A specific, accurate, and sensitive measure of total plasma malondialdehyde by HPLC.

Malondialdehyde (MDA) is one of the most commonly reported biomarkers of lipid peroxidation in clinical studies. The reaction of thiobarbituric acid (TBA) with MDA to yield a pink chromogen attributable to an MDA-TBA2 adduct is a common assay approach with products being quantified by ultraviolet-Vis assay as nonspecific TBA-reactive substances (TBARS) or chromatographically as MDA. The specificity of the TBARS assay was compared with both chromatographic assays for total plasma MDA. The levels of total plasma MDA were significantly lower than the plasma TBARS in each of the samples examined, and interestingly, the interindividual variation apparent in the level of plasma MDA was not evident in the plasma TBARS assay. Each of the four online chromatographic detectors yielded a precise, sensitive, and accurate determination of total plasma MDA, and selected-ion monitoring was the most-accurate assay (101.3%, n = 4). The online diode array detectors provided good assay specificity (peak purity index of 999), sensitivity, precision, and accuracy. This research demonstrates the inaccuracy that is inherent in plasma TBARS assays, which claim to quantify MDA, and it is proposed that the TBARS approach may limit the likelihood of detecting true differences in the level of lipid peroxidation in clinical studies.

Cytoprotective effects of phenolic antioxidants and essential fatty acids in human blood monocyte and neuroblastoma cell lines: surrogates for neurological damage in vivo.

BACKGROUND: Oxidative stress is implicated in the development of a range of neurological diseases. There is increasing interest in the neuroprotective efficacy of antioxidants in modulating such processes with at least one polyphenolic being tested as a prophylactic in Alzheimer's disease. Beneficial effects of adjunctive n-3 polyunsaturated fatty acids with combined intakes of vitamin C and E on both the positive and negative symptoms of schizophrenia have been reported. Robust in vitro systems are desirable, enabling a mechanistic investigation of the molecular mechanisms underpinning such effects and identification of further potentially efficacious nutraceuticals. MATERIALS AND METHOD: A comparative study employing a human lymphoblastoid cell line derived from a subject with early onset schizophrenia, a neuroblastoma IMR-32 cell line and the histiocytic lymphoma U937 cell line was undertaken. The cytoprotective effects of two phenols in affording protection to cellular DNA from an oxidative challenge were assessed in untreated and fatty acid treated cell lines. RESULTS AND CONCLUSION: Marked differences in the uptake of fatty acids by the cell types were found and the IMR-32 cell line was most susceptible to the oxidant challenge. Hydroxytyrosol gave significant cytoprotection in all three-cell lines and this possible neuroprotective efficacy warrants further investigation, both in vitro and in vivo.

Evaluation of quality control strategies in Scutellaria herbal medicines.

The statutory regulation of herbal medicines is under review within the United Kingdom (UK) and by 2011 all herbal medicines will require either a Product Licence or a Traditional Herbal Registration. The species Scutellaria baicalensis has been shown to possess anti-inflammatory, anti-viral and anti-tumor properties and is one of the most widely used Chinese herbal extracts in Eastern and Western medicines. The bioactivity of this herbal medicine is due to the radical scavenging activities of the flavone components of which there are more than 60. This research has characterised 5 key flavones in 18 extracts of Scutellaria using a combination of HPLC with DAD and MS detection. Employing an internal standard approach, the validated HPLC method afforded good sensitivity and excellent assay precision. Assays for the ferric reducing antioxidant power (FRAP) and total phenol determinations enabled determination of the antioxidant coefficient (PAC) of each Scutellaria extract. The potential usefulness of employing multivariate statistical analysis using a combination of the key parameters collected namely, FRAP activity, total phenol content, levels of 5 flavone biomarkers and the PAC as a means of quality evaluation of the Scutellaria herbal extracts was investigated. The PAC value was predicted by soft independent modelling of class analogy (SIMCA) as being the most discriminatory parameter and applying this ranking the herbal extracts were grouped into 3 clusters. The second most influential parameter in determining the clustering of the samples was the level of baicalin in each extract. It is proposed that the PAC value alone or in combination with a chromatographic fingerprint of key biomarkers [e.g. baicalin or (baicalin+baicalein)] may be useful indicators to adopt for the quality control of S. baicalensis.

Differentiation of opium and poppy straw using capillary electrophoresis and pattern recognition techniques.

Opium samples from four different locations and poppy straw from different plant varieties have been assayed using micellar capillary electrophoresis incorporating a sweeping technique. Individual alkaloids (morphine, codeine, papaverine, noscapine, thebaine, oripavine, reticuline and narceine) were quantitatively determined in the different samples by a validated capillary electrophoresis method. Unsupervised pattern recognition of the opium samples and the poppy straw samples using hierarchical cluster analysis (HCA) and principal component analysis (PCA), showed distinct clusters. Supervised pattern recognition using soft independent modelling of class analogy (SIMCA) was performed to show individual groupings and allow unknown samples to be classified according to the models built using the CZE assay results.

Oxidative stress in humans: validation of biomarkers of DNA damage.

Two studies have been performed to clarify the relationship between different markers of oxidative DNA damage commonly employed in molecular epidemiological studies. In the first, 8-Oxo-7,8-dihydroguanine (8-oxoGua) was induced in DNA of HeLa cells by treatment with different concentrations of photosensitizer Ro 19-8022 together with visible light. 8-OxoGua was estimated by the comet assay (alkaline single cell gel electrophoresis) with formamidopyrimidine DNA glycosylase and by HPLC with electrochemical detection. The dose-response curves indicate that the comet assay and HPLC are equally efficient at detecting induced damage. Background levels of 8-oxoGua in HeLa cells were 0.92 +/- 0.22 per 10(6) guanines by the comet assay and 2.09 +/- 0.13 per 10(6) guanines by HPLC. The second study was a small human trial, in which lymphocytes were collected for analysis of background levels of 8-oxoGua, as well as overnight and 24 h urine samples for measurement of excreted 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) by ELISA. The mean level of 8-oxoGua in lymphocytes was determined as 1.33 +/- 0.21 per 10(6) guanines by the comet assay and 3.72 +/- 1.06 per 10(6) guanines by HPLC. A strong correlation was seen between overnight and 24 h urinary 8-oxodGuo (r = 0.93, P < 0.01). Overnight urinary 8-oxodGuo concentrations correlated with 8-oxoGua in lymphocytes measured by HPLC (r = 0.85, P < 0.05) or by the comet assay (r = 0.86, P < 0.05), although individual values from HPLC and the comet assay did not correlate with each other. It is reasonable to assess oxidative stress by any of these methods.