Apolipoprotein CIII Deficiency Protects Against Atherosclerosis in Knockout Rabbits.

OBJECTIVE: Apo (apolipoprotein) CIII mediates the metabolism of triglyceride (TG)-rich lipoproteins. High levels of plasma apoCIII are positively correlated with the plasma TG levels and increase the cardiovascular risk. However, whether apoCIII is directly involved in the development of atherosclerosis has not been fully elucidated. Approach and Results: To examine the possible roles of apoCIII in lipoprotein metabolism and atherosclerosis, we generated apoCIII KO (knockout) rabbits using ZFN (zinc finger nuclease) technique. On a normal standard diet, apoCIII KO rabbits exhibited significantly lower plasma levels of TG than those of WT (wild type) rabbits while total cholesterol and HDL (high-density lipoprotein) cholesterol levels were unchanged. Analysis of lipoproteins isolated by sequential ultracentrifugation revealed that reduced plasma TG levels in KO rabbits were accompanied by prominent reduction of VLDLs (very-low-density lipoproteins) and IDLs (intermediate-density lipoproteins). In addition, KO rabbits showed faster TG clearance rate after intravenous fat load than WT rabbits. On a cholesterol-rich diet, KO rabbits exhibited constantly and significantly lower levels of plasma total cholesterol and TG than WT rabbits, which was caused by a remarkable reduction of ß-VLDLs-the major atherogenic lipoproteins. ß-VLDLs of KO rabbits showed higher uptake by cultured hepatocytes and were cleared faster from the circulation than ß-VLDLs isolated from WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. CONCLUSIONS: These results indicate that apoCIII deficiency facilitates TG-rich lipoprotein catabolism, and therapeutic inhibition of apoCIII expression may become a novel means not only for the treatment of hyperlipidemia but also for atherosclerosis.

Association of cholesterol uptake capacity, a novel indicator for HDL functionality, and coronary plaque properties: An optical coherence tomography-based observational study.

BACKGROUND: Cholesterol efflux from atherosclerotic lesion is a key function of high-density lipoprotein (HDL). Recently, we established a simple, high-throughput, cell-free assay to evaluate the capacity of HDL to accept additional cholesterol, which is herein referred to as "cholesterol uptake capacity (CUC)". OBJECTIVE: To clarify the cross-sectional relationship between CUC and coronary plaque properties. METHODS: We enrolled 135 patients to measure CUC and assess the morphological features of angiographic stenosis by optical coherence tomography (OCT). We estimated the extent of the lipid-rich plaque by multiplying the mean lipid arc by lipid length (lipid index). The extent of the OCT-detected macrophage accumulation in the target plaque was semi-quantitatively estimated using a grading system. RESULTS: Lipid-rich plaque lesions were identified in 125 patients (92.6%). CUC was inversely associated with the lipid index (R = -0.348, P < 0.0001). In addition, CUC was also inversely associated with macrophage score (R = -0.327, P < 0.0001). Conversely, neither circulating levels of HDL cholesterol nor apoA1 showed a similar relationship. CONCLUSIONS: We demonstrated that CUC was inversely related to lipid-rich plaque burden and the extent of macrophage accumulation, suggesting that CUC could be useful for cardiovascular risk stratification.

Activation of NK cells in male cancer survivors by fucoidan extracted from Cladosiphon okamuranus.

Cancer survivors are highly motivated to seek information about the use of dietary supplements and complementary nutritional therapies to improve their quality of life. Fucoidan, a sulfated polysaccharide extracted from brown marine alga, exhibits a wide range of bioactivities, including anticancer activity. As natural killer (NK) cells serve an important role in defenses against tumor cells, the present study examined the effects of fucoidan extracted from Cladosiphon Okamuranus on NK cell activity in cancer survivors. A prospective, open-label clinical study was conducted on cancer survivors treated with fucoidan via oral administration; 11 cancer survivors with a performance status of 0 or 1 participated and consumed 3 g of fucoidan for 6 months. No significant changes were observed in the mean activities of NK cells in total subjects following the ingestion of fucoidan. An analysis of each sex revealed that NK cell activity was significantly increased by the ingestion of fucoidan in male, yet not female subjects. Serum fucoidan levels were markedly increased following the ingestion of fucoidan and the peak levels ranged between 30 and 198 ng/ml. Tumor markers remained within the reference range during the trial period in subjects, in whom primary tumors were eradicated by treatment. The basal values of tumor markers were elevated in three cases; tumor markers were increased in two cases and decreased in one by the ingestion of fucoidan. These findings suggest that fucoidan enhances the activation of NK cells in male cancer survivors.

Direct Versus Calculated LDL Cholesterol and C-Reactive Protein in Cardiovascular Disease Risk Assessment in the Framingham Offspring Study.

BACKGROUND: Increases in circulating LDL cholesterol (LDL-C) and high-sensitivity C-reactive protein (hsCRP) concentrations are significant risk factors for cardiovascular disease (CVD). We assessed direct LDL-C and hsCRP concentrations compared to standard risk factors in the Framingham Offspring Study. METHODS: We used stored frozen plasma samples (-80 °C) obtained after an overnight fast from 3147 male and female participants (mean age, 58 years) free of CVD at cycle 6 of the Framingham Offspring Study. Overall, 677 participants (21.5%) had a CVD end point over a median of 16.0 years of follow-up. Total cholesterol (TC), triglyceride (TG), HDL cholesterol (HDL-C), direct LDL-C (Denka Seiken and Kyowa Medex methods), and hsCRP (Dade Behring method) concentrations were measured by automated analysis. LDL-C was also calculated by both the Friedewald and Martin methods. RESULTS: Considering all CVD outcomes on univariate analysis, significant factors included standard risk factors (age, hypertension, HDL-C, hypertension treatment, sex, diabetes, smoking, and TC concentration) and nonstandard risk factors (non-HDL-C, direct LDL-C and calculated LDL-C, TG, and hsCRP concentrations). On multivariate analysis, only the Denka Seiken direct LDL-C and the Dade Behring hsCRP were still significant on Cox regression analysis and improved the net risk reclassification index, but with modest effects. Discordance analysis confirmed the benefit of the Denka Seiken direct LDL-C method for prospective hard CVD endpoints (new-onset myocardial infarction, stroke, and/or CVD death). CONCLUSIONS: Our data indicate that the Denka Seiken direct LDL-C and Dade Behring hsCRP measurements add significant, but modest, information about CVD risk, compared to standard risk factors and/or calculated LDL-C.

Biological sources of variation of serum adiponectin among healthy individuals in comparison with related nutritional and inflammatory markers.

BACKGROUND: Serum adiponectin (AN) is a nutritional and inflammatory marker of various diseases. Its biological sources of variation (SVs) have been widely evaluated, but its relationships with other related markers remain to be studied comprehensibly by use of multivariate analyses including factor analysis. METHODS: Serum specimens from 752 well-defined healthy subjects (295 males; 457 females) obtained from Japan, Hong Kong, Taiwan, and Vietnam were tested for total AN (tAN) and high-molecular-weight AN (hmAN) together with 71 major analytes. We chose test results of 11 analytes known as nutritional and inflammatory markers: insulin, HDL-C, LDL-C, TG, ALT, GGT, UA, TTR, CRP, C3, and C4. Factor analysis (FA) was performed to elucidate commonality among the analytes. Multiple regression analysis (MRA) was performed analyte by analyte to evaluate its biological SVs including age, BMI, and levels of alcohol consumption, smoking, and exercise. RESULTS: Both serum ANs showed female predominance, reduction with increased BMI, and regional difference (lower in Southeast Asia). The ratio of hmAN to tAN was higher in females. MRA showed no clear associations of tAN or hmAN with the levels of drinking alcohol or cigarette smoking. FA revealed that both tAN and hmAN have a commonality in biological variations with HDL-C, TG, CRP, and insulin but not with ALT, GGT, and TTR. CONCLUSIONS: No apparent differences were observed between tAN and hmAN with regard to commonality or association with related analytes by FA or MRA. It appears not necessary to distinguish hmAN from tAN when interpreting test results among healthy individuals.

Changes in lipoprotein lipase and endothelial lipase mass in familial hypercholesterolemia during three-drug lipid-lowering combination therapy.

BACKGROUND: This study was performed to compare the effects of three different lipid-lowering therapies (statins, ezetimibe, and colestimide) on lipoprotein lipase and endothelial lipase masses in pre-heparin plasma (pre-heparin LPL and EL mass, respectively) from patients with familial hypercholesterolemia (FH). FH is usually treated by coadministration of these three drugs. METHODS: The pre-heparin LPL and EL masses were measured in fresh frozen plasma drawn and stored at various time points during coadministration of the three drugs from patients with heterozygous FH harboring a single mutation in the LDL receptor (n = 16, mean age 63 years). The patients were randomly divided into two groups based on the timing when ezetimibe was added. RESULTS: Plasma LPL mass concentration was significantly reduced by rosuvastatin at 20 mg/day (median = 87.4 [IQR: 71.4-124.7] to 67.5 [IQR: 62.1-114.3] ng/ml, P < 0.05). In contrast, ezetimibe at 10 mg/day as well as colestimide at 3.62 g/day did not alter its level substantially (median = 67.5 [IQR: 62.1-114.3] to 70.2 [IQR: 58.3-106.2], and to 74.9 [IQR: 55.6-101.3] ng/ml, respectively) in the group starting with rosuvastatin followed by the addition of ezetimibe and colestimide. On the other hand, the magnitude in LPL mass reduction was lower in the group starting with ezetimibe at 10 mg/day before reaching the maximum dose of 20 mg/day of rosuvastatin. Plasma EL mass concentration was significantly increased by rosuvastatin at 20 mg/day (median = 278.8 [IQR: 186.7-288.7] to 297.0 [IQR: 266.2-300.2] ng/ml, P < 0.05), whereas other drugs did not significantly alter its level. CONCLUSION: The effects on changes of LPL and EL mass differed depending on the lipid-lowering therapy, which may impact the prevention of atherosclerosis differently.

Determination of the serum metallothionein (MT)1/2 concentration in patients with Wilson's disease and Menkes disease.

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH2 terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6ng/ml and the spiked MT-1was fully recovered from the plasma. We investigated the normal range of MT1/2 (25-75%tile) in 200 healthy human serum and found it to be 27-48ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilson's disease patients, levels which were very similar to those in the Long-Evans Cinnamon (LEC) rat (model animal of Wilson's disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilson's disease.

Do insulin resistance conditions further impair the lipid and inflammatory profile in end-stage renal disease patients on hemodialysis?

BACKGROUND: Type 2 diabetes (T2DM) and chronic renal disease constitute important risk factors of atherosclerotic cardiovascular disease, associated with lipid abnormalities, and proinflammatory states. Advances in renal replacement therapy such as hemodialysis (HD) have not reduced morbi-mortality. It has not been elucidated if the concomitant presence of T2DM or metabolic syndrome with end-stage renal disease further impairs the atherogenic profiles. METHODS: We studied 122 HD patients, among which 44 presented with T2DM (HD-T2DM) and 30 with metabolic syndrome (HD-MS); 48 had neither T2DM nor metabolic syndrome (HD-C). Lipoprotein profile, including atherogenic remnant lipoproteins (RLP), and inflammation markers--high sensitivity C-reactive protein (hsCRP), adiponectin, and interleukin-6 (IL-6)--were measured. RESULTS: In all HD patients, triglycerides, free fatty acids, and RLP showed no differences between HD groups, whereas high-density lipoprotein cholesterol (HDL-C) was decreased, particularly in HD-T2DM and HD-MS, with respect to HD-C (P<0.01). Regarding inflammatory parameters, both IL-6 and hsCRP were found to be similar between HD groups. Adiponectin paradoxically shows higher values in relation to those expected for insulin resistance situations showing no differences between HD groups. CONCLUSIONS: The presence of T2DM or metabolic syndrome did not worsen atherogenic lipoprotein levels, but did reduce HDL-C. Neither was the proinflammatory profile further altered in HD patients in the presence of insulin resistance conditions.

Plasma activity of endothelial lipase impacts high-density lipoprotein metabolism and coronary risk factors in humans.

AIM: Endothelial lipase (EL) is a determinant of plasma levels of high-density lipoprotein cholesterol (HDL-C). However, little is known about the impact of EL activity on plasma lipid profile. We aimed to establish a new method to evaluate EL-specific phospholipase activity in humans. METHODS: Plasma samples were obtained from 115 patients with coronary artery disease (CAD) and 154 patients without CAD. Plasma EL protein was immunoprecipitated using an anti-EL monoclonal antibody after plasma non-specific immunoglobulins were removed by incubation with ProteinA. The phospholipase activity of the immunoprecipitated samples was measured using a fluorogenic phospholipase substrate, Bis-BODIPY FL C11-PC. RESULTS: The EL-specific phospholipase assay revealed that plasma EL activity was inversely correlated with HDL-C levels (R = -0.3088, p＜0.0001). In addition, the EL activity was associated with cigarette smoking. Furthermore, EL activity in CAD patients was significantly higher than that in nonCAD patients. Concomitantly, the HDL-C level in CAD patients were significantly lower than that in non-CAD patients. CONCLUSION: We have established a method for human plasma EL-specific phospholipase activity by combination of EL immunoprecipitation and a fluorogenic phospholipid substrate. Plasma EL activity was associated with not only plasma HDL-C levels but also the risks for CAD.

Significance of metallothionein expression in liver disease.

Hepatic metallothionein (MT) expression, with various isoforms, and varying cellular localizations is a useful marker for clinico-pathogenesis of liver diseases. In acute liver toxicity caused by cadmium, carbon tetrachloride, or acetaminophen, MT plays a protective role, via the scavenging of radical species. In chronic hepatitis C patients, hepatic MT levels appear to be a biological factor associated with the severity of HCV infection, and are associated with a better response to IFN therapy. Transgenic mice that express HBsAg in the liver show hepatocellular damage, inflammation, regeneration, hyperplasia, and, eventually, neoplasia. The MT isoform, MT-1 help mitigate HBV-induced hepatitis. Analysis of MT gene expression in the livers of chronic hepatitis B patients is useful for understanding the features of distinct liver diseases and for judging disease progression. A profound down-regulation of isoform MT-1G in hepatocellular carcinoma was observed in 63% of tumors relative to the adjacent nonmalignant liver. MT has been implicated in the control of p53 folding with zinc exchange. Therefore, it appears MT may play a role in the pathogenesis of hepatocellular carcinoma. Overall MT is linked to a variety of liver diseases.

Development of a high sensitivity ELISA for the assay of metallothionein.

We have developed a competitive ELISA using a polyclonal antibody that showed specificity to both metallothionin (MT)-1 and MT-2 isoforms in human and animal specimens. The advantage of this ELISA depends on the characteristics of the polyclonal antibody. The NH2 terminal peptide of MT with acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with liver, kidney, and brain extracts worked very well for MT1,2 in wild type mice. Extracts of MT-3 knock-out mice also react well this ELISA, as expected, very low in MT 1,2 but in MT1/2 knock-out mice. Detection limits, the ranges of linearity, and reliability coefficients of MT quantification of the ELISA were suitable for determination of MT. From the preliminary study of normal reference ranges, we found that normal MT levels were between approximately 10-30 ng/ml in human serum. We expect in the future to detect cases with low (MT deficiency) and high serum MT concentrations in patients with various diseases, such as brain/liver disorders, and cancers, using this MT ELISA.

Post-prandial remnant lipoprotein metabolism in autosomal recessive hypercholesterolaemia.

BACKGROUND: Phenotype of autosomal recessive hypercholesterolaemia (ARH), a rare lipid disorder, is known to be milder than that of homozygous familial hypercholesterolaemia (FH) with LDL receptor gene mutation. However, few data exist regarding the functional differences in ARH and FH particularly in terms of remnant-like particles' (RLP) metabolism. MATERIALS AND METHODS: Blood sampling was performed up to 6h after OFTT cream loading (50 g/body surface area) with 2-h intervals in a single ARH proband, four heterozygous FH patients with LDL receptor gene mutation and four normal controls. Plasma lipoprotein and RLP fraction were determined by HPLC system. The area under curve (AUC) of each lipoprotein including RLP fractions was evaluated. RESULTS: The AUC of TG, RLP cholesterol (RLP-C) and RLP triglyceride (RLP-TG) levels of heterozygous FH subjects was significantly higher than those of controls (466±71 mg/dL×h vs. 303±111 mg/dL×h, P<0·05; 35±7 mg/dL×h vs. 21±8 mg/dL×h, P<0·05; 124±57 mg/dL×h vs. 51±13 mg/dL×h, P<0·05, respectively). Under these conditions, those values of ARH were close to those of controls (310 mg/dL×h, 22 mg/dL×h, 23 mg/dL×h, respectively). CONCLUSION: These data demonstrate that unlike in FH, RLP clearance is preserved in ARH. The preservation of post-prandial RLP clearance may contribute to the mild phenotype of ARH compared with FH.

Comparative reactivity of remnant-like lipoprotein particles (RLP) and low-density lipoprotein (LDL) to LDL receptor and VLDL receptor: effect of a high-dose statin on VLDL receptor expression.

BACKGROUND: Comparison of the reactivity of remnant-like lipoprotein particles (RLP) and LDL particles to LDL receptor and VLDL receptor has not been investigated. METHODS: LDL receptor- or VLDL receptor-transfected ldlA-7, HepG2 and L6 cells were used. Human LDL and rabbit ß-VLDL were isolated by ultracentrifugation. Human RLP was isolated using an immunoaffinity mixed gel. The effect of statin on lipoprotein receptors was examined. RESULTS: Both LDL receptor and VLDL receptor recognized RLP. In LDL receptor transfectants, RLP, ß-VLDL and LDL all bound to LDL receptor. Cold RLP competed efficiently with DiI-ß-VLDL; however, cold LDL competed weakly. In VLDL receptor transfectants, RLP and ß-VLDL bound to VLDL receptor, but not LDL. RLP bound to VLDL receptor with higher affinity than ß-VLDL because of higher apolipoprotein E in RLP. LDL receptor expression was induced in HepG2 by the low concentration of statin while VLDL receptor expression was induced in L6 myoblasts at higher concentration. CONCLUSIONS: RLP are bound to hepatic LDL receptor more efficiently than LDL, which may explain the mechanism by which statins prevent cardiovascular risk by primarily reducing plasma RLP rather than by reducing LDL. Additionally, a high-dose of statins also may reduce plasma RLP through muscular VLDL receptor.

Adiponectin: an independent risk factor for coronary heart disease in men in the Framingham offspring Study.

OBJECTIVE: Our aim was to determine whether plasma adiponectin levels were an independent predictor of coronary heart disease (CHD) risk. METHODS AND RESULTS: Plasma adiponectin levels were measured in 3188 male and female participants from cycle 6 of the Framingham offspring Study (mean age: 57 years in both men and women; BMI: 28.5 kg/m(2) in men and 27.3 kg/m(2) in women), using a novel fully automated assay. Plasma adiponectin levels (median [25th percentile, 75th percentile]) were significantly higher in female than in male CHD-free subjects (14.8 [10.7,20.5] µg/ml versus 9.0 [7.0,12.2] µg/ml, p<0.001). Participants were followed for a mean of 7.5 years. After adjustment for age, BMI, smoking status, systolic blood pressure, treatment for hypertension, diabetes, use of cholesterol-lowering medication, total cholesterol level, high-density lipoprotein cholesterol level, and C-reactive protein levels, a higher plasma adiponectin level was a significant predictor of lower risk of future CHD events (n=117) in men (HR 0.49, p<0.0022). A similar trend was observed in women, but was no longer significant after multivariate adjustments. CONCLUSIONS: Our data indicate that plasma adiponectin levels are an independent predictor of CHD in Caucasian men initially free of CHD.

Remnant lipoproteins induced proliferation of human prostate cancer cell, PC-3 but not LNCaP, via low density lipoprotein receptor.

BACKGROUND: Hypertriglyceridemia has been shown to be one of the risk factors for prostate cancer. In this study, we investigated the effect of remnant lipoproteins on cell growth in prostate cancer cell lines. METHODS: Remnant lipoproteins were isolated as remnant like particles (RLP) from human plasma. We used RLP for TG-rich lipoproteins and low density lipoproteins (LDL) for cholesterol-rich lipoproteins respectively and examined the effect of lipoproteins on proliferation of PC-3 and LNCaP cells using MTS assays. Moreover, we studied the effect of RLP and LDL treatment on the regulation of lipoprotein receptors in prostate cancer cells to investigate the relationship between lipoprotein-induced cell proliferation and lipoprotein receptor expression using real-time PCR, Western blotting assays and siRNA. RESULTS: RLP effectively induced PC-3 cell proliferation more than LDL, whereas both RLP and LDL could not induce LNCaP cell proliferation except at a higher concentration of RLP. LDL receptor (LDLr) was expressed in both prostate cancer cells but there was a sharp difference of sterol regulation between two cells. In PC-3 cells, LDL decreased the LDLr expression in some degree, but RLP did not. Meanwhile LDLr expression in LNCaP was easily downregulated by RLP and LDL. Blocking LDLr function significantly inhibited both RLP- and LDL-induced PC-3 cell proliferation. CONCLUSIONS: This study demonstrated that RLP-induced PC-3 cell proliferation more than LDL; however, both RLP and LDL hardly induced LNCaP cell proliferation. The differences of proliferation by lipoproteins might be involved in the regulation of LDLr expression.

Cholesterol efflux from J774 macrophages and Fu5AH hepatoma cells to serum is preserved in CETP-deficient patients.

BACKGROUND: The role of CETP in the development of atherosclerosis is debatable, and few data exist regarding the total impact of CETP inhibition on cholesterol efflux. METHODS: Acceptor capacities of whole serum and HDL subfractions separated by HPLC were compared using 2 different cell systems. Subjects with CETP deficiency (2 homozygous, 1 compound heterozygous, and 5 heterozygous) were analyzed along with 10 normolipidemic controls. The fractional efflux from cholesterol-labeled Fu5AH hepatoma cells was determined to be SR-BI mediated. The efflux difference between control and liver X receptor (LXR) agonist-induced ABCA1-upregulated J774 macrophages was considered as a measure of ABCA1-mediated efflux. RESULTS: For the Fu5AH cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fraction 2 obtained from the homozygous subjects were 38% and 116% higher than the corresponding values for the controls, respectively (p<0.05). For the J774 cell system, the total acceptor capacities of whole serum and HPLC-separated HDL fractions were similar among the CETP-deficient subjects and controls. CONCLUSIONS: Serum from homozygous subjects with CETP-null defects exhibited enhanced acceptor capacity via an SR-BI dependent pathway, which is regulated by the middle HPLC-separated HDL fraction. Further, the cholesterol acceptor capacity of serum obtained from patients having complete and partial CETP deficiency was preserved via an ABCA1-dependent pathway.

Immunohistochemical staining with newly developed metallothionein fragment antibodies against NH2-terminal, middle-regional and COOH-terminal peptides in rabbits.

Metallothionein (MT) has been reported to play important physiological roles in the human body, but the distribution and significance of MT is not fully understood. In order to analyze MT expression, three kinds of polyclonal MT fragment antibodies were developed against NH2-terminal, middle regional and COOH-terminal peptide followed by human MT-IA amino acid sequence in rabbits. The characteristics of these antibodies were studied using competitive immunoassay and immunohistochemical staining. The NH2-terminal antibody (anti-MT-N) had the strongest and clearest immunoreactivity to human and mouse tissues, while COOH-terminal antibody (anti-MT-C) showed species difference because of the 4 amino acid difference in the MT fragment peptide between human and mouse. No reactivity was detected using middle regional residue antibody (anti-MT-M) both on competitive immunoassay and on immunostaining. These results suggest that anti-MT-N is the most applicable antibody to use for immunohistochemistry in human, mouse and other specimens.

Changes of heavy metal, metallothionein and heat shock proteins in Sertoli cells induced by cadmium exposure.

In this study, we examined the levels of Cadmium (Cd), iron (Fe) and zinc (Zn), which were considered to be involved in Sertoli cell damage caused by Cd exposure. We also examined metallothionein (MT), heat shock protein 70 (Hsp70) and heme oxygenase-1 (HO-1) expressions in Sertoli cells induced by Cd exposure. Evaluation by the in-air micro-particle induced X-ray emission (PIXE) method revealed that Cd and Fe distribution was increased in the cytoplasm of Sertoli cells after Cd exposure. By contrast, Zn was decreased in the cytoplasm of Sertoli cells after Cd exposure. It was suggested that the target of Cd toxicity was the cytoplasm of Sertoli cells, Fe was considered to enhance damage to Sertoli cells caused by Cd exposure. The DNA fragmentation rate was determined by ELISA after Cd exposure to Sertoli cells. It remained essentially unchanged with 2.5 microM Cd exposure of Sertoli cells; however, MT, Hsp70 and HO-1 were significantly increased by Cd exposure. As a result, Cd-induced MT was protected Sertoli cells against apoptosis, and Cd-induced HO-1 was involved in protection against oxidative stress. Incidentally, MT, Hsp70 and HO-1 showed similar responses to Cd exposure.

An apolipoprotein A-V gene SNP is associated with marked hypertriglyceridemia among Asian-American patients.

Apolipoprotein A-V (apoA-V) is an important regulator of plasma levels of triglyceride (TG) in mice. In humans, APOA5 genetic variation is associated with TG in several populations. In this study, we determined the effects of the p.185Gly>Cys (c.553G>T; rs2075291) polymorphism on plasma TG levels in subjects of Chinese ancestry living in the United States and in a group of non-Chinese Asian ancestry. The frequency of the less common cysteine allele was 4-fold higher (15.1% vs. 3.7%) in Chinese high-TG subjects compared with a low-TG group (Chi-square = 20.2; P < 0.0001), corresponding with a 4.45 times higher risk of hypertriglyceridemia (95% confidence interval, 2.18-9.07; P < 0.001). These results were replicated in the non-Chinese Asians. Heterozygosity was associated, in the high-TG group, with a doubling of TG (P < 0.001), mainly VLDL TG (P = 0.014). All eleven TT homozygotes had severe hypertriglyceridemia, with mean TG of 2,292 +/- 447 mg/dl. Compared with controls, carriers of the T allele had lower postheparin lipoprotein lipase activity but not hepatic lipase activity. In Asian populations, this common polymorphism can lead to profound adverse effects on lipoprotein profiles, with homozygosity accounting for a significant number of cases of severe hypertriglyceridemia. This specific apoA-V variant has a pronounced effect on TG metabolism, the mechanism of which remains to be elucidated.

Dietary salt restriction increases plasma lipoprotein and inflammatory marker concentrations in hypertensive patients.

BACKGROUND: Dietary salt restriction has been reported to adversely modify the plasma lipoprotein profile in hypertensive and in normotensive subjects. We investigated the effects of the low sodium intake (LSI) on the plasma lipoprotein profile and on inflammation and thrombosis biomarkers during the fasting and postprandial periods. METHODS: Non-obese, non-treated hypertensive adults (n=41) were fed strictly controlled diets. An initial week on a control diet (CD, Na=160 mmol/day) was followed by 3 weeks on LSI (Na=60 mmol/day). At admission and on the last day of each period, the 24-h ambulatory blood pressure was monitored and blood was drawn after an overnight fasting period and after a fat-rich test meal. RESULTS: The dietary adherence was confirmed by 24-h urinary sodium excretion. Fasting triglyceride (TG), chylomicron-cholesterol, hsC-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) concentrations, renin activity, aldosterone, insulin, and homeostasis model assessment insulin resistance (HOMA-IR) values were higher, but non-esterified fatty acids (NEFA) were lower on LSI than on CD. For LSI, areas under the curve (AUC) of TG, chylomicron-cholesterol, apoB and the cholesterol/apoB ratio were increased, whereas AUC-NEFA was lowered. LSI did not modify body weight, hematocrit, fasting plasma cholesterol, glucose, adiponectin, leptin, fibrinogen and factor VII (FVII), and AUC of lipoprotein lipase and of lipoprotein remnants. CONCLUSION: LSI induced alterations in the plasma lipoproteins and in inflammatory markers that are common features of the metabolic syndrome.