Publications

Developing atherosclerotic lesions in hypercholesterolemic rabbits are depleted in zinc, while iron accumulates. This study examined the influence of zinc supplementation on the development of atherosclerosis and used isotope dilution gas chromatography-mass spectrometry techniques to measure biomarkers of oxidative lipid damage in atherosclerotic rabbit aorta. Our previous method for F2-isoprostane measurement was adapted to include the quantitation of cholesterol oxidation products in the same sample. Two groups of New Zealand white rabbits were fed a high cholesterol (1% w/w) diet and one group was also supplemented with zinc (1 g/kg) for 8 weeks. Controls were fed a normal diet. Zinc supplementation did not significantly alter the increase in total plasma cholesterol levels observed in animals fed high cholesterol. However, in cholesterol-fed animals zinc supplementation significantly reduced the accumulation of total cholesterol levels in aorta which was accompanied by a significant reduction in average aortic lesion cross-sectional areas of the animals. Elevated levels of cholesterol oxidation products (5,6-α and β cholesterol epoxides, 7β-hydroxycholesterol, 7-ketocholesterol) in aorta and total F2-isoprostanes in plasma and aorta of rabbits fed a cholesterol diet were significantly decreased by zinc supplementation. Our data indicate that zinc has an antiatherogenic effect, possibly due to a reduction in iron-catalyzed free radical reactions.

Abstract Background The mechanisms underlying cognitive resilience to Alzheimer’s disease (AD) pathology are under active investigation. The association between dietary micronutrients with neuroprotective properties and cognitive resilience is currently unknown. Objective To investigate whether plasma L-ergothioneine (ET), its metabolite L-hercynine (HC), as well as their ratio (HC:ET, as an index of ET metabolism) affect the association between plasma markers of brain amyloid pathology (phosphorylated tau species) and cognitive decline. Design, setting and participants This study consisted of 259 dementia-free participants (mean age [SD] = 72 [8] years; 50% females) recruited from memory clinics and the community in Singapore from August 2010 to July 2019 as part of a larger, ongoing longitudinal cohort study on dementia, with analyses performed in February 2025. Measurements All participants of this study were dementia-free at baseline (median Clinical Dementia Rating-Sum of Boxes (CDR-SB) [IQR] scores = 0 [1]), had blood collected for measurements of plasma ET, HC, HC:ET, p-tau181 and p-tau217, and underwent annual neuropsychological assessments for up to 5 years (mean follow-up [SD] = 52 [15] months). The main cognitive outcomes were cognitive decline, defined by annual change of CDR-SB, and risk of incident cognitive decline, defined as Global CDR scores (CDR-GS) increments of ≥ 0.5 at follow-up. Linear regression analyses tested for interaction effects of plasma ET, HC and HC:ET on the association between plasma p-tau and cognitive decline, while Cox proportional hazards models were fitted to estimate hazard ratios (HRs) of incident cognitive decline. Results Plasma HC:ET significantly moderated associations between plasma p-tau181 and cognitive decline, whereby only High HC:ET attenuated the detrimental effects of plasma p-tau181 on cognitive decline (High HC:ET β = 0.0976; 95% Confidence Interval [CI] = -0.444 to 0.639 vs. Low HC:ET β = 0.849; 95% CI, 0.318 to 1.38). Among participants with high risk of amyloid pathology, those with Low HC:ET had approximately twofold increased risk of cognitive decline (Hazards ratio [HR] = 1.96; 95% CI = 1.01 to 3.79) compared to participants with High HC:ET (HR=0.87; 95% CI = 0.33 to 2.26) over the follow-up period. Conclusions Identification of plasma HC:ET as a biomarker of cognitive resilience to amyloid pathology suggests potential beneficial effects of ET metabolism. Further studies are needed to elucidate ET-mediated neuroprotective mechanisms as potential therapeutic targets in delaying or moderating AD-associated cognitive decline.

Evidence on the efficacy of high-dose coenzyme Q10 (CoQ10) in Parkinson's disease (PD) is conflicting. An open-label dose-escalation study was performed to examine the effects of CoQ10 on biomarkers of oxidative damage and clinical outcomes in 16 subjects with early idiopathic PD. Each dose (400, 800, 1200, and 2400 mg/day) was consumed daily for 2 weeks. High-dose CoQ10 was well tolerated and improvements in the total Unified Parkinson's Disease Rating Scale (median, 37 vs. 27; p=0.048) were observed following study completion. Plasma F2-isoprostanes (adjusted for arachidonate) were significantly reduced in the 400-1200 mg/day dose range, but increased at 2400 mg/day dosage. A similar pattern of change was observed with serum phospholipase A2 activities. Levels of plasma all trans-retinol, plasma total tocopherol, serum uric acid, and serum total cholesterol were unchanged despite an increase in the CoQ10 dosage. Subjects with symptomatic benefits from CoQ10 (decrease in total UPDRS >10 points) had lower baseline plasma ubiquinol (p=0.07, Mann-Whitney U test) and decreased F2-isoprostanes per unit arachidonate (p=0.04, Wilcoxon Signed-Ranks test). These results lead to the hypothesis that the therapeutic response to CoQ10 depends on baseline levels of ubiquinol and whether the dosage of CoQ10 used can ameliorate the burden of oxidative damage.

There is considerable interest in the role of metals such as iron, copper, and zinc in amyloid plaque formation in Alzheimer’s disease. However to convincingly establish their presence in plaques in vivo, a sensitive technique is required that is both quantitatively accurate and avoids isolation of plaques or staining/fixing brain tissue, since these processes introduce contaminants and redistribute elements within the tissue. Combining the three ion beam techniques of scanning transmission ion microscopy, Rutherford back scattering spectrometry and particle induced X-ray emission in conjunction with a high energy (MeV) proton microprobe we have imaged plaques in freeze-dried unstained brain sections from CRND-8 mice, and simultaneously quantified iron, copper, and zinc. Our results show increased metal concentrations within the amyloid plaques compared with the surrounding tissue: iron (85ppm compared with 42ppm), copper (16ppm compared to 6ppm), and zinc (87ppm compared to 34ppm).

: Oxidation of l ‐3,4‐dihydroxyphenylalanine ( l ‐DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of l ‐DOPA, DA, and other catecholamines to a system generating O 2 •− radical led to O 2 •− ‐dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol‐DA or ‐DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and l ‐DOPA, especially if H 2 O 2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of l ‐DOPA, DA, and 3,4‐dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of l ‐DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of l ‐DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with l ‐DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.

Vitamin C (ascorbic acid, AA) can act as an antioxidant or a pro-oxidant in vitro, depending on the absence or the presence, respectively, of redox-active metal ions. Some adults with iron-overload and some premature infants have potentially redox-active, bleomycin-detectable iron (BDI) in their plasma. Thus, it has been hypothesized that the combination of AA and BDI causes oxidative damage in vivo. We found that plasma of preterm infants contains high levels of AA and F2-isoprostanes, stable lipid peroxidation end products. However, F2-isoprostane levels were not different between those infants with BDI (138 +/- 51 pg/ml, n = 19) and those without (126 +/- 41 pg/ml, n = 10), and the same was true for protein carbonyls, a marker of protein oxidation (0.77 +/- 0.31 and 0.68 +/- 0.13 nmol/mg protein, respectively). Incubation of BDI-containing plasma from preterm infants did not result in detectable lipid hydroperoxide formation (</=10 nM cholesteryl ester hydroperoxides) as long as AA concentrations remained high. Furthermore, when excess iron was added to adult plasma, BDI became detectable, and endogenous AA was rapidly oxidized. Despite this apparent interaction between excess iron and endogenous AA, there was no detectable lipid peroxidation as long as AA was present at >10% of its initial concentration. Finally, when iron was added to plasma devoid of AA, lipid hydroperoxides were formed immediately, whereas endogenous and exogenous AA delayed the onset of iron-induced lipid peroxidation in a dose-dependent manner. These findings demonstrate that in iron-overloaded plasma, AA acts an antioxidant toward lipids. Furthermore, our data do not support the hypothesis that the combination of high plasma concentrations of AA and BDI, or BDI alone, causes oxidative damage to lipids and proteins in vivo.

Anthracycline derivatives such as doxorubicin are part of many chemotherapeutic regimens and reach peak plasma concentrations of 5 μM. We investigated the cytotoxic mechanisms of various doxorubicin concentrations in MOLT-4 ALL-cells. Concentrations of up to 100 μM doxorubicin achieved similar cytotoxic effects in cultures of MOLT-4 cells, but acted via different mechanisms. Doxorubicin induced apoptosis (maximum effect at 1 μM), which was dependent on RNA synthesis and involved oxidative stress. Concentrations higher than 3 μM did not induce apoptosis, but significantly inhibited RNA synthesis. DNA strand breaks in MOLT-4 cells occurred in the presence of 1 to 5 μM doxorubicin to a similar extent, but showed a dose-dependence at higher concentrations. There was no GC/MS-detectable oxidation of DNA bases in apoptotic cells and only 1 out of 13 DNA base oxidation products, 8-hydroxyguanine, increased significantly in the presence of as much as 100 μM doxorubicin. These results suggest that at pharmacologically relevant concentrations apoptosis and not oxidative DNA damage is the main killing mechanism of doxorubicin against ALL-cells.