CLINICAL CHEMISTRY
Bone Disorders (Osteoporosis) Nuala A. MeCarroll Department of Biochemistry, St. James’s Hospital, Dublin 8, Ireland
Osteoporosis is characterized by low bone mass (osteopenia) and microarchitectural deterioration of bone tissue leading to increased bone fragility and a consequent increase in fracture risk ( E l ) . The pathogenetic mechanisms have not yet been defined but it has been established that the disease process involves an imbalance between the processes of bone resorption and formation with a net loss of hone mass (E2). Each year in the United States 1.3 million fractures involving the vertebrae, hip, distal radius, pelvis, or proximal humerus are associated with osteoporosis (E3). Vertebral fractures are most common and can cause deformity and pain, but disability and loss of independence following hip fracture is a major public health issue with an associated cost in the United States of $7.3 billion in 1984 (E3). Ageing is a ma’or risk factor for the development of osteoporosis, and iecause the world population of elderly people is increasing a significant rise in disease prevalence is anticipated. Additional risk factors include female sex and particularly postmenopausal status, Caucasian or Asian race, chronic liver or kidney disease, excess glucocorticoidor thyroid hormones, immobilization, hypercalciuria, alcoholism, and smoking (E4). The presence of these risk factors and low bone mass does not identify those a t risk of fractures since osteoporosis with vertebral compression fractures occurs in premenopausal women and young men (E4) and fractures have occurred in individuals with above average bone mass following slight trauma but not in others with very low hone mass after severe trauma (E5). Prevention of the disease is the ultimate goal and is the focus of much epidemiological and nutritional research, but the evolving recommendations for modification of diet and life style are likely to be of benefit primarily to future generations. However, several therapeutic regimens are availablewhichcan retard bonelosa or increase hone formation (E6). These therapies can be expensive and can cause undesirable side effects; therefore, a means of identifying individuals a t increased risk for osteoporosis who are most likelyto benefit from therapyandofmonitoringtheirresponse is required. Radiological techniques for quantification of bone density have become increasingly available hut they can be expensive, provide information only on selected skeletal sites, and are relatively insensitive to changes in bone density occurring in time periods less than 12 months ( E n . Bone histomorphometry can provide a clear definition of the relative rates of hone formation and resorption a t the biopsy site and it is the only technique which can identify events a t the cellular but application of the technique is limited by the level (E8), need for a bone biopsy. Quantification of biomarkers of bone metabolism in blood or urine should provide a relatively noninvasive and inexpensive indication of bone turnover (E9) and an integrated reflection of events in the entire skeleton provided that changes associated with osteoporosis are quantitatively significant. Other less common metabolic bone diseases associated with osteopenia such as osteomalacia or osteitis fibrosa have characteristic patterns of abnormality in the serum concentrations of the calciotropic hormones parathyrin (PTH) and 25-hydroxy vitamin D, which is the immediate precursor of the active hormone 1,25-dihydroxy vitamin D, and of calcium and phosphorus, the major inorganic components of bone, and in the activity of the bone isoform of alkaline phosphatase, which is thought to reflect osteoblastic activity. Osteoporosis on the other hand has been traditionally regarded as the metabolic bone disease without associated significant or characteristic biochemical abnormalities. Recent awareness of the major public health implications ofthis common condition have resulted in a new look at some old markers of bone metabolism and application of some newer markers of bone turnover such as osteocalcin, procollagen peptides, and pyridinoline cross-links to the detection and monitoring of affected individuals. Thisreviewcoversselected reports, identifiedbyaliterature search under the subject heading “osteoporosis” and covering 388R * ANALYTICAL
CHEMISTRY. VOL. 65, NO. 12. JUNE 15, 1993
NuaIa McCanoll Is a clinical blochemlst In St. James’ Hospital. Dublin. Ireland. She received her B.Sc. degree in biochemistry from University College. Dublin. her MSc. degree in clinical biochemistry from Trinity College, Dublin. and her Ph.0. degree in
clinical chemistry from Cleveland State University. She is a Member of the Royal College of Pathologists (Chemical Pathology)andaDiplomatoftheAmericanBoard of Clinical Chemistry. From 1991 to 1992 she was a Postdoctoral Fellow in the Department of Biochemistry of the Cleveland Clinic Foundation. Her research interests include lung surfactant. disorders of porphyrin metabolism, and biochemical aspects of nutrition. the five-year period ending in October 1992,which related to calcium metabolism in osteoporosis or the applications of laboratory tests reflecting bone turnover to the identification or management of patients with osteoporosis. Some additional references were taken from these publications. Only English language publications with human subjects as the main focus were included. Some review articles covering selected aspects of osteoporosis and bone biochemistry are listed in Table E-I. A publication by the U.S. Department of Health and Human Services entitled Osteoporosis, Research. Education and Health Promotion (ElO)Drovides an overview of epidemiologic, hasic science; and’preventive aspects of osteoporosis as well as an outline of possible future researchgoals. Theroleofgrowth factors in bonemetabolism has beendiscussedelsewhere (E4,E l l , EZZ)andisnotcovered in this review. Bone Turnover. What follows is a brief overview of some aspectsof bonemetabolism whichare relevant toosteoporosis. Bone is a metabolically active organ and is continually undergoing cycles of resorption followed by formation a t specific sites called bone remodeling units (BRU) (EZ). A cycle is initiated by activation of bone lining cells, which are thought to be of osteoblastic origin, probably through the action of PTH (E4, E13). Once activated these cells may contract and expose mineralized bone to the action of osteoclasts and promote bone resorption by release of proteolytic enzymes and recruitment of osteoclasts through the release of paracrine factors (E4,E13). Over a period of about 2 weeks a discrete area of bone including mineral and organic components is resorbed through the action of osteoclasts (EZ). The osteoclasts are then replaced by osteoblasts, which fill in the resorption cavity over a 3-4-month period by laying down new matrix which is subsequently calcified. Normally the resorption and formation phases are tightly coupled so that the amount of bone resorbed is exactly balanced by newly formed bone and there is no net loss. The overall rate of bone turnover is determined by the frequency of activation of new BRU (EZ). Normally about 1 million BRU are active in the skeleton, and they may occupy up to 25% of the trabecular hone surface (E6,E14). In the period from birth through adolescence, very high rates of bone turnover are associated with longitudinal growth. The Table E-I. Review Articles on Osteoporosis and Related Subjects subject ref osteoporosis E2, E4, E6,E10, E14 hone biochemistry and cell biology E13, E57 El bone density bone histomorphometry E8 bone turnover E19, EW, E51 calcitonin E41 calcium homeostasis EZWE22, E26 osteocalcin
vitamin D growth factors
E58 E31440 Ell,E12
CLINICAL CHEMISTRY
Tabla E-11. Biochemical Markers of Bone Turnover marker source Bone Formation serum alkaline phosphataseosteoblast bone isoform serum osteocalcin osteoblast serum procollagen I extension procollagen (osteoblast) peptides (PICP) Bone Resorption serum acid phosphatase osteoclast urinary hydroxyproline collagen, diet, C l q urinary pyridinolines collagen osteocalcin,matrix Gla urine/serum y-carboxyglutamic acid protein, coagulation factors urinary hydroxylysine glycosides collagen
released during bone resorption such as hydroxyproline and collagen cross-links are thought to reflect bone resorption. Under ideal circumstances, when bone formation and resorption are in balance, bone turnover should be reflected equally by either group of markers. However there is si ificant variation in both the sensitivity and the specificity o&e individual analytes to chan es in bone metabolism and they can be variably affected %y extraosseous clearance mechanisms (E19).Of the markers listed (Table E-11) osteocalcin, procollagen extension eptides, and pyridinoline cross-links are relatively new anc r are therefore covered in greater detail in this review. Evidence from population studies that osteoporosis is associated with slight changes in the concentrations of calcium fractions and of the calciotropic hormones which could indicate pathogenetic mechanisms and preventive strategies is reviewed in the next section. CALCIUM METABOLISM
hysiologicalrole of bone remodelin in the adult is not known, gut it is an important feature of c3cium homeostasis and it may re resent a necessary process for maintaining bone strengtf (E4, E14). Bone Turnover in Osteoporosis. Bone mass increases in the early years of life until peak bone mass is attained in the second or third decade (E2,E14). Age-related bone loss begins around the fourth decade and results in loss of 35 % of cortical bone and 50% of trabecular bone in women over their lifetime, and about two-thirds of these amounts in men (E2, E14). Two patterns of bone loss have been identified; a slow age-dependent phase occurs in both men and women, and a transient accelerated phase lasting 3-6 and ossibly up to 20 years occurs in women after menopause ($2, E6). In both cases net bone loss is the result of an imbalance between resorption and formation. Age-dependent bone loss is associated with impairment of osteoblast function so that resorption cavities are incompletely filled. At the time of menopause an increased rate of bone turnover, associated with estrogen deficiency, amplifies the resor tion/formation imbalance and results in accelerated bone 08s. Oophorectomy is associated with more severe bone loss than that associated with natural menopause. Estrogen replacement therapy slows bone loss associated with both natural and surgically induced menopause (E2,E14). Recent reporta that estrogen receptors are present in cultured human osteoblasts (E15, E16)and that treatment of human osteoblast-like cells with estrogen stimulated DNA synthesis and cell growth and increased alkaline phosphatase activity (El7) suggest a direct action of estrogen on bone. It is possible that the increased bone turnover rate associated with estrogen withdrawal reflects a systemic effect of the hormone. These mechanisms sugtest that osteoporosis is an inesca able consequence of aging if the life span is long enough. It [as been suggested that osteoporosis is present if the bone mineral density lies more than two standard deviations below the mean of young adults of the same sex (E18).Using this definition it was estimated that 50% of women aged 65 and nearly 100%of those aged 80 years have osteoporosis (E18). The challenge, however, is to predict the fracture risk in an individual patient before excess bone loss has occurred and to select those in whom thera y is likely to have a beneficial effect. With this goal in minrfthe usefulness of biochemical markers of bone metabolism should be determined in longterm follow-up studies of patient populations. Most studies to date have assessed various markers according to their ability to distinguish pre- from postmenoausal women, or women identified as having osteoporosis, y a variety of criteria, from age-matched controls without osteoporosis. Few studies have focused on men with osteoporosis. Biochemical markers are usually classified according to their relationship with either bone resorption or bone formation as in Table E-11. These relationships have, in most cases, been established by histomorphometrically identified events at the cellular level (E19).They do not necessarily ersist in the presence of diseases other than that for which t f e relationshi was defined or following specific drug therapy and shoulde! reestablished for each condition. In general, roducts of osteoblasts such as alkaline phosphatase, coggen, and osteocalcin are associated with bone formation whereas products of osteoclasts such as tartrateresistant acid phosphatase and metabolites of bone matrix
P
E
Calcium. Calcium balance de ends on absorption of calcium from the gastrointestin3 tract in excess of the obligatorylossesin urine and stools and the coordinated action of the calciotropic hormones PTH, 1,25-dihydroxy vitamin D and possibly calcitonin (E20-E22). According to the calcium deficiency theory of osteoporosis, deficient calcium intake or absorption or increased calcium excretion will result in PTH-mediated net loss of calcium from bone, which contains 99% of total body calcium, to maintain plasma ionized calcium within narrow limits. Resorption of calcium cannot be achieved selectively and is therefore accom anied by breakdown of bone tissue leading to reduced bone tfensity and osteoporosis (E22). Some recently published work seems relevant to this hypothesis. Several groups reported statistically significant increases in serum total calcium (E23,E24) without changes in ionized calcium in post- compared to premenopausal women (E23E25). The total calcium increase was accounted for by a corresponding increase in the calculated concentration of the complexed fraction and was associated with increased bicarbonate concentration and increased anion gap (E23). Although the increase in complexed calcium is small in quantitative terms (0.04 mmol/L) and does not increase total calcium above the reference range, it was calculated that the resulting increased obligatory loss of this complexed fraction by glomerular filtration could account for 1%decrease in bone density per year in the postmenopausal population (E23). Si@ficantly hi her calcium/creatinine ratios in early morning urine samples kom fasting postmenopausal women on lowcalcium diets than in samples from premenopausal women support this proposed mechanism (E26). Other studies have shown that in normal individuals calcium excretion decreases at night, but to a lesser extent in women than in men (E25), but is unchanged in women with osteoporosis (E27).Serum osteocalcin and urinary deoxypyridinoline (DPD) increased at night in both pre- and ostmenopausal women, suggesting increased bone turnover,%ut the increase in DPD was greater in the osteoporotic group. As there was no change in the serum ionized calcium concentration in either group, the authors concluded that ionized calcium in the normal group was maintained by reduced renal calcium excretion and increased bone resorption but only by the latter mechanism in the osteoporotic group (E27). Taken together the results suggest greater calcium loss in urine by post- rather than premenopausal women and by women rather than men, which corresponds with the known relative rates of decrease in bone densities. The relationship of the complexed serum calcium fraction to the diurnal calcium excretion pattern and to the calciotropic hormones as well as the nature of the calcium complex awaits definition. Parathyrin (PTH).PTH is a peptide hormone which is secreted by the parathyroid glands in response to decreased plasma ionized calcium concentration and is primarily responsible for calcium homeostasis. PTH stimulates calcium resorption from bone via osteoblasts or lining cells and acts on the kidney to increase calcium reabsorption, decrease hosphorus reabsorption, and increase the activity of the laydroxylase enzyme, which converts 25-hydroxy vitamin D (25-(0H)D)to the active hormone 1,25-dihydroxyvitamin D (calcitriol, 1,25(OH)zD) which stimulates gastrointestinal calcium absorption (E21). There is much interest in possible
g
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
389R
CLINICAL CHEMISTRY
osteoporosis-related changes in PTH. Early studies showing that the increased immunoreactive serum PTH concentrations observed with aging were accompanied by increased bioactivity, and did not merely reflect reduced clearance of peptide fragments secondary to the reduction of glomerular filtration rate which accompanies aging, have been reviewed previously (E20). Technically simple two-site immunometric assays which are specific for intact PTH and allow uantification of the biologically active hormone in normal inlividuals have recently been developed (E28, E29). Application of these assays has confirmed the age-related increase observed with less specific assays and revealed differences in intraindividual variations between normal and osteoporotic subjects. Age-related increase in serum intact PTH concentrations (r = 0.48) which correlated with 1,25(OH)zDconcentrations (r = 0.44) was found in a group of women aged 20-87 years who had bone densities within a e-adjusted confidence intervals (E30). It was suggestefi that decreased intestinal responsiveness to 1,25(OH)zD with increasing age resulting in decreased intestinal calcium absorption could cause mild secondary hyperparathyroidism, increased formation of 1,25(OH)zD,and increased bone turnover. Other studies su est alterations in basal serum PTH concentrations and H response to stress in atienta with osteoporosis. Pulsatile secretion of intact PTI! with higher ulse amplitude at ni ht was demonstrated in normal men, I! ut the am litude an frequency of pulses were lower in one female a n f three male patients with osteoporosis (E31). Another group described a biphasic pattern of serum intact PTH concentrations in normal males and femaleswith troughs a t around 1000 and 2000 h (E25, E27). Women with osteoporosis tended to have lower nocturnal concentrations associated with increased urinary calcium and DPD excretion (E27).Results of challenge tests designed to decrease or increase serum ionized calcium concentrations also suggest altered arathyroid responsiveness in osteoporosis. Phosphate afministration decreased serum ionized calcium concentrations in women with and without osteoporosis, but the serum PTH increase was almost 50% less in the osteoporotic group (E32). Infusion of a recombinant preparation of the active N-terminal fraction of PTH (hPTH-(1-34)) increased the ionized calcium concentration and suppressed the serum intact PTH concentration in postmenopausal women with and without osteoporosis, but the PTH response in the group with osteoporosis appeared less sensitive to calcium changes (E33). The possibility that bone sensitivity to. PTH is altered in osteoporosis was investigated b measuring serum calcium and osteocalcin and urinary hy roxyproline concentrations in pre- and ostmenopausal women followin infusion of recombinant kterminal hPTH, but no statistic&y significant differences were found (E34, E35). Estrogen therapy for 12 weeks had no significant effect on serum intact, immunoreactive, or bioactive PTH as determined by a renal bioassay, in preprandial samples from postmenopausal women (E36). Despite the evidence of altered PTH production in osteoporosis, there is as yet no clear indication of the predictive value of these measurements in assessing fracture risk. Vitamin D. Vitamin D is derived from the skin, where its synthesis from cholesterol is dependent on ex osure to ultraviolet light, and from the diet. It is first hygoxylated in the liver to form the major circulating form 25(OH)D,which is converted to the active hormone 1,25(OH)zDby the action of renal la-hydroxylase. 1,25(OH)zDis primarilyresponsible for regulatin active calcium absorption from the gastrointestinal tract ( i 2 1 ) . Recent reviews discuss the relationship of vitamin D to osteoporosis (E37, E38) and the analysis of vitamin D and its metabolites (E39, E40). Reports of decreased renal la-hydroxylase activity and decreased intestinal calcium absorption with increasing age (E37) and in osteoporotic patients compared with agematched controls (E38) have focused attention on 25(OH)D and 1,25(OH)zD concentrations in aging and osteoporotic subjects. Some minor changes but within the reference ranges have been described in association with age or postmenopausal status (E38,E41). No significant differences were found in serum 25(OH)D, 1,25(OH)zD,or vitamin D-binding protein in a longitudinal study of women before and after menopause,
5%
d
B
990R
ANALYTICAL CHEMISTRY, VOL. 65,
NO. 12, JUNE 15,
1993
although the rate of bone loss doubled, showing that early postmenopausal bone loss is not associated with overt vitamin D deficiency (E42). Althou h concentrations of intestinal vitamin D receptors decreasecfwith increasing age in women aged between 20 and 87 years (r = -0.38), the fractional calcium absor tion did not change with age (E30). The authors suggest &at previous studies re orting reduced calcium absorption with increasin age coulxhave included older patients or atients wit! secondary diseases (E30). Fractional calcium aisor tion was weakly correlated (r = 0.36) with serum 1,25(0&D concentration, which also increased slightly with age (r = 0.32). The suggestion that increased production of the active hormone stimulated by PTH could compensate for receptor deficiency (E30)is supported by the observation that treatment of osteoporoticwomen with calcitriolcaused dose-related increases in intestinal calcium absorption (E38). Serum concentrations of 25(OH)D decreased, PTH increased, but serum 1,25(OH)zD concentrations were unchanged with increasing age in men aged 30-92 years and there was a gradual decline in bone density (E43). In women low serum concentrations of 25(OH)D were associated with low bone mass (E44, E45), but the reported mean concentrations of 25(OH)D, calcium, phosphate, and alkaliFe phosphatase could support a diagnosis of osteomalacia. Although no clearcut changes in vitamin D concentrations have been associated with osteoporosis, it has been argued that mild deficiency may be associated with calcium malabsorption sufficient to cause osteoporosis but not osteomalacia (E38). Clear distinction of these conditions associated with reduced bone density requires a bone biopsy and cannot be made at present using biochemical techniques. However, bone density was increased and the incidence of nonvertebral fractureswas significantly reduced in a group of women, aged 69-106 years, whose diets were supplemented with vitamin D and calcium (E46),suggesting that vitamin D deficiency is an important determinant of fracture risk. In this group also baseline serum 25(OH)D, calcium, and PTH concentrations were suggestive of vitamin D deficiency with secondary hyperparathyroidism. Taken together these studies ( E 4 3 4 4 6 ) su gest that quantification of serum calcium, 25(OH)D, a n t PTH concentrations could provide an indication of fracture risk. Distinction between osteoporosis and osteomalacia will require a bone biopsy. Calcitonin. Calcitonin, a peptide hormone which is secreted by thyroid C cells in response to small increases in plasma ionized calcium concentration, inhibits osteoclast activity a t pharmacological doses and has been used in the treatment of disorders associated with accelerated bone resorption such as Paget’s disease of bone 0347). However, a clear etiological or di ostic role for calcitonin in osteoporosis has not been d x e d . Conflicting reports of serum calcitonin measurements, which may have reflected methodolo ical difficulties, were comprehensively reviewed (E47). On ba!ance it was concluded that basal and stimulated serum calcitonin concentrations were unaffected by age but were lower in women than in men. Contrary to expectations, serum concentrations in osteoporoticwomen were similar to or higher than those in normal women. Further evidence against a role for calcitonin in the development of osteo orosis came from reports that patients who had undetectahe or significantly reduced serum calcitonin concentrations following th oidectom had either normal or reduced bone density in fogw-up stuiies. However, the possibility that differences in serum calcitonin concentrations may be found in osteoporotic patients with different rates of bone turnover has not been ruled out (E47). Summary. There is clear evidence that increased calcium excretion and decreased absorption are associated with female sex and increasing age, respectively, and could result in reduced bone density and increased fracture incidence secondary to calcium deficiency. Two altered PTH secretory patterns are apparent; changes in the diurnal secretion and the response to dynamic tests suggest primary abnormality a t the parathyroid gland level, but secondary hyperparathyroidism in response to subclinical vitamin D deficiency or reduced effectiveness in stimulating intestinal calcium absorption is a likely explanation for age-related changes in concentrations. At present it is difficult to relate changes in serum PTH concentration to events at the cellular level since
CLINICAL CHEMISTRY
anaboliceffecta in addition to the well-known resorptive action have been demonstrated both in clinical trials (E6,E48) and a t the cellular level (E131 and different receptor responses may be involved (E49). From presently available data it appears that quantification of calcium, 25(OH)D, and PTH concentrations could be of value in identifying elderly individuals with reduced bone density secondary to vitamin D deficiency, but the predictive value of the small changes observed at menopause either singly or in combination with markers of bone turnover awaits definition. ASSESSMENT OF BONE TURNOVER Alkaline Phosphatase. Total serum alkaline phosphatase activit in normal nonpregnant adults is about equally derived from tge bone and liver isoforms, which arise from the same gene and exhibit slight differences in heat stability and electrophoretic behavior as a result of posttranslational modifications, and from variable amounts of the intestinal variant (E19,E50, E51). The function of the enzyme in bone is unknown but it appears to be associatedwith mineralization. It is membrane bound and the mechanism of release into the circulation is unknown, but the activity within osteoblasts can be stimulated or inhibited by PTH and is also affected by other locally a c t i q factors (E13). Des ite its low sensitivity and lack of specificity as a marker of one turnover (E19, E50, E51), total serum alkaline phosphatase activity is frequently reported as an indicator of osteoblastic activity and has been used in many studies assessing the value of osteocalcin, the newer marker of osteoblastic activity. Continued use of total serum alkaline phosphatase activity measurements is an indication that quantification of the bone isoform remains technically difficult. However, results of serum bone alkaline phos hatase activity (BAP)measurements have been reportezby several groups. BAP was higher in men than women when determined usin a technique involvin recipitation of the bone isoform by wieat germ agglutinin (&A) (E52)and by polyacrylamide gel electrophoresis combined with heat inactivation (PAGE) (E53). Serum osteocalcin concentrations and BAP changed in a similar manner, remaining relatively constant in males and females a ed 30-50 years and increasing in older age groups when SAP was determined using a solid-phase immunoassay (E54),but correlation between these estimates of osteoblastic activity was found only in women when BAP was determined by the PAGE technique (E53). Osteocalcin and BAP gave concordant results when expressed as 2-scores in several disorders affecting bone turnover, such as hypoparathyroidism where bone turnover is reduced, and in conditions associated with moderate increases in bone turnover, such as hyperparathyroidism, hyperthyroidism, acromegaly, and postmenopausal osteoporosis (E54). In the latter condition, however, the relative increase in BAP was greater than the increase in osteocalcin. Significant discrepanciesbetween these markers which were observed in samples from patients with Paget's disease of bone or renal failure could have arisen because BAP and osteocalcin may reflect different aspects of osteoblast activit or because clearance mechanisms are different (E54). &though serum BAP showed some correlation (r = 0.46, P < 0.05) with histomorphometrically determined bone formation rate in normal women aged 30-73 years, there was no correlation when postmenopausal women aged 55-73 years were studied separately (E55). The latter group of women had normal bone density accordin to age-adjusted confidence intervals, and the reason for the Jscrepancy between histomorphometry and BAP is unclear. Both total alkaline phosphatase activity and BAP were significantly reduced in postmenopausal women after 1year of hormone replacement therapy (E56), suggesting possible application of this assay in monitoring treatment. More widespread application of BAP assays will no doubt follow the development of simpler methodologies. Osteocalcin (y-Carboxyglutamate Protein, Bone Gla Protein, BGP). Osteocalcin is a 5.7-kDa protein which makes up about 20% of the noncollagenous bone matrix. It is synthesized by osteoblasts and odontoblasts and appears to be specific for mineralized tissue (E57). Three y-carboxyglutamate residues, which are formed in a osttranslational vitamin K-dependent reaction which is inhifited by warfarin, account for calcium-and hydroxyapatite-binding properties,
E
and the previously used name, bone Gla protein (BGP). Although synthesized by osteoblasts, osteocalcin appears to have a role in bone resorption rather than mineralization as evidenced by chemotactic activity toward osteoclast progenitor cells, monoc es, and macrophages and premature mineralization of one in warfarin-treated rats (E57, E58). A comprehensivereview of osteocalcincovering the evidence relatin serum concentration to bone formation, metabolism, physiofogical and disease-related variations, and limitations of assay procedures was published in 1988 (E58). Osteocalcin in Osteoporosis. Serum osteocalcin concentrations are increased in osteoporosisbut to a lesser degree than in conditions associated with high bone turnover such as childhood and adolescenceor Paget's disease of bone (E19, E58), and there is significant overlap with the normal population particularly in patients with low osteoblastic activity (El9). Baseline serum osteocalcinconcentrations in women up to 3 years postmenopause correlated with the decrease in bone mineral content over a subsequent 2-year period, and initially high concentrations were suppressed to within the premenopausal range during 2 years of hormone replacement therapy (E59). It was concluded that quantification of serum osteocalcin a t the time of menopause could provide an indication of the future rate of bone loss (E59). In accordancewith the findingsfor BAP, there was significant correlation between serum osteocalcin and histomorphometrically determined bone formation rates in a group of premenopausal women (r = 0.76) but not in a group of postmenopausal women whose bone formation rates were significantly higher (E55). Despite the absence of correlation with the formation rates, serum osteocalcin and BAP concentrations were significantly increased in the older group. No significant differencewas found between serum osteocalcin concentrations in a group of men with idiopathic osteoporosis and vertebral crush fractures (mean age 65 years) and agematched normal controh (E60). Serum osteocalcin, 25(OH)D, and insulin-like growth factor-1 (IGF-1) concentrations were significantly reduced in a group of male and female patients with spinal fractures com ared with a control group with a similar age range (E61). t h e r e was significant correlation between osteocalcin concentrations and IGF-1 (r = 0.801) and 25(OH)D (r = 0.7141, suggesting a determining role for these hormones (E61). In contrast to this report, increased serum osteocalcin concentrations have been recorded in osteomalacia, which is associated with low concentrations of 25(OH)D (E19). It is clear from these reports that there is disagreement on the relationship of serum osteocalcin concentrations to osteoporosis. While these differences could be due to the inclusion of different patient groups, there is some concern about the comparability of various osteocalcin assays (E9, E58). In the last five years there have been many publications describing methodological changes and reports of serum osteocalcin measurements associated with other indicators of bone turnover in an effort to improve the predictive value of the assay. The following is a brief review of hysiological and methodological factors which could contrgute to variations in results for serum osteocalcin. Physiological and Disease-RelatedVariations in Serum Osteocalcin Concentrations. As much as one-third of newly synthesized protein is released into the circulation and the remainder is incorporated in bone (E581so that serum concentrations should ideally reflect osteoblast activity or bone formation. However, osteocalcinmay be displaced from its binding to hydroxyapatite by diphosphonate, or lead, and may bind with lower affinity to fluoroapatite formed during fluoride therapy (E58). Serum osteocalcin concentrations were significantly increased but mostly within the reference range following 3 weeks of fluoride administration to normal men, but direct assessment of osteoblast activity was not reported (E62). Without accompanying histomorphometric analysis it is not clear if these results indicate increased osteoblastic activity or displacement of preformed rotein from bone. During bone resorption, osteocalcin is &ought to undergo proteolytic degradation with release of fragments into the circulation. Circulating osteocalcin is cleared rimarily by renal filtration and metabolism. Intact osteocakn is the predominant form in normal serum but immunoreactive fragments, which are also detectable in normal urine, are present in significant concentrations in serum from patients
if
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
301R
CLINICAL CHEMISTRY
Table E-111.Osteocalcin Assays.
assay type
immunogen
RIA
human OC
RIA
ovine OC
EIA EIA EIA RIA
bovine OC bovine OC bovine OC human OC
two-site IRMA
hOC 1-12 hOC 38-49
antibody specificity
calibrator
ref population (years)
ref range mean f SD (pg/L)
no
human OC
21-63
6.62 f 2.64
ND
E70
calcium dependency
osteoporosis range (M/L)
ref
midmolecule Gla-OC or Glu-OC midmolecule Gla-OC or Glu-OC ND Gla-OC ND ND
20 mM CaClz
ovine OC
22-67
3.4 f 1.6
7.8f 1.8
E69
25 mM EDTA ND 5 mM CaClz 25 mM EDTA
bovine OC bovine OC bovine OC human OC
E73 E74 E72 E68
ND
human OC
9.53 f 7.07 3.6 f 2.19 1.4 f 0.8 25f5 20 f 6 7.04 f 8.Bb 21.45 f 13.gb
ND 10.1 f 4.6 ND 25 f 7
intact molecule
20-90 (F) 20-65 15-64 20-65 (M) 38 f 2 (F) normal
ND
E71
a Abbreviations: OC, osteocalcin; Gla-OC, 7-glutamyl OC; Glu-OC, glutamyl OC; RIA, radioimmunoassay; EM, enzyme immunoassay; IRMA, immunoradiometric assay; F, female; M, male; ND, not defined. Results with two different OC 1-12 antibodies.
with renal failure or Paget’s disease of bone (E63).However, no inverse correlation was found between serum osteocalcin concentration and creatinine clearance when a radioimmunoassay specific for the intact molecule was used (E64). Significant elevations of serum osteocalcin were found in patients with chronic renal failure only when creatinine clearance was less than 20 mL/min and could have resulted from increased bone turnover or reduced renal clearance or both (E64). There is a significant diurnal variation with peak and trough concentrations bein found at around 0400 and 1200 h, respectively (E58).lfFhe eak is independent of the nocturnal seasonal changes, increase in serum g r o w t i hormone (E65), or smoking status (E66). Serum concentrations were increased during the luteal phase of the menstrual cycle in samples drawn at 0800 h (E67).Age-relatedchanges in serum osteocalcinconcentration are most dramatic in children, with peak values in the neonatal period and associated with the adolescent growth spurt after which concentrations remain relatively constant until the fourth decade (E58).Age-related changes in older men and women are less clearly defined. Serum concentrations were constant in men aged 30-69 years (E64,E68),constant from 30 to 50 years in both sexes, and then increased (E541or decreased with age in another group of men aged 20-55 years (E69).The integrated 24-h serum osteocalcin concentration was significantly higher in a group of 20-29-year-old men and women than in a group with ages in the range 30-40 years, was higher in men than in women, and was negatively correlated w t h age (E66).However, the latter estimate could have been biased by nonuniform data distribution. Other groups have reported increasin concentrations with aging in women from the fourth decack with E64,E69). an additional rise associatedwith menopause (E19, Better discrimination of changes in osteoporosis might be achieved if reference ranges were derived in age-stratified male and female populations with both normal and reduced bone density. Osteocalcin Assays. There are many problems with quantification of osteocalcin by immunoassay as can be seen from the selected methods (E70474)summarized in Table E-111, which show a wide range of reference values, and from previously reported variations observed between laboratories (E9).Clear definition of physiological and pathological changes in serum osteocalcinconcentrations with distinction between the y-carboxylated (Gla-OC) and the non-y-carboxylated (Glu-OC)molecule and fragmentswhich may reflect changes in extraosseous clearance is required. It is essential therefore that the characteristics of the assay employed are defined. Most published methods have employedbovine osteocalcin as immunogen, tracer, and calibration standard apparently because of availability and the fact that human osteocalcin, which differs in five amino acid residues, cross-reacts in the bovine system. More recent1 ovine- (E69)and human-based (E68, E70,E71)assays have gee, described, but even within human-basedassaysthere is significant variation in the results reported for normal individuals (Table E-111). Heterogeneous immunoreactivityof circulating osteocalcin was demonstrated using two-site immunoradiometricassaysemploying the same monoclonal C-terminal antibody but different N-terminal 302R
ANALYTICAL CHEMISTRY, VOL. 85, NO. 12, JUNE 15. 1993
antibodies raised against synthetic peptides corresponding to N- and C-terminal regions of human osteocalcin (E71). Mean results obtained in normal individuals differed by a factor of 3 (E71). Calcium bindin to Gla-OC causesa conformationalchange E72). that appears to affect bindin to some antibodies (E68, Some assays therefore inclutfe calcium or EDTA in reagents (Table E-111). This calcium de endence could be ex loited in distinguishing Gla-OC and 8lu-OC (E58).Gla-8C and Glu-OC were quantified in serum using a bovine-based RIA, which detects the intact molecule, by assayin the serum before and after extraction of Gla-OC with hyfroxyapatite (E75).Using this assay, no significant difference was found between total osteocalcinconcentrations in patients on lon term warfarin therapy compared with age-matched contro% but the ratio of Glu- to Gla-OC was significantly increased in those on warfarin therapy (E75). A midmolecule human-based osteocalcin assay was used to quantify osteocalcin fr menta in urine (E63).Excretion of immunoreactive osteoc2cin fragments in urine followed a diurnal pattern similar to that described in serum for the intact molecule. Osteocalcin fragments in 24-h urine expressed as milligram equivalents of osteocalcin per gram of creatinine showed excellent correlation (r = 0.91) with the activity of the bone isoform of alkaline phosphatase in serum samples from patients with Paget’s disease of bone. It was suggestedthat quantification of osteocalcin fra menta in 24-h urine will give an inte ated estimate of daily %oneturnover which will be free of Xurnal variation (E63).It remains to be determined how such measurementsrelate to bone turnover in patients with osteoporosis. Future developments of osteocalcin methodology should include, in addition to clear definition of assay s ecificity, the pre aration and distribution of a referencestanrfard which would Kelp to improve agreement between laboratories. r-Carboxyglutamic Acid (Gla). y-Carboxy lutamate released on metabolism of osteocalcinand matrix d a protein is excreted without further metabolism but contributes only 10% to total urinary Gla (E58).The remainder is derived from the Gla-containing coagulation factors (E19,E58). Despite the small roportional contribution from bone, urine Gla was increaserfin some patients with osteoporosis (E58). No significant differences were found between results from premenopausal women and age-matched postmenopausal women when free serum Gla was quantified in serum ultrafiltrates by HPLC with external standardization (E76), although increased concentrations had reviously been found in patients with primary hyper aratKyroidism or Paget’s disease of bone (E19).No signi&ant chan e was observed following treatment with estrogen or placelo for 9 months (E76).Determination of Gla concentrations is unlikely to be of value in the management of patients with osteoporosis because of low specificity for bone metabolism. Procollagen Type I Carboxy-Terminal Extension Peptides (PICP). Type I coll en makes up 90% of bone matrix but is also present in %in and tendon and other connective tissues (E57).Newly synthesized procollagen I has both N- and C-terminalextensiontrimeric peptides, PINP and PICP, res ectively, which are removed by the action of specific p e p t i L e s prior to formation of collagen fibrils (E57,
CLINICAL CHEMISTRY
E77). Release of these peptides into the blood should provide a direct measure of the rate of collagen synthesis and osteoblastic activity. However, there is evidence that PINP remains associated with the collagen fibrils (E19,E57). No significant correlation was found between serum PICP concentrations, quantified by a double antibody radioimmunoassay, and histomorphometric indexes of bone formation in 15 women with postmenopausal osteoporosis either alone (rvalue not stated) or in combination with serum totalalkaline phos hatase activity (r = 0.36) (E77). The authors suggested that getter discrimination mi ht be achieved by correcting for metabolic clearance, whicf may be reduced in patients with liver disease (E77). Serum concentrations of PICP decreased in res onse to infusion of PTH(1-38), and the results correlatei si ificantly with the increase in ionized calcium and 1,25(0&D (r = -0.74 p d -0.58, respectively) (E78). This experiment rovided m utvo confirmationof the inhibitory effect of PTIf on collagen synthesis, which had previous1 been shown in cell culture (E78),and indicates a otentidy useful line of investigation of the relationship ktween concentrations of PICP and PTH in agin Reference ran es for serum PICP concentrations in 75 brood donors, bot! males and females aged 18-61 ears, were determined using a commercially available rac6oimmunoassay (E79). Results correlated significantly with age in men (r = -0.546) but not in women (r = -0.133) (E79) and suggest decreasing osteoblastic activity or collqen synthesis with increasing age in men. In additional studies this oup found that serum PICP concentrationscorrelated weaEfy but si ificantlywith histomorphometricallydetermined rates of K n e formation (r = 0.40) and with serum osteocalcin concentrations (r = 0.62) but not with rates of bone resorption in a group of women with postmenopausal osteoporosis (E80).Serum PICP decreased significantly (30-40% of baseline results) in women treated for 1year with estro en progestogen (E80, E81), but anabolic steroid therapy had noeffect (E80). The reductipn of serum PICP was accompanied by a similar relative reduction of total serum alkaline phosphatase activity and of osteocalcin and hydroxyprolineconcentrations in serum and urine, respectively (E81). Although changes in hydroxyproline and alkaline phos hatase or osteocalcin could not be considered specific inicators of bone resorption and formation, these results together with an accompanyingincrease in bone densit were thou ht to reflect reduced frequency of activation of B&U (E81). 80th pre- and posttreatment results for these women with severe osteoporosis were within the reference range (E73-E81) and suggest that this assay is unlikely to be of value in identifying women at risk for osteoporosis, but may be of value in monitoring therapy. The results in men and women (E7SE81)are consistent with the age-related and postmenopausal patterns of bone turnover, respectively. Hydroxyproline. The most surprising aspect of recent reports on metabolic bone disease is the number featuring hydroxyproline as an indicator of bone resorption against which newer analytes are assessed. The limitations of this test in addition to the problem of contributions from dietary gelatin have been recently reviewed and include the fact that up to 40% of urinary hydroxyproline originates in the Clq component of complement, 90% of that released from bone is metabolized in the liver, and of the 10% of urinary hydroxyproline derived from catabolism of bone collagen, a proportion results from breakdown of newly synthesized coll en and therefore does not relate to bone resorption (E19). Thzatter point is reinforced by the very poor correlation ( r = 0.04) of urinary hydrox roline (mg/day) with histomorphometrically determinefione resorption (E19) and diagnostic accurac of 52% in women with postmenopausal osteoporosis (282). Acid Phosphatase. Osteoclasts are thought to be the source of the elevated tartrate-resistant acid phosphatase activity (TRAP) found in serum from patients with Paget’s disease of bone (E19), and quantification of this enzyme activit in serum could therefore provide an indication of osteocLtic activity and bone resorption. The enzyme has received comparatively little attention as a marker of osteoclastic activity in osteoporosis. Traditional methods of quantification have depended on measurement of the fraction of acid phosphatase activity, measured at pH 5 with 4-nitrophenyl phosphate (4NPP) as
.
substrate, which is resistant to inhibition by tartrate. However, this assay is not specific for the osteoclast/tissue macrophage isoenzyme because under the usual assay conditions there is some inhibition of the ‘tartrate-resistant fraction”, the erythrocyte isoenzyme is also tartrate resistant, and the prostatic isoenzyme is incompletely inhibited (E83). There is also evidence that osteoblasts contain tartrateresistant acid phosphatase activity (E84),which could contribute to the enzyme detected in serum. A modification of the 4NPP method, with sample redilution and incubation at 37 “C to overcome inhibition y plasma components and to inactivate the erythrocyte enzyme, respectively, showed good recovery of TRAP from bovine bone matrix, and the mean activity in patients with Paget’s disease of bone was significantly increased but results overlapped with the reference range even in this high bone turnover condition (E85). Immunoassays for acid phosphatase have been described and should have improved tissue specifici . An immunoassa with specificityfor tissue macrophageaci phosphatase whicx was unaffected by erythrocyte and prostatic isoenzymes employed Sepharose-bound porcine uteroferrin antibodies to remove the isoenzyme of interest so that its activity could be determined as the difference between activities before and after treatment (E83). An antibody prepared against tartrateresistant acid phosphatase purified from the spleen of a patient with hairy cell leukemia was used to develop an ELISA technique for quantification of acid phosphatasein mass units (E86). The antibody reacted with osteoclast but not with psteoblast or normal spleen extracts or with the prostatic isoenzyme. Significantly hi her results were obtained in serum from patients with %aget’s disease of bone and hyperparathyroidism than in normal individuals (E86). TRAP determined in a group of postmenopausal women with osteoporosiswas negatively correlated with bone mineral content (r= -0.43) and was significantlyhigher but overlapped with results in a control grou without osteoporosis (E87). TRAP, which was significant& elevated within 4 weeks of bilateral oophorectomy, before increases in bone alkaline phosphatase activity or osteocalcin concentrations were detected, was reduced to normal with estrogentherapy (E56). Results appeared to be clearly above the reference range suggesting the possibility of greater sensitivity of the assay when applied to those with surgically induced menopause than in women who undergo natural menopause, although the cause of menopause in the previous group (E87) was not specified. The application of immunoassa s for acid phosphatase to the identificationof patients at r i d for osteoporosis has not yet been described. Collagen Cross-Links (Pyridinoline and Deoxypyridinoline). The identification of pyridinoline (PYD) and deoxypyridinoline (DPD) as cross-linking molecules in collagen which were released on catabolism and excreted in urine in both free and peptide-bound form (E88)has led to significant interest in these amino acids as indicators of bone turnover. These trisubstituted 3-hydroxypyridinium derivatives are formed from three hydroxyllysine residues (PYD) or two hydroxyllysine and one lysine residues (DPD) and provide stable cross-linkingbetween three collagen molecules in most connective tissues with the exception of skin and cornea (E88). PYD is the major component in all cases, but significant amounts of DPD are only present in bone and dentin (E881 as reflected by the molar ratio in bone of PYD to DPD of 3.51 compared with 1O:l in cartilage (E89). Although the total p idinoline content of bone (0.3 moVmol of coll en) is low regtive to other tissues (>1.5 mol/mol), it is pos%e that quantification of urinary DPD will provide a more s ecific index of bone turnover as dentin is unlikely to contr8ute significantly (E88).The PYD to DPD ratio remains constant in bone throughout adult life (E89), but there is evidence that the concentration of pyridinolines may be increased in calcium-deficientstates (E88). A relationship of urinary yridinolines to bone resorption is supported by significant, gut weak, correlations of fasting urinary pyridinolinefcreatinine ratios with histomo hometric indexes of bone resorption (r = 0.35 and 0.46 f o 3 Y D and and by reports that ratios of PYD DPD, respectively) (EM) to DPD in urine from normal adults, children, and patients with osteoporosis and/or fractures were approximately 3.5 (E91-E93), suggesting that bone is the major source. How-
6
Y
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
*
SWR
CLINICAL CHEMISTRY
ever, higher PYD/DPD ratios were found in other studies (E94,E95),and stron er correlations were found between the fasting urinary P f D (r = 0.77) and DPD (r = 0.80) to creatinine ratios with histomorphometricallydetermined bone formation rates than with resorption rates (E90).Several theoretical advanta es of pyridinolines over hydroxyproline have been identifiecf(E96). Unlike hydroxyproline, which is released by catabolism of newly synthesized collagen which has not been incorporated into bone, pyridinolines are present only in mature collagen and should therefore reflect only bone resorption. There is no evidence of additional metabolism of the pyridinolines released from bone or of a dietary contribution (E96).However, the effect of changes in renal function remains to be established. Quantificationof Pyridinolines. Both PYD and DPD have been quantified in acid hydrolysates of urine using reversed-phase HPLC with fluorometric detection after a preliminary extraction step using cellulose CF1 to remove fluorescent non-pyridinium components (E91,E97). Analytical problems with the technique have been described and include the absence of reference standard preparations and of a suitable internal standard and difficult in controlling for loss of pyridinolines, both free and peptiie bound, in the acid-hydrolysis ste (E96).A pyridinoline internal standard is now commercia& available (Metra Biosystems Inc., Palo Alto, CA) with the recommendation that it should be added to the hydrolyzed urine and could therefore be used to compensate for losses during the chromatomaphv steps. Reported recoveries of c a l i b r a h from hydro1Ged;r’me were 94% and 61% for PYD and 97% and 49% for DYP (E91. E97)and 90% and 915% respectively,for PYD and DPD added to unhydrolyzed urine (E94).It is not clear from descriptions that these recoveries include the hydrolysis step. An immunoassay for urinary pyridinolines which detects about 80% of total urinar PYD and DPD and shows good correlation with results ogtained by HPLC is commercially available (Metra Bios stems Inc., Palo Alta, CA). Full publications describing cgnical applications of this technique have not yet appeared, but it is likely that elimination of the hydrolysis step will allow greater comparability between laboratories. Pyridinoline Results. The analytical problems undoubtedly contribute to the differences in reference ranges reported by different groups; in 24-h urine collections from premenopausal women mean (ASD) results [nmoVmmol of creatinine (Cr)] for PYD/Cr were 37.7(*15.3) (E91)and 15.6(*10.3) (E94),and for DPD/Cr were 13.2(*8.2) (E91) and 2.3(&2.3) (E94).Despite the numerical differences there is general agreement that urinary pyridinoline excretion is increased in conditions associated with increased bone turnover, with highest results observed in samples from E91, children and patients with Paget’s disease of bone (E82, E94,E97). There is some conflict in reported chan es with age in adults. No si ificant differences were founfin 24-h urinary PYD/Cr or &?D/Cr (nmol/mmol of creatinine) between groups of males and females aged 20-70 years, but results in women aged 20-29 years were significantly higher than in women aged 30-70 years (E91).Other groups have reported significantly increased urinary pyridinoline/creatinine ratios in samples from healthy postmenopausal compared with premenopausal women (E93-E95). There is significant diurnal variation in pyridinoline excretion with increased excretion in the early morning by both premenopausal (E981 and postmenopausal women, but the increase is greater in postmenopausal women with osteoporosis than in postmenopausal women without osteoporosis (E27).The occurrence of a diurnal variation would normally be an argument in favor of 24-h urinary collections rather than spot urine samples, but results obtained on both 24-h (E82)and fasting early morning (E95)urine samples were significantly higher in women with postmenopausal osteoporosis than in premenopausal women and a similar reduction in response to hormone re lacement therapy was observed using both t es of samples (&5), suggesting that either sample may?e used with appropriate reference ranges. In a longitudinal study of healthy women it was found that urinary pyridinoline excretion was relative1 constant in the years before menopause, increased arouni6 months after menopause coincident with a significant drop in serum estradiol concentration, and decreased to almost the premenopausal range following 3 304R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
months of hormone replacement therap (E93).The value of the assays in osteoporosis is limitedrby the si ificant overlap of results with those obtained in unaffeded in%iduals (E82,E93) and indicated by diagnostic accuracies of 72 % and 77% for DPD and PYD, respectively, although this represents an improvement over the 52 % accuracy of urinary hydroxyproline (E82). Other Markers of Bone Turnover. The potential of several other bone matrix components including matrix Gla protein, osteopontin, osteonectin, and Q-HSglycoprotein as markers of bone turnover has been briefly discussed in other E19,E50,E51),but they have not yet been widely reviews (E9, applied to the investigation of osteoporosis. Hydoxyllysine glycosides which are released on metabolism of collagen are potential markers of bone resorption (E19,E50). The diagnostic accuracy of galactosylhydroxyllysine for postmenopausal osteoporosis was 81%, which is better than for urinary pyridinolines (E82).A significant rise of urinary glycosaminoglycan/creatinineratios in women after menopause suggested a possible relationship to postmenopausal osteoporosis which requires further inveeti ation (E99). Conclusions. Assessment of biochemicafmarkers of bone turnover in osteoporosisis beset bymanydiffidties. Primary problems are the heterogeneous nature of the condition and difficulty in obtainin a definitive diagnosis. Ideally, the underlying bone cond!tion should be defined by histomorphometry as well as by bone density measurements to allow correct association of biomarkers with events at the cellular level and overall bone loss. Investigation of the value of biochemical measurements, alone or in combination, in the prediction of fracture risk will require long-term patient follow-up. Significant methodological problems remain in the quantification of bone variants of acid and alkaline phosphatase and of the more recently identified markers, osteocalcin and the pyridinolines. LITERATURE CITED (El) Bouillon, R.; Burckhardt, P.; Chrlstlansen,C.; et ai. Am. J. Med. 1991, 90, 107- 110. (E2) Riggs, B. L.; Melton, L. J. N. En@. J. A M . 1988, 314, 1876-1888. (E3) Johnston, C. C., Jr. Roc. Soc. €xpti. W .A M . 1989, 101, 258-280. (E4) ReisZ, L. G. N. €I@. J. Mw. 1988, 318,618-828. W.Med. 1989, 191,272-274. (E5) Recker, R. R. Roc. Soc. -ti. (E6) R W , B. L.; MeltOn, L. J., 111. N. E@ J. M.1992, 327, 820-827. (E7) Mazess, R. B.; Barden. H. S. Roc. Soc. ExpH. M. Mw. 1989, 101, 281-271. (E8) Eriksen, E. F.; Stelnlche, T.; Mosekllde, L.; Melaen, F. E x k & o / . Metab. C h . North Am. 1989, 18, 919-955. (E9) Aula, M. CekK Tissue Int. 1989, 45,7-11. (E10) U.S. Department of Health and Human Services CJstuopmsk,Resen&, EducaMonendhtlhPromobbn:NIHPubllcatbn91-3216 National InstlMe of Arthritis and Musculoskeletal and Skin Dleeaaes: Baltimore, Md, 1991. (El 1) Canalis. E.; McCarthy, T. L.; Centrella, M. Endocnmd. Metab. Cln. Norlh Am. 1989, 18,903-918. (E12) Pfellschlfter, J.; Bonewald, L.; QNndy, 0. R. Mendb. Exp. hwmarol. 1990, 05, 371-400. (E13) Martin,T. J.; Ng. K. W.; Suda, T. Endocrlnol. Metab. Clln. NorthAm. 1989, 18,833-858. (€14) Cornpaton, J. E. Cffn. En&ctfno/. 1990, 33. 853-882. (E15) Mksen, E. F.; W a r d , D. G.; Berg, N. J.; &aham, M. L.; Mann, K. G.; Speisberg, T. C.; Riggs, B. L. Science 1988, 241,84-88. (E 18) Komm, B. S.; Terpenlng. C. M.; Benz, D. J.; Qraem,K. A,; Wlegos, A.; Korc, M.; aeene, G. L.; O’Malley, 8. W.; Haussler, M. R. Sclence 1988,24 1, 81-84. (E17) Scheven, 8. A. A.; Damn, C. A.; Hamilton, N. J.; Verhear, H. J. J.; Duursma, S. A. Blochem. Bkphys. Res. Commun. 1992, 186, 54-80. (E18) Nordln, B. E. C. CekIf.Tissue Int. 1887, 40,57-58. (El9) Delmas, P. D. E M . Metab. C//n.North Am. 1990, IO, 1-18. (E20) EasteH, R.; Riggs. 8. L. Endoc&ol. Metab. Clh. Nwth Am. 1987, 16, 829-842. (E21) AnderSOtl, J. J. 8. J. N&. Bkchem. 1991, 2,300-307. (E22) Nordin, B. E. C.; Pdley, K. J.; Need, A. G.; Morris, H. A.; Marshall, D. Am. J. C h . Nub: 1987, 45, 1295-1304. (E23) Nordin, 8. E. C.; Need, A. G.; Hartley, T. F.; Phllcox, J. C.; Wilcox, M.; Thomas, D. W. Clin. Ctmm. 1989, 35, 14-17. (E24) Sokdl, L. J.; Daweon-Hughes, 8. Cabif. Tissue Int. 1989, 44. 161-185. (E25) Calvo, M. S.; Eastell, R.; Offord, K. P.; Bergstrah, E. J.; Bwrttt, M. F. J. C h . €17&YIno/. Metab. 1991, 72,69-76. (E26) Nordln, E. E. C.; W e , H. A. Nub: Rev. 1989, 47,85-72. (€27) Eastel, R.; Calvo, M. S.; Bwritt. M. F.; Offord, K. P.; Graham, R.; Russell, 0.;Riggs. L. B. J. C h . E-. Meteb. 1992, 74, 487-494. (E28) Brown, R. C.; Aston, P. J.; Weeks, I.; Woodhead, J. S. J. Clln. €xk&o/. Metab. 1987. 65, 407-414. (E29) Endres, D. 8.;Vllianeuva. R.; Sharp, C. F.; Singer. F. R. Clln. Chem. 1991, 37,162-168. (E30) Ebellng, P. R.; Sandgren, M. E.; DlMagno, E. P.; Lane, A. W.; DeLuca, H. F.; Rlggs, B. L. J. Clln. En&ctfno/. Metab. 1892, 75,178-182.
CLINICAL CHEMISTRY (E31) Hams, H.-M.; Kaptalna, U.; KiJpmann, W.-R.; Brabant, G.; Hesch,R . D . J. Clln. Endocrlnol. Metab. 1989, 60, 843-851. (E321 Sliverberg, S. J.; Shane, E.; de la Cruz, L.; Segre, G. V.; Clemens, T. L.; Bllezklan, J. P. N. Engl. J. M.1989, 320, 277-281. (E33) Cosman, V.; Shen, B.; Herrlngton, E.; Lindsay, R. J. Clln. Endocrnd. Metab. 1991, 73, 1345-1351. (E34) Tsal, K.S.; Ebellng, P. R.; Rlggs, B. L. J. Clln. Endocrnol. Metab. 1989, 60, 1024-1027. (E3!) Jobom, C.; Ljunghall, S.; Larsson, K.; Llndh, E.; Naedn, T.; Wide, L.; Akwstrh, G.; Rastad, J. Clln. Endocrnol. 1991, 34, 335-339. (E36) Stock, J. L.; Coderre, J. A.; Posllllco, J. T. Clln. Chem. 1989, 35, 18-22. (E37) Dauca, H. F. Metabdlsm 1990, 30,3-9. (E38) Nordin, B. E. C.; Morrls, H. A. J. &/I. Bhhem. 1992, 40, 19-25. (E39) Cestellanl, W. J. Anal. Chem. 1991, 63, 202R-206R. (E40)HolM, M. F. J. N&. 1990, 120, 1464-1469. (E41) Eastell, R.; Yergey, A. L.; Vlelra, N. E.; cedel, S. L.; Kumar, R.; Rlggs, La B. J. Bone MIW. Res. 1991, 6, 125-132. (E42) Falch, J. A.; Onebro, H.; Haw, E. J. Clln. Endocrnol. Metab. 1987, 64, 036-841. ( E a ) Orwoll, E. S.; Meler, D. E. J. Clln. €ndocrnol. Meteb. 1988, 63, 12621269. (EM)Villareal, D. T.; Clvltelll, R.; Chines, A.; Avloli, L. A. J. Clln. Endocrnol. Metab. 199, 72, 826-834. (E451Khaw, K.-T.; Sneyd, M.J.; Compston, J. Br. M.J. 1992,305,273-277. (E46) Chapuy, M. C.; Arlot, M. E.; Duboeuf, F.; Bruns, J.; Crouzet, 8.; Arnaud, 8.; Delmas, P. D.; Meunier, P. J. N. Engl. J. M.1992, 327, 1837-1642. (E47) McDermott, M. T.; Kldd, 0. S. Endocr. Rev. 1987, 8, 377-390. (E48) Hesch,R.D.; Busch, U.; Rokop, M.; Delllng, G.; Rlttlngheus, E.-F. CaMf. Tlssue Inf. 1989, 44, 176-180. (E49) Hesch, R. D.; Brabent, G.; Rittlnghaus, E. F.; Atklnson, M. J.; Harms, H. CaM. Tlssue Int. 1988, 42, 341-344. (E501T o h d , J. F.; Selbel, M. J.; Sliverberg, S. J.; Robins, S. P.; Bllezlklan. J. P. Z. Rheumatd. 1991, 50, 133-141. (EN) Epsteln, S. Endocr. Rev. 1988, 0, 437-449. (E52) SPlrensen, S. Clln. Chem. 1988, 34, 1638-1640. (E53) Stelnberg, K. K.; Rogers, T. N. Ann. Clln. Lab. Scl. 1987, 17, 241-250. (E54) Dude, R. J.; OBrlen, J. F.; Katzmann, J. A.; Peterson, J. M.; Mann, K. G.; Rlg(y, 8. L. J. Clln. E ~ O l Metab. . 1988, 68, 951-957. (E55) Eastell, R.; Delmas, P. D.; Hodgson, S. F.; Erlksen, E. F.; Mann, K. 0.; Rlggs, E. L. J. Clln. EMo&nd. Metab. 1988, 67, 741-748. (EM) Steph, J. J.; Pospkhal, J.; Schrelber, V.; Kanka, J.; Mensk, J.: Presl, J.; Pacovsky, V. CaWf. Tlssue Int. 1989, 45, 273-280. (E57) Robey, P. G. Enikwhol. Metab. Clln. b M h Am. 1989, 18, 859-902. (E58)Uen, J. B.; Qundberg, C. M. Clh. orthop.Rela?.Res. 1968,226,267-291. (E59) Johansen, J. S.; Rlis, B. J.; Delmas, P. D.; Christlansen, C. Ew. J. Clln. Invest. 1988, 18, 191-195. (EBO) Resch, H.; M c h m a n n , P.; Woloszczuk, W.; Krexner, E.; Bemecker, P.; Wllivoneeder, R. Ew. J. Clln. Invest. 1992, 22, 542-545. (E611 Pun, K. K.; Leu, P.; Wong, F. H. W.; Cheng, C. L.; Pun. W. K.; Chow, S. P.; Leong, J. C. Y. Bone 1990, 71, 397-400. (E82)Dandona, P.; Coumar, A.; Gill, D. S.; Bell, J.; Thomas, M. Clln. Endocrnol. 1988, 20, 437-441. (E631 Taylor, A. K.; Llnkhart, S.; Mohan, S.; Christenson, R. A.; Singer, F. R.; BayUnk, D. J. J. Clln. Endocrnd. Metab. 1990, 70, 467-472. (E64)Johansen,J. S.; Thomsen, K.; Christlansen,C. Sad.J. Clln. Lab, Invest. 1987, 47, 345-350. (EM) Ebellng, P. R.; Butler, P. C.; Eastell, R.; R i a , R. A.; R i m , B. L. J. Clln. EMo&nol. Metab. 1991, 73. 368-372. (EM) Nlelsen, H. K.; Brlxen, K.; Mosekllde, L. Celcn. Tissue Inf. 1990. 47, 284-290.
(E67) Nlelsen, H. K.; Brlxen, K.; Bouillon, R.; Mosekllde, L. J. Uh. €ndocrtnd. Meteb. 1990, 70, 1431-1437. (E88) Bouillon. R.; Vanderschueren, D.; Van Herck,E.; Nlelsen, H. K.; Bex, M.; Heyns, W.; Van Baelen, H. Clln. Chem. 1992, 38, 2055-2060. (E89) Pastoweau, P.; Delmas, P. D. ckcn. them. 1990, 36,1620-1824. (E70) Taylor, A. K.; Linkhart, S. G.; Mohan, S.; Bayllnk, D. J. M e t a M m 1888, 37, 872-877. (E71) Deftos,L. J.; Wolfart, R. L.; Hill, C. S.; Burton, D. W. C h . Chem. 1992, 38, 2318-2321. (E72) Jaouharl, J.; Schlele, F.; Dragaccl, S.; Taralb, P.; Slest, J.P.; Henny, J.; Slest, G. Clln. Chem. 1992, 38, 1968-1974. (E73) Power, M. J.; Fottrell, P. F. Clln. Chem. 1989, 35, 2087-2092. (E74) Koyama, N.; Ohera, K.; Yokota, H.; Kurome, T.; Katayama, M.; Hlno, F.; Keto, I.; Akal, T. J. Immunol. Methods 1991, 130, 17-23. (E79 Menon, R. K.; Gill, D. S.; Thomas, M.; Kernoff, P. B. A.; Dandona, P. J. Cllt?.Et&&&. Metab. 1987, 64, 59-61. (E78) Johansen, J. S.; Delmas, P. D.; Rlls, B. J.; Glneyts, E.; Chrlstlansen, C. Bone 1991, 12, 257-260. (E77) P a m , A. M.; Simon, L. S.; Vlllaneuva, A. R.; Krane, S. M. J. Bone Mhw. Res. . .- - 1007. .... 2. 427-430. (E78) Simon, L.S.; Sbvk,D. M.; Neer, R. M.; Krane. S. M. J. Bonekllner. Res. 1988. 3 241-246. (E79) k l k k o , J.; Nleml, S.; Rlstell, L.; Ristell, J. Clln. Chem. 1990, 36, 13281332. (E80) Hasseger, C.; Jensen, L. T.; Johansen, J. S.; Rils, B. J.; Melkko, J.; Pmlenphant, J.; Rlstell, L.; Chrlstlansen, C.; Ristell, J. Metabollpm 1991, 40, 205-208. (E81) Hasllng, C.; Erlksen. E. F.; Melkko, J.; Rlstell, L.; Charles, P.; Moseklide, L.; Ristell, J. J. Bone Mhw. Res. 1991, 6 1295-1300. (E82) Benlca, P.; m o , L.; Robins, S. P.; Taybr, A. K.; Talbot, J.; Singer, F. R.; Bayllnk, D. J. Clln. Chem. 1992, 38, 2313-2318. (E83) Whkaker, K. B.; Cox, T. M.; Moss, D. M. Clln. Chem. 1989, 35, 86-89. (E84) Wergedal, J. E.; Leu, K.-H. W. Clln. Bbchem. 1992, 25, 47-53. (E851 Lau, K.-H., W.; Onlshl, T.; Wergedal, J. E.; Singer, F. R.; Bayllnk, D. J. Clln. Chem. 1987, 33,458-462. (E86) Kraenzlln, M. E.; Leu, K.-H., W.; Llang, L.; Freeman, T. K.; Singer, F. R.; Stepn, J.; Baylink, D.J. J. Clln. EMo&nol. Metab. 1990, 71, 442-451. (E87) de la Pledra, C.; Tones, R.; Rapado, A.; Curlel, M. D.; Castro, N. CaMf. Tlssue Int. 1989, 45, 58-60. (E88) Eyre, D. R.; Par, M,; Gallop, P. M. Annu. Rev. Bbchem. 1984,53, 717748. (E89)Eyre. D. R.; Dlckson, I . R.; Van Ness, K. Bkchem. J. 1988,252,495-500. (Ego) Delmas, P. D.; ScMemmer, A.; Glneyts, E.; Rlls, B.; Chrlstlansen, C. J. Bone Mlner. Res. 1991, 6, 639-644. (€91) Beadsworth, L. J.; Eyre, D. R.; Dlckson, I . R. J. Bone Mlner. Res. 1990, 5, 671-676. (E92) McLaren, A. M.; Hordon, L. D.;Bid, H. A.; Roblns, S. P. Ann. Rheum. DlS. 1992, 57, 648-851. (E93) Hassager, C.; Colwell, A.; Asslrl, A. M. A,; Eastell, R.; Russell, R. G. G.; Chrlstlansen, C. Clln. Endocrnol. 1992, 37, 45-50. (E94) Ubelhart, D.; Glneyts, E; Chapuy, M.4.; Delmas, P. D. Bone Mner. 1990, 8, 87-96. (EN) Ubelhart, D.; Schlemmer, A.; Johansen, J. S.; Gineyts, E.; Chrlstlansen, C.; DelmaS, P. D. J. Clh. E m . Metab. 1991, 72, 367-373. (E96) Eyre. D. J. C/h. €docdnol. Meteb. 1992, 74, 470A-4708. (E97) Black, D.; Duncan, A.; Robins, S. P. Anal. Bbchem. 1988, 789, 197-203. (E98) Schlemmer, A.; Hassager, C.; Jensen. S. B.; Chrlstlansen, C. J. CIVn. E n d ~ t ~ h Metab. d. 1992. 74, 476-480. (E99) Larking, P. W.; McDonald, 8. W.; Taylor, M. L.; Klrkland, A. D. Bbchem. M.Metabol. BO I .I t987,37,246-254.
.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, lQQ3 396R
