Differential association of the N-propeptide of collagen IIA (PIIANP) and collagen II C-telopeptide (CTX-II) with synovitis and erosions in early and longstanding rheumatoid arthritis.

OBJECTIVES: To determine the N-terminal propeptide of collagen IIA (PIIANP) in early and established rheumatoid arthritis (RA) and to study the association with collagen II degradation assessed by its C-telopeptide (CTX-II), x-ray status and disease activity measures. METHODS: Two cohorts of RA patients were included: A) a one-year prospective cohort including 45 patients with early, untreated RA and B) a cross-sectional study comprising 50 RA patients with advanced disease. Blood donors and healthy volunteers served as controls. PIIANP in serum and urine CTX-II were measured by ELISA. RESULTS: PIIANP did not differ from control levels at any time in patients with early RA (p=0.16 and p=0.89), but at one-year follow-up, PIIANP was decreased compared with baseline (p=0.046). In patients with longstanding RA, PIIANP was lower than in controls (p=0.002) and RA patients with a 12-month disease (p=0.01). PIIANP was unrelated to joint counts and CRP in both cohorts, but baseline PIIANP was lower among x-ray progressors than in non-progressors (p=0.04). CTX-II was persistently increased in both cohorts (p<0.001 and p<0.001). CTX-II was positively associated with joint counts and CRP but not with x-ray progression (p=0.84). There was no correlation between PIIANP and CTX-II. CONCLUSION: Declining PIIANP with increasing RA duration and persistently increased CTX-II indicate that cartilage anabolic and degradative pathways are unbalanced from clinical RA onset. Furthermore, that collagen II depletion in RA is both mediated by anti-anabolic effects unassociated with synovitis (decreased PIIANP) and by excess collagen II degradation linked to synovitis (increased CTX-II).

Urinary markers of altered collagen metabolism in fibromyalgia patients.

OBJECTIVE: To assess the metabolism of collagen in fibromyalgia (FM) patients, and to compare the occurrence of collagen metabolism markers to the severity of FM symptoms. METHODS: Morning urine was collected from 27 FM women fulfilling the American College of Rheumatology (ACR) criteria for FM, and from seven controls. FM patients completed the Fibromyalgia Impact Questionnaire (FIQ). Bone mineral density (BMD), isokinetic muscle strength in knee and elbow, and hand-grip strength were measured. Urinary concentrations of collagen type I cross-linked C-telopeptide (CTX-I) and collagen type II cross-linked C-telopeptide (CTX-II) were determined by enzyme-linked immunosorbent assay (ELISA). Pyridinoline (Pyd) and deoxypyridinoline (Dpd) were determined by liquid chromatography, and hydroxyproline (Hyp) by spectrophotometry. All concentration data were normalized to creatinine. RESULTS: Mean values in the FM group and the control group, respectively, were: urinary CTX-I 246.8 and 337.5 microg/mmol (p = 0.060); CTX-II 110.4 and 185.1 ng/mmol (p = 0.035); Pyd 56.1 and 52.3 nmol/mmol (NS); Dpd 15.1 and 14.0 nmol/mmol (NS); Pyd : Dpd ratio 4.05 and 3.96 (NS); Hyp 26.1 and 21.1 micromol/mmol (NS). Significant inverse correlations were seen between CTX-I and the intensity of fatigue, and between CTX-II and anxiety. An inverse correlation between CTX-I and muscle strength was apparent, but relied on extreme values from one patient, and no significant correlation was found between CTX-I or CTX-II and tender points or BMD in the FM group. CONCLUSIONS: Low urinary concentrations of CTX-II and CTX-I and normal levels of Pyd and Dpd were found in FM, but their relationship to the intensity of FM symptoms was unclear.

Post-translational protein modifications in type 1 diabetes: a role for the repair enzyme protein-L-isoaspartate (D-aspartate) O-methyltransferase?

AIMS/HYPOTHESIS: Post-translational modifications, such as isomerisation of native proteins, may create new antigenic epitopes and play a role in the development of the autoimmune response. Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PIMT), encoded by the gene PCMT1, is an enzyme that recognises and repairs isomerised Asn and Asp residues in proteins. The aim of this study was to assess the role of PIMT in the development of type 1 diabetes. MATERIALS AND METHODS: Immunohistochemical analysis of 59 normal human tissues was performed with a monoclonal PIMT antibody. CGP3466B, which induces expression of Pcmt1, was tested on MIN6 and INS1 cells, to assess its effect on Pcmt1 mRNA and PIMT levels (RT-PCR and western blot) and apoptosis. Forty-five diabetes-prone BioBreeding (BB) Ottawa Karlsburg (OK) rats were randomised to receive 0, 14 or 500 microg/kg (denoted as the control, low-dose and high-dose group, respectively) of CGP3466B from week 5 to week 20. RESULTS: A high level of PIMT protein was detected in beta cells. CGP3466B induced a two- to threefold increase in Pcmt1 mRNA levels and reduced apoptosis by 10% in MIN6 cells. No significant effect was seen on cytokine-induced apoptosis or PIMT protein levels in INS1 cells. The onset of diabetes in the BB/OK rats was significantly delayed (85.6+/-9.0 vs 84.3+/-6.8 vs 106.6+/-13.5 days, respectively; p<0.01 for high-dose vs low-dose and control groups), the severity of the disease was reduced (glucose 22.2+/-3.2 vs 16.9+/-2.6 vs 15.8+/-2.7 mmol; p<0.01 for high- and low-dose groups vs control group) and residual beta cells were more frequently identified (43% vs 71% vs 86%; p<0.05 for high-dose vs control group) in the treated animals. CONCLUSIONS/INTERPRETATION: The results support a role for post-translational modifications and PIMT in the development of type 1 diabetes in the diabetes-prone BB rat, and perhaps also in humans.

Sex hormones in the regulation of bone and cartilage metabolism: an old paradigm and a new challenge.

The effects of estrogen on tissues such as bone, endometrium and breast have been extensively studied, and the pleitropic effects of the female sex hormone are well established. Cartilage is not generally viewed as an estrogen responsive tissue. However, several epidemiological studies, and a few recent intervention studies supports that estrogen may have a role in osteoarthritis (OA), and recent animal studies further suggests that estrogen may be involved in regulation of cartilage turnover. Accordingly the issue of chondroprotrective properties of estrogen has received increased attention in recent scientific publications. In this review, we summarize current studies indicating a role for estrogen in the regulation of cartilage turnover and development of joint diseases. We report results from in vitro and animal studies where the effects of ovariectomy and treatment with estrogen and selective estrogen receptor modulators (SERM) on cartilage erosion have been evaluated. Furthermore, we report results from assessment of the effects of estrogen and SERM in postmenopausal women which shed new light on the interactions between estrogen and joint tissues. It still remains to be established whether estrogen or SERM could find a role in prophylaxis and/or treatment of OA, and much work lies ahead. Current data reviewed in this manuscript can be considered encouraging and they raise the hope that new treatment options for OA may become available based on estrogen and, in particular, compounds acting through the estrogen receptor. However, at present hormone replacement therapy and SERMs available in clinical practice, cannot be recommended as a therapy for arthritic disease.

A new marker for osteoarthritis: cross-sectional and longitudinal approach.

OBJECTIVE: To investigate the association between urinary concentrations of C-telopeptide fragments of type II collagen (CTX-II) and the prevalence and progression of radiographic osteoarthritis (OA) of the knee and hip. METHODS: The study population consisted of a sample of 1,235 men and women ages > or =55 years who were enrolled in the Rotterdam Study (a population-based cohort study) and who were followed up for a mean of 6.6 years. Prevalent radiographic OA was defined as a Kellgren/Lawrence score > or =2; progression of radiographic OA was defined as a decrease in joint space width. RESULTS: Subjects with a CTX-II level in the highest quartile had a 4.2-fold increased risk of having radiographic OA of the knee (95% confidence interval [95% CI] 2.5-7.0) and of the hip (95% CI 2.2-7.8) compared with subjects with a CTX-II level in the lowest quartile. We observed a substantially stronger association between CTX-II levels and radiographic OA for subjects with hip pain (odds ratio [OR] 20.4, 95% CI 2.3-185.2) than for those without hip pain (OR 3.0, 95% CI 1.5-6.0). Subjects with a CTX-II level in the highest quartile had a 6.0-fold increased risk for progression of radiographic OA at the knee (95% CI 1.2-30.8) and an 8.4-fold increased risk for progression of radiographic OA at the hip (95% CI 1.0-72.9). All of these associations were found to be independent of known risk factors for OA, such as age, sex, and body mass index. CONCLUSION: This study shows that CTX-II is associated with both the prevalence and the progression of radiographic OA at the knee and hip. Importantly, this association is independent of known clinical risk factors for OA and seems stronger in subjects with joint pain.

Type II collagen peptides for measuring cartilage degradation.

This paper describes two new immunoassays for a peptide of the triple helix of type II collagen (Coll 2-1) and its nitrated form (Coll 2-1 NO(2)). In healthy subjects aged between 20 and 65 years old, Coll 2-1 and Coll 2-1 NO(2) levels in serum were in means 125.13+/-3.71 and 0.16+/-0.08 nmol/l, respectively. These levels did not significantly vary with age. However, up to 45 years of age, Coll 2-1 NO(2) levels in women were significantly higher than in men. In patients with knee osteoarthritis (OA), Coll 2-1 in serum was found to be elevated compared to healthy controls (267.45+/-26.42 nmol/l vs 126.78+/-6.61 nmol/l). Further, we have demonstrated that an increase of the urinary levels of Coll 2-1 or Coll 2-1 NO(2) over 1 year was predictive of joint space narrowing progression in OA patients. In conclusion, these preliminary results indicate that Coll 2-1 could be a predictive marker of knee OA progression.

Osteoarthritic patients with high cartilage turnover show increased responsiveness to the cartilage protecting effects of glucosamine sulphate.

OBJECTIVE: Glucosamine sulphate has been shown in a large double-blind, placebo-controlled clinical trial to prevent structural damage and improve clinical symptoms of osteoarthritis (OA). We investigated whether early response in a newly developed biochemical marker of collagen type II degradation (CTX-II, CartiLaps ELISA) could reflect the long-term preservation of hyaline cartilage. METHODS: Study subjects comprised 212 knee OA patients participating in a clinical trial of the effects of glucosamine sulphate. Disease symptoms were assessed quarterly by WOMAC scoring and X-ray analysis was performed at baseline and after 3 years. Urine samples were obtained at baseline and after 1, 2 and 3 years for measurement in the CartiLaps assay. The measurements were corrected for creatinine. RESULTS: At baseline the patients had an average concentration of urinary CTX-II of 222.4 +/- 159.5 ng/mmol creatinine. This was significantly above the CTX-II levels measured in urine samples from 415 healthy controls (169.1 +/- 92.3 ng/mmol, p < 0.0001). There was no significant difference in the CTX-II response in the placebo group and the glucosamine treated group. However, those with high cartilage turnover presented a significant decrease in CTX-II after 12-month glucosamine treatment. Thus, three group with CTX II concentrations above normal average + 1SD decreased 15.5% after 12-month therapy. The 12 months change in CTX-II in OA patients with elevated CTX-II at baseline correlated with the change in average joint space width observed after 36 months (R = 0.43, p < 0.05). Increased baseline levels of CTX-II were associated with a worsening of the WOMAC index (p < 0.01). CONCLUSION: The data indicate that measurement of urinary collagen type II C-telopeptide fragments enables the identification of OA patients with high cartilage turnover who at the same time are most responsive to therapy with structure modifying drugs.

Increased urinary concentration of collagen type II C-telopeptide fragments in patients with osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is characterized by a progressive degeneration of articular cartilage and loss of joint function. We hypothesized that degradation of articular cartilage results in increased fragmentation of collagen type II. Thus, the concentrations of degradation products of this major cartilage matrix protein may increase in body fluids of patients with OA. METHODS: Monoclonal antibodies specific for a human collagen type II C-telopeptide (CTx-II) fragment were used in an ELISA for quantification of collagen type II fragments in urine. Clinical assessment of 88 patients with advanced OA of either hip or knee and 48 age-matched controls was performed with the Harris hip score, the Merle d'Aubigné score and a knee score. Joint space narrowing and the Kellgren and Lawrence score were assessed as radiological signs of OA. RESULTS: The concentration of CTx-II was significantly higher in OA patients compared with controls (527 vs. 190 ng/mmol, p < 0.001) whether the patients were diagnosed with hip OA (n = 51) or knee OA (n = 37). Mean CTx-II levels were higher in hip OA than in knee OA and a slight increase in levels with age was observed in the controls, but not in OA subjects. CONCLUSION: Elevation of CTx-II in urine of patients with severe OA compared with a control group suggests that collagen type II derived fragments may serve as markers for OA.

Urinary type II collagen C-telopeptide levels are increased in patients with rapidly destructive hip osteoarthritis.

OBJECTIVE: To compare type II collagen degradation using a new urinary specific marker in patients with rapidly destructive and those with slowly progressive hip OA. METHODS: Twelve patients with rapidly destructive and 28 patients with slowly progressive hip OA were included in a prospective, cross sectional case-control study. Urinary levels of C-terminal crosslinking telopeptide of collagen type II (CTX-II) as a marker of cartilage degradation were measured by an ELISA, and urinary free deoxypyridinoline (free DPD), a marker of bone resorption, was measured by high performance liquid chromatography. One x ray evaluation of the hips and urine samples was made in all patients when the diagnosis of OA was established. RESULTS: Patients with hip OA had higher mean (SD) urinary CTX-II levels than 65 healthy age matched controls (492 (232) v 342 (141), p<0.001), but no significant difference was seen for urinary free DPD (p=0.30). Increased urinary CTX-II, but not urinary free DPD, correlated significantly with decreased minimum joint space width assessed by radiograph of the hip. Mean urinary CTX-II levels were significantly higher in patients with rapidly progressive OA than in the slowly progressive group (612 (218) v 441 (221), p=0.015), whereas no significant difference of urinary free DPD was seen between the two groups (p=0.55). CONCLUSION: Patients with hip OA have increased CTX-II degradation as assessed by a new urinary marker. Increased urinary CTX-II levels are associated with rapidly destructive disease, suggesting that this marker might be useful in identifying patients with hip OA at high risk for rapid progression of joint damage.

Dose dependent effects on bone resorption and formation of intermittently administered intravenous ibandronate.

The aim of the present paper was to delineate in detail the dose-dependent effects of intermittent intravenous (IV) ibandronate treatment on the dynamics of markers of bone resorption and formation. The study included 73 healthy postmenopausal women between 50 and 70 years of age. Two groups received an IV injection of either 1 mg or 2 mg ibandronate on day 0 and 84 and one group, which received no treatment, served as control. Study duration was 168 days. Bone turnover was estimated by measuring the serum concentration of the C-terminal collagen I telopeptide (s-CTx, bone resorption) and osteocalcin (s-OC, bone formation) at 19 consecutive time-points. Serum CTx decreased rapidly reaching a nadir 7 days after drug administration. Maximal changes from baseline in the 1 and 2 mg ibandronate groups were -81% and -90%, respectively ( P<0.001). However, already 2 weeks after drug administration, s-CTx started to rise again in both treatment groups, reaching -16% and -20% by day 84, i.e. immediately before the second drug administration. In contrast, s-OC showed a slower but progressive decrease over time reaching a nadir at -35% inhibition after 5 months. On a group level, the suppression of bone resorption was greater or equal to the suppression of bone formation at all time points. However, the least significant change (LSC) analysis performed at the individual level highlighted individuals who at certain time points showed apparently greater suppression of formation than resorption, which could also contribute to the inefficacy of this dosing regime. Although the physiological relevance of this latter finding would require further analysis, the results draw attention to the need to optimize the intermittent IV dosing of ibandronate in order to approximate more closely the sustained and balanced anti-resorptive effect provided by daily oral treatment.

Oral ibandronate: changes in markers of bone turnover during adequately dosed continuous and weekly therapy and during different suboptimally dosed treatment regimens.

The aims of the present study were to investigate how changes in the cumulative dose and the frequency of dosing influence the short-term antiresorptive efficacy of oral ibandronate treatment and whether serial measurements of bone markers could provide a useful diagnostic tool for the revelation of noncompliance to established treatments with antiresorptive drugs. Study participants were 200 healthy women 50-70 years old (mean 63.1 years) with a lumbar spine BMD t-score of -1 to -5. Women were randomly allocated to receive treatment with oral ibandronate according to one of the following eight dosing regimes: (1) 2.5 mg daily for 84 days; (2) 20 mg weekly for 84 days; (3) 2.5 mg daily for 28 days + no treatment for 56 days; (4) 2.5 mg daily for 28 days + 2.5 mg weekly for 56 days; (5) 2.5 mg daily for 28 days + 2.5 mg three times weekly for 56 days; (6) 2.5 mg daily for 14 days + 2.5 mg three times weekly for 56 days; (7) 2.5 mg three times weekly for 84 days; (8) no treatment for 168 days. Study parameters were the serum concentration of the C-terminal telopeptide of collagen type I (s-CTX, resorption marker) and N-MID osteocalcin (formation marker) measured by enzyme-linked immunosorbent assay. Oral treatment with ibandronate 20 mg weekly (cumulative dose 240 mg) resulted in greater final inhibition in s-CTX and area under the curve (AUC) compared to the 2.5 mg daily treatment (cumulative dose 210 mg), indicating that as long as optimal doses are administered the frequency of dosing has secondary importance for overall efficacy. When the cumulative dose was 130 mg or less, the final degree of inhibition was still the function of the cumulative dose, but the overall efficacy estimated by the AUC was also under the influence of the frequency of dosing. These observations suggest that serial measurements of s-CTX may provide a useful diagnostic tool for the early revelation of suboptimal dosing or noncompliance to already optimized therapies with antiresorptive agents.

Cartilage turnover assessed with a newly developed assay measuring collagen type II degradation products: influence of age, sex, menopause, hormone replacement therapy, and body mass index.

BACKGROUND: Cartilage normally has a slow turnover but in arthritis increased metabolism results in degradation of the tissue. OBJECTIVE: To assess cartilage turnover in a sample of the general population by an assay measuring cartilage derived urinary collagen type II (CTX-II) C-telopeptide degradation products. METHODS: CTX-II concentrations were measured in urine samples from 615 healthy men and women aged 20-87 years, and the influence of age, sex, menopause, hormone replacement therapy (HRT), and body mass index (BMI) was assessed. RESULTS: CTX-II concentrations showed age dependent variations, with notable differences between men and women. Mean (SD) CTX-II concentration in postmenopausal women (220 (118) ng/mmol, n=25) was significantly higher than in an age matched group of premenopausal women (112 (79) ng/mmol, n=26, p<0.001). CTX-II concentration in women using HRT (118 (57) ng/mmol, n=50) was significantly lower than in an age and BMI matched group of women not receiving HRT (215 (99) ng/mmol, n=50, p<0.001). In subjects with a BMI >or=25 kg/m(2), CTX-II concentrations were significantly higher than in those with a BMI <25 kg/m(2) (185 (114) v 148 (91) ng/mmol, p<0.001). CONCLUSIONS: Cartilage turnover, as assessed by measuring urinary degradation products of CTX-II varies considerably with age, and significant differences between CTX-II levels in men and women as well as in pre- and postmenopausal women are found. Further studies are required to validate the marker for assessing cartilage degradation in arthritis.

Investigation of bone disease using isomerized and racemized fragments of type I collagen.

In the collagen type I C-telopeptide an aspartyl-glycine site within the sequence AHDGGR is susceptible to molecular rearrangement. In newly synthesized collagen this site is in the native form, denoted alpha L. During aging a spontaneous reaction occurs resulting in three age-modified forms: an isomerized form (beta L) a racemized form (alpha D), and an isomerized/racemized form (beta D). In this study, we measured the urinary excretion of the four forms of C-telopeptides (CTX) in healthy adults and in patients with bone diseases. Levels of all CTX forms were higher in healthy postmenopausal women (P<0.001) compared with premenopausal controls. Levels decreased within 3 days of bisphosphonate treatment indicating that all CTX forms reflect bone resorption. In hyperthyroidism, characterized by a generalized increased bone turnover, native (alpha L) and age-modified (beta L, alpha D and beta D) forms increased to a similar extent compared to controls, resulting in normal ratios between the alpha L and age-modified forms of CTX. Conversely, in Paget's disease and prostate cancer-induced bone metastases, conditions characterized by focal increased bone turnover, alpha L CTX levels were more elevated than those of age-related CTX forms, resulting in increased ratios between native and age-modified CTX. For example, the ratio alpha L/alpha D was increased 7-fold in Paget's disease (P<0.001) and 2-fold in prostate cancer-induced bone metastases (P<0.002). In conclusion, the study suggests that in conditions with a localized alteration in bone turnover the ratio between alpha L CTX and the age-modified forms is significantly elevated. This may provide a new diagnostic and monitoring tool for diseases such as metastatic bone cancer and Paget's disease.

Uncoupling of type II collagen synthesis and degradation predicts progression of joint damage in patients with knee osteoarthritis.

OBJECTIVE: The hallmark of osteoarthritis (OA) is the loss of articular cartilage. This loss arises from an imbalance between cartilage synthesis and cartilage degradation over a variable period of time. The aims of this study were to investigate the rates of these processes in patients with knee OA using two new molecular markers and to investigate whether the combined use of these markers could predict the progression of joint damage evaluated by both radiography and arthroscopy of the joints during a period of 1 year. METHODS: Seventy-five patients with medial knee OA (51 women, 24 men; mean +/- SD age 63 +/- 8 years, mean +/- SD disease duration 4.8 +/- 5.2 years) were studied prospectively. At baseline, we measured serum levels of N-propeptide of type IIA procollagen (PIIANP) and urinary excretion of C-terminal crosslinking telopeptide of type II collagen (CTX-II) as markers of type II collagen synthesis and degradation, respectively. Joint space width (JSW) on radiography and medial chondropathy at arthroscopy (assessed using a 100-mm visual analog scale [VAS]) were measured in all patients at baseline and in 52 patients at 1 year. Progression of joint destruction was defined as a decrease of > or =0.5 mm in JSW on radiography and as increased chondropathy (an increase in the VAS score of >8.0 units) between the baseline and 1-year evaluations. RESULTS: At baseline, compared with 58 healthy age- and sex-matched controls, patients with knee OA had decreased serum levels of PIIANP (20 ng/ml versus 29 ng/ml; P < 0.001) and increased urinary excretion of CTX-II (618 ng/mmole creatinine [Cr] versus 367 ng/mmole Cr; P < 0.001). The highest discrimination between OA patients and controls was obtained by combining PIIANP and CTX-II in an uncoupling index (Z score CTX-II - Z score PIIANP), which yielded a mean Z score of 2.9 (P < 0.0001). Increased baseline values in the uncoupling index were associated with greater progression of joint damage evaluated either by changes in JSW (r = -0.46, P = 0.0016) or by VAS score (r = 0.36, P = 0.014). Patients with both low levels of PIIANP (less than or equal to the mean - 1 SD in controls) and high levels of CTX-II (greater than or equal to the mean + 1 SD in controls) had an 8-fold more rapid progression of joint damage than other patients (P = 0.012 and P < 0.0001 as assessed by radiography and arthroscopy, respectively) and had relative risks of progression of 2.9 (95% confidence interval [95% CI] 0.80-11.1) and 9.3 (95% CI 2.2-39) by radiography and arthroscopy, respectively. CONCLUSION: Patients with knee OA are characterized by an uncoupling of type II collagen synthesis and degradation which can be detected by assays for serum PIIANP and urinary CTX-II. The combination of these two new markers could be useful for identifying knee OA patients at high risk for rapid progression of joint damage.

Circadian variation in the serum concentration of C-terminal telopeptide of type I collagen (serum CTx): effects of gender, age, menopausal status, posture, daylight, serum cortisol, and fasting.

We examined the diurnal variation in serum concentration of C-terminal telopeptide of type I collagen (serum CrossLaps, sCTx) under various conditions. The studies included a total of 100 individuals. Blood samples were collected every 3 h over 27 h. sCTx levels varied over the 24 h with a maximum at about 05:00 in the morning and a minimum of about 14:00 in the afternoon. The variation had a magnitude of about +/-40% around the 24 h mean and was similar in premenopausal and early and late postmenopausal women with normal and low bone mass. Furthermore, it was not affected by 5 days of bed-rest, by absence of a normal diurnal variation in cortisol production, or by absence of a normal light cycle (blindness). Nasal salmon calcitonin, an antiresorptive drug used for treatment of osteoporosis, was not able to break the circadian pattern whether the treatment was administered in the morning or the evening. The only parameter that showed a pronounced influence on the circadian variation was fasting, which reduced the variation significantly to about one fourth. From a practical point of view the results of this study demonstrate that samples for sCTx should be taken in the fasting state.

Effect of bisphosphonates on cartilage turnover assessed with a newly developed assay for collagen type II degradation products.

BACKGROUND: Animal studies of arthritis have suggested that bisphosphonates may have chondroprotective abilities. OBJECTIVE: To evaluate the effect of bisphosphonate treatment on cartilage degradation. METHODS: Type II collagen is almost exclusively localised in cartilage, where it is the major structural component of the tissue. Hence fragments derived from this protein should represent a specific index for cartilage degradation. The urinary concentration of collagen type II C-telopeptide degradation products (CTX-II) was measured by a new immunoassay (enzyme linked immunosorbent assay (ELISA)). The serum concentration of collagen type I C-telopeptide degradation products (CTX-I), a marker of bone degradation, was also measured by ELISA. PARTICIPANTS: Two groups were studied. The alendronate group included 63 healthy postmenopausal women aged 45-54 randomly allocated to receive three years' treatment with 1 mg, 5 mg, 10 mg, or 20 mg alendronate daily or placebo. In the third year the women receiving 20 mg were switched to placebo. The ibandronate group included 119 women at least 10 years after the menopause aged <75 randomly allocated to receive 12 months' treatment with 0.25 mg, 0.5 mg, 1.0 mg, 2.5 mg, or 5 mg ibandronate daily or placebo followed by 12 months without treatment. RESULTS: 20 mg of alendronate and 2.5 and 5 mg of ibandronate treatment produced significant decreases in urinary CTX-II to about 50% of baseline. The level reached after three months of treatment remained practically constant during the following 12-36 treatment months. When treatment was withdrawn CTX-II values returned towards baseline. Serum CTX-I also decreased rapidly within three months, but to a level of about 30% of baseline. CONCLUSIONS: The urinary excretion of CTX-II, a new marker of cartilage degradation, decreases significantly in response to bisphosphonate. This suggests that bisphosphonates may have chondroprotective effects in humans. By measurement of CTX-II it should be possible to monitor the effects of drugs that potentially inhibit cartilage destruction.

Mechanism of circadian variation in bone resorption.

The diurnal variation in bone resorption markers is poorly understood and may contain essential information about regulation of bone resorption. To explore the acute regulation of bone resorption we studied bone turnover in 14 postmenopausal women during a randomized, crossover, 24 h study of oral glucose tolerance test (OGTT), normal diet, or fasting. Whereas fasting counteracted variation in bone resorption, as measured by serum C-telopeptide fragments of collagen type 1 degradation (s-CTx), OGTT and normal diet induced a 50% reduction (p < 0.001) over 2 h. For OGTT, s-CTx reverted to baseline levels after 6 h, and for normal diet s-CTx remained suppressed during the afternoon and returned to baseline overnight. Repeated OGTT at 8:00 A.M. and 8:00 P.M. in nine postmenopausal women demonstrated that identical reductions in s-CTx could be obtained at both timepoints with an intermediate return to baseline between tests. A 2 h randomized, crossover study of OGTT and fasting in 23 men and premenopausal women similarly revealed a 50% decrease in s-CTx. A randomized, crossover 2 h study of insulin tolerance test compared with fasting in six men and premenopausal women demonstrated a 20%-30% decrease in s-CTx (p < 0.01-0.05). Nine hour follow-up of ten healthy individuals during a crossover experiment of OGTT, protein, and fat intake revealed a comparable 50% reduction in s-CTx, but distinct profiles of serum glucose and serum insulin. Bone resorption was reduced by intake of food, glucose, fat, and protein and counteracted by fasting, and this seems to have been be independent of age and gender. Both exogenous and endogenous insulin stimulation tests induced a reduction in bone resorption, but this was only partial when compared with the reduction observed during food intake.

Bone resorption in post-menopausal women with normal and low BMD assessed with biochemical markers specific for telopeptide derived degradation products of collagen type I.

Biochemical markers of bone resorption can be used clinically to predict the risk of osteoporosis-related fractures (prognostic tool) and to assess the response of an osteoporotic patient to an antiresorptive therapy (monitoring tool). Our aim was to assess the ability of four currently marketed biochemical markers of bone resorption, based on the measurement of degradation products from collage type I telopeptides to monitor the elevated resorption associated with menopause. Women (846) were stratified for menopause, age, and bone mineral density and the following markers were measured: urinary cross-linked N-telopeptides of type I collagen (NTx), the levels of breakdown products of type I collagen C-telopeptides in serum (S-CTx), and in urine, by ELISA (U-CTx-E), and RIA (U-CTx-R). Furthermore, the ratio (alpha/beta) between the alphaL form of CTx measured in the CTx RIA and the betaL form measured in the ELISA was calculated. The mean difference was calculated for each marker in women with osteopenia (Op) or osteoporosis (PMO) (WHO definition) compared with healthy premenopausal (Pre) women and postmenopausal (N Post) women with normal bone mass. Serum CTx showed the highest elevation in post- compared with premenopausal women. All marker values were significantly higher in Op and PMO subjects compared with both Pre and to N Post women. Compared with premenopausal values, the largest elevation in both Op and PMO women was observed for serum CTx. Compared with N Post, urine NTx showed the highest increase in OP subjects. The alpha/beta CTx ratio was elevated in post- compared with Pre women, but there was no difference in the ratio among N Post, Op, or PMO women. In conclusion, postmenopausal women showed elevated turnover with all bone resorption markers, but with substantial individual variation in resorption levels. Furthermore, the turnover process in postmenopausal women appears to be quantitatively different from the premenopausal stage, apparent as altered alpha/beta CTx ratios.

Collagen type II C-telopeptide fragments as an index of cartilage degradation.

We report the development of an assay for measurement of the urinary concentration of collagen type II C-telopeptide fragments. This assay was developed for providing a specific marker of joint metabolism. A monoclonal antibody, recognizing a linear six amino acid epitope from the middle region of the collagen type II C-telopeptide was used in a competitive enzyme-linked immunoassay (ELISA) format for measurement of urine samples. The technical performance and specificity of the assay was evaluated and a panel of samples from patients with rheumatoid arthritis (RA) (n = 27), osteoarthritis (OA) (n = 29), Paget's disease (n = 9), and healthy controls (n = 428) was measured in the assay. The ELISA was specific for the peptide EKGPDP derived from collagen type II C-telopeptide: it did not recognize peptides from the N-telopeptide of the molecule or from other collagen types. Collagen type II C-telopeptide fragments measured in the assay resisted seven freeze-thaw cycles and >20 h of storage at room temperature. RA and OA patients showed significant 2.33-fold (95% confidence interval [CI] 1.50-3.16) and 1.53-fold (CI 1.24-1.82) elevations in CartiLaps concentration, respectively. Paget's disease patients did not have elevated CartiLaps levels. RA patients with radiological evidence of cartilage damage had significantly higher (1.79-fold, CI 1.04-2.54) CartiLaps levels than RA patients without radiological evidence of cartilage destruction. The Cartilaps assay showed high technical precision and an ability to differentiate populations with an elevated joint metabolism from normal controls. This suggests that the assay may have clinical value in assisting in the diagnosis of joint diseases and in monitoring progression and therapy in RA and OA.

The bisphosphonate zoledronate decreases type II collagen breakdown in patients with Paget's disease of bone.

Bisphosphonates have been suggested to be partially chondroprotective in animal models of arthritis. The aim of this study was to assess the short-term effect of the bisphosphonate zoledronate on type II collagen degradation in patients with Paget's disease of bone. Twenty-six patients with active Paget's disease who were a part of a double-blind, placebo-controlled, randomized study comparing the effects of several doses of a single injection of zoledronate, a potent bisphosphonate, were studied. Type II collagen destruction was assessed by urinary levels of type II collagen C-telopeptide (CTX-II) using a new immunoassay. Bone resorption was assessed by measuring the urinary excretion of nonisomerized type I collagen C-telopeptide (alpha CTX-I). Biochemical markers were measured at baseline and 5, 10, 30, and 60 days after injection. At baseline, no significant increase of CTX-II was observed in patients with Paget's disease compared with a group of 27 gender-and age-matched controls, in contrast to the ninefold (p < 0.0001) increase of urinary alpha CTX-I. After a single intravenous injection of zoledronate (200 or 400 microg), urinary CTX-II transiently decreased by a median of 25% 5 days after the injection of zoledronate (p = 0.0023 vs. placebo), then increased to pretreatment levels after 10 days. In contrast, urinary alpha CTX-I decreased within 5 days with a maximal decrease of 51% at day 10 (p < 0.001 vs. baseline and placebo), and levels remained suppressed during the 2 months of the study. Zoledronate not only reduces bone turnover but also directly decreases type II collagen degradation in patients with Paget's disease, suggesting that bisphosphonates may have chondroprotective effects in humans. Measurement of type II collagen breakdown by a new urinary biochemical marker may be useful for in vivo assessment of the effects of drugs that potentially inhibit cartilage destruction.