Amino acid composition of collagens from single fed and starving cod killed May 1972 (residues/1000 residues) Myocommata collagen Control Starved Ala Arg Asp Glu Gly His Hyl HyPro IsLeu Leu Lys Meth Phe Pro Ser Thr Tyr Val Recovery by weight Total N g/100g
106.0 59.1 42.3 82.2 313.6 16.3 8.2 40.7 18.6 32.3 36.3 20.4 14.5 87.6 62.9 25.8 6.0 26.1 88.1~o 17.8
110.0 61.6 38.4 74.9 356.8 11.1 8.4 45.1 14.3 23.4 31.1 19.3 12.3 87.3 59.1 23.3 3.4 20.2 88.3~o 17.6
DISCUSSION The results confirm that extra collagen is laid down in the m y o c o m m a t a and skin of cod during starvat±on, but as far as can be seen the new collagen as added to and mixed with the existing structure does not differ in molecular shape (from intrinsic viscosity), amino acid composition, imino acid stabilisation (from the analytical figures and the denaturation temperature) or intramolecular crosslinking (from sedimentation analysis). The breaking strength of a
Skin collagen Control Starved 105.6 62.5 41.5 77.4 332.2 11.8 7.6 40.7 17.1 29.7 33.1 20.6 13.8 89.6 60.8 26.1 5.0 25.2 98.6~o 18.0
103.8 58.2 38.2 74.5 341.0 12.0 7.6 56.2 15.1 24.6 38.8 19.3 12.1 93.6 59.0 22.0 3.5 21.5 99.3~o 17.3
constant thickness of m y c o m m a tissue is greater, indicating rather more intermolecular crosslinking in the collagen, but skin collagens from starving and nourished fish do not differ. N o w if the connective tissues of cod thicken each winter during the depletion associated with spawning, a time must come when they are eroded in some way to bring them back to their original thickness. In this laboratory we have seen a group of what appeared to be such fish only once---a uniform haul of cod caught on the Faroe Bank in June 1966. The myo-
Table 3. Amino acid composition of collagens from fed and starving cod killed early 1974 (residues/1000 residues) Myocommata collagen Control Starved Ala Arg Asp Glu Gly His HyPro lieu Leu Lys Met Phe Pro Ser Thr Tyr Val HyPro by Leach's method (g %) Denaturation temperature (°C)
116.5 43.7 55.2 76.2 347.3 8.8 54.2 8.0 19.3 30.2 15.7 11.9 97.7 70.3 25.7 4.4 14.8
+__2.8 ± 7.3 ± 0.6 ___0.7 ___21.5 ± 0.6 ± 2.6 + 1.7 ± 4.1 ± 0.9 ± 1.6 ± 1.9 ± 1.3 ± 0.6 ± 1.0 ± 0.8 ± 1.3
111.0 + 5.4 61.0 + 17.4 54.9 ± 2.3 75.2 ± 2.3 332.8 ± 12.5 9.7 ± 2.8 51.7 ± 2.9 10.3 ± 1.8 23.8 ± 3.9 28.8 + 1.6 13.4 ± 6.2 12.8 4- 1.7 102.9 _ 9.4 67.7 ± 4.0 25.1 ± 0.9 4.8 ± 1.0 14.8 ± 0.7
Skin collagen Control Starved 106.6 ± 57.2 ± 53.0 + 74.1 ± 346.8 ± 9.1 ± 52.4 ± 8.8 ± 20.2 + 30.1 ± 16.3 ± 12.0 ± 102.8 ± 69.1 ± 24.5 ± 3.8 ± 15.2 +
2.2 8.0 1.1 2.6 23.2 0.7 4.2 2.5 4.1 0.9 2.7 1.6 7.2 1.3 0.2 0.5 0.6
108.6 ± 6.9 56.7 ± 11.1 50.8 _ 3.2 72.2 ± 2.4 332.5 _ 27.9 8.7 ___ 1.4 54.3 + 5.3 9.4 ___ 1.7 20.8 + 3.6 27.4 +__1.1 17.8 ± 4.3 12.4 ± 0.4 105.6 ± 6.5 67.2 ± 2.5 23.7 ± 1.1 3.7 +__1.3 15.2 ± 0.9
8.2 _ 0.8
8.2 _ 0.7
7.8 ± 0.1
7.4 + 0.6
13.6 + 0.6
13.6 ___0.2
12.2 + 0.3
12.7 + 0.1
Connective tissues and collagens of cod during starvation commata of these fish could not be dissected cleanly, being in an extremely soft condition, and since the muscle was still twitching the softness was not a postmortem effect. Solubility experiments showed that the fraction insoluble in both neutral sodium chloride and citrate buffer (pH 3.5) amounted to only 0.65 pg hydroxyproline/mg wet tissue compared with 4.3 pg in "normal" fish from Aberdeen Bank caught in August (Love & Lav6ty, unpublished). Our single experiment indicates that the intermolecular crosslinking can be greatly reduced under certain circumstances, and reminds us that experiments with fish collagen are likely to lead to quite different conclusions from experiments with mammalian collagen which make up the bulk of the published work on this substance. Professor V. Mohr (private communication) has also observed a seasonal variation in the crosslinking of fish collagen, and it is clear that we are not here dealing with a tissue which in mammals "serves its function for many decades with little or no turnover in the adult animal" (Piez, :1968). Collagen becomes less digestible by collagenase as cattle age (Goll et al., 1964) and has been shown to become very much less soluble in salt or dilute acid during aging in cattle (Carmichael & Lawrie, 1967a; Szeredy, 1970), rats (Nageotte & Guyon, 1934 quoted by Harkness et al., 1954; Eichhom & Butzow, 1966), pigs (Szeredy, 1970) and man (Bakerman, 1962). In 5 experiments comparing the solubility of young (ca. 45cm long) and old ( > 1 0 0 c m long) cod myocommata, J. Lav6ty (unpublished) found that in each case the NaC1 and citrate-insoluble fraction did in fact increase with age, but only fractionally as compared with the many-fold increases seen in mammals. The increase in the insoluble fraction of older fish seems inconsistent with the present finding of a decrease in mechanical strength with age. However, the increase in insolubility reflects an increase in acidstable crosslinks: there could have been a greater decrease in acid-labile crosslinks, which would reduce the overall mechanical strength but not have shown up in the solubility studies. Another pointer in Lav6ty's work is that the salt-soluble fraction, the least crosslinked material, increased slightly as the fish aged, revealing once again a very different situation from that found in mammals. The lack of obvious signs of physical aging in cod may well relate to the annual renewal of a considerable proportion of the collagen. McBride et al. (1960) noticed a seasonal variation in the collagen content of spring-spawning Pacific herring (Clupea pallasii), the highest quantity being present at the spawning time. However, a study by Hughes (1963)on spring-spawning Clupea harengus (Atlantic herring) showed a maximum amount of collagen occurring in the winter, i.e. at other than the spawning time, varying inversely with the amount of oil and so presumably reflecting the state of nutrition. Even with skins removed the musculature still showed a collagen maximum in December; this seems to be the only published report which hints at the phenomenon described in the present paper. Acknowledgements--The work described in this paper formed part of the programme of the Ministry of Agriculture, Fisheries and Food. We would like to thank Dr. A.
491
J. Bailey for reading the manuscript and making helpful suggestions. Dr Ian Mackie carried out the sedimentation analysis on our behalf. The statistical analysis of the results was done by Mr. Gordon Smith.
REFERENCES BAKERMANS. (1962) Quantitative extraction of acid soluble human skin collagen with age. Nature, Lond. 196, 375-376. BERG R. A. & PgocKoP D. J. (1973) The thermal transition of a non-hydroxylated form of collagen. Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem. biophys. Res. Commns 52, 115-120. CARMICHAELD. J. & LAWRm R. A. (1967) Bovine collagen-1. Changes in collagen solubility with animal age. J. Fd Technol. 2, 299-311. Eica-mo~ G. L. & BUTZOWJ. J. (1966) Physical chemical studies on the crosslinking of collagen with age. Int. Cong. Gerontol. Proc. 7th, 2, 5~. GAS N. (1972) Structural alterations in the white muscle of carp (Cyprinus carpio L.) during prolonged starvation. C.r. Acad. Sci. Paris 275, Ser. D, 1403-1406. GOLL D. E., HOEKSTRAW. G. & BRAYR. W. (1964) Ageassociated changes in bovine muscle connective tissue. 1. Rate of hydrolysis by collagenase. J. Fd Sci. 29, 608413. GUSTAVSOr~K. H. (1953) Hydrothermal stability and intermolecular organisation of collagens from mammalian and teleost skins. Hydroxyproline content and degree of interchain linking. Svensk Kern. Tidskr. 65, 70-76. HARKNKSSM. L. R., HARKNESSR. D. & JAr,ms D. W. (1958) The effect of a protein-free diet on the collagen content of mice. J. Physiol., Lond. 144, 307-313. HAgr,r~ESS R. D., MARKO A. M., MtnR H. M. & NEUBERGm~A. (1954) The metabolism of collagen and other proteins of the skin of rabbits. Biochem. J. 56, 558-569. HuGI-ms R. B. (1963) Chemical studies on the herring (Clupea harengus). VII.---Collagen and cohesiveness in heatprocessed herring, and observations on a seasonal variation in collagen content. J. Sci. Fd Agric. 14, 432-441. LAV~TV J. & LOVE R. M. (1972) The strengthening of cod connective tissue during starvation. Comp. Biochem. Physiol. 41A, 39-42. LEACH A. A. (1960) Notes on a modification of the Neuman and Logan method for the determination of the hydroxyproline. Biochem. J. 74, 70-71. LEwis M. S. & PIEz K. A. (1964) The characterisation of collagen from the skin of the dogfish shark, Squalus acanthias. J. biol. Chem. 239, 3336-3340. LoVE R. M. (1960) Water content of cod (Gadus callarias L.) muscle. Nature, Lond. 185, 692 only. LoVE R. M. (1970) The Chemical Biology of Fishes. Academic Press, London. LOVE R. M., ROBERTSON I. & SrRACnAN I. (1968) Studies on the North Sea cod--VI. Effects of starvation--4. Sodium and potassium. J. Sci. Fd Agric. 19, 415-422. McBRIDE J. R., MACLEOD R. A. & IDLER D. R. (1960) Seasonal variation in the collagen content of Pacific herring tissues. J. Fish. Res. Bd Can. 17, 913-918. Morro V. (1971) On the constitution and physical-chemical properties of the connective tissue of mammalian and fish skeletal muscle. Ph.D. Thesis, University of Aberdeen, 231 pp. Pn~z K. A. (1968) Cross-linking of collagen and elastin. Ann. Rev. Biochem. 37, 547-570. SOB~L H., HEwLE'rT M. J., Hostage: S. & SACKER I. M. (1967) Effect of starvation on hyaluronic acid and extractable protein in skin of mice. Am. J. Physiol. 212, 773-776.
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R.M. LOVE et al.
SZ~EDV I. (1970) Hydroxyproline contents and solubility conditions in various connective tissues---II. Examination of solubility in Ringer solution. Fleischwirtschaft SO, 481-482. Tara~,xs~tl T. & YOKOVAMAW. (1954) Physico-chemical studies on the skin and leather of marine animals--XII, The content of hydroxyproline in the collagen of differ-
ent fish skins. Bull. Jap. Soc. scient. Fish. 20, 525-529. THOMPSONM. H. & FARRAGUTR. N. (1965) Amino acid composition of the alewife (Alosa pseudoharenous). Fish. Ind. Res. 3, 47-53. YAMAGUCHIK., LAVI~TYJ. & LOW R. M. (1976) The connective tissues of fish. VIII. Comparative studies on hake, cod and catfish collagens. J. Fd Technol. (In press).