Quantification of circulating I,25-dihydroxyvitamin ... - Clinical Chemistry

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RIA for i,25-dihydroxyvitamin. D. [l,25(OH)2D1 that does not require sample ... toward the development ... but the RIA, of course, has a distinct advantage.
Clinical Chemisby 586-592 (1996)

42:4

Quantification of circulating I ,25-dihydroxyvitamin D by radioimmunoassay with an ‘251-Iabeledtracer BRUCE

W.

H0LLIs,l*

JOHN

Q.

KAMERUD,2

ANTHONY

JOSEPH

L.

KURKOWSKI,2

JACQUELINE

BEAULIEU,2

and

NAPOLI3

steroid hormone, primarily of renal origin, is affected by factors that perturb mineral and skeletal metabolism [2-4]. Additionally, circulating 1,25(OH)2D is altered in several pathophysiological states, e.g., parathyroid gland disorders [5-7],

We report here the first RIA for l,25-dihydroxyvitamm D utilizing a radioiodinated (125j) tracer. This is also the first validated RIA for i,25-dihydroxyvitamin D [l,25(OH)2D1 that does not require sample prepurification by HPLC before the binding assay. The assay involves acetonitrile extraction, treatment of the crude extract supernate with sodium periodate, extraction and purification of endogenous 1,25(OH)2D by solid-phase chromatography, and finally, quantification by RIA. Calibrators were prepared in stripped human serum and processed exactly the same as samples, eliminating the need for internal control for procedural losses of endogenous 1,25(OH)2D. The assay consists of a 2-h room temperature incubation with the primary antibody, a 20-mm incubation with a second antibody, and separation of bound from free by centrifugation. Assay results can be in hand within 5 h. The detection limit of the assay is 2.4 ng/L 1,25-dihydroxyvitamiii D3. Results compare well with those from an accepted

renal failure [I], vitamin D-dependent rickets [8, 9J, and sarcoidosis [10]. Because alterations

types I and H in circulating

1,25(OI-I)2D can provide valuable clinical information in these disorders of calcium metabolism, considerable effort has been directed toward the development of less cumbersome, more expedient assays for quantifying circulating 1,25(OH)2D [11-20]. Numerous assays for the quantification of circulating 1,25(OH)2D have been reported in the past two decades, including isotope dilution-mass fragmentography [14], bioassay [13], radioreceptor assay (RRA) [11, 12, 19, 20], cytoreceptor assay [15], and RIA [16-18]. With few exceptions [19, 20], these procedures involve exhaustive chromatography, including HPLC. In fact, the last major advance in

radioreceptor assay. Sample pretreatment with sodium periodate is absolutely essential before quantification by RIA; otherwise, concentrations of endogenous 1,25(OH)2D may be greatly overestimated.

detection of circulating 1,25(OH)2D was published a decade ago [20]: Based on RRA technology, this method greatly simplified the extraction and purification of l,25(OH)2D from serum or plasma but still used [3H]l,25(OH)2D3 and required the determination of internal recovery for each individual sample. RIA would be preferable to RRA for quantifying l,25(OH)2D, especially if 1251-labeled tracers

INDEXING TERMS: nutritional status . vitamin D and metabolites #{149} cholecalciferol #{149} calcium metabolism Vitamin D is metabolized to several compounds and, ultimately, a small portion is converted to the most biologically active metabolite, 1,25-dihydroxyvitamin D [1,25(OH),D] [J]4 This

could be incorporated into the assay scheme. RIAs for l,25(OH)2D based on 3H-labeled tracers have been described, but all of these validated RIAs for 1,25(OH)2D require sample prepurification by HPLC before final quantification by RIA [16-18]. As a result, RIAs for l,25(OH)2D have never gained widespread use in clinical diagnosis. Here we describe an RIA for quantification of circulating l,25(OH)2D that requires minimal sample prepurification,

‘Departments of Pediatrics, Biochemistry, and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. 2 !ncstar Corp., Snllwater, MN 55082. Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214. *Author for correspondence. Fax 803-792-8801; e-mail [email protected]. Nonstandard abbreviations 25(OH)D, 25-hydroxyvitamin D, including both 25-hydroxycholecakiferol [25(OH)D3) and 25-hydroxyergocalciferol [25(OH)D2l; 24,25(O1-l)2D3, 24R,25-dihydroxycholecalciferol; 25,26(OH)2D3 25S,26-dihydroxycholecalciferol; l,25(OH)2D2, lcr,25-dihydroxyergocalciferol; 1,25(OH),D,, la,25-dihydroxycholecalciferol; 1,25(OH),D, both I ,25(OH)2D, and l,25(OH)2D,. Received December 8, 1995; accepted January 16, 1996.

does not require the use of individual sample internal standards to estimate endogenous losses of 1,25(OH)2D, and, most importantly, uses an ‘251-labeled tracer. The results by this assay compared very favorably with those by RRA for l,25(OH)2D, but the RIA, of course, has a distinct advantage of speed. The RIA can be completed (results in hand) within 5 h of initiation.

586

Clinical Chemistiy

Materials and Methods REAGENTS

Crystalline cholecalciferol [vitamin D3J was obtained from Sigma Chemical Co., St. Louis, MO. Crystalline 25-hydroxycholecalciferol [2 5(OH)Dj, 24R,2 5-dihydroxycholecalciferol [24,2 5(OH),D3], 2 5S,26-dihydroxycholecalciferol [2 5,26(OH)2

D2], 1a,2 5-dihydroxycholecalciferol

[1,2 5(Ol-1)2D], I a,2 5-dihydroxyergocalciferol [1 ,25(OH),D21, 1a,2 5-dihydroxycholecalciferol-26,2 3 -lactone, 1a,24R,2 5-trihydroxycholecalciferol, and 1a,2 5S,26-trihydroxycholecalciferol were obtained from Hoffmann-La Roche, Nutley, NJ. 1,25-Dihydroxy [26,273H]cholecalciferol ([3H] l,25(OH)2D3; 160 kCi/mol) was obtained from Incstar, Stillwater, MN. Bolton-Hunter reagent (2000 MCi/mol) was obtained from New England Nuclear, Boston, MA. “Bond-Elut” C18-OH silica cartridges (500 mg) and the “Vac-Elut” cartridge rack were from Varian Instruments, Harbor City, CA. “N-Evap” evaporator, Model 112, was from Organomation, Northborough, MA. Sheep anti1,25(OH)2D3 antibody (primary antibody) was obtained from Larry Fraher, London, Ontario. Donkey anti-goat antibody (second antibody precipitating complex) was obtained from Incstar. HPLC-grade hexane, isopropanol, methylene chloride, methanol, and acetonitrile were from Fisher Chemical Co., Pittsburgh, PA. Unless otherwise noted, all other reagents were reagent grade. CLINICAL

Plasma healthy dialysis, women vitamin

SAMPLES

or serum samples were obtained from 50 apparently individuals, 11 renal patients undergoing chronic hemo5 children with extrahepatic biliary atresia, and 9 at the time of delivery. Additionally, samples from nine D2-toxic rats were obtained from R.L. Horst, Ames, IA.

METHODS

Spectroscopy. Concentrations of vitamin D and its metabolites were determined by ultraviolet spectroscopy, with absorptivity (#{128}264)values of 18 300 mol cm’ for vitamin D3 and its metabolites and 19400 mol cm for l,25(OH),D2. Preparation of radioiodinated vitamin D. 1,2 5(OH)2-24,2 5,26,27tetranor-C(23)-carboxylic acid was derivatized and radioiodinated to a specific activity of 2000 kCi/mol with [‘25I]-BoltonHunter reagent as previously described [21]. Preparationof assaycalibrators. Human serum was stripped of l,25(OH)2D by using activated charcoal. Absence of l,25(OH)2D in the stripped sera was confirmed by RRA [20]. Subsequently, crystalline 1,2 5(OH)2D2 dissolved in absolute ethanol was added to the stripped sera to final concentrations of 0, 5, 10, 20, 50, 100, and 200 ng/L. These sera, used as calibrators, were then stored at -20 #{176}C. Exiraction, pretreatment, and isolation of 1,25(OH)2D from serum orpla.rma.Into 12 X 75 mm borosilicate glass tubes we placed 0.5-0.75 mL of sample or calibrator, added an equal volume of acetonitrile, then vigorously vortex-mixed and centrifuged the solution (20 #{176}C, 2000g, 10 mm). After removing the supernate

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into clean 12 x 75 mm borosilicate glass tubes, we added one sample volume of 12 mmoL/L sodium metaperiodate and incubated the mixture for 3 0-60 mm at ambient temperature. We then applied the supernate-.-sodium periodate mix to a C1 8-OH cartridge that had been prewashed successively with 5 mL of isopropanol and 5 mL of methanol. Next, we washed the cartridge successively with 5 mL of methanol/water (70/3 0 by vol), 5 mL of hexane/methylene chloride (90/10 by vol), and 5 mL of hexane/isopropanol (99/1 by vol); placed the C18-OH cartridge into a silica cartridge previously washed successively with 5 mL of methanol, 5 mL of isopropanol, and 5 mL of hexane/isopropanol (80/2 0 by vol); and eluted 1,2 5(OH)2D onto the silica cartridge with 5 mL of hexane/isopropanol (92/8 by vol). We removed the C18-OH cartridge and washed the silica cartridge with an additional 2 mL of the 92/8 hexane/isopropanol mixture. Finally, we eluted l,25(OH)2D from the silica cartridge with 5 mL of hexane/isopropanol (80/2 0 by vol). Each C18-OH cartridge can be generated for reuse by washing with 2 mL of methanol. The silica cartridges can be reused without any further washing steps. All solvent elution on the cartridges was performed under reduced pressure [20 kPa (-150 mmHg)] and with the 24position Vac-Elut rack. We evaporated the fraction containing 1,25(OH)2D under nitrogen at 55 #{176}C, then cooled the tubes and reconstituted the residue in 50 .tL of absolute ethanol. After capping and mixing each sample, we stored them at -20 #{176}C until performing the RIA. Radioimmunoassay.The assay tubes were 12 X 75 mm borosilicate glass tubes containing 20 jiL of the ethanol-reconstituted extracted calibrators or samples. To each tube we added 50 L (50 000 counts/mm) of ‘251-labeled l,25(OH)2D derivative in an equivolume solution of ethanol and 10 mmol/L phosphate buffer (pH 7.4). We then added to each tube 0.25 mL of primary antibody diluted 200 000-fold in sodium phosphate buffer (50 mmol/L, pH 6.2) containing 1 g/L swine-skin gelatin and 3.5 mL/L polyvinyl alcohol (Mr 13 000-23 000). Nonspecific binding was estimated by using the above buffer without the antibody. We vortex-mixed the contents of the tubes and incubated them for 2 h at 20-25 #{176}C. We then added 0.5 mL of the second-antibody precipitating complex to each rube, vortexmixed, incubated at 20-25 #{176}C for 20 mm, and centrifuged (20 #{176}C, 2000g, 20 mm). After discarding the supernate, we counted the radioactivity remaining in the tubes with a gamma well-counting system. Preparation of samples for RIA after HPLCpurtfi cation. To 1.0-1.5 mL of sample in 12 x 75 mm borosilicate glass tubes we added 20 iL of [3HJ1,25(OH)2D3 in ethanol (-1000 counts/mm), mixed, and incubated the samples for 15 mm. After extraction and purification as described above, the extracts from the silica cartridges were further purified by normal-phase HPLC with a hexane/isopropanol mobile phase as described elsewhere 117]. 1,25(Ol-I)2D2 and l,25(OH),D3 comigrate on this HPLC system. The samples were collected after HPLC, dried under nitrogen, and reconstituted in absolute ethanol. Final quantification was by RIA as described earlier, with two exceptions. The

588

Hollis

Table I. Cross-reactivity with l-Iabeled

tracer

treatment

of various vItamin and S-li antibody and purification.

Steroid Vitamin 03

et al.:

125j

-J %

C 0