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Original Research| Volume 28, ISSUE 1, P12-18, January 2023

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Reduced levels of serum EPA and DHA identified in patients with non-small-cell lung cancer using a new rapid validated LC-MS/MS method

  • Yi Wang
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Tongxin Yin
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Jiaoyuan Li
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Xia Luo
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Ke Liu
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Tingting Long
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Ying Shen
    Correspondence
    Corresponding authors at: Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China.
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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  • Liming Cheng
    Correspondence
    Corresponding authors at: Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China.
    Affiliations
    Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Wuhan 430030, PR China
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Open AccessPublished:November 30, 2022DOI:https://doi.org/10.1016/j.slasd.2022.11.004

      Highlights

      • A rapid and reliable LC-MS/MS method was developed and validated for the simultaneous quantification of EPA and DHA in human serum.
      • Serum concentrations of both EPA and DHA were lower in patients with NSCLC than in healthy subjects.
      • A consecutive downward trend in serum EPA and DHA levels with NSCLC progression was observed in patients with NSCLC.

      Abstract

      Background

      Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been suggested to play roles in various diseases, yet there is little data on their changes in patients with non-small-cell lung cancer (NSCLC). A simple LC-MS/MS method for EPA and DHA determination is critical to exploring EPA and DHA level changes in NSCLC patients.

      Methods

      25 µL of serum was mixed with 25 µL of internal standard working solution, and then 450 µL of acetonitrile for protein precipitation. After vortex and centrifugation, the supernatant was directly used for LC-MS/MS analysis. The method was well validated with linearity, precision, recovery, and matrix effect. The concentrations of EPA and DHA in serum samples from 211 NSCLC patients and 227 healthy controls were determined by this LC-MS/MS method.

      Results

      Good separation and reliable quantification of EPA and DHA in serum samples were achieved by our method. Compared with healthy controls, serum EPA and DHA were significantly reduced in both adenocarcinoma and squamous cell carcinoma patients. The concentrations of EPA and DHA showed a progressive decrease in healthy controls, early- and advanced-stage NSCLC patients.

      Conclusions

      This study identified significant reductions in serum EPA and DHA in NSCLC patients through the development of an LC-MS/MS method.

      Keywords

      Abbreviations:

      ACN (acetonitrile), ADC (adenocarcinoma), ALA (α-linolenic acid), CV (coefficients of variation), DHA (docosahexaenoic acid), EPA (eicosapentaenoic acid), GC-MS (gas chromatography-mass spectrometry), IQR (interquartile range), IS (internal standard), LC-MS (liquid chromatography-mass spectrometry), LC-MS/MS (liquid chromatography-tandem mass spectrometry), LLOQ (the lower limit of quantification), LOD (the limit of detection), MeOH (methanol), MF (matrix factor), MRM (multiple reactions monitoring), NSCLC (non-small-cell lung cancer), PQC (pooled quality control), PUFA (polyunsaturated fatty acid), QC (quality control), SCC (squamous cell carcinoma), SD (standard deviation), SNR (signal-to-noise ratio)

      1. Introduction

      EPA and DHA are long-chain omega-3 PUFAs that have been proved to play essential roles in various physiological processes, including triglycerides reduction, membrane lipid composition and eicosanoid biosynthesis, as well as cell signaling cascades and gene expression [
      • Nettleton J.A.
      Omega-3 fatty acids and health.
      ]. In humans, EPA and DHA can be synthesized from the essential fatty acid ALA. However, the primary source of EPA and DHA is diet, mostly through the consumption of marine foods, whereas ALA conversion only accounts for 2% to 10%. [
      • Tur J.A.
      • Bibiloni M.M.
      • Sureda A.
      • Pons A.
      Dietary sources of omega 3 fatty acids: public health risks and benefits.
      ].
      In vitro and in vivo studies have indicated that EPA and DHA are likely to affect the proliferation, migration and invasion of various types of tumors [
      • Calviello G.
      • Di Nicuolo F.
      • Gragnoli S.
      • Piccioni E.
      • Serini S.
      • Maggiano N.
      • Tringali G.
      • Navarra P.
      • Ranelletti F.O.
      • Palozza P.
      N-3 pufas reduce vegf expression in human colon cancer cells modulating the cox-2/pge2 induced erk-1 and -2 and hif-1alpha induction pathway.
      ,
      • Hu Y.
      • Sun H.
      • Owens R.T.
      • Gu Z.
      • Wu J.
      • Chen Y.Q.
      • O'Flaherty J.T.
      • Edwards I.J.
      Syndecan-1-dependent suppression of pdk1/akt/bad signaling by docosahexaenoic acid induces apoptosis in prostate cancer.
      ]. In NSCLC cell lines, exposure treatment with DHA was suggested to reduce the proliferation of A549 cells and trigger their apoptosis [
      • Trombetta A.
      • Maggiora M.
      • Martinasso G.
      • Cotogni P.
      • Canuto R.A.
      • Muzio G.
      Arachidonic and docosahexaenoic acids reduce the growth of a549 human lung-tumor cells increasing lipid peroxidation and ppars.
      ,
      • Xia S.-H.
      • Wang J.
      • Kang J.X.
      Decreased n-6/n-3 fatty acid ratio reduces the invasive potential of human lung cancer cells by downregulation of cell adhesion/invasion-related genes.
      ]. Evidence from animal models also demonstrated that EPA and DHA inhibited NSCLC proliferation and metastasis, as well as induced the apoptosis and autophagy of tumor cells [
      • Yang P.
      • Cartwright C.
      • Chan D.
      • Ding J.
      • Felix E.
      • Pan Y.
      • Pang J.
      • Rhea P.
      • Block K.
      • Fischer S.M.
      • Newman R.A.
      Anticancer activity of fish oils against human lung cancer is associated with changes in formation of pge2 and pge3 and alteration of akt phosphorylation.
      ,
      • Kim N.
      • Jeong S.
      • Jing K.
      • Shin S.
      • Kim S.
      • Heo J.-Y.
      • Kweon G.-R.
      • Park S.-K.
      • Wu T.
      • Park J.-I.
      • Lim K.
      Docosahexaenoic acid induces cell death in human non-small cell lung cancer cells by repressing mtor via ampk activation and pi3k/akt inhibition.
      ]. However, previous observational studies primarily focusing on whether the consumption of EPA and DHA reduces the risk of NSCLC have yielded inconsistent results [
      • Vega O.M.
      • Abkenari S.
      • Tong Z.
      • Tedman A.
      • Huerta-Yepez S.
      Omega-3 polyunsaturated fatty acids and lung cancer: nutrition or pharmacology?.
      ]. Recall bias and reporting bias in dietary assessments as well as the neglect of EPA and DHA metabolism in vivo may be partly responsible. In this instance, measurements of EPA and DHA in biological samples could be more stable and repeatable, whilst providing a direct indication of the overall metabolism of dietary and endogenous fatty acids.
      Over the recent decades, the use of advanced techniques including GC-MS and LC-MS for PUFA analysis is getting prevalent owing to their high specificity and selectivity [
      • Sánchez-Avila N.
      • Mata-Granados J.M.
      • Ruiz-Jiménez J.
      • Luque de Castro M.D.
      Fast, sensitive and highly discriminant gas chromatography-mass spectrometry method for profiling analysis of fatty acids in serum.
      ,
      • Zehethofer N.
      • Pinto D.M.
      • Volmer D.A.
      Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry.
      ]. In the case of GC-MS, the sample preparation process is generally cumbersome and requires the conversion of fatty acids into suitable volatile derivatives [
      • Dyerberg J.
      • Madsen P.
      • Møller J.M.
      • Aardestrup I.
      • Schmidt E.B.
      Bioavailability of marine n-3 fatty acid formulations.
      ]. By contrast, LC-based separation methods provide the advantage of a simpler pre-treatment procedure [
      • Bromke M.A.
      • Hochmuth A.
      • Tohge T.
      • Fernie A.R.
      • Giavalisco P.
      • Burgos A.
      • Willmitzer L.
      • Brotman Y.
      Liquid chromatography high-resolution mass spectrometry for fatty acid profiling.
      ]. To date, there have been many reports of LC-MS-based methods for the quantification of EPA and DHA. However, most of these methods required a derivatization step or a drying process in sample preparation, which was not only time-consuming but may even introduce potential bias and consequently increase the analytical inaccuracy [
      • Serafim V.
      • Tiugan D.-A.
      • Andreescu N.
      • Mihailescu A.
      • Paul C.
      • Velea I.
      • Puiu M.
      • Niculescu M.D.
      Development and validation of a lc⁻ms/ms-based assay for quantification of free and total omega 3 and 6 fatty acids from human plasma.
      ,
      • Aslan M.
      • Celmeli G.
      • Özcan F.
      • Kupesiz A.
      Lc-ms/ms analysis of plasma polyunsaturated fatty acids in patients with homozygous sickle cell disease.
      ,
      • Aslan M.
      • Özcan F.
      • Aslan I.
      • Yücel G.
      Lc-ms/ms analysis of plasma polyunsaturated fatty acids in type 2 diabetic patients after insulin analog initiation therapy.
      ]. One assay developed by Zhou et al. took advantage of a simple sample preparation procedure by protein precipitation, yet the use of the analogue chlorzoxazone instead of analyte isotopes as IS may compromise the methodological performance [
      • Zhou B.
      • Lin C.
      • Xie S.
      • Zhou X.
      • Zhang F.
      • Ye X.
      • Lin F.
      • Hu L.
      • Huang A.
      Determination of four omega-3 polyunsaturated fatty acids by uplc-ms/ms in plasma of hyperlipidemic and normolipidemic subjects.
      ]. Therefore, it is essential to establish a simple and reproducible analytical method for EPA and DHA determination.
      In this study, we intended to investigate the changes in serum EPA and DHA levels in NSCLC patients by developing a rapid and reliable LC-MS/MS based quantification method.

      2. Materials & methods

      2.1 Chemical reagents

      EPA and DHA were purchased from Cambridge Isotope Laboratories (MA, USA). EPA-D5 and DHA-D5 were bought from Toronto Research Chemicals (Toronto, Canada). MeOH (HPLC grade) and ACN (HPLC grade) were obtained from Merck (Darmstadt, Germany). Formic acid (99%, LC-MS grade) was purchased from Fisher Scientific (Bremen, Germany). Deionized water was prepared by the Elga Water purification system (High Wycombe, UK).

      2.2 Sample preparation

      A mixture of stock standard solutions of EPA (8125.0 nM) and DHA (17125.0 nM) was prepared in MeOH. A 10-point calibration curve was constructed by serial dilution of the mixed standard solution with MeOH. The final concentration gradient included the following concentrations: 15.9, 31.7, 63.5, 127.0, 253.9, 507.8, 1015.6, 2031.3, 4062.5, 8125.0 nM for EPA; and 33.4, 66.9, 133.8, 267.6, 535.2, 1070.3, 2140.6, 4281.3, 8562.5, 17125.0 nM for DHA. Two levels of QC samples (63.5 nM and 2031.3 nM for EPA, 133.8 nM and 4281.3 nM for DHA) were diluted with MeOH from the mixed standard solution. Two sets of pooled human serum at different levels were served as PQC samples. IS working solution containing EPA-D5 (16.3 µM) and DHA-D5 (15.0 µM) was prepared in MeOH. All the solutions were stored at -20°C until use. Human blood samples were collected after overnight fasting and centrifuged immediately at 3,280 rpm for 5 minutes. Obtained serum specimens were frozen at -80°C until analysis.
      For sample preparation, 25 µL of each sample (blank, calibrators, QCs, PQCs, or human serum samples) was thawed at room temperature and then mixed with 25 µL of IS working solution. Subsequently, 450µL of ACN was added for protein precipitation. After vortex for 3 min and centrifugation at 14,680 rpm for 10 min, the supernatant was directly used for LC-MS/MS analysis.

      2.3 LC-MS/MS conditions

      LC-MS/MS analysis was performed on an AB Sciex ExionLC system coupled to an AB Sciex Triple Quad 5500 mass spectrometry system (CA, USA). The LC system consisted of a gradient pump, a vacuum degasser, a temperature-controlled autosampler, and a temperature-controlled column oven. Separation was achieved on a Kinetex C18 column (30*2.1 mm, 2.6 µm) with deionized water (solvent A) and 0.1% formic acid in ACN (solvent B) as mobile phases at a flow rate of 0.6 mL/min. The LC gradient flow program was set as follows: 0-2.5 min, 60-70% B; 2.5-3.2 min, 60% B. The column oven temperature was set to 50°C and the sample injection volume was 5 µL.
      Negative electrospray ionization mode was used in MS. The optimal parameters were set as follows: ion spray temperature of 450°C, ion spray voltage of -4500 V, curtain gas and collision gas of 30 psi and 9 psi respectively, ion source gas 1 (GS1) and GS2 of 40 and 50 psi respectively. The MRM transitions were 327.3/283.3 for DHA, 332.3/ 288.2 for DHA-D5, 301.3/257.2 for EPA, and 306.3/262.3for EPA-D5 respectively. Analyst 1.6.1 software (AB Sciex) was utilized for data acquisition and processing.

      2.4 Method validation

      The quantification method was validated emphasizing linearity, LLOQ, LOD, precision, accuracy and matrix effect.

      2.4.1 Linearity and analytical sensitivity

      Linearity was analyzed by a 10-point calibration curve. The calculated concentrations of the calibrators were required to be within ±15% of the expected values, except for the LLOQ within ±20%. LOD and LLOQ were defined as analyte concentrations at SNR of 3 and 10, respectively. LLOQ was determined by 5 repeated measurements of a series of low concentration calibrators and was established based on the criteria with a CV of less than 20% and accuracy within 80-120%.

      2.4.2 Accuracy and precision

      Accuracy was assessed by the recovery experiments in which three different levels of standard solution were spiked into pooled human serum in five replicates. The recovery was calculated as [(final concentration - initial concentration) / added concentration]. QCs and PQCs were used to evaluate imprecision. Six replicates of each level were assayed over six days to calculate the intra-day and inter-day precision. CVs less than 20% at LLOQ and less than 15% at the remaining tested concentrations were considered to be in compliance with the validation criteria.

      2.4.3 Matrix effect

      To evaluate the matrix effect, 10 µL of standard solutions at three different levels (507.8, 1015.6, 8125.0 nM for EPA and 1070.3, 2140.6, 17125.0 nM for DHA) were spiked into 150 µL of MeOH and 150 µL of pooled human serum, respectively. Subsequently, the signal intensities of standard substances added to the two matrices were compared at each concentration. The absolute MF of the analyte and IS was calculated as Ai/Ai’ * 100% (Ai and Ai’ represent the peak areas of the analyte and IS within and without the matrix, respectively), and the IS-normalized MF of the analyte was calculated as (MF of the analyte) / (MF of IS) * 100%. An IS-normalized MF within 85%-115% was acceptable.

      2.5 Study population and ethics statement

      To investigate the alterations of serum EPA and DHA levels in NSCLC patients, we conducted a case-control study in Chinese population. Overall, 211 NSCLC patients and 227 healthy controls were consecutively recruited between January 2017 and May 2018 at Tongji Hospital of Huazhong University of Science and Technology, Wuhan, China. All patients were histopathologically confirmed to be newly diagnosed primary NSCLC and did not receive any treatment. The histologic type of NSCLC was identified based on the results of histopathological examinations, and the tumor stage was confirmed according to the eighth edition of the tumor node metastasis staging system. A total of 227 healthy subjects were randomly enrolled from healthy people who underwent physical examinations in the same hospital. The healthy controls were matched with NSCLC cases for age, sex, smoking and drinking history, and none of them had a history of lung cancer or other types of cancer.
      This study was reviewed and approved by the Institutional Review Board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) and carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki).

      2.6 Statistical analysis

      Normality was tested by the Kolmogorov–Smirnov test. Categorical variables were presented as count and percentage in each category, and continuous variables were summarized as mean ± SD or median with IQR as appropriate. Chi-square test was used to compare categorical variables, while Students’ t test or Mann-Whitney U test was employed to compare continuous variables in two groups. For multiple comparisons, Kruskal-Wallis test followed by Dunn's multiple comparison test was performed. A two-sided P value of less than 0.05 was considered statistically significant. SPSS 25.0 (IBM Corporation, NY, USA) and GraphPad Prism 9.0 (GraphPad, CA, USA) were used for statistical analysis.

      3. Results

      3.1 Method development and optimization

      The proposed LC-MS/MS method was enabled for the synchronous quantification of serum EPA and DHA with a small sample volume of 25 µL and a short run time of 3.2 min. In order to achieve a good separation of EPA and DHA in human serum, the chromatographic conditions were optimized. The best separation of EPA and DHA was achieved on the Kinetex C18 column with deionized water and 0.1% formic acid in ACN as mobile phases. As shown in Fig. 1, the retention times for EPA and DHA were 1.14 min and 1.52 min respectively. MRM transitions regarding 327.3/283.3 and 327.3/229.2 for DHA, 301.3/257.2 and 301.3/203.3 for EPA were used for the quantification. Optimal MRM parameters were summarized in Table 1.
      Fig 1
      Fig. 1Representative Chromatograms of EPA and EPA-D5, as well as DHA and DHA-D5 from a healthy control sample (A) and a non-small-cell lung cancer patient sample (B).
      Table 1MS parameters for EPA and DHA detection.
      CompoundRetention time (min)Transition (Da)DP (volts)EP (volts)CE (volts)CXP (volts)
      EPA1.14301.300→257.200-130.000-13.000-17.000-16.000
      301.300→203.300
      DHA1.52327.300→283.300-100.000-12.000-17.000-16.000
      327.300→229.200
      EPA-D51.12306.300→262.300-160.000-9.000-17.000-15.000
      306.300→208.300
      DHA-D51.50332.300→288.200-140.000-9.000-17.000-20.000
      332.300→234.300

      3.2 Method validation

      The LC-MS/MS method was well validated in terms of linearity, analytical sensitivity, matrix effect, accuracy and precision.
      Using 10-point calibration curves, the linear correlation coefficients (r2) were 0.9981 and 0.9967 for EPA and DHA, respectively. A wide linear response over a sufficient dynamic concentration range was observed as summarized in Table 2. Analysis of a series of low concentration calibrators resulted in accuracy within 80-120% and a CV value <20% at the concentration of 63.5 and 134 nM for EPA and DHA, respectively. As a result, 63.5 and 134 nM were accepted as the LLOQ with SNR greater than 10 for EPA and DHA respectively. The concentrations of 15.9 and 16.7 nM for EPA and DHA were considered as the LOD with SNR greater than 3. Detailed results were presented in Table 2
      Table 2Linearity, LLOQ and LOD.
      AnalyteLinear range (nM)Linear equationr2LLOQ (nM)LOD (nM)
      EPA63.5-8125y=0.00272x+0.1050.998163.515.9
      DHA134-17125y=0.00328x+0.1650.996713416.7
      As shown in Table 3, the intra-day CVs were less than 7.63% and 6.16% for EPA and DHA respectively, while the inter-day CVs were less than 11.3% and 12.2%, respectively.
      Table 3Imprecision.
      AnalyteQC sampleIntra-day CV (%)Inter-day CV (%)
      EPAQCL7.639.80
      QCH4.627.56
      PQCL7.3111.3
      PQCH2.8510.4
      DHAQCL2.784.97
      QCH6.167.06
      PQCL4.029.24
      PQCH2.4912.2
      Recoveries of EPA and DHA were in the range of 78.9%-85.4% and 85.2%-90.1%, respectively (Table 4). The IS-normalized MF at three different concentrations were in the range of 1.02-1.09 for EPA and 1.01-1.09 for DHA (Table 4), which indicated that no obvious matrix effect was observed in the accurate quantification of the target analytes.
      Table 4Recovery and matrix effect.
      AnalyteSpiked concentration (nM)Recovery (%)CV (%)IS-normalized matrix factorCV (%)
      EPA507.890.19.21.095.4
      1015.685.66.01.063.3
      8125.085.24.11.021.9
      DHA1070.385.418.71.095.8
      2140.678.92.91.012.2
      17125.081.12.91.020.8

      3.3 Alterations of serum EPA and DHA levels in NSCLC patients

      The serum levels of EPA and DHA in 211 NSCLC patients and 227 healthy controls were quantified using the validated LC-MS/MS method. Among the 227 healthy subjects, EPA and DHA concentrations were observed to be higher in females than in males, although shown no significant difference (Supplementary Table 1). Likewise, serum levels of EPA and DHA tended to be higher in subjects aged ≥61 years compared to the younger groups (≤55y group and 56-60y group). The demographic and clinical characteristics of the participants were summarized in Table 5. There were no statistically significant differences between NSCLC patients and controls in terms of age, sex, smoking history and alcohol consumption history. Among the 211 patients with NSCLC, 65.9% were diagnosed with ADC, 29.9% with SCC, and the remaining 4.3% with NSCLC of undefinable histological type. According to the eighth edition of the TNM classification system, 1.8% of the NSCLC patients were categorized as stage 0, 30.3% as stage I, 7.1% as stage II, 32.2% as stage III, 26.5% as stage IV, and the remaining 1.8% of patients as unclear.
      Table 5Basic characteristics of case-control study subjects.
      NSCLC (n=211)HC (n=227)P value
      Sex0.499
       Male144 (68.2%)148 (65.2%)
       Female67 (31.8%)79 (34.8%)
      Age58.6±8.057.7±5.60.173
      Smoking history
      Data about 10 normal control subjects are absent.
      0.152
       Current/former105 (49.8%)93 (41.0%)
       None106 (50.2%)124 (54.6%)
      Drinking history
      Data about 9 normal control subjects are absent.
      0.225
       Current/former62 (29.4%)76 (33.5%)
       None149 (70.6%)142 (62.6%)
      Histological type
       Adenomatous139 (65.9%)
       Squamous63 (29.9%)
       Undefinable9 (4.3%)
      Stage
       Early stage84 (39.6%)
       Advanced stage125 (59.0%)
       Undefinable3 (1.4%)
      low asterisk Data about 10 normal control subjects are absent.
      low asterisklow asterisk Data about 9 normal control subjects are absent.
      As illustrated in Fig. 2, the median concentrations of EPA in NSCLC patients and healthy controls were 780 (536-1240) and 1000 (664-1720) nM respectively, while those of DHA were 2470 (1750-3300) nM and 4400 (3230-6080) nM, respectively. Compared with the control group, both EPA and DHA were significantly reduced in NSCLC patients (P<0.0001).
      Fig 2
      Fig. 2Alterations of serum EPA(A) and DHA (B) levels in non-small-cell lung cancer. Red thick lines indicate median concentrations, upper and lower black thin lines indicate interquartile ranges. HC, healthy controls; NSCLC, non-small-cell lung cancer; ****, P<0.0001.
      Changes in EPA and DHA levels in patients at different stages of NSCLC were shown in Fig. 3. Serum EPA and DHA concentrations were significantly decreased in both early-stage (stage 0, Ⅰ and Ⅱ) and advanced-stage (stage Ⅲ and Ⅳ) patients. Intriguingly, EPA and DHA concentrations showed a gradual decreasing trend in controls, early-stage and advanced-stage NSCLC patients, although the difference between early- and advanced-stage NSCLC was not statistically significant.
      Fig 3
      Fig. 3Changes in serum EPA (A) and DHA (B) at different stages of NSCLC. Bars represent median values with interquartile ranges. HC, healthy controls; NSCLC, non-small-cell lung cancer; *, P<0.05; ***, P<0.001; ****, P<0.0001.
      The results of subgroup analysis based on histological type were illustrated in Fig. 4. Compared with healthy controls, serum EPA and DHA levels were significantly lower in both ADC and SCC patients. However, there was no significant difference in EPA or DHA concentration between patients with ADC and SCC.
      Fig 4
      Fig. 4Change of serum EPA (A) and DHA (B) in different histological types of NSCLC. Red thick lines indicate median concentrations, upper and lower black thin lines indicate interquartile ranges. HC, healthy controls; ADC, adenocarcinoma; SCC, squamous cell carcinoma; **, P<0.01; ****, P<0.0001.

      4. Discussion

      As the predominant omega-3 PUFAs, studies have highlighted the importance of EPA and DHA in preventing as well as improving the prognosis of various tumors. In this study, we explored the changes in serum EPA and DHA concentrations in patients with NSCLC using a laboratory-developed LC-MS/MS method for the simultaneous quantification of EPA and DHA. Our method requires only a small sample volume of 25 µL, and a simple one-step sample preparation process without the need for drying and redissolution steps. Good separation of EPA and DHA in human serum was achieved by applying a 2.6 µm C18 column on the basis of a mobile phase consisting of a partial mixture of organic solvents without salt. EPA and DHA were quantitated by MRM mode with negative electrospray ionization using commercially available isotopically labeled IS. The method was further validated with consistent linearity (r2EPA=0.9981, r2DHA=0.9967), as well as satisfactory accuracy (recoveries between 78.9% and 90.1%) and precision (intra-day and inter-day CVs within 2.49% and 12.2%). Using this validated method, we observed that serum levels of both EPA and DHA were significantly decreased in NSCLC patients compared to healthy controls, although there was a visible IQR overlap between the NSCLC and HC group. Moreover, EPA and DHA concentrations showed a consecutive downward trend with the progression of NSCLC.
      The relationship of EPA and DHA with the development and progression of tumors has long been of interest. It has been extensively studied that EPA and DHA inhibit the initiation and progression of tumors in multiple ways, although the exact mechanism needs to be further corroborated. As components of the structural phospholipids in cell membranes, EPA and DHA were suggested to affect the activity of transcription factors and intracellular signal transduction pathways, including nuclear transcription factor-κB (NF-κB), the renin-angiotensin system (Ras) and protein kinase C (PKC), which in turn regulate the metabolism, growth and differentiation of cancer cells [
      • Novak T.E.
      • Babcock T.A.
      • Jho D.H.
      • Helton W.S.
      • Espat N.J.
      Nf-kappa b inhibition by omega -3 fatty acids modulates lps-stimulated macrophage tnf-alpha transcription.
      ,
      • Collett E.D.
      • Davidson L.A.
      • Fan Y.Y.
      • Lupton J.R.
      • Chapkin R.S.
      N-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic ras activation in colonocytes.
      ,
      • Murray N.R.
      • Weems C.
      • Chen L.
      • Leon J.
      • Yu W.
      • Davidson L.A.
      • Jamieson L.
      • Chapkin R.S.
      • Thompson E.A.
      • Fields A.P.
      Protein kinase c betaii and tgfbetarii in omega-3 fatty acid-mediated inhibition of colon carcinogenesis.
      ]. Additionally, it was indicated that EPA and DHA possessed inhibitory effects on inflammation, which has been identified as a hallmark of cancer, and thereby suppressing tumorigenesis [
      • Giugliano D.
      • Ceriello A.
      • Esposito K.
      The effects of diet on inflammation: emphasis on the metabolic syndrome.
      ,
      • Coussens L.M.
      • Werb Z.
      Inflammation and cancer.
      ]. In fact, EPA and DHA have been considered as immunonutrients and are commonly used in the nutritional therapy of cancer patients due to their ample biological effects, despite the fact that their actual benefits have yet to be assessed [
      • Freitas R.D.S.
      • Campos M.M.
      Protective effects of omega-3 fatty acids in cancer-related complications.
      ]. Recent findings of well-designed prospective studies have shown that the consumption of long-chain omega-3 PUFAs was robustly associated with lower mortality from total and major causes including cancer [
      • Zhang Y.
      • Zhuang P.
      • He W.
      • Chen J.N.
      • Wang W.Q.
      • Freedman N.D.
      • Abnet C.C.
      • Wang J.B.
      • Jiao J.J.
      Association of fish and long-chain omega-3 fatty acids intakes with total and cause-specific mortality: prospective analysis of 421 309 individuals.
      ,
      • Zhuang P.
      • Zhang Y.
      • He W.
      • Chen X.
      • Chen J.
      • He L.
      • Mao L.
      • Wu F.
      • Jiao J.
      Dietary fats in relation to total and cause-specific mortality in a prospective cohort of 521 120 individuals with 16 years of follow-up.
      ]. Our previous meta-analysis of observational studies among cancer patients also revealed a protective effect of dietary marine omega-3 PUFAs intake on cancer survival [
      • Wang Y.
      • Liu K.
      • Long T.
      • Long J.
      • Li Y.
      • Li J.
      • Cheng L.
      Dietary fish and omega-3 polyunsaturated fatty acids intake and cancer survival: a systematic review and meta-analysis.
      ].
      With specific reference to NSCLC, it was reported that DHA inhibited proliferation and induced apoptosis of A549 cells, as well as suppressed their invasion and metastasis through a ROS-mediated inactivation of the PI3K/Akt signaling pathway [
      • Yin Y.
      • Sui C.
      • Meng F.
      • Ma P.
      • Jiang Y.
      The omega-3 polyunsaturated fatty acid docosahexaenoic acid inhibits proliferation and progression of non-small cell lung cancer cells through the reactive oxygen species-mediated inactivation of the pi3k /akt pathway.
      ]. Another study demonstrated that RvD1, one of the DHA-initiated eicosanoids, could modulate the miR-138-5p/FOXC1 pathway in lung cancer cells, thereby decreasing lung cancer cell growth and invasion [
      • Bai X.
      • Shao J.
      • Zhou S.
      • Zhao Z.
      • Li F.
      • Xiang R.
      • Zhao A.Z.
      • Pan J.
      Inhibition of lung cancer growth and metastasis by dha and its metabolite, rvd1, through mir-138-5p/foxc1 pathway.
      ]. These evidences suggested that EPA and DHA may have beneficial effects on the prevention and prognosis of NSCLC. However, existing population-based studies on the association of EPA and DHA with NSCLC fail to reach a consensus. On the one hand, epidemiological studies regarding the consumption of EPA and DHA on the risk of NSCLC presented contradictory results. In this context, a recent umbrella review of meta-analyses concluded that there was no association between omega-3 PUFA intake and lung cancer [
      • Lee K.H.
      • Seong H.J.
      • Kim G.
      • Jeong G.H.
      • Kim J.Y.
      • Park H.
      • Jung E.
      • Kronbichler A.
      • Eisenhut M.
      • Stubbs B.
      • Solmi M.
      • Koyanagi A.
      • Hong S.H.
      • Dragioti E.
      • de Rezende L.F.M.
      • Jacob L.
      • Keum N.
      • van der Vliet H.J.
      • Cho E.
      • Veronese N.
      • Grosso G.
      • Ogino S.
      • Song M.
      • Radua J.
      • Jung S.J.
      • Thompson T.
      • Jackson S.E.
      • Smith L.
      • Yang L.
      • Oh H.
      • Choi E.K.
      • Shin J.I.
      • Giovannucci E.L.
      • Gamerith G.
      Consumption of fish and ω-3 fatty acids and cancer risk: an umbrella review of meta-analyses of observational studies.
      ]. On the other hand, findings from observational studies on the changes of EPA and DHA concentrations in NSCLC appeared to be better aligned [
      • Zuijdgeest-van Leeuwen S.D.
      • van der Heijden M.S.
      • Rietveld T.
      • van den Berg J.W.O.
      • Tilanus H.W.
      • Burgers J.A.
      • Wilson J.H.P.
      • Dagnelie P.C.
      Fatty acid composition of plasma lipids in patients with pancreatic, lung and oesophageal cancer in comparison with healthy subjects.
      ,
      • Ren J.
      • Zhang D.
      • Liu Y.
      • Zhang R.
      • Fang H.
      • Guo S.
      • Zhou D.
      • Zhang M.
      • Xu Y.
      • Qiu L.
      • Li Z.
      Simultaneous quantification of serum nonesterified and esterified fatty acids as potential biomarkers to differentiate benign lung diseases from lung cancer.
      ]. Zehethofer and colleagues measured fatty acid composition in plasma phospholipids and cholesteryl esters in 22 newly diagnosed, untreated NSCLC patients as well as 45 healthy subjects, and the results illustrated a reduction of EPA and DHA levels in NSCLC patients albeit not reaching a statistical difference [
      • Zuijdgeest-van Leeuwen S.D.
      • van der Heijden M.S.
      • Rietveld T.
      • van den Berg J.W.O.
      • Tilanus H.W.
      • Burgers J.A.
      • Wilson J.H.P.
      • Dagnelie P.C.
      Fatty acid composition of plasma lipids in patients with pancreatic, lung and oesophageal cancer in comparison with healthy subjects.
      ]. Moreover, Ren et.al also demonstrated that serum EPA and DHA concentrations in patients with lung cancer were significantly lower than healthy controls [
      • Ren J.
      • Zhang D.
      • Liu Y.
      • Zhang R.
      • Fang H.
      • Guo S.
      • Zhou D.
      • Zhang M.
      • Xu Y.
      • Qiu L.
      • Li Z.
      Simultaneous quantification of serum nonesterified and esterified fatty acids as potential biomarkers to differentiate benign lung diseases from lung cancer.
      ]. Consistently, our results provide further evidence in Chinese population for the decrease in serum EPA and DHA levels in NSCLC patients based on a quantification method with greater performance. Furthermore, we underlined a consistent decline of EPA and DHA with the advancement of NSCLC, which might suggest a protective role for them in NSCLC progression.
      In this study, EPA and DHA concentrations in patients with NSCLC were 780 and 2470 nM, respectively, which were even lower than those in vegetarians who are regarded as having nearly deficient omega-3 fatty acid levels [
      • Mann N.
      • Pirotta Y.
      • O'Connell S.
      • Li D.
      • Kelly F.
      • Sinclair A.
      Fatty acid composition of habitual omnivore and vegetarian diets.
      ]. This may suggest a possible deficiency of EPA and DHA in NSCLC patients. Nevertheless, it needs to be interpreted with caution given the different population regions as well as different EPA and DHA quantification methods [
      • Mann N.
      • Pirotta Y.
      • O'Connell S.
      • Li D.
      • Kelly F.
      • Sinclair A.
      Fatty acid composition of habitual omnivore and vegetarian diets.
      ]. The alterations of EPA and DHA in NSCLC patients might be attributed to marine food consumption as well as in vivo fatty acid metabolism, as the concentrations of EPA and DHA depend mainly on these two factors. Previous prospective cohort studies have indicated that omega-3 fatty acid intake may reduce the risk of various types of cancer including NSCLC, despite the weak evidence level [
      • Lee K.H.
      • Seong H.J.
      • Kim G.
      • Jeong G.H.
      • Kim J.Y.
      • Park H.
      • Jung E.
      • Kronbichler A.
      • Eisenhut M.
      • Stubbs B.
      • Solmi M.
      • Koyanagi A.
      • Hong S.H.
      • Dragioti E.
      • de Rezende L.F.M.
      • Jacob L.
      • Keum N.
      • van der Vliet H.J.
      • Cho E.
      • Veronese N.
      • Grosso G.
      • Ogino S.
      • Song M.
      • Radua J.
      • Jung S.J.
      • Thompson T.
      • Jackson S.E.
      • Smith L.
      • Yang L.
      • Oh H.
      • Choi E.K.
      • Shin J.I.
      • Giovannucci E.L.
      • Gamerith G.
      Consumption of fish and ω-3 fatty acids and cancer risk: an umbrella review of meta-analyses of observational studies.
      ]. Taken together with the multiple biological roles of EPA and DHA in NSCLC, a long-term low consumption of marine food might be the main reason for lower EPA and DHA levels in NSCLC patient. Apart from this, fatty acid β-oxidation (FAO) has been suggested to be a well-documented aspect of metabolic reprogramming undergoing in cancer cells that was crucial for cancer invasion and metastasis [
      • Chen M.
      • Huang J.
      The expanded role of fatty acid metabolism in cancer: new aspects and targets.
      ,
      • Li Y.-J.
      • Fahrmann J.F.
      • Aftabizadeh M.
      • Zhao Q.
      • Tripathi S.C.
      • Zhang C.
      • Yuan Y.
      • Ann D.
      • Hanash S.
      • Yu H.
      Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.
      ], which might be another reason for the decreased serum levels of EPA and DHA.
      EPA and DHA levels seems to be varied widely across populations in different regions (Supplementary Table 2). Overall, EPA and DHA levels of healthy subjects in our study showed comparable values to those previously reported in China [
      • Zhou B.
      • Lin C.
      • Xie S.
      • Zhou X.
      • Zhang F.
      • Ye X.
      • Lin F.
      • Hu L.
      • Huang A.
      Determination of four omega-3 polyunsaturated fatty acids by uplc-ms/ms in plasma of hyperlipidemic and normolipidemic subjects.
      ] except for Qingdao, a coastal city with popular seafood intake [
      • Xuan C.
      • Tian Q.-W.
      • Li H.
      • Guo J.-J.
      • He G.-W.
      • Lun L.-M.
      Serum fatty acids profile and association with early-onset coronary artery disease.
      ]. In addition, serum EPA and DHA concentrations were generally lower in the Chinese population than in the European and American populations, which may be due to the general unpopularity of fish oil supplementation in China. It is worth noting that serum levels of EPA and DHA were in a wide range in both NSCLC and HC groups. Intriguingly, the median levels of EPA and DHA in healthy subjects were found to be higher in the older group (≥61y) and the female group. These findings may be related to the age-related variations in the consumption of EPA, DHA, as well as their precursor ALA [
      • Diffenderfer M.R.
      • Rajapakse N.
      • Pham E.
      • He L.
      • Dansinger M.L.
      • Nelson J.R.
      • Schaefer E.J.
      Plasma fatty acid profiles: relationships with sex, age, and state-reported heart disease mortality rates in the United States.
      ]. It has also been presumed previously that higher levels of EPA and DHA in females were due to enhanced synthesis of EPA and DHA from ALA in pre-menopausal women [
      • Sarter B.
      • Kelsey K.S.
      • Schwartz T.A.
      • Harris W.S.
      Blood docosahexaenoic acid and eicosapentaenoic acid in vegans: associations with age and gender and effects of an algal-derived omega-3 fatty acid supplement.
      ]. Nevertheless, the explanations for these results need to be further explored in the future.
      To date, studies have placed more focus on the association of dietary EPA and DHA intake with cancer incidence or mortality. However, dietary EPA and DHA intake could not really reflect the real concentrations of EPA and DHA in the body due to the influence of various biochemical and physiological factors [
      • Serini S.
      • Calviello G.
      Long-chain omega-3 fatty acids and cancer: any cause for concern?.
      ,
      • Welch A.A.
      • Bingham S.A.
      • Ive J.
      • Friesen M.D.
      • Wareham N.J.
      • Riboli E.
      • Khaw K.T.
      Dietary fish intake and plasma phospholipid n-3 polyunsaturated fatty acid concentrations in men and women in the European prospective investigation into cancer-norfolk United Kingdom cohort.
      ]. Besides, dietary assessments are susceptible to the unavoidable recall bias as well as reporting bias, thus potentially leading to an inaccuracy of the evaluation. Hence, results from direct measurements of EPA and DHA in biological samples could be more robust and reliable compared with those based on dietary assessments. In contrast to previously reported methods for EPA and DHA quantification, the current method was simpler, more effective and more accurate (Supplementary Table 3). Taking the advantage of the reliable determination method, the reduction of EPA and DHA in patients with NSCLC was objectively confirmed. Meanwhile, the efficacy of EPA and DHA supplementation on cancer patients could be evaluated with the use of our robust quantification method.
      To our knowledge, this is the first study in Chinese population to investigate the alterations of EPA and DHA in NSCLC patients. And the method for EPA and DHA determination was simple and validated to be reliable. However, several limitations should be addressed. Firstly, there are various types of PUFAs as well as their derivatives, whereas our method only analyzed EPA and DHA which have been supposed to possess anticancer effects, and more types of PUFAs needed to be determined in the future. Additionally, albeit that we matched NSCLC patients and healthy subjects for sex, age, smoking and drinking history, our results may still be affected by other potential confounding biases. Finally, evidence from the case-control study is limited, and large-scale prospective studies along with studies on fatty acid-related metabolic pathways in NSCLC are needed in the future to elucidate the causal relationship between EPA, DHA and the development of NSCLC, as well as the potential roles that EPA and DHA might play.
      In summary, by dint of a self-developed and well-validated LC-MS/MS quantification method, this study reveals lower serum EPA and DHA levels in NSCLC patients than in healthy individuals, and a tendency for EPA and DHA concentrations to decrease progressively with the progression of NSCLC. Our findings require to be confirmed in independent replications and prospective studies.

      Funding

      This work was funded by the National Key Research and Development Plan Program of China (Grant No. 2016YFC1302702) and the National Natural Science Foundation of China (Grant No. 81572071). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

      CRediT authorship contribution statement

      Yi Wang: Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft. Tongxin Yin: Resources, Software, Validation, Visualization. Jiaoyuan Li: Conceptualization, Project administration, Supervision. Xia Luo: Resources, Software, Visualization. Ke Liu: Resources, Software, Visualization. Tingting Long: Resources, Software, Visualization. Ying Shen: Conceptualization, Methodology, Project administration, Supervision, Validation, Writing – review & editing. Liming Cheng: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

      Declaration of Competing Interest

      The authors declare that they have no conflict of interest.

      Appendix. Supplementary materials

      References

        • Nettleton J.A.
        Omega-3 fatty acids and health.
        in: Nettleton J.A. Omega-3 fatty acids and health. Springer US, Boston, MA1995: 64-76
        • Tur J.A.
        • Bibiloni M.M.
        • Sureda A.
        • Pons A.
        Dietary sources of omega 3 fatty acids: public health risks and benefits.
        Br J Nutr. 2012; 107 (Suppl): S23-S52https://doi.org/10.1017/s0007114512001456
        • Calviello G.
        • Di Nicuolo F.
        • Gragnoli S.
        • Piccioni E.
        • Serini S.
        • Maggiano N.
        • Tringali G.
        • Navarra P.
        • Ranelletti F.O.
        • Palozza P.
        N-3 pufas reduce vegf expression in human colon cancer cells modulating the cox-2/pge2 induced erk-1 and -2 and hif-1alpha induction pathway.
        Carcinogenesis. 2004; 25: 2303-2310https://doi.org/10.1093/carcin/bgh265
        • Hu Y.
        • Sun H.
        • Owens R.T.
        • Gu Z.
        • Wu J.
        • Chen Y.Q.
        • O'Flaherty J.T.
        • Edwards I.J.
        Syndecan-1-dependent suppression of pdk1/akt/bad signaling by docosahexaenoic acid induces apoptosis in prostate cancer.
        Neoplasia. 2010; 12: 826-836https://doi.org/10.1593/neo.10586
        • Trombetta A.
        • Maggiora M.
        • Martinasso G.
        • Cotogni P.
        • Canuto R.A.
        • Muzio G.
        Arachidonic and docosahexaenoic acids reduce the growth of a549 human lung-tumor cells increasing lipid peroxidation and ppars.
        Chem Biol Interact. 2007; 165: 239-250https://doi.org/10.1016/j.cbi.2006.12.014
        • Xia S.-H.
        • Wang J.
        • Kang J.X.
        Decreased n-6/n-3 fatty acid ratio reduces the invasive potential of human lung cancer cells by downregulation of cell adhesion/invasion-related genes.
        Carcinogenesis. 2005; 26: 779-784https://doi.org/10.1093/carcin/bgi019
        • Yang P.
        • Cartwright C.
        • Chan D.
        • Ding J.
        • Felix E.
        • Pan Y.
        • Pang J.
        • Rhea P.
        • Block K.
        • Fischer S.M.
        • Newman R.A.
        Anticancer activity of fish oils against human lung cancer is associated with changes in formation of pge2 and pge3 and alteration of akt phosphorylation.
        Mol Carcinog. 2014; 53: 566-577https://doi.org/10.1002/mc.22008
        • Kim N.
        • Jeong S.
        • Jing K.
        • Shin S.
        • Kim S.
        • Heo J.-Y.
        • Kweon G.-R.
        • Park S.-K.
        • Wu T.
        • Park J.-I.
        • Lim K.
        Docosahexaenoic acid induces cell death in human non-small cell lung cancer cells by repressing mtor via ampk activation and pi3k/akt inhibition.
        Biomed Res Int. 2015; 2015239764https://doi.org/10.1155/2015/239764
        • Vega O.M.
        • Abkenari S.
        • Tong Z.
        • Tedman A.
        • Huerta-Yepez S.
        Omega-3 polyunsaturated fatty acids and lung cancer: nutrition or pharmacology?.
        Nutr Cancer. 2021; 73: 541-561https://doi.org/10.1080/01635581.2020.1761408
        • Sánchez-Avila N.
        • Mata-Granados J.M.
        • Ruiz-Jiménez J.
        • Luque de Castro M.D.
        Fast, sensitive and highly discriminant gas chromatography-mass spectrometry method for profiling analysis of fatty acids in serum.
        J Chromatogr A. 2009; 1216: 6864-6872https://doi.org/10.1016/j.chroma.2009.08.045
        • Zehethofer N.
        • Pinto D.M.
        • Volmer D.A.
        Plasma free fatty acid profiling in a fish oil human intervention study using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry.
        Rapid Commun Mass Spectrom. 2008; 22: 2125-2133https://doi.org/10.1002/rcm.3597
        • Dyerberg J.
        • Madsen P.
        • Møller J.M.
        • Aardestrup I.
        • Schmidt E.B.
        Bioavailability of marine n-3 fatty acid formulations.
        Prostaglandins Leukot Essent Fatty Acids. 2010; 83: 137-141https://doi.org/10.1016/j.plefa.2010.06.007
        • Bromke M.A.
        • Hochmuth A.
        • Tohge T.
        • Fernie A.R.
        • Giavalisco P.
        • Burgos A.
        • Willmitzer L.
        • Brotman Y.
        Liquid chromatography high-resolution mass spectrometry for fatty acid profiling.
        Plant J. 2015; 81: 529-536https://doi.org/10.1111/tpj.12739
        • Serafim V.
        • Tiugan D.-A.
        • Andreescu N.
        • Mihailescu A.
        • Paul C.
        • Velea I.
        • Puiu M.
        • Niculescu M.D.
        Development and validation of a lc⁻ms/ms-based assay for quantification of free and total omega 3 and 6 fatty acids from human plasma.
        Molecules. 2019; 24https://doi.org/10.3390/molecules24020360
        • Aslan M.
        • Celmeli G.
        • Özcan F.
        • Kupesiz A.
        Lc-ms/ms analysis of plasma polyunsaturated fatty acids in patients with homozygous sickle cell disease.
        Clin Exp Med. 2015; 15: 397-403https://doi.org/10.1007/s10238-014-0293-6
        • Aslan M.
        • Özcan F.
        • Aslan I.
        • Yücel G.
        Lc-ms/ms analysis of plasma polyunsaturated fatty acids in type 2 diabetic patients after insulin analog initiation therapy.
        Lipids Health Dis. 2013; 12: 169https://doi.org/10.1186/1476-511x-12-169
        • Zhou B.
        • Lin C.
        • Xie S.
        • Zhou X.
        • Zhang F.
        • Ye X.
        • Lin F.
        • Hu L.
        • Huang A.
        Determination of four omega-3 polyunsaturated fatty acids by uplc-ms/ms in plasma of hyperlipidemic and normolipidemic subjects.
        J Chromatogr B, Anal Technol Biomed Life Sci. 2019; 1126-1127121762https://doi.org/10.1016/j.jchromb.2019.121762
        • Novak T.E.
        • Babcock T.A.
        • Jho D.H.
        • Helton W.S.
        • Espat N.J.
        Nf-kappa b inhibition by omega -3 fatty acids modulates lps-stimulated macrophage tnf-alpha transcription.
        Am J Physiol Lung Cell Mol Physiol. 2003; 284: L84-L89https://doi.org/10.1152/ajplung.00077.2002
        • Collett E.D.
        • Davidson L.A.
        • Fan Y.Y.
        • Lupton J.R.
        • Chapkin R.S.
        N-6 and n-3 polyunsaturated fatty acids differentially modulate oncogenic ras activation in colonocytes.
        Am J Physiol Cell Physiol. 2001; 280: C1066-C1075https://doi.org/10.1152/ajpcell.2001.280.5.C1066
        • Murray N.R.
        • Weems C.
        • Chen L.
        • Leon J.
        • Yu W.
        • Davidson L.A.
        • Jamieson L.
        • Chapkin R.S.
        • Thompson E.A.
        • Fields A.P.
        Protein kinase c betaii and tgfbetarii in omega-3 fatty acid-mediated inhibition of colon carcinogenesis.
        J Cell Biol. 2002; 157: 915-920https://doi.org/10.1083/jcb.200201127
        • Giugliano D.
        • Ceriello A.
        • Esposito K.
        The effects of diet on inflammation: emphasis on the metabolic syndrome.
        J Am Coll Cardiol. 2006; 48: 677-685https://doi.org/10.1016/j.jacc.2006.03.052
        • Coussens L.M.
        • Werb Z.
        Inflammation and cancer.
        Nature. 2002; 420: 860-867https://doi.org/10.1038/nature01322
        • Freitas R.D.S.
        • Campos M.M.
        Protective effects of omega-3 fatty acids in cancer-related complications.
        Nutrients. 2019; 11https://doi.org/10.3390/nu11050945
        • Zhang Y.
        • Zhuang P.
        • He W.
        • Chen J.N.
        • Wang W.Q.
        • Freedman N.D.
        • Abnet C.C.
        • Wang J.B.
        • Jiao J.J.
        Association of fish and long-chain omega-3 fatty acids intakes with total and cause-specific mortality: prospective analysis of 421 309 individuals.
        J Intern Med. 2018; 284: 399-417https://doi.org/10.1111/joim.12786
        • Zhuang P.
        • Zhang Y.
        • He W.
        • Chen X.
        • Chen J.
        • He L.
        • Mao L.
        • Wu F.
        • Jiao J.
        Dietary fats in relation to total and cause-specific mortality in a prospective cohort of 521 120 individuals with 16 years of follow-up.
        Circ Res. 2019; 124: 757-768https://doi.org/10.1161/CIRCRESAHA.118.314038
        • Wang Y.
        • Liu K.
        • Long T.
        • Long J.
        • Li Y.
        • Li J.
        • Cheng L.
        Dietary fish and omega-3 polyunsaturated fatty acids intake and cancer survival: a systematic review and meta-analysis.
        Crit Rev Food Sci Nutr. 2022; https://doi.org/10.1080/10408398.2022.2029826
        • Yin Y.
        • Sui C.
        • Meng F.
        • Ma P.
        • Jiang Y.
        The omega-3 polyunsaturated fatty acid docosahexaenoic acid inhibits proliferation and progression of non-small cell lung cancer cells through the reactive oxygen species-mediated inactivation of the pi3k /akt pathway.
        Lipids Health Dis. 2017; 16: 87https://doi.org/10.1186/s12944-017-0474-x
        • Bai X.
        • Shao J.
        • Zhou S.
        • Zhao Z.
        • Li F.
        • Xiang R.
        • Zhao A.Z.
        • Pan J.
        Inhibition of lung cancer growth and metastasis by dha and its metabolite, rvd1, through mir-138-5p/foxc1 pathway.
        J Exp Clin Cancer Res. 2019; 38: 479https://doi.org/10.1186/s13046-019-1478-3
        • Lee K.H.
        • Seong H.J.
        • Kim G.
        • Jeong G.H.
        • Kim J.Y.
        • Park H.
        • Jung E.
        • Kronbichler A.
        • Eisenhut M.
        • Stubbs B.
        • Solmi M.
        • Koyanagi A.
        • Hong S.H.
        • Dragioti E.
        • de Rezende L.F.M.
        • Jacob L.
        • Keum N.
        • van der Vliet H.J.
        • Cho E.
        • Veronese N.
        • Grosso G.
        • Ogino S.
        • Song M.
        • Radua J.
        • Jung S.J.
        • Thompson T.
        • Jackson S.E.
        • Smith L.
        • Yang L.
        • Oh H.
        • Choi E.K.
        • Shin J.I.
        • Giovannucci E.L.
        • Gamerith G.
        Consumption of fish and ω-3 fatty acids and cancer risk: an umbrella review of meta-analyses of observational studies.
        Adv Nutr. 2020; 11: 1134-1149https://doi.org/10.1093/advances/nmaa055
        • Zuijdgeest-van Leeuwen S.D.
        • van der Heijden M.S.
        • Rietveld T.
        • van den Berg J.W.O.
        • Tilanus H.W.
        • Burgers J.A.
        • Wilson J.H.P.
        • Dagnelie P.C.
        Fatty acid composition of plasma lipids in patients with pancreatic, lung and oesophageal cancer in comparison with healthy subjects.
        Clin Nutr. 2002; 21: 225-230https://doi.org/10.1054/clnu.2001.0530
        • Ren J.
        • Zhang D.
        • Liu Y.
        • Zhang R.
        • Fang H.
        • Guo S.
        • Zhou D.
        • Zhang M.
        • Xu Y.
        • Qiu L.
        • Li Z.
        Simultaneous quantification of serum nonesterified and esterified fatty acids as potential biomarkers to differentiate benign lung diseases from lung cancer.
        Sci Rep. 2016; 6: 34201https://doi.org/10.1038/srep34201
        • Mann N.
        • Pirotta Y.
        • O'Connell S.
        • Li D.
        • Kelly F.
        • Sinclair A.
        Fatty acid composition of habitual omnivore and vegetarian diets.
        Lipids. 2006; 41: 637-646
        • Lee K.H.
        • Seong H.J.
        • Kim G.
        • Jeong G.H.
        • Kim J.Y.
        • Park H.
        • Jung E.
        • Kronbichler A.
        • Eisenhut M.
        • Stubbs B.
        • Solmi M.
        • Koyanagi A.
        • Hong S.H.
        • Dragioti E.
        • de Rezende L.F.M.
        • Jacob L.
        • Keum N.
        • van der Vliet H.J.
        • Cho E.
        • Veronese N.
        • Grosso G.
        • Ogino S.
        • Song M.
        • Radua J.
        • Jung S.J.
        • Thompson T.
        • Jackson S.E.
        • Smith L.
        • Yang L.
        • Oh H.
        • Choi E.K.
        • Shin J.I.
        • Giovannucci E.L.
        • Gamerith G.
        Consumption of fish and ω-3 fatty acids and cancer risk: an umbrella review of meta-analyses of observational studies.
        Adv Nutr. 2020; 11 (Bethesda, Md): 1134-1149https://doi.org/10.1093/advances/nmaa055
        • Chen M.
        • Huang J.
        The expanded role of fatty acid metabolism in cancer: new aspects and targets.
        Precis Clin Med. 2019; 2: 183-191https://doi.org/10.1093/pcmedi/pbz017
        • Li Y.-J.
        • Fahrmann J.F.
        • Aftabizadeh M.
        • Zhao Q.
        • Tripathi S.C.
        • Zhang C.
        • Yuan Y.
        • Ann D.
        • Hanash S.
        • Yu H.
        Fatty acid oxidation protects cancer cells from apoptosis by increasing mitochondrial membrane lipids.
        Cell Rep. 2022; 39110870https://doi.org/10.1016/j.celrep.2022.110870
        • Xuan C.
        • Tian Q.-W.
        • Li H.
        • Guo J.-J.
        • He G.-W.
        • Lun L.-M.
        Serum fatty acids profile and association with early-onset coronary artery disease.
        Ther Adv Chronic Dis. 2021; 1220406223211033102https://doi.org/10.1177/20406223211033102
        • Diffenderfer M.R.
        • Rajapakse N.
        • Pham E.
        • He L.
        • Dansinger M.L.
        • Nelson J.R.
        • Schaefer E.J.
        Plasma fatty acid profiles: relationships with sex, age, and state-reported heart disease mortality rates in the United States.
        J Clin Lipidol. 2022; 16: 184-197https://doi.org/10.1016/j.jacl.2021.12.005
        • Sarter B.
        • Kelsey K.S.
        • Schwartz T.A.
        • Harris W.S.
        Blood docosahexaenoic acid and eicosapentaenoic acid in vegans: associations with age and gender and effects of an algal-derived omega-3 fatty acid supplement.
        Clin Nutr. 2015; 34: 212-218https://doi.org/10.1016/j.clnu.2014.03.003
        • Serini S.
        • Calviello G.
        Long-chain omega-3 fatty acids and cancer: any cause for concern?.
        Curr Opin Clin Nutr Metab Care. 2018; 21: 83-89https://doi.org/10.1097/mco.0000000000000439
        • Welch A.A.
        • Bingham S.A.
        • Ive J.
        • Friesen M.D.
        • Wareham N.J.
        • Riboli E.
        • Khaw K.T.
        Dietary fish intake and plasma phospholipid n-3 polyunsaturated fatty acid concentrations in men and women in the European prospective investigation into cancer-norfolk United Kingdom cohort.
        Am J Clin Nutr. 2006; 84: 1330-1339https://doi.org/10.1093/ajcn/84.6.1330