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On the site of discrimination of chicks against vitamin D2
ARCHIVES
OF
BIOCHEMISTRY
AND
130, 657-661 (1969)
BIOPHYSICS
On the Site of Discrimination D. DRESCHER: Department
of Biochemistry, Received
of Chicks
H. F. DELUCA, University
November
AND
of Wisconsin,
15, 1968; accepted
against
Vitamin
Di
ill. H. IMRIE Madison,
January
Wisconsin
63YO6
13, 1969
Chicks are capable of converting both vitamins Dz and Ds at approximately equal rates to the biologically active polar metabolite (peak IV) believed to be the metabolically active form of the vitamin. However, the peak IV metabolite from vitamin Dz has little vitamin D activity in chicks while that from D, is more biologically active than Da itself. The peak IV both from Dz and from Dz is fully antirachitic in rats suggesting that the chick discriminates against vitamin DB at the level of the peak I?metabolite. In a recent report from this laboratory, it was shown that chicks respond poorly to vitamin Ds versus D3 because Ds is rapidly deleted from blood and other tissues and excreted into the intestine via the bile (1). It was suggested that vitamin Dz or a metabolite thereof is rapidly rejected as a foreign material reducing its effective lifetime in the animal. In other experiments carried out in this laboratory, the existence of a metabolically active form of the vitamin has been established (2, 3). This metabolite is the major vitamin D-active substance in the body when truly physiologic doses of the vitamin are given (2,4). Furthermore, it is formed in large amounts before physiologic expression of the vitamin’s action is obvious (4). It not only mimics the actions of vitamin D in elevating serum calcium concentration and stimulating intestinal calcium transport, but its effects are more rapid than those of the parent vitamin (3). It is also the major vitamin Dactive substance in the nucleus where the vitamin is believed to function to initiate
events ultimately leading to increased calcium transport (4, 5). In this report it will be shown that this metabolite is formed from both Dz and Ds in chicks but the amount from Dz in the chick remains at a 10~7level whereas it is usually at high levels in the case of Da. In addition it will be shown that this metabolite fraction is fully biologically active in rats whether it is derived from Dz or DB from both chicks and rats. However the metabolite from D? is only slightly active in chicks whether it is prepared from rat or chick tissues. On the other hand, the metabolite from Ds is more biologically active in chick intestinal absorption than is the parent vitamin. These results strongly suggest that the discrimination by chicks against vitamin D, is at the level ot the suggested metabolically active form of the vitamin. ,4 preliminaq report of this work was presented before the Federated Societies for Experimental Riology and Medicine (6).
‘Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported by U.S.P.H.S. Grant AM0 5800-07 and by the Steenbock Research fund of the Wisconsin Alumni Research Fouudation. 2 Supported by NIH Training Grant GM00236 BCH from National Institute of General Medical Sciences.
Preparation of chicks. One-day-old white Leghorn cockerels were obtained from the Sunnyside Hatcheries, Oregon, Wisconsin, and housed in an incubator maintained at 37”. They were fed a purified rachitogenic diet as previously described (1). The chicks became severely rachitic on this diet and were used at 4 weeks of age. Preparation of rats. Yolmg male weanling rats
MATERIALS
657
AND
METHOl)S
658
DRESCHER,
DELUCA,
were obtained from Sprague-Dawley, Madison, Wisconsin, and were maintained in hanging wire cages. They were fed ad lib&m a rachitogenic diet (7). After 3 weeks the rats became rachitic and were suitable for the line test assay as described in the U.S. Pharmacopeia (8) as well as for the preparation of vitamin D metabolites. Biological assays. The amount of each metabolite to be tested was determined on the basis of radioactivity assuming each metabolite to possess the same specific activity as the parent vitamin Da; i.e., 122 dpm/IU for 3H Da and 98 dpm/IU for 1% Dz. Rat rickets cure test. This was performed exactly as described for the line test method in the U.S. Pharmacopeia (8). The doses were prepared in ether, dissolved in cotton seed oil (Wesson) and the ether removed with a stream of Nz. Each rat received no more than 0.1 ml of oil. Chick calcium-absorption assay. The chick bioassay used was similar to that described by Migicovsky (9) with certain modifications. Twenty-eight-day-old rachitic Leghorn cockerels weighing 180-210 g were dosed orally with 0.1 ml of the material to be assayed dissolved in Wesson oil at a concentration of 100 IU/ml. Controls were given the same volume of Wesson oil. Dosed chicks were fasted for 24 hr after which each chick was orally administered 2-3 FCi of 45Ca (Nuclear Science and Engineering Co., Pittsburgh, Penn.) in a volume of 0.2 ml. This %Ja solution was also 1% in nonradioactive CaClz. Chicks were sacrificed by ether suffocation exactly 2 hr after adCHCh
EXTRACT
OF WHOLE
CHICK
24 HOURS
7
FRACTION
NUMBER
(IO ML)
FIG. 1. Silicic acid column profile of the chloroform extract of the entire chick that received either 0.25 pg [l, 21aH vitamin D3 or 0.25 pg U 1% vitamin Dz - - - - - - 24 hr before. Chromatography was carried out as described by Lund and DeLuca (2).
AND
IMRIE TABLE
I
DISTRIBUTION OF ADMINISTERED RADIOACTIVITY FROM 0.25-pg DOSES OF 3H Da OR 1% De
AMONG: J-ITAMIN D AND ITS METABOLITE Time (hr) 1
24
Fraction III IV III I\
V;it$I&~
Vitamin Dz (% dose)
0
80.2 8.4 10.4 42.5
f z!z i f
6.8” 2.1 3.2 3.8
70.2 8.3 10.4 13.2
zk f z!z f
8.8” 3.0 4.8 2.8
a Standard deviations. There were at least 4 chicks per group. The recovered radioactivity is from the entire chick whose feathers and gastrointestinal tract had been removed. The chromatography was carried out as described previously and shown in Fig. 1. ministration of label. The tibia of each chick was then removed and dissected until clean. Bones were placed in 25X148-mm Pyrex test tubes and weighed. Radioactive vitamins DZ and D3. [I, 2JH] vitamin D3 (specific activity 26,000 dpm/IU) was prepared as previously described (10) as was [U-14C] vitamin DZ (30,000 dpm/IU) (1). For experiments dealing with the metabolic conversion of the vitamins to metabolites, 0.25 pg dissolved in 0.05 ml 95yo ethanol was administered intrajugularly to each rachitic chick. For experiments dealing with the isolation of metabolites for a study of their biological activity in both rats and chicks, the radioactive vitamins were diluted with the appropriate nonradioactive vitamin to give a specific activity of 122 dpm/IU for D3 and 98 dpm/IU for Dz. These were dissolved in ether to which was added Wesson oil. The ether was removed with a stream of Nz to give a solution containing 2 mg 3H Da or 1°C DZ in 1 ml Wesson oil. One-tenth milliliter of this solution was given orally to a rat or a chick. Extraction and chromatography of vitamin D metabolites. In the experiments concerned with the proportion of either 3H vitamin Da or r4C vitamin Dz and their metabolites in the chick after a 0.25~pg dose, chicks were killed by ether suffocation at the indicated times after dosage. The gastrointestinal tract and feathers were removed and the remaining carcass was ground in a meat grinder and then extracted with methanol-chloroform using a Waring Blendor (11). The entire chloroform extract was then chromatographed on silicic acid columns as described previously (2). Then 5 ml of concentrated nitric acid was added to each tube and bones were digested over heating coils. After cooling, each sample was brought to a volume of 25 ml with a
VITAMIN
diluent solution consisting of 30 vol water, 19 vol ethanol, and 1 vol of the detergent Triton X100. One-milliliter aliquots were then taken and placed in scintillation counting vials witch 15 ml of dioxane counting solution. r-ials were counted at 15yo gain with a window of 50-1000. Dioxane counting solution. The Wa counting solution was made by dissolving 7.0 g PPO, 300 mg dimethyl POPOP, and 150 g napthalene in 1500 ml of dioxane. Two hundred fifty milliliters of water containing 18 mg disodium EDTA was then added. Radioaclivity counting. All radioisotopes were counted in a Packard Tri-Carb liquid scintillation counter, Model 3003 (Packard Instruments Corp., LaGrange, Ill.), employing an external standard for counting efficiency determinations. Dioxane counting solution was used for counting 45Ca, while 1% and 3H were counted using toluene counting sollltion (12).
II
ANTIRACHITIC EFFECTIVENESS OF PEAK METABOLITES IN RATS~
D3
Da Peak D, Peak D2 Up Peak D2 Peak
IV from rat IV from chick IV from rat IV from chick
0.401
0
I I
2
3
INTERNATIONAL
4
5
6
7
8
9
IO
UNITS OF VITAMIN D3
FIG. 2. The relationship of and bone uptake of orally Vertical bars denote standard The exact method is described
vitamin Dt dosage administered 45Ca. error of the mean. in the t,ext.
Fig. 1) while only a small amount apas peak IV metabolites (Table I). After 24 hr, however, there is little unchanged vitamin DS or vitamin Dz. Rather large amounts of peak IV remain in the case of D, while small amounts of peak IV are found in the case of D,. Thus it is clear that peak IV is the major form of the pears
It has previously been shown in rats that 24 hr after a 0.25 pg dose of 1,2 3H vitamin Da most of the remaining radioactivity is found in the peak IV metabolite fraction. Figure 1 demonstrates that this is the case in chicks as well. This profile also serves as a reference point for the experiments to be reported in this communication. At 1 hr after injection of 0.25 pg of either 3H DS or 14C Dz, 70-80% of the dose is recoverable as unchanged vitamin D (peak
Group Vegetable oil control
osot
III,
RESULTS
TABLE
659
Ds IN CHICKS
IV
IU/ta
40 40 42 40 39 41
(1The scores represent group averages where each group consisted of at least 5 and usually 8 rats. The assays were carried out according to the U.S. Pharmacopeia (8) in which each rat was given the equivalent of 4 IU of antirachitic activity. The data are expressed as international unit,s (IU) of antirachitic activity per microgram of compound tested. Standard cholecalciferol gives 40 IU/pg.
original 3H D, administered. These results agree with previous results which showed
that the 14C from 14C D2 disappeared more rapidly from chicks than does 3H from 3H Da. The major difference lies not in the disappearance of peak III or unchanged vitamin D but rather in the rate of disappearance of peak IV. These data suggest that the chick does not discriminate between D, and Da at the level of conversion to peak IV but rather at the rate of peak IV catabolism and excretion. To test this hypothesis directly, peak IV fractions were prepared from both D, and Da from both rats and chicks. As expected, the peak IV fractions from all these sources were all fully antirachitic by the rat, line test assay (Table II) with values of 40 IU//.Lg. A biological assay was devised using rachitic chicks as the test, animal. That the assay is valid is shown by the response curve of chicks to physiologic dose of vitamin D3 (Fig. 2). The response recorded was essentially linear between l-10 IU of vitamin Da. It is also important to note t’hat the
660
DRESCHER, TABLE
ASSAY
OF
VITAMIN D METABOLITES
III
Mean f Standard deviationl”
Number of animals
I
0.22
32
-
0.93 f 1.24 f 1.36 f
0.27 0.25 0.26
14 15 8
40 40 50
1.44
f
0.43
9
57
1.02 f 0.96 f
0.18 0.25
16 11
21 16
0.83
0.13
5
6
f
-
a Standard deviations means of the expression
this will be necessary before such a conclusion can be made. DISCUSSION
f
0.77
AND IMRIE
-
% Total ‘S&/g bone
Vegetable oil control Dt (0.1 /A Ds (0.25 a) Da Peak IV (0.25 pg) from rat Da Peak IV (0.25 pg) from chick DI (10 0.25 /.& Dz Peak IV (0.25 pg) from rat Dt Peak IV (0.25 rg) from chick
IV
ACTIVITY OF PEAK IN CHICKS
I/ --
DELUCA,
were
calculated
by
The figures in parentheses represent the amount of compound given to each animal in the assay group. The data are calculated in terms of international units of antirachitic activity per microgram of compound. Standard cholecalciferol gives 40 IU of antirachitic activity per microgram.
mean response remained quite constant from experiment to experiment. When the peak IV fractions were assayed for vitamin D activity by the above-indicated assay the data shown in Table III resulted. Of great interest is the fact that the peak IV fractions from vitamin D3 derived from both rats and chicks were more active than vitamin D, itself. However, the peak IV fractions from both chicks and rats obtained from vitamin Dz were only slightly active in the chick assay. As would be expected, vitamin D, itself was also less active. It is interesting to note that peak IV from Dz isolated from chicks was less active than that isolated from rats. This suggests that the peak IV from chicks may contain some different metabolites than those from rats which may differ in their potency in chicks. However, further confirmation of
The results of the present study confirm the conclusion reached earlier that vitamin D, or a metabolite thereof is rapidly excreted thereby reducing its effective lifetime. In the course of this study, the biologically active fraction of peak IV has been positively identified as 25-hydroxycholecalciferol in the case of vitamin Dt (13, 14). The metabolite which derives from Dz has not yet been identified, however. Furthermore, strong evidence has been advanced that the 25-hydroxy derivative is the metabolically active form of the vitamin (15). Assuming the 25-hydroxy derivatives as the metabolically active form of the vitamins, it is now possible to suggest a possible mechanism for the discrimination against D, by chicks. It is apparent that the chick can convert both Dz and DS to the biologically active peak IV metabolites. Both are fully active in the rat which does not discriminate between Ds and Da. However, the same peak IV metabolite preparation from D, is only slightly effective in the chick, suggesting that the peak IV metabolite from Dz is rapidly degraded and/or excreted. Assuming the 25.hydroxy derivatives as the metabolically active form of the vitamins, it is apparent that the derivative from Dz would have only a short, lifetime in the chick thus accounting for the poor biological activity of Dz in the chick. Work is now in progress to isolate and identify the biliary excretion products of both Dz and Da in the chick. Preliminary results support the above hypothesis and it appears likely that the curious discrimination against vitamin Dz by chicks will soon be elucidated at the molecular level. REFERENCES 1.
P. F., SNELLGROVE, F., Arch Biochem. Biophys. 120, 525 (1967). LUND, J., AND DELUCA, H. F., J. Lipid Res. 7, 739 (1966). IMRIE,
M.
A. W.,
2.
H.,
NEVILLE,
AND DELUCA,
H.
\‘ITAMIN
I),
3. MORII, H., LUND, H., NEVILLE, P., AND DELUCA, H. F., Brch. Biochem. Biophvs. 120, 503 (1967). 4. DELUCA, II. F., “Vitamins and Hormones,” Vol. 25, p. 315. Academic Press, Kew York (1967). 5. STOHS, S. J., AND DELUCA, H. F., Riochemislry 6, 3338 (1967). 6. DHESCHER, D., IYRIE, M. H., AND DELUCA, II. F., Federation Proc. 27, 675 (1968). 7. STEENBOCK, H., ANDBLACK, A., J.BioZ. Chem. 64, 263 (1925). 8. U. S. Pharmacopeia, 14th Revision. Mack Publishing Co., Easton, Pennsylvania (1955).
IN
CHICKS
661
9. h~lGICOvSIrY, B. B., Can. J. Biochem. Ph!lsiol. 36, 1267 (1957). 10. NEVILLE, P., ASU DELUCA, H. F., Biochemistry 6, 2201 (1960). 11. BLIGH, E. G., AND DYER, W. J., Can J. Biothem. Physiol. 37, 911 (1959). 12. HERBERG, R. J., Anal. Chem. 32, 42 (19GO). 13. BLUNT, J. W., DELUCA, H. I?., AND SCHNOES, II. K., Chern. Conznls. No. 14, comm. 607, p. 801, July 17, 1968. 14. BLUNT, J. W., DELUCA, H. F., AND SCHNOES, H. K., Biochemislry 7,3317 (1968). 15. BLUNT, J. W., TAKAKA, Y., AND DELUCA, H. F., Proc. Natl. Acad. Sci. U. S. 61, 717 (1968).
OF
BIOCHEMISTRY
AND
130, 657-661 (1969)
BIOPHYSICS
On the Site of Discrimination D. DRESCHER: Department
of Biochemistry, Received
of Chicks
H. F. DELUCA, University
November
AND
of Wisconsin,
15, 1968; accepted
against
Vitamin
Di
ill. H. IMRIE Madison,
January
Wisconsin
63YO6
13, 1969
Chicks are capable of converting both vitamins Dz and Ds at approximately equal rates to the biologically active polar metabolite (peak IV) believed to be the metabolically active form of the vitamin. However, the peak IV metabolite from vitamin Dz has little vitamin D activity in chicks while that from D, is more biologically active than Da itself. The peak IV both from Dz and from Dz is fully antirachitic in rats suggesting that the chick discriminates against vitamin DB at the level of the peak I?metabolite. In a recent report from this laboratory, it was shown that chicks respond poorly to vitamin Ds versus D3 because Ds is rapidly deleted from blood and other tissues and excreted into the intestine via the bile (1). It was suggested that vitamin Dz or a metabolite thereof is rapidly rejected as a foreign material reducing its effective lifetime in the animal. In other experiments carried out in this laboratory, the existence of a metabolically active form of the vitamin has been established (2, 3). This metabolite is the major vitamin D-active substance in the body when truly physiologic doses of the vitamin are given (2,4). Furthermore, it is formed in large amounts before physiologic expression of the vitamin’s action is obvious (4). It not only mimics the actions of vitamin D in elevating serum calcium concentration and stimulating intestinal calcium transport, but its effects are more rapid than those of the parent vitamin (3). It is also the major vitamin Dactive substance in the nucleus where the vitamin is believed to function to initiate
events ultimately leading to increased calcium transport (4, 5). In this report it will be shown that this metabolite is formed from both Dz and Ds in chicks but the amount from Dz in the chick remains at a 10~7level whereas it is usually at high levels in the case of Da. In addition it will be shown that this metabolite fraction is fully biologically active in rats whether it is derived from Dz or DB from both chicks and rats. However the metabolite from D? is only slightly active in chicks whether it is prepared from rat or chick tissues. On the other hand, the metabolite from Ds is more biologically active in chick intestinal absorption than is the parent vitamin. These results strongly suggest that the discrimination by chicks against vitamin D, is at the level ot the suggested metabolically active form of the vitamin. ,4 preliminaq report of this work was presented before the Federated Societies for Experimental Riology and Medicine (6).
‘Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported by U.S.P.H.S. Grant AM0 5800-07 and by the Steenbock Research fund of the Wisconsin Alumni Research Fouudation. 2 Supported by NIH Training Grant GM00236 BCH from National Institute of General Medical Sciences.
Preparation of chicks. One-day-old white Leghorn cockerels were obtained from the Sunnyside Hatcheries, Oregon, Wisconsin, and housed in an incubator maintained at 37”. They were fed a purified rachitogenic diet as previously described (1). The chicks became severely rachitic on this diet and were used at 4 weeks of age. Preparation of rats. Yolmg male weanling rats
MATERIALS
657
AND
METHOl)S
658
DRESCHER,
DELUCA,
were obtained from Sprague-Dawley, Madison, Wisconsin, and were maintained in hanging wire cages. They were fed ad lib&m a rachitogenic diet (7). After 3 weeks the rats became rachitic and were suitable for the line test assay as described in the U.S. Pharmacopeia (8) as well as for the preparation of vitamin D metabolites. Biological assays. The amount of each metabolite to be tested was determined on the basis of radioactivity assuming each metabolite to possess the same specific activity as the parent vitamin Da; i.e., 122 dpm/IU for 3H Da and 98 dpm/IU for 1% Dz. Rat rickets cure test. This was performed exactly as described for the line test method in the U.S. Pharmacopeia (8). The doses were prepared in ether, dissolved in cotton seed oil (Wesson) and the ether removed with a stream of Nz. Each rat received no more than 0.1 ml of oil. Chick calcium-absorption assay. The chick bioassay used was similar to that described by Migicovsky (9) with certain modifications. Twenty-eight-day-old rachitic Leghorn cockerels weighing 180-210 g were dosed orally with 0.1 ml of the material to be assayed dissolved in Wesson oil at a concentration of 100 IU/ml. Controls were given the same volume of Wesson oil. Dosed chicks were fasted for 24 hr after which each chick was orally administered 2-3 FCi of 45Ca (Nuclear Science and Engineering Co., Pittsburgh, Penn.) in a volume of 0.2 ml. This %Ja solution was also 1% in nonradioactive CaClz. Chicks were sacrificed by ether suffocation exactly 2 hr after adCHCh
EXTRACT
OF WHOLE
CHICK
24 HOURS
7
FRACTION
NUMBER
(IO ML)
FIG. 1. Silicic acid column profile of the chloroform extract of the entire chick that received either 0.25 pg [l, 21aH vitamin D3 or 0.25 pg U 1% vitamin Dz - - - - - - 24 hr before. Chromatography was carried out as described by Lund and DeLuca (2).
AND
IMRIE TABLE
I
DISTRIBUTION OF ADMINISTERED RADIOACTIVITY FROM 0.25-pg DOSES OF 3H Da OR 1% De
AMONG: J-ITAMIN D AND ITS METABOLITE Time (hr) 1
24
Fraction III IV III I\
V;it$I&~
Vitamin Dz (% dose)
0
80.2 8.4 10.4 42.5
f z!z i f
6.8” 2.1 3.2 3.8
70.2 8.3 10.4 13.2
zk f z!z f
8.8” 3.0 4.8 2.8
a Standard deviations. There were at least 4 chicks per group. The recovered radioactivity is from the entire chick whose feathers and gastrointestinal tract had been removed. The chromatography was carried out as described previously and shown in Fig. 1. ministration of label. The tibia of each chick was then removed and dissected until clean. Bones were placed in 25X148-mm Pyrex test tubes and weighed. Radioactive vitamins DZ and D3. [I, 2JH] vitamin D3 (specific activity 26,000 dpm/IU) was prepared as previously described (10) as was [U-14C] vitamin DZ (30,000 dpm/IU) (1). For experiments dealing with the metabolic conversion of the vitamins to metabolites, 0.25 pg dissolved in 0.05 ml 95yo ethanol was administered intrajugularly to each rachitic chick. For experiments dealing with the isolation of metabolites for a study of their biological activity in both rats and chicks, the radioactive vitamins were diluted with the appropriate nonradioactive vitamin to give a specific activity of 122 dpm/IU for D3 and 98 dpm/IU for Dz. These were dissolved in ether to which was added Wesson oil. The ether was removed with a stream of Nz to give a solution containing 2 mg 3H Da or 1°C DZ in 1 ml Wesson oil. One-tenth milliliter of this solution was given orally to a rat or a chick. Extraction and chromatography of vitamin D metabolites. In the experiments concerned with the proportion of either 3H vitamin Da or r4C vitamin Dz and their metabolites in the chick after a 0.25~pg dose, chicks were killed by ether suffocation at the indicated times after dosage. The gastrointestinal tract and feathers were removed and the remaining carcass was ground in a meat grinder and then extracted with methanol-chloroform using a Waring Blendor (11). The entire chloroform extract was then chromatographed on silicic acid columns as described previously (2). Then 5 ml of concentrated nitric acid was added to each tube and bones were digested over heating coils. After cooling, each sample was brought to a volume of 25 ml with a
VITAMIN
diluent solution consisting of 30 vol water, 19 vol ethanol, and 1 vol of the detergent Triton X100. One-milliliter aliquots were then taken and placed in scintillation counting vials witch 15 ml of dioxane counting solution. r-ials were counted at 15yo gain with a window of 50-1000. Dioxane counting solution. The Wa counting solution was made by dissolving 7.0 g PPO, 300 mg dimethyl POPOP, and 150 g napthalene in 1500 ml of dioxane. Two hundred fifty milliliters of water containing 18 mg disodium EDTA was then added. Radioaclivity counting. All radioisotopes were counted in a Packard Tri-Carb liquid scintillation counter, Model 3003 (Packard Instruments Corp., LaGrange, Ill.), employing an external standard for counting efficiency determinations. Dioxane counting solution was used for counting 45Ca, while 1% and 3H were counted using toluene counting sollltion (12).
II
ANTIRACHITIC EFFECTIVENESS OF PEAK METABOLITES IN RATS~
D3
Da Peak D, Peak D2 Up Peak D2 Peak
IV from rat IV from chick IV from rat IV from chick
0.401
0
I I
2
3
INTERNATIONAL
4
5
6
7
8
9
IO
UNITS OF VITAMIN D3
FIG. 2. The relationship of and bone uptake of orally Vertical bars denote standard The exact method is described
vitamin Dt dosage administered 45Ca. error of the mean. in the t,ext.
Fig. 1) while only a small amount apas peak IV metabolites (Table I). After 24 hr, however, there is little unchanged vitamin DS or vitamin Dz. Rather large amounts of peak IV remain in the case of D, while small amounts of peak IV are found in the case of D,. Thus it is clear that peak IV is the major form of the pears
It has previously been shown in rats that 24 hr after a 0.25 pg dose of 1,2 3H vitamin Da most of the remaining radioactivity is found in the peak IV metabolite fraction. Figure 1 demonstrates that this is the case in chicks as well. This profile also serves as a reference point for the experiments to be reported in this communication. At 1 hr after injection of 0.25 pg of either 3H DS or 14C Dz, 70-80% of the dose is recoverable as unchanged vitamin D (peak
Group Vegetable oil control
osot
III,
RESULTS
TABLE
659
Ds IN CHICKS
IV
IU/ta
40 40 42 40 39 41
(1The scores represent group averages where each group consisted of at least 5 and usually 8 rats. The assays were carried out according to the U.S. Pharmacopeia (8) in which each rat was given the equivalent of 4 IU of antirachitic activity. The data are expressed as international unit,s (IU) of antirachitic activity per microgram of compound tested. Standard cholecalciferol gives 40 IU/pg.
original 3H D, administered. These results agree with previous results which showed
that the 14C from 14C D2 disappeared more rapidly from chicks than does 3H from 3H Da. The major difference lies not in the disappearance of peak III or unchanged vitamin D but rather in the rate of disappearance of peak IV. These data suggest that the chick does not discriminate between D, and Da at the level of conversion to peak IV but rather at the rate of peak IV catabolism and excretion. To test this hypothesis directly, peak IV fractions were prepared from both D, and Da from both rats and chicks. As expected, the peak IV fractions from all these sources were all fully antirachitic by the rat, line test assay (Table II) with values of 40 IU//.Lg. A biological assay was devised using rachitic chicks as the test, animal. That the assay is valid is shown by the response curve of chicks to physiologic dose of vitamin D3 (Fig. 2). The response recorded was essentially linear between l-10 IU of vitamin Da. It is also important to note t’hat the
660
DRESCHER, TABLE
ASSAY
OF
VITAMIN D METABOLITES
III
Mean f Standard deviationl”
Number of animals
I
0.22
32
-
0.93 f 1.24 f 1.36 f
0.27 0.25 0.26
14 15 8
40 40 50
1.44
f
0.43
9
57
1.02 f 0.96 f
0.18 0.25
16 11
21 16
0.83
0.13
5
6
f
-
a Standard deviations means of the expression
this will be necessary before such a conclusion can be made. DISCUSSION
f
0.77
AND IMRIE
-
% Total ‘S&/g bone
Vegetable oil control Dt (0.1 /A Ds (0.25 a) Da Peak IV (0.25 pg) from rat Da Peak IV (0.25 pg) from chick DI (10 0.25 /.& Dz Peak IV (0.25 pg) from rat Dt Peak IV (0.25 rg) from chick
IV
ACTIVITY OF PEAK IN CHICKS
I/ --
DELUCA,
were
calculated
by
The figures in parentheses represent the amount of compound given to each animal in the assay group. The data are calculated in terms of international units of antirachitic activity per microgram of compound. Standard cholecalciferol gives 40 IU of antirachitic activity per microgram.
mean response remained quite constant from experiment to experiment. When the peak IV fractions were assayed for vitamin D activity by the above-indicated assay the data shown in Table III resulted. Of great interest is the fact that the peak IV fractions from vitamin D3 derived from both rats and chicks were more active than vitamin D, itself. However, the peak IV fractions from both chicks and rats obtained from vitamin Dz were only slightly active in the chick assay. As would be expected, vitamin D, itself was also less active. It is interesting to note that peak IV from Dz isolated from chicks was less active than that isolated from rats. This suggests that the peak IV from chicks may contain some different metabolites than those from rats which may differ in their potency in chicks. However, further confirmation of
The results of the present study confirm the conclusion reached earlier that vitamin D, or a metabolite thereof is rapidly excreted thereby reducing its effective lifetime. In the course of this study, the biologically active fraction of peak IV has been positively identified as 25-hydroxycholecalciferol in the case of vitamin Dt (13, 14). The metabolite which derives from Dz has not yet been identified, however. Furthermore, strong evidence has been advanced that the 25-hydroxy derivative is the metabolically active form of the vitamin (15). Assuming the 25-hydroxy derivatives as the metabolically active form of the vitamins, it is now possible to suggest a possible mechanism for the discrimination against D, by chicks. It is apparent that the chick can convert both Dz and DS to the biologically active peak IV metabolites. Both are fully active in the rat which does not discriminate between Ds and Da. However, the same peak IV metabolite preparation from D, is only slightly effective in the chick, suggesting that the peak IV metabolite from Dz is rapidly degraded and/or excreted. Assuming the 25.hydroxy derivatives as the metabolically active form of the vitamins, it is apparent that the derivative from Dz would have only a short, lifetime in the chick thus accounting for the poor biological activity of Dz in the chick. Work is now in progress to isolate and identify the biliary excretion products of both Dz and Da in the chick. Preliminary results support the above hypothesis and it appears likely that the curious discrimination against vitamin Dz by chicks will soon be elucidated at the molecular level. REFERENCES 1.
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