[21] Cytoreceptor assay for 1,25-dihydroxyvitamin D

190

VITAMIN D GROUP

[2 1]

Acknowledgments The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of Army or the Department of Defense. The authors wish to thank Mrs. Estelle Coleman and Mrs. Judy Dubbs for help in preparation of this manuscript. This work was supported by National Research Service Award 5T32 AM 07314, Grant AM 28292, and CRC Grant RR 40 from the National Institutes of Health.

[21] C y t o r e c e p t o r A s s a y for 1 , 2 5 - D i h y d r o x y v i t a m i n

D

By STAVROS C. MANOLAGAS V i t a m i n D3 (cholecalciferol) made in the skin and vitamin D2 (ergocalciferol) ingested with food undergo two successive hydroxylations, first in the liver and then in the kidney, to form the hormone 1,25-dihydroxyvitamin D [1,25-(OH)2D]. This hormone plays an important role in mineral and skeletal homeostasis and perhaps in hematopoietic cell differentiation and immune phenomena. Like other steroid hormones, 1,25-(OH)2D acts on its target tissues by binding initially to specific intracellular receptors. Changes in the circulating levels of this hormone occur either during physiological states of increased calcium requirements or in several disease states including renal osteodystrophy, parathyroid abnormalities, rickets, osteomalacia, sarcoidosis, and perhaps old age osteoporosis. In addition, 1,25-(OH)2D3 is employed as a therapeutic modality. Hence, the measurement of the concentrations of this hormone in blood has become important for research as well as for clinical medicine. Until recently, this measurement posed a major technical problem because it required a variety of steps including column chromatography and high-performance liquid chromatography in order to isolate 1,25-(OH)2D prior to assays. The cytoreceptor assay ~,2 is a new methodological approach to the 1,25-(OH)2D measurement in blood and obviates several of the technical problems of the previous methods. This assay is based on the biological principle that 1,25-(OH)2D diffuses freely across cell membranes and is retained inside cells bound to its specific receptor while other metabolites of D are largely prevented from entering the cells because they remain bound to the serum D binding protein, j In the cytoreceptor assay, the i S. C. M a n o l a g a s a n d L . J. D e f t o s , Biochem. Biophys. Res. Commun. 95, 5 9 6 (1980). 2 S. C . M a n o l a g a s a n d L . J. D e f t o s , Lancet 2, 401 (1980).

METHODS IN ENZYMOLOGY, VOL. 123

Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

[21]

ASSAY FOR 1,25-(OH)zD3

191

quantitation of 1,25-(OH)zD is accomplished by comparing the displacement of radioactive 1,25-(OH)2D3 retained inside intact cells, by the 1,25(OH)2D o f unknown samples to the displacement of standard amounts of 1,25-(OH)2D3. The procedure described here includes minor modifications of the originally published method. 3,4 These modifications have simplified and optimized the performance of this assay. Materials and Reagents 1. 1,25-(OH)2-[26,27-3H]D3 (specific activity 160 Ci/mmol) (Amersham, Arlington Heights, IL). Kept in 1 : I toluene-ethanol at - 2 0 °. 2. Unlabeled 1,25-(OH)2D3 (Hoffman-LaRoche, Nutley, N J). Standard solution is prepared at a concentration of 1280 pg/ml in ethanol and kept stored at - 2 0 ° in sealed amber ampules. 3. Human a-globulin (Fraction IV, Miles Laboratories, Inc.). 4. Bovine serum albumin, Fraction V (Sigma, St. Louis, MO). 5. Benzene ( H P L C grade, Baker); methanol (HPLC grade, VWR); absolute ethanol (USP grade). 6. Solubilizing solution, Omnisol (WestChem, San Diego, CA). 7. Liquid scintillation cocktail, BetaMax (WestChem, San Diego, CA). 8. Disposable extraction columns, Extrelut 3 (E.M. Science, Gibbstown, N J). 9. Incubation medium is MEM culture medium with Hanks' salts (Itvine Scientific, Santa Ana, CA) adjusted to 25 mM H E P E S and pH of 7.4. The medium is filter sterilized and subaliquoted (sterilely) into 50 ml portions and stored at 4 ° . 10. Washing solution is normal saline (0.9% NaCI) enriched with 0.2% BSA (see #4) kept at 4 °. 11. Evaporating apparatus. 12. Nitrogen gas tank. 13. Solution of 1 N N a O H kept at room temperature. 14. Cells: The cells used are rat osteogenic sarcoma lines (ROS). 5 These cells are cultured on the surface of plastic flasks T175 (Falcon) at 37°C in 5% COe in air with Coon's F12 medium (Irvine Scientific), 10% fetal calf serum (GIBCO), and penicillin-streptomycin 10 p~g/ml. E v e r y 3 days confluent cells are split 1 : 2 to 1 : 3 and are subcultured in new flasks. 3 s. c. Manolagas, F. L. Culler, J. E. Howard, A. S. Brickman, and L. J. Deftos, J. Clin. Endocrinol. Metab. 56, 751 (1983). 4 S. C. Manolagas, in "Assay of Calcium-Regulating Hormones" (D. D. Bikle, ed.), p. 139, Springer-Verlag, Berlin and New York, 1978. 5 R. J. Majeska, S. B. Rodan, and G. A. Rodan, Endocrinology (Baltimore) 107, 1434(1980).

192

VITAMIN D GROUP

[2 1]

For the final subculture before harvesting, the culture medium is enriched with 10-7 M triamcinolone acetonide (Sigma). After 3 days of growth in the presence of triamcinolone, the medium is aspirated and the flasks are rinsed with 10 ml phosphate-buffered saline (PBS). The cells are then freed by 5-min treatment with 4 ml trypsin-EDTA solution in PBS buffer; this solution is prepared by 1 : 8 dilution of a concentrated preparation of trypsin-EDTA (10×, Irvine Scientific). After the 5-min treatment, fresh medium is added and the cells are transferred into plastic conical tubes where they are spun at 200 g for 5 min. The supernatant is then aspirated, and the pelleted cells are resuspended in regular medium containing 10% DMSO. While cells are being harvested from consecutive flasks the freed cells are kept at 4°; the total yield is finally pooled and mixed gently to obtain a homogeneous suspension. An aliquot of this suspension is counted with a hemocytometer to determine the total cell number and the suspension is adjusted to 5 × 10 6 cells/ml; aliquots of this are then frozen at - 7 0 ° in plastic Nunc cryotubes (Irvine Scientific) in a Revco ultralow freezer. Alternatively, cells can be frozen in liquid nitrogen. 1,25-(OH)2D Extraction Procedure 1. Aliquot 2 ml of each sample (serum or plasma) into labeled 13 x 100 mm glass test tubes. Although 2 ml represents optimal sample volume, the assay can be performed with samples as small as 1 ml. Include duplicate distilled H20 blanks and control serum samples. 2. Add 20/zl of 1,25-(OH)213H]D3 in ethanol (containing approximately 1500 cpm) to each sample and to three glass scintillation vials for recovery total activity (RTA). Vortex and let stand at least 30 min. 3. Add 230/zl absolute EtOH to RTA vials and keep for counting in 8 ml scintillation cocktail. 4. Add 50/zl of 1 N NaOH/ml sample and vortex. Bring sample volume up to 3 ml using distilled H20; vortex. 5. Apply samples to Extrelut 3 columns using glass Pasteur pipettes. Allow 5-10 min for the sample to settle into column. 6. Add 15 ml benzene to each column. Collect eluents in glass scintillation vials. 7. When columns stop dripping, remove vials and dry eluents under N2 gas in a 37° waterbath until completely dry (approximately 1 hr). Add 1 ml methanol to each vial, vortex, then dry again. 8. Reconstitute dried samples with I ml absolute EtOH and vortex well. Cap tightly and store at - 2 0 °, if not to be used in the assay immediately. The expected extraction efficiency of the Extrelut 3 column-benzene system for 1,25-(OH)2D3 is illustrated in Fig. 1. As seen in the figure, other

[21]

ASSAY FOR 1,25-(OH)zD3

193

100-

,

80, z l

60-

'~

40-

o o

20-

//J /// /// z// /// /// /// /// /// r// /// 1.25.(OH12D 3

N 24,25-(0H1203

N

25-OHD 3

Oleic Acid

FIG. 1. Extraction of vitamin D3 metabolites using Extrelut 3 minicolumns and benzene. Percentages of the total 1,25-(OH)2[~H]D~, 24,25-(OH)213H]D3, and 25-OH[3H]D3 added in the samples which were recovered in the eluent of the column are indicated. Percentage of total [3H]oleic acid recovered during this procedure is also shown. Each bar represents the mean from three determinations" vertical lines are standard deviations of the mean. major metabolites of vitamin D3 are extracted less well in this system while lipids (oleic acid) are largely retained in the column. (See note added in proof, p. 198.) Assay Procedure I. Aliquot 230 ~l of each of the ethanol-reconstituted sample extracts into one glass scintillation vial and into three glass test tubes (12 × 75 mm). The scintillation vial aliquot is used to determine recoveries from the extraction procedure. 2. M a k e serial dilutions o f the 1280 pg/ml standard with absolute ethanol in one set of glass test tubes (e.g., 0.5 ml of standard + 0.5 m! ethanol) to obtain solutions containing 64, 32, 16, 8, 4, and 2 pg of 1 , 2 5 - ( O H h D J 100/zl o f ethanol. 3. Aliquot 100/zl × 3 o f either absolute ethanol (O standard) or the standards (2, 4, 8, 16, 32, 64, 128 pg/100/zl) into 12 x 75 m m glass tubes. 4. Dry standards and samples under N2 gas at room temperature. C h e c k each tube for complete dryness. 5. While samples are drying, prepare the incubation medium by adding 0.5 mg o f human a-globulin/ml o f M E M Hanks' medium (room temperature). Add absolute ethanol to obtain a final concentration of 3% and mix well. Y o u need 0.2 ml × number of tubes.

194

VITAMIN D GROUP

[2 1]

6. Add 50/zl of the incubation medium to each tube and let sit for 15 min. 7. Place appropriate number of vials of freeze-stored cells (you need approximately 0.5 x 10 6 cells x number of assay tubes) in a 37° waterbath until thawed enough to mix contents (2-3 min). Pour immediately into a 50-ml-plastic centrifuge tube containing 6 ml incubation medium for each vial of cells to be used. Spin at 200 g for 5 min at room temperature to pellet the cells. Aspirate medium and resuspend the cell pellet in fresh incubating medium; this suspension should contain approximately 0.5 x 106 cells/100 /~1. Mix gently using a Pasteur pipette to ensure an even suspension (until visible clumps of cells have been dissolved). The cell suspension should be used within a few minutes after it is prepared and not be refrozen, otherwise the integrity of the cells cannot be maintained. 8. Add 100/xl of cell suspension to the assay tubes using a Hamilton repeating syringe and start incubation at room temperature (approximately 23°). During the incubation, maintain gentle agitation to prevent cells from settling in the bottom of the tube; this can be accomplished by placing the rack with the assay tubes on a shaking platform (approximately 150 rpm). Continue incubation for 45 min. 9. Prepare tracer by pipetting appropriate volume of the stock 1,25(OH)213H]D3 solution into a glass tube. You need -~4500 cpm per assay tube. Dry the tracer solution under N2 gas at room temperature and then resuspend into incubation medium to a final concentration of 4500 cpm/ 20/zl. 10. At the end of the first 45 min incubation add 20/zl of the tracer solution into each assay tube. Continue incubation on the shaking platform for another 30 min. 11. At the end of the second incubation, transfer tubes to an ice water bath (2°) and let cool for 15 min. 12. Add 2.5 ml of cold (4°) washing solution (normal saline + BSA) to each tube and leave in ice water bath for another 15 min. 13. Spin tubes in refrigerated centrifuge for 10 min at 1000 g to pellet the cells. Decant supernatant and blot tubes onto absorbent paper. 14. Add another 2.5 ml of washing solution to each tube and vortex gently. Spin again for 10 min at 1000 g. Decant supernatant as before and drain tubes well to minimize amount of liquid remaining. 15. Add 0.3 ml solubilizing solution (Omnisol) to each tube and vortex thoroughly. Leave at room temperature for 15 min. 16. Add 2 ml of scintillation cocktail (BetaMax) to each tube and vortex well to mix phases. Decant into scintillation vials containing 6 ml scintillation cocktail. 17. Cap vials tightly and shake content to mix. Chill vials to 4° before counting in a refrigerated beta counter. Figure 2 depicts a representative standard curve. For comparison a

[21]

ASSAYrOe 1,25-(OH)2D3

195

100-, "=""~"~,-zo.. 80-

~"~.,,.

60-

0,%

20-'°o

"'";

1'6 3'2 6'4 lh

230

4o o

Unlabeled 1,25-(OH)203 (pg)

FIG. 2. Representative standard curves of the cytoreceptor assay. Incubations were performed at either 23° (0) or 37° (©). Points indicate the displacement of 1,25-(OH)213HlD3 by standard amounts of unlabeled 1,25-(OHhD3. Each point represents the mean of triplicate determinations.

standard curve obtained at 37° is also shown; in previous descriptions of the method, we had recommended 37° for this incubation. The higher sensitivity of the curve obtained at room temperature (23°) compared to the sensitivity of the curve obtained at 37° probably reflects improved viability and integrity of the frozen-thawed cells under the former conditions; the radioactivity taken up by the cells during incubation is also increased at room temperature. Calculations

Recovery Factor Recovery factor (RF) = total radioactivity added for recovery counts/ recovered radioactivity.

Assay Calculation 1. Average all replicates to obtain mean counts. 2. Divide standard or unknown (cpm) by zero standard (cpm) and multiply by 100 to obtain percentage bound (B)/maximum binding (B0).

% B/Bo = standard (cpm) or sample (cpm) × 100 zero standard (cpm) 3. Plot concentration of standards against percentage B/Bo using semilog graph paper. 4. Tube dose for sample is read from standard curve. To obtain final value (pg/ml) multiply tube dose (pg) by the recovery factor and divide by the initial sample volume.

196

VITAMIN D GROUP

[21]

TABLE I EXPECTED RANGES FOR THE ASSAY

Mean SD Number of determinations Observed range

Normal adults

Normal children

Primary hyperparathyroidism

Renal failure (hemodialysis)

35.9 11.4 80

43.3 13.9 40

63.0 29.0 21

7.4 8.2 130

19-57

22-79

25-146

<4-16

Tube dose (pg) x RF pg/ml = initial sample volume (ml) Expected ranges for the assay are summarized in Table I. The intraassay variation has been determined with one clinical sample using one standard curve in one assay. The mean, standard deviation (SD), and coefficient of variation of nine replicates were mean = 54.1 pg/ ml, SD = 3.4, and CV = 6.28%. The interassay variation determined in four serum pools is summarized in Table II. The sensitivity of the method is 2 pg and the specificity for various metabolites of vitamin D has been determined as follows: 1,25(OH)2D3 100%; 1,25-(OH)2D2 75%; 25-OHD3 0.06%, 24,25-(OH)2D3 0.003%. Hence, the serum 1,25-(OH)2D level determined by this method represents both 1,25-(OH)2D3 and 1,25-(OH)zD2. However, greater than 95% of all vitamin D metabolites in humans occur in the D3 form, except for extremely high vitamin D2 intake. From the major metabolites of vitamin D the 25-OHD is the compound more likely to interfere with the 1,25-(OH)2D measurement in the cytoreceptor assay because it circulates at higher concentration than any other metabolite (approximately 25 ng/ ml) and has also relatively high affinity for the receptor. We have established that it requires a concentration greater than 125 ng/ml of 25-OHD to produce significant interference with the 1,25-(OH)2D measurement3; such high levels of 25-OHD, however, occur only in the rare instances of TABLE 11 1NTERASSAY VARIATION IN SERUM POOLS

Sample

Number of determinations

Mean value (pg/ml)

SD

CV (%)

1 2 3 4

21 40 22 40

76.4 38.9 46.3 39.0

11.4 6.7 7.0 6.6

14.9 17.1 15.1 16.9

[21]

ASSAY FOR 1,25-(OH)2D3

197

T A B L E 111 INTERLABORATORY REPRODUCIBILITY Cytoreceptor Lab 1

x ± SEM

HPLC Lab 2

CV (%)

Normals 28 79 40 16 19 30 41 20 49 35

9 50 41 12 19 23 28 22 72 20

31 96 50 25 17 34 37 22 48 27

42 28 10.4 31.1 9.7 20.5 20.7 13 20.1 24.8

37 ±4.6

35.7 -+5.8

29.6 +6.1

38.7 ±7.2

22.03 ±3.2

67 27 22 69 67 28 54

58 40 31 86 48 36 54

8.2 24.9 39.9 11.2 16.8 23.9 18.9

47.7 ±8.0

51.8 -+7.2

20.54 -+4.0

11 <6" 9 12 23 13

56.6 61.4 53.2 69 38 46.3

12.33 -+2.3

54 ±4.0

68 ±6.0

14 15 17 19 14 12 x -+ SEM

HPLC Lab I

27 63 41 17 22 38 47 27 53 35

71 51 57 89 73 50 85 x ± SEM

Cytoreceptor Lab 2

15.16 ±1.0

Pregnancy 64 45 45 78 63 38 69 57.4 ±5.6 Renal failure <4" <4" <4" <4" 9 6 5.16 ±0.8

<5" 17 15 <5" 19 <5" 11 ±2.7

" For values less than the detection limit we have used the detection limi! to obtain the mean ± SEM.

vitamin D intoxication. We have also established in our laboratory that other steroid hormones do not interfere with the 1,25-(OH)2D measurements even at pharmacologic levels. The interlaboratory reproducibility of the cytoreceptor assay and its comparison with HPLC assays for 1,25-(OH)2D has been tested by four

198

VITAMIN D GROUP

[2 1]

T A B L E IV C O E F F I C I E N T OF C O R R E L A T I O N a

C y t o r e c e p t o r Lab 1 Cytoreceptor Lab 2 HPLC Lab 1 HPLC Lab 2

Cytoreceptor Lab 1

Cytoreceptor Lab 2

HPLC Lab 1

HPLC Lab 2

--

r = 0.95 --

r = 0.87 r = 0.86 --

r = 0.86 r = 0.94 r = 0.81

R e p r o d u c e d from Manolagas et al. 6 with the permission of the editor of L a n c e t .

laboratories 6 (two used the cytoreceptor method and two used standard chromatographic assays). Replicates from 23 serum samples from normal individuals (n = 10), pregnant women who are known to have elevated levels (n = 7), and patients with end-stage renal failure (n = 6) were assayed in each laboratory with the respective method. The values obtained by this interlaboratory evaluation are shown in Table III. The coefficient of correlation of the 1,25-(OH)zD3 levels (n = 23) determined in each laboratory with the levels determined by each one of the other three is shown in Table IV. Acknowledgments T h e a u t h o r wishes to t h a n k Ms. D. Curran and C. Bochra for technical assistance and Dr. R. Reitz o f the E n d o c r i n e Metabolic Center, Oakland, CA, Dr. R. H o r s t of the National Animal Disease Center, A m e s , IA, and Dr. J. Haddad, University of Pennsylvania, for the interlaboratory evaluation of the method. Supported by the NIH and the Veterans Administration. NOVE ADDED~NPROOF:The single c o l u m n b e n z e n e extraction m e t h o d described here sometimes p r o d u c e s extracts that s h o w inflated values in the assay. We have recently eliminated this problem by incorporating additional extraction steps using a second c o l u m n and three ether-based reagents. The n e w procedure involves attaching a second c o l u m n containing u n b o n d e d silicic acid gel (Bio-Rad) below the original c o l u m n of d i a t o m a c e o u s earth. The sample is prepared as usual and, after equilibrating on the first column for 30 min, is extracted with 30 ml b e n z e n e u n d e r v a c u u m . T h e first column, which retains the a q u e o u s and hydrophilic phases of the matrix, is discarded. The b e n z e n e is also discarded, as the vitamin D metabolites are adsorbed onto the silicic acid in the second column. A series of solvents is then used to selectively r e m o v e the different metabolites, based on their polarity. The relatively nonpolar lipids and n o n h y d r o x y l a t e d vitamin D3 are eluted first using 4.5 ml I : 1 hexane : ether. Diethyl ether alone (6 ml) is used to remove the 25-hydroxy and m u c h of the 24,25-dihydroxy forms. Finally, 7.5 ml I : 19 methanol : ether elutes the more polar 1,25-(OHJ_,D3. Only this final eluent is collected and evaporated for use in the assay. T h e cytoreceptor a s s a y itself remains unchanged. 6 S. C. Manolagas, R. E. Reitz, R. Horst, J. H a d d a d , and L. J. Deftos, L a n c e t l , 191 (1983).