Priscilla G. Masse

Chemical and biomechanical characterization of hyperhomocysteinemic bone disease in an animal model

BackgroundClassical homocystinuria is an autosomal recessive disorder caused by cystathionine β-synthase (CBS) deficiency and characterized by distinctive alterations of bone growth and skeletal development. Skeletal changes include a reduction in bone density, making it a potentially attractive model for the study of idiopathic osteoporosis.MethodsTo investigate this aspect of hyperhomocysteinemia, we supplemented developing chicks (n = 8) with 0.6% dl-homocysteine (hCySH) for the first 8 weeks of life in comparison to controls (n = 10), and studied biochemical, biomechanical and morphologic effects of this nutritional intervention.ResultshCySH-fed animals grew faster and had longer tibiae at the end of the study. Plasma levels of hCySH, methionine, cystathionine, and inorganic sulfate were higher, but calcium, phosphate, and other indices of osteoblast metabolism were not different. Radiographs of the lower limbs showed generalized osteopenia and accelerated epiphyseal ossification with distinct metaphyseal and suprametaphyseal lucencies similar to those found in human homocystinurics. Although biomechanical testing of the tibiae, including maximal load to failure and bone stiffness, indicated stronger bone, strength was proportional to the increased length and cortical thickness in the hCySH-supplemented group. Bone ash weights and IR-spectroscopy of cortical bone showed no difference in mineral content, but there were higher Ca2+/PO43- and lower Ca2+/CO32- molar ratios than in controls. Mineral crystallization was unchanged.ConclusionIn this chick model, hyperhomocysteinemia causes greater radial and longitudinal bone growth, despite normal indices of bone formation. Although there is also evidence for an abnormal matrix and altered bone composition, our finding of normal biomechanical bone strength, once corrected for altered morphometry, suggests that any increase in the risk of long bone fracture in human hyperhomocysteinemic disease is small. We also conclude that the hCySH-supplemented chick is a promising model for study of the connective tissue abnormalities associated with homocystinuria and an important alternative model to the CBS knock-out mouse.

Bone Metabolic Abnormalities Associated with Well-Controlled Type 1 Diabetes (IDDM) in Young Adult Women: A Disease Complication Often Ignored or Neglected

Objectives: This investigation on a homogenous cohort of young adult Caucasian type 1 diabetic (IDDM) patients (1) aimed at studying the occurrence of low bone mineral density (BMD) at an early stage prior to menopause (i.e., during the first decade after peak bone mass) and (2) elucidating the possible mechanisms underlying IDDM-induced bone complication. Methods: Twenty-seven female patients with insulin-treated and well-controlled diabetes, without renal complications, and 32 well-matched healthy controls, aged between 30 and 40 years and fulfilling rigorous inclusion criteria to minimize bone-confounding factors, were enrolled. Areal BMD was evaluated by dual energy X-ray absorptiometry at axial (lumbar spine) and appendicular (femur) sites, using diagnostic WHO reference (T-scores). Osteoblast functions, bone metabolism, related key minerals, and 2 osteoclast-stimulating calciotropic hormones regulating their serum levels were assessed biochemically. Results: The number of cases with low BMD (T-score below −1.1 SD) was almost 2-fold greater (p < 0.01) in the IDDM group. BMD was significantly lower in this group for 3 lumbar sites (p < 0.01) and femur Wards triangle (p < 0.05). Bone formation was reduced, as evidenced by the suppressions of osteocalcin (OC; p < 0.01) and IGF-I (p < 0.001). However, bone alkaline phosphatase (bALP) was induced (p < 0.01), in contrast to what is usually observed in cases of reduced bone formation. Correlated total ALP activity was also significantly increased. There was no change in the specific marker of bone resorption (urinary deoxypyridinoline). Serum calcium was significantly elevated, particularly after adjustment for albumin (p < 0.001), despite lower 1,25(OH)2D3 (p < 0.001) and no elevation of PTH. All significant bone-related biochemical changes were significantly correlated with glycosylated hemoglobin, a clinical indicator of long-term glycemic control, indicating a direct effect of the disease. Conclusions: Bone loss in the IDDM group results from a decrease in bone formation rather than an increase of bone resorption. The induction of bALP is indicative of impaired osteoblast differentiation and maturation, which delayed (down-regulated) later stages of matrix mineralization, as evidenced by lower OC and BMD.

Bone Mineral Density and Metabolism at an Early Stage of Menopause When Estrogen and Calcium Supplement Are Not Used and without the Interference of Major Confounding Variables

Objectives: To measure bone mineral density (BMD) and to screen for early biochemical abnormalities in bone mineral metabolism in the first five years of natural menopause when estrogen and calcium supplement are not used and in the absence of major confounding variables. Setting: Two homogeneous and comparable groups (n = 30) of healthy pre- and postmenopausal Caucasian women living in a northern region (latitude 46° N) were recruited during the mid-Spring/Summer season in a cross-sectional design. Methods: Volumetric apparent BMAD (g/cm3) was calculated from areal BMD (g/cm2) which was evaluated by dual energy X-ray absorptiometry (Lunar®) at both axial and peripheric (femur) sites using two sets of reference values (WHO criterion expressed as T-score and absolute values of areal density) in combination to bone specific biochemical measurements. Results: BMD and BM(A)D were significantly lower in postmenopausal women for all lumbar sites, but not for Ward’s triangle and any other femoral sites whereas free deoxypyridinoline (Dpd), urinary biochemical marker of bone resorption, was markedly (p < 0.0001) greater. Their serum calcium and phosphate were significantly higher without a difference in 1,25(OH)2D3 and PTH. The prevalence of osteopenia in pre- and postmenopausal women was about 2-fold lower in both groups (26.6 and 46.9%, respectively) when lumbar (L) spine and femur neck were combined and using the criteria based on reference values of areal density instead of T-scores. Conclusions: The present study showed that the negative effects of estrogen deficiency on BMD and bone metabolism in early menopause occurred independently of the effect of major calcitropic hormones. Bone loss affects a non negligible proportion of premenopausal women. The prevalence of osteopenia in pre- and postmenopausal women varied according to the criterion used and anatomic site.

Is Serum Ferritin an Additional Cardiovascular Risk Factor for All Postmenopausal Women

Background: Most of the studies on cardiovascular disease (CVD) risk factors in menopause have focused on serum lipid(lipoprotein) abnormalities and were conducted in populations which were not well controlled for several important influential factors. Methods: Two homogenous groups of 30 apparently healthy Caucasian premenopausal women and 3–5 years postmenopausal women who were nonobese, nonsmoking and not using estrogen were compared in a well-controlled cross-sectional design. Fasting serum ferritin and plasma total homocysteine (tHcy) were evaluated concomitantly to classical serum lipid(lipoprotein) risk factors. Relationships between risk factors and the influence of other contributing variables such as diet and body weight were also examined. Results: Serum total cholesterol (p < 0.01), low-density lipoproteins (LDL; p < 0.05) and triglycerides (p < 0.05) of postmenopausal women were greater than that of their menstruating counterparts, even though they ate a CVD-preventive diet, had similar body weight and body fat distribution. Their serum ferritin was almost 3-fold greater (p < 0.0001) but was still within normal limits, except for the 38.5% of postmenopausal women who exhibited values above the 80 µg/l limit that has been associated with sharp increases in the rate of heart disease in either gender. Serum ferritin was low in one third of the postmenopausal group (as low as in the premenopausal control group, whose dietary iron intake was slightly below the nutritional recommendation). The mean plasma tHcy of the postmenopausal group was almost twice as elevated (p < 0.0001). Both ferritin and tHcy were found to be linked to serum cholesterol. The correlation between tHcy and triglycerides was also significant. Conclusion: Early menopause is not associated with blood iron overload and CVD risk factor in an important proportion of women.

Coexistence of osteoporosis and cardiovascular disease risk factors in apparently healthy, untreated postmenopausal women

This study aimed to determine whether apparently healthy, untreated postmenopausal women at risk of osteoporosis relative to nonmenopausal women are concomitantly at risk of cardiovascular disease (CVD) in terms of various aspects of lifestyle, personality, body shape and composition, and blood chemistry. Two homogeneous groups of 30 women having reached menopause for 3-5 years and 30 nonmenopausal controls, all non-estrogen users without apparent CVD risk factors, were compared in a cross-sectional design. Data related to physical activity, dietary intakes, personality type, anthropometry, and skinfold-thickness were collected. Plasma insulin-like growth factor (IGF-1) and serum lipids were measured and used as biochemical predictors of osteoporosis and CVD, respectively. Compared to nonmenopausal controls, postmenopausal women were at greater risk of bone loss given their lower plasma IGF-1, lower physical activity level, and even given their higher serum lipids, as recent literature suggests. Moreover, their dietary calcium intake fulfilled only 70% of the current recommendation, which may reduce protection against osteoporosis and CVD (particularly hypertension) as well. The two groups did not differ regarding energy intake, body weight and frame size, body mass index (BMI), waist circumference, and waist-hip ratio (WHR). However, postmenopausal subjects had more adipose tissue and differed in terms of lifestyle factors (lower dietary lipids and greater alcohol consumption). While neither group was at particular risk of CVD according to waist circumference, WHR, and serum triglycerides, postmenopausal women were at risk according to percent body adiposity and serum cholesterol. This study shows that several risk factors for osteoporosis and CVD can coexist in apparently healthy postmenopausal women after a few years of natural menopause. It emphasizes the need for a timely screening that would stress both heart and bone risk factors.

Enhancement of calcium/vitamin d supplement efficacy by administering concomitantly three key nutrients essential to bone collagen matrix for the treatment of osteopenia in middle-aged women: a one-year follow-up.

Two vitamins and proline (CB6Pro), three nutrients essential for bone collagen, were used in combination to a 1000 mg calcium/250 IU vitamin D (Ca/D) daily supplement to treat osteopenia as a preventive measure against osteoporosis later in life. Middle-aged women not using estrogen were screened for osteopenia using the WHO criteria and divided into three groups (n = 20 each): 1) placebo healthy controls with normal bone mineral density (BMD); 2) control Ca/D-treated osteopenic patients; and 3) Ca/D + CB6Pro-treated osteopenic patients. The three groups were comparable at baseline except for BMD. After one-year treatment, cortical diaphyseal BMD remained constant in each group, but trabecular bone loss persisted (at 5 lumbar sites) in osteopenic group 2. No further bone loss was detected in osteopenic group 3. A loss of 2% was evidenced in the placebo group at one lumbar site. Markers of bone formation (which increase in coupling to resorption) decreased significantly in both osteopenic groups. Although biomarkers of resorption did not change, hormone (PTH and 1,25(OH)2D3)-induced osteoclastic activity was significantly reduced. No decline in BMD occurred at any bone site in osteopenic group 3, highlighting the importance of improving the quality of bone matrix concomitantly to mineral replacement.

Loss of decorin from the surface zone of articular cartilage in a chick model of osteoarthritis

The objective of this study was to immunolocalize decorin and to assess changes as a result of pyridoxine (PN) deficiency in chick articular cartilage from femoral condyles. After maintenance on a normal diet for the first two weeks after hatching, 15 broiler chickens were deprived of this vitamin for 6 weeks. It was previously shown that the ankle joints of PN-deficient animals are swollen with effusions. They also present an abnormal gait, enlarged bony margins, and fissuring of the articular cartilages. Milder changes (no fissures) were also shown in the knee joints. Data from a previous study were suggestive that sulfated glycosaminoglycans are lost from the knee cartilage surface into synovial fluid. The current study was focused on the small proteoglycan, decorin, which coats the surface of collagen fibrils and may regulate their morphology. To examine decorin in normal and PN-deficient articular cartilage, a monoclonal antibody to an epitope on the protein core of decorin was used for immunohistochemical staining of tissue sections and for Western Blot analysis of cartilage extracts. Reduction of staining with the antibody was demonstrated in the tangential surface zone of PN-deficient cartilage, and Western Blot analysis showed reduced intensity of decorin bands compared to normal controls. These data suggest that a lack of decorin may play a role in the enlargement of collagen bundles in the tangential zone of PN-deficient articular cartilage as observed in a previous electron microscopic study.

Cardiovascular disease-risk factors in middle-aged osteopaenic women treated with calcium alone or combined to three nutrients essential to artery and bone collagen.

BACKGROUND Recent research suggests that cardiovascular disease (CVD) and bone loss are functionally interwoven. This study examined the concomitant effects of a nutritional treatment of osteopaenia on CVD-risk factors. METHODS A 1-year placebo-controlled trial was conducted on middle-aged women with normal (group A) or low (groups B and C) bone mineral density. Subjects (n = 20 per group) took daily either a placebo, calcium carbonate alone or combined to a vitamin (C and B(6))-proline capsule, respectively. Urinary pyridoxic acid (used to assess treatment compliance), plasma homocysteine, serum lipids and lipoproteins were measured before and after nutritional intervention. RESULTS Groups were comparable at baseline in most parameters of interest. No changes occurred in groups A and B. The 4%, 7% and 25% reductions of total cholesterol, LDL and triglycerides, and 14% elevation of HDL were all significant in group C. A trend toward reduction was observed for homocysteine in this group. CONCLUSIONS Vitamins C (500 mg) and B(6) (75 mg) combined with proline had consistent beneficial effects on CVD-risk factors, whereas calcium alone did not. This study also underlined the importance of considering vitamin B(6) status as a potential CVD risk factor.

Acknowledgement to the 2004 Reviewer

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