TY - JOUR
T1 - An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma
AU - Dokoh, Shigeharu
AU - Pike, J. Wesley
AU - Chandler, John S.
AU - Mancini, Jennifer M.
AU - Haussler, Mark R.
N1 - Funding Information:
The authors would like to thank Dr. Richard Gray for suggesting the use of Clin Elut columns as a selective method for 1.25-(OH),D extraction. We also thank RoseAnn Jirmejian, Christa Sitz, and John Hostettcr for assistance in preparing this manuscript. The work is supported by NIH Grant AM-15781.
PY - 1981/9/1
Y1 - 1981/9/1
N2 - We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[3H]vitamin D3 (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1-20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).
AB - We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[3H]vitamin D3 (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1-20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).
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U2 - 10.1016/0003-2697(81)90346-8
DO - 10.1016/0003-2697(81)90346-8
M3 - Article
C2 - 6272612
AN - SCOPUS:0019769401
SN - 0003-2697
VL - 116
SP - 211
EP - 222
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -