Biochemical Markers to Survey Bone Turnover

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BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER Henning W. Woitge, MD, and Markus J. Seibel, MD

Bone is a metabolically active tissue, and recent research advances have enabled us to detect disturbances of bone turnover by the quantification of specific bone-derived molecules in serum or urine. The central components of tissue homeostasis consist of three distinct types of bone cells: the bone-forming osteoblasts, the matrix-embedded osteocytes, and bone-resorbing osteoclasts. Important functions of these cells include the production of a variety of collagenous and noncollagenous bone matrix proteins and proteolytic enzymes. Some of these cell-derived components may be quantified in body fluids and may serve as more or less reliable estimates of the actual rate of bone turnover. Approximately 90% of the organic fraction of bone mass consists of collagen. The main component in bone is type I collagen, but a number of other collagens have also been identified, including type 111 and type V collagen. The noncollagenous matrix proteins present in bone are macromolecules such as osteocalcin (OC), osteopontin, and bone sialoprotein (BSP). The exact functions of the these proteins are unknown, but they likely play roles in the supramolecular organization of the bone matrix, in the adhesion of bone-resorbing cells to the matrix, and during the mineralization and degradation of bone tissue. Biochemical markers of bone turnover may be classified according to: Origin (e.g., products of bone cells, molecules derived from the organic or anorganic bone matrix) Biochemical properties (e.g., enzymatic markers of bone cells, pre-

From the Department of Medicine, Endocrinology and Metabolism, University of Heidelberg, Heidelberg, Germany ~~

RHEUMATIC DISEASE CLINICS OF NORTH AMERICA

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VOLUME 27 * NUMBER 1 FEBRUARY 2001

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cursors or degradation products of the collagenous or noncollagenous bone matrix) Function (e.g., markers of bone formation or bone resorption, adhesion molecules). Physiologically, bone formation and bone resorption are coupled to keep a steady state of overall bone turnover. Disturbances in bone turnover may be detected by an increase or decrease in the concentration of markers of bone formation or bone resorption. Thus, from a clinical point of view, the most practicable classification of bone turnover markers, although not exclusive, remains the grouping according to parameters primarily reflecting bone formation or bone resorption processes. As a result of these considerations, we here follow the more functional classification to discuss some of the biochemical features and technical aspects of the most widely used biochemical markers of bone metabolism. BIOCHEMICAL MARKERS OF BONE FORMATION Biochemical markers of bone formation are presented in Table 1. Total and Bone-Specific Alkaline Phosphatase Alkaline phosphatase is a ubiquitously distributed enzyme produced by a variety of cells from different tissues. Over 95% of the total serum activity of alkaline phosphatase (TAP) is derived from liver cells and osteoblasts. As a result, the quantification of TAP as a biochemical index is routinely used in the diagnosis and follow-up of liver and metabolic bone disease. In subjects with normal liver function, serum TAP has been shown to be a useful index of bone formation. In the presence of liver disease, however, the diagnostic usefulness of this parameter as a marker of osteoblast activity is greatly impaired because of a significant contribution of the liver-derived isoenzyme to the TAP serum pool. Consequently, a number of studies indicate that quantification of the bone-specific isoenzyme of alkaline phosphatase in serum may provide a better index of bone formation.12,46, 169, 173 The liver and bone isoenzymes of alkaline phosphatase are derived from the same gene locus and differ only with respect to their posttrans77,113,171 In years past, a variety of lational glycosylation and sial~lation.~~, techniques were developed to specifically measure the bone-derived isoenzyme of alkaline phosphatase, including heat ina~tivation,"~ wheatgerm lectin precipitation," 143 and immune electrophoresis.168None of these methods seems to provide the sensitivity, specificity, cost-effectiveness, and reliability required for routine clinical application, h0wever.4~ More recently, immunoassays using specific antibodies against human bone-specific alkaline phosphatase have been developed; so far, clinical

Immunoassay Immunoassay

Bone, soft tissue, skin Bone, soft tissue, skin

Serum

Serum

Carboxyterminal propeptide of type I procollagen Aminoterminal propeptide of type I procollagen

Bone, platelets

Serum

Osteocalcin

Immunoassay

Colorimetry, electrophoresis, precipitation, immunoassay

Serum

Analytic Method

Colorimetry

Bone-specific alkaline phosphatase

Origin

Bone, liver, intestine, kidney, placenta Bone

Serum

Specimen

Total alkaline phosphatase

Marker

Table 1. BIOCHEMICAL MARKERS OF BONE FORMATION Specificity

Ratio of 1:l between liver and bone isoenzyme in healthy adults Specific product of osteoblast; some assays show up to 20% crossreactivity with liver isoenzyme Specific product of osteoblasts; many immunoreactive forms in the circulation Specific product of proliferating osteoblasts and fibroblasts Specific product of proliferating osteoblasts and fibroblasts

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data seem to point toward an improved diagnostic validity of these new assays with regard to bone diseases and their therapeutic monitoring.12, 59, 65,173

Osteocalcin

OC, or bone GLA-protein, is one of the major components of the noncollagenous bone matrix. It is a 5-kd, hydroxyapatite-binding, osteoblast-derived protein and is secreted into the extracellular bone matrix during mineralization. Because of specific biochemical features (e.g., three vitamin K-dependent y-carboxyglutamic acid residues that are responsible for the calcium-binding properties of the molecule), OC is thought to play a role in the organization of the extracellular matrix.57* 79, 80, 99 Because the protein is expressed mainly during the phase of osteoid mineralization, a specific function during this process has also been suggested. Its precise role in bone metabolism has yet to be determined, however.16 Nevertheless, the quantification of OC in serum is considered to be a sensitive measure of osteoblast f~nction.'~ The largest part of the newly synthesized protein is incorporated into the extracellular matrix, accounting for approximately 15% of the noncollagenous protein fraction. A smaller fraction of OC is released into the circulation and may be quantified by various immunoassays. Although serum levels of immunoreactive OC have been shown to correlate with the bone formation 36 the protein is relatively unstarate as assessed by hist~morphometry,'~, ble and is rapidly metabolized on release into the circulation. The utility of the large number of existing immunoassays for its quantification in human serum is hampered by this instability and the modifications that the protein undergoes after release from the bone matrix with its impact on antibody recognition sites.I6 Type I Collagen Propeptides In bone, type I collagen is synthesized by osteoblasts and secreted as single-stranded propeptides into the matrix. These precursor molecules are characterized by a short signal sequence and terminal extension peptides.lo5After cleavage of the latter, the molecules form the characteristic triple helices of mature collagen. Serum immunoassays using polyclonal antibodies have been developed to detect the intact aminoterminal and carboxyterminal fragments of the procollagen molecules that are lZ3, 163 released into the circulation before extracellular fibril formation.104, Because carboxyterminal and aminoterminal fragments are generated from newly synthesized collagen in a stoichiometric fashion, the propeptides are considered to be quantitative measures of collagen type I production. Although they are theoretically specific and sensitive indices of bone formation, the clinical relevance of the quantification of type I

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collagen propeptides in the evaluation of metabolic bone diseases is still viewed with scepticism.ls.40, Io6 BIOCHEMICAL MARKERS OF BONE RESORPTION

Biochemical markers of bone resorption are presented in Table 2. Bone Sialoprotein

BSP is a phosphorylated 70- to 80-kd glycoprotein that accounts for 5% to 10% of the noncollagenous bone matrix?” 81 The protein is a major synthetic product of active osteo- and odontoblasts and contains an ArgGly-Asp sequence.19,54, 159 This sequence is recognized by various integrin receptors, including the a,p3 vitronectin receptor (CD51/CD61),’ls, 145 which is expressed by o s t e o ~ l a s t s It . ~ has ~ ~ also been shown that BSP improves the attachment of osteoblasts and osteoclasts to plastic surfaces,118,145 binds preferentially to the a2 chain of collagen,” nucleates hydroxyapatite crystal formation in vitr018~and seems to enhance osteoclast-mediated bone re~orpti0n.l~~ In the course of normal bone remodeling, BSP is likely to be involved in the supramolecular organization of the extracellular bone matrix and in the adhesion of bone-resorbing cells to the matrix.82 Although preferentially detected in the cells of mineralized tissues19, 47,159 BSP has been found to be ectopically expressed in the cytoplasm and on the surface of myeloma cells and tumor cells of other malignan~ i e sIn . ~studies of breast cancer patients, BSP was found to be associated with the appearance of bone metastases, suggesting that BSP may be involved in the molecular mechanisms responsible for cancer cell osteotropism.4,37 Recently, pol yclonal immunoassays have been developed to quantify immunoreactive BSP in serum.87,149 Recent studies suggest that serum levels of BSP predominantly reflect processes related to bone resorption.157,174 Children and adolescents exhibit greatly elevated BSP values, particularly during the pubertal growth Elevated BSP levels were also found in patients with primary hyperparathyroidism, Paget’s disease of bone, metastatic bone disease, or multiple myeloma.157, 172, 17* After treatment with bisphosphonates, a rapid reduction of serum BSP levels was found, similar to the reduction of the urinary hydroxypyridinium cross-links e~creti0n.l~~ Tartrate-Resistant Acid Phosphatase

Similar to the alkaline phosphatases, acid phosphatases are a family of ubiquitously occurring enzymes. Until now, five different human isoforms have been described. The tissues and cells expressing these

Collagens and collagenous proteins; galactosylhydroxylysine in high proportion in skeletal collagens Synthesized by osteoblasts and laid down in extracellular matrix, enhances osteoclastmediated bone resorption; serum levels reflect osteoclast activity

Colorimetry HPLC HPLC

Immunoassay

Type I collagen-containing tissues Bone Cartilage Soft tissue Skin Bone Soft tissue Skin Serum complement Bone Dentin

Serum Urine

Urine

HPLC = high pressure liquid chromatography

Serum

Urine

Immunoassay

Type I collagen-containing tissues

Serum (P only) Urine ( a / P )

Hydroxylysine glycosides (glycosyl-galactosylhydroxylysine and galactosyl-hydroxylysine) Bone sialoprotein

All fibrillar collagens and partly collagenous proteins, including Clq and elastin; present in newly synthesized and mature collagen

Immunoassa y

Bone Skin

Serum Immunoassa y

Collagen type I

Collagen type I

Collagens, with highest concentrations in bone; absent from cartilage or skin; present only in mature collagen Collagen type I; may be derived from newly synthesized collagen

Carboxyterminal crosslinked telopeptide of type I collagen Carboxyterminal crosslinked telopeptide of type I collagen (a-CTX, P-CTX) Aminoterminal cross-linked telopeptide of type I collagen Hydroxyproline, total and dialyzable

HPLC Immunoassay

Urine

Osteoclast and platelets; isoform 5b specific for osteoclast activity Collagens, with highest concentrations in cartilage and bone; absent from skin; present only in mature collagen

Colorimetry Immunoassay HPLC Immunoassa y

Deoxypyridinoline

Specificity

Analytic Method

Bone Blood cells Bone Cartilage Tendon Blood vessels Bone Dentin

Origin

Plasma Serum Urine

Specimen

Tartrate-resistant acid phosphatase Pyridinoline

Marker

Table 2. BIOCHEMICAL MARKERS OF BONE RESORPTION

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isoforms include the prostate, bone, spleen, platelets, erythrocytes, and macrophages. Most acid phosphatases are inhibited by + -tartrate. The only exception is the isoform 5, which has been called "tartrate-resistant acid phosphatase" (TRAP). Two subforms of this isoenzyme have been described, namely, band 5a (containing sialic acid) and band 5b (sialic Io7 acid-free). The 5b subform is synthesized and secreted by osteo~lasts~~, and can now be detected exclusively by immunoassays.21,72, 90 Until recently, the available colorimetric assays for the measurement of TRAP in serum or plasma detected both 5a and 5b isoforms. The new immunoassays may thus improve the ability to assess osteoclast activity.21,72, 90 At room temperature, TRAP loses more than 20% of its activity per hour. As a result, with the use of kinetic assays, care should be taken to stabilize the enzyme after phlebotomy (i.e., by adding citrate or sodium sulfate to the sample).', 94, 96 This seems to be unnecessary when measuring TRAP 5b serum concentrations by one of the newer immunoassays (J. M. Halleen, MD, personal communication, 1999).

Hydroxyproline and Hydroxylysine Glycosides

Hydroxyproline (OHP) and hydroxylysine are formed intracellularly during the posttranslational phase of collagen synthesis. OHP constitutes 12% to 14% of the total amino acid content of mature collagen. Approximately 90% of OHP is liberated during the degradation of bone collagen and is primarily metabolized in the liver.'O' Subsequently, OHP is excreted in the urine, where its free or peptide-bound forms may be quantified by colorimetric or high-pressure liquid chromatography (HPLC) methods.33,89 Hydroxylysine usually exists in two glycosylated forms: glycosyl-galactosyl-hydroxylysine (GGHL) and galactosyl-hydroxylysine (GHL).30Hydroxylysine is released into the circulation during the course of collagen breakdown and can also be quantified in urine by HPLC after appropriate derivatization.'" Significant amounts of urinary OHP are derived from the degradation of newly synthesized collagens.162Moreover, OHP is relatively unspecific for bone in that it can be found in other tissues such as the skinlZ9and is liberated from the degradation of elastin and Clq.140Because certain foodstuffs, (i.e., meat and other collagen-containing nutrients) contain significant amounts of OHP, measurements of the urinary amino acid have to be performed after a collagen-free diet. As a result of these disadvantages, the quantification of urinary OHP has been largely replaced by more specific techniques. In contrast to OHP, the glycosylated forms of hydroxylysine are not metabolized and are not influenced by dietary 140 Moreover, GGHL is present in skin and Clq, although GHL is more specific for bone. Thus, the GGHL:GHL ratio may allow for the recognition of tissue specificity. Although the hydroxylysines have potential as markers of

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bone resorption? 110, 112, 152 their major disadvantage is presently the absence of a convenient immunoassay format. Hydroxypyridinium Cross-Links and Related Derivatives Pyridinoline (PYD) and deoxypyridinoline (DPD), which are derivatives of 3-hydroxypyridinium, are formed during the extracellular maturation of fibrillar collagens. As multifunctional cross-links, they are considered to bridge several collagen molecules, thereby stabilizing the 116,139 During bone resorption, cross-linked colcollagen superstr~cture.~~, lagens are proteolytically broken down, leading to the release of components containing the cross-link structure. These components are then detectable in serum or urine by means of conventional chromatography or immunoassay.6,35.66,69,128,142.158 Urinary PYD and DPD show a high specificity for skeletal tissues. PYD is derived from cartilage and bone but also from other tissues, including ligaments and blood vessels. In contrast, DPD is present almost exclusively in bone. PYD and DPD are completely absent from 140 and are excreted independently of dietary influence^.^^ skin The serum and urine concentrations of DPD and, to a lesser degree, PYD are thus considered to reflect the degradation of mature skeletal collagens. In fact, experimentally and clinically, a close correlation has been shown between urinary cross-link excretion and bone resorption rates.14 The measurement of urinary or serum PYD or DPD concentrations is regarded as specific for bone resorption. The hydroxypyridinium cross-links were originally quantified by reverse-phase ion-paired HPLC.6,44 The method was later automated,12* and more recently, automated techniques have been described for the quantification of these compounds in serum.85Although cumbersome and labor-intensive, the HPLC method is still used and considered the gold standard for the measurement of total and free pyridinium crosslinks in urine. For the clinical routine, however, less expensive and simpler methods had to be developed. For this purpose, direct immunoassays for the detection of free (nonpeptide-bound) cross-links were developed.66,1 4 ~ , 15* In most clinical situations, these systems seem to produce results similar to those provided by HPLC methods.142, 156 Other approaches to measure mature collagen degradation include the development of assays for the detection of type I collagen telopeptides. The theoretic background is the fact that the cross-linking of collagen involves specific regions of the molecule, namely, the aminoterminal or carboxyterminal telopeptide. A number of assays for the measurement of both telopeptide regions have been introduced and tested extensively in a wide variety of experimental and clinical situations. Those of mention include assays for the carboxyterminal type I collagen telopeptide in serurn,l3*for the “cross-linked” aminoterminal telopeptide of type I collagen in urine (NTX),75and for the various forms of the

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

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carboxyterminal telopeptide of type I collagen in urine (a-CTX, (3CTX).'o,49 Recent developments concern the measurement of these collagen degradation products in serum. The use of urinary parameters either requires a 24-hour collection period or, in the case of untimed samples, correction for urinary creatinine. The addition of a secondary assay, however, increases the overall variability of measurements in urine. This, in addition to the fact that all bone markers exhibit significant biologic ~ariability,",'~~,'~~ has led to the development of serum assays for the C, So far, the serum and N-terminal telopeptides of type I collagen.ll,~ 5 78 assays seem to show similar clinical performance as the urinary assays.174 Whether their use does indeed reduce overall variability still needs to be shown. BIOCHEMICAL MARKERS OF BONE TURNOVER IN METABOLIC BONE DISEASE Primary Osteoporosis Diagnosis

Currently, the diagnosis of osteoporosis is based on the clinical presentation of the patient as well as on radiologic and densitometric criteria.86Further diagnostic workups may include laboratory measures and invasive procedures such as bone biopsy. These techniques are mainly implemented when the underlying causes of the disease, the current metabolic situation (i.e., low vs high bone turnover), and a thorough differential diagnosis are of interest. In this regard, biochemical markers of bone turnover are considered to provide helpful additional information. Because osteoporosis is a heterogeneous disease, it is not surprising that in untreated patients with either overt postmenopausal or agerelated osteoporosis, rates of bone turnover tend to vary over a wide range. Although most cross-sectional studies show accelerated bone turnover in a certain proportion of postmenopausal osteoporotic women, there is usually broad overlap between diseased and healthy populations (Fig. 1).22,39, 60, 93, 'lo, 155 In this context, it is important to bear in mind that research studies usually include highly selective patient populations, which may not always represent the routine clinical setting. Using a population-based data set, and therefore avoiding this selection bias, we have previously reported that none of the major biochemical markers of bone resorption provide sufficient diagnostic information to be useful in the screening for vertebral osteopenia or osteoporosis.156In another population-based study, it was shown that urinary NTX could discriminate between older individuals with normal hip bone mineral density (BMD), osteopenia, and osteoporosis.151So far, the latter data have not been reproduced by other groups, and one can safely state that a single

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1iIO5/ LD Post

UTO

€TO

-

30

DPD

Pre

Post

UTO

Figure 1. Mean urinary concentrations of the hydroxypyridinium crosslinks pyridinoline (PYD) and deoxypyridinoline (DPD) in postmenopausal (Post) healthy women and untreated (UTO) and estrogen-treated (ETO) women with postmenopausal osteoporosis compared with premenopausal (Pre) healthy women. Column inserts denote percentage change of mean value compared with normal premenopausal controls. Bars = standard error of mean (SEM); Asterisks = P values <.01 UTO levels of PYD and DPD were higher than POST ( k . 0 1 ) ; Crea = creatinine. (From Seibel MJ, Cosman F, Shen V, et al: Urinary hydroxypyridinium crosslinks of collagen as markers of bone resorption and estrogen efficacy in postmenopausal osteoporosis. J Bone Miner Res 8:881, 1993; with permission.)

marker of bone resorption is clearly inadequate to establish the diagnosis of osteoporosis or to screen for the presence or likelihood of osteoporotic fractures.156 Prediction of future Bone Loss

Measurement of bone mass is the currently accepted estimate for identifying patients at risk for osteoporosis.86Because bone mass is the net result of previous and ongoing bone remodeling, additional measures of bone turnover may improve the individual risk assessment in a patient’s workup for osteoporosis. Markers of bone turnover are neither designed nor useful to replace bone mineral density measurements in the assessment of prevalent bone mass. Nevertheless, they may be helpful in the prediction of future bone loss. Because the rate of bone turnover seems to be an important determinant of bone mass, bone loss may be assessed indirectly by molecular markers of bone turnover. A number of recently published partly prospective studies suggest that increased bone turnover is indeed associated with accelerated bone loSs.23,24, 29, 38, 61, 108, 109, 131, 132, 144,146 Certain markers of bone resorption, particularly those derived from osteoclastic collagen breakdown, have been shown to predict, at least at lo8,Io9,146 Following a small group certain skeletal sites, future bone of early postmenopausal women, previous investigations demonstrated that the combined measurement of serum TAP, OC, fasting urinary calcium, OHP, or DPD can predict 60% to 70% of the variability in

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

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measured bone 1 0 ~ s24,. 165 ~ These ~ ~ studies also suggested that the correlation between baseline markers of bone turnover and the subsequent rate of postmenopausal bone loss is consistent over a period of at least 12 years?*, 74 Similar but less optimistic results were reported by other groups using different corn bin at ion^.^^, 38 Dresner-Pollak and co-worke r have ~ ~found ~ the free cross-links and aminoterminal telopeptide of type I collagen to be strong predictors of future bone loss in elderly women. In fact, the combination of urinary NTX, serum OC, and serum parathyroid hormone explained 43% of the variability of bone loss at the total hip. Mole et allo9reported that up to 58% of the variation in the rate of bone loss in women soon after menopause could be explained by measuring urinary hydroxypyridinium cross-links in combination with an estradiol glucuronide assay and the body mass index. Recent results of the OFELY study suggest that the measurement of urinary NTX and urinary and serum CTX can predict postmenopausal forearm bone loss over a period of 4 years (Fig. 2).63In a 13-year partly retrospective study of 354 women, Ross and K n ~ w l t o n showed l~~ that a continuous relation exists between the prevalent levels of various bone biomarkserum serum oste~~alcin BAP

serum PiCP

serum PlNP

urinary NTX

urinary CTX

P=.Ol

P=.0005

P=.006

serum CTX

0 -

.0.5

-

--8

-1.0

-

”-

-1.5

-

-2.0

-

-2.5

-

-3.0

-

a,

c

L

k d

-3.5

J

P=.0016

P=.06

P=..O9

P=.OOOl

Figure 2. Midradius bone mineral density change over 4 years in 305 healthy postmenopausal women with low (hatchedbar) and high bone (solid bar) turnover at baseline. High turnover was defined as bone marker levels above the upper limit of the premenopausal range. P values refer to the difference in the rate of bone loss between the two groups of bone turnover. Bars = the mean t SEM; BAP = bone-specific alkaline phosphatase; PlCP = carboxyterminal propeptide of type I collagen; PlNP = aminoterminal propeptide of type I collagen; NTX = crosslinked aminoterminal telopeptide of type I collagen, CTX = crosslinked carboxyterminal telopeptide of type I collagen. (From Garner0 P, SornayRendu E, Duboeuf F, et al: Markers of bone turnover predict postmenopausal forearm bone loss over 4 years: the OFELY study. J Bone Miner Res 14:1614, 1999; with permission.)

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ers and the risk of rapid bone loss at the calcaneus. Thus, the odds of rapid bone loss (>2.2% per year) increased approximately twice for each SD increase in serum bone-specific alkaline phosphatase (BAP), serum OC, urinary free PYD, or DPD.146In 227 early postmenopausal women treated with either calcium alone or hormone replacement therapy (HRT) plus calcium, Chesnut et alZ0and Rosen et showed that women with high baseline rates of bone resorption were at higher risk of losing bone than women with normal turnover rates. The authors calculated that a woman with high baseline values of urinary NTX (>67 U) had a 17.3 times higher risk of bone loss if not treated with HRT.20 Despite these encouraging results, no final consensus has been reached as to whether markers of bone resorption can be used to predict bone loss and identify high-risk patients (i.e.,”fast bone losers”). In fact, several reports with negative results have been published. In a 3-year prospective study by Marcus et al,lo3bone turnover markers such as NTX, CTX, and BAP could not predict BMD changes for individual untreated or HRT-treated postmenopausal women. Also, Keen et alE8 were unable to detect any correlation between rates of bone turnover and changes in lumbar or hip BMD in a 4-year prospective study. Similarly, Cosman et a129reported that markers of bone formation and bone resorption could not accurately predict rates of bone loss at the spine or hip over a period of 3 years. Other groups7,” argue that because of the high degree of variability in urinary markers of bone resorption, predicting either bone density or changes therein for an individual patient from a single marker measurement may not be possible at all. Assessment of Fracture Risk

From a clinical point of view, the assessment of future fracture risk seems to be more relevant than predicting changes in bone mass. Many clinical studies suggest that bone mass is not the only determinant of skeletal fractures. Additional factors such as trabecular connectivity, the number of bone remodeling sites, and various other parameters of skeletal microarchitecture may contribute to the mechanical stability of bone. High bone turnover is thought to be associated with a disruption of the trabecular bone network, thus reflecting an increase in bone fragility.58,lZ0Bone resorption indices may be independent variables for estimating the future fracture risk. In a prospective and population-based sample of the Rotterdam study, including 17 incident hip fractures, urinary total PYD and DPD as well as free DPD were associated with an increased risk for sustaining a hip fracture in women over 75 years of age.167For example, the relative risk (RR) per SD increase in free DPD as determined by HPLC was 3.0. Recently, the same group published follow-up data from a larger nested case-control study performed within a prospective population-based cohort study of 7983 individuals aged 55 years and over (mean followup = 3.8 years, number of fractures = 212). The authors showed that an increased urinary excretion of free DPD predicts subsequent

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

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nonvertebral fractures, especially of the hip (odds ratio [OR] of the medium tertial = 4.9; OR of the upper tertial = 5.5) and upper humerus (OR = 4.8 and OR = 3.3, respectively). Interestingly, fracture prediction was independent of BMD and di~abi1ity.l~~ In another cohort of elderly subjects recruited from the EPIDOS study, similar results were reported for urinary CTX and free DPD in 126 hip fracture cases.62It is noteworthy that the RRs as defined by either BMD or marker measurements were not only similar but that the combined measurement of hip bone density and bone resorption markers predicted future hip fractures better than the determination of either bone density or bone markers alone. In other words, in older postmenopausal women, the RR of fracture seems to be highest in individuals with low bone mass and high rates of bone resorption (Fig. 3). Other reports include a 15-year follow-up study, in which the rate of bone loss as determined by the combination of a bone formation and bone resorption marker (i.e., serum OC and urinary CTX) was found to be equally important for the risk assessment of osteoporotic fractures as low bone mass.137Moreover, a reanalysis of data from several clinical 51

4.8

2.2

High CTX High lree D-Pyr

+ High CTX

High free D-Pvr

Figure 3. Combination of the assessment of BMD and the bone resorption rate to predict hip fracture risk in the elderly. Low BMD was defined according to the WHO guidelines, for example, by a value lower than 2.5 SD below the young adult mean (t score 5 2.5 SD). High bone resorption was defined by CTX or free D-Pyr ( = deoxypyridinoline) values higher than the upper limit (mean 2 SD) of the premenopausal range. Women with low hip BMD and high bone resorption were at higher risk of hip fracture than women with low hip BMD or high bone resorption. (From Garner0 P, Sornay-Rendu E, Chapuy MC, et al: Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Min Res 11:337,1996; with permission.)

+

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WOITGE & SEIBEL

trials suggested that in placebo-treated osteoporotic women, vertebral fracture rates increase as a direct function of either increased bone turnover or decreased vertebral BMD.136Thus, at a given level of vertebral BMD, the rate of vertebral fractures increases with the rate of bone turnover. When bone turnover was normal, however, the main determinant of vertebral fractures was vertebral BMD. Only recently, Lo Cascio et alloohave reported that urinary GHL is also a potential marker of bone fragility in postmenopausal osteoporotic women. So far, however, prospective data on the use of this marker as a potential predictor of fracture risk are not available. Primary Hyperparathyroidism

The diagnosis of primary hyperparathyroidism is based on the presence of chronic hypercalcemia, hypercalciuria, and elevated serum parathyroid hormone levels. A further clinical workup, including the use of imaging techniques such as conventional radiography and bone densitometry is usually applied to document the actual bone mass and to identify potential disease complications. The rationale for measuring biochemical markers of bone turnover in these patients is based on the fact that the knowledge of disturbances in bone turnover may modify potential therapeutic intervention strategies. In patients with asymptomatic primary hyperparathyroidism, conventional markers of bone turnover such as serum TAP or urinary OHP are usually within the normal range. In contrast, it has been shown that the urinary excretion of hydroxypyridinium cross-links is increased even in the state of asymptomatic or mild disease.154These results have been confirmed by a number of studies using other markers of bone resorp174 (Fig. 4). The acceleration, including serum BSP, CTX, and NTX140,157, tion of bone turnover even in mild cases may contribute to a significant bone loss and an increased fracture risk over the years. Thus, the initiation of an antiresorptive agent in these patients may in part be justified by measuring specific markers of bone turnover. Until now, prospective

Figure 4. Urine and serum markers of bone resorption in metabolic and malignant bone disease. Values are expressed as z scores [z = (x - mean)lSD]. The full lines represent the mean and the dotted lines represent ? 2 SD around the mean of healthy controls. ' R . 0 5 , "R.01, *"P<.OOl versus healthy controls. OPO = primary vertebral osteoporosis; PHPT = primary hyperparathyroidism;PD = Paget's disease of bone; MM = multiple myeloma; BC - = breast cancer without bone metastases; BC = breast cancer with bone metastases; U - DPD = urinary total deoxypyridinoline; U - CTX = urinary C-terminal crosslinked telopeptide of type I collagen; U - NTX = urinary N-terminal crosslinked telopeptide of type I collagen; S-BSP = serum bone sialoprotein; S-CTX = serum Cterminal crosslinked telopeptide of type I collagen; S - NTX = serum N-terminal crosslinked telopeptide of type I collagen. (From Woitge HW, Pecherstorfer M, Li Y, et al: Novel serum markers of bone resorption: clinical assessment and comparison with established urinary indices. J Bone Miner Res 14:792,1999;with permission.)

+

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

“1 30

m

U-DPD

*** A

***

U

0

N

0

**

T*

0

10

10

0

0

‘i -f

I

S-BSP

40

20

20

63

U-CTX

30

40 30

201

20 20

10

10

0

0

B

1

S-CTX

4

N

7

U-NTX

***

Q

“3

S-NTX

30

20

10

0

0

I QPQ PHPT PD

MM

BC-

BC+

QPO PHPT PD

Figure 4. See legend on opposite page

MM

BC- BC+

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WOITGE & SEIBEL

data showing improved clinical management of patients with primary hyperparathyroidism by the quantification of these parameters have not been available. Paget’s Disease of Bone

Paget’s disease of bone is a relatively common disorder of bone metabolism, affecting about 2% to 5% of the European population above the age of 50 years.l19The disease is characterized by an acceleration of bone turnover, resulting in qualitatively insufficient bone architecture.I6O, Serum TAP is currently considered to be the standard biochemical parameter for the evaluation of disease activity and therapeutic monitoring in Paget’s disease of bone.34,91, 147 Also, urinary OHP has been widely used as a marker of bone resorption in these patients.34,91*147 The clinical advantage of including the measurement of a resorption marker in the biochemical workup of patients with Paget’s disease has been difficult to demonstrate, however. An index of bone turnover superior to serum TAP should either provide a more reliable way to predict the therapeutic outcome or detect nonresponders or a relapse in disease activity at an earlier point in time. The quantification of markers with higher specificity and sensitivity for skeletal metabolism in Paget’s disease has been tested in a number of recent studies. These include the bone-specific isoenzyme of alkaline as well as the phosphatase and OC as markers of bone formation12,59,173 hydroxypyridinium cross-links and telopeptide-related epitopes of type I 176 and BSP157, 174, 176 as markers of bone resorption (see Fig. 4). In certain clinical situations (i.e., in the presence of other systemic diseases that may influence serum TAP levels), more specific parameters may indeed improve the biochemical assessment of Paget’s disease. With regard to cost-effectivenessand assay performance, none of these studies were able to demonstrate that any of the tested new markers have the potential to replace serum TAP as the marker of choice in most of these patients. BIOCHEMICAL MARKERS OF BONE TURNOVER IN SECONDARY BONE DISEASE

Table 3 summarizes the changes in serum or urine levels of commonly used biochemical markers of bone turnover in secondary bone disease with various underlying causes. Malignant Bone Disease

The development of bone metastases characterizes the spontaneous course of a variety of malignant diseases such as multiple myeloma,

U

=

AP = alkaline phosphatase; OC decreased.

=

or fl

u

or Variable

n

lt

G

-

@

u

0

Variable

0

n n u

u

e or fi

.s or U U, later fl

U

nk nk nk

TI

t)

u

= or 0

U

.s or

U

n

U

n

U

n

.s or

oc

AP

il

nk nk nk

n

n

nk

n

nk

n n

ESP

osteocalcin; BSP = bone sialoprotein; TRAP = tartrate-resistant acid phosphatase; nk

Tumor-induced bone disease Glucocorticoid-induced bone disease Rheumatoid arthritis Alcohol-induced bone disease Hypogonadism Organ transplantation Renal osteopathy Hepatic osteopathy Hyperthyroidism Mastocytosis Chronic anemia

Collagen Propeptides

Bone Formation

Table 3. SPECIFIC BIOCHEMICAL MARKERS OF BONE TURNOVER IN SECONDARY BONE DISEASE

=

not known;

n = elevated;

0

0

n n n u-n n n n or TI n n or n

Cross-Links and Derivaties

t) =

Bone Resoprtion

n n n n

unchanged;

t)

or TI nk nk nk

Variable

n n

TRAP

66

WOITGE & SEIBEL

breast cancer, prostate cancer, bronchial carcinoma, hypernephroma, and thyroid cancer. The manifestation of bone metastases is usually associated with an unfavorable prognosis and often with life-threatening complications (i.e., pathologic fractures, nerval compressions, hypercalcemia of malignan~y).~~ The therapy of advanced tumor osteolysis is difficult. The use of different diagnostic approaches to achieve an early diagnosis of bone metastases seems to be essential for an effective therapeutic intervention. Diagnostic procedures include imaging techniques such as conventional radiography, computed tomography, magnetic resonance imaging, and scintigraphic procedures as well as laboratory analyses (including serologic tumor markers) and, in selected patients, bone biopsies. With regard to the early diagnosis of bone metastases, all these methods are hampered by a relatively low ~ensitivity.~~ The development of a metastatic osteolysis is initiated by an activation of locally present osteoclasts. This process is induced by the action of local or systemic oncogenic mediators. Later in the course of the disease, proliferating tumor cell clusters add to the destruction of osseous s t r u c t ~ r e sIn . ~certain ~ cancers, however, a tumor-induced activation of osteoblasts may lead to increased formation of mineralized bone. The uncoupling of bone formation and resorption leads to local and systemic disturbances of bone turnover. Biochemically, these processes most often resemble the state of a ”high turnover” osteopathy. Primarily osteolytic metastases are characterized by an increase in bone resorption, frequently with suppressed bone formation. In contrast, osteoplastic metastases generally induce bone formation, often with simultaneously accelerated bone resorption. The quantification of specific metabolites of bone cells or the bone matrix may serve as an indicator of these tumorlZ1, lZ2,lffl, 174 induced disturbances in bone turn~ver.~, Breast Cancer In patients with breast or prostate cancer and osteoplastic metastases, the elevation of serum levels of bone formation markers such as TAP and BAP seems to be stage specific.51,lzz Moreover, overt bone metastases in breast cancer patients are associated with significantly elevated urine levels of collagen breakdown products (i.e., hydroxypyridinium cross-links, NTX) as indexes of bone re~orpti0n.l~~ We could also demonstrate that newly developed assays for the measurement of NTX and BSP in serum are highly sensitive in this group of patients (see Fig. 4).174 Nevertheless, urine and serum assays for measuring primarily the p-isomerized form of CTX did not yield elevated marker levels, suggesting a lower sensitivity of these parameters most probably as a result of the status of their racemization/isomerization. Because the degree of p-isomerization correlates with the time elapsed since synthesis of the this isoform may be underrepresented during rapid collagen breakdown (i.e., bone metastases). Measuring the native a-form of CTX

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

67

in these situations could be a more sensitive method, but this has yet to be demonstrated. Interestingly, recent data suggest that BSP may also serve as a prognostic marker for the development of bone metastases in breast cancer patients:, 37 Die1 et a137could demonstrate that in patients with initially normal serum levels for BSP, the probability of bone metastasesfree survival was significantly higher compared with that in patients with elevated serum BSP values (Fig. 5). Multiple Myeloma

Multiple myeloma is a malignant neoplasia that leads to tumorinduced osteolysis in the advanced stages. Consequently, specific markers of bone resorption seem to be the most helpful indices of bone turnover in the biochemical evaluation of the extent of bone disease. It has been shown that the measurement of urinary hydroxypyridinium cross-links is a reliable method to evaluate the degree of bone resorption in patients with untreated multiple myeloma (Fig. 6).12’ Other studies indicate that the detection of collagen degradation products or BSP in serum would further improve the ability to identify patients with increased osteoclast activity (see Fig. 4).43, 174 Interestingly, increased serum BSP levels seem to be associated with an overall unfavorable prognosis in multiple myeloma and may serve as an early marker of malignant transformation in plasma cell dyscrasias initially classified as monoclonal I.o

1

ESP c 24 nglmL

0.8

0.6 0.4 0.2

t

RR = 94.1

0 1 0

12

24

36

40

Months since Surgery Patients at risk ESP<24nglmL

359

239

I34

59

13

BSP>24ng/mL

29

25

14

a

1

Figure 5. Bone metastases-free survival in patients with primary breast cancer according to serum bone sialoprotein (=BSP) values. RR = relative risk. (From Die1 IJ, Solomayer EF, Seibel MJ, et al: Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent bone metastasis. Clin Cancer Res 5:3914,1999; with permission.)

68

WOITGE & SEIBEL

--g

IS0 -

-. 2 loo-

80

125

a2

8

1.

8

...............

0 '

I

I

I

I

I

1 i

0

20

4 J =

10

0

5 25

170.7

1

C I223 C

3

+

U'

...a ..... . . . . . . . . . . .

I

I

I

Healthy Adults

MGUS

MM I+II

_ w n

I

MM 111

Osteoporosis

Figure 6. Urinary excretion of pyridinium crosslinks in healthy adults and patients with monoclonal gammopathy of undetermined significance (MGUS), multiple myeloma (MM) and osteoporosis. Patients with smoldering myeloma (solid circre) are included in the MGUS section. h-PYD = pyridinoline (by HPLC), h-DPD = deoxypyridinoline (by HPLC), i-DPD = deoxypyridinoline(by immunoassay), MM I II = MM patients with stage I (solid circle) and stage II (open circle) disease, MM Ill = MM patients with stage 111 disease, (solid line) = Median and dotted line = upper limit of normal range. (From Pecherstorfer M, Seibel MJ, Woitge HW, et al: Bone resorption in multiple myeloma and in monoclonal gammopathy of undetermined significance: quantification by urinary pyridinium cross-links of collagen. Blood 90:3743, 1997; with permission.)

+

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

69

gammopathies of undetermined significance (H. W. Woitge, unpublished data). Treatment of Osteolytic Lesions in Patients with Metastatic Bone Disease To date, the most effective therapy of patients with metastatic bone disease is the application of bisphosphonates. Bisphosphonates act by decreasing bone resorption and bone formation in which the effect on bone formation occurs somewhat later in the course of the drug’s acti01-t.~~ Several studies have previously shown that the administration of bisphosphonates induces a rapid decline in urinary bone resorption 157 In patients with hypercalcemia of malignancy, the markers.8,26, intravenous application of 30 to 60 mg of pamidronate resulted in a rapid and significant reduction of urinary collagen breakdown products (hydroxypyridinium cross-links, NTX, CTX) and circulating levels of NTX, CTX, and BSP in serum (Fig. 7).174The parallel changes of urine and serum parameters as a result of bisphosphonate treatment are a strong hint for a similar clinical usefulness of these markers in monitoring therapeutic intervention in metastatic bone disease. In summary, the use of biochemical markers of bone turnover in malignant bone disease in combination with other diagnostic procedures may help in (1) the assessment of the presence or extent of tumorinduced bone disease in addition to imaging techniques, (2) the overall prognosis of the metastatic cancer, and (3) the therapeutic follow-up of patients with tumor-induced osteolysis. Glucocorticoid-Induced Bone Disease

The inhibitory effects of glucocorticoids on bone turnover have been known since Cushing’s first description of a higher incidence of bone fractures in patients with glucocorticoid excess.32Cortisone was first applied in the therapy of patients with rheumatoid arthritis in 194tXB3 Since then, glucocorticoids have been used as therapeutic agents in almost all fields of medicine. Curtis et aP1 first described a loss of bone mass associated with the prolonged application of oral glucocorticosteroids. Up to now, the systemic high-dose treatment with glucocorticoids has been regarded as an important risk factor for the development of bone disease.133Bone histomorphometric analyses demonstrated increased bone resorption coinciding with suppressed bone formation in patients treated with g l ~ ~ o ~ o r t i ~ o i d s . ~ ~ ~ The chronic application of glucocorticoids is the most frequent cause of an iatrogenic-induced metabolic bone disease. The annual loss of bone mass associated with glucocorticoids ranges between 5% and 15%, although significant individual differences occur.a, 134, 148 The reasons for these individual discrepancies are largely unknown, although the genetic background of the patients is likely to play a major role.

70

WOITGE & SEIBEL

--t

0-

--Q-

-8

-20 -

U-DPD U-NTX u-CTX

V

0

*

Ul

c -40 -

a

0'

-60 -

-80 0

1

2

3

4

5

6

7

5

6

7

Days

0-

n

8

-20 -

V

Q)

F -40 a 6 -60

-

-80 0

1

2

3

4

Days Figure 7. Percent change of urine (upper pane9 and serum (lower pane!, markers of bone resorption after i.v. treatment with 30-60 mg pamidronate at day 0 in patients with hypercalcemia of malignancy. Results are presented as mean f SEM. *P<.05 versus day 0, **P<.Ol versus day 0, **'P<.OOl versus day 0. U-DPD = urinary DPD, U-NTX = urinary NTX, U-CTX = urinary CTX, S-BSP = serum bone sialoprotein, S-CTX = serum CTX, and S-NTX = serum NTX. (From Woitge HW, Pecherstorfer M, Li Y, et al: Novel serum markers of bone resorption: clinical assessment and comparison with established urinary indices. J Bone Miner Res 14:792, 1999; with permission.)

Glucocorticoid-induced osteopenia resembles a "low turnover" osteoporosis. The underlying molecular and cellular mechanisms are multifactorial and not well defined. Two major pathogenic factors seem to be involved: a direct suppression of osteoblast activity by glucocorticoids

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

71

and an increased bone resorption via a parathyroid hormone-induced stimulation of osteoclasts. An additional mechanism involves the inhibition of intestinal calcium absorption during glucocorticoid treatment. Also, glucocorticoids have been shown to exert calciuric action on the kidney (Fig. 8).70 The routine biochemical measures are rarely altered in the presence of glucocorticoid-induced bone disease. Serum concentrations of calcium, phosphorus, and vitamin D metabolites are usually within normal limits, although serum levels of parathyroid hormone are sometimes mildly elevated.7o,135 The urinary excretion of calcium is often elevated after the onset of glucocorticoid therapy. This finding is most likely a result of the direct calciuric action of glucocorticoids in addition to the increased elimination of serum calcium not incorporated into the bone matrix as a result of decreased osteoblast activity. Months to years after the initiation of steroid therapy, the urinary calcium excretion returns to normal values.71 Specific biochemical markers of bone formation may serve as relatively accurate measures of decreased osteoblast activity. Within days of the application of a single-dose glucocorticoid, a reversible decrease in serum levels of OC and procollagen type I has been described.42, 117

M Parathyroid glands: PTH secretion .T

1

I

Figure 8. Glucocorticoid actions on calcium homeostasis and bone metabolism.

72

WOITGE & SEIBEL

TAP and bone-specific alkaline phosphatase serum levels remain within normal limits6*or show slightly decreased values.130J35 The short-term application of glucocorticoids is often associated with a temporary suppression of markers of bone resorption.28, lo2, In contrast, long-term or high-dose glucocorticoid therapy leads to a n~rmalization~~ or increase in the urinary excretion of parameters of collagen d e g r a d a t i ~ n This . ~ ~ uncoupling of bone formation and bone resorption may, at least in part, be responsible for the progredient loss of bone mass during glucocorticoid treatment. The clinical use of biochemical markers of bone turnover during glucocorticoid therapy includes: Pretherapeutic risk classification of an individual patient before initiation of glucocorticoid treatment Assessment of the respective actual bone turnover during followup (i.e., identification of individual responsiveness to glucocorticoids) Accelerated initiation of therapeutic intervention to avoid longterm glucocorticoid-inducedbone disease (i.e., reduction of glucocorticoid dose or induction of antiresorptive therapy) Rheumatic Diseases

Another large group of patients with secondary bone disease comprises those individuals with inflammatory and immobilizing diseases of rheumatologic disorders. In patients with rheumatoid arthritis, the inflammatory-induced local demineralization of the periarticular bone is often accompanied by generalized osteopenia or osteoporosis. The latter lZ6but is partly a result of immobilization and glucocorticoid effects102, may also be induced by the inhibitory actions of inflammatory mediators on bone formation.76,153 During increased inflammatory activity, bone resorption is often accelerated in patients with rheumatoid arthritis. The urinary excretion of OHP, hydroxylysine glycosides, and hydroxypyridinium cross-links is elevated.92, In contrast, bone formation seems to be either unaffected or slightly decreased as demonstrated by normal or low-normal serum levels of OC and procollagen type I.117,lZ7, 141, 153 SUMMARY

Molecular markers of bone turnover have gained increasing relevance in the evaluation of patients with metabolic bone diseases. Their clinical applications include the assessment of future osteoporotic fracture risk, complementation of bone density measurements, diagnosis of certain metabolic osteopathies, therapeutic decision making, and monitoring of therapeutic efficacy and patient compliance. One should be aware, however, that the results from large epidemiologic or clinical

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

73

trials are sometimes difficult to translate into the everyday clinical situation. The individual patient often has more than one disease that might affect either bone turnover or the handling of the parameters mentioned (or both). Analytic and biologic variability of bone markers can be significant and also needs to be considered when using these indices. In the scientific setting, conventional and new markers of bone turnover can help to elucidate formerly unknown mechanisms and pathways. Because the development of ever more specific and sensitive markers of bone metabolism is progressing rapidly, we are likely to witness new insights into the pathophysiology of bone diseases in the near future.

References 1. Bais R, Edwards JB: An optimized continuous-monitoring procedure for semiautomated determination of serum acid phosphatase activity. Clin Chem 22:2025, 1976 2. Behr W, Barnert J: Quantification of bone alkaline phosphatase in serum by precipitation with wheat-germ lectin: A simplified method and its clinical plausibility. Clin Chem 32:1960, 1986 3. Bellahcene A, Merville MP, Castronovo V Expression of bone sialoprotein, a bone matrix protein, in human breast cancer. Cancer Res 54:2823, 1994 4. Bellahcene A, Kroll M, Liebens F, et al: Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development. J Bone Miner Res 11:665, 1996 5. Bettica P, Moro L, Robins SP, et al: Bone-resorption markers galactosyl hydroxylysine, pyridinium crosslinks, and hydroxyproline compared. Clin Chem 38:2313, 1992 6. Black D, Duncan A, Robins SP: Quantitative analysis of the pyridinium crosslinks of collagen in urine using ion-paired reversed-phase high-performance liquid chromatography. Anal Biochem 169:197, 1988 7. Bluhmson A, Eastell R Prediction of bone loss in postmenopausal women. Eur J Clin Invest 22:764, 1992 8. Blumsohn A, Navlor KE, Assiri AMA, et a 1 Different responses on biochemical markers of bone resorption to bisphosphonate therapy in Paget disease. Clin Chem 41:1592, 1995 9. Body JJ, Cleeren A, Pot M, et al: Serum osteocalcin (BGP) in tumor-associated hypercalcemia. J Bone Miner Res 1:523, 1986 10. Bonde M, Qvist P, Fledelius C, et al: Immunoassay for quantifying type I collagen degradation products in urine evaluated. Clin Chem 40:2022, 1994 11. Bonde M, Garner0 P, Fledelius C, et al: Measurement of bone degradation products in serum using antibodies reactive with an isomerized form of an 8 amino acid sequence of the C-telopeptide of type I collagen. J Bone Miner Res 12:1028, 1997 12. Braga V, Dorizzi R, Brocco G, et al: Clinical utility of a wheat-germ precipitation assay for the determination of bone alkaline phosphatase concentrations in patients with different metabolic bone diseases. Eur J Clin Chem Clin Biochem 33:433, 1995 13. Bressot C, Meunier PJ, Chapuy MC, et a1 Histomorphometric profile, pathophysiology and reversibility of corticosteroid induced osteoporosis. Metabolic Bone Disease and Related Research 1:303, 1979 14. Brixen K, Eriksen FL: Validation of local and systemic markers of bone turnover. In Seibel MJ, Robins SP, Bilezikian JP (eds): Dynamics of Bone and Cartilage Metabolism. San Diego, Academic Press, 1999, p 427 15. Brown JP, Delmas PD, Malaval L, et a1 Serum bone Gla-protein: A specific marker for bone formation in postmenopausal osteoporosis. Lancet 1:1091, 1984 16. Calvo MS, Eyre DR, Gundberg CM: Molecular basis and clinical application of biological markers of bone turnover. Endocr Rev 17333, 1996

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17. Carpenter TO, Mackowiak SJ, Troiano N, et a1 Osteocalcin and its message: Relationship to bone histology in magnesium-deprived rats. Am J Physioi 263:E107, 1992 18. Charles P, Mosekilde L, Risteli L, et al: Assessment of bone remodeling using biochemical indicators of type I collagen synthesis and degradation: Relation to calcium kinetics. Bone and Mineral 24:81, 1994 19. Chen JK, Shapiro HS, Wrana JL, et al: Localization of bone sialoprotein (BSP) expression to sites of mineralized tissue formation in fetal rat tissues by in situ hybridization. Matrix 11:133, 1991 20. Chesnut CH 111, Bell NH, Clark GS, et al: Hormone replacement therapy in postmenopausal women: Urinary N-telopeptide of type I collagen monitors therapeutic effect and predicts response of bone mineral density. Am J Med 102:29, 1997 21. Cheung CK, Panesar NS, Haines C, et al: Immunoassay of a tartrate-resistant acid phosphatase in serum. Clin Chem 41:679, 1995 22. Cheung CK, Panesar NS, Lau E, et al: Increased bone resorption and decreased bone formation in Chinese patients with hip fracture. Calcif Tissue Int 56:347, 1995 23. Christiansen C, Riis BJ, Rodboro P: Prediction of rapid bone loss in postmenopausal women. Lancet 1:1105, 1987 24. Christiansen C, R k BJ, Rodboro P: Screening procedure for women at risk of developing postmenopausal osteoporosis. Osteoporos Int 1:35, 1990 25. Clemens JD, Herrick MV, Singer FR, et al: Evidence that serum NTx (collagen-type I N-telopeptides) can act as an immunochemical marker of bone resorption. Clin Chem 43:2058, 1997 26. Coleman RE, Purohit Or, Black C, et al: Double-blind, randomized, placebocontrolled, dose-finding study of oral ibandronate in patients with metastatic bone disease. Ann Oncol 10:311, 1999 27. Colwell A, Russell RG, Eastell R: Factors affecting the assay of urinary 3-hydroxy pyridinium crosslinks of collagen as markers of bone resorption. Eur J Clin Invest 23:341, 1993 28. Conti A, Sartorio A, Ferrero S, et al: Modifications of biochemical markers of bone and collagen turnover during corticosteroid therapy. J Endocrinol Invest 19:127, 1996 29. Cosman F, Nieves J, Wilkinson C, et al: Bone density change and biochemical indices of skeletal turnover. Calcif Tissue Int 58:236, 1996 30. Cunningham LW, Ford JD, Segrest JP: The isolation of identical hydroxylysyl glycosides from hydroxylates of soluble collagen and from human urine. J Biol Chem 2422570,1967 31. Curtis PH, Jr, Clark WS, Herndon C H Vertebral fractures resulting from prolonged cortisone and corticotropin therapy. JAMA 156:467, 1954 32. Cushing H: The basophil adenomas of the pituitary body and their clinical manifestations. Bulletin of the John Hopkins Hospital 50:137, 1935 33. Deacon AC, Hulme P, Hesp R, et a1 Estimation of whole body bone resorption rate: A comparison of urinary total hydroxyproline excretion with two radioisotopic tracer methods in osteoporosis. Clin Chim Acta 166:297, 1987 34. De la Piedra C, Rapado A, Diaz-Diego EM, et a1 Variable efficacy of bone remodeling biochemical markers in the management of patients with Paget’s disease of bone treated with tiludronate. Calcif Tissue Int 59:95, 1996 35. Delmas I’D, Gineyts E, Bertholin A, et a1 Immunoassay of pyridinoline crosslink excretion in normal adults and in Paget’s disease. J Bone Miner Res 8:643, 1993 36. Delmas PD, Malaval L, Arlot ME, et al: Serum bone Gla-protein compared to bone histomorphometry in endocrine diseases. Bone 6:339, 1985 37. Die1 IJ, Solomayer EF, Seibel MJ, et a1 Serum bone sialoprotein in patients with primary breast cancer is a prognostic marker for subsequent bone metastasis. Clin Cancer Res 5:3914, 1999 38. Dresner-Pollak R, Seibel MJ, Greenspan S, et al: Biochemical markers of bone turnover reflect femoral bone loss in elderly women. Calcif Tissue Int 59:328, 1996 39. Eastell R, Robins S, Colwell T, et a1 Evaluation of bone turnover in type I osteoporosis using biochemical markers specific for both bone formation and bone resorption. Osteoporos Int 3:255, 1993 _

I

v

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40. Ebeling PR, Peterson JM, Riggs BL: Utility of type I procollagen propeptide assays for assessing abnormalities in metabolic bone diseases. J Bone Miner Res 71243, 1992 41. Ebeling PR, Erbas 8, Hopper JL, et al: Bone mineral density and bone turnover in asthmatics treated with long-term inhaled or oral glucocorticoids. J Bone Miner Res 13:1283, 1998 42. Ekenstam E, Stalenheim G, Hallgren R The acute effect of high dose corticosteroid treatment on serum osteocalcin. Metabolism 37:141, 1988 43. Elomaa I, Virkkunen P, Risteli L, et al: Serum concentration of the cross-linked carboxyterminal telopeptide of type I collagen (ICTP) is a useful prognostic indicator in multiple myeloma. Br J Cancer 66:337, 1992 44. Eyre DR, Dickson IR, Van Ness K Collagen cross-linking in human bone and articular cartilage. Age-related changes in the content of mature hydroxypyridinium residues. Biochem J 252:495, 1988 45. Farley J, Baylink DJ: Skeletal alkaline phosphatase activity in serum [editorial]. Clin Chem 41:1551, 1995 46. Farley JR, Baylink DJ: Skeletal alkaline phosphatase activity as a bone formation index in vitro. Metabolism 35:536, 1986 47. Fisher LW, Whitson SW, Avioli LW, et al: Matrix sialoprotein of developing bone. J Biol Chem 258:12723, 1983 48. Fishman WH: Alkaline phosphatase isoenzymes: recent progress [review]. Clin Biochem 23:99, 1990 49. Fledelius C, Johnsen AH, Cloos PAC, et al: Characterization of urinary degradation products derived from type I collagen. Identification of a beta-isomerized Asp-Gly sequence within the C-terminal telopeptide (alphal) region. J Biol Chem 11:9755,1997 50. Fleisch H: Bisphosphonates: Pharmacology. Semin Arthritis Rheum 23:261, 1994 51. Francini G, Montagnani M, Petrioli R, et al: Comparison between CEA, TPA, CA 15/ 3 and hydroxyproline, alkaline phosphatase, whole body retention of 99mTc MDP in the follow-up of bone metastases in breast cancer. Int J Biol Markers 5:65, 1990 52. Fujimoto D, Moriguchi T, Ishida T, et al: The structure of pyridinoline, a collagen crosslink. Biochem Biophys Res Commun 8452, 1978 53. Fujisawa R, Nodasaka Y, Kuboki Y Further characterization of interaction between bone sialoprotein (BSP) and collagen. Calcif Tissue Int 56:140, 1995 54. Fujisawa R, Butler WT, Brunn JC, et al: Differences in composition of cell-attachment sialoproteins between dentin and bone. J Dent Res 72:1222, 1993 55. Galasko C S Mechanisms of bone destruction in the development of skeletal metastases. Nature 263:507, 1976 56. Galasko CS: The significance of occult skeletal metastases, detected by skeletal scintigraphy in patients with otherwise apparently 'early' mammary carcinoma. Br J Surg 62:694, 1975 57. Gallop PM, Lian JB, Hauschka PV: Carboxylated calcium-binding proteins and vitamin K. N Engl J Med 302:1460,1980 58. Gardsell P, Johnell 0, Nilsson BE: The predictive value of bone loss for fragility fractures in women: A longitudinal study over 15 years. Calcif Tissue Int 49:90, 1991 59. Gamero P, Delmas PD: Assessment of the serum levels of bone alkaline phosphatase with a new immunoradiometric assay in patients with metabolic bone disease. J Clin Endocrinol Metab 771046, 1993 60. Gamero P, Shih WJ, Gineyts E, et al: Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment. J Clin Endocrinol Metab 79:1693, 1994 61. Gamero P, Sornay-Rendu E, Chapuy MC, et al: Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res 11:337, 1996 62. Garnero P, Hausherr E, Chapuy MC, et al: Markers of bone resorption predict hip fracture in elderly women: The EPIDOS prospective study. J Bone Miner Res 11:1531, 1996 63. Garnero P, Somay-Rendu E, Duboeuf F, et al: Markers of bone turnover predict postmenopausal forearm bone loss over 4 years: the OFELY study. J Bone Miner Res 14:1614, 1999

76

WOITGE & SEIBEL

64. Gennari C, Civitelli R Glucocorticoid-induced osteoporosis. Clin Rheum Dis 12:637, 1986 65. Gomez B, Jr, Ardakani S, Ju J, et al: Monoclonal antibody assay for measuring bonespecific alkaline phosphatase activity in serum. Clin Chem 41:1560, 1995 66. Gomez B Jr, Ardakani S, Evans BJ, et al: Monoclonal antibody assay for free urinary pyridinium cross-links. Clin Chem 42:1168, 1996 67. Gonnelli S, Rottoli P, Cepollaro C, et al: Prevention of corticosteroid-induced osteoporosis with alendronate in sarcoid patients. Calcif Tissue Int 61:382, 1997 68. Gram J, Junker P, Nielsen HK, et al: Effects of short-term treatment with prednisolone and calcitriol on bone and mineral metabolism in normal men. Bone 23297, 1998 69. Gunja-Smith Z,Boucek RJ: Collagen cross-linking compounds in human urine. Biochem J 197759, 1981 70. Hahn TJ: Drug-induced disorders of vitamin D and mineral metabolism. Clin Endocrino1 Metab 9:107, 1980 71. Hahn TJ: Steroid and drug-induced osteopenia. In Favus MJ (ed): Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Philadelphia, Lippincott-Raven, 1995, p 250 72. Halleen JM, Karp M, Viloma S, et al: Two-site immunoassays for osteoclastic tartrateresistant acid phosphatase based on characterization of six monoclonal antibodies. J Bone Miner Res 14:464, 1999 73. Hannon R, Blumsohn A, Naylor K, et al: Response of biochemical markers of bone turnover to hormone replacement therapy: Impact of biological variability. J Bone Miner Res 13:1124, 1998 74. Hansen M: Assessment of age and risk factors on bone density and bone turnover in healthy premenopausal women. Osteoporos Int 4:123, 1994 75. Hanson DA, Weis MA, Bollen AM, et al: A specific immunoassay for monitoring human bone resorption: Quantitation of type I collagen cross-linked N-telopeptides in urine. J Bone Miner Res 7:1251,1992 76. Harris ED, Jr: Rheumatoid arthritis. Pathophysiology and implications for therapy. N Engl J Med 322:1277,1990 77. Harris H The human alkaline phosphatases: What we know and what we don’t know. Clin Chim Acta 186:133, 1989 78. Hassager C, Jensen LT, P~denphantJ, et al: The carboxy-terminal pyridinoline crosslinked telopeptide of type I collagen in serum as a marker of bone resorption: The effect of nandrolone decanoate and hormone replacement therapy. Calcif Tissue Int 5430, 1994 79. Hauschka PV, Carr SA: Calcium-dependent alpha-helical structure in osteocalcin. Biochemistry 21:2538, 1982 80. Hauschka PV, Lian JB, Cole DE, et al: Osteocalcin and matrix Gla protein: Vitamin K-dependent proteins $bone. Physiol Rev 69:990, 1989 81. Heinegard D, Oldberg A: Structure and biology of cartilage and bone matrix noncollageneous macromolecules. FASEB J 32042,1989 82. Helfrich MH, Nesbitt SA, Dorey EL, et a1 Rat osteoclasts adhere to a wide range of RGD (Arg-Gly-Asp) peptide-containing proteins, including the bone sialoproteins and fibronectin, via a beta 3 integrin. J Bone Miner Res 7335, 1992 83. Hench PS, Kendall EC, Slocumb CH, et al: The effect of a hormone of the adrenal cortex (17-hydroxy-11-dehydrocorticosterone: compound E) and of pituitary adrenocorticotropic hormone on rheumatoid arthritis: Preliminary report. Proc Staff Meet Mayo Clin 24:181, 1949 84. Hunter GK, Goldberg H A Modulation of crystal formation by bone phosphoproteins: Role of glutamic acid-rich sequences in the nucleation of hydroxyapatite by bone sialoprotein. Biochem J 302175, 1994 85. James IT,Perrett D: Automated on-line solid-phase extraction and high-performance liquid chromatographic analysis of total and free pyridinium crosslinks in serum. J Chromatogr A 798:159, 1998 86. Kanis JA, Melton LJ 111, Christiansen C, et al: The diagnosis of osteoporosis. J Bone Miner Res 9:1137,1994 87. Karmatschek M, Maier I, Seibel MJ, et a1 Improved purification of human bone

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

77

sialoprotein and development of a homologous radioimmunoassay. Clin Chem 43:2076, 1997 88. Keen RW, Nguyen T, Sobnack R, et a1 Can biochemical markers predict bone loss at the hip and spine? A 4-year prospective study of 141 early postmenopausal women. Osteoporos Int 6399, 1996 89. Kivirikko KI: Urinary excretion of hydroxyproline in health and disease. International Review of Connective Tissue Research 5:93, 1970 90. Kraenzlin ME, Lau KH, Liang L, et al: Development of an immunoassay for human serum osteoclastic tartrate-resistant acid phosphatase. J Clin Endocrinol Metab 71442, 1990 91. Krane SM, Simon L S Metabolic consequences of bone turnover in Paget's disease of bone. Clin Orthop 217:26, 1987 92. Krane SM, Conca W, Stephenson ML, et a1 Mechanisms of matrix degradation in rheumatoid arthritis. Ann NY Acad Sci 580:340, 1990 93. Kushida K, Takahashi M, Kawana K, et al: Comparison of markers for bone formation and resorption in premenopausal and postmenopausal subjects, and osteoporosis patients. J Clin Endocrinol Metab 802447, 1995 94. Lam KW, Siemens M, Sun T, et al: Enzyme immunoassay for tartrate-resistant acid phosphatase. Clin Chem 28:467,1982 95. Lam KW, Lee P, Li CY, et al: Immunological and biochemical evidence for identity of tartrate-resistant isoenzymes of acid phosphatases from human serum and tissues. Clin Chem 26:420, 1980 96. Lau KH, Onishi T, Wergedal JE, et al: Characterization and assay of tartrate-resistant acid phosphatase activity in serum: Potential use to assess bone resorption. Clin Chem 33:458, 1987 97. Laurence JR, Neville AM: The detection and evaluation of human tumor metastases. Cancer Metastasis Rev 2:351, 1983 98. Li Y, Woitge HW, Kissling C, et a1 Biological variability of serum immunoreactive bone sialoprotein. Clinical Labratory 44:553, 1998 99. Lian JB, Friedman PA: The vitamin K-dependent synthesis of gamma-carboxyglutamic acid by bone microsomes. J Biol Chem 253:6623, 1978 100. Lo Cascio V, Bertoldo F, Gambaro G, et a1 Urinary galactosyl-hydroxylysine in postmenopausal osteoporotic women: A potential marker of bone fragility. J Bone Miner Res 141420, 1999 101. Lowry M, Hall DE, Brosnan J T Hydroxyproline metabolism by the rat kidney: Distribution of renal enzymes of hydroxyproline catabolism and renal conversion of hydroxyproline to glycine and serine. Metabolism 34955, 1985 102. Lukert BP, Higgins JC, Stoskopf MM: Serum osteocalcin is increased in patients with hyperparathyroidism and decreased in patients receiving glucocorticoids. J Clin Endocrinol Metab 62:1056, 1986 103. Marcus R, Holloway L, Wells B, et a1 The relationship of biochemical markers of bone turnover to bone density changes in postmenopausal women: Results from the Postmenopausal Estrogen/Progestin Interventions (PEPI) trial. J Bone Miner Res 14:1583, 1999 104. Melkko J, Niemi S, Risteli L, et a1 Radioimmunoassay of the carboxyterminal propeptide of human type I procollagen. Clin Chem 36:1328, 1990 105. Merry AH, Harwood R, Woolley DE, et al: Identification and partial characterization of the non-collagenous amino- and carboxyl-terminal extension peptides of cartilage procollagen. Biochem Biophys Res Commun 71:83, 1976 106. Minisola S, Romagnoli E, Scarnecchia L, et a1 Serum carboxy-terminal propeptide of human type I procollagen in patients with primary hyperparathyroidism: Studies in basal conditions and after parathyroid surgery. Eur J Endocrinol 130:587, 1994 107. Minkin C: Bone acid phosphatase: Tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 34:285, 1982 108. Miura H, Yamamoto I, Yuu I, et al: Estimation of bone mineral density and bone loss by means of bone metabolic markers in postmenopausal women. Endocr J 42797,1995 109. Mole PA, Walkinshaw MH, Robins SP, et a1 Can urinary pyridinium crosslinks and urinary estrogens predict bone mass and rate of bone loss after the menopause? Eur J Clin Invest 22:767, 1992

78

WOITGE & SEIBEL

110. Moro L, Modricky C, Rovis L, et al: Determination of galactosyl hydroxylysine in urine as a means for the identification of osteoporotic women. Bone and Mineral 3:271, 1988 111. Moro L, Modricky C, Stagni N, et a1 High-performance liquid chromatographic analysis of urinary hydroxylysyl glycosides as indicators of collagen turnover. Analyst 109:1621, 1984 112. Moro L, Pozzi Mucelli RS, Gazzarrini C, et al: Urinary beta-1-galactosyl-0-hydroxylysine (GH) as a marker of collagen turnover of bone. Calcif Tissue Int 42237, 1988 113. Moss DW: Perspectives in alkaline phosphatase research [review]. Clin Chem 38:2386, 1992 114. Moss DW, Whitby LG: A simplified heat-inactivation method for investigating alkaline phosphatase isoenzymes in serum. Clin Chim Acta 61:63, 1975 115. Nordin BEC, Marshall DH, Francis RM, et al: The effects of sex steroids and corticosteroid hormones on bone. J Steroid Biochem 15:171, 1981 116. Ogawa T, Ono T, Tsuda M, et al: A novel fluor in insoluble collagen: A crosslinking moiety in collagen molecule. Biochem Biophys Res Commun 1071252, 1982 117. Oikarinen A, Autio P, Vuori J, et al: Systemic glucocorticoid treatment decreases serum concentrations of carboxyterminal propeptide of type I procollagen and aminoterminal propeptide of type 111 procollagen. Br J Dermatol 126:172, 1992 118. Oldberg A, Franzen A, Heinegard D, et al: Identification of a bone sialoprotein receptor in osteosarcoma cells. J Biol Chem 263:19433, 1988 119. Papapoulos SE: Paget’s disease of bone: Clinical, pathogenetic and therapeutic aspects. Baillieres Clin Endocrinol Metab 11:117, 1997 120. Parfitt A M Age-related structural changes in trabecular and cortical bone: Cellular mechanisms and biomechanical consequences. Calcif Tissue Int 36(suppl):S123, 1984 121. Pecherstorfer M, Seibel MJ, Woitge HW, et al: Bone resorption in multiple myeloma and in monoclonal gammopathy of undetermined significance: Quantification by urinary pyridinium cross-links of collagen. Blood 90:3743, 1997 122. Pecherstorfer M, Zimmer-Roth I, Schilling T, et a1 The diagnostic value of urinary pyridinium cross-links of collagen, serum total alkaline phosphatase, and urinary calcium excretion in neoplastic bone disease. J Clin Endocrinol Metab 80:97, 1995 123. Pedersen BJ, Bonde M: Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide. Clin Chem 40:811, 1994 124. Pedrazzoni M, Alfano FS, Gatti C, et al: Acute effects of bisphosphonates on new and traditional markers of bone resorption. Calcif Tissue Int 5725, 1995 125. Peel NF, Eastell R, Russell RG: Osteoporosis in rheumatoid arthritis-the laboratory perspective. Br J Rheumatol 30:84, 1991 126. Peretz A, Praet JP, Bosson D, et al: Serum osteocalcin in the assessment of corticosteroid induced osteoporosis. Effect of long and short term corticosteroid treatment. J Rheumatol 16:363, 1989 127. Pietschmann P, Machold KP, Wolosczuk W, et al: Serum osteocalcin concentrations in patients with rheumatoid arthritis. Ann Rheum Dis 48654, 1989 128. Pratt DA, Daniloff Y, Duncan A, et al: Automated analysis of the pyridinium crosslinks of collagen in tissue and urine using solid-phase extraction and reversed-phase high-performance liquid chromatography. Anal Biochem 207:168, 1992 129. Prockop DJ, Kivirikko KI, Tuderman L, et al: The biosynthesis of collagen and its disorders. N Engl J Med 301:13, 1979 130. Pmmmel MF, Wiersinga WM, Lips P, et al: The course of biochemical parameters of bone turnover during treatment with corticosteroids. J Clin Endocrinol Metab 72:382, 1991 131. Ravn P, Rix M, Andreassen H, et al: High bone turnover is associated with low bone mass and spinal fracture in postmenopausal women. Calcif Tissue Int 60255, 1997 132. Reeve J, Pearson J, Mitchell A, et a1 Evolution of spinal bone loss and biochemical markers of bone remodeling after menopause in normal women. Calcif Tissue Int 57105, 1995 133. Reid I R Pathogenesis and treatment of steroid osteoporosis. Clin Endocrinol (Oxf) 30:83, 1989

BIOCHEMICAL MARKERS TO SURVEY BONE TURNOVER

79

134. Reid IR, Heap SW Determinants of vertebral mineral density in patients in patients receiving long-term glucocorticoid therapy. Arch Intern Med 150:2545, 1990 135. Reid IR, Chapman GE, Fraser TRC, et al: Low serum osteocalcin levels in glucocorticoid-treated asthmatics. J Clin Endocrinol Metab 62:379, 1986 136. Riggs BL, Melton LJ 111, OFallon WM: Drug therapy for vertebral fractures in osteoporosis: Evidence that decreases in bone turnover and increases in bone mass both determine antifracture efficacy. Bone 18(suppl):197S, 1996 137. Riis BJ, Hansen MA, Jensen AM, et al: Low bone mass and fast rate of bone loss at menopause: Equal risk factors for future fracture: A 15-year follow-up study. Bone 19:9, 1996 138. Risteli J, Elomaa I, Niemi S, et al: Radioimmunoassay for the pyridinoline crosslinked carboxy-terminal telopeptide of type I collagen: A new serum marker of bone collagen degradation. Clin Chem 39:635, 1993 139. Robins SP: Fibrillogenesis and maturation of collagens. In Seibel MJ, Robins SP, Bilezikian JP (eds): Dynamics of Bone and Cartilage Metabolism. San Diego, Academic Press, 1999, p 36 140. Robins SP: Turnover of collagen and its precursors. In Viidik A, Vuust J (eds): Biology of Collagen. New York, Academic Press, 1980, p 135 141. Robins SP, Seibel MJ, McLaren AM: Collagen markers in urine in human arthritis. In Maroudas A, Kuettner K (eds): Methods in Cartilage Research. London, Academic Press, 1991, p 348 142. Robins SP, Woitge H, Hesley R, et al: Direct, enzyme-linked immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption. J Bone Miner Res 9:1643, 1994 143. Rosalki SB, Foo AY, Burlina A, et a1 Multicenter evaluation of iso-ALP test kit for measurement of bone alkaline phosphatase activity in serum and plasma. Clin Chem 39:648, 1993 144. Rosen C, Chesnut CH 111, Mallinak NJS: The predictive value of biochemical markers of bone turnover for bone mineral density in early postmenopausal women treated with hormone replacement or calcium supplementation. J Clin Endocrinol Metab 82:1904, 1997 145. Ross FP, Chappel J, Alvarez JI, et al: Interactions between the bone matrix proteins osteopontin and bone sialoprotein and the osteoclast integrin alpha v beta 3 potentiate bone resorption. J Biol Chem 268:9901, 1993 146. Ross PD, Knowlton W Rapid bone loss is associated with increased levels of biochemical markers. J Bone Miner Res 13297, 1998 147. Russell RG, Beard DJ, Cameron EC, et al: Biochemical markers of bone turnover in Paget’s disease. Metabolic Bone Disease and Related Research 3:255, 1981 148. Sambrook PN, Kelly PJ, Koegh A, et al: Bone loss after cardiac transplantation: A prospective study. J Heart Lung Transpl 13:116, 1994 149. Saxne T, Zunino L, Heinegard D: Increased release of bone sialoprotein into synovial fluid reflects tissue destruction in rheumatoid arthritis. Arthritis Rheum 3882, 1995 150. Schlemmer A, Hassager C, Jensen SB, et a1 Marked diurnal variation in urinary excretion of pyridinium cross-links in premenopausal women. J Clin Endocrinol Metab 74:476, 1992 151. Schneider DL, Barrett-Connor EL: Urinary N-telopeptide levels discriminate normal, osteopenic, and osteoporotic bone mineral density. Arch Intern Med 1571241, 1997 152. Segrest JP, Cunningham LW. Variations in human urinary 0-hydroxylysyl glycoside levels and their relationship to collagen metabolism. J Clin Invest 49:1497, 1970 153. Seibel MJ, Duncan A, Robins SP: Urinary hydroxypyridinium crosslinks provide indices of cartilage and bone involvement in arthritic diseases. J Rheumatol 16964, 1989 154. Seibel MJ, Gartenberg F, Silverberg SJ, et al: Urinary hydroxypyridinium cross-links of collagen in primary hyperparathyroidism. J Clin Endocrinol Metab 74:481, 1992 155. Seibel MJ, Cosman F, Shen V, et al: Urinary hydroxypyridinium crosslinks of collagen as markers of bone resorption and estrogen efficacy in postmenopausal osteoporosis. J Bone Miner Res 8:881, 1993 156. Seibel MJ, Woitge H, Scheidt-Nave C, et a1 Urinary hydroxypyridinium crosslinks of

80

WOITGE & SEIBEL

collagen in population-based screening for overt vertebral osteoporosis: Results of a pilot study. J Bone Miner Res 9:1433, 1994 157. Seibel MJ, Woitge HW, Pecherstorfer M, et al: Serum immunoreactive bone sialoprotein as a new marker of bone turnover in metabolic and malignant bone disease. J Clin Endocrinol Metab 81:3289, 1996 158. Seyedin SM, Kung VT, Daniloff YN, et a1 Immunoassay for urinary pyridinoline: The new marker of bone resorption. J Bone Miner Res 8:635, 1993 159. Shapiro HS, Chen J, Wrana JL, et al: Characterization of porcine bone sialoprotein: Primary structure and cellular expression. Matrix 13:431, 1993 160. Singer FR, Roodman GD: Pagefs disease of bone. In Bilezikian JP, Raisz LG, Rodan GA (eds): Principles of Bone Biology. San Diego, Academic Press, 1996, p 969 161. Siris ES: Paget’s disease of bone. J Bone Miner Res 13:1061, 1998 162. Smith R Collagen and disorders of bone. Clin Sci (Colch) 59:215, 1980 163. Taubman MB, Goldberg B, Sherr C: Radioimmunoassay for human procollagen. Science 186:1115, 1974 164. Tavassoli M, Rizo M, Yam LT Elevation of serum acid phosphatase in cancers with bone metastasis. Cancer 45:2400, 1980 165. Uebelhart D, Schlemmer A, Johansen JS, et a1 Effect of menopause and hormone replacement therapy on the urinary excretion of pyridinium crosslinks. J Clin Endocrino1 Metab 72:367, 1991 166. Uebelhart D, Gineyts EC, Chapuy MC, et a1 Urinary excretion of pyridinium crosslinks: A new marker of bone resorption in metabolic bone disease. Bone and Mineral 8237, 1990 167. Van Daele PL, Seibel MJ, Burger H, et al: Case-control analysis of bone resorption markers, disability, and hip fracture risk: The Rotterdam study. BMJ 312:482, 1996 168. Van Hoof VO, Lepoutre LG, Hoylaerts MF, et al: Improved agarose electrophoretic method for separating. alkaline phosphatase isoenzymes in serum. Clin Chem 34:1857, 1988 169. Van Straalen TP, Sanders E. Prummel ME et al: Bone alkaline uhosuhatase as indicator of bone formation. Clin Chim Acta 201:27, 1991 170. Wee1 AEAM, Seibel MJ, Hofman A, et a1 Which fractures are associated with high bone resorption in elderly women: The Rotterdam study [abstract lllO]. J Bone Miner Res 14(suppl):S160,1999 171. Weiss MJ, Henthorn PS, Lafferty MA, et a1 Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Natl Acad Sci USA 83:7182, 1986 172. Withold W, Armbruster FP, Karmatschek M, et al: Bone sialoprotein in serum of patients with malignant bone diseases. Clin Chem 43535, 1997 173. Woitge HW, Seibel MJ, Ziegler R Comparison of total and bone-specific alkaline phosphatase in patients with nonskeletal disorder or metabolic bone diseases. Clin Chem 42:1796, 1996 174. Woitge HW, Pecherstorfer M, Li Y, et a1 Novel serum markers of bone resorption: Clinical assessment and comparison with established urinary indices. J Bone Miner Res 14:792, 1999 175. Woitge HW, Scheidt-Nave C, Kissling C, et al: Seasonal variation of biochemical indexes of bone turnover: Results of a population-based study. J Clin Endocrinol Metab 83:68, 1998 176. Woitge HW, Oberwittler H, Heichel S, et al: Short- and long-term effects of ibandronate treatment on bone turnover in Pagek disease of bone. Clin Chem 46:684-690, 2000 Y

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