토양 미생물로부터 생산된 Extracellular Cholesterol Oxidase의 특성

박정수(Jeong-Su Park),정종문(Jong-Moon Jeong) 한국식품영양과학회 2008 한국식품영양과학회지 Vol.37 No.11

본 연구에서는 산업적으로 사용될 수 있는 안정하고 활성이 높은 cholesterol oxidase를 생산하는 균주를 얻기 위해 토양 미생물로부터 균주를 선별하였다. 선별된 균주에 대하여 세포외 효소 활성을 측정한 결과 BEN 115로 명명한 균주가 가장 높은 효소 활성도를 나타내었다. 이 균주는 형태학적, 생리학적 특성과 배양형태 및 G+C 함량을 분석한 결과 Nocardia속으로 확인되었다. 최적 효소 생산 조건을 조사한 결과 기존 yeast malt extract broth 조성인 0.4% yeast extract, 0.4% glucose, 1% malt extract에서 가장 높은 활성을 나타냈다. 본 균주에서 생산된 extracellular cholesterol oxidase는 SDS-PAGE와 Western blot 결과 분자량이 55, 57 kDa인 두 종류의 효소가 존재하는 것으로 나타났다. BEN 115에 대한 효소학적 특성을 연구한 결과 온도 안정성은 55℃까지 효소 활성이 유지되었고, pH 안정성은 pH 3.5~9.5의 범위까지 안정한 것으로 나타났으며 최적 온도와 최적 pH는 각각 35oC와 pH 5.5인 것으로 나타났다. 또한 detergent(Triton X-100, Triton X-114 그리고 Tween 80) 첨가 시 효소 활성이 첨가하지 않은 대조군보다 약 1.6~2.0배 증가하는 것으로 나타났다. Campesterol, sitosterol 그리고 stigmasterol에 대한 기질 특이성은 cholesterol(100%)과 상대적으로 비교 시 각각 50%, 50% 그리고 27%의 기질 특이성을 나타내었다. Cholesterol oxidase catalyses the conversion of cholesterol to 4-cholesten-3-one. This enzyme has been used for clinical assay of human serum cholesterol and for reduction of cholesterol level in foods and feeds. In order to search the microorganism which has a high extracellular and stable activity of cholesterol oxidase, soil microorganisms were screened. As a result, the one with the highest extracellular cholesterol oxidase activity was obtained and named as the BEN 115. The BEN 115 strain was identified as one of the Nocardia species based on our taxonomic studies. The cholesterol oxidase from this strain was shown to have two bands of extracellular proteins on SDS-PAGE and Western blot. Their molecular masses were estimated to be about 55 and 57 kDa, respectively. In addition, this cholesterol oxidase was considerably stable at the broad range of pH 3.5~9.5 and at the temperature of 25~55℃. The optimum pH and temperature of this cholesterol oxidase were pH 5.5 and 35℃, respectively. The activity of extracellular cholesterol oxidase could be enhanced 1.6 to 2.0 folds by the addition of nonionic detergent such as Triton X-114, Triton X-100, or Tween-80 into the culturing broth. The substrate specificities against campesterol, sitosterol and stigmasterol were measured to be 50%, 50%, and 27%, respectively, compared to the cholesterol. These results suggest that Nocardia sp. BEN 115 may be useful as a microbial source of cholesterol oxidase production.

가열조건에 따른 오징어 Cholesterol과 Cholesterol oxidative products(COPs)의 함량변화에 관한 연구

홍정훈,안덕준 한국식생활문화학회 2002 韓國食生活文化學會誌 Vol.17 No.4

The effect of cooking(boiling, steaming and baking) and drying on the cholesterol content and formation of oxidized cholesterols and acid value in squid(Japanese flying squid, Todarodes pacificus) was studied. Cholesterol content of live squid meat varied with the portion sampled. The data from spectophotometric assay ranged from 263.2 mg/100g(mantle) to 315.8 mg/ 100g(tentacle). The cholesterol levels found for squid samples analyzed by gas chromatography(GC) were lower by 7% of total cholesterol for live squid meat and 24% for processed meat than those results by spectrophotometric assay. Cooking resulted some decrease in the initial total cholesterol content of raw meat from 10%(boiling for 5 min.) to 25%(steaming for 5 min.). The amounts of cholesterol remaining after baking were 68% for microwave oven samples and 64% for convection oven samples. Drying of raw tissue caused the greater reduction in cholesterol content than cooking but brought about no significant difference in samples stored for 6 weeks at 4℃ and 20℃. Raw squid meats contained essentially no oxidized cholesterols, while the 22-hydroxychoesterol was detected in frozen meats. The additional oxidized cholesterols as cholestane-triol was indentified with 22-hydroxycholesterol in cooked samples. Sun dried meat stored at 4℃ and 20℃ for 6 weeks had the three kinds of oxidized cholesterols such as 22-hydroxycholesterol, cholesta-3,5-dien-7-one and cholestane-triol. For the boiled and steamed squids, 10% higher acid value and 5% higher acid value respectively were observed but oven cooked samples resulted in a 50% higher acid value than raw samples. Squids had a 45% higher acid value than raw one during sundrying and presrevation at 20℃ but there was not a severe difference of acid value between 4℃ and 20℃ stored samples.

흰쥐에서 수수추출물이 트랜스지방산이 함유된 지방과 콜레스테롤의 흡수에 미치는 영향

김주연(Juyeon Kim),노상규(Sang Kyu Noh),우관식(Koan-Sik Woo),서명철(Myung-Chul Seo) 한국식품영양과학회 2016 한국식품영양과학회지 Vol.45 No.6

본 연구는 수수의 섭취가 대표적인 트랜스지방산인 elaidic acid, linoelaidic acid와 cholesterol의 소장흡수에 어떤 영향을 미치는지를 조사하기 위하여 설계하였다. 소장의 지방흡수를 in vivo 상태에서 측정하기 위해 소장흡수율 측정모델을 이용하여 십이지장주입관과 림프채취관을 삽입하는 미세수술을 진행하였다. 실험동물의 십이지장으로 146.4 μ mol trielaidin, 36.8 μmol trilinoelaidin, 452.0 μmol triolein, 1.0 μCi <SUP>14</SUP>C-cholesterol, 20.7 μmol cholesterol, 396.0 μmol Na-taurocholate, PBS buffer(pH 6.4)가 포함된 지질유화액만 공급받는 동물군을 대조군, 수수추출물이 혼합된 지질유화액을 공급받는 동물군을 수수군으로 설계하였다. 지질유화액은 시간당 3 mL씩 8시간 동안 주입하면서 림프관으로 흡수되는 elaidic acid, linoelaidic acid와 각종 지방산 그리고 <SUP>14</SUP>C-cholesterol의 양을 분석하여 수수에 의한 영향을 비교하였다. 먼저 8시간 동안 분비된 림프액의 양은 대조군과 수수군 간에 유의적인 차이를 나타내지 않았다. 하지만 8시간 동안의 elaidic acid 흡수는 수수추출물의 공급으로 유의적으로 억제되는 것으로 나타났다. Linoelaidic acid 또한 마찬가지로 수수추출물 공급으로 유의적으로 그 흡수가 억제된 것으로 나타났다. 트랜스지방산을 비롯해 palmitic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, docosahexaenoic acid, <SUP>14</SUP>C-cholesterol 또한 대조군보다 수수군에서 흡수가 유의적으로 억제된 것으로 나타났다. 이상의 결과들은 수수추출물이 트랜스지방산과 cholesterol의 소장 흡수를 억제할 수 있다는 사실을 증명한다. 이번 연구를 바탕으로 수수 섭취가 트랜스 지방이나 cholesterol의 흡수를 농도 비례적으로 억제할 수 있을지, 소화흡수 과정 중 정확히 어떤 과정에 영향을 미쳤는지에 관한 연구가 앞으로 진행될 과제라고 판단된다. Excessive intake of trans fats is a risk factor for cardiovascular diseases. Previously, we showed that green tea extract lowers intestinal absorption of cholesterol and elaidic acid in rats. This study was conducted to investigate a possible role for sorghum extract on lymphatic absorption of trans fat and cholesterol in rats. Adult male rats with lymph cannulae were infused at a rate of 3.0 mL/h for 8 h via a duodenal catheter with a lipid emulsion containing 146.4 μmol of trielaidin, 36.8 μmol of trilinoelaidin, 452.0 μmol of triolein, 1.0 μCi cholesterol labeled with <SUP>14</SUP>C (<SUP>14</SUP>C-cholesterol), 20.7 μmol of cholesterol, and 396.0 μmol of Na-taurocholate without or with 100.0 mg of sorghum extract in phosphate-buffered saline buffer (pH 6.4). Lymph was collected hourly for 8 h. No significant difference was noted in lymph flow. However, the lymphatic absorption of elaidic acid and linoelaidic acid for 8h was significantly lower in rats infused with sorghum extract than in those infused with no sorghum extract. Further, lymphatic absorption of <SUP>14</SUP>C-cholesterol was reduced by sorghum extract, which was observed previously. These data indicate that sorghum extract has an inhibitory effect on the intestinal absorption of trans fat and cholesterol. The mechanism(s) by which sorghum extract lowers intestinal absorption of trans fat warrants further study.

Modified Soybean Affects Cholesterol Metabolism in Rats Similarly to a Commercial Cultivar

Elizabethe A. Esteves,Josefina Bressan,Neuza M.B. Costa,Hercia S.D. Martino,Shawn S. Donkin,Jon A. Story 한국식품영양과학회 2011 Journal of medicinal food Vol.14 No.11

The consumption of soy protein lowers blood cholesterol in humans and animals. Breeding may alter the physiological effects of soybeans, such as its cholesterol-lowering property. Our hypothesis is that breeding affects the hypocholesterolemic effect of soy by modulating the expression of key hepatic enzymes related to cholesterol and bile acid biosynthesis, as well as altering fecal neutral and acidic steroid excretion. Therefore the aim of this study was to evaluate the effect of a new Brazilian soybean cultivar (UFV-116), lacking lipoxygenases 2 and 3, compared with a commercial cultivar (OCEPAR-19), on 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and cholesterol 7α-hydroxylase (CYP7A) mRNA expression and fecal steroid output in rats. Thirty-six male rats were fed UFV-116, OCEPAR-19, or casein as the protein source, with or without addition of dietary cholesterol (0.25%). Blood and liver cholesterol, HMGR and CYP7A mRNA abundance, and fecal excretion of steroids were measured. Blood and liver cholesterol levels were lowered by both soybean cultivars, with and without cholesterol, but UFV-116 was more effective when included in the cholesterol-free diet. Both soy diets promoted lower levels of HMGR mRNA, higher levels of CYP7A mRNA, and higher excretion of fecal secondary bile acids. There was higher fecal neutral steroid output when cholesterol was added to all diets. These data show that both soybean cultivars acted similarly in lowering serum and hepatic cholesterol; therefore, breeding did not affect the hypocholesterolemic effect of the new cultivar.

Quercetin Affects Spermatogenesis-Related Genes of Mouse Exposed to High-Cholesterol Diet

Changwon Yang,Hyocheol Bae,Gwonhwa Song,Whasun Lim 한국동물생명공학회(구 한국동물번식학회) 2020 Journal of Animal Reproduction and Biotechnology Vol.35 No.1

A high-cholesterol diet can reduce male fertility. However, it is not known whether a high-cholesterol diet can regulate the expression of genes involved in sperm maturation and sperm fertilizing ability. Quercetin, a natural product, is known to have cytoprotective effects by regulating lipid metabolism in various cell types. This study aimed to confirm the expression of genes involved in sperm maturation in the testes of mice fed a high-cholesterol diet and to determine whether quercetin can reverse the genetic regulation of cholesterol. Mice were divided into groups fed a normal chow diet and a high-cholesterol diet. Mice fed the high-cholesterol diet were dose-dependently supplemented with quercetin for 6 weeks. Investigations using quantitative PCR and in situ hybridization revealed that the high-cholesterol diet alters the expression of genes associated with sperm maturation in the testes of mice, and this was reversed with the supplementation of quercetin. In addition, the high-cholesterol diet regulated the expression of genes related to lipid metabolism in the liver of mice. Under a high-cholesterol diet, quercetin can improve male fertility by regulating the expression of genes involved in sperm maturation.

Pharmacological blockade of cholesterol trafficking by cepharanthine in endothelial cells suppresses angiogenesis and tumor growth

Lyu, Junfang,Yang, Eun Ju,Head, Sarah A.,Ai, Nana,Zhang, Baoyuan,Wu, Changjie,Li, Ruo-Jing,Liu, Yifan,Yang, Chen,Dang, Yongjun,Kwon, Ho Jeong,Ge, Wei,Liu, Jun O.,Shim, Joong Sup Elsevier 2017 Cancer letters Vol.409 No.-

<P><B>Abstract</B></P> <P>Cholesterol is an important modulator of membrane protein function and signaling in endothelial cells, thus making it an emerging target for anti-angiogenic agents. In this study, we employed a phenotypic screen that detects intracellular cholesterol distribution in endothelial cells (HUVEC) and identified 13 existing drugs as cholesterol trafficking inhibitors. Cepharanthine, an approved drug for anti-inflammatory and cancer management use, was amongst the candidates, which was selected for in-depth mechanistic studies to link cholesterol trafficking and angiogenesis. Cepharanthine inhibited the endolysosomal trafficking of free-cholesterol and low-density lipoprotein in HUVEC by binding to Niemann-Pick disease, type C1 (NPC1) protein and increasing the lysosomal pH. The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes and inhibition of its downstream signaling. Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. Furthermore, cepharanthine suppressed tumor growth in vivo by inhibiting angiogenesis and it enhanced the antitumor activity of the standard chemotherapy cisplatin in lung and breast cancer xenografts in mice. Altogether, these results strongly support the idea that cholesterol trafficking is a viable drug target for anti-angiogenesis and that the inhibitors identified among existing drugs, such as cepharanthine, could be potential anti-angiogenic and antitumor agents.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A phenotypic screen identified 13 existing drugs, including cepharanthine, as cholesterol trafficking inhibitors. </LI> <LI> Cepharanthine inhibited lysosomal cholesterol trafficking by binding to NPC1 protein and increasing the lysosomal pH. </LI> <LI> The blockade of cholesterol trafficking led to a cholesterol-dependent dissociation of mTOR from the lysosomes. </LI> <LI> Cepharanthine inhibited angiogenesis in HUVEC and in zebrafish in a cholesterol-dependent manner. </LI> <LI> Cepharanthine treatment enhanced the antitumor activity of cisplatin in lung and breast cancer xenografts in mice. </LI> </UL> </P>

Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate

Chun, Yoon Sun,Shin, Sora,Kim, Yonjung,Cho, Hana,Park, Myoung Kyu,Kim, Tae-Wan,Voronov, Sergey V.,Di Paolo, Gilbert,Suh, Byung-Chang,Chung, Sungkwon Blackwell Publishing Ltd 2010 Journal of Neurochemistry Vol.112 No.5

<P><I>J. Neurochem.</I> (2010) <B>112</B>, 1286–1294.</P><P>Abstract</P><P>Ubiquitously expressed Mg<SUP>2+</SUP>-inhibitory cation (MIC) channels are permeable to Ca<SUP>2+</SUP> and Mg<SUP>2+</SUP> and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP<SUB>2</SUB>) recovered MIC current from cholesterol effect. As PIP<SUB>2</SUB> is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP<SUB>2</SUB>. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP<SUB>2</SUB>-sensitive K<SUP>+</SUP> channels: human <I>Ether-a-go-go</I> related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP<SUB>2</SUB> in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP<SUB>2</SUB> via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced &ggr;-<SUP>32</SUP>P incorporation into PIP<SUB>2</SUB> by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP<SUB>2</SUB>. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP<SUB>2</SUB>.</P>

痰飮에 應用되는 小調中湯이 高脂血症에 미치는 影響

文濬典,崔達永,朴元煥,尹相柱 대한동의병리학회 1993 동의생리병리학회지 Vol.8 No.-

연구배경 : 최근 우리나라에 있어서도 생활 양식의 서구화로 말미암아 다발하고 있는 각종 현대병의 하나인 고지혈증을 한의학적으로 치료하기 위한 방법론의 개발을 연구목적으로 하여, 한의학의 전통적 병인 개념인 담음이론을 통해 고지혈증의 한의학적 개념을 설명하고, 〈의학입문〉처방인 소조중탕을 통해 치료 약재의 개발 가능성을 실험적으로 규명하고자 한다. 방법 : 실험동물에 cholesterol현탁액을 투여하여 고지혈증을 유발시키면서, 〈의학입문〉에 수재된 소조중탕을 투여하여 혈청지질에 포함되어 있는 total-cholesterol, H. D. L-cholesterol, triglyceride, phospholipid 등의 함량변화를 관찰한다. 결과 : 血淸中 total-cholesterol과 triglyceride含量에 미치는 影響은 對照群에 比하여 매우 顯著한 有意性있는 減少가 나타났으며, 血淸中 H.D.L.-cholesterol과 phospholipid含量에 미치는 影響은 對照群에 比하여 有意性있는 減少가 나타났으며, 血淸中 total cholesterol과 HDL cholesterol의 比率은 對照群에 比하여 有意性이 認定되지 않지만 total cholesterol과 phospholipid의 比率은 對照群에 比하여 有意性이 認定된다. 결론 : 祛痰之劑인 小調中湯은 高脂血症의 改善에 有意性있는 成績을 나타내므로 高脂血症과 痰飮은 相關性이 있으며 脂質代謝異常에 따른 動脈硬化症에도 活用될 수 있는 것으로 思慮된다. In order to study the effects of Xiaotiaozhongtang on hyperlipidemia, white rats were induced by lipid metabolism disorder by the oral-injection of cholesterol. The effects of Xiaotiaozhongtang on lipid in serum were measured by the quantity of total-cholesterol and of H.D.L-cholesterol and of triglyceride and of phospholipid, by the rate of total-cholesterol vs H.D.L-cholesterol and of total-cholesterol vs phospholipid in serum. As a result, we can conclude as follows: 1. Effects of Xiaotiaozhongtang on total-cholesterol and triglyceride levels in serum of cholesterol treated rats were very noticeable. 2. Effects of Xiaotiaozhongtang on H. D. L-cholesterol and phospholipid levels in serum of cholesterol treated rats were noticeable. 3. Effect of Xiaotiaozhogtang on the rate of total-cholesterol vs H. D. L-cholesterol in serum of cholesterol treated rats was unnoticed, but on the rate of total-cholesterol vs phospholipid was noticeable. Thus,as the above-mentioned, on hyperlipidemia, the effects of Xiaotiaozhongtang can be recognized. Secondly, both hyperlipidemia and "phlegm-retention" have correlation each other. In the last place,Xiaotiaozhongtang may be used to artherosclerosis induced by, lipid metabolism disorder, hyperlipidemia.

Validity for Use of Non-HDL Cholesterol Rather than LDL Cholesterol

Kwon, Se-Young,Na, Young-Ak 대한임상검사과학회 2013 대한임상검사과학회지(KJCLS) Vol.45 No.2

NonHDL cholesterol values have been suggested as a risk marker for cardiovascular disease. NonHDL cholesterol values were calculated, using a very simple measurement [nonHDL cholesterol=serum total cholesterol-HDL cholesterol]. This formula is very useful as a screening tool for identifying dyslipoproteinemias, risk assessment, and assessing the results of hypolipidemic therapy. The data from the 2009 Korean National Health and Nutrition Examination Survey were used. Analysis was done for 1,992 subjects with lipid panels (Cholesterol, HDL, LDLdirect and Triglycerides) results. We studied the relationship between nonHDL cholesterol and LDL cholesterol. As a result, nonHDL cholesterol values were plotted against the LDL direct and calculated values. The linear regression equation for nonHDL cholesterol and direct LDL cholesterol was $nonHDLchol=23.60+1.03{\times}LDLdirect$ (p<0.0001, $r^2=0.80$) in all subjects. The subjects were classified into triglyceride values. When triglycerides are below 400 mg/dL, the linear fit to LDL direct is found to be $[nonHDLchol=17.34+1.07{\times}LDLdirect]$ (p<0.0001, $r^2=0.88$) and to the Friedewald LDL calculation is $[nonHDLchol=23.10+1.02{\times}LDLcalc]$ (p<0.0001, $r^2=0.82$). For triglycerides above 400 mg/dL, the linear fit equation is $[nonHDLchol=87.57+0.92{\times}LDLdirect]$ (p<0.0001, $r^2=0.50$) and to the LDL calculated, it is $[nonHDLchol=142.70+0.50{\times}LDLcalc]$ (p<0.0001, $r^2=0.32$). This study provides examples of the utility of nonHDL cholesterol concentrations in clinical medicine.

여대생의 HDL-콜레스테롤 수준에 따른 임상건강지표와 영양섭취 실태

정하나(Ha Na Jung),김정희(Jung Hee Kim) 대한지역사회영양학회 2008 대한지역사회영양학회지 Vol.13 No.1

Recently, a lot of epidemiological studies revealed that low HDL-cholesterol level was a better predictor of risk for coronary heart disease than total cholesterol. This study investigated the anthropometric parameters, clinical blood indices, and dietary factors influencing serum HDL-cholesterol level by using a cross-sectional study for Korean female college students. The subjects were 94 female college students. They were divided into three groups according to their serum HDL-cholesterol levels, low HDL-cholesterol (< 50 mg/dL, n = 20), medium HDL-cholesterol (50≤, < 60 mg/dL, n = 39) high serum HDL-cholesterol groups (60≤ mg/dL, n = 35). This study examined their demographic data and dietary intake throughout a questionnaire. Clinical blood indices were measured using an automatic blood chemistry analyzer (Selectra E), after 12 hours of fasting. BMI, body weight, fat mass, and waist circumferences were significantly increased according to low serum HDL-cholesterol levels. Serum lipid analysis showed a significantly higher level of TG, LDL-/ HDL-Ratio, atherogenic index in the low HDL-cholesterol group. Serum levels of GPT, uric acid and alkaline phosphatase in the low HDL-cholesterol group were significantly higher than in the other group. The average consumption of energy was 1627 kcal and 77.76% of estimated energy requirement (EER). The mean ratio of calories from carbohydrate : protein : fat was 57 : 15 : 28. The low HDL-cholesterol group was significantly higher than the other groups in eggs, fat and oils consumption. Interestingly, milk and diary products consumption of low HDL-cholesterol group was half (p < 0.05) of those of the other groups. In conclusion, serum HDL-cholesterol levels appeared to be decreased by increasing BMI, fat mass, waist circumference, and serum TG level. In addition, some dietary factors seemed to be related to serum HDL-cholesterol levels. However, further research is needed to elucidate the exact relationship between serum HDL-cholesterol level and dietary factors. (Korean J Community Nutrition 13(1) : 100~110, 2008)