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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References 1.

2.

Avioli, L. V., Lee, S. W., McDonald, J. E., Lund, J., DeLuca, H. F., J . Clin. Invest., 46: 983, 1967. Ponchon, G., Kennan, A. L., DeLuca, H. F., J . Clin. Invest., 48: -

2032,

1969.

228:

3.

Fraser, D. R., Kodicek, E., Nature,

4.

Frolick, C. A., DeLuca, H. F., Arch. Biochem. Biophys.,

764,

1970.

147:

Downloaded by [McMaster University] at 13:54 06 April 2016

143, 1971. 5.

Norman, A. W., Midgett, R. J., Myrtle, J. F., Norwicki, H. G., Biochim. Biophys. Res. Commun., i2: 1082, 1971. Raiz, L. G., Trummel, C. L., Holick, M. F., DeLuca, H. F., Science, 175: 768, 1972. Larkins, R. G., MacAuley, S. J., Rapoport, A., Martin, T. J., Tulloch, B. R., Byfield, P. G. H., Matthews, E. W., MacIntyre, I., Clin. Sci. Mol. Med., 46: 569, 1974.

8.

Norman, A. W., Henry, H., Clin. Orthopaedics, 98: 258, 1974.

9.

Belsey, R. E., DeLuca, H. F., Potts, J. T., Jr., J. Clin. Endocr. Metab., 33: 554, 1971.

10.

Haddad, J . G., Chyu, K. J . , J . Clin. Endocr. Metab.,

2 : 992,

1971. 11.

Preece, M. A., O'Riordan, J. L. H., Lawson, D. E. M., Kodicek, E., Clin. Chim. Acta, 2: 235, 1974.

12.

Haddad, J. R., Jr., Min, C., Walgate, J., Hahn, T., J . Clin. Endocr. Metab., 43: 712, 1976.

13.

O'Riordan, J. L . H., Graham, R. F., Doley, E., in Vitamin D: Biochemical, Chemical and Clinical Aspects Related to Calcium Metabolism, edited by A. W. Norman, K. Schaefer, J . W. Coburn, et al.; p . 519, Walter de Gruyter, Berlin, 1977.

14.

Root, A . W., Harrison, N. E., J . Pediatr., E : 1, 1976.

15.

MacIntyre, I., Colston, K. W., Evans, I. M. A., Lopez, E., MacAuley, S . J., Piegnoux-Deville, J., Spano, E., Vzelke, M., Clin. Endocr., 2: Suppl: 85s, 1976.

16.

Haussler, M. R., McCain, T. A . , New Eng. J. Med.,

17.

Avioli, L. V., Kidney Int.,

18.

DeLuca, H. F., Arch,

297:

13: Suppl. 8: S-36, 1978. Intern. Med., 138: 836, 1978.

1041, 1977.

309

Downloaded by [McMaster University] at 13:54 06 April 2016

VITAMIN D RECEPTOR AND ASSAY 19.

H i l l , L. F . , M a w e r , E . B . , Problems R e l a t e d t o Uremic Norman, K. S c h a e f e r , H. G . p. 755, Walter d e G r u y t e r ,

T a y l o r , C . M . , in V i t a m i n D and Bone D i s e a s e , e d i t e d by A . W . C r i g o l e i t , D. Von H e r a t h , E. R i t z , B e r l i n , 1975.

20.

S t e r n , P. H . , Hamstra, A. J . , DeLuca, H . F . , B e l l , N. H . , C l i n . Endocr. Metab., 5: 891, 1978.

21.

Brumbaugh, P. F . , H a u s s l e r , D. H . , S c i e n c e , 183: 1089, 1974.

22.

Eisman, J. A . , H a m s t r a , A. J . , K r e a m , B. E . , DeLuca, H. F . , Arch. Biochem. B i o p h y s . , 176: 235, 1976.

23.

Lambert, P. W., preparation.

24.

C o l s t o n , K. W . , Evans, I . M . A . , G a l a n t e , L , , M a c I n t y r e , I . , Moss, D . W . , Biochem. J . , 817, 1973.

25.

Norman, A . W . , M i d g e t t , R. J., M y r t l e , J . F . , N o r w i c k i , H . G . , Biochim. Biophys. Res. Commun., 36: 251, 1969.

26.

B o y l e , I . T . , M i r a v e t , L . , G r a y , R. W . , H. F . , E n d o c r i n o l o g y , 90: 605, 1972.

27.

B i c k l e , D. D . , 1974.

28.

Lambert, P. W., S y v e r s o n , B. J . , Arnaud, C. D . , J. S t e r o i d Biochem. 8: 8 2 9 , 1977.

29.

D i e z f a l u s y , E.,

30.

H a u s s l e r , M. R.,

31.

J o n e s , G.,

32.

H a u s s l e r , B., B a y l i n k , D. J . , Hughes, M. R., Brumbaugh, P. F . , Wergedal, J. E . , Shen, F. H . , N i e l s e n , R , L., C o u n t s , S . J., B u r s a k , K. M., McCain, T. A , , C l i n , E n d o c r i n o l . , 2: 151s, 1976.

33.

Gray, R. W . , W i l z , D . R . , C a l d a , A . E . , Lemann, J . , J r . , J. C l i n . Endocr. M e t a b . , 45: 299, 1977.

34.

T o f t , D . , Moudgil, V . , Lohmar, P . , M i l l e r , J . , Ann. N . Y . Acad. S c i . , 286: 2 9 , 1977.

35.

Yalow, R . S . , B e r s o n , S . A . , P r o c . of Symposium on "In V i t r o " P r o c e d u r e s w i t h R a d i o i s o t o p e s i n C l i n i c a l M e d i c i n e and R e s e a r c h , V i e n n a , I A E A , SM-124/106, 1970, p . 455.

36.

Ekins, R. P . ,

Lindmark, E . A.,

Bressler, R.,

J

H a u s s l e r , M. R . ,

T o f t , D. Q . , M a n u s c r i p t i n

134:

Rasmussen, H . ,

H o l i c k , M . F . , DeLuca,

Biochim. Biophys. A c t a ,

A c t a Endocrinol. (Suppl)

Steroids,

20:

22:

S p e l s b e r g , T. C . ,

147: 1970.

639, 1972.

DeLuca, H . F . , J. L i p i d R e s . ,

B r . Med. J . ,

362: 439,

3, 1974.

16:448,

1975.

LAMBERT ET AL.

Downloaded by [McMaster University] at 13:54 06 April 2016

310

51: 660,

37.

Scatchard, G., Ann. N. Y. Acad. Sci.,

38.

Hughes, M. R., Baylink, D. J., Jones, P. G., Haussler, M. R., J. Clin. Invest., 2: 61, 1976.

1949.

An improved method for the measurement of 1,25-(OH)2D3 in human plasma.

Endocrine Research Communications ISSN: 0093-6391 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ierc19 An Improved Method for th...
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