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Vitamin D Binding Proteins
Vitamin D Binding Proteins S. EDELSTEIN Department of Endocrinology Municipal-Governmental Medical Centre, Ichilov Hospital, Tel Aviv-Jaffo, Israel I. Introduction . . . . . . . . . . . . . . . 11. The Transporting Proteins of Vitamin L) and Its Metabolites in A. Introduction . . . . . . . . . . . . . . . B. Serum Binding Proteins for Cholecalciferol and 25-Hydroxycholecalciferol . . . . . . . . . . C. Serum Binding Proteins for 1,25-Dihydroxycholecalciferol . D. Conclusions. . . . . . . . . . . . . . . 111. Target Organ Binding Proteins . . . . . . . . . . A. Introduction . . . . . . . . . . . . . . B. Binding Proteins for 25-Hydroxycholecalciferol . . . . C. Binding Proteins for 1,25-Dihydroxycholecalciferol . . . D. Conclusions. . . . . . . . . . . . . . . IV. Binding Proteins in the Assay of Vitamin D and Its Metabolites A. Introduction . . . . . . . . . . . . . . B. Competitive Protein Binding Assays . . . . . . . C. The Assessment of Vitamin D Status . . . . . . . D. Conclusions. . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .
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Blood
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I. INTRODUCTION I n light of the recent findings that cholecalciferol is metabolized before it carries out its functions, there have been renewed attempts to study the transport of this steroid and its metabolites. Cholecalciferol, which is absorbed from the intestine or formed in the skin from its provitamin, is transported to the liver, where it is hydroxylated a t C-25. The 25hydroxycholecaliferol [25(OH)D3] formed is then transported to the kidney, where it is further hydroxylated a t C-1 to yield 1,25-dihydroxycholecaliferol [ 1,25(OH)2D3]. This more recently discovered sterol, which is regarded as the active hormonal form of the vitamin, has to be transported once again to its target organs, for example, the intestine, where it functions in initiating calcium transport. From this metabolic pathway, the immediate questions that arise are: How are the different sterols transported from one site of hydroxylation to another, and from these sites to the target organs? Is there a specific carrier protein in the blood stream for each of the metabolites, or one common protein? How does the sterol recognize its target cell? 407
408
S. EDELSTEIN
Over the past three years several studies in search of answers to these questions have been started but are still in their infancy. The purpose of this article is to summarize progress and to point out the difficulties and problems that are yet to be overcome and resolved. One practical outcome of these studies on binding-proteins was the development of competitive protein binding assays for vitamin D and its metabolites, and a review on this topic and on the assessment of vitamin D status in man will also be included.
11. THETRANSPORTING PROTEINS OF VITAMIND ITSMETABOLITES IN BLOOD
AND
A. INTRODUCTION
It is well established that cholecalciferol in blood circulates attached to proteins. In 1959, Thomas and associates studied the transport of cholecalciferol in serum of humans ingesting large doses of the vitamin. Serum proteins were separated by starch block electrophoresis, and the fractions obtained were tested for antirachitic activity. They found that the antirachitic activity was associated with al- and a,-globulins, and a small amount of the activity was associated with the albumin fractions. Similar findings were observed in vitro upon addition of 12.5 pg of the vitamin to serum. DeCrousaz et al. (1965) confirmed these findings by fractionating human serum on agar-gel electrophoresis. By staining some pherograms for lipoproteins and examining others by immunoelectrophoresis, they excluded the involvement of lipoproteins in the transport of the vitamin. Subsequently the fate of the vitamin was followed by 14C or 3H labeling. Chalk and Kodicek (1961) used for the first time 14C-labeled ergocalciferol to study the transport of vitamin D in blood. The labeled vitamin in this study was incubated in vitro with rat serum. It was possible to detect the vitamin in small regions of starch block electrophoretograms, and so determine in more detail the distribution of the labeled ergocalciferol with respect to protein. The radioactive material was found to correspond with a,-globulin and albumin. Chen and Lane (1965) gave 3H-labeled cholecalciferol to a dog and analyzed serial serum samples by ultracentrifugal flotation techniques. About 80% of the radioactivity in the serum sedimented, whereas the remaining 20% floated with the lipoprotein fraction. On addition of the vitamin to serum in vitro, some 40% of the radioactivity floated with the lipoproteins.
409
VITAMIN D BINDING PROTEINS
Rikkers and DeLuca (1967) and Rikkers e t al. (1969) have shown that the vitamin associated rapidly with rat serum proteins in vivo. The radioactivity was found associated with five protein fractions, four of which were shown to be lipoproteins, and the fifth an a-globulin. As the proportion of the radioactivity associated with the lipoproteins decreased with time, they concluded that plasma contained a specific vitamin D-binding globulin. From all the above-mentioned studies, it was well established and accepted toward the end of the 1960s that a specific a-globulin in serum is responsible for the transport of cholecalciferol.
B.
SERUM
BINDING P R OT E I NS
FOR
CHOLECALCIFEROL
AND
25-HYDROXY-
CHOLECALCIFEROL
1. Man, Rat, and Monkey
The reports of Smith and Goodman (1971) and Haddad and Chyu (1971a) were the first studies on the transport of 25(OH)D, in light of the recent findings on the metabolism of cholecalciferol. I n the first study, normal men were dosed with 3H-labeled cholecalciferol, and serial plasma samples were collected. In all the samples, almost all the radioactivity was found to be associated with proteins of density greater than 1.21. The transport protein has been partly characterized by gel filtration and gel electrophoresis and was found to be smaller than plasma albumin and to have an electrophoretic mobility slightly greater than plasma albumin. Haddad and Chyu (1971a) labeled human plasma proteins in vivo and in vitro with tritiated 25(OH)D,, and found that most of the radioactivity was associated with a protein with a-globulin mobility on polyacrylamide disc-gel electrophoresis, a molecular weight of 40,000-50,000, and a sedimentation coefficient of 3.1 S. I n another study Peterson (1971) obtained a highly purified preparation of a cholecalciferol binding protein from human plasma which had previously been incubated with “C-labeled cholecalciferol. Although these studies demonstrate clearly that both cholecalciferol and 25(OH)D, are transported in human blood bound to a specific protein, they do not answer the question whether the same or different proteins are responsible for the transport of these two sterols. I n experiments with humans (Smith and Goodman, 1971; Haddad and Chyu, 1971a), the major circulating metabolite was 25(OH)D3. Edelstein et al. (1973) have analyzed human plasma in which either radioactive labeled cholecalciferol or 25 (OH) D, was the major component. Plasma was obtained from a 60-year-old female with primary hyperparathyroidism a t 4 and 8 hours after a dose of 10 pCi of 13-H-labeled
410
S. EDELSTEIN
cholecalciferol. At these time intervals the proportions of cholecalciferol to 25(OH)D, were 5.4:l and 2:1, respectively. Cohn fractionation of these plasma samples showed that 47% of the radioactivity a t 4 hours was in fraction IV, which contains the lipoproteins; a t 8 hours the proportion in this fraction was 41%. The globulin fraction a t 4 and 8 hours conkined 20% and 25%, respectively. Another sample was obtained from a 57-year-old male with anticonvulsant osteomalacia 12 hours after he received 10 pCi of lJH-labeled cholecalciferol and 2 pCi of 4-l'C-labeled cholecalciferol. In this sample the radioactivity was due mainly to 25(OH)D,, and 50% of the radioactivity was found to be associated with the a-globulin. However, analysis of a 24-hour sample of plasma obtained from an ll-year-old male with pseudo-vitamin D deficient rickets (dosed as before) with a cholecalciferol: 25(OH)D, ratio of 6 : l showed a single peak of radioactivity after chromatography on DEAE-Sephadex, a peak that was identical to the peak of plasma protein that binds 25(OH)D3. These studies with human plasma show that although there is a significant binding to lipoproteins, this is most noticeable a t the early time-intervals after an intravenous dose of the cholecalciferol. This association with the lipoproteins decreases slowly with time, so that by 24 hours all the cholecalciferol and its metabolites are associated with the a-globulin. The binding of cholecalciferol to lipoproteins is unrelated to body stores of the vitamin. In order to establish conclusively that only one binding protein is present in plasma for both cholecalciferol and 25(OH)D,, Edelstein et al. (1973) carried out the following experiment. One group of rachitic rats was injected with 4-lT-labeled cholecalciferol, and a second group was injected with 26,27-3H-labelcd 25 (OH)D,. Serum was prepared from the first group 3 hours later, a time interval a t which the major circulating metabolite is the unchanged vitamin. Serum from the second group was prepared after 8 hours, a time interval a t which all the 25 (OH)D, present in circulation is attached to the a-globulin. The sera were mixed and chromatographed on DEAE-Sephadex. A single radioactive peak of radioactivity was observed (Fig. 1) and the 3H:14Cratio was constant throughout the peak. In other words, in rat serum a single binding protein is responsible for the transport of both cholecalciferol and 25(OH)D,. This is the case with man, pig, and monkey, and is most probably true with regard to mammals in general (Edelstein, 1974a). There have been several reports on the dissimilarity in the biological activity of ergocalciferol compared with cholecalciferol between New World and Old World monkeys (Hunt et al., 1967; Lehner e t al., 1967). Apparently, New World monkeys cannot use ergocalciferol as efficiently as cholecalciferol, and therefore it was of interest to see whether the bind-
41 1
VITAMIN D BINDING PROTEINS 0.4
16
A 0.3
I2
0.2
0
E 0 OD N
”
-: 4
.-C0
?
s X
E a
.”
n
W
0.1
4
0 10
20
30 Fraction
40
50
number
FIQ. 1. Ion-exchange chromatography on DEAE-Sephadex of mixed samples of serum of rats given a dose either of 26,27-3H-25(OH)D, or 4-14C-cliolecalciferol. 0 , E280;0, “C radioactivity; . , 3H radioactivity. According to Edelstein et al. (1973), by permission of The Biochemical Society, U.K.
ing protein of these monkeys differs from the binding protein of ariiriiala in which these two steroids have equal activity. Sera were obtained from 9 vitamin D-deficient C e b w albifrons (New World) and from a n Erythrocebus pntas (Old World), and incubated with radioactive 25 (OH) D,. Their proteins were then resolved on annlytical polyacrylamide-disc gel electrophoresis. As with other mammals and as found with baboons (Rosenstreich et al., 1971), the radioactivity in the serum of the Old World hlonkey was associated with a protein possessing a-globulin mobility. I n the New World Monkey the radioactivity was associated with a protein possessing albumin mobility. A labeled fraction with this same mobility was observed in serum obtained from the C e b m albifrons monkey 24 hours after a dose of 1 pCi of l-3H-labeled cholecalciferol. The serum that was obtained from this dosed monkey was further studied by fractionation according to Cohn’s method. It was found that 87% of the radioactivity was recovered in fraction V, the albumin fraction. An attempt to separate a carrier protein from the albumin by chromatography on Sephadex (3-200 followed by DEAE-Sephadex was unsuccessful (Edelstein e t al., 1973).
412
S. EDELSTEIN
2. Chick Unlike rat serum, chick serum contains two binding proteins, and their presence can be demonstrated readily by analysis on polyacrylamide disc gel electrophoresis of serum to which either cholecalciferol or 25 (OH)D, has been bound in vitro. If binding occurs after a dose of radioactive labeled cholecalciferol, it is found that 83% of the radioactivity attached to one protein is due to 25 (OH)D, and only 10% to cholecalciferol. The radioactivity attached to the second protein consists mainly of cholecalciferol (55%) and to a lesser extent t o 25(OH)D3 (20%) (Edelstein et al., 1972). The two proteins can be separated in a single step, using ionexchange chromatography on DEAE-Sephadex (Fig. 2 ) . We named the first protein 25-hydroxycholecalciferol-binding protein (Fig. 2, peak 1) , and the second protein cholecalciferol-binding protein (Fig. 2, peak 2 ) . By means of Cohn fractionation, (NH,) zS04 precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex, and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified (Edelstein et al., 1973). Scheme 1 outlines the purification procedure for the two proteins. In order to assess the role that each one of these proteins plays in the transport of these sterols in plasma, the following experiments have been carried out. One group of rachitic chicks was dosed with 26,27-,Hlabeled 25 (OH)D,, and serum was collected after 8 hours. A second group of chicks was given a dose of 4-lT-labeled cholecalciferol, and 3 hours 0.4
r
r
10 8
0 0
10
20 Fraction
30
40
50
60
number
Fxo. 2. Ion-exchange chromatography on DEAE-Sephadex of chick serum given dose of l-8H-cholecalciferol. -, Ezso; ..*-, 'H radioactivity. According to Edebtein et al. (19731, by permission of The Biochemical Society, U.K. R
413
VITAMIN D BINDING PROTEINS
Serum
.1
Cohn fractionation
Cohn fraction V
1
(NH4)2SO4fractionation
Proteins precipitated between 50% and 80% saturation Gel filtration on Sephadex C;-200 followed by ion-exchange chromatography on DEAE-Sephadex (0-0.6 M-NaCI gradient,). I
I
1
25Hydroxycholecalciferolbinding protein
1
Peak 2
Peak 1 Gel filtration on Sephadex G-100
C holecalciferol-binding . protein
SCHEME 1. Scheme of purification of cholecalciferol-binding protein and 25hydroxycholecalciferol-binding protein.
later, when most of the "C was still present a s the unchanged sterol, serum was again collected. The samples were mixed together, and were subjected to gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DEAE-Sephadex (Fig. 3 ) . 3H-Labeled 25 (OH)D, was bound to both of the binding proteins in approximately equal proportions, but the "C-labeled cholecalciferol was bound only to the cholecalciferol-binding protein. Analysis by thin-layer chromatography (TLC) of the lipids extracted from these two proteins showed 14C-labeled 25 (OH)D, to be distributed between the 25-hydroxycholecalciferol-binding-protein and the cholecalciferol-binding-protein in the ratio of 9 :1. These findings strongly suggest that 25 (OH)D, synthesized in the liver is released into the bloodstream bound to its specific binding protein. However, the addition of 25(OH)D, to plasma either directly into the bloodstream or in vitro results in immediate binding to these two binding proteins in approximately equal proportions. This is due most probably to the limited solubility of 25(OH)D, in water. The high-affinity for 25(OH)D, by these two proteins was demonstrated by analysis of the displacement curves (Figs. 4a and 4b) accord-
414
S. EDELSTEIN
Fraction
number
FIQ.3. Ion-exchange chromatography on DEAE-Sephadex of mixed samples of serum of chicks given a dose of either 26,27dH-25(OH)Da or 4-"C-cholecalcifero1. 0, "C radioactivity; 0, 'H radioactivity. According to Edelstein et al. (1973), by permission of The Biochemical Society, U.K.
ing to Scatchard (1949).The association constant (KRs80c.) of 25 (OH)D, with its specific binding protein was found to be 3.0 x lox liters/mole, and with the cholecalciferol-binding protein 4.3 X lo8 liters/mole. Binding could not be obtained between the 25-hydroxycholecalciferol-binding protein and cholecalciferol, indicating the high specificity of the binding sites on this protein toward the hydroxylated metabolite. However, a high K,,,,,, of cholecalciferol with its binding protein has not been obtained. This is most probably a consequence of the virtual insolubility of this sterol in water. The physical properties of the chick serum bindingproteins are summarized in Table I. 3. Toad
It was found by Bruce and Parkes (1950)that vitamin D is required by amphibians. This group of animals is the first evolutionary group to require an efficient regulation- of calcium absorption in order to maintain calcium homoeostasis. It was therefore of interest to study the transport
415
VITAMIN D BINDING PROTEINS
50
.
40 .
30 . U 3
g #
0 C
a
20. 10
0
m
’
O t
~
0
5
10
25(OH)D3
15
20 Steroid (ng)
(ng)
FIG.4. Competitive displacement of 26,27-3H-25(OH)Da from the 25(OH) D, binding protein ( a ) and of 26,27-’H-25(OH)D3 and 1,2-aH-cholecalciferol from the cholecalciferol binding protein (b) by increasing amounts of the two non25(OH)D3. According t o Edehtein et al. labeled steroids. e, Cholecalciferol; 0, (19731, by permission of The Biochemical Society, U.K. of cholecalciferol in this group as well. Vitamin D-deficient toads Xenopus lnevis were dosed with l-3H-labeled cholecalciferol, and 5 days later serum was prepared. At this time, cholecalciferol and 25(OH)D3 were present in approximately equal proportions in serum. Analytical polyacrylamide disc gel electrophoresis analysis showed the radioactivity to he concentrated near the origin, in an area which stained deeply with the lipid stain, Oil Red 0 (Edelstein et al., 1973). The radioactivity remained in the supernatant after precipitation of p-lipoproteins. The TABLE I PHYSICAL PROPERTIES OF CHICKSIERUM BINDING PROTEINS Property Sedimentation coefficient (s) Electrophoretic mobility Molecular weight (approx.) Association constant for 25(OH)D3 at 4°C
W-1)
EDTA and SH-groups as st,abiliaing agents
Cholecalciferolbinding protein
25(OH)D3binding protein
3.5 s B 60,000
3.5 s B 54,000
4.3 x 108 Yes
3 . 0 x 108 No
416
S. EDELSTEIN
serum was ,subjected to Cohn fractionation, and 80% of the radioactivity was recovered in Fraction VI-1, which contains the a-lipoproteins.
c. SERUMBINDINGPROTEINS FOR
1,25-DIHYDROXYCHOLECALCIFEROL
Although we seem to understand how cholecalciferol and 25 (OH) D, are transported in the bloodstream, nothing is yet known about the transport of the hormonal form of the vitamin, 1,25(OH)2D3.The main obstacle in studying the transport of this metabolite is its low concentration in blood. It is practically impossible to study its transport in chick serum, since it accounts for less than 5% of the total antirachitic activity of the serum and consequently is present only in extremely small amounts. In the rat, a higher proportion of 1,25 (OH)2D3may be present (Lawson et al., 1971) and therefore we have attempted to study its transport in this species. Rachitic rats were dosed with tritiated 25(OH)D3,and 16 hours later serum was prepared. TLC analysis of the lipid extract of the serum revealed that some 10% of the radioactivity was due to 1,25(OII)D3. Fractionation of the serum on Sephadex G-200 followed by ion-exchange chromatography on DEAE-Sephadex resulted in the separation of the a-globulin peak that binds cholecalciferol and %(OH) D,. The fractions within the peak were combined, and the lipids were extracted and analyzed on a silicic acid column. Both 25(OH)D3 and 1,25(OH)?D3were found to be present. When tritiated cholecalciferol was dosed 16 hours before the collection of the sera, all three metabolites, namely, cholecalciferol, 25 (OH)D,, and 1,25 (OH)D3, were found to be associated with this peak, and in the same proportions as in the lipid extract of the unfractionated serum (S. Edelstein, D. E. M. Lawson, and E . Kodicek, unpublished work). However, this finding does not rule out the possibility of the existence of a specific binding protein for 1,25(OH),D3 in blood. The reversible nature of the binding between steroids and proteins results in the accumulation of the steroids on proteins with high affinity for them, and a shift to other high affinity binding protein may have taken place. Careful examination of the fractions eluted from the DEAE-Sephadex column on which the above sera were fractionated, revealed that there exists an additional minute radioactive peak (Fig. 5, peak b) which is eluted at higher salt concentration than the a-globulin which binds cholecalciferol and 25(OH)D3 (Fig. 5, peak a ) . Similar fractionation of serum obtained a t longer time intervals, 24 and 36 hours after dosing, resulted in the increase of peak b, so that some 25-30% of the radioactivity was associated with this peak. Analysis on a silicic acid column of the lipid extract of this peak gave a similar picture of distribution of cholecalci-
417
VITAMIN D BINDlNG PROTEINS
0.4
E 0 .D N L
NaCl molarity
peak a
0.3
0.2
'
a
-
0.6-
. 30
0.4
. 20
3 5! X
0
0
C
.c
.-g
140
a1
.
Y
0
0
10
20
30
40
50
60
Fraction number
FIG.5. Ion-exchange chromatography on DEAESephadex of serum obtained from rachitic rats 16 hours after a dose of 26,27-'H-25(OH)D3. E m ; 0, 'H radioactivity.
ferol metabolites to the one obtained with peak a. I n other words, we were unable to show any preference of binding of one metabolite on the other. This may again be an artifact caused by elaborate time-consuming procedures, and therefore we have attempted to establish in vitro the affinity of this protein(s) toward the different metabolites by carrying out competitive displacement studies. Again, very flat displacement curves were obtained with 25 (OH)D, and with 1,25 (OH).D,, which leaves the entire question of the transport of 1,25(OH),D, unresolved. Nephrectomy of the rats prior to the administration of the radioactive cholecalciferol did not prevent the appearance of the second peak in plasma.
D. CONCLUSIONS
It seems that the chick is the only species so far examined that has more than one binding protein for cholecalciferol and its metabolites. In man, Old World monkey, and rat, there is only one carrier protein for both cholecalciferol and 25 (OH)D,. If two proteins are circulating in the plasma, their properties must be then w r y similar. Perhaps it is not a coincidence that the two species, chick and New World monkeys, that cannot utilize ergocalciferol as efficiently as cholecalciferol have different carrier proteins from those animals that use these two steroids equally well. It still remains to be shown whether it is albumin itself that binds the vitamin in the plasma of the New World monkeys. If the toad is typical of the amphibian group, then they do not appear
418
S. EDELSTEIN
TABLE I1 DISTRIBUTION OF BINDING PROTEINS AMONG DIFFERENT SPECIES AND THEIRELECTROPHORETIC MOBILITY Species Human Rat Pig Monkey (Erythrocebus patas)) Chick Monkey (&bus albifrons) Xenopus
Number of binding proteins
Electrophoretic mobility
1
a-Globulin
2 1 -
&Globulin Albumin a-Lipoprotein
to have iideveloped’l a specific binding protein, but rather depend on the nonspecific lipoproteins for transporting cholecalciferol and its 25-hydroxymetabolite. The electrophoretic properties of the binding proteins from the animals studied are summarized in Table 11. The transport of 1,25(OH)2D3 is a t the moment still an open question.
111. TARGET ORGAN BINDING PROTEINS A. INTRODUCTION After obtaining sufficient information about the serum binding proteins, the next step in the study of the mode of transport of vitamin D and its metabolites is the identification and characterization of binding proteins in target organs, and their interaction with the serum proteins. Recent studies on the mode of action of several steroid hormones suggest that the sterol modifies intracellular events in target cells by first complexing with soluble cytoplasmic receptor proteins, and subsequently associating with chromatin acceptor proteins (O’Malley, 1971). Accordingly, search has begun for such proteins in tissues thought to be target organs for vitamin D metabolites.
B. BINDING PROTEINS
FOR 25-HYDROXYCHOLECALCIFEROL
25-Hydroxycholecalciferol is the major circulating metabolite of vitamin D, and apparently the dominant metabolite in most tissues. Although no apparent action can be related to this metabolite in target cells, Haddad and Birge (1971) were able to show the presence of specific bind-
419
VITAMIN D BINDING PROTEINS
0
5
10
15
20
25(OH)D3 ( n g )
FIQ. 6. Competitive displacement of 26,27-3H-25(OH)D, from supernatants from different tissues by increasing amounts of nonlabeled 25(OH)D,. 0 , Bone; 0, skin; 0, muscle; V , kidney. According to Edelstein (1974a), by permission of The Biochemical Societ,y, U.K.
ing proteins for this metabolite in 105,000 g supernatants from kidney and muscle homogenates. We have prepared 105,000 g supernatants from various tissue homogenates of rachitic rats and studied their binding affinities toward 25 (OH)D, (Edelstein, 1974a). The displacement curves obtained with kidney, muscle, skin, and bone are shown in Fig. 6; the association constants toward 25 (OH)D, calculated from Scatchard plots of these curves are listed in Table 111. Flatter curves and lower association constants were obtained in our hands with supernatants from liver and intestine. The finding that kidney contains a high affinity binding protein for 25 (OH) D, is consistent with the important finding of Fraser and Kodicek (1970) that in this organ the 1-hydroxylation of 25(OH)D, takes place. However, it is not understood a t the present state of our knowledge on TABLE I11 ASSOCIATION CONSTANTS OF I ) I FFER E N T TISSUYBINDING PROTEINS TOWARD 25(OH)II3
Muscle Kidney Skin Bone
1 . 7 X loo 1 . 1 x 100 1 . 0 x 109 4 . 0 X loo
420
S. EDELSTEIN
the mode of action of vitamin D, why the other tissues should also contain such a protein. The possibility still remains that, although these proteins show high affinity toward 25 (OH)D,, in vivo, they do not bind this metabolite but another sterol with a similar structure. The binding protein in skin, for example, may well be a binding protein for cholecalciferol, which is formed in this tissuc from its provitamin, 7-dehydrocholesterol, but, because of the limited solubility of this sterol in water, it is impossible to establish its high association constant with the protein.
c.BINDINGPROTEINS
FOR 1,25-DIHYDROXYCHOLECALCIFEROL
In target cells of steroid hormones, specific receptor proteins are involved in the intracellular transport of the steroid, and a steroid-protein complex is effective in the initiation of the biochemical response of the hormone. Since 1,25 (OH) LIDS is similar in its structure, formation, and apparent mode of action to other steroid hormones, it was expected that receptor binding proteins for this metabolite will be found in its target cells. Chen and DeLuca (1973) have isolated from intestinal mucosa of rats a low density protein fraction which binds 1,25(OH),D,. This protein fraction when isolated from nephrectomized rats was found to bind only one-eighth of the radioactivity of that from intact animals after dosing with 3H-labeled 25 (OH)D,. Chemical analysis of this protein fraction has shown that some 50% of the lipid is phospholipid, which may suggest that these are membrane lipoproteins. Tsai and Norman to intestinal mucosa (1973) have studied the binding of 1,25 (OH) in an in ziitro incubation system, which enabled them to obtain saturation levels comparable to those obtained in vivo. They were able to show the presence of a cytoplasmic receptor protein for 1,25(OH)& of the size of 65,000-150,000 daltons, and that the binding of 1,25(OH)2D3to this receptor protein is an obligatory step for the subsequent localization of the hormone in the chromatin fraction. However, from the study of Brumbaugh and Haussler (1973) it seems clear that the mechanism of action of cholecalciferol is similar to the mechanism postulated for other steroid hormones. This mechanism supports the existence of a cytoplasmic hormone-receptor complex in the target cell which is accepted by the genome of the cell. As a consequence of the association of the steroid or sterol-receptor complex with the accepting sites in the nucleus, the characteristic biological response of the hormone is initiated. In order to test the specificity of the cytoplasmic receptors for 1,25(OH)2D3in intestinal mucosa, the authors have incubated mucosa homogenates with different closely related sterols, like 25 (OH)D,, chole-
VITAMIN D BINDING PROTEINS
421
calciferol or estradiol. Although it was found that 1,25(OH),D3 binds very effectively to the receptor, far more effectively than cholecalciferol or estradiol to this receptor, 25(OH)D, did bind equally well. Again 1,25(OH),D, was found to bind effectively to liver cytosol, a nontarget organ. From this it s e e m that, on the basis of in vitm binding to cytosol fractions, it is not possible to prove specificity, although displacement studies may do so, but these studies were not carried out by these authors. When such studies were carried out with regard to 1,25(OH),D3 or 25 (OH)D, and cytosol from intestinal mucosa, this fraction showed greater affinity for 25(OH)D, than for 1,25(OH),DS (S.Edelstein, unpublished work). However, when Brumhnugh and Haussler (1973) incubated whole intestinal tissue in Eagle’s medium with radioactive 1,25(OH),D,,a t 0°C: and then transferred the tissue to a fresh medium a t 37OC, the radioactive hormone was found primarily in the chromatin fraction, suggesting a temperature-dependent movement of 1,25(OH),D3 from cytosol to the nucleus, in the manner proposed for other steroid hormones. This temperature-dependent movement of 1,25(OH),D, is in contradiction with results of Tsai and Norman (1973), who did not observe such dependency in their in vitro systcm. A similar experiment with regard to 25(OH)D, would be of interest when attempting to determine the specificity of the receptor sites in intestinal mucosa toward 1,25(OH).D,. Furthermore, Brumbaugh and Haussler (1973) did demonstrate very clearly that the intestinal cytosol fraction is an obligatory requirement for the transfer of 1,25(OH),D3 to the chroinatin. 1,25(OH I .D,$did not enter the nucleus when incubated with isolated intestinal nuclei nor with boiled intestinal cytosol, and not with other nontarget tissue cytosols.
D. CONCLUSIONS From the few studies reported on the interaction of 1,25(OH),D3with intestinal mucosa preparations, it is clear that this target tissue possesses specific cytoplasmic receptor protein ( s ) for 1,25(OH),D,, and that this receptor is obligatory for the transfer of the hormone t o the nucleus. Further characterization of these initial receptor proteins, especially the chromatin receptors, is required in order to draw conclusions as to their functional role in the initiation of the physiological response to 1,25(OH),D, in the intestine. Since so far it seems that the interaction of 1,25(OH),D, with the intestine is similar to the interaction of other steroid hormones and their target cells (O’Malley et al., 1970; Tomkins and Baxter, 1971; Jcnsen et al., 1971), it may be possible that 1,25(OH)?D, acts to regulate gene expression.
422
S . EDELSTEIN
IV. BINDINGPROTEINS IN THE ASSAYOF VITAMIN D A N D ITSMETABOLITES A. INTRODUCTION During the past forty years, attempts continually have been made to develop a sensitive and accurate method for the estimation of cholecalciferol. I n general, the methods developed can be divided into two types: (1) physicochernical methods and (2) biological assays. These have been reviewed by Kodicek and Lawson (1967) ; and by Sheppard et al. (1972). At present, the available physicochemical methods are not suitable for the estimation of the small amounts of vitamin D present in animal tissue. Furthermore, these methods involve laborious and timeconsuming separation procedures in order to eliminate interference by accompanying substances, such as retinol and cholesterol. However, Sklan and Budowski (1973) have recently developed a simple separation of cholecalciferol and ergocalciferol from other sterols and retinol by argentation thin-layer chromatography, which may be very useful in the determination of vitamin D by physicochemical means. As a result, biological assays were widely used in many laboratories for the estimation of vitamin D in tissues, foodstuffs, fish oils, and pharmacological preparations. Apart from the limited sensitivity of these methods, their major disadvantage is the cost, labor, and time required for the assay. The renewed interest in vitamin D binding proteins during the past three years has led to the development of new and simple techniques for the estimation of cholecalciferol and 25(OH)D,. These are the competitive protein-binding assays, which are widely used for the estimation of many other steroid hormones. The principle of these assays is that the tested sterol is competing with a radiolabeled sterol for binding to specific sites on a protein. By separating the free molecules of the sterol from the bound ones, and comparing the percentage binding with a calibration curve, the sterol can be estimated. B. COMPETITIVE PROTEIN BINDINGASSAYS 1. ChoZecalcijerol
Belsey et al. (1971) have published the first competitive protein-binding assay for both cholecalciferol and 25(OH)D3, utilizing the serum binding protein of the rat. The sterols to be tested are extracted, sepa-
VITAMIN D BINDING PROTEINS
423
rated on columns, and then introduced to the binding assay mixture. The problem of the limited solubility of these sterols in water was overcome by inclusion in the assay system p-lipoproteins as carrier for them. The inclusion of p-lipoprotein in the assay mixture enabled these investigators to separate the free sterols from the bound sterols by selective precipitation of the p-lipoproteins only. However, several days were needed for equilibrium to be reached in a system in which the sterols are supposed to detach themselves from a somewhat low affinity binding site on the lipoprotein, and attach themselves to binding sites possessing greater affinity on the serum binding protein, and all this is an aqueous medium. Although it is possible to estimate by this method several nanograms of cholecalciferol or 2 5 ( O H ) D , , the long period of time required in order to reach equilibrium and displacement prevented the acceptance of this method for routine use. 2. 25-H ydrox~cholecalciferol Haddad and Chyu (1971b) introduced another competitive proteinbinding assay for 25(OH)D,, utilizing the kidney binding protein as the assay protein. The steroid was solubilized by including 7% absolute ethanol in the assay mixture so that the time required to reach equilibrium and displacenicnt was shortened to 11 hour. The free sterol was separated from the bound, using charcoal coated with dextran. This method provides a simple and sensitive means for the routine estimation of 25(OH)D3 levels in peripheral blood. As little as 4 ng/ml of plasma can be estimated. However, chromatography on silicic acid columns is used in this assay in order to separate 2 5 ( O H ) D , from the other plasma lipids prior to estimation. This technique we find interferes with the competitive binding and causes erratic blank values. This is due to substances produced during chromatography as a result of impurities in the silicic acid material and of the interaction of the solvents with the silicic acid (hlurphy, 1971). By using small Sephadex LH-20 columns for the separation of the 25 (OH)D, metabolite, and a partially purified vitamin D binding protein from rat serum as the assay protein, Edelstein et al. (1974) were able to develop a competitive protein-binding assay for 25 (OH) D, which eliminates these interferences and has several additional advantages. With the use of columns of Sephadex LH-20 instead of silicic ac'd, the blank values were low, erratic displacements were not observed, and larger quantities of lipids could be chromatographed. The same solvent was used throughout the chromatography, and the columns were used several times.
424
S. EDELSTEIN
25(OH)D3 Ingl
FIQ.7. A typical calibration curve for 25(OH)Da.
Diluted rat serum can be employed instead of a partially purified protein from serum. When 1:lO.OOO dilution of serum obtained from nonvitamin D-deficient rats of the Wistar strain was used in the assay, the sensitivity was increased, and as little as 50 pg per tube could be estimated. Figure 7 represents such a typical calibration curve, and an outline of the assay procedure is illustrated in Scheme 2. 3. 1,25-Dihydroxycholecalcijerol
At present, there is no method for the estimation of 1,25(OH)2D3.The development of a competitive protein-binding assay for this hormone Plasma sample
1
Extraction with chloroform and methanol
1
Chromatography on Sephadex LH-20 J.
25(OH)Da fraction
1
Equilibration of aH-25(OH)I)a and the 25(OH)Da fraction with the assay protein
1
Separation of “free” sterol with charcoal coated with dextran
1
Counting of “bound” sterol
SCHEME2. Scheme of competitive-protein-binding assay for 25(OH)Ds.
VITAMIN D BINDING PROTEINS
425
awaits further information on the properties of its specific binding proteins, like the cytoplasmic receptor protein from intestinal mucosa. Radioimmunossay may be of great advantage in the estimation of this hormone, but to date there is no such an assay for any of the metabolites of vitamin D, although research along this line is in progress. However, in the light of the facts that the formation of this hormone is controlled by a very fine and complex mechanism (Fraser and Kodicek, 1973), and that the circulating levels of this hormone under normal conditions are extremely low or may in fact be zero (S. Edelstein, D. R. Fraser, and E. Kodicek, unpublished work), the application of a method for the estimation of 1,25(OH),D, for routine clinical use is doubtful.
C. THEASSESSMENT OF VITAMIN D STATUS The method for the estimation of 25(OH)D, (Edelstein et al., 1974) was applied to the measurement of plasma levels of this metabolite in 18 normal male and female voluntcers and in 4 normal male volunteers taking daily cholecalciferol supplement of 10 pg (Edelstein, 1974b). The mean value for the normal volunteers was found to he 15.2 k 5.6 ng/ml, but as can be seen from the histogrnm illustrated in Fig. 8, 60% of the volunteers had lower 25 (OH)D, lcvels. This 25 (OH)D, value which is found in thc United Kingdom is similar to the finding of Stamp et al. (1972) but lower than the mean value of 27.3 k 11.8 found by Haddad
U.
0 0
2
25(OH)D3 ( n g / m l )
FIG.8. Histogram of 25(OH)D, values obtained for normal volunteers.
426
S. EDELSTEIN
TABLE IV I N NORMAL ADULTS
PLASMA 25(OH)I>,
Daily cholecalciferol Supplement Group
Number
(fig)
Normal Vitamin D supplement P value
18
-
4
10
a
25(OH)D3' (ng/ml) 15.2 k 5 . 6 3 5 . 9 k 15.0 <0.001
Values represent mean k SI).
and Chyu (1971b) for normal volunteers in the United States. This is due most probably to differences in dietary vitamin D intake and exposure to sunshine. Thc finding that significantly higher plasma values of 25 (OH)D,3were obtained for those who had a daily cholecalciferol supplement (Table IV) indicates that 25(OH)D, levels in blood may be useful for the nutritional assessment of vitamin D status.
D. CONCLUSIONS One practical outcome of the studies on binding proteins was the development of competitive protein binding assay for 25(OH)D,. This assay is simple, reliable, and sensitive and is suitable for routine estimation of 25(@H)D, levels in peripheral blood samples. Since the assay can detect as little as 0.2 rig or less of 25(OH)D, per tube, and since normal circulating levels of 2 5 ( O H ) D , were found to be approximately 15 ng per milliliter of plasma, very small blood samples need to be taken for analysis. Since 25 (OH)D, is the principal circulating metabolite of vitamin D in blood, with cholecalciferol accounting for only a small proportion and 1,25(OH),D, contributing less than 5% to the total antirachitic activity (Lawson et al., 1971; Mawer et al., 1971), the estimation of 25 (OH)D, is probably a useful guide in the assessment of the nutritional status of vitamin D. Attempts to develop a similar assay for cholecalciferol have so far been unsuccessful. Although specific binding proteins for cholecalciferol exist (Edelstein et al., 1972, 1973), the limited solubility of the steroid in water prevents competitive displacement from taking place. The estimation of 1,25(OH):D, awaits further information on the properties of the specific binding proteins for this hormone.
VITAMIN D BINDING PROTEINS
427
REFERENCES Belsey, R., DeLuca, H. F., and Potts, J. T. (1971). J . Clin. Endooinol. Melab. 33, 172.
Brumbaugh, P. F., and Hausslcr, M. R. (1973). Biochem. Biophys. Res. Commun. 51, 74.
Bruce, H. M., and Parkes, A. S. (1950). J . E n d o c ~ i m l 7, . 64. Chalk, K. J. I., and Kodicek, E. (1961). Biochem. J . 79, 1. Chen, P. S., and Lane, K. (1965). Arch. Biochem. Biophys. 112, 70. Chrn, T. C., and DeLuca, H. F. (1973). J . Biol. Chem. 248, 4890. DeCrousaz, P., Blane, B., and Antener, I. (1965). Helv. Odontol. Actu 9, 151. Edelstein, S. (1974a). Biochem. Soc. S y m p . , Spec. P11bl.3, 43. Edelstein, S. (1974b). Proc. Nutr. Soc. 8, 255. Edelstein, S., Lawson, D. E. M., and Kodicek, E. (1972). Biochim. Biophys. Acts 270, 570.
Edelstein, S., Lawson, D. E. M., and Kodicek, E. (1973). Biochem. J . 135, 417. Edelstein, S., Charman, M., Lawson, D. E. M., and Kodicek, E. (1974). Clin. Sci. Molec. M e d . 46, 231. Fraser, D. R., and Kodicek, E. (1970). Nature (London) 228, 764. Fraser, D. R., and Kodicek, E. (1973). Nature ( L o n d o n ) , New Biol. 241, 163. Haddad, J . G., and Birge, S. J. (1971). Biochem. Bwphys. Res. Commun. 45, 829. Haddad, J. G., and Chyu, K. J . (1971a). Biochim. Biophys. Actu 248, 471. Haddad, J. G., and Chyu, K . J. (1971b). J . Clin. Endociinol. Metab. 33, 992. Hunt, R . D., Garcia, F. G., Hrgstcd, D. M., and Iiaplinsky, N. (1967). Science 157, 943. Jensen, E. V., Numata, M., Brwher, P. I., and DeSornbre, E. R. (1971). I n “The Biochemistry of Steroid Hormone Action” ( R . M. S. Semellie, ed.), pp. 133-159. Ac,adrmic Prrss, Sew York. Kodicrk, E., and Lawson, D. E. M. (1967). I n “The Vitamins” (P. Gyorgy and W. X , Pearson, eds.), Vol. 6, pp. 211-244. Arademic Press, New York. Lawson, D. E. M., Bell, P. A,, Pelc, B., Wilson, P. W., and Kodicek, E. (1971). Biochem. J . 121, 673. Lehner, N. D. M., Bullock, B. C., Clarkson, T. B., and Lofland, H. B. (1967). Lab. Anim. Cnre 17, 483. Mawer, E. B., Lumb, G. A , , Srhaefrr, K., and Seanbury, S. W. (1971). Clin. Sci. 40, 39. Murphy, B. E. P. (1971). Nature (Londort) 232, 21. O’Mallry, B. W. (1971). M e h b . , Clin. Exp. 20, 981. O’Mallry, B. W., Sherman, M. R., and Toft, D. (1970). Proc. N n t . Acnd. Sci. U.S. 67, 501. Peterson, P. A . (1971). J .Biol.Chem. 246, 7748. Rikkers, H., and DeLuca, H. F. (1967). Amer. J. Physiol. 213, 380. Rikkers, H., Klrtziens, R., and D c L ~ c a ,H. F. (1969). Proc. Soc. E z p . B i d . M e d . 130, 1321. Rosenstrrich, S. J., Volwiler, W., and Rich. C. (1971). Amer. J . Clzn. Nutr. 24, 897. Scatchard, G . (1919). A7l7l. N . Y . Acad. Sci. 51, 660.
428
6 . EDlLSTEIN
Sheppard, A. J., Proser, A. R., and Hubbard, W. D. (1972). J . Amer. Oil Chem. Soc. 49, 619. Sklan, D., and Budowski, P. (1973). Anal. Chem. 49, 200. Smith, J. E., and Goodman, Dew. S. (1971). J . Clin. Invest. 50, 2159. Stamp, T. C. B., Round, J. M., Rowe, D. J. E., and Haddad, J. G. (1972). Brit. M e d . J . 4, 9. Thomas, W. C., Morgan, H. G., Connor, T. B., Haddock, L., Bills, C. E., and Howard, J. E. (1959). J. Clin. Invest. 38, 1078. Tomkins, G. M., and Baxter, J. D. (1971). Proc. N a t . Acad. Sci. U.S. 68,932. Tsai, H. C., and Norman, A. W. (1973). J . Biol. Chem. 248, 5967.
.
.
Blood
. 407
.
408
409 416 417 418 418 418 420 421 422 422 422 425 426 427
. . . 408
. . . . . .
. . . . . .
. . . . . .
. . . .
. . . .
. . . .
. . . .
. . . . . . . .
I. INTRODUCTION I n light of the recent findings that cholecalciferol is metabolized before it carries out its functions, there have been renewed attempts to study the transport of this steroid and its metabolites. Cholecalciferol, which is absorbed from the intestine or formed in the skin from its provitamin, is transported to the liver, where it is hydroxylated a t C-25. The 25hydroxycholecaliferol [25(OH)D3] formed is then transported to the kidney, where it is further hydroxylated a t C-1 to yield 1,25-dihydroxycholecaliferol [ 1,25(OH)2D3]. This more recently discovered sterol, which is regarded as the active hormonal form of the vitamin, has to be transported once again to its target organs, for example, the intestine, where it functions in initiating calcium transport. From this metabolic pathway, the immediate questions that arise are: How are the different sterols transported from one site of hydroxylation to another, and from these sites to the target organs? Is there a specific carrier protein in the blood stream for each of the metabolites, or one common protein? How does the sterol recognize its target cell? 407
408
S. EDELSTEIN
Over the past three years several studies in search of answers to these questions have been started but are still in their infancy. The purpose of this article is to summarize progress and to point out the difficulties and problems that are yet to be overcome and resolved. One practical outcome of these studies on binding-proteins was the development of competitive protein binding assays for vitamin D and its metabolites, and a review on this topic and on the assessment of vitamin D status in man will also be included.
11. THETRANSPORTING PROTEINS OF VITAMIND ITSMETABOLITES IN BLOOD
AND
A. INTRODUCTION
It is well established that cholecalciferol in blood circulates attached to proteins. In 1959, Thomas and associates studied the transport of cholecalciferol in serum of humans ingesting large doses of the vitamin. Serum proteins were separated by starch block electrophoresis, and the fractions obtained were tested for antirachitic activity. They found that the antirachitic activity was associated with al- and a,-globulins, and a small amount of the activity was associated with the albumin fractions. Similar findings were observed in vitro upon addition of 12.5 pg of the vitamin to serum. DeCrousaz et al. (1965) confirmed these findings by fractionating human serum on agar-gel electrophoresis. By staining some pherograms for lipoproteins and examining others by immunoelectrophoresis, they excluded the involvement of lipoproteins in the transport of the vitamin. Subsequently the fate of the vitamin was followed by 14C or 3H labeling. Chalk and Kodicek (1961) used for the first time 14C-labeled ergocalciferol to study the transport of vitamin D in blood. The labeled vitamin in this study was incubated in vitro with rat serum. It was possible to detect the vitamin in small regions of starch block electrophoretograms, and so determine in more detail the distribution of the labeled ergocalciferol with respect to protein. The radioactive material was found to correspond with a,-globulin and albumin. Chen and Lane (1965) gave 3H-labeled cholecalciferol to a dog and analyzed serial serum samples by ultracentrifugal flotation techniques. About 80% of the radioactivity in the serum sedimented, whereas the remaining 20% floated with the lipoprotein fraction. On addition of the vitamin to serum in vitro, some 40% of the radioactivity floated with the lipoproteins.
409
VITAMIN D BINDING PROTEINS
Rikkers and DeLuca (1967) and Rikkers e t al. (1969) have shown that the vitamin associated rapidly with rat serum proteins in vivo. The radioactivity was found associated with five protein fractions, four of which were shown to be lipoproteins, and the fifth an a-globulin. As the proportion of the radioactivity associated with the lipoproteins decreased with time, they concluded that plasma contained a specific vitamin D-binding globulin. From all the above-mentioned studies, it was well established and accepted toward the end of the 1960s that a specific a-globulin in serum is responsible for the transport of cholecalciferol.
B.
SERUM
BINDING P R OT E I NS
FOR
CHOLECALCIFEROL
AND
25-HYDROXY-
CHOLECALCIFEROL
1. Man, Rat, and Monkey
The reports of Smith and Goodman (1971) and Haddad and Chyu (1971a) were the first studies on the transport of 25(OH)D, in light of the recent findings on the metabolism of cholecalciferol. I n the first study, normal men were dosed with 3H-labeled cholecalciferol, and serial plasma samples were collected. In all the samples, almost all the radioactivity was found to be associated with proteins of density greater than 1.21. The transport protein has been partly characterized by gel filtration and gel electrophoresis and was found to be smaller than plasma albumin and to have an electrophoretic mobility slightly greater than plasma albumin. Haddad and Chyu (1971a) labeled human plasma proteins in vivo and in vitro with tritiated 25(OH)D,, and found that most of the radioactivity was associated with a protein with a-globulin mobility on polyacrylamide disc-gel electrophoresis, a molecular weight of 40,000-50,000, and a sedimentation coefficient of 3.1 S. I n another study Peterson (1971) obtained a highly purified preparation of a cholecalciferol binding protein from human plasma which had previously been incubated with “C-labeled cholecalciferol. Although these studies demonstrate clearly that both cholecalciferol and 25(OH)D, are transported in human blood bound to a specific protein, they do not answer the question whether the same or different proteins are responsible for the transport of these two sterols. I n experiments with humans (Smith and Goodman, 1971; Haddad and Chyu, 1971a), the major circulating metabolite was 25(OH)D3. Edelstein et al. (1973) have analyzed human plasma in which either radioactive labeled cholecalciferol or 25 (OH) D, was the major component. Plasma was obtained from a 60-year-old female with primary hyperparathyroidism a t 4 and 8 hours after a dose of 10 pCi of 13-H-labeled
410
S. EDELSTEIN
cholecalciferol. At these time intervals the proportions of cholecalciferol to 25(OH)D, were 5.4:l and 2:1, respectively. Cohn fractionation of these plasma samples showed that 47% of the radioactivity a t 4 hours was in fraction IV, which contains the lipoproteins; a t 8 hours the proportion in this fraction was 41%. The globulin fraction a t 4 and 8 hours conkined 20% and 25%, respectively. Another sample was obtained from a 57-year-old male with anticonvulsant osteomalacia 12 hours after he received 10 pCi of lJH-labeled cholecalciferol and 2 pCi of 4-l'C-labeled cholecalciferol. In this sample the radioactivity was due mainly to 25(OH)D,, and 50% of the radioactivity was found to be associated with the a-globulin. However, analysis of a 24-hour sample of plasma obtained from an ll-year-old male with pseudo-vitamin D deficient rickets (dosed as before) with a cholecalciferol: 25(OH)D, ratio of 6 : l showed a single peak of radioactivity after chromatography on DEAE-Sephadex, a peak that was identical to the peak of plasma protein that binds 25(OH)D3. These studies with human plasma show that although there is a significant binding to lipoproteins, this is most noticeable a t the early time-intervals after an intravenous dose of the cholecalciferol. This association with the lipoproteins decreases slowly with time, so that by 24 hours all the cholecalciferol and its metabolites are associated with the a-globulin. The binding of cholecalciferol to lipoproteins is unrelated to body stores of the vitamin. In order to establish conclusively that only one binding protein is present in plasma for both cholecalciferol and 25(OH)D,, Edelstein et al. (1973) carried out the following experiment. One group of rachitic rats was injected with 4-lT-labeled cholecalciferol, and a second group was injected with 26,27-3H-labelcd 25 (OH)D,. Serum was prepared from the first group 3 hours later, a time interval a t which the major circulating metabolite is the unchanged vitamin. Serum from the second group was prepared after 8 hours, a time interval a t which all the 25 (OH)D, present in circulation is attached to the a-globulin. The sera were mixed and chromatographed on DEAE-Sephadex. A single radioactive peak of radioactivity was observed (Fig. 1) and the 3H:14Cratio was constant throughout the peak. In other words, in rat serum a single binding protein is responsible for the transport of both cholecalciferol and 25(OH)D,. This is the case with man, pig, and monkey, and is most probably true with regard to mammals in general (Edelstein, 1974a). There have been several reports on the dissimilarity in the biological activity of ergocalciferol compared with cholecalciferol between New World and Old World monkeys (Hunt et al., 1967; Lehner e t al., 1967). Apparently, New World monkeys cannot use ergocalciferol as efficiently as cholecalciferol, and therefore it was of interest to see whether the bind-
41 1
VITAMIN D BINDING PROTEINS 0.4
16
A 0.3
I2
0.2
0
E 0 OD N
”
-: 4
.-C0
?
s X
E a
.”
n
W
0.1
4
0 10
20
30 Fraction
40
50
number
FIQ. 1. Ion-exchange chromatography on DEAE-Sephadex of mixed samples of serum of rats given a dose either of 26,27-3H-25(OH)D, or 4-14C-cliolecalciferol. 0 , E280;0, “C radioactivity; . , 3H radioactivity. According to Edelstein et al. (1973), by permission of The Biochemical Society, U.K.
ing protein of these monkeys differs from the binding protein of ariiriiala in which these two steroids have equal activity. Sera were obtained from 9 vitamin D-deficient C e b w albifrons (New World) and from a n Erythrocebus pntas (Old World), and incubated with radioactive 25 (OH) D,. Their proteins were then resolved on annlytical polyacrylamide-disc gel electrophoresis. As with other mammals and as found with baboons (Rosenstreich et al., 1971), the radioactivity in the serum of the Old World hlonkey was associated with a protein possessing a-globulin mobility. I n the New World Monkey the radioactivity was associated with a protein possessing albumin mobility. A labeled fraction with this same mobility was observed in serum obtained from the C e b m albifrons monkey 24 hours after a dose of 1 pCi of l-3H-labeled cholecalciferol. The serum that was obtained from this dosed monkey was further studied by fractionation according to Cohn’s method. It was found that 87% of the radioactivity was recovered in fraction V, the albumin fraction. An attempt to separate a carrier protein from the albumin by chromatography on Sephadex (3-200 followed by DEAE-Sephadex was unsuccessful (Edelstein e t al., 1973).
412
S. EDELSTEIN
2. Chick Unlike rat serum, chick serum contains two binding proteins, and their presence can be demonstrated readily by analysis on polyacrylamide disc gel electrophoresis of serum to which either cholecalciferol or 25 (OH)D, has been bound in vitro. If binding occurs after a dose of radioactive labeled cholecalciferol, it is found that 83% of the radioactivity attached to one protein is due to 25 (OH)D, and only 10% to cholecalciferol. The radioactivity attached to the second protein consists mainly of cholecalciferol (55%) and to a lesser extent t o 25(OH)D3 (20%) (Edelstein et al., 1972). The two proteins can be separated in a single step, using ionexchange chromatography on DEAE-Sephadex (Fig. 2 ) . We named the first protein 25-hydroxycholecalciferol-binding protein (Fig. 2, peak 1) , and the second protein cholecalciferol-binding protein (Fig. 2, peak 2 ) . By means of Cohn fractionation, (NH,) zS04 precipitation, gel filtration on Sephadex G-200, ion-exchange chromatography on DEAE-Sephadex, and an additional gel-filtration step on Sephadex G-100, these two binding proteins were purified (Edelstein et al., 1973). Scheme 1 outlines the purification procedure for the two proteins. In order to assess the role that each one of these proteins plays in the transport of these sterols in plasma, the following experiments have been carried out. One group of rachitic chicks was dosed with 26,27-,Hlabeled 25 (OH)D,, and serum was collected after 8 hours. A second group of chicks was given a dose of 4-lT-labeled cholecalciferol, and 3 hours 0.4
r
r
10 8
0 0
10
20 Fraction
30
40
50
60
number
Fxo. 2. Ion-exchange chromatography on DEAE-Sephadex of chick serum given dose of l-8H-cholecalciferol. -, Ezso; ..*-, 'H radioactivity. According to Edebtein et al. (19731, by permission of The Biochemical Society, U.K. R
413
VITAMIN D BINDING PROTEINS
Serum
.1
Cohn fractionation
Cohn fraction V
1
(NH4)2SO4fractionation
Proteins precipitated between 50% and 80% saturation Gel filtration on Sephadex C;-200 followed by ion-exchange chromatography on DEAE-Sephadex (0-0.6 M-NaCI gradient,). I
I
1
25Hydroxycholecalciferolbinding protein
1
Peak 2
Peak 1 Gel filtration on Sephadex G-100
C holecalciferol-binding . protein
SCHEME 1. Scheme of purification of cholecalciferol-binding protein and 25hydroxycholecalciferol-binding protein.
later, when most of the "C was still present a s the unchanged sterol, serum was again collected. The samples were mixed together, and were subjected to gel filtration on Sephadex G-200 followed by ion-exchange chromatography on DEAE-Sephadex (Fig. 3 ) . 3H-Labeled 25 (OH)D, was bound to both of the binding proteins in approximately equal proportions, but the "C-labeled cholecalciferol was bound only to the cholecalciferol-binding protein. Analysis by thin-layer chromatography (TLC) of the lipids extracted from these two proteins showed 14C-labeled 25 (OH)D, to be distributed between the 25-hydroxycholecalciferol-binding-protein and the cholecalciferol-binding-protein in the ratio of 9 :1. These findings strongly suggest that 25 (OH)D, synthesized in the liver is released into the bloodstream bound to its specific binding protein. However, the addition of 25(OH)D, to plasma either directly into the bloodstream or in vitro results in immediate binding to these two binding proteins in approximately equal proportions. This is due most probably to the limited solubility of 25(OH)D, in water. The high-affinity for 25(OH)D, by these two proteins was demonstrated by analysis of the displacement curves (Figs. 4a and 4b) accord-
414
S. EDELSTEIN
Fraction
number
FIQ.3. Ion-exchange chromatography on DEAE-Sephadex of mixed samples of serum of chicks given a dose of either 26,27dH-25(OH)Da or 4-"C-cholecalcifero1. 0, "C radioactivity; 0, 'H radioactivity. According to Edelstein et al. (1973), by permission of The Biochemical Society, U.K.
ing to Scatchard (1949).The association constant (KRs80c.) of 25 (OH)D, with its specific binding protein was found to be 3.0 x lox liters/mole, and with the cholecalciferol-binding protein 4.3 X lo8 liters/mole. Binding could not be obtained between the 25-hydroxycholecalciferol-binding protein and cholecalciferol, indicating the high specificity of the binding sites on this protein toward the hydroxylated metabolite. However, a high K,,,,,, of cholecalciferol with its binding protein has not been obtained. This is most probably a consequence of the virtual insolubility of this sterol in water. The physical properties of the chick serum bindingproteins are summarized in Table I. 3. Toad
It was found by Bruce and Parkes (1950)that vitamin D is required by amphibians. This group of animals is the first evolutionary group to require an efficient regulation- of calcium absorption in order to maintain calcium homoeostasis. It was therefore of interest to study the transport
415
VITAMIN D BINDING PROTEINS
50
.
40 .
30 . U 3
g #
0 C
a
20. 10
0
m
’
O t
~
0
5
10
25(OH)D3
15
20 Steroid (ng)
(ng)
FIG.4. Competitive displacement of 26,27-3H-25(OH)Da from the 25(OH) D, binding protein ( a ) and of 26,27-’H-25(OH)D3 and 1,2-aH-cholecalciferol from the cholecalciferol binding protein (b) by increasing amounts of the two non25(OH)D3. According t o Edehtein et al. labeled steroids. e, Cholecalciferol; 0, (19731, by permission of The Biochemical Society, U.K. of cholecalciferol in this group as well. Vitamin D-deficient toads Xenopus lnevis were dosed with l-3H-labeled cholecalciferol, and 5 days later serum was prepared. At this time, cholecalciferol and 25(OH)D3 were present in approximately equal proportions in serum. Analytical polyacrylamide disc gel electrophoresis analysis showed the radioactivity to he concentrated near the origin, in an area which stained deeply with the lipid stain, Oil Red 0 (Edelstein et al., 1973). The radioactivity remained in the supernatant after precipitation of p-lipoproteins. The TABLE I PHYSICAL PROPERTIES OF CHICKSIERUM BINDING PROTEINS Property Sedimentation coefficient (s) Electrophoretic mobility Molecular weight (approx.) Association constant for 25(OH)D3 at 4°C
W-1)
EDTA and SH-groups as st,abiliaing agents
Cholecalciferolbinding protein
25(OH)D3binding protein
3.5 s B 60,000
3.5 s B 54,000
4.3 x 108 Yes
3 . 0 x 108 No
416
S. EDELSTEIN
serum was ,subjected to Cohn fractionation, and 80% of the radioactivity was recovered in Fraction VI-1, which contains the a-lipoproteins.
c. SERUMBINDINGPROTEINS FOR
1,25-DIHYDROXYCHOLECALCIFEROL
Although we seem to understand how cholecalciferol and 25 (OH) D, are transported in the bloodstream, nothing is yet known about the transport of the hormonal form of the vitamin, 1,25(OH)2D3.The main obstacle in studying the transport of this metabolite is its low concentration in blood. It is practically impossible to study its transport in chick serum, since it accounts for less than 5% of the total antirachitic activity of the serum and consequently is present only in extremely small amounts. In the rat, a higher proportion of 1,25 (OH)2D3may be present (Lawson et al., 1971) and therefore we have attempted to study its transport in this species. Rachitic rats were dosed with tritiated 25(OH)D3,and 16 hours later serum was prepared. TLC analysis of the lipid extract of the serum revealed that some 10% of the radioactivity was due to 1,25(OII)D3. Fractionation of the serum on Sephadex G-200 followed by ion-exchange chromatography on DEAE-Sephadex resulted in the separation of the a-globulin peak that binds cholecalciferol and %(OH) D,. The fractions within the peak were combined, and the lipids were extracted and analyzed on a silicic acid column. Both 25(OH)D3 and 1,25(OH)?D3were found to be present. When tritiated cholecalciferol was dosed 16 hours before the collection of the sera, all three metabolites, namely, cholecalciferol, 25 (OH)D,, and 1,25 (OH)D3, were found to be associated with this peak, and in the same proportions as in the lipid extract of the unfractionated serum (S. Edelstein, D. E. M. Lawson, and E . Kodicek, unpublished work). However, this finding does not rule out the possibility of the existence of a specific binding protein for 1,25(OH),D3 in blood. The reversible nature of the binding between steroids and proteins results in the accumulation of the steroids on proteins with high affinity for them, and a shift to other high affinity binding protein may have taken place. Careful examination of the fractions eluted from the DEAE-Sephadex column on which the above sera were fractionated, revealed that there exists an additional minute radioactive peak (Fig. 5, peak b) which is eluted at higher salt concentration than the a-globulin which binds cholecalciferol and 25(OH)D3 (Fig. 5, peak a ) . Similar fractionation of serum obtained a t longer time intervals, 24 and 36 hours after dosing, resulted in the increase of peak b, so that some 25-30% of the radioactivity was associated with this peak. Analysis on a silicic acid column of the lipid extract of this peak gave a similar picture of distribution of cholecalci-
417
VITAMIN D BINDlNG PROTEINS
0.4
E 0 .D N L
NaCl molarity
peak a
0.3
0.2
'
a
-
0.6-
. 30
0.4
. 20
3 5! X
0
0
C
.c
.-g
140
a1
.
Y
0
0
10
20
30
40
50
60
Fraction number
FIG.5. Ion-exchange chromatography on DEAESephadex of serum obtained from rachitic rats 16 hours after a dose of 26,27-'H-25(OH)D3. E m ; 0, 'H radioactivity.
ferol metabolites to the one obtained with peak a. I n other words, we were unable to show any preference of binding of one metabolite on the other. This may again be an artifact caused by elaborate time-consuming procedures, and therefore we have attempted to establish in vitro the affinity of this protein(s) toward the different metabolites by carrying out competitive displacement studies. Again, very flat displacement curves were obtained with 25 (OH)D, and with 1,25 (OH).D,, which leaves the entire question of the transport of 1,25(OH),D, unresolved. Nephrectomy of the rats prior to the administration of the radioactive cholecalciferol did not prevent the appearance of the second peak in plasma.
D. CONCLUSIONS
It seems that the chick is the only species so far examined that has more than one binding protein for cholecalciferol and its metabolites. In man, Old World monkey, and rat, there is only one carrier protein for both cholecalciferol and 25 (OH)D,. If two proteins are circulating in the plasma, their properties must be then w r y similar. Perhaps it is not a coincidence that the two species, chick and New World monkeys, that cannot utilize ergocalciferol as efficiently as cholecalciferol have different carrier proteins from those animals that use these two steroids equally well. It still remains to be shown whether it is albumin itself that binds the vitamin in the plasma of the New World monkeys. If the toad is typical of the amphibian group, then they do not appear
418
S. EDELSTEIN
TABLE I1 DISTRIBUTION OF BINDING PROTEINS AMONG DIFFERENT SPECIES AND THEIRELECTROPHORETIC MOBILITY Species Human Rat Pig Monkey (Erythrocebus patas)) Chick Monkey (&bus albifrons) Xenopus
Number of binding proteins
Electrophoretic mobility
1
a-Globulin
2 1 -
&Globulin Albumin a-Lipoprotein
to have iideveloped’l a specific binding protein, but rather depend on the nonspecific lipoproteins for transporting cholecalciferol and its 25-hydroxymetabolite. The electrophoretic properties of the binding proteins from the animals studied are summarized in Table 11. The transport of 1,25(OH)2D3 is a t the moment still an open question.
111. TARGET ORGAN BINDING PROTEINS A. INTRODUCTION After obtaining sufficient information about the serum binding proteins, the next step in the study of the mode of transport of vitamin D and its metabolites is the identification and characterization of binding proteins in target organs, and their interaction with the serum proteins. Recent studies on the mode of action of several steroid hormones suggest that the sterol modifies intracellular events in target cells by first complexing with soluble cytoplasmic receptor proteins, and subsequently associating with chromatin acceptor proteins (O’Malley, 1971). Accordingly, search has begun for such proteins in tissues thought to be target organs for vitamin D metabolites.
B. BINDING PROTEINS
FOR 25-HYDROXYCHOLECALCIFEROL
25-Hydroxycholecalciferol is the major circulating metabolite of vitamin D, and apparently the dominant metabolite in most tissues. Although no apparent action can be related to this metabolite in target cells, Haddad and Birge (1971) were able to show the presence of specific bind-
419
VITAMIN D BINDING PROTEINS
0
5
10
15
20
25(OH)D3 ( n g )
FIQ. 6. Competitive displacement of 26,27-3H-25(OH)D, from supernatants from different tissues by increasing amounts of nonlabeled 25(OH)D,. 0 , Bone; 0, skin; 0, muscle; V , kidney. According to Edelstein (1974a), by permission of The Biochemical Societ,y, U.K.
ing proteins for this metabolite in 105,000 g supernatants from kidney and muscle homogenates. We have prepared 105,000 g supernatants from various tissue homogenates of rachitic rats and studied their binding affinities toward 25 (OH)D, (Edelstein, 1974a). The displacement curves obtained with kidney, muscle, skin, and bone are shown in Fig. 6; the association constants toward 25 (OH)D, calculated from Scatchard plots of these curves are listed in Table 111. Flatter curves and lower association constants were obtained in our hands with supernatants from liver and intestine. The finding that kidney contains a high affinity binding protein for 25 (OH) D, is consistent with the important finding of Fraser and Kodicek (1970) that in this organ the 1-hydroxylation of 25(OH)D, takes place. However, it is not understood a t the present state of our knowledge on TABLE I11 ASSOCIATION CONSTANTS OF I ) I FFER E N T TISSUYBINDING PROTEINS TOWARD 25(OH)II3
Muscle Kidney Skin Bone
1 . 7 X loo 1 . 1 x 100 1 . 0 x 109 4 . 0 X loo
420
S. EDELSTEIN
the mode of action of vitamin D, why the other tissues should also contain such a protein. The possibility still remains that, although these proteins show high affinity toward 25 (OH)D,, in vivo, they do not bind this metabolite but another sterol with a similar structure. The binding protein in skin, for example, may well be a binding protein for cholecalciferol, which is formed in this tissuc from its provitamin, 7-dehydrocholesterol, but, because of the limited solubility of this sterol in water, it is impossible to establish its high association constant with the protein.
c.BINDINGPROTEINS
FOR 1,25-DIHYDROXYCHOLECALCIFEROL
In target cells of steroid hormones, specific receptor proteins are involved in the intracellular transport of the steroid, and a steroid-protein complex is effective in the initiation of the biochemical response of the hormone. Since 1,25 (OH) LIDS is similar in its structure, formation, and apparent mode of action to other steroid hormones, it was expected that receptor binding proteins for this metabolite will be found in its target cells. Chen and DeLuca (1973) have isolated from intestinal mucosa of rats a low density protein fraction which binds 1,25(OH),D,. This protein fraction when isolated from nephrectomized rats was found to bind only one-eighth of the radioactivity of that from intact animals after dosing with 3H-labeled 25 (OH)D,. Chemical analysis of this protein fraction has shown that some 50% of the lipid is phospholipid, which may suggest that these are membrane lipoproteins. Tsai and Norman to intestinal mucosa (1973) have studied the binding of 1,25 (OH) in an in ziitro incubation system, which enabled them to obtain saturation levels comparable to those obtained in vivo. They were able to show the presence of a cytoplasmic receptor protein for 1,25(OH)& of the size of 65,000-150,000 daltons, and that the binding of 1,25(OH)2D3to this receptor protein is an obligatory step for the subsequent localization of the hormone in the chromatin fraction. However, from the study of Brumbaugh and Haussler (1973) it seems clear that the mechanism of action of cholecalciferol is similar to the mechanism postulated for other steroid hormones. This mechanism supports the existence of a cytoplasmic hormone-receptor complex in the target cell which is accepted by the genome of the cell. As a consequence of the association of the steroid or sterol-receptor complex with the accepting sites in the nucleus, the characteristic biological response of the hormone is initiated. In order to test the specificity of the cytoplasmic receptors for 1,25(OH)2D3in intestinal mucosa, the authors have incubated mucosa homogenates with different closely related sterols, like 25 (OH)D,, chole-
VITAMIN D BINDING PROTEINS
421
calciferol or estradiol. Although it was found that 1,25(OH),D3 binds very effectively to the receptor, far more effectively than cholecalciferol or estradiol to this receptor, 25(OH)D, did bind equally well. Again 1,25(OH),D, was found to bind effectively to liver cytosol, a nontarget organ. From this it s e e m that, on the basis of in vitm binding to cytosol fractions, it is not possible to prove specificity, although displacement studies may do so, but these studies were not carried out by these authors. When such studies were carried out with regard to 1,25(OH),D3 or 25 (OH)D, and cytosol from intestinal mucosa, this fraction showed greater affinity for 25(OH)D, than for 1,25(OH),DS (S.Edelstein, unpublished work). However, when Brumhnugh and Haussler (1973) incubated whole intestinal tissue in Eagle’s medium with radioactive 1,25(OH),D,,a t 0°C: and then transferred the tissue to a fresh medium a t 37OC, the radioactive hormone was found primarily in the chromatin fraction, suggesting a temperature-dependent movement of 1,25(OH),D3 from cytosol to the nucleus, in the manner proposed for other steroid hormones. This temperature-dependent movement of 1,25(OH),D, is in contradiction with results of Tsai and Norman (1973), who did not observe such dependency in their in vitro systcm. A similar experiment with regard to 25(OH)D, would be of interest when attempting to determine the specificity of the receptor sites in intestinal mucosa toward 1,25(OH).D,. Furthermore, Brumbaugh and Haussler (1973) did demonstrate very clearly that the intestinal cytosol fraction is an obligatory requirement for the transfer of 1,25(OH),D3 to the chroinatin. 1,25(OH I .D,$did not enter the nucleus when incubated with isolated intestinal nuclei nor with boiled intestinal cytosol, and not with other nontarget tissue cytosols.
D. CONCLUSIONS From the few studies reported on the interaction of 1,25(OH),D3with intestinal mucosa preparations, it is clear that this target tissue possesses specific cytoplasmic receptor protein ( s ) for 1,25(OH),D,, and that this receptor is obligatory for the transfer of the hormone t o the nucleus. Further characterization of these initial receptor proteins, especially the chromatin receptors, is required in order to draw conclusions as to their functional role in the initiation of the physiological response to 1,25(OH),D, in the intestine. Since so far it seems that the interaction of 1,25(OH),D, with the intestine is similar to the interaction of other steroid hormones and their target cells (O’Malley et al., 1970; Tomkins and Baxter, 1971; Jcnsen et al., 1971), it may be possible that 1,25(OH)?D, acts to regulate gene expression.
422
S . EDELSTEIN
IV. BINDINGPROTEINS IN THE ASSAYOF VITAMIN D A N D ITSMETABOLITES A. INTRODUCTION During the past forty years, attempts continually have been made to develop a sensitive and accurate method for the estimation of cholecalciferol. I n general, the methods developed can be divided into two types: (1) physicochernical methods and (2) biological assays. These have been reviewed by Kodicek and Lawson (1967) ; and by Sheppard et al. (1972). At present, the available physicochemical methods are not suitable for the estimation of the small amounts of vitamin D present in animal tissue. Furthermore, these methods involve laborious and timeconsuming separation procedures in order to eliminate interference by accompanying substances, such as retinol and cholesterol. However, Sklan and Budowski (1973) have recently developed a simple separation of cholecalciferol and ergocalciferol from other sterols and retinol by argentation thin-layer chromatography, which may be very useful in the determination of vitamin D by physicochemical means. As a result, biological assays were widely used in many laboratories for the estimation of vitamin D in tissues, foodstuffs, fish oils, and pharmacological preparations. Apart from the limited sensitivity of these methods, their major disadvantage is the cost, labor, and time required for the assay. The renewed interest in vitamin D binding proteins during the past three years has led to the development of new and simple techniques for the estimation of cholecalciferol and 25(OH)D,. These are the competitive protein-binding assays, which are widely used for the estimation of many other steroid hormones. The principle of these assays is that the tested sterol is competing with a radiolabeled sterol for binding to specific sites on a protein. By separating the free molecules of the sterol from the bound ones, and comparing the percentage binding with a calibration curve, the sterol can be estimated. B. COMPETITIVE PROTEIN BINDINGASSAYS 1. ChoZecalcijerol
Belsey et al. (1971) have published the first competitive protein-binding assay for both cholecalciferol and 25(OH)D3, utilizing the serum binding protein of the rat. The sterols to be tested are extracted, sepa-
VITAMIN D BINDING PROTEINS
423
rated on columns, and then introduced to the binding assay mixture. The problem of the limited solubility of these sterols in water was overcome by inclusion in the assay system p-lipoproteins as carrier for them. The inclusion of p-lipoprotein in the assay mixture enabled these investigators to separate the free sterols from the bound sterols by selective precipitation of the p-lipoproteins only. However, several days were needed for equilibrium to be reached in a system in which the sterols are supposed to detach themselves from a somewhat low affinity binding site on the lipoprotein, and attach themselves to binding sites possessing greater affinity on the serum binding protein, and all this is an aqueous medium. Although it is possible to estimate by this method several nanograms of cholecalciferol or 2 5 ( O H ) D , , the long period of time required in order to reach equilibrium and displacement prevented the acceptance of this method for routine use. 2. 25-H ydrox~cholecalciferol Haddad and Chyu (1971b) introduced another competitive proteinbinding assay for 25(OH)D,, utilizing the kidney binding protein as the assay protein. The steroid was solubilized by including 7% absolute ethanol in the assay mixture so that the time required to reach equilibrium and displacenicnt was shortened to 11 hour. The free sterol was separated from the bound, using charcoal coated with dextran. This method provides a simple and sensitive means for the routine estimation of 25(OH)D3 levels in peripheral blood. As little as 4 ng/ml of plasma can be estimated. However, chromatography on silicic acid columns is used in this assay in order to separate 2 5 ( O H ) D , from the other plasma lipids prior to estimation. This technique we find interferes with the competitive binding and causes erratic blank values. This is due to substances produced during chromatography as a result of impurities in the silicic acid material and of the interaction of the solvents with the silicic acid (hlurphy, 1971). By using small Sephadex LH-20 columns for the separation of the 25 (OH)D, metabolite, and a partially purified vitamin D binding protein from rat serum as the assay protein, Edelstein et al. (1974) were able to develop a competitive protein-binding assay for 25 (OH) D, which eliminates these interferences and has several additional advantages. With the use of columns of Sephadex LH-20 instead of silicic ac'd, the blank values were low, erratic displacements were not observed, and larger quantities of lipids could be chromatographed. The same solvent was used throughout the chromatography, and the columns were used several times.
424
S. EDELSTEIN
25(OH)D3 Ingl
FIQ.7. A typical calibration curve for 25(OH)Da.
Diluted rat serum can be employed instead of a partially purified protein from serum. When 1:lO.OOO dilution of serum obtained from nonvitamin D-deficient rats of the Wistar strain was used in the assay, the sensitivity was increased, and as little as 50 pg per tube could be estimated. Figure 7 represents such a typical calibration curve, and an outline of the assay procedure is illustrated in Scheme 2. 3. 1,25-Dihydroxycholecalcijerol
At present, there is no method for the estimation of 1,25(OH)2D3.The development of a competitive protein-binding assay for this hormone Plasma sample
1
Extraction with chloroform and methanol
1
Chromatography on Sephadex LH-20 J.
25(OH)Da fraction
1
Equilibration of aH-25(OH)I)a and the 25(OH)Da fraction with the assay protein
1
Separation of “free” sterol with charcoal coated with dextran
1
Counting of “bound” sterol
SCHEME2. Scheme of competitive-protein-binding assay for 25(OH)Ds.
VITAMIN D BINDING PROTEINS
425
awaits further information on the properties of its specific binding proteins, like the cytoplasmic receptor protein from intestinal mucosa. Radioimmunossay may be of great advantage in the estimation of this hormone, but to date there is no such an assay for any of the metabolites of vitamin D, although research along this line is in progress. However, in the light of the facts that the formation of this hormone is controlled by a very fine and complex mechanism (Fraser and Kodicek, 1973), and that the circulating levels of this hormone under normal conditions are extremely low or may in fact be zero (S. Edelstein, D. R. Fraser, and E. Kodicek, unpublished work), the application of a method for the estimation of 1,25(OH),D, for routine clinical use is doubtful.
C. THEASSESSMENT OF VITAMIN D STATUS The method for the estimation of 25(OH)D, (Edelstein et al., 1974) was applied to the measurement of plasma levels of this metabolite in 18 normal male and female voluntcers and in 4 normal male volunteers taking daily cholecalciferol supplement of 10 pg (Edelstein, 1974b). The mean value for the normal volunteers was found to he 15.2 k 5.6 ng/ml, but as can be seen from the histogrnm illustrated in Fig. 8, 60% of the volunteers had lower 25 (OH)D, lcvels. This 25 (OH)D, value which is found in thc United Kingdom is similar to the finding of Stamp et al. (1972) but lower than the mean value of 27.3 k 11.8 found by Haddad
U.
0 0
2
25(OH)D3 ( n g / m l )
FIG.8. Histogram of 25(OH)D, values obtained for normal volunteers.
426
S. EDELSTEIN
TABLE IV I N NORMAL ADULTS
PLASMA 25(OH)I>,
Daily cholecalciferol Supplement Group
Number
(fig)
Normal Vitamin D supplement P value
18
-
4
10
a
25(OH)D3' (ng/ml) 15.2 k 5 . 6 3 5 . 9 k 15.0 <0.001
Values represent mean k SI).
and Chyu (1971b) for normal volunteers in the United States. This is due most probably to differences in dietary vitamin D intake and exposure to sunshine. Thc finding that significantly higher plasma values of 25 (OH)D,3were obtained for those who had a daily cholecalciferol supplement (Table IV) indicates that 25(OH)D, levels in blood may be useful for the nutritional assessment of vitamin D status.
D. CONCLUSIONS One practical outcome of the studies on binding proteins was the development of competitive protein binding assay for 25(OH)D,. This assay is simple, reliable, and sensitive and is suitable for routine estimation of 25(@H)D, levels in peripheral blood samples. Since the assay can detect as little as 0.2 rig or less of 25(OH)D, per tube, and since normal circulating levels of 2 5 ( O H ) D , were found to be approximately 15 ng per milliliter of plasma, very small blood samples need to be taken for analysis. Since 25 (OH)D, is the principal circulating metabolite of vitamin D in blood, with cholecalciferol accounting for only a small proportion and 1,25(OH),D, contributing less than 5% to the total antirachitic activity (Lawson et al., 1971; Mawer et al., 1971), the estimation of 25 (OH)D, is probably a useful guide in the assessment of the nutritional status of vitamin D. Attempts to develop a similar assay for cholecalciferol have so far been unsuccessful. Although specific binding proteins for cholecalciferol exist (Edelstein et al., 1972, 1973), the limited solubility of the steroid in water prevents competitive displacement from taking place. The estimation of 1,25(OH):D, awaits further information on the properties of the specific binding proteins for this hormone.
VITAMIN D BINDING PROTEINS
427
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Brumbaugh, P. F., and Hausslcr, M. R. (1973). Biochem. Biophys. Res. Commun. 51, 74.
Bruce, H. M., and Parkes, A. S. (1950). J . E n d o c ~ i m l 7, . 64. Chalk, K. J. I., and Kodicek, E. (1961). Biochem. J . 79, 1. Chen, P. S., and Lane, K. (1965). Arch. Biochem. Biophys. 112, 70. Chrn, T. C., and DeLuca, H. F. (1973). J . Biol. Chem. 248, 4890. DeCrousaz, P., Blane, B., and Antener, I. (1965). Helv. Odontol. Actu 9, 151. Edelstein, S. (1974a). Biochem. Soc. S y m p . , Spec. P11bl.3, 43. Edelstein, S. (1974b). Proc. Nutr. Soc. 8, 255. Edelstein, S., Lawson, D. E. M., and Kodicek, E. (1972). Biochim. Biophys. Acts 270, 570.
Edelstein, S., Lawson, D. E. M., and Kodicek, E. (1973). Biochem. J . 135, 417. Edelstein, S., Charman, M., Lawson, D. E. M., and Kodicek, E. (1974). Clin. Sci. Molec. M e d . 46, 231. Fraser, D. R., and Kodicek, E. (1970). Nature (London) 228, 764. Fraser, D. R., and Kodicek, E. (1973). Nature ( L o n d o n ) , New Biol. 241, 163. Haddad, J . G., and Birge, S. J. (1971). Biochem. Bwphys. Res. Commun. 45, 829. Haddad, J. G., and Chyu, K. J . (1971a). Biochim. Biophys. Actu 248, 471. Haddad, J. G., and Chyu, K . J. (1971b). J . Clin. Endociinol. Metab. 33, 992. Hunt, R . D., Garcia, F. G., Hrgstcd, D. M., and Iiaplinsky, N. (1967). Science 157, 943. Jensen, E. V., Numata, M., Brwher, P. I., and DeSornbre, E. R. (1971). I n “The Biochemistry of Steroid Hormone Action” ( R . M. S. Semellie, ed.), pp. 133-159. Ac,adrmic Prrss, Sew York. Kodicrk, E., and Lawson, D. E. M. (1967). I n “The Vitamins” (P. Gyorgy and W. X , Pearson, eds.), Vol. 6, pp. 211-244. Arademic Press, New York. Lawson, D. E. M., Bell, P. A,, Pelc, B., Wilson, P. W., and Kodicek, E. (1971). Biochem. J . 121, 673. Lehner, N. D. M., Bullock, B. C., Clarkson, T. B., and Lofland, H. B. (1967). Lab. Anim. Cnre 17, 483. Mawer, E. B., Lumb, G. A , , Srhaefrr, K., and Seanbury, S. W. (1971). Clin. Sci. 40, 39. Murphy, B. E. P. (1971). Nature (Londort) 232, 21. O’Mallry, B. W. (1971). M e h b . , Clin. Exp. 20, 981. O’Mallry, B. W., Sherman, M. R., and Toft, D. (1970). Proc. N n t . Acnd. Sci. U.S. 67, 501. Peterson, P. A . (1971). J .Biol.Chem. 246, 7748. Rikkers, H., and DeLuca, H. F. (1967). Amer. J. Physiol. 213, 380. Rikkers, H., Klrtziens, R., and D c L ~ c a ,H. F. (1969). Proc. Soc. E z p . B i d . M e d . 130, 1321. Rosenstrrich, S. J., Volwiler, W., and Rich. C. (1971). Amer. J . Clzn. Nutr. 24, 897. Scatchard, G . (1919). A7l7l. N . Y . Acad. Sci. 51, 660.
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6 . EDlLSTEIN
Sheppard, A. J., Proser, A. R., and Hubbard, W. D. (1972). J . Amer. Oil Chem. Soc. 49, 619. Sklan, D., and Budowski, P. (1973). Anal. Chem. 49, 200. Smith, J. E., and Goodman, Dew. S. (1971). J . Clin. Invest. 50, 2159. Stamp, T. C. B., Round, J. M., Rowe, D. J. E., and Haddad, J. G. (1972). Brit. M e d . J . 4, 9. Thomas, W. C., Morgan, H. G., Connor, T. B., Haddock, L., Bills, C. E., and Howard, J. E. (1959). J. Clin. Invest. 38, 1078. Tomkins, G. M., and Baxter, J. D. (1971). Proc. N a t . Acad. Sci. U.S. 68,932. Tsai, H. C., and Norman, A. W. (1973). J . Biol. Chem. 248, 5967.