The regulation of rat liver calciferol-25-hydroxylase.

Abstract Rats treated with vitamin D3 show decreased levels of liver calciferol-25-hydroxylase activity. The extent of the depression and its persistence are related to the dose of vitamin D3. The decrease in enzyme activity measured in vitro cannot be accounted for by dilution of the labeled substrate by unlabeled vitamin D3 remaining in the liver. The in vitro decrease is reflected in vivo by a decrease in the rate and extent of appearance of 3H-labeled 25-hydroxyvitamin D3 in the blood and liver following a dose of 3H-labeled vitamin D3 administered to rats pretreated with unlabeled vitamin D3. These data show the existence of a mechanism which regulates the activity of the rat liver calciferol-25-hydroxylase.

decreased levels of The extent of the depression and its persistence are related to the dose of vitamin DS. The decrease in enzyme activity measured in vitro cannot be accounted for by dilution of the labeled substrate by unlabeled vitamin Da remaining in the liver. The in vitro decrease is reflected in viva by a decrease in the rate and extent of appearance of 3H-labeled 25-hydroxyvitamin Da in the blood and liver following a dose of aH-labeled vitamin D3 administered to rats pretreated with unlabeled vitamin DB. These data show the existence of a mechanism which regulates the activity of the rat liver calciferol-25-hydroxylase.
In recent years it has hccomc clear that the metabolism of vitamin D to more polar biologically active metabolites is an important aspect of the vitamin's function.
In 1966 Lund and DeLuca (1) demonstrated the existence of biologically act,ive I)olar metabolites of vitamin D3 (D,),l one of which proved to bc the major metabolite circulating in blood. Blunt ef al. (2) identified this metabolite as 25.hydroxyvitamin Da (25.OH-D3). Subsequent work (3) established not only its potent antirachitic activity but also its ability to stimulate bone calcium mobilization and intestinal calcium transport more rapidly than vitamin Da. The difference in time of response to the two compounds was considered to be th& time needed for conversion of vitamin D3 t,o 2%OH-D3.
In an effort to determine the location of the calciferol-25-hydroxylase, Ponchon et al. (4) studied the metabolism of t3H]D3 in rats whose livers had been surgically disconnected from their circulatory system. These rats produced no 25-OH-D3 in 4 hours, while sham operated control rats had as much as 20% 25OH-D3 in their blood after 4 hours.
Since these rats did not live for more than 4 to 6 hours, a study was carried out (5) which showed the conversion of vitamin D3 to 25.OH-D3 in the isolated, perfused liver and in liver homogenates (but not kidney or intestine) In this report evidence for the existence of a mechanism responsible for regulating the activity of rat liver calciferol-25hydroxylase will be presented.

Animals-Male
albino rats (Holtzman Co., Madison, Wis.) were maintained in hanging wire cages and fed ad libitum a low vitamin D diet of Steenbock (7), modified by using skim milk powder and butter instead of whole milk.
Rats weighing 200 to 250 g were used for each experiment.
Vitamin 03 Compounds-The [I , 2-aH]D3 used (specific activity 1509 or 1262 dpm per pmole) was prepared in our laboratory according to the method of Neville and DeLuca (8). Crystalline vitamin Ds and 25-OH-D3 were gifts of the Philips-Duphar Co. of Weesp, The Netherlands.
Rat Liver ZZomogenate Incubations-The rat livers were removed and chilled immediately in ice-cold 0.25 M sucrose. A 25% homogenate in 0.25 M sucrose was prepared using a Potter-Elvehjem homogenizer.
To a 5-ml aliquot of this homogenate in a small homogenizing vessel were added 25 ~1 of 95% EtOH containing 260 pmoles of [l ,2-3H]D3 (4 i.u.). The substrate was mixed with the homogenate by three additional strokes with a Potter-Elvehjem homogenizer and the mixture was poured into a 125.ml Erlenmeyer flask for incubation.
The remaining contents of the homogenizing vessel were rinsed into the incubation flask first with 2.5 ml of buffer-cofactor solution (0.1 M K2HPOI, 0.4 1nM TPN, 160 mM nicotinamide, 20 mM ATP, 22.4 MM glucose adjusted to pH 7.4), and then with 2.5 ml of salt solution (5 mM MgC12, 0.1 M KCl) to give a final incubation volume of 10 ml. Incubations were carried out for 2 hours at 37" at 120 oscillations per min at which time the reactions were terminated by addition of 25 ml of MeOH plus 12.5 ml of CHC13.
E&m&on Procedure-Extractions were carried out essentially according to the method of Bligh and Dyer (9). After the reactions were terminated, the resulting single phase was allowed to extract overnight at 4". The extracts were filtered into separatory funnels to remove precipitated protein.
The incubation flasks and precipitated protein were rinsed with 12.5 ml of CHC18, which was then added to the extract.
To each separatory funnel were added 10 ml of water to complete separation of the phases. When both phases had cleared, the CHC13 layer was collected.
The water-methanol layer was re-extracted with 20 ml of CHCl,. The CHCl, extract was diluted to 50 ml with 100% EtOH and aliquots were removed for determination of total radioactivity. The remainder was evaporated to dryness with a flash evaporator and the residue dissolved in 1 ml of 500/;, CHCll in Skellysolve B for application to columns.
Liver and serum samples collected from in V~VO studies with 3H-vitamin D3 were extracted in an analogous manner.
Twenty-five per cent liver homogenates were prepared with 0.25 M sucrose and then extracted.
Serum samples were brought to 10 ml with distilled water and extracted.
In this case, samples were not filtered and 10 ml of saturated NaCl were used instead of water to prevent emulsification of serum extracts.

Column
Chromatography-All lipid extracts were applied to columns (2 x 30 cm) containing 20 g of Sephadex LH-20 equilibrated with 50% CHC& in Skellysolve B (10). The samples were eluted with the same solvent.
A maximum of six such columns were run simultaneously on a time-operated fraction collector.
The flow rate for each column was adjusted to deliver exactly 2.5-to 3.0-ml fractions every 2 min. Forty such fractions were collected directly in 5-ml counting vials. The solvents were evaporated under a stream of air, and the resulting residues were redissolved in 4 ml of toluene counting solution (2 g of 2,5-diphenyloxazole and 100 mg of 1,4-bis[2-(4-methyl-5phenyloxazolyl)]benzene per liter of toluene). The 3H radioactivity in the vials was determined with a Packard Tri-Carb model 3375 liquid scintillation counter.
After each group of determinations the columns were stripped with 200 ml of 65% CHC13 in Skellysolve 13. They were regenerated by washing with 50 ml of 50% CHC13 in Skellysolve 13 before applying a new sample.
Silicic acid column chromatography was carried out as described by Ponchon and DeLuca (11) while liquid-liquid partition chromatography followed the procedure of Blunt et al.
(2). Calciferol-%-hydroxylase Assay-The basic properties of the rat liver homogenate calciferol-25-hydroyylase system were studied in order to set up an assay for the enzyme.
That the product produced is 25-OH-D3 was established by co-chromatography of the peak of radioactivity produced on incubation of vitamin D3 with rat liver homogenates together with synthetic 25.OH-I), in three different chromatographic systems (Fig. I). TWO of these chromatographic procedures were used to isolate 1.3 mg of 25-OH-Da in pure form (a), while the relatively new Sephadex LH-20 method has potent resolving powers (10). Maximum 25.hydroxylation was shown to require the presence of added TPN and ATP (Fig. 2). The tissue concentration used for routine work (1.25 g of liver per 10 ml of reaction mixture) was chosen since it is within the range which shows a linear relationship between enzyme activity and tissue concentration.
The pH maximum was determined to be 6.9, using 50 mM phosphate buffer to maintain the pII.
Since the reaction using 25 mM phosphate buffer as already described (pHinit = 7.2, pHfina1 = 6.8) proceeds slightly better than the one with 50 mM phosphate buffer, pH 6.9, 25 rnM phosphate buffer was used routinely.
The substrate concentration chosen gives a measure of initial reaction velocity at 2 hours even in the most active preparations (5).  As shown in Fig. 3, animals which had received a prior dose of vitamin DB had very little in vitro 25.hydroxylase activity. The change in enzyme activity as a function of time following three dose levels of vitamin D3 is shown in Fig. 4. The 25hydroxylase activity at each dose level fell rapidly from 15 min to 1 hour after administration of the vitamin and then remained low. The extent of the decrease depended upon the amount of vitamin Da administered, and was greatest for the highest dose level. -1 third incubation was prepared adding 0.26 nmole of ["HID, substrate of the same specific activity as the ["HID, predose. Control liver homogenate incubations were prepared from LIP treated animals and from animals treated with 0.65 nmole of unlabeled vitamin DS 12 hours prior to killing.

RESULTS
As shown in Table II, liver homogenates from [3H]D3-treated rats showed the same decrease in 25-hydroxylase activity as did the liver homogenates of animals treated with unlabeled vitamin Ds. Decrease in &OH-Da Production in Vivo in Response to Prior Vitamin 03 Administration-Since in vitro calciferol-25.hydroxylase activity decreases in response to a dose of vitamin &, an effort was made to determine whether a corresponding decrease in calciferol-25-hydroxylation could be observed in vivo. Rats were injected with 0.65 nmole of unlabeled vitamin Ds intrajugularly.
Ten hours later when both in vitro enzyme activity and in vivo pool sizes of unlabeled vitamin 1)s were low (see Fig. 4 and Table I), the rats were injected with a second 0.65nmole dose of t3H]D8. Control animals received only the [3H]D3 dose. The animals were killed at various times following the 3H-labeled vitamin Da dose. Lipid extracts of the sera and livers were chromatographed on Sephadex LH-20 columns to determine metabolite levels. Indeed, the rate and extent of appearance of 25-OH-[3H]D3 in the blood and liver of animals by guest on March 23, 2020 http://www.jbc.org/ Downloaded from pretreated with 0.65 nmole of vitamin D3 were considerably lower than in the case of the untreated animals (Fig. 5). Yet from Fig. 6 it is apparent that the predosed animals still showed a rapid uptake of 13H]D, into their livers.
Ry adding together the amounts of vitamin Da and 25-OH-Da known to be present in the liver due to the lo-hour predose and the amounts of 3H-metabolites present in the liver 15 min after As shown in Fig. 7, dilution of the label was small and the metabolite levels alone compared with those of the control animals (no vitamin D8 predose) could not explain the reduced rate of 25-OH-D3 production observed in the pretreated animals. DISCUSSIOX This report shows the existence of a mechanism which regulates the level of liver calciferol-25.hydroxylase activity according to the amount of vitamin D3 presented to the rat. After a rat receives a dose of vitamin D1, the calciferol-25-hydroxylase activity as measured in vitro quickly falls (Fig. 4). The enzyme activity slowly returns to initially observed levels, with the amount of time required for return to control levels increasing as the dose of vitamin DS increases. Since the extent to which the 25-hydroxylase activity falls also increases as the dose increases, possibly this mechanism operates in vivo to maintain a low level of 25-OH-D3 in the blood even when large amounts of vitamin D are being ingested.
Certainly the daily requirement for vitamin D3 is many times less than the amount required to produce toxicity symptoms.
The decrease in calciferol-25-hydroxylase activity observed in vitro cannot be explained by dilution of labeled substrate used in the assay system by unlabeled vitamin D3 present in the rat liver tissue, since that amount is too small to significantly dilute the pH]D$ added to the assay mixture (Table I).
Also, when dilution of substrate radioactivity is eliminated by predosing animals with t3H]D3 instead of unlabeled vitamin Da, the same decrease in calciferol-25-hydroxylase activity is observed (Table II).
A decrease in the rate and estent of appearance of 25-OH-D3 in the liver and blood of vitamin D-treated rats compared to control rats (Fig. 5) indicates that the in vitro decrease in calciferol-2j-l1)-dros~lase activity has significance in vivo. The liver vitamin I& and 25-OH-D3 levels of the treated and control rats 15 min after injection of 0.65 nmole of [3H]D3 are very comparable (Fig. i), and yet the treated animals do not put 25-OH-D3 into the blood at the rate of control animals.
Recent attempts to bring about a decrease in in viva 25.OH-D3 production and in vitro 25.hydroxylase activity by pretreatment of rats with unlabeled 25.OH-D3 have shown that calciferol-25. hydrosylase activity is not sensitive to 25-OH-D3 injected into the blood stream of the rat. These data indicate that metabelites of 25.OH-D3 are not responsible for the decrease in the enzyme activity.
It is interesting to follow the 25.OH-Da levels in the liver (Table I) and blood (Fig. 5) of rats which have received a single intrajugular injection of 0.65 nmole of [3H]DS. Upon injection, vitamin D3 is very quickly taken up from the blood by the liver. The 25.OH-D3 formed is immediately released into the blood; it does not accumulate in the liver, but remains at very low concentrations there, while the 25.OH-D3 concentration in the blood increases to a plateau level. Thus at 15 min after dosing, the 25-OH-D3 level in the liver is if anything higher than the 25. OH-D8 level measured 10 hours after dosing (Table I). Yet the calciferol-25.hydroxylase activity of the liver 10 hours after dosing is much less than that measured 15 min after dosing. These data imply that product inhibition cannot explain the 2973 decrease in enzyme activity observed in response to a dose of vitamin Ds. Attempts are currently being made to determine the mechanism whereby pretreatment of rats with vitamin Da decreases the calciferol-25-hydroxylase activity.
Meanwhile, this phenomenon must be taken into account by investigators attempting to measure the vitamin D 25-hydroxylase of the liver.