Volume 28, Issue 4 (1991)
287
Osteocalcin: Diagnostic Methods and Clinical Applications Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nanyang Technological University on 08/20/15 For personal use only.
Michael J. Power and Patrick f . fottrefl
ABSTRACT Osteocalcin is a small (M, 5800), very interesting bone specific protein, synthesized by osteoblasts and measured in plasma as a biochemical indicator of bone formation. Many immunoassays for osteocalcin have been developed, including radio- and enzymoimmunoassays, with the use of monoclonal and polyclonal antibodies. These are used in many different clinical settings, including bone, kidney, and liver diseases. However, there is a wide range of published values for plasma osteocalcin concentrations in control and patient samples and this has hindered a more widespread adoption of osteocalcin measurement by clinicians. This review discusses how various immunoassays for osteocalcin may contribute to the wide variation of published values and suggests approaches for the development of standardized assays. For example, epitope specificity and immunoreactivity with multiple forms of osteocalcin and osteocalcin peptides in plasma are discussed. It also includes a recent update on interesting clinical applications of osteocalcin. Key Words: osteocalcin (Oc), Bone-Gla protein (BGP), immunoassays, monoclonal, concentrations, osteoblasts.
1. STRUCTURE, IMMUNOREACTIVITY, BIOSYNTHESIS, METABOLISM, AND FUNCTIONS OF OSTEOCALCIN Osteocalcin (Oc), or bone-Gla protein (BGP), is the most abundant noncollagenous protein in mature human bone, where it constitutes 1 to 2% of the total protein.'-3 Synthesized by osteoblasts, it is incorporated into the bone matrix.'-3 Although its exact function is not known, the presence of three gamma carboxyglutamicacid residues in human Oc at positions 17, 21, and 24 confer on it a very strong ability to bind hydr~xyapatite.~.~ Vitamin K, is essential for its biosynthesis, which is stimulated by 1,25 (OH),D,.5-8 The concentration detectable in serum may represent the part of newly synthesized Oc that is not adsorbed but is instead released directly into the circulation. Oc has received much attention as a possible marker for bone turnover. Its concentration in serum increased in patients with metabolic bone disease characterized by increased bone t ~ r n o v e rIt. ~is~a~ ~ more specific index of bone metabolism than the more established indices, such as total serum alkaline phosphatase activity or urinary hydroxyproline. Serum Oc measurements are used in various clinical situations, including metabolic bone diseases, renal disorders, hyperthyroidism, and diseases related to excess glucocorticoids.
P. F. Fottrell, B.Sc., M.Sc., Ph.D., D.Sc., M. J. Power, B.Sc., Ph.D., Dept. ofBiochemistry, University College, Galway, Ireland.
288
Critical Reviews in Clinical Laboratoly Sciences
__
__
5 10 15 Tyr Gln Trp Leu Gly A h Pro Val Pro Tyr Pro Asp Pro Asp His __ __ __ Hyp Ala __ __ __
--
--
Asp pro Gly
1
Human Calf Sheep
Tyr Leu
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nanyang Technological University on 08/20/15 For personal use only.
Swordfish
Human
calf Sheep Swordfish
35 Glu Leu Ala Asp His ne
__ __
__ __
--
Met
__ __
__ --
__
__ __
--
Thr Ala --
Sheep
--
__
__
__ __
Hyp Ala
--
Gly Asp Leu Thr --
__
__
__
__
__
Leu Gln
40 45 Gly Phe Gln Glu Ala Tyr A r g Arg Phe
__
Swordfish
calf
--
Ala Thr Arg
49 Tyr Gly Pro Val --__ _--__
Human
__
__
._
__
__
__ __ __ __ __ Ile
Val Ala
__
__ __
_- __ _- __
--
--
Ile
-
__
__
Ala Tyr
__
--
--
ne
Gln Phe
FIGURE 1. Amino acid sequences of human, bovine, ovine, and swordfish osteocalcin, showing Gla-residues (underlined) and position of disulfide bond.
Radioimmunoassays (RIAs) for quantifying Oc in serum have been and are available commercially. Most involve the use of radioiodinated bovine Oc as label and polyclonal antisera raised against purified bovine Oc. More recently, sensitive enzymoimmunoassays for Oc with use of rnonocl~nal'~ and p o l y ~ l o n a l " - ' ~ antibodies ~ ' ~ ~ ' ~ have been published. Although Oc has considerable clinical potential for the management of patients with metabolic bone disorders, it is difficult at present to interpret serum concentrations because of the wide ranges of published normal and clinical values. Possible explanations for the O,~' published variations in Oc concentrations have been suggested by us13 and ~ t h e r s ' ~ . ~and include factors involved in the immunoassay for Oc and in the selection of control and patient populations. For example, the extent to which various antisera recognize multiple forms of Oc in human serum may be a contributing factor. This review discusses the various factors involved in the development of immunoassays for Oc that may contribute to assay variation and makes recommendations that could improve the clinical interpretation of Oc values. These aspects of Oc have not been discussed in detail in previous r e v i e w ~ . ' . ~ . ~ ~ . ~ ~
A. Structure and lmmunoreactivity of Oc Over 90% of the primary structure has been conserved between human, ovine, and calf
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Volume 2 8 , Issue 4 (1991)
289
FIGURE 2. The proposed calcium-induced three-dimensional structure for intact bovine osteocalcin.’ Letters refer to the one letter code for the amino acids and the subscripts to their positions. The backbone is represented by the n b b n structure. Formal electronic charges are indicated as they would exist at pH 7 to 8. Probable metal binding sites are represented by M,, M,, and M,. The single disulfide bond, between positions 23 and 29, is indicated as are the locations of the two calcium-induced alpha helices, i.e., the “Gla helix” from residues 16 to 25, and the “Asp-Glu helix” between residues 30 and 41.
Oc, and 50% between human and swordfish Oc5 (see Figure 1). The conservation of primary sequences in the 400 million years in evolutionary time between swordfish and human Oc indicates that it has an important function in bone metabolism. This conservation of primary amino acid sequence is useful for assay development because antisera raised against bovine and ovine Oc apparently cross-react well with human Oc. 11-15 Human Oc is usually measured with the use of purified bovine Oc to generate antibodies, labels, and standards, although several immunoassays have been developed recentlyz5-*’with use of purified ovine Oc as standard, label, and for generating antisera. Conformational aspects of Oc structure have been studied with the use of circular dichroism, UVhisible spectroscopy, nuclear magnetic resonance spectroscopy, and chemical and immunochemical t e c h n i q ~ e s There . ~ ~ ~are~ two ~ ~ ~antiparallel ~ a-helical structures that represent 40% of the overall structure and are stabilized by a disulfide bond between positions 23 and 29 (Figure 2). The a-helical structure is calcium-dependent and does not exist in calcium-deplete Oc molecules. This structure forces the more polar amino acids at the Cand N-terminal ends of Oc to the outside edges, resulting in a more energetically stable structure. The nonpolar amino acids at the C-terminal end may stabilize the calcium-induced a-helical structure.23Likewise, the N-terminal peptide may be important for stabilization of the C-terminal peptide. The latter peptide is the proposed location of an antigenic site.” Many antisera used in the measurement of Oc do not detect the presence of the a-helical structure because most react independently of calcium. Some antisera, however, are calciumdependent25*z9-31 and may detect the a-helical structure, although the exact location of the antigenic site for these antisera has not been determined. The remainder of the molecule is in the P-sheet format, and it appears that the C-terminal and N-terminal ends are in close proximity to each other. The C-terminal and N-terminal peptides may therefore overlap with a resultant sharing of an antigenic site (see Figure 2 ) . This could contribute to the variability in the detection of the antigenic site(s) by different antisera.
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Critical Reviews in Clinical Laboratory Sciences
The following features of the structure and calcium-bindingproperties of Oc may influence its immunoreactivity:
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nanyang Technological University on 08/20/15 For personal use only.
1.
2.
3.
4.
5.
Peptide bonds between residues 19-20 and 43-44 in human and bovine Oc are susceptible to tryptic hydrolysis. The resultant peptides may be the main products of Oc breakdown in the liver, kidney, and plasma.32The immunoreactivity of these peptides with antisera raised against intact Oc varies and is another possible contributory factor to the wide range of published plasma Oc values. Iodination of tyrosine (of which there are 5 in bovine and 4 in human Oc) may be at or near the antigenic site(s) and may influence immunoreactivity. All the Gla-residues in human and bovine Oc may not be ~arboxylated,~ which may reflect the vitamin K status of the donor. Because these Gla-residues induce an ahelical structure in the presence of calcium,28the location of the antigenic site will be influenced by the degree of carboxylation of the Gla-residues. The reduction of the disulfide bond between amino acids 23 and 29 in human or bovine Oc does not apparently influence the immunoreactivity of Oc, when measured by some antisera.’ When calcium ions are complexed by Oc, only two of the 6 to 9 possible coordination sites on the calcium ion are i n ~ o l v e d . ~Calcium ~ * ~ ~bound - ~ ~ to Oc may therefore interact with other Oc molecules to form a cross-link between two Oc molecules, or between Oc and other calcium-binding proteins or complexes.
B. Biosynthesisof Oc The mechanism involved in the biosynthesis and secretion of human Oc by osteoblasts is similar to that for other secretory proteins (see Figure 3). The one copy of the Oc gene in the human genome is located on chromosome number 1,38 which also has the gene for alkaline phosphatase, another biochemical marker for bone metabolism. The Oc gene has four exons and three i n t r ~ n sand , ~ ~codes for a protein with a molecular weight of about 13,500 Da.39.40This protein, pre-pro-Oc, includes the native Oc molecule (exons 3 and 4; 49 amino acids), the membrane signal sequence (exon l), and the propeptide (exon 2), which is recognized by the carboxylating enzymes. This constitutes the entire sequence of the mRNA for Oc. It is the propeptide, together with certain segments of the native Oc molecule itself, which have the recognition sites for the microsomal carboxylatingenzymes.22 Also associated with the Oc gene are the reputed recognition sites for the Oc gene regulators, 1,25 (OH),D, and CAMP.^'^ Potential sites for the binding of several hormone-receptor complexes have been detected, including those for estrogens and thyroid hormone^.^^.^ After the Oc gene in transcribed into mRNA and following removal of the introns, the mRNA is translated at the rough endoplasmic reticulum. The membrane leader sequence directs the remainder of its synthesis into the lumen of the rough endoplasmic reticulum where a carboxylating enzyme system recognizes appropriate glutamic acid residues to form y-carboxyglutamic acid. In bovine Oc the proline at position 9 may be converted to hydroxyproline in the lumen of the rough endoplasmic reticulum. This latter conversion is interesting because bovine Oc is relatively unique in its requirement for three vitamins (D, K, C) for its optimum biosynthesis. Finally, the recognition sequence is cleaved and native Oc is secreted into the external medium. The antibody that recognizes native rat Oc also reacts with ~ r e - p r o - O c , ~suggesting ~ * ~ ~ , ~that the antigenic site is not located near the prepro-segment of Oc . Studies in vitro and in vivo showed that Oc biosynthesis is increased six-fold by 1,25 (OH)2D3,6.7 although there may be substantial synthesis in its absence.40Some osteoblastic cell lines do not synthesize Oc unless 1,25 (OH),D, is present in the m e d i ~ m . A ~ CAMP~.~~ binding site has been located close to the Oc gene,424 but its precise function is unknown.
Volume 28, Issue 4 (1991)
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PE- pro- osteocalcin
signal peptidase
vitK
1
osteoblast cytog-srn
carboxylase
\
propeptide cleavage
bone mineral
\
+
\
blood plasma
FIGURE 3. Biosynthesis of osteocalcin in the human osteoblast cell.23
An apparent increase in Oc biosynthesis has been reported following stimulation of osteoblasts by agents that stimulate adenylate cyclase activity,**but in contrast others detected a decrease after osteoblastic tim mu la ti on.^^ The mRNA for Oc is stabilized by CAMP,^^ which could account for the stimulation of Oc biosynthesis detected by some groups. Carboxylation of the glutamic acid residues is apparently not essential for secretion of Oc. However, the efficiency of secretion may be enhanced by carboxylation because Oc does not accumulate in the medium of vitamin K-depleted cells grown in tissue culture,
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although considerable Oc accumulates inside the ce11.47,48 However, in vivo, there may be oversecretion of Oc in warfarin-treated rat^,'^.'^ and cows,57but, on the other hand, undersecretion has been reported in human beings and sheep treated with ~ a r f a r i n .Further ~~.~~ studies are required to define the role of Gla-residues in the secretion of Oc. Calcitonin, parathyroid hormone, or the serum calcium concentration do not apparently directly influence Oc bio~ynthesis.’.~~ With respect to the possible role of 24,25 (OH),D,, it has been suggested by some researchers that it has no effe~t,4~.” whereas others reported that it increased biosynthesis and promoted accumulation of Oc in hydroxyapatite.60Several growth factors and hormones have been reported to influence Oc biosynthesis, although their mechanism of action or physiological significance is not known. They include transforming growth factors p,6’ interleukin-1,62 insulin-like growth factor 1,63 fibroblast growth factor, both acidic and basic forms (FGFa and b),63 interferon y,@ retinoic a ~ i d , ~gluco’ ~ ~ r t i ~ ~ i d ~ ,thyroid ~ ~ . hormones ~ ~ . ~ ~(T3 . ~and ~ .T4) ~ ,68 ~ ,insulin,69 ~ ~ and e s t r ~ g e n . ~ - ~ & ~ ~ It is interesting and relevant to the phenotypic characterization of the osteoblastic cell lineage that the cell lines that synthesize Oc do not produce other vitamin K-dependent bone proteins, such as matrix Gla-protein. However, prolonged exposure of several osteoblast cell lines to pharmacological concentrations of 1,25 (OH),D, induces the biosynthesis of matrix Gla-protein at the expense of Oc bio~ynthesis.~~ The physiological significance of this is not known. Matrix Gla-protein biosynthesis appears to precede Oc biosynthesis in the mineralization process in bone, and its biosynthesis is a good parameter for differentiating K is also a cofactor for between the various stages of osteoblastic d e ~ e l o p m e n tVitamin .~~ posttranslational carboxylation of matrix Gla-protein, and its biosynthesis is also increased by 1,25 (OH),D,.’3,74 Matrix Gla-protein is soluble in aqueous buffers only at relatively dilute concentrations (kg/ml range) compared with Oc (mg/ml range).**This relatively low solubility in aqueous solutions is not apparent from the predominance of hydrophilic amino acids in matrix Gla-protein. It is, however, one of the main reasons why matrix Gla-protein was not investigated to the same extent as Oc until relatively recently.?’
C. Metabolism of Oc Plasma Oc represents, almost exclusively, the activity of osteoblasts of bone with a very small contribution from the odontoblasts in teeth.76 Sixty to ninety percent of the newly synthesized Oc combines with hydroxyapatite in the mineralized phase of bone and the remainder goes into the plasma.55This distribution may vary with vitamin D status of the individual, and more specifically the relative amounts of 1,25 (OH),D, and 24,25 (OH),D3 in the circulation.60Likewise, in individuals with vitamin K deficiency, or undergoing treatment with warfarin, the percentage distribution of Oc between bone and plasma will vary because more Oc will escape into the circulation due to the reduced numbers of Glaresidues. Species differences may exist, however, because plasma Oc concentrations increased in vitamin K-deficient rat^'^.'^ and COWS,'^ but decreased in human being^^^.^^ and sheep.59These differences may be related to species variation in the rate of excretion of Oc from the cell rather than the influence of Gla-residues on its excretion. Oc is rapidly metabolized in liver and k i dn e ~ ~ , .and ’ ~ this is reflected in its relatively short half-life in the circulation (approximately 5 min).5.23 Injected iodinated Oc accumulates in the bone r n a t r i ~ , probably ~ ~ ~ ~ ’ as a result of adsorption from plasma to the surface of the hydroxyapatitecrystals. Oc peptides have not been detected in bone extracts, possibly because relatively little Oc is broken down while associated with the hydroxyapatite surface, and intact Oc has been detected in prehistoric bone matrix from fossilized bone^.^^.^^ Recent results,30however, suggest that some breakdown of Oc may occur in bone extracts, although the extent to which this occurs in the intact bone before extraction is not known. In some studies”’78relatively little Oc breakdown was detected in plasma, although we and others found that breakdown occurs.‘6v’9*80 This disparity may be due to a difference in
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Volume 28, Issue 4 (1991)
293
the ability of various antisera to detect Oc-related peptides, or multiple forms of Oc, in plasma. In kidney and liver to a lesser extent, Oc is hydrolyzed by mitochondrial, microsomal, and supernatant fraction^.^' Hydrolysis is inhibited by EDTA and it was inferred therefore that metallo-proteases are involved. But another possible explanation is that if calcium is chelated by EDTA, Oc would assume a more random or P-pleated sheet format that would ‘render it more susceptible to proteolytic breakdown.
D. Functions of Oc The precise functions of Oc are not known, although several possible roles have been The most characteristic feature of Oc and matrix Gla-protein compared with other bone-matrix proteins is the presence of Gla-residues. These residues have an essential role in blood clotting proteins such as prothrombin8’ and it is likely that they also have an important function in Oc. Purified Oc from warfarin-treated rats does not contain Gla-residues and does not inhibit hydroxyapatite crystals formation from b r u ~ h i t eAlthough .~ this suggests a role for Oc in the regulation of bone mineralization, it is difficult to isolate its role from that of matrix Gla-protein, which is similarly affected by warfarin.75 It has been reported that relatively low concentrations of Oc are present in bone matrix at the onset of minera l i z a t i ~ n , although ~ ~ . ~ ~ this has not been confirmed by other^.^'-^^ Apparently, Oc concentrations are highest in bone matrix following completion of the mineralization process,86 and this suggests that its main function is in the maturation phase of bone mineral formation, or at a later stage in the resorptive phase of bone metabolism. Some s t ~ d i e s ~have ~ - ~suggested ’ that the chemotactic ability of the Gla-containing proteins in bone from warfarin-treated rats could be due to matrix Gla-protein or other Gla-containing proteins, rather than Oc. There is, however, good published evidence” for a chemotactic role for Oc based on studies with peptides with amino acid sequences homologous with Oc but not with matrix Gla-protein. Another possible role for Oc is in the bone-resorptive processes. Some evidence for this emerged from studies that showed that there was poor resorption of bone matrix from warfarin-treated rats when placed subcutaneously into vitamin K-replete animal^.^^-*^ Also, Oc biosynthesis was induced in these animals at high concentrations of 1,25(0h)2D,,6 which inhibits bone formation in v i v ~ . ~ ’It. ~has been suggested, therefore, that Oc may function as a “messenger” for 1,25(OH),D3 and would influence osteoclasts through its action on osteoblasts by acting as a type of “coupling factor” between these two cell lines. Its main influence on osteoclasts could be to increase the rate of differentiation and/or to increase osteoclastic activity. 92-94 Other proposed functions for Oc include inhibition of leukocyte elastase% and growth factor a ~ t i v i t y , although ~ ~ - ~ ~ the physiological significance of these roles is not known.
II. PLASMA Oc A. Origin of Plasma Oc Plasma Oc is derived from de novo biosynthesis by o~teoblasts~.~ and is not released from bone matrix by resorptive processes. Evidence for this comes from experiments that showed that in warfarin-treated rats serum Oc changed within 6 h from being fully carboxylated to a noncarboxylated form. In contrast, Oc in bone matrix remained totally carboxylated over the same period.55Carboxylated and noncarboxylated Oc may be distinguished by the reduced ability of the latter to bind hydroxyapatite.” When vitamin K, was administered to the aforementioned warfarin-treated rats, carboxylation of Oc resumed in osteoblasts and the carboxylated form was detected in plasma. Additional evidence for the osteoblastic origin of Oc emerges from in vitro studies with cell lines derived from bone that secrete Oc, and respond to various physiological stimuli.6 Northern blot analysis has not detected mRNA
Critical Reviews in Clinical Laboratory Sciences
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Table 1 Published Concentrations of Osteocalcin (ng/ml) in Plasma From Healthy Subjects
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Osteocalcin (ng/ml) Mean f Ref.
Standard
Antibody
n
INC INC INC INC INC INC
INC INC INC INC INC INC
150
11 11
11 11
Kit data 272 273
Seragen n.d. 12
Seragen n.d. 12
173 58 59 196
Seragen CIS INC INC
Seragen CIS INC INC
274 26
11
11
Home (Ov)
n.d.
156 152 275
156 Peptide n.d.
156 Peptide n.d.
88 147 20
193 132
n.d. 132
n.d. 132
20 207 113 135
104 Poser Peptide 104
104 Poser Peptide 104
194 344 72 31
137 249 270 172 27 1 16 11
17 47 28 30 28 25 30 62 47 10 242 107 62 22 6 10 10
232 207 72 Note:
Peptide Poser Peptide
Peptide Poser Peptide
18 13 18
Age
25-55
287
Osteocalcin: Diagnostic Methods and Clinical Applications Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nanyang Technological University on 08/20/15 For personal use only.
Michael J. Power and Patrick f . fottrefl
ABSTRACT Osteocalcin is a small (M, 5800), very interesting bone specific protein, synthesized by osteoblasts and measured in plasma as a biochemical indicator of bone formation. Many immunoassays for osteocalcin have been developed, including radio- and enzymoimmunoassays, with the use of monoclonal and polyclonal antibodies. These are used in many different clinical settings, including bone, kidney, and liver diseases. However, there is a wide range of published values for plasma osteocalcin concentrations in control and patient samples and this has hindered a more widespread adoption of osteocalcin measurement by clinicians. This review discusses how various immunoassays for osteocalcin may contribute to the wide variation of published values and suggests approaches for the development of standardized assays. For example, epitope specificity and immunoreactivity with multiple forms of osteocalcin and osteocalcin peptides in plasma are discussed. It also includes a recent update on interesting clinical applications of osteocalcin. Key Words: osteocalcin (Oc), Bone-Gla protein (BGP), immunoassays, monoclonal, concentrations, osteoblasts.
1. STRUCTURE, IMMUNOREACTIVITY, BIOSYNTHESIS, METABOLISM, AND FUNCTIONS OF OSTEOCALCIN Osteocalcin (Oc), or bone-Gla protein (BGP), is the most abundant noncollagenous protein in mature human bone, where it constitutes 1 to 2% of the total protein.'-3 Synthesized by osteoblasts, it is incorporated into the bone matrix.'-3 Although its exact function is not known, the presence of three gamma carboxyglutamicacid residues in human Oc at positions 17, 21, and 24 confer on it a very strong ability to bind hydr~xyapatite.~.~ Vitamin K, is essential for its biosynthesis, which is stimulated by 1,25 (OH),D,.5-8 The concentration detectable in serum may represent the part of newly synthesized Oc that is not adsorbed but is instead released directly into the circulation. Oc has received much attention as a possible marker for bone turnover. Its concentration in serum increased in patients with metabolic bone disease characterized by increased bone t ~ r n o v e rIt. ~is~a~ ~ more specific index of bone metabolism than the more established indices, such as total serum alkaline phosphatase activity or urinary hydroxyproline. Serum Oc measurements are used in various clinical situations, including metabolic bone diseases, renal disorders, hyperthyroidism, and diseases related to excess glucocorticoids.
P. F. Fottrell, B.Sc., M.Sc., Ph.D., D.Sc., M. J. Power, B.Sc., Ph.D., Dept. ofBiochemistry, University College, Galway, Ireland.
288
Critical Reviews in Clinical Laboratoly Sciences
__
__
5 10 15 Tyr Gln Trp Leu Gly A h Pro Val Pro Tyr Pro Asp Pro Asp His __ __ __ Hyp Ala __ __ __
--
--
Asp pro Gly
1
Human Calf Sheep
Tyr Leu
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nanyang Technological University on 08/20/15 For personal use only.
Swordfish
Human
calf Sheep Swordfish
35 Glu Leu Ala Asp His ne
__ __
__ __
--
Met
__ __
__ --
__
__ __
--
Thr Ala --
Sheep
--
__
__
__ __
Hyp Ala
--
Gly Asp Leu Thr --
__
__
__
__
__
Leu Gln
40 45 Gly Phe Gln Glu Ala Tyr A r g Arg Phe
__
Swordfish
calf
--
Ala Thr Arg
49 Tyr Gly Pro Val --__ _--__
Human
__
__
._
__
__
__ __ __ __ __ Ile
Val Ala
__
__ __
_- __ _- __
--
--
Ile
-
__
__
Ala Tyr
__
--
--
ne
Gln Phe
FIGURE 1. Amino acid sequences of human, bovine, ovine, and swordfish osteocalcin, showing Gla-residues (underlined) and position of disulfide bond.
Radioimmunoassays (RIAs) for quantifying Oc in serum have been and are available commercially. Most involve the use of radioiodinated bovine Oc as label and polyclonal antisera raised against purified bovine Oc. More recently, sensitive enzymoimmunoassays for Oc with use of rnonocl~nal'~ and p o l y ~ l o n a l " - ' ~ antibodies ~ ' ~ ~ ' ~ have been published. Although Oc has considerable clinical potential for the management of patients with metabolic bone disorders, it is difficult at present to interpret serum concentrations because of the wide ranges of published normal and clinical values. Possible explanations for the O,~' published variations in Oc concentrations have been suggested by us13 and ~ t h e r s ' ~ . ~and include factors involved in the immunoassay for Oc and in the selection of control and patient populations. For example, the extent to which various antisera recognize multiple forms of Oc in human serum may be a contributing factor. This review discusses the various factors involved in the development of immunoassays for Oc that may contribute to assay variation and makes recommendations that could improve the clinical interpretation of Oc values. These aspects of Oc have not been discussed in detail in previous r e v i e w ~ . ' . ~ . ~ ~ . ~ ~
A. Structure and lmmunoreactivity of Oc Over 90% of the primary structure has been conserved between human, ovine, and calf
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Volume 2 8 , Issue 4 (1991)
289
FIGURE 2. The proposed calcium-induced three-dimensional structure for intact bovine osteocalcin.’ Letters refer to the one letter code for the amino acids and the subscripts to their positions. The backbone is represented by the n b b n structure. Formal electronic charges are indicated as they would exist at pH 7 to 8. Probable metal binding sites are represented by M,, M,, and M,. The single disulfide bond, between positions 23 and 29, is indicated as are the locations of the two calcium-induced alpha helices, i.e., the “Gla helix” from residues 16 to 25, and the “Asp-Glu helix” between residues 30 and 41.
Oc, and 50% between human and swordfish Oc5 (see Figure 1). The conservation of primary sequences in the 400 million years in evolutionary time between swordfish and human Oc indicates that it has an important function in bone metabolism. This conservation of primary amino acid sequence is useful for assay development because antisera raised against bovine and ovine Oc apparently cross-react well with human Oc. 11-15 Human Oc is usually measured with the use of purified bovine Oc to generate antibodies, labels, and standards, although several immunoassays have been developed recentlyz5-*’with use of purified ovine Oc as standard, label, and for generating antisera. Conformational aspects of Oc structure have been studied with the use of circular dichroism, UVhisible spectroscopy, nuclear magnetic resonance spectroscopy, and chemical and immunochemical t e c h n i q ~ e s There . ~ ~ ~are~ two ~ ~ ~antiparallel ~ a-helical structures that represent 40% of the overall structure and are stabilized by a disulfide bond between positions 23 and 29 (Figure 2). The a-helical structure is calcium-dependent and does not exist in calcium-deplete Oc molecules. This structure forces the more polar amino acids at the Cand N-terminal ends of Oc to the outside edges, resulting in a more energetically stable structure. The nonpolar amino acids at the C-terminal end may stabilize the calcium-induced a-helical structure.23Likewise, the N-terminal peptide may be important for stabilization of the C-terminal peptide. The latter peptide is the proposed location of an antigenic site.” Many antisera used in the measurement of Oc do not detect the presence of the a-helical structure because most react independently of calcium. Some antisera, however, are calciumdependent25*z9-31 and may detect the a-helical structure, although the exact location of the antigenic site for these antisera has not been determined. The remainder of the molecule is in the P-sheet format, and it appears that the C-terminal and N-terminal ends are in close proximity to each other. The C-terminal and N-terminal peptides may therefore overlap with a resultant sharing of an antigenic site (see Figure 2 ) . This could contribute to the variability in the detection of the antigenic site(s) by different antisera.
290
Critical Reviews in Clinical Laboratory Sciences
The following features of the structure and calcium-bindingproperties of Oc may influence its immunoreactivity:
Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by Nanyang Technological University on 08/20/15 For personal use only.
1.
2.
3.
4.
5.
Peptide bonds between residues 19-20 and 43-44 in human and bovine Oc are susceptible to tryptic hydrolysis. The resultant peptides may be the main products of Oc breakdown in the liver, kidney, and plasma.32The immunoreactivity of these peptides with antisera raised against intact Oc varies and is another possible contributory factor to the wide range of published plasma Oc values. Iodination of tyrosine (of which there are 5 in bovine and 4 in human Oc) may be at or near the antigenic site(s) and may influence immunoreactivity. All the Gla-residues in human and bovine Oc may not be ~arboxylated,~ which may reflect the vitamin K status of the donor. Because these Gla-residues induce an ahelical structure in the presence of calcium,28the location of the antigenic site will be influenced by the degree of carboxylation of the Gla-residues. The reduction of the disulfide bond between amino acids 23 and 29 in human or bovine Oc does not apparently influence the immunoreactivity of Oc, when measured by some antisera.’ When calcium ions are complexed by Oc, only two of the 6 to 9 possible coordination sites on the calcium ion are i n ~ o l v e d . ~Calcium ~ * ~ ~bound - ~ ~ to Oc may therefore interact with other Oc molecules to form a cross-link between two Oc molecules, or between Oc and other calcium-binding proteins or complexes.
B. Biosynthesisof Oc The mechanism involved in the biosynthesis and secretion of human Oc by osteoblasts is similar to that for other secretory proteins (see Figure 3). The one copy of the Oc gene in the human genome is located on chromosome number 1,38 which also has the gene for alkaline phosphatase, another biochemical marker for bone metabolism. The Oc gene has four exons and three i n t r ~ n sand , ~ ~codes for a protein with a molecular weight of about 13,500 Da.39.40This protein, pre-pro-Oc, includes the native Oc molecule (exons 3 and 4; 49 amino acids), the membrane signal sequence (exon l), and the propeptide (exon 2), which is recognized by the carboxylating enzymes. This constitutes the entire sequence of the mRNA for Oc. It is the propeptide, together with certain segments of the native Oc molecule itself, which have the recognition sites for the microsomal carboxylatingenzymes.22 Also associated with the Oc gene are the reputed recognition sites for the Oc gene regulators, 1,25 (OH),D, and CAMP.^'^ Potential sites for the binding of several hormone-receptor complexes have been detected, including those for estrogens and thyroid hormone^.^^.^ After the Oc gene in transcribed into mRNA and following removal of the introns, the mRNA is translated at the rough endoplasmic reticulum. The membrane leader sequence directs the remainder of its synthesis into the lumen of the rough endoplasmic reticulum where a carboxylating enzyme system recognizes appropriate glutamic acid residues to form y-carboxyglutamic acid. In bovine Oc the proline at position 9 may be converted to hydroxyproline in the lumen of the rough endoplasmic reticulum. This latter conversion is interesting because bovine Oc is relatively unique in its requirement for three vitamins (D, K, C) for its optimum biosynthesis. Finally, the recognition sequence is cleaved and native Oc is secreted into the external medium. The antibody that recognizes native rat Oc also reacts with ~ r e - p r o - O c , ~suggesting ~ * ~ ~ , ~that the antigenic site is not located near the prepro-segment of Oc . Studies in vitro and in vivo showed that Oc biosynthesis is increased six-fold by 1,25 (OH)2D3,6.7 although there may be substantial synthesis in its absence.40Some osteoblastic cell lines do not synthesize Oc unless 1,25 (OH),D, is present in the m e d i ~ m . A ~ CAMP~.~~ binding site has been located close to the Oc gene,424 but its precise function is unknown.
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PE- pro- osteocalcin
signal peptidase
vitK
1
osteoblast cytog-srn
carboxylase
\
propeptide cleavage
bone mineral
\
+
\
blood plasma
FIGURE 3. Biosynthesis of osteocalcin in the human osteoblast cell.23
An apparent increase in Oc biosynthesis has been reported following stimulation of osteoblasts by agents that stimulate adenylate cyclase activity,**but in contrast others detected a decrease after osteoblastic tim mu la ti on.^^ The mRNA for Oc is stabilized by CAMP,^^ which could account for the stimulation of Oc biosynthesis detected by some groups. Carboxylation of the glutamic acid residues is apparently not essential for secretion of Oc. However, the efficiency of secretion may be enhanced by carboxylation because Oc does not accumulate in the medium of vitamin K-depleted cells grown in tissue culture,
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Critical Reviews in Clinical Laboratory Sciences
although considerable Oc accumulates inside the ce11.47,48 However, in vivo, there may be oversecretion of Oc in warfarin-treated rat^,'^.'^ and cows,57but, on the other hand, undersecretion has been reported in human beings and sheep treated with ~ a r f a r i n .Further ~~.~~ studies are required to define the role of Gla-residues in the secretion of Oc. Calcitonin, parathyroid hormone, or the serum calcium concentration do not apparently directly influence Oc bio~ynthesis.’.~~ With respect to the possible role of 24,25 (OH),D,, it has been suggested by some researchers that it has no effe~t,4~.” whereas others reported that it increased biosynthesis and promoted accumulation of Oc in hydroxyapatite.60Several growth factors and hormones have been reported to influence Oc biosynthesis, although their mechanism of action or physiological significance is not known. They include transforming growth factors p,6’ interleukin-1,62 insulin-like growth factor 1,63 fibroblast growth factor, both acidic and basic forms (FGFa and b),63 interferon y,@ retinoic a ~ i d , ~gluco’ ~ ~ r t i ~ ~ i d ~ ,thyroid ~ ~ . hormones ~ ~ . ~ ~(T3 . ~and ~ .T4) ~ ,68 ~ ,insulin,69 ~ ~ and e s t r ~ g e n . ~ - ~ & ~ ~ It is interesting and relevant to the phenotypic characterization of the osteoblastic cell lineage that the cell lines that synthesize Oc do not produce other vitamin K-dependent bone proteins, such as matrix Gla-protein. However, prolonged exposure of several osteoblast cell lines to pharmacological concentrations of 1,25 (OH),D, induces the biosynthesis of matrix Gla-protein at the expense of Oc bio~ynthesis.~~ The physiological significance of this is not known. Matrix Gla-protein biosynthesis appears to precede Oc biosynthesis in the mineralization process in bone, and its biosynthesis is a good parameter for differentiating K is also a cofactor for between the various stages of osteoblastic d e ~ e l o p m e n tVitamin .~~ posttranslational carboxylation of matrix Gla-protein, and its biosynthesis is also increased by 1,25 (OH),D,.’3,74 Matrix Gla-protein is soluble in aqueous buffers only at relatively dilute concentrations (kg/ml range) compared with Oc (mg/ml range).**This relatively low solubility in aqueous solutions is not apparent from the predominance of hydrophilic amino acids in matrix Gla-protein. It is, however, one of the main reasons why matrix Gla-protein was not investigated to the same extent as Oc until relatively recently.?’
C. Metabolism of Oc Plasma Oc represents, almost exclusively, the activity of osteoblasts of bone with a very small contribution from the odontoblasts in teeth.76 Sixty to ninety percent of the newly synthesized Oc combines with hydroxyapatite in the mineralized phase of bone and the remainder goes into the plasma.55This distribution may vary with vitamin D status of the individual, and more specifically the relative amounts of 1,25 (OH),D, and 24,25 (OH),D3 in the circulation.60Likewise, in individuals with vitamin K deficiency, or undergoing treatment with warfarin, the percentage distribution of Oc between bone and plasma will vary because more Oc will escape into the circulation due to the reduced numbers of Glaresidues. Species differences may exist, however, because plasma Oc concentrations increased in vitamin K-deficient rat^'^.'^ and COWS,'^ but decreased in human being^^^.^^ and sheep.59These differences may be related to species variation in the rate of excretion of Oc from the cell rather than the influence of Gla-residues on its excretion. Oc is rapidly metabolized in liver and k i dn e ~ ~ , .and ’ ~ this is reflected in its relatively short half-life in the circulation (approximately 5 min).5.23 Injected iodinated Oc accumulates in the bone r n a t r i ~ , probably ~ ~ ~ ~ ’ as a result of adsorption from plasma to the surface of the hydroxyapatitecrystals. Oc peptides have not been detected in bone extracts, possibly because relatively little Oc is broken down while associated with the hydroxyapatite surface, and intact Oc has been detected in prehistoric bone matrix from fossilized bone^.^^.^^ Recent results,30however, suggest that some breakdown of Oc may occur in bone extracts, although the extent to which this occurs in the intact bone before extraction is not known. In some studies”’78relatively little Oc breakdown was detected in plasma, although we and others found that breakdown occurs.‘6v’9*80 This disparity may be due to a difference in
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the ability of various antisera to detect Oc-related peptides, or multiple forms of Oc, in plasma. In kidney and liver to a lesser extent, Oc is hydrolyzed by mitochondrial, microsomal, and supernatant fraction^.^' Hydrolysis is inhibited by EDTA and it was inferred therefore that metallo-proteases are involved. But another possible explanation is that if calcium is chelated by EDTA, Oc would assume a more random or P-pleated sheet format that would ‘render it more susceptible to proteolytic breakdown.
D. Functions of Oc The precise functions of Oc are not known, although several possible roles have been The most characteristic feature of Oc and matrix Gla-protein compared with other bone-matrix proteins is the presence of Gla-residues. These residues have an essential role in blood clotting proteins such as prothrombin8’ and it is likely that they also have an important function in Oc. Purified Oc from warfarin-treated rats does not contain Gla-residues and does not inhibit hydroxyapatite crystals formation from b r u ~ h i t eAlthough .~ this suggests a role for Oc in the regulation of bone mineralization, it is difficult to isolate its role from that of matrix Gla-protein, which is similarly affected by warfarin.75 It has been reported that relatively low concentrations of Oc are present in bone matrix at the onset of minera l i z a t i ~ n , although ~ ~ . ~ ~ this has not been confirmed by other^.^'-^^ Apparently, Oc concentrations are highest in bone matrix following completion of the mineralization process,86 and this suggests that its main function is in the maturation phase of bone mineral formation, or at a later stage in the resorptive phase of bone metabolism. Some s t ~ d i e s ~have ~ - ~suggested ’ that the chemotactic ability of the Gla-containing proteins in bone from warfarin-treated rats could be due to matrix Gla-protein or other Gla-containing proteins, rather than Oc. There is, however, good published evidence” for a chemotactic role for Oc based on studies with peptides with amino acid sequences homologous with Oc but not with matrix Gla-protein. Another possible role for Oc is in the bone-resorptive processes. Some evidence for this emerged from studies that showed that there was poor resorption of bone matrix from warfarin-treated rats when placed subcutaneously into vitamin K-replete animal^.^^-*^ Also, Oc biosynthesis was induced in these animals at high concentrations of 1,25(0h)2D,,6 which inhibits bone formation in v i v ~ . ~ ’It. ~has been suggested, therefore, that Oc may function as a “messenger” for 1,25(OH),D3 and would influence osteoclasts through its action on osteoblasts by acting as a type of “coupling factor” between these two cell lines. Its main influence on osteoclasts could be to increase the rate of differentiation and/or to increase osteoclastic activity. 92-94 Other proposed functions for Oc include inhibition of leukocyte elastase% and growth factor a ~ t i v i t y , although ~ ~ - ~ ~ the physiological significance of these roles is not known.
II. PLASMA Oc A. Origin of Plasma Oc Plasma Oc is derived from de novo biosynthesis by o~teoblasts~.~ and is not released from bone matrix by resorptive processes. Evidence for this comes from experiments that showed that in warfarin-treated rats serum Oc changed within 6 h from being fully carboxylated to a noncarboxylated form. In contrast, Oc in bone matrix remained totally carboxylated over the same period.55Carboxylated and noncarboxylated Oc may be distinguished by the reduced ability of the latter to bind hydroxyapatite.” When vitamin K, was administered to the aforementioned warfarin-treated rats, carboxylation of Oc resumed in osteoblasts and the carboxylated form was detected in plasma. Additional evidence for the osteoblastic origin of Oc emerges from in vitro studies with cell lines derived from bone that secrete Oc, and respond to various physiological stimuli.6 Northern blot analysis has not detected mRNA
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Table 1 Published Concentrations of Osteocalcin (ng/ml) in Plasma From Healthy Subjects
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Osteocalcin (ng/ml) Mean f Ref.
Standard
Antibody
n
INC INC INC INC INC INC
INC INC INC INC INC INC
150
11 11
11 11
Kit data 272 273
Seragen n.d. 12
Seragen n.d. 12
173 58 59 196
Seragen CIS INC INC
Seragen CIS INC INC
274 26
11
11
Home (Ov)
n.d.
156 152 275
156 Peptide n.d.
156 Peptide n.d.
88 147 20
193 132
n.d. 132
n.d. 132
20 207 113 135
104 Poser Peptide 104
104 Poser Peptide 104
194 344 72 31
137 249 270 172 27 1 16 11
17 47 28 30 28 25 30 62 47 10 242 107 62 22 6 10 10
232 207 72 Note:
Peptide Poser Peptide
Peptide Poser Peptide
18 13 18
Age
25-55
