TY - JOUR
T1 - Bioassay of 1,25-dihydroxyvitamin D in human plasma purified by partition, alkaline extraction, and high-pressure chromatography
AU - Stern, Paula H.
AU - Phillips, Thomas E.
AU - Mavreas, Thalia
N1 - Funding Information:
This work was supported by NIH Research Grants AM11262 and NIH Research Career Development Award SK04 AM70210 (to P.H.S.). A number of individuals contributed materials which made this work possible. We thank Dr. Norman Bell of the Indiana University Medical School, and Drs. Frederic Coe and Murray Favus of Michael Reese Hospital, Chicago, for supplying normal and pathologic human sera. We thank Dr. John Babcock of the Upjohn Company for 25OH-vitamin Da and Dr. Milan Uskokovic of Hoffman LaRoche for l-25-(OH)2D,. We especially thank Dr. Hector DeLuca of the University of Wisconsin for generously providing the 1,25-(08),[23,24-‘HID3 used to monitor recoveries in some of the studies as well as for 1,25-(OH),D,, 1,25-(OH),D,, 24,25-(OH),D,, 25.26(OH),D3, and 1,24,25-(OH),D,. We are grateful to Dr. Samuel Lucas, currently at Battelle
PY - 1980/2
Y1 - 1980/2
N2 - A method is described which permits the assay of 1,25-dihydroxyvitamin D in 1 ml of normal human plasma. Samples are extracted with benzene or isopropyl ether. The organic phase is then back-extracted with 0.1 m Na2HPO4, pH 10.5. The final purification step involves high-pressure liquid chromatography on μPorosil with a solvent system of 13% isopropanol in hexane. Recovery through the entire purification procedure averages 76.8%, the purified extract is assayed for 1,25-dihydroxyvitamin D by determining its effect on the resorption of fetal rat bones in vitro. 1,25-Dihydroxyvitamin Da standards in the 4- to 25 pg/ml range gave responses which fitted a logarithmic dose-response curve with an average r2 of 0.986. Repeat assays with normal human plasma gave a coefficient of variation ( SD x) between experiments of 16.1 ± 0.3%. Normal human serum contains 33.4 ± 6.4 pg/ml (x ± SD) 1,25-dihydroxyvitamin D by this assay. Values averaging less than normal are found in plasmas of patients with renal failure and in patients with pseudohypoparathyroidism. Average values greater than normal were found in patients with hyperparathyroidism. 1,25-Dihydroxyvitamin D was not detectable in plasma of anephric patients.
AB - A method is described which permits the assay of 1,25-dihydroxyvitamin D in 1 ml of normal human plasma. Samples are extracted with benzene or isopropyl ether. The organic phase is then back-extracted with 0.1 m Na2HPO4, pH 10.5. The final purification step involves high-pressure liquid chromatography on μPorosil with a solvent system of 13% isopropanol in hexane. Recovery through the entire purification procedure averages 76.8%, the purified extract is assayed for 1,25-dihydroxyvitamin D by determining its effect on the resorption of fetal rat bones in vitro. 1,25-Dihydroxyvitamin Da standards in the 4- to 25 pg/ml range gave responses which fitted a logarithmic dose-response curve with an average r2 of 0.986. Repeat assays with normal human plasma gave a coefficient of variation ( SD x) between experiments of 16.1 ± 0.3%. Normal human serum contains 33.4 ± 6.4 pg/ml (x ± SD) 1,25-dihydroxyvitamin D by this assay. Values averaging less than normal are found in plasmas of patients with renal failure and in patients with pseudohypoparathyroidism. Average values greater than normal were found in patients with hyperparathyroidism. 1,25-Dihydroxyvitamin D was not detectable in plasma of anephric patients.
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U2 - 10.1016/0003-2697(80)90311-5
DO - 10.1016/0003-2697(80)90311-5
M3 - Article
C2 - 7356156
AN - SCOPUS:0018900319
VL - 102
SP - 22
EP - 30
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -