Phyto-oestrogen excretion and rate of bone loss in postmenopausal women.

OBJECTIVE: The hypothesis was tested that the rate of postmenopausal bone loss is inversely associated with long-term urinary excretion of phyto-oestrogens, as a marker of habitual dietary intake. DESIGN: Secondary analysis of a 10-year follow-up study (1979 1989) among postmenopausal women in the Netherlands. SUBJECTS: From the original population of 154 women, 32 women were selected with an annual rate of radial bone loss of < or = 0.5% over the first 5 years of the study and 35 women with a rate of > or = 2.5% per year. METHODS: The isoflavonoids genistein, daidzein and equol, and the lignan enterolactone were determined by gas chromatography mass spectrometry in aggregate samples from annually collected urine samples. Cortical bone density of the radius had previously been measured annually by single-photon absorptiometry. RESULTS: Excretion of isoflavonoids did not differ between both groups, although in multivariate analysis equol excretion was weakly positively associated with rate of bone loss in the 5 years after the menopause. Enterolactone excretion was significantly higher in the group with high rate of bone loss. This positive association remained in multivariate linear regression analysis after adjustment for age, years since menopause, body mass index and intake of calcium, vegetable protein and dietary fibre. CONCLUSIONS: Enterolactone excretion is likely to be an indicator of consumption of grains and legumes; it is not clear whether the observed positive association with rate of bone loss is a causal one. Our results do not support a preventive effect of low, unsupplemented dietary intake of phyto-oestrogens on postmenopausal cortical bone loss. However, no conclusions can be drawn about effects of higher doses of phyto-oestrogens.

Selective hydrolysis of milk proteins to facilitate the elimination of the ABBOS epitope of bovine serum albumin and other immunoreactive epitopes.

Milk proteins are hydrolyzed to prevent immunological reactions, but immunoreactive epitopes, including the ABBOS epitope of bovine serum albumin (BSA), can still be detected in commercially available milk protein hydrolysates. We used lactococcal cell-envelope proteinase (CEP) for the hydrolysis of the individual milk proteins and of mixtures thereof, or for the hydrolysis of sodium caseinate (contaminated with whey proteins). CEP exclusively degraded casein, leaving the four major whey proteins intact. This property facilitated the removal of the intact whey proteins from the casein fragments by ultrafiltration. Depending on the molecular mass of the whey protein to be removed, membranes with cutoff values between 3 and 30 kDa were used, resulting in casein hydrolysates free of protein fragments with cross-reactive whey-protein-specific IgE (immunoglobulin E) or ABBOS antibody-binding sites. Even the casein itself was degraded in such a way by CEP that cross-reactive casein-specific IgE antibody-binding sites could be eliminated. The product could find application in infant formulas for therapeutic and preventive treatment of children with cow's milk allergy; in addition, the preventive use of such formulas in children genetically susceptible to the development of insulin-dependent diabetes mellitus (IDDM) should be considered if a relationship between the consumption of BSA and IDDM were to become more apparent. The method is also applicable for preparing casein-free whey protein preparations.

Incomplete elimination of the ABBOS epitope of bovine serum albumin under simulated gastrointestinal conditions of infants.

OBJECTIVE: Because the bovine serum albumin residues 126-144 (ABBOS) have been reported to be responsible for the autoimmune reaction directed against pancreatic islet cells, it was our aim to study the potential survival of the ABBOS epitope during digestion in the gastrointestinal tract. RESEARCH DESIGN AND METHODS: Either nontreated (commercially available) or heat-treated bovine serum albumin (BSA) was hydrolyzed in vitro with pepsin at a pH of 2.0, 3.0, and 4.0 and subsequently with pancreatic enzymes at a pH of 7.5. Cross-reactivity between the ABBOS peptide and the BSA hydrolysates was determined by competitive enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antibody raised against the ABBOS peptide in BSA. RESULTS: Biochemical findings clearly showed that the degradation of BSA during simulated physiological digestion depended on its conformation and on the pH of its pepsin-catalyzed hydrolysis. Raising the pH of the pepsin-catalyzed hydrolysis from 2.0 to 4.0 decreased the efficiency of the process, especially when BSA had first been denatured by heat treatment. As a consequence, a large proportion of the cross-reactive anti-ABBOS antibody-binding sites was still intact in the final hydrolysates. CONCLUSIONS: The present results suggest that the ABBOS epitope of BSA will not be completely eliminated during digestion under conditions that prevail in the stomach of infants (pH 3-4).

Detection by competitive enzyme-linked immunosorbent assay of a bovine serum albumin peptide (ABBOS) in infant formulas based on hydrolyzed cow's milk protein.

OBJECTIVE: Our aim was to determine whether currently available products based on hydrolyzed milk protein and a small peptide (ABBOS) in bovine serum albumin (BSA) (positions 126-144) share common antigenic sites. The commercial products are primarily developed to reduce cow's milk protein-associated allergenicity, but whether they also could be used in intervention trials to elucidate the role of BSA in the etiology of IDDM is not known. RESEARCH DESIGN AND METHODS: A sensitive competitive enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antibody raised against the ABBOS peptide in BSA was developed. With this method, we determined cross-reactivity between the ABBOS peptide and commercially available products: four milk protein hydrolysates and eight infant formulas based on hydrolyzed milk protein. The products were further characterized by physicochemical techniques. RESULTS: Hydrolyzed milk protein products are found to inhibit competitively the binding of ABBOS peptide to antibody. The number of residual reactive sites varied considerably among products and was not strongly related to the degree of hydrolysis (DH) or the molecular mass distribution of the hydrolysates. CONCLUSIONS: The presence of possible immunoreactive peptides in infant formulas based on hydrolyzed cow's milk protein cannot be adequately predicted by the DH or molecular mass distribution of the hydrolysates. Its specific determination is needed to ensure infant formulas free of cross-reactive ABBOS antibody binding sites for use in ongoing and forthcoming intervention trials to elucidate the role of BSA as a possible environmental triggering agent of IDDM.

Raising the pH of the pepsin-catalysed hydrolysis of bovine whey proteins increases the antigenicity of the hydrolysates.

BACKGROUND: Hypersensitivity to cow milk protein is frequently observed in infancy. Since the pH in the infant's stomach is relatively high (pH 3-4) compared with adults (pH 2), an incomplete digestion of the milk proteins is expected to occur. OBJECTIVE: The determination of the degree of hydrolysis by pepsin of the four main proteins of bovine whey, i.e. alpha-lactalbumin (alpha La), beta-lactoglobulin (beta Lg), bovine serum albumin (BSA) and bovine immunoglobulin G (B-IgG), in the pH range 2.0-4.0 and of the antigenic properties of the resulting hydrolysates. METHODS: Whey proteins were successively hydrolysed with pepsin at pH values ranging from 2.0 to 4.0 and with pancreatic enzymes at pH 7.5 using a pH-stat. The resulting hydrolysates were characterized by their degree of hydrolysis, and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, gel permeation chromatography and immunologically by competitive enzyme-linked immunosorbent assay. RESULTS: In general, the degree of hydrolysis, the gel electrophoretic patterns, the contents of peptides of molecular mass > 5 kDa and the residual human-immunoglobulin E and G antigenicities of the hydrolysates did not differ much whether the pepsin incubation was done at pH 2.0 or 3.0. Pepsin incubation at pH 4.0, however, resulted in a decreased hydrolysis and enhanced residual antigenicity of alpha La, BSA and B-IgG, but not of beta Lg. CONCLUSION: The poor and slow degradation of the antigenic epitopes of whey proteins when pepsin digestion occurs under conditions that prevail in the stomach of infants could be of much importance for the development of cow milk hypersensitivity. The immature gastrointestinal mucosal barrier of infants allows large antigenic fragments of these proteins to pass into the systemic circulation.

Residual antigenicity of hypoallergenic infant formulas and the occurrence of milk-specific IgE antibodies in patients with clinical allergy.

BACKGROUND: Milk protein hydrolysates are frequently used in milk substitutes for children with cow's milk allergy. However, cases of hypersensitivity to commercially available hypoallergenic infant formulas based on milk protein hydrolysates have been reported. OBJECTIVE: Our purpose was to determine the immunologic response of milk protein-specific IgE and IgG in the serum of patients allergic to cow's milk against four commercially available hypoallergenic milk protein hydrolysates and eight infant formulas. METHODS: Antibody levels in patients' serum and milk protein-specific residual antigenicity of the hypoallergenic products were determined by indirect and competitive ELISA. RESULTS: Patients allergic to cow's milk had IgE and IgG antibodies to several protein fractions of cow's milk; intraindividual and interindividual variation in the concentrations of these antibodies was considerable. In general, IgE and IgG residual antigenicity of individual milk proteins in the hypoallergenic products was lower compared with that of the intact milk protein, but immunoreactive epitopes could still be detected in all products. Their number varied considerably among the individual milk proteins and also differed among products. CONCLUSIONS: The individual sensitization pattern of the patient allergic to cow's milk and the milk protein-specific residual antigenicity might be considered as possible laboratory predictors of adverse reactions to hypoallergenic products. Their determination could be a useful preclinical screening test for pediatricians to select a formula adapted to the individual patient.

Urinary excretion of magnesium and calcium as an index of absorption is not affected by lactose intake in healthy adults.

The effect of lactose on the urinary excretion of Mg and Ca, as an index of absorption, was studied in a double-blind, crossover study during three 1-week periods. Twenty-four healthy, lactose-tolerant, adult volunteers maintained their habitual diets with the exception that all lactose-containing dairy products in the diet were replaced by 600 g/d of three specially prepared dairy products. These products were based on either lactose-enriched cow's milk or lactose-enriched, lactase (EC 3.2.1.23)-treated cow's milk, with or without added Mg, and were given in turn during 1 week. Lactose intake was increased by 127 mmol/d (46 g/d) while taking the lactose-enriched products. While taking the Mg-enriched products, Mg intake was increased by 2.8 mmol/d (69 mg/d) which was equivalent to 17% of the habitual Mg intake. Apart from the lactose and Mg intake, nutrient intake was comparable during the three dietary periods. Urinary excretions of Mg and Ca were used as indicators for their absorption. Mg supplementation significantly increased urinary Mg excretion by 0.97 mmol/d (equivalent to an increase of 18%, P < 0.001), indicating that urinary Mg excretion is a valid indicator for intestinal Mg absorption. Hydrolysis of lactose did not affect urinary excretion of Mg and Ca, which implies that lactose intake does not affect the absorption of Mg and Ca in healthy adults.

Perimenopausal increase in serum cholesterol: a 10-year longitudinal study.

The relative contribution of menopause to the age-related increase in serum total cholesterol concentration in women is not exactly known. Cross-sectional studies suggest a considerable contribution of menopause, whereas in short-term longitudinal studies, only a small increase was observed around menopause. In a 10-year follow-up study (1979-1989), serum total cholesterol concentration and dietary intake were measured annually in 167 healthy perimenopausal women (initially aged 49-56 years) who lived in the mixed rural/industrial community of Ede, the Netherlands. The longitudinal design enabled us to study the "natural course" of serum total cholesterol concentration and the influence of dietary fat intake during and after cessation of ovulation. For data analysis, three menopausal cohorts were created based on years relative to menopause: 2 years before ("perimenopausal"), 2 years after ("early postmenopausal"), and 6 years after ("late postmenopausal"). In each cohort, the analysis was conducted on data from 4 years of follow-up. The results show that from 2 years before to 6 years after menopause, serum total cholesterol concentration increased on average by at least 1.1 mmol/liter (19%). Thereafter, only a minor increase was observed. The rise was significantly lower in women who increased their intake of polyunsaturated fatty acids as compared with women who reduced their intake during that period. It is concluded that cessation of ovulation appears to be associated with a major increase of 19% in serum total cholesterol concentration during an 8-year period around menopause. This increase may be reduced by increasing the intake of polyunsaturated fatty acids.

Body weight and/or endogenous estradiol as determinants of cortical bone mass and bone loss in healthy early postmenopausal women.

The objective was to study the independent relationships of body mass index and endogenous estradiol to cortical bone mineral density and the rate of cortical bone loss at the radius in healthy early postmenopausal women. Fifty-one healthy early postmenopausal women (aged 58-66 years) participated. The women were a subset of a population participating in a 10-year longitudinal study to elucidate the influence of dietary calcium on the rate of cortical bone loss. Cortical bone mineral density at the radius, body weight and body height were measured annually (1979-89). Concentrations of sex steroids were measured in serum samples collected during the last year of follow-up (1989). Endogenous estradiol levels, although significantly positively correlated with body mass index, were not independently related to bone mass indices of the radius. Body mass index, on the other hand, was found to be positively related to cortical bone mineral density and negatively to the rate of bone loss, even after adjustments had been made for confounding factors. Our results suggest that the level of total estradiol is not an important determinant of cortical bone mass indices in healthy early postmenopausal women. Other factors of overweight such as mechanical loading may be important.

Habitual dietary calcium intake and blood pressure change around the menopause: a longitudinal study.

In a 10-year follow-up study, blood pressure and dietary intake were measured annually in 167 healthy perimenopausal normotensive women. Their initial ages ranged between 49 and 56 years and habitual calcium intake between 560 and 2580 mg/day (mean 1110 mg/day); they lived in the mixed rural/industrial community of Ede, the Netherlands. The longitudinal design provided an opportunity to study the 'natural history' of blood pressure and the effect of dietary calcium during and after the period of ovarian failure. For data analysis, person-time experience was divided into three menopausal periods. Based on years relative to menopause three menopausal cohorts were created starting 2 years before, 2 years after and 6 years after menopause, each was followed for 4 years. Changes in systolic (SBP) and diastolic blood pressure (DBP) during the menopausal periods were adjusted for change in body mass index and other relevant variables in multiple regression analysis. An average decline in SBP of 6 mm Hg was observed in the period of 2 years before menopause to 6 years after menopause, and an increase of almost 5 mm Hg in the period between 6 and 10 years after menopause. A significant change in DBP was not observed. Neither changes in, nor the absolute level of, calcium intake showed any relevant association with blood pressure change. Ovarian failure seems to reverse temporarily the increase in blood pressure due to aging. The results do not suggest that a habitual calcium intake exceeding 800-1000 mg/day (the current Recommended Daily Allowance for adults) is effective in preventing hypertension during the peri- and postmenopausal period.

Bioavailability of magnesium and calcium from cow's milk and soya-bean beverage in rats.

The milk components lactose and casein enhance the apparent absorption of magnesium and possibly also of calcium, whereas phytate, which occurs in soya-bean products, has an inhibitory effect. This implies that soya-bean beverage v. cow's milk could lower bioavailability of Mg and Ca. This hypothesis was tested in two experiments with growing rats. Feeding soya-bean beverage v. cow's milk consistently lowered body-weight gain, enhanced bone turnover, as measured by increased plasma alkaline phosphatase (EC 3.1.3.1) activity and increased urinary hydroxyproline excretion, and decreased Mg and Ca concentrations in the femur. Because the mineral compositions of the soya-bean beverage and the cow's milk were different, the intake of Mg was higher in rats fed on soya-bean beverage, whereas that of Ca was higher in rats fed on cow's milk. Supplementation of the soya-bean beverage either with phosphorus and Ca or with P, Ca and methionine, to concentrations identical to those in milk, restored growth and bone mineralization. When using diets carefully balanced for Mg, Ca, P, sodium, potassium and methionine, soya-bean beverage v. cow's milk in the diets decreased apparent absorption and urinary excretion of Mg and Ca. Hydrolysis of lactose in milk decreased absorption and urinary excretion of Mg; it did not significantly affect Ca absorption but lowered urinary Ca excretion. The present study shows that soya-bean beverage v. milk depresses Mg and Ca bioavailability, as would be predicted on the basis of reported effects of their purified components.

Interaction of calcium and phosphate decreases ileal magnesium solubility and apparent magnesium absorption in rats.

We tested the hypothesis that increased intakes of calcium and phosphate lower magnesium solubility in the intestinal lumen, causing a decreased magnesium absorption. In in vitro experiments at a constant magnesium concentration, increasing calcium concentrations reduced magnesium solubility. This effect did not occur in the absence of phosphate. Increasing phosphate concentrations decreased the solubility of magnesium in the presence, but not in the absence, of calcium. These results suggest that the formation of an insoluble calcium-magnesium-phosphate complex determines magnesium solubility. To extend this concept to in vivo conditions, rats were fed purified diets containing a constant concentration of magnesium (16.4 mumol/g) but different concentrations of calcium (25, 100 or 175 mumol/g) and phosphate (58, 103 or 161 mumol/g). Increased intakes of calcium decreased magnesium solubility in the ileal lumen and lowered magnesium absorption. The latter result occurred only if the dietary phosphate concentration was at least 103 mumol/g. Increasing dietary phosphate concentrations reduced both magnesium solubility in the ileum and magnesium absorption, but only if the dietary calcium concentration was at least 100 mumol/g. These results support those obtained in vitro. We conclude that increased intakes of calcium and phosphate decrease magnesium absorption by the formation of an insoluble calcium-magnesium-phosphate complex in the intestinal lumen.

Development and evaluation of an index to predict early postmenopausal bone loss.

An index to predict individual postmenopausal bone loss is presented. The index is developed by means of data from a 10-year prospective Norwegian study in which bone mass of the distal forearm was measured annually in 73 women. All the women were 47 years old and premenopausal at inclusion. Independent risk factors for postmenopausal bone loss were identified by applying multivariate regression analysis on anthropometric, biochemical, nutritional, and life-style variables measured at menopause. The analysis identified low body weight, reduced renal phosphate reabsorption, and smoking as significant independent risk factors, and by means of these three factors a predictive index for postmenopausal bone loss was developed. This index was validated by using data from a 10-year longitudinal Dutch study, in which bone mass of the proximal radius was measured annually in 86 women, aged between 49 and 57 years and perimenopausal at inclusion. We defined women with the highest index score as "high-risk persons." According to this definition approximately 25% of the perimenopausal women were classified as high-risk persons, and the estimated sensitivity/specificity/positive predictive power were 36%, 89%, and 74%, respectively, when used to select women with a postmenopausal bone loss above average. We conclude that the index may be helpful in identifying healthy perimenopausal women in whom bone mass measurements should be considered.

Inhibitory effect of dietary soybean protein vs. casein on magnesium absorption in rats.

The effects of casein and soybean protein on magnesium absorption and magnesium concentration in the femur were investigated in rats. Purified diets containing either casein or soybean protein and three concentrations of added magnesium (0.82, 1.64 or 2.46 mmol/100 g diet) were used. The isonitrogenous diets were carefully balanced for the different mineral concentrations in the protein preparations. Absolute and percent magnesium absorption and urinary magnesium excretion were significantly decreased in rats fed soybean protein when compared with casein, irrespective of the dietary concentration of added magnesium. The magnesium content of femur was significantly lower in rats fed soybean protein, but this effect was seen only when the diet contained 0.82 mmol magnesium/100 g diet. The addition of sodium phytate to the casein diets, to a concentration identical to that in the diets containing soybean protein as provided by the soybean protein preparation, produced similar effects on magnesium absorption as the diets containing soybean protein. These results indicate that soybean protein, when compared with casein, decreases magnesium absorption through its phytate component.

Habitual dietary calcium intake and cortical bone loss in perimenopausal women: a longitudinal study.

During an 8-year follow-up study, the effect of habitual dietary calcium intake on cortical bone loss in 154 healthy perimenopausal women was examined. Dietary calcium intake, determined by the cross-check dietary history method, and cortical bone mineral content of the radius were measured annually. Habitual dietary calcium intake was calculated as the mean of the estimated daily dietary calcium intake during the follow-up period. The women were classified according to their habitual calcium intake: those with an intake below 800 mg/day (n = 28), between 800 and 1350 mg/day (n = 95), and above 1350 mg/day (n = 31). The results show a continuous significant loss of cortical bone in all groups, amounting yearly to 1.3 +/- 0.25, 1.5 +/- 0.10, and 1.9 +/- 0.23% (mean +/- SE) for the groups with a low, medium, and high habitual calcium intake, respectively (P less than 0.01). The differences among the three groups did not reach statistical significance (P = 0.11). Body mass index was found to be positively correlated with the negative changes in cortical bone mineral density (r = 0.32, P less than 0.01), even after adjustments had been made for confounding factors. It is concluded that a habitual calcium intake exceeding 800 mg/day (the current Recommended Daily Allowance for adults) is ineffective in preventing cortical bone loss during early menopause. Body mass index is of major importance for the perimenopausal bone loss.

Milk: does it affect blood pressure? A controlled intervention study.

In a double-blind trial, the effect on blood pressure of supplementation of normal milk (1180 mg Ca2+, 1650 mg K+ and 110 mg Mg2+ d-1) vs. 'mineral-poor' milk (95 mg Ca2+, 580 mg K+ and 10 mg Mg2+ d-1) was studied. Young healthy normotensive female students consumed one of the two supplements while on a low calcium diet (less than 500 mg Ca2+ d-1) for a period of 6 weeks. In both the normal milk- and 'mineral-poor' milk-supplemented groups systolic blood pressure decreased slightly. However, this decrease was persistently greater in the milk-supplemented group. The individual mean systolic blood pressure change during normal milk treatment (-4.1%) was significantly greater (P = 0.03) than that during 'mineral-poor' milk treatment (-1.3%). An effect of normal milk supplementation on diastolic blood pressure could not be demonstrated. The results of the present study indicate a small hypotensive effect of milk consumption, which is attributable to its content of essential minerals.

Relationship between the calcium-to-protein ratio in milk and the urinary calcium excretion in healthy adults--a controlled crossover study.

Over the years, doubts have arisen concerning the use of milk as a calcium source in the prevention of osteoporosis, particularly because of potential offsetting effects of protein and phosphorus. Thus, a new milk product with a higher calcium content and lower contents of protein, phosphorus, and energy was developed. A controlled crossover study was done to determine the way in which substitution of the new milk product (860 mL) for normal milk (1000 mL) in the diet of healthy adults affected the urinary excretion of calcium and hydroxyproline. Short-term consumption of the product significantly lowered 24-h urinary calcium excretion by approximately 0.65 +/- 0.19 mmol/d (means +/- SEM, p less than 0.001). The ratio of fasting urinary hydroxyproline to creatinine did not decrease, indicating no significant reduction of bone resorption in our subjects. Elderly people in particular might benefit from the new product because it reduces their calcium loss as well as their intake of protein, phosphorus, energy, and liquid volume.

Relation of axial bone mass to habitual calcium intake and to cortical bone loss in healthy early postmenopausal women.

A group of 60 healthy early postmenopausal women participating in an ongoing study on the effect of habitual calcium intake on the rate of cortical bone loss at the radius, were subjected to additional skeletal measurements at the lumbar spine and femoral neck. The women were between 58 and 64 years of age, and 3 to 10 years postmenopausal. No correlations were found between habitual calcium intake (range 560 to 2580 mg/day) and either bone mineral content of the radius, the lumbar spine and the femoral neck, or spine deformity index. Body mass index was found to be positively correlated with bone mass indices of the radius (decrease of BMD and BMD) and femoral neck (BMC), but not with of the lumbar spine (BMC, BMD and SDI), even after adjustments had been made for confounding factors. Although the rate of cortical bone loss at the radius correlated significantly with bone mineral content of lumbar spine and femoral neck, the error in predicting bone mass of the lumbar spine or the femoral neck from longitudinal measurements of cortical bone at the radius was high. The rate of cortical bone loss did not correlate with the spine deformity index. We conclude that in healthy women in early menopause, the bone mineral content of both the appendicular and the axial skeleton are not influenced by habitual calcium intake. A higher body mass index has a protective effect on the appendicular skeleton but appears to be less protective to the axial skeleton. Longitudinal measurements of cortical bone mass are of limited value to predict bone density of the appendicular and axial skeleton.