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Introduction

Osteoporosis is a well-known disease and caused 9 million osteoporotic fractures worldwide in the year 2000 (1). Osteoporotic fractures occurring at the spine and the forearm are associated with significant morbidity, but the most serious consequences arise in patients with hip fractures. The mortality rate is 20% in the first year following hip fracture (2). Among those who survive this period, 1 in 5 requires nursing home care (3). Thus, osteoporosis is associated with not only morbidity, but also decline in the quality of life. Osteoporosis is classified as primary osteoporosis induced by menopause or aging and secondary osteoporosis. Well-known causes of secondary osteoporosis include, endocrine disorders, such as Cushing's syndrome, hypogonadism, hyperthyroidism, hyperparathyroidism and diabetes mellitus (4). Gastrointestinal diseases, such as inflammatory bowel disease (5), rheumatoid arthritis (6) and myeloma bone disease (7), also induce osteoporosis. In addition, the incidence of osteoporosis following gastrectomy has become a clinical issue. Although numerous studies have examined bone metabolic disorders following gastrectomy since it was first reported by Sarasin (8), the pathophysiology and the treatment of these disorders have not been fully elucidated. Based on experimental results using rat models, general nutritional deficiencies (9), calcium malabsorption (10), vitamin D deficiency (11), loss of gastric acid (12) and secondary hyperparathyroidism (13) have been suggested as possible causes of bone metabolic disorder following gastrectomy. However, fully understanding the morbidity and developing clinical therapies for skeletal disorders is critical for improving patient quality of life.

Recent epidemiological studies have shown that long-term therapy with proton pump inhibitors (PPIs) significantly increases the risk of osteoporosis and pathological hip fracture in patients with gastroesophageal reflux disease (14). It is thought that PPIs reduce the production and secretion of hydrochloric acid in stomach, increase the pH in the stomach and inhibit absorption of insoluble calcium in the small intestine, thus leading to malabsorption of calcium phosphate and bone metabolism disorder (15,16). PPIs also reportedly inhibit bone resorption by osteoclasts (17,18). However, the irreversible PPI mediated by the PPIs requires a specific pH environment. PPIs are all prodrugs that require two sequential protonation steps for activation (19). In the first step, a pyridine radical is activated, and in the second step, a benzimidazole radical is activated. The first step is required for accumulation of the PPI in the intracellular secretory canaliculus, and the second step is necessary for binding of the PPI with the proton pump. The pKa in the second step does not significantly differ (<1) among the various PPIs (such as rabeprazole, lansoprazole and omeprazole), whereas the pKa for the first step with rabeprazole (4.53) is higher compared to that for lansoprazole (3.83) and omeprazole (4.06) (20). This suggests that rabeprazole can efficiently combine with the proton pump to produce an immediate effect (21). For the two sequential protonation steps, the proton pump inhibitory action of the PPIs is extremely site-specific. In addition to the secretory canaliculi of the gastric parietal cells, the osteoclastic resorption vacuole may be the only other place in which proton pump inhibition by PPIs is known to occur. Clinical studies have supported the theory that the short-term use of a PPI reduces bone resorption markers (18,22). There are conflicting data regarding the effect of PPIs on bone metabolism, with little known concerning the effects of PPIs on osteoclasts and bone resorption.

Therefore, the present study analyzed the effect of PPIs on bone metabolism following total gastrectomy (TG) in a rat model of osteoporosis. Using the rat TG model, poor calcium absorption that is observed during gastric anacidity was reproduced and the confounding antisecretory activity of the PPI was excluded to determine the specific effect of a PPI on osteoclasts.

Materials and methods

Ethical approval

All the procedures performed in the studies involving animals adhered to the Standard Guidelines for Animal Experiments at Kanazawa University (Kanazawa, Ishikawa, Japan; date of issue: May 18, 2012; registration number: AP-122484).

Animals

In total, 75 male Wistar rats (Charles River Laboratories Japan, Inc., Kanagawa, Japan) that were 12 weeks of age were used for the experiments. The rats were housed three to a cage, and were maintained at a room temperature of 22±3°C and humidity of 55±5% with a 12-h light-dark cycle. The rats were provided a standard solid chow, CRF-1 (Charles River Laboratories Japan, Inc.) and tap water. The animal welfare committee of Kanazawa University approved the experiments.

Drugs

The effects of the PPI rabeprazole on bone metabolism were compared with those of minodronic acid, a bisphosphonate clinically administered in the treatment of patients with osteoporosis. Minodronic acid was obtained from Astellas Pharma, Inc. (Tokyo, Japan), and rabeprazple was obtained from Eisai Co., Ltd. (Tokyo, Japan).

Experimental design

The rats were randomly divided into the following four groups: i) Sham-surgery (n=15); ii) TG control (n=20); iii) TG plus rabeprazole (30 mg/kg) administered three times per week (n=20); iv) TG plus minodronic acid (0.04 mg/kg/day) (n=20). Beginning 4 weeks after surgery, rabeprazole was administered subcutaneously three times per week for 18 weeks to the rats in the TG plus rabeprazole group, and minodronic acid was administered subcutaneously daily for 18 weeks to the rats in the TG plus minodronic acid group. The rationale for the dose of rabeprazole used was based on prior studies, which showed that a subcutaneous dose of rabeprazole at 30 mg/kg to rats reduced acid secretion by 100% within 4 h, with the return of acid secretion to normal levels at 3 days (23,24).

Surgery

After 24 h of fasting, the rats were anesthetized with intraperitoneal injections of medetomidine, midazolam and butorphanol. TG using the reconstructed Roux-en-Y method was performed through an upper middle incision. The duodenal stump was closed with sutures. The jejunum was amputated ~6 cm distal to the ligament of Treitz. The esophageal stump was anastomosed to the anal side of amputated jejunum in an end-to-side manner. The jejunojejunostomy was performed in a side-to-side manner. Intestinal anastomosis was performed with interrupted full-thickness stitches using 7-0 monofilament suture. The rats had free access to water and food beginning 24 h after surgery.

Autopsy

Blood samples were obtained and the animals were sacrificed by exsanguination under isoflurane anesthesia 22 weeks after the surgery. The serum samples were immediately separated from the blood by centrifugation at 1,000 × g for 10 min, and the serum was frozen and stored at −80°C until used for analysis. Femurs were isolated for evaluation, and the soft tissue was removed. Femurs were wrapped in saline-soaked gauze and stored at −80°C until used for analysis.

Bone morphometry and density

The right femur was fixed in 10% neutral buffered formalin, degreased in 100% ethanol, re-fixed in cyanuric chloride (Wako Pure Chemical Industries, Osaka, Japan) and decalcified with formic acid. Thin sections of the right femur were made with a sliding microtome (Leica SM-2000R; Leica Biosystems, Nussloch, Germany). The sections were stained with hematoxylin and eosin (H&E) stain. The amount and width of each trabecular bone, correlated with bone strength, was traced distal to epiphyseal line using a bio-imaging navigator (Biorevo BZ-9000; Keyence, Osaka, Japan). The range of calcified bone was extracted. The area of the calcified bone in the traced range was calculated using analytical software (Hybrid Cell Count Software; Keyence).

Bone strength

The bending strength of the left femur was measured with a three-point bending test using a mechanical testing machine (AG-X; Shimadzu, Kyoto, Japan). The specimen was placed horizontally in the loading section of the machine. The center of the diaphysis was pressed with 50 kgf; the distance from the support point was 15 mm. The breaking strength in Newtons (N) was used as a measure of bone strength.

Serum biochemistry

Biochemical measurements, including serum calcium, phosphorus, total protein and albumin were measured with an auto-analyzer (Hitachi 7180; Hitachi High-Technologies, Tokyo, Japan).

Bone metabolism

Serum tartrate-resistant acid phosphatase 5b (TRACP-5b), a bone resorption marker, and bone-specific alkaline phosphatase (BAP), a bone formation marker, were used as biochemical markers of turnover and, measured using an enzyme immunoassay (Rat TRACP-5b ELISA; Cusabio Biotech Co., Ltd., Wuhan, China).

Statistical analysis

Continuous variables are expressed as mean ± standard deviation. Comparisons between groups were made using the Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.

Results

Final sample numbers

Subsequent to being assigned to the experimental groups, 1 rat died in the TG plus rabeprazole group, and 3 rats died in the TG plus minodronic acid group. All 4 rats succumbed to ileus; none from drug toxicity. No rats died in the sham-surgery or TG control groups. Of the 75 assigned animals, 71 rats survived 22 weeks post-surgery and were included in the study (15 in the sham group, 20 in the TG control group, 19 in the TG plus rabeprazole group, and 17 in the TG plus minodronic acid group).

Body weights

Changes in the body weights of the rats in each experimental group are shown in Fig. 1. Body weights of the rats in the TG group were reduced in weeks 1–2. The body weights of the rats in the sham group increased gradually throughout the study period, whereas those in the TG groups remained nearly constant after week 8. No significant differences in the body weights were observed among the three TG groups.

Figure 1. Changes in body weight of the rats 22 weeks after surgery in each experimental group. The body weights of the rats in the sham group increased gradually throughout the study period, whereas those in the TG groups remained nearly constant after week 8. No significant differences were observed in body weights between the three TG groups. TG, total gastrectomy.

Bone mineral density (BMD) of the femur

Twenty-two weeks after TG, the rate of the calcified bone area in the traced are in the TG control group (50.0±8.1%) was lower than that in the sham group (69.0±5.6%) (Fig. 2). The rates in the TG plus rabeprazole (56.5±7.5%) and TG plus minodronic acid (59.0±6.0%) groups were significantly higher than that in the TG control group (P<0.05). These results indicated that rabeprazole inhibited the TG-induced BMD decrease with an effect comparable to that of minodronic acid (Fig. 2).

Figure 2. Bone mineral density of the femoral bone. Twenty-two weeks after surgery, the rate of the calcified bone area in the traced area in the TG control group (50.0±8.1%) was lower than that in the sham group (69.0±5.6%). The rates in the TG plus rabeprazole (56.5±7.5%) and TG plus minodronic acid (59.0±6.0%) groups were significantly higher than that in the TG control group (*P<0.05). TG, total gastrectomy.

Bone strength

Femoral bending strength was markedly decreased in the TG control group (152.34±24.01 N•m) compared with that in the sham group (251.99±23.14 N•m). This effect was significantly ameliorated and also counteracted with minodronic acid treatment (190.50±26.95 N•m), but not by the rabeprazole treatment (151.67±22.41 N•m) (Fig. 3).

Figure 3. Femoral bending strength in the different groups. Femoral bending strength was markedly decreased in the TG control group (152.34±24.01 N•m) compared with that in the sham group (251.99±23.14 N•m; *P<0.05). Femoral bending strength in the TG plus minodronic acid group (190.50±26.95 N•m) was higher than that in the TG control group (*P<0.05). Whereas, femoral bending strength in the TG plus rabeprazole group (151.67±22.41 N•m) is equal with that in the TG control group. TG, total gastrectomy.

Serum biochemistry

Compared with that in the sham group, the serum calcium levels were reduced in rats with TG, while the serum phosphorus levels remained unchanged by TG or by the administration of either minodronic acid or rabeprazole. Minodronic acid significantly decreased the serum calcium level compared with the level in the TG control group (P<0.05). By contrast, rabeprazole did not affect serum calcium levels (Fig. 4).

Figure 4. (A) Serum calcium and (B) phosphorus levels in the different groups. Compared with that in the sham group, the serum calcium levels were reduced in rats with TG (*P<0.05). Minodronic acid significantly decreased the serum calcium level compared with the level in the TG control group (*P<0.05). By contrast, rabeprazole did not affect serum calcium levels. The serum phosphorus levels remained unchanged by TG or by the administration of either minodronic acid or rabeprazole. TG, total gastrectomy.

Bone metabolism

No significant differences in serum TRACP-5b levels were observed between the four groups (Fig. 5A). The serum BAP level was reduced in rats with TG compared with that in the sham group. The administration of minodronic acid or rabeprazole did not ameliorate this serum BAP decrease (Fig. 5B).

Figure 5. (A) Serum TRACP-5b and (B) BAP levels in the different groups. No significant differences were observed in the serum TRACP-5b levels between the four groups. The serum BAP level was reduced in rats with TG compared with that in the sham group (*P<0.05). The administration of minodronic acid or rabeprazole did not ameliorate this serum BAP decrease. TRACP-5b, tartrate-resistant acid phosphatase 5b; BAP, bone-specific alkaline phosphatase; TG, total gastrectomy.

Bone morphology

The morphology of the femoral metaphysis stained with H&E stain was observed microscopically (Fig. 6). The trabecular sponge-like network in the metaphysis (arrowhead in Fig. 6) and trabecular separation (asterisks in Fig. 6) were compared across the four groups. The width (bidirectional arrow in Fig. 6) in the TG control group was thinner than that in the sham group. By contrast, the width was wider in the rabeprazole and minodronic acid-treated groups compared to the TG control group.

Figure 6. Micrographs of the femur, focusing on the metaphysis of the distal end (H&E stain). The morphology of the femoral metaphysis stained with H&E stain was observed microscopically. The trabecular sponge-like network in the metaphysis (arrowhead) and trabecular separation (asterisks) were compared across the four groups. The width (bidirectional arrow) in the TG control group was thinner than that in the sham group. By contrast, the width was wider in both the rabeprazole and minodronic acid treated groups than in the TG control group. TG, total gastrectomy; H&E, hematoxylin and eosin.

Discussion

The present study demonstrated the effects of rabeprazole on bone metabolic disorders in gastrectomized rats. Minodronic acid almost completely blocked the TG-induced decreases in bone density and bone strength. Rabeprazole also inhibited the TG-induced decrease in bone density.

PPIs reduce gastric acid secretion, and are thus widely used in conditions such as gastroesophageal reflux, Zollinger-Ellison syndrome, dyspepsia and peptic ulcer disease (25). For years, PPIs were considered safe, without any major complications during long-term use (26). However, Yang et al (27) conducted a nested case-control study using the General Practice Research Database and examined the risk of hip fractures associated with PPI use. The study reported that the risk of hip fracture was markedly increased among long-term users of high-dose PPI therapy. PPIs were recently identified as an independent risk factor for osteoporotic fracture (14,27–31). PPIs reportedly increase the risk of osteoporotic fracture by causing hypochlorhydria, reducing intestinal calcium absorption and subsequently inducing a negative calcium balance (26,30). However, existing studies provide conflicting information regarding the direct effects of PPIs on calcium absorption. Hansen et al (32) administered omeprazole (40 mg/day) to menopausal women for 30 days and reported that 30 days of continuous PPI therapy did not alter calcium absorption, suggesting that PPI-associated hypochlorhydria does not reduce calcium absorption.

However, several in vitro studies have reported that PPIs inhibit the vacuolar-ATPase of osteoclasts and reduce their activity (33,34). Sheraly et al (35) examined the potential of PPIs to prevent osteoclast-mediated resorption of calcium phosphate cements in vivo. The study reported that the PPIs (pantoprazole and high-dose omeprazole) produced a delay in osteoclast resorption. Ohta et al (22) administered rabeprazole (10 mg/day for 8 weeks) to 22 non-osteoporotic patients presenting with upper gastrointestinal symptoms and investigated the effect of rabeprazole on bone metabolism. They reported that rabeprazole did not affect BAP, but significantly decreased type I collagen cross-linked N-telopeptides. However, in humans and rat models, the effect of PPIs on bone metabolism appears complicated as PPIs have contradictory effects: Inhibition of calcium absorption by gastric acid suppression versus inhibition of osteoclasts. The association between PPI-associated hypochlorhydria and the decrease in calcium absorption remains controversial.

In the present study, the TG rat model was used to exclude the PPI-induced effect on gastric acid secretion and examine the PPI-induced inhibition of bone resorption mediated by osteoclasts in skeletal metabolism. The results demonstrated that rabeprazole ameliorated the reduction in bone density induced by TG at the distal end of the femur, indicating that rabeprazole may control osteoclastic bone resorption, similar to bisphosphonate.

However, rabeprazole did not ameliorate the reduction in bone strength at the femoral diaphysis. This may be due to the improvement in bone density by rabeprazole, which was milder than that of the bisphosphonate. The difference between cancellous bone and cortical bone may also be one of the causes. Iwamoto et al (36) examined the influence of TG on cortical and cancellous bones in rats. The study measured the bone mineral content and density and the mechanical strength of the femoral distal metaphysis and diaphysis. The TG-induced osteopenia and deterioration in bone strength were more severe at skeletal sites rich in cancellous bone (distal metaphysis) compared with those rich in cortical bone (diaphysis). The present study measured bone density at the femoral distal metaphysis, rich in cancellous bone. However, bone strength was measured at the femoral diaphysis, rich in cortical bone and thus less affected by gastrectomy or medication than cancellous bone. This may be one reason that administration of rabeprazole did not appear to affect bone strength in the present study.

The serum BAP level in the TG groups was significantly lower than that in the sham group, whereas the serum TRACP-5b levels in all groups were similar. This result indicates that the bone metabolic disorder induced by TG is more dependent on suppressing the bone formation compared to on increasing bone resorption. Although in the group with TG plus the bisphosphonate the serum TRACP-5b level appeared lower than those in the other TG groups, the individual variability was large and this difference was not statistically significant. It is plausible that feedback occurred and altered the level of this marker, however, this change could not be captured as the present study examined only chronic associations. TRACP-5b is not considered a reflection of bone metabolism in this particular model.

As aforementioned, osteoporosis following gastrectomy has become a clinical issue. In patients receiving proximal gastrectomy or pylorus preserving gastrectomy, which preserves gastric acid secretion, reflux of gastric acid could be another cause of esophagitis. PPIs are typically effective in these patients (37,38). Certain epidemiological studies suggested that PPIs induce skeletal metabolism disorders. However, due to the effect of PPIs on osteoclasts, the administration of PPI may improve TG-induced bone metabolic disorders. The present study used the TG model to reproduce the poor calcium absorption that occurs during gastric anacidity. As the effect of calcium malabsorption due to rabeprazole-induced gastric acid suppression could be excluded, the specific effects of rabeprazole on osteoclasts could be examined. Rabeprazole inhibited the TG-induced bone density decrease, suggesting that the administration of a PPI is at least not an exacerbating factor in bone metabolic disorders.

Acknowledgements

The authors thank Dr Seiji Naganuma, Department of Pathology, Kochi University, for the expert advice on the pathological bone density measurement method.

References