Saponin Formosanin C-induced Ferritinophagy and Ferroptosis in Human Hepatocellular Carcinoma Cells.

Ferroptosis, a recently discovered form of iron-dependent cell death, requires an increased level of lipid-reactive oxygen species (ROS). Ferritinophagy, a ferritin degradation pathway, depends on a selective autophagic cargo receptor (NCOA4). By screening various types of natural compounds, formosanin C (FC) was identified as a novel ferroptosis inducer, characterized by attenuations of FC-induced viability inhibition and lipid ROS formation in the presence of ferroptosis inhibitor. FC also induced autophagic flux, evidenced by preventing autophagic marker LC3-II degradation and increasing yellow LC3 puncta in tandem fluorescent-tagged LC3 (mRFP-GFP) reporter plasmid (ptfLC3) transfected cells when combined with autophagic flux inhibitor. It is noteworthy that FC-induced ferroptosis and autophagic flux were stronger in HepG2 cells expressing higher NCOA4 and lower ferritin heavy chain 1 (FTH1) levels, agreeing with the results of gene expression analysis using CTRP and PRISM, indicating that FTH1 expression level exhibited a significant negative correlation with the sensitivity of the cells to a ferroptosis inducer. Confocal and electron microscopy confirmed the pronounced involvement of ferritinophagy in FC-induced ferroptosis in the cells with elevated NCOA4. Since ferroptosis is a non-apoptotic form of cell death, our data suggest FC has chemotherapeutic potential against apoptosis-resistant HCC with a higher NCOA4 expression via ferritinophagy.

Chemopreventive Potential of Ethanolic Extracts of Luobuma Leaves (Apocynum venetum L.) in Androgen Insensitive Prostate Cancer.

Luobuma (Apocynum venetum L. (AVL)) is a popular beverage in Asia and has been reportedly to be associated with the bioactivities such as cardiotonic, diuretic, antioxidative, and antihypertensive. However, its biofunction as chemoprevention activity is seldom addressed. Herein, we aimed to characterize the anti-androgen-insensitive-prostate-cancer (anti-AIPC) bioactive compounds of Luobuma, and to investigate the associated molecular mechanisms. Activity-guided-fractionation (antioxidative activity and cell survivability) of Luobuma ethanolic extracts was performed to isolate and characterize the major bioactive compounds using Ultra Performance Liquid Chromatography (UPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), and Nuclear Magnetic Resonance (NMR). Plant sterols (lupeol, stigamasterol and ß-sitosterol) and polyphenolics (isorhamnetin, kaempferol, and quercetin) were identified. Lupeol, a triterpene found in the fraction (F8) eluted by 10% ethyl acetate/90% hexane and accounted for 19.3% (w/w) of F8, inhibited the proliferation of PC3 cells. Both lupeol and F8 induced G2/M arrest, inhibition of ß-catenin signaling, regulation of apoptotic signal molecules (cytochrome c, Bcl-2, P53, and caspase 3 and 8), and suppression DNA repair enzyme expression (Uracil-DNA glycosylase (UNG)). To our knowledge, our study is the first report that lupeol inhibited the expression of UNG to elicit the cytotoxicity against androgen-insensitive-prostate-cancer cells. Collectively, Luobuma, which contains several antitumor bioactive compounds, holds the potential to be a dietary chemopreventive agent for prostate cancer.

Effects of high-dose phytoestrogens on circulating cellular microparticles and coagulation function in postmenopausal women.

BACKGROUND/PURPOSE: Estrogen in hormone replacement therapy causes homeostatic changes. However, little is known regarding the safety of high-dose phytoestrogen on coagulation and hematological parameters in healthy postmenopausal women. This study evaluated the effects of high-dose soy isoflavone (300 mg/day) on blood pressure, hematological parameters, and coagulation functions including circulating microparticles in healthy postmenopausal women. METHODS: The original study is a 2-year prospective, double-blind, placebo-controlled study. In total, 431 postmenopausal women (from 3 medical centers) were randomly assigned to receive either high-dose isoflavone or placebo for 2 years. At baseline, 6 months, 1 year, and 2 years after treatment, blood pressure, body weight, liver function tests, hematological parameters, and lipid profiles were measured. The 1(st) year blood specimens of 85 cases of 144 eligible participants (from one of the three centers) were analyzed as D-dimer, von Willebrand factor antigen, factor VII, plasminogen activator inhibitor type 1, and circulating cellular microparticles, including the measurement of monocyte, platelet, and endothelial microparticles. RESULTS: In the isoflavone group, after 1 year, the changes in liver function tests, hematological parameters, and coagulation tests were not different from those of the control. Triglyceride levels were significantly lower after 6 months of isoflavone treatment than the placebo group, but the difference did not persist after 1 year. Endothelial microparticles increased steadily in both groups during the 1-year period but the trend was not affected by treatment. CONCLUSION: The results of the present study indicate that high-dose isoflavone treatment (300 mg/day) does not cause hematological abnormalities or activate coagulation factors.

Nutrition and Health Survey of Taiwan Elementary School Children 2001-2002: research design, methods and scope.

The "Nutrition and Health Survey of Taiwan's Elementary School Children (2001-2002)" was to investigate the nutritional status, influential dietary and non-dietary factors, health and development, and school performance, as well as the inter-relationships among these factors. The survey adopted a two-staged stratified, clustered probability sampling scheme. Towns and districts in Taiwan with particular ethnic and geographical characteristics were designated into 13 strata including Hakka areas, mountain areas, eastern Taiwan, the Penghu Islands, 3 northern regions, 3 central regions and 3 southern regions. Eight schools were selected from each stratum using the probabilities proportional to sizes method. Twenty-four pupils were randomly selected within each school. The survey included face-to-face interviews and health examinations. Taking seasonal effects into consideration, the face-to-face interviews were evenly allocated into each of the two semesters. A total of 2,419 face-to-face interviews and 2,475 health examinations were completed. Interview data included household information, socio-demographics, 24-hour dietary recall, food frequency, dietary and nutritional knowledge, attitudes and behaviors, physical activity, medical history, oral health, pubertal development, and bone health. Health exam data included anthropometry, blood pressure, physical fitness, bone density, dental health, and blood and urine collection. SUDAAN was used to adjust sampling design effect. There were no significant differences in sibling rank and parental characteristics between respondents and non-respondents, which indicates that our survey is representative and unbiased. The results of this survey will increase our understanding on the nutrition and health status of schoolchildren and can be used to shape public health policy in Taiwan.

Thiamin and riboflavin status of Taiwanese elementary schoolchildren.

In The Nutrition and Health Survey of Taiwan Elementary School Children (NAHSIT Children 2001~2002), erythrocyte activity coefficients of transketolase (ETKAC) and glutathione reductase (EGRAC) were chosen as indices for assessing the functional status of thiamin and riboflavin nutriture. Mean values of the ETKAC for boys and girls, both of which were in a normal range, were 1.07+/-0.00 and 1.06+/-0.01, respectively. The respective prevalence rates of marginal and deficient thiamin states were 10.4% and 7.8% for boys and 9.3% and 7.3% for girls. Mean values of the EGRAC were 1.18+/-0.00 for boys and 1.19+/-0.01 for girls, both of which showed an increasing trend with age. The respective rates of marginal and deficient riboflavin states were as high as 32.5% and 3.5% for boys and 35.9% and 4.5% for girls. The marginal and deficient riboflavin statuses of Taiwanese schoolchildren were associated with a low frequency of dairy food consumption and an elevated risk of anemia.

Evaluation of folate status by serum and erythrocyte folate levels and dietary folate intake in Taiwanese schoolchildren.

The folate status and dietary folate intake of Taiwanese schoolchildren was investigated by analysis of both serum and red blood cell (RBC) folate levels and dietary folate intake in 1105 boys and 958 girls aged 6-13 years sampled from the Nutrition and Health Survey in Taiwan Elementary School Children 2001-2002 (NAHSIT Children 2001-2002). Mean serum folate levels were 18.3+/-8.8 nmol/L (8.1+/-3.9 ng/mL) in boys and 20.3+/-9.7 nmol/L (9.0+/-4.3 ng/mL) in girls. Mean RBC folate levels were 700+/-320 nmol/L (308+/-141 ng/mL) in boys and 751+/-347 nmol/L (331+/-153 ng/mL) in girls. The prevalence of serum folate deficiency was 1.4% in boys and girls, and the prevalence of marginal serum folate deficiency (7-14 nmol/L) was 31.1% in boys and 25.8% in girls. In addition, 8.5% of boys and 7.4% girls had RBC folate deficiency (RBC folate < 318 nmol/L), and 17% of children had marginal RBC folate deficiency (RBC folate of 318-454 nmol/L). Our results suggesting that Taiwanese schoolchildren have poor folate status especially during periods of rapid growth and development such as the transition from childhood to early adolescence (boys at age 12-12.9, girls at age 11-12.9). The average estimated folate intakes were 269+/-9 microg/d in boys and 259+/-9 microg/d in girls, and 42% of Taiwanese schoolchildren had a dietary folate intake below 2/3 of the RDA, indicating a poor dietary folate intake in this population. This study shows that the folate status of Taiwanese schoolchildren is currently inadequate and strategies are needed for improvement.

Magnesium deficiency and its lack of association with asthma in Taiwanese elementary school children.

The purpose of this study was to investigate magnesium nutritional status and its association with asthma in elementary school children (1277 boys and 1109 girls) participating in the Nutrition and Health Survey in Taiwan (NAHSIT children 2001-2002). Dietary magnesium intake was based on 24-hour dietary recalls. Serum magnesium was measured. Average magnesium intake was 247 mg and 228 mg for the boys and girls, respectively, which is equivalent to 135-123% of the relevant Taiwanese Dietary Reference Intakes (DRIs). Mean serum magnesium concentration was 0.87 mmol/L and 0.86 mmol/L, respectively. The prevalence of serum magnesium <0.8 mmol/L was 10.5-13.5% in both gender in our sample of elementary school children. In Taiwan, asthma was diagnosed in about 5.8% and 4.7% of boys and girls, respectively. In this study, there were no associations between asthma prevalence, dietary magnesium and serum magnesium concentration. In about 40% of our sample of school children, however, dietary magnesium may be suboptimal. This deficit should be targeted though improved intake of magnesium-rich foods such as whole grains, green vegetables and soybean products, particularly in Taiwanese school children whose dietary intakes are below the DRIs and/or who have low serum magnesium levels.

Elderly Nutrition and Health Survey in Taiwan (1999-2000): research design, methodology and content.

The purpose of the Elderly Nutrition and Health Survey in Taiwan (1999-2000) was to assess the diet, nutrition and health of persons aged 65 and above in Taiwan. A multi-staged, stratified, clustered probability sampling scheme was used in the survey. The survey population was stratified into a total of 13 strata. The four strata of "Hakka areas", "Mountain areas", " Eastern areas", and "PengHu islands" were unique in their ethnicity or geographic locations. The remaining areas of Taiwan were stratified into "Northern", "Central", and "Southern" parts with these 3 strata, then each subdivided into a further 3 strata based on population density. The household interview of the survey was arranged such that effect of seasonal variation was taken into account. A total of 1,937 persons completed the interview and 2,432 persons completed the health exam. The following data were collected: (1) Interview data : household information, basic demographics, 24 hour dietary recall, food frequency and habit, knowledge, attitudes and practice, medical history, 36-item Short Form for generic health status, and physical activity. (2) Health exam data: blood sample for measurement of nutritional biochemical indicators and complete clinical chemistry profile, urine sample for urinary electrolytes, anthropometric measurements, ECG, blood pressure, body temperature, pulmonary function, and an osteoporosis assessment. Data from the survey were analyzed using SUDAAN to adjust for the design effect and to obtain unbiased estimates of the mean, standard error and confidence intervals. Survey respondents were slightly younger compared to non-respondents; however, after weighting and adjustment with SUDAAN, the education levels and ethnicity of respondents and non-respondents were similar indicating lack of bias. We anticipate that the results of this survey will be of benefit in understanding the nutritional status of the elderly, the relationship between nutrition and health, and factors influencing elderly persons' nutritional status. Furthermore, this information could be used in the development of public health nutrition policy aimed at improving the nutrition and health of the elderly in Taiwan.

Prevalence of thiamin and riboflavin deficiency among the elderly in Taiwan.

The purpose of this study was to perform biochemical assessments of the nutritional status of thiamin and riboflavin in the 2379 elderly persons (1213 males, 1166 females) participating in the Elderly Nutrition and Health Survey in Taiwan (1999-2000) (Elderly NAHSIT). Through analysis of the data we aimed to investigate possible factors related to the prevalence of vitamin deficiency. Activity coefficients of erythrocyte transketolase and glutathione reductase (ETKAC and EGRAC) were the chosen biochemical indicators for thiamin and riboflavin status. The results showed that 14.7% of men and 11.9% of women were marginally thiamin deficient, and 16.5% of men and 14% of women were thiamin deficient. The prevalence rates of marginal riboflavin deficiency were 25.7% for males and 20.1% for females, and the deficiency rates were 6.6% for elderly males and 4.1% for elderly females. Although the average dietary thiamin and riboflavin intakes reached 146%-164% of Taiwan RDAs, the percentage of senior citizens whose thiamin or riboflavin dietary intakes were less than EARs (equivalent to 83.3% of RDAs) was around 30% for males and 40% for females. Some contributing factors to the significant prevalence of thiamin and riboflavin deficiencies are discussed in this article.

Association between dietary folate-rich food intake and folate status of elderly Taiwanese.

To investigate the relationship between folate status and dietary folate intake in the Taiwanese elderly, we analyzed plasma folate levels and dietary folate intake in 725 males and 705 females aged 65-90 years, sampled from the Elderly Nutrition and Health Survey in Taiwan (1999-2000) (Elderly NAHSIT). Results showed that the mean plasma folate levels were 22.9+/-1.4 nmol/L (10.1+/-0.6 ng/ml) for males and 29.5+/-1.6 nmol/L (13.0+/-0.7 ng/ml) for females. The average plasma folate concentrations of males from all age groups were significantly lower than those of females (P<0.0001). None of the study subjects had a plasma folate below 7 nmol/L (3 ng/ml). However, 18.6% of males and 12.1% of females had marginal folate deficiency, with plasma folate between 7-14 nmol/L (3-6 ng/ml). This suggests that elderly males have a poorer folate status than elderly females in the Taiwanese population. The percentage of marginal folate deficiency tended to increase with age among females (P trend=0.0137). The average estimated folate intakes were 379+/-18 microg/d in males and 351+/-27 microg/d in females. However, 45.5% of males and 48.8% of females had a dietary folate intake below 2/3 of the RDA of 400 microg/d. Our results indicated that dietary folate intake is positively correlated with plasma folate levels (r=0.10, P<0.05). In addition, dietary folate intake increased with increased intakes of vegetables, mushrooms and fruit. A lower intake of fruit appeared to be responsible for the higher prevalence of marginal folate deficiency among females over the age of 80 years.

Association of B vitamins status and homocysteine levels in elderly Taiwanese.

To investigate the relationship between homocysteine (Hcy) and B vitamins status in the Taiwanese elderly population, an analysis was made of the plasma Hcy levels in elderly persons. The study sample was taken from the Elderly Nutrition and Health Survey in Taiwan (1999-2000) (Elderly NAHSIT) and included 1094 males and 1135 females aged 65-90 years. The results showed that average plasma Hcy was 13.3+/-0.6 micromol/ L for males and 10.6+/-0.7 micromol/L for females. The average plasma Hcy levels of males from all age groups were significantly higher than those of females, and significantly increased with age (P<0.0001). The overall prevalence of hyperhomocysteinemia (Hcy>15 micromol/L) was 23.4% for elderly males and 11.2% for elderly females, and this also increased with age (P<0.0001). In subjects with normal renal function, folate, vitamin B2, B6, and B12 status were significantly lower in males with hyperhomocysteinemia, while only folate and vitamin B12 were significantly lower in females with hyperhomocysteinemia. Further analysis suggested that folate, vitamin B6 or B12 insufficiency were associated with hyperhomocysteinemia in both sexes, while vitamin B2 insufficiency was significantly associated only in males. In elderly persons with adequate folate, vitamin B6, and B12 status, there was no significant association between vitamin B2 and hyperhomocysteinemia. This association occurred only in those who had concurrent poor folate, vitamin B6, or B12 status. The strength of the association between vitamin B12 insufficiency and hyperhomocysteinemia was not affected by simultaneous vitamin B2 or B6 insufficiency, but increased about 3-fold when combined with folate. This suggests that poor folate and vitamin B12 status has a synergistic effect on the risk of hyperhomocysteinema in the elderly, as did a poor folate and vitamin B6 status. Therefore, maintaining adequate vitamin B12 status and avoiding multiple B vitamin insufficiency, especially that of folate and vitamin B12 or B6, should be emphasized as an important measure for reducing plasma Hcy levels among elderly Taiwanese.

Magnesium status and association with diabetes in the Taiwanese elderly.

The average dietary intake of magnesium is below recommended dietary allowances in many affluent Western countries. Prolonged low magnesium intake tends to result in hypomagnesaemia which might increase the risk of chronic diseases in elderly people. A national population-based cross-sectional nutrition survey, the Elderly Nutrition and Health Survey in Taiwan (1999-2000), was used to investigate the magnesium status and association with diabetes in the Taiwanese elderly. Dietary magnesium intake was based on 24-hour dietary recalls. Blood biochemical parameters including plasma magnesium and blood glucose were also measured. Average magnesium intake was 250 mg in men and 216 mg in women, which is equivalent to 68-70% of relevant Taiwanese Dietary Reference Intakes. The mean plasma magnesium concentration was 0.903 mmol/L in men and 0.906 mmol/L in women. The prevalence of a plasma magnesium level of <0.7 mmol/L was 0.7-0.9% in the elderly, and that of <0.8 mmol/L was 8.0-9.1%. Elderly vegans had a significantly lower magnesium intake than ovo-lacto vegetarians and non-vegetarians. Diabetic men and women had significantly higher blood glucose levels than non-diabetics. The risk of diabetes was elevated 3.25 times at plasma magnesium levels<0.863 mmol/L. There was an inverse association between plasma magnesium concentration and the prevalence of diabetes. However, no association was found between diabetes and low dietary magnesium. Taiwanese elderly persons had suboptimal levels of dietary magnesium intake, which although may be sufficient to avoid overt magnesium deficiency, may not be sufficient to reduce the risk of diabetes in the elderly. Further prospective study is required to help explain the differential results between dietary and plasma magnesium levels.

Iron status of the Taiwanese elderly: the prevalence of iron deficiency and elevated iron stores.

Iron status, prevalence of iron deficiency and elevated iron stores, and the effect of gastrointestinal ulceration on iron status in free-living Taiwanese elderly persons were all assessed in a nationally representative, cross-sectional nutrition survey--the Elderly NAHSIT. The survey included blood measurements of iron indices. Data were collected from 1202 elderly men and 1152 elderly women aged 65 years and older. Multiple iron measures, including serum ferritin (SF), transferrin saturation (Tsat), and hemoglobin were used to evaluate the prevalence of iron deficient erythropoiesis (ID) and iron deficiency anemia (IDA). Despite no routine practice of iron fortification in Taiwan, elderly subjects had a low prevalence of ID and IDA. The prevalence of ID was 2.3% in men and 1.4% in women. The prevalence of IDA was 2.5% in men and 2.0% in women. In contrast, 15.7% of men and 9.8% of women had elevated iron stores as diagnosed by SF>300 microg/L. Subjects with a history of gastrointestinal ulceration had significantly lower serum ferritin than those without ulcers, but the prevalence of anemia, ID and IDA was unaffected. In conclusion, elderly people in Taiwan are an iron-replete population with a high prevalence of elevated iron stores and a low prevalence of iron deficiency.

Mitochondrial cysteine desulfurase iron-sulfur cluster S and aconitase are post-transcriptionally regulated by dietary iron in skeletal muscle of rats.

Cysteine desulfurase IscS is required for cellular iron-sulfur protein maturation in eukaryotes and prokaryotes. In this study, we examined the effect of dietary iron intake on the expression in rat skeletal muscle of IscS in relation to 2 iron-sulfur proteins, cytosolic aconitase (c-aconitase) and mitochondrial aconitase (m-aconitase). Three groups of male weanling Wistar rats were used; 1 group was fed an iron-deficient diet (D), and the other 2 groups were pair-fed (P) or freely fed (C) a control (35 mg Fe/kg diet) diet for 1 or 2 wk. At the end of wk 1 and 2, the mitochondrial IscS protein levels in the skeletal muscle of iron-deficient rats had decreased to 45 and 50% of those of the control and pair-fed rats, respectively, whereas the IscS mRNA levels did not differ among the 3 groups, indicating that iron deficiency affected the expression of IscS protein at the post-transcriptional level. Iron deficiency caused a 55-76% reduction in c-aconitase activity and an approximately 50% reduction in the c-aconitase protein level. The m-aconitase activity and protein level in iron-deficient rats also declined to 50 and 58-64% of the control levels, respectively. Our results indicate that dietary iron modulates mitochondrial IscS protein and aconitase at the post-transcriptional level, and mitochondrial IscS may be associated with this regulation of aconitase in skeletal muscle.

The hypoglycemic effects of soy isoflavones on postmenopausal women.

OBJECTIVES: Soy isoflavones have many effects similar to those of estrogen and have become popular among postmenopausal women as an alternative for hormone replacement therapy (HRT). The purpose of this study was to determine the effects of soy isoflavones on glucose, insulin, and lipid profiles in postmenopausal Taiwanese women. METHODS: We conducted a randomized, double-blind, active placebo-controlled clinical trial to compare the effects of isoflavones with estrogen replacement therapy (ERT) on blood glucose, insulin, and lipid profiles in postmenopausal Taiwanese women. Thirty postmenopausal Taiwanese women were randomly assigned to two groups, and each received treatment for 6 months. The isoflavone group received 100 mg isoflavone soft capsules, 300 mg calcium, and a blank vitamin capsule per day. The estrogen active control group received 0.625 mg conjugated estrogen, 300 mg calcium, and blank isoflavone soft capsules per day. We measured baseline levels of fasting blood sugar, insulin, lipid profiles, and isoflavone concentrations prior to the study and repeated the same measurements every 3 months for a total duration of 6 months. RESULTS: Two-way ANOVA revealed that fasting glucose (p < 0.001) and insulin (p < 0.005) levels were significantly affected by estrogen and isoflavone treatments after 6 months. For the isoflavone group, the average blood genistein concentration was 6-10 times higher than those of the estrogen group. Within the same periods, the fasting blood glucose was reduced to 83% and 85% of the baseline levels, and insulin was reduced to 56% and 67% of the baseline levels, respectively, in the estrogen and isoflavone groups. CONCLUSIONS: Soy isoflavones (100 mg) and 0.625 mg conjugated estrogen equally lower fasting blood glucose and insulin levels in postmenopausal women.

Role of hypolipidemic drug clofibrate in altering iron regulatory proteins IRP1 and IRP2 activities and hepatic iron metabolism in rats fed a low-iron diet.

In addition to reducing the expression of transferrin and ceruloplasmin genes, hypolipidemic peroxisome proliferators may alter iron homeostasis in the liver. Therefore, this study investigates the effects of clofibrate on proteins related to liver iron metabolism in a rat model using a 2 x 2 experimental design: two dose levels of clofibrate in diet (0 and 0.5%) and two dietary iron levels (35 ppm as normal level and 15 ppm as low-iron diet). Twenty-four Wistar rats were assigned to the four diets and fed for 6 weeks. Subsequent measurements of iron parameters in the blood and the liver indicated that, in addition to mild anemia and the reduction in serum iron and total iron-binding capacity, clofibrate treatment altered IRP1 and IRP2 activities differentially and increased mitochondrial aconitase both at activity and protein levels. At both normal and low-iron intakes, clofibrate caused a 50% reduction in serum iron and TIBC with a corresponding reduction in transferrin mRNA. The RNA-binding activities of IRP1 were selectively activated by clofibrate treatment even though liver iron concentration was not depleted. The RNA-binding activity of IRP2 was selectively activated by the low iron intake and correlated with an increase of transferrin receptor mRNA, while clofibrate treatment offset the effects of the low iron intake.