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Lack of vitamin D3 synthesis in Tilapia mossambica from cholesterol and acetate
Camp. Biochem. Physiol. Vol. 114A, No. 1, pp. 21-25, 1996 Copyright 0 1996 Elsevier Science Inc.
ISSN 0300-9629/96/$15.00 SSDI 0300-9629(95)02025-F
ELSEVIER
Lack of Vitamin D, Synthesis in Tilapia moss~~&ca from Cholesterol and Acetate D. Sunita Rao ad
N. Raghuramulu
NATIONAL INSTITUTE OF NUTRITION, JAMAI OSMANIA, P.0
HYDEEUBAD--500 007,
INDIA
ABSTRACT. Vitamin D synthesis in fish was studied using cholesterol and acetate as precursors. Crude liver homogenate preparation of Til@ia mossambica (Tilapia) incubated with 4-14C cholesterol was found not to result in the formation of radiolabelled vitamin D,. Intravenous injection of 4-14C cholesterol
also did not result in the formation of 14C vitamin Dj. Intraperitoneal injection of 14C acetate was found not to have the label in vitamin D3 fraction of the whole fish. Thus, the results suggest the absence of a nonphotochemical pathway of vitamin D synthesis in fish or, even if it exists, cholesterol may not be the substrate. In addition, fish may not be able to synthesize secosteroid directly from 2-carbon units like acetate, to make vitamin D,. COMP BIOCHEM PHYSIOL 114A;1:21-25,
KEY WORDS.
Acetate,
1996.
cholesterol,
liver, nonphotochemical,
INTRODUCTION About 65 years ago, Bills (2) reported that at least a portion of the vitamin D of cod liver oil probably originates from nondietary sources. Subsequent studies reported that the nondietary source may not be related to solar energy, because all traces of irradiation are absorbed by sea water in the first few meters of depth (1,9). Further, Bills demonstrated an increase in the vitamin D content of the livers of catfish maintained in darkness and on a vitamin D-free diet for 6 months. Although a number of parameters in these investigations were left undefined, the results strongly suggested, as Bills (2,3) pointed out, that fish may have the ability to produce vitamin D by a nonphotochemical process. Later, several workers examined the possibility of a nonphotochemical pathway for vitamin D synthesis in fish (5,6,8,10,12). However, the findings are equivocal. Most of these workers failed to demonstrate vitamin D, formation in fish using 7-dehydrocholesterol (7-DHC) as precursor, indicating that this may not be a proper compound. However, the role of cholesterol and acetate as precursors for vitamin D, were not explored using good analytical tools. Hence, in the present study, 14C cholesterol and 14C acetate were explored as precursors for vitamin D formation in Tilapia. It was observed earlier that Tilapia, a freshwater fish, contained significant amounts of vitamin D (11).
Address reprint requests to: Dr. N. Raghuramulu, Sr. Deputy Director, National Institute of Nutrition, Hyderabad-500 007, A.P, India. Received 14 July 1994; revised 29 May 1995; accepted 8 June 1995.
secosteroid,
MATERIALS Chemicals
sterol, synthesis tilapia
AND METHODS
4-14C cholesterol (spec. act. 55 mCi/mmol) was purchased from Radiochemical Centre, Amersham, U.K. and 1,2 14C acetate (spec. act. 41.9 mCi/mmol) was purchased from Bhabha Atomic Research Centre, Bombay, India. The details of all the other chemicals used and HPLC analysis was as described earlier (11).
Fish Sam@es Tilapia were supplied by the Department of Fisheries, Tank Bund, Hyderabad, Andhra Pradesh, India.
Incubation of 4-14C Cholesterol with Liver Homogenate of Tilapiu Livers were excised from four advanced fingerlings of Tilapia (appr. body wt. 200-250 g) and a 10% homogenate was prepared by mixing 2 g of the pooled liver with 20 ml of 0.02 M potassium phosphate buffer, pH 7.4 containing 0.1 mM EDTA. The liver homogenate (2 ml) was incubated with 75 nCi of 4-14C cholesterol in 50 ~1 of ethanol at 25°C for 1 h in darkness. Similar incubation was carried out with liver homogenate previously heated at 100°C for 30 min and served as control. The incubated homogenates were mixed with 40 ml of chloroform: methanol (1: 1) and lipids were extracted by the method of Bligh and Dyer (4). The extracted lipids were then examined for vitamin D, and its metabolites by HPLC.
D. Sunita Rao and N. Raghuramulu
22
Intruvenous Injection 4 4-14C Cholesterol
to Tilapiu ‘I-DHC & Cholwtarol
4-14C cholesterol (75 nCi in 100 ~1 ethanol) was injected into advanced fingerling Tilapia (2 fish-approx. body wt. 200-250 g) through the caudal vein and the fish were kept in an aerated water tank in a dark room for 4 h at 25°C.
region
After 4 h, the livers of the fish were excised, homogenized, and processed as mentioned above for the analysis of vitamin D and its metabolites.
Intraperitoneal
Injection of 14C Acetate to Tilapiu
14C acetate (1 FCi in 100 ~1) was injected into Tilapia (4 fish-approx body wt. 20 g) intraperitoneally, and the fish were kept in an aerated water tank in a dark room at 25°C and fed commercial food containing all the nutrients except vitamin Dj until they were killed at 6 h (2 fish) and 4 days (2 fish) after the injections. The lipids were extracted from the whole fish by the method of Sugisaki et al. (10). The lipid was hydrolyzed in 2.5 ml of 15% KOH (w/v)
Fhtion volume bd) FIG. 1. Elution pattern of the liver sample of
sambica (incubated
with 14C cholesterol)
Tilapia mos-
on Sephadex
LH-20 column.
in ethanol, and the neutral products were removed by extraction with diethyl ether. The hydrolyzed lipid was then treated with digitonin in ethanol to remove sterols. The digitonide was removed by centrifugation and the ethanol layer was extracted with carbon tetrachloride. The combined solvent layers were dried under vacuum below 30°C and subjected to HPLC for vitamin D metabolites. Vitamin D and its metabolites scribed earlier (11).
were examined
as de-
About 70% of the label was recovered in the lipid extract. Figure 1 shows the elution pattern of the vitamin D metabolites in liver sample of Tilapia (after incubation with 4-14C cholesterol) on Sephadex LH-20 column. About 12.6% of the label loaded was recovered in the region corresponding to vitamin DJ, whereas 87.1% of the label was found in the region of cholesterol and 7-DHC, and only 0.3% of region. profiles of the vitamin
jected radioactivity was found in the liver. From the label loaded on the Sephadex LH-20 column, about 7.8% was recovered in the vitamin D, region, and around 90.3% in the combined fraction of 7-DHC and cholesterol region; no radioactivity was observed in the region corresponding to 25-OH-D,. The HPLC profiles of the vitamin D3 fraction from the Sephadex LH-20 column loaded with the liver homogenate
RESULTS Conversion of 4-14C Cholesterol to Vitamin D3 by the Liver Homogenate 4 Tilapia In Vitro
the label was in the 25-OH-D, Figure 2 shows the HPLC
column from lipid extracts of liver of Tilapia given intravenous injection of 4-14C cholesterol. About 2% of the in-
D,
(panel A) and 25-OH-D3 fractions (pane1 B) as obtained above from the Sephadex LH-20 column. It can be seen that no peaks corresponding to standard radiolabelled vitamin Dj and 25-OH-D3 were observed. Similarly, no peaks of 14C vitamin D, or 14C 25-OH-D3 were observed in the control sample.
of Tilapia is shown in Fig. 4. Similar to the in vitro study, no peak corresponding to 14C vitamin D, was observed (pane1 b). Endogenous vitamin Dj was detected in the liver sample. The elution pattern of the liver sample with added standard vitamin D, and monitored at 265 nm is given in panel c. Figure 5 shows the HPLC profiles of the 25-OH-D, tion of liver homogenate. No [14C] 25-OH-D, found (panel b). Al so, no endogenous 25-OH-D, detected in the sample.
frac-
peak was peak was
Conversion of 14C Acetate to Vitamin D, in Tilapiu In Vivo The radiolabelled HPLC profile of the lipid extracts of Tilapis injected with 14C acetate (6 hr and 4 days) showed absence of radiolabelled vitamin D, and 25-OH-D, peaks (Fig. 6).
Conversion of 4-14C Cholesterol to Vitamin D, in Tilapia In Vivo
DISCUSSION
Figure 3 shows the pattern of separation of vitamin D,, ‘I-DHC, cholesterol, and 25-OH-D, on Sephadex LH-20
A nonphotochemical pathway for vitamin D synthesis has been considered by several authors. Although Blondin et
Vitamin D Biosynthesis in Tifupiu mossambica
0
23
0
FIG. 2. Reverse-phase HPLC profiles of the vitamin D, (A) and 25OH-D, (B) fractions (obtained from the Sephadex LH-20 column) of liver of Thpia mossambica incubated with 4-“C cholesterol (dotted line). Solid line gives the profile for standard [‘HI vitamin Da and 25OH-D,.
al. (5,7) provided direct evidence for the formation of vitamin D (albeit to a very small extent) from 14C 7-DHC on incubation for 4 hr in dark with liver homogenates of Atlantic striped bass Ruccus sax&s, Sugisaki et al. (10) could not demonstrate any significant formation of 14C vitamin D in goldfish injected with 14C 7-DHC even after a longer duration of 7 days. Whatever little vitamin D claimed to have been formed by earlier workers could not be established, due to nonavailability of appropriate sensitive and
7-DHC & Cholesterol resion
specific analytical tools. Takeuchi
et al. ( 12), using specific
and sensitive techniques, failed to show the formation of any vitamin D using 7-DHC as a precursor in Big eye tuna, Yellowtail and Chub mackerel. These results suggest that this ‘steroid’ may be an inappropriate compound for vitamin D formation in fish. It has been shown that the sterol biosynthesis
in fish
(Bass) follows the same pathway as in mammals, and the principal sterol found in fish liver was cholesterol (6). Hence, it was considered that cholesterol may be an appropriate precursor for vitamin D formation. However, we found that no 14C vitamin D3 could be detected in Tilapia injected with 14C cholesterol in dark for 15 hr, whereas exposure to UV light resulted in 14C vitamin D, and 25 OH-D, (data not shown). Similarly, Sugisaki et al. (10) could not show formation of radioactive vitamin D in dark, even after a longer duration of 8 days in goldfish. Here, vitamin D, if formed might have been converted to other compounds during 8 days. Hence, in the present study, the formation of vitamin D3 from 14C cholesterol by the liver homogenate in Tilapia was studied with a lesser time period (1 hr) and found that fish was unable to form 14C vitamin D3 in vitro. Fish being a cold blooded animal, the in vitro formation of 14C vitamin D, may not occur if the incuba-
FIG. 3. Elution pattern of the liver sample of Tiktpiamossambica given intravenous injection of 14C cholesterol on Sephadex LH-20 cohunn.
tion conditions are not ideal, and also liver may not be the site for vitamin D, formation. Thus, the in viva formation of vitamin D was examined. Moreover, even if vitamin D is synthesized elsewhere in fish, it may come to the liver for storage. Because 14C cholesterol was given via the intravenous route and liver was examined for 14C vitamin D3, 4 hr should have been sufficient for the detection of
24
D. Sunita Rao and N. Raghuramulu
1
3
5
7
9
0
0
8
12
00
Retention time (min 1
FIG. 4. Reverse-phase HPLC profiles of vitamin D, fraction (obtained from the Sephadex LH-20 column) of the liver of Ttiapia mossambica injected with 4-14C cholesterol. (a) Standard radiolabelled profile. (b) Sample radiolabelled elution profile (dotted line). Solid line is the elution pattern monitored at 265 nm. (c) Coelution with standard vitamin D,.
vitamin D3 in liver, if formed. However, the finding was in line with the in vitro observation indicating that cholesterol may be an inappropriate intermediate in vitamin D, formation. Thus, it appears that the formation of vitamin D from a steroid (cholesterol or 7-DHC) may not occur. The breakdown of bond between C-9 and C-10 of the ‘B ring’ of the perhydro-1,2 cyclopenteno phenanthrene ring system to result in a secosteroid (vitamin D,) needs a lot of energy which, in biological systems, may not be possible. Further, Sugisaki et al. (10) observed that goldfish were unable to make vitamin D, from 14C mevalonate in dark over a loday period. Again, this may be because mevalonate is an
intermediate in the biosynthesis of sterols, it should depend on ‘B ring’ cleavage to form vitamin D. But earlier studies, along with the present study, have shown that fish were unable to cleave the ‘B ring’ of the steroid in dark, which may be possible only by exposure to UV light as was observed in the present study. Therefore, we suggest that no substrate that has to go through the steroid pathway can serve as a precursor for nonphotochemical vitamin D, formation in fish. The next question we addressed was whether or not fish can form the secosteroid directly without involving the steroid pathway. In this context, acetate, which is the source of all the carbon atoms of vitamin D in higher ani-
!2 . 500 -? L 400 w
.
.g 300 > ._ w 5 200 ._ % a
too
1
2
3
4
5 Retention
0
time
3
hid
FIG. 5. Reverse-phase HPLC profiles of 25-OH-D, fraction (obtained from the Sephadex LH-20 column) of liver of Tifapia mossambica injected with 14C cholesterol. (a) Standard radiolabelled profile. (b) Sample radiolabelled elution profile (dotted line). Solid line gives the UV monitored (265 nm) profile.
Vitamin
D Biosynthesis
in Tilupia mossumbica
25
0
0 1
3
5
ReCmtion
time
?
1
9
2
3 Retention
[nin)
b time
5
h,in)
FIG. 6. Reverse-phase HPLC profiles of radiolabelled vitamin D, (A) and 25OH-D, (B) fractions obtained from Sephadex LH-20 column of INapia mossambica (whole fish) given intraperitoneal injection of 14C acetate and kept in dark for 6 hr (closed circles) or 4 days (open circles). Solid line is elution profile of radiolabelled standards.
mals,
may
compound, tion
be
the
however,
of labelled
more
appropriate
was found
vitamin
precursor.
not to result
D. Thus,
photochemical process’ for vitamin be operating in fish.
it appears
Even
this
4.
in the formathat,
D formation
a ‘nonmay not
5.
6.
We thank Dr. M. Raghunath, fur his useful comments in the prepamtion of thismanustipt. We thank Dr. Vinodini Reddy, fbrrner Director of the Institute, and Dr. Muhtab S. Bamji, former Director Grade
Scientist, for their constant encouragement and useful suggestions throughout this study. Our thanks ure due to the Commissioner and Staff Department of Fisheries, Government of Andhra Pradesh, Hyderabad for the supply of fish. The financial support of the h&m Council of Medical Research, New Delhi, India is gratefully ucknowl-
7.
8.
edged. 9. References Atkins, W.R.G.; Poole, H.H. Photoelectric measurement of the penetration of light of various wave lengths into the sea and the physiological bearing of the results. Phil. Trans. R. Sot. B222:129-164;1933. Bills, C.E. Antiricketic substances VI. The distribution of vitamin D, with some notes on its possible origin. J. Biol. Chem. 72:751-758;1927. Bills, C.E. Vitamin D group. In: Serbell, W.H., Jr.; Harris, R.S., eds. The Vitamins, Vol. 2, New York: Academic Press; 1954:132-210.
10.
11.
12.
Bligh, E.G.; Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917; 1959. Blondin, G.A.; Kulkami, B.D.; Nes, W.R. Concerning the nonphotochemical biosynthesis of vitamin D, in fish. J. Am. Chem. Sot. 86:2528-2529;1964. Blondin, G.A.; Scott, J.L.; Hummer, J.K.; Kulkami. B.D.; Nes, W.R. The biosynthesis of squalene and sterols in fish. Comp. Biochem. Physiol. 17:391-407;1966. Blondin, G.A.; Kulakami, B.D.; Nes, W.R. A study of the origin of vitamin D from 7-Dehydrocholesterol in fish. Comp. Biochem. Physiol. 20:379-390;1967. Holick, M.F. Phylogenetic and evolutionary aspects of vitamin D from phytoplankton to humans. In: Vertebrate Endocrinology Fundamentals and Biomedical Implications, vol. 3, New York: Academic Press; 1989:7-43. Hulburt, E.O. Penetration of ultra-violet light into pure water and sea-water. J. Opt. Sot. Am. 17:15-22;1928. Sugisaki, N.; Welcher, M.; Monder, C. Lack of vitamin Dj synthesis by goldfish (Carussiw uuxuus L). Comp. Biochem. Physiol. 49B:647-653;1974. Sunita Rao, D.; Raghuramulu, N. Vitamin D and its related parameters content in fresh-water wild fishes. Comp. Biothem. Physiol. 111(2):191-198;1995. Takeuchi, A.; Okano, T.; Tanda, M.; Kobayashi, T. Possible origin of extremely high contents of vitamin Dj in some kinds of fish liver. Comp. Biochem. Physiol. lOOA(2):483487;1991.
ISSN 0300-9629/96/$15.00 SSDI 0300-9629(95)02025-F
ELSEVIER
Lack of Vitamin D, Synthesis in Tilapia moss~~&ca from Cholesterol and Acetate D. Sunita Rao ad
N. Raghuramulu
NATIONAL INSTITUTE OF NUTRITION, JAMAI OSMANIA, P.0
HYDEEUBAD--500 007,
INDIA
ABSTRACT. Vitamin D synthesis in fish was studied using cholesterol and acetate as precursors. Crude liver homogenate preparation of Til@ia mossambica (Tilapia) incubated with 4-14C cholesterol was found not to result in the formation of radiolabelled vitamin D,. Intravenous injection of 4-14C cholesterol
also did not result in the formation of 14C vitamin Dj. Intraperitoneal injection of 14C acetate was found not to have the label in vitamin D3 fraction of the whole fish. Thus, the results suggest the absence of a nonphotochemical pathway of vitamin D synthesis in fish or, even if it exists, cholesterol may not be the substrate. In addition, fish may not be able to synthesize secosteroid directly from 2-carbon units like acetate, to make vitamin D,. COMP BIOCHEM PHYSIOL 114A;1:21-25,
KEY WORDS.
Acetate,
1996.
cholesterol,
liver, nonphotochemical,
INTRODUCTION About 65 years ago, Bills (2) reported that at least a portion of the vitamin D of cod liver oil probably originates from nondietary sources. Subsequent studies reported that the nondietary source may not be related to solar energy, because all traces of irradiation are absorbed by sea water in the first few meters of depth (1,9). Further, Bills demonstrated an increase in the vitamin D content of the livers of catfish maintained in darkness and on a vitamin D-free diet for 6 months. Although a number of parameters in these investigations were left undefined, the results strongly suggested, as Bills (2,3) pointed out, that fish may have the ability to produce vitamin D by a nonphotochemical process. Later, several workers examined the possibility of a nonphotochemical pathway for vitamin D synthesis in fish (5,6,8,10,12). However, the findings are equivocal. Most of these workers failed to demonstrate vitamin D, formation in fish using 7-dehydrocholesterol (7-DHC) as precursor, indicating that this may not be a proper compound. However, the role of cholesterol and acetate as precursors for vitamin D, were not explored using good analytical tools. Hence, in the present study, 14C cholesterol and 14C acetate were explored as precursors for vitamin D formation in Tilapia. It was observed earlier that Tilapia, a freshwater fish, contained significant amounts of vitamin D (11).
Address reprint requests to: Dr. N. Raghuramulu, Sr. Deputy Director, National Institute of Nutrition, Hyderabad-500 007, A.P, India. Received 14 July 1994; revised 29 May 1995; accepted 8 June 1995.
secosteroid,
MATERIALS Chemicals
sterol, synthesis tilapia
AND METHODS
4-14C cholesterol (spec. act. 55 mCi/mmol) was purchased from Radiochemical Centre, Amersham, U.K. and 1,2 14C acetate (spec. act. 41.9 mCi/mmol) was purchased from Bhabha Atomic Research Centre, Bombay, India. The details of all the other chemicals used and HPLC analysis was as described earlier (11).
Fish Sam@es Tilapia were supplied by the Department of Fisheries, Tank Bund, Hyderabad, Andhra Pradesh, India.
Incubation of 4-14C Cholesterol with Liver Homogenate of Tilapiu Livers were excised from four advanced fingerlings of Tilapia (appr. body wt. 200-250 g) and a 10% homogenate was prepared by mixing 2 g of the pooled liver with 20 ml of 0.02 M potassium phosphate buffer, pH 7.4 containing 0.1 mM EDTA. The liver homogenate (2 ml) was incubated with 75 nCi of 4-14C cholesterol in 50 ~1 of ethanol at 25°C for 1 h in darkness. Similar incubation was carried out with liver homogenate previously heated at 100°C for 30 min and served as control. The incubated homogenates were mixed with 40 ml of chloroform: methanol (1: 1) and lipids were extracted by the method of Bligh and Dyer (4). The extracted lipids were then examined for vitamin D, and its metabolites by HPLC.
D. Sunita Rao and N. Raghuramulu
22
Intruvenous Injection 4 4-14C Cholesterol
to Tilapiu ‘I-DHC & Cholwtarol
4-14C cholesterol (75 nCi in 100 ~1 ethanol) was injected into advanced fingerling Tilapia (2 fish-approx. body wt. 200-250 g) through the caudal vein and the fish were kept in an aerated water tank in a dark room for 4 h at 25°C.
region
After 4 h, the livers of the fish were excised, homogenized, and processed as mentioned above for the analysis of vitamin D and its metabolites.
Intraperitoneal
Injection of 14C Acetate to Tilapiu
14C acetate (1 FCi in 100 ~1) was injected into Tilapia (4 fish-approx body wt. 20 g) intraperitoneally, and the fish were kept in an aerated water tank in a dark room at 25°C and fed commercial food containing all the nutrients except vitamin Dj until they were killed at 6 h (2 fish) and 4 days (2 fish) after the injections. The lipids were extracted from the whole fish by the method of Sugisaki et al. (10). The lipid was hydrolyzed in 2.5 ml of 15% KOH (w/v)
Fhtion volume bd) FIG. 1. Elution pattern of the liver sample of
sambica (incubated
with 14C cholesterol)
Tilapia mos-
on Sephadex
LH-20 column.
in ethanol, and the neutral products were removed by extraction with diethyl ether. The hydrolyzed lipid was then treated with digitonin in ethanol to remove sterols. The digitonide was removed by centrifugation and the ethanol layer was extracted with carbon tetrachloride. The combined solvent layers were dried under vacuum below 30°C and subjected to HPLC for vitamin D metabolites. Vitamin D and its metabolites scribed earlier (11).
were examined
as de-
About 70% of the label was recovered in the lipid extract. Figure 1 shows the elution pattern of the vitamin D metabolites in liver sample of Tilapia (after incubation with 4-14C cholesterol) on Sephadex LH-20 column. About 12.6% of the label loaded was recovered in the region corresponding to vitamin DJ, whereas 87.1% of the label was found in the region of cholesterol and 7-DHC, and only 0.3% of region. profiles of the vitamin
jected radioactivity was found in the liver. From the label loaded on the Sephadex LH-20 column, about 7.8% was recovered in the vitamin D, region, and around 90.3% in the combined fraction of 7-DHC and cholesterol region; no radioactivity was observed in the region corresponding to 25-OH-D,. The HPLC profiles of the vitamin D3 fraction from the Sephadex LH-20 column loaded with the liver homogenate
RESULTS Conversion of 4-14C Cholesterol to Vitamin D3 by the Liver Homogenate 4 Tilapia In Vitro
the label was in the 25-OH-D, Figure 2 shows the HPLC
column from lipid extracts of liver of Tilapia given intravenous injection of 4-14C cholesterol. About 2% of the in-
D,
(panel A) and 25-OH-D3 fractions (pane1 B) as obtained above from the Sephadex LH-20 column. It can be seen that no peaks corresponding to standard radiolabelled vitamin Dj and 25-OH-D3 were observed. Similarly, no peaks of 14C vitamin D, or 14C 25-OH-D3 were observed in the control sample.
of Tilapia is shown in Fig. 4. Similar to the in vitro study, no peak corresponding to 14C vitamin D, was observed (pane1 b). Endogenous vitamin Dj was detected in the liver sample. The elution pattern of the liver sample with added standard vitamin D, and monitored at 265 nm is given in panel c. Figure 5 shows the HPLC profiles of the 25-OH-D, tion of liver homogenate. No [14C] 25-OH-D, found (panel b). Al so, no endogenous 25-OH-D, detected in the sample.
frac-
peak was peak was
Conversion of 14C Acetate to Vitamin D, in Tilapiu In Vivo The radiolabelled HPLC profile of the lipid extracts of Tilapis injected with 14C acetate (6 hr and 4 days) showed absence of radiolabelled vitamin D, and 25-OH-D, peaks (Fig. 6).
Conversion of 4-14C Cholesterol to Vitamin D, in Tilapia In Vivo
DISCUSSION
Figure 3 shows the pattern of separation of vitamin D,, ‘I-DHC, cholesterol, and 25-OH-D, on Sephadex LH-20
A nonphotochemical pathway for vitamin D synthesis has been considered by several authors. Although Blondin et
Vitamin D Biosynthesis in Tifupiu mossambica
0
23
0
FIG. 2. Reverse-phase HPLC profiles of the vitamin D, (A) and 25OH-D, (B) fractions (obtained from the Sephadex LH-20 column) of liver of Thpia mossambica incubated with 4-“C cholesterol (dotted line). Solid line gives the profile for standard [‘HI vitamin Da and 25OH-D,.
al. (5,7) provided direct evidence for the formation of vitamin D (albeit to a very small extent) from 14C 7-DHC on incubation for 4 hr in dark with liver homogenates of Atlantic striped bass Ruccus sax&s, Sugisaki et al. (10) could not demonstrate any significant formation of 14C vitamin D in goldfish injected with 14C 7-DHC even after a longer duration of 7 days. Whatever little vitamin D claimed to have been formed by earlier workers could not be established, due to nonavailability of appropriate sensitive and
7-DHC & Cholesterol resion
specific analytical tools. Takeuchi
et al. ( 12), using specific
and sensitive techniques, failed to show the formation of any vitamin D using 7-DHC as a precursor in Big eye tuna, Yellowtail and Chub mackerel. These results suggest that this ‘steroid’ may be an inappropriate compound for vitamin D formation in fish. It has been shown that the sterol biosynthesis
in fish
(Bass) follows the same pathway as in mammals, and the principal sterol found in fish liver was cholesterol (6). Hence, it was considered that cholesterol may be an appropriate precursor for vitamin D formation. However, we found that no 14C vitamin D3 could be detected in Tilapia injected with 14C cholesterol in dark for 15 hr, whereas exposure to UV light resulted in 14C vitamin D, and 25 OH-D, (data not shown). Similarly, Sugisaki et al. (10) could not show formation of radioactive vitamin D in dark, even after a longer duration of 8 days in goldfish. Here, vitamin D, if formed might have been converted to other compounds during 8 days. Hence, in the present study, the formation of vitamin D3 from 14C cholesterol by the liver homogenate in Tilapia was studied with a lesser time period (1 hr) and found that fish was unable to form 14C vitamin D3 in vitro. Fish being a cold blooded animal, the in vitro formation of 14C vitamin D, may not occur if the incuba-
FIG. 3. Elution pattern of the liver sample of Tiktpiamossambica given intravenous injection of 14C cholesterol on Sephadex LH-20 cohunn.
tion conditions are not ideal, and also liver may not be the site for vitamin D, formation. Thus, the in viva formation of vitamin D was examined. Moreover, even if vitamin D is synthesized elsewhere in fish, it may come to the liver for storage. Because 14C cholesterol was given via the intravenous route and liver was examined for 14C vitamin D3, 4 hr should have been sufficient for the detection of
24
D. Sunita Rao and N. Raghuramulu
1
3
5
7
9
0
0
8
12
00
Retention time (min 1
FIG. 4. Reverse-phase HPLC profiles of vitamin D, fraction (obtained from the Sephadex LH-20 column) of the liver of Ttiapia mossambica injected with 4-14C cholesterol. (a) Standard radiolabelled profile. (b) Sample radiolabelled elution profile (dotted line). Solid line is the elution pattern monitored at 265 nm. (c) Coelution with standard vitamin D,.
vitamin D3 in liver, if formed. However, the finding was in line with the in vitro observation indicating that cholesterol may be an inappropriate intermediate in vitamin D, formation. Thus, it appears that the formation of vitamin D from a steroid (cholesterol or 7-DHC) may not occur. The breakdown of bond between C-9 and C-10 of the ‘B ring’ of the perhydro-1,2 cyclopenteno phenanthrene ring system to result in a secosteroid (vitamin D,) needs a lot of energy which, in biological systems, may not be possible. Further, Sugisaki et al. (10) observed that goldfish were unable to make vitamin D, from 14C mevalonate in dark over a loday period. Again, this may be because mevalonate is an
intermediate in the biosynthesis of sterols, it should depend on ‘B ring’ cleavage to form vitamin D. But earlier studies, along with the present study, have shown that fish were unable to cleave the ‘B ring’ of the steroid in dark, which may be possible only by exposure to UV light as was observed in the present study. Therefore, we suggest that no substrate that has to go through the steroid pathway can serve as a precursor for nonphotochemical vitamin D, formation in fish. The next question we addressed was whether or not fish can form the secosteroid directly without involving the steroid pathway. In this context, acetate, which is the source of all the carbon atoms of vitamin D in higher ani-
!2 . 500 -? L 400 w
.
.g 300 > ._ w 5 200 ._ % a
too
1
2
3
4
5 Retention
0
time
3
hid
FIG. 5. Reverse-phase HPLC profiles of 25-OH-D, fraction (obtained from the Sephadex LH-20 column) of liver of Tifapia mossambica injected with 14C cholesterol. (a) Standard radiolabelled profile. (b) Sample radiolabelled elution profile (dotted line). Solid line gives the UV monitored (265 nm) profile.
Vitamin
D Biosynthesis
in Tilupia mossumbica
25
0
0 1
3
5
ReCmtion
time
?
1
9
2
3 Retention
[nin)
b time
5
h,in)
FIG. 6. Reverse-phase HPLC profiles of radiolabelled vitamin D, (A) and 25OH-D, (B) fractions obtained from Sephadex LH-20 column of INapia mossambica (whole fish) given intraperitoneal injection of 14C acetate and kept in dark for 6 hr (closed circles) or 4 days (open circles). Solid line is elution profile of radiolabelled standards.
mals,
may
compound, tion
be
the
however,
of labelled
more
appropriate
was found
vitamin
precursor.
not to result
D. Thus,
photochemical process’ for vitamin be operating in fish.
it appears
Even
this
4.
in the formathat,
D formation
a ‘nonmay not
5.
6.
We thank Dr. M. Raghunath, fur his useful comments in the prepamtion of thismanustipt. We thank Dr. Vinodini Reddy, fbrrner Director of the Institute, and Dr. Muhtab S. Bamji, former Director Grade
Scientist, for their constant encouragement and useful suggestions throughout this study. Our thanks ure due to the Commissioner and Staff Department of Fisheries, Government of Andhra Pradesh, Hyderabad for the supply of fish. The financial support of the h&m Council of Medical Research, New Delhi, India is gratefully ucknowl-
7.
8.
edged. 9. References Atkins, W.R.G.; Poole, H.H. Photoelectric measurement of the penetration of light of various wave lengths into the sea and the physiological bearing of the results. Phil. Trans. R. Sot. B222:129-164;1933. Bills, C.E. Antiricketic substances VI. The distribution of vitamin D, with some notes on its possible origin. J. Biol. Chem. 72:751-758;1927. Bills, C.E. Vitamin D group. In: Serbell, W.H., Jr.; Harris, R.S., eds. The Vitamins, Vol. 2, New York: Academic Press; 1954:132-210.
10.
11.
12.
Bligh, E.G.; Dyer, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917; 1959. Blondin, G.A.; Kulkami, B.D.; Nes, W.R. Concerning the nonphotochemical biosynthesis of vitamin D, in fish. J. Am. Chem. Sot. 86:2528-2529;1964. Blondin, G.A.; Scott, J.L.; Hummer, J.K.; Kulkami. B.D.; Nes, W.R. The biosynthesis of squalene and sterols in fish. Comp. Biochem. Physiol. 17:391-407;1966. Blondin, G.A.; Kulakami, B.D.; Nes, W.R. A study of the origin of vitamin D from 7-Dehydrocholesterol in fish. Comp. Biochem. Physiol. 20:379-390;1967. Holick, M.F. Phylogenetic and evolutionary aspects of vitamin D from phytoplankton to humans. In: Vertebrate Endocrinology Fundamentals and Biomedical Implications, vol. 3, New York: Academic Press; 1989:7-43. Hulburt, E.O. Penetration of ultra-violet light into pure water and sea-water. J. Opt. Sot. Am. 17:15-22;1928. Sugisaki, N.; Welcher, M.; Monder, C. Lack of vitamin Dj synthesis by goldfish (Carussiw uuxuus L). Comp. Biochem. Physiol. 49B:647-653;1974. Sunita Rao, D.; Raghuramulu, N. Vitamin D and its related parameters content in fresh-water wild fishes. Comp. Biothem. Physiol. 111(2):191-198;1995. Takeuchi, A.; Okano, T.; Tanda, M.; Kobayashi, T. Possible origin of extremely high contents of vitamin Dj in some kinds of fish liver. Comp. Biochem. Physiol. lOOA(2):483487;1991.