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,1
*
Department of Biological Sciences,
College of Veterinary Medicine and
**
Department of Biochemistry and Molecular Biology, Mississippi State University, Mississippi State, MS 39762; and
Institute of Nutrition, Directorate of Fisheries, Bergen, Norway
1To whom correspondence should be addressed. E-mail: rkb1{at}ra.msstate.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • fish • vitamin A • transport • adaptation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). First, retinyl esters, the principal form in the diet, are hydrolyzed by pancreatic and apical membrane–associated enzymes to release retinol (2
), which is absorbed across the apical membrane of enterocytes in a free form (3
,4
) or in association with proteins that can bind retinol and other lipid molecules (5
,6
). After transapical absorption, intracellular retinol bound to a cellular retinol binding protein type II (CRBPII)2
is esterified by lecithin retinol acyltransferase (LRAT), whereas free retinol is esterified by acyl-CoA acyltransferase (ARAT) (1
). The esterified retinol is then processed into chylomicrons and exported into the systemic circulation (7
).
Regulation of retinol systemic availability is likely to be complex. Each of the steps associated with the absorption and transfer of dietary retinol to the systemic circulation can be modulated. Because retinol is an essential nutrient, when dietary levels are below or barely meet requirements, the processes facilitating retinol availability should be up-regulated. However, high dietary levels can be toxic to fish (8
) and other vertebrates (9
). Based on the complex relationship between dietary levels and rates of absorption of the essential amino acid methionine, which is also toxic in excess (10
), the a priori expectation was that at high dietary levels, the processes facilitating retinol availability should be down-regulated to reduce toxic effects. In agreement with this expectation, the circulating concentration of retinol when rats were fed a diet containing 1000-fold the retinol requirement was less than when the dietary level was at the requirement. However, the excessive intake of retinol increased the serum concentrations of retinol palmitate, which is less toxic (11
).
The present study examined the relationship between the transapical absorption of free retinol and dietary intake. In light of the evidence for the presence of a transporter for retinol, we speculated that rates of absorption would be modulated in a manner similar to that for other essential, but potentially toxic nutrients (e.g., methionine). The approach involved measuring in vitro rates of retinol absorption by the proximal small intestine of juvenile sunshine bass (hybrid of Morone chrysops x M. saxatilis) after 6 wk of feeding diets with concentrations of retinol that were below, within the range of requirement or exceeded by up to 50-fold the reported requirements for several species of fish (12
). This study was a component of a larger project to determine the retinol requirement of sunshine bass under cultivation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Three experimental diets were prepared to contain retinol amounts that were less than, approximately equal to and in excess of requirements by adding synthetic all-trans retinol acetate (Sigma Chemical, St. Louis, MO) as the source of retinol [1 retinol equivalent = 1 µg retinol] to a basal diet (Table 1
). Analysis of the diets revealed actual levels as fed to be 568, 1657 and 40,244 µg/kg dry diet of trans retinol (dehydrogenated retinol was not detected).
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Fish and their care.
The MSU Institutional Animal Care and Use Committee approved all aspects of the research involving animals. Juvenile sunshine bass were obtained from the Small Fry Fish Farm (Wilmot, AR) and acclimated to laboratory conditions for 2 mo, during which time they were fed a commercial trout diet (Zeigler Bros., Gardeners, PA).
After the acclimation period, the fish were randomly distributed to 110-L glass aquariums (n = 20/aquarium with a total biomass of 
150 g) that had flow-through circulation (0.9 L/min). Water temperature was maintained at 25.1 ± 0.4°C and hardness at 110 mg/L by adding a solution of calcium chloride to the incoming water. Water temperature was recorded daily and hardness was measured once each week. A diurnal cycle of 14 h light and 10 h dark was imposed using overhead fluorescent lights.
For the first week after distribution to the aquariums, the fish were fed a retinol-free basal diet. Before starting the experimental feeding, 15 fish were randomly sampled, killed with an overdose of ethyl 3-aminobenzoate (MS-222; Argent Laboratory, Redmond, WA), and body mass was recorded for each fish. At the start of the feeding trial, the fish were anesthetized (100 mg/L MS-222), and the total initial body mass was recorded for each tank. After the weighing, the fish were treated with acriflavin (Sigma Chemical; 3 mg/L) to reduce bacteriological infection (13
). Each of the three experimental diets was fed to three aquariums of fish for 6 wk. The fish were fed to satiation (based on observation of consumption) twice per day (at 0900 and 1600 h), and the amount of diet fed was recorded.
Sample collection.
At the end of the feeding period, individual body mass and total tank biomass were recorded. A total of 12 fish were randomly sampled at 1000–1100 h without feeding from each of the three treatments (4 fish from each of the three aquariums) and killed for measuring transapical rates of retinol absorption. An additional 9 fish (3 fish from each of the three aquariums) were used to collect tissues for analyses of retinol levels in selected tissues, including the small intestine.
Retinol absorption by the proximal small intestine.
The entire postgastric alimentary canal was removed immediately after death and placed in cold (2–4°C) fish Ringers aerated with a gas mixture of 95% O2 and 5% CO2. A segment (3–5 cm) of proximal small intestine, extending from the pyloric ceca was isolated for measuring retinol absorption. In other vertebrates, retinol absorption is higher in the jejunum than in the ileum (4
,5
).
Following a published protocol (14
), the proximal segment of small intestine was everted and two sleeves (1 cm in length) from each were secured by silk ligatures on stainless steel rods that approximated the diameter (1–2 mm), thereby yielding a snug fit. The mounted tissues were stored in cold, aerated fish Ringers until 45 min after death at which time both sleeves were placed in 25°C aerated Ringers for 4 min. One of the sleeves was then incubated for 5 min in 22°C aerated Ringers which contained tracer 3H-labeled all trans retinol (3.57 10-5 mmol/L). The second tissue was incubated in Ringers that contained the tracer retinol in the presence of 0.05 mmol/L unlabeled all-trans retinol. Polyethylene glycol (MW = 4000) labeled with 14C was added to each incubation solution to correct for retinol in the fluid adhering to the tissue. After the incubation, the tissues were removed, placed in tared vials, mass recorded and solubilized (Solvable; Packard, Meriden, CT). Scintillant (Ultima Gold; Packard) was added and radioactivity measured by liquid scintillation counting. Rates of retinol absorption by the tissues were normalized to tissue mass.
Retinol concentrations in the diets and the proximal small intestine tissue.
The proximal small intestines (extending 2 cm from the pyloric ceca) of three fish from each aquarium were rinsed with distilled water, snap-frozen in liquid nitrogen, pooled and stored at -20°C in the dark until analyzed. The tissues were again rinsed in distilled water to remove all visible residues before they were homogenized (Ultra Turrax, IKA Labortechnik, Staufen, Germany). Concentrations of all-trans retinol and dehydro-retinol were measured in the experimental diets and in the tissues using a modified HPLC procedure (15
) that has been validated for fish tissues (16
). The analytical system consisted of a Shimadzu LC-9 A pump, a Shimadzu SIL-6B/9 A autoinjector, a Shimadzu SPD-2 A UV-detector at 325 nm, an integrator (Turbochrom, version 4.0, Perkin-Elmer, Boston, MA), and a Hichrom Hypersil column (4.6 x 150 mm, 3 µm particle size). The mobile phase was a 90:10 mixture of hexane and isopropanol.
Chemicals.
Unless otherwise indicated, reagents used to prepare solutions were purchased from Sigma Chemical. The radiolabeled compounds were obtained from Perkin-Elmer (14C polyethylene glycol) and Amersham Pharmacia Biotech (Buckinghamshire, UK; 3H retinol).
Statistical analysis.
The data were analyzed by one-way ANOVA (SAS Institute, Cary, NC) to search for a diet effect. Duncan’s multiple range test was used to identify specific treatment differences. For all comparisons, P < 0.05 was accepted as the critical level of significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The initial body mass of the fish was 7.64 ± 0.04 g. After the 6-wk feeding period, body mass did not differ among fish fed the diets containing retinol levels that were below (26.98 ± 0.98 g), approximating (28.89 ± 0.30 g), and exceeding (28.82 ± 1.92 g) requirements. None of the fish exhibited obvious signs of retinol deficiency or toxicity, both of which cause increased mortality, reduced growth, impaired skeletal formation, reduced mucous secretion and hemorrhages of the eyes, fins and skin. Deficiency also causes blindness and exophthalmia in fish.
Rates of retinol absorption.
Rates of absorption at tracer concentration for fish fed the diet with the lowest level of retinol were less than in fish fed the diets with the required and excessive levels of retinol, which did not differ (Fig. 1
). Rates of retinol absorption at 0.05 mmol/L (tracer + unlabeled retinol) were significantly higher with each increase in dietary retinol. The addition of 0.05 mmol/L unlabeled retinol to the incubation solution reduced (P < 0.05) the accumulation of tracer [fmol/(mg · min)] retinol by 32% (no difference among treatments), indicating that a portion of absorption was via a saturable pathway.
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Concentrations of all-trans retinol in small intestine tissue were 0.21 ± 0.03 and 0.49 ± 0.21 pmol/g for fish fed the diets with low and required levels of trans retinol, respectively, with both significantly less than the 338 ± 89 pmol/g for fish fed the diet with excess trans retinol (P < 0.001). Dehydro-retinol concentrations in the small intestine tissues were 0.11 ± 0.04, 0.91 ± 0.04 and 454 ± 109 pmol/g for fish fed the low, required and excess levels (P < 0.001).
The relationship between tissue and diet retinol concentrations was visually obvious when the tissues used to measure rates of absorption were solubilized. Specifically, the solubilized small intestine tissues of fish fed the high retinol diet were a distinct yellow color, whereas those of fish fed the two other diets were colorless.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The present study measured transapical rates of retinol absorption, not trans-tissue, which would have involved the additional steps of intracellular esterification, packaging into chylomicrons and export into the systemic circuit. Moreover, because unesterified retinol was used, the need to hydrolyze retinyl esters before absorption was avoided, which could be a rate-limiting step when measuring in vitro retinol absorption using intact tissues.
The presence of a saturable component of transapical retinol absorption, hence carrier-mediated transport, by the small intestine of juvenile sunshine bass is consistent with findings for various animal models (3
,4
,5
,6
,18
). Although the carriers have not yet been characterized, the transport process is specific for trans-retinol (19
). Because the incubation solutions did not include protein, little, if any, of the measured rates of retinol uptake would have been via the endocytosis of retinol-protein complexes (5
,6
). As a consequence, it was not possible to evaluate the relative contribution of the two pathways of apical retinol absorption, or whether the two pathways had different responses to the varying dietary retinol levels.
The direct relationship between dietary levels of retinol and rates of absorption is similar to that for rates of sugar transport and dietary levels of carbohydrate (20
). This is perplexing in that the increased transapical absorption of free retinol when dietary intake is excessive would appear to increase systemic availability and the risk of toxic effects. In addition, the higher rates of absorption were measured despite already high tissue concentrations. However, the lack of increase in circulating concentrations of retinol of rats fed diets with 1000x the requirement (11
) indicates that transapical absorption is not the only determinant. Instead, an increase in the less toxic form, retinol palmitate (11
), suggests that regulation of retinol systemic availability occurs largely after transapical absorption.
The higher concentration of retinol measured in the proximal small intestine of fish fed the excessive levels of retinol was indicative of accumulation. Intracellular retinol binding protein type II (CRBPII) plays a critical role in mediating the systemic availability of retinol (21
). Although CRBPII is one of the more abundant cytoplasmic proteins in enterocytes, and may be important for preventing toxic effects, at high dietary intake, retinol-binding capacities can be saturated (11
), and this can alter the pathway of systemic availability (22
). We did not quantify CRBPII levels, but the higher rates of retinol absorption by fish fed the diet with excessive retinol would be consistent with higher concentrations of CRBPII (23
) and higher tissue levels of retinol. Moreover, higher rates of retinol absorption in the jejunum correspond to higher concentrations of CRBPII (24
).
Despite its name, expression of CRBPII is modulated mainly by the amount and composition of fat in the diet (21
), with synthesis particularly responsive to unsaturated fatty acids (25
–28
). The oil mixture added to the three diets was high in unsaturated fatty acids and would have stimulated CRBPII synthesis similarly in all three groups of fish. Varying the levels of dietary retinol may influence expression of CRBPII (29
), but to a lesser magnitude than fat (28
). It remains unclear whether the addition of an excessive amount of retinol to the diet, which increased the lipid fraction by < 0.5 g (0.4%), would have up-regulated synthesis of CRBPII, thereby causing the higher rates of absorption.
Intracellular retinol bound to CRBPII is a substrate for LRAT, which esterifies fatty acids to retinol, whereas unbound intracellular retinol is esterified by ARAT (1
). Thereafter, the retinol is packaged in chylomicrons and exported to the systemic circuit. Although systemic availability of retinol will be affected by changes in the postabsorption esterification, packaging into chylomicrons and export, because of the relatively short incubation time used to measure apical absorption (5 min), it is likely the majority of the absorbed retinol would have remained inside the enterocytes. Furthermore, any retinol that would have left the enterocytes would have remained in the tissue, been detected and would have contributed to the reported rates of absorption.
The complex regulation of the systemic availability of retinol varies among species (30
). This may reflect adaptations to the varying levels of retinol in the natural diets consumed during the evolution of different species. For most species, retinol depletion is more likely than the chronic exposure to potentially toxic levels, and this may influence the capacities to adaptively modulate systemic availability to reduce systemic levels when the diet contains excessive levels. Moreover, any changes that influence the systemic availability of retinol are likely to influence availability of other nutrients, including carotenoids (18
). Although the present study does not provide conclusive evidence for the adaptive modulation of transapical absorption of retinol, the data corroborate previous findings indicating that systemic availability of retinol can be regulated by modulating the various processes associated with transferring retinol from the lumen of the small intestine to the blood.
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FOOTNOTES |
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Manuscript received 22 February 2002. Initial review completed 10 April 2002. Revision accepted 5 June 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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