GENERAL AND COMPARATIVE ENDOCRINOLOGY 78, 74-79 (19%)

Effects of Vitamin D,, 25(OH) Vitamin D,, 24,25(0H), Vitamin D,, and
1,25(OH), Vitamin D, on the in Vitro Intestinal Calcium Absorption in
the Marine Teleost, Atlantic Cod (Gadus morhua)

KRISTINA SUNDELL AND BJ~RN TH. BJ~RNSSON

Department of Zoophysiology, University of Giiteborg, P.O. Box 250 59, MOO 31 Giiteborg, Sweden
Accepted June 9, 1989

The role of vitamin D,, 25(OH) vitamin D,, 24,25(0H), vitamin D,, and 1,25(0H)* vitamin
D,, in the regulation of calcium absorption across the intestine in the marine teleost, Gadus
morhua, was investigated. The intestine was perfused, in vitro, both vascularly and through
the intestinal lumen, and the calcium it&x was measured using “Ca. Vitamin D, and its
metabolites were tested in perfusate concentrations of 10 ng . ml- ‘. 25(OH)D3 increased the
intestinal calcium uptake by 65%, while 24,25(0H),Ds decreased it by 36%. Vitamin D, and
1,25(OH),D,, on the other hand, did not affect the calcium infhrx across the intestinal
mucosa. This indicates that 25(OH)D, and 24,25(OH),D, may be active regulators of cal-
cium transport across the intestine of Atlantic cod. o 1990AC&& PMS. !IIC.

Teleost fish, particularly marine species, (Nahm et al., 1979; Avioli et al., 1981; Hol-
have large hepatic stores of vitamin D, ick et al., 1982; Marcocci et al., 1982).
(Bills, 1927) and this vitamin is required in Transport proteins for 25(OH)D, and
the diet for normal growth (Bamett et al., 24,25(OH),D, are found in plasma and ex-
1979). istence of the enzyme 25(OH) vitamin D-
Mammals and birds obtain vitamin D, 1-hydroxylase has been demonstrated in
through metabolism of 7-dehydrocholes- kidney and liver of both freshwater and ma-
terol in the skin or from dietary sources. rine teleost fish (Henry and Norman, 1975;
The vitamin normally undergoes two hy- Hay and Watson, 1976, 1977; Kenny ef al.,
droxylations, one taking place in the liver 1977; Hayes et al., 1986). On the other
to form 25(OH) vitamin D, (25(OH)D,) and hand, several studies have failed to detect
one in the kidney to form either 24,25(OH), either metabolism of vitamin D, and
vitamin D, (24,25(OH),D,) or 1,25(OH), vi- 25(OH)D, or circulating levels of any vita-
tamin D, (1,25(OH),D,). The latter form is min D, metabolite in rainbow trout and
considered to be an active hormone which goldfish (Oizumi and Monder, 1972; Hollis
stimulates intestinal calcium and phosphate et al., 1977; Barnett et al., 1979, 1982).
uptake, as well as bone calcium resorption In mammals, the intestine is the main
(Fraser, 1980; DeLuca and Schnoes, 1983), target organ for the hormonal action of
while the physiological role of 24,25- 1,25(OH),D,, where it increases calcium
(OH),D, still remains unclear (Norman et uptake (Halloran and DeLuca, 1981). A
al., 1982; DeLuca, 1988). similar effect is found in freshwater teleosts
The metabolism of vitamin D, in teleosts where 1,25(OH),D, enhances intestinal cal-
is poorly understood and data on plasma cium uptake in eel, tilapia, and goldfish
levels of vitamin D, metabolites are contra- (Char-tier et al., 1979; Flik et al., 1982; Fen-
dictory. Circulating levels of vitamin D,, wick, 1984). Vitamin D, has a similar effect
25(OH)D,, and 1,25(OH),D, have been in eel and goldfish, whereas 24,25(OH),D,
demonstrated in rainbow trout and eel does not affect the intestinal calcium ab-
74
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 VITAMIN D3 AND INTESTINAL 13~ ABSORPTION 75

sorption in tilapia and freshwater eel (Flik (basal flux), and then for 1 hr with the substance
et al., 1982; Fenwick, 1984). present. The Jz was calculated for the basal-flux pe-
riod and for the period during treatment, separately,
No comparable studies have been per- using the following formula:
formed in marine teleosts. In a previous
study, Sundell and Bjdmsson (1988) re-
ported that one part of the calcium uptake
across the intestinal mucosa in the marine where V = volume of vascular perfusate in each frac-
teleost, Atlantic cod (Gadus morhua), was tion (ml), X = body weight (g), and Y = sampling
period (mm).
saturable and could be blocked by chlor- Statistical analysis. A nested one-way analysis of
promazine (low4 M). Thus, it is possible variance was used to test for significant differences
that this part of the intestinal calcium influx between and within treatment groups (Sokal and
is under hormonal control. The present Rohlf, 1%9). Further, the effects of each metabolite on
study was designed to test the effects of the calcium uptake across the intestinal mucosa were
tested using Wilcoxon matched-pairs signed ranks
vitamin D, and its major metabolites,
test. Data are presented as means + SEM.
25(OH)D,, 24,25(OH)2D3, and 1,25(OH),
D,, on the calcium uptake across the intes- RESULTS
tinal mucosa in this species.
The effects of vitamin D, and its metab-
MATERIALS AND METHODS olites on the calcium uptake across the in-
testinal mucosa (Jp), in the Atlantic cod,
Fish. Atlantic cod, G. morhua, were captured off
the Swedish west coast and acclimated in recirculat- are presented in Fig. 1.25(OH)D, increased
ing, filtered, and aerated seawater at lo”, for at least 1 the JF by 65% (P < 0.05), from 1.27 + 0.38
week prior to experiments. The fish were of both sexes to 2 10 + 0 76 Fmol Ca*’ - hr-’ . kg-‘,
with a mean body weight of 578.5 ? 22.6 g and were whereas vitamin D, and 1,25(OH),D, did
not fed in the laboratory. not affect the Jp (P > 0.05). 24,25(OH),D,
Perfusion procedure. The fish were killed with a
blow to the head and immediately injected with 0.5 ml decreased the JFt by 36% (P < 0.05),
heparin . kg-’ (5000 IU . ml-‘) into the caudal ves- from 1.69 + 0.48 to 1.09 * 0.35 p,mol
sels.
The intestine was perfused through the coeliac ar-
tery with a cod Ringer’s solution and through the in-
testinal lumen with a balanced salt solution as de-
scribed by Sundell and Bjomsson (1988).
To measure the influx of calcium from the intestinal
lumen to the circulation (Jp), 45Ca (sp act 1.14 f 0.10
MBq . (pm01 Ca*+)- i; 45Ca,p) was added to the intes-
tinal perfusate (salt solution perfusing the intestinal
lumen). The vascular perfusate was collected in 5-rnin
fractions from an oufflow catheter in the intestinal
vein. In order to allow the system to reach a steady
D E
flux of calcium, collection of vascular perfusate did
not start until 1 hr after addition of “Ca. The amount FIG. 1. Effects of vitamin Da and various metabo-
of 4sCa (dpm) in each fraction (4sCar) was measured in lites on calcium absorption across the mucosa of in
a liquid scintillation counter (LKB Wallac, 1217 Rack- vitro-perfused intestinal preparations in the Atlantic
beta). cod, Gadus morhua. For each group of preparations
Experimental design. Vitamin D,, 25(OH)D,, the basal calcium influx is measured for 1 hr (open
1 ,25(OH)*Ds, and 24,25(OH),Ds, diluted in 95% etha- bars), followed by a calcium influx measurement dur-
nol or ethanol only, were added to the perfusion media ing 1 hr of treatment (crosshatched bars). The treat-
to make a fmal concentration of 10 ng . ml-’ (598 ~1 ments are A, ethanol; B, vitamin D,; C, 25(OH)D,D,;
ethanol . 300 ml perfusate- ‘). Each substance was ad- D, 24,25(OH),D,; and E, 1,25(OH),D,. Values are
ministered to eight preparations. Furthermore, each means f standard error of the mean. N = 8 in all
group served as its own control: the intlux of 4sCa was groups. *Statistically different (P < 0.05) from the
measured for 1 hr without addition of the substance basal calcium influx within the group.
76 SUNDELL AND BJdRNSSON

Ca*’ * hr-’ * kg- ‘. In the ethanol-treated show a low phylogenic level compared with
group, the Jp was 0.77 & 0.21 before and birds. The present study demonstrates,
0.68 f 0.17 pmol Ca*’ * hr-’ . kg-’ dur- however, that vitamin D, metabolites have
ing treatment (P > 0.05). clearly differentiated effects on the intesti-
Thus, the basal Jp varies from 0.77 to nal calcium regulation in the Atlantic cod.
1.69 pmol Ca*+ - hr- ’ * kg-’ for the dif- The stimulation of the cod intestinal cal-
ferent treatment groups. This is due to a cium uptake by 25(OH)D, can either be a
seasonal variation, where the Jp is higher direct effect of the metabolite or an indirect
in prespawning than postspawning Atlantic effect through its further metabolism. The
cod (Sundell and Bjiirnsson, 1988). Mostly possibility of further metabolism is indi-
postspawning fish were used in the study, cated by the conversion of 25(OH)D, to
but four and one preparations from pre- several more polar metabolites in freshwa-
spawning fish were treated with 24,25- ter- and seawater-adapted rainbow trout
(OH),D3 and 1,25(OH)*D3, respectively. (Hayes et al. 1986) and observations
No difference in response was observed be- (HPLC) of at least one metabolite more po-
tween pre- and postspawning fish. lar than 25(OH)D, in the plasma of Atlantic
cod (M. Sturen and K. Sundell, unpub-
DISCUSSION lished results). However, as both 25(OH)D,
and 24,25(OH),D, have rapid effects on the
Among the vitamin D, metabolites exam- Atlantic cod intestinal calcium influx, it is
ined in the present study, two had signifi- probable that these effects are direct. This
cant but opposing effects: 25(OH)D, in- is in line with Char-tier et al. (1979) who
creased the intestinal calcium uptake suggested a direct regulatory role of vita-
whereas 24,25(OH),D, decreased it. Previ- min D, on the eel intestinal calcium irdlux
ously, the effects of vitamin D, and its ma- and Yanda and Ghazarian (1981) who could
jor metabolites on intestinal calcium ab- not detect further metabolism of vitamin D,
sorption have not been examined in marine and 25(OH)D, in the intestinal mucosa of
teleosts, and the present results show a dif- rainbow trout. Although the existence of
ferent pattern compared with that obtained circulating 25(OH)D, in marine teleosts
in freshwater teleost species. In freshwater has not been confirmed by direct measure-
eel, vitamin D3 and 1,25(OH),D, increased ments, this is indicated by the presence of a
the intestinal calcium uptake, whereas specific transport protein for 25(OH)D, in
24,25(OH)*D, had no effect, both when us- the plasma of Atlantic cod (Hay and Wat-
ing vitamin D3 pretreatment (7 days) (Fen- son, 1976).
wick et al., 1984) and when the intestine Both in freshwater eel and tilapia the in-
was exposed to the metabolites during in creased intestinal calcium uptake was
vitro perfusion (Char-tier et al., 1979). The partly or entirely inhibited by chlorproma-
intestinal calcium infhrx also increased by zine (Flik et al., 1982; Fenwick, 1984; Fen-
1,25(OH),D, in tilapia and by vitamin D, in wick et al., 1984), a substance known to
goldfish (Flik et al., 1982; Fenwick, 1984). inhibit calcium-dependent ATP hydrolysis
The freshwater eel responded more (Ghisjen et al., 1980). Vitamin D, was
strongly to unmodified vitamin D3 than to therefore suggested to act, at least partly,
other metabolites tested (Char-tier et al., via an active uptake mechanism in eel in-
1979; Fenwick et al., 1984). On the basis of testine (Fenwick et al., 1984). The intesti-
these findings and previous results showing nal calcium uptake in Atlantic cod contains
ontogenetic specificity of vitamin D, me- a saturable part (60%) that can be inhibited
tabolism and action in chickens, Fenwick et by chlorpromazine (SundelI and Bjiimsson,
al. (1984) suggested that teleosts might 1988). This suggests that marine teleosts
 VITAMIN D3 AND INTESTINAL Ca2+ ABSORPTION 77

can respond to a vitamin D, regulatory sys- The present study concludes that 25-
tem in a manner similar to that proposed for (OH)D, and 24,25(OH),D, are active in reg-
freshwater teleosts. However, the vitamin ulating calcium influx across the intestinal
D, system may also act by changing the mucosa of the Atlantic cod. 25(OH)D, in-
permeability of the intestinal mucosa, as creases calcium absorption whereas
suggested for mammals (DeLuca and 24,25(OH),D, decreases it, both which can
Schnoes, 1983). be important regulatory mechanisms in a
Since marine teleosts are exposed to a marine teleost. It is further concluded that
large concentration gradient of calcium di- the active metabolites of vitamin D3 are dif-
rected inward, the necessity of an endo- ferent in the marine Atlantic cod compared
crine system to increase calcium uptake with freshwater species since vitamin D,
can be questioned. However, the intestinal and 1,25(OH)2D3, which are active in fresh-
calcium uptake of Atlantic cod increases water teleost species, did not affect the in-
during sexual maturation (Sundell and testinal calcium influx in the Atlantic cod.
Bjiimsson, 1988), probably due to the in-
creased calcium demand during this period ACKNOWLEDGMENTS
(Bjiimsson and Haux, 1985), and this pro-
cess might be controlled by 25(OH)D,. The The authors express their appreciation to Docent
role in fish would thus be analogous to that Michael Sturen, Department of Veterinary Medicine,
in humans and hens, where 1,25(OH),D, is Swedish University of Agriculture and Forestry Sci-
considered to play an important role in cal- ences, Skara, for supplying vitamin Ds, 25(OH)Ds,
cium mobilization during pregnancy and 24,25(OH)*Ds, and l,ZS(OH),D,. This work was sup
ported by the Swedish Natural Science Research
egg laying, respectively (Spanos et al., Council and the Hierta-Retzius Foundation.
1976; Kumar et al., 1977).
The physiological role of 24,25(OH),D,
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