Glucocorticosteroid-induced osteoporosis in adult primiparous Göttingen miniature pigs: effects on bone mineral and mineral metabolism.

Information on the pathophysiology of glucocorticoid-induced osteoporosis (GIO) is limited, since its clinical picture often reflects a combined effect of glucocorticoids (GC) and the treated systemic disease (i.e., inflammation and immobility). In 50 healthy adult (30-mo-old) primiparous Göttingen minipigs, we studied the short-term (8 mo, n = 30) and long-term (15 mo, n = 10) effect of GC on bone and mineral metabolism longitudinally and cross-sectionally compared with a control group (n = 10). All animals on GC treatment received prednisolone orally at a dose of 1.0 mg x kg body wt(-1) x day(-1) for 8 wk and thereafter at 0.5 mg/kg body wt(-1) x day(-1). In the short term, GC reduced bone mineral density (BMD) at the lumbar spine by -47.5 +/- 5.1 mg/cm(3) from baseline (P < 0.001), which was greater (P < 0.05) than the loss [not significant (NS)] in the control group of -11.8 +/- 12.6 mg/cm(3). Calcium absorption decreased from baseline by -2,488 +/- 688 mg/7 days (P < 0.001) compared with -1,380 +/- 1,297 mg/7 days (NS) in the control group. Plasma bone alkaline phosphatase (BAP) decreased from baseline by -17.8 +/- 2.2 U/l (P < 0.000), which was significantly different (P < 0.05) from the value of the control group of -1.43 +/- 4.8 U/l. In the long term, the loss of BMD became more pronounced and bone mineral content (BMC), trabecular thickness, mechanical stability, calcium absorption, 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), and parathyroid hormone tended to be lower compared with the control group. There was a negative association between the cumulative dose of GC and BMD, which was associated with impaired osteoblastogenesis. In conclusion, the main outcomes after GC treatment are comparable to symptoms of GC-induced osteoporosis in human subjects. Thus the adult Göttingen miniature pig appears to be a valuable animal model for GC-induced osteoporosis.

Influence of estradiol on vascular endothelial growth factor expression in bone: a study in Göttingen miniature pigs and human osteoblasts.

Ovariectomy (OVX) in animal models is an accepted method to simulate postmenopausal osteoprosis. Vascular endothelial growth factor (VEGF) has been recently shown to play an important role during endochondral bone formation, hypertrophic cartilage remodeling, ossification, and angiogenesis. We hypothesized that reduced VEGF expression in bone contributes to OVX-induced bone loss and tested it in a miniature pig model and in vitro using human osteoblasts. Seventeen primiparous sows (Göttingen miniature pigs) were allocated to two experimental groups when they were 30 months old: a control group (n = 9) and an OVX group (n = 8). After 15 months, VEGF levels in lumbar vertebrae were measured by enzyme-linked immunosorbent assay and verified by Western blot analysis. VEGF and its receptor (VEGFR) were localized by immunohistochemistry. Expression of VEGF mRNA was analyzed by real-time reverse-transcription polymerase chain reaction. Differently sulfated glycosaminoglycans were localized in subchondral bone histochemically. Osteoblasts were immunopositive for VEGF. VEGF concentration in the vertebra was 27% lower in OVX miniature pigs. VEGFR-2 could be immunostained on osteoblasts. VEGF mRNA and protein were detectable in the lumbar vertebrae of all animals. In subchondral trabecular bone of OVX animals, significantly more islands of mineralized cartilage containing chondroitin 4- and 6-sulfate or keratan sulfate occurred compared to the control group. The occurrence of remnants of mineralized cartilage in subchondral bone of the OVX group may be caused by a delayed bone turnover due to low VEGF levels. In vitro experiments revealed an increase of VEGF in the supernatant of osteoblasts after incubation with estradiol. In conclusion, estrogen seems to be a key factor for regulation of VEGF expression in bone. Loss of VEGF due to menopause may be a reason for reduction of bone density.

Ibandronate treatment reverses glucocorticoid-induced loss of bone mineral density and strength in minipigs.

The Göttingen minipig is one of the few large animal models that show glucocorticoid (GC)-induced bone loss. We investigated whether GC-induced loss of bone mineral density (BMD) and bone strength in minipigs can be recovered by treatment with the bisphosphonate ibandronate (IBN). 40 primiparous sows were allocated to 4 groups when they were 30 months old: GC treatment for 8 months (GC8), for 15 months (GC15), GC treatment for 15 months plus IBN treatment for months 8-15 (GC&IBN), and a control group without GC treatment. Prednisolone was given at a daily oral dose of 1 mg/kg body weight for 8 weeks and thereafter 0.5 mg/kg body weight. IBN was administered intramuscularly and intermittently with an integral dose of 2.0 mg/kg body weight. BMD of the lumbar spine (L1-3) was assessed in vivo by Quantitative Computed Tomography (QCT) at months 0, 8, and 15. Blood and urine samples were obtained every 2-3 months. After sacrificing the animals lumbar vertebrae L4 were tested mechanically (Young's modulus and ultimate stress). Histomorphometry was performed on L2 and mineral content determined in ashed specimens of T12 and L4. In the GC&IBN group, the GC associated losses in BMD of -10.5%+/-1.9% (mean+/-standard error of the mean, p<0.001) during the first 8 months were more than recovered during the following 7 months of IBN treatment (+14.8%+/-1.2%, p<0.0001). This increase was significantly larger (p<0.0001) than the insignificant +2.1%+/-1.2% change in group GC15. At month 15, the difference between groups GC&IBN and GC15 was 22% (p<0.01) for BMD, 48% (p<0.05) for Young's modulus, and 31% (p<0.14) for ultimate stress; bone-specific alkaline phosphatase showed trends to lower values (p<0.2) while deoxypyridinoline was comparable. This minipig study demonstrates that GC-induced impairment of bone strength can be effectively and consistently treated by IBN. GC&IBN associated alterations in BMD and bone turnover markers can be monitored in vivo using QCT of the spine and by biochemical analyses, reflecting the changes in bone strength.

Effects of prebiotics on mineral metabolism.

Nondigestible oligosaccharides (NDOs) have been found to stimulate absorption of several minerals and to improve mineralization of bone. Hence, these substances are potential ingredients for "functional foods." In addition to a nutritional effect, functional foods have physiologic and psychological benefits that result in improved health or reduced risk of chronic disease. Most of the scientific evidence for the functional effects of NDOs is based on animal experiments in which NDOs increased the availability of calcium, magnesium, zinc, and iron. This stimulatory effect of some NDOs is assumed to be mainly due to their prebiotic character. A prebiotic is defined as a substrate or food ingredient that is nondigestible for the host but is fermented selectively by some of the intestinal microflora. Thus, it stimulates the growth and activity of bacteria with beneficial consequences for the host's health. Recently, these findings were confirmed in human studies for some NDOs. The effects seem to be specific for the type of carbohydrate and are likely related to the rate of fermentation by the intestinal flora and appear to depend on the ingested dose. Contradictory results of the effect of prebiotics in literature may be due to the experimental design because the effect of NDOs depends on the dose, the time of administration, the content of calcium in the diet, the part of the skeleton investigated, and the age of the subjects studied.

Effects of bioactive substances in milk on mineral and trace element metabolism with special reference to casein phosphopeptides.

Bioactivity of phosphopeptides yielded after tryptic hydrolysis of casein (CPP) was reported more than 50 years ago when CPP were found to improve calcium balance in rachitic newborns. Several investigations have been carried out to study the effects of CPP mainly on calcium metabolism but also on other minerals like iron and zinc. Most of the experiments were in vitro studies or short-term experiments like the effects of CPP after single meals or their effect on mineral disappearance from intestinal everted sac or ligated loop. Investigations on calcium balance were also mainly short term, i.e. 3-4 weeks, and mainly done in rats. A few experiments have been carried out in minipigs, an animal model that is closer to the human than the rat. Studies in human were rare and short term. To date a variety of other peptides have been isolated after enzymatic hydrolysis, and some have been investigated for bioactivity, with equivocal findings. Bioactivity of phosphopeptides seemed to be more obvious when investigations were done in vitro or short term. Results were less clear in metabolic balance studies, especially under physiological conditions. The composition of the basal diet, i.e. content of calcium and phytate, or the protein source had a significant impact on the effect of phosphopeptides. It was concluded that phosphopeptides revealed positive effects on mineral solubility and absorbability, and bone mineralisation under certain experimental conditions. Accordingly they could have a beneficial effect on bone health for some groups of the population.

Effect of ovariectomy on bone histology and plasma parameters of bone metabolism in nulliparous and multiparous sows.

To investigate the suitability of the pig as animal model for postmenopausal osteoporosis, effects of ovariectomy (OVX) on bone metabolism and histology were studied in two groups of sows (9 months, nulliparous or 35 months, multiparous). A standard diet of about 1.5% calcium (Ca) was fed till sacrifice at either 12 or 20 months post OVX when mineral content and histology were studied in representative bone specimens of proximal tibia, iliac crest and lumbar vertebrae. At 4, 8, 12, and 18 months post OVX, total and bone-specific alkaline phosphatase (APt, APb) calcidiol, calcitriol and parathyroid hormone (PTH) were measured in plasma. In young sows OVX did not significantly affect plasma variables except for calcitriol, which was higher at 4 months post OVX. No significant differences between OVX or control animals were observed in the variables of bone chemical and histological analyses, neither 12 nor 20 months post OVX. In multiparous sows OVX significantly increased PTH plasma concentrations at 8 months post OVX and plasma calcitriol, APt and APb at 12 months post OVX. All effects were moderate and transient. OVX did not significantly affect the variables of bone chemical and histological analyses neither 12 nor 20 months post OVX. Although undoubtedly the clinical-chemical changes observed were not accompanied by any histomorphometric signs of osteopenia/osteoporosis, it must be left to future experiments as to whether this resulted from the ample calcium supply provided. This possibility is supported by recent observations showing that porcine osteopenia could be induced by OVX in animals maintained on only 0.75% dietary calcium but not on higher (0.9%) Ca regimens.

Plasma amino acids and cholesterol following consumption of dietary casein or soy protein in minipigs.

Numerous investigators have claimed that protein-induced differences in plasma cholesterol are mediated by differences in amino acid composition. We have explored whether the venous postprandial amino acid profile reflects differences in the amino acid composition of the protein consumed. Six adult Göttingen miniature pigs were fed a semisynthetic diet based on either casein or soy protein isolate. Frequent blood sampling was performed over a whole day after consumption of each diet for 6 wk. Postprandial plasma amino acid concentrations reached their maxima within the first 3 h. A group of eight protein amino acids (Met, Arg, Tyr, Val, Trp, Leu, Lys and Cys) exhibited the most marked and significant protein-dependent differences during this early postprandial phase, whereas Thr and His showed less marked differences. With one exception (Ser) all protein amino acids exhibited venous plasma concentration changes in qualitative accordance with their content in the dietary protein consumed. In quantitative terms, however, venous plasma amino acid changes were less marked than expected from the amino acid composition of the dietary proteins. We conclude that neither the considerable number of amino acids showing differences as reported herein nor the multitude of contradictory reported by others concerning single amino acids affecting serum cholesterol favor the hypothesis that one or several amino acid(s) cause protein-induced hypercholesterolemia.

Response of hormones modulating plasma cholesterol to dietary casein or soy protein in minipigs.

To elucidate the mechanism mediating the effect of dietary casein or soy protein on serum cholesterol concentrations we followed the endocrine response to the intake of these dietary proteins. The hormones analyzed were those known to modulate serum cholesterol concentration. A 7-wk crossover nutrition study was performed with six adult Göttingen minipigs consuming semisynthetic diets based on either 20 wt% casein or soy isolate. At d 42 and 49, concentrations of six hormones were determined in 22 blood samples taken over the whole day. There were no significant differences in insulin, glucagon, the insulin/glucagon ratio, hydrocortisone or triiodothyronine among dietary groups. In the late postprandial phase (5 h after the meal and later) there were significantly higher growth hormone concentrations in soy-fed animals. At all times of the day, total and free thyroxine concentrations were higher after soy feeding than after casein feeding. On average, total and free thyroxine concentrations were 34 and 26% higher with soy protein feeding than with casein feeding. Our data agree with other reports of protein-dependent changes of thyroid hormones and may explain why different dietary proteins have different effects on serum cholesterol levels in sensitive species.

Difference of plasma amino acids following casein or soy protein intake: significance for differences of serum lipid concentrations.

There were significant differences of postprandial plasma concentrations for 8 amino acids (Cys, Val, Met, Leu, Tyr, Lys, Trp, and Arg) depending on whether pigs consumed a meal containing casein or isolated soy protein. The postprandial plasma amino acid pattern conformed with the amino acid composition of the dietary protein (except for Ser). The data, however, do not allow to conclude unambiguously, whether specific amino acids are responsible for the difference of serum cholesterol following casein or soy protein intake. Significant differences between casein- and soy-fed rats were observed regarding total and free plasma thyroxine and triiodothyronine concentrations. This observation can explain the accompanying different serum cholesterol concentrations. The different thyroid hormone concentrations were not paralleled by differences in TSH levels suggesting that dietary proteins affect thyroid function at the thyroid gland.