Abstract

Plasmalogens are membrane glycerophospholipids with diverse biological functions. Reduced plasmalogen levels have been observed in metabolic diseases; hence, increasing their levels might be beneficial in ameliorating these conditions. Shark liver oil (SLO) is a rich source of alkylglycerols that can be metabolized into plasmalogens. This study was designed to evaluate the impact of SLO supplementation on endogenous plasmalogen levels in individuals with features of metabolic disease. In this randomized, double-blind, placebo-controlled cross-over study, the participants (10 overweight or obese males) received 4-g Alkyrol® (purified SLO) or placebo (methylcellulose) per day for 3 weeks followed by a 3-week washout phase and were then crossed over to 3 weeks of the alternate placebo/Alkyrol® treatment. SLO supplementation led to significant changes in plasma and circulatory white blood cell lipidomes, notably increased levels of plasmalogens and other ether lipids. In addition, SLO supplementation significantly decreased the plasma levels of total free cholesterol, triglycerides, and C-reactive protein. These findings suggest that SLO supplementation can enrich plasma and cellular plasmalogens and this enrichment may provide protection against obesity-related dyslipidemia and inflammation.

Supplementary key wordsAbbreviations: BH (Benjamini-Hochberg), COH (cholesterol), hsCRP (high-sensitivity C-reactive protein), LPC(O) (lysoalkylphosphatidylcholine), PC (phosphatidylcholine), PC(O) (alkyl phosphatidylcholine), PC(P) (alkenyl phosphatidylcholine), PE(O) (alkyl phosphatidylethanolamine), PE(P) (alkenyl phosphatidylethanolamine), SLO (shark liver oil), TG(O) (monoalkyl-diacylglycerol)Metabolic disease refers to a group of complex chronic conditions including obesity, type 2 diabetes, cardiovascular disease, and certain forms of cancer (1Paul S. Lancaster G.I. Meikle P.J. Plasmalogens: A potential therapeutic target for neurodegenerative and cardiometabolic disease.). These disorders share some common pathogenic features including altered lipid metabolism or dyslipidemia, which often leads to lipid accumulation at diverse cellular/tissue locations. Such aberrant lipid accumulation alters cell and/or tissue function, inducing events such as oxidative stress and inflammation that contribute to disease pathogenesis. Lipidomic profiling provides the opportunity to identify novel lipid signatures in metabolic diseases and explore their relationship with disease pathogenesis (2Sphingolipids and phospholipids in insulin resistance and related metabolic disorders.). Using this approach, a deficit of circulating plasmalogens has been identified as a feature of metabolic disease that is independent of age, sex, and BMI in multiple population and clinical cohorts (3Alshehry Z.H. Mundra P.A. Barlow C.K. Mellett N.A. Wong G. McConville M.J. Simes J. Tonkin A.M. Sullivan D.R. Barnes E.H. Nestel P.J. Kingwell B.A. Marre M. Neal B. Poulter N.R. et al.Plasma lipidomic profiles improve on traditional risk factors for the prediction of cardiovascular events in type 2 diabetes mellitus., 4Meikle P.J. Wong G. Barlow C.K. Weir J.M. Greeve M.A. MacIntosh G.L. Almasy L. Comuzzie A.G. Mahaney M.C. Kowalczyk A. Haviv I. Grantham N. Magliano D.J. Jowett J.B. Zimmet P. et al.Plasma lipid profiling shows similar associations with prediabetes and type 2 diabetes., 5Meikle P.J. Wong G. Tsorotes D. Barlow C.K. Weir J.M. Christopher M.J. MacIntosh G.L. Goudey B. Stern L. Kowalczyk A. Haviv I. White A.J. Dart A.M. Duffy S.J. Jennings G.L. et al.Plasma lipidomic analysis of stable and unstable coronary artery disease., 6Weir J.M. Wong G. Barlow C.K. Greeve M.A. Kowalczyk A. Almasy L. Comuzzie A.G. Mahaney M.C. Jowett J.B. Shaw J. Curran J.E. Blangero J. Meikle P.J. Plasma lipid profiling in a large population-based cohort., 7Moxon J.V. Liu D. Wong G. Weir J.M. Behl-Gilhotra R. Bradshaw B. Kingwell B.A. Meikle P.J. Golledge J. Comparison of the serum lipidome in patients with abdominal aortic aneurysm and peripheral artery disease.).Plasmalogens are a subclass of glycerophospholipids, primarily present as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species (8High plasmalogen and arachidonic acid content of canine myocardial sarcolemma: a fast atom bombardment mass spectroscopic and gas chromatography-mass spectroscopic characterization.). They consist of a vinyl ether–linked fatty alcohol at the sn1 position and an acyl-linked fatty acid at the sn2 position of the glycerol backbone (Fig. 1C). Plasmalogens are often esterified with polyunsaturated fatty acids such as arachidonic acid (20:4) and docosahexaenoic acid (22:6), whereas the vinyl ether–linked residue is usually saturated (e.g., O-16:0 or O-18:0) or monounsaturated (O-18:1) (9Plasmalogens: biosynthesis and functions.).

Fig. 1Structure of alkylglycerols and alkenyl phosphatidylethanolamine (plasmalogen). Alkylglycerols (A and B) are present in shark liver oil and can be metabolized into plasmalogens (C) in humans.

Plasmalogens have diverse biological functions. They are important constituents of the plasma membrane and can modulate its biophysical properties (10The biophysical properties of ethanolamine plasmalogens revealed by atomistic molecular dynamics simulations.). They are also considered as endogenous antioxidants because of their vinyl ether linkage, which is highly susceptible to attack by reactive oxygen species, and this could therefore be helpful in protecting other biomolecules from oxidative damage (11Zoeller R.A. Nagan N. Gaposchkin D.P. Legner M.A. Lieberthal W. Plasmalogens as endogenous antioxidants: somatic cell mutants reveal the importance of the vinyl ether., 12Plasmalogens: targets for oxidants and major lipophilic antioxidants.). In addition, plasmalogens may regulate cholesterol (COH) metabolism (13Munn N.J. Arnio E. Liu D. Zoeller R.A. Liscum L. Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport., 14Honsho M. Abe Y. Fujiki Y. Dysregulation of Plasmalogen Homeostasis Impairs Cholesterol Biosynthesis.) and immune responses (15Wallner S. Grandl M. Konovalova T. Sigrüner A. Kopf T. Peer M. Orsó E. Liebisch G. Schmitz G. Monocyte to macrophage differentiation goes along with modulation of the plasmalogen pattern through transcriptional regulation., 16Facciotti F. Ramanjaneyulu G.S. Lepore M. Sansano S. Cavallari M. Kistowska M. Forss-Petter S. Ni G. Colone A. Singhal A. Peroxisome-derived lipids are self antigens that stimulate invariant natural killer T cells in the thymus., 17Hu C. Zhou J. Yang S. Li H. Wang C. Fang X. Fan Y. Zhang J. Han X. Wen C. Oxidative stress leads to reduction of plasmalogen serving as a novel biomarker for systemic lupus erythematosus., 18Ifuku M. Katafuchi T. Mawatari S. Noda M. Miake K. Sugiyama M. Fujino T. Anti-inflammatory/anti-amyloidogenic effects of plasmalogens in lipopolysaccharide-induced neuroinflammation in adult mice.).Plasmalogens are present in all mammalian tissues; however, their abundance varies across tissues and cell types such that levels are relatively high in the brain, heart, kidney, skeletal muscle, and certain immune cell types but lower in the liver and small intestine (9Plasmalogens: biosynthesis and functions., 19Braverman N.E. Moser A.B. Functions of plasmalogen lipids in health and disease.). Plasmalogen biosynthesis involves a complex metabolic pathway through the peroxisome and endoplasmic reticulum (20Plasmalogens the neglected regulatory and scavenging lipid species.) (Fig. 2). The rate-limiting steps occur in the peroxisome but can be bypassed through oral administration of alkylglycerols (Figs. 1A and 2). These alkylglycerols can be incorporated directly into the biosynthetic pathway (21Brites P. Ferreira A.S. da Silva T.F. Sousa V.F. Malheiro A.R. Duran M. Waterham H.R. Baes M. Wanders R.J. Alkyl-glycerol rescues plasmalogen levels and pathology of ether-phospholipid deficient mice.) (Fig. 2) and lead to an increase in circulating and tissue plasmalogens (21Brites P. Ferreira A.S. da Silva T.F. Sousa V.F. Malheiro A.R. Duran M. Waterham H.R. Baes M. Wanders R.J. Alkyl-glycerol rescues plasmalogen levels and pathology of ether-phospholipid deficient mice., 22Das A.K. Holmes R.D. Wilson G.N. Hajra A.K. Dietary ether lipid incorporation into tissue plasmalogens of humans and rodents.). Although alkylglycerols are present in our diet, the levels in typical western diets are insufficient to significantly boost our plasmalogen levels. Shark liver oil (SLO), a dietary supplement rich in alkylglycerols in the form of monoalkyl-diacylglycerols (TG(O)) (Fig. 1B), could be used to increase endogenous plasmalogen levels (Fig. 2). SLO has been used to treat a number of conditions, including lung inflammation (23An update on the therapeutic role of alkylglycerols.), alimentary tract diseases (24Molina S. Moran-Valero M.I. Martin D. Vázquez L. Vargas T. Torres C.F. De Molina A.R. Reglero G. Antiproliferative effect of alkylglycerols as vehicles of butyric acid on colon cancer cells.), lymphadenopathy (25Bartfai E. Orsiere T. Duffaud F. Villani P. Pompili J. Botta A. Studies on the genotoxic effects of crude liver oils from 3 species of Mediterranean sharks by means of in vitro micronucleus test using human lymphocytes.), cancer (26Iagher F. de Brito Belo S.R. Souza W.M. Nunes J.R. Naliwaiko K. Sassaki G.L. Bonatto S.J. de Oliveira H.H. Brito G.A. de Lima C. Kryczyk M. de Souza C.F. Steffani J.A. Nunes E.A. Fernandes L.C. Antitumor and anti-cachectic effects of shark liver oil and fish oil: comparison between independent or associative chronic supplementation in Walker 256 tumor-bearing rats.)s and dermatitis (27Nowicki R. Barańska-Rybak W. Shark liver oil as a supporting therapy in atopic dermatitis.) and to help with wound healing (23An update on the therapeutic role of alkylglycerols.). SLO supplementation also improved immune function in surgical patients (28Palmieri B. Pennelli A. Di Cerbo A. Jurassic surgery and immunity enhancement by alkyglycerols of shark liver oil.). However, the mechanistic basis of the beneficial effects observed with SLO supplementation is not well defined, possibly because of the lack of proper understanding of the impact of SLO alkylglycerols on endogenous lipid metabolism.

Fig. 2Plasmalogen biosynthesis and modulation by alkylglycerol precursors. Dietary alkylglycerols can bypass the rate-limiting peroxisomal biosynthetic steps (red pathway). Metabolites are shown in red and black and enzymes are shown in blue. AADHAP-R, alkyl/acyl-DHAP-reductase; AAG3P-AT, alkyl/acyl-glycero-3-phosphate acyltransferase; ADHAP-S, alkyl DHAP synthase; AG kinase, alkylglycerol kinase; CoA, coenzyme A; CoA-IT, coenzyme A–independent transacylase; C-PT, choline phosphotransferase; i-phospholipase A2, calcium-independent phospholipase A2; Δ1-desaturase, plasmanylethanolamine desaturase; DHAP, dihydroxyacetone phosphate; DHAP-AT, DHAP acyltransferase; E-PT, ethanolamine phosphotransferase; Far1/2, fatty acyl-CoA reductase 1 or 2; GPC, glycerophosphocholine; GPE, glycerophosphoethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine N-methyltransferase; PH, phosphohydrolase; PLC, phospholipase C.

Here, we report on the characterization of the alkylglycerols contained within SLO and the effects of SLO supplementation on the plasma and cellular lipidome in overweight or obese individuals.

Materials and methods Study design for SLO supplementation in humansIn this double-blind, placebo-controlled crossover study, participants (n=10) were overweight or obese (BMI in the range of 28–40 kg/m2) adult males (aged 25–60 years) with no signs of cardiovascular disease or diabetes. Among the 10 participants, only four met the definition of having the metabolic syndrome according to the strict International Diabetes Federation criteria (29Alberti K.G. Zimmet P. Shaw J.E. Metabolic syndrome – a new world-wide definition. A consensus statement from the International Diabetes Federation.); however, all the participants had at least two features of metabolic syndrome. Written informed consent was obtained from all study participants before the commencement of the study. This study was performed in accordance with the ethical principles set forth in the Declaration of Helsinki and received approval from the Alfred Hospital Ethics Committee (approval number: 436/15).Participants were randomized into placebo or treatment arms and received 4-g Alkyrol® (purified SLO; Eurohealth, Ireland) per day or placebo (methylcellulose) for 3 weeks followed by a 3-week washout phase and were then crossed over to 3 weeks of the alternate placebo/Alkyrol® treatment. Methylcellulose was chosen as a placebo to avoid possible confounding effects of an oil-based placebo. Both Alkyrol® and methylcellulose capsules were prepared to have similar visual appearance. Participants were instructed to keep their dietary composition and food intake constant during the two treatment phases. Fasting blood samples were collected at the start and end of each intervention (Fig. 3).

Fig. 3Study design for shark liver oil supplementation in humans. Participants were recruited into the study and asked to attend an initial screening. At the screening visit, participants underwent a medical examination to assess their eligibility. Eligible participants were recalled, within three weeks, where they were randomized to take either Alkyrol® (shark liver oil gel caps) or placebo for three weeks. At the three-week visit, the participants discontinued the treatment/placebo for a three-week washout period. At visit 4, the participants commenced the alternative treatment for 3 weeks. At visit 5, the participants underwent the same medical examination as visit 1 to assess any change throughout the study period. Fasting blood samples from each participant were collected at the initial screening and at the start and end of each intervention.

 Isolation of plasma and white blood cells from whole blood

Participants’ blood samples were collected in K3-EDTA tubes and centrifuged at 1,711 g for 15 min at room temperature. The top plasma layer was aspirated, 1 μl of 100 mM butylhydroxytoluene per milliliter of plasma was added, and the plasma was stored at −80°C. The buffy layer was mixed with 8 ml of PBS and layered on top of 5 ml of Ficoll-Paque and centrifuged at 400 g for 30 min at room temperature with the lowest brake. The resulting upper layer (containing plasma and platelets) was discarded, and the thin cloudy layer of white blood cells was collected and transferred to a fresh tube. PBS (8 ml) was added, and the sample was centrifuged at 250 g for 10 min at room temperature with the highest brake. The cells were then resuspended in 1.5 ml of PBS and centrifuged at 100 g for 10 min at room temperature. After centrifugation, the supernatant was discarded and the white blood cell pellet was suspended in 400 μl PBS and stored at −80°C.

 Clinical measurements

The fasting plasma levels of glucose, COH, triglycerides, HDL-C, LDL-C, insulin, and high-sensitivity C-reactive protein (hsCRP) were measured using commercially available kits on a COBAS Integra 400 Plus blood chemistry analyzer (Roche Diagnostics, Australia) following standard procedures. Remnant COH was estimated as the total COH minus LDL-C minus HDL-C, non–HLD-C was calculated as total COH minus HDL-C, and homeostatic model assessment for insulin resistance was calculated as fasting insulin (mIU/l) multiplied by fasting glucose (mmol/l) and then divided by 22.5. The measurement of tumor necrosis factor alpha, monocyte chemoattractant protein-1, and vascular cell adhesion protein 1 levels in plasma was performed by Cardinal Bioresearch, Queensland, Australia.

 Complete blood count and flow cytometryComplete blood count was performed on a Sysmex XS-1000i automated haematology analyser following manufacturer’s guidelines. Monocyte subpopulations (classical (CD14++CD16−), intermediate (CD14+CD16+), and nonclassical (CD14dimCD16++)) were identified by staining peripheral blood cells with anti-human antibodies (BD Pharmingen, San Diego, CA) specific for CD56 (MY31, PE), CD2 (RPA-2.10, PE), CD19 (HIB19, PE), NKp46 (9E2/NKp46, PE), CD15 (W6D3, PE), HLA-DR (TU36, FITC), CD14 (M5E2, Pacific Blue), and CD16 (3G8, PE-CY7). Briefly, 100 μl of whole blood was added to 5 ml of the lysis buffer (BD Pharm Lyse) and then incubated in the dark for 5 min. The sample was then added to 10 ml of the wash buffer (9:1 ratio of PBS and fetal bovine serum) and centrifuged at 300 g for 5 min at 4°C. The resulting pellet was then resuspended in the wash buffer, placed in an Eppendorf tube, and centrifuged at (300 g, 5 min, room temperature). Antibodies were then added to the samples and incubated for 30 min in the dark. The samples were washed with PBS and centrifuged (300 g, 5 min, room temperature) before transferring the cells to a fluorescence activated cell sorting tube. Finally, the cells were analyzed using the BD FACSCanto II flow cytometer. The following gating strategy was used to define the various monocyte populations: white blood cells were initially gated based on size and granularity (forward scatter and side scatter). To identify monocytes, cells were gated on the basis of being HLA-DR+ and cell-linage marker (CD56, CD2, CD19, NKp46, and CD15) negative. HLA-DR+ cells were subsequently assessed for CD14 and CD16 expression, with classical monocytes being defined as CD14++CD16−, intermediate monocytes being defined as CD14+CD16+, and nonclassical monocytes being defined as CD14dimCD16++, as described previously (30Al-Sharea A. Lee M.K.S. Moore X.-L. Fang L. Sviridov D. Chin-Dusting J. Andrews K.L. Murphy A.J. Native LDL promotes differentiation of human monocytes to macrophages with an inflammatory phenotype.). The flow cytometry data were analyzed using the BD FACSDiva software. Lipidomic analysis Characterization of TG(O) species in SLOAlkyrol® was diluted 1:50,000 in chloroform:methanol (1:1) and infused into a QTRAP 4000 triple quadrupole mass spectrometer (AB Sciex) using a Harvard syringe pump at a flow rate of 20 μl/min, and a Q1 scan in positive-ion mode (mass range: 300–1,000 Da) was performed. The most abundant molecular species in SLO were then identified based on the peak intensity from the Q1 spectrum. For relative quantification of these species, Alkyrol® was diluted 10,000 times in chloroform:methanol (1:1) and 10 μl of diluted Alkyrol® was then mixed with 10 μl of internal standard mix (supplemental Table S1) and 40 μl of water-saturated butanol and 40 μl of methanol with 10 mM ammonium formate. The resultant mixture was then analyzed using the method described in the LC/MS/MS section. Characterization of alkylglycerol composition in SLOAlkylglycerols are present in Alkyrol® as TG(O), that is, consisting of one alkyl chain at the sn1 position and two acyl chains at sn2 and sn3 positions. The 1-O-alkylglycerol composition of Alkyrol® was determined after an alkaline hydrolysis of the acyl chains. In brief, Alkyrol® was diluted 10,000 times with chloroform:methanol (1:1) and 10 μl of the diluted samples was mixed with 100 μl of 0.8 M sodium hydroxide in methanol and then incubated at 37°C for 2 h. Then, 10 μl of 8 M formic acid was added to stop the hydrolysis reaction. Next, 10 μl of internal standard mix (supplemental Table S1) was added, and lipids were extracted following Folch extraction procedure (31Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.) and finally reconstituted with 50 μl of water-saturated butanol and 50 μl of methanol with 10 mM ammonium formate. The extract was then analyzed using the method described in the LC/MS/MS section. Extraction of lipids from plasma and white blood cellsLipids were extracted using a single-phase chloroform:methanol (2:1) extraction protocol as described previously (5Meikle P.J. Wong G. Tsorotes D. Barlow C.K. Weir J.M. Christopher M.J. MacIntosh G.L. Goudey B. Stern L. Kowalczyk A. Haviv I. White A.J. Dart A.M. Duffy S.J. Jennings G.L. et al.Plasma lipidomic analysis of stable and unstable coronary artery disease.). Briefly, 10 μl of plasma or 20 μl of white blood cell pellet (suspended in PBS) was combined with 20 volumes (200 or 400 μl) of chloroform:methanol (2:1) and 10 μl of the internal standard mix (supplemental Table S1) and then vortexed. Samples were mixed in a rotary mixer for 10 min, sonicated for 30 min, and then allowed to stand for 20 min at room temperature. Samples were then centrifuged (16,000 g, 10 min, 20°C), and the supernatant was dried under a stream of nitrogen at 40oC. The extracted lipids were finally resuspended with 50 μl of water-saturated butanol and 50 μl of methanol containing 10 mM ammonium formate. LC/MS/MSAnalysis of lipids were performed on an Agilent 1200 HPLC system coupled to an AB Sciex QTRAP 4000 triple quadrupole mass spectrometer using scheduled multiple reaction monitoring experiments described previously (32Huynh K. Barlow C.K. Jayawardana K.S. Weir J.M. Mellett N.A. Cinel M. Magliano D.J. Shaw J.E. Drew B.G. Meikle P.J. High-throughput plasma lipidomics: Detailed mapping of the associations with cardiometabolic risk factors.). LC separation was performed on a 2.1 × 100 mm C18 Poroshell column (Agilent) at 400 μl/min. The following gradient conditions were used: 10% B to 55% B over 3 min, then to 70% B over 8 min, to 89% B over 0.1 min, and finally to 100% B over 3.3 min. The solvent was then held at 100% B for 1 min. Equilibration was as follows: the solvent was decreased from 100% B to 10% B over 0.1 min and held for an additional 4.5 min. The solvent system consisted of solvent A: 50% water/30% acetonitrile/20% isopropanol (v/v/v) containing 10 mM ammonium formate and solvent B: 1% water/9% acetonitrile/90% isopropanol (v/v/v) containing 10 mM ammonium formate. The conditions for the MS/MS of each lipid class are provided in supplemental Table S1.The concentrations of individual lipid species were calculated by taking a ratio of the area under the curve of the lipid of interest to the area under the curve of the internal standard of the corresponding lipid class (supplemental Table S1) and then multiplying the said ratio by the amount of internal standard added into the sample. Response factors were also applied for some lipid species (supplemental Table S2) to better estimate true lipid concentrations as described previously (6Weir J.M. Wong G. Barlow C.K. Greeve M.A. Kowalczyk A. Almasy L. Comuzzie A.G. Mahaney M.C. Jowett J.B. Shaw J. Curran J.E. Blangero J. Meikle P.J. Plasma lipid profiling in a large population-based cohort.). Lipid class concentrations were calculated from the sum of individual species within that class. TG(O) species were measured both as single ion monitoring and neutral loss of specific fatty acyl/alkyl chains. As single ion monitoring measurements captured more diverse species, they were used for calculation of the concentration of total TG(O). Statistical analysis

Lipidomics data were either used as concentrations or as concentrations normalized to the total PC concentration. Zero values (i.e., values below the detection limit) and values more than 4.5 standard deviations below the mean of the considered lipid (i.e., extreme low outliers due to measurement errors around the detection limit) were set to missing. Values were log10-transformed before analyses. All missing values were then single-imputed using sample-wise k- nearest neighbor imputation (using k=5, given that only 10 participants were available at each time point). Modeling results for the lipids with imputed values may thus be considered as overconfident, although these results aligned well with results for other species in these classes.

For each lipid species or class as well as for clinical measures, blood cell count, monocyte subpopulations, and inflammatory markers, we posited linear mixed models explaining (log10) levels by an overall intercept, a treatment effect (either none or placebo/SLO at visits V3 and V5), and a carryover effect (either none or placebo/SLO at visit V4 only), with a random intercept for each participant. Contrasts for treatment and carryover effects were designed with across-group averaging vectors for use in the type-III ANOVAs below. The treatment contrast then compared placebo with baseline and SLO with placebo, whereas the carryover contrast compared placebo with none and SLO with none. For each species or total, the inclusion or exclusion of treatment or carryover effects was done by an ad hoc forward stepwise feature selection process: first, treatment was considered, and only included if the type-III ANOVA P value for that term was below 0.10, and then, carryover was considered in a similar way. Our modeling thus allowed carryover effects to be estimated independently of treatment effects. We allowed this as we reasoned that not all long-term effects (ie, at visit V4) of supplementation would necessarily reflect the direct effect of supplementation seen at visit V3: longer response times (ie, time taken for changes to be visible in the lipidome greater than the 3 weeks between visits V2 and V3), compensatory mechanisms, slow metabolism modifications, behavioral changes, and more might impact any carryover effect in a way unrelated to the treatment effect seen at V3. The similar pre-SLO and pre-placebo plasma ether lipid levels in the participants (supplemental Fig. S1) irrespective of their SLO treatment order (first or second) indicate that the 3-week washout period was sufficient.

We then extracted the beta coefficients, 95% confidence intervals, and corresponding post hoc P values from each model. We applied Benjamini-Hochberg (BH) multiple testing correction to said P values (because of the selection process above, the impact of the multiple testing correction was thus reduced for model terms that were less frequently included) across lipid species and classes separately. As the outcome was on a log scale, beta coefficients (and their confidence intervals) were transformed into fold changes by a power transformation (fc=10beta).

The mean percentage change of the alkenyl chain composition of plasma alkenyl phosphatidylethanolamine (PE plasmalogen or PE(P)) after Alkyrol® and placebo treatments were compared with repeated measures ANOVA using the combined data from the two intervention arms (visit 2 to 3 and visit 4 to 5), taking into account treatment (as a between-subject variable) and treatment order.

Corrected P values less than 0.05 were considered statistically significant. All analyses were performed in R (v3.5), in particular using packages lme4 (v1.1.21) and lmerTest (3.1.0) for the linear mixed modeling.

Results Co