Lower blood glucose, hyperglucagonemia, and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice.

Glucagon, the counter-regulatory hormone to insulin, is secreted from pancreatic alpha cells in response to low blood glucose. To examine the role of glucagon in glucose homeostasis, mice were generated with a null mutation of the glucagon receptor (Gcgr(-/-)). These mice display lower blood glucose levels throughout the day and improved glucose tolerance but similar insulin levels compared with control animals. Gcgr(-/-) mice displayed supraphysiological glucagon levels associated with postnatal enlargement of the pancreas and hyperplasia of islets due predominantly to alpha cell, and to a lesser extent, delta cell proliferation. In addition, increased proglucagon expression and processing resulted in increased pancreatic glucogen-like peptide 1 (GLP-1) (1-37) and GLP-1 amide (1-36 amide) content and a 3- to 10-fold increase in circulating GLP-1 amide. Gcgr(-/-) mice also displayed reduced adiposity and leptin levels but normal body weight, food intake, and energy expenditure. These data indicate that glucagon is essential for maintenance of normal glycemia and postnatal regulation of islet and alpha and delta cell numbers. Furthermore, the lean phenotype of Gcgr(-/-) mice suggests glucagon action may be involved in the regulation of whole body composition.

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.

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.

Clinical evaluation of the Serum CrossLaps One Step ELISA, a new assay measuring the serum concentration of bone-derived degradation products of type I collagen C-telopeptides.

The Serum CrossLaps One Step ELISA is a sandwich assay using two monoclonal antibodies specific for a beta-aspartate form of the epitope EKAHDGGR derived from the carboxy-terminal telopeptide region of type I collagen alpha1-chain. Our objective was to assess the clinical value of the Serum CrossLaps assay for monitoring antiresorptive therapy in osteoporosis treatment. Samples obtained from postmenopausal women treated with different doses of cyclic or continuous hormone replacement therapy (HRT) with an estrogen analog (tibolone) or with a bisphosphonate (ibandronate) were measured in the Serum CrossLaps One Step ELISA at baseline and at various time points during therapy. The corresponding urine samples were measured in the urine CrossLaps ELISA and corrected for creatinine excretion. The serum CrossLaps measurements and corresponding urinary CrossLaps measurements were highly correlated (r >0.8 for all studies). The serum and urine CrossLaps measurements showed a significant decrease among the women treated with clinically relevant doses of either of the antiresorptive agents. Furthermore, the annual percentage change in bone mineral density (BMD) correlated with the measured changes in CrossLaps concentration. The serum CrossLaps assay showed a specificity of 83-100% and a sensitivity of 59-83% for assessing BMD changes. The corresponding values for the creatinine-corrected urinary measurements were 83-92% specificity and 68-79% sensitivity. We conclude that performance of the convenient Serum CrossLaps One Step ELISA is at least equivalent to that of the urine text for follow up of antiresorptive treatment in osteoporosis. Further studies are needed to optimize its use in this and other clinical applications.

Serum CrossLaps One Step ELISA. First application of monoclonal antibodies for measurement in serum of bone-related degradation products from C-terminal telopeptides of type I collagen.

We have developed a two-site ELISA for measurement in serum of bone-related degradation products derived from C-terminal telopeptides of type I collagen. The assay is based on the application of two highly specific monoclonal antibodies against the amino acid sequence of AHD-beta-GGR, where the aspartic acid residue (D) is beta-isomerized. In a one-step incubation procedure, the degradation products containing cross-linked diisomerized EKAHD-beta-GGR peptides are captured by a biotinylated antibody and a peroxidase-conjugated antibody. The generated complex is then bound to the streptavidin surface via the biotin conjugate. Desalted urinary antigens are used for standardization, and parallelism is observed with serum samples. Results are obtained in <2.5 h, and both inter- and intraassay imprecision are <8%. The serum CrossLaps concentration was 1748+/-740 pmol/L (mean +/- SD) in premenopausal women (n = 65) and 2952+/-1325 pmol/L in a group of healthy postmenopausal women (n = 169). The Serum CrossLaps One Step ELISA was capable of detecting a highly significant (P <0.001) effect of hormone replacement therapy in a retrospective study involving 22 postmenopausal women.

Decreased beta-isomerization of the C-terminal telopeptide of type I collagen alpha 1 chain in Paget's disease of bone.

In Paget's disease of bone, the normal lamellar bone is replaced by a woven structure with an irregular arrangement of collagen fibers. In this study, we investigated whether the degree of beta-isomerization within C-telopeptide of alpha 1 chain of type I collagen was altered in Paget's disease compared with other bone diseases with no alteration of bone structure. In Paget's disease (n = 26), but not in patients with primary hyperparathyroidism (n = 6) or hyperthyroidism (n = 17), the urinary excretion of nonisomerized (alpha) fragments derived from degradation of type I collagen C-telopeptide (CTX) was markedly increased compared with beta-isomerized CTX (+ 13-fold vs. + 3.5-fold over controls) resulting in an urinary alpha CTX/beta CTX ratio 3-fold higher than in controls (2.6 +/- 1.0 vs. 0.8 +/- 0.3, p < 0.001). In five pagetic patients in complete remission, as demonstrated by normal total alkaline phosphatase activity, the alpha CTX/beta CTX ratio was normal. The immunohistochemistry of normal and pagetic human bone sections showed a preferential distribution of alpha CTX within woven structure, while lamellar bone was intensely stained with an anti-beta CTX antibody, suggesting a lower degree of beta-isomerization of type I collagen in the woven pagetic bone. In collagenase digest of human bone specimens, we found a lower proportion of beta-isomerized type I collagen molecules in pagetic bone (40% of beta CTX) than in normal bone taken from trabecular (68%) and cortical compartments (71%). In conclusion, we found that in Paget's disease the alpha CTX/beta CTX ratio in bone and in urine is markedly increased. This altered beta isomerization can be accurately detected in vivo by measuring urinary degradation products arising from bone resorption.

Measurement of bone degradation products in serum using antibodies reactive with an isomerized form of an 8 amino acid sequence of the C-telopeptide of type I collagen.

An enzyme-linked immunosorbent assay for measuring type I collagen degradation products in serum (S-ELISA) was developed. The assay uses a high affinity polyclonal antibody which reacts with an isomerized form of an 8 amino acid sequence of the C-telopeptides of type I collagen (EKAHD-beta-GGR). Cross-reactivity to a nonisomerized synthetic peptide form of the 8 amino acid sequence is less than 0.2%. Values obtained in a group of premenopausal women (age, 33.3 +/- 3.11 years) were 69 +/- 24 ng/ml(n = 22). In a group of early postmenopausal women (age, 51.8 +/- 1.88 years) values obtained were 125 +/- 43 ng/ml (n = 46), which represents an increase of 81% (p < 0.001). Values found in untreated patients with Paget's disease were 234 +/- 95 ng/ml (n = 15), and for primary hyperparathyroidism we found 335 +/- 82 ng/ml (n = 10). Intravenous administration of a bisphosphonate (Pamidronate) to Paget's disease patients for 3 days was reflected in the S-ELISA by a decrease in the values of 55% when compared with values before treatment (n = 15). Following treatment with another bisphosphonate (Alendronate) for 6 months, values were decreased to 48 +/- 19 ng/ml (n = 12), which corresponds to a 62% decrease. Clinical results presented in this context support that the assay is a sensitive and specific index of bone resorption. It may, therefore, prove useful in the follow up of treatment of patients with metabolic bone diseases and in the clinical investigation of osteoporosis.

Circadian variation in bone resorption is not related to serum cortisol.

Serum osteocalcin, serum procollagen type I carboxyterminal propeptide (sPICP), and the urinary excretion of pyridinium crosslinks (biochemical markers of bone formation and resorption) all exhibit a circadian variation with a peak during the night. This study was performed to investigate the influence of the endogenous circadian rhythm in cortisol on the biochemical markers of bone turnover. Participants included 11 patients substituted with hydrocortisone due to either hypopituitarism (n = 7) or bilateral adrenalectomy (n = 4). Their daily tablet intake of hydrocortisone was divided in four equal doses in order to abrogate the known circadian variation in cortisol. 24 healthy postmenopausal women served as controls. The study design was performed over 24 h, with blood samples taken every 3 h, and urine collected in 3 h aliquots. Urinary pyridinium crosslinks (Pyr/ Cr, D-Pyr/Cr), serum osteocalcin (sOC), and serum PICP were measured. Patients without a circadian variation in cortisol had normal circadian variation in the urinary excretion of pyridinium crosslinks and sPICP, but no circadian rhythm in serum osteocalcin. We conclude that the etiology of the circadian rhythm in the biochemical markers of bone turnover is still unknown. This study indicates that the circadian variation in sOC can be controlled by the endogenous circadian variation in serum cortisol, whereas this hormone does not control the circadian variation in either the serum PICP or the urinary excretion in pyridinium crosslinks.

Characterization of urinary degradation products derived from type I collagen. Identification of a beta-isomerized Asp-Gly sequence within the C-terminal telopeptide (alpha1) region.

The heterogeneity of urinary degradation products of C-terminal telopeptides derived from the alpha1 chain of human type I collagen was investigated and characterized. The urinary fragments characterized in this study consisted of two cross-linked (X) amino acid sequences derived from the C-terminal telopeptide (alpha1) of type I collagen. Fragments containing the sequence EXAHDGGR, with a DG site being either nonisomerized (Asp-Gly) or beta-isomerized (betaAsp-Gly), were identified. Pyridinoline was detected among the pyridinium cross-links, but there was a dominance of deoxypyridinoline and a cross-link containing pyridinoline having a molecular weight identical with that of galactosyl pyridinoline. A nonfluorescent cross-link was also found. The concentration of fragments derived from the C-terminal telopeptide region of type I collagen containing the sequence Asp-Gly (alphaCTX) and/or betaAsp-Gly (betaCTX) was measured by enzyme-linked immunosorbent assays in urine and in collagenase digests of trabecular and cortical bone of young and old origin. It was shown that the urinary ratio between such fragments, alphaCTX/betaCTX, was higher in children compared with adults and that the ratio decreased with increasing age of bone. The results indicated that the C-terminal telopeptide fragments derived from type I collagen excreted into urine originated mainly from bone. In conclusion, it is demonstrated for the first time that the C-terminal telopeptide alpha1 chain of type I collagen contains an Asp-Gly site prone to undergo beta-isomerization and that the degree of beta-isomerization of this linkage apparently increases with increasing age of bone. These findings indicate that the ratio alphaCTX/betaCTX might be clinically important in diagnosing metabolic bone diseases.

Development of a monoclonal antibody to urinary degradation products from the C-terminal telopeptide alpha 1 chain of type I collagen. Application in an enzyme immunoassay and comparison to CrossLaps ELISA.

A monoclonal antibody MAbA7 was raised against a synthetic peptide having a sequence (EKAHDGGR) specific for a part of the C-telopeptide alpha 1 chain of type I collagen. MAbA7 was labelled with horseradish peroxide and used in a competitive one-step enzyme-linked immunosorbent assay (ELISA) for measurement of urinary type I collagen degradation products. The assay was technically evaluated and preliminary clinical data are presented. The measuring range was 200-7000 micrograms l-1 with a detection limit of 25 micrograms l-1. Within-run and total CVs were 5.5 and 8.0%, respectively. Analytical recovery averaged 96.6% +/- 5.3 (mean +/- 1SD). Values obtained in the ELISA were highly correlated (r = 0.93) to values obtained by a commercially available assay (CrossLaps ELISA) known to measure urinary degradation products derived from the C-telopeptide of type I collagen reflecting the rate of bone resorption. Investigation of the urinary fragments responsible for the immunological response in the two assays revealed, however, that they are not identical. Values obtained in urine samples from postmenopausal women (n = 108) and patients with Paget's disease (n = 6) increased 43% (p < 0.01) and 28-fold (p < 0.001), respectively, when compared to a premenopausal level (n = 50). A decrease in the urinary concentrations of 67% (p < 0.01) was seen after 6 months in urine samples from postmenopausal women (n = 13) receiving hormone replacement therapy (HRT) compared to a group receiving placebo (n = 9). Likewise, the urinary concentrations decreased 88% (p < 0.001) in early postmenopausal women receiving bisphosphonate therapy (n = 11) for a period of 9 months compared to a group receiving placebo (n = 12). These results suggest that the monoclonal antibody and the new assay may be useful for further investigations of the physiological and clinical importance of type I collagen degradation.

Coated-tube radioimmunoassay for C-telopeptides of type I collagen to assess bone resorption.

We present a coated-tube RIA that is useful for assessment of bone resorption. The assay uses a monoclonal antibody raised against a linear 8-amino-acid sequence (EKAHDGGR) derived from the C-telopeptides of type I collagen. Within-run and total CVs were 4.4% and 5.3-6.2%, respectively, at concentrations of 1-7 mg/L (n = 4-20). Analytical recovery was 98% +/- 8% and dilution 97% +/- 7%. Values obtained in a group of 36 premenopausal women were 227 +/- 89.6 mg/mol creatinine. In a group of 141 postmenopausal women, the values obtained were 429 +/- 225 mg/mol creatinine, a highly significant increase of 89% (P <0.001) over the premenopausal value. In a double-blind placebo-controlled clinical study of these postmenopausal women receiving five different doses of a bisphosphonate, a significant decrease of RIA-measured C-telopeptide values was seen in all bisphosphonate-treated groups, after just 3 months. Values in urine samples from postmenopausal women assayed with the RIA (gamma) and the CrossLaps(TM) ELISA (x) agreed well: slope = 0.98 (95% confidence interval, 0.94-1.01), intercept = 0.34 (0.25-0.43) mg/L, and Sylx = 0.93 mg/L (n = 678). We conclude that this RIA represents a valuable tool for assessing bone resorption.

A simple enzyme-linked immunosorbent assay of human osteocalcin.

A heterologous ELISA for measurement of osteocalcin (bone Gla-protein) is described, involving biotinylated bovine osteocalcin and polyclonal antibodies. The log-linear range was 2.3-37.5 micrograms/L. Between-run (total) and within-run CVs (n = 10) were 5.7-6.4 and 5.9-6.1%, respectively; analytical recoveries ranged from 92% to 108%. Comparison of our method (x) with an RIA (y) yielded y = 1.10 x -0.01 microgram/L, Sylx = 1.4 micrograms/L, r = 0.958 (n = 167). Serum osteocalcin in healthy premenopausal women (n = 29) was 8.7 +/- 3.3 micrograms/L (mean +/- SD) and 11.8 +/- 4.5 micrograms/L in early-postmenopausal women (n = 24). The assay was evaluated in a double-blind placebo-controlled study of healthy early-postmenopausal women, treated for 12 months with either (a) estrogen valerate plus medroxyprogesterone acetate (n = 18), (b) 17 beta-estradiol and desogestrel (n = 22), or (c) placebo (n = 17). Serum osteocalcin decreased significantly (P < 0.001) with either therapy, but increased (P < 0.05) with placebo.