MaxSimil and Vitamin K2: Determining Their Bioavailability
Condition: Healthy
Study type: Interventional

Summary: 
      The benefits of a diet enriched with omega-3 fatty acids are multiple and confirmed by
      several clinical studies. Supplementation with vitamin K, a fat-soluble vitamin, can increase
      or maintain bone density in postmenopausal women and reduce the risk of fracture. In
      addition, some studies show that vitamin K may promote the absorption of omega-3 fatty acids.
      Fish oil, rich in omega-3, is one of the world's favorite forms of omega-3 supplements.
      However, many people suffer from gastrointestinal discomfort when ingesting fish oil
      capsules. To minimize these discomforts and improve plasmatic omega-3 bioavailability,
      Neptune Wellness Solutions has developed a patented formulation of fish oil called MaxSimil®,
      where omega-3s are in the monoglyceride (MAG) form, a predigested omega-3 form. This
      formulation has been tested in humans in a double-blind controlled-randomized pharmacokinetic
      (PK) pilot study with crossover design. PK is defined as a monitoring of omega-3 levels in
      the blood by frequent blood sampling over a period of 24 hours following the ingestion of a
      single dose of omega-3. The results obtained showed that MaxSimil® omega-3s are 3 times more
      absorbed in the blood than the comparison formulation, a source of omega-3 in the ethyl ester
      (EE) form.

      Although this first study confirms a greater bioavailability of MaxSimil®, a complementary PK
      study is necessary to confirm these results and to correct an important methodological bias.
      In fact, the pilot study did not include a comparator group where omega-3s were in the
      triglyceride (TG) form, the most widely omega-3 form currently consumed, but rather use an EE
      form, which have lower bioavailability than TG form. This may therefore have biased the study
      from the point of view of the comparator and thus give the impression that the comparator had
      been deliberately chosen to be less bioavailable than the MaxSimil®.

      In order to confirm the superiority of MaxSimil® (omega-3 MAG form), both in terms of
      bioavailability and incidence of side effects, the aim of this study is to redo a PK study
      using this time two comparators, the two main forms of omega-3 currently used (TG and EE
      forms), as well as a supplementation with vitamin K2 (a form of vitamin K). Our hypothesis is
      that MaxSimil® will be associated with a better omega-3 bioavailability and a lower incidence
      of side effects than the other two forms (TG and EE), and possibly also with a better vitamin
      K bioavailability.
    

Detailed description: 
      Long Chain-Polyunsaturated fatty acids (LC-PUFA) are needed to support normal physiological
      functions: Unlike saturated and monounsaturated fatty acids, synthesis of eicosapentaenoic
      acid (EPA, 20:5 omega-3) and docosahexaenoic acid (DHA, 22:6 omega-3) from its omega-3 PUFA
      precursor, alpha-linolenic acid (18:3 omega-3), is extremely limited in humans. Thus, it is
      recommended that DHA be obtained from dietary sources such as fish and seafood. Intake of EPA
      and DHA from fish normally correlates positively with the concentrations of EPA and DHA in
      plasma. However, recent data suggest that EPA levels are approximately twice higher in plasma
      lipids of the elderly as compared to young individuals, suggesting that potential alterations
      in EPA incorporation and utilization occur during aging. Similar results were obtained with a
      DHA-enriched supplement where the increase of DHA in plasma total lipids was 42% higher in
      the elderly compared to the young. At sufficient levels of cellular content, the LC-PUFA
      influence the physical nature of cell membranes and membrane protein-mediated responses,
      lipid-mediator generation, cell signalling, and gene expression in many different cell types.
      Through these mechanisms, ARA (arachidonic acid, 20:4 omega-6) and DHA influence both cell
      and tissue responses to external signals, and thereby their physiology. Therefore, imbalances
      in LC-PUFA homeostasis potentially induce dysfunctions in the physiology of organs.

      Aging of the Canadians and their nutrition: Canada's population is expected to age more
      rapidly in the coming years. Senior citizens have become more numerous than children in 2015.
      A major concern about old age, both the individual and society, is a decline in health,
      especially if this means a loss of self-sufficiency and independence. Increasing research for
      promoting healthy aging is ongoing but there are physiological modifications occurring during
      aging that might change bioavailability of LC-PUFA. For instance, intake of EPA and DHA
      concentrated in fish normally correlates positively with the concentrations of EPA and DHA in
      plasma. However, recent data from our laboratories suggest that EPA levels are approximately
      twice higher in plasma lipids of the elderly as compared to young individuals, suggesting
      that potential alterations in EPA incorporation and utilization occur during aging. DHA
      response to a DHA-rich supplement was significantly higher in the elderly as compared to the
      young. While on DHA-rich supplement, ARA was not decreased in the young and the elderly, but
      similarly to EPA, ARA remained significantly higher in the elderly compared to the young. The
      investigators also have publish data suggesting that 40 years old is the age where the EPA
      and DHA levels in the plasma are higher than expected, and this was independent to their
      dietary omega-3 fatty acid intake. Hence, these results are important indications that the
      metabolism of LC-PUFA is modified by age and this observation lead to the idea that their
      uptake and usage by organs and tissue can be compromised.

      13C-DHA in humans: Tracing metabolism of carbon 13 (13C)-labelled fatty acids may provide
      some insight into possible aging-related changes in fatty acid metabolism in humans. The
      investigators recently used 13C-DHA to trace its metabolism in six young and six elderly
      participants. The investigators found that, in the elderly, 13C-DHA was 4 times higher in
      plasma triglycerides and free fatty acids at 4 h post-dose, beta-oxidation was 1.9 times
      higher whereas apparent retro-conversion of 13C-DHA to other 13C-omega-3 fatty acids was 2.1
      times higher 24 h and 7 d after tracer intake compared to the young. Hence, because DHA seems
      to remain transiently for longer periods of time in the blood of the elderly compared to the
      young, it may thus indicate that efficiency to remove DHA from the blood is lower in the
      elderly than in the young, resulting in lower incorporation of DHA in the membrane of cells
      that serve to initiate signalization. This observation is potentially at the root of altered
      signalization in the elderly compared to the young.

      Bioavailability of EPA and DHA esterified in TG, PL or as an ethyl ester (EE): Most of fish
      oil supplements on the market are EPA and DHA esterified in TG and to a lower level in EE and
      PL. There has been a lot of study evaluating whether one form of esterification enhances
      bioavailability of EPA and DHA. In pharmacology, "Bioavailability" is defined as a
      subcategory of absorption and it is the portion of the administered dose of a drug or in this
      case, EPA+DHA that reaches the systemic circulation. By definition, an intravenous drug is
      100% bioavailable but when a drug is administered orally, its bioavailability is usually
      decreased due to incomplete absorption or first-pass metabolism. Hence, in the review paper
      of Ghasemifard et al., they reviewed 21 papers that evaluated whether omega-3 fatty acid were
      more bioavailable when given in the form of EE, TG, non-esterified form or as a PL. Four
      studies were rejected because there were no control group, eight studies were evaluating
      pharmacokinetics (PK) defined as a follow up between 8 h up to 72 h of a single oral dose of
      the omega-3 fatty acid product whereas nine studies evaluated the long term intake
      (pharmacodynamic, PD) of a repeated daily dose of omega-3 fatty acids over a period of 2
      weeks and up to 6 months. Although the methodologies of the studies differ, some conclusion
      about the PK was that bioavailability of EPA+ DHA was higher when given in the forms of
      non-esterified fatty acid > TG >>> EE. Lower absorption of the EE form might be because
      pancreatic hydrolysis of EPA and DHA of the EE form compared to the TG form is 10-50 times
      lower. Hence, the esterification form of omega-3 fatty acids might change their short-term
      bioavailability and this information is particularly relevant in the context of metabolic
      diseases that can either affect fat absorption or to an aging population where there are
      dyslipidemias that might change omega-3 fatty acid bioavailability.

      Is MAG-Omega-3 form a better bioavailable source of EPA+DHA?: One study in rats reported
      bioavailability of DHA esterified in TG, PL or MAG (2-mono-acylglycerol) in plasma,
      erythrocytes, retina and brain tissue. They reported that after giving DHA in the different
      forms to rats for 35 days, DHA given in the MAG and PL form were 23-50% more concentrated in
      plasma total lipids and erythrocytes compared to TG-DHA. In the retina and the brain, DHA
      increase to the same levels whatsoever esterification form it was provided.

      In conditions of lipid malabsorption, MAG enriched with EPA + DHA enhance their delivery to
      circulatory system in humans. In humans with cystic fibrosis, a disease recognized as having
      fat malabsorption, especially the long chain fatty acids such as DHA, it was reported that
      providing a MAG-DHA supplementation for one month was efficient to increase levels of DHA in
      the erythrocytes. This pilot study indicates that MAG-DHA supplementation corrects
      erythrocyte ARA/DHA imbalance and may exert anti-inflammatory properties. However, whether
      MAG-DHA is more efficient in participants without malabsorption problems remains to be
      established.

      Side effects associated to fish oil intake: In a meta-analysis evaluating the safety and
      tolerability of prescribed omega-3 fatty acid supplements, it was reported that only three
      minor side effects were associated to fish oil intake: fishy taste, fishy burp and nausea.
      Although these sides effects seem limited to gastro-intestinal discomforts, it can largely
      compromise short- and long-term adherence to fish oil intake. One protective microorganism to
      gastroesophageal reflux is the presence of Helicobacter Pylori (H. Pylori). Moreover, it was
      recently suggested that omega-3 fatty acid supplementation inhibit H. Pylori, by inhibiting
      the synthesis of vitamin K that is required by this microorganism for its own survival. A
      pre-digested form of omega-3 fatty acid such as a MAG form with added vitamin K might limit
      these gastro-intestinal discomforts, and this is something the investigators want to
      investigate in this research project.

      What is Vitamin K2?: Vitamins are essential for different physiological functions of the
      body. Vitamin K is a fat-soluble vitamin. Its discovery was mainly driven by its role on
      blood coagulation but other roles of this vitamin include regulation of calcium metabolism in
      tissues, cell growth and proliferation. There are three main forms for vitamin K: K1, K2 and
      K3. Vitamin K2 is referred as menaquinone since it shares a naphthoquinone ring and a side
      chain with variable length. Most of menaquinones are synthesized by bacteria. Since omega-3
      fatty acid intake seems to inhibit vitamin K2 synthesis by bacteria such as H. Pylori, the
      investigators decided to add vitamin K2 in the MAG-omega-3 formulation to evaluate whether
      this formulation could decrease side effects of fish oil supplement consumption.

      Rationale of this study: Over the 20th century, consumption of linoleic acid (18:2 n-6)
      increased from 2.79% to 7.21% of energy and this is largely due to our dependence on new food
      production methodologies, including soybean oil. This has created over decades a diet that is
      deficient in long chain omega-3 fatty acids. Recently, and because of the enthusiasm around
      consumption of fish oil, there is increasing concerns about fish sustainability. These
      critical issues leads us to have a sustainable source of omega-3 fatty acids, that is the
      most bioavailable and to recommend lower doses of EPA+DHA to be consumed by the population to
      have similar health effects. At the same time, having a source of omega-3 fatty acid with
      limited side effects would also contribute to a better adherence of the population to their
      consumption and perhaps decrease capsule waste. The plasma DHA pool is critical to bring DHA
      to the brain and other organs and it is dynamic, constantly exchanging FA with organs and
      tissues. Numerous studies have assessed the absorption, bioavailability and accretion of DHA
      in the plasma with the conclusion that non-esterified DHA > TG >>> EE. Recently, there has
      been enthusiasm for MAG-Omega-3 formulations since it was shown to improve EPA and DHA
      bioavailability in humans with lower intestinal lipid absorption. However, whether EPA and
      DHA are better absorbed and bioavailable in humans without any issues with intestinal lipid
      absorption remain to be established. Therefore, this project will test a MAG-Omega-3
      formulation enriched with vitamin K2, since the investigators hypothesized that this
      formulation will be the most absorbed and bioavailable with the lowest side effects in men
      and women without any disease such as intestinal lipid absorption issues.
    

Intervention name(s): Omega-3 + vitamin K2 (TG form of omega-3); Omega-3 + vitamin K2 (EE form of omega-3); Omega-3 + vitamin K2 [MaxSimil (MAG form of omega-3)]
Intervention type(s): Dietary Supplement; Dietary Supplement; Dietary Supplement
Intervention description(s): The intervention is a randomized double bond cross over design testing the pharmacokinetics of a monoglyceride formulation compared to a triglyceride and an ethyl ester form.
Treatments are randomly assigned on days 0, 7 and 14 of the clinical study. Blood samples will be collected at time 0, 1, 2, 4, 5, 6, 8, 9, 10, 12 and 24 hours. Each participant will perform all three treatments, with a minimum of 6 days between treatments. A questionnaire will document the side effects felt by participants during the omega-3 supplement taking day.; The intervention is a randomized double bond cross over design testing the pharmacokinetics of a monoglyceride formulation compared to a triglyceride and an ethyl ester form.
Treatments are randomly assigned on days 0, 7 and 14 of the clinical study. Blood samples will be collected at time 0, 1, 2, 4, 5, 6, 8, 9, 10, 12 and 24 hours. Each participant will perform all three treatments, with a minimum of 6 days between treatments. A questionnaire will document the side effects felt by participants during the omega-3 supplement taking day.; The intervention is a randomized double blind cross over design testing the pharmacokinetics of a monoglyceride formulation compared to a triglyceride and an ethyl ester form.
Treatments are randomly assigned on days 0, 7 and 14 of the clinical study. Blood samples will be collected at time 0, 1, 2, 4, 5, 6, 8, 9, 10, 12 and 24 hours. Each participant will perform all three treatments, with a minimum of 6 days between treatments. At each blood draw time points, a questionnaire will be administered to the particpant to monitor if they experiencec side effects with the dietary supplement they ingested in the morning.
Intervention MeSH terms: Vitamins; Vitamin K; Vitamin K 2
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: Double-blind controlled-randomized pharmacokinetic (PK) pilot study with crossover design (10 men and 10 women), with a minimum of 6 days between treatments
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Omega-3 fatty acids; Vitamin K2; Bioavailability; Side effects; Monoacylglyderols