Effect of fluid mechanical stresses and plasma constituents on aggregation of LDL

LDL aggregates when exposed to even moderate fluid mechanical stresses in the laboratory, yet its half-life in the circulation is 2-3 days, implying that little aggregation occurs. LDL may be protected from aggregation in vivo by components of plasma, or by a qualitative difference in flows. Previous studies have shown that HDL and albumin inhibit the aggregation induced by vortexing. Using a more reproducible method of inducing aggregation and assessing aggregation both spectrophotometrically and by sedimentation techniques, we showed that at physiological concentrations, albumin is the more effective inhibitor, and that aggregation is substantially but not completely inhibited in plasma. Heat denatured and fatty-acid-stripped albumin were more effective inhibitors than normal albumin, supporting the idea that hydrophobic interactions are involved. Aggregation of LDL in a model reproducing several aspects of flow in the circulation was 200-fold slower, but was still inhibited by HDL and albumin, suggesting similar mechanisms are involved. Within the sensitivity of our technique, LDL aggregation did not occur in plasma exposed to these flows.jlr Thus, as a result of the characteristics of blood flow and the inhibitory effects of plasma components, particularly albumin, LDL aggregation is unlikely to occur within the circulation.

The aluminium content of breast tissue taken from women with breast cancer

The aetiology of breast cancer is multifactorial. While there are known genetic predispositions to the disease it is probable that environmental factors are also involved. Recent research has demonstrated a regionally specific distribution of aluminium in breast tissue mastectomies while other work has suggested mechanisms whereby breast tissue aluminium might contribute towards the aetiology of breast cancer. We have looked to develop microwave digestion combined with a new form of graphite furnace atomic absorption spectrometry as a precise, accurate and reproducible method for the measurement of aluminium in breast tissue biopsies. We have used this method to test the thesis that there is a regional distribution of aluminium across the breast in women with breast cancer. Microwave digestion of whole breast tissue samples resulted in clear homogenous digests perfectly suitable for the determination of aluminium by graphite furnace atomic absorption spectrometry. The instrument detection limit for the method was 0.48 μg/L. Method blanks were used to estimate background levels of contamination of 14.80 μg/L. The mean concentration of aluminium across all tissues was 0.39 μg Al/g tissue dry wt. There were no statistically significant regionally specific differences in the content of aluminium. We have developed a robust method for the precise and accurate measurement of aluminium in human breast tissue. There are very few such data currently available in the scientific literature and they will add substantially to our understanding of any putative role of aluminium in breast cancer. While we did not observe any statistically significant differences in aluminium content across the breast it has to be emphasised that herein we measured whole breast tissue and not defatted tissue where such a distribution was previously noted. We are very confident that the method developed herein could now be used to provide accurate and reproducible data on the aluminium content in defatted tissue and oil from such tissues and thereby contribute towards our knowledge on aluminium and any role in breast cancer.

Development of a liquid chromatographic time-of-flight mass spectrometric method for the determination of unlabelled and deuterium-labelled alpha-tocopherol in blood components

A method is described for the analysis of deuterated and undeuterated alpha-tocopherol in blood components using liquid chromatography coupled to an orthogonal acceleration time-of-flight (TOF) mass spectrometer. Optimal ionisation conditions for undeuterated (d0) and tri- and hexadeuterated (d3 or d6) alpha-tocopherol standards were found with negative ion mode electrospray ionisation. Each species produced an isotopically resolved single ion of exact mass. Calibration curves of pure standards were linear in the range tested (0-1.5 muM, 0-15 pmol injected). For quantification of d0 and d6 in blood components following a standard solvent extraction, a stable-isotope-labelled internal standard (d3-alpha-tocopherol) was employed. To counter matrix ion suppression effects, standard response curves were generated following identical solvent extraction procedures to those of the samples. Within-day and between-day precision were determined for quantification of d0- and d6-labelled alpha-tocopherol in each blood component and both averaged 3-10%. Accuracy was assessed by comparison with a standard high-performance liquid chromatography (HPLC) method, achieving good correlation (r(2) = 0.94), and by spiking with known concentrations of alpha-tocopherol (98% accuracy). Limits of detection and quantification were determined to be 5 and 50 fmol injected, respectively. The assay was used to measure the appearance and disappearance of deuterium-labelled alpha-tocopherol in human blood components following deuterium-labelled (d6) RRR-alpha-tocopheryl acetate ingestion. The new LC/TOFMS method was found to be sensitive, required small sample volumes, was reproducible and robust, and was capable of high throughput when large numbers of samples were generated. Copyright (C) 2003 John Wiley Sons, Ltd.