Biochemical markers of bone turnover I: Theoretical considerations and clinical use in osteoporosis

Biochemical Markers of Bone Turnover I: Theoretical Considerations and Clinical Use in Osteoporosis PIERRE

D.

DELMAS,

M.D.,

Ph.D.,

Lyon Cedex, France

The recent development of noninvasive techniques to measure bone mass and bone turnover represents a major advance in the diagnosis and management of osteoporosis. The rate of formation or degradation of the bone matrix can be assessed either by measuring a prominent enzymatic activity of the bone-forming or resorbing cells, such as alkaline and acid phosphatase activity, or by measuring bone matrix components released into the circulation during formation or resorption. Recent studies have shown that the appropriate combination of the most efficient markers of bone resorption and formation will provide a powerful tool in the clinical investigation of osteoporosis.

T

he recent development of a noninvasive technique to measure bone mass and bone turnover represents a major advance in the diagnosis and management of osteoporosis. In contrast to metabolic bone diseases (such as Paget’s disease of bone or renal osteodystrophy) characterized by dramatic changes of bone turnover, osteoporosis is a condition in which subtle modifications of the bone remodeling activity can lead to a substantial loss of bone mass after a long period of time. This explains why most conventional markers may well be normal in any given individual patient who has recently experienced menopause, whereas such a patient may actually have well characterized vertebral osteoporosis. Consequently, there have been efforts to develop more sensitive biochemical markers of bone turnover. The rate of formation or degradation of the bone matrix can be assessed either by measuring a prominent enzymatic activity of the bone-forming or resorbing cells, such as alkaline and acid phosphatase activity, or by measuring bone matrix components released into the circulation during formation or resorption. They have been separated into markers of formation and resorption (Table I), but it should be borne in mind that in disease states in which both events are coupled and in balance, either of these markers will reflect the overall rate of bone turnover. As discussed below, these markers are of unequal specificity and sensitivity, and some of them have not yet been fully investigated. Recent studies have shown that the appropriate combination of the most efficient markers of bone resorption and formation will provide a powerful tool in the clinical investigation of osteoporosis.

BIOCHEMICALMARKERSOF BONE FORMATION Total and Bone Alkaline Phosphatase

From the Department of Medicine, Claude Bernard University; and the INSERM Research Unit 234, HBpital E. Hernot, Lyon Cedex, France. Requests for reprints should be addressed to: Pierre D. Delmas, M.D., the INSERM Research Unit 234, HBpital E. Hernot, 69437 Lyon Cedex 03, France.

The skeletal alkaline phosphatase is an enzyme localized in the membrane of the osteoblasts that is released into the circulation by an unclear mechanism. Serum total alkaline phosphatase activity is the most commonly used marker of bone formation, but it lacks sensitivity and specificity. Nevertheless, several studies have shown that its activity increases with aging in adults, especially in women

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TABLE I Biochemical Markers of Bone Turnover Formation

Resorption 1

Most efficient marken: Osteocalcin (BGP) Bone alkalinephosphatase

Most efficient markers, Pyr and dPyr collagencrosslinks and related peptides

Other markers: Total alkaline phosphatase Procollagen I extension peptides

Ofher markers: Urinary hydroxyproline Urinary hydroxylysine glycosides Plasma tartrate-resistant acid phosphatase

Osteocalcin

‘r = pyridinoline; dPyr = deoxypyridinoline.

after menopause. In patients with vertebral osteoporosis, values are either normal or slightly elevated and poorly correlated with bone formation determined by iliac crest histomorphometry [1,2]. Also, a moderate increase of serum alkaline phosphatase is ambiguous, as it may reflect a mineralization defect in elderly patients or the effect of one of the numerous medications that have been shown to increase the hepatic isoenzyme of alkaline phosphatase. In an attempt to improve the specificity and the sensitivity of serum alkaline phosphatase measurement, techniques have been developed to differentiate the bone and the liver isoenzymes, which, because they are coded by a single gene, differ only by posttranslational modifications. These techniques, which rely on the use of differentially effective activators and inhibitors, separation by electrophoresis, and wheat germ lectin precipi-

Intact osteocalcin 49

1

Potential fragments 4344

1 N-teminaCMi

1

49

C-terminal

43

18 20 Mid

N-terminal

49

20 Mid-C-terminal

1. Circulating immunoreactive forms of osteocalcin. Fragments can be released in vitro by trypsin digestion, because of an Arg-Arg bond in positions 19-20 and 43-44. In vivo studies using a battery of monoclonal antibodies recognizing different epitopes on the molecule have confirmed the existence of such circulating fragments (see text).

Figure

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tation, have slightly enhanced the sensitivity of this marker, but most of them are indirect and/or technically cumbersome. A real improvement should be obtained by using a monoclonal antibody recognizing preferentially the bone isoenzyme, a reagent that has recently become available [3].

Osteocalcin, also called bone Gla-protein (BGP), is a small noncollagenous protein that is specific for bone tissue and dentin, but its precise function remains unknown. Osteocalcin is predominantly synthesized by the osteoblasts and incorporated into the extracellular matrix of bone, but a fraction of newly synthesized osteocalcin is released into the circulation, where it can be measured by radioimmunoassay [4,5]. Circulating osteocalcin has a short elimination half-life and is rapidly cleared by the kidney [6,7]. Serum osteocalcin correlates with skeletal growth at the time of puberty and is increased in a variety of conditions characterized by increased bone turnover (primary and secondary hyperparathyroidism, hyperthyroidism, acromegaly, Paget’s disease of bone, etc.); it is decreased in hypothyroidism, hypoparathyroidism, and in glucocorticoid-treated patients (reviewed in [8]). We have recently characterized the various immunoreactive forms of circulating osteocalcin, using a battery of monoclonal antibodies that recognize three distinct epitopes of human osteocalcin (Figure 1). In normal adult serum, the intact 49 amino acid molecule represents 36% of the total immunoreactivity. A large N-terminal midfragment, resulting from proteolytic cleavage of the newly synthesized osteocalcin at the 43-44 amino acid bond, represents approximately 30%, and three smaller fragments (1-19, 20-43, 29-49) represent the last third of the immunoreactivity. This pattern of fragment distribution was similar in osteoporotic patients and in Pagetic patients, suggesting that the fragments result from enzymatic degradation rather than from being released from bone matrix during resorption [9,10]. These fragments circulate but are also generated in vitro within a few hours. Thus, depending on the epitope recognized by the antibody, some polyclonal antisera (but also monoclonal antibodies) may see, in addition to the intact molecule, fragments of osteocalcin [ll-131, explaining why different assays provide different results with the same samples [14]. In most cases, however, serum osteocalcin is a valid marker of bone turnover when resorption and formation are coupled and is a specific marker of bone formation whenever formation and resorption are uncoupled [1,14]. A sandwich assay using monoclonal antibodies recognizing well-characterized

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epitopes of human osteocalcin are likely to improve the sensitivity of the assay (Figure 2). Procollagen I Extension Peptides

During the extracellular processing of collagen I, there is a cleavage of the amino terminal (p toll-IN) and carboxy terminal (p toll-I-C) extension peptides prior to the fibril formation. These peptides, also referred to as the procollagen carboxy-terminal propeptide (PICP) and amino-terminal propeptide (PINP), circulate in blood where they might represent useful markers of bone formation, as collagen is by far the most abundant organic component of bone matrix. Serum PICP levels are weakly correlated with histological bone formation (r = 0.36-0.50) in patients with vertebral osteoporosis 1151. Menopause induces a significant but marginal (t-20%) increase in serum PICP, which is not correlated with the subsequent rate of bone loss measured by densitometry [16]. The reasons for this lack of sensitivity are not clear. The in vivo metabolism of this peptide is unknown, although it has been shown in the rat that PICP is bound and internalized by the endothelial cells of the liver through the mannose receptor [17]. Characterization of the circulating immunoreactive forms of PICP may help to clarify these uncertainties. There is still little information about circulating PINP, which is released into the circulation during collagen synthesis but also incorporated into bone matrix where it has been identified as the 24,000 Dalton phosphoprotein of bone. An assay for PINP using a synthetic peptide as an immunogen has beenrecentlyreported, which provided surprisingand quite disappointing-results in several metabolic bone diseases [18]. BIOCHEMICAL MARKERS OF BONE RESORPTION Fasting Urinary Calcium and Hydroxyproline

Fasting urinary calcium measured on a morning sample and corrected by creatinine excretion is certainly the cheapest assay of bone resorption. It is useful to detect a marked increase of bone resorption but lacks sensitivity. As half of human collagen resides in bone, where its turnover is probably faster than in soft tissues, excretion of hydroxyproline in urine is regarded as a marker of bone resorption. Actually, the Clq fraction of the complement contains significant amounts of hydroxyproline and could account for up to 40% of urinary hydroxyproline. Hydroxyproline is present in urine in three forms: free hydroxyproline, small hydroxyprolinecontaining peptides that are dialyzable and represent >90% of the total urinary excretion of this amino acid, and a small number of nondialyzable

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polypeptides containing hydroxyproline [19]. Colorimetric assay of hydroxyproline is usually performed on a hydrolyzed urine sample and therefore reflects the total excretion of the amino acid. As a consequence of its tissue origin and metabolism pattern, urinary hydroxyproline is poorly correlated with bone resorption assessed by calcium kinetics or bone histomorphometry, and there is an obvious need for a more sensitive marker of bone resorption. Urinary Pyridinium Crosslink

Pyridinoline (Pyr) and deoxypyridinoline (dPyr)also called, respectively, hydroxylysylpyridinoline and lysylpyridinoline-are the two nonreducible pyridinium crosslinks present in the mature form of collagen [SO]. The concentration of Pyr and dPyr in connective tissues is very low and varies dramatically with tissue type. Pyr is widely distributed in the type I collagen of bone and in the type II collagen of cartilage and in smaller amounts in other connective tissues except skin, whereas large amounts of dPyr have only been found in bone. Pyr and dPyr are likely to be released from bone matrix during its degradation by the osteoclasts, and available data suggest that they are not metabolized in vivo. They are excreted in urine in free form (approximately 40%) and in peptide-bound form (60%), and the total amount can be measured by fluorimetry after reversed phase high-performance liquid chromatography (HPLC) of a cellulose-bound extract of hydrolyzed urine. Urinary Pyr and dPyr

4321-

Glucocorticoid treatment

OHyperparathymidism

*

Paget’s disease

p< 0.05 vs RIA

Figure 2. Serum osteocalcin in patients on chronic glucocorticoid therapy and in patients with primary hyperparathyroidism and Paget’s disease of bone. Serum osteocalcin was measured both with a conventional bovine radioimmunoassay (RIA) and with a human immunoradiometric assay (IRMA), as previously published [9]. Data are expressed in Z scores, i.e., in standard deviation from the sex- and agealusted normal mean established in 300 normal adults. In the three metabolic bone diseases, discrimination from normal was significantly greater with the IRMA than with the RIA.

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TABLE II Pyridinoline

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Crosslinks: Questions to Be Addressed

* Pyr and dPyr concentrations in cortical and trabecular bone * Contribution of other connective tissues to urinary excretion a Metabolism and clearance in humans l

Importance of urine collection time: 24 hours versus first/second morning void

l

Significance of urinary and serum PyridPyr containing peptides versus free PyrldPyr excretion, and validation of related immunoassay

r = pyrrdmoline; dPyr = deoxypyridinoline.

are increased by 50-100% at the time of menopause and return to premenopausal levels with estrogen therapy [21]. In patients with vertebral osteoporosis, the urinary crosslink levels, especially of dPyr, are correlated with bone turnover measured by calcium kinetics [22] and bone histomorphometry [23]. Pyr and dPyr appear to be more sensitive than hydroxyproline as markers in Paget’s disease of bone; are also significantly increased in patients with primary hyperparathyroidism, malignant hypercalcemia, and hyperthyroidism; and appear to be a sensitive index of bone metabolism in hypothyroid patients treated with L-thyroxine [24-261. The measurement of urinary crosslinks appear to have several potential advantages over hydroxyproline: they are relatively specific for bone turnover, they do not appear to be metabolized in vivo prior to their urinary excretion, and the absence of intestinal absorption of Pyr and dPyr contained in gelatin allows the collection of urine without any food restriction. Pyr and dPyr excretion undergoes a circadian rhythm, with a peak during the night and a nadir during the afternoon, a pattern similar to the rhythm of osteocalcin that probably reflects a nocturnal increase of bone turnover and resorption

L=l. All the data obtained so far have been with the HPLC assay of total Pyr and dPyr excretion. More convenient techniques are required for this marker to attain broad clinical use. Immunoassays are currently being developed, using antibodies directed against either free pyridinoline [28] or one of the two crosslinking domains of type I collagen, i.e., the N-telopeptide to helix [29] and the C-telopeptide to helix domains [30]. Preliminary results are quite promising, although each of these assays needs to be validated in defined clinical conditions, such as osteoporosis. Table II lists the questions that need to be addressed for a better understanding of the significance of this marker. Other Markers of Bone Resorption

Hydroxylysine is another amino acid unique to collagen and proteins containing collagen-like se-

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quences, and although it is much less abundant than hydroxyproline, it is a potential marker of collagen degradation [31]. The relative proportion and total content of galactosyl hydroxylysine and glucosylgalactosyl hydroxylysine vary in bone and soft tissues, which suggests that their urinary excretion might be a more sensitive marker of bone resorption than urinary hydroxyproline. This marker deserves further evaluation in osteoporosis, but its development is limited by the current HPLC technique. In normal plasma, tartrate-resistant acid phosphatase (TRAP) corresponds to plasma isoenzyme 5, which originates partly from bone, as osteoclasts contain a TRAP that is released into the circulation. Plasma TRAP is increased in a variety of metabolic bone disorders with increased bone turnover after oophorectomy and in vertebral osteoporosis [321. The lack of specificity of plasma TRAP activity for the osteoclast, its instability in frozen samples, and the presence of enzyme inhibitors in serum are potential drawbacks that should trigger the development of an immunoassay using monoclonal antibodies specifically directed against the bone isoenzyme of TRAP.

BONETURNOVERIN OSTEOPOROSIS Superimposed on the effect of aging, menopause induces a dramatic increase of bone turnover that peaks l-3 years after cessation of ovarian function and slows down thereafter for the next 8-10 years, a pattern that is also illustrated by cross-sectional studies after oophorectomy [32]. Serum osteocaltin, urinary Pyr crosslinks, as well as other markers of bone turnover, are significantly increased after the menopause and return to premenopausal levels within a few months of hormone replacement therapy [21,33]. Serum osteocalcin is correlated with the spontaneous rate of bone loss assessed by repeated measurements of the bone mineral eontent of the radius and the lumbar spine, i.e., the’ higher the bone turnover rate, the higher the rate of bone loss [33]. The combination of a single measurement of serum osteocalcin, urinary hydroxyproline, and dPyr can predict the rate of bone loss over 2 years with an r value of 0.77 [21]. Whether slow and fast losers of bone do so for a prolonged period of time after the menopause is debated, but a recent long-term study suggests that the rate of bone loss measured over 12 years is increased in postmenopausal women classified as rapid losers from the initial bone marker measurements [34]. Thus, despite identical bone mass at baseline, women who were diagnosed as fast losers at the initial biochemical measurement had lost 50% more

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bone 12 years later than those diagnosed as slow losers (total bone loss 26.6% vs 16.6%; p CO.001). The combination of bone mass measurement and assessment of bone turnover by a battery of specific markers is likely to be helpful in the future for the screening of patients at risk for osteoporosis who should be treated. In patients with untreated vertebral osteoporosis, there is a wide scatter beyond the normal range of individual values of biochemical markers of bone turnover, which reflects the histological heterogeneity of the disease. Recognizing this variable level of bone turnover might be important for choosing the optimal therapy. Indeed, a subgroup of osteoporotic patients with high turnover showed a significant increase of spinal bone mineral density after 1 year of calcitonin therapy, contrasting with no increase of bone mass despite the same therapy in those with a low bone turnover [35]. Biochemical markers should also be useful in monitoring patients on chronic corticosteroid therapy, which inhibits osteoblastic activity, as reflected by subnormal serum osteocalcin levels [361. Despite the importance of hip fracture as a major health problem, few studies have been devoted to potential bone turnover abnormalities in those patients. In a large group of patients studied immediately after hip fractures, we have found increased urinary excretion of crosslinks and decreased serum osteocalcin levels when comparing to agematched healthy elderly, suggesting that increased bone resorption and decreased bone formation might be an important determinant of the low bone mass that characterizes patients with hip fracture [37]. Osteocalcin contains three residues of gamma Kcarboxy-glutamic acid (Gla), a vitamin dependent amino acid. The level of circulating noncarboxylated osteocalcin is significantly increased in elderly women and decreases with vitamin K treatment [38,39]. We have recently shown, in a prospective study in a cohort of elderly institutionalized women followed for 18 months, that serum noncarboxylated osteocalcin measured at baseline was significantly higher in those who subsequently sustained a hip fracture (Table III). Thus, in those women with a subnormal level of circulating noncarboxylated osteocalcin, the relative risk of hip fracture was increased fivefold [40]. The level of the gamma carboxylation of osteocalcin appears to reflect the poor nutritional status of elderly patients with hip fracture, through unclear mechanisms that deserve further investigation. Presently, measurement of noncarboxylated osteocalcin is the first marker of bone fragility associated with hip fracture in the elderly.

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TABLE Ill Baseline Biochemical Characteristics SerumParameter Osteocalcin (ngimt) Carboxylated Noncarboxylated Alkaline Phosphatase (Ill/liter) PTH (pg/mt) 25.OHD (ngimt) Creatinine(mgiliter)

Hip Fracture

No Hip Fracture

6.4 k 3.2 1.62 i- 1.16

0.94i- 0.93

a2 A 22 62 t 27

80 t 35 50 i- 30

1428

9.2 2 4.4

5.4 t 2.8

p Value NS

a1 NS NS

17 i 12

NS

7.9 i 2.3

NS

,

Data are from a populatron of 195 elderly instrtutionalized women, divided into those who subsequently sustained a hip fracture during an 18 month follow-up and those who did not fracture [40]. 25OHD = 25.hydroxyvitamrn D; NS = nonsignifican’r; PTH = parathyroid hormone,

REFERENCES 1. Brown JP, Delmas PD, Malavai L, eta/. Serum bone Gla-protein: a specrfrc marker for bone formation in postmenopausal osteoporosis. Lancet 1984; i: 1091-3. 2. Podenphant J, Johansen JS, Thomsen K, et al. Bone turnover in sprnal osteoporosis. J Bone Miner Res 1987; 2: 497-503. 3. Hill CS, Wolfert RL. The preparation of monoclonal antibodies which react preferentially with human bone alkaline phosphatase and not liver alkaline phosphatase. Clin Chim Acta 1986; 186: 315-20. 4. Price PA, Parthemore JG, Deftos U. New biochemical marker for bone metabolism. J Clan Invest 1980, 66: 878-83. 5. Delmas PD, Stenner D, Wahner HW, et al. Effect of renal function on plasma levels of bone Gla-protein. J Clin invest 1983; 71: 1316-21. 6. Price PA, Williamson MK, Lothringer JW. New biochemical marker for bone metabolism. J Biol Chem 1981; 256: 12760-6. 7. Delmas PD, Wilson DM, Mann KG, et al. Orrgin of vitamin K-dependent bone protein found in plasma and its clearance by kidney and bone. J Clin Endocrinol Metab 1983; 57: 1028-30. 8. Delmas PD. Biochemical markers of bone turnover for the clinical assessment of metabolic disease. Endocrinol Metab Clin North Am 1990; 19; 1: 1-18. 9. Garner0 P, Grimaux M, Demiaux B, Preaudat C, Seguin P, Delmas PD. Measurement of serum osteocalcm with a human-specific two-site immunoradiometric assay. J Bone Miner Res 1992; 12: 1389-98. 10. Garner0 P, Grimaux M, Bore1 0, Seguin P, Delmas PD. lmmunoreactive forms of circulating human osteocalcrn. J Bone Miner Res 1992; 7: S155. 11. Gundberg C, Weinstein RS. Multiple immunoreactive forms in uremic serum. J Clin Invest 1986, 77: 1762-7. 12. Tracy RP, Andrianorivo A, Riggs BL, Mann KG. Comparison of monoclonal and polyclonal antibody-based immunoassays for osteocalcin. A study of sources of variation in assay results. J Bone Miner Res 1990; 5: 451-61. 13. Taylor AK, Linkart S, Mohan S, Chrinstenson RA, Singer FR, Baylink D. Multrple osteocaicin fragments in human urine and serum as detected by a midmolecule osteocalcin radioimmunoassay. J Clin Endocrinol Metab 1990; 70: 467-72. 14. Delmas PD, Demraux B, Malaval L, eta/. Serum bone Gla-protein (osteocalcin) in primary hyperparathyroidism and in malignant hypercalcemia. Comparison with bone histomorphometry. J Clin invest 1986; 77 985-91. 15. Parhtt AM, Simon LS, Villanueva AR, et al. Procollagen Type I carboxy-terminal extensron peptide in serum as a marker of collagen biosynthesis in bone. Correlation with iliac bone formation rates and comparison with total alkaline phosphatase. J Bone Miner Res 1987, 2: 427-36. 16. Hassager C, Fabbri-Mabelli G, Christiansen C. The effect of the menopause and hormone replacement therapy on serum carboxyterminal propeptide of type I collagen. Osteoporosis Int 1993; 3: 50-2. 17. Smedsrod B, Melkko J, Ristelli L, Risteili J. Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor In hver endothelial cells. Biochem J 1990; 271: 345-50. 18. Ebeling PR, Peterson JM, Riggs BL. Utility of type I procollagen propeptide assays for assessing abnormalities in metabolic bone diseases. J Bone Miner Res 1992; 7: 1243-50.

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19. Prockop OJ, Kivirrkko KI. Hydroxyproline and the metabolism of collagen. In: Gould ES, ed. Treatise on Collagen. New York: Academic Press, 1968; 215-46. 20. Eyre DR. Collagen crosslinking amino-acids. Methods Enzymol 1987; 144: 11539. 21. Uebelhart D, Schlemmer A, Johansen J, Gineyts E, Christiansen C, Delmas PD. Effect of menopause and hormone replacement therapy on the urinary excretion of pyridinium crosslinks. J Clin Endocrinol Metab 1991, 72: 367-73. 22. Eastell R, Hampton L, Colwell A. Urinary collagen crosslinks are highly correlated with radioisotopic measurements of bone resorption. In: C. Christiansen and K. Overgaard, eds. Proceedings of the Third International Symposium on Osteoporosis, Osteopress, Aalborg, Denmark, 1990; 469-70. 23. Delmas PD, Schlemmer A, Gineyts E, Riis B, Christiansen C. Urinary excretion of pyridinoline crosslinks correlates with bone turnover measured on iliac crest biopsy in patients with vertebral osteoporosis. J Bone Miner Res 1991; 6: 639-44. 24. Uebelhart D, Gineyts E, Chapuy MC, Delmas PD. Urinary excretion of pyridinium crosslinks: a new marker of bone resorption in metabolic bone disease. Bone Miner 1990; 8: 87-96. 25. Robins SP, Black D, Paterson CR, Reid DM, Duncan A, Seibel MJ. Evaluation of urinary hydroxypyridinium crosslink measurements as resorption markers in metabolic bone disease. Eur J Clin Invest 1991; 21: 310-5. 26. Harvey RD, MC Hardy KC, Reid IW. Measurement of bone collagen degradation in hyperthyroidism and during thyroxine replacement therapy using pyridinium cross-links as specific urinary markers. J Clin Endocrinol Metab 1991; 72: 1189-94. 27. Eastell R, Calvo MS, Burritt MF, Offord KP, Russell RGG, Riggs BL. Anormalities in circadian patterns of bone resorption and renal calcium conservation in type I osteoporosis. J Clin Endocrinol Metab 1992; 74: 487-94. 28. Seyedin S, Zuk R, Kung V, Daniloff Y, Shepard K. An immunoassay to urinary pyridinoline: the new marker of bone resorption. J Bone Miner Res 1993 8: 635-42. 29. Hanson DA, Weiss MAE, Bollen AM, Maslan SL, Singer FR, Eyre DR. A specific immunoassay for monitoring human bone resorption: quantitation of type I collagen cross-linkgd N-telopeptides in urine. J Bone Miner Res 1992; 7: 1251-8.

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30. Ristelli J, Elomaa I, Niemi S, Novamo A, Ristelli L. Radioimmunoassay for the pyridinoline cross-linked carboxyterminal telopeptide of type I collagen: a new serum marker of bone collagen degradation. Clin Chem 1993; 39: 635-40. 31. Krane SM, Kantrowitz FG, Byrne M, Pinnel SR, Singer FR. Urinary excretion of hydroxylysine and its glycosides as an index of collagen degradation. J Clin Invest 19n; 59: 819-27. 32. Stepan JJ, Pospichal J, Presl J, et al. Bone loss and biochemical indices of bone remodeling in surgically induced postmenopausal women, Bone 1987; 8: 279-84. 33. Johansen JS, Riss BJ, Delmas PD, et al. Plasma BGP: an indicator of spontaneous bone loss and effect of estrogen treatment in postmenopausal women. Eur J Clin Invest 1988; 18: 191-5. 34. Hansen MA, Kirsten 0, Riss BJ, Christiansen C. Role of peak bone mass and bone loss in postmenopausal osteoporosis: 12 years study. Br Med J 1991; 303: 961-4. 35. Civitelli R, Gonnelli S, Zacchei F, et a/. Bone turnover in postmenopausal porosis Effect of calcitonin treatment. J Clin Invest 1988; 82: 1268-74.

osteo-

36. Garrel DR, Delmas PD, Welsh C, et al. Effect of moderate physical training on prednisone-induced protein wasting: a study of whole bone and bone protein metabolism. Metabolism 1988; 37: 257-62. 37. Akesson K, Vergnaud P, Gineyts E, Delmas PD, Obrant K. Increased bone resorption and decreased bone formation in elderly women with hip fracture. Bone Miner 1992; 17(Sl): 184. 38. Knapen MHJ, Hamulyak K, Vermeer C. Increased bone resorption and decreased bone formation in elderly women with hip fracture. Ann Intern Med 1989; 111: 1001-5. 39. Plantalech L, Guillaumont M, Leclercq M, Delmas PD. Impaired carboxylation serum osteocalcin in elderly women. J Bone Miner Res 1991; 6: 1211-6.

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40. Szulc P, Chapuy MC, Meunier PJ, Delmas PD. Serum undercarboxylated osteocalcin is a marker of the risk of hip fracture in elderly women. J Clin Invest 1993; 91: 1769-74.

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