Thirty-one healthy, resistance trained males (22.1 ± 5.0 yrs, 180 ± 0.1 cm, 86.1 ± 13.0 kg, 18.1 ± 6.4% body fat) were informed of study protocol approved by the Institutional Review Board at Baylor University prior to participation. Training status was self-reported, and individuals who lacked at least one year of experience prior to study were excluded. In addition, subjects were excluded if they: 1) had any history of metabolic, hypertension, hepatorenal, musculoskeletal, autoimmune, or neurologic disease; 2) were currently taking thyroid, antihyperlipidemic, hypoglycemic, anti-hypertensive, or androgenic medications; and 3) had taken nutritional supplements that may affect muscle mass [i.e., creatine, hydroxy-beta-methylbutyrate (HMB)] and/or anabolic/catabolic hormone levels [i.e. androstenedione, dihydroepiandrosterone (DHEA), or other prohormones] within three months of the starting the study.
Eligible subjects were familiarized to the study protocol via a verbal and written explanation of the study design. Subjects signed an informed consent statement and completed personal and medical histories while also completing a Wingate anaerobic capacity test prior to baseline testing. Subjects were instructed to refrain from strenuous exercise for 48 hours and fast for 10 hours prior to baseline testing (i.e., day 0) which occurred 3–4 days following familiarization to allow for recording of dietary intake. This study employed a double-blind, placebo-controlled, parallel study design whereby subjects were matched evenly into clusters according to age and body mass.
During days 0, 25, and 50 each subject reported to the laboratory after a 10-hour fast. Height was measured using standard anthropometry and total body mass was measured using a calibrated Healthometer digital strain gauge electronic scale (Bridgeview, IL) with a precision of ± 0.02 kg. Body composition was then determined using a calibrated Hologic Discovery W dual-energy x-ray absorptiometer (DXA) device (Hologic Inc., Bedford, MA), while blood pressure and resting heart rate was determined using standard procedures. Subjects then donated approximately 25 ml of fasting blood using standard venipuncture techniques for hematological, clinical chemistry panels and later cytokine and hormone analysis. Two 10 ml serum separation vacutainer tubes and one 5 ml K3 EDTA vacutainer tube were inserted into the vacutainer holder for blood collection in succession using multiple sample phlebotomy techniques. Whole blood was immediately analyzed for a complete blood count while serum vacutainer tubes were centrifuged at room temperature for 15 min at 1,500 g, the serum supernatant was transferred into microcentrifuge tubes, and the serum samples were stored at -20°C for subsequent hormonal and metabolite analyses. On days 0 and 50 only, subjects donated approximately 60 mg of skeletal muscle from the vastus lateralis using the Bergstrom biopsy technique. Upper and lower body strength were assessed using standard 1RM testing procedures  with a bench press and 35° hip sled machine (Nebula, Versailles, OH). Test-retest reliability of these strength tests on resistance-trained subjects in our laboratory have yielded a high reliability for the bench press (r = 0.94) and leg press (r = 0.91). After determination of hip sled 1RM, subjects rested 10 minutes before completing a 30 seconds Wingate anaerobic capacity test on a computerized cycle ergometer (Lode Excalibur, Lode, Groningen, The Netherlands) to assess lower body anaerobic power. This test consisted of having each subject sprint in an all out fashion on the bicycle ergometer for 30 seconds against a standard workload of 0.075 kg·kg-1 of body weight. Test-retest reliability for absolute peak power and mean power in our laboratory have also yielded high reliability values (r = 0.69 and r = 0.95, respectively, P < 0.05).
Percutaneous muscle biopsies
Muscle biopsies were taken on days 0 and 50 prior to all strength testing to avoid potential myofibrillar disruption due to exercise . Subjects were instructed to refrain from exercise 48 hours prior to each muscle biopsy. Muscle was extracted from the lateral portion of the vastus lateralis midway between the patella and iliac crest of the dominant leg using a 5 mm biopsy needle with applied suction . Briefly, 1.5 ml of 1.0% Lidocaine HCl was injected subcutaneously prior to making a small pilot incision. Using double-chop procedures and applied suction, the specimen first had all visible fat and connective tissue removed prior to being flash frozen in liquid nitrogen. All samples were subsequently stored at -80°C until later analyses.
Supplementation protocol and dietary monitoring
In a double-blind fashion, subjects ingested four 250 mg capsules containing a corn oil placebo or AA (X-Factor, Molecular Nutrition, Jupiter, FL) over 50 days following baseline testing. Supplements were prepared in capsule form and packaged in generic bottles by Molecular Nutrition. Compliance was monitored by having subjects return empty supplement bottles after 25 and 50 days of supplementation. In accordance with previous guidelines and in an effort to ensure energy and protein intake were adequate to facilitate muscle hypertrophy, all subjects were instructed to increase caloric intake by approximately 500 kcal·day-1 while also maintaining an estimated protein intake of 2 g·kg-1·day-1 when compared to baseline dietary analysis . Subjects were provided a commercially-available meal replacement powder (Lean Body, Labrada Nutrition, Houston, TX) containing approximately 290 kilocalories, 24 g of carbohydrate, 45 g of protein and 1 g of fat per serving in an attempt to accommodate the above mentioned energy and protein requirements. Depending on baseline protein intake, subjects were told to ingest 1 to 2 packets of the meal replacement supplement in the morning and/or immediately following each workout . Additionally, subjects were instructed to avoid regular consumption of foods known to be high in ω-3 fatty acids including fish oil, flaxseed oil, cold water fish, olive oil, sesame oil, peanut butter, N-acetyl-cysteine, conjugated linoleic acid, as well as anti-inflammatory medications including acetaminophen, ibuprofen, aspirin and other non-steroidal anti-inflammatory drugs . Dietary intake as well as linoleic (18:2, ω-6), linolenic (18:3, ω-3), and AA intake were monitored with 4-day dietary recalls at days 0, 25 and 50 and assessed using the Food Processor III Nutrition Software (ESHA Nutrition Research, Salem, OR).
Over a 50-day period, subjects completed a 4 day·week-1 split-body, linear periodization resistance-training program. Upper body lifts included bench press, lat pull, shoulder press, seated rows, shoulder shrugs, chest flies, biceps curls, and triceps press-downs while lower body lifts included leg press, back extension, step ups, leg curls, leg extension, heel raises, and abdominal crunches twice per week. Subjects performed 3 sets of 10 repetitions with as much weight as they could lift per set (i.e., 60–80% of 1RM). Rest periods between exercises did not exceed 3 minutes, while the rest between sets did not exceed 2 minutes. Training was conducted at the university's student life center, documented in training logs, and signed off by designated staff members to verify compliance and monitor progress. This protocol has been shown in prior research to promote significant gains in muscular strength, muscular endurance, and fat free mass .
Serum and whole blood analyses
Serum and whole blood samples were used to evaluate clinical safety during the supplementation protocols. Serum samples were assayed for comprehensive metabolic panels including glucose, total protein, blood urea nitrogen (BUN), creatinine, BUN/creatinine ratio, uric acid, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), lactate dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGT), albumin, globulin, sodium, chloride, calcium, carbon dioxide, total bilirubin, alkaline phosphatase (ALP), triglycerides, cholesterol, HDL and LDL using a Dade Dimension XL clinical chemistry system (Dade Behring Inc., Newark, DE). Complete blood cell counts including red cell counts, hemoglobin, hematocrit, mean cell volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, white blood cell counts, neutrophils, lymphocytes, monocytes, eosinophils, and basophils were analyzed via flow cytometry using the Cell-DYN 1800 (Abbott Laboratories, Abbott Park, IL). Test to test reliability (within and between) of performing these assays ranged from 2 to 6% for individual assays with an average coefficient of variation (C
) of 3.0%. Samples were run in duplicate to verify results if the observed values were outside control values and/or clinical norms according to standard procedures.
Subsequent serum samples were later assayed for cortisol (CORT), free testosterone (fTEST), total testosterone (tTEST), interleukin-6 (IL-6), prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). Commerical enzyme immunoabsorbent assays were used to analyze serum concentrations of PGF2α, PGE2, and IL-6 (Cayman Chemical, Ann Arbor, MI) and CORT, fTEST, and tTEST (Diagnostic Systems Laboratories, Webster, TX). The C
of performing these EIA-based assays ranged from 3.0 to 5.0%.
Total RNA isolation
Total cellular RNA was extracted from the homogenate of biopsy samples with a monophasic solution of phenol and guanidine isothiocyanate contained within the TRI-reagent (Sigma Chemical Co., St. Louis, MO) [26–30]. Total RNA concentrations from each sample were determined spectrophotometrically with an optical density of 260 nm (OD260), with final concentration adjusted to 200 ng·μl-1 by diluting the crude total RNA extracts into DEPC-treated nuclease-free H2O. This procedure has been shown to yield un-degraded RNA, free of DNA and proteins as indicated by prominent 28S and 18S ribosomal RNA bands, as well as an OD260/OD280 ratio of approximately 2.0 [26–30]. The RNA samples were stored at -80°C until later analyses.
Reverse transcription and clonal DNA synthesis
The standardized solutions of total cellular RNA were reverse transcribed to synthesize clonal DNA (cDNA) as described previously [26–30]. In short, a reverse transcription reaction mixture [i.e., 1 μl of total cellular RNA, 4 μl 5× reverse transcription buffer, a dNTP mixture containing dATP, dCTP, dGTP, and dTTP, MgCl2, RNase inhibitor, an oligo(dT)15 primer, 10 μL of nuclease-free H2O and 1 U·μl-1 MMLV reverse transcriptase enzyme (Bio-Rad, Hercules, CA)] were incubated at 42°C for 40 minutes, heated to 85°C for 5 minutes, and then quick-chilled on ice yielding the cDNA product. Starting cDNA template concentrations were standardized to 200 ng·μl-1 prior to real-time polymerase chain reaction (RT-PCR) amplification by detecting crude cDNA synthesized products spectrophotometrically at a wavelength of 260 nm and diluting them in nuclease-free H2O. The standardized cDNA solutions were frozen at -80°C until real-time RT-PCR was performed.
Anti-sense and sense oligonucleotide primer pairs were constructed using commercially available Beacon Designer software (Bio-Rad, Hercules, CA) from known mRNA sequences published in the GenBank nucleotide database  and commercially synthesized (Integrated DNA Technologies, Coralville, IA). The following 5' sense and 3' anti-sense oligonucleotide primers were used to isolate the three adult MHC isoforms (Type I, IIa, and IIx): Type I MHC mRNA (5' primer: bases 776–796, 3' primer: bases 1398-1378, GenEMBL AC X06976), Type IIa MHC mRNA (5' primer: bases 1785–1805, 3' primer: bases 2440-2420, GenEMBL AC AF111784), Type IIx MHC mRNA (5' primer: bases 1138–1158, 3' primer: bases 1746-1726, GenEMBL AC AF111785). These primers amplify fragments of 141, 145, and 148 base pairs, respectively, for Type I, IIa, IIx MHC. β-actin was used as an external reference standard for detecting relative change in the quantity of target mRNA due to its consideration as a constitutively expressed housekeeping gene , These β-actin primers amplify a PCR fragment of 135 base pairs. Two hundred ng of cDNA was added to each of the four PCR reactions for MHC Type I, -IIa, and -IIx, and β-actin. Specifically, each PCR reaction contained the following mixtures: 2 μl of cDNA template was added along with 12.5 μl of 2× SYBR Green Supermix (Bio-Rad, Hercules, CA) [100 mM KCl mixture, 40 mM Tris-HCl, 0.4 mM of each dNTP, 50 U·ml-1 of iTaq DNA polymerase, 6.0 mM MgCl2, SYBR Green I, 20 nM flourescein], 1.5 μl of sense and anti-sense primers and 7.5 μl nuclease-free dH2O]. Each PCR reaction was amplified with a thermal cycler (Bio Rad, Hercules, CA) and the amplification sequence involved a denaturation step at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, and extension at 72°C for 60 seconds [27, 33, 34]. RT-PCR was performed over 40 cycles with emitted fluorescence from the SYBR green fluorophore being measured after each cycle. An emission of fluorescence occurs due to the integration of the SYBR green into the double-stranded cDNA produced during the PCR reaction. All CT values were assessed in the linear portion of amplification and a DNA melting curve analysis was performed after amplification to assure that the single gene products were amplified in absence of primer-dimers. Quantification of all mRNA was expressed relative to β-actin expression. A comparison of CT value ratios [Day 0 (MHC mRNA CT/β-actin mRNA CT) versus Day 50 (MHC mRNA CT/β-actin mRNA CT)] were used to compare changes in basal gene expression between the AA and PLA groups. Agarose gel electrophoresis using 25 μl aliquots of the finalized PCR reaction mixtures was performed in 1.5% agarose gels [1 μg·ml-1] using 1× Tris-Boric acid-EDTA (TBE) buffer and illuminated with a UV transilluminator (Chemi-Doc XRS, Bio-Rad, Hercules, CA) to verify positive amplification of target mRNA (data not shown) [33, 34]. The C
for MHC I, IIa, and IIx were 2.06%, 3.18%, and 2.73%, respectively .
Total muscle protein quantitation
Total protein remaining from the total RNA isolation procedure was isolated with isopropanol, ethanol, and 0.3 M guanidine hydrochloride. Myofibrillar protein was isolated with 0.1% sodium dodecyl sulfate (SDS) , prior to having protein content determined spectrophotometrically using a Bradford assay at a wavelength of 595 nm. A standard curve was generated using (r2 = 0.98, P < 0.001) bovine serum albumin as the standard and represented relative to muscle wet weight . Each protein sample was subsequently diluted to 50 μg of protein per 30 μl SDS buffer for subsequent immunoblotting. The C
for myofibrillar protein was 2.03% .
MHC protein isoform quantitation
The composition of MHC protein isoforms within each muscle homogenate sample was determined by automated SDS-PAGE using Experion Pro260 chips (Bio-Rad, Hercules, CA). Approximately 6 μl aliquots of each sample were pipetted into each sample well on the microchip. Each unknown sample was prepared from 4 μl of the protein dilution from each subject (or 4 μl of the molecular weight ladder), 2 μl of sample buffer with β-mercaptoethanol, and 84 μl of de-ionized water. Based upon the findings of Gazith and colleagues , all three MHC isoforms were expected to migrate in the 200–210 kiloDaulton region within the polyacrylamide gel relative to the molecular weight ladder. The gels were digitally visualized by the Experion software (Bio-Rad, Hercules, CA) and MHC concentrations in each sample were assessed by comparing the arbitrary density of each MHC isoform to the arbitrary densities of molecular weight markers with known concentrations. The C
of protein bands ≥10 kD were ≤1.1%.
PGF2α (FP) and PGE2 (EP3) receptor quantitation was performed at room temperature by extracting total muscle protein from the homogenate and slot-blotting 50 μg of total protein onto nitrocellulose membranes using a Bio-Dot protein blotting system (Bio-Rad, Hercules, CA). The blotted membranes were incubated with blocking solution for 1 hour on an orbital rocker, decanted and membranes were incubated with a TTBS wash solution for 5 minutes for a total of three washes. The membranes were incubated with specific anti-FP receptor and anti-EP3 receptor polyclonal antibodies (Cayman Chemical, Ann Arbor, MI), diluted to 4 μg·ml-1, for 1 to 2 hours on an orbital rocker. Primary antibody solutions were then decanted and the membranes washed with TTBS solution for 5 minutes on an orbital rocker for a total of three washes. The TTBS wash solution was decanted and the membranes were incubated with a secondary biotinylated goat anti-rabbit antibody solution (Bio-Rad, Hercules, CA) for 1 hour on an orbital rocker. Continuing, the secondary biotinylated goat anti-rabbit antibody solution was decanted and the membranes incubated in TTBS wash solution for a total of three washes at 5 minutes per wash. The membranes were incubated with a streptavidin-biotinylated alkaline phosphatase complex solution (Bio-Rad, Hercules, CA) for 1 hour on an orbital rocker. Finally, the streptavidin-biotinylated alkaline phosphatase complex solution was decanted and the membranes washed three times with TTBS solution at 5 minutes per wash on an orbital shaker. Color development solution containing BCIP/NBT (Bio-Rad, Hercules, CA) was added and color development was monitored over 30 – 60 minutes. The color development was stopped by incubating the membrane in double distilled H2O for 10 minutes on an orbital rocker. Blotted membranes were digitized by way of densitometry using a Chemi-Doc XRS imaging system (Bio-Rad, Hercules, CA) and band density was expressed in integrated density units relative to muscle weight.
Statistical analyses were performed using SPSS (version 14.0, SPSS Inc., Chicago, IL). Whole blood, serum, performance, and body composition variables were analyzed using 2 × 3 (group × testing session) analysis of variance (ANOVA) with repeated measures univariate tests. MHC protein, FP and EP3 receptor protein levels were analyzed using separate 2 × 2 (group × testing session) ANOVA with repeated measures. Additionally, in the case of significant main effect for group, one-way ANOVAs with repeated measures on testing sessions was performed for each group to assess any differences between tests. Independent t-tests were used to analyze the changes in MHC mRNA expression after 50 days of supplementation. In addition to raw score analysis, delta score analysis (i.e. day 0 values subtracted from day 25 and/or 50) was performed for variables that exhibited extraneous variation between groups on day 0. As mentioned with raw scores, a 2 × 3 (group × testing session) analysis of variance (ANOVA) with repeated measures univariate tests was used to analyze delta scores for body composition, performance variables, and hormone concentrations whereas a 2 × 2 repeated measures ANOVA was used to analyze all intramuscular delta scores. In circumstances where equal variances within groups could not be assumed, the Hunyhs-Feldt epsilon correction factor was used to adjust within group F-ratios. In circumstances where statistical trends appeared to exist (i.e., P = 0.05 to 0.10), effect sizes were also reported as partial Eta squared (ηp2). Partial Eta squared effect sizes were determined to be weak (ηp2 ≤ 0.01), medium (ηp2 = 0.06), strong = (ηp2 = 0.14) as previously described . Significance for all statistical analyses was determined using an alpha level of 0.05. It should be noted that an a priori power analysis of the design indicated that an n-size of 15 participants per treatment would yield a high power (> 0.8) for criterion variable delta values of 0.75 to 1.25. It should also be noted that post hoc outlier analysis using box plots was performed in circumstances where there were significant group × time interactions to ensure there were no outliers present. All data are reported as means ± standard deviations (SD).