The Influence of Food Matrix Delivery System on the Bioavailability of Vitamin D3
The Influence of Food Matrix Delivery System on the Bioavailability of Vitamin D3
This study investigates the influence of different food matrices on the bioavailability of
 vitamin D.

 Although most vitamin D comes from skin synthesis in response to sun exposure, dietary intake
 is also important - especially during winter time where there is no endogenous production of
 vitamin D in Denmark. A way to maintain an adequate vitamin D status is to supplement either
 as tablets/droplets or as fortified food. However, there seems to be an inter-individual
 variation in response to supplementation.

 This study aims to investigate whether this variation in absorption of vitamin D may depend
 on delivery system.
BACKGROUND The current project is a part of the vitamin D fortification with enhanced
 bioavailability study program (acronym: DFORT) which is an interdisciplinary project
 including research groups from Denmark, Spain, and the Netherlands supported by the Danish
 Innovation Foundation. The overall aim of DFORT is to develop more efficient strategies for
 vitamin D fortification by studying the influence of the delivery matrix on the
 bioavailability of vitamin D. DFORT is organized into four scientific work packages (WP).

 The first two WPs have aimed to study whether complex formation (nano-encapsulation) of
 vitamin D with different proteins may enhance the stability of vitamin D (WP 1 lead by prof.
 Daniel Otzen, AU-iNANO) and the effect of complex formation in real food systems including
 investigations on the stability during storage, light- and heat-exposure (WP 2 lead by
 associate professor Trine Kastrup Dalsgaard, AU-FOOD). WP 1+2 have shown that vitamin D can
 be stabilized by complex formation with whey protein and that the encapsulation may cause
 less oxidative degradation thereby improving the stability of vitamin D in different food
 systems.

 In the current study (WP 3 lead by prof. Lars Rejnmark, AU-Health), the bioavailability of
 vitamin D in different food matrices (including complex formation with whey protein) will be
 studied in humans. Biological samples will be collected in WP 3 allowing for metabolomics
 studies on possible associations between vitamin D supplementation through different food
 matrices and metabolic phenotype (WP 4 lead by prof. Hanne C. Bertram, AU-FOOD).

 Although most of the total body vitamin D is synthesized in the skin after exposure to UV
 light (wavelength of 290-315 nm), most individuals require at least some dietary vitamin D to
 maintain a replete vitamin D status. This is especially true during wintertime. With a
 latitude of 56°N in Denmark, there is no endogenous synthesis of vitamin D in the months
 extending from October to April, which means that the inhabitants have to rely on food
 sources in order to maintain a replete vitamin D status. Cholecalciferol (vitamin D3 [D3]) is
 the main dietary source of vitamin D, but it is only present in a limited number of food
 items (such as fatty fish) making it difficult to achieve the recommended intake of 10 µg D3
 per day.

 Vitamin D status may be improved in response to an increased intake of vitamin D in terms of
 either supplementation with tablets or food fortification. Numerous studies have shown
 increased 25-hydroxy vitamin D (25OHD) levels in response to an increased intake of vitamin
 D. It is generally assumed that mean 25OHD concentrations increase by 0.7 nmol/L in response
 to an increased long-term intake of 1 µg vitamin D per day although the relative increase per
 microgram supplemented may be higher if baseline levels are low. Despite this well-known
 dose-response relationship in groups of people, several studies have documented that the
 change in serum 25OHD levels in response to vitamin D supplementation varies widely.

 Several reasons may account for the inter-individual variation in response to vitamin D
 supplementation. In gross terms, the variation may be due to dosing inaccuracies
 (inconsistencies between claimed and actual values of vitamin D) and variation in
 bioavailability of vitamin D.

 Inconsistencies between claimed and measured values of vitamin D content in vitamin D tablets
 and food-fortified products may be due to inconsistencies in dose used for fortification or
 to the instability of the vitamin per se. Discrepant results have been reported on the
 stability of vitamin D in different food matrices and when exposed to different
 physiochemical hazards. Some investigators have reported vitamin D to be unstable whereas
 others have found it to be remarkable stable when exposed to oxidation, light, and to acid
 and alkali.

 Only few studies have searched for factors responsible for the inter-individual variation in
 25OHD levels in response to vitamin D supplementation. These studies have suggested that body
 composition (including fat mass content), genetic variants of the vitamin D binding protein
 (VDBP), and the ratio of serum 24,25-dihydroxy vitamin D (24,25(OH)2D) to 25OHD may
 contribute to variation in serum 25OHD levels. However, in a recent study only 47% of the
 variations in the response to vitamin D supplementation could be explained by accounting for
 factors of known importance to changes in 25OHD levels.

 In addition to the above-mentioned indices, factors of importance to the intestinal
 absorption of vitamin D as well as the food matrix by which vitamin D supplementation is
 provided may contribute to inter-individual variations in 25OHD responses. However, only few
 studies are available on the bioavailability of vitamin D from different food matrices and
 the intestinal absorption of vitamin D, including the intraluminal fate, and molecular
 mechanisms facilitating the absorption are still only partially understood.

 As vitamin D is a fat-soluble molecule, it has generally been assumed that vitamin D is
 absorbed in the small intestine by simple passive diffusion with vitamin D being incorporated
 into the micelle and transported by chylomicrons via lymph veins to the liver. This is in
 alignment with studies showing an increased risk of low 25OHD levels in patients with fat
 malabsorption. Accordingly, it has been suggested that ingestion of vitamin D with a meal
 rich in fat may increase the release of bile, allowing an increased incorporation of vitamin
 D in the bile salt micelle thereby improving the bioavailability of vitamin D. However,
 discrepant results have been reported, on whether the composition of the food matrices (and
 its fat content) by which vitamin D is ingested influence its bioavailability.

 In a randomized, controlled trial by Raimundo et al., the mean change in 25OHD levels two
 weeks after the treatment with a single large oral dose of 50,000 IU D3 was larger, when the
 meal had at least 15 g of fat compared to a fat-free meal. In contrast, the fat content of
 the food matrices was not found to influence the time-concentration profile as measured by
 vitamin D2 levels in plasma 2, 4, 8, 12, 48, and 72 h after ingestion of a single dose of
 25,000 IU D2 added to either whole milk, skim milk or dissolved in 0.1 mL corn oil and
 applied to toast. However, both of these studies are limited by the use of very high
 (pharmacological) doses of vitamin D, which may override any physiological effects of the
 composition of the food matrices.

 A lack of an effect of the fat content of the food by which vitamin D is ingested is also
 supported by studies on vitamin D fortification of orange juice. Comparing the
 bioavailability of vitamin D added to orange juice or supplemented as capsules showed a
 similar increase in 25OHD concentrations in response to 11 weeks of supplementation with 1000
 IU vitamin D per day and the increase was significant compared to placebo. The fact that
 vitamin D may be sufficiently absorbed following a fat-free meal (such as orange juice) may
 be explained by recent findings on the mechanism by which vitamin D is absorbed. It seems
 that vitamin D is not only absorbed by simple passive diffusion (by incorporation into the
 micelle), as cholesterol membrane transporters, such as SR-BI, CD36, or NPC1L1, have been
 shown to be involved in the absorption. Differences in expression levels and the existence of
 functional polymorphisms in the genes encoding these proteins may also contribute to the
 large inter-individual variation in postprandial responses to vitamin D.

 Only very few studies are available on the time-plasma concentration profile of vitamin D
 after intake of an oral dose. Denker et al. studied the pharmacokinetic profile of vitamin D3
 after administration of a single D3 dose of either 2800 or 5600 IU, showing that plasma D3
 levels increased steadily after the intake and peaked at 9±2.3 h with concentrations
 returning to near baseline values by 72 h. It is unknown whether the food matrix (including
 complex formation of vitamin D by encapsulation with whey proteins) affects the
 bioavailability of vitamin D as assessed by the plasma-time concentration profiles and
 whether this may influence the inter-individual variability in response to vitamin D
 supplementation.

 The importance of calcium intake, and especially calcium intake from milk products and
 tablets (supplements) has been investigated in a number of studies, showing discrepant
 results. A Cochrane meta-analysis has suggested an overall beneficial effect of increased
 calcium intake from milk products and calcium supplements. However, a recent trial has
 suggested an increase in blood pressure in the hours following intake of 1000 mg of calcium
 citrate compared with placebo. It has so far not been investigated whether milk intake causes
 similar effects on indices of cardiovascular health, including blood pressure and arterial
 stiffness.

 AIM The overall aim of the study is to investigate the influence of different food matrices
 (including complex-formation with whey proteins) on the bioavailability of vitamin D, as
 assessed by maximum concentration profiles (Cmax) and the time-concentration curve of D3 in
 plasma and thereby whether the inter-individual variation in the absorption of vitamin D may
 depend on delivery system.

 Co-primary (null-) hypothesis:

 -  The food matrix by which D3 is delivered does not affect Cmax of D3 as determined 10h
 post-dosing.

 -  The absorption profile (time-concentration curve in terms of Area Under the Curve from
 0h to 12h [AUC0-12h]) does not differ according to the food matrix by which D3 is
 delivered.

 Secondary (null-)hypotheses

 -  Compared with vitamin D provided as droplets, the absorption of D3 is not enhanced by
 delivery through each of the tested food matrices (i.e., increased Cmax).

 -  Compared with vitamin D added to juice, the absorption of D3 is not enhanced by whey
 protein complex-bound D3 (i.e., increased Cmax).

 -  Treatments do not affect plasma levels of parathyroid hormone (PTH) and ionized calcium.

 -  The variability to vitamin D supplementation in terms of Cmax is lower if vitamin D is
 complex-bound to whey proteins as compared to the other tested supplementation methods.

 -  Arterial stiffness as assessed by tonometry is not affected by milk intake.

 Explanatory hypotheses In order to allow for further investigations on indices of importance
 to responses to vitamin D supplementation, data will be collected on body composition,
 genetic polymorphisms, cholesterol status, and habitual dietary habits.

 MATERIALS AND METHODS

 STUDY DESIGN The study is performed as a multiple cross-over study using a balanced
 latin-square design. This design allows for each participant to function as her own control
 thereby counterbalancing risk of an adverse influence on results of the order of treatment or
 other factors such as effect of period, as well as inter-individual variations attributable
 to e.g., genetic variations, body weight etc. By randomization, each participant will be
 allocated to receive all the five treatment regimes in a pre-specified order with a 10-21
 days wash-out period in-between each of the treatment arms.

 The treatment sequences are:

 Treatment sequence 1: A B E C D

 Treatment sequence 2: B C A D E

 Treatment sequence 3: C D B E A

 Treatment sequence 4: D E C A B

 Treatment sequence 5: E A D B C

 Treatment sequence 6: D C E B A

 Treatment sequence 7: E D A C B

 Treatment sequence 8: A E B D C

 Treatment sequence 9: B A C E D

 Treatment sequence 10: C B D A E

 PROCEDURES FOR HANDLING VITAMIN D SUPPLEMENTATION The supplement will be acquired
 commercially and stored at the Osteoporosis Clinic, Aarhus University Hospital and kept away
 from other medication and supplementation. Sub-investigator is responsible for correct
 handling and dispensing of vitamin D supplement, as well as securing that the supplement will
 only be used as described in the protocol and that the participants are instructed to take it
 correct.

 PROCEDURES FOR RANDOMIZATION Randomization will be done using a computer generate list.
 Treatments will not be blinded for the investigator. In terms of comparing juice with or
 without whey proteins bound-complexes, a single-blind design will be applied, as participants
 will not be told which of the treatments they are receiving. Each treatment sequence will be
 allocated to the same number of patients - e.g. 3 participants will be in treatment sequence
 1, 3 in treatment sequence 2 etc.

 POPULATION Thirty participants will be recruited from the general background population by
 direct mailing using a list of randomly selected individuals living in the area of Aarhus
 generated by "Research services" at Statens Serum Institut. The study will be performed
 during wintertime (November-April).

 WITHDRAWAL AND DROPOUT Any participant can at any point drop out of the study without any
 explanation and will not have to go through a final examination. The investigator can
 withdraw a participant if this seems necessary for the participant's safety. Dropouts and
 withdrawals will be noted and explained in the CRF.

 Withdrawal will happen in case of one of the following criteria is fulfilled:

 -  Change in vitamin D supplementation

 -  Ionized calcium ≥1.40 mmol/L

 -  Disease or new medication that will influence the study

 -  Serious adverse effects/symptoms that is expected to be caused by vitamin D
 supplementation Diseases that occur within 7 days of treatments can be a possible cause
 of participation in the study. Sub-investigator can in this time frame be contacted in
 order to investigate whether it is a cause of the vitamin D supplementation. In case it
 is, the symptoms or disease will be followed until it is cured or have become chronic.

 EXAMINATIONS Participants will be examined 5 times over a time period of 6 to 12 weeks. At
 each visit, the participants will arrive fasting before 9am and will stay at the department
 until blood sampling at 12 hours is taken. Hereafter, the participants is free to go home and
 come back the following day for the 24 hours blood sampling and delivering the urine samples
 or stay the night at the hospital. During the 12 hours at the department, the participants
 will get standardized food.

 Basic health information and questionnaires:

 Participants will answer questionnaires regarding their general health as well as dietary
 habits and sun exposure.

 Biochemistry:

 Blood samples will be collected at different time points (0, 2, 4, 6, 8, 10, 12, and 24
 hours).

 All measurements will be performed when all material from all 30 participants have been
 collected in order to avoid variation in results. Blood samples will be stored in a biobank
 for a maximum of 15 years after the end of the study.

 Urine samples:

 Urine will be collected in 3 batches at the first day of each dosing i.e., from 0-4 hours,
 from 4-8 hours and from 8-24 hours.

 All measurements will be performed when all material from all 30 participants have been
 collected.

 Bone scans:

 Dual-Energy X-ray absorptiometry (DXA) and High-Resolution peripheral Quantitative Computed
 Tomography (HRpQCT):

 DXA scanning with the Hologic QDR Discovery scanner. Bone mineral density (BMD) will be
 measured in lumbar spine (L1-L4), femoral neck, and the distal forearm. Furthermore, total
 body composition will be determined, including fat- and lean-tissue mass.

 A HRpQCT bone scan of the distal radius and tibia will be performed using an Xtreme
 CT-scanner (SCANCO Medical AG, Switzerland). This will allow for assessment of volumetric BMD
 for cortical and trabecular bone, bone structure and geometry (including cortical and
 trabecular thickness, trabecular separation etc.) and bone strength.

 Blood pressure measurements and tonometry:

 Blood pressure and measurements of arterial stiffness (tonometry) are performed twice in each
 participant in relation to treatment regimes "C" and "D".

 On both occasions, measurements are performed in the morning with the participant in the
 fasting state. After the measurements are performed, the participant will be provided the
 intervention together with a breakfast meal. After this, the participant will be fasting
 until next measurement is performed four hours later.

 Office blood pressure (BP) is measured in a sitting position after 5 minutes of rest on the
 right upper arm using a digital automatic BP monitor. Three BP readings will be performed
 with 2 minutes of rest in-between. The average of the last two measurements is recorded.

 Arterial stiffness and pulse wave velocity (PWV) will be assessed by tonometry using the
 SphygmoCor system (Xcel; AtCor Medical, Sydney, NSW, Australia). For measurements of
 carotid-to-femoral PWV, an inflated femoral cuff placed on the right upper thigh combined
 with carotid applanation tonometry will be used. Measurements are performed in a quiet room.
 The participant will be resting for 10 minutes in a supine position prior to test start.
 Brachial BP is measured on the right upper arm and two consecutive BP readings are performed.
 If BP readings do not differ by > 5 mmHg, the last one is recorded. If BP readings differ by
 > 5 mmHg, four BP readings are obtained. The average of the last two measurements is
 recorded. AIx is assessed as the ratio of wave reflection amplitude to central pulse
 pressure. The mean of two measurements are used in the analyses. Carotid-femoral PWV is
 assessed as the distance travelled divided by the transit time using the direct
 carotid-to-cuff distance as measured with a non-stretchable tape (infantometer). A minimum of
 two measurements is performed. If measurements differs < 0.5 m/s the average of the two
 measurements is used for analyses. If PWV differs by > 0.5 m/s a third measurement is
 obtained and the median value is used for analyses. According to general recommendations, the
 direct carotid-to-cuff distance mean PWV is multiple with 0.8.

 PERSPECTIVES The study will provide insight into the bioavailability of vitamin D3
 supplementation, including sources of variation. Since Denmark is a country with low latitude
 and high prevalence of vitamin D insufficiency and fortification of food items is not common
 or legislated, this study may lead to way to fortifying food items in Denmark.
Inclusion Criteria:

 -  Postmenopausal

 -  Caucasian

 -  Total plasma 25-hydroxy vitamin D < 50 nmol/L

 -  Understand oral and written Danish

 -  Able to consent

 Exclusion Criteria:

 -  Known allergic reaction/intolerance to Vitamin D supplementation / milk products /
 juice

 -  Known chronic kidney disease (creatinine > 90 µmol/L), previous kidney transplantation
 or known kidney artery stenosis

 -  Known liver disease

 -  Known gastrointestinal malabsorption

 -  Current malignant disease

 -  Hypercalcemia (ionised calcium ≥ 1.33 mmol/L)

 -  Treatment with diuretics, lithium or current use of steroids

 -  Current use of calcium and/or vitamin D supplementation

 -  Planned travel during the intervention period to areas where sun exposure is expected

 -  Use of solarium

 -  Treatment with beta-blockers

 -  Overt cardiovascular disease such as known severe heart failure (NYHA III-IV),
 previous major heart surgery, pacemaker, arrhythmias (e.g. atrial fibrillations or
 flutter, second- and third-degree atrioventricular block)