Endocrine Research Communications
ISSN: 0093-6391 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ierc19
An Improved Method for the Measurement of 1,25-(OH)2D3 in Human Plasma Phillip W. Lambert, David O. Toft, Stephen F. Hodgson, Elizabeth A. Lindmark, Bonnie J. Witrak & Bernard A. Roos To cite this article: Phillip W. Lambert, David O. Toft, Stephen F. Hodgson, Elizabeth A. Lindmark, Bonnie J. Witrak & Bernard A. Roos (1978) An Improved Method for the Measurement of 1,25-(OH)2D3 in Human Plasma, Endocrine Research Communications, 5:4, 293-310, DOI: 10.1080/07435807809061094 To link to this article: http://dx.doi.org/10.1080/07435807809061094
Published online: 07 Aug 2009.
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ENDOCRINE RESEARCH COMMUNICATIONS, 5 ( 4 ) , 293-310 (1978)
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AN IMPROVED METHOD FOR THE MEASUREMENT OF 1,25-(0H)2D3 IN HUMAN PLASMA Phillip W. Lambert,*t David 0. Toft,* Stephen F. Hodgson,* Elizabeth A. Lindmark,* Bonnie J. Witrak,* and Bernard A. Roost *Department of Molecular Medicine and Endocrine Research Unit, Mayo Clinic, Rochester, Minn. 55901 and !Endocrinology and Mineral Metabolism, Veterans Administration Hospital, Case Western Reserve University, Cleveland, Ohio 44106
Abstract Here we report a highly sensitive and convenient ligand binding assay for the determination of 1,25(OH) D in small volumes of human plasma. This . of3vitamin D and its metabolices using method involves: (1) extraction 2 phases by centrifugation; (2) methanol-methylene chloride with separation gel chromatography and high pressure liquid chromatography for the quantitative isolation of 1,25-(OH)2D ; and (3) a sensitive ligand binding assay for 1,25-(OH) D employing cytoso? receptor from the intestinal mucosa of rachitic chicks. 2U?ing modified rachitogenic chick diets allows early ( < 4 wks) harvesting of active receptor for 1,25-(OH) D in high yield. The method 2 3 includes a rapid and effective procedure for stable and long-term storage of the active cytosol receptor. A convenient dextran-charcoal means is used for the separation of receptor bound from free 1,25-(OH) D resulting in the 2 3 achievement of a lower (( 5%) background (i.e., nonspecific binding) than reported for other 1,25-(OH)2D3 assays. Analysis of this receptor shows it to be a saturable, single classlpf binding sites with a dissociation constant The final recovery of 1,25-(OHI2D follow(Kd) of approximately 3.7 x 10 ing extraction and chromatography is 80 5 3% and triplicate determinazions can be made on a 3 ml plasma sample. The ligand binding assay routinely detects 5 5pg of 1,25-(OH) D per assay tube and the inter- and intraassay 2 3 variation, based on repeated determinations of 1,25-(OH)2D3 id pooled normal human plasma, is < 5%. Preliminary studies indicate that our methodology will permit measurement of plasma 1,25-(OH) D levels in all normal subjects and in levels may be below or above normal pathophysiologic states where 1,25-(0Hf in human plasma obtained from both values. 1,25-(0H)2D3 values (pg/ml 5 normals and patients with various untreated calcium homeostatic disorders were: 1.2; primary normals = 33.5 5 1.8; end-stage chronic renal failure = 5.1 hypoparathyroidism = 18.3 +_ 2.8; primary hyperparathyroidism = 61.4 2 7.1; and 8.4. hyperthyroidism with associated hypercalcemia = 42.1
02
.
2
Sh?
293 Copyright 0 I Y 7 Y by Marcel Dekker, Inc. All Rights Reserved. Neither this work iior any part may be reproduced
or transmitted in any form or by any means. electronic or mechanical, including photocopying, microfilming. and recording. or by any information storage and retrieval system, without permission in writing from the publisher.
LAMBERT ET A L .
294
Introduction Fecent basic advances in vitamin D metabolism (1-8), coupled with the development of ligand binding assays for some of the physiologically important vitamin D metabolites (9-13), have aided our efforts in understanding the pathophysiology involved in various
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mineral metabolic disorders (14-18).
These efforts, however, have
until recently been hampered by the lack of sensitive means for the specific measurement of the low circulating levels (pg) of 1,25-
(OH)2D3, the biologically active form of vitamin D (3-5).
In addition
to two bioassay methods (19-20), sensitive receptor assays have recently been reported that employ either intestinal mucosal chromatin-cytosol preparations (21) or cytosol receptor (22) of rachitic chicks. We have developed a ligand binding assay for 1,25-(OH) D in 2 3 small volumes of human plasma employing a cytosol receptor from rachitic chick intestinal mucosa. from that of Brumbaugh
The assay method reported here differs
&.
(21) and Eisman
g.
(22) in several
important aspects, These differences, detailed in Methods and Discussion, result in an assay for plasma 1,25-(OH)2D3 which we believe is more sensitive, rapid, and convenient than previously reported.
Materials Solvents.
The solvents used were methanol, methylene chloride,
chloroform, n-hexane, and isopropanol (Burdick and Jackson Laboratories Inc., Muskegon, MI.).
All solvents were of spectroanalyzed
grade and were filtered through 1
pore size fluoropore filters
(Yillipore Corp., Bedford, MA) and then degassed prior to use.
295
VITAMIN D RECEPTOR AND ASSAY Biological samples. Human plasma was obtained from 59 healthy volunteers (20 to 50 years old) and patients with calcium homeostatic disorders. All biological samples not immediately used were stored at -76OC. Sterols.
Reference compounds obtained commercially in crys-
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talline form were:
vitamin D3 (Grand Island Biological C o . , Grand
Island, N.Y.); 250HD3, 24,25-(OH)*D3, and 1,25-(OH) 2D3 (a generous gift from Dr. M. Uskokovic of Hoffman - LaRoche Inc., Nutley, N.J.). The following radioactive sterols were obtained commercially: [ 1,2-
D3 (12.3 Ci/mmol; Amersham-Searle Corp,, Arlington 3 Heights, Ill.) ; [23,24- HI-250HD3 (110 Ci/mmol., ; Amersham-Searle 3H]-vitamin
3
Corp., Arlington Heights, Ill,); [23,24- H]-24,25-(OH)2D3
and
3 [23,24- HI- 1,25-(OH)
D (110 Ci/mmol.) were biosynthesized in vitro 2 3 3 with high yield from [23,24- HI-250HD3 utilizing modifications (23)
of previously reported techniques (24-27).
All sterols were quanti-
tated by micro-U.V. spectrophotometry after purification by high pressure liquid chromatography (HPLC).
Until use, all sterols were
stored under N2 in deoxygenated absolute ethanol in sealed ampules at -76OC.
Methods Extraction of plasma, Prior to extraction, all human plasma samples were stored at -76OC, All glassware was siliconized to reduce the problem of inert binding of sterols (28).
Human plasma
(3-4 m l ) was added to a 30 ml polypropylene centrifuge tube at 0-5OC. 3 5 pg (2700 dpm) of [23,24- H]-1,25-(OH)2D3
(110 Cilmmol) in 20 X of
95% ethanol served as an interns1 standard for monitoring recovery
296
LAMBERT ET AL.
through extraction and chromatography. Three volumes of cold methanolmethylene chloride (2~1,v/v) was added, flushed with N2, and vortexed for 1 min.
One volume of cold methylene chloride was then added, The extract was
flushed with N2, and vortexed an additional min. centrifuged at 23,300 g for 10 min
at 0-5OC in a Sorvall HB-4 swing-
ing bucket rotor. The lower organic phase was siphoned off via
a
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teflon tube and the aqueous phase was reextracted with 2 volumes of cold methybene chloride. The combined organic phases were N 2 evaporatedand stored in 95% ethanol under N2 at -76OC until chromatography. Chromatography. To employ a more rapid and less tedious gel chromatography method, a modification of a previously reported technique was used (28).
The dried plasma extract was resolubilized in
100 X of n-hexane:chloroform:methanol
(9:1:1,
v/v) (22,28) and applied
with 2 additional 100 A washes to a Sephadex LH-20 column (0.9 x 1 5 cm) equilibrated with the same solvent system. a flow rate of 1.3 ml/min.
Elution was carried out at
Although there was good resolution of
vitamin D and 250HD with this system, there was some overlap of 3 3 the 24,25(OH),D3
and 1,25(0Hl2D3 peaks (Figure 1).
Therefore, these
latter two metabolite peaks, eluting between 20 and 50 mls, were pooled, N2 evaporated, and stored in 95% ethanol under N2 at -76OC until subjected to further purification by high pressure liquid chromatography (HPLC). The combined 24,25(0H)2D3 and 1,25(0H),D3
peaks from LH-20
chromatography were resolubilized in 30 X of HPLC grade chloroform and applied with a 20 1 wash to a HPLC system slightly modified from previously published methods (22,28). two
The HPLC system consisted of
0.4 x 30 an u-porosil columns (Waters Associates, Inc., Milford,
297
VITAMIN D RECEPTOR AND ASSAY
600 ,--
rr)
c l
rr)
a c
500-
I
c3
0
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(u
(u h
rr)
I
0
5
10
15
25
20
30 35 40 45
50
Elution Volume (ml) FIGURE 1 Sephadex LH-20 g e l chromatography p t j o f i l e of t h e o r g a n i c s o l v e n t e x t r a c t of normal human plasma c o n t a i n i n g [ H ] l a b e l e d v i t a m i n D3 and m e t a b o l i t e m a r k e r s . Chromatography w a s c a r r i e d o u t on a 0 . 9 x 15 cm c lumn w i t h a s o l v e n t s y s t e m of n-hexane-CHC13-CH OH ( 9 : 1 : 1 , v / v ) . The [ Hi-dpm a r e p l o t t e d a s a f u n c t i o n of e l u t i o n vo?umes f o r v i t a m i n D3, 250HD3, 24,25(OH)2D3, and 1,25-(OH)2D3.
s
Ma.) and a C0:PELL PAC guard column (Whatman I n c . , C l i f t o n , N.J.) i n
tandem, and an i s o c r a t i c s o l v e n t system of n-hexane:
(88:12, v / v ) a t 1 . 5 ml/min and 900 p s i .
isopropanol
As shown i n F i g u r e 2 , t h e
1,25(OH)*D3 peak e l u t e d from t h e HPLC a t a r e t e n t i o n t i m e of 2 1 t o 27 min
and w a s c l e a r l y s e p a r a t e d from o t h e r v i t a m i n D3 m e t a b o l i t e s
t h a t eluted earlier. Cytosol receptor preparation. c y t o s o l from r a c h i t i c c h i c k s ,
We used i n t e s t i n a l mucosal
he-day-old
f a s t i n g white leghorn
LAMBERT ET AL.
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298
B
2000
T-T 1500
E Q V 7
1000
, I
-
Y
500
I
I
u
0
5
10
15
20
25
30
Retention Time ( m i d FIGURE 2 Elution profile of 1,25-(OH) D and vitamin D metabolites on normal 2 3 phase HPLC. Panel A represents the elution profile of the U.V. absorbing material after 150 ng of each of the unlabeled standard 3 vitamin D metabolit s were applied to the HPLC. The elution of [ HI5 24,25-(0H) D and [ H]-1,25-(OH),D3 that had been added to and then 3 extracted plasma, chromatographed on LH-20, and applied to HPLC is shown in panel B. A normal phase isocratic system of n-hexaneisopropanol (88:12, v/v) with 2 p-Porosil columns in tandem with a C0:PELL PAC guard column (Whatman Inc., Clifton, N . J . ) was used at a flow rate of 1.5 ml/min and 900 psi.
gram
VITAMIN D RECEPTOR AND ASSAY
299
cockerels, housed in cages maintained at 23-30°C without W light, were provided ad libitum a 0.6% calcium, 0.4% phosphate, vitamin D-deficient, purified soy-protein diet (custom diet, Teklad Mills, Madison, Wisc.) and deionized water until sacrifice by decapitation at 4 to 6 wks.
The duodenal loops were rapidly removed and irri-
gated with 20 cc of cold normal saline, and the mucosa was harvested
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by scraping at 0-5OC.
The mucosa was gently washed 4 times with 6
to 8 volumes (mllgm) of cold normal saline, centrifuging each time at 1000 g for 5 min
at 0-5OC.
The mucosal pellet was resuspended in
3 volumes of buffer (0.05M Tris-HC1, 0.15M KC1, 12 mM thioglycerol, pH 7.4 at 23OC) and homogenized in an ice bath with 4 periods (5 sec each with 1CLsec
rests between periods) of a polytron homogenizer
(Brinkman, Lugeru, Switzerland).
The homogenate was ultracentrifuged
in 2.0 x 0.5 inch Beckman polyallomer tubes at 233,000 g for 1 hr at After removal of the lipid layer, the cytosol supernatant
0-5OC.
was rapidly frozen with acetone-dry ice and stored under N2 at -76OC. 1,25-(OH),D, L
receptor assay.
Immediately before the assay
>
the cytosol receptor preparation was thawed and diluted with buffer
(0.05 M Tris-HC1, 0.15M KC1, 12mM Thioglycerol, pH 7.4 at 23OC) to yield a final protein concentration of 0.75 mg/ml.
Each assay mixture
3 consisted of 0.75 mg of cytosol protein; 2700 dpm of [23,24- HI-1, 25(OH) D in 20 X of ethanol; and nonradioactive 1,25-(OH) D standard 2 3 2 3 or the HPLC peak of the 1,25(OH)2D plasma sample extract in 20 X of ethanol.
Standard curves were carried out in triplicate over a range
of 5-200 pg using synthetic 1,25-(OH)2D3,
Human plasma samples were
also performed in triplicate. Nonspecific binding was assessed by the inclusion of an excess of 1,25-(OH)2D3 curve.
3
[23,24- H]-1,25-(OH)2D3
(5 pg) in the standard
was initially added to the plasma
LAMBERT ET AL.
300
sample to monitor recovery, To compensate for this additional label, 3 an appropriate reduction in the [23,24- HI-1 ,25(OHI2D3 added to each assay tube was made to equalize the total radioactivity at the time of the assay. The assays were performed in siliconized 7 5 x 12 mm glass tubes with incubation at 25OC for 1 hr at 120 oscillations/min. The tubes
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were then placed in an ice bath for 10-15 min.
Separation of bound
from free 1,25-(OH) D was done by a modification of standard dextran2 3 charcoal techniques (see dextran-charcoal-human serum preparation below).
250 X of the dextran-charcoal-human plasma mixture was added
to each assay tube, The tubes were vortexed, allowed to stand in an ice bath 30 min with mixing at 1C-min trifuged at 1000 g for 10 min 1,25-(0H)2D3)
at 0-5OC.
intervals, and then were cen3 The supernatant (bound [ HA-
was decanted into a 20 ml scintillation vial, and 10 ml
of scintillator solution (Aquasol-2, New England Nuclear, Boston, Ma.) was added for determination of the
3H
dpm.
Dextran-charcoal-human plasma preparation, This modification of standard dextran-charcoal preparations ( 2 9 ) employed the addition of human plasma to deal with the problem of sterol binding by char-
coal, A 0.5% (w/v) charcoal (Norrit Carbon Decolorizing Neutral, Fischer Scientific
Co.)
and 0.05% dextran (Dextran T-70, Pharmacia
Fine Chemicals) mixture was made up in double distilled water, mixed, and allowed to stand for 5 min
with occasional stirring. The mixture
was centrifuged at 250 g for 10 min
at 0-5OC, and the precipitate
was resuspended in buffer (0.05M Tris-HC1, 0.15M KC1, 12mM thioglycerol, pH 7 . 4 at 23OC) with the addition of normal human plasma to yield a 1.0% charcoal, 0.1% dextran, and 5 % plasma concentration. The mixture was magnetically stirred for 12-16 hrs at 0-5OC and centrifuged at
VITAMIN D RECEPTOR AND 250 g for 10 min
301
ASSAY
at 0-5OC.
The precipitate was resuspended in Tris
buffer to yield a final 5% charcoal and 0.5% dextran concentration.
Results-Discussion Extraction of plasma.
The use of a methanol-methylene chloride
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extraction resulted in a greater recovery of 1,25(OH)2D3 from the plasma than obtained with previously published methods that emplov either methanol-chloroform (30) or methylene chloride (22).
The mean
+ SE
2 3
-
% recovery following extraction of radioactive 1,25-(OH) D
added to the plasma as an internal marker was 98
0.8 % (n = 150).
Chromatography. The Sephadex LH-20 gel chromatography of radioactive vitamin D3, 25-OH-D3, 24,25(OH)2D3, and 1,25-(0H)*D3
from
a methanol-methylene-chloride extract of 3 ml of human plasma is shown in Figure 1.
There was an effective separation of the pooled
24,25-(0H) 2 D3 and 1,25-(OH),D3
peaks from the other vitamin D3
metabolites.
We and others have demonstrated that using additional chromatographic steps beyond the initial preparative gel chromatography, particularly HPLC, eliminates problems of interference in ligand binding assays for 1,25-(OH)2D3 (22,28,31).
The HPLC isocratic normal
phase system detailed in the methodology and shown in Figure 2 is one of several possible approaches (22,32,33).
In our HPLC system,
labeled 1,25-(0H)2D3 previously extracted from plasma and chromatographed on LH-20 coeluted with unlabeled 1,25(OH) D standard applied 2 3 directly to the HPLC system, Using HPLC provided a clear separation of 1,25(OH)2D3
from 24,25-(0H)2D3
recovery of radioactive 1,25-(OH)
and the other metabolites.
The final
D added to the plasma following ex-
2 3
traction, LH-20 chromatography, and HPLC was 80 5 3% (n
=
59).
LAMBERT ET AL.
302
Cytosol receptor preparation. The vitamin D-deficient diet described in Methods is highly rachitogenic; this allowed harvesting o f cytosol receptor from intestinal mucosa after 4-6 wks, in contrast
to
the
9-12
wks
needed
with
a
standard rachitogenic
diet
containing normal levels of calcium and phosphate (1.18% Ca and 0.8%
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PO4, A . O . A . C .
rachitogenic test diet, Teklad Mills).
A l s o , males
yielded equivalent or even greater amounts of cytosol receptor than did the more expensive female chicks, The extensive washing of the intestinal mucosa with normal saline prior to homogenization was essential for minimizing serum protein contamination (22) and maintaining stability of the receptor preparation. The efficiency of mucosal cell disruption, as assessed by phase contrast microscopy and yield of receptor judged by total specific binding activity recovered, was
greater with the polytron
homogenizer than with the Potter-Elvehjeh teflon-glass homogenizer
(90% vs 75% cell disruption, respectively). A s previously noted by other investigators (221,
the addition
of KC1 to the homogenate and assay buffer was critical for the specific binding
of
C3H]-1 ,25-(OH)2D3
to the cytosol receptor. Maximum
specific binding was achieved with 0.15 M KC1. Rapid freezing of the cytosol with acetone-dry ice and storing under N2 at -76OC conveniently and effectively insured stable storage of the receptor preparation, Maximum specific binding was greater
when the receptor preparation had been stored frozen rather than lyophilized. A s demonstrated with other steroid cytosol receptors ( 3 4 1 , the addition of a reducing agent (thioglycerol) to the buffer system enhanced the storage stability of the receptor preparations.
The
VITAMIN D RECEPTOR AND ASSAY
303
receptor preparations showed no decrease in maxi.mum specific binding after 4 mos
of storage in a frozen state at -76OC.
1,25-(OH),D, L
receptor assay. A series of assays performed with L
varying amounts of labeled 1,25(OH) D and cytosol protein concentra2 3 tions determined the level of each factor that was necessary for an
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optimal specific binding range of 50-60% of the total ligand (35,36).
0.75 mg of cytosol protein and 2700 dpm (
5 pg) of [ 3H]-1,25-
(OH)2D3/assay tube consistently resulted in maximum specific binding. Increasing the amount of protein and/or dpm per assay tube resulted in a decrease in the sensitivity of the assay. The dextran-charcoal technique described in the methods for the
3 separation of receptor bound from free [ H]-1,25-(OH)2D3
was l e s s
tedious and more effective than separation by either of the previously described methods employing a chromatin binding technique (21) or use of polyethylene glycol (22).
With the dextran-charcoal method of sep-
aration, a low and reproducible background (i.e.,nonspecific binding) of
5 5%
was achievable.
Specificity of the cytosol receptor, Specificity studies showed a high affinity of the receptor for 1,25(OH)2D3.
Addition
of 5 ng of vitamin D3, 250HD3, 24,25-(OH)2D3, or laOHD3 did not reduce 1,25-(OH),D3
binding; 25-OH-D and 24,25-(0H)2D3 3
showed a
1000-fold lower magnitude of affinity for the cytosol receptor than
1,25-(0H)2D3
(Figure 3 ) .
Since 1,25-(OH)2D2
was not available it
could not be shown if the method accurately determined both 1,25-
(OH)2D2 and 1,25-(0H)2D3
(i.e., 1,25-(OH)2D).
Analysis of binding.
The relationship of binding to hormone
concentration was examined over a 12.7-fold concentration range (0.039 to 0.495 pmoles/tube) of total 1,25-(OH)2D3.
The binding
LAMBERT ET AL.
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304
.-u
'c V W
' lOpg
50 lOOpg
'
I
I I 1111'
I
I
I I I1ld
In9 long lOOng Metabolite Added / Assay Tube
b.4
FIGURE 3
Comparison of the ability of 1,25-(0H)*D3 and other vitamin D metabolites or analogues to compete for the cytosol rece or binding sites. Ligand binding assays were performed using f5H]-l ,25-(O€02D3 and varying amounts of competing 1,25-(OH) D3 ( 0 1, vitamin D ( A ), 250HD3( X ) , 24,25-(OH)2D3 ( 0 ) , and l ~ 0 H 6( ~ ) Specific ginding (i-e,,total binding minus nonspecific binding) expressed as a % of maximum specific binding is plotted against the amount of the metabolitefassay tube on a logarithmic scale.
.
sites were saturable (Figure 4A) and a Scatchard plot (37) of these data (Figure 4 B ) yielded a straight line indicating the presence of a single class of binding sites with a dissociation constant of approximately 3 . 7 x 10-l'~. Receptor assay precision.
pg 1,25-(OH)2D3/assay
The assay routinely detected 5 5
tube, a value of equal or greater sensitivity
than previously reported with ligand binding assays for 1,25-(OH)*D
305
VITAMIN D RECEPTOR AhD ASSAY
. 0” (u c-
I -0 w
.05 .04
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.03
.02
U
0
m
.o 1
m 0.1
0
0.2 0.3 0.4
0.5
0 .02 .04 .06 .08 .lo
Total 1,25-(OH), D,
Bound 1,25-(OH), D,,
pmoles/tube
pmoles/tube FIGURE 4
K i n e t i c d a t a f o r t h e 1,25-(OH) D c y t o s o l r e c e p t o r from r a c h i t i c c h i c k 3 i n t e s t i n a l mucosa. A. S a t u t a z i o n of t h e c y t o s o l r e c e p t o r s y s t e m w i t h 1,25-(OH) D I n c r e a s i n g amounts of 1,25-(OH) D s t a n d a r d were 2 3’ 3 i n c u b a t e d w i t h t h e c y t o s o l r e c e p t o r s y s t e m a s j e s c r i b e d i n t h e Methods and t h e amount of bound 1,25-(OH) D ( l a b e l e d arid u n l a b e l e d ) w a s d e t e r 2.3 mined. B. The e q u i l i b r i u m d i s s o c i a t i o n c o n s t a n t (Kd) w a s c a l c u l a t e d a c c o r d i n g t o S c a t c h a r d ’ s f o r m u l a : bound / unbound = 1/Kd (n- bound ) .
(21,22).
A r e p r e s e n t a t i v e s t a n d a r d c u r v e f o r t h e 1,25-(0HI2D3 c y t o -
sol r e c e p t o r a s s a y i s shown i n F i g u r e 5 .
The i n t e r - and i n t r a a s s a y
v a r i a t i o n of r e p e a t e d 1,25-(0H)2D3 measurements i n p o o l e d normal human blood bank plasma was < 5 % . Human v a l u e s of 1,25-(OH),D
,
c
mated f o r normal human plasma w a s 34.5
The 1,25-(0H),D3
2
v a l u e esti-
2.5 pg/ml (mean
5
SEM) and w a s
i n agreement w i t h v a l u e s p r e v i o u s l y r e p o r t e d by l i g a n d b i n d i n g a s s a y (22,28,38).
These plasma samples were o b t a i n e d t h r o u g h o u t t h e y e a r
306
LAMBERT ET A L .
E a 1800
1600 D
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1200
1000
800 600 400
200 0
0
40
1,25-(OH),
80
120 160 200
D, p g / assay tube FIGURE 5
1,25-(OH) D receptor assay standard curve. Specific binding (i.e., 2.3 total binding minus nonspecific binding) is expressed as a % maximum specific binding and is plotted against the amount of unlabeled 1125(OH) D added per assay tube on a logarithmic scale. Points repre2 sent tie mean SEM derived from 105 separate receptor assay standard curves.
from 59 normal individuals of both sexes with a mean age of 32 years. In addition, the following values (pg/ml+ SEM) were obtained from plasma in untreated patients with various calcium homeostatic disorders:
(1)
end-stage chronic renal failure = 5.1 f. 1.2 (n
=
10);
307
VITAMIN D RECEPTOR AND ASSAY (2)
primary hypoparathyroidism = 18.3
hyperparathyroidism = 6 1 . 4 5 7 . 1 with associated hypercalcemia
=
_+.
1 6
( n = 19) 42.1
+_
8.4
(n = 4 ) ;
and ( 4 )
(3)
primary
hyperthyroidism
(n = 4 ) .
In summary, the new techniques detailed in Methodology include: (1)
rapid and effective extraction techniques employing methanol-
methylene chloride; ( 2 )
modified rachitogenic diets allowing rapid
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and more efficient harvesting of specific, high affinity cytosol receptor for 1,25-(OH)
D from chick intestinal mucosa; (3) stable
2 3
and rapid storage of the cytosol receptor in a frozen state; and ( 4 ) rapid'and efficient separation of receptor bound from free 1,
25-(OH)
D by the use of dextran-charcoal-human plasma with notably
2 3
low (< 5%) nonspecific binding.
These techniques in conjunction
with minor modifications of previously reported Sephadex LH-20 and HPLC chromatography systems allow specific and sensitive determinations of 1,25-(OH)
D in small volumes ( 3 ml) of human plasma. 2 3
This
methodology provides an effective means o f studying both physiologic and pathophysiologic fluctuations in circulating levels of 1,25-(OH) 2D 3 '
Acknowledgments We gratefully acknowledge the secretarial help of M s . Geraldine Montgomery and Ms. Julya M. Taylor, the aid of Drs. D. C . Purnell, D. L. Hoffman, D. A. Scholz, and D. M. Wilson for assistance in patient recruitment, and the technical advice of Dr. T. C. Spelsberg. The authors also wish to thank Dr. C. D. Arnaud for his assistance and support in the initial stages of this work.
This research was supported in part by
the Veterans Administration and by the National Institutes of Health.
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