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The effect of pyridoxine deficiency on aminotransferase activity in liver and white muscle of rainbow trout (Salmo gairdneri Richardson)
Comp. Biochem. PMsiol.. Vol. 6111. pp. 385 to 389 0 Pergamon Prex.~ Ltd 1978. Printed in Great Brilain
0305-0491/78/1015-0385502.00;0
THE EFFECT OF PYRIDOXINE DEFICIENCY ON AMINOTRANSFERASE ACTIVITY IN LIVER AND WHITE MUSCLE OF RAINBOW TROUT (SALMO GAIRDNERI RICHARDSON) KARL JURSS Department of Biology, Subdepartment Animal Physiology, Wilhelm Pieck University Rostock, 25 Rostock, East Germany
(Received 3 April 1978) Abstract--l. Aspartate aminotransferase activity in the white muscle of rainbow trout dropped significantly after the animals had received a pyridoxine-deficient diet for 7 days. 2. Alanine aminotransferase activity in the white muscle of rainbow trout dropped significantly after the animals had been fed on a pyridoxine-deficient diet for 21 days. 3. Alanine and aspartate aminotransferase activity in the liver of rainbow trout did not drop significantly until the animals had received a pyridoxine-deficient diet for 28 days. 4. After rainbow trout have received a pyridoxine-deficient diet for 35 days, feeding with complete diet for 7 days is sufficient to restore the aminotransferase activities to the levels observed in control animals.
INTRODUC~ON
Alanine aminotransferase (E.C.2.6.1.2) and aspartate aminotransferase (E.C.2.6.1.1) activities in teleost organs can be influenced by the temperature (Braun et aL, 1970; Mayerle & Butler, 1971 ; Mester & lordachescu, 1972; Iordachescu et al., 1973; Litzbarski, 1974), salinity (Jiirss, 1977) and pollution (Bell, 1968; Holmberg et al. 1972; Racicot et al., 1975) of the environment, and by the quantity (Storer, 1967; Larson & Lewander, 1973; Wittenberger & Giurgea, 1973; Inui & Yokote, 1974; Jiirss & Nicolai, 1976) and quality (Ogino, 1965; Takeda & Yone, 1971; Cowey et al., 1974; Smith et al., 1974; De la Higuera et aL 1977; Jiirss, 1978) of the feed. The pyridoxine content of the feed is especially important in this respect. Pyridoxine (vitamin B6) is the precursor of pyridoxal-5-phosphate, the coenzyme of alanine and aspartate transferases (Braunstein, 1960). An inadequate pyridoxine supply can thus lead to reduced aminotransferase activity in the animal organism. Feeding for several weeks on a pyridoxine-deficient diet led to reduced aspartate and alanine aminotransferase levels in the livers of Cyprinus carpio (Ogino, 1965) and Chrysophrys major (Takeda & Yone, 1971). Reduced alanine aminotransferase activity levels were observed only in the erythrocytes and muscle but not in the liver of rainbow trout which had received a pyridoxine-deficient diet for 9 weeks (Smith et aL, 1974). In contrast, our experiments involving a different pyridoxine-deficient diet reduced the aspartate and alanine aminotransferase activity levels to 66 and 52%, respectively, in the liver and to 38 and 78%, respectively, in the muscle of rainbow trout after the animals had received our diet for only 3 weeks (Jtirss, 1978). The maximum time for which rainbow trout can be fed on a pyridoxine-deficient diet without modifi-
cation of the aminotransferase activity occurring is unknown. Furthermore, no precise data have been published regarding the time required to reverse such modifications by feeding with a complete diet. Our experiments were designed to determine how the aminotransferase activity changes in time in rainbow trout fed first on a pyridoxine-deficient and subsequently on a complete diet.
385
MATERIALS AND METHODS Rainbow trout were brought to Rostock from a fish farm in plastic containers with continuous aeration. The animals were kept in well-ventilated 5001. basins (30 animals per basin) containing fiat tap water which was changed at weekly intervals. The rainbow trout were fed once daily with pellets. The experiments commenced on 3rd May after acclimation for 18 days. The 60 rainbow trout receiving the pyridoxine-deficient diet were fed ad libitum. The controls (64 rainbow troutl subsequently received a corresponding amount of complete diet. After the 35th day of the experiment, the experimental animals previously fed on the pyridoxine-deficient diet received the complete diet (reversal of the modification). Feeding took place at 11 a.m., 5 days a week. The diets used were based on data published by Halver (1957). The composition of the basic diet is shown in Table 1. The basic diet was stored at -18~C and every week an appropriate amount was defrosted and divided into two portions, of which one was mixed with an appropriate quantity of vitamin mixture according to Halver (1957) (completc diet), whereas the other was mixed with the same vitamin mixture except that the pyridoxine hydrochloride was omitted fpyridoxinedeficient diet). Both diets werc then stored again at - 18 C in portions corresponding to one daily ration. These wcrc fed to the animals immediately aftcr a brief defrosting period. The rainbow trout were exposed to light at a natural cycle during the 42 days of the experiment and tbc watcr temperature was held at 16-17 C. Rainbow trout were slaughtered after 7. 14. 21. 28. 35. 38. and 42 days. roughly 24 hr after the previous feeding.
386
KARL J~RSS
Table I. Composition of basal diet Vitamin-free casein Gelatin Sunflower oil Soluble starch Cellulose powder Mineral mixture* I-Tryptophan l-Methionin Aqua destillata
The arithmetic means and standard deviations were calculated for each experimental group. Differences between means were checked by the Student t-test or, where variances differed significantly, after Welch (Clauss & Ebner, 1967).
65 12 9 5 4 4 0.5 0.5 200
RESULTS Of the rainbow trout used in the experiment, five fed on complete diet and three fed on pyridoxinedeficient diet died during the experiment. On the 34th day of the experiment, the animals fed on the pyridoxine-deficient diet gave the impression that they sometimes missed the feed and that they fed more slowly than the controls. Nevertheless, the animals in the two groups did not differ from each other in appearance throughout the experiment. The relative liver index of the animals fed on the pyridoxine-deficient diet did not reach a significantly higher (P < 0.01) value (2.06 +_ 0.58) than that of the animals receiving the complete diet (1.41 + 0.18) until after 35 days. The fact that light patches of various sizes were observed on two of the nine livers taken from animals fed on the pyridoxine-deficient diet after the 28th day of the experiment is worthy of note. Such patches were found on four of nine livers taken from the experimental group of rainbow trout after the 35th day. The AIT and AspT activities in the livers of rainbow trout which had received the pyridoxine-deficient diet were not significantly lower until after the 28th day of the experiment (Table 2), The drop was greatest in the case of AIT (Fig. 1). The differences between the aminotransferase activity levels of the animals in the experimental and control groups increased in the course of the following 7 days. Nevertheless, the animals fed on the pyridoxine-deficient diet needed only three feedings (3 days) on complete diet to eliminate all differences in AIT and AspT activity levels between them and the controls, although
* USP Xlli was supplemented with the following (in mg/100g of salt mixture): AICI 3, 18: ZnSO4, 357: CuCI, 11: MnSO4. 80" KJ, 17: COCI2, 105. The whole liver and a piece of the lateral tail musculature were taken from these animals and held at -20"C until they could be analysed (less than 14 days). After defrosting, the tissue samples were homogenized with ice-cold distilled water in a glass homogenizer to obtain a 4°% homogenate, the red muscle layer having been previously removed from the muscle samples. The crude extract was then centrifuged at 22,000.q for 30rain in a refrigerated centrifuge with a temperature of +4"C. The extract concentrations required for measuring the enzyme activity were obtained from the supernatant liquor. Liver extract concentrations of 0.3°(, were used to measure the aspartate aminotransferase activity (AspT) and alanine aminotransferase activity tA1T) in the liver. Extract concentrations of 1 and 4061 were used to determine the AspT and AIT activities in the white muscle. AspT and AIT activities were measured by means of Fermognost aminotransferase equipment for the u.v. test (VEB Arzneimittelwerke Dresden). The volume of the measuring sample was 1.4 ml for 0,1 ml of tissue extract. The reduction in extinction of the NADH was measured after 10 min initial incubation at 25°C and starting by the addition of oxoglutarate. Measurements were performed at 340 nm using the VSU 2-P (VEB Carl Zeiss Jena). Soluble protein was determined according to the method of Lowry et al. (1951) using human serum albumin as a standard.
Table 2. Aminotransferase activities in the liver of Salmo oairdneri Diet group Pyridoxine-deficient diet Time of test (days)
No. of fish
7
6
14
8
21
7
28
8
35
9
38
7
42
6
Weight of fish (g) 39.3 ( + 9,5) 45. I (+_ 7.2) 51.4 (_ 3.1) 50.8 (+ 13.9) 55.4 (+ 10.1) 55.0 (+ 17.2) 65.3 (+ 9,0)
"Significantly different Significantly different ' Significantly different Significantly different
Complete diet
AIT AspT (mU/mg protein) 320 ( ___74.3) 326 (___ 139) 257 (_ 104) 192~ (+ 45.4) 175" (+__ 54) 322 (___ 142) 250 (+_ 68.7) from from from from
"(P J (P f (P h (p
< < < <
0.05). 0.05). 0.01). 0.002).
268 ( + 78.2) 269 (__. 49.6) 261 (+ 58.4) 219 ~ (+ 36.5) 226 g (+ 46,5) 312 (+_ 61.3) 338 (+ 36.6)
No. of fish 6 8 7 8 9 7 6
Weight of fish (g) 49.3 (+ 7.5) 40.0 ( _ 5.4) 49.1 (+__ 10.0) 53.2 (+_. 7.4) 50.0 (+ 8.0) 56.4 (+ 7.2) 61.3 (+ 14.9)
AlT AspT (mU/mg protein) 350 (+ 81.5) 341 (_+120) 329 (+ 68,6) 296 ~ (+ 111) 358t (+_ 136) 387 (+ 124) 279 (+ 74.8)
310 (_ 52.4) 296 (+ 58.6) 292 (+ 42.9) 277a (___ 63.9) 321 h (+ 61.3) 309 (+ 20.5) 330 (+_ 27.2)
The effect of pyridoxine deficiency on aminotransferase activity • AtT • AspT (Liver)
387
I-I ArT
I~ AspT (White muscle)
,oo
~--~'1
"
£-
.~_ 5C
.
'
7'
'
14
'
21
'
28
Days
.
.
.
'11 3~
'
38
42
'
Fig. I. Percentage drop in time of aminotransferase activities in the liver and white muscle of rainbow trout fed on a pyridoxine-deficient diet. The corresponding values shown in the diagram for animals fed on a complete diet are 100~o in each case. The arrow indicates when animals originally fed on the pyridoxine-deficient diet started to receive the complete diet. activity could "be observed until after 21 days in the muscle from pyridoxine-deficient animals. Thus, the muscle of rainbow trout shows the most sensitive AspT response to feeding on a pyridoxinedeficient diet. Furthermore, the AspT in the muscle of pyridoxine-deficient animals also reached the lowest value relative to that of the controls (24%) (Fig. 1). The AIT and AspT activities in the muscle of rainbow trout which had been fed for 35 days on a pyridoxine-deficient diet did not appear to have returned to normal in all rainbow trout after feeding
the AIT activities in the livers of the experimental animals still differed from those of the controls by some 17~ (Fig, 1). After feeding with complete diet for 7 days (five feedings), the livers of the animals originally fed on the pyridoxine-deficient diet showed AIT and AspT activity levels which did not differ appreciably from those of the controls (Fig. 1). The AspT level in the white muscle of rainbow trout fed on pyridoxine-deficient diet for 5 days is only 72~ that of animals fed on complete diet (Table 3, Fig. 1). In contrast, no significant reduction in AIT
Table 3. Aminotransferase activities in the white muscle of Salmo gairdneri Diet group Complete diet
Pyridoxine-deficient diet Time of test (days)
No. of fish
7
9
14
9
21
7
28
8
35
9
38
7
42
6
Weight of fish (g)
AIT AspT (mU/mg protein)
43.5 (_ 10.9) 45.2 (_+ 6.7) 51.4 (-)- 3.1) 50.8 ( _ 13.9) 55.4 (_+ 10.1) 55.0 (_+ 17.2) 65.3 (_ 9.0)
Significantly different Significantly different ' Significantly different Significantly different
70.8 (__+ 14.7) 58.4 (+ 10.6) 48.6' (_ 7.9) 56.5 i (_+ 23.0) 37.4" (_+ 12.7) 57.1 (+ 28.2) 54.6 (+ 14.4) from from from from
b(p < 0.02). a (p < 0.001 ). r (p < 0.02). h(p < O.OOI).
229 J (+ 62.8) 186~ (+ 55.1) 136~ (_+ 33.6) 135)` (_+ 64.2) 80.5" (_+ 31.5) 185 (4- 103) 256 (+ 38.3)
No. of fish 9 9 7 9 9 7 6
Weight of fish (g)
AIT AspT (mU/mg protein)
46.0 (+ 10.0) 42.3 (_+ 8.6) 49.1 (_+ 10.0) 53.8 (+ 7.1) 50,0 (__ 8.0) 56.4 (+ 7.2) 61.3 (+ 14.9)
Significantly different k Significantly different "'Significantly different "Significantly different
from from from from
80.0 (+ 25.0) 70.9 (+ 14.7) 72.3f (+ 18.4) 81.9 i (_+ 16.0) 81.5" (+ 13.7) 64.0 (+ 15.1) 55.2 (+ 12.4) i(p r (p "(P "(P
< < < <
0.02). 0.001 ). 0.001 ). 0.001 ).
318" (+ 68.9) 287 a (+ 51) 342 h (+ 71.0) 357 ~ (___ 76.4) 337 ~' (+ 91.2) 260 (+44.5) 254 (+ 58.9)
388
KARL Ji3 RSS
for 3 days on the complete diet. The differences in the responses shown by the different animals are expressed by the substantial standard deviations obtained for the AIT and AspT (Table 3) in the muscle of animals which had been previously fed on the pyridoxine-deficient diet. After feeding for 5 days on complete diet, however, no differences could be found between the AIT and AspT activities in the white muscle from the experimental and control groups. DISCUSSION
The rainbow trout used by Smith et al. were 100g heavier than ours, and also the animals were held at a lower temperature (10°C) than that used in our experiments. Both the AIT in muscle and liver tissues and the AspT in the liver assume almost normal values after three feedings on complete diet (Fig. 1). This does not apply to the AspT in muscle tissue which did not reach the levels found in muscle tissue from the control animals until the animals formerly fed on the pyridoxine-deficient diet had received the complete diet for 5 days. The AspT level in muscle tissue, which exhibited the greatest drop of all in the course of the 35 day feeding period with pyridoxine-deficient diet (Fig. 1), thus also requires longer to normalize than the AIT level in muscle and liver tissue and than the AspT level in the liver, The AIT and AspT levels drop at roughly equal rates in the livers of Cyprinus carpio (Ogino, 1965) and Chrysophrys major (Takedo & Yone, 1971) due to pyridoxine deficiency. According to our earlier findings (Jiirss, 1978), and the results presented here, at least the AspT responds very differently in the liver and in muscle tissue (Fig. 1). Different A1T and AspT responses induced by pyridoxine deficiency have also been observed in birds (Brin et al., 1954) and mammals (Stielau et al., 1965). These differences in the AspT response in Salmo 9airdneri when fed on a pyridoxine-deficient diet may be due to the prevalence of different isoenzymes in the muscle and liver.
Pyridoxine deficiency in salmonids has frequently been a subject of extensive discussion (McLaren et al., 1947; Halver, 1972; Ashley, 1972; Snieszko, 1972: Steffens, 1974). We shall therefore restrict ourselves to discussing the effects of a pyridoxine-deficient diet on amonotransferase activity in teleosts. The early significant reduction in AspT in the white muscle after rainbow trout have been fed for 5 days on a pyridoxine-deficient diet (Table 3, Fig. 1) appears to be noteworthy. The liver AspT level exhibited no such drop until after the 28th day of the experiment. A roughly comparable reduction in the AIT in both the muscle and liver of rainbow trout can be observed after 14 days. The response of the AspT activity level in the muscle to feeding with a pyridoxine-deficient diet is also more sensitive than that of the traasketolase activity (coenzyme: thiamine pyrophosphate) in the kidAcknowledgements--The author wishes to express his ney when rainbow trout are fed on a thiamine-defigratitude to Mrs. K, Gries for her technical assistance durcient diet. The kidney transketolase activity in rain- ing these studies. bow trout does not drop significantly until the animals have been fed on a thiamine-deficient diet for REFERENCES 22 days (Jiirss & Tiemann, 1976). ASHLEYL. M. (1972) In Fish Nutrition (Edited by HALVER The reduction of the AspT activity to 40% and of J. E.), pp. 439-53Z Academic Press, New York. the A1T activity to 67°/0 observed in the white muscle BELL G. R. (1968) Distribution of transaminases (aminoafter the animals had received a pyridoxine-deficient transferases) in the tissues of Pacific salmon (Oncorhynchus), with emphasis on the properties and diagnostic diet for 21 days is fully compatible with the drops o/ respectively, observed previously in use of glutamic-oxalacetic transaminase. J. Fish. Res. Bd to 38 and 78/o, Can. 25. 1247-1268. muscle tissue (Ji.irss, 1978). BRAUN K., Kt3NNEMAN~H. & LAUDIENH. (1970) Der EinIn our previous studies, livers with bright patches fluff yon Temperaturanderungen auf Enzyme der Fischwere observed after only 21 days in nine of I0 trout muskulatur. Versuche mit Rhodeus amarus. Mar, Biol. fed on a pyridoxine-deficient diet. The relative liver 7, 59--70. index was also significantly greater in these animals, BRAUNSTEINA. E. (1960) In The Enzymes, Vol. 2 (Edited and their A1T and AspT activities in the liver were by BOVER P. D., LARDY H. & MVRBXCK K.), pp. 113-185. Academic Press, New York. significantly reduced. In subsequent experiments, rainbow trout fed on a pyridoxine-deficient diet did BRIN M., OLSON R. E. & STAREF. J. (1954) Metabolism of cardiac muscle. VIII Pyridoxine deficiency. J. biol. not show similar changes until 14 days later (Table 2, Chem. 210. 435-444. Fig. 1). Diets. water temperatures and animal size were the same in both experiments, so that the differ- CLAUSS G. & EaNER H. (1967) Grundlagen der Statistik fur Psychologen Pfidayogen und Soziologen. Verlag Volk ent response of the liver is probably due to some und Wissen, Berlin. seasonal effect. The first experiments were performed COWEYC. B., BROWND. A., At)ROy J. W. & SHANKSA. M. in November-December, whereas the experiments on (1974) Studies on the nutrition of marine flatfish. The which this paper is based were performed in Mayeffect of dietary protein content on certain cell components and enzymes in the liver of Pleuronectes plaJune. tessa. Mar. Biol. 28. 207-213. Smith et al. (1974) found, after feeding rainbow trout on a pyridoxine-deficient diet for 63 days, that DE LA HIGUERA M,, MURILLO A., VARELA G. & ZAMORA S. (1977) The influence of high dietary fat levels on protein the AIT in the haemolysate and muscle tissue had utilization by the trout (Sahno gairdneri). Comp. Biochem. dropped to 46 and 77~o, respectively, whereas the liver Physiol. 56A, 37-41. A1T rose to 116~o. These figures are confirmed neither HALVER J. E. (1957) Nutrition of salmonoid fishes III. by our present results (Table 3, Fig. 1) nor by the Water-soluble vitamin requirements of chinook salmon, results obtained earlier (Jiirss, 1978). The discrepanJ. Nutr. 62. 225-243. cies in these results may be due to the different con- HALVERJ. E. (1972) In Fish Nutrition (Edited by HALVER J. E.), pp. 30-103. Academic Press, New York. ditions under which the experiments were performed.
The effect of pyridoxine deficiency on aminotransferase activity HOLMBERG B., JENSEN S., LARSSON A., LEWANDER K. & OLSSON M. (1972) Metabolic effects of technical pentachlorophenol (PCP) on the eel An#uilla anguilla L. Comp. Biochem. Physiol. 43B, 171-183. INUI Y. & YOKOTE M. (1974) Gluconeogenesis in the eel I. Gluconeogenesis in the fasted eel. Bull, Freshwater Res. Lab. 24, 33-46. IORD,I~CHESCU l)., NICULESCU S. & MESTER R. (1973) The influence of adaptation temperature on the behaviour of partially purified L-alanine: 2-oxoglutarate aminotransferase from the liver of pond loach Misgurnus lossills L.). Rev. roum. Biol.-Zool. 18, 295-303. JLrRSS K. (1977) Einflufl yon osmotischer Adaptation auf die Aminotransferaseaktivit/iten in Leber und Muskel junger Regenbogenforellen (Salmo gairdneri). Zool. Jb. Physiol. 81, 327-334. JURSS K. (1978) Die Wirkungen yon Pyridoxinmangel und Hunger auf Aminotransferase-Aktivit~iten in der Leber und im Muskel der Regenbogenforelle (Sahno gairdneri). Zool. Jb. Physiol. 82, 141-149. JURSS K. & NICOLAI B. (1976) Biochemische Ver~nderungen in Leber und Muskel der Regenbogenforelle (Salmo gairdneri) bei Hunger. Zool. Jb. Physiol. 80, 101-109. Jugss K. & TIEMANN U. (1976) Thiaminversorgung und Transketolaseaktivit~it in der Niere der Regenbogenforelle, Salmo gairdneri. Zool. Jb. Physiol. 80, 465-475. LARSSON A. & LEWANDER K. (1973) Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A, 367-374. LITZBARSKI H. (1974) Der Einflufl der Wassertemperatur auf das Blutbild des Karpfens (Cyprinus carpio). Z. Binnenfisch. 21, 272-275. LOWRY O. H., ROSEBROUGH N. J., FARR A. L. t~ RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAYERLE J. A. & BUTLER D. G. (1971) Effects of temperature and feeding on intermediary metabolism in North American eels (Anguilla rostrata Lesuer). Comp. Biochem. Physiol. 40A. 1087-1095.
389
MCLAREn B. A.. KELLER E., O'DONNEL D. J, & ELVEHJEM C. A. 0947) The nutrition of rainbow trout I. Studies of vitamin requirement. Arch Biochem. Biophys. 15, 169-178. MESTER R. & JORDACHESCU D, (1972) L'effet de l'acclimatation aux temperatures basses sur la L-alanine: 2 oxoglutarate aminotransf6rase du muscle squelettique chez les loches d'6tang (MisgurnusJbssilis L.). C.r. hebd. S~anc. Acad. Sci., Paris D, 275. 1083-1086. OGINO C. (1965) B vitamins requirements of carp, Cyprinus carpio I. Deficiency symptoms and requirement of vitamin B6. Bull. Jap. Soc. scient, Fish. 31, 546-551. RAOCOT J. G., GAUDET M. & LERAV C. (1975) Blood and liver enzymes in rainbow trout (Sahno gairdneri Rich.) with emphasis on their diagnostic use: study of CCI4 toxicity and a case of Aeromonas infection. J. Fish. Biol. 7, 825-835. SMITH C. E., BRIN M. & HALVER J. E. (1974) Biochemical, physiological, and pathological changes in pyridoxinedeficient rainbow trout (Salmo gairdneri). J. Fish. Res. Bd Can. 31, 1893-1898. SNIESZKO.S.F. (1972) In Fish Nutrition (Edited by HALVER J. E.), pp. 404-437. Academic Press, New York. STEEFENS W. (1974) Der Vitaminbedarf der Regenbogenforelle (Salmo gairdneri). Int. Rev. ges. Hydrobiol. 59, 255-282. STIELAU W. J., FREEDLAND R. A. & MEYER J. H. (1965) Effects of B vitamin deficiences and of starvation on liver enzyme activities of growing rats. J. Nutr. 87, 109-116. STORER J. H, (1967) Starvation and the effects of cortisol in the goldfish (Carassius auratus L.). Comp. Biochera. Physiol. 20, 939-948. TAKEDA T. & YONE (1971) Studies of red sea bream--II. Comparison of vitamin B6 requirement level between fish fed a synthetic diet and fish fed beef liver during prefeeding period. Rep. Fish. Res. Lab. Kyushu Univ. !. 37-47. WITTENBERGER C. (~ GIURGEA R. (1973) Transaminase activities in muslces and liver of the carp, Rev. roum. Biol.Zool. 18, 441 444.
0305-0491/78/1015-0385502.00;0
THE EFFECT OF PYRIDOXINE DEFICIENCY ON AMINOTRANSFERASE ACTIVITY IN LIVER AND WHITE MUSCLE OF RAINBOW TROUT (SALMO GAIRDNERI RICHARDSON) KARL JURSS Department of Biology, Subdepartment Animal Physiology, Wilhelm Pieck University Rostock, 25 Rostock, East Germany
(Received 3 April 1978) Abstract--l. Aspartate aminotransferase activity in the white muscle of rainbow trout dropped significantly after the animals had received a pyridoxine-deficient diet for 7 days. 2. Alanine aminotransferase activity in the white muscle of rainbow trout dropped significantly after the animals had been fed on a pyridoxine-deficient diet for 21 days. 3. Alanine and aspartate aminotransferase activity in the liver of rainbow trout did not drop significantly until the animals had received a pyridoxine-deficient diet for 28 days. 4. After rainbow trout have received a pyridoxine-deficient diet for 35 days, feeding with complete diet for 7 days is sufficient to restore the aminotransferase activities to the levels observed in control animals.
INTRODUC~ON
Alanine aminotransferase (E.C.2.6.1.2) and aspartate aminotransferase (E.C.2.6.1.1) activities in teleost organs can be influenced by the temperature (Braun et aL, 1970; Mayerle & Butler, 1971 ; Mester & lordachescu, 1972; Iordachescu et al., 1973; Litzbarski, 1974), salinity (Jiirss, 1977) and pollution (Bell, 1968; Holmberg et al. 1972; Racicot et al., 1975) of the environment, and by the quantity (Storer, 1967; Larson & Lewander, 1973; Wittenberger & Giurgea, 1973; Inui & Yokote, 1974; Jiirss & Nicolai, 1976) and quality (Ogino, 1965; Takeda & Yone, 1971; Cowey et al., 1974; Smith et al., 1974; De la Higuera et aL 1977; Jiirss, 1978) of the feed. The pyridoxine content of the feed is especially important in this respect. Pyridoxine (vitamin B6) is the precursor of pyridoxal-5-phosphate, the coenzyme of alanine and aspartate transferases (Braunstein, 1960). An inadequate pyridoxine supply can thus lead to reduced aminotransferase activity in the animal organism. Feeding for several weeks on a pyridoxine-deficient diet led to reduced aspartate and alanine aminotransferase levels in the livers of Cyprinus carpio (Ogino, 1965) and Chrysophrys major (Takeda & Yone, 1971). Reduced alanine aminotransferase activity levels were observed only in the erythrocytes and muscle but not in the liver of rainbow trout which had received a pyridoxine-deficient diet for 9 weeks (Smith et aL, 1974). In contrast, our experiments involving a different pyridoxine-deficient diet reduced the aspartate and alanine aminotransferase activity levels to 66 and 52%, respectively, in the liver and to 38 and 78%, respectively, in the muscle of rainbow trout after the animals had received our diet for only 3 weeks (Jtirss, 1978). The maximum time for which rainbow trout can be fed on a pyridoxine-deficient diet without modifi-
cation of the aminotransferase activity occurring is unknown. Furthermore, no precise data have been published regarding the time required to reverse such modifications by feeding with a complete diet. Our experiments were designed to determine how the aminotransferase activity changes in time in rainbow trout fed first on a pyridoxine-deficient and subsequently on a complete diet.
385
MATERIALS AND METHODS Rainbow trout were brought to Rostock from a fish farm in plastic containers with continuous aeration. The animals were kept in well-ventilated 5001. basins (30 animals per basin) containing fiat tap water which was changed at weekly intervals. The rainbow trout were fed once daily with pellets. The experiments commenced on 3rd May after acclimation for 18 days. The 60 rainbow trout receiving the pyridoxine-deficient diet were fed ad libitum. The controls (64 rainbow troutl subsequently received a corresponding amount of complete diet. After the 35th day of the experiment, the experimental animals previously fed on the pyridoxine-deficient diet received the complete diet (reversal of the modification). Feeding took place at 11 a.m., 5 days a week. The diets used were based on data published by Halver (1957). The composition of the basic diet is shown in Table 1. The basic diet was stored at -18~C and every week an appropriate amount was defrosted and divided into two portions, of which one was mixed with an appropriate quantity of vitamin mixture according to Halver (1957) (completc diet), whereas the other was mixed with the same vitamin mixture except that the pyridoxine hydrochloride was omitted fpyridoxinedeficient diet). Both diets werc then stored again at - 18 C in portions corresponding to one daily ration. These wcrc fed to the animals immediately aftcr a brief defrosting period. The rainbow trout were exposed to light at a natural cycle during the 42 days of the experiment and tbc watcr temperature was held at 16-17 C. Rainbow trout were slaughtered after 7. 14. 21. 28. 35. 38. and 42 days. roughly 24 hr after the previous feeding.
386
KARL J~RSS
Table I. Composition of basal diet Vitamin-free casein Gelatin Sunflower oil Soluble starch Cellulose powder Mineral mixture* I-Tryptophan l-Methionin Aqua destillata
The arithmetic means and standard deviations were calculated for each experimental group. Differences between means were checked by the Student t-test or, where variances differed significantly, after Welch (Clauss & Ebner, 1967).
65 12 9 5 4 4 0.5 0.5 200
RESULTS Of the rainbow trout used in the experiment, five fed on complete diet and three fed on pyridoxinedeficient diet died during the experiment. On the 34th day of the experiment, the animals fed on the pyridoxine-deficient diet gave the impression that they sometimes missed the feed and that they fed more slowly than the controls. Nevertheless, the animals in the two groups did not differ from each other in appearance throughout the experiment. The relative liver index of the animals fed on the pyridoxine-deficient diet did not reach a significantly higher (P < 0.01) value (2.06 +_ 0.58) than that of the animals receiving the complete diet (1.41 + 0.18) until after 35 days. The fact that light patches of various sizes were observed on two of the nine livers taken from animals fed on the pyridoxine-deficient diet after the 28th day of the experiment is worthy of note. Such patches were found on four of nine livers taken from the experimental group of rainbow trout after the 35th day. The AIT and AspT activities in the livers of rainbow trout which had received the pyridoxine-deficient diet were not significantly lower until after the 28th day of the experiment (Table 2), The drop was greatest in the case of AIT (Fig. 1). The differences between the aminotransferase activity levels of the animals in the experimental and control groups increased in the course of the following 7 days. Nevertheless, the animals fed on the pyridoxine-deficient diet needed only three feedings (3 days) on complete diet to eliminate all differences in AIT and AspT activity levels between them and the controls, although
* USP Xlli was supplemented with the following (in mg/100g of salt mixture): AICI 3, 18: ZnSO4, 357: CuCI, 11: MnSO4. 80" KJ, 17: COCI2, 105. The whole liver and a piece of the lateral tail musculature were taken from these animals and held at -20"C until they could be analysed (less than 14 days). After defrosting, the tissue samples were homogenized with ice-cold distilled water in a glass homogenizer to obtain a 4°% homogenate, the red muscle layer having been previously removed from the muscle samples. The crude extract was then centrifuged at 22,000.q for 30rain in a refrigerated centrifuge with a temperature of +4"C. The extract concentrations required for measuring the enzyme activity were obtained from the supernatant liquor. Liver extract concentrations of 0.3°(, were used to measure the aspartate aminotransferase activity (AspT) and alanine aminotransferase activity tA1T) in the liver. Extract concentrations of 1 and 4061 were used to determine the AspT and AIT activities in the white muscle. AspT and AIT activities were measured by means of Fermognost aminotransferase equipment for the u.v. test (VEB Arzneimittelwerke Dresden). The volume of the measuring sample was 1.4 ml for 0,1 ml of tissue extract. The reduction in extinction of the NADH was measured after 10 min initial incubation at 25°C and starting by the addition of oxoglutarate. Measurements were performed at 340 nm using the VSU 2-P (VEB Carl Zeiss Jena). Soluble protein was determined according to the method of Lowry et al. (1951) using human serum albumin as a standard.
Table 2. Aminotransferase activities in the liver of Salmo oairdneri Diet group Pyridoxine-deficient diet Time of test (days)
No. of fish
7
6
14
8
21
7
28
8
35
9
38
7
42
6
Weight of fish (g) 39.3 ( + 9,5) 45. I (+_ 7.2) 51.4 (_ 3.1) 50.8 (+ 13.9) 55.4 (+ 10.1) 55.0 (+ 17.2) 65.3 (+ 9,0)
"Significantly different Significantly different ' Significantly different Significantly different
Complete diet
AIT AspT (mU/mg protein) 320 ( ___74.3) 326 (___ 139) 257 (_ 104) 192~ (+ 45.4) 175" (+__ 54) 322 (___ 142) 250 (+_ 68.7) from from from from
"(P J (P f (P h (p
< < < <
0.05). 0.05). 0.01). 0.002).
268 ( + 78.2) 269 (__. 49.6) 261 (+ 58.4) 219 ~ (+ 36.5) 226 g (+ 46,5) 312 (+_ 61.3) 338 (+ 36.6)
No. of fish 6 8 7 8 9 7 6
Weight of fish (g) 49.3 (+ 7.5) 40.0 ( _ 5.4) 49.1 (+__ 10.0) 53.2 (+_. 7.4) 50.0 (+ 8.0) 56.4 (+ 7.2) 61.3 (+ 14.9)
AlT AspT (mU/mg protein) 350 (+ 81.5) 341 (_+120) 329 (+ 68,6) 296 ~ (+ 111) 358t (+_ 136) 387 (+ 124) 279 (+ 74.8)
310 (_ 52.4) 296 (+ 58.6) 292 (+ 42.9) 277a (___ 63.9) 321 h (+ 61.3) 309 (+ 20.5) 330 (+_ 27.2)
The effect of pyridoxine deficiency on aminotransferase activity • AtT • AspT (Liver)
387
I-I ArT
I~ AspT (White muscle)
,oo
~--~'1
"
£-
.~_ 5C
.
'
7'
'
14
'
21
'
28
Days
.
.
.
'11 3~
'
38
42
'
Fig. I. Percentage drop in time of aminotransferase activities in the liver and white muscle of rainbow trout fed on a pyridoxine-deficient diet. The corresponding values shown in the diagram for animals fed on a complete diet are 100~o in each case. The arrow indicates when animals originally fed on the pyridoxine-deficient diet started to receive the complete diet. activity could "be observed until after 21 days in the muscle from pyridoxine-deficient animals. Thus, the muscle of rainbow trout shows the most sensitive AspT response to feeding on a pyridoxinedeficient diet. Furthermore, the AspT in the muscle of pyridoxine-deficient animals also reached the lowest value relative to that of the controls (24%) (Fig. 1). The AIT and AspT activities in the muscle of rainbow trout which had been fed for 35 days on a pyridoxine-deficient diet did not appear to have returned to normal in all rainbow trout after feeding
the AIT activities in the livers of the experimental animals still differed from those of the controls by some 17~ (Fig, 1). After feeding with complete diet for 7 days (five feedings), the livers of the animals originally fed on the pyridoxine-deficient diet showed AIT and AspT activity levels which did not differ appreciably from those of the controls (Fig. 1). The AspT level in the white muscle of rainbow trout fed on pyridoxine-deficient diet for 5 days is only 72~ that of animals fed on complete diet (Table 3, Fig. 1). In contrast, no significant reduction in AIT
Table 3. Aminotransferase activities in the white muscle of Salmo gairdneri Diet group Complete diet
Pyridoxine-deficient diet Time of test (days)
No. of fish
7
9
14
9
21
7
28
8
35
9
38
7
42
6
Weight of fish (g)
AIT AspT (mU/mg protein)
43.5 (_ 10.9) 45.2 (_+ 6.7) 51.4 (-)- 3.1) 50.8 ( _ 13.9) 55.4 (_+ 10.1) 55.0 (_+ 17.2) 65.3 (_ 9.0)
Significantly different Significantly different ' Significantly different Significantly different
70.8 (__+ 14.7) 58.4 (+ 10.6) 48.6' (_ 7.9) 56.5 i (_+ 23.0) 37.4" (_+ 12.7) 57.1 (+ 28.2) 54.6 (+ 14.4) from from from from
b(p < 0.02). a (p < 0.001 ). r (p < 0.02). h(p < O.OOI).
229 J (+ 62.8) 186~ (+ 55.1) 136~ (_+ 33.6) 135)` (_+ 64.2) 80.5" (_+ 31.5) 185 (4- 103) 256 (+ 38.3)
No. of fish 9 9 7 9 9 7 6
Weight of fish (g)
AIT AspT (mU/mg protein)
46.0 (+ 10.0) 42.3 (_+ 8.6) 49.1 (_+ 10.0) 53.8 (+ 7.1) 50,0 (__ 8.0) 56.4 (+ 7.2) 61.3 (+ 14.9)
Significantly different k Significantly different "'Significantly different "Significantly different
from from from from
80.0 (+ 25.0) 70.9 (+ 14.7) 72.3f (+ 18.4) 81.9 i (_+ 16.0) 81.5" (+ 13.7) 64.0 (+ 15.1) 55.2 (+ 12.4) i(p r (p "(P "(P
< < < <
0.02). 0.001 ). 0.001 ). 0.001 ).
318" (+ 68.9) 287 a (+ 51) 342 h (+ 71.0) 357 ~ (___ 76.4) 337 ~' (+ 91.2) 260 (+44.5) 254 (+ 58.9)
388
KARL Ji3 RSS
for 3 days on the complete diet. The differences in the responses shown by the different animals are expressed by the substantial standard deviations obtained for the AIT and AspT (Table 3) in the muscle of animals which had been previously fed on the pyridoxine-deficient diet. After feeding for 5 days on complete diet, however, no differences could be found between the AIT and AspT activities in the white muscle from the experimental and control groups. DISCUSSION
The rainbow trout used by Smith et al. were 100g heavier than ours, and also the animals were held at a lower temperature (10°C) than that used in our experiments. Both the AIT in muscle and liver tissues and the AspT in the liver assume almost normal values after three feedings on complete diet (Fig. 1). This does not apply to the AspT in muscle tissue which did not reach the levels found in muscle tissue from the control animals until the animals formerly fed on the pyridoxine-deficient diet had received the complete diet for 5 days. The AspT level in muscle tissue, which exhibited the greatest drop of all in the course of the 35 day feeding period with pyridoxine-deficient diet (Fig. 1), thus also requires longer to normalize than the AIT level in muscle and liver tissue and than the AspT level in the liver, The AIT and AspT levels drop at roughly equal rates in the livers of Cyprinus carpio (Ogino, 1965) and Chrysophrys major (Takedo & Yone, 1971) due to pyridoxine deficiency. According to our earlier findings (Jiirss, 1978), and the results presented here, at least the AspT responds very differently in the liver and in muscle tissue (Fig. 1). Different A1T and AspT responses induced by pyridoxine deficiency have also been observed in birds (Brin et al., 1954) and mammals (Stielau et al., 1965). These differences in the AspT response in Salmo 9airdneri when fed on a pyridoxine-deficient diet may be due to the prevalence of different isoenzymes in the muscle and liver.
Pyridoxine deficiency in salmonids has frequently been a subject of extensive discussion (McLaren et al., 1947; Halver, 1972; Ashley, 1972; Snieszko, 1972: Steffens, 1974). We shall therefore restrict ourselves to discussing the effects of a pyridoxine-deficient diet on amonotransferase activity in teleosts. The early significant reduction in AspT in the white muscle after rainbow trout have been fed for 5 days on a pyridoxine-deficient diet (Table 3, Fig. 1) appears to be noteworthy. The liver AspT level exhibited no such drop until after the 28th day of the experiment. A roughly comparable reduction in the AIT in both the muscle and liver of rainbow trout can be observed after 14 days. The response of the AspT activity level in the muscle to feeding with a pyridoxine-deficient diet is also more sensitive than that of the traasketolase activity (coenzyme: thiamine pyrophosphate) in the kidAcknowledgements--The author wishes to express his ney when rainbow trout are fed on a thiamine-defigratitude to Mrs. K, Gries for her technical assistance durcient diet. The kidney transketolase activity in rain- ing these studies. bow trout does not drop significantly until the animals have been fed on a thiamine-deficient diet for REFERENCES 22 days (Jiirss & Tiemann, 1976). ASHLEYL. M. (1972) In Fish Nutrition (Edited by HALVER The reduction of the AspT activity to 40% and of J. E.), pp. 439-53Z Academic Press, New York. the A1T activity to 67°/0 observed in the white muscle BELL G. R. (1968) Distribution of transaminases (aminoafter the animals had received a pyridoxine-deficient transferases) in the tissues of Pacific salmon (Oncorhynchus), with emphasis on the properties and diagnostic diet for 21 days is fully compatible with the drops o/ respectively, observed previously in use of glutamic-oxalacetic transaminase. J. Fish. Res. Bd to 38 and 78/o, Can. 25. 1247-1268. muscle tissue (Ji.irss, 1978). BRAUN K., Kt3NNEMAN~H. & LAUDIENH. (1970) Der EinIn our previous studies, livers with bright patches fluff yon Temperaturanderungen auf Enzyme der Fischwere observed after only 21 days in nine of I0 trout muskulatur. Versuche mit Rhodeus amarus. Mar, Biol. fed on a pyridoxine-deficient diet. The relative liver 7, 59--70. index was also significantly greater in these animals, BRAUNSTEINA. E. (1960) In The Enzymes, Vol. 2 (Edited and their A1T and AspT activities in the liver were by BOVER P. D., LARDY H. & MVRBXCK K.), pp. 113-185. Academic Press, New York. significantly reduced. In subsequent experiments, rainbow trout fed on a pyridoxine-deficient diet did BRIN M., OLSON R. E. & STAREF. J. (1954) Metabolism of cardiac muscle. VIII Pyridoxine deficiency. J. biol. not show similar changes until 14 days later (Table 2, Chem. 210. 435-444. Fig. 1). Diets. water temperatures and animal size were the same in both experiments, so that the differ- CLAUSS G. & EaNER H. (1967) Grundlagen der Statistik fur Psychologen Pfidayogen und Soziologen. Verlag Volk ent response of the liver is probably due to some und Wissen, Berlin. seasonal effect. The first experiments were performed COWEYC. B., BROWND. A., At)ROy J. W. & SHANKSA. M. in November-December, whereas the experiments on (1974) Studies on the nutrition of marine flatfish. The which this paper is based were performed in Mayeffect of dietary protein content on certain cell components and enzymes in the liver of Pleuronectes plaJune. tessa. Mar. Biol. 28. 207-213. Smith et al. (1974) found, after feeding rainbow trout on a pyridoxine-deficient diet for 63 days, that DE LA HIGUERA M,, MURILLO A., VARELA G. & ZAMORA S. (1977) The influence of high dietary fat levels on protein the AIT in the haemolysate and muscle tissue had utilization by the trout (Sahno gairdneri). Comp. Biochem. dropped to 46 and 77~o, respectively, whereas the liver Physiol. 56A, 37-41. A1T rose to 116~o. These figures are confirmed neither HALVER J. E. (1957) Nutrition of salmonoid fishes III. by our present results (Table 3, Fig. 1) nor by the Water-soluble vitamin requirements of chinook salmon, results obtained earlier (Jiirss, 1978). The discrepanJ. Nutr. 62. 225-243. cies in these results may be due to the different con- HALVERJ. E. (1972) In Fish Nutrition (Edited by HALVER J. E.), pp. 30-103. Academic Press, New York. ditions under which the experiments were performed.
The effect of pyridoxine deficiency on aminotransferase activity HOLMBERG B., JENSEN S., LARSSON A., LEWANDER K. & OLSSON M. (1972) Metabolic effects of technical pentachlorophenol (PCP) on the eel An#uilla anguilla L. Comp. Biochem. Physiol. 43B, 171-183. INUI Y. & YOKOTE M. (1974) Gluconeogenesis in the eel I. Gluconeogenesis in the fasted eel. Bull, Freshwater Res. Lab. 24, 33-46. IORD,I~CHESCU l)., NICULESCU S. & MESTER R. (1973) The influence of adaptation temperature on the behaviour of partially purified L-alanine: 2-oxoglutarate aminotransferase from the liver of pond loach Misgurnus lossills L.). Rev. roum. Biol.-Zool. 18, 295-303. JLrRSS K. (1977) Einflufl yon osmotischer Adaptation auf die Aminotransferaseaktivit/iten in Leber und Muskel junger Regenbogenforellen (Salmo gairdneri). Zool. Jb. Physiol. 81, 327-334. JURSS K. (1978) Die Wirkungen yon Pyridoxinmangel und Hunger auf Aminotransferase-Aktivit~iten in der Leber und im Muskel der Regenbogenforelle (Sahno gairdneri). Zool. Jb. Physiol. 82, 141-149. JURSS K. & NICOLAI B. (1976) Biochemische Ver~nderungen in Leber und Muskel der Regenbogenforelle (Salmo gairdneri) bei Hunger. Zool. Jb. Physiol. 80, 101-109. Jugss K. & TIEMANN U. (1976) Thiaminversorgung und Transketolaseaktivit~it in der Niere der Regenbogenforelle, Salmo gairdneri. Zool. Jb. Physiol. 80, 465-475. LARSSON A. & LEWANDER K. (1973) Metabolic effects of starvation in the eel, Anguilla anguilla L. Comp. Biochem. Physiol. 44A, 367-374. LITZBARSKI H. (1974) Der Einflufl der Wassertemperatur auf das Blutbild des Karpfens (Cyprinus carpio). Z. Binnenfisch. 21, 272-275. LOWRY O. H., ROSEBROUGH N. J., FARR A. L. t~ RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MAYERLE J. A. & BUTLER D. G. (1971) Effects of temperature and feeding on intermediary metabolism in North American eels (Anguilla rostrata Lesuer). Comp. Biochem. Physiol. 40A. 1087-1095.
389
MCLAREn B. A.. KELLER E., O'DONNEL D. J, & ELVEHJEM C. A. 0947) The nutrition of rainbow trout I. Studies of vitamin requirement. Arch Biochem. Biophys. 15, 169-178. MESTER R. & JORDACHESCU D, (1972) L'effet de l'acclimatation aux temperatures basses sur la L-alanine: 2 oxoglutarate aminotransf6rase du muscle squelettique chez les loches d'6tang (MisgurnusJbssilis L.). C.r. hebd. S~anc. Acad. Sci., Paris D, 275. 1083-1086. OGINO C. (1965) B vitamins requirements of carp, Cyprinus carpio I. Deficiency symptoms and requirement of vitamin B6. Bull. Jap. Soc. scient, Fish. 31, 546-551. RAOCOT J. G., GAUDET M. & LERAV C. (1975) Blood and liver enzymes in rainbow trout (Sahno gairdneri Rich.) with emphasis on their diagnostic use: study of CCI4 toxicity and a case of Aeromonas infection. J. Fish. Biol. 7, 825-835. SMITH C. E., BRIN M. & HALVER J. E. (1974) Biochemical, physiological, and pathological changes in pyridoxinedeficient rainbow trout (Salmo gairdneri). J. Fish. Res. Bd Can. 31, 1893-1898. SNIESZKO.S.F. (1972) In Fish Nutrition (Edited by HALVER J. E.), pp. 404-437. Academic Press, New York. STEEFENS W. (1974) Der Vitaminbedarf der Regenbogenforelle (Salmo gairdneri). Int. Rev. ges. Hydrobiol. 59, 255-282. STIELAU W. J., FREEDLAND R. A. & MEYER J. H. (1965) Effects of B vitamin deficiences and of starvation on liver enzyme activities of growing rats. J. Nutr. 87, 109-116. STORER J. H, (1967) Starvation and the effects of cortisol in the goldfish (Carassius auratus L.). Comp. Biochera. Physiol. 20, 939-948. TAKEDA T. & YONE (1971) Studies of red sea bream--II. Comparison of vitamin B6 requirement level between fish fed a synthetic diet and fish fed beef liver during prefeeding period. Rep. Fish. Res. Lab. Kyushu Univ. !. 37-47. WITTENBERGER C. (~ GIURGEA R. (1973) Transaminase activities in muslces and liver of the carp, Rev. roum. Biol.Zool. 18, 441 444.