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Abstract

Background: Vitamin E supplementation might represent an efficient strategy to increase the vitamin E content in milk. The present study aimed to evaluate the impact of supplementation with 800 IU RRR-alpha-tocopherol on the alpha-tocopherol content of milk and the factors associated with the increase in vitamin E. Methods: Randomized clinical trial with 79 lactating women from Brazil, who were assigned to the control group, or to the supplemented group (800 IU of RRR-alpha-tocopherol). Milk and serum were collected between 30 and 90 days after delivery (collection 1), and on the next day (collection 2). Alpha-tocopherol was analyzed using high-performance liquid chromatography. Results: In the supplemented group, the alpha-tocopherol content in serum and milk increased after supplementation (p < 0.001). In the multivariate analysis, only alpha-tocopherol in milk (collection 1) was associated with the level of this vitamin in milk after supplementation (β = 0.927, p < 0.001), and binary logistic regression showed that the dietary intake was the only determinant for the greater effect of supplementation in milk. Conclusion: The pre-existing vitamin level in milk and diet are determinants for the efficacy of supplementation in milk, suggesting that in populations with vitamin E deficiency, high-dose supplementation can be used to restore its level in milk.

Abstract

A novel application of the liquid chromatography method combined with the triple quadrupole tandem mass spectrometry method was developed for the quantification of vitamin K1 and two forms of vitamin K2 (menaquinone-4, menaquinone-7) in human serum. Total chromatography time for each run was 9 min. Time required for the sample pretreatment procedures was approximately 4 h. The coefficients of variation (CVs) of intra-assay were 10.4%, 3.2 % and 2.3% for vitamin K1 in three levels of quality control samples; were 14.3%, 3.2% and 6.7% for menaquinone-4; and were 11.1%, 6.0% and 7.0% for menaquinone-7. The inter-assay CVs were 12.8%, 11.3% and 7.4% for vitamin K1; were 15.2%, 9.2% and 8.7% for menaquinone-4; and were 13.2%,11.1% and 7.2% for menaquinone-7. No interference was found between K1, menaquinone-4 and menaquinone-7, nor any deuterated internal standards. This method was then used to determine reference values for Caucasian populations of central European origin. Samples were measured from 191 healthy volunteers (51.2 ± 16.2 years (mean ± SD)) and the values concerning K1 were 0.044–1.357 ng/mL for women and 0.030–1.214 ng/mL for men. The values for menaquinone-4 and menaquinone-7 did not exhibit any differences between women and men, and were 0.050–1.598 and 0.074–0.759 ng/mL, respectively.

Subject and study design

Volunteers were determined to be healthy according to their medical history, physical examination and standard laboratory procedures. Samples were measured from 191 volunteers (51.2 ± 16.2 years (mean ± SD)), with 149 participants being female and 42 male. Ethical approval for this study was obtained from Ethics committee of University Hospital Motol (Ref.No.: EK-1422/13). Written informed consent was obtained from the participants at recruitment prior to their inclusion in the study.

Results

Our LC–MS/MS method for analysis of K1, MK-4 and MK-7 in human serum has been successfully validated following the criteria of the . Calibration curves were established by plotting the peak area ratios K1, MK-4 and MK-7 against IS at 10-point concentrations. Pooled serum samples exposed to UV light were spiked with 0.03, 0.05, 0.1, 0.3, 0.6, 1.0, 1.5, 2.0, 4.0 and 10.0 ng/mL to obtain calibration curves. The assays were linear across the whole range of concentrations with mean correlation coefficients R2 of 0.980 for K1, 0.994 for MK-4 and 0.978 for MK-7. The first point of calibration curves (0.03 ng/mL) corresponds to the lower limit of quantification (LLOQ). Limits of detection (LoD) were determined by a further dilution of the first calibration point (LLOQ). Concentrations of 0.0019 ng/mL for K1 and MK-7 and 0.00375 ng/mL for MK-4 were obtained.

Results

We measured the vitamin K levels in a healthy Central European population. The group of patients consisted of 191 volunteers, 22% of them male, ages 51 ± 16 years (mean ± SD). Serum levels of K1, MK-4 and MK-7 from the healthy Central European population were: 0.03–2.19 (0.20) ng/mL, 0.05–2.79 (0.19) ng/mL, and 0.05–1.39 (0.09) ng/mL, respectively (min—max (median)).

Abstract

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, β-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic β-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

2.1. MK-4 Enhanced GSIS in Isolated Mouse Pancreatic Islets and Rat INS-1 Cells

To confirm the effect of MK-4 on GSIS, isolated islets and INS-1 cells were used in the present study. To determine the dosage of MK-4 for INS-1 cells, we analyzed the cell survival rate under 0–100 µM MK-4 treatment for 24 h by using the WST-1 assay (), which suggested that the concentration of 3 µM MK-4 was harmless to the cells. Accordingly, MK-4 at 1 and 3 µM was used in INS-1 cells for the subsequent experiments. We then evaluated the effect of MK-4 on GSIS in INS-1 cells, as shown in . MK-4 enhanced insulin levels in both basal (2.8 mM) and high (16.8 mM) glucose-stimulated INS-1 cells in a dose-dependent manner. We used mouse pancreatic islets isolated from genetically diabetic KK-Ay mice subsequently. The isolated pancreatic islets were stimulated with 2.8 mM Glc and MK-4 for 1 h, and 20 µM MK-4 was found to enhance GSIS about two fold compared with the 0 µM MK-4 group; thus, MK-4 amplified GSIS in the isolated mouse pancreatic islets of KK-Ay mice (). Moreover, we also found that MK-4 enhanced GSIS in C57BL/6J mice (). Hence, MK-4 enhanced insulin secretion in both isolated islets and INS-1 cells under glucose stimulation, and pancreatic β-cells were the target of MK-4.

2.2. MK-4 Increased cAMP Levels in INS-1 Cells

To investigate the mechanism by which MK-4 regulates GSIS in β-cells, INS-1 cells were used in the following experiments. Initially, intracellular cAMP was measured in INS-1 cells because increased cAMP levels promote nutrient-induced insulin secretion in enteroendocrine cells. As shown in , the level of cAMP tended to increase with the lower dose (1 µM MK-4), and was significantly increased in the group treated with 3 µM MK-4 for 1 h. These results revealed that MK-4 might enhance insulin secretion by regulating the cAMP-dependent pathway in INS-1 cells.

2.3. MK-4 Amplified GSIS by Regulating the cAMP/Epac-Dependent Pathway but not the cAMP/PKA-Dependent Pathway in INS-1 Cells

To verify the effect of MK-4 on cAMP-dependent pathways, the cAMP/PKA pathway was investigated first (). However, through the cAMP response element (CRE)-reporter gene assay, it was found that luciferase activity did not change with either 1 or 3 µM of MK-4 treatment (A). Not unexpectedly, treatment with the protein kinase A (PKA) inhibitor H89 also did not affect the influence of MK-4 on GSIS (B), which showed that MK-4 might not regulate PKA activity in INS-1 cells. Therefore, we analyzed another cAMP-dependent pathway, the cAMP/Epac2 pathway of GSIS, in INS-1 cells. We blocked the cAMP/Epac2 pathway using the exchange protein directly activated by the cAMP 2 (Epac2) inhibitor ESI-05. As shown in , the insulinotropic effect of MK-4 might have been abolished by the Epac2 inhibitor, suggesting that MK-4 amplified GSIS through the regulation of the cAMP/Epac-dependent pathway but not the cAMP/PKA-dependent pathway in INS-1 cells.

Abstract

Vitamin D2 deficiency is one of the most common micronutrient insufficiencies among children. Few foods, mainly those derived from animal sources, naturally contain this vitamin. The basidiomycete mushroom Pleurotus ostreatus could be used as an innovative and sustainable ingredient for food fortification with vitamin D2. This study was aimed at exploring children’s acceptance of a whole-cereal-based product (breadsticks) combined with increasing concentrations of P. ostreatus powder rich in vitamin D2. The food neophobia trait (fear of trying unfamiliar and new food) on sample acceptability was also investigated. One hundred and three children (47 girls and 56 boys, aged 9–11 years) were recruited, and breadstick-liking was studied in relation to gender and neophobic traits. Results showed that the samples enriched in vitamin D2 were well accepted by children even if liking decreased with increasing concentration of mushroom powder. Generally, neophilic subjects gave higher liking scores compared with the neophobic ones, especially for the modified samples. New, well-accepted fortified products could be developed using an adequate concentration of mushroom powder to deal with the increasing vitamin D2 deficiency among children.

2.1. Participants

One hundred and three children (47 girls and 56 boys, aged 9–11 years) were recruited in the Milan area (Italy) via primary schools. Parents received full information about the research study and provided written informed consent for their children’s participation. Children without a signed informed consent were not involved in the study. Children who suffered from food allergies or did not like the tested samples, which could negatively affect hedonic responses, performed alternative activities so that they did not feel excluded from the test activities. The study was performed in adherence with the principles established by the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the University of Milan (protocol n°19/18).

2.2. Stimuli

A commercial strain of P. ostreatus deposited at the mycotheca of the Società Agricola IoBoscoVivo (Vergiate, Varese, Italy) was used. This strain was cultivated on wood of Populus tremula in a greenhouse at room temperature. After one year of incubation, about 10 kg of body fruits were collected, cut into 4-mm slices, spread on an oven rack in a single layer, and air-dried at 37 °C for 48 h. At the end of the process, about 1 kg of dried mushroom was obtained and all the product was ground to a fine powder using a Thermomix TM 31 (Vorwerk Contempora S.r.l., Italy). The production of breadsticks added with P. ostreatus was performed at the Società Agricola IoBoscoVivo (Vergiate, Varese, Italy), according to the addition levels decided on by the sensory group at the University of Milan, based on preliminary trials. The breadstick ingredients were whole-wheat flour, sunflower oil, yeast, and salt. The experimental samples were prepared by adding different increasing concentrations of P. ostreatus powder (B2 = 2%, B4 = 4%, and B6 = 6%) to the standard formulation (B0). A hidden control of the unmodified sample (Bhc = 0%) was also included to verify that children were able to assign a comparable score to the two samples without mushroom powder addition (B0 and Bhc). The nutritional composition and the energy content of the developed samples, which were provided by Società Agricola IoBoscoVivo (Vergiate, Varese, Italy), are reported in .

2.4. Experimental Procedure

The liking evaluation was performed at school in the classroom in the presence of a teacher and an experimenter in a single day. During the evaluation, each child was seated at their own table and received the questionnaire, which was extensively explained by the experimenters. The questionnaire consisted of an evaluation of food neophobia followed by the evaluation of breadstick liking. The questionnaire was self-completed by the children. To increase ecological validity, the breadsticks were offered as a snack and liking was assessed during the mid-morning break. Each of the five formulations, coded with three-digit numbers, were randomly presented to each child in blind condition. Children were instructed to drink a sip of water between each sample. The experimenters monitored the children to ensure that they did not influence each other during the evaluation. The session took approximately 30 min and children received a small award (e.g., pencil case) for their participation.

3.2. Liking Assessment

A significant sample effect was found on liking scores (F(4; 404) = 12.38, p < 0.0001). As reported in , B0 and Bhc samples obtained comparable scores (MB0 = 5.5 ± 0.2; MBhc = 5.3 ± 0.2). Children equally liked these samples, which were significantly preferred to samples with increasing added concentrations of P. ostreatus powder. Samples enriched with mushroom powder at 2% and 4% obtained comparable liking scores to each other (MB2 = 4.9 ± 0.2; MB4 = 4.9 ± 0.1) while the sample with the highest concentration of P. ostreatus powder received the lowest liking scores (MB6 = 4.3 ± 0.2). However, all the vitamin D2-enriched samples were well accepted by the children (liking scores were still up the middle of the scale).

2.3. Determination of Vitamin D2 Content

Vitamin D2 was extracted according to the procedure of Sławinska and collaborators [] and identified by the HPLC procedure by Huang and colleagues []. Analysis was performed on breadsticks enriched with 2%, 4%, and 6% of P. ostreatus powder and on the control breadstick after processing and on the breadstick enriched with 6% of P. ostreatus after storage at room temperature for six months.

3.1. Vitamin D2 Content and Product Design

As shown in , the addition of P. ostreatus from 2% to 6% significantly increased the level of vitamin D2 from not detectable to 28.4 µg in 100 g. Breadsticks enriched with 2%, 4%, and 6% can provide 32%, 54%, and 95% of the recommended daily amount of vitamin D2 per single dose (50 g). The 6% enriched breadsticks were also analyzed after being stored for six months at room temperature and found to have the same vitamin D2 content as just after production. According to the USDA database, milk and orange juice fortified with vitamin D provide 30% of the Recommended Dietary Allowance (RDA), while 20% and 10% of RDA is provided by fortified yogurt and cereal breakfast, respectively (). Hence, all the enriched breadsticks could be of interest to improve the dietary intake of vitamin D.

Methods

We measured baseline and 4-month serum concentrations of vitamin D3, 25-hydroxyvitamin D3 [25(OH)D3], 25-hydroxyvitamin D2, 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3], 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3], and 4β,25-dihydroxyvitamin D3 [4β,25(OH)2D3] in 52 adults randomized to receive a total of four oral bolus doses of 2.5 mg vitamin D2 (n = 28) or vitamin D3 (n = 24) over four months. Metabolite-to-parent compound ratios were calculated to estimate hydroxylase activity. Pairwise before vs after comparisons were made to evaluate effects of vitamin D2 and vitamin D3 on metabolism of vitamin D. Mean postsupplementation metabolite-to-parent ratios were then compared between groups.

Trial design and participants

As previously described, we conducted a double-blind, randomized placebo-controlled trial that enrolled a total of 340 men and women aged 30 to 75 years who had been identified as being at increased risk of developing type 2 diabetes mellitus in London and Cambridge, United Kingdom (). Full details of inclusion and exclusion criteria are described in the published protocol (). Eligible participants were randomly allocated to one of three groups on a 1:1:1 basis within four strata defined by age (30 to 50 or 51 to 75 years) and sex, with a block size of six within each stratum. One group received four monthly oral bolus doses of 2.5 mg vitamin D2: each dose was presented as 5 mL Sterogyl solution (Desma Pharma, Paris, France) containing 0.5 mg vitamin D2 per milliliter in ethanol. The second group received four monthly oral bolus doses of 2.5 mg vitamin D3: each dose was presented as 5 mL Vigantol oil (Merck Serono, Darmstadt, Germany) containing 0.5 mg vitamin D3 per milliliter in Miglyol oil (Caesar & Loretz, Hilden, Germany). The third group received four monthly oral doses of placebo (Miglyol oil). The order of treatments within each block was determined by a computer-generated pseudo-random sequence, generated by the study medication manufacturer (Nova Laboratories, Leicester, UK). Neither the participants, the investigators, nor the laboratory staff knew the treatment allocation. Each participant was followed-up for a total of four months from their first visit; serum samples were collected at baseline and at the end of the study. Baseline and four-month serum samples taken from a subset of 28 participants and allocated to vitamin D2 and 25 participants allocated to vitamin D3 were sent for determination of concentrations of vitamin D3 and its metabolites as detailed below. The subset of participants contributing samples to the current study were selected on the basis that they were all recruited in London; that they had each received four directly observed doses of vitamin D2 or vitamin D3; and that they were the 28 samples in each group having the greatest volume of serum available at both baseline and four-month follow-up to be sent for further analysis. For the vitamin D3 group, only 25 samples had sufficient sample volume for analysis. Ethical approval for the trial was provided by the Charing Cross Medical Ethics Committee (ref 09/H0711/85) and the Cambridge Local Research Ethics Committee (ref 04/Q0108/19), and written informed consent was obtained from all participants. The trial was registered under the numbers EudraCT 2009-011264-11 and ISRCTN86515510 on 23 October 2009.

Influence of vitamin D2 on metabolism of vitamin D3

To characterize effects of vitamin D2 on metabolism of D3, we conducted pairwise statistical analyses comparing circulating concentrations of vitamin D3 and its metabolites in 28 individuals before vs after oral administration of four monthly doses of 2.5 mg vitamin D2. Results are presented in . Administration of vitamin D2 had no statistically significant effect on serum concentrations of vitamin D3 (P = 0.07), but it did reduce mean serum concentrations of 25(OH)D3 (43.2% decrease, P < 0.001; ), 24R,25(OH)2D3 (37.5% decrease, P = 0.007), 1α,25(OH)2D3 (53.4% decrease, P < 0.001; ) and 4β,25(OH)2D3 (42.0% decrease, P = 0.03). Administration of vitamin D2 reduced molar ratios of 25(OH)D3 to vitamin D3 (76.2% decrease, P = 0.003) and 1α,25(OH)2D3 to 25(OH)D3 (52.2% decrease, P = 0.02), but increased the molar ratio of 24R,25(OH)2D3 to 25(OH)D3 (24.5% increase, P = 0.04). No statistically significant effect of vitamin D2 on the molar ratio of 4β,25(OH)2D3 to 25(OH)D3 was seen (P = 0.97).

Discussion

Our findings are consistent with reports that administration of vitamin D2 reduces serum concentrations of 25(OH)D3 (–), and that this phenomenon is associated with an increase in the ratio of 24R,25(OH)2D3 to 25(OH)D3 and a decrease in the ratio of 1α,25(OH)2D3 to 25(OH)D3 in the circulation. Interestingly, serum 25(OH)D2 concentrations were markedly elevated in all study participants receiving vitamin D2, consistent with induction of the vitamin D2 25-hydroxylation pathway, although there was an opposite effect on 25(OH)D3 and rate of formation in the same treated individuals (), raising the possibility that different enzymes might catalyze 25-hydroxylation of the two forms of vitamin D. Alternatively, the decline of 25(OH)D3 following administration of vitamin D2 may reflect competition of vitamin D2 for the same 25-hydroxylation pathway as vitamin D3. Although changes in metabolite-to-parent ratios may reflect alteration in rates of conversion of one metabolite to another, they could also be explained by removal of vitamin D and its metabolites from the circulation (e.g., via direct excretion or disposition into depots such as adipose tissue and muscle). Further investigations to compare the effects of different forms of vitamin D on expression and activity of the enzymes responsible for metabolizing vitamin D3 are needed to resolve the question of whether changes in metabolite-to-parent ratios truly reflect changes in activity of cytochrome P450 enzymes. Such studies would potentially require liver and renal biopsies: both are invasive procedures and their inclusion in a study protocol could raise issues relating to ethics and acceptability to participants.

Abstract

Myriocin is an antibiotic derived from Mycelia sterilia, and is a potent inhibitor of serine palmitoyltransferase, the enzyme involved in the first step of sphingosine synthesis. Myriocin, inhibiting ceramide synthesis, has a great potential for treatment of diseases characterized by high ceramide levels in affected tissues, such as retinitis pigmentosa (RP). Drug delivery to the retina is a challenging task, which is generally by-passed through intravitreal injection, that represents a risky invasive procedure. We, therefore, developed and characterized an ophthalmic topical nanotechnological formulation based on a nanostructured lipid carrier (NLC) and containing myriocin. The ocular distribution of myriocin in the back of the eye was assessed both in rabbits and mice using LC-MS/MS. Moreover, rabbit retinal sphingolipid and ceramides levels, after myriocin-NLC (Myr-NLC) eye drops treatment, were assessed. The results demonstrated that Myr-NLC formulation is well tolerated and provided effective levels of myriocin in the back of the eye both in rabbits and mice. We found that Myr-NLC eye drops treatment was able to significantly decrease retinal sphingolipid levels. In conclusion, these data suggest that the Myr-NLC ophthalmic formulation is suitable for pharmaceutical development and warrants further clinical evaluation of this eye drops for the treatment of RP.

Ocular pharmacokinetics and tolerability

Our experimental protocol complied with the statements of the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and visual research. New Zealand (NZ) albino rabbits (weight 2.0–2.5 kg) were purchased from Envigo s.r.l (San Piero al Natisone, Udine, Italy). Rabbit can be considered a reliable animal model for translational ocular pharmacokinetics studies (Durairaj, ). Rabbits were housed in standard conditions for one week before the experiment (light-controlled room, controlled temperature 22 ± 1 °C and humidity 10–30%). Rabbits were randomly assigned to two experimental groups (treated and vehicle): n = 10 per experimental group, 2 rabbits and 4 retinas per time point. The two experimental groups of rabbits received ocular topical administration, 30 µl × 2 times within 5 min of the interval, of Myr-NLC (NLC1, drug loading 0.68 mg/ml) and vehicle (unloaded NLC), respectively. Rabbits were sacrificed at 30, 60, 120, 180, 240 min after ocular topical administration of the myriocin-NLC and unloaded NLC. Rabbits were sacrificed by intravenous administration of 0.3 ml/kg of Tanax© (Intervet, Milano, Italy), after sedation with an intramuscular administration of 10 mg/kg of Zoletil© (Virbac, Milano, Italy). Eyes were enucleated and ocular tissues (vitreous and retina) were collected. Tissue samples were stored at −80 C° until quantitative analysis of myriocin. Ocular distribution of myriocin was determined also in the retina of C57BL6J mice after topical administration of myriocin-NLC formulation (NLC1, drug loading 0.68 mg/ml). In order to increase formulation corneal residency, three 5 µl administrations within 2 min of interval were carried out. The amount of topical administered myriocin was 15 µg. C57BL6J mice (n = 4) per group were sacrificed 30, 60, 180 and 240 min after ocular topical administration of myriocin-NLC formulation; a separate set of animals (n = 12) was used to determine myriocin in control mice (vehicle). Mice were sacrificed by cervical dislocation; the eye enucleated and retina collected and stored at −80 °C until analysis.

Ocular pharmacokinetics and tolerability

The following pharmacokinetics parameters were determined in rabbit and mouse eye tissues: peak eye tissue concentration (Cmax), and time of peak of eye tissue concentration (Tmax). The area under the curve (AUC0-240) of eye tissues was extrapolated from the concentration of myriocin [myriocin] vs. time curves. Ocular safety of unloaded NLC and Myr-NLC formulations were evaluated by a modified Draize’s test in a separate set of rabbits (n = 6) (Leonardi et al., ; Giannavola et al., ). Formulations were topically administered (30 μl) in the eye every 30 min for 6 h (12 treatments). Scores were assigned accordingly to previous studies, and eyes were examined at 10 min and 6 h after Myr-NLC administration by a slit lamp (4179 T Sbisà, Florence, Italy). Observations were made by two independent observers in a masked way.

Ocular pharmacokinetics and tolerability

Our experimental protocol complied with the statements of the Association for Research in Vision and Ophthalmology (ARVO) for the use of animals in ophthalmic and visual research. New Zealand (NZ) albino rabbits (weight 2.0–2.5 kg) were purchased from Envigo s.r.l (San Piero al Natisone, Udine, Italy). Rabbit can be considered a reliable animal model for translational ocular pharmacokinetics studies (Durairaj, ). Rabbits were housed in standard conditions for one week before the experiment (light-controlled room, controlled temperature 22 ± 1 °C and humidity 10–30%). Rabbits were randomly assigned to two experimental groups (treated and vehicle): n = 10 per experimental group, 2 rabbits and 4 retinas per time point. The two experimental groups of rabbits received ocular topical administration, 30 µl × 2 times within 5 min of the interval, of Myr-NLC (NLC1, drug loading 0.68 mg/ml) and vehicle (unloaded NLC), respectively. Rabbits were sacrificed at 30, 60, 120, 180, 240 min after ocular topical administration of the myriocin-NLC and unloaded NLC. Rabbits were sacrificed by intravenous administration of 0.3 ml/kg of Tanax© (Intervet, Milano, Italy), after sedation with an intramuscular administration of 10 mg/kg of Zoletil© (Virbac, Milano, Italy). Eyes were enucleated and ocular tissues (vitreous and retina) were collected. Tissue samples were stored at −80 C° until quantitative analysis of myriocin. Ocular distribution of myriocin was determined also in the retina of C57BL6J mice after topical administration of myriocin-NLC formulation (NLC1, drug loading 0.68 mg/ml). In order to increase formulation corneal residency, three 5 µl administrations within 2 min of interval were carried out. The amount of topical administered myriocin was 15 µg. C57BL6J mice (n = 4) per group were sacrificed 30, 60, 180 and 240 min after ocular topical administration of myriocin-NLC formulation; a separate set of animals (n = 12) was used to determine myriocin in control mice (vehicle). Mice were sacrificed by cervical dislocation; the eye enucleated and retina collected and stored at −80 °C until analysis.

Abstract

Omega 3-docosahexaenoic acid (DHA) and vitamin E Delta-tocotrienol (Delta-T3) are extensively studied as protective nutrients against cancer development. Little is known about the biological mechanisms targeted by these bioactive molecules on lipid droplet (LD) biogenesis, an important breast cancer aggressiveness marker, and the occurrence of lipophagy in breast cancer cells. The aim of this study was to investigate the effect of DHA, Delta-T3 and DHA plus Delta-T3 co-treatment in LD biogenesis and lipophagy process in triple negative breast cancer cell line MDA-MB-231. Cells were treated with 50 μM DHA and/or 5 μM Delta-T3. Our results demonstrated that DHA can trigger an increase in LD biogenesis and co-treatment with Delta-T3 was able to reduce this LD biogenesis. In addition, we showed that a higher cytoplasmic LD content is associated with a higher breast cancer cells malignance and proliferation. Reduction of cytoplasmic LD content by silencing ADRP (adipose differentiation-related protein), a structural LD protein, also decreased cell proliferation in MDA-MB-231 cells. Treatment with DHA and Delta-T3 alone or co-treatment did not reduce cell viability. Moreover, we showed here that DHA can trigger lipophagy in MDA-MB-231 cells and DHA plus Delta-T3 co-treatment was able to enhance this lipophagy process. Our findings demonstrated that co-treatment with DHA plus Delta-T3 in MDA-MB-231 cells could reduce LD biogenesis and potentiate lipophagy in these cells, possibly having a positive impact to inhibit breast cancer malignancy. Therefore, suitable doses of DHA and Delta-T3 vitamin E isoform supplementation can be a prominent tool in therapeutic treatments against breast cancer.

Abstract

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder and the leading cause of dementia. The disease progression is associated with the build-up of amyloid plaques and neurofibrillary tangles in the brain. However, besides the well-defined lesions, the AD-related pathology includes neuroinflammation, compromised energy metabolism, and chronic oxidative stress. Likewise, the blood–brain barrier (BBB) dysfunction is suggested to be a cause and AD consequence. Accordingly, therapeutic targeting of the compromised BBB is a promising disease-modifying approach. We utilized a homozygous triple-transgenic mouse model of AD (3×Tg-AD) to assess the effects of L-norvaline on BBB integrity. We scrutinized the perivascular astrocytes and macrophages by measuring the immunopositive profiles in relation to the presence of β-amyloid and compare the results with those found in wild-type animals. Typically, 3×Tg-AD mice display astroglia cytoskeletal atrophy, associated with the deposition of β-amyloid in the endothelia, and declining nitric oxide synthase (NOS) levels. L-norvaline escalated NOS levels, then reduced rates of BBB permeability, amyloid angiopathy, microgliosis, and astrodegeneration, which suggests AD treatment agent efficacy. Moreover, results undergird the roles of astrodegeneration and microgliosis in AD-associated BBB dysfunction and progressive cognitive impairment. L-norvaline self-evidently interferes with AD pathogenesis and presents a potent remedy for angiopathies and neurodegenerative disorders intervention.

2.1. Norvaline Improves the BBB Integrity in the 3×Tg-AD Mice

Tibbling et al. (1977) pointed out that CNS albumin is derived from peripheral sources. Accordingly, the albumin index can indicate the rate of BBB disruption and facilitate the efficacy of routine clinical work []. Subsequently, several groups demonstrated elevated levels of CSF albumin in patients with AD [,]. We applied a traditional Evans blue assay to assess the albumin presence in the brain tissue. The mean measured supernatant optical density (OD620) per brain weight from WT control animals was 0.4 ± 0.02 (B), which accords with the results from other groups that utilized the same protocol []. Remarkably, the mean OD620 of the supernatant from seven-month-old 3×Tg-AD controls increased up to 0.62 ± 0.04 and still was substantially lower than OD620 = 0.9 obtained from 15-month-old 3×Tg-AD mice [], which points to gradual progressive severe age-dependent BBB defects in this particular AD model. Remarkably, L-norvaline treatment significantly (p < 0.01) improved the BBB integrity in the transgenic mice, as reflected by a reduction (by 31%) in the mean OD620 (0.43 ± 0.066). Of note, we did not observe any significant effect of the treatment upon the BBB permeability in the WT animals (). The two-way ANOVA test was used to reveal a significant (F(1,20) = 10.47 p = 0.0041) interaction (accounting for 14.21% of the total variance) between the genotype and the type of treatment, and their mutual effect upon the BBB permeability, which further indicates L-norvaline efficacy.

2.2.1. Norvaline Reduces Endothelial Amyloidosis

We applied an immunohistochemistry assay to analyze the effect of L-norvaline upon the rate of Aβ deposition in the 3×Tg-AD mice brain microvessels’ walls. Previously, we have shown a severe Aβ positivity decline in the cortices [] and hippocampi [] following the L-norvaline treatment. Here, we evidenced considerable levels of APP/Aβ in the cerebral microvessels’ ECs (A), and demonstrated a substantial reduction in the levels of Aβ positivity following the treatment (B).

2.2.2. Norvaline Diminishes the Rate of Astrodegeneration in the 3×Tg-AD Mice

Here, we analyze the GFAP immunopositive surface area and optical density (C) in the vicinity of hippocampal penetrating blood microvessels (A) (about 10 µm in diameter). The relative thickness of the astroglia end-feet (B) displayed significant genotype and treatment-related differences (D). The two-way ANOVA test revealed a significant (F(1,36) = 15.7 p = 0.0003) interaction (accounting for 13.94% of the total variance) between the genotype and the type of treatment, and their mutual effect upon the astroglia end-feet thickness. Remarkably, L-norvaline did not affect this parameter in WT mice but demonstrated a significant (p < 0.001) influence upon 3×Tg-AD animals with an increase (by about 42%) (D).

2.2.2. Norvaline Diminishes the Rate of Astrodegeneration in the 3×Tg-AD Mice

Imaris® software has been applied for large-scale analysis of 3D astrocyte morphology. We reconstructed individual cells in the CA1 area (A inset) and revealed a significant (by about 50%) increase (from 20.39 ± 1.55 to 29.79 ± 3.01) in the number of astrocytic protoplasmic processes following the treatment (E). The analysis of the staining intensity (C) and cell volume (D) also demonstrated an increase in the L-norvaline treated group; however, the effect did not reach statistical significance.

Abstract

The effect of hempseed in the diet of laying hens was evaluated at 0, 30, 60 and 90 g/kg concentrations. The aim of the study was to determine the effect of dietary treatment on the performance of hens, the physical characteristics of egg quality, the concentrations of α- and γ-tocopherol and the carotenoid and cholesterol contents of egg yolks, together with the breaking strength of tibial measurements. In light of the obtained results, our study aimed to address the optimal dietary level of hempseed in laying hen diets. Lohmann Brown hens (n = 240) were divided into 4 dietary treatment groups (6 cages per treatment) with 10 hens per cage. The experiment lasted for 12 weeks. The level of nutrients in all diets (wheat-corn) was well balanced. The dietary metabolisable energy was adjusted using rapeseed oil. The addition of 30 g/kg of hempseed to the diet significantly increased (P˂0.001) egg production and egg mass. Alpha-tocopherol increased significantly (P = 0.002) only in the case of the 60 g/kg hempseed level (101 mg/kg dry matter (DM) versus 83 mg/ kg DM in the control group). In contrast, the level of γ-tocopherol increased gradually from 11 mg/kg DM in the control to 29, 39 and 43 mg/kg DM at the 30, 60 and 90 g/kg levels of dietary hempseed, respectively. The concentrations of beta carotene, zeaxanthin and lutein in egg yolks were not influenced by the dietary treatment. Hempseed at 90 g/kg (P = 0.036) decreased egg shell thickness without affecting its strength. The addition of hempseed decreased (P˂0.001) the cholesterol concentration in the egg yolks in all experimental groups. The addition of 30, 60 and 90 g hempseed increased (P˂0.001) the breaking strength of the tibia to 354, 352 and 350 N, respectively, compared to 297 N in the control group. The highest level of hempseed in the diet positively influenced the Ca concentration in the tibia (P = 0.021). The concentration of P in the tibia was negatively affected in the 60 to 90 g/kg hempseed treatments (P˂0.001). Eggs are a significant source of α-tocopherol. Based on our results, there is a possibility for enrichment of egg yolks with γ-tocopherol, with all of its associated health benefits, by the addition of hempseed to the diet of laying hens. Another significant benefit of hempseed is its effect on the breaking strength of the tibia, which can help with crucial problems in the commercial breeding of laying hens.

Hens, husbandry and diets

Two hundred forty 52-week-old Lohmann Brown (Egg Production Company in Kosičky, Kosičky, Czech Republic) hens were randomly assigned to 4 treatments with 6 replicate cages and 10 hens per cage. The hens were housed in three-floor enriched cages in the same air-conditioned facility. Each cage was 7,560 cm2 in area. The cage equipment conformed to the European Council Directive 1999/74 EC (European Union 1999). The light cycle was 16 h of light and 8 h of darkness, and the light intensity was approximately 10 lux in the central storey. The control diet had no hempseed addition, and the experimental groups received diets supplemented with 30, 60 and 90 g of hempseed per kg of diet. The ingredients and nutrient composition of the diets are listed in . All diets were formulated to contain similar levels of metabolisable energy (11.6 MJ/kg) and crude protein (165 g/kg). A vitamin-mineral premix incorporated into the diet did not contain tocopherol. Feed and fresh water were supplied ad libitum. The experiment lasted 12 weeks. The Ethical Committee of the Institute of Animal Science approved the study protocol and the use of animals in the experiment.

Results

As the hempseed concentration increased, the amount of α-tocopherol, β-carotene, lutein and zeaxanthin increased accordingly (). The amount of γ-tocopherol is almost twenty times higher than that of α-tocopherol in hempseed, which was correspondingly increased in the mixed feed. In the control diet, with no hempseed added, α-tocopherol was higher, which was also observed in the diet enriched with 30 g/kg hempseed. When 60 g/kg hempseed was added, both tocopherols were almost equally present, and in the case of the 90 g/kg hempseed diet, γ-tocopherol was present at a higher concentration when compared with α-tocopherol. Regarding the tocopherol concentrations in egg yolks, eggs of hens fed with the control feed contained mostly α-tocopherol at 83 mg/kg DM; the amount of γ-tocopherol was 11.3 mg/kg DM (). The ratio between the two tocopherols was 7.35. With the increase in hempseed in the diet, the ratio decreased from 3.2 to 2.6 to 2. Between groups, there was a significant difference in both the α- and γ-tocopherol concentrations (P = 0.002 a P < 0.001, respectively). There were no significant differences in the amounts of β-carotene, lutein and zeaxanthin between treatments. In contrast, all experimental diets significantly decreased (P < 0.001) the cholesterol concentration in egg yolks. There was no effect of the hempseed concentration on cholesterol concentration.

Conclusion

In conclusion, dietary hempseed supplementation increased γ-tocopherol in egg yolks and the breaking strength of tibia and decreased cholesterol content in egg yolks. In terms of performance and bone quality, the most suitable dose for laying hens is 30 g/kg of hemp seed in the diet.

Abstract

Thermometry via magnetic resonance imaging (MRI) would provide a powerful noninvasive window into physiological temperature management. Cobalt-59 nuclear spins demonstrate exceptional temperature dependence of their NMR chemical shifts, yet the insight to control this dependence via molecular design is lacking. We present the first systematic evidence that encapsulation of this spin system amplifies the temperature sensitivity. We tested the temperature dependence of the 59Co chemical shift (Δδ/ΔT) in a series of five low-spin cobalt(iii) complexes as a function of increasing encapsulation within the 1st coordination sphere. This study spans from [Co(NH3)6]Cl3, with no interligand connectivity, to a fully encapsulated dinitrosarcophagine (diNOsar) complex, [Co(diNOsar)]Cl3. We discovered Δδ/ΔT values that span from 1.44(2) ppm °C–1 in [Co(NH3)6]Cl3 to 2.04(2) ppm °C–1 in [Co(diNOsar)]Cl3, the latter among the highest for a molecular complex. The data herein suggest that designing 59Co NMR thermometers toward high chemical stability can be coincident with high Δδ/ΔT. To better understand this phenomenon, variable-temperature UV-Vis, 59Co NMR relaxation, Raman spectroscopic, and variable-solvent investigations were performed. Data from these measurements highlight an unexpected impact of encapsulation – an increasingly dynamic and flexible inner coordination sphere. These results comprise the first systematic studies to reveal insight into the molecular factors that govern Δδ/ΔT and provide the first evidence of 59Co nuclear-spin control via vibrational means.

Results and discussion

Precise determination of the sensitivity of the 59Co NMR peak to temperature (Δδ/ΔT) was achieved via linear regression of the temperature-dependent data (see , S8, and Table S2). These analyses revealed Δδ/ΔT values for K3[Co(CN)6] and 1–5, respectively, of 1.44(1), 1.44(2), 1.38(1), 1.30(2), 1.71(1), and 2.04(2) ppm °C–1. These values are within the ranges of sensitivity reported for the few 59Co NMR thermometers,–, but it's worth noting that, to the best of our knowledge, the Δδ/ΔT of 5 is eclipsed only by Co(acac)3, a molecule that is completely unsuitable for aqueous (e.g. physiological) applications., Most importantly (and surprisingly), these data indicate that the highest sensitivity to changes in temperature is held by the completely encaged complex 5.

Results and discussion

The values of Δδ/ΔT follow an opposing trend to the initial hypothesis, in that 5, with the highest degree of encapsulation, displays the strongest Δδ/ΔT. Complex 4, with the second highest degree of encapsulation, displays the second highest sensitivity of our studied complexes. Yet, a comprehensive trend for all complexes on the basis of encapsulation is not indicated by these data. For example, in 1 and K3[Co(CN)6], the ligand donor atoms are not connected in any manner. Yet, these species demonstrate higher Δδ/ΔT than both 2 and 3, which contain bidentate chelates. Furthermore, the collected data show that electronic structure considerations alone (specifically, Δo) do not govern sensitivity. Here, neither the trend in Δo extracted from UV-Vis (3 < 5 < 1 < 4 < 2 < K3[Co(CN)6]) nor that from the 25 °C 59Co NMR (3 < 1 < 4 < 2 < 5 < K3[Co(CN)6]) reproduce the trend in Δδ/ΔT (see Fig. S9 and Table S3).