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Different metabolism of EPA, DPA and DHA in humans: A double-blind cross-over study
Prostaglandins, Leukotrienes and Essential Fatty Acids xxx (xxxx) xxxx
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Original research article
Different metabolism of EPA, DPA and DHA in humans: A double-blind cross-over study Xiao-fei Guoa,1, Wen-feng Tongb,1, Yue Ruanb, Andrew J. Sinclairc,d, Duo Lia,b,
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a
Institute of Nutrition and Health, Qingdao University, Qingdao, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou, China c Faculty of Health, Deakin University, Geelong, Australia d Department of Nutrition, Dietetics and Food, Monash University, Melbourne, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Eicosapentaenoic acid Docosapentaenoic acid Docosahexaenoic acid Blood lipid fraction Metabolomics
This study aimed to compare eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) incorporated into red blood cells (RBC) phospholipids (PL), plasma PL, plasma triglyceride (TAG), and plasma cholesteryl ester (CE) fractions, and the metabolomics profiles in a double-blind cross-over study. Twelve female healthy subjects randomly consumed 1 g per day for 6 days of pure EPA, DPA, or DHA. The placebo treatment was olive oil. The fasting venous blood was taken at days 0, 3 and 6, and the RBC PL and plasma lipid fractions were separated for fatty acid determination using thin layer chromatography followed by gas chromatography. Plasma metabolites were analyzed by UHPLC-Q-Exactive Orbitrap/MS. Supplemental EPA significantly increased the concentrations of EPA in RBC PL (days 3 and 6). For subjects consuming the DPA supplement, the concentrations of both DPA and EPA were significantly increased in RBC PL over a 6-day period, respectively. For plasma PL fraction, EPA and DPA supplementation significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. Supplemental DHA significantly increased the concentrations of DHA in plasma PL at day 6. For plasma TAG fraction, supplementation with EPA and DPA significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. After DHA supplementation, significant increases in the concentrations of DHA were found relative to baseline at both days 3 and 6. For plasma CE fraction, EPA supplementation significantly increased the concentrations of EPA (days 3 and 6) and DPA (days 6), respectively. Supplemental DPA significantly increased the concentrations of EPA at day 6. Meanwhile, the concentrations of DHA were significantly increased over a 6-day period of intervention after subjects consuming the DHA supplements. There were a total of 922 plasma metabolites identified using metabolomics analyses. Supplementation with DPA and DHA significantly increased the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DHA = 0.021) in comparison with olive oil group. Additionally, supplementation with EPA (P = 0.007) and DHA (P = 0.005) significantly reduced the levels of linoleyl carnitine, compared with olive oil group. This study shows that DPA might act as a reservoir of n-3 LCP incorporated into blood lipid fractions, metabolized into DHA, and retroconverted back to EPA. Metabolomics analyses indicate that supplemental EPA, DPA and DHA have shared and differentiated metabolites. The differences of these metabolic biomarkers should be investigated in additional studies.
1. Introduction Considerable research supporting the beneficial biological effects of n-3 long-chain polyunsaturated fatty acids (LCP) have shown that these LCP contribute to preventing and improving chronic disease outcomes, including metabolic disease, cardiovascular diseases (CVD), non-
alcoholic fatty liver disease and some kinds of cancers [1–3]. A large proportion of these studies have focused on fish oils, which commonly contain three kinds of n-3 LCP, namely eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). Increasing literature has revealed that these three n-3 LCP have independent and shared biological benefits [4,5]. With the development
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Corresponding author at: Institute of Nutrition and Health, Qingdao University, Qingdao, China. E-mail addresses: [email protected], [email protected] (D. Li). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.plefa.2019.102033 Received 19 September 2019; Received in revised form 11 November 2019; Accepted 11 November 2019 0952-3278/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xiao-fei Guo, et al., Prostaglandins, Leukotrienes and Essential Fatty Acids, https://doi.org/10.1016/j.plefa.2019.102033
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4 treatment-arms as follows: (1) Control group consumed 2 ml olive oil; (2) EPA group consumed 2 ml EPA-olive oil mixture (1:1; W/W); 3) DPA group consumed 2 ml DPA-olive mixture (1:1; W/W) and 4) DHA group consumed 2 ml DHA-olive oil mixture (1:1; W/W). During the period of intervention, 2 ml oil was spread on the bread using an injection syringe. The subjects consumed the bread containing oil within 3 min in the presence of research staff. At the beginning, the fasting blood samples were collected, and the subjects consumed bread with 2 ml olive oil as breakfast for 6 consecutive days. Then, the subjects were randomized into 3 groups by computer-generated random numbers, and received EPA-olive oil, DPA-olive oil or DHA-olive oil with a 4-week period of washout between the three n-3 LCP treatments. The fasting blood samples were collected at days 0, 3 and 6, respectively.
of bulk chromatographic separations, there is ample research on the metabolism of purified EPA and DHA, whereas few studies have been conducted on pure DPA, due to a lack of availability [5]. As an intermediary product of EPA and DHA, DPA is mainly found in fish, marineorigin products and ruminant meats. Considering the difficulty in separation and purification of DPA from fish oil, few studies using pure DPA have been carried out to evaluate its biological benefits [6-8]. To date, extensive evidence from in vitro and in vivo studies has shown the beneficial roles of DPA in improving blood lipid profiles, inflammation, mental health, fat oxidation, and inhibiting platelet aggregation [6,9]. However, the knowledge is insufficient in relation to the metabolism of pure DPA in human studies, compared with EPA and DHA. A 6 weeks clinical trial has reported that supplemental seal oil (340 mg EPA, 230 mg DPA and 450 mg DHA) significantly increased the proportion of DPA in red blood cells (RBC) [10]. Since seal oil contained a significant amount of EPA and DHA, the incorporation of DPA in RBC is still ambiguous. Compared with EPA, pure DPA (in free fatty acid form) incorporated into blood lipids was investigated in a short-time period of cross-over study [6]. The previous findings demonstrated that DPA might be as a reservoir of n-3 LCP [6]. Two other studies using pure DPA and EPA revealed significant differences between DPA and EPA in chylomicron metabolism [8] and in the plasma lipid mediators produced [7]. Since the three kinds of n-3 LCP incorporated into human lipoproteins are varied substantially, it is important and necessary to explore these heterogeneities. No human trial to date has comprehensively compared the incorporation of these three n-3 LCP (DPA, EPA and DHA) into blood lipid fractions or the plasma metabolomics profiles following consumption of these LCP. Therefore, we conducted this study investigating the incorporation of the three n-3 LCP into blood lipid fractions, including phospholipid (PL) in RCB, and PL, triglyceride (TAG) and cholesteryl ester (CE) fractions in plasma. Additionally, the plasma metabolomics profiles in response to the three n-3 LCP were analyzed using ultra-high-performance liquid chromatography (UHPLC)-Q-Exactive Orbitrap/mass spectrometry (MS) in positive ionization mode (ESI+).
2.4. Fatty acid determination After 10 h fasting, blood samples were collected into EDTA-containing vacuum tubes for laboratory analyses. Plasma was separated by centrifugation (4000 rmp for 10 min at 4 °C) and stored at −40 °C refrigerator for further analysis. After removal of plasma, sodium chloride-washed RBC were aliquoted and stored at −40 °C in a refrigerator. Total of plasma lipids were extracted from plasma as described previously [11]. Briefly, 850 μL of plasma was extracted using 10 mL of chloroform/methanol (1:1; V/V) containing 10 mg/L of butylated hydroxytoluene (BHT) and reference internal standards, including phosphatidyl choline (PC)−17:0, triglyceride-17:0, cholesteryl ester-17:0 (Avanti Polar Lipids, USA). The neutral lipid classes were separated by thin layer chromatography (TLC), and the PL, TAG and CE fractions were scraped from the TLC plate and transmethylated with 0.9 mol/L concentrated sulfuric acid in methanol, and then the fatty acid methyl esters (FAME) were separated and determined by gas chromatography (GC-2014, Shimadzu, Japan) equipped with an AOC20i auto injector. The FAME peak was recognized in accordance with known external standards (Supleco, Bellefonte, USA). Total RBC lipids were extracted from 500 μL of RBC with chloroform/methanol (1:1; V/ V) containing 10 mg/L BHT and PC-17:0 as an internal standard. The PL fraction of red blood cell lipids was separated by TLC, and scraped from the TLC plate for transmethylation with 0.9 mol/L concentrated sulfuric acid in methanol. The resulting FAME were isolated and then separated and identified using gas chromatography as reported above.
2. MATERIALS and methods 2.1. Materials EPA (98%; W/W), DPA (72%; W/W), and DHA (97%; W/W) in ethyl ester form were kindly provided by Bizen Chemical Co., LTD (Okayama, Japan). The three n-3 LCP were diluted in olive oil for dietary supplementation, as follows: 80 g of each n-3 LCP were mixed with 80 g of olive oil in 200 ml glass bottles. The blended oils were sub-packed into 10 ml brown bottle glasses, sealed with nitrogen and stored at −4 °C refrigerator.
2.5. Plasma metabolomics determination The plasma samples both at baseline and at end of the intervention were prepared for metabolomics analysis with Thermo Scientific UltiMate 3000 UHPLC system coupled to Q Exactive equipped with a HESI source. A mixture of 150 μL plasma sample and 600 μL methanol was vigorously vortexed and centrifuged for 10 min at 12,000 rpm to remove particulates. The supernatant was filtered through a 0.22 μm membrane and transferred into autosampler vial, and then 5 μL of the solution was injected into the column (ACQUITY UPLC BEH C18 Column, 1.7 μm, 2.1 mm × 150 mm) for detection. The mobile phases consisted of eluent A (0.1% formic acid-water, V/V) and eluent B (0.1% formic acid-acetonitrile, V/V), and the elution gradient was described in previous study [12]. During sequence analysis, a quality control sample was prepared by combining each plasma sample to verify the reproducibility of analytes and the stability of the equipment. The original data were transferred into Compound Discover 2.1 data preprocessing. Features were classified as non-detected with the peak area less than 5000. Besides, features were conducted further analysis if they were existed in the quality samples with coefficient of variations less than 20% in the quality control sample. The identification of metabolites were based on accurate mass according to ChemSpider, Human Metabolome database, KEGG and mzCloud.
2.2. Subjects Female healthy subjects were recruited by internet advertisement at Zhejiang University. Written informed consent was obtained from individual subjects after approval of the clinical trial by College of Biosystems Engineering and Food Science at Zhejiang University, China (ZJU-BEFS-2015015). Subjects completed a medical questionnaire in the preliminary screening, and a 3-day dietary record during intervention. Subjects were instructed to restrict n-3 fatty acids intake during the period of supplementation, by excluding fish oil supplements, fatty fish, flax products, nuts, beef and lamb. The participants were also requested to maintain ordinary lifestyles and a constant body weight. 2.3. Procedures This study was a randomized double-blind cross-over study by using 2
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2.6. Statistical analysis For determination of fatty acid incorporated in blood lipid fractions, analysis of variance (ANOVA) for repeated measurements was performed to evaluate the effects of supplements, time, and the interaction of supplements and time. Meanwhile, Bonferroni's post hoc test was performed between the groups. Regarding plasma metabolomics analyses, principal component analysis (PCA) was implemented to visualize the clustering of the quality control samples. The metabolic data between baseline and endpoint of the specific intervention were discriminated by using supervised multivariate partial least squares discriminant analysis (PLS-DA). The peak areas of each feature were log10-transformed and performed to PCA and PLS-DA. The quality of the PLS-DA model was evaluated using R2Y (goodness of fit) and Q2Y (goodness of prediction) parameters, and Q2Y ≥ 0.4 was regarded as an acceptable criterion [13]. The selection of the metabolites was according to the variable influence on projection (VIP) value by using PLS-DA. The metabolite was selected for further analysis if the VIP value was higher than 2.0. The differentiated metabolites between baseline and endpoint plasma samples were assessed by paired Student's t-test or Wilcoxon (Mann-Whitney). For the acquired features in the positive-ionization mode, one-way ANOVA followed by Bonferroni's post hoc test was implemented to identify significant fold changed features between groups. A P-value of ≤ 0.05 was regarded as significant difference. The PCA and PLS-DA were conducted with SIMCA 14.1, and other statistical analysis was performed using Stata 13.0 for Windows (Stata Corp, College Station, TX, USA). 3. Results A total of 15 subjects were enrolled at Zhejiang University. They were free of dyslipidemia, metabolic syndrome, type 2 diabetes, and CVD, and took no medications. Due to personal reasons, three subjects withdrew from the trial. Finally, twelve healthy female subjects, aged between 18 and 28 years, finished this study. The included individuals had a mean age of 22 ± 2 years, and a BMI of 20.4 ± 1.5 kg/m2 (Table 1). No subject had reported abnormal physical reactions after n3 LCP intervention throughout the trial. According to a 3-day food record, none of the subjects consumed sea food or related products during the intervention periods. 3.1. Concentration of PL fatty acids in red blood cells After EPA supplementation, the concentrations of EPA were significantly increased relative to baseline at both days 3 and 6 (Fig. 1). For subjects consuming DPA, there were significant increases in the concentrations of DPA at day 6, compared with baseline (Fig. 1). Besides, in the DPA-treated group there were also significant increases in the concentrations of EPA at day 6, compared with baseline. There was Table 1 Characteristics of the included subjects. Variables
Values
Age, y BMI, Kg/m2 Weight (Kg) Height (cm) Total cholesterol (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) Triglycerides (mmol/L) Fasting glucose (mmol/L)
22 ± 2 20.4 ± 1.5 52.6 ± 4.8 160 ± 6 2.78 ± 0.71 0.82 ± 0.25 1.32 ± 0.41 0.51 ± 0.08 3.44 ± 0.52
Fig. 1. Fatty acid concentration in red blood cell phospholipids (PL) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group. Abbreviation: OO, olive oil; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid.
Data are expressed as mean ± standard deviation (SD) (n = 12 per group). Abbreviations: HDL-C, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol. 3
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Fig. 3. Fatty acid concentration in plasma triglyceride (TAG) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect within each group, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group.
Fig. 2. Fatty acid concentration in plasma phospholipids (PL) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group.
4
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no significant increase in the concentration of DHA in the DHA supplemented group (Fig. 1). 3.2. Plasma PL fatty acids After EPA intervention, there were significant increases in the concentrations of EPA and DPA relative to baseline at both days 3 and 6, respectively (Fig. 2). Similarly, supplemental DPA showed significant increase in the concentrations of DPA and EPA relative to baseline at both days 3 and 6. Additionally, significant increases in the concentrations of DHA were found at day 6 compared with baseline for subjects consuming DHA (Fig. 2). 3.3. Plasma TAG fatty acids After EPA supplementation, the concentrations of EPA and DPA were significantly increased relative to baseline at both days 3 and 6 (Fig. 3). There were significant increases in the concentrations of DPA and EPA relative to baseline at both days 3 and 6 for subjects consuming the DPA supplements. Meanwhile, there were significant increases in the concentrations of DHA at day 3 compared with baseline for subjects consuming the DPA supplement. After DHA supplementation, there were significant increases in the concentrations of DHA relative to baseline at both days 3 and 6 (Fig. 3). 3.4. Plasma CE fatty acids There were significant increases in the concentrations of EPA relative to baseline at both days 3 and 6 for subjects consuming the EPA supplements, and the concentrations of DPA were significantly increased at day 6 compared with baseline (Fig. 4). After DPA supplementation, there were significant increases in the concentrations of EPA relative to baseline at days 3 and 6 (Fig. 4). For subjects consuming the DHA supplement, the concentrations of DHA were significantly increased relative to baseline at day 6 (Fig. 4). 3.5. Plasma metabolomics profiles Quality control samples mixed from each plasma sample were clustered, indicating the reproducibility of the instrument and the stability of original data acquisition. Based on untargeted metabolomics detection, a total of 922 features were obtained from positive ionization mode. There were clear separations between baseline and endpoint in response to olive oil, EPA, DPA and DHA interventions, respectively (Supplementary Figs. 1–4). The values of R2Y and Q2Y in PLS-DA models were all over 0.6, suggesting relatively high predictability of these models (Table 2). Three metabolites were found to be statistically significant differences between groups (Table 3). Of these, supplemental DPA and DPA groups showed significant increases in the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DPA = 0.021), compared with olive oil group. In addition, supplementation with EPA and DHA groups were associated with reduced levels of linoleyl carnitine (P for EPA = 0.007; P for DHA = 0.004) in comparison with olive oil group (Table 3). Fig. 4. Fatty acid concentration in plasma cholesteryl ester (CE) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group.
4. Discussion Incorporation of EPA, DPA and DHA in blood lipid fractions was systematically investigated in the present study. As intermediary product of EPA and DHA, a short period of DPA supplementation could retro-convert back to EPA and further metabolize into DHA. Supplemental EPA showed significant increases in the concentrations of DPA in plasma PL, TAG and CE fractions. Non-significant retro-conversion of DHA to DPA was found in blood lipid fractions after DHA supplementation. Recently, Metherel et al. have questioned whether
retro-conversion actually happens in humans. They suggest that rather than retro-conversion, the DHA is preventing forward metabolism of EPA to DHA [14]. Consistent with the evidence of previous study, DPA might be as a reservoir of EPA and DHA [6]. Metabolomics analyses 5
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TAG. The results were consistent with the previous published data [6]. The tissues of animal models also supported that DPA as a pool of EPA and DHA [17,18]. It has been reported that supplementation with EPA and DHA for 7 days significantly increased the composition of EPA and DHA in plasma TAG, respectively [19]. Another study showed that the composition of DHA in plasma TAG significantly increased as a result of DHA supplementation over 14 weeks; however, non-significant difference was found in relation to the composition of DPA [20]. Further study needs to explore whether a longer period of DHA supplementation could increase the concentration of DPA in plasma TAG. The present showed supplementation with EPA significantly increased the concentrations of EPA, and which were further elongated to DPA in plasma CE. Using an EPA-rich fish oil as intervention, the concentration of EPA also significantly increased in plasma CE within 30 days [21]. The concentrations of DHA were significantly increased after DHA supplementation over 6 days. However, the increase of DHA in the plasma CE was limited relative to the amount supplied. The finding of the current study was in keeping with the data of BronsgeestSchoute et al. [22], Vidgren et al. [20], and Katan et al. [21]. Who also found that DHA was not efficiently incorporated into plasma CE. As an important sphingolipid metabolite, extensive research has shown that sphingosine 1-phosphate is a potent lipid which participates in a variety of physiological processing, such as cardiac development, diabetes mellitus, inflammation, immunity, tumorigenesis, and cell proliferation [23–25]. Also, it has been shown to regulate a diverse set of biological responses by binding to G protein-coupled receptors [26]. The present findings indicated that supplemental DPA and DHA significantly increased the levels of sphingosine 1-phosphate compared with olive oil group, suggesting that sphingosine 1-phosphate might be a biomarker in response to n-3 LCP supplementation. 15-deoxy-Δ12,14prostaglandin A1 is a synthetic prostaglandin A1 analog. It shares common structural feature in comparison with 15-deoxy-Δ12,14-prostaglandin J2. Although no clinical trials have reported the biological activity of 15-deoxy-Δ12,14-prostaglandin A1, extensive studies found that 15-deoxy-Δ12,14-prostaglandin J2 has exerted anti-tumor activities and anti-inflammatory effects [27,28]. Thus, further studies are warranted to confirm whether the circulating 5-deoxy-Δ12,14-prostaglandin A1 are associated with chronic diseases in observational studies and clinical trials. Linoleyl carnitine, a long-chain acylcarntine, is shown to be an indispensable intermediate involved in transporting fatty acids from cytosol to mitochondrial matrix for beta-oxidation. A previous clinical trial indicated that the levels of plasma long-chain acylcarnitines were significant higher in subjects with obesity and type 2 diabetes, compared with lean subjects [29]. Also, a population-based prospective cohort study showed that plasma long-chain acylcarnitines were positively associated with risk of diabetes [30]. Additionally, the levels of linoleyl carnitine were significantly higher in patients with chronic liver failure, compared with healthy individuals. Similarly, the levels of linoleyl carnitine were significantly higher in non-alcoholic fatty liver patients with type 2 diabetes in comparison with control group [31]. The accumulation of long-chain acylcarnitines in plasma derived from incomplete fatty acid oxidation might be responsible for dysregulation of fatty acid oxidation in mitochondria [30]. Therefore,
Table 2 Summary of parameters from PLS-DA models for detecting metabolomics profiles. Intervention
R2Y
Q2Y
Olive oil EPA DPA DHA
0.665 0.626 0.611 0.607
0.999 0.995 0.991 0.983
Abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoic Acid; DPA, docosapentaenoic acid; PLS-DA, partial least squares discriminant analysis. R2Y indicated the goodness of fit, and Q2Y indicated goodness of prediction.
indicated that supplemental DPA and DHA were positively associated with increased levels of sphingosine 1-phosphate and 15-deoxy-Δ12,14prostaglandin A1, compared with olive oil group. Additionally, the levels of linoleyl carnitine were showed to be reduced in EPA and DHA groups in comparison with olive group. In our study, DPA supplementation significantly increased the concentrations of DPA and further retro-conversed to EPA in RBC PL at day 6 compared with baseline. However, another short-time period of study indicated that DPA intervention did not significantly increase the composition of DPA in the RBC PL [6]. The inconsistent results might be attributed to two reasons. First, DPA in ethyl ester form was used in the present study, compared with DPA as free fatty acid in the previous study.6 They might have different bioavailability when incorporating in RBC. Besides, the previous study measured the composition of DPA in the RBC PL [6]. By contrast, our study calculated the concentrations of DPA in the RBC PL using C17:0 PC as a reference internal standard. Supplementation with EPA, DPA and DHA for 6 days, revealed there were significant increases in the concentrations of EPA, DPA and DHA compared with the baseline values in plasma PL fractions, respectively. Meanwhile, EPA was metabolized into DPA, and the retro-conversion of DPA to EPA was found after supplemental DPA. Similar results indicated that a high dose of pure EPA supplementation (4 g per day) for 6 weeks significantly increased the composition of EPA as well as DPA in plasma PL [15]. Supplemental seal oil, which contain a higher proportion of DPA than fish oils, showed a significant increase in the proportion of DPA in plasma PL after 6 weeks intervention [16]. Meanwhile, there was a significant increase in the composition of EPA as well as DHA [16]. In current study, no pronounced change was found regarding concentrations of DHA in plasma PL as a result of DPA supplementation. Consistent with our result, the composition of DHA was not significantly increased after DPA supplementation over a 7-day trial in a previous study [6]. The metabolism of DPA to DHA needs an elongation to 24:5n-3 and desaturation to 24:6n-3, and involves betaoxidation to yield DHA [8]. The level of DHA might be significantly increased in the plasma PL over an extended period as a result of DPA supplementation. The baseline concentrations of the three kinds of n-3 LCP in plasma TAG fraction were apparently lower compared with plasma PL and CE fractions. DPA acts as a reservoir of EPA and DHA. It could be metabolized into DHA and further retro-converted back to EPA in plasma
Table 3 Characteristics of identified metabolites with significant fold changes (P < 0.05) in EPA, DPA and DHA groups compared with olive group. Mode
Detected Mass (m/ z)
Retention time (min)
Formula
HESI+ HESI+
299.282 318.219
16.297 13.547
C18H37NO2 C20H30O3
HESI+
423.334
15.870
C25H45NO4
Putative identification
Sphingosine 1-phosphate 15-deoxy-Δ12,14prostaglandin A1 Linoleyl carnitine
P-ANOVAa
EPA Fold change
Pb
DPA Fold change
Pb
DHA Fold change
Pb
1.23 ± 0.05 1.17 ± 0.11
0.064 0.113
1.30 ± 0.30 1.24 ± 0.29
0.025 0.034
1.34 ± 0.21 1.26 ± 0.26
0.029 0.021
0.012 0.011
0.85 ± 0.18
0.007
0.93 ± 0.09
0.107
0.84 ± 0.16
0.005
0.004
Abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoic Acid; DPA, docosapentaenoic acid; HESI+, positive-ion mode. P-ANOVAa, one-way ANOVA comparing fold changes during interventions between groups. Pb, post hoc Bonferroni's test for comparing fold changes of EPA, DPA and DHA groups with the olive oil group. 6
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the circulating linoleyl carnitine level might be an independent biomarker in predicting type 2 diabetes and liver-related diseases, and that this finding needs to be replicated in other studies. The limitations of this study should be acknowledged. First, because pure DPA has not been readily available, the sample-size is relatively small. Although cross-over study design was adopted to eliminate individual variation, the selective and inherent biases might affect the present findings. Second, the DPA was only 72% pure, and it contained other PUFAs, including 4.8% EPA, 4.8% 21:5n-3, 6% 22:5n-6 and 8% DHA [32]. Thus, these other PUFAs might have played roles in fatty acid metabolism. Besides, supplementation with the three n-3 LCP lasted only for 6 days. A longer period of supplementation might have contributed to identify more abundant biomarkers. In conclusion, this cross-over study demonstrated that the three n-3 LCP were differentially incorporated into RBC and plasma lipid fractions. In the context of this study, DPA as a reservoir could be metabolized into DHA, and further retro-conversed back to EPA. Supplementation with EPA was efficiently incorporated into plasma lipid fractions, whereas incorporation of DHA into plasma lipid fractions was limited within a short period of supplement. Several identified metabolites following n-3 LCP supplementation are associated with inflammation and fatty acid oxidation. Whether these metabolites could be as biomarkers in predicting chronic diseases warrant further investigation.
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
Sources of support
[18]
This work is supported by the National Basic Research Program of China (973 Program: 2015CB553604); by National Natural Science Foundation of China (NSFC: 81773433); by the Key Scientific Research Projects in Shandong Provence China (2017YYSP007); and by the 2018 Chinese Nutrition Society (CNS) Nutrition Research Foundation-DSM Research Fund (CNS-DSM2018A30). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[19]
[20]
[21]
Declaration of Competing Interest There are no conflicts to declare.
[22]
Acknowledgments
[23]
We thank all the participants for their contributions in the present study.
[24]
[25]
Supplementary materials [26]
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2019.102033.
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Original research article
Different metabolism of EPA, DPA and DHA in humans: A double-blind cross-over study Xiao-fei Guoa,1, Wen-feng Tongb,1, Yue Ruanb, Andrew J. Sinclairc,d, Duo Lia,b,
⁎
a
Institute of Nutrition and Health, Qingdao University, Qingdao, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou, China c Faculty of Health, Deakin University, Geelong, Australia d Department of Nutrition, Dietetics and Food, Monash University, Melbourne, Australia b
A R T I C LE I N FO
A B S T R A C T
Keywords: Eicosapentaenoic acid Docosapentaenoic acid Docosahexaenoic acid Blood lipid fraction Metabolomics
This study aimed to compare eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) incorporated into red blood cells (RBC) phospholipids (PL), plasma PL, plasma triglyceride (TAG), and plasma cholesteryl ester (CE) fractions, and the metabolomics profiles in a double-blind cross-over study. Twelve female healthy subjects randomly consumed 1 g per day for 6 days of pure EPA, DPA, or DHA. The placebo treatment was olive oil. The fasting venous blood was taken at days 0, 3 and 6, and the RBC PL and plasma lipid fractions were separated for fatty acid determination using thin layer chromatography followed by gas chromatography. Plasma metabolites were analyzed by UHPLC-Q-Exactive Orbitrap/MS. Supplemental EPA significantly increased the concentrations of EPA in RBC PL (days 3 and 6). For subjects consuming the DPA supplement, the concentrations of both DPA and EPA were significantly increased in RBC PL over a 6-day period, respectively. For plasma PL fraction, EPA and DPA supplementation significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. Supplemental DHA significantly increased the concentrations of DHA in plasma PL at day 6. For plasma TAG fraction, supplementation with EPA and DPA significantly increased the concentrations of EPA and DPA at both days 3 and 6, respectively. After DHA supplementation, significant increases in the concentrations of DHA were found relative to baseline at both days 3 and 6. For plasma CE fraction, EPA supplementation significantly increased the concentrations of EPA (days 3 and 6) and DPA (days 6), respectively. Supplemental DPA significantly increased the concentrations of EPA at day 6. Meanwhile, the concentrations of DHA were significantly increased over a 6-day period of intervention after subjects consuming the DHA supplements. There were a total of 922 plasma metabolites identified using metabolomics analyses. Supplementation with DPA and DHA significantly increased the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DHA = 0.021) in comparison with olive oil group. Additionally, supplementation with EPA (P = 0.007) and DHA (P = 0.005) significantly reduced the levels of linoleyl carnitine, compared with olive oil group. This study shows that DPA might act as a reservoir of n-3 LCP incorporated into blood lipid fractions, metabolized into DHA, and retroconverted back to EPA. Metabolomics analyses indicate that supplemental EPA, DPA and DHA have shared and differentiated metabolites. The differences of these metabolic biomarkers should be investigated in additional studies.
1. Introduction Considerable research supporting the beneficial biological effects of n-3 long-chain polyunsaturated fatty acids (LCP) have shown that these LCP contribute to preventing and improving chronic disease outcomes, including metabolic disease, cardiovascular diseases (CVD), non-
alcoholic fatty liver disease and some kinds of cancers [1–3]. A large proportion of these studies have focused on fish oils, which commonly contain three kinds of n-3 LCP, namely eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and docosahexaenoic acid (DHA). Increasing literature has revealed that these three n-3 LCP have independent and shared biological benefits [4,5]. With the development
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Corresponding author at: Institute of Nutrition and Health, Qingdao University, Qingdao, China. E-mail addresses: [email protected], [email protected] (D. Li). 1 Both authors contributed equally to this work. https://doi.org/10.1016/j.plefa.2019.102033 Received 19 September 2019; Received in revised form 11 November 2019; Accepted 11 November 2019 0952-3278/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xiao-fei Guo, et al., Prostaglandins, Leukotrienes and Essential Fatty Acids, https://doi.org/10.1016/j.plefa.2019.102033
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4 treatment-arms as follows: (1) Control group consumed 2 ml olive oil; (2) EPA group consumed 2 ml EPA-olive oil mixture (1:1; W/W); 3) DPA group consumed 2 ml DPA-olive mixture (1:1; W/W) and 4) DHA group consumed 2 ml DHA-olive oil mixture (1:1; W/W). During the period of intervention, 2 ml oil was spread on the bread using an injection syringe. The subjects consumed the bread containing oil within 3 min in the presence of research staff. At the beginning, the fasting blood samples were collected, and the subjects consumed bread with 2 ml olive oil as breakfast for 6 consecutive days. Then, the subjects were randomized into 3 groups by computer-generated random numbers, and received EPA-olive oil, DPA-olive oil or DHA-olive oil with a 4-week period of washout between the three n-3 LCP treatments. The fasting blood samples were collected at days 0, 3 and 6, respectively.
of bulk chromatographic separations, there is ample research on the metabolism of purified EPA and DHA, whereas few studies have been conducted on pure DPA, due to a lack of availability [5]. As an intermediary product of EPA and DHA, DPA is mainly found in fish, marineorigin products and ruminant meats. Considering the difficulty in separation and purification of DPA from fish oil, few studies using pure DPA have been carried out to evaluate its biological benefits [6-8]. To date, extensive evidence from in vitro and in vivo studies has shown the beneficial roles of DPA in improving blood lipid profiles, inflammation, mental health, fat oxidation, and inhibiting platelet aggregation [6,9]. However, the knowledge is insufficient in relation to the metabolism of pure DPA in human studies, compared with EPA and DHA. A 6 weeks clinical trial has reported that supplemental seal oil (340 mg EPA, 230 mg DPA and 450 mg DHA) significantly increased the proportion of DPA in red blood cells (RBC) [10]. Since seal oil contained a significant amount of EPA and DHA, the incorporation of DPA in RBC is still ambiguous. Compared with EPA, pure DPA (in free fatty acid form) incorporated into blood lipids was investigated in a short-time period of cross-over study [6]. The previous findings demonstrated that DPA might be as a reservoir of n-3 LCP [6]. Two other studies using pure DPA and EPA revealed significant differences between DPA and EPA in chylomicron metabolism [8] and in the plasma lipid mediators produced [7]. Since the three kinds of n-3 LCP incorporated into human lipoproteins are varied substantially, it is important and necessary to explore these heterogeneities. No human trial to date has comprehensively compared the incorporation of these three n-3 LCP (DPA, EPA and DHA) into blood lipid fractions or the plasma metabolomics profiles following consumption of these LCP. Therefore, we conducted this study investigating the incorporation of the three n-3 LCP into blood lipid fractions, including phospholipid (PL) in RCB, and PL, triglyceride (TAG) and cholesteryl ester (CE) fractions in plasma. Additionally, the plasma metabolomics profiles in response to the three n-3 LCP were analyzed using ultra-high-performance liquid chromatography (UHPLC)-Q-Exactive Orbitrap/mass spectrometry (MS) in positive ionization mode (ESI+).
2.4. Fatty acid determination After 10 h fasting, blood samples were collected into EDTA-containing vacuum tubes for laboratory analyses. Plasma was separated by centrifugation (4000 rmp for 10 min at 4 °C) and stored at −40 °C refrigerator for further analysis. After removal of plasma, sodium chloride-washed RBC were aliquoted and stored at −40 °C in a refrigerator. Total of plasma lipids were extracted from plasma as described previously [11]. Briefly, 850 μL of plasma was extracted using 10 mL of chloroform/methanol (1:1; V/V) containing 10 mg/L of butylated hydroxytoluene (BHT) and reference internal standards, including phosphatidyl choline (PC)−17:0, triglyceride-17:0, cholesteryl ester-17:0 (Avanti Polar Lipids, USA). The neutral lipid classes were separated by thin layer chromatography (TLC), and the PL, TAG and CE fractions were scraped from the TLC plate and transmethylated with 0.9 mol/L concentrated sulfuric acid in methanol, and then the fatty acid methyl esters (FAME) were separated and determined by gas chromatography (GC-2014, Shimadzu, Japan) equipped with an AOC20i auto injector. The FAME peak was recognized in accordance with known external standards (Supleco, Bellefonte, USA). Total RBC lipids were extracted from 500 μL of RBC with chloroform/methanol (1:1; V/ V) containing 10 mg/L BHT and PC-17:0 as an internal standard. The PL fraction of red blood cell lipids was separated by TLC, and scraped from the TLC plate for transmethylation with 0.9 mol/L concentrated sulfuric acid in methanol. The resulting FAME were isolated and then separated and identified using gas chromatography as reported above.
2. MATERIALS and methods 2.1. Materials EPA (98%; W/W), DPA (72%; W/W), and DHA (97%; W/W) in ethyl ester form were kindly provided by Bizen Chemical Co., LTD (Okayama, Japan). The three n-3 LCP were diluted in olive oil for dietary supplementation, as follows: 80 g of each n-3 LCP were mixed with 80 g of olive oil in 200 ml glass bottles. The blended oils were sub-packed into 10 ml brown bottle glasses, sealed with nitrogen and stored at −4 °C refrigerator.
2.5. Plasma metabolomics determination The plasma samples both at baseline and at end of the intervention were prepared for metabolomics analysis with Thermo Scientific UltiMate 3000 UHPLC system coupled to Q Exactive equipped with a HESI source. A mixture of 150 μL plasma sample and 600 μL methanol was vigorously vortexed and centrifuged for 10 min at 12,000 rpm to remove particulates. The supernatant was filtered through a 0.22 μm membrane and transferred into autosampler vial, and then 5 μL of the solution was injected into the column (ACQUITY UPLC BEH C18 Column, 1.7 μm, 2.1 mm × 150 mm) for detection. The mobile phases consisted of eluent A (0.1% formic acid-water, V/V) and eluent B (0.1% formic acid-acetonitrile, V/V), and the elution gradient was described in previous study [12]. During sequence analysis, a quality control sample was prepared by combining each plasma sample to verify the reproducibility of analytes and the stability of the equipment. The original data were transferred into Compound Discover 2.1 data preprocessing. Features were classified as non-detected with the peak area less than 5000. Besides, features were conducted further analysis if they were existed in the quality samples with coefficient of variations less than 20% in the quality control sample. The identification of metabolites were based on accurate mass according to ChemSpider, Human Metabolome database, KEGG and mzCloud.
2.2. Subjects Female healthy subjects were recruited by internet advertisement at Zhejiang University. Written informed consent was obtained from individual subjects after approval of the clinical trial by College of Biosystems Engineering and Food Science at Zhejiang University, China (ZJU-BEFS-2015015). Subjects completed a medical questionnaire in the preliminary screening, and a 3-day dietary record during intervention. Subjects were instructed to restrict n-3 fatty acids intake during the period of supplementation, by excluding fish oil supplements, fatty fish, flax products, nuts, beef and lamb. The participants were also requested to maintain ordinary lifestyles and a constant body weight. 2.3. Procedures This study was a randomized double-blind cross-over study by using 2
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2.6. Statistical analysis For determination of fatty acid incorporated in blood lipid fractions, analysis of variance (ANOVA) for repeated measurements was performed to evaluate the effects of supplements, time, and the interaction of supplements and time. Meanwhile, Bonferroni's post hoc test was performed between the groups. Regarding plasma metabolomics analyses, principal component analysis (PCA) was implemented to visualize the clustering of the quality control samples. The metabolic data between baseline and endpoint of the specific intervention were discriminated by using supervised multivariate partial least squares discriminant analysis (PLS-DA). The peak areas of each feature were log10-transformed and performed to PCA and PLS-DA. The quality of the PLS-DA model was evaluated using R2Y (goodness of fit) and Q2Y (goodness of prediction) parameters, and Q2Y ≥ 0.4 was regarded as an acceptable criterion [13]. The selection of the metabolites was according to the variable influence on projection (VIP) value by using PLS-DA. The metabolite was selected for further analysis if the VIP value was higher than 2.0. The differentiated metabolites between baseline and endpoint plasma samples were assessed by paired Student's t-test or Wilcoxon (Mann-Whitney). For the acquired features in the positive-ionization mode, one-way ANOVA followed by Bonferroni's post hoc test was implemented to identify significant fold changed features between groups. A P-value of ≤ 0.05 was regarded as significant difference. The PCA and PLS-DA were conducted with SIMCA 14.1, and other statistical analysis was performed using Stata 13.0 for Windows (Stata Corp, College Station, TX, USA). 3. Results A total of 15 subjects were enrolled at Zhejiang University. They were free of dyslipidemia, metabolic syndrome, type 2 diabetes, and CVD, and took no medications. Due to personal reasons, three subjects withdrew from the trial. Finally, twelve healthy female subjects, aged between 18 and 28 years, finished this study. The included individuals had a mean age of 22 ± 2 years, and a BMI of 20.4 ± 1.5 kg/m2 (Table 1). No subject had reported abnormal physical reactions after n3 LCP intervention throughout the trial. According to a 3-day food record, none of the subjects consumed sea food or related products during the intervention periods. 3.1. Concentration of PL fatty acids in red blood cells After EPA supplementation, the concentrations of EPA were significantly increased relative to baseline at both days 3 and 6 (Fig. 1). For subjects consuming DPA, there were significant increases in the concentrations of DPA at day 6, compared with baseline (Fig. 1). Besides, in the DPA-treated group there were also significant increases in the concentrations of EPA at day 6, compared with baseline. There was Table 1 Characteristics of the included subjects. Variables
Values
Age, y BMI, Kg/m2 Weight (Kg) Height (cm) Total cholesterol (mmol/L) HDL-cholesterol (mmol/L) LDL-cholesterol (mmol/L) Triglycerides (mmol/L) Fasting glucose (mmol/L)
22 ± 2 20.4 ± 1.5 52.6 ± 4.8 160 ± 6 2.78 ± 0.71 0.82 ± 0.25 1.32 ± 0.41 0.51 ± 0.08 3.44 ± 0.52
Fig. 1. Fatty acid concentration in red blood cell phospholipids (PL) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group. Abbreviation: OO, olive oil; EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid.
Data are expressed as mean ± standard deviation (SD) (n = 12 per group). Abbreviations: HDL-C, high-density lipoprotein cholesterol; LDLC, low-density lipoprotein cholesterol. 3
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Fig. 3. Fatty acid concentration in plasma triglyceride (TAG) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect within each group, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group.
Fig. 2. Fatty acid concentration in plasma phospholipids (PL) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group.
4
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no significant increase in the concentration of DHA in the DHA supplemented group (Fig. 1). 3.2. Plasma PL fatty acids After EPA intervention, there were significant increases in the concentrations of EPA and DPA relative to baseline at both days 3 and 6, respectively (Fig. 2). Similarly, supplemental DPA showed significant increase in the concentrations of DPA and EPA relative to baseline at both days 3 and 6. Additionally, significant increases in the concentrations of DHA were found at day 6 compared with baseline for subjects consuming DHA (Fig. 2). 3.3. Plasma TAG fatty acids After EPA supplementation, the concentrations of EPA and DPA were significantly increased relative to baseline at both days 3 and 6 (Fig. 3). There were significant increases in the concentrations of DPA and EPA relative to baseline at both days 3 and 6 for subjects consuming the DPA supplements. Meanwhile, there were significant increases in the concentrations of DHA at day 3 compared with baseline for subjects consuming the DPA supplement. After DHA supplementation, there were significant increases in the concentrations of DHA relative to baseline at both days 3 and 6 (Fig. 3). 3.4. Plasma CE fatty acids There were significant increases in the concentrations of EPA relative to baseline at both days 3 and 6 for subjects consuming the EPA supplements, and the concentrations of DPA were significantly increased at day 6 compared with baseline (Fig. 4). After DPA supplementation, there were significant increases in the concentrations of EPA relative to baseline at days 3 and 6 (Fig. 4). For subjects consuming the DHA supplement, the concentrations of DHA were significantly increased relative to baseline at day 6 (Fig. 4). 3.5. Plasma metabolomics profiles Quality control samples mixed from each plasma sample were clustered, indicating the reproducibility of the instrument and the stability of original data acquisition. Based on untargeted metabolomics detection, a total of 922 features were obtained from positive ionization mode. There were clear separations between baseline and endpoint in response to olive oil, EPA, DPA and DHA interventions, respectively (Supplementary Figs. 1–4). The values of R2Y and Q2Y in PLS-DA models were all over 0.6, suggesting relatively high predictability of these models (Table 2). Three metabolites were found to be statistically significant differences between groups (Table 3). Of these, supplemental DPA and DPA groups showed significant increases in the levels of sphingosine 1-phosphate (P for DPA = 0.025, P for DHA = 0.029) and 15-deoxy-Δ12,14-prostaglandin A1 (P for DPA = 0.034; P for DPA = 0.021), compared with olive oil group. In addition, supplementation with EPA and DHA groups were associated with reduced levels of linoleyl carnitine (P for EPA = 0.007; P for DHA = 0.004) in comparison with olive oil group (Table 3). Fig. 4. Fatty acid concentration in plasma cholesteryl ester (CE) for subjects consuming olive oil, EPA, DPA or DHA for a consecutive 6 days. Data are expressed as percentage mean ± standard deviation (SD) (n = 12 per group). The superscripts with capital letters represent intervention effect, and different letters indicate significant difference within each group. The superscripts with small letters represent time effect within each group.
4. Discussion Incorporation of EPA, DPA and DHA in blood lipid fractions was systematically investigated in the present study. As intermediary product of EPA and DHA, a short period of DPA supplementation could retro-convert back to EPA and further metabolize into DHA. Supplemental EPA showed significant increases in the concentrations of DPA in plasma PL, TAG and CE fractions. Non-significant retro-conversion of DHA to DPA was found in blood lipid fractions after DHA supplementation. Recently, Metherel et al. have questioned whether
retro-conversion actually happens in humans. They suggest that rather than retro-conversion, the DHA is preventing forward metabolism of EPA to DHA [14]. Consistent with the evidence of previous study, DPA might be as a reservoir of EPA and DHA [6]. Metabolomics analyses 5
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TAG. The results were consistent with the previous published data [6]. The tissues of animal models also supported that DPA as a pool of EPA and DHA [17,18]. It has been reported that supplementation with EPA and DHA for 7 days significantly increased the composition of EPA and DHA in plasma TAG, respectively [19]. Another study showed that the composition of DHA in plasma TAG significantly increased as a result of DHA supplementation over 14 weeks; however, non-significant difference was found in relation to the composition of DPA [20]. Further study needs to explore whether a longer period of DHA supplementation could increase the concentration of DPA in plasma TAG. The present showed supplementation with EPA significantly increased the concentrations of EPA, and which were further elongated to DPA in plasma CE. Using an EPA-rich fish oil as intervention, the concentration of EPA also significantly increased in plasma CE within 30 days [21]. The concentrations of DHA were significantly increased after DHA supplementation over 6 days. However, the increase of DHA in the plasma CE was limited relative to the amount supplied. The finding of the current study was in keeping with the data of BronsgeestSchoute et al. [22], Vidgren et al. [20], and Katan et al. [21]. Who also found that DHA was not efficiently incorporated into plasma CE. As an important sphingolipid metabolite, extensive research has shown that sphingosine 1-phosphate is a potent lipid which participates in a variety of physiological processing, such as cardiac development, diabetes mellitus, inflammation, immunity, tumorigenesis, and cell proliferation [23–25]. Also, it has been shown to regulate a diverse set of biological responses by binding to G protein-coupled receptors [26]. The present findings indicated that supplemental DPA and DHA significantly increased the levels of sphingosine 1-phosphate compared with olive oil group, suggesting that sphingosine 1-phosphate might be a biomarker in response to n-3 LCP supplementation. 15-deoxy-Δ12,14prostaglandin A1 is a synthetic prostaglandin A1 analog. It shares common structural feature in comparison with 15-deoxy-Δ12,14-prostaglandin J2. Although no clinical trials have reported the biological activity of 15-deoxy-Δ12,14-prostaglandin A1, extensive studies found that 15-deoxy-Δ12,14-prostaglandin J2 has exerted anti-tumor activities and anti-inflammatory effects [27,28]. Thus, further studies are warranted to confirm whether the circulating 5-deoxy-Δ12,14-prostaglandin A1 are associated with chronic diseases in observational studies and clinical trials. Linoleyl carnitine, a long-chain acylcarntine, is shown to be an indispensable intermediate involved in transporting fatty acids from cytosol to mitochondrial matrix for beta-oxidation. A previous clinical trial indicated that the levels of plasma long-chain acylcarnitines were significant higher in subjects with obesity and type 2 diabetes, compared with lean subjects [29]. Also, a population-based prospective cohort study showed that plasma long-chain acylcarnitines were positively associated with risk of diabetes [30]. Additionally, the levels of linoleyl carnitine were significantly higher in patients with chronic liver failure, compared with healthy individuals. Similarly, the levels of linoleyl carnitine were significantly higher in non-alcoholic fatty liver patients with type 2 diabetes in comparison with control group [31]. The accumulation of long-chain acylcarnitines in plasma derived from incomplete fatty acid oxidation might be responsible for dysregulation of fatty acid oxidation in mitochondria [30]. Therefore,
Table 2 Summary of parameters from PLS-DA models for detecting metabolomics profiles. Intervention
R2Y
Q2Y
Olive oil EPA DPA DHA
0.665 0.626 0.611 0.607
0.999 0.995 0.991 0.983
Abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoic Acid; DPA, docosapentaenoic acid; PLS-DA, partial least squares discriminant analysis. R2Y indicated the goodness of fit, and Q2Y indicated goodness of prediction.
indicated that supplemental DPA and DHA were positively associated with increased levels of sphingosine 1-phosphate and 15-deoxy-Δ12,14prostaglandin A1, compared with olive oil group. Additionally, the levels of linoleyl carnitine were showed to be reduced in EPA and DHA groups in comparison with olive group. In our study, DPA supplementation significantly increased the concentrations of DPA and further retro-conversed to EPA in RBC PL at day 6 compared with baseline. However, another short-time period of study indicated that DPA intervention did not significantly increase the composition of DPA in the RBC PL [6]. The inconsistent results might be attributed to two reasons. First, DPA in ethyl ester form was used in the present study, compared with DPA as free fatty acid in the previous study.6 They might have different bioavailability when incorporating in RBC. Besides, the previous study measured the composition of DPA in the RBC PL [6]. By contrast, our study calculated the concentrations of DPA in the RBC PL using C17:0 PC as a reference internal standard. Supplementation with EPA, DPA and DHA for 6 days, revealed there were significant increases in the concentrations of EPA, DPA and DHA compared with the baseline values in plasma PL fractions, respectively. Meanwhile, EPA was metabolized into DPA, and the retro-conversion of DPA to EPA was found after supplemental DPA. Similar results indicated that a high dose of pure EPA supplementation (4 g per day) for 6 weeks significantly increased the composition of EPA as well as DPA in plasma PL [15]. Supplemental seal oil, which contain a higher proportion of DPA than fish oils, showed a significant increase in the proportion of DPA in plasma PL after 6 weeks intervention [16]. Meanwhile, there was a significant increase in the composition of EPA as well as DHA [16]. In current study, no pronounced change was found regarding concentrations of DHA in plasma PL as a result of DPA supplementation. Consistent with our result, the composition of DHA was not significantly increased after DPA supplementation over a 7-day trial in a previous study [6]. The metabolism of DPA to DHA needs an elongation to 24:5n-3 and desaturation to 24:6n-3, and involves betaoxidation to yield DHA [8]. The level of DHA might be significantly increased in the plasma PL over an extended period as a result of DPA supplementation. The baseline concentrations of the three kinds of n-3 LCP in plasma TAG fraction were apparently lower compared with plasma PL and CE fractions. DPA acts as a reservoir of EPA and DHA. It could be metabolized into DHA and further retro-converted back to EPA in plasma
Table 3 Characteristics of identified metabolites with significant fold changes (P < 0.05) in EPA, DPA and DHA groups compared with olive group. Mode
Detected Mass (m/ z)
Retention time (min)
Formula
HESI+ HESI+
299.282 318.219
16.297 13.547
C18H37NO2 C20H30O3
HESI+
423.334
15.870
C25H45NO4
Putative identification
Sphingosine 1-phosphate 15-deoxy-Δ12,14prostaglandin A1 Linoleyl carnitine
P-ANOVAa
EPA Fold change
Pb
DPA Fold change
Pb
DHA Fold change
Pb
1.23 ± 0.05 1.17 ± 0.11
0.064 0.113
1.30 ± 0.30 1.24 ± 0.29
0.025 0.034
1.34 ± 0.21 1.26 ± 0.26
0.029 0.021
0.012 0.011
0.85 ± 0.18
0.007
0.93 ± 0.09
0.107
0.84 ± 0.16
0.005
0.004
Abbreviations: EPA, eicosapentaenoic acid; DHA, docosahexaenoic Acid; DPA, docosapentaenoic acid; HESI+, positive-ion mode. P-ANOVAa, one-way ANOVA comparing fold changes during interventions between groups. Pb, post hoc Bonferroni's test for comparing fold changes of EPA, DPA and DHA groups with the olive oil group. 6
Prostaglandins, Leukotrienes and Essential Fatty Acids xxx (xxxx) xxxx
X.-f. Guo, et al.
the circulating linoleyl carnitine level might be an independent biomarker in predicting type 2 diabetes and liver-related diseases, and that this finding needs to be replicated in other studies. The limitations of this study should be acknowledged. First, because pure DPA has not been readily available, the sample-size is relatively small. Although cross-over study design was adopted to eliminate individual variation, the selective and inherent biases might affect the present findings. Second, the DPA was only 72% pure, and it contained other PUFAs, including 4.8% EPA, 4.8% 21:5n-3, 6% 22:5n-6 and 8% DHA [32]. Thus, these other PUFAs might have played roles in fatty acid metabolism. Besides, supplementation with the three n-3 LCP lasted only for 6 days. A longer period of supplementation might have contributed to identify more abundant biomarkers. In conclusion, this cross-over study demonstrated that the three n-3 LCP were differentially incorporated into RBC and plasma lipid fractions. In the context of this study, DPA as a reservoir could be metabolized into DHA, and further retro-conversed back to EPA. Supplementation with EPA was efficiently incorporated into plasma lipid fractions, whereas incorporation of DHA into plasma lipid fractions was limited within a short period of supplement. Several identified metabolites following n-3 LCP supplementation are associated with inflammation and fatty acid oxidation. Whether these metabolites could be as biomarkers in predicting chronic diseases warrant further investigation.
[8]
[9]
[10]
[11]
[12]
[13] [14]
[15]
[16]
[17]
Sources of support
[18]
This work is supported by the National Basic Research Program of China (973 Program: 2015CB553604); by National Natural Science Foundation of China (NSFC: 81773433); by the Key Scientific Research Projects in Shandong Provence China (2017YYSP007); and by the 2018 Chinese Nutrition Society (CNS) Nutrition Research Foundation-DSM Research Fund (CNS-DSM2018A30). The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[19]
[20]
[21]
Declaration of Competing Interest There are no conflicts to declare.
[22]
Acknowledgments
[23]
We thank all the participants for their contributions in the present study.
[24]
[25]
Supplementary materials [26]
Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2019.102033.
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