Plasma allantoin measurement by isocratic liquid chromatography with tandem mass spectrometry: method evaluation and application in oxidative stress biomonitoring.

BACKGROUND: Allantoin in human plasma is a specific biomarker of oxidative stress. We describe a sensitive method to measure plasma allantoin using isocratic liquid chromatography and mass spectrometry (LC-MS/MS). METHODS: Direct injection of deproteinized plasma into the LC-MS/MS system was performed. The method was technically evaluated. Results on 200 healthy and 35 Type 2 diabetic Chinese subjects were compared. RESULTS: Dose-response of allantoin was linear to at least 21 pmol (20 µmol/l in plasma); LOD was 0.16 pmol; recovery 99.2-100.2% at 1-5 µmol/l; accuracy, 98.5-100.8%; within-day and between-day CVs (n=6), <4.0% (at 5.00-40 µmol/l) and <2.0% (at 1-5 µmol/l), respectively. Plasma allantoin in diabetic patients was ~8-fold higher than in healthy subjects; mean (SD): 8.82 (7.26) and 1.08 (0.86) µmol/l, respectively (p<0.0001). Allantoin was slightly higher in healthy men than in age- and BMI-matched women: 1.21 (0.99) µmol/l, n=88 compared to 0.97 (0.74) µmol/l, n=112; p<0.001. No association with age was seen. Gender difference was also seen in the diabetes patients: men, n=14, 11.57 (8.57) µmol/l; women, n=21, 6.99 (5.75) µmol/l, p<0.05. CONCLUSIONS: Based on 95th percentiles of the healthy subjects, plasma allantoin of >2.2 µmol/l in women and >3.1 µmol/l in men indicates increased oxidative stress. Allantoin in diabetes subjects is clearly and markedly increased. The method will facilitate future studies of oxidative stress in human biomonitoring studies.

Comparison of catechin profiles in human plasma and urine after single dosing and regular intake of green tea (Camellia sinensis).

Green tea (Camellia sinensis) catechin profiles in plasma and urine following single dosing and regular ingestion of green tea are not clear. We performed a placebo-controlled intervention study with sixteen healthy volunteers to determine changes in total and free catechins after a single dose and following 1 week of twice-daily green tea. Blood and urine samples were collected before (fasting) and after (60 and 120 min for blood; 90 and 180 min for urine) drinking 200 ml of 1.5% (w/v) green tea or water (n 8 each), and fasting samples were again collected after 7 d of 150 ml of 1% (w/v) supplemental green tea or water twice daily. After a 4-week washout, subjects were crossed onto the other treatment and procedures repeated. Plasma results at 1 h post-ingestion showed elevated (P < 0.05) mean epigallocatechin gallate (EGCG; 310 (SD 117) nmol/l; all in free form), epigallocatechin (EGC; 192 (SD 67) nmol/l; 30% free) and epicatechin gallate (ECG; 134 (SD 51) nmol/l; 75% free). Fasting plasma after 7 d of regular intake showed increased (P < 0.05) EGCG (80 v. 15 nmol/l at baseline) and ECG (120 v. 40 nmol/l), with > or =90% of both in their conjugated forms. Total EGC was < 10 nmol/l. Post-ingestion conjugation and renal loss of EGC and epicatechin were rapid and high, but were negligible for EGCG and ECG. In the green tea consumed, the content was EGCG > EGC > ECG, and the acute plasma response mirrored this. However, after chronic consumption there was almost no EGC found in fasting plasma, some EGCG was present, but a rather high level of ECG was maintained.

Colour additives in snack foods consumed by primary school children in Hong Kong.

The objective of the present study was to assess synthetic colours in common snack foods consumed by children and the accuracy of labelling. Dietary exposure to synthetic colours was estimated using food frequency questionnaire data obtained from primary school children in Hong Kong. The concentration of synthetic colours in food items consumed was determined by HPLC with photodiode array detection. Dietary exposure to synthetic colours for an average primary school student was considerably lower than the acceptable daily intake for their age. Estimates fell below the maximum acceptable daily intake established by the Food and Agriculture Organization/World Health Organization (FAO/WHO) and European Food Safety Authority (ESFA). However, data from HPLC analyses showed that several synthetic colours, which were labelled as being present in the food, were not detected and vice versa.

Urine 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a specific marker of oxidative stress, using direct, isocratic LC-MS/MS: Method evaluation and application in study of biological variation in healthy adults.

BACKGROUND: Urine 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a specific biomarker of oxidative stress. We evaluated a modified LC-MS/MS assay for urine 8-oxodG and determined biological variation in healthy adults. METHOD: Untreated urine was injected into an isocratic LC-MS/MS system (positive-ion MRM mode). Urine 8-oxodG in 51 healthy volunteers was measured; within- and between-day variations in 23 healthy volunteers were investigated. RESULTS: Dose-response was linear to 452 nmol/l; limit of detection=2.3 nmol/l; within-run and between-run CVs were <3.0% and <4.7%, respectively; recovery=97%-101%; accuracy=97.7-103.5%. Urine 8-oxodG (median, mean [SD]): 1.70, 1.70[0.60]nmol/mmol creatinine (n=51). Men had higher (p=0.027) concentrations than women matched for age and body mass index: mean [SD]: 1.90[1.60]; n=26 vs. 1.50[0.55]; n=25. Within- and between-day variations were wide but random. No significant differences were seen overall across time-points within 1 day or at the same time-point across 5 consecutive days. CONCLUSIONS: The method has advantages of speed and relative simplicity as it does not require sample pre-treatment for 8-oxodG extraction, the use of internal standard or gradient LC elution and has high linearity, specificity, precision and recovery. Biological variation in urine 8-oxodG is wide, but no within- or between-day differences at the group concentration were seen in healthy adults.

Fasting plasma zeaxanthin response to Fructus barbarum L. (wolfberry; Kei Tze) in a food-based human supplementation trial.

Age-related macular degeneration (AMD) is a common disorder that causes irreversible loss of central vision. Increased intake of foods containing zeaxanthin may be effective in preventing AMD because the macula accumulates zeaxanthin and lutein, oxygenated carotenoids with antioxidant and blue light-absorbing properties. Lycium barbarum L. is a small red berry known as Fructus lycii and wolfberry in the West, and Kei Tze and Gou Qi Zi in Asia. Wolfberry is rich in zeaxanthin dipalmitate, and is valued in Chinese culture for being good for vision. The aim of this study, which was a single-blinded, placebo-controlled, human intervention trial of parallel design, was to provide data on how fasting plasma zeaxanthin concentration changes as a result of dietary supplementation with whole wolfberries. Fasting blood was collected from healthy, consenting subjects; fourteen subjects took 15 g/d wolfberry (estimated to contain almost 3 mg zeaxanthin) for 28 d. Repeat fasting blood was collected on day 29. Age- and sex-matched controls (n 13) took no wolfberry. Responses in the two groups were compared using the Mann-Whitney test. After supplementation, plasma zeaxanthin increased 2.5-fold: mean values on day 1 and 29 were 0.038 (sem 0.003) and 0.096 (sem 0.009) micromol/l (P<0.01), respectively, for the supplementation group; and 0.038 (sem 0.003) and 0.043 (sem 0.003) micromol/l (P>0.05), respectively, for the control group. This human supplementation trial shows that zeaxanthin in whole wolfberries is bioavailable and that intake of a modest daily amount markedly increases fasting plasma zeaxanthin levels. These new data will support further study of dietary strategies to maintain macular pigment density.

Reflex tear ascorbate in Hong Kong Chinese subjects: method comparison and biological variation.

BACKGROUND: Tear ascorbate is important for corneal health. A rapid and simple method for measurement of ascorbate in tears is needed, and adequate knowledge of physiological variation of tear ascorbate is important to facilitate comparative studies of the effect of, for example, contact lens wear and environmental conditions and stresses. However, there are currently no data on physiological variation of tear ascorbate. This study validated a simple and speedy method for tear ascorbate and investigated between-eye and between-day variation in tear ascorbate in healthy young adults. METHODS: Yawn-induced reflex tears were collected from 32 healthy Hong Kong Chinese subjects and measured by both high-performance liquid chromatography (HPLC) and by an enzyme-linked colorimetric method known as FRASC (total ferric reducing (antioxidant) activity and ascorbate concentration measurement). For between-eye variation, yawn reflex tears were collected from each eye of the same 32 healthy subjects, and ascorbate was measured using HPLC; in a separate experiment for between-day variation, tears were collected on two separate days from 14 subjects, and ascorbate was measured by FRASC. RESULTS: Both HPLC and FRASC showed high precision, and results obtained using FRASC were not statistically different from those using HPLC; mean +/- SD were, respectively, 18.5 +/- 4.4 microM and 18.5 +/- 4.8 microM for HPLC and FRASC methods (p = 0.943). No significant between-eye difference in tear ascorbate was found (p = 0.386), and no significant between-day variation was found overall: mean +/- SD ascorbate was 20.0 +/- 6.2 microM on day 1 and 19.3 +/- 6.8 microM on day 2 (p = 0.772). However, between-day variation was large in seven of 14 subjects. CONCLUSION: FRASC is an acceptable alternative to HPLC for measurement of tear ascorbate. Tears for ascorbate investigation can be collected from either eye or, if necessary, from both eyes and pooled. However, tear ascorbate may vary widely from day to day in the same individual. The reasons for this variation require further study but may relate to differences in ascorbate supply or demand within the precorneal tear layer.