Biomedical subjects

Y T Szeto

Publications and source records attributed to Y T Szeto.

7 recordsLinked to original sources

PubMed2005

A buccal cell model comet assay: development and evaluation for human biomonitoring and nutritional studies.

The comet assay is a widely used biomonitoring tool for DNA damage. The most commonly used cells in human studies are lymphocytes. There is an urgent need to find an alternative target human cell that can be collected from normal subjects with minimal invasion. There are some reports of buccal cells, collected easily from the inside of the mouth, being used in studies of DNA damage and repair, and these were of interest. However, our preliminary studies following the published protocol showed that buccal cells sustained massive damage and disintegrated at the high pH [O. Ostling, K.J. Johanson. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123 (1984) 291-298] used, but that at lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, the aims of this study were to develop a protocol than enabled buccal cell lysis and DNA damage testing in the comet assay, and to use the model to evaluate the potential use of the buccal cell model in human biomonitoring and nutritional study. Specifically, we aimed to investigate intra- and inter-individual differences in buccal cell DNA damage (as strand breaks), the effect of in vitro exposure to both a standard oxidant challenge and antioxidant treatment, as well as in situ exposure to an antioxidant-rich beverage and supplementation-related effects using a carotenoid-rich food. Successful lysis was achieved using 0.25% trypsin for 30 min followed by proteinase K (1mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis (in 0.01 M NaOH, 1mM EDTA, pH 9.1, 18 min at 12 V), a satisfactory comet image was obtained, though inter-individual variation was quite wide. Pre-lysis exposure of cells to a standard oxidant challenge (induced by H2O2) increased DNA strand breaks in a dose related manner, and incubation of cells in Trolox (a water soluble Vitamin E analogue) conferred significant protection (P<0.05) against subsequent oxidant challenge. Exposure of buccal cell in situ (i.e. in the mouth) to antioxidant-rich green tea led to an acute decrease in basal DNA strand breaks. In a controlled human intervention trial, buccal cells from 14 subjects after 28 days' supplementation with a carotenoid-rich berry (Fructus barbarum L.) showed a small but statistically significant (P<0.05) decrease in DNA strand breaks. These data indicate that this buccal cell comet assay is a feasible and potentially useful alternative tool to the usual lymphocyte model in human biomonitoring and nutritional work.

Antioxidants↗

PubMed2004

Effects of a long-term vegetarian diet on biomarkers of antioxidant status and cardiovascular disease risk.

OBJECTIVE: We compared plasma biomarkers of antioxidant status, oxidative stress, inflammation, and risk for coronary heart disease in long-term vegetarians and age- and sex-matched omnivores. METHODS: Thirty vegetarians (mean age +/- standard deviation: 44.2 +/- 9.0 y) were recruited. The subjects had been vegetarian for 5 to 55 y (21.8 +/- 12.2 y). The control group comprised 30 adults selected by age-stratified sampling from a community health project (mean age: 44.0 +/- 9.2 y). Fasting plasma total antioxidant status (ferric-reducing antioxidant power), ascorbic acid (AA), alpha-tocopherol (total and lipid standardized), malondialdehyde, total cholesterol, triacylglycerol, uric acid (UA), and high-sensitivity C-reactive protein (hsCRP) were measured. RESULTS: Plasma AA was significantly higher in the vegetarians than in the omnivores (90.5 +/- 21.0 and 61.8 +/- 17.0 microM; P < 0.001). The vegetarians had lower concentrations of triacylglycerol, UA, and hsCRP. Plasma total and lipid-standardized alpha-tocopherol concentrations were also lower in the vegetarians: 22.0 +/- 5.9 and 27.0 +/- 7.9 microM versus 3.76 +/- 0.57 and 4.23 +/- 0.58 microM per millimoles per liter of total cholesterol plus triacylglycerol, respectively. There was a significant inverse correlation between AA and UA (r = -0.343, P < 0.01; n = 60) and between AA and hsCRP (r = -0.306, P < 0.05; n = 55). Plasma ferric-reducing antioxidant power and malondialdehyde did not differ significantly between groups; however, the contribution of AA to the total antioxidant capacity of plasma was approximately 50% greater in the vegetarians. CONCLUSIONS: A long-term vegetarian diet is associated with markedly higher fasting plasma AA concentrations and lower concentrations of TAG, UA, and hsCRP. Long-term vegetarians have a better antioxidant status and coronary heart disease risk profile than do apparently healthy omnivores. Plasma AA may act a useful marker of overall health status.

Adult↗

PubMed2002

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure.

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.

Antioxidants↗

PubMed2002

Glycohaemoglobin measurement: methodological differences in relation to interference by urea.

The aim of this study was to investigate the agreement between a cation-exchange HPLC method and a boronate affinity method of measuring glycohaemoglobin (HbA1c), with particular reference to the effect of elevated urea concentration. HbA1c was measured by both methods in samples from 75 subjects who were classified as diabetic with normal (n=36) or abnormal (n=12) renal function, and non-diabetic with normal (n=8) or abnormal (n=19) renal function. Urea was found to cause a clinically significant interference in the HPLC method at a level > or =17.0 mmol/l. Each increase of 1 mmol/l urea in serum was associated with an absolute increase of 0.04% in the HbA1c value as measured by the HPLC method. The boronate affinity method for HbA1c did not appear to be affected by elevated urea concentration. There was significant correlation (r=0.97, p<0.001) between HbA1c results obtained by the two methods, however, results obtained by the boronate affinity method were generally lower. The discrepancy between results obtained by the two methods was particularly marked in uraemic samples from diabetic subjects, as the HPLC/boronate affinity difference increased as the HbA1c increased and also as the urea concentration increased. Results indicate that blood from diabetic patients with renal failure may give erroneously high HbA1c values by HPLC. Results also highlight the importance of choosing appropriate clinical samples and statistical techniques when evaluating or comparing test methods.

Chromatography, Affinity↗

PubMed2001

Plasma ascorbic acid: measurement, stability and clinical utility revisited.

AIMS: To compare plasma ascorbic acid results by the colorimetric FRASC (Ferric Reducing/Antioxidant and Ascorbic Acid) assay and a reference HPLC method; to re-examine plasma ascorbic acid stability, and anticoagulant effect. DESIGN AND METHODS: For method comparison, 31 plasma samples were tested by both methods. For stability, matching EDTA, heparin, citrate and fluoride/oxalate plasma, stored under different conditions of time and temperature, was measured. RESULTS: FRASC is an acceptable alternative to HPLC for plasma ascorbic acid: precision, limit of detection and recovery were similar, and results by the two methods were indistinguishable: mean (95% CI) difference:1.8 (-1.1-4.6; n = 31) micromol/L. Ascorbic acid was most stable in heparinized plasma. Marked loss (p < 0.05) in EDTA plasma occurred within 30 min of blood collection. CONCLUSIONS: FRASC offers a speedy and reliable alternative to HPLC for plasma ascorbic acid. Heparin is proposed as the anticoagulant of choice; loss of ascorbic acid is rapid in EDTA plasma ex vivo.

Adult↗

PubMed1999

Total antioxidant capacity of teas by the ferric reducing/antioxidant power assay.

This study aimed to compare in vitro antioxidant power of different types of tea (Camellia sinensis). The ferric reducing/antioxidant power (FRAP) assay was used to measure the total antioxidant power of freshly prepared infusions of 25 types of teas. Results showed that different teas had widely different in vitro antioxidant power and that the antioxidant capacity was strongly correlated (r = 0. 956) with the total phenolics content of the tea. Expressed as micromol of antioxidant power/g of dried tea leaves, values ranged as 132-654 micromol/g for black ("fermented") teas, 233-532 micromol/g for Oolong ("semifermented") teas, and 272-1144 micromol/g for green ("nonfermented") teas. One cup of tea of usual strength (1-2%), therefore, can provide the same potential for improving antioxidant status as around 150 mg of pure ascorbic acid (vitamin C).

Antioxidants↗

PubMed1999

Consumption of green tea causes rapid increase in plasma antioxidant power in humans.

Green tea contains polyphenolic antioxidants that have shown anticarcinogenic properties in animal and in vitro experimental studies. Current data regarding absorption and bioavailability of tea antioxidants in humans, however, are conflicting. In this study, plasma and urine antioxidant power after ingestion of green tea was measured using the ferric reducing/antioxidant power (FRAP) assay (US patent pending) to assess absorption, systemic distribution, and renal excretion of green tea antioxidants in healthy adults. Results showed that absorption of green tea antioxidants was rapid, with peak increase in plasma FRAP of around 4% at 40 minutes after ingestion: mean increase was 44 +/- 9 (SE) mumol/l. Excretion of polyphenolic antioxidants was also fast, peaking at 60-90 minutes, with significant correlation between urinary FRAP values and urinary total phenolic concentrations (r = 0.845, p < 0.001). In control studies, no increase in plasma or urine FRAP values was seen after intake of water. Although the amount of antioxidants absorbed was relatively small and the increase in plasma antioxidant power was of short duration, results demonstrate that some potentially anticarcinogenic polyphenolic antioxidants in green tea enter the systemic circulation soon after ingestion and cause a significant increase in plasma antioxidant status. This increase may, in turn, lower oxidative damage to DNA and so decrease risk of cancer.

Adult↗