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.

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.

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.

Changes in the carboxyl-terminal propeptide of type I procollagen and other markers of bone formation upon five days of bed rest.

This study was performed in order to investigate the influence of skeletal unloading on the serum concentration of the carboxyl-terminal propeptide of type I procollagen (sPICP) and other markers of bone formation. Blood samples were taken every third hour from nine healthy premenopausal women (22-29 years) in two 24 h studies, before and at the end of five days of bed rest. Furthermore, a set of samples were taken 12 h apart after three days of bed rest. We measured sPICP, the serum concentration of intact and N-terminal-Mid fragment osteocalcin (sOC), and the serum concentration of alkaline phosphatase (sAP). During the five days of bed rest a gradual increase in sOC was observed, while sPICP gradually decreased. sAP was unchanged. Five days of best rest resulted in the following overall changes in the 24 h mean values: sPICP: -14% (p = 0.002); sOC: +9% (p = 0.009); sAP: -1% (not significant). The circadian patterns did not change significantly after bed rest. It is puzzling that the changes in the bone formation markers are of different magnitude, and for sPICP and sOC even in opposite directions. The increase in sOC may be caused by an increase in OC secretion by the osteoblasts or a release of bone-incorporated OC from resorbing sites; the accompanying decrease in sPICP may indicate that bone formation is actually transiently decreased after short term bed rest.

Circadian rhythm in type I collagen formation in postmenopausal women with and without osteopenia.

A circadian rhythm in the serum concentration of the procollagen type I carboxyl-terminal propeptide (sPICP) has previously been demonstrated in premenopausal women. This study was performed to investigate the circadian rhythm in sPICP in healthy and osteopenic postmenopausal women. Blood samples were taken every third hour for 27 h from three groups of women: 12 early postmenopausal women (aged 55 +/- 2 years; mean +/- SD); 12 late postmenopausal women (aged 73 +/- 1 years); and 12 osteopenic but otherwise healthy late postmenopausal women (aged 73 +/- 1 years). A circadian rhythm in sPICP was found in all three groups, as shown by cosinor analysis (p = 0.000003-0.03). The circadian rhythm in sPICP was significantly different between the osteopenic group and the age-matched healthy group (p < 0.008). The amplitude of the circadian rhythm in sPICP was about twice as high in the osteopenic group, and the time of the maximum tended to be about 3 h later, as compared with the age-matched healthy group. The plasma concentration of osteocalcin, as measured by a recently developed two-site enzyme-linked immunosorbent assay, also showed a circadian rhythm in all three groups (p = 0.0001-0.05), with no significant differences between groups. In conclusion, we have found a significant circadian rhythm in sPICP in both early and late postmenopausal women. In osteopenic women the nightly peak in sPICP is larger and persists later into the night as compared with non-osteopenic women.

Two-dimensional gel analysis of human endometrial proteins: cyclic changes in the expression of specific proteins during the normal menstrual cycle.

High resolution two-dimensional (2-D) gel electrophoresis was used to compare the patterns of [35S]methionine-labelled cellular proteins in endometrial tissue from healthy, normally menstruating women. Samples of endometrial tissue were incubated with [35S]methionine for 20 h, and total cell lysates were processed for 2-D gel electrophoresis. Using this technique it was possible to study proteins with iso-electric points (pI) ranging from 3.5 to 11 and relative molecular weights (M(r)) ranging from 10,000 to 300,000 Da. The fluorograms were compared by computer-aided analysis whereby a total of 1095 [35S]-labelled proteins were resolved on the iso-electric focusing gels (IEF, pI 3.5-7) and 488 on the non-equilibrium pH gradient electrophoresis (NEPHGE) gels (pI 6.5-11). Of the proteins on the IEF gels, 125 showed differential expression during the menstrual cycle. Of these, 36 were maximally expressed in proliferative phase endometrium, 26 in the interval phase and 63 in secretory and/or late secretory phase endometrium. Correspondingly, on the NEPHGE gels a total of 61 proteins exhibited cyclical variation, of which 30 were more prominent in proliferative phase, 13 in interval phase and 18 in secretory phase endometrium. This study shows that 2-D gel electrophoresis is eminently suited to the identification of proteins whose expression varies in a cyclical manner during the menstrual cycle. Further investigations should be carried out to isolate and characterize these proteins with the aim of establishing useful markers for specific endometrial phases of the menstrual cycle.

Posture, age, menopause, and osteopenia do not influence the circadian variation in the urinary excretion of pyridinium crosslinks.

This study was performed to investigate whether the circadian variation in urinary pyridinium crosslinks is related to physical activity, age, the menopause, and asymptomatic osteopenia. We measured urinary pyridinoline/creatinine (Pyr/Cr) and deoxypyridinoline/creatinine (D-Pyr/Cr) in 9 healthy premenopausal women in two 27 h studies, before and at the end of 5 days of total bed rest. Both Pyr/Cr and D-Pyr/Cr showed highly significant circadian variations, with the peak at night and the nadir during the day (p < 0.001). The 5 days of complete bed rest produced no changes in the circadian pattern, but a general increase of 28% was observed in pyridinium crosslinks. A group of 12 healthy, early postmenopausal women (aged 55 +/- 2 years), 12 healthy, elderly postmenopausal women (aged 73 +/- 1 years), and 12 elderly osteopenic but otherwise healthy women (aged 73 +/- 1 years) were also studied for 27 h. All three groups showed highly significant (p < or = 0.001) circadian variations in the urinary excretion of pyridinium crosslinks. As expected, both Pyr/Cr (p < 0.05) and D-Pyr/Cr (p < 0.001) increased at the time of menopause, but the circadian variations in Pyr/Cr and D-Pyr/Cr were similar in all groups studied. We conclude that the circadian variation in the urinary excretion of pyridinium crosslinks is independent of physical factors. Furthermore, the circadian variation in pyridinium crosslinks was not related to age, menopausal status, or asymptomatic osteopenia.

Purification of human procollagen type I carboxyl-terminal propeptide cleaved as in vivo from procollagen and used to calibrate a radioimmunoassay of the propeptide.

We purified human procollagen type I carboxyl-terminal propeptide (PICP) that had been cleaved as in vivo from procollagen. PICP in serum-free medium from cultured human fetal fibroblasts was purified by thiophilic adsorption chromatography, low-pressure gel filtration, and HPLC gel filtration. The purity and homogeneity of the protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequencing showed that the sequences of the alpha 1 and alpha 2 chains of this PICP were identical to those of the PICP produced in vivo. The monocomponent PICP thus purified was used as calibrator in a simple equilibrium-type RIA of PICP with polyclonal antibodies raised in rabbits. The measuring range is 0.15-3.75 nmol/L, and the assay detection limit is 0.03 nmol/L. The within-run and total CVs are 2% and 4%, respectively. The reference interval for the plasma concentration of PICP in healthy women of ages > 30 years is 0.36-1.44 nmol/L (geometric mean 0.72 nmol/L, n = 154).