Quantification of derivatized phenylalanine and tyrosine in dried blood spots using liquid chromatography with tandem spectrometry for newborn screening of phenylketonuria.

Phenylketonuria (PKU) is an autosomal genetic disorder caused by a deficiency of the phenylalanine hydroxylase (PAH) enzyme. The lack of PAH results in the inability of phenylalanine (PHE) to transform into tyrosine (TYR). Consequently, this leads to the accumulation of PHE in the blood samples of newborns causing metabolic diseases such as irreversible neurological problems. An analysis was required for determining the values of PHE and TYR in blood samples from newborn babies. In this study, therefore, we developed a derivatized method to monitor PHE and TYR in plasma samples using liquid phase chromatography linked with quadrupole mass spectrometry. Accessible formaldehyde isotopes and cyanoborohydride were used to react with PHE and TYR amino groups to generate h2-formaldehyde-modified PHE and TYR (as standards) and d2-formaldehyde-modified PHE and TYR (as internal standards). We used tandem mass spectrometry for multiple reaction monitoring. We demonstrated a derivatized method suitable for the PKU screening of newborns. The recoveries for PHE and TYR were 85% and 90%, respectively. Furthermore, we compared the values of PHE and TYR in different human plasma sample storage methods, including direct plasma and dried blood spots, and the results showed no significant difference.

Application of response surface methodology and quantitative NMR for the optimum extraction, characterization, and quantitation of Antrodia cinnamomea triterpenoids.

Antrodia cinnamomea (AC) is a treasured Asian medicinal mushroom, which has attracted attention due to recent research on its effectiveness in targeting a variety of serious ailments such as cancer and liver diseases. Among different A. cinnamomea constituents, triterpenoids are regarded as the most therapeutically attractive components because of their anti-inflammatory and cytotoxic activities. In the present study, we proposed a mathematical and statistical extraction protocol to evaluate the concentrations of total ergostane and lanostane triterpenoid derivatives from the ethanolic extract of the wild fruiting bodies of A. cinnamomea (EEAC) by utilizing response surface methodology (RSM) and quantitative NMR (qNMR) approaches. The optimum response surface model showed that the variations of the investigated response variables reached more than 90%, suggesting that the developed model is accurate in explaining response variability. Furthermore, the EEAC major characteristic triterpenoids were quantified through the comparison of the HPLC-tandem MS results with those of the qNMR results. The precision of the used techniques was also evaluated. The experimental design of the EEAC optimum extraction procedure obtained by using RSM and qNMR enabled accurate characterization and quantitation of A. cinnamomea triterpenoids.

Fluorescence detection of apolipoprotein E gene polymorphisms based on oligonucleotide ligation and magnetic separation.

Alzheimer's disease is a progressive neurodegenerative condition that causes brain cell death and is the leading cause of dementia. Most patients with Alzheimer's disease are diagnosed with late-onset Alzheimer's disease (LOAD), with apolipoprotein E (APOE) genotypes being highly associated with the frequency of LOAD risk. A fluorescence detection system coupled with oligonucleotide ligation and magnetic separation was developed to identify two single-nucleotide polymorphisms (SNPs) for the APOE gene and recognize APOE alleles for LOAD. The system utilized a fluorescence probe with one base-discriminating nucleoside for SNP (F probe) and a perfectly complementary biotin-modified sequence against the target DNA (P probe). When the F and P probes matched the target DNA sequences, DNA ligation occurred, and ligation products were produced. Streptavidin magnetic beads were subsequently employed to remove the ligation products, and a decrease in fluorescence intensity was observed in the supernatant compared to when there was no target DNA. This system detected two SNPs of APOE alleles, namely rs429358 and rs7412. The results indicated that the R-values ((F0 - F1)/F0) for rs429358 were 0.92 ± 0.002 for the T/T target, 0.47 ± 0.004 for the T/C target and 0.11 ± 0.004 for the C/C target, respectively. The R-values for rs7412 were 0.73 ± 0.009 for the C/C target, 0.42 ± 0.001 for the C/T target and 0.16 ± 0.007 for the T/T target, respectively. F0 and F1 represent the fluorescence intensity of the F probe without and with target DNA, respectively. Based on fluorescence intensity, the fluorescence detection system was able to identify the genotypes of the APOE gene accurately to evaluate the risk of Alzheimer's disease.

A Study of the Relationship between Phthalate Exposure and the Occurrence of Adult Asthma in Taiwan.

Although phthalate esters contribute to airway remodeling by increasing bronchial cells' migration and proliferation, the relationship between human exposure to phthalates and asthma is not understood. We measured phthalate exposure in the human body and evaluated its effect on asthma. Asthma (n = 123) and asthma-free (n = 139) participants were, respectively, recruited from an asthma clinic and the community in Taiwan. The urine levels of six phthalate metabolites were determined by liquid chromatography tandem mass spectrometry. Compared with the controls, male asthma patients had higher means of mono-(2-ethylhexyl) phthalate (MEHP) (116.3 nmol/g), monobutyl phthalate (MBP) (850.3 nmol/g) and monoethyl phthalate (MEP) (965.8 nmol/g), and female patients had greater MBP (2902.4 nmol/g). Each 10-fold increase in the level of these phthalate metabolites was correspondingly associated with a 5.0-, 5.8-, 4.2- and 5.3-fold risk of contracting asthma. Male asthma patients were identified to have a higher proportion of MEHP exposure (32.5%) than the controls (25.3%). In asthma patients, an increase in urine MEHP levels and the total phthalate metabolite concentration were notably linked to increased risks of emergency room visits and being hospitalized. For the occurrence and acute clinical events of adult asthma, phthalate exposures and MEHP retention may contribute to higher risks of contracting this respiratory disorder.

Hydrogels: Properties and Applications in Biomedicine.

Hydrogels are crosslinked polymer chains with three-dimensional (3D) network structures, which can absorb relatively large amounts of fluid. Because of the high water content, soft structure, and porosity of hydrogels, they closely resemble living tissues. Research in recent years shows that hydrogels have been applied in various fields, such as agriculture, biomaterials, the food industry, drug delivery, tissue engineering, and regenerative medicine. Along with the underlying technology improvements of hydrogel development, hydrogels can be expected to be applied in more fields. Although not all hydrogels have good biodegradability and biocompatibility, such as synthetic hydrogels (polyvinyl alcohol, polyacrylamide, polyethylene glycol hydrogels, etc.), their biodegradability and biocompatibility can be adjusted by modification of their functional group or incorporation of natural polymers. Hence, scientists are still interested in the biomedical applications of hydrogels due to their creative adjustability for different uses. In this review, we first introduce the basic information of hydrogels, such as structure, classification, and synthesis. Then, we further describe the recent applications of hydrogels in 3D cell cultures, drug delivery, wound dressing, and tissue engineering.

Stability and Transformation of Metabolic Syndrome in Adolescents: A Prospective Assessment in Relation to the Change of Cardiometabolic Risk Factors.

Underlying pathophysiological mechanisms drive excessive clustering of cardiometabolic risk factors, causing metabolic syndrome (MetS). MetS status may transform as adolescents transition to young adulthood. This study investigated the latent clustering structure and its stability for MetS during adolescence, and assessed the anthropometric and clinical metabolic determinants for MetS transformation. A community-based representative adolescent cohort (n = 1516) was evaluated for MetS using four diagnostic criteria, and was followed for 2.2 years to identify new-onset MetS. The clustering structure underlying cardiometabolic parameters was stable across adolescence; both comprised a fat-blood pressure (BP)-glucose three-factor structure (total variance explained: 68.8% and 69.7% at baseline and follow-up, respectively). Among adolescents with MetS-negative at baseline, 3.2-4.4% had incident MetS after 2.2 years. Among adolescents with MetS-positive at baseline, 52.0-61.9% experienced MetS remission, and 38.1-48.0% experienced MetS persistence. Increased systolic BP (SBP) was associated with a high MetS incidence risk, while decreased levels of SBP and glucose were associated with MetS remission. Compared with adolescents with a normal metabolic status at baseline, those with an initial abdominal obesity and increased triglycerides level had a 15.0- and 5.7-fold greater risk for persistent abnormality, respectively. Abdominal obesity and low high-density lipoprotein cholesterol are two abnormal MetS components that highly persist during adolescence, and are the intervention targets for reducing the future risk of cardiometabolic disorders.

New Cytotoxic Terpenoids from Soft Corals Nephthea chabroli and Paralemnalia thyrsoides.

A novel cytotoxic diterpenoid, chabrolin A (1) (possessing an unprecedented terpenoid skeleton), as well as three new cytotoxic sesquiterpenoids, parathyrsoidins E-G (2-4), were isolated by cytotoxicity-guided fractionation from soft corals Nephtheachabroli and Paralemnalia thyrsoides. The structures of the new compounds were determined by extensive analysis of spectroscopic data.

Euphol from Euphorbia tirucalli Negatively Modulates TGF-ß Responsiveness via TGF-ß Receptor Segregation inside Membrane Rafts.

Transforming growth factor-ß (TGF-ß) responsiveness in cultured cells can be modulated by TGF-ß partitioning between lipid raft/caveolae- and clathrin-mediated endocytosis pathways. Lipid rafts are plasma membrane microdomains with an important role in cell survival signaling, and cholesterol is necessary for the lipid rafts' structure and function. Euphol is a euphane-type triterpene alcohol that is structurally similar to cholesterol and has a wide range of pharmacological properties, including anti-inflammatory and anti-cancer effects. In the present study, euphol suppressed TGF-ß signaling by inducing TGF-ß receptor movement into lipid-raft microdomains and degrading TGF-ß receptors.

Betel chewing and arecoline affects eotaxin-1, asthma and lung function.

BACKGROUND: Betel nut is commonly used in many countries. Despite evidence suggesting an association with asthma, few studies have investigated the connection between betel nut use and asthma; thus, the underlying mechanism for the association with asthma is also unclear. The aim of this study was to investigate the association between betel chewing and asthma as well as the associations of plasma arecoline (a biomarker for exposure) and eotaxin-1 (a potential mediator) with asthma and lung function. METHODS: We recruited 600 hospital-based asthmatic patients and 1200 age- and gender-matched community controls in southern Taiwan. To clarify the mechanism of action for eotaxin-1 in the association between betel chewing and asthma, we also designed an in vitro experiment to study the functional associations between arecoline exposure and eotaxin-1 levels. RESULTS: A significant association was found between asthma and current betel chewing (adjusted odds ratio 2.05, 95% CI = 1.12-3.76), which was independent of potential confounders but was attenuated following adjustment for eotaxin-1. Arecoline and eotaxin-1 levels were positively correlated (Spearman r = 0.303, p = 0.02), while arecoline and arecaidine were negatively correlated with lung function. Functionally, arecoline alone does not induce eotaxin-1 release in vitro from dermal and gingival fibroblasts. However, in the presence of IL-4 and TNF-alpha, arecoline at 100 µg/ml induced more eotaxin-1 release than arecoline at 0 µg/ml (2700±98 pg/ml vs 1850±142 pg/ml, p = 0.01 in dermal fibroblast cells, and 1489±78 pg/ml vs 1044±95 pg/ml, p = 0.03 in gingival fibroblast cells, respectively). CONCLUSION: Betel chewing is associated with asthma in this population, with arecoline induction of eotaxin-1 supported as a plausible causal pathway.

Design, synthesis, biological evaluation and molecular modeling studies of 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes as a new class of potent antitumor agents.

A series of novel enediyne-containing molecules, 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes, were synthesized and displayed significant IC50 values of 10(-7) to 10(-6) M against various cancer cell lines. Of these compounds, 1-(2-pyridinyl)-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diyne (8) demonstrated the greatest growth inhibition activity. Compound 8 also arrested cancer cells in the G2/M phase and induced apoptosis via activation of Caspase-3. In addition to the G2/M block, compound 8 caused microtubule depolymerization at low concentrations and markedly decreased tumor size in xenographic studies.

Chewing areca nut increases the risk of coronary artery disease in Taiwanese men: a case-control study.

BACKGROUND: Areca nut chewing has been reported to be associated with obesity, metabolic syndrome, hypertension, and cardiovascular mortality in previous studies. The aim of this study was to examine whether chewing areca nut increases the risk of coronary artery disease (CAD) in Taiwanese men. METHODS: This study is a hospital-based case-control study. The case patients were male patients diagnosed in Taiwan between 1996 and 2009 as having a positive Treadmill exercise test or a positive finding on the Thallium-201 single-photon emission computed tomography myocardial perfusion imaging. The case patients were further evaluated by coronary angiography to confirm their CAD. Obstructive CAD was defined as a ≥ 50% decrease in the luminal diameter of one major coronary artery. The patients who did not fulfill the above criteria of obstructive CAD were excluded.The potential controls were males who visited the same hospital for health check-ups and had a normal electrocardiogram but no history of ischemic heart disease or CAD during the time period that the case patients were diagnosed. The eligible controls were randomly selected and frequency-matched with the case patients based on age. Multiple logistic regression analyses were used to estimate the odds ratio of areca nut chewing and the risk of obstructive CAD. RESULTS: A total of 293 obstructive CAD patients and 720 healthy controls, all men, were analyzed. Subjects who chewed areca nut had a 3.5-fold increased risk (95% CI = 2.0-6.2) of having obstructive CAD than those without, after adjusting for other significant covariates. The dose-response relationship of chewing areca nut and the risk of obstructive CAD was also noted. After adjusting for other covariates, the 2-way additive interactions for obstructive CAD risk were also significant between areca nut use and cigarette smoking, hypertension and dyslipidemia. CONCLUSIONS: Long-term areca nut chewing was an independent risk factor of obstructive CAD in Taiwanese men. Interactive effects between chewing areca nut and cigarette smoking, hypertension, and dyslipidemia were also observed for CAD risk. Further exploration of their underlying mechanisms is necessary.

1-(2-((Z)-6-(2-(Trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)piperidin-2-one as a new potent apoptosis agent.

Compounds 4a-f, 5a-f and 6-9, showed significant growth inhibition activity against human tumor cell lines. Of these compounds, 1-(2-((Z)-6-(2-(trifluoromethyl)phenyl)hexa-3-en-1,5-diynyl)phenyl)piperidin-2-one (8) displayed the most potent growth inhibition activity. Compound 8 also arrested cancer cells in G2/M phase and induced apoptosis via activation of caspase-3 and -9. According to western-blotting analysis, compound 8 can up-regulate Bax, down-regulate Bcl-2 and XIAP, as well as promote cytochrome c release.

2-(6-aryl-3(Z)-hexen-1,5-diynyl)anilines as a new class of potent antitubulin agents.

Compounds 2a- h and 6 displayed significant GI 50 values of 10(-7)-10(-6) M against various cancer cell lines. Of these compounds, 2-(6-(2-trifluoromethylphenyl))-3(Z)-hexen-1,5-diynyl)aniline (2c) showed the most potent growth inhibition activity. Compound 2c also arrested cancer cells in the G2/M phase and in low concentration reduced a significant percentage of MDA-MB-231/ATCC breast cancer tetraploid cells. In addition to the G2/M block, compound 2c caused microtubule depolymerization and induced apoptosis via activation of the caspase family.

Fluorimetric liquid chromatographic analysis of amantadine in urine and pharmaceutical formulation.

A simple and sensitive liquid chromatographic method is described for the analysis of amantadine and memantine. The method is based on the derivatization of amantadine and memantine extracted from alkalified samples with (2-naphthoxy)acetyl chloride at mild conditions. The resulting derivatives were analyzed by isocratic HPLC with a fluorimetric detector (lambdaex, 227 nm; lambdaem, 348 nm). The linear range for the determination of amantadine or memantine spiked in urine (1.0 ml) was 1.0-10.0 nmol with a detection limit of about 0.2 nmol (S/N = 3; injected sample 20 microl). Only amantadine preparations are available on our local market, and application of the method to the analysis of amantadine in formulation and in the urine of a dosed subject was demonstrated and proved feasible. Quantitation of AT in tablets or capsules is capable in the linear range of 2.0-50.0 microM. Toluene was used as the solvent for extracting amantadine or memantine in samples and the resulting toluene extract was directly subjected to subsequent derivatization without solvent replacement leading to a simpler analytical procedure.

(2-Naphthoxy)acetyl chloride, a simple fluorescent reagent.

In continuing the search for fluorescent reagents for analytical derivatization in chromatography, we found a simple chemical, (2-naphthoxy)acetyl chloride, with potential fluorophore/chromophore characteristics for the highly sensitive detection of analytes with an amino function. The reagent has an auxochrome (a substituted alkoxy moiety) attached to the fluorophoric/chromophoric naphthalene system, resulting in favorable spectrophotometric properties. The reagent can be easily prepared from (2-naphthoxy)acetic acid and has been used in organic synthesis; it is initially introduced as a fluorescent reagent to derivatise amantadine and memantine (amino pharmaceuticals) as model analytes. The resulting naphthoxy derivatives of the drugs can be analyzed at sub-microM levels by HPLC with fluorimetric detection (excitation wavelength 227 nm, emission wavelength 348 nm). Application of the reagent to the fluorimetric derivatization of important biological amines for sensitive detection can be expected.