Quantitation of vitamin D and its metabolites and their plasma concentrations in five species of animals

ANALYTICAL

BIOCHEMISTRY

Quantitation

116, 189-203

of Vitamin Concentrations

R. L.

HoRsT,*,’

E. T.

(1981)

D and Its Metabolites in Five

Species J. L.

LITTLEDIKE,*

RILEY,*

and Their

Plasma

of Animals’ AND

J. L.

NAPOLI?

*National Animal Disease Center, Agricultural Research, Science and Education Administration. U. S. Department 01‘ Agriculture, Ames, Iowa 500/O and tDepartment of Biochemistry, The University of Texas Health Science Center, Dallas, Texas 75235 Received

January

13, 1981

Chromatographic methods suitable for the resolution of 24.25-dihydroxyvitamin D,, 24,25dihydroxyvitamin D2, 2Shydroxyvitamin D,-26,23 lactone, and 25.26-dihydroxyvitamin D1 arc described. These four metabolites comigrated in high-pressure liquid chromatography on silicic acid columns developed in 1 I:89 isopropanol:hexane. Adequate resolution was achieved by subjecting the four-metabolite complex to high-pressure liquid chromatography column developed in 2:98 isopropanol:methylene chloride. This additional chromatographic step, coupled with modifications of assay procedures previously described, allowed for the estimation of plasma concentrations of vitamin DZr vitamin D,, 25-hydroxyvitamin DI, 25.hydroxyvitamin D,, 24.25.dihydroxyvitamin D,, 24,25-dihydroxyvitamin D,, 25.26 dihydroxyvitamin Dzr 25.26.dihydroxyvitamin D,, 25-hydroxyvitamin D,-26,23 lactone, and I ,25-dihydroxyvitamin D (1,25-dihydroxyvitamin DZ plus 1,25-dihydroxyvitamin D,). The samples automatically were introduced onto the high-pressure liquid chromatography columns with a Waters 710A “intelligent” processor. The metabolites were automatlcally collected with the aid of a programmable timer that advanced a fraction collector at predetermined intervals. The assays were used to determine the plasma vitamin D and vitamin D metabolite concentrations in five species of adult farm animals.

Vitamin D3 undergoes several hydroxylation steps before its activity is expressed as enhanced calcium and phosphorus absorption from the intestine and calcium and phosphorus resorption from bone. A vitamin D metabolite produced in the kidney. 1,25dihydroxyvitamin D [ I ,25-(OH)zD],4 is cur’ Mention of a trade name, proprietary product, or vendor does not constitute a guarantee or warranty by the U. S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may be suitable. ’ To whom correspondence should be addressed. ’ Lack of identifying subscript indicates both vitamin D1 and vitamin D,. 4 Abbreviations used: 1,25-(OH)>D. I ,25-dihydroxyvitamin D; 25-OHD,, 2Shydroxyvitamin D3; 24,25(OH),D,. 24,25-dihydroxyvitamin D,; 25,26-(OH)2D,, 25.26.dihydroxyvitamin D,; 25.OHDjm26,23 lactone, 25-hydroxyvitamin D,-26,23 lactone; 25.OHD2-26,23 lactone, 25-hydroxyvitamin D,-26.23 lactone; 25,26(OH)>D2. 25.26.dihydroxyvitamin Dz; 25-OHD,, 25-

rently believed to be the form of vitamin D responsible for this activity (l-6). Other metabolites of vitamin D, however, have also been demonstrated in the plasma of normal animals and humans. The compound 25-hydroxyvitamin Dj (25-OHD3), a liver metabolite, is the major circulating form of vitamin D in normal mammals ( 1). This metabolite acts as a precursor for at least two additional dihydroxylated metabolites, 24,25-dihydroxyvitamin D3[ 24,25-(OH)2D3] and 25,26-dihydroxyvitamin D3[25,26-(OH)zD3], which may be active under specific circumstances (3). Although both 24,25-(OH)?D3 and 25,26-(OH)ZD3 can be produced in the hydroxyvitamin Dz; hplc, high-pressure liquid chromatography; 24,25-(OH),D, 24,25-dihydroxyvitamin D; 24,25-(OH)2DZ, 24,25-dihydroxyvitamin D,; PBG buffer, 0.05 M phosphate buffer (pH 7.5) containing 0.01% gelatin and 0.01 B merthiolate; PTH. parathyroid hormone.

189

0003-2697/81/130189-15502.00/O Copyright G. 198 I by Academic Press. Inc. All rights of reproduction in any form reserved.

190

HORST

kidney, extrarenal sources of both the 24and the 26-hydroxylase have been demonstrated (2,7,8). Recently a new metabolite, 25-hydroxyvitamin D3-26,23 lactone (25OHD3-26,23 lactone), was discovered (9) and shown to require the presence of the kidney for production (10). All of the aforementioned vitamin D, hydroxylations have been shown to also take place with vitamin Dz as the metabolic precursor ( 1 l-l 3) with the exception of 25-hydroxyvitamin D,-26,23 lactone (25-OHD*-26,23 lactone) and 25,26dihydroxyvitamin Dz [ 25,26-(OH)2DZ]. This report will demonstrate the isolation and purification of 25,26-(OH)*D2 from vitamin D,-toxic pig plasma. To date, radioligand binding, spectral assays, and bioassays have been described for the quantitation of some of the physiologically important vitamin D metabolites (7,8,14-20). Accurate quantitation of the vitamin D metabolites requires extensive and time-consuming chromatographic techniques. With few exceptions during the development of these techniques, little attention was given to the migration of vitamin Dz metabolites in relation to the migration of vitamin D3 metabolites. This problem has resulted mainly from the lack of vitamin Dz metabolite standards. Horst et al. (7), Shepard et al. (8) and Jones (20) have described methods for the separation and individual quantitation of vitamin Dz, vitamin Dj, 25hydroxyvitamin D2 (25-OHD& and 25OHD3 by high-pressure liquid chromatography (hplc). Heretofore, no techniques have been described for the complete resolution and individual quantitation of dihydroxylated vitamin D, metabolites. Most of the multiple assay techniques described assume very near migration or comigration of the Dz and D3 metabolites on hplc and other chromatographic procedures and also assume their equal potency on radioligand binding assays (7,8,18,19). The metabolite most difficult to isolate in pure form for radioligand binding assay has been 24,25(OH)*D3. Horst et al. (21) and Taylor et al.

-kl

AL. .

(19) have shown that the inability to adequately purify this metabolite leads to falsely high values in normal and anephric humans. The best results have been attained when 24,25-(OH),D, has been isolated by hplc and quantitated by radioligand binding assay (7,8). As we shall demonstrate in this report, previous methods are probably unsuitable for the determination of 24,25-dihydroxyvitamin D [ 24,25-(OH),D] because of the possible presence of 24,25-dihydroxyvitamin D2 [24,25-(OH),D,] and 25,26(OH)2D2. We will also demonstrate that 24,25-(OH)2D2 and 25,26-(OH)*D* are not equivalent to vitamin D, derivatives in their binding affinity to vitamin D-binding proteins in rat plasma. MATERIALS

AND METHODS

Apparatus All hplc was carried out with a Waters Associates Model LC-204 chromatograph fitted with a Model 6000-A pumping system and a Model 440 uv fixed-wavelength (254nm) detector. The samples and standards were automatically introduced onto the hplc columns with a No. 710A Waters “intelligent” sampling processor (WISP 7 1OA). The procedure involved transferring the samples into a limited-volume insert (Waters Associates, part No. 72704) with a final volume of 150 ~1. The introduction of the samples onto the column with a total volume of 150 ~1 resulted in minimal peak spreading (compared to smaller volumes) with negligible sample losses. (Dead volume of insert was 6 ~1.) A fraction collector that required moving collection tubes at specified times was used for collecting the individual metabolites from the hplc columns. Therefore, a programmable timer (Fig. 1) was built which would sequence through a set of programmed time intervals, causing relay closure and movement of the fraction collector at the end of each time interval. The timer would continuously repeat this sequence and

VITAMIN

01 METABOLITE

QIJANTITATION

191

192

HORST

could be set to initial conditions by a relay closure originating from the WISP 710A. Each time interval could be easily and accurately programmed to the nearest second by placing a switch in the program mode. A display of the step and time interval was provided so that the operator could observe where the timer was in sequence. The availability of integrated circuits and integrated circuit memories made the design of this timer straightforward and inexpensive. A variable-wavelength Beckman DB spectrophotometer was used to scan the vitamin D metabolites in ethanol. Mass spectra were obtained with a Varian CH-7 mass spectrometer at 70 eV with a direct probe inlet at 90°C above ambient.

Solvents Diethylether was purchased from J. T. Baker, Inc., Phillipsburg, New Jersey. All other solvents used in the extraction, conventional chromatography, and hplc were purchased from Burdick and Jackson, Muskegon, Michigan.

Preparation and Isolation of 24,25(OHj2Db 25,2~5-(OH)~D~, 25,26-(OHjzD3, and 25-OHDj-26,23 Lactone Both 24,25-(OH)*D2 and 25,26-(OH)2Dz were isolated and purified from the plasma of pigs dosed weekly for 3 weeks with 40 X lo6 IU (I g) of vitamin DZ. To isolate the metabolites, the plasma was extracted with methylene chloride:methanol (1:2). The resulting lipid extract was spiked with [3H]25-OHD3 and [‘HI-l ,25-(OH)*D3, dried down, and placed on a 2.2 X 50-cm Sephadex LH-20 column developed in 35:65 hexane:chloroform. The 24,25-(OH)*D* and 25,26-(OH),D, were assumed to be more polar than 25-OHD, and at least as polar as 1,25-(OH)zD3. Therefore, the eluant immediately after the elution of [3H]-25-OHD3 up to and including the [3H]-1,25-(OH),D3 region was collected, [3H]-25-OHD3 was added again, and the solvent was evaporated. The residue was chromatographed on a 1.1

ET AL. X 60-cm Sephadex LH-20 column developed in 1: 1:9 chloroform:methanol:hexane. The eluant between the [ 3H]-25-OHD, and [ 3H]1,25-(OH),D, was collected and the solvent evaporated. The residue was subjected to hplc with a Zorbax Sil column (0.45 X 25 cm, DuPont) eluted at a flow rate of 2.0 ml/ min with 8:92 isopropanol:hexane. Two-milliliter fractions were collected, and radioligand binding analysis was performed for 24,25-(OH)zD3-like activity. Binding activity appeared in the 6- to lo-ml and the 12to 18-ml fractions (Fig. 2). The binding activity in the 6- to lo-ml region was found to be 25-OHD,. The activity in the 12- to 18ml region migrated with 24,25-(OH)zD3. The 12- to 18-ml region was rechromatographed in toto on an hplc Zorbax Sil column (0.45 X 25 cm). The vitamin D2 metabolites were eluted with 2:98 isopropanol:methylene chloride at a flow rate of 1.5 ml/min. The resulting chromatogram showed 24,25-(OH)*D,-like binding activity associated with two major uv peaks, I and II (Fig. 3). The two peaks were collected individually and further purified by hplc with a Zorbax Sil silicic acid column developed with 6:94 isopropanol:hexane. Peak I was confirmed to be 24,25-(OH)zD2. The mass spectrum (Fig. 4) agreed well with that obtained by Jones et al. (13) for chemically synthesized 24(R),25-(OH),D,. Peak II was confirmed to be 25,26-(OH)2D2 from mass spectra taken from the original material (Fig. 5). The compound 25,26-(OH)*D3 was prepared similarly to 25,26-(OH)2Dz except that plasma from vitamin D3-toxic pigs was used. The mass spectrum (Fig. 6) agreed well with that of chemically synthesized 25,26-(OH)2Dz (22). Further confirmation was achieved from the uv spectrum of each metabolite showing the cis-triene chromophore (absorption maximum at 264 nm and minimum at 228 nm), which is typical of vitamin D and its metabolites (Fig. 7). The compound 25OHD,-26,23 lactone was prepared from plasma taken from vitamin D3toxic pigs, as previously described (23).

VITAMIN

0

D METABOLITE

4

8

12

193

QUANTITATION

16

20

24

28

32

ml FIG. 2. The uv and competitive protein-binding analysis of material isolated from vitamin D,-toxic pig plasma after hplc on a Zorbax Sil column (0.45 X 25 cm) developed in 8:92 isopropanol:hexane at a flow rate of 2.0 ml/min. Collection of 2-ml aliquots of column eluant for 24,25-dihydroxyvitamin Dlike binding activity was begun at the time of sample injection. The hplc analysis was performed after lipid extraction of the plasma and prepurification on two Sephadex LH-20 columns as described in the text. The elution positions of 25-hydroxyvitamin D, and 24,25-dihydroxyvitamin D1 are indicated.

Dz, which was synthesized as previously reported (24). The product recovered from the reaction mixture was 90% pure as determined by hplc analysis on a Zorbax Sil ODS column (0.46 X 25 cm, DuPont) eluted with 5:95 water:methanol at a flow rate of 2.0 ml/ min. The [3cu-3H]-vitamin D2 was purified to homogeneity by hplc before use. The [ 3a-3H]-vitamin D, metabolites were

Preparation of [3H]-Vitamin D3 and [3H]Vitamin D: and Their Metabolites The [3H]-vitamin D3 metabolites used in this analysis were prepared as previously described (7,8,23). The compound [ 3a-‘HIvitamin Dz (2.3 Ci/mmol) was prepared by the sodium boro[ ‘HIhydride reduction of the a-tricarboxyliron complex of 3-ketovitamin

1.6

IO

20

30

40

50

60

70

1

ml FIG. 3. The uv and competitive protein-binding analysis of the l2- 18 ml fraction (see Fig. 2) combined and rechromatographed by hplc on a Zorbax Sil column (0.45 X 25 cm) developed in 2:98 isopropanol:methylene chloride at a flow rate of I .5 ml/min. Collection of I .5-ml aliquots of column eluant for 24.25-dihydroxyvitamin D-like binding activity was begun at the time of sample injection. The elution positions of 25.hydroxyvitamin D, and 24,25-dihydroxyvitamin DJ are indicated,

194

tlORST

118

100

ET AL.

136

150

200

250

300

350

m/e F'IG. 4. Mass spectrum of 24,25-dihydroxyvitamin Dz (peak I, Fig. 3) obtained from vitamin D1treated pigs. The molecular ion at nz/e 428 and peaks at m/e 27 I (Mf. side chain), 253 (27 I. HZO). I36 (A rings + carbons 6 and 7), and I I8 (I 36. H,O) establish the structure as a side-chain dihydroxylated vitamin Dz compound. This conclusion is reinforced by the peak at m/e 370. which corresponds to cleavage between carbons 24 and 25. resulting in the loss of carbons 25-27 from the parent molecule.

produced in vivo in vitamin D-deficient rats given [ 3cu-‘HI-vitamin D, (2.3 Ci/mmol) orally. A total of 10 rats were given 0.2 mCi of [3H]-vitamin D2 per day for 3 successive days. Twenty-four hours after the last dose, the rats were exsanguinated. The plasma lipids were extracted ( 16) and the [‘HI-vitamin Dz metabolites separated on a 1 X 60-cm Sephadex LH-20 column developed in 9: 1: 1 hexane:chloroform:methanol. The identity of the [3H]-25-OHDz, [3H]-24,25-(OH)zD2, and [‘HI-25,26-(OH),D2 was confirmed by comigration with original standards on hplc. Each metabolite was purified to homogeneity by hplc before use.

Animals

and Blood Samples

The age and species of blood donors are given in Table 1. All the animals received supplementation to meet National Research Council requirements for calcium, phosphorus, and vitamin D (as vitamin D,). The chickens and turkeys were randomly selected from the laying flocks. The pigs, cattle, and sheep were nonlactating, nonpregnant females. The turkeys and chickens were housed inside, with no exposure to direct sunlight. The sheep, cattle, and pigs were housed outside on concrete or in a drylot, with access to sheltered areas for protection from bad

-H20, I’ 118

FIG. 5. Mass spectrum of 25.26-dihydroxyvitamin D1 (peak II, Fig. 3) isolated from vitamin Dztreated pigs. The molecular ion at m/e 428 indicates a dihydroxylated vitamin Dz derivative. The peaks at m/e 271 (M’, side chain) and 253 (271, H,O) demonstrate that the hydroxy groups are on the side chain, whereas the peaks at m/e 136 and I I8 are characteristic of the vitamin D cis-triene structure.

VITAMIN

D METABOLITE

195

QLJANTITATION

1 367

416

253

365

FIG. 6. Mass spectrum of 25.26.dihydroxyvitamin D, isolated from vitamin D,-treated pigs. The intense molecular ion at m/e 416 is diagnostic of a dihydroxylated vitamin D, derivative. Loss of 31 amu from the molecular ion (m/e 385) indicates that a primary alcohol is present. and the peaks at m/e 253 and 27 I show clearly that the hydroxy groups are in the side chain. The base peak at m/e 136 and the large peak at m/e I 18 ( 136, H,O) are characteristic of the vitamin D cis-triene structure. This spectrum is virtually identical with that of synthetic 25.26-dihydroxyvitamin D, (19).

weather. Blood samples (30 ml) were taken during October from five adult animals from each of the five species, making a total of 25 blood samples. The blood was taken in heparinized ( IO W/ml of blood) syringes, and the red cells were separated from the plasma in a refrigerated (4°C) centrifuge. The plasma was harvested and stored frozen (- 1S°C) until analyzed. Assay

Procedures

Figure 8 shows a flow diagram for the multiple assay procedures. A well-mixed plasma sample was placed in a 50-ml screwcap tube. To each plasma sample (3-5 ml) and to counting vials (in duplicate) was added 1000 cpm of the following radioactive standards for monitoring of the analytical recoveries of the assay: [3H]-vitamin D3, [ ‘HI-25OHD3, [3H]-25-OHD2, [ 3H]-24,25( OH)?D3, [ 3H]-24,25-(OH)2Dz, [ 3H]-25,26(OH)?D3, [ ‘HI-25OHD3-26,23 lactone, and [ 3H]- 1,25-(OH),D;. After vortex mixing of the plasma samples, the lipids were extracted by adding 3.0 volumes of peroxide-free ether, capping the tube, and shaking it horizontally for 5 min at 120 oscillations/min. After shaking, the aqueous layer was allowed to separate for 1 to 2 min
layer was poured into a 25 X 150-cm tube for drying. The aqueous layer was thawed and the above procedure repeated. The final extraction involved denaturing the plasma proteins by adding 4 volumes of I:3 methanohmethylene chloride and shaking for 3 to 5 min. This step generally resulted in an emulsion, which was broken by adding 1 volume of methanol. The tube was shaken again for 15 s. The upper aqueous layer was removed by aspiration and the lower methylene chloride layer was washed twice with 0.1 M phosphate (pH 10.5). The washed methylene chloride layer was combined with 6

oJ l’l 300

280

260

240

220

WAVE LENGTH (nm) FIG. 7. The uv scans taken in ethanol of 25,26-dihydroxyvitamin Dz (&). 24,25-dihydroxyvitamin D, (---), .and 25.26.dihydroxyvitamin D, (~ - -) isolated from prg plasma.

196

HORST

TABLE AGES

ANU

Species

OF BLOOD Age

DONORS (months)”

Turkey Chicken

IO-12 IO-12

cow Sheep

48-72 36-48

Pig

36-60

animals

per

age group

within

AI

7.0-ml fraction) was collected as fraction 2 (25-OHD); and finally 12.0 ml (7.0-I 9.0-ml fraction) was collected as fraction 3 (the dihydroxylated metabolites of vitamin D and 25-OHD3-26,23 lactone).

I

Species

’ Five

ET

Resolution and Quantitation of the Three Vitamin D and Vitamin D Metabolite Fractions

each

species,

Fraction 1 (vitamin D). The fraction containing the vitamin D was dried under N, and chromatographed on a Lipidex 5000 (Packard) column (0.6 X 14.5 cm) in 5:95 chloroform:hexane. The sample was applied in 0.5 ml of column solvent, which was followed by a 0.5-ml wash. Thereafter, the first 6.0 ml was discarded. The next 4.0 ml of column eluant was collected, and it contained the vitamin D. The eluant from the Lipidex 5000 column containing the vitamin D was dried under N2 and prepared for hplc by the addition of 150 ~1 of 0.25:99.75 isopropanol:methylene chloride. The sample was chromatographed on a Zorbax Sil column (0.45 X 25 cm) de-

the ether fraction and dried under Nz. This final 1:3 methanokmethylene chloride extraction was needed to facilitate the removal of 25OHDJ-26.23 lactone and 25OHD. The resulting lipid extract was chromatographed on a 0.6 X 15.5-cm Sephadex LH20 column developed in 1: 1:9 chloroform:methanol:hexane (Fig. 9). The column eluant was divided into three fractions (vitamin D, 25-OHD, and dihydroxyvitamin D). After the lipid residue was applied with two 0.5-ml washes, 2.5 ml of solvent was added to the column and collected as fraction 1 (vitamin D). The next 4.5 ml (2.5-

PLASMAi3-5mlIt3H

METABOLITES 1

DIETHYLETHER (2x1 t METHANOL METHYLENE CHLORIDE II 31 I BACKWASH w/OIb NoPo4lpHlO 51

II I9

t SEPHADEX LH-20 CHLOROFORM METHANOL

1 YlTAMlN D t LlPlDEX 5000 15 95CHtOROf~:HEXANE

(025

1

t I

HPLC ON ZOABAX SIL 99.75 ISOPROPANOL:METHYLENECHtOFIDEl

WANTlTATE

HEXANEI

1 25.OHD SEPHADEX LH-20 150 50 HEXANE CHLOROFORM 1 OR LlPlDEX 5000 11090 CHLOROF+ORM HEXANEI HPLCON ZORGAY Sit I4 96 ISOPROPANOL HEXANE t QUANTITATE BOTH 028D3 FORMS BY"" ABSORBANCE

BOTH Dz8DI

Ill

DlHYDROXYVlTAMlN D t HPLC ON LOREAX SIL 89 ISOPROPANOL 'HEXANEI

OUANTITATE BOTH D28Ds FORMS SIMULTANEOUSLY BY COMPETITIVE PROTEIN BINDING ASSAY

1

I

I 9UbNTlTATE BOTH @D, FOAMS SIMULTANEOUSLY BY CDMPETITIVE PROTEIN BINDING ASSAY

1 HPLC ON ZDREAX SIL (2 98 ,SOPRDPANOL:METHYLENE t 25-0HD1-26,23

LACTONE

t 24.25.IOH12D1

I Q"ANT,TATED

FIG. 8. Flow diagram D and its metabolites.

of the

purification

and

ultimate

,ND,",D"ALLY

quantitative

"SING

t COMPETITIVE

steps

CHLORIDE1 t 24,25-10H12D, I PROTEIN

used

25,26-10H12D2 iI BINDING

for

ASSAY

the assay

of vitamin

VITAMIN

D METABOLITE

197

QUANTITATION

o(&y-y&, , , , , / 0

2

FIG. 9. The their 20

metabolites column

4

6

8 IO 12 14 I6 I8 2u 25 30 35 40 45 ELUTION VOLUME(ml)

elution from developed

of vitamin DZr vitamin D3. and a 0.6 X 15.5~cm Sephadex LHin

I:]:9

FIG.

a hplc

0 2

4 6 8 IO 12 I4 I6 I8 20 22 24 26 28 ml

IO. Elution Zorbax Sil

of vitamin D2 and vitamin D, column (0.45 X 25 cm) developed

0.25:99.75 isopropanol:methylene of 2.0 ml/min.

chloride

at a flow

from in rate

chloroform:metha-

nol:hexanc.

veloped in 0.25:99.75 isopropanol:methylene chloride with a flow rate of 2.0 ml/min. Vitamin D, and vitamin D3 comigrated in this system and eluted between 7 and 9 min (Fig. 10). The vitamin D fraction was dried under Nz, and the vitamin Dz and vitamin D3 were quantitated individually by reversephase chromatography as previously described (8,25). Alternatively, as depicted in Fig. 8, the vitamin D from the Zorbax Sil column developed in isopropanol:methylene chloride (0.25:99.75) could be measured directly by a competitive protein-binding assay with a l/50,000 dilution of sheep pl.asma in 0.01% bovine serum albumin or gelatin and [jH]-25-OHD, as radioactive tracer. When samples contained ~1 .O rig/ml either vitamin Dz or vitamin D3, this method was utilized. Further details and verification of this competitive protein-binding assay are described in another publication (26). Fraction 2 (25-OHD). Methods for purification and quantitation of 25-OHD, and 25-OHD3 were previously described (7,8,16). Fraction 3 (dihydroxyvitamin D metabolites and 2.5-OHDj-26.23 lactone). The dihydroxyvitamin D metabolite-containing fraction from the initial Sephadex LH-20 column was subjected to hplc on a Zorbax Sil column developed in 11:89 isopropanol:hexane at a flow rate of 2.0 ml/

min. The elution profile of the metabolites is shown in Fig. 11. The 24,25-(OH)zD complex, consisting of 24,25-(OH)2D2, 24,25(OH)*D,, 25,26-(OH)?D2, and 25-OHD,26,23 lactone, migrated in the 8.0-l 1.5 ml region of this column, whereas 25,26(OH),D, and 1.25-(OH)zDj migrated in the 16- 18 ml and 26-30 ml regions, respectively. For complete resolution of the four metabolites in the 24,25-(OH)zD complex, the fraction was collected and resubjected to hplc on a Zorbax Sil column (0.45 X 25 cm) developed in 2:98 isopropanol:methylene chloride at a flow rate of 1.5 ml/min. This hplc system resolved the 24,25-(OH),D complex into 25-OHD,-26,23 lactone ( 1822 ml), 24,25-(OH)*D* (38-43 ml), 24,25(OH)2D3 (57-63 ml), and 25,26-(OH),D, (64-70 ml) (Fig. 12). These metabolites could also be resolved by reversephase hplc on a Zorbax ODS column developed in 25:75 co4 5 003 5 @ 002 s

OCII ‘3 I 0

4

FIG. I I. Elution metabolites from cm) developed of 2.0 ml/min.

8

12

I6 ml

of polar vitamin hplc Zorbax Sil

in I I :89 isopropanol:hexane

20

24

D, and column

28

32

Dz

vitamin (0.45

X 25

at a flow

rate

198

HORST 004

1

J

25.0HD3-26.23

0

LACTONE

8 25-O,+

5 IO I5 20 25 30 35 40 45 50 55 60 65 70 75 ml

FIG. 12. Elution of polar vitamin D, and vitamin D, metabolites from hplc Zorbax Sil column (0.45 X 2.5 cm) developed in 2:98 isopropanol:methylene chloride at a flow rate of I.5 ml/min.

water:methanol at a flow rate of 2.0 ml/mm (Fig. 13). We have had difficulty in evaporating the samples for preparation in the binding assays because of the excessive amounts of water in the eluant. Also, the recoveries of the metabolites were usually 1530% lower with this system than with the previous one. Radioligand binding assays for dihydroxy metabolites and 2.5-OHD,-26,23 lactone. After their final purification, all the metabolites to be measured by competitive protein-binding assays were collected from the hplc column individually in 13 X lOOmm culture tubes. The solvent was dried under Nz, and 200 ~1 of 100% ethanol was added to each tube. Three 25~1 fractions were taken for binding analysis, and two 25 ~1 fractions were taken for recovery estimates. The 1,25-(OH),D was quantitated by a modification of the procedure of Eisman et al. ( 14) as previously described (7,8). The 24,25-(OH)zD3, 24,25-(OH),D,, 25,25,26-(OH)2D2, and 25WOWJL OHD,-26,23 lactone were measured by radioligand binding assays with rat plasma vitamin D-binding protein. The rat plasma was diluted l/5000 (v/v) in 0.05 M phosphate buffer (pH 7.5) containing 0.01% gelatin and 0.01% merthiolate (PBG buffer). Each assay mixture was placed in a 12 X 75-mm borosilicate glass tube and consisted of (1) 0.5 ml of l/5000 dilution of vitamin D-deficient rat plasma in PBG

--I21

AL.

buffer, (2) 6000-8000 cpm of [23,24-3H]25-OHD, in 20 ~1 of 100% ethanol, and (3) standards or unknowns in 25 ~1 of ethanol. Standard curves for 24,25-(OH)2D2, 24,2525,26-(OH),D,, and 25,26(OHM&, (OH),D, were constructed over the range 0.1-6.4 ng. The standard curve for 25OHDj-26,23 lactone was constructed over the range 0.05-3.2 ng. After 1 to 2 h incubation of standards and unknowns at 4°C bound steroid was separated from free by adding 0.2 ml of a mixture of cold 1.0% Norite A charcoal and 0.1% Dextran T-70 in PBG buffer to each tube. After 30 min at 4°C the tubes were spun at 1OOOgfor 10 min in a refrigerated centrifuge. A portion (0.5 ml) of the supernatant was removed for quantitation of the bound [ ‘HI-25-OHD3. RESULTS

Extraction

AND

DISCUSSION

of Plasma

Repetitive extractions with peroxide-free diethylether followed by plasma protein denaturation with 1:3 methanol:methylene chloride yielded recoveries of 90% or more of [3H]-vitamin D3, [3H]-vitamin Dz, and their [ 3H]-metabolites. The diethylether alone was insufficient to remove the 25OHD,-26,23 lactone and 25-OHD, from the plasma (generally ~20% of each metabolite was removed from cow and sheep plasma), hence the requirement for the protein denaturation step. In our laboratory, recoveries

I

"1 003

25-D”D,-26.23 LACTDNE

1

0

4 8

12 16 20 24 20 32 36 40 44 48 52 56 60 64 ml

FIG. 13. Elution of polar vitamin D2 and vitamin D, metabolites from hplc Zorbax ODS column (0.45 X 25 cm) developed in 25:75 water:methanol at a flow rate of 2.0 ml/min.

VITAMIN

D METABOLITE

of 1,25-(OH),D were more consistent and higher with this extraction procedure than with methylene chloride:methanol ( 1:2) alone. Resolution and Quantitation Metabolites

of Vitamin

199

QUANTITATION

2500

D

Vitamin D and 25-OHD. With our extraction procedure, the plasma lipid extract could be suitably prepared for hplc-uv analysis or competitive protein bindng analysis of vitamin Dz, vitamin D3, 25-OHD,, and 25-OHD3, as previously described (7,8,16,25,26). The recoveries of [3H]-vitamin D3, [3H]-25-OHD,, and t3H]-25-OHD3 in the 25 plasma samples assayed for this report were (X rfr SD) 72.1 rt 10.6, 68.5 k 3.0, and 68.3 +- 5.2, respectively. The least detectable concentration of each sterol was 1 rig/ml and 0.2 ng if the competitive protein binding assay was used. 24.25-(OH)?D Complex, 25,26-(OH),D,, and 1,25-(OH)?D. The 24,25-(OH)zD complex, which includes 25-OHD,-26,23 lactone, 24,25-(OH)2D2, 24,25-(OH),:D,, and 25,26-(OH)*D*, was separated from 25,26(OH)2D3 and 1,25-(OH),D by hplc on a Zorbax Sil column developed in 11:89 isopropanol:hexane with a flow rate of 2.0 ml/ min. The 25,26-(OH)zDJ and 1,25-(OH)*D were collected for analysis by radioligand binding assays. The least detectable concentrations of these two metabolites were 0.2 rig/ml and 7 pg/ml, respectively. Resolution of the 24,25-(OH)*D complex into its four individual metabolites required thle introduction of a new solvent system heretofore undescribed in the vitamin D literature. The solvent system (2:98 isopropanol:methylene chloride) coupled with the Zorbax Sil column provided the proper chemistry and selectivity to separate 25-OHDj-26!23 lactone, 24,25-(OH)2D2, 24,25-(OH)*D3, and 25,26-(OH)>D? into individual and conveniently collectable fractions for quantitation by radioligand binding assay, The recoveries of these metabolites for the 25 samples assayed for this report were (X f SD): [3H]-

500 I,,,

0

01

I

I

02

04

I

08

I

I

16

32

$4

ng 24,25-(OHl,D, FIG. 14. Inlluence of the addition rat plasma competitive protein-binding dihydroxyvitamin D,. See text for NSB, nonapecitic binding.

of gelatin on the assay for 24.25assay conditions.

25-0HD3-26,23 lactone, 48.5 k 6.2; 24,25(OH),Dz, 63.4 -+ 5.6; [ 3H]-24,25-(OH)zD3, 52.4 :k 8.1; 25,26-(OH),D,, 61.6 -t 8.7; [ 3H]-25,26-(OH),D,, 63.7 t 8.6; and [-‘HI1,25-(OH)*D7, 81.3 k 10.5. The radioligand binding assay buffer (PBG buffer) was modified from previous reports (7,8,16). The phosphate buffer (pH 7.5) was modified to contain 0.01% gelatin, and, to prevent bacterial growth, we added 0.01% merthiolate. The addition of the gelatin resulted in higher specific binding (Fig. 14) at the l/5000 dilution of rat plasma/PBG buffer (v/v) used in this assay. Figure 15 shows the binding curves for each metabolite quantitated with the rat plasma binding protein. As previously described (7,8,16), 24,25-(OH)>D3 and 25,26(OH):zD, were equipotent at displacing [‘HI25-OHD3 from rat plasma binding protein. Also, as previously described ( 15) 25-OHD,26,23 lactone was five times more potent at displacing [ 3H]-25-OHD, than any other metabolite. Heretofore, standard preparations of 24,25-(OH)2D2 and 25,26-(OH),Dz were unavailable for testing in our radioligand binding assay. We have isolated these two vitamin Dz metabolites from vitamin DZtoxic pig plasma, and their isolation has led to the observation that they are only onehalf lto one-third as potent at displacing [ 3H]-:25-OHD, from the rat plasma binding

200

HORST

ET AL.

100

O--O 24,25-iOH1203 m- -4 25,x-(ON* 03 A-4 25-OH03 H 25-OHOz-26,23 LACTCM 0-O 24,25 (OH1202 A-A 25,26-~oHl~ o*

90 80 70 60 co$50

40 30 20 IO 0 0

01

02

0.4

0.8

1.6

32

64

ng OF METABOLITE FIG. petitive

15. Competitive protein-binding

displacement of vitamin D2 and vitamin assay. See text for assay conditions.

as their D3 counterparts, 24,25-(OH)ZD3 and 25,26-(OH)*Dj (Fig. 15). A similar finding was observed when 24,25-(OH)2D2 was compared to 24,25-(OH)*D3 in the rat plasma competitive protein binding assay (27). This is rather surprising, since 25-OHD? and 25-OHD3 have been shown to be recognized equally by the rat plasma binding protein (28). Therefore, the assumption that 24,25-(OH)2D2 and 24,25-(OH)zD3, as well as 25,26-(OH)*D* and 25,26-(OH)zD3, are indistinguishable by the rat plasma binding protein (7,8,16,18) is in error. In view of the above results, 24,25(OH)*D3 and 25,26-(OH)2Dj could be quantitated in a common assay with 24,25(OH),D, used for constructing the standard curve. However, the other metabolites, 25OHD,--26,23 lactone, 24,25-(OH)2D2, and 25,26-(OH)*D2, could be quantitated only when measured against standards prepared from each metabolite; i.e., 25-OHD,-26,23 lactone was measured against standard preparations of 25-OHD3-26,23 lactone, 24,25(OH)*D2 against standard preparations of 24,25-(OH)*D*, etc. By using this assay approach, we found that the lower units of detection of metabolites in the 24,25-(OH)zD complex were: 25-OHD,-26,23 lactone, 0.05

Dj metabolites

in the rat plasma

com-

rig/ml; 24,25-(OH)2D2, 0.3 rig/ml; 24,25(OHLD3, 0.2 rig/ml; and 25,26-(OH)zD2, 0.35 rig/ml. Plasma concentrations of vitamin Dz, vitamin Dj, and their metabolites in farm animals. The plasma concentrations of vitamin Dz, vitamin D3, and their metabolites in normal adult turkeys, chickens, cows, sheep, and pigs are shown in Table 2. There were considerable differences between species as to the major circulating forms of vitamin D. Vitamin Dz, for example, was undetectable by competitive protein binding assay (26) in plasma of all the animals, with the exception of sheep. Vitamin Dj, however, was present in all animals. The observation that vitamin DZ was the major form of vitamin D in sheep was interesting because the sheep were continuously exposed to sunlight and also were given a vitamin Dj supplement, but they still had vitamin D2 and 25-OHD, as their major circulating form of vitamin D and 25-OHD. The vitamin DZ in natural foodstuffs appears to supply the majority of vitamin D to sheep, as the heavy coating of wool and lanolin make the sheep apparently inefficient utilizers of the photochemical conversion of 7-dehydrocholesterol to vitamin D,. In contrast, the cows also were continuously exposed to sun-

VITAMIN

D METABOLITE TABLE

VITAMIN

D, AND

VITAMIN

D,

2

METABOUTES

A.ND THEIR

201

QCJANTITATION

IN FIVE SPECIES OF ANIMALS Species

Sterol

Turkey

Vitamin D:” (rig/ml) Vitamin indml)

ND

cow

ND

Sheep

ND

0.52

Pig

+

0.35

ND

D,

25-OH Dz (w/ml) 25-OHD, (w/ml)

3.4 2

0.2

2.3 t 0.3

I.7 *

0.7

0.6 +

0.5

10.2 k

4.1

0.8 k

0.7

1.6 k

5.8 *

1.7

13.9 i

3.7

0.8 If

0.3

18.2 k

7.9

*

7.1

12.9 k

5.3

0.1 f

0.2

6.4 +

I.0

2.6 +

1.3

4.4 k

0.7

0.5 *

0.2

1.0 *

0.3

24.25m(OH)zDzh (rig/ml)

I.??

ND

?5,26-(OH)*D, (w/ml)

0.6 k

1.5

k 3.9

36.9

ND

0.1

25%(OH)>D>” (w/ml)

26.23

25.3

ND

24,25-(OH),D, (w/ml)

25-OI1D9 (rig/ml)

Chicken

2.3 + 0.2

ND

0.4

ND

I.1 *

0.2

2.5 *

1.5 f

0.5

ND

1.0

74.9

*

21.2

ND

20.2

t

10.3

ND

4.1 *

0.3

2.0 -+

I.2

lactone’ ND

ND

1.25.(OH)lD 51.8

(pg/ml) ’ ND

(not determinable)

= ~0.2

+ 31.3

21.2

rig/ml.

b ND

i

2.1

38.0

= 10.2

light and received dietary vitamin D, supplementation, but they had vitamin D, and 25-OHDJ as their major circulating form of vitamin D and 25-OHD. Vitamin I), from the cow feedstuffs did, however, contribute to the vitamin D pool, since plasma 25 OHD2 concentrations were in the range 610 rig/ml. Therefore, it appears that continuous exposure of a species of animal to sunlight is not a sound basis for assuming that animals will have high circulating levels of vitamin D3 as a result of enhanced conversion of 7-dehydrocholesterol to vitamin D3. In combined data from all species, there was significant (p < .05) positive correlation (r = 0.80) between the plasma concentrations of vitamin D and 25-OHD. The rela-

q/ml.

t

10.4 ’ ND

35.9 = ~0.1

i

30.0

60.3

r

7.2

rig/ml

tionship followed the regression line y = 5.03(x) + 18.4, where y equals the plasma 25-OHD concentrations. There were, however, apparent species differences in the conversion of 25-OHD to 24,25-(OH),D or turnover rates of 24,25-(OH),D. The relationship between 25-OHD and 24,25(OH),D concentrations in turkey, cow, and chicken plasma followed the regression line y = 0.08(x) + 0.15 (r = 0.80, p < .05), where y equals the plasma 24,25-(OH)2D concentrations. The same relationship in sheep and pig plasma, however, followed the regression line y = 0.18(x) + 5.4 (r = 0.71, p < .05). The compound 25-OHD,-26,23 lactone (lactone) is a recently discovered vitamin D, metabolite (9) that has no known physiolog-

202

HORST

ical function. Horst and Littledike ( 10) have shown that the kidney is required for the bioproduction of lactone. Of the five species tested in our experiment, only the chicken and pig had detectable plasma lactone. Other species, such as the cow, have been shown to produce this metabolite only when massiveamounts of vitamin D3 were injected intramuscularly (23). DeLuca (1) has postulated that 25,26-(OH)ZD3 is the immediate precursor to lactone. If this is indeed the case, then it is interesting that chickens with plasma 25,26-(OH)2D concentrations of 1.1 & 0.2 rig/ml had detectable plasma lactone, whereas old cows with 25,26-(0H)2D) concentrations of 2.50 ? 0.95 had lactone below the detectable limits of the assay. This could indicate a high affinity of 25,26-(OH)*D for the lactone enzyme(s) in chickens and pigs or that someother vitamin D metabolite acts as precursor to the lactone. Recently, Napoli and Horst (submitted for publication) have shown that 25,26-(OH)2D) is a doubtful precursor to lactone. The presence of lactone in plasma, however, does represent an important analytical problem. Lipid extracts “purified” for 24,25(OH)*D analysis with Sephadex LH-20 and 35:65 hexane:chloroform as eluting solvents (l&29) have the potential of being contaminated with lactone (23). A similar problem exists, but to a lesser extent, in preparation of 24,25-(OH),D for analysis by hplc on Zorbax Sil columns developed in lo:90 isopropanol:hexane (7,8,16). The addition of the Zorbax Sil column developed in 2:98 isopropanol:methylene chloride represents a powerful system for resolving 25-OHD326,23 lactone from 24,25-(OH)zD. The samples must, however, be free of 25-OHD3, since this metabolite comigrates with 25OHD,-26,23 lactone in this system (Fig. 12). For determination of total 24,25(OH)*D activity, 24,25-(OH)*D2 standards must be available, since this compound is completely resolved from 24,25-(OH)zD3 (Fig. 12). The compound 25,26-(OH)?D3 was found

ET AI

in the plasma of all species, but 25,26(OH)zDz was found only in sheep. There was a significant (p < .05) correlation (r = 0.85) between the 25-OHD and 25,26-(OH),D concentrations when the data from all species were combined. The relationship followed the regression line y = 0.06(x) - 0.39, where y is the plasma 25,26-(OH)zD concentration. Unlike the 24,25-(OH),D-to-25OHD ratio, the 25,26-(OH)2D-to-25-0HD ratio was very consistent between species. Therefore, the concentrations of substrate (25-OHD) for 25,26-(OH)*D synthesis appear to have more influence on the plasma concentrations of 25,26-(OH),D than other factors such as plasma parathyroid hormone (PTH), Pi, or Ca concentrations. The compound 1,25-(OH),D is an active form of vitamin D (1). It stimulates Ca and Pi absorption from the intestine alone and Ca and Pi resorption from bone in the presence of PTH ( 1). The plasma concentrations of this metabolite are under the control of PTH and possibly Pi. High concentrations of PTH and low concentrations of kidney Pi have been shown to stimulate the lcu-hydroxylase (1,30). Of all the animals, the pigs had the highest plasma concentrations of l,25-(OH)zD, whereas the adult chicken had the lowest plasma concentrations of 1,25-(OH),D. CONCLUSIONS

We have demonstrated multiple assay techniques for the separation and quantitation of vitamin Dz and vitamin D3 and their major metabolites in plasma from several speciesof farm animals. A major emphasis of this report was the demonstration that extensive purification is needed to measure 24,25-(OH)2Dj accurately in plasma samples. In procedures previously described (7,8), it is highly probable that 24,25(OH),D, fractions were contaminated with 25-OHD,-26,23 lactone, 24,25-(OH)2D2, and 25,26-(OH),Dz. Furthermore, the assumption that 24,25-(OH)2Dz and 25,26-

VITAMIN

D METABOLITE

(OH)2D2 bind to the vitamin D,-binding protein equally as well as their D3 counterparts is invalid. Therefore, separation and individual quantitation of these metabolites are required before their blood concentrations can be determined accurately. Through the use of our multiple assay techniques, we have shown that I( 1) 25OHD3-26,23 lactone was present only in chick and pig plasma, (2) unsheared sheep were not efficient utilizers of the photochemical conversion of vitamin D3 to 25-OHD3, and (3) the conversion of 25-OHD to 24,25(OH)2D was more efficient in some farm animal species than in others.

The authors gratefully acknowledge the fine technical assistance of Mrs. Cynthia Hauber and Mrs. Rebecca Lyon. They would also like to acknowledge the assistance of Dr. Peter J. Matthews in collecting blood samples from the animals.

REFERENCES I. DeLuca, H. F. (1979) Nutr. Rev. 37, 16lll93. 2. Garabedian, M.. Pavlovitch. H.. Fellot. C.. and Balsan. S. ( 1974) Proc. Nat. Acad. Sri. USA 71, 554-557. 3. Norman, A. W. (1979) Vitamin D: The Calcium Homeostatic Steroid Hormone, Academic Press. New York. 4. Kodicek. E. (1974) Lancer 1, 325-329. 5. Napoli, J. L.. and DeLuca. H. F. (I 979) in Berger’s Medicinal Chemistry (Wolff. M. E., ed.), 4th ed., Part II. pp. 705-750, Wiley, New York. 6. Haussler. M. R., and McCain, T. A. (1977). Nens Eng.

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974-983.

7. Worst. R. L.. Shepard. R. M.. Jorgensen, and DeLuca, H. F. ( 1979) J. Lah. Clin. 93, 2777285. 8. Shepard. R. M., Horst, R. L., Hamstra. A. DeLuca. H. F. (1979) Biochem. J. 182, 9. Wichman, J. K.. DeLuca. H. F., Schnoes.

Horst,

N. A., Med.

IO. Hoist,

55-69.

H. K.,

R. M.,

Biochemistry

E. T. (1980) Biochem. I49- I 54. H. F., Schnoes. H. K., and Blunt,

Res.

Commun.

90,

I I. Suda, T., DeLuca. J. W. (1969) Biochemistry 12. Jones, G.. Schnoes, H. K., 13.

14. IS.

16.

18.

and Jorgensen,

18, 4775-4780.

R. L.. and Littledike,

Biophys.

8, 3515-3520.

and DeLuca. H. F. ( 1975) Bioc~hemisrry 14. 1250 1256. Jones. Cl.. Rosenthal. A.. Segcv. D.. Mazur, Y., Frolow, F., Halfon. Y., Rabinovich, D.. and Shakked. Z. (1974) Biochemistry 18, lO941101. Eisman, J. A., Hamstra. A. J., Kream, B. E.. and DeLuca, H. F. ( 1976) Science 193, IO2 I 1023. Lambert, P. W., Syverson, B. F., Arnaud. C. D., and Spelsberg, T. C. ( 1977) J. Sleroid Biochem. 8, 929-937. Horst. R. L., Shepard. R. M., Jorgensen, N. A.. and DeLuca. H. F. ( 1979) Arch. Biochem. Biophys. 192. 512 -523. Lumb. Cl. A.. Mawer. E. B., and Stanbury. S. W. (1971) .4mrr. J. Med. 50, 421-441. Haddad. J. G., Min. C.. Mendelsohn, M.. Slatopolsky, E.. and Hahn. T. J. ( 1977) .4rch. Biochem. Biophys.

19. Taylor,

182,

390

C. M., Hughes,

Biochem.

Biophys.

39.5.

S. E., and desilva. Res.

Commun.

89.

P. ( 1976) 286-293.

20. Jones, G. (1978) Clin. (‘hem. 24, 287-298. 21. Horst, R. L., Littledike, E. T., Gray, R. W.. and Napoli, J. L. (1981) J. C/in. fm’est. 67. 274-280. 22. Suda. T., DeLuca, H. F.. Schnoes. H. K.. Tanaka. Y., and Holick, M. F. (I 970) Biochemistry 9, 4776-4780. 23. Horst, R. L. (1979) Biochem. Biophys. ReS. Cornmun. 89, 286-293. 24. Barton, D. H. R., and Datin. H. R. (1976) J. Chem. !joc. Perkin I 829-83 I. 25. Horst, R. L.. and Littledike. E. T. (1979) J. DairJa !ici. 62, l746- I75 I. 26. Horst. R. L.. Reinhardt, T. A.. Beitz, D. C.. and ILittledike. E. T. (1981) Steroids 37, 581-591. 27. Jones. G.. Byrnes, B.. Palna. F., Segev, D.. and Mazur, Y. (1980) J. Clin. Endocrinol. Metah. so, 7733775. 28. Preece. M. A.. O’Riordan. J. L. H., Lawson. D. E. M., and Kodicek. E. (I 974) C/in. Chem. .Acta

J.. and

R. L., Shepard,

N. A. (1979)

I 7.

ACKNOWLEDGMENTS

203

QUANTITATION

29.

Hollis. (1977)

30. Gray,

54,

235-242.

B. W..

Burton,

J. H., and Draper,

30, 285-293. R. W. ( I98 I ) Fed. Proc.

H. Ii.

Sferoids

40, 899 (Abstract).