A simplified assay for serum 25-hydroxycalciferol

A simplified assay for serum 25-hydroxycalciferol

Clin. Biochem. 13 (3) 106-108 (1980) A Simplified Assay for Serum 25-Hydroxycalciferol E.E. DELVIN, M. DUSSAULT AND F.H. GLORIEUX Genetics Unit, Shri...

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Clin. Biochem. 13 (3) 106-108 (1980)

A Simplified Assay for Serum 25-Hydroxycalciferol E.E. DELVIN, M. DUSSAULT AND F.H. GLORIEUX Genetics Unit, Shriners Hospital and the Departments of Pediatrics and Surgery, McGill University, Montreal, Quebec (Accepted February 15, 1980) The measurement of serum 25-hydroxycalciferol (25-0H D) has proved to be a reliable index of the vitamin D nutritional status in man providing the renal 25-hydroxycalciferol-1 a-hydroxylase is functional. Since it is known that 25 OHD binds to a-globulins for its transport to the kidney, we have developed a competitive binding assay using an a-globulin enriched fraction (Cohn's fraction IV) as the ligand protein. A detection limit of O.l ng 2 5 - O H D / t u b e was achieved and the non specific binding amounted to less than 3% of the initial binding. The intraand inter-assay variations were 8.9 and 8.4% respectively. The reference values ( n m o l / l ± 2 s.d.) obtained from samples of 40 children were 50.5 -~ 21.5 with extremes of 34.8 and 95.2. The method thus proved to be a reliable and sensitive tool to assess serum 25-OHD levels.

ERGOCALCIFEROL (VITAMIN D2) A N D CHOLECALCIFEROL (VITAMIN D.~) grouped under the generic n a m e of calciferol (vitamin D), are both hydroxylated in liver to yield 25-hydroxyergocalciferol (25-OHD~) and 25-hydroxycholecalciferol (25-0HD3) respectively '''~). Their bulk measurement as 25-hydroxycalciferol (25O H D ) has proved to reasonably reflect the vitamin D repletion status in m a n providing that the renal 25hydroxyvitamin D-la-hydroxylase is functionaU 3~. In most of the radioligand assays currently available, serum from rachitic rat '') or m a n '~' or rat kidney cytosol extract '~' are used as the source of binding protein. Their preparation or isolation hinder the use of this assay in clinical chemistry laboratories. It has been well shown that 2 5 - O H D binds to ~-globulins '7~ for its transport to the kidney where it is further metabolized '8'.This property and the fact that serum fractionation according to the method of Cohn et al.<9) yields a fraction enriched in ~-globulins prompted us to develop an assay for 2 5 - O H D using an ~-globulin enriched fraction as the source of ligand. MATERIALS AND METHODS Chemicals

Chemists (Cincinnati, OH. USA). All solvents used for chromatography were of spectroscopy grade and were purchased from British Drug House (Montreal, Que. Canada). Samples For the reference group, morning fasting samples were collected between November and April from 40 infants (8-48 months) without specified vitamin D supplementation and having no overt liver, kidney or bone diseases. Serum samples were also obtained from 36 patients affected by bone diseases, some of which were receiving pharmacological doses of vitamin D~. Extraction and chromatography To each aliquot of serum (0.5 ml) were added 1500 dpm of (3H)-25-OHDa to monitor the recovery of 25-OHD.~ and a 30-45 rain equilibration was allowed. The lipoproteins were precipitated ~H~ by the addition of 20 ~l of 1% heparin in saline (Abbott Laboratories, Montreal, Que. Canada) and 10 ~l of 1 M MnCl2 solution. A f t e r a 20 minute equilibration period, the samples were extracted 3 times with 4 volumes of diethyl ether. The combined extracts were dried under N2, redissolved in n-hexane and purified by column chromatography. Short (0.5 x 4 cm) silicic acid (minus 325 mesh, Bio-Rad Laboratories, Richmond, CA, USA) columns were washed and equilibrated with n-hexane. Samples were applied in a 2 ml volume and a 4 ml fraction was afterward collected. The 25-OHD, which eluted in 4 ml of 50% ether in n-hexane, was redissolved in absolute ethanol. Duplicate aliquots (50 t~l) were taken for its quantitation and recovery. Competitive binding assays A 60 ~ g / m l a-globulin enriched fraction solution was prepared in a 0.05 M NaH2PO4 buffer adjusted at pH 8.6 with 1.0 M NaOH. To each tube were added in sequence either 50 ~1 of absolute ethanol containing increasing concentrations of 25-OHD3 for the standard curve or 50 F1 of the purified extracts. Then 20 ~tl of (3H)-25-OHD3 was added to each tube so as to contain 10000 DPM. A f t e r mixing, 1 ml of the binding protein solution was added and the whole was incubated for 1 hour at 4°C. A f t e r incubation, 150 ~1 of a Dextran T-20 (5%) Nuchar charcoal (8%) suspension in 0.05 M KH2PO4 buffer adjusted to pH 8.6 with 0.1 M NaOH was added. A f t er 15 min at room temperature the tubes were centrifuged at 4000 g × 15 min at 4°C. An aliquot (500 ~1) of the supernatant was taken for

Crystalline 25-hydroxycholecalciferol (25-OHD~) and 25-hydroxyergocalciferol (25-0HD2) kindly supplied by the Upjohn Company (Kalamazoo, MI., USA) were dissolved in absolute ethanol and their concentration adjusted at 10 F g / m l using 18,200 and 19,400 as molar coefficients of extinction respectively (265 rim). The substance 25hydroxy (26,27-'~H-methyl)-cholecalciferol [5-15 Ci/mmol, (3H)-25-OHD3], obtained from Amersham Searle (Oakville, Ont. Canada) was periodically verified for its purity by column chromatography according to the method of Holick and DeLuca ~1°~. Bovine a-globulin enriched fraction (Cohn fraction IV) was obtained from Sigma Chemicals (St. Louis, MO, U S A ) , Dextran T-20 from Pharmacia Fine Chemicals (Montreal, Que. Canada) and Nuchar charcoal from Matheson, Coleman and Bell MF

K,, (XI0-]OM) Binding capacity (pmol/mg protein)

Correspondence: E,E. Delvin Genetics U n i t - - Shriners Hospital 1529 Cedar Ave. Montr4al, P. Qu6. Canada H3G 1A6

One milliliter aliquots of cytosol extract, equivalent to 100 - 120 ~g of protein, or Cohn fraction IV (60 ~g) were added to increasing amounts of 25-OHD3 in the presence of a constant amount of (aH)-25-OHDa. The data derived from Scatchard plots (~2) are mean -4- s.d. •) No significant difference (n = 3) b) significant difference p < 0.001 (n = 3)

TABLE 1 APPARENT BINDINGAFFINITYAND CAPACITYOF KIDNEY CYTOSOL]C EXTRACTAND BOVINE COHN FRACTIONIV FOR 25-(OH)D3 Kidney cytosol

Cohn fr IV

4.0 -4- 0.8 3.9 -4- 0.2

2.8 -4- 0.7") 9.8 -4- 0.6 b)

S I M P L I F I E D ASSAY FOR SERUM 25-HYDROXYCALCIFEROL

107

~

140120-

40z

~i

100-

ca

80-

~

60-

~

40-

20_z

e

~leeL/~ 0.23 +0.93x ry :: 0.99 p< 0.001

,. . . . , . . , 20 40 60

I 80

; 100

,''; 120 140

SERUM 25(OH)D (ng/ml) TEST METHOD Fig. 2 - - C o m p a r i s o n o f the observed s e r u m 2 5 - O H D levels u s i n g the test m e t h o d (abscissa) a n d the c o m p a r a t i v e m e t h o d ( o r d i n a t e ) [s). The li~ear r e g r e s s i o n line w a s obtained by the least squares method. :It

RESULTS AND DISCUSSION

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1

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2

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5

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10

25 (OH) D3 (ng tube) Fig. 1 - - C a l i b r a t i o n c u r v e s f o r 25-OHD:~ u s i n g the rachitic r a t k i d n e y cytosol e x t r a c t (o-o) and the bovi~e Cohn's f r a c t i o n I V (0 - O) as the sources of ligand. E a c h p o i n t is the m e a n of 10 s e p a r a t e a s s a y s ( ± 1 s.d.). The o r d i n a t e (log scale) is e x p r e s s e d as tire perce~Ttage o]" the ('~H)-25-OHDa bi~ding in absence of unlabelled secosteroid. T h e p e r c e n t a g e s ( m e a n ~ S D ) o f total C P M added to the i n c u b a t i o n m e d i u m , bound in the 0 n g tube were 30.0 + 2.~ and 31.0 ~ 2.1 f o r the cytosol e x t r a c t a v d ~-globulb~ respectively.

counting. The non-specific binding consisted of a tube containing 500 ng of 25-0HD3 in presence of the binding protein and the blank of only aH-25-OHDa in 50 ~l of absolute alcohol and 1 ml of buffer. This assay was compared to that reported by Haddad and Chyu (") using a rachitic rat kidney cytosol receptor protein. All radioactivity was monitored in a Packard Tri-Carb (Downers Grove, IL., USA) liquid scintillation spectrometer using AQUASOL (New England Nuclear Canada Ltd., Montreal, Canada) as the scintillation fluid. TABLE 2 CONCENTRATION(10-9 M) OF INHIBITORSREQUIREDFOR h 50% DISPLACEMENTOF (aH)-25-OHDa Inhibitor 25-OHDa . . . . . . . . . . . . . . 25-OHD2 . . . . . . . . . . . . . . Vitamin D3 . . . . . . . . . . . . . 24(r)25(OH)2D3 . . . . . . . . . 7-dehydrocholesteml . . . . Ergosterol . . . . . . . . . . . . . . Cholesterol . . . . . . . . . . . . . Dihydrotachysterol . . . . . a--tocopherol . . . . . . . . . . . . Retinol acetate . . . . . . . . .

Cytosol extract

> > > >

1.5 2~) 50 2.5 2.5 x 105 2.5 x 10s 2.5 x 105 2.5 x 104 N.D. N.D.

a--globulin

> > > > >

2.0 2.7 > 200 > 100 2.5 x 105 2.5 x 105 2.5 x l0 s 2.5 x 104 2.5 x 104 N.D.

The displacement of labeled 4H-25-OHDa was performed as described in the Materials and Methods section. N.D.: no disDlacement at a concentration ~reater than 10-aM.

The overall recovery of (3H)-25-OHD3 a f t e r the e t h e r e x t r a c t i o n s and silicic acid column chromatog r a p h y a v e r a g e d 90.1% ( ± S D : 1.6%, n ---- 48) and was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t of 25-hydroxy-(3a-3I-I)-ergocalciferol. The p r e c i p i t a t i o n of l i p o p r o t e i n s with h e p a r i n and MnC12('') p r i o r to the e x t r a c t i o n yielded cleaner e x t r a c t s which could r e a d i l y be dissolved in the c h r o m a t o g r a p h y solvent. The disp l a c e m e n t of (3H)-25-OHD~ f r o m a r a c h i t i c r a t k i d n e y cytosol e x t r a c t a n d the bovine a - g l o b u l i n f r a c t i o n by i n c r e a s i n g c o n c e n t r a t i o n s of c r y s t a l l i n e 25-0HD3 is shown in Fig. 1. The non-specific b i n d i n g , elicited in presence of 1.0 x 10~M non radioactive 25-OHD,~, cont r i b u t e d to 8% of the i n i t i a l b i n d i n g (zero 25-OHD~ c o n c e n t r a t i o n ) with the k i d n e y e x t r a c t a n d was negligible for the a - g l o b u l i n f r a c t i o n . The a p p a r e n t association c o n s t a n t s (K.) and m a x i m a l b i n d i n g capacities (MBC) calculated f r o m Scatchard plots ''2) for both b i n d i n g p r o t e i n s are shown in Table 1. The K, were not s t a t i s t i c a l l y d i f f e r e n t f r o m each o t h e r a n d were of the same order of m a g n i t u d e as those r e p o r t e d for other sources of b i n d i n g p r o t e i n s "3""). The a-glob u l i n MBC was however s i g n i f i c a n t l y h i g h e r t h a n t h a t of the cytosol e x t r a c t t h e r e b y s h o w i n g its h i g h e r degree of p u r i t y . U n l i k e H a d d a d et al. ('5), we observed a slightly b u t s i g n i f i c a n t l y lesser a f f i n i t y of the k i d n e y cytosol b i n d i n g p r o t e i n for 25-0HD2 t h a n for 25-0HD5. A s i m i l a r d i f f e r e n c e a n d of the same m a g n i t u d e was also observed f o r the a - g l o b u l i n f r a c t i o n (Table 2 ) . T h e r e f o r e it is likely t h a t in v i t a m i n D2 t r e a t e d pat i e n t s the high s e r u m 25-OHD levels could be u n d e r evaluated by a p p r o x i m a t e l y 10%. F o r p r a c t i c a l p u r poses, however t h i s d e v i a t i o n f r o m the t r u e value h a s no real consequence. W h e r e a s the r a t k i d n e y p r o t e i n b i n d s equally well 25-0HD5 a n d 2 4 r , 2 5 - d i h y d r o x y cholecalciferol (24r,25- (OH) 5), as a l r e a d y r e p o r t e d ('", the a - g l o b u l i n p r e p a r a t i o n has lost its a f f i n i t y for t h i s d i h y d r o x y l a t e d v i t a m i n D m e t a b o l i t e ( T a b l e 2 ) . The

108

DELVIN, D U S S A U L T AND G L O R I E U X

p r o p o s e d m e t h o d t h e r e f o r e o f f e r s an a d d i t i o n a l ass u r a n c e of m e a s u r i n g t r u e values o f 25-OHD in s i t u a t i o n s w h e r e t h e s e p a r a t i o n of 2 5 - 0 H D f r o m 24, 2 5 ( O H ) 2 D is n o t i n s u r e d ''7''8). O t h e r s t e r o l s t e s t e d as well as v i t a m i n s A a n d E h a d little or no e f f e c t on t h e b i n d i n g of (~H)-25-OHD3 ( T a b l e 2). T h e i n t r a - a s s a y v a r i a t i o n a s s e s s e d by m e a s u r i n g t h e s a m e s e r u m pool (n ---- 10) w i t h a m e a n 25-OHD c o n c e n t r a t i o n of 54.2 nmol/1 was 8.9 %. The i n t e r - a s s a y v a r i a t i o n in 10 s e p a r a t e a s s a y s was 8.4% a t a m e a n 2 5 - 0 H D value of 37.2 nmol/1. To t e s t the a c c u r a c y of t h e method, a l i q u o t s (500 /zl) of a s e r u m pool w e r e s p i k e d w i t h e i t h e r 100 nmol/1 or 250 nmol/1 of 25OHD.~. T h e r e c o v e r i e s a v e r a g e d 105.2% a t t h e f o r m e r c o n c e n t r a t i o n ( v a r i a t i o n : 9.8%, n = 10) a n d 100.9% a t t h e l a t t e r ( v a r i a t i o n : 7.6%, n ---- 10). The r e l a t i o n s h i p b e t w e e n 25-OHD s e r u m levels o b t a i n e d f r o m s a m p l e s m e a s u r e d s i m u l t a n e o u s l y w i t h t h e t e s t and t h e c o m p a r a t i v e '8~ m e t h o d s is shown in F i g . 2. The s t r a i g h t line o b t a i n e d follows t h e e q u a t i o n : Y = 0.23 q- 0.93X ( r ---- 0.99, p < 0.001) and c o n t a i n s w i t h i n i t s w e i g h t e d 95% confidence i n t e r v a l , the line of i d e n t i t y , t h e r e b y s h o w i n g t h e equivalence of t h e two methods. S e r a f r o m 40 h e a l t h y M o n t r e a l c h i l d r e n (8-48 m o n t h s ) collected between N o v e m b e r and A p r i l h a d a m e a n 2 5 - 0 H D level of 50.5 ( - - 2 SD = 21.5) nmol/1 w i t h e x t r e m e s of 34.8 a n d 95.2. These levels a r e w i t h i n r a n g e s r e p o r t e d by o t h e r s f o r a N o r t h A m e r i c a n p o p u l a t i o n t a k i n g in account the seasonal variations"*'~°'. T h e r e f o r e in our opinion t h e s e r e s u l t s w a r r a n t t h e use o f t h i s m e t h o d f o r a s s e s s i n g v i t a m i n D n u t r i t i o n a l or liver m e t a b o l i c s t a t u s . ACKNOWLEDGEMENTS W e a r e g r a t e f u l to Dr. Glenville J o n e s ( H o s p i t a l f o r S i c k Children, T o r o n t o ) f o r p r o v i d i n g us w i t h 25hydroxy-3a-3H)-ergocalciferol. W e w i s h to t h a n k Ms. Denyse B i s s o n n e t t e f o r h e r excellent s e c r e t a r i a l work. T h i s w o r k was s u p p o r t e d by the S h r i n e r s of N o r t h America. REFERENCES

1. Jones, G., Schnoes, H.K., DeLuca, H.F. An in vitro study of vitamin D2 hydroxylases in the chick. J. Biol. Chem. 251, 24 (1976). 2. Horsting, M., DeLuca, H.F. In vitro production of 25-hydroxycholecalciferol. Biochem. Biophys. Res. Commun. 36, 251 (1969). 3. Stamp, T.C.B. Factors in human vitamin D nutrition and in the production and cure of classical rickets. Proc. Nutr. Soc. 34, 119 (1975).

4. Belsey, R., DeLuca, H.F. Potts, J.T. Jr. Competitive binding assay for vitamin D and 25-hydroxyvitamin D. J. Clin. Endocrinol. 33, 554 (1971). 5. Bayard, F., Bec, P., Louvet, J.P. Measurement of plasma 25-hydroxycholecalciferol in man. Eur. J. Clin. Invest. 2, 195 (1972). 6. Haddad, J.G., Chyu, K.J. Competitive protein binding radio-assay for 25-hydroxycholecalciferol. J. Clin. Endocrinol. 33, 992 (1971). 7. Haddad, J.G., Walgate, J. 25-hydroxyvitamin D transport in human plasma: Isolation and p a r t i a l characterization of calciferol-binding protein. J. Biol. Chem. 251, 4803 (1976). 8. DeLuca, H.F. Vitamin D metabolism and function. Arch. Intern. Med. 138, 836 (1978). 9. Cohn, E.J., Strong, L.E., Hughes, W.L. Jr., Mulford, D.J., Ashworth, J.N., Melin, M., Taylor, H.L. A system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. J. Amer. Chem. Soc. 68, 459 (1946). 10. Holick, M.F., DeLuca, H.F. A .new chromatographic system for vitamin D3 and its metabolites: Resolution of a new vitamin D3 metabolite. J. Lipid Res. 12, 460 (1971). 11. Burstein, M., Scholnick, H.R., Morfin, R. Rapid method for the isolation of lipoproteins from human serum by precipitation with polyanions. J. Lipid Res. 11, 583 (1970). 12. Scatchard, G. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660 (1949). 13. Haddad, J.G., Hillman, L., Rojanasathit, S. Human serum binding capacity and a f f i n i t y for 25-hydroxycholecalciferol and 25-hydroxyergocalciferol. J. Clin. Endocrinol. Metab. 43, 86 (1976). 14. Rojanasathit, S., Haddad, J.G. Ontogeny and effect of vitamin D deprivation on r a t serum 25-hydroxyvitamin D binding protein. Endocrinology 100, 642 (1977). 15. Haddad, J.G., Chyu, K.J., Hahn, T.J., Stamp, T.C.B. Serum concentrations of 25-Hydroxyvitamin D in sexlinked hypophosphatemic vitamin D-resistant rickets. J. Lab. Clin. Med. 81, 22 (1973). 16. Haddad, J.G., Min, C., Walgate, J., Hahn, T.J. Competition by 24,25-dihydroxycholecalciferol in the competitive protein binding radio-assay of 25-hydroxycalciferol. J. Clin. Endocrinol. Metab. 43, 712 (1976). 17. Belsey, R.E., DeLuca, H.F., Potts, J.T. Jr. A rapid assay for 25-OH-vitamin D3 without p r e p a r a t i v e chromatography. J. Clin. Endocrinol. Metab. 38, 1046 (1974). 18. Offermann, G., Dittmar, F. A direct protein-binding assay for 25-hydroxycalciferol. Horm. Metab. Res. 6, 534 (1974). 19. Arnaud, S.B., Matthusen, M., Gilkinson, J.B., Goldsmith, R.S. Components of 25-hydroxyvitamin D in serum of young children in upper midwestern United States. Amvr. J. Clin. Nutr. 30, 1082 (1977). 20. Roginsky, M.S., Moo, H.F., Abesamis, C. Seasonal variations in serum 25-hydroxycholecalciferol. Lancet 1, 1235 (1974).