|Year : 2018 | Volume
| Issue : 3 | Page : 91-97
Lessening hepatic injury in cholestatic liver by optimal dietary docosahexaenoic acid supplementation in rats
Guan-Yeu Diau, Shih-Ming Kuo, Chieh-Wen Lin
Department of Surgery, Division of Pediatric Surgery, National Defense Medical Center, School of Medicine, Tri-Service General Hospital, Taipei, Taiwan
|Date of Submission||03-Sep-2017|
|Date of Decision||11-Oct-2017|
|Date of Acceptance||16-Nov-2018|
|Date of Web Publication||21-Jun-2018|
Dr. Guan-Yeu Diau
325, Chenggung Rd., Section 2, Neihu District, Taipei City 11490
Source of Support: None, Conflict of Interest: None
Background: Dietary adjuvant management for the cholestatic liver disease before/after surgery is an important clinical issue. This study investigated the possibility for the dietary supplementation of docosahexaenoic acid (DHA) to treat cholestasis liver through the bile duct ligation (BDL) rat model.
Materials and Methods: Thirty-six male Wistar rats were divided into four groups (N: no BDL; BL, 1P, 5P: received BDL) and consumed either a regular diet (N, BL) or of DHA-enriched diet (1P: at 1% and 5P: at 5% weight percentage) for 4 weeks. The liver fatty acids (FAs) profiles, serum aspartate transaminase (AST), alanine aminotransferase (ALT), total bilirubin, alkaline phosphatase, interleukin-2 (IL-2), interferon γ (INF-γ), and pathological examination with H and E, Masson, CD56 (natural killer cell), CD68 (macrophage) were examined.
Results: The DHA dietary supplement increased liver DHA after BDL. Liver DHA N 8.09 ± 0.60% and BL 8.41 ± 0.55% were the lowest than the supplemented groups 1P 12.57 ± 1.16%, 5P 18.36 ± 2.00% (P = 0.000). However, liver arachidonic acid (20:4n-6) 1P 23.13 ± 2.19% was the highest than N 18.86 ± 4.31%, BL 17.13 ± 3.07%, 5P 18.78 ± 1.76% (P = 0.001). The serum AST (U/L) in N 147.4 ± 28.2 and 1P 155.9 ± 35.1 were lower than B 317.1 ± 195.8, 5P 326.9 ± 141. 8 (P = 0.006). The serum alkaline phosphatase (U/L) showed the same trend N 49.8 ± 5.4, 1P 67.6 ± 21.1 were lower than the BL 172.2 ± 108.1, 5P 171.1 ± 149.1 (P = 0.017). Pathological examination with H and E, Masson revealed the fibrosis was prominent in BL, 5P. However, there were no significant differences in serum ALT, total bilirubin, IL-2, INF-γ, and immunohistochemical stain for the CD56, CD68.
Conclusions: The results suggested that optimal dietary supplementing of DHA (1P) had less destruction and liver enzymes released after the BDL. However, higher enriched DHA (5P) could not benefit from this dietary treatment. The body weight did not increase even with this enriched high FAs diet after BDL for 4 weeks.
Keywords: Bile duct ligation, cholestasis liver disease, docosahexaenoic acid, hepatic injury
|How to cite this article:|
Diau GY, Kuo SM, Lin CW. Lessening hepatic injury in cholestatic liver by optimal dietary docosahexaenoic acid supplementation in rats. Formos J Surg 2018;51:91-7
|How to cite this URL:|
Diau GY, Kuo SM, Lin CW. Lessening hepatic injury in cholestatic liver by optimal dietary docosahexaenoic acid supplementation in rats. Formos J Surg [serial online] 2018 [cited 2020 Sep 20];51:91-7. Available from: http://www.e-fjs.org/text.asp?2018/51/3/91/234878
| Introduction|| |
There is an unmet, compelling need for a safe and effective dietary adjuvant for the patient suffered from the cholestasis liver disease. Dietary adjuvant treatment for the cholestatic liver disease is still a major challenge for surgeons. However, the effective immunomodulation diets contribute to the pre- and post-operative care are still limited at the present time. Docosahexaenoic acid (DHA) (22:6n-3) is the most unsaturated fatty acid (FA) in mammalian tissue, and it is found at a particularly high concentration in the sperm,,, cerebral cortex , and retina. It concentrated mainly in the serine and ethanolamine phosphoglycerides, especially at the sn-2 position., There is a large portion of FA metabolism occurred in the liver. The DHA tissue contents in the liver might affect its function and outcome by cholestasis.
To investigate the impaction of liver damage by cholestasis, we used the bile duct ligation (BDL) rat model to simulate the damage. For the detrimental effects by cholestasis, a considerable amount of oxygen radicals generated. Recent studies provided evidence that the oxygen free radicals can cause cellular damage, such as the biliary atresia in pediatric patients. DHA reported to have the ability to reduce these free radicals. Its definite mechanisms are still elusive. The reported mechanisms involved in the affecting cytokine production, leukotriene level, cellular populations, function of the immune cells,,, increase anti-oxidative enzymes,,,, protective effects for apoptosis,,, replenish the lost cell membrane,, decrease blood viscosity,, and influence on the inflammatory gene expression,,, etc.
Modulation of the immune system might be one of the crucial roles for the successfully treatment of cholestasis liver disease. The DHA reported to have the ability of immunomodulation. This might be one of the mechanisms that attenuate the detrimental effects after the liver destruction. Literatures reported DHA could downregulation of the proliferation of CD4 cells,,, inhibit the lymphocyte function,, decrease CD8 expression,, modulate mitogen-activated protein kinase, decrease natural killer (NK) cell function,, affect the immunoglobulin status, decreased cytokine production and inflammatory markers,,, inhibit antigen-presenting function of lymphocyte, and modulate gene expression, etc. This study examined the immune reactions through the pathological examination of the liver tissue (H and E, Masson, CD56, CD68). Furthermore, the cytokines affected by the DHA administration after the cholestasis, for example, interleukin-2 (IL-2) production is still controversial. Most literature supported the DHA reduced the amount of IL-2 in the serum;, however, some shown no obvious effects at all., Based on our understanding, the changes after the DHA supplement in cholestasis were still not examined up to now. The serum concentration of IL-2 and interferon γ (INF-γ) was checked through enzyme-linked immunosorbent assay (ELISA).
The single cell DHA were used instead of the traditional DHA manufactured from the fish oil. Long chain polyunsaturated (LCP) FAs are abundant in the deep sea fish. Fish oil was also a well-known source for the essential n-3 FA DHA production. It might have the possibility of contamination by various chemicals and heavy mental from deep-sea large fish sources oil. Recently, single cell oil from cultured microalgae (Crypthecodinium cohni) has been used as an alternative source of DHA in North America.,,
We proposed that supplementing dietary DHA could decrease the rat liver damage by the BDL and provided enough energy supply to this mishap situation.
| Materials and Methods|| |
Experimental animals and diet
All institutional and national guidelines for the care and use of laboratory animals were followed and compliance with the ethical standard. Experiments evaluated and passed the ethical standard (issued # Institutional Animal Care and Use Committee [IACUC]-09-107) by Animal Experimentation of IACUC and performed in accordance with the procedures outlined in the guidelines for IACUC, National Defense Medical Center. Male Wistar rats (250 g) divided into four groups (N, BL, 1P, 5P): N and BL (n = 18) consumed a regular diet based on a purified rodent diet (AIN 93G, Dyets, PA, USA) and 1P and 5P (n = 18) consumed a supplemented single cell DHA powder (DHASCO ®, Martek Bioscience, Columbia, MD, USA) at various strength for 4 weeks after BDL (BL, 1P, 5P received BDL). 1P: 1% DHA powder + 99% rodent regular diet powder. 5P: 5% DHA powder + 95% rodent regular diet powder. The purified rodent diet was grounded into powder. Then, it could mix with the DHA powder without any difficulty. This single-cell source DHA is chosen because they are used in the manufacturing of many North American products. The rats gave food and water ad libitum, and food consumption and body weight monitored throughout the experimental period.
Bile duct ligation
The bile duct ligation (BDL) surgery performed according to the method described by Gross. Male Wistar rats (250 g) anesthetized with Zoletil (5–7 mg/kg) injection intramuscularly. The common bile duct identified and double ligated using 5-O silk thread and divided in the middle between those two ligatures to ensure the complete transaction of the bile duct. Two days after surgery, the presence of bilirubin in the urine turned it a deep yellow color, indicated successful ligation. Details described as cited.
The liver evaluated for the histopathologic examination at the end of the experiment. Sections approximately 4 mm thick were stained with hematoxylin and eosin (H and E) staining for routine histology.
The Masson's trichrome staining for collagen to evaluate the fibortic change of the liver.
The immunohistochemical staining was performed for CD56 (NK cells) and CD68 (macrophage) in some occasions to evaluate the possible cellular response by our dietary manipulation.
Lipid extraction and fatty acids analyses
Total lipids will be extracted from the liver use the method of Bligh and Dyer  modified for the water content of the tissue to ensure a single-phase solvent mixture. FA methyl ester analyzed using a (gas chromatography-17A, Shimadzu Co, Tokyo, Japan) with a BPX 70 capillary column (60 m × 0.32 mm I. D. × 0.25 μm film; SGE, Austin, TX, USA) and H2 as carrier gas. Details were described as cited.
Serum liver enzyme profiles analyses
The serum level of aspartate transaminase (AST), alanine aminotransferase (ALT), total bilirubin and alkaline phosphatase examined at the end of this study. Serum concentration of these parameter checked by the clinical, pathological laboratory in the Tri-Service General Hospital.
IL-2 and IFN-γ serum levels examined at the end of this study. Serum concentrations of these cytokines were checked by colorimetric sandwich ELISA. (R and D Systems Inc., Minneapolis, MN, USA). Details were described as cited.
All results were expressed as means and standard deviations. Student's t-test and one-way ANOVA used for statistical analysis. Duncan's multiple-range test employed to indicate the statistical significance at P < 0.05.
| Results|| |
Characteristics of the animals were presented in [Table 1]. There were no significant differences in any parameter associated within groups statistically at the beginning of the experiment. However, the body weight and weight gain of the unreceived BDL (N) is significant increased than the received BDL groups (BL, 1P, 5P). This suggested that BDL could stress the rat health and decrease the body weight gain. 1P and 5P group did not increase the body weight compared with BL group. This revealed that even with enriched high-energy DHA dietary manipulation did not affect the body weight gain after BDL. This phenomena contrast to our previous study (in preparation, not published yet) through the burn rat model that higher enriched DHA diet increased the weight gain.
FA compositions of the diet (RD) and fortified formula (1P, 5P) details were listed in [Table 2]. Dietary 20:4n-6 were 0.87 ± 0.00%, 0.79 ± 0.28%, and 0.81 ± 0.18% of total FAs, respectively. The 22:6n-3 were 2.52 ± 0.24%, 14.52 ± 0.02%, and 27.54 ± 0.24% separately.
The DHA dietary supplementation increased liver DHA after a 4-week feeding in 1P, 5P [Table 3]. N 8.09 ± 0.60%, BL 8.41 ± 0.55% were the lowest than the supplemented groups 1P 12.57 ± 1.16%, 5P 18.36 ± 2.00%. However, the 20:4n-6 concentrations revealed the 1P was the highest than N, BL, 5P. This results contrast to the general theory that the DHA competes with the enzyme system to reduce the ascorbic acid (AA) level in 1P.
The serum AST (U/L) N 147.4 ± 28.2 and 1P 155.9 ± 35.1 were lower than the 5P 326.9 ± 141. 8, BL 317.1 ± 195.8. The serum alkaline phosphatase (U/L) showed the same trend N 49.8 ± 5.4 and 1P 67.6 ± 21.1 were lower than the 5P 171.1 ± 149.1, BL 172.2 ± 108.1 [Table 4]. This suggested that only relative optimal concentration (1P) decrease some of the liver enzymes release after BDL. However, there were no significant differences in serum ALT, bilirubin, IL-2, INF-γ.
Pathological examination with H and E, Masson stain of the liver tissue revealed the fibrosis was prominent in BL, 5P [Figure 1]. However, there were no significant differences for the CD56, CD68.
The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee of the institute. Informed written consent was obtained from all patients prior to their enrollment in this study.
| Discussion|| |
The result revealed the enriched DHA diet increased DHA tissue level in the liver after BDL. However, the tissue level of 20:4n-6 (arachidonic acid [AA]) increased a lot in the most effective 1P. There were no significant differences between the N, BL, and 5P. This trend was not universally compatible with most of literatures and traditional theory. The previous knowledge was that n-3 FAs bearing its biological function by competing with the n-6 FAs through the same enzyme system. If n-3 FAs administration increased, the n-6 FAs should decrease. It is well known that AA (20:4n-6, AA) is oxygenated and further transformed into a variety of products, for example, Plasma prostaglandin E2, thromboxane A4, leukotriene A4, which mediate or modulate the inflammatory cascades. Our data revealed that 1P had the highest tissue AA unexpectedly. Usually, the serum AA rose, especially in the situation of liver cirrhosis, for example, cystic fibrosis, hepatitis B. The AA also exhibited an inverse relationship to serum bilirubin. As a result, the AA in 1P should not so high theoretically. One of the possible explanations is that the DHA supplementation increased the lipid peroxidation. In human, the LCP-supplemented formulae improved LCP status in severe cholestasis infant, but might also enhance the lipid peroxidation. This peroxidation resulted in the overproduction of FAs in both the AA and DHA. The 5P could possibly attenuate the AA more than 1P. This explained why there was the highest tissue AA content in 1P. Based on the result of this experiment, another tentative speculation was that AA might have a more crucial impact on the BDL rat. However, the total n-6/n-3 ratio was consisted with the traditional enzyme system competition theory. As a result, this unusual increased AA might imply that AA stands for more important role than other FAs only in the optimal dosing (1P) situation.
The impaired cholestasis liver might benefit from the DHA predominately at 1P. However, a lot of metabolism of the FAs, for example, lipogenesis, β-oxidation, and esterification was performed at the liver. We do not know if the administration of the DHA might cause more burdens under this situation. Furthermore, some of the liver enzyme profile, for example, the bilirubin and AST were not affected by the dietary manipulation. The destructive parameters liver enzymes were not universally showed beneficial effect. The question that less or increase burden of the live was not answered in this preliminary study. The FAs compositions between N and BL revealed that BDL only has effect on increase 18:0, total n-3 FAs, decrease total n-9 FAs, total n-6/n3 ratio. Most of the FAs composition did not have significant differences after BDL. This implied that the liver function was not so bad after a 4-week BDL. However, the body weight loss after the BDL was significant. One of the possibilities was that there might be a huge FAs pooling in the body. The cholestasis liver might take the FAs back from other body components.
The body weight gain did not increase in the DHA supplemented group. The FAs are efficient energy resources. It could produce more energy than the carbohydrate and protein per weight. The results of this experiment suggested that even with higher dietary enrichment of DHA (5P), it could only partially compensate the basic energy requirement after BDL. Lots of the metabolism of FAs were accomplished in the liver. One of the possibilities of the body weight failed to gain might be due to the liver function severely compromised by the BDL. Furthermore, the disease state by cholesteric liver might also decrease the foot intake. The daily food intake was not recorded in this experiment. This possibility could not be answered this time. It was also possible these rodents did not like the diets we prepared. The taste of DHA powder might be not so delicious for them. This powder was directly packed into capsules and sold by stores for human product. This result was quite different from the literatures and our previous study using rat model in burn injury, which revealed that the higher concentration dietary DHA would increase the body weight gain significantly. This suggested that there were differences among the various species and models of diseases.
The serum immunological and inflammatory parameters, for example, serum IL-2, INF-γ did not show significant differences statistically. The previous literature revealed DHA impact on the immune system were controversial.,,,,, Up to now, the controversies existed and varied with different researchers. Furthermore, the histobiochemical stain for the NK cells and macrophage, which stand for a very important inflammation response after the BDL, did not revealed significant difference among various groups. Further studies would be advisable and necessary to search the possibility mechanisms about the changes after BDL.
The safety issue regarding the high dietary DHA supplementation is also a major clinical concern., Immunomodulation might also be possible when the toxicity of the DHA reached especially in 5P. However, there were no observable side effects, for example, bleeding tendency in those rats. It was not clear that the failure of body weight gain came from BDL or dietary manipulation is this survey. Even with the relative lower toxicity of DHA, the administration of dietary DHA in the cholestasis liver patient should be more cautiously and need more strong clinical trials to prove its safety and effectiveness.
Another rare clinical situation is the cholestasis liver occurred in the neonate, for example, biliary atresia. Unlike AA, the DHA is an essential FA which is also vulnerable to limitations in the supply of precursor from food. It is not very clear to what extent would the compromised liver function in the progression of biliary atresia. However, under the assumption that the metabolism of the liver should not as good as the normal one, dietary administration these essential FAs seemed reasonable. This is especially important for the pediatric group who need the FAs to build up body mass, especially for the important organs, e.g. brain, retina. Further study is necessary to make this approach more practical. Treatment for the biliary atresia is still mostly relying on surgery. The baby suffered from biliary atresia might also mimic the situation of BDL. However, the ligature of bile duct and anesthesia was extreme difficult, especially in the very tiny unweaning breast milk-fed rat. Furthermore, the dietary manipulation for breastfeeding through the mother rat was also very difficult. The artificially fortified mother rat milk and route of feeding are very hard to achieve. This is the future target for our team to struggle on.
| Conclusions|| |
The results of this experiment concluded that only optimal dietary supplementing of DHA (1P) could reduce the tissue destruction, decrease serum AST, alkaline phosphatase after BDL. However, the higher enriched DHA (5P) did not have this beneficial effect at all. The dietary administration of various enriched DHA did not have any effect on the body weight gain after BDL for 4-weeks. Finally, the DHA enriched dietary did not have any observable side effect after BDL.
The authors thank the input and help by Dr. Shyi-Jou, Chen, Chief of Department of Pediatics, Tri-Service General Hospital, Taipei, Taiwan, ROC.
Financial support and sponsorship
This work was supported by Ministry of National Defense Medical Affairs Bureau grant DOD100-I-02, Taiwan, ROC.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Lin DS, Connor WE, Wolf DP, Neuringer M, Hachey DL. Unique lipids of primate spermatozoa: Desmosterol and docosahexaenoic acid. J Lipid Res 1993;34:491-9.
Connor WE, Lin DS, Wolf DP, Alexander M. Uneven distribution of desmosterol and docosahexaenoic acid in the heads and tails of monkey sperm. J Lipid Res 1998;39:1404-11.
Lenzi A, Gandini L, Maresca V, Rago R, Sgrò P, Dondero F, et al.
Fatty acid composition of spermatozoa and immature germ cells. Mol Hum Reprod 2000;6:226-31.
Breckenreidge WC, Gombos G, Morgan IG. The docosahexaenoic acid of the phospholipids of synaptic membranes, vesicles and mitochondria. Brain Res 1971;33:581-3.
Hoffman DR, Birch EE, Birch DG, Uauy RD. Effects of supplementation with omega 3 long-chain polyunsaturated fatty acids on retinal and cortical development in premature infants. Am J Clin Nutr 1993;57:807S-812S.
Benolken RM, Anderson RE, Wheeler TG. Membrane fatty acids associated with the electrical response in visual excitation. Science 1973;182:1253-4.
Svennerholm L. Distribution and fatty acid composition of phosphoglycerides in normal human brain. J Lipid Res 1968;9:570-9.
Huang L, Si XM, Feng JX. NF-kappaB related abnormal hyper-expression of iNOS and NO correlates with the inflammation procedure in biliary atresia livers. Pediatr Surg Int 2010;26:899-905.
Collison LW, Collison RE, Murphy EJ, Jolly CA. Dietary n-3 polyunsaturated fatty acids increase T-lymphocyte phospholipid mass and acyl-coA binding protein expression. Lipids 2005;40:81-7.
Almallah YZ, El-Tahir A, Heys SD, Richardson S, Eremin O. Distal procto-colitis and n-3 polyunsaturated fatty acids: The mechanism(s) of natural cytotoxicity inhibition. Eur J Clin Invest 2000;30:58-65.
Di Stasi D, Bernasconi R, Marchioli R, Marfisi RM, Rossi G, Tognoni G, et al.
Early modifications of fatty acid composition in plasma phospholipids, platelets and mononucleates of healthy volunteers after low doses of n-3 polyunsaturated fatty acids. Eur J Clin Pharmacol 2004;60:183-90.
Arrington JL, McMurray DN, Switzer KC, Fan YY, Chapkin RS. Docosahexaenoic acid suppresses function of the CD28 costimulatory membrane receptor in primary murine and jurkat T cells. J Nutr 2001;131:1147-53.
Choi-Kwon S, Park KA, Lee HJ, Park MS, Lee JH, Jeon SE, et al.
Temporal changes in cerebral antioxidant enzyme activities after ischemia and reperfusion in a rat focal brain ischemia model: Effect of dietary fish oil. Brain Res Dev Brain Res 2004;152:11-8.
Sarsilmaz M, Songur A, Ozyurt H, Kuş I, Ozen OA, Ozyurt B, et al.
Potential role of dietary omega-3 essential fatty acids on some oxidant/antioxidant parameters in rats' corpus striatum. Prostaglandins Leukot Essent Fatty Acids 2003;69:253-9.
Hossain MS, Hashimoto M, Gamoh S, Masumura S. Antioxidative effects of docosahexaenoic acid in the cerebrum versus cerebellum and brainstem of aged hypercholesterolemic rats. J Neurochem 1999;72:1133-8.
Hossain MS, Hashimoto M, Masumura S. Influence of docosahexaenoic acid on cerebral lipid peroxide level in aged rats with and without hypercholesterolemia. Neurosci Lett 1998;244:157-60.
Akbar M, Kim HY. Protective effects of docosahexaenoic acid in staurosporine-induced apoptosis: Involvement of phosphatidylinositol-3 kinase pathway. J Neurochem 2002;82:655-65.
Siddiqui RA, Jenski LJ, Neff K, Harvey K, Kovacs RJ, Stillwell W, et al.
Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process. Biochim Biophys Acta 2001;1499:265-75.
Siddiqui RA, Jenski LJ, Harvey KA, Wiesehan JD, Stillwell W, Zaloga GP, et al.
Cell-cycle arrest in Jurkat leukaemic cells: A possible role for docosahexaenoic acid. Biochem J 2003;371:621-9.
Tully AM, Roche HM, Doyle R, Fallon C, Bruce I, Lawlor B, et al.
Low serum cholesteryl ester-docosahexaenoic acid levels in Alzheimer's disease: A case-control study. Br J Nutr 2003;89:483-9.
Kimura S, Tamayama M, Minami M, Hata N, Saito H. Docosahexaenoic acid inhibits blood viscosity in stroke-prone spontaneously hypertensive rats. Res Commun Mol Pathol Pharmacol 1998;100:351-61.
Hashimoto M, Tanabe Y, Fujii Y, Kikuta T, Shibata H, Shido O, et al.
Chronic administration of docosahexaenoic acid ameliorates the impairment of spatial cognition learning ability in amyloid beta-infused rats. J Nutr 2005;135:549-55.
Tanabe Y, Hashimoto M, Sugioka K, Maruyama M, Fujii Y, Hagiwara R, et al.
Improvement of spatial cognition with dietary docosahexaenoic acid is associated with an increase in fos expression in rat CA1 hippocampus. Clin Exp Pharmacol Physiol 2004;31:700-3.
Nogusa Y, Yanaka N, Sumiyoshi N, Kaseda Y, Tsuboyama-Kasaoka N, Ezaki O, et al.
Short-term feeding of fish oil down-regulates the expression of pyruvate dehydrogenase E1 alpha subunit mRNA in mouse brain. Biosci Biotechnol Biochem 2005;69:301-6.
Marcheselli VL, Hong S, Lukiw WJ, Tian XH, Gronert K, Musto A, et al.
Novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression. J Biol Chem 2003;278:43807-17.
Scherer JM, Stillwell W, Jenski LJ. Spleen cell survival and proliferation are differentially altered by docosahexaenoic acid. Cell Immunol 1997;180:153-61.
Sasaki T, Kanke Y, Nagahashi M, Toyokawa M, Matsuda M, Shimizu J, et al.
Dietary docosahexaenoic acid can alter the surface expression of CD4 and CD8 on T cells in peripheral blood. J Agric Food Chem 2000;48:1047-9.
Kew S, Mesa MD, Tricon S, Buckley R, Minihane AM, Yaqoob P, et al.
Effects of oils rich in eicosapentaenoic and docosahexaenoic acids on immune cell composition and function in healthy humans. Am J Clin Nutr 2004;79:674-81.
Fowler KH, Chapkin RS, McMurray DN. Effects of purified dietary n-3 ethyl esters on murine T lymphocyte function. J Immunol 1993;151:5186-97.
Denys A, Hichami A, Khan NA. Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase enzyme activity in human T-cells. Mol Cell Biochem 2002;232:143-8.
Yamashita N, Maruyama M, Yamazaki K, Hamazaki T, Yano S. Effect of eicosapentaenoic and docosahexaenoic acid on natural killer cell activity in human peripheral blood lymphocytes. Clin Immunol Immunopathol 1991;59:335-45.
Purasiri P, Mckechnie A, Heys SD, Eremin O. Modulation in vitro
of human natural cytotoxicity, lymphocyte proliferative response to mitogens and cytokine production by essential fatty acids. Immunology 1997;92:166-72.
Hung P, Kaku S, Yunoki S, Ohkura K, Gu JY, Ikeda I, et al.
Dietary effect of EPA-rich and DHA-rich fish oils on the immune function of sprague-dawley rats. Biosci Biotechnol Biochem 1999;63:135-40.
Verlengia R, Gorjão R, Kanunfre CC, Bordin S, de Lima TM, Martins EF, et al.
Effects of EPA and DHA on proliferation, cytokine production, and gene expression in raji cells. Lipids 2004;39:857-64.
Pischon T, Hankinson SE, Hotamisligil GS, Rifai N, Willett WC, Rimm EB, et al.
Habitual dietary intake of n-3 and n-6 fatty acids in relation to inflammatory markers among US men and women. Circulation 2003;108:155-60.
Hughes DA, Pinder AC. N-3 polyunsaturated fatty acids inhibit the antigen-presenting function of human monocytes. Am J Clin Nutr 2000;71:357S-60S.
Socha P, Koletzko B, Jankowska I, Pawłowska J, Demmelmair H, Stolarczyk A, et al.
Long-chain PUFA supplementation improves PUFA profile in infants with cholestasis. Lipids 2002;37:953-7.
Arrington JL, Chapkin RS, Switzer KC, Morris JS, McMurray DN. Dietary n-3 polyunsaturated fatty acids modulate purified murine T-cell subset activation. Clin Exp Immunol 2001;125:499-507.
Thies R, Kleinebudde P. Melt pelletization of a hygroscopic drug in a high shear mixer. Part 3. Effects of binder variation. Chem Pharm Bull (Tokyo) 2001;49:140-6.
Huang MC, Chao A, Kirwan R, Tschanz C, Peralta JM, Diersen-Schade DA, et al.
Negligible changes in piglet serum clinical indicators or organ weights due to dietary single-cell long-chain polyunsaturated oils. Food Chem Toxicol 2002;40:453-60.
Burns RA, Wibert GJ, Diersen-Schade DA, Kelly CM. Evaluation of single-cell sources of docosahexaenoic acid and arachidonic acid: 3-month rat oral safety study with an in utero
phase. Food Chem Toxicol 1999;37:23-36.
Ratledge C. Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimie 2004;86:807-15.
Gross JB Jr. Reichen J, Zeltner TB, Zimmermann A. The evolution of changes in quantitative liver function tests in a rat model of biliary cirrhosis: Correlation with morphometric measurement of hepatocyte mass. Hepatology 1987;7:457-63.
Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37:911-7.
Diau GY, Loew ER, Wijendran V, Sarkadi-Nagy E, Nathanielsz PW, Brenna JT, et al.
Docosahexaenoic and arachidonic acid influence on preterm baboon retinal composition and function. Invest Ophthalmol Vis Sci 2003;44:4559-66.
Drzymała-Czyż S, Szczepanik M, Krzyżanowska P, Duś-Żuchowska M, Pogorzelski A, Sapiejka E, et al
. Serum phospholipid fatty acid composition in cystic fibrosis patients with and without liver cirrhosis. Ann Nutr Metab 2017;71:91-8.
Arain SQ, Talpur FN, Channa NA, Ali MS, Afridi HI. Serum lipid profile as a marker of liver impairment in hepatitis B cirrhosis patients. Lipids Health Dis 2017;16:51.
Socha P, Koletzko B, Pawlowska J, Socha J. Essential fatty acid status in children with cholestasis, in relation to serum bilirubin concentration. J Pediatr 1997;131:700-6.
[Table 1], [Table 2], [Table 3], [Table 4]