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Clinical Chemistry 44: 2126-2132, 1998;
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(Clinical Chemistry. 1998;44:2126-2132.)
© 1998 American Association for Clinical Chemistry, Inc.

Enzymes and Protein Markers

Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation

Thomas G. Rosano1,a, Robert T. Peaston2, Henry G. Bone3,2, Henning W. Woitge4, Roger M. Francis5, and Markus J. Seibel4

1 Department of Pathology and Laboratory Medicine, Division of Laboratory Medicine, Albany Medical College, Albany, NY 12208.

2 Clinical Biochemistry, Freeman Hospital, Newcastle upon Tyne NE7 7DN, United Kingdom.

3 Henry Ford Hospital, Detroit, MI 48202.

4 Department of Medicine, Division of Endocrinology and Metabolism, University of Heidelberg, Bergheimerstrasse 58, D-69115 Heidelberg, Germany.

5 Musculo Skeletal Unit, Freeman Hospital, Newcastle upon Tyne NE7 7DN, United Kingdom.
a Author for correspondence. Fax 518-262-4337.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We evaluated an automated chemiluminescence immunoassay (CLIA) developed for the measurement of urinary free deoxypyridinoline (DPD). The new DPD method by CLIA is based on the competition of DPD with particle-bound pyridinoline for a limited amount of monoclonal mouse anti-DPD antibody. Total imprecision (CV) was 3.2–9.0% at 30–270 nmol/L. Regression analysis of urinary DPD concentration (second morning-void) measured by CLIA (y) and enzyme immunoassay (EIA) for adult volunteers (n = 449) with and without bone disease revealed a best fit equation of: y = 1.08 ± 0.03x - 1.15 ± 0.98 nmol/L (r = 0.964, Sy||x = 14 nmol/L). CLIA and EIA methods were correlated with HPLC measurement of urinary free DPD (r = 0.846 and 0.871, respectively). For healthy adults, the creatinine-normalized excretion of DPD (mean ± SD) measured by CLIA for 61 men (4.1 ± 1.2 µmol DPD/mol creatinine) and 76 premenopausal women (5.3 ± 1.8 µmol DPD/mol creatinine) did not differ significantly (P >0.05) from DPD excretion measured by EIA, and both immunoassays showed a significant gender difference (P <0.001) in reference intervals. In a clinical trial, DPD excretion (µmol DPD/mol creatinine) measured by CLIA differed substantially from the reference population for 54 untreated pagetic (12.7 ± 8.0 SD), 255 untreated osteoporotic (7.5 ± 4.1), 21 osteomalacic (12.4 ± 8.5), 17 primary hyperparathyroid (9.4 ± 4.4), and 14 secondary hyperparathyroid (9.2 ± 5.1) patients. Clinical sensitivities of the CLIA and EIA methods range from 38% to 80% in bone disorders and limit the use of the DPD measurement in disease detection. DPD excretion after pamidronate treatment in a subgroup of the pagetic patients fell dramatically as assessed by CLIA or EIA. We conclude that the automated CLIA method for DPD is a convenient and reliable method that may aid in the evaluation and management of bone disease and is applicable to high volume testing in the routine clinical laboratory.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Population aging and the development of more effective antiresorptive therapy for bone disorders, particularly osteoporosis, have increased the need for noninvasive biochemical markers of bone resorption as an adjunct or alternative to bone mineral density and morphometry studies (1). Traditional biochemical markers of bone resorption, including urinary calcium and hydroxyproline, lack both clinical sensitivity and specificity. More recent advances in biochemical techniques have led to the use of pyridinium cross-links of collagen as clinical markers of bone resorption. The deoxypyridinoline (DPD)1 cross-link has the greatest specificity for bone, and several recent studies have confirmed urinary DPD as a marker of bone resorption in Paget disease (2), postmenopausal osteoporosis (3)(4)(5)(6), hyperparathyroidism (7)(8), rheumatoid arthritis (9)(10), prostate cancer (11)(12)(13)(14)(15), and other malignant bone diseases (16)(17)(18)(19)(20). Preliminary studies in patients with osteoporosis also indicate that urinary free DPD measurement may predict the risk of fracture when used alone and in conjunction with densitometry (21)(22). Urinary measurement of DPD has also been used to evaluate response to treatment in metabolic bone disease (23)(24)(25)(26)(27)(28) and has been correlated with radiolabeled calcium and bone scan data (4)(15).

Routine measurement of DPD in the clinical laboratory was initially limited by the nonspecificity of immunoassay methods and the use of technically demanding chromatographic methods. An initial immunoassay for pyridinoline cross-links, using polyclonal antiserum that recognized the free form of both DPD and pyridinoline, revealed increased urinary concentrations of the cross-link compounds in metabolic osteopathies but did not provide tissue-specific information and therefore lacked clinical specificity (29). HPLC methods allowed both a direct assessment of free DPD concentration and a posthydrolysis determination of total DPD. Because the measurement of free and total DPD are highly correlated in nonpathological and disease states (30)(31), simpler immunoassays have been developed to selectively measure the free DPD component. Generation of a mouse monoclonal anti-DPD antibody led to the development and commercialization of an enzyme immunoassay (EIA) method (Pyrilinks®-D, Metra Biosystems, Inc.) for free DPD in urine (32). Chiron Diagnostics has recently developed an automated chemiluminescence immunoassay (CLIA) for urinary free DPD, using the same monoclonal antibody utilized in the commercial EIA method. The CLIA method, performed on the ACS:180 Chemiluminescent Immunoassay System, is designed for rapid, high-volume testing in the routine clinical laboratory. The purpose of the current study is to evaluate the analytical characteristics of the automated method and to examine clinical performance in a large number of patients with metabolic bone disorders.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
reference population
Adult reference intervals for DPD by CLIA and EIA were determined with urine samples (second morning-void) collected from 137 healthy adult volunteers ranging in age from 25 to 55 years, including 61 men and 76 premenopausal women. Eligibility criteria included informed consent (institution approved), admission of good health, regular menstruation in women, abstinence from medications affecting renal or bone function, and absence of current or prior bone, cancer, renal, or infectious disease.

patients
For evaluation of clinical performance and correlation of DPD concentration by CLIA and EIA, urine samples were obtained, by institutionally approved informed consent, from 312 patients with Paget disease, osteoporosis, osteomalacia, primary hyperparathyroidism, or secondary hyperparathyroidism. Posttreatment urine samples were collected in a subset of patients with Paget disease to evaluate the effect of antiresorptive therapy. For comparison of immunoassay and HPLC measurements of DPD, urine samples were collected from an additional cohort of 513 healthy males, healthy females, and patients with Paget disease, primary hyperparathyroidism, secondary hyperparathyroidism, or postmenopausal osteoporosis. A diagnosis of osteoporosis was made using the World Health Organization (WHO) criteria (33) when the lumbar spine or femoral neck bone mineral density measurement was more than 2.5 SD below the mean for young adults (T score <2.5). The majority of patients had postmenopausal or idiopathic osteoporosis; however, a few had underlying secondary causes of osteoporosis (34). Although some patients had a past history of fragility fractures, no symptomatic fractures were reported in the preceding 6 months. The diagnosis of Paget disease of bone was based on the finding of typical radiographic features of that disorder, confirmed by clinical and biochemical findings. The diagnosis of primary hyperparathyroidism was based on persistent hypercalcemia with persistent increase or nonsupression of the serum intact parathyroid hormone. In addition, clinical evaluation excluded other hypercalcemic disorders. Secondary hyperparathyroidism was diagnosed in patients with increased parathyroid hormone, without increased serum Ca, in the presence of established causes of secondary hyperparathyroidism, such as renal insufficiency, renal Ca wasting, or hypovitaminosis D. Urine specimens for reference and clinical subjects were collected during the second morning-void, and urine aliquots were stored under stable conditions (35) at -20 °C until analyzed.

determination of urinary free dpd by clia
The automated CLIA method for DPD developed by Chiron Diagnostics is a competitive immunoassay using direct chemiluminescent technology. DPD in the urine sample competes with pyridinoline, which is covalently coupled to paramagnetic particles in the solid phase, for a limited amount of mouse monoclonal anti-DPD antibody. The anti-DPD antibody is bound to polyclonal goat anti-mouse antibody labeled with an acridinium ester. The assay was performed on ASC:180 automated chemiluminescent immunoassay system in accordance with the manufacturer's protocol. Urine pools supplemented with DPD concentrations covering the analytical range of the method were used to assess analytical precision. Pool aliquots were stored at -20 °C and thawed on the day of analysis. Precision was evaluated according to guidelines established by the National Committee for Clinical Laboratory Standards (36).

determination of urinary free dpd by eia
The EIA method for DPD (Pyrilinks-D assay, Metra Biosystems, Inc.) is a competitive EIA performed in a microtiter well strip format, utilizing a monoclonal anti-DPD antibody coated on the strip to capture DPD. DPD in the sample competes with conjugated DPD-alkaline phosphatase for the antibody, and the enzyme activity is detected with p-nitrophenyl phosphate substrate and measurement of reaction product at 405 nm. Data reduction for the EIA method was performed with a Metrafit, Ver. 1.10, computer program. DPD measurements by CLIA and EIA were performed with the same anti-DPD mouse monoclonal antibody, and results are expressed in nanomoles or normalized for the urinary concentration of creatinine, which is determined by an alkaline picrate method.

determination of urinary free dpd by hplc
Urinary concentration of free DPD was measured by an automated reversed-phase, ion-pair HPLC technique as described earlier (37)(38). Unhydrolyzed urine was extracted by an automated procedure before gradient chromatography with 170–300 mL/L acetonitrile. The column eluate was monitored fluorometrically, and free DPD was quantified by external and internal standardization techniques (39). The mean imprecision (CV) of the HPLC assay for free DPD was 3.3% (intraassay) and 7.8% (interassay).

data reduction
Urinary DPD excretion was expressed in µmol DPD/mol creatinine. Nonparametric statistics were used to establish reference intervals for DPD excretion, and the gender-specific 90th percentiles were used to assess abnormal results in patients with metabolic bone disease. Clinical sensitivity was defined as the percentage of affected individuals in each disease category with DPD excretion concentrations exceeding the 90th percentile for the gender-specific reference populations. Linear regression was performed to assess correlation and agreement between methods, and a two tailed t-test was used to determine probability of difference between groups. A P value <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The relative light units for the method calibration and for analysis of the reference material in five consecutive analytical runs performed over a 10-day period are displayed in Fig. 1 . The precision of the DPD measurement by CLIA was determined by replicate analysis of thawed aliquots from five control pools that were supplemented with DPD concentrations that extended over the analytical range of the method. Each control pool was analyzed in triplicate in 15 analytical runs. The precision profile displayed in Fig. 2 shows total assay CV of 3.2–9.4%, with acceptable reproducibility at concentrations as low as 30 nmol/L.



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  		Figure 1. Calibration curve stability for DPD analysis by CLIA.

({bullet}), master curve; ({circ}), analytical run 1; ({blacksquare}), analytical run 2; ({square}), analytical run 3; ({diamond}), analytical run 4; (), analytical run 5; ({circ}), analytical run 6.



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  		Figure 2. Total precision profile for DPD measurement by CLIA.

Each control point represents the total CV plotted against the mean DPD concentration determined by triplicate analysis in 15 analytical runs.

Adult reference intervals were determined by the CLIA and EIA methods (Table 1 ). The creatinine-normalized intervals (two-tailed 90th percentile) measured by CLIA for men (1.7–5.9 µmol DPD/mol creatinine) and women (3.1–8.1 µmol DPD/mol creatinine) showed a significant gender difference (P <0.001). The DPD reference intervals by EIA for men (1.9–5.6 µmol DPD/mol creatinine) and women (3.0–7.6 µmol DPD/mol creatinine) did not differ significantly from CLIA intervals determined in the same cohort (P >0.05).


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  		Table 1. Nonparametric 90th percentile intervals for DPD1 by CLIA and EIA.

Correlation of urinary DPD concentration by CLIA and EIA for reference and clinical samples is shown in Fig. 3 . Regression analysis of data for 449 subjects revealed an r of 0.964 and a best-fit equation: y = 1.08 ± 0.01x - 1.15 ± 0.98. In an additional set of 513 clinical samples, correlation of urinary free DPD concentrations by CLIA (y) and HPLC (x) measurements revealed an r of 0.846 and a best fit equation: y = 0.88 ± 0.02x 4.75 ± 1.72 (Fig. 4 ). The regression equation for EIA (y) and HPLC (x) data analysis was: y = 0.73 ± 0.02x 11.2 ± 1.26 (r = 0.871).



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  		Figure 3. Correlation of DPD concentrations determined by CLIA and EIA.



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  		Figure 4. Correlation of DPD measurement by CLIA and HPLC.

The clinical performance of the CLIA and EIA methods was also evaluated in bone disease groups (Fig. 5 ). Mean urinary DPD excretion, determined by CLIA and reported as µmol DPD/mol creatinine (± SD), differed significantly from the reference populations for patients with untreated Paget disease of bone (12.7 ± 8.0, P <0.001, n = 54), untreated osteoporosis (7.5 ± 4.1, P <0.001, n = 255), osteomalacia (12.4 ± 8.5, P <0.001, n = 21), primary hyperparathyroidism (9.4 ± 4.9, P <0.005, n = 17), and secondary hyperparathyroidism (9.2 ± 5.1, P <0.01, n = 14). In a parallel assessment of DPD excretion measured by EIA (Fig. 5 ), mean urinary DPD excretion also differed significantly from the reference population for untreated pagetic (13.1 ± 8.7, P <0.001), osteoporotic (7.7 ± 3.8, P <0.001), osteomalacia (12.9 ± 10.1, P <0.001), primary hyperparathyroid (9.1 ± 3.6, P <0.001), and secondary hyperparathyroid (8.6 ± 5.8, P <0.05) patients.



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  		Figure 5. Box plot of DPD concentrations measured by CLIA and EIA in reference population and patients with bone disorders.

The boxes represents the 25th to 75th percentile limits, with the median value represented by the horizontal line. ({bullet}), values 1.5- to 3-fold greater or less than the 25th to 75th pecentile limits. ({circ}), values more than 3-fold greater than the 25th to 75th pecentile limits.

The clinical sensitivity of DPD measurement by CLIA and EIA in bone disorders, using gender-specific 90th percentile reference limits determined in healthy men and women, is compared in Fig. 6 . Clinical sensitivity of the CLIA method ranged from 80% for pagetic to 38% for osteoporotic patients and compared closely with the clinical sensitivity of the EIA method.



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  		Figure 6. Comparison of clinical sensitivity of DPD measured by CLIA and EIA methods.

DPD was also measured pre- and posttherapy in a subset of pagetic patients (n = 23), using the CLIA and EIA methods. Fig. 7 shows the effect of pamidronate therapy on normalized DPD excretion in the pagetic patients. The posttreatment DPD concentrations (µmol DPD/mol creatinine, mean ± SD) by CLIA (7.6 ± 3.8) and by EIA (7.8 ± 5.1) were significantly lower (P <0.05) than the corresponding pretreatment DPD concentrations by CLIA (13.6 ± 10.1) and by EIA (14.3 ± 10.6). The greatest posttreatment reduction in DPD excretion occurred in patients with increased baseline excretion concentrations.



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  		Figure 7. DPD excretion concentrations measured by CLIA in pagetic patients before and after pamidronate therapy.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Because of the socioeconomic impact within the aging population of the Western industrialized countries, metabolic bone diseases have recently gained increasing attention both in the general public and in clinical medicine. However, diagnosis and therapeutic monitoring of these disorders are often hampered by the lack of useful measures of disease activity and progression. In the diagnosis of osteoporosis, for example, conventional clinical and laboratory indicators are often within reference values or ambiguous, and radiographic evaluation is effective only at the late stage of the disease. Thus, vertebral and hip fractures are still the most common primary manifestation of osteoporotic bone disease, accounting for >90% of all hip and spine fractures among elderly women (40). Consequently, there is a great need for validation and availability of noninvasive biochemical markers of bone resorption for use in routine clinical practice.

In this study we show that the analytical performance of an automated CLIA for urinary free DPD is comparable with an established EIA method (32). The total assay precision is <10% at DPD concentrations as low as 30 nmol/L, and DPD measurement by CLIA correlates closely EIA measurement over the full range of nonpathological and pathologic DPD concentrations. Reference intervals are statistically indistinguishable between the immunoassays, and both methods showed gender-specific reference intervals that are modestly higher in women. The correlation of CLIA with HPLC is weaker than immunoassay comparisons. This observation, which has been reported by others (41), may be attributable to assay differences in the discrimination of free DPD components or differences in calibration material arising from a lack of standardization in reference material.

In the evaluation of creatinine-normalized excretion of DPD in disease groups, mean DPD measurements by CLIA is significantly increased in Paget disease, osteoporosis, osteomalacia, and hyperparathyroidism of primary and secondary etiology. Although clinical sensitivity is greatest in detection of bone resorption in pagetic patients, the concentration of clinical sensitivity for each of the diseases would limit the use of the DPD measurement in disease detection. In osteoporotic patients, for example, <50% of the patients demonstrate DPD excretion concentrations greater than the 90th percentile of the gender-specific reference intervals. A significant reduction in normalized DPD excretion was observed in pagetic patients treated with biphosphonates, indicating a clinical value in monitoring DPD response to antiresorptive therapy.

In summary, measurement of urinary free DPD by the automated CLIA method is rapid, reliable, and provides equivalent results to an established manual immunoassay method. Adaptability of the new automated method in the routine clinical laboratory should allow the wider application of urinary free DPD measurement in the monitoring of patients with bone pathology and metabolic bone disease.


   Acknowledgments
 
We thank Nayana Parikh of the Henry Ford Hospital Bone and Mineral Laboratory, Patricia Ortega, formerly of the Bone and Mineral Division of the Henry Ford Hospital, and Marian Dybas of the Albany Medical Center Clinical Chemistry Laboratory for assistance in this study.


   Footnotes
 
2 Present address: Michigan Bone and Mineral Clinic, Detroit, MI 48236.

1 Nonstandard abbreviations: DPD, deoxypyridinoline; EIA, enzyme immunoassay; and CLIA, chemiluminescence immunoassay.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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H. W. Vesper, L. M. Demers, R. Eastell, P. Garnero, M. Kleerekoper, S. P. Robins, A. K. Srivastava, G. R. Warnick, N. B. Watts, and G. L. Myers
Assessment and Recommendations on Factors Contributing to Preanalytical Variability of Urinary Pyridinoline and Deoxypyridinoline
Clin. Chem., February 1, 2002; 48(2): 220 - 235.
[Abstract] [Full Text] [PDF]

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N. B. Watts
Clinical Utility of Biochemical Markers of Bone Remodeling
Clin. Chem., August 1, 1999; 45(8): 1359 - 1368.
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