388
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[41]
active on androstenedione is different from that active on dehydroepiandrosterone. T M Thus, in testing out specificities of different preparations of cytochrome P-450 it is preferable to use more than one steroid substrate. In conclusion, the use of steroid hormones as substrates in experimental work involving liver microsomal cytochrome P-450 is encouraged owing to the unique possibilities to obtain detailed and clear information on catalytic specificity of cytochrome P-450 preparations. Acknowledgment This work was supported by grants from the Medical Research Council (No. 03X-2819).
[41] D e t e r m i n a t i o n
of Vitamin D Metabolites
By JOHN A. EISMAN and HECTOR F. DELUCA In 1966 it was first reported that vitamin D3 must be metabolically altered before it carries out its functions in calcium and phosphate homeostasis and bone calcification. 1'2 It is now known that vitamin D is first hydroxylated to yield 25-hydroxyvitamin D3 (25-OH-D3) largely if not entirely in the liver. 3"4 In 1971 it was further discovered that 25-OHD3 must be converted to 1,25-dihydroxyvitamin D3 [1,25-(OH)~D3] in the kidney prior to expression of biological activity. 5"6 This hydroxylation to yield 1,25-(OH)2Da was found to be regulated by dietary and serum calcium concentration, 7 apparently through parathyroid hormone intervention, s'9 and by serum phosphate concentration.I° When produc1 j. Lund and H. F. DeLuca, J. Lipid Res. 7, 739 (1966). 2 H. Morii, J. Lund, P. F. Neville, and H. F. DeLuca, Arch. Biochem. Biophys. 120, 508 (1%7). 3 G. Ponchon, A. L. Kennan, and H. F. DeLuca, J. Clin. Invest. 48, 2032 (!%9). 4 E. B. Olson, Jr., J. C. Knutson, M. H. Bhattacharyya, and H. F. DeLuca, J. Clin. Invest. 57, 1213 (1976). 5 M. F. Holick, H. K. Schnoes, H. F. DeLuca, T. Suda, and R. J. Cousins, Biochemistry 10, 2799 (1971). 6 D. R. Fraser and E. Kodicek, Nature (London) 228, 764 (1970). 7 I. T. Boyle, R. W. Gray, and H. F. DeLuca, Proc. Natl. Acad. Sci. U.S.A. 68, 2131 (1971). 8 M. Garabedian, M. F. Holick, H. F. DeLuca, and I. T. Boyle, Proc. Natl. Acad. Sci. U.S.A. 69, 1673 (1972). 9 D. R. Fraser and E. Kodicek, Nature (London), N e w Biol. 241, 163 (1973). ~0 y . Tanaka and H. F. DeLuca, Arch. Biochem. Biophys. 154, 566 (1973).
[41]
389
DETERMINATION OF VITAMIN D METABOLITES
tion of 1,25-(OH)2D3 is curtailed, another metabolite, 24(R),25-dihydroxyvitamin D3 [24(R),25-(OH)2Dz], is formed. 7,H Other metabolites of vitamin D3, including 25,26-dihydroxyvitamin D3 [25,26-(OH)2D3] ~2 and 1,24(R),25-trihydroxyvitamin D3 [,124(R),25-(OH)3D3] 13 have been isolated and identified. While they possess some biological activity, their physiological roles have not been adequately defined. Figure 1 summarizes the known pathways of vitamin D3 metabolism. =a Essentially identical pathways and functions have been demonstrated for vitamin D2.14
The levels of three metabolites of vitamin D in biological samples are of greatest interest. These metabolites are 25-OH-D3, 24(R),25-(OH)zD3, and 1,25-(OH)2D3. Since both vitamin D2 and vitamin D3 are used by man, measurement of 25-OH-D2, 24(R),25-(OH)2D2, and 1,25-(OH)2Dz are also of interest, and to facilitate further discussion, 25-OH-D, 24(R),25-(OH)2D, and 1,25-(OH)2D levels will refer to a sum of the appropriate vitamin D2 and D3 metabolite levels.
~
vet
HO" v
HO~ " ~ " 25 -OH -03
D3
OHH
2) LOW Pi . Kidney
25, ~6-(OH) 2 03
OH
OH
real Ca
Jl
~,~., HO"~
(24R)-24, 25-(OH) t 03
NOv c.
OH H OH
~Jl j~ ~,, CH2 HO,~ C}'I2
•
= 2) Norr~ll P] Kidney
CH2 ~OH
(24 RpI, 24, 25- (OH)3 O:~
x~,.. CH2
HO ' ~ O H 1,25 -(OH) 2 O 3
FIG. 1. Known pathways of vitamin D metabolism. Note especially that the proven functional forms of vitamin D are 25-OH-Dz and 1,25-(OH)2D.~. The other metabolites are known, but their exact functions, if any, remain unknown. " M. F. Holick, H. K. Schnoes, H. F. DeLuca, R. W. Gray, I. T. Boyle, and T. Suda, Biochemistry 11, 4251 (1972). 12 T. Suda, H. F. DeLuca, H. K. Schnoes, Y. Tanaka, and M. F. Holick, Biochemistry 9, 4776 (1970). 13 M. F. Holick, A. Kleiner-Bossaller, H. K. Schnoes, P. M. Kasten, 1. T. Boyle, and H. F. DeLuca, J. Biol. Chem. 248, 6691 (1973). 14 H. F. DeLuca and H. K. Schnoes, Annu. Rev. Biochem. 45, 631 (1976).
390
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[41]
25-OH-D concentration in plasma is of considerable interest to clinicians inasmuch as it is believed to reflect the vitamin D status of the patient and on occasion reflects a defect in hepatic 25-hydroxylation. 1,25-(OH)zD is of most interest inasmuch as it is believed to be the form of vitamin D active on intestine, bone, and kidney. Although of less interest, 24,25-(OH)zD levels may provide some insight since they may reflect an inactivation pathway. Several in vivo bioassays of vitamin D metabolites have been devised utilizing intestinal calcium transport stimulation, antirachitic activity, and bone mineral mobilization. 1~ These assays, which are time consuming and do not preclude the possibility of metabolic transformations, have now given way to in vitro assays. In addition, the bioassays are described in detail in a previous volume. 15 This presentation will describe competitive binding assays for 1,25-(OH)2D and 24,25-(OH)2D and a high-pressure liquid chromatography assay method for 25-OH-D. The high-pressure liquid chromatographic methods are extremely effective in separating vitamin D metabolites and analogs TM and form the purification basis of the assays developed. A competitive binding assay for 25-OH-D has also been described in a previous volume ~ and is conveniently used for 25-OH-Da determination. A similar method of Haddad and Chyu is also widely used. ~7 However, neither binding assay approaches the accuracy and reliability of the high-pressure liquid method described in this section. 18'19 This method is currently being adopted for the determination of 24(R),25(OH)2D. The only modifications required are the preparation of [~-I]24(R),25-(OH)2D and the use of different fractions from the Sephadex columns used for prepurification of the 24(R),25-(OH)zDa prior to application to high-pressure liquid chromatography as described for 25OH-Da. Obviously the elution position of 24(R),25-(OH)2Da must be determined with standard 24(R),25-(OH)2Da. Preliminary results suggest normal levels of 24(R),25-(OH)2Da in human serum to be about 1-2 ng/ ml. Unfortunately, the authors have not completed their development of this method to meet the publication deadline. Finally, it should be noted that the development of an ethyl acetate extraction method and modified chromatography will permit the analysis of all three metabolites on a single 5-ml serum sample. Again, develop15 M. F. Holick and H. F. DeLuca, this series, Vol. 36, p. 512. 16 G, Jones and H. F. DeLuca, J. Lipid Res. 16, 448 (1975). iT j. G. Haddad and K. J. Chyu, J. Clin. Endocrinol. 33, 992 (1971). 18 j. A. Eisman, A. J. Hamstra, B. E. Kream, and H. F. DeLuca, Arch. Biochern. Biophys. 176, 235 (1976). 19 j. A. Eisman, A. J. Hamstra, B. E. Kream, and H. F. DeLuca, Science 193, 1021 (1976).
[41]
DETERMINATION OF VITAMIN D METABOLITES
391
ment of this method is not yet complete. However, the 1,25-(OH)2Da and 25-OH-Da assays described here will provide the basic methods that will be adapted to the refined and combined system for all three metabolites. Competitive Binding Assay for 1,25-(OH)2D Binding protein is prepared from the duodena of 9-12-week-old White Leghorn chickens (Northern Hatcheries, Beaver Dam, Wisconsin) raised from 1-day-old on a vitamin D-deficient diet previously described, ls'2° Chickens are killed by cervical dislocation. The duodenum is removed, emptied, and rinsed with ice-cold phosphate buffer (50mM potassium phosphate, 50 mM potassium chloride, 1 mM dithiothreitol, pH 7.4 at 25°). The mucosa is separated from serosa, minced in five volumes (ml/g) of iced phosphate buffer and centrifuged at 2000 g for 10 min at 4 °. The mucosal pellet is resuspended and centrifuged in five volumes of fresh buffer. The twice washed pellet is then homogenized in two volumes of buffer with 2-3 strokes of a Potter-Elvehjem Teflon-glass homogenizer and centrifuged for 1 hr at 40,000 rpm (226,600 g) in a type 50 Titanium rotor in a Beckman L5-50 refrigerated ultracentrifuge to yield a postmicrosomal cytosol. The fine lipid coat is removed, and the cytosol, which contains about 12 mg of protein per milliliter, is pooled, divided into portions, lyophilized, and stored under nitrogen at - 2 0 ° until used. Plasma samples are measured into 125-ml separatory funnels, and 3500 dpm (70 pg) of [3H]l,25-(OH)zD3 ~ are added in 50/zl of absolute ethanol. Six volumes of dichloromethane are then added, and the funnel is shaken at 4 cps for 5 min in a horizontal shaker after carefully venting to release pressure. The dichloromethane phase is collected, and the aqueous phase is reextracted twice with three volumes of dichlorome~0 j. Omdahl, M. Holick, T. Suda, Y. Tanaka, and H. F. DeLuca, Biochemistry' 10, 2935 (1971). 21 Preparation of [3H]l,25-(OH)2D3. Homogenates (20% w/v) of kidneys from vitamin Ddeficient chicks fed a low-calcium diet ]J. L. Omdahl, R. W. Gray, I. T. Boyle, J. Knutson, and H. F. DeLuca, Nature (London), New Biol. 237, 63 (1972)] are prepared in 0.25 M sucrose-15 mM Tris-chloride, pH 7.4, at 0 °. Two-hundred milligrams of tissue are incubated in a total volume of 1.5 ml containing 0.19 M sucrose, 15 mM of Tris acetate, pH 7,4, 1.9 mM magnesium acetate, 25 mM succinate and 1.3 nmol of [3H]25OH-D3 for 10 min at 37 ° under an atmosphere of oxygen. The mixture is extracted with 2 parts methanol, 1 part chloroform. Another 1 part CHCI3 and 1 part H20 are added, and the chloroform layer is recovered and dried on anhydrous MgSO~. The extract is dried under reduced pressure and chromatographed on a 1 × 60 cm column of Sephadex LH20 packed and developed in chloroform:Skellysolve B 65:35. The [3H]l,25-(OHhD~ elutes at 200-250 ml.
392
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[41]
thane with vigorous shaking for 1 min each time. The combined dichloromethane layers are dried on a flash evaporator and redissolved in 0.5 ml of the column solvent for the succeeding chromatographic step. The plasma extract is applied in toto to a 0.7 × 9 cm, 3,5 ml column of Sephadex LH-20 (Pharmacia, Piscataway, New Jersey) [column solvent in Skellysolve B: chloroform:methanol (9: 1: 1)] and twice rinsed in with 0.25 ml of column solvent. The first 8 ml of elution volume are discarded, and the next 12 ml are collected. This fraction, which contains more than 90% of both 1,25-(OH)~D~ and 1,25-(OH)2D3 but relatively little other lipid, is dried in a stream of nitrogen gas and resuspended in 30/zl of high-pressure liquid chromatography solvent. The partially purified plasma extract is applied to a Waters Associates (Milford, Massachusetts) 0.4 × 30 cm high-pressure liquid chromatography column of microparticulate silicic acid and is developed in 10% isopropanol in hexane (a mixture of hexane isomers) with a flow rate of 1.8-2 ml/min at 800-1000 psi. 1,25-(OH)2D2 and 1,25-(OH)2D3 standards, which are used to calibrate the high-pressure liquid chromatography system for each fresh batch of solvent, are monitored by their absorbance at 254 nm. The 1,25-(OH)2D region is collected, dried under a stream of nitrogen, and redissolved in 250/zl of absolute ethanol. Three 50-/zl portions are placed in 11 × 75 mm polypropylene tubes (Walter Sarstedt Co., Princeton, New Jersey) and are stored at - 2 0 ° for assay. Two further 50-/zl portions are placed in 5-ml minivials with 4 ml of a dioxane-based scintillation solution. These samples are counted to determine recovery, which in the authors' laboratory has been 68 -+ 2% (mean -- SEM, n = 20). The p-dioxane scintillation solution contains 10% naphthalene and 0.5% 2,5-diphenyloxazole in p-dioxane and results in 38% counting efficiency in a Beckman LS-100C ambient-temperature scintillation counter. Quadruplicate standards of crystalline 1,25-(OH)zD3 are dissolved in absolute ethanol and pipetted into assay tubes to produce a range of 1,25-(OH)2D3 amounts of 0, 5, 10, 20, 50, and 100 pg and 5 ng in 50/~1 of ethanol. [zI-I] 1,25-(OH)2D3 (70 pg, 3500 dpm) is added to each standard tube. [aH]l,25-(OH)2D3 is also added to each sample tube to bring the total tritium content up to 3500 dpm, including the residual [aI-I]1,25-(OH)2D3 used for the estimation of recovery. One milliliter of reconstituted cytosol diluted in phosphate buffer to 1-1.5 mg/ml ~ is added to each tube in an iced water bath. The tubes are incubated at 25 ° for 1 hr in a 2z Lyophilized cytosol is dissolved in 25 mM phosphate buffer, pH 7.4, to give 0.4 rng of protein per milliliter.
[41]
DETERMINATION
OF VITAMIN
D METABOLITES
393
shaking water bath and then returned to the iced water bath. To separate bound from unbound [all] 1,25-(OH)2D3, 1 ml of 40% (w/v) polyethylene glycol 6000 (average molecular weight 6000-7500, J. T. Baker Chemical Co., Phillipsburgh, N e w Jersey) in distilled water is added to each tube, which is vigorously mixed and then centrifuged at 9000 g for 1 hr in the HS-4 head of a Sorvall RC5 refrigerated centrifuge. The supernatant is
decanted; the tip of the tube, which contains the protein pellet, is cut off and immersed in 4 ml of the dioxane scintillation fluid, and the tritium content is determined. The 3H in the pellet to which 5 ng of 1,25(OH)2Dz was added is considered to be nonspecifically bound [3H]l,25-
(OH)2D3 and is subtracted from all samples. The corrected tritium content of the pellet is plotted against the unlabeled 1,25-(OH)2D:~ content of the standard sample (Fig. 2). The corrected tritium content of
700
600
500 O. "0 c 0 f13
400
(3. C.)
300
20(
0
5
,0
50
~00 5000
1,25-{OH)zD3 pgl Tube
FiG. 2. A standard curve for the determination of 1,25-(OH)~D3 using 9.3 Citmmol 1,25(OH)~D3. With this specific activity material, as little as 10 pg of 1,25-(OH)zD3 can be determined. By using 78 Ci/mmol [3H]1,25-(OH)2D3, as little as 2 pg can be detected by the present method.
394
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[41]
the pellets from a sample is read from this curve to yield a picogram 1,25-(OH)2D3 value, which is then further corrected for the recovery and the initial plasma volume to yield a value picograms of 1,25-(OH)2Da/ml. Both 1,25-(OH)~D2 and 1,25-(OH)2Da are recovered during preparation of the sample and measured equally by the assay, so that the value determined by this assay is actually both dihydroxylated vitamins and should be expressed as 1,25-(OH)~D pg/ml or, more conveniently, as picomolar 1,25-(OH)~D. The normal values of 50- to 60-year-old adults by this assay are 33 -+ 2 pg/ml (mean --- SEM, n = 30) or 79 --. 3 pM. Plasma from rachitic chicks or patients with end-stage renal failure have values of
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[41]
active on androstenedione is different from that active on dehydroepiandrosterone. T M Thus, in testing out specificities of different preparations of cytochrome P-450 it is preferable to use more than one steroid substrate. In conclusion, the use of steroid hormones as substrates in experimental work involving liver microsomal cytochrome P-450 is encouraged owing to the unique possibilities to obtain detailed and clear information on catalytic specificity of cytochrome P-450 preparations. Acknowledgment This work was supported by grants from the Medical Research Council (No. 03X-2819).
[41] D e t e r m i n a t i o n
of Vitamin D Metabolites
By JOHN A. EISMAN and HECTOR F. DELUCA In 1966 it was first reported that vitamin D3 must be metabolically altered before it carries out its functions in calcium and phosphate homeostasis and bone calcification. 1'2 It is now known that vitamin D is first hydroxylated to yield 25-hydroxyvitamin D3 (25-OH-D3) largely if not entirely in the liver. 3"4 In 1971 it was further discovered that 25-OHD3 must be converted to 1,25-dihydroxyvitamin D3 [1,25-(OH)~D3] in the kidney prior to expression of biological activity. 5"6 This hydroxylation to yield 1,25-(OH)2Da was found to be regulated by dietary and serum calcium concentration, 7 apparently through parathyroid hormone intervention, s'9 and by serum phosphate concentration.I° When produc1 j. Lund and H. F. DeLuca, J. Lipid Res. 7, 739 (1966). 2 H. Morii, J. Lund, P. F. Neville, and H. F. DeLuca, Arch. Biochem. Biophys. 120, 508 (1%7). 3 G. Ponchon, A. L. Kennan, and H. F. DeLuca, J. Clin. Invest. 48, 2032 (!%9). 4 E. B. Olson, Jr., J. C. Knutson, M. H. Bhattacharyya, and H. F. DeLuca, J. Clin. Invest. 57, 1213 (1976). 5 M. F. Holick, H. K. Schnoes, H. F. DeLuca, T. Suda, and R. J. Cousins, Biochemistry 10, 2799 (1971). 6 D. R. Fraser and E. Kodicek, Nature (London) 228, 764 (1970). 7 I. T. Boyle, R. W. Gray, and H. F. DeLuca, Proc. Natl. Acad. Sci. U.S.A. 68, 2131 (1971). 8 M. Garabedian, M. F. Holick, H. F. DeLuca, and I. T. Boyle, Proc. Natl. Acad. Sci. U.S.A. 69, 1673 (1972). 9 D. R. Fraser and E. Kodicek, Nature (London), N e w Biol. 241, 163 (1973). ~0 y . Tanaka and H. F. DeLuca, Arch. Biochem. Biophys. 154, 566 (1973).
[41]
389
DETERMINATION OF VITAMIN D METABOLITES
tion of 1,25-(OH)2D3 is curtailed, another metabolite, 24(R),25-dihydroxyvitamin D3 [24(R),25-(OH)2Dz], is formed. 7,H Other metabolites of vitamin D3, including 25,26-dihydroxyvitamin D3 [25,26-(OH)2D3] ~2 and 1,24(R),25-trihydroxyvitamin D3 [,124(R),25-(OH)3D3] 13 have been isolated and identified. While they possess some biological activity, their physiological roles have not been adequately defined. Figure 1 summarizes the known pathways of vitamin D3 metabolism. =a Essentially identical pathways and functions have been demonstrated for vitamin D2.14
The levels of three metabolites of vitamin D in biological samples are of greatest interest. These metabolites are 25-OH-D3, 24(R),25-(OH)zD3, and 1,25-(OH)2D3. Since both vitamin D2 and vitamin D3 are used by man, measurement of 25-OH-D2, 24(R),25-(OH)2D2, and 1,25-(OH)2Dz are also of interest, and to facilitate further discussion, 25-OH-D, 24(R),25-(OH)2D, and 1,25-(OH)2D levels will refer to a sum of the appropriate vitamin D2 and D3 metabolite levels.
~
vet
HO" v
HO~ " ~ " 25 -OH -03
D3
OHH
2) LOW Pi . Kidney
25, ~6-(OH) 2 03
OH
OH
real Ca
Jl
~,~., HO"~
(24R)-24, 25-(OH) t 03
NOv c.
OH H OH
~Jl j~ ~,, CH2 HO,~ C}'I2
•
= 2) Norr~ll P] Kidney
CH2 ~OH
(24 RpI, 24, 25- (OH)3 O:~
x~,.. CH2
HO ' ~ O H 1,25 -(OH) 2 O 3
FIG. 1. Known pathways of vitamin D metabolism. Note especially that the proven functional forms of vitamin D are 25-OH-Dz and 1,25-(OH)2D.~. The other metabolites are known, but their exact functions, if any, remain unknown. " M. F. Holick, H. K. Schnoes, H. F. DeLuca, R. W. Gray, I. T. Boyle, and T. Suda, Biochemistry 11, 4251 (1972). 12 T. Suda, H. F. DeLuca, H. K. Schnoes, Y. Tanaka, and M. F. Holick, Biochemistry 9, 4776 (1970). 13 M. F. Holick, A. Kleiner-Bossaller, H. K. Schnoes, P. M. Kasten, 1. T. Boyle, and H. F. DeLuca, J. Biol. Chem. 248, 6691 (1973). 14 H. F. DeLuca and H. K. Schnoes, Annu. Rev. Biochem. 45, 631 (1976).
390
M I C R O S O M AELECTRON L TRANSPORT AND CYT P-450
[41]
25-OH-D concentration in plasma is of considerable interest to clinicians inasmuch as it is believed to reflect the vitamin D status of the patient and on occasion reflects a defect in hepatic 25-hydroxylation. 1,25-(OH)zD is of most interest inasmuch as it is believed to be the form of vitamin D active on intestine, bone, and kidney. Although of less interest, 24,25-(OH)zD levels may provide some insight since they may reflect an inactivation pathway. Several in vivo bioassays of vitamin D metabolites have been devised utilizing intestinal calcium transport stimulation, antirachitic activity, and bone mineral mobilization. 1~ These assays, which are time consuming and do not preclude the possibility of metabolic transformations, have now given way to in vitro assays. In addition, the bioassays are described in detail in a previous volume. 15 This presentation will describe competitive binding assays for 1,25-(OH)2D and 24,25-(OH)2D and a high-pressure liquid chromatography assay method for 25-OH-D. The high-pressure liquid chromatographic methods are extremely effective in separating vitamin D metabolites and analogs TM and form the purification basis of the assays developed. A competitive binding assay for 25-OH-D has also been described in a previous volume ~ and is conveniently used for 25-OH-Da determination. A similar method of Haddad and Chyu is also widely used. ~7 However, neither binding assay approaches the accuracy and reliability of the high-pressure liquid method described in this section. 18'19 This method is currently being adopted for the determination of 24(R),25(OH)2D. The only modifications required are the preparation of [~-I]24(R),25-(OH)2D and the use of different fractions from the Sephadex columns used for prepurification of the 24(R),25-(OH)zDa prior to application to high-pressure liquid chromatography as described for 25OH-Da. Obviously the elution position of 24(R),25-(OH)2Da must be determined with standard 24(R),25-(OH)2Da. Preliminary results suggest normal levels of 24(R),25-(OH)2Da in human serum to be about 1-2 ng/ ml. Unfortunately, the authors have not completed their development of this method to meet the publication deadline. Finally, it should be noted that the development of an ethyl acetate extraction method and modified chromatography will permit the analysis of all three metabolites on a single 5-ml serum sample. Again, develop15 M. F. Holick and H. F. DeLuca, this series, Vol. 36, p. 512. 16 G, Jones and H. F. DeLuca, J. Lipid Res. 16, 448 (1975). iT j. G. Haddad and K. J. Chyu, J. Clin. Endocrinol. 33, 992 (1971). 18 j. A. Eisman, A. J. Hamstra, B. E. Kream, and H. F. DeLuca, Arch. Biochern. Biophys. 176, 235 (1976). 19 j. A. Eisman, A. J. Hamstra, B. E. Kream, and H. F. DeLuca, Science 193, 1021 (1976).
[41]
DETERMINATION OF VITAMIN D METABOLITES
391
ment of this method is not yet complete. However, the 1,25-(OH)2Da and 25-OH-Da assays described here will provide the basic methods that will be adapted to the refined and combined system for all three metabolites. Competitive Binding Assay for 1,25-(OH)2D Binding protein is prepared from the duodena of 9-12-week-old White Leghorn chickens (Northern Hatcheries, Beaver Dam, Wisconsin) raised from 1-day-old on a vitamin D-deficient diet previously described, ls'2° Chickens are killed by cervical dislocation. The duodenum is removed, emptied, and rinsed with ice-cold phosphate buffer (50mM potassium phosphate, 50 mM potassium chloride, 1 mM dithiothreitol, pH 7.4 at 25°). The mucosa is separated from serosa, minced in five volumes (ml/g) of iced phosphate buffer and centrifuged at 2000 g for 10 min at 4 °. The mucosal pellet is resuspended and centrifuged in five volumes of fresh buffer. The twice washed pellet is then homogenized in two volumes of buffer with 2-3 strokes of a Potter-Elvehjem Teflon-glass homogenizer and centrifuged for 1 hr at 40,000 rpm (226,600 g) in a type 50 Titanium rotor in a Beckman L5-50 refrigerated ultracentrifuge to yield a postmicrosomal cytosol. The fine lipid coat is removed, and the cytosol, which contains about 12 mg of protein per milliliter, is pooled, divided into portions, lyophilized, and stored under nitrogen at - 2 0 ° until used. Plasma samples are measured into 125-ml separatory funnels, and 3500 dpm (70 pg) of [3H]l,25-(OH)zD3 ~ are added in 50/zl of absolute ethanol. Six volumes of dichloromethane are then added, and the funnel is shaken at 4 cps for 5 min in a horizontal shaker after carefully venting to release pressure. The dichloromethane phase is collected, and the aqueous phase is reextracted twice with three volumes of dichlorome~0 j. Omdahl, M. Holick, T. Suda, Y. Tanaka, and H. F. DeLuca, Biochemistry' 10, 2935 (1971). 21 Preparation of [3H]l,25-(OH)2D3. Homogenates (20% w/v) of kidneys from vitamin Ddeficient chicks fed a low-calcium diet ]J. L. Omdahl, R. W. Gray, I. T. Boyle, J. Knutson, and H. F. DeLuca, Nature (London), New Biol. 237, 63 (1972)] are prepared in 0.25 M sucrose-15 mM Tris-chloride, pH 7.4, at 0 °. Two-hundred milligrams of tissue are incubated in a total volume of 1.5 ml containing 0.19 M sucrose, 15 mM of Tris acetate, pH 7,4, 1.9 mM magnesium acetate, 25 mM succinate and 1.3 nmol of [3H]25OH-D3 for 10 min at 37 ° under an atmosphere of oxygen. The mixture is extracted with 2 parts methanol, 1 part chloroform. Another 1 part CHCI3 and 1 part H20 are added, and the chloroform layer is recovered and dried on anhydrous MgSO~. The extract is dried under reduced pressure and chromatographed on a 1 × 60 cm column of Sephadex LH20 packed and developed in chloroform:Skellysolve B 65:35. The [3H]l,25-(OHhD~ elutes at 200-250 ml.
392
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[41]
thane with vigorous shaking for 1 min each time. The combined dichloromethane layers are dried on a flash evaporator and redissolved in 0.5 ml of the column solvent for the succeeding chromatographic step. The plasma extract is applied in toto to a 0.7 × 9 cm, 3,5 ml column of Sephadex LH-20 (Pharmacia, Piscataway, New Jersey) [column solvent in Skellysolve B: chloroform:methanol (9: 1: 1)] and twice rinsed in with 0.25 ml of column solvent. The first 8 ml of elution volume are discarded, and the next 12 ml are collected. This fraction, which contains more than 90% of both 1,25-(OH)~D~ and 1,25-(OH)2D3 but relatively little other lipid, is dried in a stream of nitrogen gas and resuspended in 30/zl of high-pressure liquid chromatography solvent. The partially purified plasma extract is applied to a Waters Associates (Milford, Massachusetts) 0.4 × 30 cm high-pressure liquid chromatography column of microparticulate silicic acid and is developed in 10% isopropanol in hexane (a mixture of hexane isomers) with a flow rate of 1.8-2 ml/min at 800-1000 psi. 1,25-(OH)2D2 and 1,25-(OH)2D3 standards, which are used to calibrate the high-pressure liquid chromatography system for each fresh batch of solvent, are monitored by their absorbance at 254 nm. The 1,25-(OH)2D region is collected, dried under a stream of nitrogen, and redissolved in 250/zl of absolute ethanol. Three 50-/zl portions are placed in 11 × 75 mm polypropylene tubes (Walter Sarstedt Co., Princeton, New Jersey) and are stored at - 2 0 ° for assay. Two further 50-/zl portions are placed in 5-ml minivials with 4 ml of a dioxane-based scintillation solution. These samples are counted to determine recovery, which in the authors' laboratory has been 68 -+ 2% (mean -- SEM, n = 20). The p-dioxane scintillation solution contains 10% naphthalene and 0.5% 2,5-diphenyloxazole in p-dioxane and results in 38% counting efficiency in a Beckman LS-100C ambient-temperature scintillation counter. Quadruplicate standards of crystalline 1,25-(OH)zD3 are dissolved in absolute ethanol and pipetted into assay tubes to produce a range of 1,25-(OH)2D3 amounts of 0, 5, 10, 20, 50, and 100 pg and 5 ng in 50/~1 of ethanol. [zI-I] 1,25-(OH)2D3 (70 pg, 3500 dpm) is added to each standard tube. [aH]l,25-(OH)2D3 is also added to each sample tube to bring the total tritium content up to 3500 dpm, including the residual [aI-I]1,25-(OH)2D3 used for the estimation of recovery. One milliliter of reconstituted cytosol diluted in phosphate buffer to 1-1.5 mg/ml ~ is added to each tube in an iced water bath. The tubes are incubated at 25 ° for 1 hr in a 2z Lyophilized cytosol is dissolved in 25 mM phosphate buffer, pH 7.4, to give 0.4 rng of protein per milliliter.
[41]
DETERMINATION
OF VITAMIN
D METABOLITES
393
shaking water bath and then returned to the iced water bath. To separate bound from unbound [all] 1,25-(OH)2D3, 1 ml of 40% (w/v) polyethylene glycol 6000 (average molecular weight 6000-7500, J. T. Baker Chemical Co., Phillipsburgh, N e w Jersey) in distilled water is added to each tube, which is vigorously mixed and then centrifuged at 9000 g for 1 hr in the HS-4 head of a Sorvall RC5 refrigerated centrifuge. The supernatant is
decanted; the tip of the tube, which contains the protein pellet, is cut off and immersed in 4 ml of the dioxane scintillation fluid, and the tritium content is determined. The 3H in the pellet to which 5 ng of 1,25(OH)2Dz was added is considered to be nonspecifically bound [3H]l,25-
(OH)2D3 and is subtracted from all samples. The corrected tritium content of the pellet is plotted against the unlabeled 1,25-(OH)2D:~ content of the standard sample (Fig. 2). The corrected tritium content of
700
600
500 O. "0 c 0 f13
400
(3. C.)
300
20(
0
5
,0
50
~00 5000
1,25-{OH)zD3 pgl Tube
FiG. 2. A standard curve for the determination of 1,25-(OH)~D3 using 9.3 Citmmol 1,25(OH)~D3. With this specific activity material, as little as 10 pg of 1,25-(OH)zD3 can be determined. By using 78 Ci/mmol [3H]1,25-(OH)2D3, as little as 2 pg can be detected by the present method.
394
MICROSOMAL ELECTRON TRANSPORT AND CYT P-450
[41]
the pellets from a sample is read from this curve to yield a picogram 1,25-(OH)2D3 value, which is then further corrected for the recovery and the initial plasma volume to yield a value picograms of 1,25-(OH)2Da/ml. Both 1,25-(OH)~D2 and 1,25-(OH)2Da are recovered during preparation of the sample and measured equally by the assay, so that the value determined by this assay is actually both dihydroxylated vitamins and should be expressed as 1,25-(OH)~D pg/ml or, more conveniently, as picomolar 1,25-(OH)~D. The normal values of 50- to 60-year-old adults by this assay are 33 -+ 2 pg/ml (mean --- SEM, n = 30) or 79 --. 3 pM. Plasma from rachitic chicks or patients with end-stage renal failure have values of
