2025 Volume 74 Issue 1 Pages 1-11
Omega-3 long-chain polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are widely used as supplements and pharmaceuticals because of their beneficial effects on human health. Triacylglycerols (TAG) and glycerophospholipids (GPL) comprise the primary chemical structures of DHA/EPA in marine sources. Furthermore, DHA/EPA-enriched glycerophospholipids (DHA/EPA-GPL) and lysoglycerophospholipids (DHA/EPA-LysoGPL) consumed through food and supplements are more effective than TAG in promoting health, which may be attributed to a specific underlying mechanism. However, the specific effects of DHA/EPA bound to GPL structure have been still unclear. The aim of this review is to clarify the significance of the binding of DHA/EPA to GPL in promoting the health benefits of DHA/EPA-GPL and DHA/EPA-LysoGPL. Additionally, the potential use of fishery by-products as sources of DHA/EPA-GPL and DHA/EPA-LysoGPL has been discussed.
Omega-3 (ω-3) long-chain polyunsaturated fatty acids (PUFA) ̶especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ̶have been globally recognized as being important for human health, particularly after the discovery that the incidence of cardiovascular disease (CVD) is significantly low among Greenland Inuit who consume large quantities of these fatty acids1) , 2) . Similarly, epidemiological studies on Japanese islanders have reported an association between blood EPA level and CVD development3) ,4) . Moreover, ω-3 PUFA are effective in improving cardiac function5) , decreasing blood triacylglycerol (TAG) content6) , lowering blood pressure7) , relieving depression8) , and enhancing cognitive function9) . Additionally, various international guidelines recommend the use of ω-3 PUFA to lower blood TAG levels in individuals with high blood TAG levels. For example, the American Heart Association recommends the intake of up to 4 g/day of DHA/EPA or EPA alone for treating hypertriglyceridemia10) . In contrast, the Food and Drug Administration (FDA) advises consumers not to exceed a total intake of 3 g/day of DHA/EPA and no more than 2 g/day from dietary supplements because excessive DHA/EPA consumption increases blood clotting time11) .
TAG and glycerophospholipids (GPL) comprise the primary chemical structures of DHA/EPA in marine foods, and most commercially available DHA/EPA supplements contain the TAG form. Recent studies have suggested that DHA/EPA-GPL from certain dietary sources may provide greater health benefits than that of DHA/EPA-TAG. Evidence from human clinical trials suggests that the bioavailability of DHA/EPA-enriched GPL (DHA/EPA-GPL) may be higher than that of DHA/EPA-enriched TAG (DHA/EPA-TAG) 12) ,13) ,14) . However, these findings remain controversial15) . Several animal and clinical studies have reported that DHA/EPA-GPL is effective in improving brain function16) , inducing antitumor activity17) , regulating lipid and glucose metabolism18) ,19) , exerting anti-inflammatory effects20) , and enhancing exercise training and performance21) . Certain individuals have reported no benefits from consuming DHA/EPA (TAG form) supplements whereas others have reported positive effects after consuming DHA/EPA in their diet; a possible underlying reason may be that a proportion of the DHA/EPA in seafood is present in the GPL form22) . Regarding DHA/EPA-enriched lysoglycerophospholipids (DHA/EPA-LysoGPL) , there have been many studies on the efficiency of supplying DHA to the brain. Dietary DHA/EPA-lysophosphatidylcholine (LysoPC) can improve behavior in mice by regulating APOE4, a gene linked to age-related cognitive decline and neurodegenerative diseases23) . Additionally, there have been reports regarding its effects on angiogenesis24) and the opening of tight junctions in small intestinal epithelial cell monolayers25) . In contrast, the glycerophosphate structure of GPL, which does not contain DHA/EPA, may exert a better health effect than that of TAG26) . Several animal and clinical studies have reported that GPL, which does not contain DHA/EPA, is effective in improving brain function27) and liver function28) , increasing oxidative stability29) , regulating lipid metabolism30) ,31) , and enhancing sports performance32) . To evaluate the health benefits of DHA/EPA-GPL and DHA/EPA-LysoGPL, it is essential to consider not only the effects of DHA/EPA but also those of GPL. However, the specific effects of DHA/EPA bound to GPL structure have been still unclear. Several comprehensive reviews have been published on the structure, characteristics, sources, extraction, quantification, and health effects of marine phospholipids26) ,33) ,34) ,35) ,36) , but no reviews focus on the health effects from the perspective of the structure of DHA/EPA bound to GPL. In addition, the development of the aquaculture industry has increased the focus on high-value utilization of fishery by-products including those that may be used as sources of DHA/EPA-GPL. This review explains the significance of the binding of DHA/EPA to GPL in expressing the health benefits of DHA/EPA-GPL and DHA/EPA-LysoGPL. Furthermore, the use of fishery by-products as potential sources of DHA/EPA-GPL has been discussed in-depth.
Phospholipids are important components of cellular lipids as they are essential for biological signal transduction and maintenance of cell structure and function. These compounds are amphiphilic and contain both hydrophobic (acyl, alkyl, or alkenyl groups) and hydrophilic (phosphate groups or polar heads) components. Based on their molecular structures, phospholipids are classified as GPL, sphingomyelins, and phosphonolipids37) . GPL are formed by the esterification of the fatty acids at the sn-1 and sn-2 positions of the glycerol backbone and a phosphate group at the sn-3 position (Fig. 1A) 38) . GPL are further categorized as phosphatidylcholine (PC) , phosphatidylethanolamine (PE) , phosphatidylserine (PS) , phosphatidylinositol (PI) , and phosphatidic acid depending on the polar head group bound to the phosphate group at the sn-3 position. Ether-type GPL are characterized by a vinyl ether or ether bond at the sn-1 position of the glycerol backbone39) . Lysoglycerophospholipids (LysoGPL) lack one acyl chain and contain only one acyl chain in the glycerol backbone (Fig. 1B) . Based on the position of the fatty acid bond, they are further classified into 1-acyl-LysoGPL (acyl chain bound at sn-1 position) and 2-acyl-LysoGPL (acyl chain bound at sn-2 position) . GPL derived from marine sources primarily contain DHA/EPA at the sn-2 position and saturated fatty acids (SFA) such as stearic acid (SA) and palmitic acid (PA) at the sn-1 position40) .
Chemical structures of typical glycerophospholipids (A) and lysoglycerophospholipids (B) from marine sources.
Studies on phospholipids have primarily focused on the physiological significance of cell membrane phospholipids, which includes the production of bioactive substances such as eicosanoids and their roles as intracellular signaling molecules as in the case of PI, diacylglycerol, and LysoGPL. Additionally, other studies have highlighted the significant nutritional and physiological functions of dietary GPL40) . Particularly, DHA/EPA-GPL has garnered attention because of its high bioavailability12) and health benefits that surpass those exerted by DHA/EPA-TAG.
The studies that have demonstrated the health benefits of DHA/EPA-GPL evaluated the effects of DHA/EPA-GPL by comparing them with DHA/EPA-TAG and ordinary GPL (such as plant-derived GPL) , which lack DHA/EPA. Hence, whether the beneficial effects of DHA/EPA-GPL are due to esterification of the glycerophosphate structure of DHA/EPA or the combination of the glycerophosphate structure and DHA/EPA remains unclear. Therefore, we investigated the health benefits specific to DHA/EPA-GPL, which cannot be replicated by the DHA/EPA-TAG combination and ordinary GPL.
First, we prepared DHA/EPA-GPL from squid and evaluated its effects on serum and liver lipid levels in rats41) . Our findings showed that DHA/EPA-GPL intake led to reduction of serum cholesterol level by the glycerophosphate structure and reduction of serum TAG level by DHA/EPA. Specifically, the cholesterol-lowering effect of DHA/EPA-GPL was linked to increased excretion of fecal neutral sterols and elevated expression of the liver ATP-binding cassette transporter (Abc) g5 and Abcg8 genes. Furthermore, these effects were a result of the combined effect of the glycerophosphate structure and DHA/EPA.
Second, we aimed to identify the health benefits specific to DHA/EPA-GPL by comparing the health benefits exerted by DHA/EPA-GPL with those exerted by a DHA/EPA-TAG and soybean GPL (DHA/EPA-TAG+SoyGPL) combination, which has a fatty acid and phospholipids composition similar to that of DHA/EPA-GPL42) . Rats fed the DHA/EPA-GPL and DHA/EPA-TAG+SoyGPL diets showed reduced TAG and cholesterol levels in the serum and liver, and the effects were equivalent. However, an analysis of total lipid fatty acid composition of the serum and organs showed that the DHA/EPA-GPL group had a higher serum DHA ratio and lower white adipose tissue (WAT) DHA ratio than that of the DHA/EPA-TAG+SoyGPL group (Figs. 2A and 2B) 43) . Other studies have also shown that DHA/EPA-TAG was more effective than DHA/EPA-GPL in increasing the WAT DHA ratio44) . In contrast, the DHA ratio did not differ significantly between the heart and the liver (Figs. 2C and 2D) . Therefore, we inferred that difference in the DHA ratio in specific organs related with the binding or non-binding of DHA/EPA to GPL or TAG. Subsequently, a mouse study that used a similar approach reported that the effect of DHA/EPA-GPL intake on lowering atherogenic indices was superior to that observed for the combination of DHA/EPA-TAG and egg GPL45) . Overall, these findings suggest that the effect exerted by DHA/EPA-GPL cannot be entirely replicated by substituting this combination with the DHA/EPA-TAG combination or ordinary PL because the binding of DHA to either GPL or TAG exerts different effects on the DHA ratio in serum and WAT.
Relative DHA compositions in serum (A) , epididymal WAT (B) , liver (C) , and heart (D) .Data are presented as mean±SEM. Different letters indicate significant differences atp<0.05, using Tukey’s multiple comparison test. These figures are reconstructed from the figure in the reference 43. Please refer to reference 43 regarding detailed experimental conditions. EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; GPL, glycerophospholipids; SEM, standard error of means; TAG, triacylglycerols; WAT, white adipose tissue.
The effects of DHA/EPA-GPL are not entirely replicated by the DHA/EPA-TAG and ordinary GPL combination. This indicates the crucial need for DHA/EPA to be bound to GPL. The specific effect of DHA/EPA-GPL may be attributed to its ability to sustain this form during absorption from the small intestine into the lymph (Fig. 3) .
The estimated pathway of the metabolic fate of DHA-TAG, DHA-PC, and DHA-LysoPC in the small intestine. (A) DHA present in TAG in marine sources is mainly located at the sn-2 position of TAG. DHA-TAG is cleaved by pancreatic lipase at the sn-1 and sn-3 positions to produce free FAs and DHA-MAG. These are then re-esterified into TAG in the small intestinal epithelial cells and taken into the inner of the CM. (B) DHA present in PC in marine sources is mainly at the sn-2 position of PC. DHA-PC is cleaved by PLA2 at the sn-2 position to produce 1-acyl-LysoPC and free DHA. On the other hand, there is also a pathway that cleaves the sn-1 position FA to produce DHA-LysoPC. The DHA-LysoPC is re-esterified into DHA-PC in the small intestinal epithelial cells and then incorporated into the outer of CM and HDL. Dietary DHA-LysoPC can be directly absorbed into small intestinal epithelial cells. The most absorbed DHA is incorporated into the TAG in lymph, regardless of the dietary DHA forms (DHA-TAG, DHA-PC, and DHA-LysoPC) . Although not shown in Fig. 3, LysoPLA degrades DHA-LPC to free DHA and GPC in the small intestinal lumen and epithelial cells.CM, chylomicrons; DHA, docosahexaenoic acid; FA, fatty acid; GPC, glycerophosphocholine; HDL, high-density lipoproteins; LysoPC, lysophosphatidylcholine; LysoPLA, lysophospholipase A; MAG, 2-monoacylglycerol; PC, phosphatidylcholine; PLA2, phospholipase A2; P-Choline: phosphorylcholine.
Most marine sources of fatty acids such as fish and fish roe contain GPL with DHA/EPA at the sn-2 position. The fatty acid at the sn-2 position in GPL is hydrolyzed in the intestine by pancreatic phospholipase A2 (PLA2) and released as a free fatty acid, which is re-esterified into TAG within the small intestinal epithelial cells, followed by incorporation into chylomicrons and secretion into the lymph46) .
In contrast, fish oil supplements primarily contain TAG with DHA/EPA at the sn-2 position47) . The consumed TAG is hydrolyzed to 2-monoacylglycerol and two free fatty acids by pancreatic lipases48) , which enter the bile micelles that aid in the passage of non-polar lipids through the water barrier of the small intestine surface to reach the intestinal microvilli49) . After the hydrolyzed products are absorbed by the small intestinal epithelial cells, they are re-esterified into TAG, followed by their incorporation into chylomicrons and secretion into the lymph50) . Notably, the chemical form of DHA bound to the sn-2 position of GPL or TAG in the lymph is the same in both absorption pathways because both types of DHA are re-esterified into TAG in the small intestinal epithelial cells.
However, a study on the absorption of DHA-PC, DHA-TAG, and free DHA in rats showed that DHA-PC administration resulted in a significantly higher DHA level in the phospholipid fraction of the lymph than that observed after DHA-TAG and free DHA administration51) . Furthermore, the simultaneous administration of free DHA and LysoPC mimicked the digestion products of DHA-PC, although DHA-PC administration did not produce the same results52) . Additionally, dietary DHA-GPL is more resistant than other types of GPL to pancreatic PLA2. Hence, other enzymes with PLA1 activity in pancreatic juice may be responsible for producing DHA-LysoGPL51) . These findings suggest that DHA-LysoPC is partially hydrolyzed from DHA-PC at the sn-1 position and re-esterified into DHA-GPL in small intestinal epithelial cells. Moreover, the lipoproteins that take up the re-esterified GPL and TAG in small intestinal epithelial cells differ from each other52) . Studies using piglets53) have shown that DHA in dietary DHA-GPL gets incorporated into high-density lipoproteins (HDL) , whereas DHA in dietary DHA-TAG gets incorporated into low-density lipoproteins (LDL) 54) . Furthermore, GPL and TAG are incorporated into the outer and inner parts of chylomicrons and HDL particles, respectively55) . These differences may also contribute to differences in target tissues and extent of delivery to the respective tissues.
The assumption that absorption of DHA/EPA in a bound state to GPL mediates the specific effects exerted by DHA/EPA-GPL leads to the expectation that a greater effect may be elicited if consumed in the DHA/EPA-LysoGPL form, which is absorbed directly in the digestive tract. 1-DHA-LysoPC was approximately five times more abundant than free DHA in the lymph phospholipids recovered from chylomicrons and HDL46) . Furthermore, the chemical structure and nutritional efficacy of DHA have been extensively studied in terms of its transport and accumulation in the brain because neurons cannot synthesize DHA, which necessitates its consumption through diet and transportation from the plasma to the brain through the blood-brain barrier (BBB) 22) . The major facilitator superfamily domain-containing protein 2A (Mfsd2a) primarily transports DHA across the BBB, and Mfsd2a specifically transports DHA in the LysoPC form56) . This has been substantiated in a study where the brains of mice that were orally administered DHA-LysoPC showed significantly higher DHA content, whereas those of mice that were administered free DHA did not show an increase in DHA content57) . The DHA-TAG in LDL readily forms free DHA bound to albumin via the lipase54) ,58) ,59) . Some of the DHA-LysoPC reconstituted into GPL in the small intestinal epithelial cells are incorporated into HDL and readily form DHA-LysoPC in the blood via the enzyme lecithin-cholesterol acyltransferase, which is an endothelial lipase that is abundant in the BBB and shows phospholipase A1 (PLA1) activity54) ,60) . Thus, DHA-LysoPC may be a superior chemical structure for DHA transport into the brain and is a subject of extensive study.
The use of fishery by-products as sources of DHA/EPA-LysoGPL is effective. Most GPL prepared from fishery by-products bind to DHA/EPA at the sn-2 position and to SFA at the sn-1 position61) . Therefore, removing the SFA at the sn-1 position enabled the preparation of LysoGPL while increasing the DHA/EPA concentration. Our collaborators have previously reported that GPL prepared from fishery by-products are partially hydrolyzed using sn-1,3-specific lipase (Rhizopus oryzae lipase) 61) ,62) . Additionally, we found that PLA1 activity against GPL increased DHA/EPA and reduced SFA concentrations compared with that observed for squid phospholipids before partial hydrolysis (Table 1) 63) . DHA/EPA-LysoGPL prepared from squid reduced serum and liver TAG levels by increasing acyl-CoA oxidase (ACOX) activity and inhibiting fatty acid synthase activity in the liver compared with that observed for soybean oil alone. Additionally, DHA/EPA-LysoGPL prepared from squid exhibited lower serum cholesterol content than that of soybean oil, which could be attributed to a certain degree to the reduction in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase mRNA expression in the liver. Further purification of DHA/EPA-LysoGPL prepared from squid (using activated carbon, ion-exchange resin, and silica gel purification) yielded a DHA/EPA-LysoPC-rich oil with a 79.3 mol% DHA and 66.5% (w/w) LysoPC composition64) . Furthermore, intake of DHA/EPA-LysoPC-rich oil reduced TAG levels in the serum and liver of rats, which may be partially attributed to the enhancement of carnitine palmitoyltransferase-2 and ACOX activities and suppression of acetyl-CoA carboxylase and glucose-6-phosphate dehydrogenase activities. Moreover, increased DHA level in rat hippocampus following the intake of DHA/EPA-LysoPC-rich oil may be due to the elevation of DHA level in serum LysoPC (Fig. 4) . Moreover, compared with the effect elicited by untreated krill oil, krill oil pretreated with Mucor meihei lipase induced a 5- and 70-fold increase in DHA and EPA concentrations, respectively, in the brains of mice30) ,44) . Additionally, we successfully prepared DHA/EPA-LysoGPL and -LysoPC on a pilot plant scale from the by-products of squid processing, and we believe that this model is applicable to other fishery by-products.
Fatty acid and phospholipid compositions of DHA/EPA-LysoGPL and -LysoPC rich oil prepared from squid by-products.
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Relative DHA compositions in hippocampus (A) and serum total lipid (B) , PC (C) , and LysoPC (D) .Data are presented as mean±SEM. *p<0.05 (Student’s t-test) .DHA, docosahexaenoic acid; LysoPC, lysophosphatidylcholine; PC, phosphatidylcholine. These figures are reconstructed from the figure in the reference 64. Please refer to the reference 64 regarding detailed experimental condition.DHA, docosahexaenoic acid; LysoPC, lysophosphatidylcholine; PC, phosphatidylcholine; SEM, standard error of means.
Several studies have demonstrated that the health impact elicited by DHA/EPA-GPL and DHA/EPA-LysoGPL is superior to that elicited by the more commonly used DHA/EPA-TAG22) ,35) ,55) . Particularly, DHA-LysoPC is necessary for delivering DHA to the brain, as it is capable of transporting DHA through phospholipids to the bloodstream46) . However, most current findings on DHA-LysoPC have been obtained through in vitro and animal studies. Therefore, future studies are required to determine the significance of DHA-LysoPC in human beings.
The increasing demand for sustainable and healthy food sources has caused the re-evaluation of unused resources in the seafood industry where only an estimated 50-60% of marine animal catches are used for direct human consumption. Additionally, the production and disposal of by-products cause problems related to industrial logistics, environmental impact, and human health65) ,66) ,67) . Amidst paramount issues of overfishing, waste generation and disposal, and environmental sustainability, the use of fishery by-products has emerged as a logical and appealing solution. Hence, the rational use of these by-products provides a feasible and appropriate solution to both nutritional and environmental issues. Furthermore, identifying new ways to commercialize these raw materials may result in positive economic benefits68) .
In this context, we used the internal organs of scallops, which are the largest fishery by-product in Japan, and successfully produced scallop oil (SCO) containing DHA/EPA-GPL. SCO contains approximately 30% (w/w) EPA, 10% (w/w) DHA, and 10-20% (w/w) phospholipids. The safety of SCO has been confirmed through bacterial reverse mutation tests, micronucleus tests69) , and studies of single and repeated doses in rodents70) ,71) . Studies on type II diabetes/obese mice showed that SCO significantly lowered serum TAG and liver cholesterol levels compared with the effect elicited by lard72) ,73) . Additionally, phospholipids in SCO (DHA/EPA-GPL) may significantly influence the cholesterol-lowering effect74) . Studies on human participants showed that the consumption of 1.2 g/day of SCO could be beneficial for reducing serum TAG and malondialdehyde-LDL levels in individuals with higher TAG levels75) . Therefore, SCO could address the issues related to the stable supply of DHA/EPA-GPL and effective utilization of scallop internal organs. However, this project did not progress much further owing to problems related to funding, raw material supply, and manufacturing costs. Therefore, in addition to exploring the benefits of DHA/EPA-GPL from fishery by-products, it is essential to create profitability through a rational and comprehensive approach to the commercialization and reuse of fishery by-products as a source of DHA/EPA-GPL. We hope that continued efforts would lead to progress in the commercialization of oils containing DHA/EPA-GPL from fishery by-products and establishment of feasible and suitable solutions that address nutritional and environmental concerns.
The research works presented in this review have been performed with help of collaborators and students in our research group. The author thanks Prof. Munehiro Yoshida, Prof. Kenji Fukunaga (Kansai University) , Prof. Koretaro Takahashi, Prof. Kazuo Miyashita, Prof. Masashi Hosokawa (Hokkaido University) , Dr. Toshihiro Nagao (Osaka Research Institute of Industrial Science and Technology) , and Dr. Takeya Yoshioka (Hokkaido Industrial Technology Center) for their guidance and support. This work was partially supported by “A Scheme to Revitalize Agriculture and Fisheries in Disaster Area through Deploying Highly Advanced Technology” from the Ministry of Agriculture, Forestry and Fisheries of Japan and the Adaptable and Seamless Technology Transfer Program through Target driven R& (A-STEP) from the Japan Science and Technology Agency (JST) grant number: JPMJTR174C.
The author declares that there are no conflicts of interest.
