Printer Friendly

Assessment and recommendations on factors contributing to preanalytical variability of urinary pyridinoline and deoxypyridinoline.

Biochemical bone markers can provide a valuable tool in the management of metabolic bone diseases. Their most recognized application in clinical practice is for monitoring treatment for osteoporosis as an adjunct to bone mineral density measurements. Other applications that have been investigated include their use as a diagnostic tool for bone diseases other than osteoporosis and as predictive markers for bone loss and the risk of bone fracture (1-3). Biochemical markers are available to assess both bone formation and bone loss (resorption). Because most metabolic bone diseases are characterized by an increase in bone resorption, these particular biochemical markers are of special interest. Our focus in this review will be exclusively on the pyridinium cross-links pyridinoline (PYD;10 hydroxylysylpyridinoline) and deoxypyridinoline (DPD; lysylpyridinoline) because they are part of an extensive laboratory standardization program at the CDC designed to improve the measurement of biochemical markers and risk factors associated with selected chronic diseases. A description of other bone markers, including other resorption markers, such as type I collagen telopeptide breakdown products, is provided in several reviews (4-7).

One of the main issues hampering the interpretation of PYD and DPD results for clinical use is preanalytical and analytical variation (8). The effects of analytical variability can be minimized through standardization of results of laboratory measurements by controlling imprecision through the use of good laboratory practices and by validating proper method calibration through appropriate traceability to reference methods, using suitable reference materials. These strategies have already demonstrated their benefit for other analytes, such as cholesterol, and are currently being implemented for PYD and DPD, as stated above, as well as for other bone markers (9).

Despite extensive reports on preanalytical variability for various analytes, no overview has been published to date that addresses in detail this source of variability and its contribution to unreliable PYD and DPD measurements. Furthermore, there has been no previous effort to establish guidelines for minimizing or controlling the effects of preanalytical variability on PYD and DPD results. Preanalytical variability for other biochemical bone markers is acknowledged, but the expert panel convened to consider this topic focused exclusively on preanalytical variability in pyridinium cross-links measurements, with the primary objective of presenting guidelines that can facilitate comparability and interpretation of PYD and DPD results. Evaluating the clinical applicability or performance of these markers and defining clinical decision levels is beyond the scope of this review and have been addressed in other reviews (3, 10). Likewise, comparing the clinical utility of the various formation and resorption markers will not be addressed here. No recommendation of pyridinium cross-links measurements over other resorption markers is implied.

Preanalytical variability includes variation from specimen collection and handling, as well as biologic variation of the individual being tested. Biologic variation is composed of an intraindividual (within-subject, both within-day and among-day) component and an interindividual (between-subjects) component. Recognizing and controlling factors that contribute to preanalytical variability is crucial for the interpretation of bone marker results. Identifying true biologic change by taking into consideration an individual's biologic variability is the basis for the concept of least significant change for monitoring patients during treatment, as proposed by Hannon et al. (11). To compare data between patients or assess patient data within a reference interval requires that factors affecting the biologic variability be identified and controlled or minimized consistently.

Most data have been generated using urine, and only limited results are available from other specimen types (12-15). Therefore, because of the paucity of reviewed literature on specimens other than urine, this review will address only urinary PYD and DPD. Pyridinium cross-links can be measured as total PYD and DPD (sum of free and peptide bound) after acidic hydrolysis or as free, non-peptide-bound molecules only. HPLC methods and immunoassays that measure free as well as total pyridinium cross-links in urine have been developed and are reported to correlate well (16, 17). Differences in the response to medications or other events have been observed between free pyridinium cross-links and total cross-links (18-22). However, the observed differences are generally only in magnitude or in the time point of the change, rather than suggesting different clinical interpretations. PYD and DPD are reported to change in parallel in various conditions with the exception of arthritis, in which there is a relatively greater increase in PYD (23). Thus, for the purpose of this review, distinctions between free and total pyridinium cross-links, or between PYD and DPD, were made only if they were regarded as necessary for general understanding.

LITERATURE SEARCH

We searched the literature for scientific articles published until January 2001 that dealt with PYD and/or DPD (keywords used: PYD, DPD, PYR, DPYR, pyridinoline, deoxypyridinoline, lysylpyridinoline, and hydroxylysylpyridinoline). We limited the search to English as the language, urine as specimen, and humans as subjects, except in the case of stability, when the search was expanded to other species. A second search was performed on the topic of creatinine measurement. Both searches were done in MEDLINE, Current Contents, The Cochran Database of Systematic Reviews, and CANCERLIT (1969-2000). A total of 599 articles were identified as potentially relevant, and additional references from these articles were examined. We also included additional literature that had not been identified by the electronic search but was found to be appropriate.

Assessment of Biological Variability

INTRAINDIVIDUAL VARIABILITY

Circadian cycle. Both PYD and DPD measured in urine follow a circadian or diurnal cycle with a peak in the early morning and nadir in the late afternoon to early evening. The magnitude of the diurnal change, i.e., nadir concentration divided by peak concentration, expressed as a percentage is listed in Table 1. The median change was 73% for PYD (range, 57-78%) and 70% for DPD (range, 53-75%). The authors of one study (24) observed a night/day difference of greater magnitude (nadir/peak ratio of 57% for PYD and 54% for DPD) than the others; in that study, nine specimens were collected over 24 h. The authors of another study, which involved one nighttime and one daytime collection, observed a night/day difference in peptide-bound DPD but not in free DPD (25). The magnitude of the daily cycle is expected to be higher as the number of collections during the cycle increases; fewer but longer collection intervals will smooth changes during each collection and not detect the full amplitude. Because sources of variability other than diurnal variation are reported in terms of distribution (CV), comparisons of diurnal variation with other sources of variation are difficult. One study (26) used a statistical package to convert amplitude to distribution. The authors found that a 70% (nadir/peak) amplitude corresponded to a CV of 10.4%. The mean of nadir/peak values for PYD and DPD was 69%. Therefore, the proportion of variability attributable to diurnal variation can be assumed to approximate a CV of 10%.

Although the excretion of cross-links in urine varies by condition, the nadir/peak ratio seems to be reasonably consistent (24, 27). Postmenopausal and elderly osteopenic women demonstrated similar relative amplitudes in their diurnal patterns. The diurnal cycle is reportedly unaffected by hormonal changes during the menstrual cycle (24). Although differences in amplitude of the diurnal cycle between men and women have been reported, the differences were not substantial (28). Children demonstrated a diurnal pattern similar to that of adults (29). After 5 days of bed rest, the total cross-link excretion was reported to increase, but the relative amplitude of the cycles remained the same (24). Consistent with the large increases from afternoon to early morning, wake/sleep cycle (nyctohermal) differences are expected; in fact, daytime concentrations were reported to be ~78% of night concentrations (30).

Day-to-day variability. Even with morning collections at the same time each day for each person, longer-term biologic variability contributes to the overall variation. The reported day-to-day variation (Table 2) averaged 15.7% for PYD (range, 12-21% in five collection conditions/studies) and 17.4% for DPD (range, 5-24% in six combinations). Weekly variation was similar, averaging 15.8% for PYD and 17.1% for DPD. One study with three collection conditions was eliminated from the DPD series because the reported variations were much lower than those in the other studies, perhaps because of the small number of collections (two in 2 weeks) (31). In another study, the monthly variation in DPD monitored over 5 months in both males and females averaged 16.7%. The reported variations for DPD were consistently higher than those for PYD. One collection per day over 5 days and up to 21 days showed approximately the same variability as collections made weekly over 5 weeks. Similar results were obtained when samples were collected monthly over 5 months. Consequently, collection of daily specimens over 5 days seems a reasonable approximation of biologic variation. In fact, one study reported that three collections over 3-5 days captured the full range of biologic variation (32).

INTERINDIVIDUAL VARIABILITY

The only study specifically intended to assess intersubject variability of urinary pyridinium cross-links reported intersubject variabilities of 14.2% (PYD) and 13.0% (DPD) for postmenopausal healthy women and 18.7% (PYD) and 37.8% (DPD) for postmenopausal osteoporotic women (8). This study included only a small number of postmenopausal women. To obtain information across different studies and groups of subjects, we calculated the biologic variability based on the following equation:

[CV.sub.B.sup.2] = [CV.sub.T.sup.2] = [CV.sub.A.sup.2] (1)

In which [CV.sub.T] is the total variability calculated from mean concentrations and standard deviations, and [CV.sub.A] is the analytical variability (intraassay variability) reported for each study. We considered only studies using first morning void (FMV), second morning void (SMV), or 24-h urines. If the analytical variability was missing, we assumed a variability of 10%, which is the average analytical variability mentioned by the authors cited in Table 3. On the basis of the obtained biologic variability, the intersubject variability ([CV.sub.inter]) was estimated using the following equation:

[CV.sub.inter.sup.2] = [CV.sub.B.sup.2] = [CV.sub.intra.sup.2] (2)

with an intraindividual variation ([CV.sub.intra]) of 16% as discussed previously.

As shown in Table 3, the average interindividual variability was highest in children [DPD, 48% (range, 23-82%); PYD, 35% (range, 10-40%)], followed by postmenopausal women [DPD, 40% (range, 27-54%); PYD, 36% (range, 22-61%)] and premenopausal women [DPD, 34% (range, 8-98%); PYD, 26% (range, 12-63%)]. This estimate is not applicable to men, for whom negative values were obtained with the available data. Within the group of children, the highest variability was observed between the ages of 10 and 18 years. Within the group of menopausal and postmenopausal women, no correlation between age groups and variability was found. There were substantial differences in the variability as well as in the mean concentrations in the different studies even when data were grouped for methods and collection mode.

Factors Affecting Urinary PYD and DPD Excretion

The following factors are reported to affect urinary PYD and DPD excretion and therefore may contribute to the inter- and intrasubject variability.

INFLUENCE OF AGE, SEX, GEOGRAPHIC EFFECTS, AND RACE

Of the three studies investigating premenopausal women, the changes in the urinary excretion of PYD and DPD in women 20-50 years of age were small and reached a maximum of 10-15% at age 50 compared with age 20, with the highest excretion rates at age 20-29 (33-35). When this age range was excluded, no significant changes were observed in the age group 30-50. Usually menopause is defined as an absence of menses for at least 12 months. However, before menopause there is a poorly defined period of perimenopausal status, which could last several months or years and is characterized by irregular menses and subtle estrogen deficiency associated with increased follicle-stimulating hormone. The only study investigating the influence of the perimenopausal status on urinary pyridinoline cross-links excretion reported no differences between premenopausal women and perimenopausal women (36). Two cross-sectional studies compared total PYD and DPD concentrations of premenopausal women and very recently menopausal women of similar ages. They showed 30-55% higher pyridinium cross-links excretions rates in postmenopausal women than in premenopausal women, with a larger increase in DPD vs PYD (36, 37). One study suggested that there was no change in free DPD excretion through menopause (36). These data have been confirmed by a longitudinal study of women who became postmenopausal during follow-up (38). After menopause, changes in the excretion rates of urinary PYD appear small (33) with a slight increase of ~8-15% during age 50-80 in the urinary excretion of total PYD (33, 39), but not of total DPD (33). For urinary free PYD and DPD, the increase is reported to be larger (49-70% for free PYD and 50% for free DPD from age 50 to age 80). This increase does not seem to be related to decreased creatinine excretion resulting from decreased muscle mass (35).

The available studies on urinary pyridinium cross-links in men report concordant results with high excretion rates in young men at age 20, which decrease and reach a nadir at age 50-60 (34, 40, 41). After the age of 60, the urinary excretion of free PYD and free and total DPD increased slightly with age, by ~ 20-35%.

Pyridinium cross-link concentrations in children are reported to be 5- to 10-fold higher than in adults and to change drastically from birth to cessation of bone growth at 20-25 years of age. The pattern of change during childhood is described consistently among different investigators, with the highest concentrations reported in newborns and relatively constant or slightly decreasing concentrations from early childhood (age 3-5 years) to start of puberty. No information is given whether these small changes are statistically significant (42-45). Increasing pyridinium cross-link concentrations are observed with progressing pubertal stage, leading to a peak followed by a decrease to normal adult excretion rates (29, 42, 44, 46-49).

Higher concentrations of total and free pyridinium cross-links have been observed in preterm newborns than in term newborns (50-52). The high concentrations observed in newborns declined in the first 4-8 weeks after birth (51). During puberty, concentrations increase to a peak and then decrease to adult concentrations (42, 43, 45, 53). In studies in which the pubertal stage was assessed by categorizing participants in Tanner stages, for girls the highest concentrations were observed at Tanner stages 1-3, and in boys the highest concentrations were observed at Tanner stage 3-5 (18, 30, 46, 54, 55). No sex-specific differences have been reported in children before puberty.

GEOGRAPHIC EFFECTS AND EFFECTS OF RACE

No study exists investigating urinary pyridinium cross-links concentrations in different countries with assays performed in the same laboratory, using the same method and the same type of urine samples. Therefore, a metaanalysis (one-sided ANOVA) of reported concentrations of healthy premenopausal women from different regions [England (30), Germany (43), France (34, 56), Sweden (57), Japan (37, 58-60), Australia (36), South Africa (61), Taipei (62), and Italy (63)] was performed. The studies were grouped according to type of sample collection, and a recently reported interlaboratory variability (64) was taken into consideration as analytical variability. Significant differences were found only between Japan and all other countries.

The available studies investigating the effect of race on urinary PYD and DPD excretion rates compare African and Caucasian individuals only (26, 61, 65, 66). The largest study performed in premenopausal and postmenopausal women from South Africa (61) observed nonsignificant differences in urinary free PYD in native African women compared with Caucasian women (2% and 9% lower for premenopausal and postmenopausal, respectively) (61). Similar findings are reported in a recent study for DPD in Caucasian and Afro-Caribbean men (65). Other studies, with fewer participants, reported slightly but significantly lower urinary PYD in African-American compared with Caucasian-American women (26, 66).

INFLUENCE OF THE MENSTRUAL CYCLE, CONTRACEPTION, PREGNANCY, AND LACTATION

Of the three studies investigating changes during menstrual cycle of apparently healthy menstruating women, two found no significant changes in urinary DPD (67, 68). The third study observed a slight increase in urinary DPD excretion rates of ~5% from early follicular phase to the luteal phase peak before falling back to baseline during luteal phase (69). Investigations in women with abnormal cycles described controversial findings. Authors reported either increased concentrations (25% for DPD; PYD not analyzed) in the case of hypothalamic amenorrhea (70) or decreased concentrations (20% PYD; 35% DPD) in amenorrheic women compared with eumenorrheic women and sedentary controls (71). The hypothalamic amenorrheic women in both studies had low estrogen concentrations. No data are available on urinary DPD and PYD in oral contraceptive users. During pregnancy, pyridinium cross-links increase substantially, especially during the third trimester, with concentrations generally being two or more times higher than in nonpregnant women and in the first trimester (72-74). DPD concentrations are reported to increase further with uterine involution in the first weeks postpartum (75). During lactation, PYD gradually returns to prepregnancy concentrations (74).

SEASONAL VARIATIONS

Of the studies investigating seasonal variations (76, 77), one study was cross-sectional, with relatively large group sizes of elderly men and women; the second study was a longitudinal study of only 20 women, using a crossover design complicated by supplementation with vitamins D and K; and the third study was performed cross-sectionally in groups of young women (78). Urinary excretion of pyridinium cross-links was higher in the winter (October 1 to April 30) than in the summer. There were, however, differences in the magnitude of these changes, which varied from ~5% to 25%; in many cases, these were not statistically significant. In addition, the changes were not always synchronized with seasonal changes in the concentrations of vitamin D metabolites and parathyroid hormone.

INFLUENCE OF PHYSICAL ACTIVITY

As indicated in one study, the type of exercise seems to influence urinary cross-links concentrations (79). Increased excretion rates (significant and nonsignificant) have been reported by authors investigating the effect of running (80-83). In other types of sports [gymnastics (84), endurance exercise (85), aerobic exercise (86), weight lifting (87), resistance exercise (88, 89)] or physical activity [occupational walking (90, 91)], either no differences or decreased excretion rates (significant and nonsignificant) were reported, with changes ranging from 8% to 40%. Studies investigating the effect of immobilization from prolonged bed rest (4 days or longer) consistently found increased concentrations for urinary PYD and DPD (92-94). One study with bed rest over 20 days found increasing concentrations with a peak after 10 days followed by a decrease. Another study reported excretion rates that remained increased 5 days after bed rest was terminated. The reported changes ranged from 20% to 44% for PYD and 27% to 44% for DPD. Weightlessness as experienced during space flight has been reported to increase pyridinium cross-links significantly (95, 96).

INFLUENCE OF DIET

Extreme fasting over 4 days had no significant effect on pyridinium cross-links concentrations (97). However, in severely malnourished children, concentrations were decreased to approximately one-third of those after recovery from malnourishment (98). One cross-sectional study investigated the influence of different diets in women 45-55 years of age with regard to fruit and vegetable intake, using a food-frequency questionnaire. The potassium, magnesium, and phosphate intake was negatively correlated with PYD excretion, and potassium, magnesium, [beta]-carotene, and fiber intake was negatively correlated with DPD excretion. Statistical calculations indicated that magnesium intake accounted for 12% of the variability (99). Another study investigated high sodium intake (250 mmol/day) and found increased DPD excretion rates (27%) in postmenopausal women (100). However, other studies investigating the effect of sodium intake, using different diets with lower sodium concentrations, found no significant effects on excretion rates of pyridinium cross-links (101, 102). No effects were reported for phosphate supplementation [1500 mg (103)], high protein intake [2.7 g protein per kg of body weight per day (104)], zinc supplementation (105), and increased milk consumption [586 mL/day (106)]. Diets low in copper (0.7 mg/day) significantly increased excretion rates in DPD and PYD (30% and 25%, respectively) compared with a medium copper diet (107). Vitamin D deficiency resulting from vitamin D-deficient diets or malabsorption reportedly caused increased pyridinium cross-links excretion rates (108-111). Concentrations were approximately two- to threefold higher in deficient postmenopausal women than in healthy controls.

Calcium supplementation ([greater than or equal to] 600 mg/day) decreased urinary pyridinium cross-links excretion rates in men and women (premenopausal and postmenopausal). The degree of observed changes as well as the time point in which significant changes were observed differed for immunoreactive free and total pyridinium cross-links (111, 112). Changes up to ~33% as a result of calcium supplementation have been reported (104). The effects of calcium supplementation on diurnal variability are reportedly controversial. Evening calcium administration, but not morning calcium administration, seems to suppress the nocturnal increase in cross-links concentrations (113, 114). However, another study described no effect of evening administration on the nighttime increase in PYD (115). Increased pyridinium cross-links have been reported in alcoholics and abstainers even 5 years after alcohol withdrawal (116). No association between smoking and pyridinium cross-links excretion rates and no difference in PYD and DPD excretion rates in smokers were found regardless of fast or slow loss in lung function (117, 118).

INFLUENCE OF DISEASES AND MEDICATION

Evidently, urinary pyridinium cross-links are affected by metabolic disorders of bone, such as osteoporosis, and change with treatments to cure these disorders (11, 119-122). However, several other diseases, conditions, and drugs are known to affect urinary PYD and DPD excretion rates. Some of these conditions affect bone metabolism directly, whereas others may affect the clearance of pyridinium cross-links or affect other cross-links containing tissue rather than bone. A profound increase in bone turnover (concentrations 100% or higher than in healthy controls) occurs in patients with hyperthyroidism (123-126), hyperparathyroidism (127), Paget disease (128), Ehlers-Danlos syndrome (129, 130), multiple myeloma, hypercalcemia of malignancy, and certain cancers, particularly if they are associated with bone metastases (14, 131-133). In addition, fractures are known to cause increased PYD and DPD concentrations that remain increased up to 1 year after occurrence of fracture (134-136). More modest or inconsistent observations are reported for other diseases, such as diabetes mellitus (137-141) or arthritis (142, 143). Other diseases and conditions that are reported to cause increased pyridinium cross-links concentrations are liver dysfunction (144), renal osteodystrophy (153), Camurati-Engelmann disease (146), spinal cord injury (147), bone marrow transplantation (148), gastrointestinal diseases related to nutrition and mineral metabolism (149), cystic fibrosis (150), scleroderma (151), growth hormone/receptor deficiencies and other growth disorders, growth hormone treatment (152), hyperprolactinemia in amenorrheic patients with estrogen deficiency (153), myelomeningocele (154), and seronegative spondylarthropathy (155). Decreased pyridinium cross-links concentrations are reported in fibromyalgia (156), severe burns (157), and acute lymphoblastic leukemia in children (158). Excretion rates may vary with disease stage and severity or drug dosage (111). Although this listing is not exhaustive, it shows the range of conditions in which PYD and DPD excretion can be altered. In this context it should be mentioned that the drug sulfasalazine has been reported to interfere with DPD in a HPLC assay (159).

INFLUENCE OF OTHER PREANALYTICAL FACTORS AND ANALYTICAL FACTORS ON PYD AND DPD MEASUREMENT

Urine collection. To measure pyridinoline excretion, several urine collection types have been used, including uncontrolled spot samples, 24-h urine collection, FMV, and SMV. Of the three studies directly comparing different types of sample collection, the 24-h concentrations were significantly lower by ~17% than the FMV and SMV concentrations (31). This can be explained by the diurnal variation in pyridinium cross-links excretion, as discussed above. In the studies in which both FMV and SMV were measured, FMV concentrations were nonsignificantly higher (~4%) than SMV concentrations. With regard to variability of each type of collection, a SMV collection showed slightly less variation than a 24-h collection, which was in turn less variable than a FMV. Two studies reported modestly higher biologic variation for women than for men (160, 161). A direct comparison of biologic variation in free and total PYD and DPD did not appear to show significant differences (162).

Stability of calibrators and samples. PYD and DPD dissolved in water are very sensitive to ultraviolet (UV) light. The rate of degradation depends on the pH of the solution and the wavelength of the light source used for exposure. The highest degradation rate is at the wavelength of maximum absorbance. When UV light was used at wavelengths of 254 and 365 nm, the degradation rate was higher at high pH (163). Complete degradation of the aqueous calibrator was observed within 6 h of exposure to a UV lamp (164). Pyridinium cross-links in urine are much less sensitive to UV light (163, 165). A 1-cm layer of urine absorbs >99% of UV light (163). No effect on urinary cross-links was observed with laboratory light (fluorescent lights and filtered daylight) (164, 166). In addition, no effect of daylight was found on urine stored in a large container (850 mL) for a whole day during the summer. However, a small amount of degradation was observed if the urine was stored in a small (2 mL) container (163). The effect of UV light on the degradation of cross-links was greater for free PYD and DPD than for total pyridinolines (free and peptide bound) with a decrease of 80% in free DPD and a decrease of 60% in total DPD after 3 days of exposure.

The degradation in urine was also pH dependent, with greater degradation at higher pH (163, 164, 166). The half-life of pyridinium cross-links was ~1 h at 50 [degrees]C and 6 h at 37 [degrees]C (167). No degradation was observed at or below 20 [degrees]C (down to -70 [degrees]C) for up to 9 months (165, 167). Findings concerning freeze-thaw stability are controversial. The authors of one study found no degradation of cross-links in urine after 10 freeze-thaw cycles (167), whereas the authors of another study found degradation after 5 freeze-thaw cycles (166).

Creatinine measurement. Urinary concentrations of PYD and DPD are frequently expressed as molar ratios with the creatinine concentration. Considering the importance of creatinine in defining cross-links excretion rates, examining factors affecting creatinine concentrations in urine is essential. Several endogenous and exogenous compounds influence assay results. These effects vary depending on the type of creatinine assay used (alkaline picric acid assay, enzymatic assay, or HPLC assay). The presence of high salt concentrations; fluorescein (168); carbonyl compounds (169), in particular acetoacetic acid and ketoacids (170); glucose (171); and several drugs (172), including dopamine (173), cephalosporins (174, 175), and trimethoprim (176), cause a moderate to high increase in creatinine concentration. In contrast, high concentrations of bilirubin (177) produce negative interference. Several lifestyle and pathologic conditions influence the creatinine concentration, including strenuous exercise [30-50% vs baseline (178)], stress [5-10% vs control (179)], dietary intake of meat or polyunsaturated fat [10-30% vs control (180)], time of menstrual cycle [10-15% vs baseline (181)], pregnancy [5-20% vs baseline (182)], age, infection, trauma, and renal insufficiency [20-100% vs control (183, 184)].

Daily excretion of creatinine follows a circadian rhythm with a 14% higher value in the late afternoon (185), compared with the 24-h mean. When creatinine is measured in 24-h urine, the intraindividual variation is generally 11-15% (186-192), whereas it is 29-31% for FMV samples (190, 192) and 36-45% in random urine samples (190, 192, 193). In these studies, the intersubject variability was 13-28% for 24-h urine samples, 26% for FMV, and 33-36% in random urine. The 24-h creatinine output in men is significantly higher than in women (194).

Relatively few studies have described the stability of creatinine in urine specimens, and most of them are based on the short-term stability of creatinine in urine stored 1-30 days. Creatinine is generally stable in urine stored at 4 [degrees]C for at least 5-7 days (195, 196), although some studies indicated no significant change in creatinine concentration when urine was stored refrigerated for 30 days (197). Data on creatinine stability in urine stored after the addition of acid or alkali are controversial. The authors of one study found some loss of creatinine when acidified urine was stored frozen (-15 [degrees]C) (198), whereas the authors of another study found no significant change in creatinine under these conditions (195). No data are available about the effect of freeze-thaw cycles on the creatinine concentration. In our hands, up to five-freeze thaw cycles did not significantly change the creatinine concentration (A.K. Srivastava, unpublished observation).

Discussion

Urinary PYD and DPD concentrations can be affected substantially by several preanalytical factors. These factors need to be recognized and controlled before collecting a specimen to minimize variability and facilitate data interpretation. Minimized variability leads to consistent signal-to-noise ratios and reference intervals, and consequently to constant limits of detection for abnormal pyridinium cross-links excretion rates or significant changes in the excretion rates.

The within-day or diurnal variability of pyridinium excretion, as one of the important sources of variability, is ~10%, and the among-day variation for DPD and PYD is 16% within an individual. On the basis of these data, specimen collections made at random times during the day would be expected to increase the variation from 16% to ~19% compared with serial collections made at the same time of day. Therefore, collecting serial urine specimens from a patient as close as practical to the same time each day could eliminate the within-day component of variation. To reduce the among-day variability, multiple collections (e.g., three over 3-5 days) analyzed in duplicate (as individual samples or pooled) may improve the reliability of clinical decision-making, as has been shown in a similar approach to minimize the effect of biologic variability in blood lipid testing (199). Among the different types of urine collection, the 24-h collection offers the advantage of allowing assessment of an integrated daily excretion, but it is complex and inconvenient. A SMV or FMV collection may provide the best signal-to-noise ratio because of the high pyridinium cross-links concentrations in the morning. The SMV might have some advantage in clinical practice in that patients who urinate frequently during the night might better accommodate the SMV and may even provide it during the office visit.

The average among-subject biologic variability was 35% for premenopausal women, 42% for postmenopausal women, and 45% for children (average of PYD and DPD excretion rates) with a high variability between different studies. The observed high inconsistency in the biologic variability across studies is probably attributable to the heterogeneity of the individuals investigated in the studies (age ranges, definition of menopausal status, different exclusion criteria). However, as indicated by some studies using well-characterized participants, biologic variation as low as 14-18% in healthy individuals can be achieved. To minimize biologic variability, individuals need to be grouped according to their age and gender, which are two factors that have a profound affect on biologic variability.

Because of the rapid changes during childhood, children of the same age or within a narrow age range (2 years) should be used for comparison purposes. In case of pubertal children, it might be more suitable to compare children of the same pubertal status (i.e., categorized in Tanner stages) than using children of the same age. In adult premenopausal women, the smallest variability was observed from age 30 until the start of menopause. The available data on perimenopausal women and on menopausal transition are not sufficient to make any general statements about variability in PYD and DPD excretion rates. For postmenopausal populations, it would be suitable to express postmenopausal excretion rates per age group rather than by postmenopausal years because age is an easier and more reliable variable to collect than years since menopause. Until the start of puberty, no sex-specific differences have been reported. The most pronounced differences between men and women are observed at the start of menopause. Therefore, concentrations for both genders need to be assessed and interpreted separately.

Profound changes are also observed during the third trimester of pregnancy (73, 200, 201). In addition, certain diseases and medications, such as fractures, hyperparathyroidism, hyperthyroidism, or certain types of cancers, substantially impact urinary PYD and DPD concentrations. To properly interpret results obtained from crosslinks measurements, a detailed characterization of the patient and exact diagnosis of all diseases and conditions are necessary. Other factors affecting biologic variability with only modest effects or inconsistent findings reported are menstrual cycle, physical activity, diet, seasonal variation, and geographic differences. These factors seem to be important when small differences in urinary pyridinium cross-links excretion rates need to be detected.

The small changes in urinary PYD and DPD concentrations during the menstrual cycle with lower concentrations in the follicular phase and increased concentrations in luteal phase are consistent with findings obtained with other bone markers (202). The available data on the impact of irregular menstrual cycles indicate that the excretion rates of pyridinium cross-links in women with abnormal cycles might be different from the excretion rates in women with regular cycles. No data are available on the impact of oral contraceptives. However, studies with other bone markers point to possibly lower pyridinium cross-links excretion rates in oral contraceptive users than in nonusers (203). Data available on the impact of physical activity are somewhat scattered, with most changes being nonsignificant. The study designs are too different to find any correlations across the studies. However, the reported magnitudes of changes (up to 40%) are substantial and need to be confirmed in further studies. Data on immobilization consistently show increases in PYD, DPD, and creatinine concentrations with prolonged bed rest. Consequently, excretion rates of immobilized individuals may need to be considered separately from those for nonimmobilized individuals.

Long-term effects of diets or impaired dietary behavior have been reported in situations such as severe malnutrition or anorexia nervosa, with decreased excretion rates in the first and increased excretion rates in the latter case. In supplementation studies, significant effects were reported with vitamin D, calcium, and copper supplementation. For calcium, it was shown that the time of supplementation affects diurnal variation. Patients' diets should be carefully observed, not only in view of the effect on pyridinium cross-links concentrations but also in view of creatinine concentrations, which are known to be affected by diet. Therefore, nutritional status and use of dietary supplements need to be assessed carefully. The small number of relevant published reports in the area of seasonal variation is insufficient to clearly assess its impact. The reported magnitude of changes, 10-20%, might be significant and needs to be confirmed in further studies. The available data on geographic differences and differences attributable to race are very limited and cannot completely be separated from those of lifestyle or nutrition. However, studies using other bone markers and more appropriate study designs found geographic differences and differences attributable to race (204, 205).

In addition to factors that affect the biologic variability of urinary PYD and DPD measurement, other components of preanalytical variability, such as handling of samples and calibrators and creatinine correction, are of importance. The reported susceptibility of pyridinium cross-links to light requires special precautions when handling samples and calibrators. Because urinary PYD and DPD concentrations usually are corrected for creatinine, variability in creatinine measurements impacts pyridinium cross-links results. The available data indicate that a considerable part of variability can derive from the biologic variability in creatinine excretion. Creatinine measurements should not be used to adjust cross-links concentrations in patients with renal insufficiency, acute infection, early phase of injury, or trauma because creatinine output is not proportional to muscle mass under these conditions. In this context, it needs to be noted that creatinine correction in children can have several limitations (206-208) and therefore needs to be considered carefully.

CONCLUSION/RECOMMENDATIONS

Variability in urinary PYD and DPD measurements from preanalytical factors contributes substantially to problems in data interpretation. However, this variability can readily be minimized and/or standardized by applying the following suggested recommendations:

1. Specimens should be collected at a specific time of day to avoid diurnal variability. SMV urine seems to be the most practical urine type and therefore should be used for routine measurements, except for special applications or situations requiring a different type of urine.

2. Excretion rates from the same type of urine collection should be used for data comparisons.

3. Samples and calibrators should not be exposed to direct sunlight.

4. Collected samples should be stored at 2-6 [degrees]C if they are analyzed on the same day or frozen at -20 [degrees]C if they are analyzed after >24 h. No preservatives should be added to the sample, except for 24-h urines. Here, the sample should be stabilized with a weak acid (e.g., boric acid) and/or other chemicals to avoid changes from microbial contamination. In such a case, specifications of the assay in use need to be taken into consideration to avoid loss of assay performance.

5. Reference intervals should be established using well-characterized, healthy premenopausal women after assessment of menses and plasma follicle-stimulating hormone concentrations. The age range of 30-45 years would probably be the most adequate.

a. Perimenopausal women should not be included in the population used to establish reference intervals for urinary pyridinium cross-links.

b. For men, separate reference intervals should be defined.

c. For children, reference intervals should be reported by age group not older than 2 years; during puberty, Tanner stages should be mentioned.

6. Abnormalities in menstrual cycle should be identified, and results should be interpreted carefully in such patients.

7. Factors and conditions affecting creatinine excretion should be recognized and included in data interpretation.

8. Use of dietary supplements should be assessed, not only in view of vitamin D and calcium intake, but also in view of other vitamins and minerals.

9. Current and previous diseases and conditions need to be taken into consideration. Data obtained from immobilized patients should be interpreted with caution. Data from immobilized patients probably should be compared with data from ambulatory individuals rather than with healthy individuals described under number 5.

This work was partly funded by the CDC Office of Women's Health.

Received August 14, 2001; accepted November 15, 2001.

References

(1.) Looker AC, Bauer DC, Chesnut CH 3rd, Gundberg CM, Hochberg MC, Klee G, et al. Clinical use of biochemical markers of bone remodeling: current status and future directions [Review]. Osteoporos Int 2000;11:467-80.

(2.) Garnero P, Hausherr E, Chapuy MC, Marcelli C, Grandjean H, Muller C, et al. Markers of bone resorption predict hip fracture in elderly women: the EPIDOS Prospective Study. J Bone Miner Res 1996;11:1531-8.

(3.) Miller PD, Baran DT, Bilezikian JP, Greenspan SL, Lindsay R, Riggs BL, et al. Practical clinical application of biochemical markers of bone turnover: consensus of an expert panel [Review]. J Clin Densitom 1999;2:323-42.

(4.) Hart SM, Eastell R. Biochemical markers of bone turnover [Review]. Curr Opin Nephrol Hypertens 1999;8:421-7.

(5.) Seibel MJ, Baylink DJ, Farley JR, Epstein S, Yamauchi M, Eastell R, et al. Basic science and clinical utility of biochemical markers of bone turnover-a Congress report. Exp Clin Endocrinol Diabetes 1997;105:125-33.

(6.) Souberbielle JC, Cormier C, Kindermans C. Bone markers in clinical practice [Review]. Curr Opin Rheumatol 1999;11:312-9.

(7.) Garnero P, Delmas PD. Biochemical markers of bone turnover. Applications for osteoporosis [Review]. Endocrinol Metab Clin North Am 1998;27:303-23.

(8.) Beck-Jensen JE, Sorensen HA, Kollerup G, Jensen LB, Sorensen OH. Biological variation of biochemical bone markers. Scand J Clin Lab Invest 1994;54:36-9.

(9.) Risteli J, Demers LM, Eastell R, Garnero P, Hoyle N. Committee for markers of bone turnover and bone disease (C-MBTBD) [Abstract]. Clin Chem Lab Med 1999;37:S109-10.

(10.) Delmas PD, Eastell R, Garnero P, Seibel MJ, Stephan J. The use of biochemical markers of bone turnover in osteoporosis. Committee of Scientific Advisors of the International Osteoporosis Foundation. Osteoporos Int 2000;6:2-17.

(11.) Hannon R, Blumsohn A, Naylor K, Eastell R. Response of biochemical markers of bone turnover to hormone replacement therapy: impact of biological variability. J Bone Miner Res 1998; 13:1124-33.

(12.) Woitge HW, Pecherstorfer M, Li Y, Keck AV, Horn E, Ziegler R, et al. Novel serum markers of bone resorption: clinical assessment and comparison with established urinary indices. J Bone Miner Res 1999;14:792-801.

(13.) Rodriguez-Arnao J, James I, Jabbar A, Trainer PJ, Perrett D, Besser GM, et al. Serum collagen crosslinks as markers of bone turn-over during GH replacement therapy in growth hormone deficient adults. Clin Endocrinol (Oxf) 1998;48:455-62.

(14.) Siddiqi A, Burrin JM, Noonan K, James I, Wood DF, Price CP, et al. A longitudinal study of markers of bone turnover in Graves' disease and their value in predicting bone mineral density. J Clin Endocrinol Metab 1997;82:753-9.

(15.) Sarno M, Powell H, Tjersland G, Schoendorfer D, Harris H, Adams K, et al. A collection method and high-sensitivity enzyme immunoassay for sweat pyridinoline and deoxypyridinoline cross-links. Clin Chem 1999;45:1501-9.

(16.) James IT, Walne AJ, Perrett D. The measurement of pyridinium crosslinks: a methodological overview [Review]. Ann Clin Biochem 1996;33:397-420.

(17.) Robins SP, Woitge H, Hesley R, Ju J, Seyedin S, Seibel MJ. Direct, enzyme-linked immunoassay for urinary deoxypyridinoline as a specific marker for measuring bone resorption. J Bone Miner Res 1994;9:1643-9.

(18.) Blumsohn A, Hannon RA, Wrate R, Barton J, al-Dehaimi AW, Colwell A, et al. Biochemical markers of bone turnover in girls during puberty. Clin Endocrinol (Oxf) 1994;40:663-70.

(19.) Blumsohn A, Naylor KE, Assiri AM, Eastell R. Different responses of biochemical markers of bone resorption to bisphosphonate therapy in Paget disease. Clin Chem 1995;41:1592-8.

(20.) Garnero P, Gineyts E, Arbault P, Christiansen C, Delmas PD. Different effects of bisphosphonate and estrogen therapy on free and peptide-bound bone cross-links excretion. J Bone Miner Res 1995;10:641-9.

(21.) Popp-Snijders C, Lips P, Netelenbos JC. Intra-individual variation in bone resorption markers in urine. Ann Clin Biochem 1996;33: 347-8.

(22.) Robins SP. Collagen crosslinks in metabolic bone disease [Review]. Acta Orthop Scand 1995;Suppl 266:171-5.

(23.) Seibel M, Duncan A, Robins SP. Urinary hydroxypyridinium crosslinks provide indices of cartilage and bone involvement in arthritic diseases. J Rheumatol 1989;16:964-70.

(24.) Schlemmer A, Hassager C, Pedersen BJ, Christiansen C. Posture, age, menopause, and osteopenia do not influence the circadian variation in the urinary excretion of pyridinium crosslinks. J Bone Miner Res 1994;9:1883-8.

(25.) Aoshima H, Kushida K, Takahashi M, Ohishi T, Hoshino H, Suzuki M, et al. Circadian variation of urinary type I collagen crosslinked C-telopeptide and free and peptide-bound forms of pyridinium crosslinks. Bone 1998;22:73-8.

(26.) Cosman F, Morgan DC, Nieves JW, Shen V, Luckey MM, Dempster DW, et al. Resistance to bone resorbing effects of PTH in black women. J Bone Miner Res 1997;12:958-66.

(27.) Eastell R, Calvo MS, Burritt MF, Offord KP, Russell RG, Riggs BL. Abnormalities in circadian patterns of bone resorption and renal calcium conservation in type I osteoporosis. J Clin Endocrinol Metab 1992;74:487-94.

(28.) Stone PJ, Beiser A, Gottlieb DJ. Circadian variation of urinary excretion of elastin and collagen crosslinks. Proc Soc Exp Biol Med 1998;218:229-33.

(29.) Fujimoto S, Kubo T, Tanaka H, Miura M, Seino Y. Urinary pyridinoline and deoxypyridinoline in healthy children and in children with growth hormone deficiency. J Clin Endocrinol Metab 1995;80:1922-8.

(30.) Eastell R, Simmons PS, Colwell A, Assiri AM, Burritt MF, Russell RG, et al. Nyctohemeral changes in bone turnover assessed by serum bone Gla-protein concentration and urinary deoxypyridinoline excretion: effects of growth and ageing. Clin Sci 1992;83: 375-82.

(31.) Leino A, Impivaara O, Kaitsaari M. Measurements of deoxypyridinoline and hydroxyproline in 24-h, first morning, and second morning urine samples. Clin Chem 1996;42:2037-9.

(32.) Ginty F, Flynn A, Cashman K. Inter and intra-individual variations in urinary excretion of pyridinium crosslinks of collagen in healthy young adults. Eur J Clin Nutr 1998;52:71-3.

(33.) Ohishi T, Takahashi M, Kushida K, Horiuchi K, Ishigaki S, Inoue T. Quantitative analyses of urinary pyridinoline and deoxypyridinoline excretion in patients with hyperthyroidism. Endocr Res 1992;18:281-90.

(34.) Delmas PD, Gineyts E, Bertholin A, Garnero P, Marchand F. Immunoassay of pyridinoline crosslink excretion in normal adults and in Paget's disease. J Bone Miner Res 1993;8:643-8.

(35.) Melton LJ, Khosla S, Atkinson EJ, O'Fallon WM, Riggs BL. Relationship of bone turnover to bone density and fractures. J Bone Miner Res 1997;12:1083-91.

(36.) Ebeling PR, Atley LM, Guthrie JR, Burger HG, Dennerstein L, Hopper JL, et al. Bone turnover markers and bone density across the menopausal transition. J Clin Endocrinol Metab 1996;81: 3366-71.

(37.) Kawana K, Kushida K, Takahashi M, Ohishi T, Denda M, Yamazaki K, et al. The effect of menopause on biochemical markers and ultrasound densitometry in healthy females. Calcif Tissue Int 1994;55:420-5.

(38.) Hassager C, Colwell A, Assiri AM, Eastell R, Russell RG, Christiansen C. Effect of menopause and hormone replacement therapy on urinary excretion of pyridinium cross-links: a longitudinal and cross-sectional study. Clin Endocrinol (Oxf) 1992;37: 45-50.

(39.) Yoshihara K, Nemoto S, Nagata M. Urinary excretion level of hydroxylysylpyridinoline as an index of bone resorption. Biol Pharm Bull 1994;17:840-2.

(40.) Fatayerji D, Eastell R. Age-related changes in bone turnover in men. J Bone Miner Res 1999;14:1203-10.

(41.) Szulc P, Garnero P, Munoz F, Marchand F, Delmas PD. Crosssectional analysis of age-related changes of bone turnover in men: the MINOS-Study [Abstract]. J Bone Miner Res 1999;14: S308.

(42.) Marowska J, Kobylinska M, Lukaszkiewicz J, Talajko A, Rymkiewicz-Kluczynska B, Lorenc RS. Pyridinium crosslinks of collagen as a marker of bone resorption rates in children and adolescents: normal values and clinical application. Bone 1996;19: 669-77.

(43.) Rauch F, Schonau E, Woitge H, Remer T, Seibel M. Urinary excretion of hydroxy-pyridinium cross-links of collagen reflects skeletal growth velocity in normal children. Exp Clin Endocrinol 1994;102:94-7.

(44.) Rauch F, Rauch R, Woitge HW, Seibel MJ, Schonau E. Urinary immunoreactive deoxypyridinoline in children and adolescents: variations with age, sex and growth velocity. Scand J Clin Lab Invest 1996;56:715-9.

(45.) Shaw NJ, Dutton J, Fraser WD, Smith CS. Urinary pyridinoline and deoxypyridinoline excretion in children. Clin Endocrinol (Oxf) 1995;42:607-12.

(46.) Mora S, Prinster C, Proverbio MC, Bellini A, de Poli SC, Weber G, et al. Urinary markers of bone turnover in healthy children and adolescents: age-related changes and effect of puberty. Calcif Tissue Int 1998;63:369-74.

(47.) Husain SM, Mughal Z, Williams G, Ward K, Smith CS, Dutton J, et al. Urinary excretion of pyridinium crosslinks in healthy 4-10 years olds. Arch Dis Child 1999;80:370-3.

(48.) Acil Y, Brinckmann J, Notbohm H, Muller PK, Batge B. Changes with age in the urinary excretion of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP). Scand J Clin Lab Invest 1996;56:275-83.

(49.) Beardsworth LJ, Eyre DR, Dickson IR. Changes with age in the urinary excretion of lysyl- and hydroxylysylpyridinoline, two new markers of bone collagen turnover. J Bone Miner Res 1990;5: 671-6.

(50.) Tsukahara H, Watanabe Y, Hirano S, Tsubokura H, Kimura K, Mayumi M. Assessment of bone turnover in term and preterm newborns at birth: measurement of urinary collagen crosslink excretion. Early Hum Dev 1999;53:185-91.

(51.) Tsukahara H, Miura M, Hori C, Hiraoka M, Nosaka K, Hata K, et al. Urinary excretion of pyridinium cross-links of collagen in infancy. Metabolism 1996;45:510-4.

(52.) Naylor KE, Eastell R, Shattuck KE, Alfrey AC, Klein GL. Bone turnover in preterm infants. Pediatr Res 1999;45:363-6.

(53.) Rauch F, Middelmann B, Rosmalen F, Seibel MJ, Schonau E. Free deoxypyridinoline in urine and serum-results in children and adolescents. Exp Clin Endocrinol Diabetes 1996;104:396-9.

(54.) Conti A, Ferrero S, Giambona S, Sartorio A. Urinary free deoxypyridinoline levels during childhood. J Endocrinol Invest 1998; 21:318-22.

(55.) Libanati C, Baylink DJ, Lois-Wenzel E, Srinvasan N, Mohan S. Studies on the potential mediators of skeletal changes occurring during puberty in girls. J Clin Endocrinol Metab 1999;84:2807-14.

(56.) Uebelhart D, Gineyts E, Chapuy MC, Delmas PD. Urinary excretion of pyridinium crosslinks: a new marker of bone resorption in metabolic bone disease. Bone Miner 1990;8:87-96.

(57.) Akesson K, Vergnaud P, Gineyts E, Delmas PD, Obrant KJ. Impairment of bone turnover in elderly women with hip fracture. Calcif Tissue Int 1993;53:162-9.

(58.) Gorai I, Taguchi Y, Chaki O, Nakayama M, Minaguchi H. Specific changes of urinary excretion of cross-linked N-telopeptides of type I collagen in pre- and postmenopausal women: correlation with other markers of bone turnover. Calcif Tissue Int 1997;60: 317-22.

(59.) Kushida K, Takahashi M, Kawana K, Inoue T. Comparison of markers for bone formation and resorption in premenopausal and postmenopausal subjects, and osteoporosis patients. J Clin Endocrinol Metab 1995;80:2447-50.

(60.) Ohishi T, Takahashi M, Kawana K, Aoshima H, Hoshino H, Horiuchi K, et al. Age-related changes of urinary pyridinoline and deoxypyridinoline in Japanese subjects. Clin Invest Med 1993; 16:319-25.

(61.) Daniels ED, Pettifor JM, Schnitzler CM, Moodley GP, Zachen D. Differences in mineral homeostasis, volumetric bone mass and femoral neck axis length in black and white South African women. Osteoporos Int 1997;7:105-12.

(62.) Lin JS, Kao JT, Tsai KS. Type I collagen and procollagen fragments in patients with metabolic bone diseases. J Formos Med Assoc 1996;95:523-9.

(63.) Mariconda M, Pavia M, Colonna A, Angelillo IF, Marsico O, Sanzo F, et al. Appendicular bone density, biochemical markers of bone turnover and lifestyle factors in female teachers of Southern Italy. Eur J Epidemiol 1997;13:909-17.

(64.) Seibel MJ, Lang M, Auler B, Kissling C, von Schickfus A, Rohle G. Standardization trial of biochemical bone markers of bone turnover [Abstract]. J Bone Miner Res 1999;14:S161.

(65.) Henry YM, Eastell R. Ethnic and gender differences in bone mineral density and bone turnover in young adults: effect of bone size. Osteoporos Int 2000;11:512-7.

(66.) Aloia JF, Mikhail M, Pagan CD, Arunachalam A, Yeh JK, Flaster E. Biochemical and hormonal variables in black and white women matched for age and weight. J Lab Clin Med 1998;132:383-9.

(67.) Chiu KM, Ju J, Mayes D, Bacchetti P, Weitz S, Arnaud CD. Changes in bone resorption during the menstrual cycle. J Bone Miner Res 1999;14:609-15.

(68.) Schlemmer A, Hassager C, Risteli J, Risteli L, Jensen SB, Christiansen C. Possible variation in bone resorption during the normal menstrual cycle. Acta Endocrinol 1993;129:388-92.

(69.) Gorai I, Taguchi Y, Chaki O, Kikuchi R, Nakayama M, Yang BC, et al. Serum soluble interleukin-6 receptor and biochemical markers of bone metabolism show significant variations during the menstrual cycle. J Clin Endocrinol Metab 1998;83:326-32.

(70.) Adami S, Zamberlan N, Castello R, Tosi F, Gatti D, Moghetti P. Effect of hyperandrogenism and menstrual cycle abnormalities on bone mass and bone turnover in young women. Clin Endocrinol (Oxf) 1998;48:169-73.

(71.) Zanker CL, Swaine IL. Bone turnover in amenorrhoeic and eumenorrhoeic women distance runners. Scand J Med Sci Sports 1998;8:20-6.

(72.) Yamaga A, Taga M, Minaguchi H. Changes in urinary excretions of C-telopeptide and cross-linked N-telopeptide of type I collagen during pregnancy and puerperium. Endocrine 1997;44:733-8.

(73.) Yamaga A, Taga M, Minaguchi H, Sato K. Changes in bone mass as determined by ultrasound and biochemical markers of bone turnover during pregnancy and puerperium: a longitudinal study. J Clin Endocrinol Metab 1996;81:752-6.

(74.) Cross NA, Hillman LS, Allen SH, Krause GF, Vieira NE. Calcium homeostasis and bone metabolism during pregnancy, lactation, and postweaning: a longitudinal study. Am J Clin Nutr 1995;61: 514-23.

(75.) Naylor KE, Iqbal P, Fledelius C, Fraser RB, Eastell R. The effect of pregnancy on bone density and bone turnover. J Bone Miner Res 2000;15:129-37.

(76.) Woitge HW, Scheidt-Nave C, Kissling C, Leidig-Bruckner G, Meyer K, Grauer A, et al. Seasonal variation of biochemical indexes of bone turnover: results of a population-based study. J Clin Endocrinol Metab 1998;83:68-75.

(77.) Zittermann A, Scheld K, Stehle P. Seasonal variations in vitamin D status and calcium absorption do not influence bone turnover in young women. Eur J Clin Nutr 1998;52:501-6.

(78.) Douglas AS, Miller MH, Reid DM, Hutchison JD, Porter RW, Robins SP. Seasonal differences in biochemical parameters of bone remodelling. J Clin Pathol 1996;49:284-9.

(79.) Matsumoto T, Nakagawa S, Nishida S, Hirota R. Bone density and bone metabolic markers in active collegiate athletes: findings in long-distance runners, judoists, and swimmers. Int J Sports Med 1997;18:408-12.

(80.) Hetland ML, Haarbo J, Christiansen C. Low bone mass and high bone turnover in male long distance runners. J Clin Endocrinol Metab 1993;77:770-5.

(81.) Isdale A, Helliwell PS. Athletes and osteoarthritis-is there any relationship [Letter]? Br J Rheumatol 1991;30:67-8.

(82.) Welsh L, Rutherford OM, James I, Crowley C, Comer M, Wolman R. The acute effects of exercise on bone turnover. Int J Sports Med 1997;18:247-51.

(83.) Stacey E, Korkia P, Hukkanen MV, Polak JM, Rutherford OM. Decreased nitric oxide levels and bone turnover in amenorrheic athletes with spinal osteopenia. J Clin Endocrinol Metab 1998; 83:3056-61.

(84.) Nickols-Richardson SM, O'Connor PJ, Shapses SA, Lewis RD. Longitudinal bone mineral density changes in female child artistic gymnasts. J Bone Miner Res 1999;14:994-1002.

(85.) Eliakim A, Raisz LG, Brasel JA, Cooper DM. Evidence for increased bone formation following a brief endurance-type training intervention in adolescent males. J Bone Miner Res 1997;12: 1708-13.

(86.) Welsh L, Rutherford OM. Hip bone mineral density is improved by high-impact aerobic exercise in postmenopausal women and men over 50 years. Eur J Appl Physiol Occup Physiol 1996;74: 511-7.

(87.) Ashizawa N, Fujimura R, Tokuyama K, Suzuki M. A bout of resistance exercise increases urinary calcium independently of osteoclastic activation in men. J Appl Physiol 1997;83:1159-63.

(88.) Fujimura R, Ashizawa N, Watanabe M, Mukai N, Amagai H, Fukubayashi T, et al. Effect of resistance exercise training on bone formation and resorption in young male subjects assessed by biomarkers of bone metabolism. J Bone Miner Res 1997;12: 656-62.

(89.) Brown SJ, Child RB, Day SH, Donnelly AE. Indices of skeletal muscle damage and connective tissue breakdown following eccentric muscle contractions. Eur J Appl Physiol Occup Physiol 1997;75:369-74.

(90.) Hoshino H, Kushida K, Yamazaki K, Takahashi M, Ogihara H, Naitoh K, et al. Effect of physical activity as a caddie on ultrasound measurements of the Os calcis: a cross-sectional comparison. J Bone Miner Res 1996;11:412-8.

(91.) Weiss M, Yogev R, Dolev E. Occupational sitting and low hip mineral density. Calcif Tissue Int 1998;62:47-50.

(92.) Nishimura Y, Fukuoka H, Kiriyama M, Suzuki Y, Oyama K, Ikawa S, et al. Bone turnover and calcium metabolism during 20 days bed rest in young healthy males and females. Acta Physiol Scand 1994;Suppl 616:27-35.

(93.) Lueken SA, Arnaud SB, Taylor AK, Baylink DJ. Changes in markers of bone formation and resorption in a bed rest model of weightlessness. J Bone Miner Res 1993;8:1433-8.

(94.) Fiore CE, Pennisi P, Ciffo F, Scebba C, Amico A, Di Fazzio S. Immobilization-dependent bone collagen breakdown appears to increase with time: evidence for a lack of new bone equilibrium in response to reduced load during prolonged bed rest. Horm Metab Res 1999;31:31-6.

(95.) Caillot-Augusseau A, Lafage-Proust MH, Soler C, Pernod J, Dubois F, Alexandre C. Bone formation and resorption biological markers in cosmonauts during and after a 180-day space flight (Euromir 95). Clin Chem 1998;44:578-85.

(96.) Smith SM, Nillen JL, Leblanc A, Lipton A, Demers LM, Lane HW, et al. Collagen cross-link excretion during space flight and bed rest. J Clin Endocrinol Metab 1998;83:3584-91.

(97.) Grinspoon SK, Baum HB, Kim V, Coggins C, Klibanski A. Decreased bone formation and increased mineral dissolution during acute fasting in young women. J Clin Endocrinol Metab 1995;80:3628-33.

(98.) Branca F, Robins SP, Ferro-Luzzi A, Golden MH. Bone turnover in malnourished children. Lancet 1992;340:1493-6.

(99.) New SA, Robins SP, Campbell MK, Martin JC, Garton MJ, Bolton-Smith C, et al. Dietary influences on bone mass and bone metabolism: further evidence of a positive link between fruit and vegetable consumption and bone health? Am J Clin Nutr 2000; 71:142-51.

(100.) Evans CE, Chughtai AY, Blumsohn A, Giles M, Eastell R. The effect of dietary sodium on calcium metabolism in premenopausal and postmenopausal women. Eur J Clin Nutr 1997;51: 394-9.

(101.) Ginty F, Flynn A, Cashman KD. The effect of dietary sodium intake on biochemical markers of bone metabolism in young women. Br J Nutr 1998;79:343-50.

(102.) Lietz G, Avenell A, Robins SP. Short-term effects of dietary sodium intake on bone metabolism in postmenopausal women measured using urinary deoxypyridinoline excretion. Br J Nutr 1997;78:73-82.

(103.) Karkkainen M, Lamberg-Allardt C. An acute intake of phosphate increases parathyroid hormone secretion and inhibits bone formation in young women. J Bone Miner Res 1996;11:1905-12.

(104.) Shapses SA, Robins SP, Schwartz EI, Chowdhury H. Short-term changes in calcium but not protein intake alter the rate of bone resorption in healthy subjects as assessed by urinary pyridinium cross-link excretion. J Nutr 1995;125:2814-21.

(105.) Clark PJ, Eastell R, Barker ME. Zinc supplementation and bone growth in pubertal girls [Letter]. Lancet 1999;354:485.

(106.) Cadogan J, Eastell R, Jones N, Barker ME. Milk intake and bone mineral acquisition in adolescent girls: randomised, controlled intervention trial. BMJ 1997;315:1255-60.

(107.) Baker A, Harvey L, Majask-Newman G, Fairweather-Tait S, Flynn A, Cashman K. Effect of dietary copper intakes on biochemical markers of bone metabolism in healthy adult males. Eur J Clin Nutr 1999;53:408-12.

(108.) Kamel S, Brazier M, Picard C, Boitte F, Samson L, Desmet G, et al. Urinary excretion of pyridinolines crosslinks measured by immunoassay and HPLC techniques in normal subjects and in elderly patients with vitamin D deficiency. Bone Miner 1994;26: 197-208.

(109.) Keaveny AP, Freaney R, McKenna MJ, Masterson J, O'Donoghue DP. Bone remodeling indices and secondary hyperparathyroidism in celiac disease. Am J Gastroenterol 1996;91:1226-31.

(110.) Fardellone P, Sebert JL, Garabedian M, Bellony R, Maamer M, Agbomson F, et al. Prevalence and biological consequences of vitamin D deficiency in elderly institutionalized subjects. Rev Rhum Engl Ed 1995;62:576-81.

(111.) Kamel S, Brazier M, Rogez JC, Vincent O, Maamer M, Desmet G, et al. Different responses of free and peptide-bound cross-links to vitamin D and calcium supplementation in elderly women with vitamin D insufficiency. J Clin Endocrinol Metab 1996;81:3717-21.

(112.) Rubinacci A, Melzi R, Zampino M, Soldarini A, Villa I. Total and free deoxypyridinoline after acute osteoclast activity inhibition. Clin Chem 1999;45:1510-6.

(113.) Blumsohn A, Herrington K, Hannon RA, Shao P, Eyre DR, Eastell R. The effect of calcium supplementation on the circadian rhythm of bone resorption. J Clin Endocrinol Metab 1994;79: 730-5.

(114.) Scopacasa F, Horowitz M, Wishart JM, Need AG, Morris HA, Wittert G, et al. Calcium supplementation suppresses bone resorption in early postmenopausal women. Calcif Tissue Int 1998;62:8-12.

(115.) Sairanen S, Tahtela R, Laitinen K, Karonen SL, Valimaki MJ. Nocturnal rise in markers of bone resorption is not abolished by bedtime calcium or calcitonin. Calcif Tissue Int 1994;55:349-52.

(116.) Nyquist F, Ljunghall S, Berglund M, Obrant K. Biochemical markers of bone metabolism after short and long time ethanol withdrawal in alcoholics. Bone 1996;19:51-4.

(117.) Hopper JL, Seeman E. The bone density of female twins discordant for tobacco use. N Engl J Med 1994;330:387-92.

(118.) Gottlieb DJ, Stone PJ, Sparrow D, Gale ME, Weiss GL, O'Connor GT. Urinary desmosine excretion in smokers with and without rapid decline of lung function. Am J Respir Crit Care Med 1996;154:1290-5.

(119.) Braga de C, Hannon R, Eastell R. Monitoring alendronate therapy for osteoporosis. J Bone Miner Res 1999;14:602-8.

(120.) Watts NB. Clinical utility of biochemical markers of bone remodeling [Review]. Clin Chem 1999;45:1359-68.

(121.) Eyre DR. Bone biomarkers as tools in osteoporosis management [Review]. Spine 1997;22:17S-24S.

(122.) Price CP, Thompson PW. The role of biochemical tests in the screening and monitoring of osteoporosis [Review]. Ann Clin Biochem 1995;32:244-60.

(123.) Oikawa M, Kushida K, Takahashi M, Ohishi T, Hoshino H, Suzuki M, et al. Bone turnover and cortical bone mineral density in the distal radius in patients with hyperthyroidism being treated with antithyroid drugs for various periods of time. Clin Endocrinol (Oxf) 1999;50:171-6.

(124.) Lakatos P, Foldes J, Horvath C, Kiss L, Tatrai A, Takacs I, et al. Serum interleukin-6 and bone metabolism in patients with thyroid function disorders. J Clin Endocrinol Metab 1997;82:7881.

(125.) Langdahl BL, Loft AG, Moller N, Weeke J, Eriksen EF, Mosekilde L, et al. Is skeletal responsiveness to thyroid hormone altered in primary osteoporosis or following estrogen replacement therapy? J Bone Miner Res 1997;12:78-88.

(126.) Knudsen N, Faber J, Sierbaek-Nielsen A, Vadstrup S, Sorensen HA, Hegedus L. Thyroid hormone treatment aiming at reduced, but not suppressed, serum thyroid-stimulating hormone levels in nontoxic goitre: effects on bone metabolism amongst premenopausal women. J Intern Med 1998;243:149-54.

(127.) Hoshino H, Kushida K, Takahashi M, Kawana K, Denda M, Yamazaki K, et al. Characteristics of biochemical markers in patients with metabolic bone disorders. Endocr Res 1998;24: 55-64.

(128.) Rosano TG, Peaston RT, Bone HG, Woitge HW, Francis RM, Seibel MJ. Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation. Clin Chem 1998;44:2126-32.

(129.) Pasquali M, Dembure PP, Still MJ, Elsas LJ. Urinary pyridinium cross-links: a noninvasive diagnostic test for Ehlers-Danlos syndrome type VI [Letter]. N Engl J Med 1994;331:132-3.

(130.) Pasquali M, Still MJ, Vales T, Rosen RI, Evinger JD, Dembure PP, et al. Abnormal formation of collagen cross-links in skin fibroblasts cultured from patients with Ehlers-Danlos syndrome type VI. Proc Assoc Am Physicians 1996;109:33-41.

(131.) Alvarez L, Guanabens N, Peris P, Monegal A, Bedini JL, Deulofeu R, et al. Discriminative value of biochemical markers of bone turnover in assessing the activity of Paget's disease. J Bone Miner Res 1995;10:458-65.

(132.) Pecherstorfer M, Seibel MJ, Woitge HW, Horn E, Schuster J, Neuda J, et al. Bone resorption in multiple myeloma and in monoclonal gammopathy of undetermined significance: quantification by urinary pyridinium cross-links of collagen. Blood 1997;90:3743-50.

(133.) Seibel MJ, Gartenberg F, Silverberg SJ, Ratcliffe A, Robins SP, Bilezikian JP. Urinary hydroxypyridinium cross-links of collagen in primary hyperparathyroidism. J Clin Endocrinol Metab 1992;74: 481-6.

(134.) Ingle BM, Hay SM, Bottjer HM, Eastell R. Changes in bone mass and bone turnover following ankle fracture. Osteoporos Int 1999;10:408-15.

(135.) Ingle BM, Hay SM, Bottjer HM, Eastell R. Changes in bone mass and bone turnover following distal forearm fracture. Osteoporos Int 1999;10:399-407.

(136.) Peichl P, Rintelen B, Kumpan W, Broll H. Increase of axial and appendicular trabecular and cortical bone density in established osteoporosis with intermittent nasal salmon calcitonin therapy. Gynecol Endocrinol 1999;13:7-14.

(137.) Bjorgaas M, Haug E, Johnsen HJ. The urinary excretion of deoxypyridinium cross-links is higher in diabetic than in nondiabetic adolescents. Calcif Tissue Int 1999;65:121-4.

(138.) Cakatay U, Telci A, Kayali R, Akcay T, Sivas A, Aral F. Changes in bone turnover on deoxypyridinoline levels in diabetic patients. Diabetes Res Clin Pract 1998;40:75-9.

(139.) Cloos C, Wahl P, Hasslacher C, Traber L, Kistner M, Jurkuhn K, et al. Urinary glycosylated, free and total pyridinoline and free and total deoxypyridinoline in diabetes mellitus. Clin Endocrinol (Oxf) 1998;48:317-23.

(140.) Rosato MT, Schneider SH, Shapses SA. Bone turnover and insulin-like growth factor I levels increase after improved glycemic control in noninsulin-dependent diabetes mellitus. Calcif Tissue Int 1998;63:107-11.

(141.) Valerio G, Franzese A, Esposito-Del PA, Formicola S, Di Maio S, Contaldo F, et al. Increased urinary excretion of collagen crosslinks in type 1 diabetic children in the first 5 years of disease. Horm Res 1999;51:173-7.

(142.) Suzuki M, Takahashi M, Miyamoto S, Hoshino H, Kushida K, Miura M, et al. The effects of menopausal status and disease activity on biochemical markers of bone metabolism in female patients with rheumatoid arthritis. Br J Rheumatol 1998;37: 653-8.

(143.) Takahashi M, Suzuki M, Naitou K, Miyamoto S, Kushida K. Comparison of free and peptide-bound pyridinoline cross-links excretion in rheumatoid arthritis and osteoarthritis. Rheumatology (Oxford) 1999;38:133-8.

(144.) Guanabens N, Pares A, Alvarez L, Martinez deOsaba M, Monegal A, Peris P, et al. Collagen-related markers of bone turnover reflect the severity of liver fibrosis in patients with primary biliary cirrhosis. J Bone Miner Res 1998;13:731-8.

(145.) Ha SK, Park CH, Seo JK, Park SH, Kang SW, Choi KH, et al. Studies on bone markers and bone mineral density in patients with chronic renal failure. Yonsei Med J 1996;37:350-6.

(146.) Hernandez MV, Peris P, Guanabens N, Alvarez L, Monegal A, Pons F, et al. Biochemical markers of bone turnover in Camurati-Engelmann disease: a report on four cases in one family. Calcif Tissue Int 1997;61:48-51.

(147.) Roberts D, Lee W, Cuneo RC, Wittmann J, Ward G, Flatman R, et al. Longitudinal study of bone turnover after acute spinal cord injury. J Clin Endocrinol Metab 1998;83:415-22.

(148.) Ebeling PR, Thomas DM, Erbas B, Hopper JL, Szer J, Grigg AP. Mechanisms of bone loss following allogeneic and autologous hemopoietic stem cell transplantation. J Bone Miner Res 1999; 14:342-50.

(149.) Bjarnason I, Macpherson A, Mackintosh C, Buxton-Thomas M, Forgacs I, Moniz C. Reduced bone density in patients with inflammatory bowel disease. Gut 1997;40:228-33.

(150.) Stone PJ, Konstan MW, Berger M, Dorkin HL, Franzblau C, Snider GL. Elastin and collagen degradation products in urine of patients with cystic fibrosis. Am J Respir Crit Care Med 1995;152: 157-62.

(151.) Stone PJ, Korn JH, North H, Lally EV, Miller LC, Tucker LB, et al. Cross-linked elastin and collagen degradation products in the urine of patients with scleroderma. Arthritis Rheum 1995;38: 517-24.

(152.) Bachrach LK, Marcus R, Ott SM, Rosenbloom AL, Vasconez O, Martinez V, et al. Bone mineral, histomorphometry, and body composition in adults with growth hormone receptor deficiency. J Bone Miner Res 1998;13:415-21.

(153.) Shaarawy M, El-Dawakhly AS, Mosaad M, El-Sadek MM. Biomarkers of bone turnover and bone mineral density in hyperprolactinemic amenorrheic women. Clin Chem Lab Med 1999;37: 433-8.

(154.) Quan A, Adams R, Ekmark E, Baum M. Bone mineral density in children with myelomeningocele [Abstract]. Pediatrics 1998; 102:E34.

(155.) MacDonald AG, Birkinshaw G, Durham B, Bucknall RC, Fraser WD. Biochemical markers of bone turnover in seronegative spondylarthropathy: relationship to disease activity. Br J Rheumatol 1997;36:50-3.

(156.) Sprott H, Muller A, Heine H. Collagen crosslinks in fibromyalgia. Arthritis Rheum 1997;40:1450-4.

(157.) Klein GL, Herndon DN, Goodman WG, Langman CB, Phillips WA, Dickson IR, et al. Histomorphometric and biochemical characterization of bone following acute severe burns in children. Bone 1995;17:455-60.

(158.) Crofton PM, Ahmed SF, Wade JC, Stephen R, Elmlinger MW, Ranke MB, et al. Effects of intensive chemotherapy on bone and collagen turnover and the growth hormone axis in children with acute lymphoblastic leukemia. J Clin Endocrinol Metab 1998; 83:3121-9.

(159.) Peel N, al-Dehaimi A, Colwell A, Russell G, Eastell R. Sulfasalazine may interfere with HPLC assay of urinary pyridinium crosslinks [Letter]. Clin Chem 1994;40:167-8.

(160.) Panteghini M, Pagani F. Biological variation in urinary excretion of pyridinium crosslinks: recommendations for the optimum specimen. Ann Clin Biochem 1996;33:36-42.

(161.) Ju HS, Leung S, Brown B, Stringer MA, Leigh S, Scherrer C, et al. Comparison of analytical performance and biological variability of three bone resorption assays. Clin Chem 1997;43:1570-6.

(162.) Abbiati G, Bartucci F, Longoni A, Fincato G, Galimberti S, Rigoldi M, et al. Monitoring of free and total urinary pyridinoline and deoxypyridinoline in healthy volunteers: sample relationships between 24-h and fasting early morning urine concentrations. Bone Miner 1993;21:9-19.

(163.) Sakura S, Fujimoto D, Sakamoto K, Mizuno A, Motegi K. Photolysis of pyridinoline, a cross-linking amino acid of collagen, by ultraviolet light. Can J Biochem 1982;60:525-9.

(164.) Blumsohn A, Colwell A, Naylor K, Eastell R. Effect of light and gamma-irradiation on pyridinolines and telopeptides of type I collagen in urine. Clin Chem 1995;41:1195-7.

(165.) Casserly UM, O'Rorke A, Power MJ, Fottrell PF. Validation of a high-performance liquid chromatographic assay for lysylpyridinoline in urine: a potential biomarker of bone resorption. Anal Biochem 1996;242:255-60.

(166.) Walne AJ, James IT, Perrett D. The stability of pyridinium crosslinks in urine and serum [Letter]. Clin Chim Acta 1995;240: 95-7.

(167.) Gerrits MI, Thijssen JH, Van Rijn HJ. Determination of pyridinoline and deoxypyridinoline in urine, with special attention to retaining their stability. Clin Chem 1995;41:571-4.

(168.) Koumantakis G, Wyndham L. Fluorescein interference with urinary creatinine and protein measurements [Letter]. Clin Chem 1991;37:1799.

(169.) Kroll MH, Roach NA, Poe B, Elin RJ. Mechanism of interference with the Jaffe reaction for creatinine. Clin Chem 1987;33:1129-32.

(170.) Blank DW, Nanji AA. Ketone interference in estimation of urinary creatinine: effect on creatinine clearance in diabetic ketoacidosis. Clin Biochem 1982;15:279-80.

(171.) Lo SC, Tsai KS. Glucose interference in Jaffe creatinine method: effect of calcium from peritoneal dialysate [Letter]. Clin Chem 1994;40:2326-7.

(172.) Young DS. Effects of drugs on clinical laboratory tests, 3rd ed. Washington: AACC Press, 1990:122-32.

(173.) Weber JA, van Zanten AP. Interferences in current methods for measurements of creatinine. Clin Chem 1991;37:695-700.

(174.) Grotsch H, Hajdu P. Interference by the new antibiotic cefpirome and other cephalosporins in clinical laboratory tests, with special regard to the "Jaffe" reaction. J Clin Chem Clin Biochem 1987; 25:49-52.

(175.) Saah AJ, Koch TR, Drusano GL. Cefoxitin falsely elevates creatinine levels. JAMA 1982;247:205-6.

(176.) Naderer O, Nafziger AN, Bertino JS Jr. Effects of moderate-dose versus high-dose trimethoprim on serum creatinine and creatinine clearance and adverse reactions. Antimicrob Agents Chemother 1997;41:2466-70.

(177.) Dorwart WV. Bilirubin interference in kinetic creatinine determination [Letter]. Clin Chem 1979;25:196-7.

(178.) Calles-Escandon J, Cunningham JJ, Snyder P, Jacob R, Huszar G, Loke J, et al. Influence of exercise on urea, creatinine, and 3-methylhistidine excretion in normal human subjects. Am J Physiol 1984;246:E334-8.

(179.) Scrimshaw NS, Habicht JP, Piche ML, Cholakos B, Arroyave G. Protein metabolism of young men during university examinations. Am J Clin Nutr 1966;18:321-4.

(180.) Delanghe J, De Slypere JP, De Buyzere M, Robbrecht J, Wieme R, Vermeulen A. Normal reference values for creatine, creatinine, and carnitine are lower in vegetarians [Letter]. Clin Chem 1989; 35:1802-3.

(181.) Bisdee JT, Garlick PJ, James WPT. Metabolic changes during menstrual cycle. Br J Nutr 1989;61:641-50.

(182.) Davison JM, Noble MCB. Serial changes in 24 h creatinine clearance during normal menstrual cycles and the first trimester of pregnancy. Br J Obstet Gynaecol 1981;88:10-7.

(183.) Schiller WR, Long CL, Blakemore WS. Creatinine and nitrogen excretion in seriously ill and injured patients. Surg Gynecol Obstet 1979;149:561-6.

(184.) Mitch WE, Collier VU, Walser M. Creatinine metabolism in chronic renal failure. Clin Sci 1980;58:327-35.

(185.) Faucheux B, Kuchel O, Cuche JL, Messerli FH, Buu NT, Barbeau A, et al. Circadian variations of the urinary excretion of catecholamines and electrolytes. Endocr Res Commun 1976;3: 257-72.

(186.) James GD, Sealey JE, Alderman M, Ljungman S, Mueller FB, Pecker MS, et al. A longitudinal study of urinary creatinine and creatinine clearance in normal subjects. Race, sex, and age differences. Am J Hypertens 1988;1:124-31.

(187.) Shephard MD, Penberthy LA, Fraser CG. Short- and long-term biological variation in analytes in urine of apparently healthy individuals. Clin Chem 1981;27:569-73.

(188.) Gowans EM, Fraser CG. Biological variation in analyte concentrations in urine of apparently healthy men and women. Clin Chem 1987;33:847-50.

(189.) Howey JE, Browning MC, Fraser CG. Selecting the optimum specimen for assessing slight albuminuria, and a strategy for clinical investigation: novel uses of data on biological variation. Clin Chem 1987;33:2034-8.

(190.) Ricos C, Jimenez CV, Hernandez A, Simon M, Perich C, Alvarez V, et al. Biological variation in urine samples used for analyte measurements. Clin Chem 1994;40:472-7.

(191.) Bingham SA, Williams R, Cole TJ, Price CP, Cummings JH. Reference values for analytes of 24-h urine collections known to be complete. Ann Clin Biochem 1988;25:610-9.

(192.) Sebastian-Gambaro MA, Liron-Hernandez FJ, Fuentes-Arderiu X. Intra- and inter-individual biological variability data bank [Review]. Eur J Clin Chem Clin Biochem 1997;35:845-52.

(193.) Bennett S, Wilkins HA. Within-person variation in urinary sodium, potassium and creatinine concentrations, and their relationship to changes in the blood pressure of adult male Gambians. J Trop Med Hyg 1993;96:267-73.

(194.) Kesteloot HE, Joossens JV. Relationship between dietary protein intake and serum urea, uric acid and creatinine, and 24-hour urinary creatinine excretion: the BIRNH Study. J Am Coll Nutr 1993;12:42-6.

(195.) Miki K, Sudo A. Effect of urine pH, storage time, and temperature on stability of catecholamines, cortisol, and creatinine. Clin Chem 1998;44:1759-62.

(196.) Rock RC, Walter WG, Jennings CD. Nitrogen metabolites and renal function. In: Tietz, N. ed. Textbook of clinical chemistry. Philadelphia: WB Saunders, 1998:1278.

(197.) Spierto FW, Hannon WH, Gunter EW, Smith SJ. Stability of urine creatinine. Clin Chim Acta 1997;264:227-32.

(198.) Soliman SA, Abdel-Hay MH, Sulaiman MI, Tayeb OS. Stability of creatinine, urea and uric acid in urine stored under various conditions. Clin Chim Acta 1986;160:319-26.

(199.) Cooper CR, Smith SJ, Myers GL, Sampson EJ, Magid E. Estimating and minimizing effects of biologic sources of variation by relative range when measuring the mean of serum lipids and lipoproteins. Clin Chem 1994;40:227-32.

(200.) Katznelson L, Finkelstein JS, Schoenfeld DA, Rosenthal DI, Anderson EJ, Klibanski A. Increase in bone density and lean body mass during testosterone administration in men with acquired hypogonadism. J Clin Endocrinol Metab 1996;81:4358-65.

(201.) Rodin A, Duncan A, Quartero HW, Pistofidis G, Mashiter G, Whitaker K, et al. Serum concentrations of alkaline phosphatase isoenzymes and osteocalcin in normal pregnancy. J Clin Endocrinol Metab 1989;68:1123-7.

(202.) Gorai I, Chaki O, Nakayama M, Minaguchi H. Urinary biochemical markers for bone resorption during the menstrual cycle. Calcif Tissue Int 1995;57:100-4.

(203.) Garnero P, Sornay-Rendu E, Delmas PD. Decreased bone turnover in oral contraceptive users. Bone 1995;16:499-503.

(204.) Cohen FJ, Eckert S, Mitlak BH. Geographic differences in bone turnover: data from a multinational study in healthy postmenopausal women. Calcif Tissue Int 1998;63:277-82.

(205.) Kleerekoper M, Nelson DA, Peterson EL, Flynn MJ, Pawluszka AS, Jacobsen G, et al. Reference data for bone mass, calciotropic hormones, and biochemical markers of bone remodeling in older (55-75) postmenopausal white and black women. J Bone Miner Res 1994;9:1267-76.

(206.) Lacour B. Creatinine et fonction renale. Nephrologie 1992;13: 73-81.

(207.) Forbes GB, Bruining GJ. Urinary creatinine excretion and lean body mass. Am J Clin Nutr 1976;29:1359-66.

(208.) Hadj-Aissa A, Cochat P, Dubourg L, Wright C, Pozet N. La mesure de la fonction renale chez l'enfant. Arch Pediatr 1994;1:273-80.

(209.) McLaren AM, Isdale AH, Whiting PH, Bird HA, Robins SP. Physiological variations in the urinary excretion of pyridinium crosslinks of collagen. Br J Rheumatol 1993;32:307-12.

(210.) Sairanen S, Tahtela R, Laitinen K, Loyttyniemi E, Valimaki MJ. Effects of short-term treatment with clodronate on parameters of bone metabolism and their diurnal variation. Calcif Tissue Int 1997;60:160-3.

(211.) Schlemmer A, Hassager C, Alexandersen P, Fledelius C, Pedersen BJ, Kristensen IO, et al. Circadian variation in bone resorption is not related to serum cortisol. Bone 1997;21:83-8.

(212.) Fradinger EE, Rodriguez G, Bogado C, Zanchetta JR. Effect of antiresorptive therapy on day-to-day variation of urinary free deoxypyridinoline excretion [Letter]. Clin Chem 1998;44:2224-5.

(213.) Beck-Jensen JE, Kollerup G, Sorensen HA, Pors NS, Sorensen OH. A single measurement of biochemical markers of bone turnover has limited utility in the individual person. Scand J Clin Lab Invest 1997;57:351-9.

(214.) Cheng S, Kovanen V, Heikkinen E, Suominen H. Serum and urine markers of type I collagen metabolism in elderly women with high and low bone mineral density. Eur J Clin Invest 1996;26:186-91.

(215.) Gfatter R, Braun F, Herkner K, Kohlross C, Hackl P. Urinary excretion of pyridinium crosslinks and N-terminal crosslinked peptide in preterm and term infants. Int J Clin Lab Res 1997; 27:238-43.

[10] Nonstandard abbreviations: PYD, pyridinoline (lysylhydroxypyridinoline); DPD, deoxypyridinoline (lysylpyridinoline); FMV, first morning void; SMV second morning void; and UV, ultraviolet.

Hubert W. Vesper, [1] * Laurence M. Demers, [2] Richard Eastell, [3] Patrick Garnero, [4] Michael Kleerekoper, [5] Simon P. Robins, [6] Apurva K. Srivastava, [7] G. Russell Warnick, [8] Nelson B. Watts, [9] and Gary L. Myers [1]

[1] Centers for Disease Control and Prevention, Atlanta, GA 30341-3724.

[2] Milton S. Hershey Medical Center, Hershey, PA 17033.

[3] Northern General Hospital, Sheffield S5 7AU, United Kingdom.

[4] INSERM Research Unite 403 & Synarc, F-69003 Lyon, France.

[5] Wayne State University, Detroit, MI 48201.

[6] The Rowett Research Institute, Aberdeen AB21 9SB, United Kingdom.

[7] J.L. Pettis Veterans' Affairs Medical Center, Loma Linda, CA 92357.

[8] Pacific BioMetrics Research Foundation, Issaquah, WA 98027.

[9] Emory University, Atlanta, GA 30322.

* Author for correspondence. Fax 770-488-4192; e-mail hav2@cdc.gov.
Table 1. Diurnal variation in PYD and DPD.

PYD, (a) % DPD, (a) % Population

78 (b) 75 (b) 9 young, healthy adults
 70 18 premenopausal women
57 54 9 healthy premenopausal women
78 9 healthy postmenopausal women
74 31 young adult women
 70 38 healthy adults
72 9 healthy postmenopausal women
68 (b) 65 (b) 24 postmenopausal women
Median: 73% Median: 70%

PYD, (a) % Assay Reference

78 (b) HPLC (209)
 HPLC (113)
57 HPLC (24)
78 Immunoassay (115)
74 Immunoassay (26)
 Immunoassay (161)
72 Immunoassay (210)
68 (b) HPLC (211)
Median: 73%

(a) Nadir/peak x 100%.

(b) Nadir-to-peak amplitudes estimated from plots.

Table 2. Long-term biologic variation.

 CV, %
 No. of samples/
 PYD DPD duration

Day-to-day

 16 24 21/21 days
 12 5/10 days
 12 13 8-12/8-19 days
 15 17 5/5 days
 14 14 5/5 days
 21 24 5/5 days
 Mean 16 17

Week-to-week

 21 22 5/5 weeks
 18 24 5/5 weeks
 14 16 4/4 weeks
 6 2/2 weeks
 7 2/2 weeks
 5 2/2 weeks
 17 4/4 weeks
 17 4/4 weeks
 19 4/4 weeks
 12 14 5/5 weeks
 Mean 16 13 (17) (b)

Month-to-month

 16 8/5 months
 17
 Mean 17

 Sample
 type Population

Day-to-day

 24-h 3 postmenopausal women
 SMV 11 healthy postmenopausal women
 20 osteoporotic postmenopausal
 women on alendronate
 FMV 4 children
 FMV 40 adult men and women
 24-h 40 adult men and women
 FMV 17 men and women
 Mean

Week-to-week

 24-h 6 men and women
 24-h 6 men and women
 SMV 11 healthy postmenopausal women
 20 osteoporotic postmenopausal women
 24-h 45 women
 FMV 45 women
 SMV 45 women
 FMV 8 men and 6 women
 SMV 8 men and 6 women
 24-h 8 men and 6 women
 FMV 30 women
 Mean

Month-to-month

 FMV Men
 Women
 Mean

 Assay Reference

Day-to-day

 HPLC (209)
 Immunoassay (212)
 HPLC (42)
 Immunoassay (16)
 Immunoassay (16)
 HPLC (32)
 Mean

Week-to-week

 HPLC (a) (162)
 HPLC (162)
 HPLC (8)
 Immunoassay (31)
 Immunoassay (31)
 Immunoassay (31)
 Immunoassay (33)
 Immunoassay (33)
 Immunoassay (33)
 HPLC (213)
 Mean

Month-to-month

 Immunoassay (161)
 Mean

(a) Non-peptide-bound cross-links measured by HPLC.

(b) Week-to-week mean CV deleting outliers from one study (31).

Table 3. Biologic variability.

 Age
 range or
 24-h mean age,
Group Assay sample years n

Premenopausal women HPLC 24-h 20-29 7
 HPLC 24-h 20-49 15
 HPLC 24-h 23-46 20
 HPLC FMV 36-39 40
 IA FMV 35-55 134
 HPLC FMV 36-39 40
 HPLC SMV 22-55 36
 HPLC SMV 33-44 17
 IA SMV 32-60 164
 Mean (SD)

Postmenopausal women HPLC 24-h 78 18
 HPLC 24-h 52-60 19
 HPLC 24-h 50-75 17
 HPLC FMV 48-57 55
 IA FMV 82.5 292
 HPLC SMV 42-77 45
 HPLC SMV 69-89 77
 IA SMV 41-67 156
 Mean (SD)

Men HPLC 24-h 20-70 12
Children HPLC 24-h 4-10 25
 HPLC 24-h 10.1-12.0 19
 HPLC 24-h 12.1-14 21
 HPLC 24-h 14.1-16.0 14
 HPLC 24-h 16.1-18.0 9
 HPLC 24-h 2-15 12
 HPLC FMV Preterm 18
 HPLC FMV Term 25
 Mean (SD)

 Mean, nmol/ [CV.sub.B], %
 mmol Cre (a)

Group PYD DPD PYD DPD

Premenopausal women 422 (b) 146 (b) 16 22
 31 8 24 18
 15 2 65 99
 20 4 20 35
 NA 4 -- (d) 25
 20 4 20 35
 25 5 23 25
 43 7 24 50
 42 NA 46 -- (d)
 Mean (SD) 30 (17) 39 (26)

Postmenopausal women 35 8 28 32
 36 7 36 50
 60 15 50 49
 30 7 30 35
 NA 6 -- (d) 32
 31 6 63 56
 58 9 28 46
 52 NA 45 -- (d)
 Mean (SD) 40 (13) 43 (10)

Men 260 81 7 8
Children 159 44 26 31
 80 50 43 83
 69 50 35 34
 69 29 59 80
 67 17 73 82
 1963 345 19 28
 1640 227 29 36
 1263 434 30 37
 Mean (SD) 39 (18) 51 (25)

 [CV.sub.inter], %

Group PYD DPD Reference

Premenopausal women -- (c) 15 (49)
 18 8 (27)
 63 98 (56)
 12 31 (37)
 -- (d) 19 (2)
 12 31 (37)
 17 20 (57)
 17 47 (57)
 44 -- (d) (63)
 Mean (SD) 26 (20) 34 (28)

Postmenopausal women 23 27 (214)
 33 47 (56)
 47 46 (27)
 25 31 (37)
 -- (d) 28 (2)
 61 54 (58)
 22 43 (57)
 42 -- (d) (63)
 Mean (SD) 36 (14) 40 (11)

Men -- (c) -- (c) (49)
Children 21 27 (43)
 40 82 (43)
 31 30 (43)
 57 78 (43)
 72 81 (43)
 10 23 (49)
 24 32 (215)
 26 33 (215)
 Mean (SD) 35 (20) 48 (27)

(a) Cre, creatinine; IA, immunoassay; NA, not analyzed.

(b) nmol * [L.sup.-1] * 24 [h.sup.-1].

(c) CVB < [CV.sub.inter].

(d) Data not available.
COPYRIGHT 2002 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2002 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Vesper, Hubert W.; Demers, Laurence M.; Eastell, Richard; Garnero, Patrick; Kleerekoper, Michael; Ro
Publication:Clinical Chemistry
Date:Feb 1, 2002
Words:13636
Previous Article:Remnant lipoproteins: measurement and clinical significance.
Next Article:Methods for measurement of LDL-cholesterol: a critical assessment of direct measurement by homogeneous assays versus calculation.
Topics:


Related Articles
Short-term urine deoxypyridinoline biological variability in the first 5 years after menopause.
Interlaboratory variation of biochemical markers of bone turnover.
Standardization of bone marker nomenclature.
Serum galactosyl hydroxylysine as a biochemical marker of bone resorption.
Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation.
Response of several markers of bone collagen degradation to calcium supplementation in postmenopausal women with low calcium intake.
Acute effects of fracture on bone markers and vitamin K.
Evidence that serum NTx (collagen-type I N-telopeptides) can act as an immunochemical marker of bone resorption.
Urine pyridinium cross-links determination by Beckman cross links kit.

Terms of use | Privacy policy | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters