Analysis of sex steroids in human tears using LC-MS and GC-MS: Considerations and developments to improve method sensitivity and accuracy.

Sex steroids play a role in regulation of tear film function and may exert their action locally at the ocular surface. However, measurement of sex steroids in tears is difficult due to small-volume tear samples and very low concentrations of the hormones. This short communication highlights what has been achieved to date in the analysis of tear sex steroids using ultra-performance LC-MS (UPLC-MS) as previously published, and reports further and more recent investigations toward optimising mass spectrometry method sensitivity and accuracy. The published UPLC-MS method successfully measured progesterone, androsterone glucuronide and 5α-androstane-3α,17ß-diol in pooled basal tears of postmenopausal women, and fourteen sex steroid standards in methanol. Limitations included sub-optimal limits of detection (LOD) and lower limits of quantification (LLOQ) for some analytes (particularly oestrogens), exclusion of sample matrix effects and no use of internal standards. This update reports on further experiments carried out to improve sensitivity and accuracy. Sample matrix effects, internal standard spiking, and derivatisation with dansyl chloride and oximes were investigated. Dansylation significantly improved the LOD and LLOQ of oestrogens and their metabolites, by a factor of 10 for oestradiol and a factor of 5 for oestrone, but sensitivity of this updated method is not sufficient however for analysis of these oestrogens in human tears. Using gas chromatography-mass spectrometry (GC-MS) as an alternative technique to LC-MS, improved sensitivity for derivatised oestradiol is reported. This work demonstrates the need to develop higher sensitivity methods and points researchers towards specific MS ionisation techniques for future analysis of sex steroids in tears, in order to progress current understanding of the role of sex steroids in tear function and dry eye.

Evidence for altered cholesterol metabolism in Huntington's disease post mortem brain tissue.

AIMS: Cholesterol plays an essential role in membrane structure and function, being especially important in the brain. Alteration of brain cholesterol synthesis and metabolism has been demonstrated in several Huntington's disease (HD) mouse and cell models; however, less is known about these alterations in human tissue. This study aimed to identify alterations to cholesterol synthetic and metabolic pathways in human HD brain tissue. METHODS: A broad range of cholesterol synthetic precursors, metabolites and oxidation products were measured by gas chromatography-tandem mass spectrometry in five regions of human post mortem HD brain and compared with age- and sex-matched control tissues. The level of enzymes that regulate cholesterol homeostasis, cholesterol 24-hydroxylase and delta(24)-sterol reductase were investigated by Western blotting and qPCR in putamen. RESULTS: The most significant changes were localized to the putamen, where a 60% decrease in 24(S)-hydroxycholesterol, 30% increase in cholesterol and 100-200% increase in synthetic precursors (lathosterol, zymosterol and desmosterol) was detected. The enzymes cholesterol 24-hydroxylase and delta(24)-sterol reductase were also significantly decreased in HD putamen as compared with control tissues. Free radical-generated cholesterol oxidation products 7-keto cholesterol and 7ß-hydroxycholesterol were also increased by 50-70% in HD putamen. CONCLUSION: Human HD brain has significantly decreased cholesterol metabolism and disrupted cholesterol homeostasis. Our data also indicate that lipid oxidative stress accompanies HD pathology.

Phospholipid Profiles Are Selectively Altered in the Putamen and White Frontal Cortex of Huntington's Disease.

Huntington's disease (HD) is a genetic, neurodegenerative illness that onsets in late adulthood as a series of progressive and terminal cognitive, motor, and psychiatric deficits. The disease is caused by a polyQ mutation in the Huntingtin gene (HTT), producing a polyglutamine expansion in the Huntingtin protein (HTT). HTT interacts with phospholipids in vitro; however, its interactions are changed when the protein is mutated in HD. Emerging evidence suggests that the susceptibility of brain regions to pathological stimuli is influenced by lipid composition. This study aimed to identify where and how phospholipids are changed in human HD brain tissue. Phospholipids were extracted using a modified MTBE method from the post-mortem brain of 13 advanced-stage HD patients and 13 age- and sex-matched controls. Targeted precursor ion scanning mass spectrometry was used to detect phospholipid species. In the white cortex of HD patients, there was a significantly lower abundance of phosphatidylcholine (PC) and phosphatidylserine (PS), but no difference in phosphatidylethanolamine (PE). In HD putamen, ester-linked 22:6 was lower in all phospholipid classes promoting a decrease in the relative abundance of ester polyunsaturated fatty acids in PE. No differences in phospholipid composition were identified in the caudate, grey cortex or cerebellum. Ether-linked PE fatty acids appear protected in the HD brain, as no changes were identified. The nature of phospholipid alterations in the HD brain is dependent on the lipid (subclass, species, and bond type) and the location.

The long and the short of Huntington's disease: how the sphingolipid profile is shifted in the caudate of advanced clinical cases.

Huntington's disease is a devastating neurodegenerative disorder that onsets in late adulthood as progressive and terminal cognitive, psychiatric and motor deficits. The disease is genetic, triggered by a CAG repeat (polyQ) expansion mutation in the Huntingtin gene and resultant huntingtin protein. Although the mutant huntingtin protein is ubiquitously expressed, the striatum degenerates early and consistently in the disease. The polyQ mutation at the N-terminus of the huntingtin protein alters its natural interactions with neural phospholipids in vitro, suggesting that the specific lipid composition of brain regions could influence their vulnerability to interference by mutant huntingtin; however, this has not yet been demonstrated in vivo. Sphingolipids are critical cell signalling molecules, second messengers and membrane components. Despite evidence of sphingolipid disturbance in Huntington's mouse and cell models, there is limited knowledge of how these lipids are affected in human brain tissue. Using post-mortem brain tissue from five brain regions implicated in Huntington's disease (control n = 13, Huntington's n = 13), this study aimed to identify where and how sphingolipid species are affected in the brain of clinically advanced Huntington's cases. Sphingolipids were extracted from the tissue and analysed using targeted mass spectrometry analysis; proteins were analysed by western blot. The caudate, putamen and cerebellum had distinct sphingolipid changes in Huntington's brain whilst the white and grey frontal cortex were spared. The caudate of Huntington's patients had a shifted sphingolipid profile, favouring long (C13-C21) over very-long-chain (C22-C26) ceramides, sphingomyelins and lactosylceramides. Ceramide synthase 1, which synthesizes the long-chain sphingolipids, had a reduced expression in Huntington's caudate, correlating positively with a younger age at death and a longer CAG repeat length of the Huntington's patients. The expression of ceramide synthase 2, which synthesizes very-long-chain sphingolipids, was not different in Huntington's brain. However, there was evidence of possible post-translational modifications in the Huntington's patients only. Post-translational modifications to ceramide synthase 2 may be driving the distinctive sphingolipid profile shifts of the caudate in advanced Huntington's disease. This shift in the sphingolipid profile is also found in the most severely affected brain regions of several other neurodegenerative conditions and may be an important feature of region-specific cell dysfunction in neurodegenerative disease.

Up-regulation of endoplasmic reticulum stress-related genes during the early phase of treatment of cultured cortical neurons by the proteasomal inhibitor lactacystin.

Inhibition of proteasome degradation pathway has been implicated in neuronal cell death leading to neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. We and others demonstrated that treatment of cortical neurons with the proteasomal inhibitor lactacystin leads to apoptosis. We discovered by microarray analysis that lactacystin treatment modulates the expression of both potentially neuroprotective as well as pro-apoptotic genes in neurons. However, the significance of the genes which upon transcriptional modulation contributed to proteasomal inhibition-induced apoptosis, remained unidentified. By employing microarray analysis to decipher the time-dependent changes in transcription of these genes in cultured cortical neurons, we discovered different groups of genes were transcriptionally regulated in the early and late phase of lactacystin-induced cell death. In the early phase, several neuroprotective genes such as those encoding the proteasome subunits and ubiquitin-associated enzymes, as well as the heat-shock proteins (HSP) were up-regulated. However, the pro-apoptotic endoplasmic reticulum (ER) stress-associated genes were also up-regulated at the early phase of lactacystin-induced neuronal cell death. In the late phase, genes encoding antioxidants and calcium-binding proteins were up-regulated while those associated with cholesterol biosynthesis were down-regulated. The data suggest that ER stress may participate in mediating the apoptotic responses induced by proteasomal inhibition. The up-regulation of the neuroprotective antioxidant genes and calcium-binding protein genes and down-regulation of the cholesterol biosynthesis genes in the later phase are likely consequences of stimulation of the pro-apoptotic signaling pathways in the early phase of lactacystin treatment.

Changes in cholesterol biosynthetic and transport pathways after excitotoxicity.

The present study was carried out to elucidate changes in the gene expression and activity of cholesterol biosynthetic enzymes and transporters in the rat hippocampus after kainate excitotoxicity. Significantly increased cholesterol level was detected in the degenerating hippocampus, reaching double normal levels at 1 week after kainate injury. RT-PCR analyses of hippocampal homogenates showed significantly decreased mRNA expression of the transcription factor controlling cholesterol biosynthesis SREBP-2, and the rate-controlling enzyme HMG-CoA (3-hydroxy-3-methyl-glutaryl-CoA) reductase at all time points after kainate injection; and decreased lanosterol synthase and CYP51 at 1 and 2 weeks post-kainate injection respectively. GC-MS analyses showed a significant increase in cholesterol biosynthetic precursors lanosterol, desmosterol and 7-dehydrocholesterol at 1 day after kainate injection presumably reflecting biosysnthesis in injured neurons, and significant decreases in precursors at 1 and 2 weeks post-kainate injection, at time of gliosis in the degenerating hippocampus. Levels of cholesterol autooxidation including 7 ketocholesterol and cholesterol epoxides were elevated in the kainate lesioned hippocampus. Furthermore, loss of expression of the cholesterol transporter, ABCA1 was detected in neurons, but increased expression in astrocytes was detected after kainate lesions. The results suggest that increased cholesterol biosynthesis and loss of ABCA1 expression in injured neurons might result in increase in cholesterol in the degenerating hippocampus. The increased cholesterol might predispose to increased formation of cholesterol oxidation products which have been shown to be toxic to neurons.

Cholesteryl ester levels are elevated in the caudate and putamen of Huntington's disease patients.

Huntington's disease (HD) is an autosomal dominant neurodegenerative illness caused by a mutation in the huntingtin gene (HTT) and subsequent protein (mhtt), to which the brain shows a region-specific vulnerability. Disturbances in neural cholesterol metabolism are established in HD human, murine and cell studies; however, cholesteryl esters (CE), which store and transport cholesterol in the brain, have not been investigated in human studies. This study aimed to identify region-specific alterations in the concentrations of CE in HD. The Victorian Brain Bank provided post-mortem tissue from 13 HD subjects and 13 age and sex-matched controls. Lipids were extracted from the caudate, putamen and cerebellum, and CE were quantified using targeted mass spectrometry. ACAT 1 protein expression was measured by western blot. CE concentrations were elevated in HD caudate and putamen compared to controls, with the elevation more pronounced in the caudate. No differences in the expression of ACAT1 were identified in the striatum. No remarkable differences in CE were detected in HD cerebellum. The striatal region-specific differences in CE profiles indicate functional subareas of lipid disturbance in HD. The increased CE concentration may have been induced as a compensatory mechanism to reduce cholesterol accumulation.

A metabolite profiling approach to identify biomarkers of flavonoid intake in humans.

Flavonoids are phytochemicals that are widespread in the human diet. Despite limitations in their bioavailability, experimental and epidemiological data suggest health benefits of flavonoid consumption. Valid biomarkers of flavonoid intake may be useful for estimating exposure in a range of settings. However, to date, few useful flavonoid biomarkers have been identified. In this study, we used a metabolite profiling approach to examine the aromatic and phenolic profile of plasma and urine of healthy men after oral consumption of 200 mg of the pure flavonoids, quercetin, (-)-epicatechin, and epigallocatechin gallate, which represent major flavonoid constituents in the diet. Following enzymatic hydrolysis, 71 aromatic compounds were quantified in plasma and urine at 2 and 5 h, respectively, after flavonoid ingestion. Plasma concentrations of different aromatic compounds ranged widely, from 0.01 to 10 micromol/L, with variation among volunteers. None of the aromatic compounds was significantly elevated in plasma 2 h after consumption of either flavonoid compared with water placebo. This indicates that flavonoid-derived aromatic compounds are not responsible for the acute physiological effects reported within 2 h in previous human intervention studies involving flavonoids or flavonoid-rich food consumption. These effects are more likely due to absorption of the intact flavonoid. Our urine analysis suggested that urinary 4-ethylphenol, benzoic acid, and 4-ethylbenzoic acid may be potential biomarkers of quercetin intake and 1,3,5-trimethoxybenzene, 4-O-methylgallic acid, 3-O-methylgallic acid, and gallic acid may be potential markers of epigallocatechin gallate intake. Potential biomarkers of (-)-epicatechin were not identified. These urinary biomarkers may provide an accurate indication of flavonoid exposure.

Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample.

Oxidized lipids such as F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs) are widely believed to be involved in multiple diseases. Usually, each product is measured individually in separate blood samples. In this study we describe a method allowing us to measure F2-IsoPs, HETEs, COPs, and arachidonate using a single sample. Plasma (1 ml) samples from healthy volunteers were diluted with heavy isotopic standards, hydrolyzed in alkali with organic solvent, and then subjected to anionic-exchange solid-phase extraction (SPE). After the SPE column was washed, hexane and hexane/ethyl acetate portions were collected and combined for COPs measurement. Thereafter the column was loaded with hexane/ethanol/acetic acid and fractions were collected for total F2-IsoPs, total HETEs, and arachidonate measurement. All compounds in the eluates were measured by gas chromatography-mass spectrometry. The efficiency of SPE and reproducibility for all compounds measured were high. Levels of total F2-IsoPs (0.45+/-0.26 ng/ml (n=157)), total HETEs (34.06+/-16.35 ng/ml (n=21)), total arachidonate (68.36+/-24.45 microg/ml (n=33)), and COPs (7-ketocholesterol, 12.25+/-6.56 ng/ml; 7beta-hydroxycholesterol, 6.32+/-3.46 ng/ml; 7alpha-hydroxycholesterol, 15.06+/-7.06 ng/ml; 24-hydroxycholesterol, 41.39+/-18.22 ng/ml; and 27-hydroxycholesterol, 29.08+/-16.79 ng/ml (n=26)) were recorded in healthy subjects (age range 20 to 66 years; average male to female ratio 1:1).

Changes in cytochrome P450 side chain cleavage expression in the rat hippocampus after kainate injury.

Our previous study showed an increase in total cholesterol level of the hippocampus after kainate-induced injury, but whether this is further metabolized to neurosteroids is not known. The first step in neurosteroid biosynthesis is the conversion of cholesterol to pregnenolone by the enzyme cytochrome P450 side chain cleavage (P450scc). This study was carried out to elucidate the expression of this enzyme in the kainate-lesioned rat hippocampus. A net decrease in P450scc protein was detected in hippocampal homogenates by Western blots at 2 weeks post-kainate injection (time of peak cholesterol concentration after kainate injury). Immunohistochemistry showed decreased labeling of the enzyme in neurons, but increased expression in a small number of astrocytes. The level of pregnenolone was also analyzed using a newly developed gas chromatography-mass spectrometry (GC-MS) method, optimized for the rat hippocampus. A non-significant tendency to a decrease in pregnenolone level was detected 2 weeks post-lesion. This is in contrast to a large increase in oxysterols in the lesioned hippocampus at this time (He et al. 2006). Together, they indicate that increased cholesterol in the kainate lesioned hippocampus is mostly metabolized to oxysterols, and not neurosteroids.

The identification of antioxidants in dark soy sauce.

Soy sauce is a traditional fermented seasoning in Asian countries, that has high antioxidant activity in vitro and some antioxidant activity in vivo. We attempted to identify the major antioxidants present, using the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay as a guide. 3-Hydroxy-2-methyl-4H-pyran-4-one (maltol) was one of several active compounds found in an ethyl acetate extract of dark soy sauce (DSS) and was present at millimolar concentrations in DSS. However, most of the antioxidant activity was present in colored fractions, two of which (CP1 and CP2) were obtained by gel filtration chromatography. Their structural characteristics based on nuclear magnetic resonance (NMR) and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis suggest that carbohydrate-containing pigments such as melanoidins are the major contributors to the high antioxidant capacity of DSS.

Sterol Analysis by Quantitative Mass Spectrometry.

Analysis of sterols by mass spectrometry is a fundamental technique allowing for both qualitative and quantitative characterization of sterol molecular lipid species. Lipids are isolated from matrix or matrices by homogenization and solvent extraction, and converted into species amenable for ionization either by derivatization or adduct formation. Chromatogaphy (either gas or liquid phase) can assist with the resolution of sterols. Tandem mass spectrometry allows the precise identification of sterol lipid species, while comparison to internal standards added during extraction enables accurate quantification.

Lovastatin modulates increased cholesterol and oxysterol levels and has a neuroprotective effect on rat hippocampal neurons after kainate injury.

This study was carried out to elucidate the effect of a brain-permeable statin (lovastatin) on cholesterol and oxysterol levels of the hippocampus after neuronal injury induced by the excitotoxin, kainic acid. Increased immunolabeling to cholesterol and the oxysterol biosynthetic enzyme, cholesterol 24-hydroxylase, was observed in the rat hippocampus after kainate lesions. This was accompanied by increased levels of cholesterol, 24-hydroxycholesterol (product of cholesterol 24-hydroxylase enzymatic activity), and 7-ketocholesterol in homogenates of the degenerating hippocampus as detected by gas chromatography/mass spectrometry. Hippocampi from rats or organotypic slices that had been treated with kainate plus lovastatin showed significantly lower levels of cholesterol, 24-hydroxycholesterol, and 7-ketocholesterol compared with those that had been treated with kainate only. Lovastatin also modulated hippocampal neuronal loss after kainate treatment in vivo and in vitro. The level of 24-hydroxycholesterol detected in vivo after kainate treatment (>50 microM) was found to be neurotoxic in hippocampal slice cultures. These results suggest that brain-permeable statins such as lovastatin could have a neuroprotective effect by limiting the levels of oxysterol in brain areas undergoing neurodegeneration.

Effect of tea phenolics and their aromatic fecal bacterial metabolites on intestinal microbiota.

Tea is rich in polyphenols and other phenolics that have been widely reported to have beneficial health effects. However, dietary polyphenols are not completely absorbed from the gastrointestinal tract and are metabolized by the gut microflora so that they and their metabolites may accumulate to exert physiological effects. In this study, we investigated the influence of the phenolic components of a tea extract and their aromatic metabolites upon bacterial growth. Fecal homogenates containing bacteria significantly catalyzed tea phenolics, including epicatechin, catechin, 3-O-methyl gallic acid, gallic acid and caffeic acid to generate aromatic metabolites dependent on bacterial species. Different strains of intestinal bacteria had varying degrees of growth sensitivity to tea phenolics and metabolites. Growth of certain pathogenic bacteria such as Clostridium perfringens, Clostridium difficile and Bacteroides spp. was significantly repressed by tea phenolics and their derivatives, while commensal anaerobes like Clostridium spp., Bifidobacterium spp. and probiotics such as Lactobacillus sp. were less severely affected. This indicates that tea phenolics exert significant effects on the intestinal environment by modulation of the intestinal bacterial population, probably by acting as metabolic prebiotics. Our observations provide further evidence for the importance of colonic bacteria in the metabolism, absorption and potential activity of phenolics in human health and disease. The bioactivity of different phenolics may play an important role in the maintenance of gastrointestinal health.

Therapeutic Effects of Anthocyanins and Environmental Enrichment in R6/1 Huntington's Disease Mice.

BACKGROUND: Huntington's disease (HD) is a progressive neurodegenerative disease with no effective treatment or cure. Environmental enrichment has been used to slow processes leading to ageing and neurodegenerative diseases including HD. Phenolic phytochemicals including anthocyanins have also been shown to improve brain function in ageing and neurodegenerative diseases. OBJECTIVE: This study examined the effects of anthocyanin dietary supplementation and environmental enrichment on behavioural phenotypes and brain cholesterol metabolic alterations in the R6/1 mouse model of HD. METHODS: R6/1 HD mice and their wild-type littermate controls were randomised into the different experimental conditions, involving either environmentally enriched versus standard housing conditions, or anthocyanin versus control diet. Motor dysfunction was assessed from 6 to 26 weeks using the RotaRod and the hind-paw clasping tests. Gas chromatography - tandem mass spectrometry was used to quantify a broad range of sterols in the striatum and cortex of R6/1 HD mice. RESULTS: Anthocyanin dietary supplementation delayed the onset of motor dysfunction in female HD mice. Environmental enrichment improved motor function and the hind paw clasping phenotype in male HD mice only. These mice also had lower levels of cholesterol oxidation products in the cortex compared to standard-housed mice. CONCLUSION: Both anthocyanin supplementation and environmental enrichment are able to improve the motor dysfunction phenotype of R6/1 mice, however the effectiveness of these interventions was different between the two sexes. The interventions examined did not alter brain cholesterol metabolic deficits that have been reported previously in this mouse model of HD.

Human fecal water content of phenolics: the extent of colonic exposure to aromatic compounds.

Phenolic compounds are not completely absorbed in the small intestine and so enter the colon, where they might exert physiological effects. To identify phenolics that are present in normal human colon, fecal water was prepared from 5 free-living volunteers with no dietary restrictions and analyzed by gas chromatography-mass spectrometry. Daily measurements were also performed on a single individual to examine the variation more closely. Levels of polyphenols were variable between individuals. Naringenin and quercetin had mean concentrations of 1.20 and 0.63 microM. All other flavonoids examined were present < or =0.17 microM. Simple phenolic and other aromatic acids were present at much higher concentrations. The major components were phenylacetic acid, 479 microM; 3-phenylpropionic acid, 166 microM; 3-(4-hydroxy)-phenylpropionic acid, 68 microM; 3,4-dihydroxycinnamic acid, 52 microM; benzoic acid, 51 microM; 3-hydroxyphenylacetic acid, 46 microM; and 4-hydroxyphenylacetic acid, 19 microM. Other phenolic acids ranged from 0.04 to 7 microM. Decreased dietary phenolic intake caused a decrease in polyphenol and monophenolic acid concentration in fecal water 24 h later. This study is the first to measure the range of aromatic compounds in human fecal water and demonstrates that phenolic acid concentrations are high. The biological effects of phenolics may play an important role in colon function.

Vitamin C protects against hypochlorous Acid-induced glutathione depletion and DNA base and protein damage in human vascular smooth muscle cells.

Hypochlorous acid (HOCl), generated by myeloperoxidase released from activated macrophages, is thought to contribute to vascular dysfunction and oxidation of low density lipoproteins (LDLs) in atherogenesis. We have previously shown that HOCl exposure can cause chlorination and oxidation of isolated DNA and that vitamin C protects human arterial smooth muscle cells against oxidized LDL-mediated damage. We report in the present study that vitamin C attenuates HOCl-induced DNA base and protein damage and depletion of intracellular glutathione (GSH) and ATP in human arterial smooth muscle cells. Cells were pretreated in the absence or presence of 100 micromol/L vitamin C (24 hours) and then exposed to HOCl (0 to 500 micromol/L, 0 to 60 minutes) in the absence of vitamin C. Intracellular GSH and ATP levels were depleted by HOCl treatment, and gas chromatography-mass spectroscopy revealed a concentration- and time-dependent increase in DNA base oxidation and protein damage (measured as 3-chlorotyrosine). Pretreatment of smooth muscle cells with vitamin C significantly reduced the extent of HOCl-induced DNA and protein damage and attenuated decreases in intracellular ATP and GSH. Our findings suggest that physiological levels of vitamin C provide an important antioxidant defense against HOCl-mediated injury in atherosclerosis.

A high-throughput and sensitive methodology for the quantification of urinary 8-hydroxy-2'-deoxyguanosine: measurement with gas chromatography-mass spectrometry after single solid-phase extraction.

8-hydroxy-2'-deoxyguanosine (8OHdG) is a widely used biomarker for the measurement of endogenous oxidative DNA damage. A sensitive method for the quantification of 8OHdG in urine by single solid-phase extraction and GC-MS (gas chromatography with MS detection) using selective ion monitoring is described in the present study. After solid-phase extraction, samples are freeze-dried, derivatized by trimethylsilylation and analysed by GC-MS. The urinary 8OHdG was quantified using heavy isotope dilution with [18O]8OHdG. The recovery of 8OHdG after the solid-phase extraction ranged from 70 to 80% for a wide range of urinary 8OHdG levels. Using 1 ml of urine, the limit of quantification was >2.5 nM (2.5 pmol/ml) and the calibration curve was linear in the range 2.5-200 nM. This method was applied to measure 8OHdG in urine samples from 12 healthy subjects. The intra- and inter-day variations were <9%. Urinary 8OHdG levels in spot urine samples from four healthy subjects were also measured for 1 week and, again, the variation was small. The presence of H2O2 in urine did not cause artifactual formation of 8OHdG. Since this assay is simple, rapid, sensitive and reproducible, it seems suitable to be used as a routine methodology for the measurement of urinary excretion of 8OHdG in large population studies.

Fatty acid composition of the anterior cingulate cortex indicates a high susceptibility to lipid peroxidation in Parkinson's disease.

BACKGROUND: Oxidative stress contributes to Parkinson's disease (PD) etiology. Although previous studies have focused on sources of free radical formation in brain regions affected by PD, less is known regarding changes in lipid composition and the implications for susceptibility to peroxidation. OBJECTIVE: To assess fatty acid profiles from control and PD tissues that are susceptible to PD pathology but devoid of severe destruction. METHODS: We used gas chromatography methods to assess fatty acid profiles from control (n = 10) and PD (n = 9) postmortem tissues. We focused on the anterior cingulate cortex (ACC), a region that accumulates alpha-synuclein, but does not undergo severe destruction, and compared this to the occipital cortex, a region that is pathologically spared. RESULTS: Our data indicate a significant 33% increase in the proportion of polyunsaturated fatty acids (mol%) present in the PD ACC as compared to control ACC. Increases in highly unsaturated 22:5n-6 and 22:6n-3 fatty acids were particularly pronounced (109% and 73%, respectively). Calculation of a peroxidation index (accounting for total fatty acyl double bounds) indicated a 44% increase in susceptibility of the PD ACC to lipid peroxidation compared to control ACC. Such differences were not detected in the occipital cortex from the same donors. Assessment of F2-isprostane levels confirmed that PD tissue lipids were more oxidized than controls. CONCLUSIONS: The global composition of fatty acids in the PD ACC is altered in a way that increases susceptibility to peroxidation in a region-specific manner. This has important implications for PD, supporting the oxidative stress hypothesis of PD pathogenesis.

Brain Cholesterol Synthesis and Metabolism is Progressively Disturbed in the R6/1 Mouse Model of Huntington's Disease: A Targeted GC-MS/MS Sterol Analysis.

BACKGROUND: Cholesterol has essential functions in neurological processes that require tight regulation of synthesis and metabolism. Perturbed cholesterol homeostasis has been demonstrated in Huntington's disease, however the exact role of these changes in disease pathogenesis is not fully understood. OBJECTIVE: This study aimed to comprehensively examine changes in cholesterol biosynthetic precursors, metabolites and oxidation products in the striatum and cortex of the R6/1 transgenic mouse model of Huntington's disease. We also aimed to characterise the progression of the physical phenotype in these mice. METHODS: GC-MS/MS was used to quantify a broad range of sterols in the striatum and cortex of R6/1 and wild type mice at 6, 12, 20, 24 and 28 weeks of age. Motor dysfunction was assessed over 28 weeks using the RotaRod and the hind-paw clasping tests. RESULTS: 24(S)-Hydroxycholesterol and 27-hydroxycholesterol were the major cholesterol metabolites that significantly changed in R6/1 mice. These changes were specifically localised to the striatum and were detected at the end stages of the disease. Cholesterol synthetic precursors (lathosterol and lanosterol) were significantly reduced in the cortex and striatum by 6 weeks of age, prior to the onset of motor dysfunction, as well as the cognitive and affective abnormalities previously reported. Elevated levels of desmosterol, a substrate of delta(24)-sterol reductase (DHCR24), were also detected in R6/1 mice at the end time-point. Female R6/1 mice exhibited a milder weight loss and hind paw clasping phenotype compared to male R6/1 mice, however, no difference in the brain sterol profile was detected between sexes. CONCLUSION: Several steps in cholesterol biosynthetic and metabolic pathways are differentially altered in the R6/1 mouse brain as the disease progresses and this is most severe in the striatum. This provides further insights into early molecular mediators of HD onset and disease progression and identifies candidate molecular targets for novel therapeutic approaches.