Components of biological variation of biochemical markers of bone turnover in Paget’s bone disease

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Components of Biological Variation of Biochemical Markers of Bone Turnover in Paget’s Bone Disease ´ S,3 P. PERIS,4 N. GUAN ˜ ABENS,2,4 A. MONEGAL,4 F. PONS,5 and L. ALVAREZ,1,2 C. RICO 1 A. M. BALLESTA Services of 1Clinical Biochemistry, 4Rheumatology, and 5Nuclear Medicine, Hospital Clinic, University of Barcelona, Barcelona, Spain 2 Metabolic Bone Diseases, Institut D’Investigacions Biome`diques August Pi i Sunyer, Barcelona, Spain 3 Service of Biochemistry, Hospital General Vall d’Hebron, Barcelona, Spain

Introduction

The aims of this study were to evaluate the components of biological variation of the new markers of bone turnover in patients with Paget’s bone disease and to compare the results with data obtained in healthy subjects. Fifteen patients with Paget’s disease in a stable period of the disease and 12 healthy premenopausal women were included for a 1 year follow-up study. Within- and between-subject biological variation, indices of individuality, and critical differences were evaluated for the following biochemical markers: in serum, total (tAP), and bone (bAP) alkaline phosphatases, procollagen type I N-terminal propeptide (PINP) and ␤-carboxyterminal telopeptide of type I collagen (sCTx); in urine, hydroxyproline (Hyp), and amino (NTx) and ␤-carboxyterminal (CTx) telopeptides of collagen type I. Serum markers of bone turnover showed lower biological variability than urinary markers. Within-subject biological variation was higher in pagetic patients than in healthy subjects for all serum markers. In both groups, bAP presented the lowest within-subject biological variation. In pagetic patients, all markers presented indices of individuality of <0.6, indicating their usefulness for patient monitoring. Critical differences were lower for serum markers than for urinary markers. Among pagetic patients, serum bAP and PINP showed the lowest critical differences with values close to 30%, whereas urinary CTx presented the highest critical differences (near 70%). Conversely, in healthy subjects, tAP was the marker with the lowest critical differences, being two-fold higher in pagetic patients. This study confirms the lower sensitivity of urinary markers to detect significant changes and indicates that data obtained on biological variations from healthy populations cannot always be extrapolated to pathological conditions. In addition, serum bAP and PINP seem to be the markers that best reflect a significant change in activity of Paget’s disease. (Bone 26:571–576; 2000) © 2000 by Elsevier Science Inc. All rights reserved.

Biochemical markers of bone turnover have proven to be of value for assessing bone metabolism. Recently, new specific markers have been developed, including immunoassays for serum bone alkaline phosphatase (bAP), procollagen type I Nterminal propeptide (PINP), and ␤-telopeptide carboxyterminal of type I collagen, and for telopeptides carboxy-(CTx) and aminoterminal (NTx) of type I collagen in urine. These markers have shown higher diagnostic efficacy than the traditional serum total alkaline phosphatase (tAP) and urinary hydroxyproline (Hyp).8,11 An important issue is whether the new markers provide early and reliable information about bone turnover and response to therapy of metabolic bone diseases. The usefulness of bone markers in the evaluation of disease activity and in the monitoring of antiresorptive therapy in Paget’s bone disease has been demonstrated in previous studies.1,9 Nevertheless, many investigators and users have pointed out problems concerning the variability of these markers, especially those determined in urine, which are further increased by the far-from-optimal standardization of creatinine corrections.3,17,21,25 Fraser and Harris proposed a widely used model to study the components of biological variation, the within- and betweensubject variability.14 This model is based on healthy subjects, generally volunteers, under strict protocol-controlled conditions (e.g., same time of day, same life conditions for the period of the study, same person doing blood withdrawal, same analytical procedure). No particular conditions regarding number of subjects, frequency of sampling, and time duration of sampling is advocated. Analytical and biological components of variation must be calculated using the analysis of variance (ANOVA) test or the formulas proposed by these investigators. Most studies have evaluated the biological variation from data obtained from healthy subjects.12,13,31,33,35 However, it is not clear that biological variation components derived from healthy subjects can be extrapolated to pathological situations.31 In this sense, it is unknown whether the biological variation of biochemical markers of bone turnover has the same behavior in healthy subjects than in patients with Paget’s disease. Hypothetically, if biochemical markers of bone turnover show higher variation in patients with Paget’s disease than in healthy subjects, a change between two consecutive observations based on values obtained from healthy subjects will not reflect a modification in Paget’s disease activity. Therefore, the aims of the present study were: (1) to calculate the within- and between-subject biological variation, the indices

Key Words: Amino (NTx) and carboxyterminal (CTx) telopeptides; Bone alkaline phosphatase (bAP); Procollagen type I N-terminal propeptide (PINP); Monitoring; Within-subject variability; Critical differences.

Address for correspondence and reprints: Luisa Alvarez, M.D., Servicio Bioquı´mica Clı´nica, Hospital Clinic, C/Villarroel 170, 08036 Barcelona, Spain. E-mail: [email protected] © 2000 by Elsevier Science Inc. All rights reserved.

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of individuality, and the critical differences between serial results of the new biochemical markers of bone turnover in patients with stable Paget’s disease; (2) to compare these results with data obtained in healthy subjects to determine whether extrapolation to pathological situations can be done; and (3) to evaluate if the knowledge of the biological variability of such markers can improve the monitoring of Paget’s disease activity. Materials and Methods Subjects Twelve healthy premenopausal women, ages 28 – 49 years (mean ⫾ SD: 40 ⫾ 6.2 years), among the healthy staff of the Departments of Rheumatology and Clinical Biochemistry, volunteered to participate in a 1 year follow-up study during 1998 – 1999. All subjects stated they were free of disease at the time of the study and not taking any medication that could affect bone turnover. Fifteen patients with stable and asymptomatic Paget’s disease (ten men and five women, mean ⫾ SD: 63 ⫾ 10 years) were included in the study. Seven patients had a monostotic lesion and eight patients had poliostotic disease. The diagnosis of the disease was documented by X-ray and bone scan in all patients. Liver and renal function tests were normal in all patients and none had been treated with any medication before and/or during the study. The biological components of variation of biochemical markers of bone turnover in patients with Paget’s disease were obtained in a stable period of the disease as verified by quantified bone scintigraphy; that is, there were no significant changes in activity between two bone scans made during the study period. Bone scintigraphy was performed 2 h after intravenous injection of 740 MBq (20 mCi) of 99mtechnetium-hydroxymethylene diphosphonate. To provide a more accurate interpretation, semiquantitative analysis was performed.2 From two to five samples (median of four samples) were obtained from all healthy subjects and pagetic patients over a period of 1 year. The difference in time between the first and last sample of each subject was 1 year in all cases. All subjects gave informed consent for their participation, and the study was approved by the ethics committee of the hospital. Biochemical Methods Blood and second morning urine from each subject were collected between 8:00 and 10:00 A.M., after an overnight fast. The samples were divided into aliquots and stored at ⫺20°C until assayed. The samples were analyzed in real time—that is, at different times for each individual patient. Serum tAP activity was measured by a spectrophotometric kinetic assay, according to the recommendations of the Scandinavian Committee for Clinical Chemistry and Clinical Physiology, using a DAX-72 analyzer (Bayer Diagnostics Technicon, Tarrytown, NY). Serum bAP was assayed by immunoradiometric assay using a Tandem Ostase (Hybritech, Liege, Belgium). Serum PINP determination was made by radioimmunoassay using a kit from Orion Diagnostica (Espoo, Finland). Serum ␤-CTx (sCTx) and urinary ␤-CTx and NTx were measured by enzyme immunoassays (Serum CrossLaps One-step ELISA, CisBio International, Gif-sur-Yvette, France and CrossLaps ELISA, Osteomark, Ostex, Seattle, WA). Urinary Hyp was measured by high-performance liquid chromatography. Urine determinations were expressed in relation to creatinine excretion. Measurement of urinary creatinine was performed using a Cobas Mira S

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analyzer with an assay based on the modified Jaffe method (Roche Diagnostics Kit, Basel, Switzerland). Analytical Imprecision Serum tAP was controlled using Precinorm U and Precipath U (Roche Diagnostics). Serum bAP, PINP, and sCTx were controlled with control material supplied by each respective manufacturer’s kit at normal and pathological levels. Day-to-day imprecision was assessed during 10 days using the control materials just mentioned. For urinary markers, due to the lack of control materials, day-to-day imprecision was obtained from 74 human specimens. As with serum markers, they were also obtained at normal (n ⫽ 38) and pathological (n ⫽ 36) levels. Analytical imprecision (sa) was calculated using the following formula: (s a2) ⫽ 兺(d 2 ⫺ d 1) 2/ 2n where d2 and d1 are the results obtained from urine samples analyzed twice, and n is the number of urine samples analyzed. Analytical imprecision was expressed by means of the coefficient of variation (CVa): CVa ⫽ 100 ⴱ s a /mean where mean is the average of control materials results for serum markers and average of the d2 results for urinary markers. Components of Biological Variation All the calculations detailed in what follows were applied to the results obtained for the analysis of the seven biochemical markers of bone turnover determined in both groups: healthy subjects and patients with Paget’s bone disease. According to the model of Fraser and Harris,14 the ANOVA test was used to estimate within-subject plus analytical variation 2 (si⫹a ) expressed as the weighted mean of variances from all subjects studied in each group. Within-subject (intraindividual) biological variation (s2i ) was calculated by a subtraction step from the two previous variables: 2 ⫺ s a2 s 2i ⫽ s i⫹a

The within-subject variation is the fluctuation of values around the homeostatic set point from time to time in the average subject, and indicates how much a given quantity can change in a single person. Between-subject (interindividual) biological variation (s2g) was obtained by subtracting from the total variation (s2total) the within-subject plus analytical variation: 2 2 s g2 ⫽ s total ⫺ s i⫹a

where s2total is the total variance of all data from all subjects. The between-subject variation is the variance of homeostatic set points among subjects and describes the variation between persons in a group. The within- and between-subject biological variations were expressed as coefficients of variation (CVi [%] and CVg [%], respectively). The individuality index (II) was calculated as the ratio: II ⫽ CVi⫹a/CVg This index provides information about the usefulness of population-based reference ranges. If the index is low (⬍0.6), an abnormal value for a person is likely to appear inside the

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Figure 1. Fluctuations of the ratios of each result in relation to the first value in pagetic patients for some of the markers analyzed (serum total alkaline phosphatase, tAP; procollagen type I N-terminal propeptide, PINP; urinary hydroxyproline, Hyp; and aminoterminal telopeptide of type I collagen, NTx). The hatched area represents the amplitude of ratio fluctuations in healthy subjects.

reference interval; conversely, the marker is useful for monitoring purposes. A high index (⬎1.4) is able to separate abnormal and normal values.14 The critical differences (CD) at a significant level of p ⬍ 0.05 were calculated according to the following formula: CD ⫽ 1.96 ⴱ √2 ⴱ √(CV2i ⫹ CVa2) The CD, also called the least significant change and reference change values, is the minimum difference between two successive measurements in an individual that can be considered to reflect a true biological change. Evaluation of Stability of Disease The stability of the disease in the pagetic patients was evaluated by bone scan, as previously indicated, and was also verified by means of a graphic system from the laboratory results. For each marker, a ratio of each result in relation to the first value was obtained to minimize interindividual variation and to facilitate the detection of relative changes among patients.10,26,27 This ratio was analyzed individually for each subject both patients and controls. Fluctuation of ratios for various bone markers are shown in Figure 1. Disease stability was confirmed when the ratio fluctuations were similar in pagetic patients and in healthy subjects. Results of bone markers are expressed as mean ⫾ SD. p ⬍ 0.05 was considered statistically significant. Results Stability of Disease Patients with Paget’s disease remained stable during the study period. No significant changes were observed in scan activity indices or in bone markers at the end of the study, in relation to baseline values (mean ⫾ SD: tAP [U/L], 562 ⫾ 425 vs. 584 ⫾ 424; bAP [ng/mL], 72 ⫾ 62 vs. 70 ⫾ 61; PINP [ng/mL], 176 ⫾ 79 vs. 172 ⫾ 86; sCTx [pmol/L], 5545 ⫾ 1645 vs. 6283 ⫾ 1741; Hyp [nmol/L 䡠 mg creatinine], 193 ⫾ 106 vs. 199 ⫾ 84; CTx [␮g 䡠 mmol/L creatinine], 469 ⫾ 241 vs. 498 ⫾ 326; NTx [nmol/L BCE 䡠 mmol/L creatinine], 250 ⫾ 126 vs. 257 ⫾ 146; p ⫽ n.s.). The ratio plots for all healthy subjects showed fluctuations between 0.8 and 1.2 for serum markers and between

0.5 and 1.7 for urinary markers. Ratio plots for some of these markers in Figure 1 show that fluctuations in bone markers of pagetic patients fell within the ratio fluctuations range of healthy subjects, demonstrating that there were no positive or negative trends that could indicate modification of the patient status. The only exception is the PINP plot, which showed various points outside the ratio fluctuation range. Analytical Imprecision Analytical imprecision was calculated using data from the internal quality control protocol that analyzes control material at two levels (normal and pathological) on the day that the analytical procedure was performed. An average was calculated for monthly analytical coefficients of variation for each control material. The values obtained for analytical imprecision throughout the study are shown in Table 1. Among markers of bone formation, serum tAP showed the lowest coefficients of variation at both normal and pathological levels (3.1% and 1.5%, respectively), whereas serum bAP showed the highest (8.8% and 7.5%). Among markers of bone resorption, the lowest coefficients of variation were obtained for Hyp at normal levels (4.8%), followed by CTx at both levels, normal and pathological (6.2% and 6.3%, respectively). Components of Biological Variation Table 2 shows the components of biological variation of the markers found in the study and are expressed in terms of coefficient of variation (%). Serum markers of bone turnover showed a lower within-subject variability than urinary markers. Pagetic patients showed higher within-subject biological variation than healthy subjects in bone formation markers, especially in tAP, whereas within-subject biological variation in resorption markers were similar in both groups. Serum CTx, however, also showed a high within-subject biological variation. In addition, bAP was the marker with the lowest within-subject biological variation in both groups. Except for sCTx, patients with Paget’s disease showed a higher between-subject variability in all biochemical markers when compared with healthy subjects. The indices of individuality (II) and critical differences (CD) are shown in Table 3. In pagetic patients, all markers showed an II of ⬍0.6. Except for Hyp and CTx, similar results were obtained

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Table 1. Day-to-day analytical imprecision Normal levels

Pathological levels

Marker

Mean

SD

CV (%)

Mean

SD

CV (%)

tAP (U/L) bAP (ng/mL) PINP (ng/mL) sCTx (pmol/L) Hyp (nM/mg creatinine) CTx (␮g/mM creatinine) NTx (nM BCE/mM creatinine)

144.0 23.8 35.2 2807.0 82.5 169.0 36.0

4.5 2.1 2.0 258.0 4.0 10.6 3.3

3.1 8.8 5.6 9.2 4.8 6.2 9.2

557.0 89.3 80.6 ND ND 352.0 124.0

8.2 6.7 5.9 ND ND 22.1 7.8

1.5 7.5 7.4 ND ND 6.3 6.3

KEY: BCE, bone collagen equivalents; CV, coefficient of variation; ND, not determined; tAP, total alkaline phosphatase; bAP, bone alkaline phosphatase; PINP, procollagen type I N-terminal propeptide; sCTx, serum ␤-carboxyterminal telopeptide of type I collagen; Hyp, hydroxyproline; CTx, ␤-carboxyterminal telopeptide of type I collagen; NTx, aminoterminal telopeptide of type I collagen.

in healthy subjects. These markers showed II values of 0.92 and 0.74, respectively. The CD was lower for serum than for urinary markers. Urinary CTx showed the highest CD, whereas serum bAP and PINP were the markers with the lowest CD in pagetic patients. It should be pointed out that the CD of serum tAP was twofold higher in pagetic patients than in healthy subjects. Critical differences were slightly higher in pagetic patients than in healthy subjects for bone formation markers, whereas the CD of resorption markers was higher in healthy subjects than in pagetic patients. Discussion This study shows the usefulness of the analysis of biological variation of bone markers for improving the knowledge of bone cell activity in Paget’s disease and for assessing the activity of the disease and the individual’s biochemical response to therapy. Therefore, our data show differences between healthy subjects and pagetic patients in the biological variability of some markers, particularly in tAP. The analytical imprecision found in this study for tAP reached the analytical goal proposed by Fraser et al.,15 indicating the appropriateness of the analytical procedure used in our laboratory for this marker. Quality specifications are still not available for the other markers. The study of the biological variation of biochemical markers of bone turnover was performed in a group of patients with stable Paget’s disease. The results obtained are reliable, because the stability was confirmed by means of quantified bone scan and by means of a graphic system in the laboratory. Although from some pagetic patients we had only two samples available during the 1 year period, the ratio fluctuations of these patients for all markers were in the same range as those observed in the other patients. For this reason, there is no evidence that the components of

biological variation found in these patients would be different from those found in the other patients. Despite the lack of data dealing with this type of study, the majority of the studies used graphic models to demonstrate stability. We can plot using differences or using ratios to show the various results found for each subject studied. In both cases, each analytical result was compared with the previous one. Ratio plots are the most practical when various analytes are included in the study, because the same scale can be used. The graphic system allows us to verify the stability of disease status in the group of patients for all markers, except for PINP. Presently, we do not have a convincing explanation for this finding. Nevertheless, because previous studies have indicated a high sensitivity of this marker in assessing Paget’s disease activity,2,29 PINP levels may be influenced by slight changes in bone turnover. Knowledge about intraindividual variations for markers of bone turnover are of particular interest when they are used for monitoring response to treatments. The standard criterion for monitoring is to select those markers with the highest sensitivity to detect the minimal changes, and data derived from studies on biological variation may be used for this purpose. Thus, Harris and Brown23 pointed out that the distributions of within-subject variances could be useful in this type of evaluation. In the present study, serum markers of bone turnover showed lower variability than urinary markers. This is a common finding in studies of biochemical constituents. Markers of bone turnover are submitted to a diurnal variation with high values during early morning and low values during the afternoon,24,34 reflecting the circadian rhythm of bone resorption. The greater variation of urinary markers is not only explainable by pulse liberation of the hormones that regulates bone metabolic activity, because use of the second morning void urine minimizes this component of variation. The lower serum variability could also be attributed to the small size of the markers

Table 2. Components of biological variation in healthy subjects and pagetic patients Healthy subjects

Paget’s

Marker

Mean

CVi (%)

CVg (%)

Mean

CVi (%)

CVg (%)

tAP (U/L) bAP (ng/mL) PINP (ng/mL) sCTx (pmol/L) Hyp (nM/mg creatinine) CTx (␮g/mM creatinine) NTx (nM BCE/mM creatinine)

139.7 10.5 33.8 3001.0 63.9 128.1 35.0

4.5 3.4 6.2 9.3 19.3 25.6 17.4

28.1 37.4 18.4 38.9 22.1 36.3 37.6

586.0 136.0 176.5 5976.0 200.4 484.2 248.0

12.4 4.9 10.0 12.4 18.5 24.4 15.8

66.2 77.5 41.6 23.6 45.7 59.3 51.2

KEY: CVi and CVg, coefficient of variations of within-subject and between-subject biological variation, respectively. For other abbreviations see Table 1.

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Table 3. Indices of individuality (II) and critical differences (CD) in healthy subjects and pagetic patients II Marker tAP (U/L) bAP (ng/mL) PINP (ng/mL) sCTx (pmol/L) Hyp (nM/mg creatinine) CTx (␮g/mM creatinine) NTx (nM BCE/mM creatinine)

CD

Healthy

Paget’s

Healthy

Paget’s

0.19 0.33 0.46 0.33 0.92 0.74 0.53

0.18 0.14 0.15 0.49 0.38 0.35 0.30

15.20 26.30 23.10 36.20 55.10 73.10 54.60

34.60 29.60 29.40 36.30 51.60 68.80 44.70

For abbreviations see Table 1.

rapidly released in urine and the elapsed time from its liberation in early morning. In addition, the renal metabolism of the telopeptides, with a rate-limiting step in their degradation, contributes to the variability of these markers in urine.7,30 Another explanation for such differences may be related in part to the matrix effects of the two biological fluids, and to the variability of the creatinine measurement used to express the results of the urine markers.21 Other investigators have found this greater variability in urinary markers, some of whom reported even higher values of within-subject biological variability than those obtained in this study.4,5,16,17,20 –22,26 The distinct results with regard to between-subject biological variability likely depend on the subject population and the length of study period.17,21,32 Moreover, in the present series, bone formation markers showed a greater within-subject variability in the pagetic patients than in the healthy subjects, whereas the within-subject variability of bone resorption markers was similar in both healthy subjects and pagetic patients. Although the reasons for these findings are presently unknown, osteoblastic dysfunction, which has been associated with Paget’s disease of bone, could play a role.28 Moreover, when we take into consideration all of the bone markers analyzed in this study, serum bAP and PINP seemed to be the best markers for monitoring Paget’s disease activity. These markers showed the lowest intraindividual biological variation and low analytical day-to-day imprecision; consequently, they presented the lowest CD, with values near 30%. Nevertheless, follow-up study of a larger group of pagetic patients on therapy is necessary to further confirm our hypothesis. On the other hand, among resorption markers, sCTx showed the lowest biological variability and CD in pagetic patients. Theoretically, this finding makes this marker one of the most useful bone resorption markers for monitoring of Paget’s disease. Nevertheless, as demonstrated previously by Garnero et al.19 there is alteration in the degree of ␤-isomerization of collagen type I molecules in Paget’s disease, which can be modified with the disease treatment.19 It is known that sCTx measures the ␤-isomerized fragments derived from the carboxyterminal telopeptides of type I collagen.6 This fact, combined with the specific conditions of the present study (i.e., our patients were in a stable period of the disease), emphasize the necessity of evaluating the usefulness of this marker in patients on therapy. Except for sCTx, bone formation and resorption markers showed higher between-subject variability in patients with Paget’s disease than healthy subjects. Such differences reflect the variable increase of the markers depending on the extent and the activity of the disease. Markers of bone turnover showed strong individuality (a low II). This indicates that these markers have little diagnostic value but can be useful for patient monitoring. To evaluate the significance of changes in serial results for patient monitoring, the use of critical differences is highly recommended.14 As expected, in

the present study, serum bone markers showed lower critical differences than urinary markers. It should be pointed out that, in healthy subjects, the CD of tAP was twofold lower than in pagetic patients. Although the CD values of the other markers did not show great differences between the two populations studied, the fact that tAP is one of the most commonly used markers in the assessment and monitoring of Paget’s disease activity make this finding of special interest. Indeed, as we hypothesized previously, our results indicate that a change in tAP values between two consecutive measurements in pagetic patients should be higher than that expected from the healthy population in order to evaluate clinical response. Pagetic patients, therefore, must have a change of ⬎35% of their previous tAP value to reflect a modification of disease activity, whereas in healthy subjects this change should be approximately 15%. This finding leads us to believe that data on biological variation of markers of bone turnover, derived from healthy subjects, may not always be extrapolated to pathological situations, confirming that CD should be established for individual laboratories and for populations appropriate for the patients being monitored.21 It should be pointed out that the pagetic patients included in this study had mild and moderate disease with a mean serum tAP value of 586 U/L. We do not know if the biological variability of bone markers in patients with more active or inactive disease would be different. However, our results have some limitations; for instance, it would probably have been better to compare the biological variability of bone markers of pagetic patients to that of healthy subjects of similar age and gender. At present, there are not enough data to state that age and gender influence the biological variability of bone markers. In conclusion, this study confirms the lower sensitivity of urinary markers to detect significant changes and indicates that data obtained on biological variations from healthy populations can not always be extrapolated to pathological conditions. In addition, we showed that serum bAP and PINP seem to be the more sensitive markers for monitoring Paget’s bone disease, although a study with patients on therapy is necessary to support these findings.

References 1. Alvarez, L., Guan˜abens, N., Peris, P., Monegal, A., Bedini, J. L., Deulofeu, R., Martı´nez de Osaba, M. J., Mun˜oz-Go´mez, J., Rivera-Fillat, F., and Ballesta, A. M. Discriminative value of biochemical markers of bone turnover in assessing the activity of Paget’s disease. J Bone Miner Res 10:458 – 465; 1995. 2. Alvarez, L., Peris, P., Pons, F., Guan˜abens, N., Herranz, R., Monegal, A., Bedini, J. L., Deulofeu, R., Martı´nez de Osaba, M. J., Mun˜oz-Go´mez, J., and Ballesta, A. M. Relationship between biochemical markers of bone turnover and bone scintigraphic indices in assessment of Paget’s disease activity. Arthritis Rheum 40:461– 468; 1997. 3. Beck Jensen, J. E., Sorensen, H. A., Kollerup, G., Jensen, L. B., and Sorensen,

576

4.

5. 6.

7. 8. 9. 10.

11. 12. 13. 14. 15.

16.

17.

18.

19.

20.

21.

22.

L. Alvarez et al. Bone marker variability in Paget’s disease O. H. Biological variation of biochemical bone markers. Scand J Clin Lab Invest 54(Suppl. 219):36 –39; 1994. Blumsohn, A., Hannon, R. A., Al-Dehaimi, A. W., and Eastell, R. Short-term intraindividual variability of markers of bone turnover in healthy adults. J Bone Miner Res 9(Suppl. 1):S153; 1994. Braga de Castro Machado, A., Hannon, R., and Eastell, R. Monitoring alendronate therapy for osteoporosis. J Bone Miner Res 14:602– 607; 1999. Christgau, S., Rosenquist, C., Alexandersen, P., Bjarnason, N. N., Ravn, P., Fledelius, C., Herling, C., Qvist, P., and Christiansen, C. Clinical evaluation of the serum CrossLaps one step ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen Ctelopeptides. Clin Chem 44:2290 –2300; 1998. Colwell, A. and Eastell, R. The renal clearance of free and conjugated pyridinium cross-links of collagen. J Bone Miner Res 11:1976 –1980; 1996. Delmas, P. D. Clinical use of biochemical markers of bone remodelling in osteoporosis. Bone 13:517–521; 1992. Delmas, P. D. and Meunier, P. J. The management of Paget’s disease of bone. N Engl J Med 8:558 –566; 1997. Edler, V., Eyben, F., Lindegaard, E., Billabjerg, O., and Hyltoft Petersen, P. Serum lactate dehydrogenase isoenzyme: An early indicator of relapse in patients with testicular germ cell tumors. Acta Oncol 35:925–929; 1995. Eriksen, E. F., Brixen, K., and Charles, P. New markers of bone metabolism: Clinical use in metabolic bone disease. Eur J Endocrinol 132:251–263; 1995. Fraser, C. G. The application of theoretical goals based on biological variation data in proficiency testing. Arch Pathol Lab Med 112:404 – 415; 1988. Fraser, C. G. Biological variation in clinical chemistry. An update: collated data, 1988 –1991. Arch Pathol Lab Med 116:916 –923; 1992. Fraser, C. G., and Harris, E. K. Generation and application of data on biological variation in clinical chemistry. Crit Rev Clin Lab Sci 27:409 – 437; 1989. Fraser, G., Hyltoft Petersen, P., Ricos, C., and Haeckel, R. Quality specifications. In: Haeckel, R., Ed. Evaluation Methods in Laboratory Medicine. Weinheim: VCH; 1993; 87–100. Garnero, P., Darte, C., and Delmas, P. D. A model to monitor the efficacy of alendronate treatment in women with osteoporosis using a biochemical marker of bone turnover. Bone 24:603– 609; 1999. Garnero, P. and Delmas, P. D. Variability and response of urinary resorption markers to hormone replacement therapy. J Bone Miner Res 14:470 – 471; 1999. Garnero, P., Fledelius, C., Gineyts, E., Serre, C., Vignot, E., and Delmas, P. D. Decreased ␤-isomerization of the C-terminal telopeptide of type 1 collagen ␣1 chain in Paget’s disease of bone. J Bone Miner Res 12:1407–1415; 1997. Garnero, P., Gineyts, E., Schaffer, A. V., Seaman, J., and Delmas, P. D. Measurement of urinary excretion of non-isomerized and ␤-isomerized forms of type 1 collagen breakdown products to monitor the effects of the bisphosphonate Zoledronate in Paget’s disease. Arthritis Rheum 41:354 –360; 1998. Garnero, P., Hih, W. J., Gineyts, E., Karpf, D. B., and Delmas, P. D. Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to Alendronate treatment. J Clin Endocrinol Metab 79:1693–1700; 1994. Hannon, R., Blumsohn, A., Naylor, K., and Eastell, R. Response of biochemical markers of bone turnover to hormone replacement therapy: Impact of biological variability. J Bone Miner Res 13:1124 –1133; 1998. Hannon, R. and Eastell, R. Variability and response of urinary resorption markers to hormone replacement therapy. J Bone Miner Res 14:471– 472; 1999.

Bone Vol. 26, No. 6 June 2000:571–576 23. Harris, E. K. and Brown, S. S. Temporal changes in the concentrations of serum constituents in healthy men. Ann Clin Biochem 16:169; 1979. 24. Hassager, C., Risteli, J., Risteli, L., Jensen, S. B., and Christiansen, C. Diurnal variation in serum markers of type 1 collagen synthesis and degradation in healthy premenopausal women. J Bone Miner Res 7:1307–1311; 1992. 25. Ju, H. S. J., Leung, S., Brown, B., Stringer, M. A., Leigh, S., Scherrer, C., Shepard, K., Jenkins, D., Knudsen, J., and Cannon, R. Comparison of analytical performance and biological variability of three bone resorption assays. Clin Chem 43:1570 –1576; 1997. 26. Kancir, C. B., Petersen, P. H., and Wandrup, J. The effects of plasma volume variations on the calcium concentration during epidural anaesthesia. Acta Anaesthesiol Scand 31:338 –342; 1987. 27. Lassen, J. F., Kjelden, J., Anonsen, S., Hyltoft Petersen, P., and Brandslund, I. Interpretation of serial measurements of international normalized ratio for prothrombin times in monitoring oral anticoagulant therapy. Clin Chem 41: 1171–1176; 1995. 28. Meunier, P. Bone histomorphometry and skeletal distribution of Paget’s disease of bone. Sem Arthritis Rheum 23:219 –221; 1994. 29. Pons, F., Alvarez, L., Peris, P., Guan˜abens, N., Vidal-Sicart, S., Monegal, A., Pavia, J., Ballesta, A. M., Mun˜oz-Go´mez, J., and Herranz, R. Quantitative evaluation of bone scintigraphy in the assessment of Paget’s disease activity. Nucl Med Commun 20:525–528; 1999. 30. Randall, A. G., Kent, G. N., Garcia-Webb, P., Bhagat, C. I., Pearce, D. J., Gutteridge, D. H., Prince, R. L., Stewart, G., Stuckey, B., Will, R. K., Retallack, R. W., Price, I. R., and Ward, L. Comparison of biochemical markers of bone turnover in Paget disease treated with Pamidronate and a proposed model for the relationships between measurements of the different forms of pyridinoline cross-links. J Bone Miner Res 11:1176 –1184; 1996. 31. Rico´s, C., Alvarez, V., Cava, F., Garcia-Lario, J. V., Herna´ndez, A., Jime´nez, C. V., Minchinela, J., Perich, C., and Simo´n, M. Current databases on biological variation: Pros, cons and progress. Scand J Clin Lab Invest. In press. 32. Rico´s, C., Jime´nez, C. V., Herna´ndez, A., Simo´n, M., Perich, C., Alvarez, V., Minchinela, J., and Macia, M. Biological variation in urine samples used analyte measurements. Clin Chem 40:472– 477; 1994. 33. Ross, J. W. Evaluation of precision. In: Werner, M., Ed. Handbook of Clinical Chemistry. Vol. 1. Boca Raton, FL: CRC; 1989; 391– 442. 34. Schlemmer, A., Hassager, C., Jensen, S. B., and Christiansen, C. Marked diurnal variation in urinary excretion of pyridinium cross-links in premenopausal women. J Clin Endocrinol Metab 74:476 – 480; 1992. 35. Sebastian-Gambaro, M. A., Liro´n-Herna´ndez, P. J., and Fuentes-Arderiu, X. Intra- and interindividual biological variation data bank. Eur J Clin Chem Clin Biochem 35:845– 852; 1997. 36. Woitge, H. W., Pecherstorfer, M., Li, Y., Keck, A. V., Horn, E., Ziegler, R., and Seibel, M. Novel serum markers of bone resorption: Clinical assessment and comparison with established urinary indices. J Bone Miner Res 14:792– 801; 1999.

Date Received: November 25, 1999 Date Revised: January 5, 2000 Date Accepted: February 7, 2000