Biofortification of Chicken Eggs with Vitamin K-Nutritional and Quality Improvements.

National nutrition surveys have shown that over half of all adults in Ireland, the United Kingdom (UK), and the United States of America (USA) have low vitamin K intakes. Thus, dietary strategies to improve vitamin K intakes are needed, and vitamin K biofortification of food may be one food-based approach. The primary aim of our study was to establish whether increasing the vitamin K3 content of hen feed can increase the vitamin K content of eggs, and the secondary aims were to examine the effects on hen performance parameters, as well as egg and eggshell quality parameters. A 12 week hen feeding trial was conducted in which Hyline chickens were randomized into four treatment groups (n = 32/group) and fed diets containing vitamin K3 (as menadione nicotinamide bisulfite) at 3 (control), 12.9, 23.7, and 45.7 mg/kg feed. Vitamin K1, menaquinone (MK)-4, MK-7, and MK-9 were measured in raw whole eggs via a liquid chromatography tandem mass spectrometry method. MK-4 was the most abundant form of vitamin K (91-98%) found in all eggs. Increasing the vitamin K3 content of hen feed over the control level significantly (p < 0.001) enhanced the MK-4 content of eggs (mean range: 46-51 µg/100 g, representing ~42-56% of US Adequate Intake values). Vitamin K biofortification also led to significant (p < 0.05) increases in the yellowness of egg yolk and in eggshell weight and thickness, but no other changes in egg quality or hen performance parameters. In conclusion, high-quality vitamin K-biofortified eggs can be produced with at least double the total vitamin K content compared to that in commercially available eggs.

Poor bioavailability of vitamin D2 from ultraviolet-irradiated D2-rich yeast in rats.

Ultraviolet-irradiated yeast (Saccharomyces cerevisiae) can be used to biofortify bakery products with vitamin D, but in bread, it was not effective in increasing serum 25-hydroxyvitamin D [25(OH)D] in humans, possibly because of the low digestibility of the yeast matrix. We investigated the effects of vitamin D2-rich intact yeast cells and their separated fraction, yeast cell walls, which we hypothesized to provide vitamin D2 in a more bioavailable form, on serum 25(OH)D and its metabolites in growing female Sprague-Dawley rats (n = 54) compared to vitamin D2 and D3 supplements (8 treatment groups: 300 or 600 IU vitamin D/d, and a control group, 8-week intervention). The D3 supplement groups had the highest 25(OH)D concentrations, and the vitamin D2 supplement at the 600-IU dose increased 25(OH)D better than any yeast form (P < .001 for all, analysis of covariance, adjusted for body weight). There were no significant differences between the yeast forms at the same dose (P > .05). Serum 24,25-dihydroxyvitamin D (a vitamin D catabolite) concentrations and the trend in the differences between the groups were in line with 25(OH)D (P < .001 for all). The 24,25-dihydroxyvitamin D to 25(OH)D ratio between the D2 supplement and the yeast groups did not differ (P > .05). These findings do not support the hypothesis: the ability of the different ultraviolet-treated vitamin D2-containing yeast forms to increase 25(OH)D did not differ, and the poor bioavailability of vitamin D2 in the yeasts compared D3 or D2 supplements could not be explained by the increased vitamin D catabolism in the yeast-treated groups.

A Mediterranean-like dietary pattern with vitamin D3 (10 µg/d) supplements reduced the rate of bone loss in older Europeans with osteoporosis at baseline: results of a 1-y randomized controlled trial.

Background: The Mediterranean diet (MD) is widely recommended for the prevention of chronic disease, but evidence for a beneficial effect on bone health is lacking. Objective: The aim of this study was to examine the effect of a Mediterranean-like dietary pattern [NU-AGE (New Dietary Strategies Addressing the Specific Needs of the Elderly Population for Healthy Aging in Europe)] on indexes of inflammation with a number of secondary endpoints, including bone mineral density (BMD) and biomarkers of bone and collagen degradation in a 1-y multicenter randomized controlled trial (RCT; NU-AGE) in elderly Europeans. Design: An RCT was undertaken across 5 European centers. Subjects in the intervention group consumed the NU-AGE diet for 1 y by receiving individually tailored dietary advice, coupled with supplies of foods including whole-grain pasta, olive oil, and a vitamin D3 supplement (10 µg/d). Participants in the control group were provided with leaflets on healthy eating available in their country. Results: A total of 1294 participants (mean ± SD age: 70.9 ±4.0 y; 44% male) were recruited to the study and 1142 completed the 1-y trial. The Mediterranean-like dietary pattern had no effect on BMD (site-specific or whole-body); the inclusion of compliance to the intervention in the statistical model did not change the findings. There was also no effect of the intervention on the urinary biomarkers free pyridinoline or free deoxypyridinoline. Serum 25-hydroxyvitamin D significantly increased and parathyroid hormone decreased (P < 0.001) in the MD compared with the control group. Subgroup analysis of individuals with osteoporosis at baseline (site-specific BMD T-score ≤ -2.5 SDs) showed that the MD attenuated the expected decline in femoral neck BMD (n = 24 and 30 in MD and control groups, respectively; P = 0.04) but had no effect on lumbar spine or whole-body BMD. Conclusions: A 1-y intervention of the Mediterranean-like diet together with vitamin D3 supplements (10 µg/d) had no effect on BMD in the normal age-related range, but it significantly reduced the rate of loss of bone at the femoral neck in individuals with osteoporosis. The NU-AGE trial is registered at clinicaltrials.gov as NCT01754012.

Interactions between Vitamin D Status, Calcium Intake and Parathyroid Hormone Concentrations in Healthy White-Skinned Pregnant Women at Northern Latitude.

Adverse effects of low vitamin D status and calcium intakes in pregnancy may be mediated through functional effects on the calcium metabolic system. Little explored in pregnancy, we aimed to examine the relative importance of serum 25-hydroxyvitamin D (25(OH)D) and calcium intake on parathyroid hormone (PTH) concentrations in healthy white-skinned pregnant women. This cross-sectional analysis included 142 participants (14 ± 2 weeks' gestation) at baseline of a vitamin D intervention trial at 51.9 °N. Serum 25(OH)D, PTH, and albumin-corrected calcium were quantified biochemically. Total vitamin D and calcium intakes (diet and supplements) were estimated using a validated food frequency questionnaire. The mean ± SD vitamin D intake was 10.7 ± 5.2 µg/day. With a mean ± SD serum 25(OH)D of 54.9 ± 22.6 nmol/L, 44% of women were <50 nmol/L and 13% <30 nmol/L. Calcium intakes (mean ± SD) were 1182 ± 488 mg/day and 23% of participants consumed <800 mg/day. The mean ± SD serum albumin-adjusted calcium was 2.2 ± 0.1 mmol/L and geometric mean (95% CI) PTH was 9.2 (8.4, 10.2) pg/mL. PTH was inversely correlated with serum 25(OH)D (r = -0.311, p < 0.001), but not with calcium intake or serum calcium (r = -0.087 and 0.057, respectively, both p > 0.05). Analysis of variance showed that while serum 25(OH)D (dichotomised at 50 nmol/L) had a significant effect on PTH (p = 0.025), calcium intake (<800, 800⁻1000, ≥1000 mg/day) had no effect (p = 0.822). There was no 25(OH)D-calcium intake interaction effect on PTH (p = 0.941). In this group of white-skinned women with largely sufficient calcium intakes, serum 25(OH)D was important for maintaining normal PTH concentration.

Effects of vitamin D supplementation on markers for cardiovascular disease and type 2 diabetes: an individual participant data meta-analysis of randomized controlled trials.

Background: Evidence from randomized controlled trials (RCTs) for the causal role of vitamin D on noncommunicable disease outcomes is inconclusive. Objective: The aim of this study was to investigate whether there are beneficial or harmful effects of cholecalciferol (vitamin D3) supplementation according to subgroups of remeasured serum 25-hydroxyvitamin D [25(OH)D] on cardiovascular and glucometabolic surrogate markers with the use of individual participant data (IPD) meta-analysis of RCTs. Design: Twelve RCTs (16 wk to 1 y of follow-up) were included. For standardization, 25(OH)D concentrations for all participants (n = 2994) at baseline and postintervention were re-measured in bio-banked serum samples with the use of a certified liquid chromatography-tandem mass spectrometry method traceable to a reference measurement procedure. IPD meta-analyses were performed according to subgroups of remeasured 25(OH)D. Main outcomes were blood pressure and glycated hemoglobin (HbA1c). Secondary outcomes were LDL, HDL, and total cholesterol and triglycerides; parathyroid hormone (PTH); fasting glucose, insulin, and C-peptide; and 2-h glucose. In secondary analyses, other potential effect modifiers were studied. Results: Remeasurement of 25(OH)D resulted in a lower mean 25(OH)D concentration in 10 of 12 RCTs. Vitamin D supplementation had no effect on the main outcomes of blood pressure and HbA1c. Supplementation resulted in 10-20% lower PTH concentrations, irrespective of the 25(OH)D subgroups. The subgroup analyses according to achieved 25(OH)D concentrations showed a significant decrease in LDL-cholesterol concentrations after vitamin D supplementation in 25(OH)D subgroups with <75, <100, and <125 nmol of -0.10 mmol/L (95% CI: -0.20, -0.00 mmol/L), -0.10 mmol/L (95% CI: -0.18, -0.02 mmol/L), and -0.07 mmol/L (95% CI: -0.14, -0.00 mmol/L), respectively. Patient features that modified the treatment effect could not be identified. Conclusions: For the main outcomes of blood pressure and HbA1c, the data support no benefit for vitamin D supplementation. For the secondary outcomes, in addition to its effect on PTH, we observed indications for a beneficial effect of vitamin D supplementation only on LDL cholesterol, which warrants further investigation. This trial was registered at www.clinicaltrials.gov as NCT02551835.

Estimation of the maternal vitamin D intake that maintains circulating 25-hydroxyvitamin D in late gestation at a concentration sufficient to keep umbilical cord sera ≥25-30 nmol/L: a dose-response, double-blind, randomized placebo-controlled trial in pregnant women at northern latitude.

Background: In the absence of dose-response data, Dietary Reference Values for vitamin D in nonpregnant adults are extended to pregnancy. Objective: The aim was to estimate vitamin D intake needed to maintain maternal 25-hydroxyvitamin D [25(OH)D] in late gestation at a concentration sufficient to prevent newborn 25(OH)D <25-30 nmol/L, a threshold indicative of increased risk of nutritional rickets. Design: We conducted a 3-arm, dose-response, double-blind, randomized placebo-controlled trial in Cork, Ireland (51.9oN). A total of 144 white-skinned pregnant women were assigned to receive 0, 10 (400 IU), or 20 (800 IU) µg vitamin D3/d from ≤18 wk of gestation. Vitamin D metabolites at 14, 24, and 36 wk of gestation and in cord sera, including 25(OH)D3, 3-epi-25(OH)D3, 24,25(OH)2D3, and 25(OH)D2 were quantified by liquid chromatography-tandem mass spectrometry. A curvilinear regression model predicted the total vitamin D intake (from diet and antenatal supplements plus treatment dose) that maintained maternal 25(OH)D in late gestation at a concentration sufficient to maintain cord 25(OH)D at ≥25-30 nmol/L. Results: Mean ± SD baseline 25(OH)D was 54.9 ± 10.7 nmol/L. Total vitamin D intakes at the study endpoint (36 wk of gestation) were 12.1 ± 8.0, 21.9 ± 5.3, and 33.7 ± 5.1 µg/d in the placebo and 10-µg and 20-µg vitamin D3 groups, respectively; and 25(OH)D was 24.3 ± 5.8 and 29.2 ± 5.6 nmol/L higher in the 10- and 20-µg groups, respectively, compared with placebo (P < 0.001). For maternal 25(OH)D concentrations ≥50 nmol/L, 95% of cord sera were ≥30 nmol/L and 99% were >25 nmol/L. The estimated vitamin D intake required to maintain serum 25(OH)D at ≥50 nmol/L in 97.5% of women was 28.9 µg/d. Conclusions: Thirty micrograms of vitamin D per day safely maintained serum 25(OH)D concentrations at ≥50 nmol/L in almost all white-skinned women during pregnancy at a northern latitude, which kept 25(OH)D at >25 nmol/L in 99% and ≥30 nmol/L in 95% of umbilical cord sera. This trial was registered at www.clinicaltrials.gov as NCT02506439.

The use of synthetic and natural vitamin D sources in pig diets to improve meat quality and vitamin D content.

This study investigated the effects of synthetic and natural sources of vitamin D biofortification in pig diets on pork vitamin D activity and pork quality. One hundred and twenty pigs (60 male, 60 female) were assigned to one of four dietary treatments for a 55 d feeding period. The dietary treatments were (1)50 µg vitamin D3/kg of feed; (2)50 µg of 25-hydroxvitamin D3/kg of feed (25-OH-D3); (3)50 µg vitamin D2/kg of feed; (4)50 µg vitamin D2-enriched mushrooms/kg of feed (Mushroom D2). The pigs offered the 25-OH-D3 diet exhibited the highest (P < 0.001) serum total 25-hydroxyvitamin D concentration and subsequently exhibited the highest (P < 0.05) Longissimus thoracis (LT) total vitamin D activity. Mushroom D2 and 25-OH-D3 supplementation increased pork antioxidant status. The vitamin D2-enriched mushrooms improved (P < 0.05) pig performance, carcass weight and LT colour. In conclusion, 25-OH-D3 is the most successful source for increasing pork vitamin D activity, while Mushroom D2 may be a new avenue to improve animal performance and pork quality.

Prevalence of vitamin D deficiency and insufficiency among schoolchildren in Greece: the role of sex, degree of urbanisation and seasonality.

The current study was aiming to report the prevalence of suboptimal vitamin D status among schoolchildren in Greece and investigate the role of sex, urbanisation and seasonality on vitamin D status. A sample of 2386 schoolchildren (9-13 years old) from four distinct prefectures was examined. The prevalence of 25-hydroxyvitamin D (25(OH)D) concentration <30 and <50 nmol/l (vitamin D deficiency and insufficiency respectively) was 5·2 and 52·5 %, respectively. Girls had a higher prevalence of 25(OH)D<30 (7·2 v. 3·2 %) and 50 nmol/l (57·0 v. 48·0 %) than boys (P<0·001). The highest prevalence rates of 25(OH)D<30 and 50 nmol/l (9·1 and 73·1 %, respectively) were observed during spring (April to June), whereas the lowest (1·5 and 31·9 %, respectively) during autumn (October to December). The prevalence of 25(OH)D<50 nmol/l was higher in urban/semi-urban than rural regions, particularly during spring months (74·6 v. 47·2 %; P<0·001). Female sex, urban/semi-urban region of residence and spring months were found to increase the likelihood of vitamin D deficiency and insufficiency, with the highest OR observed for spring months (7·47; 95 % CI 3·23, 17·3 and 5·14; 95 % CI 3·84, 6·89 for 25(OH)D<30 and 50 nmol/l respectively). In conclusion, despite the southerly latitude, the prevalence of low vitamin D status among primary schoolchildren in Greece is comparable to or exceeds the prevalence reported among children and adolescents on a European level. Sub-populations at highest risk are girls in urban/semi-urban areas during spring months, thus indicating the need for effective initiatives to support adequate vitamin D status in these population groups.

The positive impact of general vitamin D food fortification policy on vitamin D status in a representative adult Finnish population: evidence from an 11-y follow-up based on standardized 25-hydroxyvitamin D data.

Background: A systematic vitamin D fortification of fluid milk products and fat spreads was started in 2003 in Finland to improve vitamin D status. Objective: We investigated the effects of the vitamin D fortification policy on vitamin D status in Finland between 2000 and 2011.Design: Serum 25-hydroxyvitamin D [S-25(OH)D] concentrations of a nationally representative sample comprising 6134 and 4051 adults aged ≥30 y from the Health 2000 and Health 2011 surveys, respectively, were standardized according to the Vitamin D Standardization Program with the use of liquid chromatography-tandem mass spectrometry. Linear and logistic regression models were used to assess the change in S-25(OH)D concentrations.Results: Between 2000 and 2011, the mean S-25(OH)D increased from 48 nmol/L (95% CI: 47, 48 nmol/L) to 65 nmol/L (95% CI: 65, 66 nmol/L) (P < 0.001). The prevalence of vitamin D supplement users increased from 11% to 41% (P < 0.001). When analyzing the effect of fortification of fluid milk products, we focused on supplement nonusers. The mean increase in S-25(OH)D in daily fluid milk consumers (n = 1017) among supplement nonusers was 20 nmol/L (95% CI: 19, 21 nmol/L), which was 6 nmol/L higher than nonconsumers (n = 229) (14 nmol/L; 95% CI: 12, 16 nmol/L) (P < 0.001). In total, 91% of nonusers who consumed fluid milk products, fat spreads, and fish based on Finnish nutrition recommendations reached S-25(OH)D concentrations >50 nmol/L in 2011.Conclusions: The vitamin D status of the Finnish adult population has improved considerably during the time period studied. The increase is mainly explained by food fortification, especially of fluid milk products, and augmented vitamin D supplement use. Other factors, such as the difference in the ultraviolet radiation index between 2000 and 2011, may partly explain the results. When consuming vitamin D sources based on the nutritional recommendations, vitamin D status is sufficient [S-25(OH)D ≥50 nmol/L], and supplementation is generally not needed.

Improved accuracy of an tandem liquid chromatography-mass spectrometry method measuring 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D metabolites in serum using unspiked controls and its application to determining cross-reactivity of a chemiluminescent microparticle immunoassay.

Measurement of serum 25-hydroxyvitamin D [25(OH)D] is considered the best indicator of vitamin D status. Two minor vitamin D metabolites are common interferences encountered in 25(OH)D assays. The first is 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], which if not chromatographically resolved from 25-hydroxyvitamin D3 [25(OH)D3], can overestimate 25(OH)D concentrations. The second is 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], which can cross-react with the antibodies in 25(OH)D immunoassays. Our aim was to develop an LC-MS/MS method capable of detecting both 3-epi-25(OH)D3 and 24R,25(OH)2D3 in serum without the use of a derivatization agent. We report an isotope dilution LC-MS/MS method, with electrospray ionization in the positive mode, that can simultaneously detect 24R,25(OH)2D3, 25(OH)D3, 3-epi-25(OH)D3, and 25-hydroxyvitamin D2. The method employs a cost-effective liquid-liquid extraction using only 150µL of sera and a total run time of 10min. Method performance was assessed by using quality controls made from pooled sera as an alternative to sera spiked with analytes. Biobanked samples, originally analyzed by chemiluminescent microparticle immunoassay (CMIA), were re-analyzed with this method to determine the contribution of 24R,25(OH)2D3 cross-reactivity to 25(OH)D measurement bias. The CMIA over-estimation of 25(OH)D measurements relative to LC-MS/MS was found to depend on both 25(OH)D and 24R,25(OH)2D3 concentrations.

Validation study to compare effects of processing protocols on measured N (ε)-(carboxymethyl)lysine and N (ε)-(carboxyethyl)lysine in blood.

Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured N (ε)-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, N (ε)-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.

Effect of inhibitor compounds on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in model foods.

The possible adverse effects on health of diet-derived advanced glycation endproducts (AGEs) and advanced lipoxidation endproducts (ALEs) is of current interest. This study had the objective of determining the effects of the addition of AGE/ALE inhibitors and different types of sugar and cooking oil on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in model foods (sponge cakes). The cake baked using glucose produced the highest level of CML (2.07±0.24 mmol/mol lysine), whereas the cake baked using fructose produced the highest concentration of CEL (25.1±0.15 mmol/mol lysine). There were no significant differences between CML concentrations formed in the cakes prepared using different types of cooking oil, but significant differences (P<0.001) were observed between the cakes prepared using different proportions of cooking oil. The cakes containing oil generated greater concentrations of CML than sucrose. α-Tocopherol and rutin did not inhibit CML and CEL formation. In contrast, ferulic acid and thiamin, thiamin monophosphate, and thiamin pyrophosphate reduced CML and CEL formation.