Effect of PEA on Muscle Recovery Following Resistance Exercise
Condition: Muscle Recovery; Muscle Soreness
MeSH terms: Myalgia
Study type: Interventional

Summary: 
      This study evaluates the effects of short term palmitoylethanolamide (PEA) supplementation on
      functional responses (i.e. muscle soreness and performance parameters) to strenuous eccentric
      exercise, and investigateq the systemic and muscle molecular mechanisms through which PEA
      impacts on these functional responses.
    

Detailed description: 
      Study design The protocol entails a double-blind, placebo-controlled, crossover trial design
      in which non-elite male athletes (training load >5h/wk, n=14) are enrolled. Participants
      undergo 2 experimental sessions in which they receive PEA or placebo in a randomized order.
      The two sessions are separated by a 3-week wash out period to allow a full recovery from the
      eccentric exercise bout. The scientific rationale to use a within-subjects design is based on
      the assumption of reduced data variability and increased statistical power compared to a
      between-subjects design. Additionally, a double-blind placebo controlled trial is used to
      prevent treatment bias and placebo effects.

      Participants are randomly allocated to receive PEA or appearance-matched placebo supplements
      twice a day for a total of 6 days. Three days before the start of supplementation (D-3),
      baseline measurements are collected during which participants report to the laboratory in a
      fasted and rested state and mark their perceived muscle soreness on a visual analogue scale
      (VAS). Next, blood samples and muscle biopsies are collected, after which maximal isometric,
      eccentric and concentric voluntary contractions and jump height (Counter Movement Jump and
      Squat Jump) are assessed. Furthermore, participants complete the Pittsburgh Sleep Quality
      Index (PSQI) to evaluate sleep quality and quantity of the previous month. Three days after
      the collection of baseline measurements (D0), participants return to the laboratory in a
      rested and fasted state. After collection of a fasted blood sample, they receive their first
      supplement with a light standardized breakfast (~500 kcal, 60% carbohydrates, 20% protein,
      20% fat). Afterwards, they rate their perceived muscle soreness and fill in the St. Mary's
      Hospital questionnaire (to evaluate their sleep quality and quantity from the previous
      night). One hour after supplement intake, participants perform a unilateral eccentric
      exercise (EE) bout consisting of 24 sets of 10 eccentric contractions of the knee extensors.
      Measurements of maximal voluntary contractions and jump height are repeated 24, 48, 72 and
      120h following EE. Additionally, blood samples are collected 24, 48, 72, 96 and 120h
      following EE, and muscle biopsies are collected 48h following EE. Furthermore, participants
      rate their perceived muscle soreness, complete a food diary and fill in the St. Mary's
      Hospital questionnaire (daily), for the entire duration of the experimental protocol.

      Screening Volunteers are screened to assess their eligibility to participate in the study
      based on in- and exclusion criteria . During the screening, body mass is measured using a
      digital balance with an accuracy of 0.1 kg and body height is measured with an accuracy of 1
      cm. Furthermore, a medical questionnaire is obtained to assess participants' general health.
      When volunteers are deemed eligible according to the screening results and the in- and
      exclusion criteria, they are included in the study.

      Familiarization To prevent any learning effect during the intervention trial, all
      participants are familiarized with the test equipment at least one week before the start of
      the trial. Participants are invited to perform 3 submaximal isometric, eccentric and
      concentric voluntary contractions and 3 counter movement jumps and squat jumps as described
      in detail below (see paragraph 76.3 on page 12). Participants are instructed about the proper
      technique of the movements and are supervised by a staff member at all times. During the
      familiarization, individual settings on the isokinetic dynamometer are determined and will be
      used in all subsequent measurements. Additionally, participants are instructed on how to
      complete their food diaries.

      Randomization: Participants are randomly allocated to one of the supplemental groups (PEA
      orplacebo) during the first session. After a wash out period of 3 weeks participants are
      crossed over to the other supplement group. Randomization is performed by a researcher within
      our lab who is not involved in any of the experimental procedures. As such, the participants
      as well as the researchers performing the measurements are unaware of the order in which
      participants receive the different supplements. The randomization sequence will be generated
      by a member of the research group that is not involved in the current study using an online
      tool (RANDOM.ORG - Integer Set Generator). Identity concealment codes are used in the
      randomization list which is in the safe custody of the same member of the research group that
      is not involved in the current study.

      Blinding: To avoid bias, treatment arms will be blinded to Investigators/Trial
      staff/participants, as follows: the supplement will be manufactured, packaged and labelled in
      such a way that the visual appearance, nor the smell and/or touch of the supplement can be
      distinguished from the placebo. Supplements are delivered to the participants by a member of
      the research group that is not involved in any of the experimental procedures or analyses.
      Furthermore, to maintain the blind, the supplement and placebo will follow the same
      administration route and process.

      Unblinding: After all participants have completed the Trial and all wet-lab analyses are
      completed, the database will be locked and the collected Trial data will be unblinded to
      allow statistical analysis of the Trial data. The PI will provide the designated researchers
      with the unblinded treatment information for all participants after completing the data
      analysis.

      In case of an emergency in a blinded Trial, the Investigator has the sole responsibility for
      determining if premature unblinding of a participant's treatment assignment is warranted.
      Participant safety must always be the first consideration in making such a determination. The
      date and reason for unblinding must be recorded, as applicable.

      Muscle damaging eccentric exercise (EE) protocol Participants perform a 5-minute warm up on
      the cycle ergometer at 100W. Afterwards, they take place on the isokinetic dynamometer where
      their upper body is strapped to the chair to stabilize their trunk and their right leg is
      strapped to the lever arm. The EE protocol consists of 24 sets of 10 isokinetic unilateral
      eccentric contractions of the knee extensors at an angular velocity of -30⁰/s and a range of
      motion from 180⁰ to 60⁰ (where 180⁰ refers to full extension). In between sets, 60 seconds of
      rest is provided. Participants are verbally encouraged to maximally resist the downward
      rotation of the lever arm during the eccentric phase of the movement. No resistance is
      applied during the concentric phase of the movement. This eccentric exercise protocol showed
      to effectively induce muscle damage as indicated in previous studies.

      Supplements Participants receive PEA and placebo supplements on two separate occasions in a
      randomized order. The PEA supplements contain 350 mg of Levagen+ (315 mg of PEA and 35 mg of
      excipients) and the placebo supplements contain 350 mg of maltodextrine. Levagen+ consists
      for 90% of palmitoylethanolamide and for 10% of a mixture of coconut oil (fractionated),
      polyglycerol polyricinoleate, citrus oil, olive oil, lecithin, dl-alpha tocopheryl acetate,
      and silicon dioxide to improve the bioavailability of PEA. Both PEA and placebo supplements
      are packaged in non-see-through hard capsules to assure that there are no differences in
      appearance or taste. Participants are instructed to ingest their supplement twice a day (in
      the morning and one hour before bedtime) for 6 consecutive days starting after collection of
      baseline measures.

      Physical activity and Dietary control The participants are asked to maintain their habitual
      activity patterns (e.g. biking to class). However, they are instructed not to perform any
      moderate to strenuous exercise during and 72h before the start of the experimental session.
      Furthermore, no exercise of any kind is allowed 24h before the collection of muscle biopsies.

      Participants receive a standardized meal (1200 kcal; 180 g CHO, 33 g fat, 45 g protein) on
      the evening before EE as well as on the evening before muscle biopsy collection. On testing
      days, when participants have to report to the lab in a fasted state, a breakfast (500 kcal)
      is provided upon completion of all measurements. The same breakfast is provided 1 hour before
      the start of EE. Furthermore, participants complete a food diary daily, starting after
      baseline measurements, in order to check for possible dietary variation between conditions.
      Dietary analyses are performed using a web-platform for dietary recording and analysis of
      macronutrient and micronutrient composition (https://mijn.voedingscentrum.nl/nl/eetmeter/).
      Participants are not allowed to consume alcoholic or caffeinated beverages during the
      experimental sessions.

      Blood samples Blood (2x10 mL) is sampled from an arm vein (Venoject system), and plasma or
      serum is immediately separated by centrifuging. Plasma and serum samples are distributed in
      different 1 mL aliquots and stored at -20°C until usage for biochemical assays at a later
      date. Blood samples are collected at baseline, immediately before EE and 24, 48, 72, 96 and
      120h following EE in a fasted state.

      Plasma samples are analysed by appropriate specific Enzyme-Linked Immunosorbent Assay (ELISA)
      for CK (marker for muscle damage), IL-6 and TNFalpha (both markers of systemic inflammation).
      PEA concentrations are assessed by high-performance liquid chromatography with tandem mass
      spectrometry. Additional assays on blood samples may be performed if judged relevant after
      the primary analyses have been performed. After all analyses have been performed, any
      residual sample is stored for an additional 3 years period before being permanently
      destroyed.

      Muscle biopsies Muscle biopsies are collected at baseline and 48h following EE from the
      dominant leg in a fasted state. Hence, a total of 4 biopsies per subject are taken throughout
      the study. Biopsies are taken by an experienced physician (Dr. Ruud Van Thienen) from the m.
      vastus lateralis of the trained leg using a Bergström needle with a 5 mm diameter as
      previously described (29). In short, a small incision (~5mm) is made through the skin and
      underlying fascia under local anaesthesia (Lidocaïne, 1ml) to facilitate smooth penetration
      of the needle into the muscle belly. Once the needle is placed within the muscle belly, a
      tube and syringe are attached to the upper extremity of the needle which is used to create a
      vacuum by which the muscle is drawn into the needle. At this point, a smooth twisting
      downward movement of the inner part of the needle cuts through the tissue to obtain the
      muscle sample. After cutting of the tissue (~150-300 mg of muscle) the samples are freed from
      any visual blood and connective tissue before being frozen in liquid nitrogen. The incision
      that was made through the skin will be closed using adhesive strips (steristrips) and
      bandaged with a waterproof plaster. Participants will wear the plaster for 5 days before they
      are allowed to take it off. During these 5 days participants are allowed to take a shower but
      they will be instructed not to immerse or plunge the plaster (i.e. taking a bad or swim).
      Participants will be warned about the fact that the muscle biopsy procedure leaves a small
      scar of ~5mm that most likely will be invisible after one or two years. Furthermore, the
      local anaesthesia assures that there is no pain sensation during the procedure, but
      mechanical sensors in the muscles remain sensitive. As such, participants may experience the
      feeling of pressure but not pain. After the procedure the muscle may feel stiff for a day or
      two.

      Muscle biopsies are biochemically analysed within our lab (Exercise Physiology research lab,
      KU Leuven) for parameters of muscle inflammation, stress, regeneration and anabolism.

      Maximal voluntary contraction (MVC) of the knee extensors The isometric, eccentric and
      concentric knee extensor torque of the trained leg are measured using an isokinetic
      dynamometer (cfr. (28)) at baseline and 24, 48, 72 and 120h following EE. In short,
      participants are seated with their upper body strapped to the chair to stabilize the trunk
      and their trained leg strapped to the lever arm. For maximal isometric voluntary contraction
      peak, the knee joint is placed at an angle of 90⁰. Participants are instructed to maximally
      contract their knee extensors and hold it for 3 seconds. Maximal eccentric and concentric
      voluntary contraction are measured at an angular velocity of 30⁰/s and a range of motion
      between 180-60⁰ (where 180⁰ refers to full extension). Each movement is performed three times
      with 60 seconds of rest between attempts. The maximal peak torque value obtained between
      attempts is used for analysis.

      Counter movement jump Participants perform a counter movement jump (CMJ) on a force platform
      (SMARTJUMP, Fusion sport, Nottingham, UK) at baseline and 24, 48, 72 and 120h following EE.
      Participants are asked to take an upright position (knee angle of 180⁰) with their feet on
      hip width before they initiate a downward movement to squat position (knee angle of 90⁰)
      after which they jump vertically for maximum height. Participants are instructed to place
      their hands on their hips and to keep their body erect throughout the jump. CMJ is performed
      three times with 60 seconds of rest between attempts. The highest jump recorded is used for
      analysis.

      Squat jump Participants perform a squat jump (SJ) on a force platform at baseline and 24, 48,
      72 and 120h following EE. Participants are asked to start at a squat position (knee angle of
      90⁰) and hold this for 3 seconds before they jump vertically for maximum height. Participants
      are instructed to place their hands on their hips and to keep their body erect throughout the
      jump. SJ is performed three times with 60 seconds rest between attempts. The highest jump
      recorded is used for analysis.

      Muscle soreness Muscle soreness of the trained leg is assessed at baseline, immediately
      before EE and 24, 48, 72, 96 and 120h following EE using a visual analogue scale (VAS).
      Participants are asked to hold a squat position for 3 seconds with their hands on their hips
      and to mark their perceived muscle soreness on a scale that is represented by a 100mm line of
      which the far-left represents 'no pain' while the far-right represents 'extreme pain'.

      Sleep quality and quantity At baseline, participants' habitual sleep hygiene is evaluated
      using the Pittsburgh Sleep Quality Index . In addition, participants complete the St. Mary's
      Hospital questionnaire daily to evaluate their sleep quality and quantity from the previous
      night, starting on the night before baseline measurements. The St. Mary's Hospital
      questionnaire showed to be a reliable tool for assessing the previous night's sleep of a
      subject.
    

Intervention name(s): PEA; PLACEBO
Intervention type(s): Dietary Supplement; Dietary Supplement
Intervention description(s): PEA supplementation; Maltodextrin supplementation
Other intervention name(s): Levagen+
Allocation: Randomized
Intervention model: Crossover Assignment
Intervention model description: Double-blind, placebo-controlled, crossover trial design
Masking: Double (Participant, Investigator)
Primary purpose: Basic Science
Keywords: PEA; Skeletal muscle