Absorption and blood clearance of labelled carotenoids ([14C]astaxanthin, [3H]Canthaxanthin and [3H]zeaxanthin) in mature female rainbow trout (Oncorhynchus mykiss)

• 1.1. Using one force-fed meal, eight mature female rainbow trout received [¹⁴C]astaxanthin ([¹⁴C]Ax) with [³H]canthaxanthin ([³H]Cx; N = 3) or with [³H]zeaxanthin ([³H]Zx; N = 5).
• 2.2. Approximately 200 μl of blood were collected via caudal puncture every 24 hr for 4 days. After 96 hr, the fish were killed and pyloric caeca (P.C.) from the duodenal intestine (D.I.) section, ileal intestine (I.I.), and posterior intestine (P.I.) were dissected out.
• 3.3. In the blood, Ax levels were higher than Cx followed by Zx levels.
• 4.4. This corresponds to their respective absorption by the trout as was confirmed by their relative concentrations in P.C., I.I. and P.I.
• 5.5. However, blood clearance was similar for all three compounds. [¹⁴C]Phoenicoxanthin ([¹⁴C]Px) was detected as a reduced metabolite of [¹⁴C]Ax in all gut sections.

     

Traced studies on metabolism of astaxanthin in Atlantic salmon (Salmo salar)

The present studies were performed to investigate the metabolism of astaxanthin (Ax) in Atlantic salmon, especially in the liver of salmon. The investigations were undertaken in vivo salmon that were fed a diet containing 60 ppm 15, 15' 14C-labelled Ax prior to sacrifice. The samples of blood, bile, liver, gastrointestinal tract and contents, muscle, skin, remaining carcass and feces were taken for scintillation counting. The highest radioactivity (71.36%) of 14C-labelled Ax was found in the gastrointestinal contents and feces, 7.13% in the bile and 10.68% in the samples of liver, muscle, and skin at the end of the experiments. The metabolites of 14C-labelled Ax were extracted from the bile of the salmon and analyzed using thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Predominant 14C-labelled Ax and its cis-isomers were found and no conjugation of 14C-labelled Ax was observed. These results indicate that 14C-labelled Ax was not conjugated into larger colorless compound in Atlantic salmon liver.     

Intraperitoneal and dietary administration of astaxanthin in rainbow trout (Oncorhynchus mykiss)--plasma uptake and tissue distribution of geometrical E/Z isomers

Astaxanthin enters circulation in salmonid fishes upon intraperitoneal injection (IP) of small doses. Blood uptake and tissue distribution of geometrical E/Z astaxanthin isomers were determined in tissues and plasma of duplicated groups of rainbow trout (Oncorhynchus mykiss, initial weight 550 g) some of which were administered high doses of astaxanthin by IP in a trial lasting for 8 weeks. Doses of 10 (IP10), 50 (IP50) or 100 mg (IP100) astaxanthin (Lucantin Pink, BASF, Germany), respectively, dispersed in phosphate buffered saline were tested in comparison with diets containing 10 (Control) or 60 (Fed 60) mg astaxanthin kg(-1). Astaxanthin concentrations in all examined tissues and plasma were significantly higher in IP50 and IP100 than in controls and Fed 60 (p<0.05). In IP50, 11 mg astaxanthin kg(-1) muscle was detected after 4 weeks, compared to 4 mg kg(-1) in rainbow trout fed 60 mg kg(-1). Concentrations up to 80 and 100 mg astaxanthin kg(-1) were detected in liver and kidney after IP, respectively, whereas fish only fed astaxanthin contained about 2 mg astaxanthin kg(-1). No increase in muscle astaxanthin concentration was found between 4 and 8 weeks in fish given IP, and the muscle astaxanthin concentration in IP50 and IP100 were similar. Muscle concentration and injected dose were curvilinearly correlated and the proportion of ingested dose retained by the muscle was negatively correlated with the amount of injected astaxanthin. Plasma and muscle concentrations of astaxanthin were highly correlated (p<0.0001). Astaxanthin Z-isomers accumulated selectively in the various tissues after IP, whereas all-E-astaxanthin was preferably absorbed into plasma when administered via the diet. There was a selective uptake of all-E-astaxanthin in the muscle of all fish. Mortality was not affected by treatment, but a dose-dependent reduction in SGR was evident after IP. In conclusion, a more rapid and higher uptake of astaxanthin in plasma, muscle, kidney and liver of rainbow trout takes place after IP compared to when astaxanthin is fed via the diet.     

Metabolism of [11-3H]retinyl acetate in liver tissues of vitamin A-sufficient, -deficient and retinoic acid-supplemented rats

A study was conducted on the incorporation of [11-3H]retinyl acetate into various retinyl esters in liver tissues of rats either vitamin A-sufficient, vitamin A-deficient or vitamin A-deficient and maintained on retinoic acid. Further, the metabolism of [11-3H]retinyl acetate to polar metabolites in liver tissues of these three groups of animals was investigated. Retinol metabolites were analyzed by high-performance liquid chromatography. In vitamin A-sufficient rat liver, the incorporation of radioactivity into retinyl palmitate and stearate was observed at 0.25 h after the injection of the label. The label was further detected in retinyl laurate, myristate, palmitoleate, linoleate, pentadecanoate and heptadecanoate 3 h after the injection. The specific radioactivities (dpm/nmol) of all retinyl esters increased with time. However, the rate of increase in the specific radioactivity of retinyl laurate was found to be significantly higher (66-fold) than that of retinyl palmitate 24 h after the injection of the label. 7 days after the injection of the label, the specific radioactivity between different retinyl esters were found to be similar, indicating that newly dosed labelled vitamin A had now mixed uniformly with the endogenous pool of vitamin A in the liver. The esterification of labelled retinol was not detected in liver tissues of vitamin A-deficient or retinoic acid-supplemented rats at any of the time point studied. Among the polar metabolites analyzed, the formation of [3H]retinoic acid from [3H]retinyl acetate was found only in vitamin A-deficient rat liver 24 h after the injection of the label. A new polar metabolite of retinol (RM) was detected in liver of the three groups of animals. The formation of 3H-labelled metabolite RM from [3H]retinyl acetate was not detected until 7 days after the injection of the label in the vitamin A-sufficient rat liver, suggesting that metabolite RM could be derived from a more stable pool of vitamin A.     

Absorption and fate of labelled canthaxanthin 15, 15'-3H2 in rainbow trout (Salmo gairdneri Rich.)

1. Using one force fed meal, absorption of labelled canthaxanthin 15,15'-3H2 was studied by collecting blood via caudal punction at regular intervals and measuring radioactivity in various tissues and organs of rainbow trout. 2. Canthaxanthin absorption showed a large variability between individuals irrespective of their sex. A rapid increase in the radioactivity linked to canthaxanthin 15,15'-3H2 is observed in the blood followed by a slow decrease. 3. Twenty-four and 72 hours after ingestion of labelled canthaxanthin by rainbow trout, the radioactivity was widely distributed. From the total radioactivity given, most was found 24 hours after the meal in the pyloric caeca, ovary and skin. Seventy-two hours after diet, the radioactivity increased in the muscle, liver and kidney. On the other hand, the percentage of radioactivity of intestine and caeca declined. 4. Data from absorption curve indicates that 0.86% of radioactivity ingested is present in the blood, suggesting that canthaxanthin is not readily transferred from the digestive tract. 5. The metabolic clearance of canthaxanthin (lambda = 0.0246/hr +/- 0.005) which would correspond to a half-life of 28 hours gives a preliminary idea of the digestive and metabolic utilization of canthaxanthin by the rainbow trout.     

Disruption of the lecithin:retinol acyltransferase gene makes mice more susceptible to vitamin A deficiency

Lecithin:retinol acyltransferase (LRAT) catalyzes the esterification of retinol (vitamin A) in the liver and in some extrahepatic tissues, including the lung. We produced an LRAT gene knock-out mouse strain and assessed whether LRAT-/- mice were more susceptible to vitamin A deficiency than wild type (WT) mice. After maintenance on a vitamin A-deficient diet for 6 weeks, the serum retinol level was 1.34 +/- 0.32 microM in WT mice versus 0.13 +/- 0.06 microM in LRAT-/- mice (p < 0.05). In liver, lung, eye, kidney, brain, tongue, adipose tissue, skeletal muscle, and pancreas, the retinol levels ranged from 0.05 pmol/mg (muscle and tongue) to 17.35 +/- 2.66 pmol/mg (liver) in WT mice. In contrast, retinol was not detectable (<0.007 pmol/mg) in most tissues from LRAT-/- mice after maintenance on a vitamin A-deficient diet for 6 weeks. Cyp26A1 mRNA was not detected in hepatic tissue samples from LRAT-/- mice but was detected in WT mice fed the vitamin A-deficient diet. These data indicate that LRAT-/- mice are much more susceptible to vitamin A deficiency and should be an excellent animal model of vitamin A deficiency. In addition, the retinol levels in serum rapidly increased in the LRAT-/- mice upon re-addition of vitamin A to the diet, indicating that serum retinol levels in LRAT-/- mice can be conveniently modulated by the quantitative manipulation of dietary retinol.     

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss)

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.     

Survey of fructose 1,6-bisphosphatase isoenzyme in rat organs and ontogenic expression of the enzyme in rat fetus

1. Among eleven tissues of rat, the liver type of fructose 1,6-bisphosphatase (FBPase) subunit was detected in the liver, kidney, testis, pancreas and lung by Western blot analysis using anti-(liver FBPase) or anti-(muscle FBPase) serum. 2. The muscle type of the enzyme subunit was detected only in the pancreas other than skeletal muscle. Both types of the enzyme subunit were found in the pancreas. 3. Neither anti-(liver FBPase) nor anti-(muscle FBPase) serum detected the band of enzyme subunit on the blots of the extracts of brain, heart, small intestinal mucosa, spleen and placenta. 4. FBPase is present in fetal rat liver at least as early as the 14th day of gestation. 5. In agreement with the increase in immunological staining density, the level of the enzyme activity in fetal liver increased exponentially during fetal development. 6. The muscle enzyme was not detected until the fetus reached the 19th day of gestation.     

Oxidative stress generated by dietary Zn-deficiency: studies in rainbow trout (Oncorhynchus mykiss).

To date, there is scarce information on the metabolic and biochemical repercussions of Zn-deficiency in fish. In this work, the effect of dietary Zn-deficiency on the diet utilization and the metabolism of activated oxygen species in rainbow trout (Oncorhynchus mykiss) has been studied. Fish were randomly separated in different lots according to their Zn-starvation and diet intake. In crude extracts of liver, gut and muscle, total and isoenzymatic superoxide dismutase and catalase activities were analysed. Lipid peroxidation was also determined in the same tissues. Western blotting was performed using antibodies against manganese- and copperzinc-containing superoxide dismutase. Lots fed on the Zn-deficient diet and with low intake showed significantly lower weight gain and feed-conversion efficiency indexes than control trouts. However, these parameters returned to control values when trouts were recovered by feeding them a control diet ad libitum. In control trouts, three independent copperzinc superoxide dismutase isozymes were detected in liver, whereas only one isozyme was present in the other lots. However, by Western blotting analysis the presence of a manganese superoxide dismutase was found in liver from all lots except in control trouts. Catalase activity and lipid peroxidation values were mainly detected in liver and gut, respectively, and both parameters increased in all lots with respect to the control group. Our results thus suggest that in rainbow trout an oxidative stress appears to occur as a consequence of Zn-deficiency.     

Effect of dietary carbohydrate source on soluble protein glucose concentration and enzyme activity (aldolase) of European eels (Anguilla anguilla L.)

This study was undertaken to determine the influence of dietary carbohydrate sources: wheat meal, bread meal, soluble corn starch, native potato starch and sorghum meal, on soluble protein, enzyme activity (aldolase) and glucose concentration in muscle and liver of European eels (Anguilla anguilla). There was less soluble protein in both muscle and liver of eels fed 30% wheat meal or bread meal than the other experimental groups. However, eels fed 30% bread meal or soluble corn starch had a higher glucose concentration in muscle and liver than the other experimental groups. High enzyme activity (aldolase) was found in the liver of eels fed 30% wheat meal, bread meal or soluble starch.     

Synthesis and metabolism of all-trans-[11-3H]retinyl beta-glucuronide in rats in vivo.

All-trans-[11-3H]retinyl beta-glucuronide (all-trans-[11-3H]ROG) was synthesized from [3H]retinol by an improved synthetic procedure. After its intraperitoneal injection into rats, ROG is initially found as the predominant labelled component in the serum, but then is distributed to the liver, intestine, kidney and other organs of the body. Esters of vitamin A, which constituted the major metabolite of ROG, were detected in the liver as well as in other tissues. Of the labelled vitamin A esters derived from tritiated ROG in the liver and intestine, about 50% contained 5,6-epoxyretinol, which was characterized by its chromatographic behaviour, formation of an acetyl ester and lack of reactivity with diazomethane. Thus ROG, although converted to retinol in vivo, might also act physiologically in an intact form.     

Carbohydrate energy reserves and effects of food deprivation in male and female rainbow trout

We investigated the effects of nutritional state on carbohydrate, lipid, and protein stores in the heart, liver, and white skeletal muscle of male and female rainbow trout. For fed animals we also partitioned glycogen into fractions based on acid solubility. Fish (10-14 months-old, ~400-500 g) were held at 14 °C and either fed (1% of body weight, every other day) or deprived of food for 14 days. Under fed conditions, glycogen was increased 54% in ventricles from males compared with females, and elevated in the liver (87%) and white muscle (70%) in sexually-maturing versus immature males. Acid soluble glycogen predominated over the acid insoluble fraction in all tissues and was similar between sexes. Food deprivation 1) selectively reduced glycogen and free glucose in male ventricles by ~30%, and 2) did not change glycogen in the liver or white muscle, or triglyceride, protein or water levels in any tissues for both sexes. These data highlight sex differences in teleost cardiac stores and the metabolism of carbohydrates, and contrast with mammals where cardiac glycogen increases during fasting and acid insoluble glycogen is a significant fraction. Increased glycogen in the hearts of male rainbow trout appears to pre-empt sex-specific cardiac growth while storage of acid soluble glycogen may reflect a novel strategy for efficient synthesis and mobilization of glycogen in fishes.     

Strategies for maintaining Na⁺ balance in zebrafish (Danio rerio) during prolonged exposure to acidic water

Teleost fish store lipids among several tissues primarily as triacylglycerol (TG). Upon metabolic demand, stored TGs are hydrolyzed by hormone-sensitive lipase (HSL). In this study, two distinct cDNAs encoding HSL were isolated, cloned, and sequenced from adipose tissue of rainbow trout. The full-length cDNAs, designated HSL1 and HSL2, were 2562-bp and 2887-bp in length, respectively, and share 82% nucleotide identity. Phylogentic analysis suggests that the two HSLs derive from paralogous genes that may have arisen during a teleost-specific genome duplication event. Quantitative real-time PCR revealed that HSL1 and HSL2 were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. HSL1 and HSL2 mRNAs were detected in the brain, spleen, pancreas, kidney, gill, intestine, heart, and white muscle, but were most abundant in the red muscle, liver, and adipose tissue. HSL1 mRNA was more abundant than HSL2 mRNA in the adipose tissue, whereas HSL2 mRNA was more abundant than HSL1 mRNA in the liver. Short term fasting (4 weeks) increased HSL1 and HSL2 mRNA expression in the adipose tissue, but only HSL1 mRNA levels increased in the liver and the red muscle. During a prolonged fast (6 weeks), there was continued elevation of HSL1 and HSL2 mRNA levels in the liver and muscle; HSL mRNA expression in mesenteric fat declined, coincident with depletion of mesenteric fat mass. Refeeding fish reduced HSL expression to levels seen in continuously fed fish. These findings indicate that the pattern of HSL expression is consistent with the diverse lipid storage pattern of fish and suggest that distinct mechanisms serve to regulate differential expression of the two HSLs in tissues and during a progressive fast.     

Fatty acid, carotenoid and vitamin A composition of tissues of free living gulls

The aim of this study was to investigate fatty acid and carotenoid profile as well as vitamin A (retinol and retinol esters) content in gull (Larus fucus) tissues. Palmitic (16:0) and stearic (18:0) fatty acids were major saturates in all the tissues studied. Oleic acid (18:1n-9) was the major monounsaturate in the tissue phospholipids varying from 11.9% (liver) up to 18.2% (lung). Arachidonic acid (20:4n-6) was the major unsaturate in the phospholipid fraction in all the tissues. Liver contained the highest total carotenoid concentration which was 5 and 7 fold higher compared to kidney and pancreas. In the liver beta-carotene was major carotenoid. In contrast, in all other tissues beta-carotene was minor fraction with lutein being major carotenoid. Zeaxanthin, canthaxanthin, beta-cryptoxanthin and echinenone were also identified in the gull tissues. Liver and kidney were characterised by the highest vitamin A concentrations (1067.5 and 867.5 microg/g, respectively). Retinol comprised from 55.3% (pancreas) down to 8% (kidney) of the total vitamin A but was not detected in the abdominal fat. Retinyl palmitate was the major retinyl ester in the liver, kidney and heart (44.2; 38.1 and 46.0% of total retinyl esters). In muscles and abdominal fat retinyl stearate was the major retinyl ester fraction. Therefore high proportions of beta-carotene were found in gull liver and peripheral tissues were enriched by lutein and zeaxanthin compared to the liver, a very high concentration of retinyl esters in the kidney was observed and tissue-specificity in retinyl ester proportions in peripheral tissues was found.     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.     

Fatty‐acid profiles of white muscle and liver in stream‐maturing steelhead trout Oncorhynchus mykiss from early migration to kelt emigration

The profiles of specific fatty acids (FA) in white muscle and liver of fasting steelhead trout Oncorhynchus mykiss were evaluated at three periods during their prespawning migration and at kelt emigration in the Snake–Columbia River of Washington, Oregon and Idaho, to improve the understanding of energy change. Twenty‐seven FAs were identified; depletion of 10 of these was positively correlated in liver and white muscle of prespawning O. mykiss. To observe relative changes in FA content more accurately over sampling intervals, the lipid fraction of tissues was used to normalize the quantity of individual FA to an equivalent tissue wet mass. Saturated and monounsaturated FAs were depleted between upstream migration in September and kelt emigration in June, whereas polyunsaturated FAs were more conserved. Liver was depleted of FAs more rapidly than muscle. Three FAs were detected across all sampling intervals: 16:0, 18:1 and 22:6n3, which are probably structurally important to membranes. When structurally important FAs of O. mykiss are depleted to provide energy, physiological performance and survival may be affected.     

Branched-chain amino acid aminotransferase activity in the tissues of lake trout

The enzyme branched-chain amino acid aminotransferase (BCAT) was found in five tissues of fingerling lake trout, Salvelinus namaycush, (listed in order of decreasing tissue specific activity): posterior kidney, skeletal muscle, gill, liver, and anterior kidney. This pattern is consistent with that found in other animals. The results of this study seem to indicate that BCAT in the liver of lake trout has a higher specific activity than that of the rat and that the specific activity is higher in both the liver and skeletal muscle than it is in these organs of the chick.