ARCHIVES
OF
BIOCHEMISTRY
A Sensitive,
AND
176, 235-243 (1976)
BIOPHYSICS
Precise, and Convenient Method for Determination 1,2!5Dihydroxyvitamin D in Human Plasma’
JOHN A. EISMAN,”
of
ALAN J. HAMSTRA, BARBARA E. KREAM, HECTOR F. DELUCA~
AND Department
of Biochemistry,
College of Agricultural and Life Sciences, University Madison, Wisconsin 53706 Received
March
of Wisconsin-Madison,
5, 1976
A new, highly sensitive and relatively convenient method has been developed for the determination of 1,25-dlhydroxyvitamin D, and 1,25-dihydroxyvitamin D, in blood plasma. The method involves a simplified and more speoific extraction procedure, new rapid and effective methods of purification, and a competitive binding assay using intestinal cytosol from rachitic chicks. The method also includes a procedure for stabilizing the cytosol binding protein and a convenient procedure for the separation of bound from free 1,25dihydroxyvitamin D, with the use of polyethylene glycol. The recovery of 1,25dihydroxyvitamin D3 during extraction and purification is 68% and triplicate determinations can be made on a 5-ml plasma sample. With this method, rachitic chick plasma, plasma from anephric patients, and plasma from patients suffering severe endstage renal failure show no detectable 1,25-dihydroxyvitamin D, while normal human values have been found to be 29 f 2 pg/ml.
In recent years, studies of the metabolism and action of vitamin D have shown that the 1,25dihydroxylated derivative of the vitamin is the major, if not sole, metabolically active form of the vitamin. 1,25Dihydroxyvitamin D3 (1,25-(OH),D,)4 stimulates intestinal calcium transport (l4) and enhances intestinal absorption of phosphate (5). This metabolite also stimulates bone mineral resorption with release 1 This work was supported by Grant AM-14881 and contract NIAMDD 72-2226 from the National Institutes of Health, contract NAS2-8752 from the National Aeronautics and Space Administration and the Proctor & Gamble Company. * Recipient of the C. J. Martin Travelling Fellowship from the National Health and Medical Research Council of Australia. 3 To whom all correspondence should be addressed. 4 Abbreviations used: 1,25-(OH12D3, 1,25-dihydroxyvitamin Da; 25-OH-D,, 25-hydroxyvitamin D,; 1,25-(OH),D,, 1,2&dihydroxyvitamin D,. PIPES, piperazine-Nfl-bis(2-ethane sulfonic acid); MOPS, (2N-morpholinolpropane sulfonic acid; MES, (2-Nmorpholino) ethane sulfonic acid.
of bone calcium and phosphate into the circulation (4, 6, 7). Studies in tissue culture on isolated fetal bone have shown 1,25-(OH),D,-induced resorption while other metabolites of vitamin D are effect,ive at much higher concentrations in this system (8, 9). The 1-hydroxylation of the vitamin occurs in renal tissue (10,ll) after 25-hydroxylation has occurred in the liver (12). It is the renal hydroxylation which is tightly regulated in a complex manner by serum calcium (13, 14), parathyroid hormone (15, 16), and serum phosphate concentrations (17, 18). These relationships have been discovered primarily using radioactive tracers in vitamin D-depleted animals (13-18) and in vitro measurement of the hydroxylases (19-22). The former approach has revealed concentrations ranging from 200 to 700 pg of 1,25-(OH)zD3 per milliliter of plasma after relatively short periods of vitamin D replacement (i.e., l-2 weeks). More recently, Brumbaugh et al. (23-25) have used a chromatin binding assay to meas235
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
EISMAN
236
ure the level of 1,25-(OHjzD3 in rat and human plasma without the need for radioactive tracer administration. They have reported similar values for rats but their studies on human plasma have yielded 1,25-(OH),D, values one order of magnitude less than those found in rats. We have developed a competitive binding assay for 1,25-(OH),D, in biological materials particularly for measurement of plasma levels. We have used chick intestinal cytosol as the source of binding protein, as does the method of Brumbaugh et al.; however, the chromatin binding step has been eliminated. This is possible because of stabilization of the cytosol binding protein and a convenient method of separation of bound from free 1,25-(OH),D,. Additional improvements include a simplified method of extraction and purification of plasma 1,25-(OH),D, which eliminates high background values and interfering substances. The presently derived method allows triplicate determinations on 5 ml of plasma and reveals a normal value for human plasma of 29 -+ 2 pg/ml. MATERIALS Crystalline 25OH-D, was obtained from the Upjohn Company (Kalamazoo, Mich.), crystalline vitamin D, was obtained from Philips-Duphar (Weesp, The Netherlands), and crystalline 1,25(OH),D, was obtained from Hoffmann-LaRoche (Nutley, N. J.). [26,27-3H125-OH-D3 (9.3 Ci/mmol) was synthesized as described previously (26) and [26,27-3H11,25-(OH)2D3 was prepared from this compound by incubation with chicken kidney homogenates (27). 1,25-(OH),D, was prepared in this laboratory as described previously (28). Sephadex LH-20 was obtained from the Pharmacia Fine Chemicals Company (Piscataway, N.J.). Polyethylene Glycol 6000 (average molecular weight, 6000-7500) was obtained from the J. T. Baker Chemical Company (Phillipsburg, N.J.). All solvents and reagents were of analytical grade. Skellysolve B was redistilled (67-69”(Z) before use. High-pressure liquid chromatography was performed on a DuPont 830 LC apparatus fitted with a Waters U-6-K injection port (Waters Associates, Milford, Mass.) (29). Polyallomer centrifuge tubes were obtained from Beckman Instruments, Inc. (Palo Alto, Calif.). METHODS Extraction of plasma (or serum). To five milliliters of plasma (or serum) is added 0.25 ml of 95%
ET AL
6000
c
FIG. 1. Chromatography of 25-OH-D, and 1,25(OH),D, on Sephadex LH-20 in Skellysolve B:chloroform:methanol (9:l:l). [26,27-3H125-OH-D, (10,000 cpm) and 126,27-3H11,25-(0H),D, (5000 cpm) were applied to a 0.7 x 9 cm column of Sephadex LH20 and chromatographed. One-milliliter fractions were collected. ethanol containing 70 pg (3500 dpm) of [26,27SH11,25-(OH),D, (9.3 Ci/mmol) in a 125-ml separatory funnel. Thirty milliliters of dichloromethane (methylene chloride) was added to the separatory funnel, vigorously shaken, vented, and then shaken for 5 min on a horizontal shaker at 4 cps. The phases were allowed to separate and the dichloromethane phase was collected. The aqueous phase was washed with two additional l&ml portions of dichloromethane; the dichloromethane fractions were pooled and evaporated to dryness on a rotary evaporator under reduced pressure. Purification of dichloromethane extract. The dichloromethane plasma extract was chromatographed on a small Sephadex LH-20 column (0.7 x 9 cm) in a new solvent system of Skellysolve B:chloroform:methanol (9:1:1P (Fig. 1). This resulted in very little lipid contamination of the 1,25(OH),D, region of elution. The sample was applied in 0.5 ml of the column solvent and rinsed with an additional 0.5 ml. Thereafter, the next 7.75 ml of elution was discarded and the next 7.5 ml was collected as the 1,25-(OH),D, region. This 1,25-(OH),D, region was subjected in toto to high-pressure liquid chromatography on a Waters lo-pm silicic acid column (0.4 x 30 cm) with a flow rate of 1.8 ml/min of 10% isopropanol in twice distilled Skellysolve B at a pressure of 800 psi (Fig. 2). 3 Developed mann-LaRoche
by Dr. James Hamilton of the HoffCompany, Nutley, N.J.
ASSAY
OF PLASMA
1,25-DIHYDROXYVITAMIN
FIG. 2. High-pressure liquid chromatography of standard 25-OH-D, (60 ng), 1,25-(OH),D, (80 ng), and 1,25-(OH),D, (110 ng) in a Waters 0.4 x 30 cm 10 pm silicic acid column. The column was developed in a solvent system of 10% isopropanol in twice distilled Skellysolve B at 850 psi, giving a flow rate of 1.8 ml/min. Optical density was recorded at 254 nm. The sample was applied in 20 ~1 of the solvent and rinsed into the injector (Waters U-K-6) with a further 20 ~1. The 1,25-(OH),D, eluted at 20-23 ml; this position was checked with a 1,25-(OH),D, standard for each run of samples. The 1,25-(OHj2D3 region was collected, evaporated under a stream of dry nitrogen, and resuspended in a small volume of 95% ethanol for assay and for counting to estimate recovery. Preparation of binding protein. White Leghorn chickens were raised from the day of hatching on a vitamin D-deficient, purified soy protein diet (1) until sacrifice by decapitation at 9-12 weeks. The duodenal loops were removed, emptied, and rinsed with 5 ml of ice-cold buffer (0.05 M potassium phosphate, pH 7.4, 0.05 M potassium chloride). This buffer was used throughout preparation and assay procedures. All subsequent steps were performed either on ice or at 4°C. The mucosa was scraped from the serosa and minced in 5 vol (ml/g) of the buffer. The minced mucosa was centrifuged at 2000g for 10 min and the supernatant was discarded. The pellet was resuspended twice in 5 vol of fresh buffer and recentrifuged and the supernatant was discarded. The final mucosal pellet was resuspended and homogenized in 2 vol of the buffer with two passes of a Potter-Elvehjem Teflon-glass homogenizer maintained at 0°C with an ice bath. The homogenate was centrifuged at 50,000 rpm (226,600g) for 45 min in a number 50 titanium head of a Beckman L2 ultracentrifuge. The tine lipid layer was removed with a Pasteur pipet and the clear cytosol was collected. This cytosol was frozen in 4- to 6-ml portions in an
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237
acetone-dry ice bath, lyophilized, and stored under N, gas at -20°C until use. The yield of binding protein was 20 mg/g wet wt of duodenal mucosa, and the binding activity was stable for several weeks under these conditions. Cytosol binding protein prepared in this manner was invariably active. Immediately prior to use, the lyophilized cytosol was reconstituted with distilled water and diluted to yield a final protein concentration of l-l.3 mg/ml. Protein concentrations were determined by a Biuret method with a bovine serum albumin standard. Competitive binding assay. Each assay mixture consists of 1 mg of cytosol protein in 1 ml of buffer, 3500 dpm (70 pg) of 126,27-3H11,25-(OH),D, in 50 pl of 95% ethanol, and varying amounts of nonradioactive 1,25-(OH12D3 in an additional 50 ~1 of 95% ethanol. Standard curves were constructed over a useful range of lo-140 pg using crystalline 1,25(OH),D, (see Fig. 3). Nonspecific binding was determined by the inclusion of an assay tube to which was added 5 ng of 1,25-(OH)1D, to effectively displace any [3H]1,25-(OH)2D3 from specific binding sites. Samples for assay were introduced in 50 ~1 of 95% ethanol as was the crystalline 1,25-(OH),D, used to produce the standard curve. To compensate in the assay for the [3Hll,25-(OH),D, added to measure recovery during extraction and chromatography, an appropriate reduction in the [3H11,25-(OH),D, added to each assay tube was made so that all tubes (samples and standards) had the same total radioactivity at the time of final assay. The standard curve incubations were carried out in quadruplicate and the sample incubations were performed in triplicate. The assays were carried out in polyallomer test tubes (‘/la x 2Q-in.) in a shaking water bath at 25°C for 1 h and then were immersed in an iced water bath. One milliliter of icecold 40% polyethylene glycol was added to each tube which was vortexed vigorously to produce a slightly turbid solution. The tubes were centrifuged at 4800g in the HS-4 head of a Sorvall RC-5 refrigerated centrifuge at 4°C for 30 min. The supernatant was discarded and the tip of the test tube, which contained the small pellet, was cut off into a 5-ml scintillation vial. Three milliliters of a p-dioxane-based scintillator solution was added to the vial and shaken vigorously. This scintillator solution, containing 10% naphthalene and 0.5% 2,5-diphenyloxazole in p-dioxane, provided 37.5% efficiency in an ambient temperature Beckman LS-1OOC scintillation counter. Optimum incubation conditions. The effect of pH on 13H11,25-(OH&D, binding was examined by preparing a series of cytosol solutions from the same pool of intestinal mucosa at different pH’s and in different buffers (Fig. 4). Piperazine-NJ-bis(2ethane sulfonic acid) (PIPES), (2 ,N-morpholino) sulfonic acid (MOPS), (2-N-morphopropane linolethane sulfonic acid (MES), and Tris buffers
238
EISMAN
ET AL. b)
a) 60C 1ii-1.25~iOHl2D, sound
I” Pellet
I L
0
I
s
I
I
a
I
50
I
I
I
.*.”
100
l,25-(OH)2D3pgltube
5000
IO
I
I
,
203050
1
100
1.25~(OH),D,pg/tube
FIG. 3. Competitive binding assay standard curve. [26,27-3H11,25-(OH)2D3 bound in the pellet (cpmltube) is plotted against amount of competing unlabeled 1,25-(OH),D, added per tube on (a) a linear scale and (b) a logarithmic scale. Points are mean 2 SEM of triplicate determinations.
PH
FIG. 4. The
effect of pH on [26,27-3H11,25(OH),D, binding to cytosol protein. Specific binding (total binding minus nonspecific binding) was determined using cytosol prepared and incubated in the buffers at the pH indicated. Values are expressed as specific counts per minute bound per milligram of protein and are means of duplicate determinations. (0.025-0.05 M) were used to cover the pH range from 5.8 to 9.2 at 25°C. The buffers contained 0.15 M potassium chloride and 0.001 M ethylene diaminetetraacetic acid and were used throughout the cytosol
preparation after the first wash of the intestinal mucosa. The preparation apart from the changes in pH was identical in each case. Maximum binding and nonspecific binding were determined in duplicate for each cytosol with a 1 h of incubation at 25°C. The specific binding was expressed as specific counts per minute bound per milligram of protein, as shown in Fig. 4. The effect of divalent cations on binding was determined for magnesium and calcium ions. Cytosol was prepared in the usual way in a 0.05 M potassium phosphate (pH 7.4) buffer made 0.001 M with respect to EDTA. Various amounts of calcium and magnesium chloride were added to produce a range of free cation concentrations from 0 to 10 mM. Specific binding was determined as described above. Similarly, the effect of potassium chloride on binding was determined in the presence and absence of the divalent cations mentioned. Time and temperature dependence. The specific binding of [3H11,25-(OH1,D, to the cytosol binding protein was determined at three temperatures from 4 to 37°C over a period of time from 0 to 21 h, as shown in Fig. 5. Specificity. Competitive binding assays were carried out using various amounts of vitamin D,, 25OH-D,, 1,25-(OH),D,, and 1,25-(OH),D, to compete with radioactive 1,25-(OH),D,. These assays, which were designed to elucidate the specificity of the
ASSAY
OF PLASMA
1,25-DIHYDROXYVITAMIN
D
239
.
FIG. 5. Effect of temperature on [26,27-3H]1,25-(OH)2D3 binding to cytosol protein. Specific binding was determined at pH 7.4 after various periods of incubation at the temperatures indicated. Values are expressed as specific counts per minute bound per milligram of protein and are means of duplicate determinations. binding protein, were otherwise identical with the assays described above. Extracts of plasma from vitamin D-deficient chickens were examined at various stages of puritication for interference with the competitive binding assay. The chickens used for this experiment were at least 12 weeks old and showed the physical evidence of severe rachitic bone disease.
RESULTS
Extraction of plasma. The dichloromethane extraction recovered 91.4 + 6.0% (mean f SEM) of radioactive 1,25-(OHjzD3 added to a plasma sample. The first extraction recovered 80% of the added radioactivity and the second and third extractions of the aqueous layer recovered an additional 7 and 5%, respectively. Validity of in vitro recovery estimations. It is possible that the recovery of radioactive 1,25-(OHjzD3 added to the plasma in vitro might differ from the recovery of 1,25(OH),D, produced and released into the plasma in vivo. To examine this possibility radioactive [26,27-3H125-OH-D, (9.3 Ci/ mmol) was administered by wing vein to three vitamin D-deficient chickens. Sixteen hours after dosing, the chickens were bled by heart puncture and the pooled plasma was divided into four equal portions. Two portions were extracted as de-
scribed in the two phase system using dichloromethane and two portions were extracted with the standard one-phase extraction using chloroform:methanol:aqueous (25:50:16). In this method additional chloroform (25 parts) was added to produce two phases and the aqueous phase was “washed” twice with two further additions of chloroform (25 parts). Each extract was chromatographed on Sephadex LH-20 in chloroform:Skellysolve B (75:25) and the 1,25-(OHj2D3 region was rechromatographed on Sephadex LH-20 in Skellysolve B:chloroform:methanol (9:l:l). The radioactivity recovered in the 1,25-(OH),D, region was 382 +- 30 dpm/ml plasma (mean lr SEM) for the dichloromethane extractions and 254 ? 30 dpm/ml plasma for the chloroform:methanol extraction method. Purification of the dichloromethane extract. The chromatographic profile of radioactive 1,25-(OH)*D3 and 25-OH-D, on Sephadex LH-20 in the Skellysolve B:chloroform:methanol (9:l:l) solvent system is shown in Fig. 1. This chromatographic system separated 25-OH-D, from 1,25(OH),D, even on the small 0.7 x 9 cm columns when both samples were applied in 0.5 ml of solvent. However, this chromatographic system was somewhat sensitive to the amount of lipid applied. The
240
EISMAN ET AL.
lipid from 5 ml of plasma extracted by the standard chloroform:methanol method resulted in poor chromatography with a phase separation in the application solvent, hence plasma extracted in this way had to be partially purified in a chloroform:Skellysolve B (7525) solvent system on a Sephadex LH-20 column. When plasma was extracted by the dichloromethane method, the amount of lipid was much less and the lipid extracted from 510 ml of plasma could be applied directly to the Skellysolve B:chloroform:methanol Sephadex LH-20 chromatography column. High-pressure liquid chromatography of vitamin D metabolites on microparticulate silica columns was carried out as has been described recently (23). The solvent system of 10% isopropanol in twice distilled Skellysolve B used in these experiments separates all known metabolites of vitamin D. 1,25-(OHl,D, elutes slightly ahead of 1,25(OH),D,, but the region collected for assay included both 1,25-dihydroxylated vitamins (Fig. 2). Recovery. Overall recovery of 1,25(OHl,D, from plasma after extraction and purification through the two column procedures prior to assay was 68 ? 1.2% (mean -+ SEMI. Incubation conditions. The specific binding of [3H11,25-(OH)2D3 to the cytosol binding protein and precipitation by polyethylene glycol had a pH optimum in the range of 7-8, as shown in Fig. 4. There was no difference in the specific binding about the pH optimum using the various sulfonic acid derivative buffers or potassium phosphate, so the latter was used for all assays thereafter. Calcium and magnesium were not required for maximum specific binding and, in fact, a high concentration of calcium ion (0.01 M) halved specific binding. Maximum specific binding was achieved in the presence of 0.05-0.1 M potassium chloride, so 0.05 M potassium chloride was added to the 0.05 M potassium phosphate buffer (pH 7.4) used in all routine assays. Time and temperature dependence. The specific binding of [3H11,25-(OH)zD3 at 4°C increased up to the longest time examined, 21 h. At 37°C the binding was greatest at
0.5 h and rapidly decreased thereafter. The specific binding at 25°C had a broad maximum at 1 h, which was greater than achieved at either 4 or 37°C (Fig. 5). The slow decrease in specific binding at 25°C and rapid decrease at 37°C are presumably the result of enzymatic degradation in the crude intestinal cytosol binding protein preparation. Determination of saturation of cytosol binding protein. A series of competitive binding assays are performed with each batch of intestinal cytosol to determine the optimum concentration of cytosol protein for use in the assay. Incubations containing 10 pg and 5 ng of unlabeled 1,25(OH),D, were compared to those incubated with no unlabeled metabolite at protein concentrations in the range of 1.0-l .5 mgl incubation. The difference between the maximum binding (no added unlabeled metabolite) and nonspecific binding (5 ng added unlabeled metabolite) was considered to be the specific binding. Statistical considerations indicate that at saturation, those incubations with 10 pg of unlabeled 1,25-(OH),D, added should show a 12.5% decrease in specific binding because of the 12.5% decrease in specific activity of the labeled 1,25-(OH),D, by the unlabeled metabolite. Therefore, the highest protein concentration, which showed this 12.5% decrease in specific binding with 10 pg of unlabeled metabolite, was used for that batch of cytosol preparation. Binding activity decreased about 50% in cytosol stored in solution overnight at 4°C. However, during prolonged storage of the lyophilized cytosol under nitrogen gas and at -2O”C, there was only a gradual decrease in the specific binding of the order of lo-20% over l-2 months. This change was the result of an increase in nonspecific binding and a decrease in maximum specific binding. Nonspecific binding, which includes any contamination of the pellet with supernatant, is reproducible and usually about 10% of total radioactivity. The stability of the cytosol binding protein was determined by measuring the specific binding as a function of storage time. The washing of the mucosa prior to homogenization is essential for maximum stability.
ASSAY
OF PLASMA
1.25DIHYDROXYVITAMIN
Other important factors are cold temperatures during homogenization, lyophilization prior to storage, and the N, atmosphere during storage. Extracts of plasma from vitamin D-deficient chickens before and after Sephadex LH-20 chromatography interfered with the competitive binding assay, causing a variable decrease in the maximum binding and a variable increase in the nonspecific binding. However, high-pressure liquid chromatography on the microparticulate silica column effectively removed these interfering compounds, so that plasma from vitamin D-deficient chickens gave assay values which were identical with zero nonradioactive 1,25-(OH),D, in the standard assay. Specificity of the binding protein. 1,25(OH),D, and 1,25-(OH),D, produced indistinguishable competition curves in the binding assay, as shown in Fig. 6. The binding assay thus would estimate 1,25dihydroxylated vitamin derived from either vitamin D2 or D,.
The competition curve with 25OH-D3 showed a lower affinity by three orders of magnitude (Fig. 71, while vitamin D, did not compete even at the level of 1 Fg/ incubation. Estimation of 1,25-(OH)9, level in plasma samples. Samples and standards were run in the same assay using the same batch of cytosol. The radioactivity bound in the pellet was plotted against the amount of unlabeled standard 1,25(OH),D, added to the incubation (Fig. 3a). This produced a straight line on a semilogarithmic plot (Fig. 3b). The amount of 1,25-(OHjzD3 required to produce the reduction of [3H]1,25-(OH),D, bound to the pellet seen in the sample incubations was read from the graph or determined from a linear regression equation. Each value was corrected for the recovery percentage estimated after its extraction and purification. Normal human values. The value estimated for normal human serum was 29 ? 2 pg/ml (mean 4 SEM, n = 20). These serum samples were derived from normal persons of both sexes with a mean age of 55
\\
0 IoPg
1OOPO Mctabolite
I, 25-(OH),
0
pg/ tube
FIG. 6. Competition of 1,25-(OH),D, and 1,25(OH),D, for cytosol protein binding sites. A competitive binding assay was performed using 126,273H]1,25-(OH),D, and varying amounts of competing unlabeled 1,25-(OH)PD2 (0) and 1,25-(OH)zD, (A). [26,27-3H]1,25-(OH)ZD3 counts per minute bound in the pellet is plotted against amount of unlabeled competing metabolite. Points are mean 2 SEM of triplicate determinations.
241
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long
1 ng Added,
100 ng
c IN
tube
FIG. 7. Comparison of ability of 25OH-D, and 1,25-(OH)ZD, to compete for cytosol protein binding sites. Competitive binding assays were performed using [26,27-3H]1,25-(OH)2D3 and varying amounts of competing unlabeled 1,25-(OH12D3 (0) and 25-OHD, (A). Specific binding (total binding minus nonspecific binding) at each metabolite concentration is expressed as a percentage of the maximum specific binding and is plotted against amount of metabolite per tube on a logarithmic scale. Points are mean f SEM of triplicate determinations.
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EISMAN
years (kindly supplied by Dr. B. L. Riggs, Mayo Clinic). Assays of plasma from three persons with chronic renal failure and three persons with bilateral nephrectomy showed no detectable 1,25-(OH),D,. Plasma from one patient with chronic renal failure had a value of 13 pglml but a plasma sample taken 3 months later from the same patient also showed no detectable 1,25WW,. Precision of the assay. Fresh plasma from two people was obtained from the Red Cross Blood Center and frozen in portions. These plasma samples were extracted and purified on eight occasions and each was assayed in triplicate. The concentrations of 1,25-(OH)zD3 determined were 30 * 3 and 28 + 2 pg/ml (mean f SEM). The coefficient of variation of triplicate determinations of the tritium counts bound in the pellet was 2.4 ? 0.5% (mean + SEM, n = 14). DISCUSSION
The present report describes a new sensitive competitive binding assay for 1,25(OH)2D, in biological materials and a new extraction and purification scheme for 1,25-(OH)zD3 and 1,25-(OH),D, in human and animal blood. The extraction and purification scheme is relatively rapid and simple, although it utilizes an expensive and elaborate highpressure liquid chromatography instrument, as previously described (29). The solvent flow rate and pressure used in these experiments could easily be achieved on a much simpler and less expensive high-pressure liquid chromatography system than that used in these experiments. The extraction of plasma from chickens dosed with 13H125-OH-D, showed that the dichloromethane extraction recovered plasma 1,25-(OH),D, as well as, if not better than, the commonly used chloroformmethanol extraction. This supported the reliability of the recovery estimation using in vitro addition of [3H]1,25-(OH),D3 to plasma samples prior to extraction. The lower recovery of 1,25-(OH)zD3 using the chloroform-methanol extraction is unexplained as yet, but may be related to
ET AL.
breakdown of 1,25-(OH)*D3 in the aqueous phase following protein denaturation by methanol. The overall recovery of 68% for 1,25(OH),D, from plasma after extraction and purification combined with the 10 pg sensitivity per assay shows that human plasma levels of 1,25-(OH),D, can be assayed in triplicate with a 5-ml plasma sample. The lower sensitivity of the chromatin binding assay and their lower recovery during extraction and purification presumably accounts for the 20-ml plasma requirement of the assay of Brumbaugh et al. (23-25). The equal sensitivity of the assay to 1,25-(OH),D, and 1,25-(OH),D, has important practical significance in human studies, where the 1,25-dihydroxylated metabolite could and probably would be derived from both vitamin D, and vitamin D, under normal circumstances. The low sensitivity of the assay to 25-OH-D, has little practical significance as any 25-OH-D3 extracted from a sample would be completely eliminated during the purification steps. However, the marked difference in sensitivity is consistent with the minimal biological activity of physiological doses of 25OH-D, on intestinal calcium transport in nephrectomized animals (30). This agreement supports the view that the cytosol binding protein may be a physiological receptor for 1,25-(OH),D, in the intestine as has been suggested by Brumbaugh and Haussler (25). The assay method reported here differs from that of Brumbaugh et al. (23-25) in several important aspects. This assay is nearly twice as sensitive as the assay they have described. Furthermore, the use of polyethylene glycol precipitation to separate bound from free [3H11,25-(OH),D, eliminates the need for the chromatin binding step of their assay. The extensive washing of the mucosa prior to homogenization stabilizes the binding protein so that it can be stored for long periods, thus eliminating the need for fresh preparation of binding protein for each assay. Furthermore, the washing increases the specific binding of [3H11,25-(OH)2D3 as has been shown in studies in this laboratory using
ASSAY
OF PLASMA
1,25-DIHYDROXYVITAMIN
sucrose density gradient centrifugation.6 These differences result in an assay which is more rapid, more sensitive, and more convenient than that previously described. The mean value estimated in this report of 29 pglml is considerably lower than the 40 pg/ml most recently reported by Brumbaugh et al. (24). This difference may reflect the average age of the blood donors, which was rather high in our studies. The zero values found in anephric patients and those in chronic renal failure are consistent with the renal locus of the l-hydroxylase enzyme and confirms the removal of interfering substances during the extraction and purification processes. ACKNOWLEDGMENTS We gratefully acknowledge helpful suggestions from Drs. 0. N. Miller, J. Hamilton, and R. Dixon of the Hoffmann-LaRoche Company.
REFERENCES 1. OMDAHL, J., HOLICK, M., SUDA, T., TANAKA, Y., AND DELUCA, H. F. (1971) Biochemistry 10, 2935-2940. 2. HAUSSLER, M. R., BOYCE, D. W., LITTLEDIKE, E. T., AND RASMUSSEN, H. (1971). Proc. Nut. Acad. Sci. USA 68, 177-181. 3. MYRTLE, J. F., AND NORMAN, A. W. (1971) Science 171, 79-82. 4. TANAKA, Y., AND DELUCA, H. F. (1971) Arch. Biochem. Biophys. 146, 574-578. 5. CHEN, T. C., CASTILU), L., KORYCKA-DAHL, M., AND DELUCA, H. F. (1974) J. Nutr. 104, 10561060. 6. CASTILLO, L., TANAKA, Y., AND DELUCA, H. F. (1975) Endocrinology 97, 995-999. 7. TANAKA, Y., AND DELUCA, H. F. (1974) Proc. Nat. Acad. Sci. USA 71, 1040-1044. 8. RAISZ, L. G., TRUMMEL, C. L., HOLICK, M. F., AND DELUCA, H. F. (1972) Science 175, 768769. 9. REYNOLDS, J. J., HOLICK, M. F., AND DELUCA, 6 Kream, B. E., Reynolds, R. D., Knutson, J. C., Eisman, J. A., and DeLuca, H. F., submitted for publication.
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H. F. (1973) Calc. Tiss. Res. 12, 295-301. 10. FRASER, D. R., AND KODICEK, E. (1970) Nature (London) 228, 764-766. 11. GRAY, R., BOYLE, I., AND DELUCA, H. F. (1971) Science 172, 1232-1234. 12. PONCHON, G., AND DELUCA, H. F. (1969). J. N&r. 99, 157-167. 13. BOYLE, I. T., GRAY, R. W., OMDAHL, J. L., AND DELUCA, H. F. (1972) in Endocrinology 1971 (Taylor, S., ed.), pp. 468-476, Wm. Heinemann Medical Books, London. 14. BOYLE, I. T., GRAY, R. W., AND DELUCA, H. F. (1971) Proc. Nut. Acad. Sci. USA 68, 21312134. 15. GARABEDIAN, M., HOLICK, M. F., DELUCA, H. F., AND BOYLE, I. T. (1972) Proc. Nat. Acad. Sci. USA 69, 1673-1676. 16. FRASER, D. R., AND KODICEK, E. (1973) Nature New BioZ. 241, 163-166. 17. TANAKA, Y., AND DELUCA, H. F. (1973) Arch. Biochem. Biophys. 154, 566-574. 18. TANAKA, Y., FRANK, H., AND DELUCA, H. F. (1973) Science 181, 564-566. 19. OMDAHL, J. L., GRAY, R. W., BOYLE, I. T., KNUTSON, J., AND DELUCA, H. F. (1972) Nuture New Biol. 237, 63-64. 20. OMDAHL, J. L., AND DELUCA, H. F. (1971) Science 174, 949-951. 21. OMDAHL, J. L., AND DELUCA, H. F. (1972) J. Biol. Chem. 247, 5520-5526. 22. HENRY, H. L., MIDGETT, R. J., AND NORMAN, A. W. (1974) J. Biol. Chem. 249, 7584-7592. 23. BRUMBAUGH, P. F., HAUSSLER, D. H., BRESSLER, R., AND HAUSSLER, M. R. (1974). Science 183, 1089-1091. 24. BRUMBAUGH, P. F., HAUSSLER, D. H., BURSAC, K. M., AND HAUSSLER, M. R. (1974) Biochemistry 13, 4091-4097. 25. BRUMBAUGH, P. F., AND HAUSSLER, M. R. (1975) J. Biol. Chem. 250, 1588-1594. 26. SUDA, T., DELUCA, H. F., AND HALLICK, R. B. (1971) Anal. Biochem. 43, 139-146. 27. BOYLE, I. T., MIRAVET, L., GRAY, R. W., HOLICK, M. F., AND DELUCA, H. F. (1972) Endocrinology 90,605-608. 28. JONES, G., SCHNOES, H. K., AND DELUCA, H. F. (1975) Biochemistry 14, 1250-1256. 29. JONES, G., AND DELUCA, H. F. (1975) J. Lipid Res. 16, 448-453. 30. STERN, P. H., TRUMMEL, C. L., SCHNOES, H. K., AND DELUCA, H. F. (1975) Endocrinology 97, 1552-1558.
OF
BIOCHEMISTRY
A Sensitive,
AND
176, 235-243 (1976)
BIOPHYSICS
Precise, and Convenient Method for Determination 1,2!5Dihydroxyvitamin D in Human Plasma’
JOHN A. EISMAN,”
of
ALAN J. HAMSTRA, BARBARA E. KREAM, HECTOR F. DELUCA~
AND Department
of Biochemistry,
College of Agricultural and Life Sciences, University Madison, Wisconsin 53706 Received
March
of Wisconsin-Madison,
5, 1976
A new, highly sensitive and relatively convenient method has been developed for the determination of 1,25-dlhydroxyvitamin D, and 1,25-dihydroxyvitamin D, in blood plasma. The method involves a simplified and more speoific extraction procedure, new rapid and effective methods of purification, and a competitive binding assay using intestinal cytosol from rachitic chicks. The method also includes a procedure for stabilizing the cytosol binding protein and a convenient procedure for the separation of bound from free 1,25dihydroxyvitamin D, with the use of polyethylene glycol. The recovery of 1,25dihydroxyvitamin D3 during extraction and purification is 68% and triplicate determinations can be made on a 5-ml plasma sample. With this method, rachitic chick plasma, plasma from anephric patients, and plasma from patients suffering severe endstage renal failure show no detectable 1,25-dihydroxyvitamin D, while normal human values have been found to be 29 f 2 pg/ml.
In recent years, studies of the metabolism and action of vitamin D have shown that the 1,25dihydroxylated derivative of the vitamin is the major, if not sole, metabolically active form of the vitamin. 1,25Dihydroxyvitamin D3 (1,25-(OH),D,)4 stimulates intestinal calcium transport (l4) and enhances intestinal absorption of phosphate (5). This metabolite also stimulates bone mineral resorption with release 1 This work was supported by Grant AM-14881 and contract NIAMDD 72-2226 from the National Institutes of Health, contract NAS2-8752 from the National Aeronautics and Space Administration and the Proctor & Gamble Company. * Recipient of the C. J. Martin Travelling Fellowship from the National Health and Medical Research Council of Australia. 3 To whom all correspondence should be addressed. 4 Abbreviations used: 1,25-(OH12D3, 1,25-dihydroxyvitamin Da; 25-OH-D,, 25-hydroxyvitamin D,; 1,25-(OH),D,, 1,2&dihydroxyvitamin D,. PIPES, piperazine-Nfl-bis(2-ethane sulfonic acid); MOPS, (2N-morpholinolpropane sulfonic acid; MES, (2-Nmorpholino) ethane sulfonic acid.
of bone calcium and phosphate into the circulation (4, 6, 7). Studies in tissue culture on isolated fetal bone have shown 1,25-(OH),D,-induced resorption while other metabolites of vitamin D are effect,ive at much higher concentrations in this system (8, 9). The 1-hydroxylation of the vitamin occurs in renal tissue (10,ll) after 25-hydroxylation has occurred in the liver (12). It is the renal hydroxylation which is tightly regulated in a complex manner by serum calcium (13, 14), parathyroid hormone (15, 16), and serum phosphate concentrations (17, 18). These relationships have been discovered primarily using radioactive tracers in vitamin D-depleted animals (13-18) and in vitro measurement of the hydroxylases (19-22). The former approach has revealed concentrations ranging from 200 to 700 pg of 1,25-(OH)zD3 per milliliter of plasma after relatively short periods of vitamin D replacement (i.e., l-2 weeks). More recently, Brumbaugh et al. (23-25) have used a chromatin binding assay to meas235
Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
EISMAN
236
ure the level of 1,25-(OHjzD3 in rat and human plasma without the need for radioactive tracer administration. They have reported similar values for rats but their studies on human plasma have yielded 1,25-(OH),D, values one order of magnitude less than those found in rats. We have developed a competitive binding assay for 1,25-(OH),D, in biological materials particularly for measurement of plasma levels. We have used chick intestinal cytosol as the source of binding protein, as does the method of Brumbaugh et al.; however, the chromatin binding step has been eliminated. This is possible because of stabilization of the cytosol binding protein and a convenient method of separation of bound from free 1,25-(OH),D,. Additional improvements include a simplified method of extraction and purification of plasma 1,25-(OH),D, which eliminates high background values and interfering substances. The presently derived method allows triplicate determinations on 5 ml of plasma and reveals a normal value for human plasma of 29 -+ 2 pg/ml. MATERIALS Crystalline 25OH-D, was obtained from the Upjohn Company (Kalamazoo, Mich.), crystalline vitamin D, was obtained from Philips-Duphar (Weesp, The Netherlands), and crystalline 1,25(OH),D, was obtained from Hoffmann-LaRoche (Nutley, N. J.). [26,27-3H125-OH-D3 (9.3 Ci/mmol) was synthesized as described previously (26) and [26,27-3H11,25-(OH)2D3 was prepared from this compound by incubation with chicken kidney homogenates (27). 1,25-(OH),D, was prepared in this laboratory as described previously (28). Sephadex LH-20 was obtained from the Pharmacia Fine Chemicals Company (Piscataway, N.J.). Polyethylene Glycol 6000 (average molecular weight, 6000-7500) was obtained from the J. T. Baker Chemical Company (Phillipsburg, N.J.). All solvents and reagents were of analytical grade. Skellysolve B was redistilled (67-69”(Z) before use. High-pressure liquid chromatography was performed on a DuPont 830 LC apparatus fitted with a Waters U-6-K injection port (Waters Associates, Milford, Mass.) (29). Polyallomer centrifuge tubes were obtained from Beckman Instruments, Inc. (Palo Alto, Calif.). METHODS Extraction of plasma (or serum). To five milliliters of plasma (or serum) is added 0.25 ml of 95%
ET AL
6000
c
FIG. 1. Chromatography of 25-OH-D, and 1,25(OH),D, on Sephadex LH-20 in Skellysolve B:chloroform:methanol (9:l:l). [26,27-3H125-OH-D, (10,000 cpm) and 126,27-3H11,25-(0H),D, (5000 cpm) were applied to a 0.7 x 9 cm column of Sephadex LH20 and chromatographed. One-milliliter fractions were collected. ethanol containing 70 pg (3500 dpm) of [26,27SH11,25-(OH),D, (9.3 Ci/mmol) in a 125-ml separatory funnel. Thirty milliliters of dichloromethane (methylene chloride) was added to the separatory funnel, vigorously shaken, vented, and then shaken for 5 min on a horizontal shaker at 4 cps. The phases were allowed to separate and the dichloromethane phase was collected. The aqueous phase was washed with two additional l&ml portions of dichloromethane; the dichloromethane fractions were pooled and evaporated to dryness on a rotary evaporator under reduced pressure. Purification of dichloromethane extract. The dichloromethane plasma extract was chromatographed on a small Sephadex LH-20 column (0.7 x 9 cm) in a new solvent system of Skellysolve B:chloroform:methanol (9:1:1P (Fig. 1). This resulted in very little lipid contamination of the 1,25(OH),D, region of elution. The sample was applied in 0.5 ml of the column solvent and rinsed with an additional 0.5 ml. Thereafter, the next 7.75 ml of elution was discarded and the next 7.5 ml was collected as the 1,25-(OH),D, region. This 1,25-(OH),D, region was subjected in toto to high-pressure liquid chromatography on a Waters lo-pm silicic acid column (0.4 x 30 cm) with a flow rate of 1.8 ml/min of 10% isopropanol in twice distilled Skellysolve B at a pressure of 800 psi (Fig. 2). 3 Developed mann-LaRoche
by Dr. James Hamilton of the HoffCompany, Nutley, N.J.
ASSAY
OF PLASMA
1,25-DIHYDROXYVITAMIN
FIG. 2. High-pressure liquid chromatography of standard 25-OH-D, (60 ng), 1,25-(OH),D, (80 ng), and 1,25-(OH),D, (110 ng) in a Waters 0.4 x 30 cm 10 pm silicic acid column. The column was developed in a solvent system of 10% isopropanol in twice distilled Skellysolve B at 850 psi, giving a flow rate of 1.8 ml/min. Optical density was recorded at 254 nm. The sample was applied in 20 ~1 of the solvent and rinsed into the injector (Waters U-K-6) with a further 20 ~1. The 1,25-(OH),D, eluted at 20-23 ml; this position was checked with a 1,25-(OH),D, standard for each run of samples. The 1,25-(OHj2D3 region was collected, evaporated under a stream of dry nitrogen, and resuspended in a small volume of 95% ethanol for assay and for counting to estimate recovery. Preparation of binding protein. White Leghorn chickens were raised from the day of hatching on a vitamin D-deficient, purified soy protein diet (1) until sacrifice by decapitation at 9-12 weeks. The duodenal loops were removed, emptied, and rinsed with 5 ml of ice-cold buffer (0.05 M potassium phosphate, pH 7.4, 0.05 M potassium chloride). This buffer was used throughout preparation and assay procedures. All subsequent steps were performed either on ice or at 4°C. The mucosa was scraped from the serosa and minced in 5 vol (ml/g) of the buffer. The minced mucosa was centrifuged at 2000g for 10 min and the supernatant was discarded. The pellet was resuspended twice in 5 vol of fresh buffer and recentrifuged and the supernatant was discarded. The final mucosal pellet was resuspended and homogenized in 2 vol of the buffer with two passes of a Potter-Elvehjem Teflon-glass homogenizer maintained at 0°C with an ice bath. The homogenate was centrifuged at 50,000 rpm (226,600g) for 45 min in a number 50 titanium head of a Beckman L2 ultracentrifuge. The tine lipid layer was removed with a Pasteur pipet and the clear cytosol was collected. This cytosol was frozen in 4- to 6-ml portions in an
D
237
acetone-dry ice bath, lyophilized, and stored under N, gas at -20°C until use. The yield of binding protein was 20 mg/g wet wt of duodenal mucosa, and the binding activity was stable for several weeks under these conditions. Cytosol binding protein prepared in this manner was invariably active. Immediately prior to use, the lyophilized cytosol was reconstituted with distilled water and diluted to yield a final protein concentration of l-l.3 mg/ml. Protein concentrations were determined by a Biuret method with a bovine serum albumin standard. Competitive binding assay. Each assay mixture consists of 1 mg of cytosol protein in 1 ml of buffer, 3500 dpm (70 pg) of 126,27-3H11,25-(OH),D, in 50 pl of 95% ethanol, and varying amounts of nonradioactive 1,25-(OH12D3 in an additional 50 ~1 of 95% ethanol. Standard curves were constructed over a useful range of lo-140 pg using crystalline 1,25(OH),D, (see Fig. 3). Nonspecific binding was determined by the inclusion of an assay tube to which was added 5 ng of 1,25-(OH)1D, to effectively displace any [3H]1,25-(OH)2D3 from specific binding sites. Samples for assay were introduced in 50 ~1 of 95% ethanol as was the crystalline 1,25-(OH),D, used to produce the standard curve. To compensate in the assay for the [3Hll,25-(OH),D, added to measure recovery during extraction and chromatography, an appropriate reduction in the [3H11,25-(OH),D, added to each assay tube was made so that all tubes (samples and standards) had the same total radioactivity at the time of final assay. The standard curve incubations were carried out in quadruplicate and the sample incubations were performed in triplicate. The assays were carried out in polyallomer test tubes (‘/la x 2Q-in.) in a shaking water bath at 25°C for 1 h and then were immersed in an iced water bath. One milliliter of icecold 40% polyethylene glycol was added to each tube which was vortexed vigorously to produce a slightly turbid solution. The tubes were centrifuged at 4800g in the HS-4 head of a Sorvall RC-5 refrigerated centrifuge at 4°C for 30 min. The supernatant was discarded and the tip of the test tube, which contained the small pellet, was cut off into a 5-ml scintillation vial. Three milliliters of a p-dioxane-based scintillator solution was added to the vial and shaken vigorously. This scintillator solution, containing 10% naphthalene and 0.5% 2,5-diphenyloxazole in p-dioxane, provided 37.5% efficiency in an ambient temperature Beckman LS-1OOC scintillation counter. Optimum incubation conditions. The effect of pH on 13H11,25-(OH&D, binding was examined by preparing a series of cytosol solutions from the same pool of intestinal mucosa at different pH’s and in different buffers (Fig. 4). Piperazine-NJ-bis(2ethane sulfonic acid) (PIPES), (2 ,N-morpholino) sulfonic acid (MOPS), (2-N-morphopropane linolethane sulfonic acid (MES), and Tris buffers
238
EISMAN
ET AL. b)
a) 60C 1ii-1.25~iOHl2D, sound
I” Pellet
I L
0
I
s
I
I
a
I
50
I
I
I
.*.”
100
l,25-(OH)2D3pgltube
5000
IO
I
I
,
203050
1
100
1.25~(OH),D,pg/tube
FIG. 3. Competitive binding assay standard curve. [26,27-3H11,25-(OH)2D3 bound in the pellet (cpmltube) is plotted against amount of competing unlabeled 1,25-(OH),D, added per tube on (a) a linear scale and (b) a logarithmic scale. Points are mean 2 SEM of triplicate determinations.
PH
FIG. 4. The
effect of pH on [26,27-3H11,25(OH),D, binding to cytosol protein. Specific binding (total binding minus nonspecific binding) was determined using cytosol prepared and incubated in the buffers at the pH indicated. Values are expressed as specific counts per minute bound per milligram of protein and are means of duplicate determinations. (0.025-0.05 M) were used to cover the pH range from 5.8 to 9.2 at 25°C. The buffers contained 0.15 M potassium chloride and 0.001 M ethylene diaminetetraacetic acid and were used throughout the cytosol
preparation after the first wash of the intestinal mucosa. The preparation apart from the changes in pH was identical in each case. Maximum binding and nonspecific binding were determined in duplicate for each cytosol with a 1 h of incubation at 25°C. The specific binding was expressed as specific counts per minute bound per milligram of protein, as shown in Fig. 4. The effect of divalent cations on binding was determined for magnesium and calcium ions. Cytosol was prepared in the usual way in a 0.05 M potassium phosphate (pH 7.4) buffer made 0.001 M with respect to EDTA. Various amounts of calcium and magnesium chloride were added to produce a range of free cation concentrations from 0 to 10 mM. Specific binding was determined as described above. Similarly, the effect of potassium chloride on binding was determined in the presence and absence of the divalent cations mentioned. Time and temperature dependence. The specific binding of [3H11,25-(OH1,D, to the cytosol binding protein was determined at three temperatures from 4 to 37°C over a period of time from 0 to 21 h, as shown in Fig. 5. Specificity. Competitive binding assays were carried out using various amounts of vitamin D,, 25OH-D,, 1,25-(OH),D,, and 1,25-(OH),D, to compete with radioactive 1,25-(OH),D,. These assays, which were designed to elucidate the specificity of the
ASSAY
OF PLASMA
1,25-DIHYDROXYVITAMIN
D
239
.
FIG. 5. Effect of temperature on [26,27-3H]1,25-(OH)2D3 binding to cytosol protein. Specific binding was determined at pH 7.4 after various periods of incubation at the temperatures indicated. Values are expressed as specific counts per minute bound per milligram of protein and are means of duplicate determinations. binding protein, were otherwise identical with the assays described above. Extracts of plasma from vitamin D-deficient chickens were examined at various stages of puritication for interference with the competitive binding assay. The chickens used for this experiment were at least 12 weeks old and showed the physical evidence of severe rachitic bone disease.
RESULTS
Extraction of plasma. The dichloromethane extraction recovered 91.4 + 6.0% (mean f SEM) of radioactive 1,25-(OHjzD3 added to a plasma sample. The first extraction recovered 80% of the added radioactivity and the second and third extractions of the aqueous layer recovered an additional 7 and 5%, respectively. Validity of in vitro recovery estimations. It is possible that the recovery of radioactive 1,25-(OHjzD3 added to the plasma in vitro might differ from the recovery of 1,25(OH),D, produced and released into the plasma in vivo. To examine this possibility radioactive [26,27-3H125-OH-D, (9.3 Ci/ mmol) was administered by wing vein to three vitamin D-deficient chickens. Sixteen hours after dosing, the chickens were bled by heart puncture and the pooled plasma was divided into four equal portions. Two portions were extracted as de-
scribed in the two phase system using dichloromethane and two portions were extracted with the standard one-phase extraction using chloroform:methanol:aqueous (25:50:16). In this method additional chloroform (25 parts) was added to produce two phases and the aqueous phase was “washed” twice with two further additions of chloroform (25 parts). Each extract was chromatographed on Sephadex LH-20 in chloroform:Skellysolve B (75:25) and the 1,25-(OHj2D3 region was rechromatographed on Sephadex LH-20 in Skellysolve B:chloroform:methanol (9:l:l). The radioactivity recovered in the 1,25-(OH),D, region was 382 +- 30 dpm/ml plasma (mean lr SEM) for the dichloromethane extractions and 254 ? 30 dpm/ml plasma for the chloroform:methanol extraction method. Purification of the dichloromethane extract. The chromatographic profile of radioactive 1,25-(OH)*D3 and 25-OH-D, on Sephadex LH-20 in the Skellysolve B:chloroform:methanol (9:l:l) solvent system is shown in Fig. 1. This chromatographic system separated 25-OH-D, from 1,25(OH),D, even on the small 0.7 x 9 cm columns when both samples were applied in 0.5 ml of solvent. However, this chromatographic system was somewhat sensitive to the amount of lipid applied. The
240
EISMAN ET AL.
lipid from 5 ml of plasma extracted by the standard chloroform:methanol method resulted in poor chromatography with a phase separation in the application solvent, hence plasma extracted in this way had to be partially purified in a chloroform:Skellysolve B (7525) solvent system on a Sephadex LH-20 column. When plasma was extracted by the dichloromethane method, the amount of lipid was much less and the lipid extracted from 510 ml of plasma could be applied directly to the Skellysolve B:chloroform:methanol Sephadex LH-20 chromatography column. High-pressure liquid chromatography of vitamin D metabolites on microparticulate silica columns was carried out as has been described recently (23). The solvent system of 10% isopropanol in twice distilled Skellysolve B used in these experiments separates all known metabolites of vitamin D. 1,25-(OHl,D, elutes slightly ahead of 1,25(OH),D,, but the region collected for assay included both 1,25-dihydroxylated vitamins (Fig. 2). Recovery. Overall recovery of 1,25(OHl,D, from plasma after extraction and purification through the two column procedures prior to assay was 68 ? 1.2% (mean -+ SEMI. Incubation conditions. The specific binding of [3H11,25-(OH)2D3 to the cytosol binding protein and precipitation by polyethylene glycol had a pH optimum in the range of 7-8, as shown in Fig. 4. There was no difference in the specific binding about the pH optimum using the various sulfonic acid derivative buffers or potassium phosphate, so the latter was used for all assays thereafter. Calcium and magnesium were not required for maximum specific binding and, in fact, a high concentration of calcium ion (0.01 M) halved specific binding. Maximum specific binding was achieved in the presence of 0.05-0.1 M potassium chloride, so 0.05 M potassium chloride was added to the 0.05 M potassium phosphate buffer (pH 7.4) used in all routine assays. Time and temperature dependence. The specific binding of [3H11,25-(OH)zD3 at 4°C increased up to the longest time examined, 21 h. At 37°C the binding was greatest at
0.5 h and rapidly decreased thereafter. The specific binding at 25°C had a broad maximum at 1 h, which was greater than achieved at either 4 or 37°C (Fig. 5). The slow decrease in specific binding at 25°C and rapid decrease at 37°C are presumably the result of enzymatic degradation in the crude intestinal cytosol binding protein preparation. Determination of saturation of cytosol binding protein. A series of competitive binding assays are performed with each batch of intestinal cytosol to determine the optimum concentration of cytosol protein for use in the assay. Incubations containing 10 pg and 5 ng of unlabeled 1,25(OH),D, were compared to those incubated with no unlabeled metabolite at protein concentrations in the range of 1.0-l .5 mgl incubation. The difference between the maximum binding (no added unlabeled metabolite) and nonspecific binding (5 ng added unlabeled metabolite) was considered to be the specific binding. Statistical considerations indicate that at saturation, those incubations with 10 pg of unlabeled 1,25-(OH),D, added should show a 12.5% decrease in specific binding because of the 12.5% decrease in specific activity of the labeled 1,25-(OH),D, by the unlabeled metabolite. Therefore, the highest protein concentration, which showed this 12.5% decrease in specific binding with 10 pg of unlabeled metabolite, was used for that batch of cytosol preparation. Binding activity decreased about 50% in cytosol stored in solution overnight at 4°C. However, during prolonged storage of the lyophilized cytosol under nitrogen gas and at -2O”C, there was only a gradual decrease in the specific binding of the order of lo-20% over l-2 months. This change was the result of an increase in nonspecific binding and a decrease in maximum specific binding. Nonspecific binding, which includes any contamination of the pellet with supernatant, is reproducible and usually about 10% of total radioactivity. The stability of the cytosol binding protein was determined by measuring the specific binding as a function of storage time. The washing of the mucosa prior to homogenization is essential for maximum stability.
ASSAY
OF PLASMA
1.25DIHYDROXYVITAMIN
Other important factors are cold temperatures during homogenization, lyophilization prior to storage, and the N, atmosphere during storage. Extracts of plasma from vitamin D-deficient chickens before and after Sephadex LH-20 chromatography interfered with the competitive binding assay, causing a variable decrease in the maximum binding and a variable increase in the nonspecific binding. However, high-pressure liquid chromatography on the microparticulate silica column effectively removed these interfering compounds, so that plasma from vitamin D-deficient chickens gave assay values which were identical with zero nonradioactive 1,25-(OH),D, in the standard assay. Specificity of the binding protein. 1,25(OH),D, and 1,25-(OH),D, produced indistinguishable competition curves in the binding assay, as shown in Fig. 6. The binding assay thus would estimate 1,25dihydroxylated vitamin derived from either vitamin D2 or D,.
The competition curve with 25OH-D3 showed a lower affinity by three orders of magnitude (Fig. 71, while vitamin D, did not compete even at the level of 1 Fg/ incubation. Estimation of 1,25-(OH)9, level in plasma samples. Samples and standards were run in the same assay using the same batch of cytosol. The radioactivity bound in the pellet was plotted against the amount of unlabeled standard 1,25(OH),D, added to the incubation (Fig. 3a). This produced a straight line on a semilogarithmic plot (Fig. 3b). The amount of 1,25-(OHjzD3 required to produce the reduction of [3H]1,25-(OH),D, bound to the pellet seen in the sample incubations was read from the graph or determined from a linear regression equation. Each value was corrected for the recovery percentage estimated after its extraction and purification. Normal human values. The value estimated for normal human serum was 29 ? 2 pg/ml (mean 4 SEM, n = 20). These serum samples were derived from normal persons of both sexes with a mean age of 55
\\
0 IoPg
1OOPO Mctabolite
I, 25-(OH),
0
pg/ tube
FIG. 6. Competition of 1,25-(OH),D, and 1,25(OH),D, for cytosol protein binding sites. A competitive binding assay was performed using 126,273H]1,25-(OH),D, and varying amounts of competing unlabeled 1,25-(OH)PD2 (0) and 1,25-(OH)zD, (A). [26,27-3H]1,25-(OH)ZD3 counts per minute bound in the pellet is plotted against amount of unlabeled competing metabolite. Points are mean 2 SEM of triplicate determinations.
241
D
long
1 ng Added,
100 ng
c IN
tube
FIG. 7. Comparison of ability of 25OH-D, and 1,25-(OH)ZD, to compete for cytosol protein binding sites. Competitive binding assays were performed using [26,27-3H]1,25-(OH)2D3 and varying amounts of competing unlabeled 1,25-(OH12D3 (0) and 25-OHD, (A). Specific binding (total binding minus nonspecific binding) at each metabolite concentration is expressed as a percentage of the maximum specific binding and is plotted against amount of metabolite per tube on a logarithmic scale. Points are mean f SEM of triplicate determinations.
242
EISMAN
years (kindly supplied by Dr. B. L. Riggs, Mayo Clinic). Assays of plasma from three persons with chronic renal failure and three persons with bilateral nephrectomy showed no detectable 1,25-(OH),D,. Plasma from one patient with chronic renal failure had a value of 13 pglml but a plasma sample taken 3 months later from the same patient also showed no detectable 1,25WW,. Precision of the assay. Fresh plasma from two people was obtained from the Red Cross Blood Center and frozen in portions. These plasma samples were extracted and purified on eight occasions and each was assayed in triplicate. The concentrations of 1,25-(OH)zD3 determined were 30 * 3 and 28 + 2 pg/ml (mean f SEM). The coefficient of variation of triplicate determinations of the tritium counts bound in the pellet was 2.4 ? 0.5% (mean + SEM, n = 14). DISCUSSION
The present report describes a new sensitive competitive binding assay for 1,25(OH)2D, in biological materials and a new extraction and purification scheme for 1,25-(OH)zD3 and 1,25-(OH),D, in human and animal blood. The extraction and purification scheme is relatively rapid and simple, although it utilizes an expensive and elaborate highpressure liquid chromatography instrument, as previously described (29). The solvent flow rate and pressure used in these experiments could easily be achieved on a much simpler and less expensive high-pressure liquid chromatography system than that used in these experiments. The extraction of plasma from chickens dosed with 13H125-OH-D, showed that the dichloromethane extraction recovered plasma 1,25-(OH),D, as well as, if not better than, the commonly used chloroformmethanol extraction. This supported the reliability of the recovery estimation using in vitro addition of [3H]1,25-(OH),D3 to plasma samples prior to extraction. The lower recovery of 1,25-(OH)zD3 using the chloroform-methanol extraction is unexplained as yet, but may be related to
ET AL.
breakdown of 1,25-(OH)*D3 in the aqueous phase following protein denaturation by methanol. The overall recovery of 68% for 1,25(OH),D, from plasma after extraction and purification combined with the 10 pg sensitivity per assay shows that human plasma levels of 1,25-(OH),D, can be assayed in triplicate with a 5-ml plasma sample. The lower sensitivity of the chromatin binding assay and their lower recovery during extraction and purification presumably accounts for the 20-ml plasma requirement of the assay of Brumbaugh et al. (23-25). The equal sensitivity of the assay to 1,25-(OH),D, and 1,25-(OH),D, has important practical significance in human studies, where the 1,25-dihydroxylated metabolite could and probably would be derived from both vitamin D, and vitamin D, under normal circumstances. The low sensitivity of the assay to 25-OH-D, has little practical significance as any 25-OH-D3 extracted from a sample would be completely eliminated during the purification steps. However, the marked difference in sensitivity is consistent with the minimal biological activity of physiological doses of 25OH-D, on intestinal calcium transport in nephrectomized animals (30). This agreement supports the view that the cytosol binding protein may be a physiological receptor for 1,25-(OH),D, in the intestine as has been suggested by Brumbaugh and Haussler (25). The assay method reported here differs from that of Brumbaugh et al. (23-25) in several important aspects. This assay is nearly twice as sensitive as the assay they have described. Furthermore, the use of polyethylene glycol precipitation to separate bound from free [3H11,25-(OH),D, eliminates the need for the chromatin binding step of their assay. The extensive washing of the mucosa prior to homogenization stabilizes the binding protein so that it can be stored for long periods, thus eliminating the need for fresh preparation of binding protein for each assay. Furthermore, the washing increases the specific binding of [3H11,25-(OH)2D3 as has been shown in studies in this laboratory using
ASSAY
OF PLASMA
1,25-DIHYDROXYVITAMIN
sucrose density gradient centrifugation.6 These differences result in an assay which is more rapid, more sensitive, and more convenient than that previously described. The mean value estimated in this report of 29 pglml is considerably lower than the 40 pg/ml most recently reported by Brumbaugh et al. (24). This difference may reflect the average age of the blood donors, which was rather high in our studies. The zero values found in anephric patients and those in chronic renal failure are consistent with the renal locus of the l-hydroxylase enzyme and confirms the removal of interfering substances during the extraction and purification processes. ACKNOWLEDGMENTS We gratefully acknowledge helpful suggestions from Drs. 0. N. Miller, J. Hamilton, and R. Dixon of the Hoffmann-LaRoche Company.
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