Effect of 2 dosages of prepartum cholecalciferol injection on blood minerals, vitamin D metabolites, and milk production in multiparous dairy cows: A randomized clinical trial.

The objective of the present study was to evaluate the effect of 2 dosages of prepartum cholecalciferol injection on blood minerals, vitamin D metabolites, and milk production. Cows entering their second or greater lactation (n = 158) were randomly assigned to a control group (CON) or one of 2 treatment groups receiving either 6 × 106 IU (6VitD) or 12 × 106 IU (12VitD) cholecalciferol intramuscularly on d 275 ± 1.2 (SD) of gestation. Concentrations of serum total Ca (tCa), phosphate, and Mg were determined on 1, 2, 3, 5, 7, and 10 d in milk (DIM). For a subsample of 30 cows entering the third lactation (n = 10/group), these samples were analyzed for cholecalciferol, 25-hydroxycholecalciferol (25-OHD3), and 24,25-dihydroxycholecalciferol (24,25-[OH]2D3). In these cows, we also determined 1,25-dihydroxycholecalciferol (1,25-[OH]2D3), the biologically most active metabolite, on 1, 2, 3, and 5 DIM. Repeated measures ANOVA was performed to evaluate the effect of different dosages of cholecalciferol on blood minerals, vitamin D metabolites, and milk yield over the first 5 test days after calving. Binary outcomes such as retained placenta and metritis were analyzed using a chi-squared test. Although the 12VitD treatment increased tCa concentrations on 1, 2, and 3 DIM compared with CON, administration of 6VitD increased tCa concentrations only on 1 DIM. Compared with CON cows and 6VitD cows, 12VitD cows had greater serum phosphate concentration during the first 10 DIM. Furthermore, 6VitD cows had greater serum phosphate concentrations compared with CON cows. On the contrary, 12VitD cows had lower serum Mg concentrations during the first 10 DIM compared with CON and 6VitD cows. Cholecalciferol was increased by the treatment and decreased quickly until 10 DIM. In respect to 25-OHD3, the 6VitD treatment resulted in a 4.1-fold increase in comparison to the CON group, while a 6.5-fold increase was observed in 12VitD animals. The vitamin D metabolite 24,25-(OH)2D3 increased linearly with 25-OHD3 serum levels, resulting in the highest concentrations in the 12VitD group. An increase of 1,25-(OH)2D3 until 3 DIM was observed in all cows. However, this rise was most pronounced in the CON group. The incidence of retained placenta was 1.9%, 11.5%, and 29.6%, and that of metritis was 11.5%, 15.4%, and 31.5% for CON, 6VitD, and 12VitD cows, respectively. Although none of the treated cows exerted clinical signs of hypocalcemia, one cow in CON incurred clinical hypocalcemia. Cows of the 12VitD group had a lower milk yield over the first 5 monthly test days compared with the control and 6VitD group (42.2 ± 0.5, 42.0, ± 0.5 and 40.7 ± 0.5 kg for control cows, 6VitD cows and 12VitD cows, respectively). Although no negative side effects were observed in 6VitD cows, we do not recommend the general application of 6 × 106 IU cholecalciferol before calving as positive effects on calcium homeostasis were marginal and restricted to the first DIM. The present findings confirm that the application of 12 × 106 IU cholecalciferol negatively affected milk production on this farm.

Vitamin D is not linked to folate status and mRNA expression of intestinal proton-coupled folate transporter.

PURPOSE: In vitro studies discovered intestinal proton-coupled folate transporter (PCFT) as a vitamin D hormone-responsive gene. In vivo effects of vitamin D on PCFT and folate status are currently not available. METHODS: Three experiments were conducted. At first, vitamin D receptor knockout (VDR(-/-)) mice and corresponding wild-type (WT) mice were compared for their plasma and hepatic folate concentration and PCFT mRNA expression in intestinal mucosa. In a second experiment with rats, we analyzed the folate status of offspring in response to a maternal vitamin D-adequate (1,000 IU/kg) or vitamin D-deficient (0 IU/kg) diet that was fed for 11 weeks. Finally, the plasma folate concentration of healthy individuals was studied at baseline (in winter) and in response to an oral treatment for 8 weeks with 2,000 IU vitamin D3 per day or a placebo, respectively. RESULTS: Here, we show that folate status and intestinal PCFT mRNA abundance did not differ between the VDR(-/-) and the WT mice. No effect of vitamin D on folate status was also found in rat dams and their offspring, and plasma folate levels of individuals did not change in response to vitamin D. CONCLUSIONS: Current data from studies with model animals and humans provide no indication for a vitamin D effect on intestinal uptake and status of folate.

Maternal and newborn vitamin D status and its impact on food allergy development in the German LINA cohort study.

BACKGROUND: Vitamin D levels are known to be associated with atopic disease development; however, existing data are controversial. The aim of this study was to investigate whether corresponding maternal and cord blood vitamin D levels are associated with atopic outcomes in early infancy. METHODS: Within the LINA cohort study (Lifestyle and environmental factors and their Influence on Newborns Allergy risk), 25(OH)D was measured in blood samples of 378 mother-child pairs during pregnancy and at birth. Information about children's atopic manifestations during the first 2 years of life was obtained from questionnaires filled out by the parents during pregnancy and annually thereafter. Cord blood regulatory T cells (Treg) were detected by methylation-specific PCR using a Treg-specific demethylated region in the FOXP3 gene. RESULTS: The median maternal 25(OH)D(3) level was 22.19 ng/ml (IQR 14.40-31.19 ng/ml); the median cord blood 25(OH)D(3) 10.95 ng/ml (6.99-17.39 ng/ml). A high correlation was seen between maternal and cord blood 25(OH)D(3) levels, both showing a seasonal distribution. Maternal and cord blood 25(OH)D(3) was positively associated with children's risk for food allergy within the first 2 years. Further, higher maternal 25(OH)D(3) resulted in a higher risk for sensitization against food allergens at the age of two. Cord blood 25(OH)D(3) levels were negatively correlated with regulatory T cell numbers. CONCLUSION: Our study demonstrates that high vitamin D levels in pregnancy and at birth may contribute to a higher risk for food allergy and therefore argues against vitamin D supplement to protect against allergy.

Effects of fish oil and conjugated linoleic acids on carnitine homeostasis in laying hens.

1. The effects of n-3 polyunsaturated fatty acids (PUFA) and conjugated linoleic acids (CLA) on genes involved in carnitine homeostasis were compared in laying hens. Three groups of laying hens were fed on a control diet or a diet with either 3% of fish oil or CLA for 4 weeks. 2. Feed intake and egg production rate did not differ between the three groups. Diets with fish oil or CLA had only a weak effect on mRNA levels of PPARα target genes (ACO, CPT-I) in the liver and did not influence mRNA concentrations of the most important carnitine transporter OCTN2, enzymes of involved in carnitine synthesis (TMLD, TMABA-DH, BBD) or concentrations of carnitine in plasma, liver and total egg contents. 3. Hens fed the CLA diet had lower concentrations of free and total carnitine in egg yolk but higher concentrations of carnitine in albumen than control hens (P < 0·05), whereas the amount of free and total carnitine in whole egg did not differ. 4. In conclusion, the study showed that feeding fish oil or CLA causes only a weak activation of PPARα in tissues of laying hens that probably explained the lack of effect on carnitine homeostasis. The results contrast with those in humans and mice that show a significant effect of synthetic PPARα agonists on carnitine homeostasis in humans and mice.

Effects of tryptophan supplementation on plasma tryptophan and related hormone levels in heifers and dairy cows.

This study was conducted to investigate the effects of rumen-protected tryptophan (125 g tryptophan per day) in heifers and dairy cows. Blood samples from dairy cows and heifers were collected for 24h in 3-h intervals on the day before tryptophan supplementation, on day 2, 5 and 7 of tryptophan supplementation, and in heifers additionally on d 14 after tryptophan supplementation was ceased. Plasma tryptophan, melatonin, serotonin, and prolactin concentrations were determined. Tryptophan plasma concentrations on d 5 were augmented at day (11:00 h) and nighttime (02:00 h), (P<0.05) in response to tryptophan supplementation in heifers by 119% and in dairy cows by 47%, respectively, as compared with d 0. Melatonin increased (P<0.05) in response to tryptophan supplementation in heifers, but not in cows. The effect of tryptophan supplementation on plasma tryptophan and melatonin was reversible as demonstrated in heifers on d 14 after cessation of tryptophan supplementation. Serotonin and prolactin in plasma did not respond to tryptophan supplementation. However, milk yield during morning milking increased significantly in tryptophan supplemented cows on d 1, 3 and 4 as compared to the day before tryptophan supplementation. Additional blood samples were taken during afternoon milking in cows at 1-min intervals for the analyses of oxytocin and prolactin on the day before the start and on d 7 of tryptophan supplementation. Milk flow curves were recorded during milking. No effect of tryptophan supplementation on the milking related release of oxytocin and prolactin and on any characteristic of milk flow was observed. In conclusion, tryptophan supplementation caused increased plasma tryptophan in cows and heifers and plasma melatonin in heifers. However, plasma serotonin, prolactin and oxytocin release in cows remained unchanged by tryptophan supplementation. Milk yield at morning milking increased slightly and transiently in response to tryptophan supplementation.

Effect of dietary methionine on plasma and liver cholesterol concentrations in rats and expression of hepatic genes involved in cholesterol metabolism.

Methionine has been shown to increase plasma cholesterol in animals. In the present study, mechanisms were investigated by which methionine could alter cholesterol metabolism. In the first experiment, forty growing rats were fed four casein-based diets differing in methionine content (2.6, 3.5, 4.5 or 6.0 g/kg) for 14 d. In the second experiment, isolated rat hepatocytes were incubated in media supplemented with 50, 100 or 200 micromol/l methionine. Dietary methionine tended to increase plasma homocysteine concentrations in the rats (P=0.058). A weak positive correlation between circulating homocysteine and plasma cholesterol was observed (R2 0.27, P<0.01). Rats fed 3.5 g/kg or more of methionine had higher concentrations of cholesterol in their plasma, in lipoprotein fractions of density (rho; kg/l) 1.0061.063, and in liver than rats fed 2.6 g/kg methionine. Rats fed 6 g/kg methionine had a higher hepatic expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol-7alpha-hydroxylase than rats fed less methionine. The phosphatidylcholine:phosphatidylethanolamine ratio in rat liver increased with rising dietary methionine concentration; the relative mRNA concentrations of phosphatidylethanolamine N-methyltransferase and cystathionine beta-synthase remained unaffected. Hepatocytes incubated in media supplemented with 100 or 200 micromol/l methionine had a higher cholesterol synthesis than hepatocytes incubated in a medium supplemented with 50 micromol/l methionine; the LDL uptake in hepatocytes was independent of the methionine concentration of the medium. In conclusion, the present study suggests that dietary methionine induces hypercholesterolaemia at least in part via an enhanced hepatic cholesterol synthesis.

Concentrations of oxysterols in meat and meat products from pigs fed diets differing in the type of fat (palm oil or soybean oil) and vitamin E concentrations.

The aim of this study was to find out whether concentrations of oxysterols in pig meat are affected by dietary polyunsaturated fatty acids and vitamin E. 48 growth-finishing pigs were fed diets with either palm oil or soybean oil and vitamin E concentrations of 15, 40 or 200 mg/kg. Concentrations of oxysterols were analyzed in fresh and heat-processed (180 °C, 20 min) meat (M. longissimus dorsi) and in boiled sausage prepared from meat and back fat of the animals. Concentrations of oxysterols in fresh muscle were below 5 nmol/g dry matter; they were independent of the dietary fat type and vitamin E concentration. Heating caused a large increase of oxysterol concentration (up to 55 nmol/g dry matter). This effect was reduced by increasing dietary vitamin E concentration but was independent of the dietary fat. Sausage from pigs fed soybean oil had higher concentrations of oxysterols than sausage from pigs fed palm oil; vitamin E reduced concentrations of oxysterols in sausage from pigs fed soybean oil, but not in sausage from pigs fed palm oil.

Effects of a dietary thermally oxidized fat on thyroid morphology and mRNA concentrations of thyroidal iodide transporter and thyroid peroxidase in rats.

BACKGROUND/AIM: Recent studies demonstrated that feeding oxidized fats increases the concentrations of total and free thyroxine in blood of rats and pigs. This finding suggested that oxidized fats affect the function of the thyroid gland. This study investigates the effects of a thermally oxidized dietary fat on the morphology of the thyroid gland and on the expression of proteins (Na(+)/I(-) symporter, thyroid peroxidase) involved in the synthesis of thyroid hormones in rats at different dietary iodine concentrations. METHODS: An experiment was conducted with 48 growing male Sprague-Dawley rats which were allotted to four groups of 12 animals each. According to a bifactorial experimental design, the rats received semisynthetic diets with 10% of either a fresh or an oxidized fat, with low (50 microg/kg) or adequate (400 microg/kg) iodine concentrations, over a period of 38 days. The oxidized fat was prepared by heating sunflower oil at a temperature of 55 degrees C for a period of 5 weeks. The oxidized fat had much higher concentrations of lipid peroxidation products than the fresh fat as assessed by determining the peroxide concentrations (877 vs. 33 mEq O(2)/kg) and those of thiobarbituric acid reactive substances (25 vs. 0.7 micromol/kg). RESULTS: Rats fed the diets containing oxidized fat had higher concentrations of total and free thyroxine in plasma, a greater height of thyroid epithelial cells, a smaller diameter of thyroid follicle lumen, a lower Na(+)/I(-) symporter mRNA concentration, and a higher thyroid peroxidase mRNA concentration than rats fed the fresh fat (p < 0.05 for all effects). The concentrations of triiodothyronine and thyrotropin were not different between rats fed the fresh fat and those fed the oxidized fat. The dietary iodine supply also had significant effects on some of the parameters analyzed. There were no interactions between type of fat and dietary iodine concentrations. CONCLUSION: The rat model used here shows that dietary oxidized fats affect the morphology and the function of the thyroid gland, irrespective of the dietary iodine supply.

Requirement of tryptophan in relation to the supply of large neutral amino acids in laying hens.

The present study was undertaken to find out whether the tryptophan requirement of laying hens is influenced by the supply of large neutral amino acids (LNAA). A factorial experiment was performed in which the dietary tryptophan concentration was varied at six different levels (1.0, 1.25, 1.5, 1.75, 2.0, and 2.5 g tryptophan/kg diet). As the second factor, the dietary concentrations of LNAA (isoleucine, valine, leucine, phenylalanine, and tyrosine) were varied at two levels. The first level provided an adequate supply of these amino acids; at the second level the concentrations of these amino acids were 40% higher than at the first level. The tryptophan requirement was estimated by a broken-line model and an exponential model of regression analysis. The tryptophan intake required for optimum (100% of maximum in the broken-line model, 95% of the maximum in the exponential model) egg production and daily egg mass was lower in hens fed the diets with high LNAA concentrations (145 and 155 mg/hen per day, respectively, in average of both models) than in hens fed the diets with adequate concentrations of LNAA (184 and 198 mg/hen per day, respectively, in average of both models). In contrast, the tryptophan requirement for optimum BW gain was lower in hens fed the diets with adequate LNAA concentrations (178 mg tryptophan per day) than in hens fed the diets with a high concentration of LNAA (212 mg tryptophan per day). In conclusion, the study suggests that an interaction between dietary LNAA and tryptophan exists in laying hens.

Fibroblasts in mechanically stressed collagen lattices assume a "synthetic" phenotype.

Fibroblasts are subjected to changes of the mechanical force balance during physiological as well as pathological situations, such as wound healing, development of hypertrophic scars, and fibrogenesis. However, the molecular response and the changes in fibroblast gene expression upon mechanical stimulation remain poorly understood. As an in vitro model, human dermal fibroblasts were cultured within a three-dimensional network of fibrillar collagen either under high (stressed) or low tension (relaxed). cDNA microarray technology in combination with Northern blot analysis led to identification of mechano-responsive genes coding for extracellular matrix proteins, fibrogenic growth factors, protease inhibitors, components of focal adhesions, and the cytoskeleton. Application of biaxial strain to fibroblasts cultured on flexible silicone membranes revealed that the type of strain as well as the properties of the substrate induced different patterns of gene regulation. The transcriptional profile of mechanically induced genes in collagen lattices suggests that mechanical stimuli lead to a "synthetic" fibroblast phenotype characterized by induction of connective tissue synthesis while simultaneously inhibiting matrix degradation.

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.

The determination of phospholipase D activity in emulsion systems.

Although phospholipase D (PLD) is often used in emulsion systems consisting of buffer and a nonpolar organic solvent, most activity assays have been designed to work in purely aqueous milieu. Here a method is described for the determination of PLD activity in emulsion systems. The assay is based on the transphosphatidylation of phosphatidylcholine with 1-butanol in dichloromethane/buffer with the subsequent densitometric quantification of the products after their separation by HPTLC and staining with a CuSO4/H3PO4 reagent. The method is particularly appropriate for the determination of enzymes such as PLD from Streptomyces sp. that prefer the exchange of the head group in glycerophospholipids to their hydrolysis. Since the application of an organic solvent in the PLD assay allows the determination of the enzyme in analytes insoluble in aqueous media, the method can also be used to determine PLD activity in the presence of high concentrations of phospholipids.

The interfacial pressure is an important parameter for the rate of phospholipase D catalyzed reactions in emulsion systems.

Phospholipase D (PLD) is widely used for the transformation of phospholipids, which is preferably performed in aqueous-organic emulsion systems. The influence of the organic solvent on the reaction rates has been studied on the hydrolysis of phosphatidylcholine (PC) and its transesterification with glycerol by two types of PLD (cabbage and Streptomyces sp.). The initial rates determined by quantitative HPTLC show great differences in dependence on the solvent used with a similar tendency for both reactions and both PLDs. Since the polymorphism of the PC aggregates was assumed to be responsible for these effects, the critical concentration of micelle formation, the size of the aggregates, the water content of the organic phase, and the interfacial tension were determined in the different reaction systems. As result the interfacial pressure in the reaction systems influencing the package density of the PC aggregates is suggested to regulate the enzymatic activity.