## 4 Discussion  
PlsEtn has been reported to exhibit physiological and nutritional functions in the liver [5; 13; 15]. In this study, dietary PlsEtn from ascidian suppressed continuous APAP-induced hepatic injury, as evidenced by the prevention of body and liver weight loss, reduction in hepatic oxidative stress, and impairment of lipid metabolism. In contrast, dietary PtdEtn, another EtnGpl subclass, ameliorated APAP-induced lipid oxidation in the liver but did not suppress the decrease in hepatic total fatty acid content and the loss of liver weight. Additionally, we found that PlsEtn suppressed the expression of apoptosis-related proteins in the liver, and the extent of suppression by PlsEtn was greater than that by PtdEtn, as shown in Figure 1. In previous studies, dietary PtdEtn suppresses hepatic steatosis and inflammatory response in rodents fed a high-fat diet [29; 30]. Liver levels of PtdEtn and its metabolites, including phosphatidylcholine (PtdCho) and phosphatidylserine, decrease to induce mitochondrial dysfunction in non-alcoholic fatty liver disease [31]. Thus, dietary PtdEtn may maintain their hepatic levels and protect the liver. Dietary PlsEtn also alleviates hepatic impairments in rodents fed a high-fat diet [15]; however, the concentration of PlsEtn in the liver is much lower compared to PtdEtn, and PlsEtn is not directly converted to PtdEtn due to the structural variation at the \(sn\)-1 position. Therefore, it is proposed that the hepatoprotective mechanism of dietary PlsEtn differs from that of PtdEtn. Dietary PlsEtn increased the hepatic levels of DHA and the DHA/ARA ratio. Intracellular DHA suppresses oxidative and inflammatory stress, both by itself and via its metabolites [32], and the DHA/ARA ratio exhibits protective properties [33]. ARA is metabolized by oxidizing enzymes, resulting in ARA-derived eicosanoids that induce inflammation [34]. Conversely, DHA and EPA competitively inhibit ARA metabolism to suppress inflammation, and their metabolites, docosanoids and EPA-derived eicosanoids, exert anti-inflammatory effects. In the liver of mice expressing the gene encoding \(n\)-3 desaturase, an increase in DHA and EPA results in elevated levels of docosanoids and EPA-derived eicosanoids [35]. In this study, the basal diet contained nearly equivalent levels of DHA and EPA as the AM and PL diets. Glycerophospholipids are resynthesized preferentially with PUFAs following digestion [9; 36]. Although PUFA absorption from glycerophospholipids in the small intestine is similar to that from triacylglycercrols, PUFA accumulation in the liver is more efficient with glycerophospholipid intake than with triacylglycerollicerol intake [37]. Dietary PlsEtn may not markedly influence the total plasmalogen level in the liver. The intracellular concentration of total EntGpl, comprising PlsEtn and PtdEtn, is meticulously regulated [38]. In mice with a hepatocyte-specific peroxisomal defect, which is crucial for plasmalogen biosynthesis, the hepatic PlsEtn levels are similar to those observed in the control group [39]. This suggests that the hepatic PlsEtn level is maintained through provision via plasma from/to other organs. Conversely, in mice subjected to diets deficient in \(n\)-3 PUFAs, the administration of PlsEtn or PtdCho enriched with EPA results in elevated hepatic DHA levels of both PlsEtn and PtdCho [40]. Furthermore, dietary PlsEtn significantly increases the hepatic concentration of DHA within PtdCho, a predominant phospholipid in the liver, compared to dietary PtdCho. When considered in conjunction with the previous paragraph, dietary PlsEtn can modify the fatty acid composition of phospholipids, including PIsEtn, in the liver. PIsEtn serves as a carrier of \(n\)-3 PUFAs when consumed alongside \(n\)-3 PUFA sources such as fish oil, and it exhibits hepatotoxicity effects, potentially by enhancing membrane functions and generating bioactive metabolites.