Effect of 6,9,12,15-Hexadecatetraenoic Acid (C16:4 n -1)-Ethyl Ester on Lipid Content and Fatty Acid Composition in the Blood and Organs of Mice

the isolation of a ） -methyl ester ME ） of with EPA-EE, a major n- 3 PUFA found in fish oil. In this study, the HDTA-EE was concentrated to approximately 10 ％ by precision distillation of anchovy oil-EE and 98 ％ by preparative-scale high-performance liquid chromatography. Abstract: The effects of 6,9,12,15-hexadecatetraenoic acid (C16:4 n -1, HDTA), an n -1 polyunsaturated fatty acid (FA), on plasma and liver lipid content and distribution in blood and tissues were investigated. Mice were fed experimental diets containing 10% HDTA or eicosapentaenoic acid in ethyl ester form based on corn oil for four weeks. Dietary HDTA intake lowered plasma triacylglycerol content without affecting plasma total cholesterol content. HDTA barely accumulated in the epididymal white adipose tissue (eWAT), while C18:4 n -1, an HDTA metabolite, was detected in small amounts (< 1% of total FAs) in the plasma, liver, and eWAT.


Introduction
Fish oil is rich in n-3 polyunsaturated fatty acids PUFA , such as eicosapentaenoic acid EPA and docosahexaenoic acid DHA . The intake of EPA and DHA has beneficial effects on human health, including anti-arrhythmic 1 , antiinflammatory 2 , and anti-hypertriglyceridemic 3 effects. In addition to EPA and DHA, fish oil is composed of uncommon n-3 PUFA such as 6,9,12,15-octadecatetraenoic acid C18:4n-3 and 4,7,10,13-hexadecatetraenoic acid C16:4n-3 4 , which are physiologically relevant. Previous studies reported that C18:4n-3 has inhibitory effects on platelet aggregation 5 and the metabolism of C18:4n-3 in cultured NIH-3T3 cells 6 , while C16:4n-3 and C18:4n-3 have been reported to inhibit the production of leukotriene B4, leukotriene C4, and 5-hydroxyeicosatetraenoic acid in mouse mast cells 7 . A trace amount of C16:4n-3 induced by platinum-based chemotherapy was resistant to antitumor activity in mice, and fish oil containing C16:4n-3 neutralized the antitumor activity of platinum-based chemotherapy in various mouse models 8 . Fish oils derived from sardine and herring contain approximately 1 to 3 of 6,9,12, 15-hexadecatetraenoic acid C16:4n-1, HDTA , an isomer of C16:4n-3 4 . HDTA is uniquely characterized by the pres-ence of a double bond at the terminal carbon in its chemical structure Fig. 1A . A previous study reported the isolation of HDTA from a fatty acid FA -methyl ester ME prepared from fish oil using high-speed counter-current chromatography 9 . Thus far, only one patent report has investigated the physiological effects of HDTA and described anti-inflammatory effects of HDTA against colitis, carrageenan-induced edema, and arthritis in a mouse model 10 . The metabolism and health-promoting functions of the n-1 PUFA are relatively unknown to date and would constitute an interesting area of research with substantial implications for advancing oleochemistry. This study evaluated the effect of dietary HDTA-ethyl ester EE on lipid content and FA composition in the blood and organs of mice compared with EPA-EE, a major n-3 PUFA found in fish oil. In this study, the HDTA-EE was concentrated to approximately 10 by precision distillation of anchovy oil-EE and 98 by preparative-scale high-performance liquid chromatography.

Materials
HDTA-EE 98 and EPA-EE 98 were provided by Bizen Chemical Co., Ltd. Akaiwa, Japan . Corn oil-EE was prepared by NaOH-ethanol solution from triacylglycerol TAG -form corn oil Merck KGaA, Darmstadt, Germany . The experimental diet components were purchased from Oriental Yeast Co. Ltd. Tokyo, Japan and Fujifilm Wako Pure Chemical Co. Osaka, Japan . All other reagents were of reagent grade, and were obtained from Merck KGaA and Nacalai Tesque, Inc. Kyoto, Japan .

Analysis of HDTA-EE
HDTA-EE was dissolved in n-hexane 2 , w/v and analyzed via gas chromatography GC /mass spectrometry MS GC/MS to confirm its chemical structure, using a GC/MS GCMS-QP2010/PARVUM2, Shimadzu Co., Kyoto, Japan system equipped with an Omegawax ® column 30 m 250 μm 0.25 μm; Merck KGaA . The GC system parameters were set as follows: injector and interface temperatures of 250 , column temperature of 120 , gradual heating to 240 at a rate of 2 /min, held at 240 for 10 min, and 1 mL/min helium as a carrier gas. MS system parameters were set as follows: ion source temperature, 200 ; electronic ionization EI , 70 eV; full scan m/z 45-350 amu. Detailed total-ion chromatograms and EI mass spectra of HDTA-EE are shown in Figs. 1B and 1C.

Animal diet and care
All animal experiments were conducted in accordance with the Guide for the Care and Use of Experimental Animals issued by the Prime Minister s Office of Japan and were reviewed and approved by the Animal Ethics Committee of Kansai University Approval No. 1704 .
The experimental diet was prepared based on the AIN-93G composition 11 so that HDTA-EE and EPA-EE each had an FA composition of 10 mol HDTA and EPA diets, respectively . Corn oil-EE was added to the control diet to adjust the EE content to be approximately equal to that in the HDTA and EPA diets. Details of the ingredients and FA compositions of the experimental diets are shown in Table  1.
Four-week-old male C57BL/6J mice obtained from Japan SLC, Inc. Hamamatsu, Japan were kept in individual cages, housed at 21-23 with a 12 h light-dark cycle lights on: 08:00-20:00 , and allowed free access to tap water and the prepared diets. Following an acclimatization period of seven days, 18 mice were divided into three dietary groups: control, HDTA, and EPA. Food consumption, water intake, and body weight BW were recorded every two days. After four weeks, the mice were fasted overnight, weighed, and euthanized using isoflurane Intervet K.K., Osaka, Japan between 09:00 and 11:00. Blood was collected from the inferior vena cava with anticoagu-lants, and plasma and red blood cells RBC were separated by centrifugation 2,000 g for 15 min. The liver, kidney, spleen, heart, brain, interscapular brown adipose tissue BAT and abdominal white adipose tissue WAT from the epididymis, mesentery, and perinephric and inguinal regions were quickly removed, weighed, and rinsed with cold saline. The tissues were frozen in liquid nitrogen and stored at 80 until further analyses. A portion of the liver was stored in RNA-Later Storage Solution Merck KGaA for quantitative real-time PCR.

Analysis of lipid parameters in plasma and liver
Plasma biochemical parameters aspartate aminotransferase AST , alanine aminotransferase ALT , urea nitrogen UN , TAG, phospholipids PL , total cholesterol, high-density lipoprotein HDL cholesterol, and non-HDL cholesterol were measured using an Olympus AU5431 automatic analyzer Olympus Co., Tokyo, Japan by a com- mercial service Japan Medical Laboratory, Osaka, Japan . Total liver lipids were extracted using the chloroform/ methanol/water extraction method as described by Bligh and Dyer 12 and then dissolved in 2-propanol. Liver TAG content in total liver lipids dissolved in 2-propanol was determined using the Triglyceride-E-Test Fujifilm Wako Pure Chemical Co. . Liver PL content was measured using phosphorus analyses as described by Rouser et al. 13 . Liver cholesterol content was determined by GC as described by Kaneda et al. 14 .

Analysis of fatty acid composition
The total lipids from the experimental diets, plasma, RBC, liver, epididymal WAT eWAT , and brain were extracted using the chloroform/methanol/water extraction method described above 12 . The FA compositions of the total lipids were determined using a GC system GC-2014; Shimazu Co. with an Omegawax ® column after the methylation of FA with boron-trifluoride-methanol as described in our previous report 15 . Identification of each FA was carried out using a standard mixture of FA-ME Supelco 37 Component FAME Mix, Merck KGaA .
2.6 RNA isolation and real-time quantitative reverse transcription PCR Liver samples, which were stored in RNA-Later Storage Solution, were homogenized using a bead beater-type homogenizer MicroSmash MS-100R, TOMY SEIKO CO., LTD., Tokyo, Japan . Total RNA was isolated and purified using the TRIzol ® reagent Thermo Fisher Scientific Inc, Massa-  Table S1. Gene expression levels were standardized to the expression level of glyceraldehyde-3-phosphate dehydrogenase Gapdh and expressed as the fold-change in gene expression relative to the control group.

Statistical analysis
Data are presented as mean values and standard errors of the mean SEM . The differences between multiple groups were evaluated using analysis of variance ANOVA and Tukey s multiple comparison test. Statistical significance and tendency were set as p 0.05, and 0.05 ≤ p 0.10, respectively. Analyses were performed using Graph-Pad Prism7 ver. 7.0d, GraphPad Software, California, USA . Table 2 shows growth parameters, organ weights, plasma biochemical parameters, and liver lipid contents. There were no significant differences in growth parameters and organ weights among the three groups.

Growth parameters, organ weights, and biochemical parameters in plasma and liver
HDTA intake decreased plasma TAG content compared to the control group. The EPA group showed reduced plasma PL, total cholesterol, HDL cholesterol, and non-HDL cholesterol contents compared to the control group. The EPA diet significantly reduced liver TAG content compared to the HDTA group, whereas it tended to be lower than that in the control group p 0.06 . There were no significant differences in plasma AST, ALT, UN, liver PL, or cholesterol levels among the experimental groups.

Fatty acid composition in the blood and organs
C18:1n-9 proportions compared to the control and EPA groups. The EPA diet decreased the C18:3n-3 and C20:4n-6 proportions compared to the control and HDTA diets, as well as the C18:1n-7 and C20:1n-9 proportions compared to the control diet. Moreover, the C20:5n-3 and C22:6n-3 proportions in the EPA group were higher than those in the control and HDTA groups. In the brain, the EPA group had a higher C20:3n-6 proportion than the HDTA group, which lowered the C20:4n-6 proportion compared to the control and HDTA groups. Figure 4 shows the relative expression levels of genes related to FA metabolism in the liver. The expression of stearoyl-coenzyme A desaturase-1 Scd-1 in the liver of mice fed the EPA diet was lower than that in the control group. There were no significant differences in sterol regulatory element binding transcription factor 1 Srebf1 , fatty acid synthase Fas , acetyl-coenzyme A carboxylase alpha Acaca , acyl-coenzyme A oxidase 1 Acox1 , carnitine palmitoyltransferase 1a Cpt1a , fatty acid desaturase Fads 1, Fads2, or elongation of very long-chain fatty acids Elovl 2, Elovl3, Elovl5, Elovl6, and Elovl7 among the three groups.

Discussion
In this study, HDTA intake decreased plasma TAG content compared to the control group. The reduction in plasma TAG content in the HDTA group was notably more substantial than that in the EPA group Table 2 . EPA intake has a lowering effect on plasma TAG content due to its suppression of FA synthesis and the enhancement of FA oxidation through regulation of liver X receptor and peroxisome proliferator-activated receptor-α 17,18 . Thus, EPA is offered as a pharmaceutical compound for patients with hypertriglyceridemia. HDTA intake was observed to have a more potent plasma TAG-lowering effect than EPA intake under these experimental conditions. As this study was conducted using normal mice, further studies should evaluate this effect using a mouse model of hyperlipidemia. Conversely, TAG content in the liver was increased in the HDTA group compared to that in the EPA group; however, it was not significantly different from that in the control group Table 2 . To elucidate the mechanism of HDTA-induced decrease in plasma TAG content, mRNA expression levels of genes related to FA metabolism in the liver were measured; however, there were no significant changes in the expression levels of genes associated with FA synthesis and oxidases Srebf1, Fas, Acaca, Acox1, and Cpt1a . A previous study reported that fasting time significantly affected lipid metabolism, especially through regulation of enzymes involved in liver FA biosynthesis in rodents 19 ; the results indicated that in order to elucidate the mechanisms of lipid reduction induced by dietary food components, rodents should not be fasted on the last day before dissection 19 . In our study, we dissected the mice after a period of overnight fasting to assess plasma biochemical parameters and organ FA compositions, which were not affected by diet. This study therefore did not allow us to evaluate the effect of HDTA on FA metabolism in the liver, and further studies should be conducted in non-fasting conditions.
To evaluate the distribution of HDTA and its metabolites in the blood and organs, we measured FA compositions of the total lipids in the plasma, RBC, liver, eWAT, and brain Fig. 3 . We detected two unknown FA peaks RTs 32.8 and 42.7 min, respectively that were detected only in mice  Table S1.
fed the HDTA diet and not in mice fed the control and EPA diets Fig. 2A . From the GC/MS data, two unknown peaks were identified as HDTA C16:4n-1 and C18:4n-1 Figs. 2B and 2C, respectively . HDTA was detected only in 0.6 0.0 mol of eWAT in the HDTA group Fig. 3D . In addition, the C18:4n-1 was detected in 0.3 0.1, 0.9 0.1, and 0.5 0.1 mol of plasma, liver, and eWAT in the HDTA group, respectively. However, EPA in the plasma, RBC, liver, and eWAT in the EPA group were 6.1 1.5, 5.6 0.3, 4.7 0.5, and 1.0 0.1 mol , respectively. EPA was not detected in the brains of mice in the EPA group. The mammalian central nervous system is very low in EPA, which suggests that in the brain EPA may be a precursor to DHA synthesis 20 . In the plasma, RBC, liver, and eWAT, HDTA proportions in the HDTA group were much lower than EPA proportions in the EPA group. HDTA intake accumulates in small amounts 1 of total FAs in the plasma, liver, and eWAT as HDTA and/or C18:4n-1. Dietary palmitic acid C16:0 , which has the same carbon chain length as HDTA, is incorporated in the eWAT TAG of rats 21 . In addition, the intake of diet containing about 17 of total FAs in diet palmitoleic acid C16:1n-7 was accumulated in 3.5 of total FAs of the liver in low-density lipoprotein receptor-deficient mice 22 . Previous studies have administered unusual FAs to laboratory animals and investigated their distribution 23,24 . Diet containing about 7 of total FAs in diet conjugated linoleic acid administration was found to be incorporated in various tissues kidney, brain, testis, liver, lung, spleen, and eWAT to reach a proportion of 0. 5 -5.5 of total FAs in rats 23 , while the intake of about 10 of total FAs in diet sciadonic acid, a nonmethylene-interrupted PUFA, accumulated in 5.3 and 2.1 wt of total FAs in the serum and liver TAG of rats 24 . Odd-numbered long-chain FAs are more likely to accumulate in the mammalian body than even-numbered long-chain FAs 25 , whereas dietary medium-chain FAs MCFA are absorbed from the intestinal tract to the portal vein, from where they are taken into the liver and rapidly undergo β-oxidation 26 . Previous reports showed that mice fed a diet containing lauric acid 3 wt of diet showed no lauric acid in liver TAG and mice fed a diet containing medium-chain triglyceride were decreased serum TAG content 27,28 . Since there was little accumulation of HDTA in the plasma, liver, and eWAT even when the mice were fed a 10 HDTA diet of total FAs in our study, it can be considered that HDTA undergoes β-oxidation as well as MCFA, which may have decreased the plasma TAG content. However, HDTA may not be directly β-oxidized because C18:4n-1, a 2-carbon chain elongation of HDTA, is detected as a metabolite of HDTA. HDTA has four double bonds for its carbon chain length C16 , and since mouse do not contain such FAs, HDTA may have rapidly elongated the carbon chain, which may affect the stability of the cell membrane if it is incorporated into PL. In addition, it is also possible that the absorption rate of HDTA in the gastrointestinal tract is low. HDTA, an n-1PUFA, may not be a substrate for cluster of differentiation 36, which is important for FA uptake in the apical side of enterocyte 29 . Further studies are therefore necessary to clarify the metabolism and absorption of HDTA.

Conclusion
In this study, we investigated the effects of HDTA, which has a double bond at the n-1 position, on the lipid content and FA composition in the blood and organs of mice. HDTA intake has a potent plasma TAG-lowering function. In addition, HDTA hardly accumulated in the plasma, RBC, liver, and eWAT, and a proportion of the HDTA was metabolized to C18:4n-1. This study shows, for the first time, the effects of HDTA on the plasma and liver lipid contents and FA composition in vivo.