A double-blind dose-ranging study of risedronate in Japanese patients with osteoporosis (a study by the Risedronate Late Phase II Research Group).

To determine the clinical recommended dosage regimen of risedronate for the treatment of involutional osteoporosis in Japanese patients, dose-response relationships for the efficacy and safety of this drug were investigated using a multi-center, randomized, double-blind, parallel group comparative design with four dose levels of risedronate (placebo, 1 mg, 2.5 mg and 5 mg per day). A total of 211 patients diagnosed with involutional osteoporosis according to the criteria proposed by the Japanese Society for Bone and Mineral Research were randomized and received one of the four doses once daily for 36 weeks. All patients were supplemented with 200 mg of calcium daily in the form of calcium lactate. The primary efficacy endpoint was the percent change in bone mineral density of the lumbar spine (L2-L4 BMD) determined by dual-energy X-ray absorptiometry (DXA) from baseline to the time of final evaluation. Changes in biochemical markers of bone turnover and safety profile were also compared. Percent changes in L2-L4 BMD at final evaluation in the placebo, and 1-, 2.5-, and 5-mg risedronate groups were 0.79+/-5.30, 2.71+/-4.93, 5.29+/-3.96, and 5.15+/-4.25% (mean+/-SD), respectively. A linear dose-response relationship was obtained up to a dose of 2.5 mg, whereas no further increase in BMD was observed at 5 mg. The decrease in bone turnover markers, including N-terminal osteocalcin, phosphorus, and urinary deoxypyridinoline, also showed a linear dose-response relationship up to a dose of 2.5 mg. Alkaline phosphatase level decreased linearly up to a dose of 5 mg. Risedronate was well tolerated in this 36-week study with 1- to 5-mg doses. Neither the overall incidence of adverse events nor the percentage of patients without problem in overall safety assessment differed significantly among the dose groups including the placebo group. Based on these results, a once-daily dose of 2.5 mg of risedronate, which is half that used in Caucasians, is recommended for the treatment of involutional osteoporosis in Japanese patients.

A comparison of the effect of risedronate and etidronate on lumbar bone mineral density in Japanese patients with osteoporosis: a randomized controlled trial.

To demonstrate the clinical benefit of 2.5 mg daily risedronate in the treatment of involutional osteoporosis, the effect of risedronate on bone mineral density (BMD) of the lumbar spine was compared with that of etidronate, selected as a representative of the bisphosphonates currently marketed in Japan. In this multicenter, randomized, double-masked, active (etidronate) controlled comparative study, a total of 235 Japanese patients with involutional osteoporosis were randomized to receive either treatment with 2.5 mg/day of risedronate for 48 weeks or intermittent treatment with etidronate (4 cycles of 2 weeks of treatment with 200 mg/day followed by 10-week medication-free periods). All patients received 200 mg of calcium supplement daily in the form of the calcium lactate. Bone mineral density of the lumbar spine (L2-L4 BMD) was determined at 12, 24, 36 and 48 weeks by dual-energy X-ray absorptiometry. The primary endpoint was the percent change in L2-L4 BMD from baseline to the time of final evaluation. Changes in biochemical markers of bone turnover and safety profiles were also compared. A significant increase in L2-L4 BMD was observed at 12 weeks after initiation of therapy in both the risedronate (2.8%) and etidronate (1.8%) groups. The increase in L2-L4 BMD at the time of final evaluation in the risedronate group (4.9%) was significantly greater ( p = 0.002) than that in the etidronate group (3.1%). The changes in bone resorption markers (urinary total deoxypyridinoline and N-terminal telopeptide of type I collagen) from baseline to 48 weeks were -37.6% and -41.3% for risedronate and -22.5% and -26.6% for etidronate, respectively. New vertebral fractures or deterioration of existing fractures were observed in 2.8% (3/106) of the patients in the etidronate group, while no such cases (0/101) were observed in the risedronate group. No significant difference in the incidence of adverse events was found between two treatments. Daily oral risedronate (2.5 mg) exhibited efficacy superior to that of intermittent cyclical etidronate (200 mg) in increasing L2-L4 BMD, and was well tolerated by Japanese patients with involutional osteoporosis.

Clinical use of nafarelin in the treatment of leiomyomas. A review of the literature.

OBJECTIVE: To review the efficacy and safety of nafarelin in the treatment of leiomyomas. STUDY DESIGN: A literature review of published clinical trials was conducted. Six studies, including a total of 602 patients with leiomyomas, were reviewed. Patients received intranasal nafarelin, 50-400 micrograms twice daily for three to six months. Vaginal bleeding patterns, leiomyoma and uterine size, surgical conditions and adverse effects were assessed. RESULTS: Nafarelin consistently suppressed estrogen production, reduced leiomyoma and uterine size, and controlled menorrhagia. The significant reduction in uterine bleeding and amenorrhea resulting from administration of nafarelin was associated with a rise in mean hemoglobin concentrations. In addition, nafarelin improved hematologic parameters in women with and without anemia. Nafarelin was well tolerated, although hot flushes were the most commonly reported adverse events. Measured bone mineral density decreased significantly during treatment, although by six to nine months post-treatment, it increased to values not significantly different from baseline. The adverse effects of nafarelin were generally reversible after treatment withdrawal. CONCLUSION: Nafarelin treatment of women with symptomatic leiomyomas effectively decreases uterine bleeding; improves hematologic parameters; manages symptoms of menometrorrhagia, dysmenorrhea and pelvic discomfort; reduces uterine and myoma size; and is well tolerated. Reduction in bone mineral density occurs, but levels return to, or near, baseline levels within six months after treatment.

Biological action of keratinocyte growth factor in BeWo cells, a human choriocarcinoma cell line.

Previously, we reported that the expression of keratinocyte growth factor (KGF) is enhanced in secretory phase endometrial and decidual cells in early pregnancy as compared with the expression of KGF in proliferative phase endometrial cells, in humans. In order to clarify the role of KGF in embryo-endometrial interaction, we analyzed the in vitro effect of KGF on the human chorionic gonadotropin (hCG) secretion and on DNA synthesis in chorionic villi which are in close contact with the endometrium/decidua in the early stage of pregnancy. In this study, we used the BeWo cell line, a human choriocarcinoma cell line that possesses the biological features of secreting various placental hormones including hCG. Furthermore, we investigated the expression of KGF receptor (KGF-R) in these cells. KGF significantly stimulated hCG secretion in cultured BeWo cells but did not affect [3H]-thymidine incorporation. KGF-R mRNA was detected in BeWo cells by reverse transcriptase-polymerase chain reaction. These results suggest that the expression of KGF, which is induced in endometrial/decidual cells by progesterone, plays an important role in the embryo-endometrial/ decidual interaction by stimulating hCG secretion rather than affecting cell proliferation.

A double-masked multicenter comparative study between alendronate and alfacalcidol in Japanese patients with osteoporosis. The Alendronate Phase III Osteoporosis Treatment Research Group.

To evaluate the efficacy and safety of alendronate, a double-masked, active (alfacalcidol) controlled comparative study for 48 weeks was carried out in a total of 210 Japanese patients with osteoporosis. The doses of alendronate and alfacalcidol were 5 mg/day and 1 microgram/day, respectively. The lumbar bone mineral density (LBMD) values observed at 12, 24, 36 and 48 weeks after the initiation of alendronate treatment were 3.53 +/- 0.53%, 5.37 +/- 0.62%, 5.87 +/- 0.74% and 6.21 +/- 0.59% (mean +/- SE), respectively, higher than the baseline value. Corresponding values in the alfacalcidol group were 1.50 +/- 0.43%, 0.69 +/- 0.63%, 1.12 +/- 0.60% and 1.36 +/- 0. 63%, respectively. There was a significant difference between the two groups at each time point (p<0.05 or p<0.001). The bone turnover markers were depressed during treatment in the alendronate group: -32.2% for alkaline phosphatase, -53.7% for N-terminal osteocalcin and -45.0% for urinary deoxypyridinoline compared with the corresponding baseline values. On the contrary, no notable changes in these parameters were observed in the alfacalcidol group. Treatment with alendronate caused a transient decrease in serum calcium concentrations associated with an increase in the serum level of intact parathyroid hormone. In contrast, treatment with alfacalcidol resulted in a tendency of these parameters to change in the opposite direction. No difference in fracture incidence between the two groups was observed. The overall safety of alendronate was comparable to that of alfacalcidol. In conclusion, although it was a relatively short-term study of 48 weeks, the results of the present study indicate that alendronate at the daily dose of 5 mg was effective in increasing LBMD and that no serious drug-related adverse events were observed in the alendronate-treated patients. Alendronate is more efficacious than alfacalcidol in increasing bone mineral density, although the mechanisms of the actions of the two drugs are apparently different.

Early postmenopausal bone loss is prevented by estrogen and partially by 1alpha-OH-vitamin D3: therapeutic effects of estrogen and/or 1alpha-OH-vitamin D3.

A total of 79 Japanese women who were within 5 years of menopause were randomly assigned 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] 1.0 microg/day, conjugated estrogens 0.625 mg/day, a combination of both, or control (no treatment). Lumbar spine and proximal femur bone mineral density (BMD) and biochemical indices were monitored over 2 years. In the 1alpha(OH)D3-treated group, there was a nonsignificant decrease in lumbar spine BMD compared with controls, and no significant loss in the femoral neck compared with controls. In the estrogen-treated group, there was a nonsignificant increase in spine BMD (+2.17% in the first year and +1.71% in the second year), and no loss in femoral neck BMD. The combination of conjugated estrogens +1alpha(OH)D3 was more effective in increasing BMD in the spine (+3. 68% in the first year and +3.63% in the second year) and femur (+2. 56% in the first year and +4.44% in the second year) BMD. There was a significant difference in lumbar spine BMD in both the first and second years between the combination-treated group and the 1alpha(OH)D3-treated and control groups (P < 0.01). Serum osteocalcin (OC) significantly decreased in the combination-treated group (-23.8% in the first year) and the estrogen-treated group (-37. 6% and -41.2% at 6 and 18 months, respectively), and serum alkaline phosphatase (Alp) decreased significantly in the first year in the combination-treated (-31.5%), estrogen-treated (-27.3%), and 1alpha(OH)D3-treated (-7.9%) groups, whereas serum OC increased (+45. 4% in the first year) in women without treatment. The results of this study indicate that early postmenopausal bone loss in the femoral neck is prevented by conjugated estrogens, 1alpha(OH)D3, or both, whereas bone loss in the spine is not prevented by 1alpha(OH)D3. Estrogen proves effective in preventing early postmenopausal bone loss by markedly inhibiting bone turnover. Moreover, a synergistic bone-sparing effect can be expected when estrogen is administered concomitantly with 1alpha(OH)D3 rather than when used alone.

Effects of glucose and related substrates on the recovery of the electrical activity of gonadotropin-releasing hormone pulse generator which is decreased by insulin-induced hypoglycemia in the estrogen-primed ovariectomized rat.

We investigated the effect of glucose and its related substrates on the recovery of pulsatile luteinizing hormone (LH) secretion which was suppressed by insulin in estrogen-primed ovariectomized rats. We also examined the effect of glucose on the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator which was suppressed by insulin. The intravenous (i.v.) injection of insulin (5 units/rat) suppressed the pulsatile LH secretion for 3 h in estrogen-primed ovariectomized rats. This suppressive effect of insulin on the LH secretion was rapidly reversed by the i.v. injection of glucose and mannose but not by the injection of lactate and saline. Fructose could recover the LH secretion suppressed by insulin, but took a longer time than glucose did. By monitoring the electrical activity of the GnRH pulse generator, we found that i.v. injection of insulin suppressed the pulsatile LH secretion by decreasing the activity of the GnRH pulse generator. Again, the i.v. injection of glucose, but not saline, immediately recovered the decrease in the electrical activity of the GnRH pulse generator. Fructose could recover the activity of the GnRH pulse generator, but it took a longer time than glucose did. We suggest that glucose availability, but not simply a metabolic state such as the ATP level, is an essential factor for maintaining the electrical activity of the GnRH pulse generator which is responsible for pulsatile LH secretion.

Early and late postmenopausal bone loss is associated with BsmI vitamin D receptor gene polymorphism in Japanese women.

To determine whether vitamin D receptor (VDR) gene polymorphisms are associated with bone mineral density (BMD) and bone loss in the Japanese population, VDR BsmI RFLPs were analyzed in 191 postmenopausal Japanese women by comparing B allele and b allele DNA sequences, and a point mutation was confirmed. We examined VDR BsmI restriction fragment length polymorphism (RFLP) with an amplification refractory mutation system (ARMS) using this point of mutation. The frequency of VDR BsmI alleles in the Japanese population was significantly different from that in whites. The bb genotype was identified in 79.6%, of the subjects, the Bb genotype in 19.3%, and the BB genotype was in only 1.1%. We find no significant differences in lumbar spine baseline BMD between the bb genotype and the Bb genotype. In both early and late postmenopausal periods, serial measurements of vertebral BMD revealed that subjects with the Bb genotype lost BMD faster than those with the bb genotype (P = 0.001). We conclude that there is a significant relationship between RFLPs of BsmI VDR and the annual rates of bone loss during early and late postmenopausal periods in the Japanese population.

A heterogeneity in serum osteocalcin N-terminal fragments in Paget's disease: a comparison with other biochemical indices in pre- and postmenopause.

An osteocalcin (OC) nitrogen (N)-terminal sandwich enzyme immunoassay that employs anit-N-20 (amino acids 1-20) polyclonal and monoclonal antibodies has been developed. This assay has demonstrated the existence of a heterogeneity in the OC N-terminal fragments observed in the serum of a patient with Paget's disease and a normal child. The elevation of the serum OC N-terminal value that occurs in Paget's disease was considered to be comparable to the serum alkaline phosphatase (ALP) elevations that also occur. The size of the peaks corresponding to N-terminal OC fragments in Paget's serum decreased 3 months after bisphosphonate treatment. Serum levels of OC N-terminals, and other biochemical indices, were determined for 67 premenopausal and 181 postmenopausal Japanese women. Serum OC N-terminal levels increased significantly (41.2%) in postmenopausal women compared with age-matched premenopausal women. These results strongly suggest that serum OC N-terminal levels reflect bone turnover rates when bone resorption is dominant.

Differential gene expression of TGF-beta isoforms and TGF-beta receptors during the first trimester of pregnancy at the human maternal-fetal interface.

PROBLEM: The transforming growth factor (TGF)-beta s are multifunctional cytokines, and they play a role in the controlled growth of trophoblasts. Moreover they are thought to be important in maternal-fetal interaction during early gestation. METHOD OF STUDY: Human decidual and villous tissues in the first trimester were Northern blotted and amplified by reverse transcription-polymerase chain reaction to measure the expression of TGF-beta 1, -beta 2, and -beta 3 and their receptors, types I and II, at the first trimester of pregnancy. In addition, their cell-specific expression at the maternal-fetal interface was determined by in situ hybridization. RESULTS: Each isoform of TGF-beta was expressed in both decidual and villous tissues. Because most TGF-beta 1 gene expression was found in villous tissues, TGF-beta 2 mRNA was expressed preferentially in the decidual tissues. TGF-beta 3 transcripts were expressed in the nonpregnant endometrium. CONCLUSIONS: The results suggest that each isoform of TGF-beta plays some specific role in decidualization and placentation. Furthermore, it is predicted that they regulate the maternal-fetal interaction at early gestation.

Effects of GnRH on protein kinase C activity, Ca2+ mobilization and steroidogenesis of human granulosa cells.

Gonadotropin-releasing hormone (GnRH) and its agonists have been known to directly affect steroid hormone production in human granulosa cells. In this study, we examined effects of GnRH on Ca2+ mobilization, protein kinase C activity and steroidogenesis of human granulosa cells. Human granulosa cells were harvested by aspiration of follicles during oocyte retrieval for IVF. Test substances, that is, GnRH agonist ([D-Ser(TBu)6]-LHRH-EA), 12-O-tetradecanoyl-phorbol-13-acetate (TPA), A23187, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and nordihydroguaiaretic acid (NDGA) were added to HTF medium with human granulosa cells. At the end of a 5-h incubation period, progesterone and estradiol in the medium were extracted and measured. GnRH agonist (10(-9)M and 10(-8)M) significantly stimulated (P < 0.05) progesterone and estradiol accumulation in the medium. TPA (10(-7)M, 10(-8)M and 10(-9)M) also significantly stimulated (P < 0.05) progesterone accumulation. A23187 (10(-6)M) significantly stimulated progesterone accumulation (P < 0.05) and significantly enhanced (P < 0.05) the stimulatory effect of TPA on progesterone production. H-7 (10(-3)M, 10(-4)M and 10(-5)M) and NDGA (10(-3)M, 10(-4)M and 10(-5)M) significantly inhibited (P < 0.05) the effect of GnRH agonist on progesterone and estradiol production by the granulosa cells. The granulosa cells were loaded with 4 microM Fura 2-AM, and the ratio of the intensities of fluorescent emission at 510 nm with excitation at 340 and 380 nm was calculated at 100-ms intervals. GnRH agonist (10(-7)M and 10(-6)M) increased (P < 0.05) [Ca2+]i significantly as compared with the control. These results suggested that the effects of GnRH on progesterone and estradiol production in human granulosa cells were mediated partially by calcium mobilization and partially by C-kinase activity, and protein kinase C, [Ca2+]i and lipoxygenase were suspected of being important intracellular messages for GnRH in human granulosa cells.

A placebo-controlled, single-blind study to determine the appropriate alendronate dosage in postmenopausal Japanese patients with osteoporosis. The Alendronate Research Group.

Alendronate (4-amino-1-hydroxybutylidene-1,1-bisphosphonate) is a potent inhibitor of bone resorption. The efficacy and safety of 36 weeks of treatment with alendronate were evaluated in Japanese women with osteoporosis, osteoporotic osteopenia or artificial menopause. The bone mineral density (BMD) of the lumbar vertebrae, markers of bone and calcium metabolism and clinical symptoms were monitored. A total of 113 randomly selected patients with osteoporosis or osteopenia were enrolled in the study, of whom 12 were excluded from the analyses because of lack of data. As a result, 101 patients were evaluated for the safety of the drug. Since eight patients were excluded from the efficacy analysis, 93 were evaluated. The incidence of adverse effects in the placebo (P), alendronate 2.5 mg/day (L) and alendronate 10 mg/day (H) groups increased with increasing dose of alendronate, being 6.1, 14.3 and 18.2%, respectively. The most common adverse effects were gastrointestinal symptoms, none of which was serious. Lumbar BMD increased after 36 weeks of drug administration to 5.21%, 5.64% and -0.90% in the L, H and P groups, respectively (P < 0.001, L vs. P and H vs. P). Serum alkaline phosphatase activity, serum osteocalcin and urinary deoxypyridinoline excretion were significantly decreased in a dose-related manner. Serum calcium and phosphorus were also significantly decreased after alendronate administration. Serum intact PTH was transiently increased. The present results indicate that alendronate effectively decreases bone turnover in a dose-related manner and increases lumbar BMD at a dosage of 2.5 mg/day, the lowest dose used in this study, in Japanese patients with osteoporosis.

Hyperprolactinemic recurrent miscarriage and results of randomized bromocriptine treatment trials.

OBJECTIVE: To evaluate hyperprolactinemia in the pathogenesis of recurrent spontaneous abortion. DESIGN: Randomized trial. SETTING: Miscarriage clinic, Yokohama City University Hospital, Yokohama, Japan. PATIENT(S): From a group of 352 women with recurrent spontaneous abortion, we identified 64 patients with a prolactin disorder that was not associated with any other etiologic abnormalities, including ovarian or endocrinologic disturbances such as luteal phase dysfunction, polycystic ovaries, hypersecretion of LH, galactorrhea, or thyroid hormone disorders. INTERVENTION(S): Restoration of prolactin levels with bromocriptine. MAIN OUTCOME MEASURE(S): Successful pregnancy (live birth). RESULT(S): The percentage of successful pregnancies was higher in the bromocriptine-treated group than in the group that was not treated with bromocriptine (85.7% versus 52.4%, P < .05). Serum prolactin levels during early pregnancy (5-10 weeks of gestation) were significantly higher in patients who miscarried (31.8-55.3 ng/mL) than in patients whose pregnancies were successful (4.6-15.5 ng/mL, P < .01 or P < .05). CONCLUSION(S): Appropriate circulating levels of prolactin may play an important role in maintaining early pregnancy, especially in cases of hyperprolactinemic recurrent miscarriage.

Differences in bone resorption after menopause in Japanese women with normal or low bone mineral density: quantitation of urinary cross-linked N-telopeptides.

The objective of this study was to examine the value of NTx, a urinary cross-linked N-telopeptides of type I collagen, as a marker of bone resorption. We assessed changes in pre- and postmenopausal bone resorption by evaluating the correlation of NTx with L2-4 bone mineral density (BMD) in a total of 1100 Japanese women, aged 19-80 years [272 premenopausal (45.2 +/- 6.2 years) and 828 postmenopausal (59.5 +/- 6.2 years)]. Postmenopausal women were divided into three groups based on the range of BMD (normal, osteopenic, and osteoporotic). Within each group, subjects were further segregated according to years since menopause (YSM). NTx values were then evaluated for each group. Our results showed that BMD was significantly decreased (P < 0.05) and NTx was significantly increased (P < 0.01) after menopause in age-matched analysis. Consistent with a previous report, NTx was inversely correlated with BMD for the entire cohort of study subjects (r = -0.299), although NTx correlated better with premenopausal than postmenopausal BMD (r = -0.240 versus r = -0.086). This may have been due to the fact that elevated values of NTx were exhibited over the entire range of BMD present in the postmenopausal women, suggesting that NTx might respond faster to the estrogen withdrawal than BMD. In all postmenopausal women, regardless of the range of BMD, the increase in NTx reached a peak within 5 YSM. After 11 YSM, however, NTx remained elevated in the osteoporotic group but it decreased in the osteopenic group, and showed no significant change in the group of postmenopausal women with normal BMD. These findings suggest that bone resorption is dramatically increased within 5 years after menopause but remains increased only in osteoporotic women.

The effect of hormone replacement therapy in postmenopausal women on urinary C-telopeptide and N-telopeptide of type I collagen, new markers of bone resorption.

To analyze the inhibitory effect of hormone replacement therapy (HRT) on bone resorption, we measured urinary excretion of C-telopeptide (CTX) and N-telopeptide (NTX) of type I collagen as new markers of bone resorption and assessed their correlation with bone mineral density, in comparison with urinary pyridinoline (Pyr), deoxypyridinoline (D-Pyr) and hydoxyproline (Hpr). CTX and NTX, in addition to Pyr, D-Pyr, and Hpr, in urinary samples from 33 postmenopausal women with climacteric symptoms who were treated with 0.625 mg conjugate equine estrogen and 2.5 mg medroxyprogesterone acetate for 12 months were measured using ELISA for each telopeptide. Bone mineral density in the lumbar spine was also measured by dual energy X-ray absorptiometry. Similar to Pyr, D-Pyr, and Hpr, urinary excretions of CTX and NTX significantly decreased during the continuous administration of conjugate equine estrogen and medroxyprogesterone acetate for 12 months. The magnitudes of the reduction of CTX and NTX were significantly greater than those of Pyr, D-Pyr, and Hpr. CTX and NTX correlated well with Pyr, D-Pyr, and Hpr, and there was a good correlation between CTX and NTX. Unlike Pyr, D-Pyr, and Hpr, the pretreatment values of CTX and NTX correlated negatively with bone mineral density at baseline. These results suggest that, among the markers for bone resorption, the changes in CTX and NTX could be greater than those in Pyr, D-Pyr, and Hpr during HRT in postmenopausal women.

Specific expression of PP5/TFPI2 mRNA by syncytiotrophoblasts in human placenta as revealed by in situ hybridization.

Placental protein 5 (PP5) is a placenta-derived glycoprotein with serine proteinase-inhibiting activity. To date its physiological functions have not been well elucidated. Recently, cDNA sequence analysis revealed that PP5 belongs to the Kunitz-type proteinase inhibitor family and it is identical to tissue factor pathway inhibitor-2 (TFPI-2), homologous to TFPI. Northern blot analysis demonstrated that placental tissue is extremely rich in the transcripts. This study localized PP5/TFPI-2 mRNA in placental tissues at three different gestational periods using in situ hybridization. PP5/TFPI-2 mRNA was specifically detected in syncytiotrophoblast at any gestational period examined, suggesting that syncytiotrophoblast is the principal production site of PP5/TFPI-2 in developing placental tissues. This mRNA expression pattern of PP5/TFPI-2 is quite different from that of TFPI, which is mainly found in vascular endothelial cells. The results indicated possible roles of PP5/TFPI-2 in the trophoblast differentiation and in the maintenance of intervillous blood flow. Also, Northern analysis demonstrated no or little expression of PP5/TFPI-2 in four choriocarcinoma cell lines, in contrast to its abundant expression in syncytiotrophoblast.

Changes in urinary excretion of type-I collagen cross-linked C-telopeptide and N-telopeptide in perimenopausal women.

In order to assess the dynamics of bone turnover during menopausal transition, we measured the urinary excretion of C-telopeptide (CTX) and N-telopeptide (NTX) of type-I collagen, new markers of bone resorption, using two ELISAs which recognize the corresponding cross-linked peptide of type-I collagen. CTX and NTX levels in postmenopausal women were significantly higher than those in premenopausal women, confirming the enhancement of bone resorption after menopause. The measurement of CTX and NTX, which is convenient and specific for bone resorption compared with conventional bone resorption markers, pyridinoline and deoxypyridinoline, might be useful in the management of postmenopausal women.

Differential expression of trypsin in human ovarian carcinomas and low-malignant-potential tumors.

It is widely recognized that matrix metalloproteinases and serine proteinases play an important role in cancer invasion and metastasis. We have reported that trypsin is synthesized in ovarian carcinomas as well as in some other types of cancers. In general, ovarian cancers easily tend to invade, metastasize, and spread widely into the peritoneal cavity. However, low-malignant-potential (LMP, borderline tumor) ovarian tumors are known to have limited malignant potential for progression, although microinvasion and distant metastasis have been reported among them. To analyze the relationship between varied degrees of trypsin expression and malignant behavior of ovarian tumors, immunohistochemical studies with monoclonal antibodies to human trypsin and clinicopathologic analysis were performed in human ovarian carcinomas, low-malignant-potential tumors, and benign cystadenomas. Thirteen (44.8%) cases of 29 ovarian carcinomas showed prominent trypsin expression, while only 2 (18.2%) cases of 11 LMP ovarian tumors demonstrated low levels of expression. Benign tumors and normal ovaries did not show any positivity for trypsin. These data suggest that tumor-derived heterotropic trypsin may be associated with ovarian tumors in parallel with malignant potential or behavior such as invasiveness or metastasis. At least in some ovarian carcinomas, prominent stromal invasion or metastasis might require the acquisition of or association with tumor-derived trypsin production.

Purification and characterization of bone-specific alkaline phosphatase from a human osteosarcoma cell line.

Bone-specific alkaline phosphatase (bone ALP) levels are considered to reflect osteoblastic activity and can therefore be used as a marker of bone formation. However, bone ALP is difficult to distinguish from other ALP isoforms since the kidney, liver, and bone isoenzymes are encoded by the same gene and only differ because of post-translational modification of their carbohydrate side chains. The aim of this study was to purify and separate bone ALP which could be used to raise specific antisera against human bone ALP, from Saos-2, a human osteogenic sarcoma cell line. The procedure involved two steps. The first step, cultivation of 10(5) Saos-2 cells, yielded approximately 1 U ALP. Subsequent butanol extraction achieved 1.82-fold purification. For the second step, separating bone ALP, we used serial lectin affinity chromatography to distinguish between the carbohydrate side chains of the various ALP isoforms. A sample of the butanol extract was fractionated into three peaks (I, II, and III) by concanavalin A. Peaks II and III were subsequently identified as types IIa and IIIb bone ALP using pea lectin and wheat germ agglutinin columns, respectively. The specific activity of bone ALP was measured using commercial kits. Since bone ALP accounted for at least 84% of the total ALP activity after the final separation, this method appears more convenient and reproducible than others using bone or Pagetic sera. The bone ALP purified in this study could be used to raise monoclonal antibodies against bone-specific ALP.

Serum soluble interleukin-6 receptor and biochemical markers of bone metabolism show significant variations during the menstrual cycle.

We examined sequential changes of bone-resorbing cytokines and bone metabolic markers and the effect of ovarian hormones on bone metabolism during the menstrual cycle in 10 healthy Japanese women, aged 22-43 yr, with normal ovarian function. Serum soluble interleukin-6 receptor (sIL-6R) showed a significant variation; a rise during the early and late follicular periods followed by a fall during the early luteal period (P = 0.0423, P = 0.0334) and an increase during the mid and late luteal periods. There were significant changes in the levels of markers of bone formation: a rise in serum bone-specific alkaline phosphatase (ALP) during the mid and late follicular (P = 0.0265) periods and a fall in serum carboxyl-terminal propeptide of type I procollagen (PICP) during the midluteal period (P = 0.0161). As for the levels of bone resorption markers, urinary type I collagen C-telopeptide breakdown products (CTx) and free deoxypyridinoline (D-Pyr) decreased significantly during the early and midfollicular periods, urinary free D-Pyr and serum pyridinoline cross-linked carboxyl-terminal telopeptide of type I collagen (ICTP) (P = 0.0440) increased significantly during the early luteal period, and urinary CTx, free D-Pyr, and serum ICTP decreased significantly during the late luteal period (P = 0.0170-0.0008). The serum PTH level was significantly higher during the follicular than the luteal period (P = 0.0132). Serum sIL-6R significantly correlated with urinary CTx (r = 0.190, P < 0.05) and serum ALP (r = 0.209, P < 0.05) and serum estradiol with intact osteocalcin (r = 0.309, P < 0.0005) and serum ALP (r = 0.181, P < 0.05). These observations strongly suggest that cyclic variations in the levels of bone formation and resorption markers and of a bone-resorbing cytokine may be modulated by cyclic changes in serum steroid hormones during the menstrual period. In addition, the specific days of biochemical events in the menstrual cycle are crucial for evaluating osteoclastic and osteoblastic activities in pre- and perimenopausal women or in women starting GnRH agonist therapy.