K. Kawana

The Maillard protein cross-link pentosidine in urine from diabetic patients

SummaryThe Maillard protein cross-link pentosidine is a fluorescent condensation product of lysine, arginine and ribose. It accumulates in human tissues with age, and the accumulation process is accelerated in the tissues of diabetic patients. Using SP-Sephadex C-25 in the pretreatment for HPLC, we examined levels of pentosidine in urine without hydrolysis (free form) and levels of pentosidine in urine after hydrolysis (total forms), from 23 diabetic patients and 21 control subjects. The mean percentages of the values of free form per total forms (±SD) were 89±15% in diabetic patients, 88±16% in control subjects and 89±15% in total populations of diabetic patients and control subjects. There was a significant correlation between the values of free form and total forms in diabetic patients (r=0.938, p=0.0001), in control subjects (r=0.820, p<0.02) and in total populations of diabetic patients and control subjects (r=0.951, p=0.0001). The mean level of pentosidine per mol creatinine (±SD) was significantly elevated in urine from diabetic patients as compared to the level in control subjects (8.8±4.3 μmol/mol creatinine vs 4.2±1.4 μmol/mol creatinine, p=0.0001 in free form; 10.1±5.3 μmol/mol creatinine vs 4.7±1.4 μmol/mol creatinine, p=0.0001 in total forms). These results demonstrate that urinary pentosidine, especially in free form, could be a useful marker for the assessment of diabetes and diabetic complications.

The effect of menopause on biochemical markers and ultrasound densitometry in healthy females.

Urinary pyridinoline (pyr) and deoxypyridinoline (dpyr) are new markers for bone resorption, and serum osteocalcin reportedly indicates osteoblastic activity. Recently, a new ultrasound bone densitometer instrument has been developed that measures ultrasonic properties of the os calcis, namely, the speed of sound (SOS), broadband ultrasound attenuation (BUA), and stiffness index. The effects of menopause on biochemical markers and ultrasound densitometry were investigated in 40 healthy females, 36–39 years, with regular menstruation, and in 117 healthy perimenopausal females, 47–57 years, who were divided into a premenopausal group and a postmenopausal group. Significantly elevated values of pyr, dpyr, and serum osteocalcin were found for the postmenopausal group as a whole compared with the premenopausal group. We examined postmenopausal groups 48–57 years of age stratified into 2-year intervals (within 2 years of the menopause, 2–4 years postmenopause and 4–6 years postmenopause). Elevated values of urinary pyr, dpyr, and serum osteocalcin were evident even in the first 2 years postmenopause compared with the premenopausal group, and these higher values were exhibited until 6 years after menopause. We found a significant decrease in SOS, BUA, and stiffness index of the postmenopausal group as a whole, compared with those of the premenopausal group. SOS, BUA, and stiffness index of the group within 2 years of menopause significantly decreased compared with those of the premenopausal group. The Z-scores of the increase in biochemical markers and the decrease in stiffness index in the postmenopausal group were approximately 0.7–1.3 compared with the premenopausal group. The results suggest that these biochemical markers and ultrasound densitometry are potentially sensitive parameters of postmenopausal bone change.

Urinary bone resorption markers in patients with metabolic bone disorders.

Recently, urinary pyridinoline and deoxypyridinoline have been commonly employed as bone resorption markers. We studied these markers in 17 patients with hyperthyroidism, 15 undergoing long-term anticonvulsant drug therapy, and 28 with postmenopausal osteoporosis. Both markers had significantly higher levels than those in age-matched control groups. Values of urinary pyridinoline and deoxypyridinoline correlated well with urinary hydroxyproline levels in patients with hyperthyroidism (r = 0.856, p < 0.001 for pyridinoline and hydroxyproline; r = 0.919, p < 0.001 for deoxypyridinoline and hydroxyproline); however, poor correlations were observed, especially between urinary deoxypyridinoline and urinary hydroxyproline (r = 0.357, NS) in patients with postmenopausal osteoporosis. To compare the discriminatory ability of urinary pyridinoline and deoxypyridinoline, receiver operating characteristic (ROC) curves were generated for each of these patient groups using data from age-matched healthy females as the control group. The areas under the curves for both markers were 100.0% in hyperthyroidism. The areas under the curves for pyridinoline in patients undergoing long-term anticonvulsant drug therapy (mean +/- SE; 98.1 +/- 2.8%) and postmenopausal osteoporosis (77.9 +/- 5.7%) were significantly higher than those for deoxypyridinoline in anticonvulsant drug therapy (92.4 +/- 3.3%) and in osteoporosis (64.9 +/- 4.3%). Using data from premenopausal healthy females as the control group, areas under ROC curves for urinary pyridinoline (100.0%) and deoxypyridinoline (94.8 +/- 5.9%) were significantly higher than those for urinary hydroxyproline (73.8 +/- 9.4%) in patients undergoing long-term anticonvulsant drug therapy. In patients with postmenopausal osteoporosis, those for urinary pyridinoline (97.0 +/- 2.8%) were also significantly higher than those for urinary hydroxyproline (74.0 +/- 6.4%).(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of the crosslinks, pyridinoline, deoxypyridinoline and pentosidine, in human aorta with dystrophic calcification

Pyridinoline and, its minor analogue deoxypyridinoline, are trifunctional crosslinks of mature collagen in the connective tissues. Pentosidine, a new type of fluorescent crosslink, is possibly one of the senescent crosslinks but its function and metabolism are still unclear. In this study, we quantitated the crosslinks, pyridinoline, deoxypyridinoline and pentosidine, in human aorta which were obtained from 21 autopsy cases. In each case, the existence of dystrophic calcification in the aorta and complications (diabetes, chronic renal failure and hypertension) were examined. The determination of the content of the three crosslinks was carried out using high performance liquid chromatography (HPLC) analysis. In calcified lesions, the amount of deoxypyridinoline/collagen showed a decrease and the amount of deoxypyridinoline/pyridinoline showed a prominent decrease compared to those in non-calcified lesions (deoxypyridinoline/collagen, P < 0.005; deoxypyridinoline/pyridinoline, P < 0.0001). In non-calcified lesions without complications, the amount of pentosidine/pyridinoline and that of pentosidine/deoxypyridinoline significantly increased with age (pentosidine/pyridinoline, r = 0.704, P < 0.05; pentosidine/deoxypyridinoline, r = 0.624, P < 0.05). This result suggests a possible relationship between dystrophic calcification and crosslink formation of collagen in human aorta.

Comparison of serum and urinary C-terminal telopeptide of type I collagen in aging, menopause and osteoporosis.

BACKGROUND Urinary C-terminal telopeptide of type I collagen (u-CTx) has been reported to be a sensitive biochemical marker of bone turnover. There have been two assays for urinary CTx, which are alpha-CTx and beta-CTx. A newly developed immunoassay for serum CTx (s-CTx) is now available for assessment of bone resorption. We evaluated the effects of aging, menopause, and osteoporosis on the measurements of serum CTx and compared them to urinary CTx assays. METHODS In 79 premenopausal healthy women, 80 postmenopausal healthy women, 61 osteoporotic patients with vertebral fractures and 34 osteoporotic patients with hip fractures, s-CTx and urinary beta-CTx (u-betaCTx) were measured by ELISAs, and urinary alpha-CTx (u-alphaCTx) was measured by an RIA. RESULTS In all subjects, s-CTx significantly correlated with both u-alphaCTx (r=0.54) and u-betaCTx (r=0.51). There was no significant difference among s-CTx, u-alphaCTx and u-betaCTx in the T-scores of the postmenopausal group over the premenopausal group. These findings indicate that the value of s-CTx, as well as urinary CTxs, reflected the increase of bone resorption associated with menopause with a high degree of sensitivity. Patients with vertebral fractures had moderately increased concentrations of bone resorption markers compared to age-matched healthy postmenopausal women (T-score; s-CTx: 0.8, u-alphaCTx: 0.9, u-betaCTx: 0.7), whereas bone resorption markers in hip fracture patients were greatly increased compared to healthy postmenopausal women (T-score; s-CTx: 1.1, u-alphaCTx: 1.3 u-betaCTx: 1.3). The T-scores of u-CTxs against the postmenopausal group in vertebral fracture group and in hip fracture group were not significantly different from those of s-CTx. CONCLUSIONS s-CTx, as well as urinary CTxs, reflects the increase of bone resorption in patients with vertebral fractures and hip fractures.

CIRCULATING LEVELS OF VITAMIN K1, MENAQUINONE-4, AND MENAQUINONE-7 IN HEALTHY ELDERLY JAPANESE WOMEN AND PATIENTS WITH VERTEBRAL FRACTURES AND PATIENTS WITH HIP FRACTURES

Recently, vitamin K has become increasingly of interest in the bone metabol-ism field because of its role as a cofactor in the carboxylation of osteocalcin. Although the role of osteocalcin is not clear, noncarboxylated osteocalcin is one risk factor in hip fractures. It has been reported that the circulating levels of vitamin K1 in osteoporotic patients were significantly lower than those of age-matched control subjects. In this study, we measured circulating levels of vitamin K1, menaquinone-4 (MK-4) and menaquinone-7 (MK-7) in 23 normal healthy women aged 52–93 years (mean ± SD: 80.1 ± 3.5), 13 female patients with vertebral fractures aged 66–93 years (80.3 ± 7.8) and 38 female patients with hip fractures aged 76–87 years (79.8 ± 9.2), (all Japanese), in order to make sure whether these vitamin K levels were different in these three groups. Serum circulating levels of MK-4 was undetectable in most subjects (only one out of 74). Appreciable numbers from these three groups had undetectable levels of MK-7 (52% of the control group, 23% of the vertebral fracture group and 24% of the hip fracture group). Eight subjects from the normal control group (35%) and five patients from the vertebral group (38%) had undetectable levels of vitamin K1. We did not find a significant difference in the measurable levels of vitamin K1, MK-4 and MK-7 in patients with vertebral fractures or patients with hip fractures compared to age-matched normal controls. Undetectable levels of measured vitamin K1, MK-4 and MK-7 in most of subjects may significantly affect the results.

The effect of tamoxifen on bone metabolism and skeletal growth is different in ovariectomized and intact rats.

Abstract. The effects of tamoxifen (TAM) treatment on bone metabolism and skeletal growth were studied in sexually mature intact or ovariectomized (OVX) rats. Experiment 1 was designed to observe the effects of TAM on bone metabolism and skeletal growth in intact rats and included two groups: (1) intact plus vehicle and (2) intact plus TAM. Experiment 2 was designed to investigate the effects of TAM on OVX rats and included the other two groups: (3) OVX plus vehicle and (4) OVX plus TAM. Serum calcium osteocalcin and urinary pyridinoline (Pyr) and deoxypyridinoline (Dpyr) were measured serially before and after TAM treatment for 6 weeks in order to monitor bone turnover. Bone mineral density (BMD) and bone mineral content (BMC) of excised right femora and lumbar vertebrae were determined by dual energy X-ray absorptiometry (DXA). To examine the effect of TAM on skeletal growth, the conventional parameters of femora and the histology of right tibiae were also measured. TAM did not induce significant change in the biochemical markers in intact rats during the 6-week experiment. Bone mass and skeletal growth were not changed by TAM treatment in intact rats. However, TAM treatment reduced the increase in serum osteocalcin and urinary pyridinium cross-links from 1 week to 6 weeks postovariectomy in the OVX rats. TAM inhibited the skeletal growth in OVX rats, because TAM treatment shortened femoral length and decreased the cell number in the growth plate in OVX rats in this study. Our findings indicate that TAM exerts an effect of estrogen agonist on bone metabolism and skeletal growth in OVX rats, however, it does not affect them in intact rats.

Characteristics of biochemical markers in patients with metabolic bone disorders.

Biochemical markers of bone turnover are expected to have some different characteristics among bone metabolic disorders. We compared bone formation markers: serum total alkaline phosphatase (s-Alp), serum osteocalcin (s-OC) and serum carboxy-terminal propeptide of type I collagen (s-PICP); and bone resorption markers: serum carboxy-terminal telopeptide of type I collagen (s-ICTP), urinary pyridinoline (u-Pyr) and urinary deoxypyridinoline (u-Dpyr) to examine which marker is the most suitable and reliable to evaluate bone turnover in patients with osteoporosis (n = 29), osteomalacia (n = 10), primary hyperparathyroidism (n = 6) and renal osteodystrophy (n = 21). The value of s-Alp in the osteomalacia group was significantly higher than those in the normal control group and the osteoporosis group (p < 0.001), and T-score of s-Alp was significantly higher than those of s-OC and s-PICP in the osteomalacia group. The values of u-Pyr and u-Dpyr in the primary hyperparathyroidism group were significantly higher than those in the other groups (p < 0.001). S-PICP, which are not dependent upon renal function, was much higher in the renal osteodystrophy group than in all other groups. In the osteoporosis group, T-score of s-ICTP was significantly higher than those of s-OC. Thus, s-Alp was a good marker in osteomalacia, u-Pyr and u-Dpyr in primary hyperparathyroidism, s-PICP in renal osteodystrophy, and s-ICTP in osteoporosis.

The effect of cyclosporin A administration on bone metabolism in the rat evaluated by biochemical

We investigated the effects of cyclosporin A (CsA), an immunosuppressive agent, on bone remodeling in 6 rats compared to 6 controls, using a histomorphometric technique and biochemical markers for bone metabolism. With an oral daily dose of 15 mg CsA/kg of body weight for 28 days, the trabecular bone volume in CsA administered rats was significantly lower than that in control rats, which indicates bone loss in CsA rats. In CsA rats, bone resorption increased, and urinary pyridinoline (Pyr) significantly increased on day 28 compared with control rats. In contrast, bone formation assessed by serum osteocalcin and osteoid volume had no remarkable changes. These results suggest that administration of CsA for 28 days induces bone loss due to uncoupling between bone resorption and bone formation.

Analysis of urinary pyridinoline and deoxypyridinoline in patients undergoing long-term anticonvulsant drug therapy.

Urinary pyridinoline and deoxypyridinoline were analyzed in 23 patients undergoing long-term anticonvulsant drug therapy and compared with those in an age-matched control group, which consisted of 218 healthy premenopausal women. Values of urinary pyridinoline and deoxypyridinoline in the patient group were significantly higher than those in the control group. However, mean serum levels of alkaline phosphatase in the patient group were within the control range. Our data demonstrate that bone resorption is accelerated by long-term anticonvulsant drug therapy and there may be an imbalance between bone resorption and bone formation in the patient group.