Probiotics and the immunological response to infant vaccinations; a double-blind randomized controlled trial.

OBJECTIVES: To examine the effect of a combination of probiotics on the antibody response to pneumococcal and pertussis vaccination in healthy Danish children, aged 8-14 months, at the time of starting day care. Moreover, the cytokine response to lipopolysaccharide of whole blood was assessed. METHODS: A total of 290 children were randomly allocated to receive a combination of Bifidobacterium animalis ssp. lactis and Lactobacillus rhamnosus GG daily for a 6-month intervention period, and blood samples were drawn at the start and end of the study. Specific antibody response towards Streptococcus pneumoniae serotypes and Bordetella pertussis toxin, as well as endotoxin-induced interleukin-6 (IL-6) and interferon-γ (IFN-γ) production in blood were analysed by Luminex and ELISA. RESULTS: There was no significant difference between the average individual changes from baseline to end of study in antibody concentrations for S. pneumoniae for both the probiotics (340.4% ± 11.2%) and the placebo group (382.9% ± 10.4%) (p 0.525), nor for B. pertussis toxin in the two groups (probiotics 190.1% ± 12.6% versus placebo 238.8% ± 1.1%, p 0.340). The average individual change in IL-6 concentration was significantly lower in the probiotics versus the placebo group (2.9% ± 10.3% versus 33.7% ± 9.0%, p 0.024), whereas there was no difference in IFN-γ concentration (0.0% ± 0.2% versus -0.2% ± 0.1%, p 0.279). CONCLUSIONS: The probiotic intervention did not affect the antibody response against S. pneumoniae and B. pertussis toxin in healthy Danish children.

Associations between adiposity, hormones, and gains in height, whole-body height-adjusted bone size, and size-adjusted bone mineral content in 8- to 11-year-old children.

UNLABELLED: We examined fat-independent associations of hormones with height and whole-body bone size and mineral content in 633 school children. IGF-1 and osteocalcin predict growth in height, while fat, osteocalcin, and in girls also, IGF-1 predict growth in bone size. Leptin and ghrelin are inversely associated with bone size in girls. INTRODUCTION: Obesity causes larger bone size and bone mass, but the role of hormones in this up-regulation of bone in obesity is not well elucidated. We examined longitudinal associations between baseline body fat mass (FM), and fat-independent fasting levels of ghrelin, adiponectin, leptin, insulin, insulin-like growth factor-I (IGF-1), osteocalcin, and intact parathyroid hormone, and subsequent changes in height and in whole-body height-adjusted bone area "BAheight" and size-adjusted bone mineral content "BMCsize" in 8- to 11-year-olds. METHODS: Analyses were carried out separately for boys (n = 325) and girls (n = 308) including data from baseline, 3 and 6 months from OPUS School Meal Study. RESULTS: In both sexes: gain in BAheight was positively associated with baseline FM (≥2.05 cm(2)/kg, both p ≤ 0.003). Furthermore, gain in height was positively associated with baseline IGF-1 (≥0.02 cm/ng/ml, p = 0.001) and osteocalcin (≥0.13 cm/ng/ml, p ≤ 0.009); and gain in BAheight was positively associated with baseline osteocalcin (≥0.35 cm(2)/ng/ml, p ≤ 0.019). In girls only, gain in BAheight was also positively associated with baseline IGF-1 (0.06 cm(2)/ng/ml, p = 0.017) and inversely associated with both baseline ghrelin (-0.01 cm(2)/pg/ml, p = 0.001) and leptin (-1.21 cm(2)/µg/ml, p = 0.005). In boys, gain in BMCsize was positively associated with osteocalcin (0.18 g/ng/ml, p = 0.030). CONCLUSIONS: This large longitudinal study suggests that in 8- to 11-year-old children, IGF-1 and osteocalcin predict growth in height, while FM, osteocalcin, and in girls also, IGF-1 predict growth in BAheight. Fat-independent inverse associations of leptin and ghrelin with BAheight in girls' are contrary to proposed growth-stimulating effects of leptin. Osteocalcin in boys predicts gain in BMCsize.

Effect of increased intake of skimmed milk, casein, whey or water on body composition and leptin in overweight adolescents: a randomized trial.

BACKGROUNDS: Dairy proteins may support muscle protein synthesis and improve satiety in adults. However, there are limited studies using exact measures of body composition, especially in adolescents. OBJECTIVES: This study investigates the effect of milk proteins and water on body composition and leptin in overweight adolescents. METHODS: Subjects (n = 193) aged 12-15 years were randomized to drink 1 L d(-1) of skimmed milk, whey, casein (all milk-based drinks 35 g protein L(-1) ) or water for 12 weeks. Twenty participants dropped out. A pre-test control group of 32 adolescents was examined 12 weeks before start of intervention. Outcomes included leptin and dual-energy X-ray absorptiometry scanning. The effects of the milk-based drinks on body composition and leptin were compared with baseline, pre-test control and water. RESULTS: Lean mass index (LMI) increased compared to baseline (all 95% confidence intervals 0.05-0.50 kg m(-2) , all P ≤ 0.009) and the pre-test control group (0.044-0.247 kg m(-2) , P ≤ 0.002) for all four test drinks. Fat mass index (FMI) increased only for milk-based drink groups compared with baseline (0.15-0.67 kg m(-2) , P < 0.001) and also compared with water (0.029-0.255 kg m(-2) , P ≤ 0.011). For pre-test control, there was no change in FMI or LMI. Leptin increased in the casein (1.016-3.246 ng mL(-1) , P < 0.001; 0.952-3.294 ng mL(-1) , P < 0.001) and whey groups (0.135-2.273 ng mL(-1) , P = 0.027; 0.069-2.322, P = 0.038) compared with water and pre-test control group, respectively. CONCLUSIONS: Although milk proteins increased LMI in overweight adolescents, there was a concurrent increase in FMI and leptin, whereas water only resulted in increased LMI. Thus, increased water intake may be beneficial for body composition in overweight adolescents.

Maternal obesity and offspring dietary patterns at 9 months of age.

BACKGROUND/OBJECTIVES: Differences in the quality of complementary feeding between infants of obese and nonobese mothers have not been examined sufficiently. The aim of this paper was to compare dietary patterns, foods, nutrients and energy intakes of 9-month-old Danish infants in a cohort comprising obese mothers (SKOT II, n=184; SKOT, Danish abbreviation of small children's diet and well-being) with a cohort consisting mainly of nonobese mothers (SKOT I, n=329). SUBJECTS/METHODS: Dietary intake was assessed by 7-day records, and dietary patterns were identified by principal component analysis. RESULTS: SKOT I was characterized by a lower maternal body mass index (BMI) and a higher social class than SKOT II in relation to parental education and occupation. Infants in SKOT II had lower scores on a Health-Conscious Food pattern reflected at the food group level, for example, with lower intake of the food groups Fruit and Vegetable but higher intake of WheatBreadNoWholegrain in SKOT II compared with SKOT I. Moreover, SKOT II had shorter durations of breastfeeding, earlier introductions of complementary feeding, higher energy intake from protein but lower energy intakes from monounsaturated fatty acids and polyunsaturated fatty acids at 9 months. SKOT II had higher weight-for-age and length-for-age z-scores, but no differences in BMI z-scores, as compared with SKOT I at 9 months. CONCLUSIONS: Infants of obese mothers from a lower social class seem to have a less healthy diet and higher weight and length z-scores at 9 months. Therefore, the promotion of healthy complementary feeding might be beneficial for the prevention of health implications, such as obesity, later in life for these infants.

IGF-I at 9 and 36 months of age ­ relations with body composition and diet at 3 years ­ the SKOT cohort.

OBJECTIVE: High infancy levels of insulin-like growth factor-I (IGF-I) have been associated with increased linear growth and fat-free mass (FFM) but also with risk of obesity. This paper examines how IGF-I at 9 and 36 months relates to diet and body composition. DESIGN: Healthy term infants from the prospective cohort study, SKOT, were examined at 9 and 36 months with anthropometry, bioelectrical impedance (36 months), 7-day food records and blood analysis of IGF-I and IGFBP-3 by chemiluminescent immunometric assay. RESULTS: IGF-I at 36 months (n = 229) was positively correlated with 9 months values and values were considerably higher in girls (43%). Children breastfed at 9 months had lower IGF-I concentrations at 9 months but reached the same IGF-I concentrations at 36 months as infants not breastfed at 9 months. IGF-I at 36 months was positively associated with height, weight, BMI, predicted FFM and FFM index (FFM/height (kg/m2)). Although there also was a positive association with predicted fat mass (FM) there was no association with FM index (FM/height (kg/m2)). Further, a negative association with skin fold thickness was observed. A change in IGF-I from 9­36 months was positively related to FFM and FFM index but not BMI, FM and FM index. No associations were seen between IGF-I and current intake of milk, meat or protein energy percentage, but both fat and saturated fat energy percentage were negatively associated with IGF-I. CONCLUSION: IGF-I concentrations were positively associated with growth but not with adiposity at this age. However, the higher tempo of growth may influence age at adiposity rebound and thereby later risk of obesity. Milk and protein intake at 36 months did not influence IGF-I but there was a negative association with intake of fat and saturated fat. The implications of this finding for development of obesity need further exploration.

Amount and quality of dietary proteins during the first two years of life in relation to NCD risk in adulthood.

During late infancy many infants have a protein intake, which is more than three times as high as the physiological need. Several observational studies have shown an association between a high-protein intake (>15 energy %) early in life and an increased risk of developing obesity and thereby non-communicable diseases (NCDs) later in life. This effect was supported by a recent intervention study with infant formulas with two levels of protein, showing that a higher protein intake during the first year of life resulted in a higher body mass index (BMI) at age 2 years. It is also plausible that an important reason for the slower growth in breast-fed infants is the lower content of protein in breastmilk, but other qualities of breastmilk could also play a role. A high intake of protein, especially dairy protein, stimulates the growth factors insulin-like growth factor (IGF-I) and insulin, and it has been suggested that the lower risk of NCDs in breast-fed infants is mediated through a regulation of IGF-I. A low quality of protein, as in cereal-based diets with no animal foods as often seen in low-income countries, may contribute to undernutrition, which can also result in an increased risk of NCDs later in life. In conclusion, there is some evidence that a high protein intake during the complementary feeding period is associated with increased risk of NCDs and that avoidance of a high protein intake could reduce the risk of obesity. In low-income countries, emphasis should be on providing sufficient amounts of high-quality protein to improve survival, growth and development.

Thin newborns are more insulin resistant at 10 years of age.

AIMS: To investigate the relation between ponderal index or birth weight and insulin resistance in late childhood. METHODS: An observational study of 92-term appropriate-for-gestational age infants was carried out. Weight and length were measured at birth and at 9 months and duration of breast feeding was noted at 9 months. Follow-up examinations at 10 years of age included measurement of weight, height, pubertal status, fasting insulin and glucose concentrations. RESULTS: Ponderal index at birth was negatively (B±SE=-0.05±0.02; p=0.025) and current BMI was positively (B±SE=0.14±0.02; p<0.001) associated with insulin resistance measured as homeostasis model assessment (HOMA) at 10 years of age adjusted for gender and pubertal stage. Current BMI and ponderal index at birth were still significant after adjusting for weight at 9 months. Birth weight and weight at 9 months was not correlated with HOMA (p=0.58) adjusted for current BMI, gender and pubertal stage. HOMA was higher in the tertile with the lowest ponderal index than in the two remaining tertiles (p=0.024). CONCLUSION: Relative thinness at birth, but neither birth weight nor weight gain from 0-9 months, was associated with higher insulin resistance at 10 years of age in this cohort with a low prevalence of overweight at 10 years of age and normal birth weight.

Determinants of blood glucose and insulin in healthy 9-month-old term Danish infants; the SKOT cohort.

AIMS: Insulin secretion is important for early regulation of growth, but high insulin concentration is also a risk factor for insulin resistance later in life. It is therefore important to better understand how insulin and glucose are associated with early diet and growth. The aim of this study was to examine blood glucose and insulin concentration in relation to anthropometric measurements, growth, breastfeeding practice and complementary feeding in 9-month-old infants. METHODS: This was a cross-sectional study (SKOT cohort), examining 312 healthy term infants from the age of 9 months. Of these, 265 infants had data on insulin and glucose and were included in this study. Measurements include weight, length, skinfold thickness, waist circumference, 7-day food records, 2-h fasting venous glucose and insulin analysis, and questionnaire. RESULTS: At 9 months of age there was a strong negative association between number of breastfeedings per day and insulin concentration (P=0.0015). Insulin concentration was positively associated to waist circumference (P=0.042) and change in Z-score for weight-for-age between 5 and 9 months (P=0.004). Glucose concentration was positively associated to subscapular skinfold (P=0.002) and sum of skinfolds thicknesses (P=0.006). CONCLUSION: At 9 months, breastfeeding still had a strong negative effect on insulin concentrations, which were positively associated with weight gain and current waist circumference, while glucose concentrations were associated with subcutaneous fat. These results are of interest in disentangling the association between early growth and later risk of disease.

Does vitamin D supplementation of healthy Danish Caucasian girls affect bone turnover and bone mineralization?

INTRODUCTION: A high peak bone mass may be essential for reducing the risk of osteoporosis later in life and a sufficient vitamin D level during puberty may be necessary for optimal bone accretion and obtaining a high peak bone mass. Dietary intake and synthesis during winter of vitamin D might be limited but the effect of vitamin D supplementation in adolescence on bone mass is not well established. OBJECTIVE: To investigate the effect of supplementation with 5 and 10 microg/day vitamin D(3) for 12 months in 11- to 12-year-old girls on bone mass and bone turnover as well as the possible influence of VDR and ER genotype on the effect of the supplementation. METHODS: The girls (n=221) were randomized to receive either 5 microg or 10 microg vitamin D(3) supplementation per day or placebo for 12 months. Whole body and lumbar spine bone mass measured by DXA and pubertal status were determined at baseline and after 12 months whereas physical activity and dietary intake of calcium and vitamin D were assessed at baseline. Serum (S) 25-hydroxyvitamin D (25OHD), S-osteocalcin, S-parathyroid hormone, S-calcium, S-inorganic phosphate, urinary (U) pyridinoline (Pyr) and deoxpyridinoline (Dpyr) were measured at baseline and after 6 and 12 months. RESULTS: The S-25OHD concentration increased (p<0.001) relative to the baseline values in the groups receiving either 5 microg/day (mean+/-SD; 11.0+/-10.3 nmol/l, baseline 41.9+/-17.6 nmol/l) or 10 microg/day (13.3+/-11.8 nmol/l, baseline 44.4+/-16.6 nmol/l) vitamin D(3) for 12 months compared to placebo (-3.1+/-9.8 nmol/l, baseline 43.4+/-17.1 nmol/l). There was no effect of vitamin D-supplementation on biomarkers for bone turnover or on whole body or spine bone mineral augmentation. However, vitamin D supplementation increased whole body bone mineral density (BMD) (p=0.007) and bone mineral content (BMC) (p=0.048) in the FF VDR genotype but not in the Ff or ff VDR genotypes. CONCLUSION: Supplementation with vitamin D (5 or 10 microg/day) over 12 months increased the S-25OHD concentration but there was no effect on indices of bone health in the entire group of girls. However, there was an effect on BMD for a subgroup with the FF VDR genotype indicating an influence of genotype.

The effects of whole milk and infant formula on growth and IGF-I in late infancy.

BACKGROUND/OBJECTIVES: High protein intake has been associated with increased growth. This may be linked to increased concentrations of insulin-like growth factor I (IGF-I), which seems to be influenced by the diet, especially its protein component. The short-term effects of high protein intake in late infancy are not known. The objective was to investigate the effects of high protein intake in the form of whole milk (WM) on growth and IGF-I from 9 to 12 months of age. SUBJECTS/METHODS: Healthy infants (n=83) were randomized to receive either WM or infant formula and fish oil or no fish oil (2 x 2 design). Anthropometric variables, IGF-I concentrations, serum urea nitrogen (SUN) and diet were recorded before and after the intervention. RESULTS: Intake of WM significantly increased the protein energy percentage (PE%; P< or =0.001) and SUN (P=0.01), whereas there was no effect on size. The milk intervention increased IGF-I in boys (P=0.034) but not in girls. Intake of fish oil had no effect on the outcomes. Including all infants in the analysis there was a significant correlation between weight and IGF-I at 12 months (r=0.316, P=0.017), and PE% was positively associated with IGF-I after adjusting for sex and breastfeeding at both 9 (r=0.329, P=0.015) and 12 months (r=0.272, P=0.044). CONCLUSIONS: Randomization to WM had no overall effect on growth. However, the positive effect of WM on IGF-I in boys and the positive association between PE% intake and IGF-I at 9 and 12 months is consistent with the hypothesis that a high milk intake stimulates growth.

Structure of heparin fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to alpha 2-macroglobulin-receptor/low-density-lipoprotein-receptor-related protein by heparin fragments.

Heparin-derived deca- and octa-saccharides were subjected to affinity chromatography on lipoprotein lipase-Sepharose and the fractions eluted at high salt concentration were analysed by strong-anion-exchange chromatography. Two high-affinity decasaccharides were isolated and the structure determined by one- and two-dimensional 1H-n.m.r. spectroscopy. The affinities of 3H-labelled low-molecular-mass heparin and size-fractionated deca-, octa-, and hexa-saccharides for lipoprotein lipase immobilized on microtitre plates were determined from saturation curves. From competition experiments the affinities of unlabelled heparins and pure deca- and hexa-saccharide fragments were determined. The binding was size- and charge-dependent, but structural dependency was also indicated. Thus substitution of a 2-O-sulphated L-iduronic acid with D-glucuronic acid was less important than the sulphation pattern of the D-glucosamine residue for affinity for lipoprotein lipase. Heparin inhibits binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein. The effects of size, charge and structure for this inhibition were studied. The ability of the heparin fragments to inhibit binding correlated with their affinity for lipoprotein lipase. This indicates that the inhibition of the binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein by heparin is exclusively mediated by binding of heparin to lipoprotein lipase.

Isolation and characterization of hexasaccharides derived from heparin. Analysis by HPLC and elucidation of structure by 1H NMR.

Four hexasaccharides representing major structural sequences of heparin were isolated and characterized after degradation of heparin by heparinase. The structures were determined from two-dimensional 1H NMR spectroscopy including TOCSY (total correlated spectroscopy), COSY (correlated spectroscopy), and ROESY (rotating frame nuclear Overhauser enhancement spectroscopy) methods, providing new data on hexasaccharides. One of the hexasaccharides, the last eluting component from anion exchange chromatography, was derived from the tri-sulfated repeating disaccharide, alpha-L-idopyranosyluronic acid 2-sulfate-(1-->4)-2-amino-2-deoxy-D-glucopyranose 6,N-disulfate, and having the structure delta UAp2S-(1)-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S. The second hexasaccharide contained a nonsulfated D-glucuronic acid unit instead of the L-iduronic acid adjacent to the reducing end, and having the structure delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-beta-D- GlcAp-(1-->4)-alpha-D-GlcNp2S6S. The last two hexasaccharides were obtained in lower yield and they have not been isolated and characterized before. The structure of the third saccharide corresponded to a trimer of the repeating disaccharide except for the lack of a 6-O-sulfate group at the reducing end glucosamine residue; deltaUAp2S-(1-->4)-alpha-D-Glcnp2S6S-(1-->4)-alpha-L- IdoAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L-IdoAp2S -(1-->4)-alpha- D-GlcNp2S. The fourth and last hexasaccharide were less sulfated and the following structure was established delta UAp2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- Idop2S-(1-->4)-alpha-D-GlcNp2S6S-(1-->4)-alpha-L- IdoAp-(1-->4)-alpha-D-GlcNpAc6S. Analysis of the ROESY spectra revealed conformational difference of the glucosidic linkage alpha-L-IdoAp-(1-->4)-alpha-D-GlcNp between the hexasaccharides and longer heparin chains.

Binding of low molecular weight heparin (Tinzaparin sodium) to bovine endothelial cells in vitro.

Heparinase depolymerized low molecular weight (LMW) heparin (Tinzaparin sodium, Logiparin) was radiolabelled by catalytic tritiation to high specific radioactivity and the binding to fetal bovine heart endothelial (FBHE) cells was studied at 4 degrees C and 37 degrees C. The binding was found to be time dependent and saturable. Two classes of binding sites could be distinguished from Scatchard analysis at both temperatures: One with high affinity (KD = 0.027 microM at 4 degrees C, KD = 0.012 microM at 37 degrees C) and another with very low affinity (KD = 69 microM at 4 degrees C and 37 microM at 37 degrees C). The binding reversibility was affected by the temperature indicating internalization of a fraction of the bound LMW heparin. At 4 degrees C only 11% of the specifically bound heparin was bound irreversibly. At 37 degrees C the non displaceable fraction accounted for 28% of the specifically bound LMW heparin. This work demonstrates that tinzaparin sodium binds specifically to endothelial cells. This binding may be useful in interpreting pharmacokinetic properties of this low molecular weight heparin.