JOURNAL OF BONE AND MINERAL RESEARCH Volume 7, Number 11, 1992 Mary Ann Liebert, Inc., Publishers

A Specific Immunoassay for Monitoring Human Bone Resorption: Quantitation of Type I Collagen Cross-linked N-Telopeptides in Urine DENNIS A. HANSON,' MARY ANN E. WEIS,' ANNE-MARIE BOLLEN,' SHOSHANA L. MASLAN,2 FREDERICK R. SINGER,3 and DAVID R. EYRE',4

ABSTRACT Peptides of low molecular weight that contain pyridinoline cross-links were isolated from adolescent human urine. A fraction was selected that was enriched in the N-telopeptide-to-helix intermolecular cross-linking domain of bone type I collagen. Mouse monoclonal antibodies were generated against these urinary peptides conjugated to a carrier protein as immunogen. A high-affinity antibody was identified that specifically bound to the trivalent peptides derived from the N-telopeptide-to-helix pyridinoline cross-linking site in type I collagen of human bone. This was confirmed by the direct isolation from human bone collagen of similar fragments recognized selectively by the antibody. A sensitive inhibition ELISA was established on microtiter plates that could quantify the bone-derived peptides in human urine. The assay, which can be run directly on untreated urine, was thoroughly tested against samples from normal subjects and from patients with metabolic bone disease. For example, strong correlations with urinary hydroxyproline and total pyridinoline cross-links were found in patients with Paget's disease of bone. The method shows considerable promise as a rapid and specific index of human bone resorption rates, with greatly improved specificity and convenience over total pyridinoline analysis. Potential applications include the study of normal metabolism, the diagnosis and monitoring of bone disease, and evaluating the effectiveness of antiresorption therapies.

INTRODUCTION reliable method for monitoring bone resorption rates in humans or animals based on a biochemical marker. Urinary hydroxyproline has long been used for this purpose, but it is mainly useful clinically as a n index of the greatly increased rates of bone resorption seen, for example, in Paget's disease and severe hyperparathyroidism. ( I ) Sources of hydroxyproline in addition to bone resorption, including the diet, turnover of soft connective tissues, C l q and other serum proteins, and degradation of the N-propeptides from collagen biosynthesis, are all thought to contribute t o urinary hydroxyproline. ( 2 ) In addition, much of the free hydroxyproline that arises

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from collagen degradation is oxidized in the liver, further blunting the value of this metabolite as an index of bone breakdown. w4) Hydroxylysine glycosides may be a better urinary m a r k e ~ ' ~but . ~ ) suffer from similar problems and are difficult to assay. Recently, the pyridinoline cross-linking amino acids of collagen, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), which are excreted in urine both as the free amino acids and in peptide-bound form, have proved to be the best available biochemical markers of bone resorption. ('-I3) Their assay in urine by high-performance liquid chromatography (HPLC), however, is inconveniently complex, requiring acid hydrolysis and cleanup procedures, which can result in low and variable yields.(141An immu-

'Department of Orthopaedics, University of Washington, Seattle. 'Department of Comparative Medicine, University of Washington, Seattle. 'Bone Center, Cedars-Sinai Medical Center and School of Medicine, University of California, Los Angeles. 'Department of Biochemistry, University of Washington, Seattle. 1251

HANSON ET AL.

1252

noassay for pyridinoline has been described, (I5) but the Peptides from human bone cartilage polyclonal antiserum recognized only free hydroxylysylMidshaft cortical bone from a human femur (male, aged pyridinoline, not the lysylpyridinoline (deoxypyridinoline) 25, banked tissue, Northwest Tissue Center) was powdered variant or the peptide-bound residues. in a Spex mill under liquid N2, defatted in CHCI, and Since the pyridinoline cross-linking residues are excreted CHJOH (3:l vol/vol), and decalcified in 0.5 M EDTA, primarily in peptide-bound form, we set out to identify a 0.05 M Tris-HCl, pH 7.5, at 4°C for 1 week. The collagecross-linked fragment of collagen type I that was excreted nous matrix was digested with bacterial collagenase (Sigma as a reproducible fraction of total bone-derived pyridinotype 1A; substrate to enzyme, 50:l wt/wt) at 37°C for 24 lines and so could act as a quantitative measure of the sysh. Cross-linked peptides containing pyridinoline residues temic rate of bone resorption. The aim was to produce a were isolated from this digest by chromatography. monoclonal antibody with suitable properties for developing a specific immunoassay for monitoring bone resorption rates clinically. Here we describe the development and mo- Molecular sieve chromatography lecular basis of this new assay with examples of results of Urinary cross-linked peptides and cross-linked peptides its application to normal urine and urine from patients from bone tissue collagen were independently fractionated with Paget's disease of bone. on a column (90 x 2.5 cm) of Bio-Gel P10 (200-400 mesh, Bio-Rad) eluted with 10% (vol/vol) acetic acid at 12 rnl/h.(I6)Collected fractions (4 ml) were assayed for peptide absorbance at 280 nm and pyridinoline fluorescence (excitation 297 nm, emission 395 nm). Eventually, fraction MATERIALS AND METHODS aliquots were also monitored for cross-linked N-telopepUrinary source of collagen peptides tides of type I collagen using the inhibition enzyme-linked Urine (20 liters) was collected from a normal adolescent immunosorbent assay (ELISA) assay we developed (see male (aged 17) during a period of rapid skeletal growth later). and stored at 4°C. The urine was diluted 5 x , adjusted to 2% (vol/vol) trifluoroacetic acid, and passed through a seReversed-phase HPL C ries of conditioned C18 Sep-Paks (Millipore). Bound pepSelected pools of urinary peptides and collagenase-detides were eluted with 20% (vol/vol) acetonitrile. The eluent was diluted twice with 0.1 M NH,HCO, and passed rived peptides from Bio-Gel P10 chromatography were through a conditioned QMA anion-exchange Sep-Pak further resolved by reversed-phase HPLC on a macropor(Millipore). The cartridge was washed with 0.1 M NaCl in ous C8 column (Brownlee RP300, 4.6 mm x 25 cm), elut20'70 (vol/vol) acetonitrile, and retained peptides were ing with a gradient of acetonitrile and 1-propanol (3: 1 vol/ vol) containing 0.1 To (vol/vol) trifluoroacetic acid.(16'Elueluted with 1% (vol/vol) trifluoroacetic acid and dried.

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FIG. 1. (a) Molecular diagram of the site of N-telopeptide intermolecular cross-linking in type I collagen. The structure of the predominant pyridinoline-containing peptide found in human urine is shown, which is derived from this domain as an end product of bone resorption. (b) Molecular sieve chromatography of pyridinoline-containing peptides concentrated from human urine. A sample (75 mg) of enriched peptides was eluted from a Bio-Gel P10 column (BioRad; 90 x 2.5 cm) in 10% (vol/vol) acetic acid, monitoring for pyridinoline fluorescence. The pool identified by the bar was shown to be enriched in the cross-linked type I collagen N-telopeptides of bone (data not shown) and was taken for antibody production.

1253

IMMUNOASSAY FOR BONE RESORPTION ent was monitored for peptide absorbance (220 nm) and pyridinoline fluorescence (297 nm excitation, 395 nm emission).

Monoclonal antibody production Balb/C mice were immunized in Freund's complete adjuvant with the cross-linked N-telopeptide fraction of urinary peptides, conjugated to keyhole limpet hemocyanin (KLH) with glutaraldehyde. (I7) A conventional immunization protocol was followed.(") Animals were selected for a final boost and fusion of spleen cells based on high serum titers to the peptide immunogen conjugated to bovine serum albumin (BSA) in an ELISA screen. Standard methods of fusion with mouse myeloma cells (NS-1) were employed. (I9) Hybridoma supernatants were screened by ELISA on microtiter plates using various fractions of urinary peptide (conjugated to BSA) to select for desirable monoclonal antibodies that recognized the cross-linked Ntelopeptides containing pyridinoline residues from human bone type I collagen. Of several candidate monoclonal antibodies (MAb), the product of hybridoma 1H 11 was selected for assay development on the basis of its high affinity, specificity, and desirable subclass (IgG, x ) .

Characterization of 1HI 1 binding properties Ascites fluid was produced in Balb/C mice from the 1H11 hybridoma cell line. The antibody was tested in 96well microtiter plates for its ability to bind to type I and type I1 collagen molecules and denatured a-chains, CNBrderived peptides, bacterial collagenase-derived peptides, and the purified urinary peptides using both solid-phase ELISA and inhibition ELISA formats. Various urinary peptide fractions were conjugated to BSA with glutaraldehyde and coated on the microtiter plate wells. Competing antigen in test samples was incubated in solution with appropriate dilutions of 1H11 in the well. Bound l H l l was detected by a secondary antibody, goat antimouse IgG conjugated to peroxidase, using tetramethylbenzidine (TMB) as the chromogenic substrate. Developed plates were read at 450 nm on a microplate reader.

Peptide m icrosequencing A Porton Model 2090E instrument with on-line reversed-phase HPLC detection of phenylthiohydantoin (PTH) amino acid derivatives was used to help identify the molecular origin of cross-linked peptides.

Electrospray mass spectrometry Individual peptides purified by affinity chromatography on 1H1I-Sepharose and then reversed-phase HPLC were also structurally identified by the molecular mass of their ions on electrospray ionization mass spectrometry using a Sciex triple quadrupole instrument (Model API 111).

Analysis of pyridinoline cross-links H P and LP were quantified after acid hydrolysis by reversed-phase HPLC in the ion-pairing agent n-heptafluorobutyric acid using fluorescence detection. (201 For urine samples, equal volumes of urine and 12 M HCI were heated in a sealed tube at 108°C for 24 h to hydrolyze peptide-bound pyridinolines. Dried hydrolysates were eluted from a Bio-Gel P2 column(16)to separate the pool of pyridinolines from the bulk of urinary metabolites.

Inhibition ELISA of urine samples using MAb l H l l The working concentrations of solid-phase antigen, 1H11 ascites, and enzyme-linked secondary antibody were determined by checkerboard titrations. Polystryrene mi-

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Immunoaffinity chromatography An immunoaffinity column was made from the purified MAb 1H11 linked to agarose beads. Sepharose (CNBr activated, Pharmacia) was covalently linked according to the manufacturer's instructions to MAb 1H11 that had been purified by binding to a protein G-Sepharose column (protein G-Sepharose 4 Fast Flow, Pharmacia). Adolescent urine was diluted in phosphate-buffered saline (PBS) and was passed dropwise through a column (0.9 x 13 cm) of the 1Hll-Sepharose. After washing with phosphate-buffered saline, bound peptides were eluted with 50% saturated (NH,),SO. in 1@lo (vol/vol) trifluoroacetic acid. Peptides were extracted and concentrated from the eluent using a conditioned C18 Sep-Pak (Millipore) then fractionated by reverse-phase HPLC as described earlier.

100 200 300 400 500 600 700 800 ELUTION VOLUME (rnl)

FIG. 2. Molecular sieve chromatography of pyridinolinecontaining peptides from a bacterial collagenase digest of human bone collagen. The sample (100 mg) was eluted from a Bio-Gel P10 column as in Fig. 1. The elution profiles of peptide absorbance and pyridinoline fluorescence (upper) and immunoreactivity with MAb 1H11 (lower) are shown. The elution positions of the major pools of type I collagen cross-linked C-telopeptides (250-320 ml) and Ntelopeptides (400-470 ml) were established by reversedphase HPLC and microsequence analysis.

1254

HANSON ET AL.

crotiter plates (Nunc Maxisorp) were coated with 100 p1 per well of urinary peptide-BSA conjugate diluted in 0.05 M carbonate and bicarbonate, pH 9.6, sealed and incubated overnight at 4°C. The following day, plates were washed twice with ELISA wash buffer (0.15 M NaCl containing 0.05% vol/vol Tween 20), resealed, and stored at 4°C for up to 1 month. On the day of an assay, plates were taken out, washed twice with the buffer, and patted dry. Titrations of samples were performed directly on the coated assay plate. All samples, urine and standards, were diluted twice by adding 100 pl sample to 100 pl double-strength assay buffer (0.29 M NaCI, 0.01 M KCI, 0.162 M Na,HPO,, 3 mM KH,P04, 0.002% wt/vol thimerosol, and 0.1070 vol/vol Tween 20, pH 7.4). Serial dilutions were made by transferring 100 pl from this initial well into 100 pl assay buffer ( I : ] dilution of double strength buffer in water) in the next well, and so on. After all dilutions were complete, leaving 100 pl per well of each diluted sample, 100 pl per well of 1H11 ascites diluted in PBS was quickly added directly to each sample. Plates were then incubated for 1 h at room temperature. Plates were washed three times and patted dry, and 100 p1 per well was added of goat antimouse secondary antibody conjugated to horseradish peroxidase (Jackson Immuno Research Laboratories) diluted in assay buffer. Plates were again incubated for 1 h at room temperature and then washed five times and patted dry. Color was developed by adding 100 pl per well of 0.1 mg/ml of tetramethylbenzi-

dine (Pierce) in 0.1 M Na acetate, pH 5.0 and 0.01% wt/vol H1O,. The reaction was stopped with 100 p1 per well of 1.5 N HISO,, and absorbance was read at 450 nm using a Titertek Multiskan-plus plate reader.

Urine collections Urine samples for ELISA analysis were either 2 h fasting or 24 h collections from prior clinical studies on Paget's disease of bone or spot urine samples, stored frozen at -20°C before analysis.

Creatinine and hydroxyproline assays Creatinine was quantified in urine samples by the Jaffe procedure using a reagent kit (Sigma) and hydroxyproline by a published

RESULTS A peptide fraction from normal adolescent urine (Fig. la) that was enriched in pyridinoline-containing fragments of the N-telopeptide cross-linking domain of type I collagen (Fig. Ib) was selected as the immunogen for generating monoclonal antibodies. This fraction was characterized by extensive chromatographic and structural analyses (not shown). Similar peptides were identified previously

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ELUTION TIME (min.) FIG. 3. Reversed-phase HPLC elution profile and structural identification of the major peptides recovered from adolescent human urine by immunoaffinity on a column of MAb 1H1I-Sepharose. Peptides containing pyridinoline crosslinks were detected by their natural fluorescence. Structural identities were established by amino-terminal microsequencing and molecular mass determination on electrospray mass spectrometry.

1255

IMMUNOASSAY FOR BONE RESORPTION

in the urine of patients with severe Paget’s disease of bone(22)and in urine of rapidly growing children (unpublished findings). Several mouse hybridomas were identified that secreted MAb recognizing these urinary peptides. One of these MAb (1HI 1) was chosen for immunoassay development because of its high binding affinity and specificity for cross-linked N-telopeptides of human bone type I collagen. It did not bind to native molecules or intact a-chains of pepsin-extracted collagen type I from human bone, collagen type I1 from human cartilage, or CNBr peptides or tryptic peptides of these collagens plated out in microplate wells either directly or conjugated to BSA (data not shown). Nor did l H l l bind to these collagen chains or peptides when they were competed in solution against the conjugated urinary antigen as the solid phase in an inhibition ELISA format. However, I H l l bound to peptides generated by bacterial collagenase digestion of human bone collagen. The immunoreactive peptides in the collagenase digest were shown to be restricted to the cross-linked N-telopeptide pool separated by molecular sieve chromatography (Fig. 2). The nature of the peptide epitope recognized by MAb 1H11 was further investigated by immunoaffinity chromatography. Figure 3 shows the elution profile on reversephase HPLC of components bound by a 1H11-Sepharose affinity column from adolescent urine. Peptides under the major peaks of pyridinoline fluorescence were characterized by N-terminal microsequence analysis and by electrospray mass spectrometry (ESMS). Microsequencing results and mass spectra (data not shown) established the chemical structures shown in Fig. 3 against each pyridinoline-containing peptide peak. All were peptide fragments derived from the N-telopeptide cross-linking domain of human type I collagen. The a2(I) N-telopeptide is present in all the structures and hence appears to be an essential part of the epitope recognized by 1H11. Interestingly, a peptide that consists of two a2(I) N-telopeptides linked to a pyridinoline residue was found (elution position 27 minutes, Fig. 3), in addition to the more abundant heteromeric peptides, which all were derived from pyridinoline-linked al(I) and al(I) N-telopeptides. For each of the peptide peaks, molecular ions were present on ESMS that corresponded to the masses of both HP- and LP-containing cross-linked forms (not shown). The ratios of these ions indicated HP/LP ratios of 2.0-2.5:l for the al(I)N to a2(I)N telopeptides and 1 : l for the a2(I)N to a2(I)N fragments, in agreement with the HP/LP ratios measured by reverse-phase HPLC after acid hydrolysis of material under each peptide peak. The epitope recognized by l H l l was therefore equally represented in the HP-containing and the LP-containing forms of each of the indicated cross-linked peptide structures. The isolated free pyridinolines, moreover, did not bind to the antibody. It was also established that the epitope was not simply a linear peptide sequence by synthesizing the individual a#) and crl(I) N-telopeptide sequences and showing that these did not compete in the inhibition assay. The epitope recognized therefore appears to be a conformational feature of the specific cross-linked peptide sequences, but is not necessarily dependent on the pyri-

dinoline nature of the cross-linking residue itself (unpublished observations). Figure 4 shows inhibition curves using MAb l H l l in a quantitative ELISA format. Results are compared for serial dilutions of a standard of purified cross-linked Ntelopeptides from urine, of collagenase-generated peptides from human bone matrix, and of adolescent human urine. The curves are parallel, indicating that the antibody recognizes the same epitope in untreated urine as in the peptides prepared from urine or bone tissue. The assay can be calibrated quantitatively by the content of pyridinoline residues (HP plus LP) in the peptide standard or, as shown in Fig. 4, by the amount of bone collagen from which peptides were generated by collagenase digestion. (The yield of immunoreactive peptides per unit mass of human bone collagen on exhaustive collagenase digestion was found to be highly reproducible.) Dilutions of mouse ascites fluid exceeding 10 million-fold still gave significant optical densities in this assay format, indicating a high binding affinity of I H l l for its complex peptide epitope. The quantitative ELISA assay has been applied to urine from normal subjects of all ages and from patients with various metabolic bone disorders. The assay requires as little as 50 p1 urine and no cleanup steps and takes 2 h or less to acquire final data. Even the most dilute samples (CO.1 mg/ml of creatinine) can be quantified with the present assay sensitivity. To evaluate the stability of the peptide antigen, urine samples were analyzed after multiple freeze-thaw cycles with no loss of immunoreactivity. Even urine that was routinely acidified with HCI (to pH 2 or less) and stored at -20°C for years could be neutralized and analyzed with no apparent effect on the measured yield of cross-linked N-telopeptides. The peptide epitope is therefore very stable. Table 1 compares the quantitative results of the assay

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FIG. 4. Inhibition curves using the ELISA method for serial twofold dilutions of three samples. The standard was an enriched fraction of cross-linked N-telopeptides from normal adolescent urine. The scale of antigen units on the lower axis refers to the quantity of bone collagen in the collagenase digest (bone digest plot); the other two inhibition curves (the peptide standard and untreated adolescent urine) are positioned arbitrarily relative to the lower axis.

1256

HANSON ET AL.

TABLE1. COMPARISON OF CROSS-LINKED N-TELOPEPTIDE CONCENTRATIONS IN URINEOF NORMAL CHILDREN AND ADULTS~ ~~

~~~~

Study groups

Children, years 0- 1 2-5 6-10 11-15 16-20 Premenopausal females (mean age 30 years; range 25-40 years) Postmenopausal females (c3 years after menopause; mean age 51 years; range 42-58 years) Males (mean age 30 years; range 24-40 years) Males (mean age 5 5 years; range 45-65 years)

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Meanb

Standard deviationb

Rangeb

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1639 689 497 429 192 36

915 294 667 215 147 10

102-4769 34-1752 90-1356 34-2158 34-780 10-89

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aNTX units by immunoassay. Data were collected on spot urine samples taken during the day or aliquots from 24 h collections. The immunoassay measurements (picomoles type 1 collagen per micromole creatinine) were calibrated using a standard of bacterial collagenase-digested human bone collagen and are expressed as equivalent moles of bone type I collagen (bone collagen equivalents [BCE]) normalized to creatinine (CT). bPicomoles BCE per pmole CT. clncrease over premenopausal adult females and adult males was highly significant (p < 0.01 by twotailed comparison of means).

applied to urine of children of increasing age and to adult females and males. Concentrations are expressed normalized to creatinine. In comparing males and premenopausal females with early postmenopausal females, a mean twofold elevation is seen postmenopausally, consistent with the previous finding of an increased bone resorption rate based on measurements of total urinary pyridinolines by HPLC.'I3) Figure 5 shows the relative consistency day to day for an individual premenopausal female subject monitored for 60 days by immunoassay of spot urine samples taken each day, with no restrictions on diet or time of sampling. The high outlying value came from a very dilute urine sample and consequently is probably not accurate. Measurements on serial samples collected over 24 h periods from individual subjects (results not shown) revealed a consistent trend to higher values at night (particularly in growing children and young adults), but daytime values after the morning void were quite consistent. Figure 6 plots the urinary concentrations of cross-linked Ntelopeptide against hydroxyproline (Fig. 6a) and total pyridinolines (Fig. 6b) for 23 different Paget's disease patients who differed widely in clinical severity. The correlation with urinary hydroxyproline was excellent but was not quite as good, however, with urinary pyridinolines. We believe this reflects the general imprecision of the complex HPLC assay for pyridinolines, including variable destructive losses during acid hydrolysis of urine (unpublished observations). Figure 7 plots the longitudinal decrease in excretion rates of urinary hydroxyproline and cross-linked N-telopeptides over a 6 month period from the initiation of oral bisphos-

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FIG. 5. A plot of serial daily measurement of the concentration of cross-linked N-telopeptides expressed per micromole creatinine in spot urine collections from a normal female (44 years, premenopausal). phonate therapy in an individual with severe Paget's disease of bone. The immunoassay data show a greater suppression of bone resorption (ratio of initial to final values) than the hydroxyproline data, consistent with the belief that hydroxyproline comes from other sources and is not a specific measure of bone resorption, particularly when resorption rates are low.

DISCUSSION The results presented here trace the development of a

1257

IMMUNOASSAY FOR BONE RESORPTION

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400

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600

FIG. 6. Plots of urinary cross-linked N-telopeptide concentrations against hydroxyproline (a) and pyridinolines (b) for 23 patients with Paget’s disease of bone. Urine samples (24 h) were collected on a low-gelatin diet. The inset in each graph shows the plot and correlation coefficient ( r 2 )for the same data set minus the high outlying point. (The predicted 99% confidence limits on the population correlation coefficients for the data in the inset in a are 0.80-0.97 and in the inset in b are 0.38-0.91.)

(N-telopeptide to helix and C-telopeptide to fragments from the N-telopeptide-to-helix site were the best candidates. First, they were the most abundant discrete peptide fragments containing hydroxylysylpyridinoline and lysylpyridinoline that were consistently recovered from urine samples. Second, this end of the molecule proved to be the source of about 60% of the lysylpyridinoline (i.e., deo~ypyridinoline(~~’1 in human bone collagen. Only 40% was contributed by the C-telopeptide-to-helix site (unpublished observations). Third, the N-telopeptide interactions that formed pyridinolines at this molecular site in bone collagen turned out to be predominantly a , ( [ )to 0 60 120 1 80 a2(I)and a2(I)to a#), a feature that distinguishes bone DAY type I collagen from other tissue type I collagens. The a,(I)N to CY,(I)N telopeptide product was much less abunFIG. 7. Monitoring the suppression of bone resorption during bisphosphonate therapy in one patient with Paget’s dant in bone collagen than predicted theoretically (unpubdisease treated orally with 30 mg risedronate per day from lished observations). In contrast, at the other end of the days 0-56. The rate of bone resorption was assessed in molecule, the C-telopeptide-to-helix cross-linking site, only fasting 2 h urine samples by measuring the concentration one interaction, aI(I)C to aI(I)C, is possible because the of cross-linked N-telopeptides and hydroxyproline relative a2(I)C telopeptide lacks a cross-linking lysine residue. (”) to creatinine. This C-telopeptide structure is therefore common to all tissues in which type I collagen is cross-linked by pyridinoline new immunoassay for monitoring bone resorption rates in residues and so on theoretical grounds will provide a less human subjects by urine analysis. A rational approach was specific index of bone breakdown. taken in designing the assay. We knew that the excretion Measuring the excretion rate in urine of the cross-linked rate of the pyridinoline cross-linking amino acids in urine N-telopeptide antigen appears to provide a direct and recorrelated well with bone resorption. (‘*-I4) Their assay by producible measure of bone resorption rate. The results HPLC, however, is tedious and prone to error and is a with patients with Paget’s disease strongly support this. poor prospect for a routine clinical test. The target peptides therefore behave as limit digestion Because the pyridinolines were known to be excreted products resulting from the endogenous proteolytic degrapredominantly in peptide-bound form,(’.e1we set out to dation of bone collagen fibrils. The cross-linked N-teloidentify a specific pyridinoline-containing fragment of peptides presumably resist further proteolysis (in bone, type I collagen in urine that could provide a quantitative liver, and kidney) because they present compact structures. index of the total pyridinoline pool. From studies of cross- Quantitative data based on the new ELISA method can be linked peptides in Paget’s disease urine”” and normal ado- expressed as absolute units of peptide, as equivalent moles lescent urine, we established that of the two pyridinoline- of bone-specific pyridinolines, or simply as equivalent forming sites within the bone type I collagen molecule moles of bone collagen. These last units have the merit of

HANSON ET AL.

1258 being most readily interpretable in terms of mass of bone resorbed. In summary, this immunoassay shows considerable promise as a convenient, noninvasive index of the systemic rate of human bone resorption. It has potential application in the study of normal subjects and in the diagnosis and therapeutic monitoring of patients in whom bone metabolism is believed to be disturbed. For example, in a study of early postmenopausal women followed by serial sample analysis for 9 months, the immunoassay was able to distinguish high-resorbing subjects with a much greater longitudinal consistency than urinary hydroxyproline or pyridinolines measured by HPLC on the same samples.(26’

ACKNOWLEDGMENTS This work was supported by research grants from Ostex International, Inc., Seattle, WA 98134 and the National Institutes of Health (AR36794, AR37318, and RR05543). We thank Lowell Ericsson for the mass spectral analyses, Ms. Shao Ping for superb technical assistance, and Ms. Kae Pierce for preparing the manuscript. This work was presented in part at the annual meeting of the American Society of Bone and Mineral Research in San Diego, August 1991 (J Bone Miner Res 6(Suppl 1):Abstract 669).

11. Robins SP, Stewart P, Astbury C. Bird HA 1986 Measure-

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22.

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Address reprint requests to: Dr. David R . Eyre Department of Orthopaedics RK-10 University of Washington Seattle, WA 98195 Received for publication March 17, 1992; in revised form June 3, 1992; accepted June 10, 1992.