Koretaro Takahashi

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm2 with 50 μM PC, and 17.0 and 23.0 nmol/h/cm2 with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)

Preparation of therapeutic phospholipids through porcine pancreatic phospholipase A2-mediated esterification and lipozyme-mediated acidolysis

Highly unsaturated fatty acid-containing phospholipids (HUFA-PL), which serve to increase the deformability of human red blood cells, were prepared through porcine phospholipase A2-mediated esterification of the lysophosphatidylcholine, which was derived from soy phosphatidylcholine (PC), and by Lipozyme-mediated acidolysis. Through these processes, phospholipase A2, with formamide as a water mimic, enhanced the incorporation of HUFA into positionsn-2 of PC and suppressed hydrolysis of the synthesized PL. On the other hand, Lipozyme-mediated acidolysis between positionsn-1 of soy PC and HUFA was enhanced by a combination of water and propylene glycol. Simultaneously, the recovered PL products showed decreased hydrolysis of newly synthesized health-beneficial HUFA-PL.

The protective effect of eicosapentaenoic acid-enriched phospholipids from sea cucumber Cucumaria frondosa on oxidative stress in PC12 cells and SAMP8 mice.

Alzheimers disease (AD) is a common neurodegenerative disorders, in which oxidative stress plays an important role. The present study investigated the effect of eicosapentaenoic acid-enriched phospholipids (EPA-enriched PL) from the sea cucumber Cucumaria frondosa on oxidative injury in PC12 cells induced by hydrogen peroxide (H2O2) and tert-butylhydroperoxide (t-BHP). We also studied the effect of EPA-enriched PL on learning and memory functions in senescence-accelerated prone mouse strain 8 (SAMP8) in vivo. Pretreatment with EPA-enriched PL resulted in an enhancement of survival in a dose-dependent manner in H2O2 or t-BHP damaged PC12 cells. EPA-enriched PL pretreatment could also reduce the leakage of lactate dehydrogenase (LDH), and increase the intracellular total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity compared with the H2O2 or t-BHP group. The down-regulated Bcl-2 mRNA level and up-regulated Bax, Caspase-9, and Caspase-3 mRNA expression induced by H2O2 or t-BHP could be restored by EPA-enriched PL pretreatment. These results demonstrated that EPA-enriched PL exhibited its neuroprotective effects by virtue of its antioxidant activity, which might be achieved by inhibiting the mitochondria-dependent apoptotic pathway. The neuroprotective effect of EPA-enriched PL was also verified in vivo test: the EPA-enriched PL administration prevented the development of learning and memory impairments in SAMP8 mice. Our results indicated that EPA-enriched PL could offer an efficient and novel strategy to explore novel drugs or functional food for neuronprotection and cognitive improvement.

Growth inhibition and induction of apoptosis of colon cancer cell lines by applying marine phospholipid.

Polyunsaturated fatty acids (PUFAs) exhibit beneficial biological functions in carcinogenic processes. We examined the effects of PUFAs in the acid and phospholipid forms on three colon cancer cell lines (HT-29, Caco-2, and DLD-1). Docosahexaenoic acid (DHA) and eicosapentaenoic (EPA) in both acid and phospholipid forms showed growth inhibition effects on experimental colon cancer cell lines. But these PUFAs had the strongest growth-inhibitory effect on HT-29 than Caco-2 and DLD-1. Combined application of PUFAs and sodium butyrate (NaBt) increased the growth inhibition. Growth inhibition was apparently caused by increased lipid peroxidation. DHA or EPA in combination with NaBt significantly increased caspase-3 activity compared to control. DHA and DHA-rich phosphatidylcholine decreased Bcl-2 level in HT-29 and Caco-2 cells.

GROWTH INHIBITION AND INDUCTION OF DIFFERENTIATION AND APOPTOSIS MEDIATED BY SODIUM BUTYRATE IN CACO-2 CELLS WITH ALGAL GLYCOLIPIDS

SummaryGlycolipids should have potential effects as antitumor agents. However, very few studies have examined this property of digalactosyl diacylglycerol (DGDG) and sulfoquinovosyl diacylglycerol (SQDG) on colon cancer cells. Cell viability was determined every 24 h with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay up to 72 h. Alkaline phosphatase activity was measured for assessing cell differentiation. Apoptosis was tested with enzyme-linked immunosorbent assay analysis. Growth of Caco-2 cells was inhibited apparently at 48 h after addition of SQDG and at 72 h with DGDG. Alkaline phosphatase activity of Caco-2 cells obviously increased in combination with DGDG or SQDG and sodium butyrate (NaBT) at 72 h, indicating that DGDG and SQDG enhanced cell differentiation induced with NaBT. An increased enrichment factor was found when the cell was treated in combination with DGDG or SQDG and NaBT. These results strongly suggest that DGDG and SQDG should be considered as the leading compounds of potentially useful colon cancer chemotherapy agents when NaBT is combined.

A new concept for determining triglyceride composition of fats and oils by liquid chromatography

The matrix concept was used to characterize the chromatographic rules in the elution of molecular species of triglyceride. To prove the hypothesis experimentally, cacao butter, palm oil, linseed oil, olive oil, rapeseed oil and triglyceride of “Ogonori” (Gracilaria verrucosa) were examined. A matrix model was suggested to help determine the individual molecular species of naturally occurring triglycerides.

Marine phosphatidylcholine suppresses 1,2-dimethylhydrazine-induced colon carcinogenesis in rats by inducing apoptosis

In vitro and animal studies indicate that n-3 polyunsaturated fatty acids (PUFAs) suppress carcinogenesis. This study presents a new insight on effectiveness of marine phospholipids for suppression of colon carcinogenesis. The purpose of this study was to investigate growth inhibition and apoptosis inducing effects of n-3 PUFA in the form of marine phosphatidylcholine (PC) on chemically induced (1,2-dimethylhydrazine) colon cancer in rats. Growth inhibition of Caco-2 cells was determined by colorimetric sodium 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) dye reduction assay. For animal studies, the rats were fed 5 different diets containing docosahexaenoic acid (DHA)-ethyl ester, eicosapentaenoic acid (EPA)-ethyl ester, squid meal PC (rich in DHA), starfish PC (rich in EPA), and corn oil. The 1,2-dimethylhydrazine (30 mg/kg) or saline was injected 48 hours before the experiment. Rats were anesthetized, and apoptotic as well as mitotic cells in crypt were counted based on morphological criteria in isolated crypts. Squid meal and starfish PC potently inhibited the growth of Caco-2 cells. The experimental diets containing n-3 PUFA suppressed colon cancer in rats. Rats that consumed diets containing DHA-ethyl ester, EPA-ethyl ester, squid meal PC, and starfish PC showed increased apoptosis (P < .01) and suppressed proliferation. These results suggest that marine PC-containing diets might be an effective dietary protective factor against colon cancer.

A mathematical model for the prediction of triglyceride molecular species by high performance liquid chromatography

The physicochemical and theoretical relationships between the traditional equivalent carbon number (ECN) and a proposed elution theory for triglyceride molecular species that has been expressed as a matrix model were demonstrated by multiple regression analysis. It was concluded that the ECN expression has two independent variables, that is, the total acyl carbon number (CN) and the total double bonds (DB), and one dependent variable, the relative retention time (RRT). In the proposed elution equation, there are six independent variables, including the carbon numbers and number of double bonds in each acyl group to be considered.

Application of water mimics on preparation of eicosapentaenoic and docosahexaenoic acids containing glycerolipids

To obtain enhanced incorporation of highly unsaturated fatty acids and recovery of glycerolipid products, organic solvents with high dielectric constants (water mimics) were substituted for part of the essential water for lipase activation to study their effect on acidolysis and transesterification. In acidolysis/transesterification of fish oil triglycerides and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), Lipozyme IM-60 with ethylene glycol as a water mimic enhanced the incorporation of EPA and suppressed the hydrolysis of synthesized glycerolipid. On the other hand, transesterification between soy phosphatidylcholine and EPA was enhanced by a water and propylene glycol combination. In a nonaqueous medium that contained appropriate amounts of water and organic solvents (water mimics), Lipozyme IM-60 increased transesterification of EPA into soy phosphatidylcholine. Simultaneously, the recovered glycerolipid products showed decreased hydrolysis of newly synthesized EPA- and DHA-containing glycerolipids.

Polyunsaturated fatty glyceride syntheses by microbial lipases

The degree of glyceride syntheses by lipase TOYO (Chromobacterium viscosum) and lipase OF (Candida cylindracea) using individual free fatty acids C18∶1, C18∶2, C18∶3, C18∶4, C20∶4, C20∶5 and C22∶6 were compared. Lipase TOYO incorporated each of the fatty acids into glycerol at levels of greater than 89%. Lipase OF incorporated most of the fatty acids at levels above 70% (docosahexaenoic acid incorporation was 63%). It was concluded that these two lipases are feasible for producing glycerides from unsaturated fatty acids.