European Journal of Clinical Investigation (1979) 9,433-438

Transport of vitamin D sterols in human plasma: effect of excess vitamin D, 25 hydroxyvitamin D and 1,25 dihydroxyvitamin D JUSTIN SILVER & MENAHEM FAINARU, Lipid Research Laboratory, Department of Medicine B, Hebrew University Medical School, Hadassah Hospital, Jerusalem, Israel Received 8 January 1979 and in revised form 3 July 1979

Abstract. Vitamin D and its more active metabolites, 25 hydroxyvitamin D (25-OH-D) and 1,25-dihydroxyvitamin D (1,25-(OH)z-D), are transported in human plasma on a specific binding protein (DBP), which has been shown to have an cr-globulin electrophoretic mobility. Since the concentration of DBP in normal human plasma is approximately 5 pmol/l, whereas that of all the vitamin D metabolites is less than 0.2 pmol/l, DBP is less than 3% saturated under physiological conditions. We have studied the transport of the above-mentioned metabolites in human plasma in vitro at normal and saturating concentrations. Human plasma was incubated with increasing amounts of vitamin D metabolites together with their radiolabelled tracers. Ultracentrifugation was used to isolate plasma lipoproteins (density, d < 1.21 g/ml) and agarose gel electrophoresis of lipoprotein-free plasma ( d > 1.21 g/ml) to separate DBP ( a globulin) from albumin. The recovery of the tracer in plasma proteins was always more than 80%. At physiological concentrations [3H]25-OH-Dbound almost exclusively to DBP (98%), [3H]vitamin D or ['4C]vitamin D bound both to DBP and to lipoproteins (40%), and L3H]1,25-(OH)z-D bound to DBP (62%), to lipoproteins (1 5%) and also to albumin (23%). When the concentration of vitamin D metabolites was increased, DBP became saturated. The binding capacity of DBP was similar for all three sterols, about 5 pmol/l plasma, or one mole of sterol per mole of protein, but the saturating concentration was different for the three sterols (vitamin D > 1,25-(OH)2-D> 25-OH-D). 25-OH-D had the greatest affinity for DBP, and it completely displaced both vitamin D and 1,25(OH)z-D from DBP at higher concentrations. All sterols bound to both plasma lipoproteins and albumin: vitamin D preferentially to lipoproteins and both 25-OH-D and 1,25-(OH)z-D to albumin. A similar binding pattern for vitamin D in plasma was observed previously by us in a child with vitamin D toxicity. The increased binding of vitamin D to lipoproteins and especially to albumin may help Correspondence: Dr J. Silver, Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital, P.O.B. 12000, Jerusalem, Israel. 0014-2972/79/ 1200-0433$02.00 01979 Blackwell Scientific Publications

explain the pathogenesis of toxicity in hypervitaminosis D, where the plasma levels of the more active metabolites are insufficient to account for the clinical signs.

Key words. Vitamin D, 25(0H)vitamin D, 1,25(OH)2 vitamin D, lipoproteins, vitamin D binding protein (Gc),albumin. Introduction Vitamin D and its biologically more active metabolites 25-hydroxyvitamin D (25-OH-D) and 1,25-dihydroxyvitamin D (1,25-(OH)2-D) are transported in plasma bound to a specific carrier protein (DBP) which has been purified and shown to be identical to the group specific component (G,) [ 141. DBP has an a-globulin electrophoretic mobility [5, 61, a molecular weight of 52-59 x 10' [I-3, 51 and a concentration in plasma of about 5 pmol/l [ 1, 31. Although under normal conditions this globulin is less than 3% saturated by the metabolites of vitamin D [ I , 31, vitamin D is also transported on plasma lipoproteins [6, 71. In a child with vitamin D toxicity we observed an increased binding of vitamin D to both plasma lipoproteins and albumin, which became normal on clinical recovery [8]. This different binding pattern may help explain the pathogenesis of vitamin D toxicity as has been shown for vitamin A toxicity [9]. Therefore we have studied the binding to plasma of high concentrations of vitamin D and its metabolites. Methods Plasma. Blood was drawn from five fasting normal male volunteers (3WO years old), with 0.2% (w/v) disodium-ethylenediaminetetra-acetic acid as anticoagulant. All subjects were normolipaemic: cholesterol, 3-5 mmol/l; triglycerides, 0.7-1.7 mmol/l. Plasma was separated by centrifugation in a Sorvall centrifuge at 10,000g for 10 min at 4°C. Materials. Crystalline vitamin D3 was purchased from Sigma (St Louis, Mo., U.S.A.) and crystalline 25-OH-D and 1,25-(OH)2-D were gifts of Drs Gun-

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JUSTIN SILVER & MENAHEM FAINARU

caga and Donnalek of Hoffman-La Roche (Basel, Switzerland). [4-I4C]Vitamin-D3 (36 mCi/mmol), [ I-3H]vitamin-D3 (12 Ci/mmol) and 25 hydroxy[26,27-methyL3H]cholecalciferol ([3H]25-OH-D)( 1 1.3 Ci/mmol) were obtained from The Radiochemical Centre, Amersham, Bucks., England. 1,25-Dihydroxy(26,27-meth~l-[~H] cholecalciferol ([3H]1,25(OH)*D) was synthesized by Dr S. Edelstein (Weizmann Institute of Science, Rehovoth, Israel [lo]). The purity of the four radiolabelled vitamin D sterols was checked by thin-layer chromatography [ 1 I], and shown to be more than 95%. Incubation procedure. Individual plasma samples (1.5 ml) were incubated in duplicate for 16 h at 4°C with one of the following amounts of radiolabelled vitamin D sterol dissolved in 30 pl ethanol: 0.3 nmol [14C]vitaminD, 2.25 pmol [3H]vitamin D, 6.6 pmol [3H]25-OH-D or 4.8 pmol ['H]1,25-(OH)*-D. In some experiments ['4C]vitamin D was added together with one of the tritiated metabolites and various amounts of unlabelled vitamin-D sterols (0-150 nmol in 50 pl ethanol). Ultracentrifugation. The incubated samples were brought to a density (d) of 1.21 g/ml with potassium bromide and centrifuged using a 40.3 rotor in a Beckman L-3-50 ultracentrifuge for 48 h at 110,000 g and 4°C. The tubes were sliced at the clear zone and the lipoproteins aspirated from the top. In some experiments the individual lipoproteins were isolated by sequential ultracentrifugation at d = 1.006, I .063 and 1.21 g/ml in a 40.3 Beckman rotor at 110,000 g and 4°C.

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The lipoprotein fractions ( 1.6 ml) and the lipoprotein-free plasma (-4.8 ml) were dialysed against six changes of 0.154 mol/l sodium chloride for 48 h at 4°C. Agarose gel electrophoresis. Portions of lipoproteinfree plasma ( d > 1.21 g/ml) were electrophoresed in slabs of 1% (w/v) agarose gel (B.D.H., Poole, Dorset, England) in 0.05 mol/l sodium barbital buffer (pH 8.6) on glass plates as detailed elsewhere (121. In brief, 100 pl samples were applied in troughs (1 x 15 mm), and electrophoresis was carried out for 5-6 h at 4°C until the tracking dye reached a 55 mm front. The slabs were cut longitudinally, and the central strips ( 1 2 mm wide) removed and sliced into 2 mm sections for radioassay. The remainder was fixed in methanol: acetic acid : water (5: 1 : 5 by vol.), stained in 0.2% (w/v) Amidoschwarz and destained in the fixing solution. Preparation ofsamplesfor radioassay. Aliquots (0.1 ml) of the incubated plasma, lipoprotein fractions or lipoprotein-free plasma ( d > 1.21 g/ml) were solubilized in 0.5 ml Soluene 350 (a quarternary ammonium chloride solubilizer from Packard Instrument Co. Inc., Downers Grove, Ill., U.S.A.). This method proved to be as efficient as and much simpler than the commonly used Folch extraction. The 2 mm wide agarose slab slices were incubated with 1 ml Soluene 350 in scintillation vials for 18 h at 60°C. To all solubilized samples was added 10 ml of scintillation fluid (containing 0.4% (w/v) 2,5 diphenyloxazole (PPO) and 0.01% (w/v) 2,2'-p-phenylenbis (4-methyl-5-phenyloxazole) (POPOP) in toluene). Radioactivity was assayed in a Packard Model 3380 liquid scintillation spectrometel: (Packard Instrument Co., Downers Grove, Ill. U.S.A.) after the samples had been kept in the dark for 24 h at

Table 1. Recovery of radiolabelled sterols added t o human plasma. Vitamin D metabolites were incubated with 1.5 ml of normal fasting plasma for 16 h at 4 C. and separated into density fractions of A 1.21 g/ml (lipoproteins) and d> 1.21 g/ml (lipoprotein-free plasma). The lipoprotein-free plasma ( 100 1'1) was subjected to agarose gel electrophoresis and resolved into two radioactive peaks with c( globulin and albumin mobility respectively [12]. The recovery for radiolabelled sterols was more than 80':,, in each procedure. The recovery data include all experiments in the study and are expressed as mean SEM (number of experiments in parentheses). Tracer

Procedure Ultracentrifugation*

A gar o se electrophoresist

Type

Concentration (nmol/l)

(" 6)

(" 0.5). Agarose gel electropherograms of lipoprotein free plasma (d> 1.2 1 g/ml) revealed two radioactive peaks with x globulin and albumin mobilities [ 121. The recovery in those peaks was more than 90% of the applied radioactivity (calculations were corrected for the 20% loss in the slab used for staining) (Table I). In this procedure as with the ultracentrifugation the addition of various amounts of the three unlabelled vitamin D compounds did not alter the recovery (P>O.5). Since the recovery in all experimental procedures was constant, all results are expressed as percentages of the total radioactivity recovered, and not of that originally applied.

c

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Distribution of radiolabelledvitamin D-sterols in normal human plasma

The distribution of the added radioactive vitamin-D metabolites among plasma proteins was unique for the particular sterol (Table 2). 25-OH-D was bound almost exclusively to x globulin and over half the vitamin D and 1,25-(OH)z-D was also recovered in this fraction. The remainder of the vitamin-D was recovered in lipoproteins, whereas 1,25-(OH)z-D was found both in lipoproteins and albumin (Table 2). There was no appreciable difference between the binding of [3H]vitamin D (1.5 nmol/l) and ['4C]vitamin D (200 nmol/l) ( P > 0.5). Table 2. Distribution of labelled vitamin D metabolites among plasma proteins. The experimental procedure was the same as described in Table I , but in these experiments only the radioactive sterols were added. The results are expressed as the percentage (mean+SEM) (number of experiments in parentheses) of the total radioactivity recovered in the plasma proteins. Tracer added

Type

Lipoproteins Concentration ( d < 1.21 g/ml) ('X) (nmoljl)

['4C]Vitamin D [3H]Vitamin D [3H]25-OH-D [3H]l .25-(OH)z-D

200 1.5 4.4 3.2

38.3+ 1.9(20) 40.3 + 1.4 (10) 2.0+0.4(20) 15.3+1.1 (12)

r*

globulin

Albumin

(2,)

(X)

61.7k2.1 (20) 59.5 & I .8 (10) 98.0+0.4(20) 62.2+2.4(12)

< O . I (20) < O . I (10) 40.1 (20) 23.0?2.0(12)

I

I

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JUSTIN SILVER & MENAHEM FAINARU

250H D

0-0

80

1

20

Y

0.5 1

0.1

5

30 0.l as 1 r m o l I I plasma

10

5

30

10

Figure 2. [‘4C]Vitamin-D (200 nmolil) distribution in plasma proteins.

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0 10m

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t

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-

4m[

LIPOPROTEINS / I 0

l J - b i = s ! l x h ~ ~ ~ 01 0.5 1 5 10 30

0.1

0.5 1

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Amol /I plasma Figure 3. [3H]2S-OH-D (4.4 nmolil) distribution in plasma proteins.

I o-.-.-.

- 100 ,z

a

w .W

.-

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o------o

80-

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Vitamin D

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A L B U M I N --o

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TRANSPORT O F VITAMIN D STEROLS IN HUMAN PLASMA When the individual lipoproteins were isolated vitamin D was associated with all three major classes. Its distribution (mean percentage k SEM, n = 5) was: 28 k 6.2, 34 k 5.8 and 37 ? 6.9 in very low density lipoproteins ( d < 1.006), low density lipoproteins ( d = 1.006-1.063) and high density lipoproteins (d = 1.063-1.2 1 g/ml) respectively. Effect of excess of vitamin D metabolites on their own distribution among plasma proteins At increased concentrations of the vitamin D metabolites (0-80 pmol/l plasma) the a globulin became saturated and the binding to lipoproteins and albumin increased (Fig. 1). The binding capacities of a globulin for vitamin D, 25-OH-D and 1,25-(0H)~-Dwere similar: respectively 5.6 k 0.3, 5.0 k 0.2 and 4.8 ? 0.8 pmol sterol/l plasma (meankSEM, n = 6 ; P>O.5), but the amount of added sterol leading to saturation was different. 25-OH-D saturated the a globulin at the lowest concentration and vitamin-D at the highest (Fig. 1, top). The remainder of the added sterols was bound both to lipoproteins and albumin. Vitamin D was preferentially bound to lipoproteins whereas 25-OH-D and 1,25-(OH)*-D were bound more to albumin (Fig. 1). Effect of excess vitamin D on binding to lipoprotein classes With increasing concentrations of added vitamin D there was a corresponding increase in binding to all lipoprotein classes. There was, however, more binding to HDL than to LDL or VLDL. At a vitamin D concentration of 26 pmol/l the amount bound to HDL (8.5 f 0.9) was about twice and four-fold that bound to LDL (4.9 k 0.5) and VLDL (2.2 f0.4) respectively (pmol/l, mean k SEM, n = 5). Competitive efSect of excess vitamin D metabolites on their binding to plasma proteins 25-OH-D (5 pmol/l plasma) completely displaced both vitamin D and 1,25-(OH)z-Dfrom the a globulin, whilst both vitamin D and 1,25-(OH)2-Deven in large excess ( > 30 pmol/l plasma) had no effect on the binding of 25-OH-D to M globulin (Figs. 2-4). Vitamin D and 1,25-(OH)2-Ddisplaced from the a globulin bound both to lipoproteins and albumin. Vitamin D was displaced more to lipoproteins (Fig. 2) and 1,25-(OH)*-D more to albumin (Fig. 4), which was similar to their binding when added in excess (Fig. I).

Discussion We have used ultracentrifugation and electrophoresis to study the binding of radiolabelled vitamin D and its active metabolites to plasma proteins. Agarose electrophoresis has been shown to be reproducible, and specific in its ability to identify and measure the binding of vitamin D compounds to a globulin (DBP) and albumin [ 121). The three radioactive vitamin D sterols are known to behave identically to their unlabelled compounds both biologically and biochemically in the

437

radioligand assays [ 13-1 51. Therefore we assume that the results obtained from our experiments using radiolabelled sterols reflect the true binding of natural vitamin D metabolites. The physiological plasma concentrations of both vitamin D (5-70 nmol/l) [16, 17 and 25-hydroxyvitamin D (3CL70 nmol/l) [17, 181 are much higher than the amounts of tracers we added. Moreover, DBP is normally less than 3% saturated by vitamin D sterols [ l , 3, 19, 201; hence the addition of less than 5 nmol/l plasma of radiolabelled sterols ([3H] vitamin D, [3H]25-OH-D or [3H]1,25-(0H)*-D) is unlikely to affect their physiological binding in normal plasma. However, because of practical difficulties the experiments were not performed under strict physiological conditions. Low temperature needed for prolonged incubations, high salt concentrations essential for lipoprotein separation, and low ionic strength (0.05 M) and high pH (8.2) essential for protein electrophoresis, all may influence these in vitro results, that could not be performed in vivo for obvious reasons. We have shown that at physiological concentrations the three sterols have different binding affinities for the plasma proteins. 25-OH-D binds exclusively to the a globulin (DBP), as shown also by other investigators [21, 221. Vitamin D is bound mainly to a globulin (about 60%) and the rest is recovered with lipoproteins, as has also been shown in vivo [6, 71, and in vitro [23]. 1,25-(0H)z-D is also bound mainly to LY globulin (62%) and to lipoproteins (1 5%) but in addition appreciably to albumin (23%). The competitive binding studies reported here demonstrate a different affinity of DBP for the vitamin D sterols. Its affinity for 25-OH-D was higher than for vitamin D or 1,25-(OH)z-D. 25-OH-D completely displaced the other two sterols from a globulin, whereas both vitamin D and 1,25-(0H)z-D, even in large excess, had no effect on the binding of 25-OH-D to LY globulin. This differential affinity of DBP for the three vitamin D compounds has been reported previously by Haddad & Walgate, using purified DBP [3]. On the other hand, the binding capacity of the a-globulin was similar for the three sterols studied (approximately 5 pmol/l plasma), and is also similar to that reported by Bouillon for 25-OH-D 1 mole/mole [ 1, 201. However, the concentrations of the three sterols needed to saturate DBP were different and correlated with the respective affinities. An increased binding of vitamin D and 1,'25-(OH)z-D but not 25-OH-D to lipoproteins and albumin was observed at concentrations lower than those required to saturate the LY globulin. Vitamin D bound preferentially to lipoproteins, whereas 25-OH-D and 1,25-(OH)2-D bound more to albumin. This difference in binding to plasma proteins other than DBP may be explained by the different polarity of vitamin D compounds [7]. The capacity of both albumin and the various lipoprotein classes to bind vitamin D sterols was enormous and saturation was never reached. Among the lipoproteins HDL seemed to have the highest affinity for vitamin D, which may reflect its high protein to lipid ratio [24].

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JUSTIN SILVER & MENAHEM FAINARU

A comparable displacement of vitamin D to lipoproteins and albumin has been reported by us in a child with vitamin D toxicity, which reverted to normal on clinical recovery [8]. Initially only 25% of the patient’s vitamin D was bound to c1 globulin, and the remainder to plasma lipoproteins (60%) and albumin (15%). When the child recovered the partition of endogenous vitamin D reverted to normal, 60% on c1 globulin and 40% on lipoproteins. Thomas et al. [25], using starch gel electrophoresis and a bioassay, have also shown that the serum of patients treated with large doses of calciferol has antiricketic activity in both a globulin and albumin. This alteration in the normal binding of vitamin D in situations of vitamin D excess may help one understand the clinical manifestations of vitamin D toxicity. In hypervitaminosis D, the level of 1,25-(O&-D is normal whereas that of 25-OH-D is elevated [26], but sometimes no higher than the level observed in lifeguards or patients on long-term vitamin D therapy, who show no signs of toxicity [13]. Vitamin D itself normally has no physiological activity in plasma [ 181, and we wish to speculate that its toxicity in hypervitaminotic states may be explained by its abnormal binding to albumin or excessive binding to lipoproteins. This hypothesis is in agreement with activity on calcium absorption in tied rat duodenal loops [27] and cultures of chick embryo intestine [28] of directly applied vitamin D. Furthermore, this hypothesis may be analogous to one for vitamin A toxicity: Smith & Goodman [9] showed that vitamin A is displaced from the vitamin A binding globulin to lipoproteins, which may then nonspecifically deliver vitamin A to cell membranes thus causing toxicity. Acknowledgments We are grateful to Dr S. Edelstein from the Department of Biochemistry, Weizmann Institute of Science, Rehovot, for generously providing [3H]1,25-(OH)2-D and to Drs Guncaga and Donnalek of Hoffman-La Roche for supplying crystalline 25-OH-D and 1,25-(OH)*-D.We would like to thank Henry Matzner and Tuvia Hadar for their excellent technical assistance. This work was supported by grants from the Israeli Ministry of Health, and from the joint research fund of Hadassah and the Hebrew University. References 1 Bouillon R., van Baelen H., Rombauts W. & de Moor P. (1976) The purification and characterisation of the human-serum bind-

2

3 4 5

ing protein for 25-Hydroxycholecalciferol (Transcalciferin) identity with group-specific component. Ezir J Eiochem 66,285-291. Imawari M., Kida K. &Goodman De W.S. (1976) The transport of vitamin D and its 25-hydroxy metabolite in human plasma. Isolation and partial characterization of vitamin D and 25hydroxyvitamin D binding protein. J Clin Invest 58,5 14-523. Haddad J.G. & Walgate J. (1976) 25-Hydroxyvitamin D transport in human plasma. J Biol Chem 251,4803-4809. Daiger S.P., Schanfield M.S. & Cavalli-Sforza L.L. (1975) Group-specific component (G,) proteins bind vitamin D and 25-hydroxyvitamin D. Proc Natl Acad Sci USA 72, 2076--2080. Peterson P.A. (1971) Isolation and partial characterization of a

human vitamin D-binding plasma protein. J Biol Chem 246, 7748-7754. 6 Smith J.E. &Goodman DeW.S. (1971) The turnover and transport ofvitamin D and o f a polar metabolite with the propertiesof 25-hydroxycholecalciferol in human plasma. J Clin Invest SO, 2 159-2 167. 7 Edelstein S . (1974) Vitamin D-binding proteins. Biochem Soc Spec Puhl3,43-54. 8 Silver J., Shvil Y. & Fainaru M. (1978) Vitamin D transport in an infant with vitamin D toxicity. Er Med Jii, 93. 9 Smith F.R. & Goodman DeW.S. (1976) Vitamin A transport in human vitamin A toxicity. N Engl J Med 294,805-808. 10 Fraser D.R. & Kodicek E. (1970) Unique biosynthesis by kidney of a biologically active vitamin D metabolite. Nature 228, 764766. 1 I Lawson D.E.M., Bell P.A., Pelc B., Wilson P.W. & Kodicek E. (I97 I ) Synthesis of [I ,2-3H2]cholecalciferol and metabolism of [4-I4C, I ,2-3H& and [4-14C, I-’HI-cholecalciferoI in rachitic rats and chicks. Biochem J 121,673482. 12 Fainaru M. & Silver J. (1979 A method for studying plasma transport of vitamin D applicable to hypervitaminosis D. Clin Chim Acta 91,303-307. 13 Haddad J.G. & Chyu K.J. (1971) Competitive protein-binding radioassay for 25-hydroxycholecalciferol.J Clin Endocrinol33, 992-995. 14 Edelstein S.,Charman M., Lawson D.E.M. & Kodicek E. (1974) Competitive protein-binding assay for 25-hydroxycholecalciferol. Clin Sci Mol Med 46,23 1-240. 15 Brumbaugh P.F., Haussler D.H.. Bursac K.M. & Haussler M.R. (1974) Filter assay for la,25-dihydroxyvitamin D3. Utilization of the hormone’s target tissue chromatin receptor. Biochemistry 13, 4091-4097. 16 Bouillon R., van Kerckhove P. & De Moor P. (1976) Measurement of 25-hydroxyvitamin D3 in serum. Clin Chem 22,364368. 17 Jones G. (1978) Assay of vitamins Dz and D3 and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography. Clin Chem 24,287-298. 18 Haussler M.R. & McCainT.A. (1977) Basicand clinicalconcepts related to vitamin D metabolism and action. N Engl J Med 297, 104I - 1050. 19 Haddad J.G. & Walgate J. (1976) Radioimmunoassay of the binding protein for vitamin D and its metabolites in human serum. Concentrations in normal subjects and patients with disorders of mineral homeostasis. J Clin Invesr 58, I21 7-1222. 20 Bouillon R., Van Baelen H. & De Moor P. (1977) The measurement of the vitamin D-binding protein in human serum. J Clin Endocrinol Metah 45,225-23 I. 21 Edelstein S.. Lawson D.E.M. & Kodicek E. (1973) The transporting proteins of cholecalciferol and 25-hydroxycholecalciferol in serum of chicks and other species. Partial purification and characterization of the chick proteins. Biochem J 135, 41 7-426. 22 Haddad J.G. & Chyu K.J. (1971) 25-Hydroxycholecalciferolbinding globulin in human serum. Biochim Biophys Acta 248, 471-48 I . 23 Chen P.S. & Lane K . (1965) Serum protein binding of vitamin D3. Arch Biochem Biophys Il2,70-75. 24 Eisenberg S. (1976) Lipoprotein metabolism and hyperlipemia. Atherosclerosis Reuiews (ed. by R. Paoletti and A. M. Gotto), Vol. I , pp. 23 61. Raven Press. New York. 25 Thomas M.C., Morgan H.G.. Connor T.B., Haddock L., Bills C.E. & Howard J.E. (1959) Studies of antiricketic activity in sera from patients with disorders of calcium metabolism and preliminary observations on the mode of transport of vitamin D in human serum. J Clin Invest 38, 1078-1085. 26 Hughes M.R., Baylink D.J., Jones P.G. & Haussler M.R. (1976) Radioligand receptor assay for 25-hydroxyvitamin D2iD3 and la.25-dihydroxyvitamin D2/D3. J Clin Invest 58,61--70. 27 Schachter D.. Kowarski S. & Finkelstein J.D. (1964) Vitamin Dj: direct action on the small intestine of the rat. Science 143, 143-144. 28 Corradino R.A. (1973) Embryonic chick intestine in organ culture: a unique system for the study of the intestinal calcium absorptive mechanism. J Cell Biol58,64-78.

Transport of vitamin D sterols in human plasma: effect of excess vitamin D, 25 hydroxyvitamin D and 1,25 dihydroxyvitamin D.

European Journal of Clinical Investigation (1979) 9,433-438 Transport of vitamin D sterols in human plasma: effect of excess vitamin D, 25 hydroxyvit...
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