Elevated plasma level of homocysteine is being increasingly associated with many diseases. There is a significant interest in the development of methods to determine the total homocysteine in biologically relevant tissues. Over the years, researchers use various methods to determine the exact concentrations of homocysteine in these tissues. However, the precise method used in many studies earlier was questionable. We have reviewed various methodologies for the measurement of homocysteine. We list the commonly used methodologies currently in use to determine homocysteine levels. Through extensive literature search, we have come up with the most popular as well as the newest measurement modalities and listed them with a brief discussion of each of the methodology. In conclusion, we have presented the historical perspective of homocysteine measurement in biological fluids in this manuscript. Thus, the precise understanding of its concentration in biological fluids coupled with its importance in health and disease should justify a newer but reliable technique in the area of ongoing research in homocysteine.
Optical methods using phosphorescence quenching by oxygen are suitable for the measurement of oxygen concentration within cells. In cells, however, the dyes such as Pt-porphyrins interact with biological components so that their optical properties are changed. Therefore, the absolute oxygen concentration determination in cells is difficult. To suppress this interaction, we focussed on porphyrin-cored dendrimers (dendrimer-porphyrins) and synthesized 2nd–4th generation dendrimer-porphyrins with various surface functional groups (G2–G4, ARG, αGLU and γGLU). These dendrimer-porphyrins showed oxygen sensing property and the change of their spectroscopic properties by biomolecules was supressed. Additionally, the dendrimer-porphyrins were accumulated in cells even in the presence of serum, so oxygen concentration imaging without the effect of serum starvation was also achieved.
The reaction properties of the thiol compounds, cysteine (Cys), N-acetyl-l-cysteine (NAC), the reduced form glutathione (GSH), and homocysteine (HCS) were compared. The main purpose of this study was to find a thiol-based anti-oxidant suitable for biological experiments and to provide clear reasoning for its selection. The availability of thiol compounds to generate superoxide by reducing molecular oxygen (O2) at a hyperthermal temperature was discussed. An oxidative atmosphere, i.e., superoxide generation by the hypoxanthine-xanthine oxidase reaction, hydroxyl radical generation by X-ray irradiation, or direct one-electron oxidation by ferricyanide, was prepared in a reaction mixture containing 0.1 mM TEMPOL and 1 mM test compound, and the EPR signal decay of TEMPOL was observed. A reaction mixture containing 0.1 mM TEMPOL and 1 mM thiol compound was incubated at 44°C, and the EPR signal decay of TEMPOL was observed. Thiols could function as H-donors to the oxoammonium cation and produce the hydroxylamine form of TEMPOL in an oxidative atmosphere. Thiols could also irreversibly react with the oxoammonium cation. GSH and Cys could reduce O2 to form superoxide/hydroperoxyl radical at hyperthermal temperatures, but HCS and NAC could not reduce O2. GSH and Cys may cause reductive stress, whereas NAC is a simple tractable antioxidant.
Nitidine chloride (NC), a natural, bioactive, phytochemical alkaloid derived from the roots of Zanthoxylum nitidum, has been reported to exhibit anti-tumor activity against various types of cancer. However, the potential therapeutic role of NC in human cervical cancer has not yet been studied. We are the first to report that NC acts as a potential apoptosis-inducing agent for human cervical cancer in vitro. NC treatment of human cervical cancer cell lines induced caspase-mediated apoptosis, thereby reducing cell viability. Phospho-kinase proteome profiling using a human phospho-kinase array revealed that NC treatment phosphorylated Checkpoint kinase 2 (Chk2) at Thr68, which activates Chk2 in both cell lines. We also found that NC significantly affected the p53/Bim signaling axis, which was accompanied by mitochondrial membrane depolarization and cytochrome c release from the mitochondria into the cytosol. In addition, NC profoundly increased phosphorylation of the histone variant H2AX at Ser139, a typical marker of DNA damage. Taken together, these results provide in vitro evidence that NC can increase Chk2 activation, thereby acting as an attractive cell death inducer for treatment of human cervical cancer.
It has been reported that activation of NF-E2 p45-related factor-2 (NRF2), a transcription factor, induces a variety of antioxidant enzymes, and plays an important role in preventing carcinogenesis. AHCC is a standardized extract of cultured Lentinula edodes mycelia and it has been demonstrated to improve cancer. However, the effects of AHCC on NRF2 have not been examined, and the effects on intestinal adenoma development are not yet fully understood. We first investigated the effects of AHCC (1–5 mg/ml) on NRF2 activity in human colon cancer cell lines by a luciferase reporter gene assay, and found NRF2 transcriptional activities were increased ~12.6-fold. In addition, AHCC dose-dependently increased HO-1 and NQO-1 mRNA levels, and decreased interleukine-6 mRNA levels. Next, we administered 1,000 ppm AHCC for 8 weeks in the diet of Apc mutant Min mice, and found that AHCC significantly reduced the total number of intestinal polyps to 57.7% and to 67.6% of the control value in male and female Min mice, respectively, with suppression of interleukine-6 in the polyp part. These data suggest that AHCC possesses an ability to suppress cellular oxidative stress through activation of NRF2, thereby lowering intestinal polyp development in Min mice.
AKR1A, an aldo-keto reductase, is involved in the synthesis of ascorbic acid as well as the reduction of a variety of aldehyde compounds. AKR1A−/− mice produce considerably less ascorbic acid (about 10%) compared to AKR1A+/+ mice and require ascorbic acid supplementation in order to breed. To elucidate the roles played by AKR1A in spatial memory, AKR1A−/− male mice were weaned at 4 weeks of age and groups that received ascorbic acid supplementation and no supplementation were subjected to a Morris water maze test. Juvenile AKR1A−/− mice that received no supplementation showed impaired spatial memory formation, even though about 70% of the ascorbic acid remained in the brains of the AKR1A−/− mice at day 7 after weaning. To the contrary, the young adult AKR1A−/− mice at 13–15 weeks of age maintained only 15% of ascorbic acid but showed no significant difference in the spatial memory compared with the AKR1A+/+ mice or ascorbic acid-supplemented AKR1A−/− mice. It is conceivable that juvenile mice require more ascorbic acid for the appropriate level of formation of spatial memory and that maturation of the neural system renders the memory forming process less sensitive to an ascorbic acid insufficiency.
Histatin, a salivary protein, affects oral homeostasis through preservation of tooth integrity and protection against caries and fungal infections. However, the effects of histatin in the generation of oxidative stress induced by reactive oxygen species and in the oral cavity remain unclear. In this study, the effects of histatin on direct reactive oxygen species scavenging activity were examined using electron spin resonance. We demonstrated, for the first time, that histatin exhibits antioxidant activity against hydroxyl radicals generated by Fenton’s reaction by metal chelation or binding. The direct antioxidant effects of histatin, along with its antimicrobial activity, may be important in the oral protection of salivary proteins.
Both nutritive and non-nutritive sweeteners may influence energy and glucose metabolism differently. The hypothesis that sucrose, fructose, aspartame, and sucralose intake differently modulate energy and glucose metabolism was tested in an estrogen-deficient animal model. At 30 min after giving aspartame and sucralose (10 mg/kg body weight), an oral glucose tolerance test (OGTT) was conducted with glucose, sucrose, and fructose in ovariectomized (OVX) rats. After OGTT, they were continuously fed high fat diets including either 10% corn starch (Control), 10% sucrose (Sucrose), 10% fructose (Fructose), 0.05% aspartame + 9.95% starch (Aspartame) or 0.05% sucralose + 9.95% starch (Sucralose) for 8 week. During 30 min after acute administration of aspartame and sucralose, serum glucose concentrations increased despite slightly increased serum insulin levels before glucose infusion. However, glucose tolerance was not significantly different among the groups. In chronic study, serum glucose concentrations were lowest and insulin highest at the overnight-fasted state in Aspartame and Sucralose. Postprandial serum glucagon-like peptide-1 (GLP-1) and insulin levels were higher in Aspartame and Sucralose than Control. Hepatic insulin signaling (pAkt → pGSK-3β) and phosphoenolpyruvate carboxykinase (PEPCK) expression were lower in Sucralose and Aspartame than the Fructose. Serum acetate levels produced by gut microbiota were higher were lower in the fructose group than Aspartame and Sucralose groups. In conclusion, aspartame and sucralose with a meal might be preferable sweeteners to fructose and sucrose in estrogen deficient rats, and possibly post-menopausal women; however, this needs to be confirmed in human studies.
Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice has been reported. Recent research has revealed that several intestinal bacteria metabolize quercetin. We hypothesize that the difference in composition of intestinal microbiota affects quercetin absorption from the intestine. Germ-free BALB/cA female mice (18 weeks old) were randomly divided into four groups and orally administered with fecal suspension from four human individuals (HF1, HF2, HF3, HF4) to produce the human microbiota-associated mice. All mice were fed the 0.05% quercetin-containing pelleted diet for four weeks. Significant differences were observed in plasma total cholesterol and cecal microbiota among the four groups. Plasma quercetin concentration was significantly higher in the HF3 group than in the HF1 group. The plasma isorhamnetin/quercetin ratio showed significant negative correlation with visceral fat levels (r = −0.544, p = 0.013). Positive correlation was observed between the Log10 Enterobacteriaceae count and the plasma quercetin metabolites. Principal component analysis showed that all groups were distributed in different regions by using the correlation diagram with the second and third principal component. This study indicates that intestinal microbiota of human microbiota-associated mice inoculated with different fecal suspensions react to dietary quercetin in different ways.
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a major hereditary small vessel disease caused by mutations in NOTCH3. The variations in progression and severity among patients suggest that the CADASIL phenotype is modified by some genetic and environmental factors. Recent studies have shown the potential roles of gut microbiota in human diseases. We hypothesized that gut microbiota modifies the disease phenotype. We performed gut microbial meta 16S rRNA analysis of fecal samples from 15 CADASIL patients and 16 controls. The microbial α- and β-diversities and taxonomy were compared between CADASIL patients and controls and between CADASIL patients with and without an ischemic stroke history. No significant difference in α- or β-diversity was observed in either case-control or subgroup comparisons. In the taxonomic microbial analysis, there was a significant increase in abundance of 6 genera and significant decrease in 2 genera in CADASIL patients compared with controls. There was a significant decrease in abundance of 2 genera in CADASIL patients with compared with those without stroke. This is the first study on CADASIL focusing on gut microbiota. Our findings suggest that gut microbiota modifies the onset and progression of CADASIL.
Calcitriol [1,25(OH)2D3] is usually investigated in studies on the preventive effect of activated vitamin D against interstitial pneumonia. Although cholecalciferol (vitamin D3) can be easily obtained in the diet and has a longer half-life than calcitriol, there have been few investigations of its effect on interstitial pneumonia. We used human pulmonary fibroblast cell lines (HPFCs) and a mouse model of bleomycin-induced pulmonary fibrosis to evaluate whether vitamin D3 was activated in the lungs and had a preventive effect against interstitial pneumonia. Expression of the vitamin D receptor gene and genes for enzymes metabolizing vitamin D was evaluated in two HPFCs, and the suppressive effect of vitamin D3 on induction of inflammatory cytokines was also assessed. Gene expression of the vitamin D receptor and vitamin D-metabolizing enzymes was observed in both human pulmonary fibroblast cell lines. Vitamin D3 suppressed bleomycin-induced expression of inflammatory cytokines and fibrosis markers by the HPFCs. In mice, symptoms of bleomycin-induced pulmonary fibrosis were improved and expression of fibrosis markers/fibrosis inducers was decreased by a high vitamin D3 diet. Vitamin D3 is activated locally in lung tissues, suggesting that high dietary intake of vitamin D3 may have a preventive effect against interstitial pneumonia.
The aim of the present study was to examine whether dietary salt restriction guidance is beneficial for dietary salt restriction and lowering of home blood pressure in patients with diabetes with excessive salt intake. We performed an intervention trial of 37 people with type 2 diabetes and excessive salt intake. National registered dietitians provided dietary salt restriction guidance to each patient at the start of the study. All participants were instructed to perform triplicate morning and evening home blood pressure measurements using home blood pressure telemonitoring system. Daily salt intake at 2 months and 6 months was significantly lower than that at baseline; the difference was 0.8 [95% confidence interval (CI): 0.2–1.4, p = 0.009] g and 0.7 (95% CI: 0.1–1.3, p = 0.009) g, respectively. Morning systolic blood pressure at 2 months and 6 months was significantly lower than that at baseline; the difference was 2.7 (95% CI: 0.2–5.1, p = 0.034) mmHg and 5.8 (95% CI: 0.5–11.1, p = 0.034) mmHg, respectively. This intervention study revealed, for the first time, that dietary salt restriction guidance provided by a national registered dietitian is beneficial for reducing daily salt intake and home blood pressure in people with diabetes with excessive salt intake.