Daya Naidoo

Improved method for plasma malondialdehyde measurement by high-performance liquid chromatography using methyl malondialdehyde as an internal standard

Measurement of malondialdehyde (MDA) is an important contribution to the assessment of oxidative stress. We report a sensitive and reproducible high-performance liquid chromatography (HPLC) method for measurement of plasma MDA in the assessment of lipid peroxidation. Using methyl malondialdehyde (Me-MDA) as an internal standard with reversed-phase HPLC and UV detection and derivatisation with 2,4 dinitrophenylhydrazine (DNPH), we obtained maximum MDA values with 60-min incubation of 10% plasma with 1 M NaOH at 60 degrees C. The dilution of the plasma and a longer incubation time in the alkaline hydrolysis step greatly improved recovery of MDA from its bound form. Ratios of peak height of MDA/Me-MDA were linear over a range of 0-100 microM with correlation coefficients >0.99. The recovery was 88.5%. Within and between run variations were <4 and <7%, respectively. The mean MDA value measured in 20 healthy volunteers was 13.8 microM (+/-1.32).

Relationship of homocysteine, folic acid and vitamin B12 with depression in a middle-aged community sample.

BACKGROUND Case control studies have supported a relationship between low folic acid and vitamin B112 and high homocysteine levels as possible predictors of depression. The results from epidemiological studies are mixed and largely from elderly populations. METHOD A random subsample of 412 persons aged 60-64 years from a larger community sample underwent psychiatric and physical assessments, and brain MRI scans. Subjects were assessed using the PRIME-MD Patient Health Questionnaire for syndromal depression and severity of depressive symptoms. Blood measures included serum folic acid, vitamin B12, homocysteine and creatinine levels, and total antioxidant capacity. MRI scans were quantified for brain atrophy, subcortical atrophy, and periventricular and deep white-matter hyperintensity on T2-weighted imaging. RESULTS Being in the lowest quartile of homocysteine was associated with fewer depressive symptoms, after adjusting for sex, physical health, smoking, creatinine, folic acid and B12 levels. Being in the lowest quartile of folic acid was associated with increased depressive symptoms, after adjusting for confounding factors, but adjustment for homocysteine reduced the incidence rate ratio for folic acid to a marginal level. Vitamin B12 levels did not have a significant association with depressive symptoms. While white-matter hyperintensities had significant correlations with both homocysteine and depressive symptoms, the brain measures and total antioxidant capacity did not emerge as significant mediating variables. CONCLUSIONS Low folic acid and high homocysteine, but not low vitamin B12 levels, are correlates of depressive symptoms in community-dwelling middle-aged individuals. The effects of folic acid and homocysteine are overlapping but distinct.

Pharmacokinetics of Ciprofloxacin in the Human Eye: a Clinical Study and Population Pharmacokinetic Analysis

ABSTRACT Ciprofloxacin, a fluoroquinolone antibiotic active against a wide variety of bacteria, is one of a few antibiotics which enters the human eye after oral administration. However, little is known about its pharmacokinetics in the human eye. One or two oral doses of 750 mg of ciprofloxacin (at a 12-h interval) were administered to 48 patients at various times prior to ocular surgery. Clotted blood, aqueous, and vitreous were collected at surgery, and the concentrations of ciprofloxacin were assayed by high-performance liquid chromatography. Our data were combined with those of others, and a population pharmacokinetic analysis was conducted. The concentrations of ciprofloxacin in both aqueous and vitreous were lower than those in serum and peaked at a later time. The pharmacokinetics of ciprofloxacin in aqueous and vitreous were fitted to a compartmental model in which the antibiotic was transferred into and out of the two compartments (aqueous and vitreous) by first-order processes. Population pharmacokinetic software, P-Pharm, was used to calculate the mean half-lives of the loss of ciprofloxacin from aqueous and vitreous, which were 3.5 and 5.3 h, respectively. At steady state, the mean ratios of then concentrations in aqueous and vitreous to the concentrations in serum were 23 and 17%, respectively. After the administration of one or two doses of 750 mg of ciprofloxacin, the concentrations in both aqueous and vitreous in a number of patients were lower than the MICs at which 90% of isolates are inhibited (0.5 mg/liter) for common intraocular bacterial pathogens. Simulations of concentrations in the eye after the administration of higher doses (1,500 mg of ciprofloxacin as a single dose, two doses of 750 mg 2 h apart, and 750 mg every 6 h) indicated that in approximately 20% of patients the concentrations would still be below 0.5 mg/liter. Although oral ciprofloxacin may be a beneficial adjunctive therapy, the use of oral ciprofloxacin alone may not be adequate for perioperative prophylaxis or for treatment of bacterial endophthalmitis.

High dose intravitreal ganciclovir injection provides a prolonged therapeutic intraocular concentration.

BACKGROUND: Although intravitreal high dose ganciclovir has previously been found to provide excellent control of cytomegalovirus (CMV) retinitis, little was known about the vitreous concentrations of ganciclovir after a 2 mg intravitreal injection. METHODS: Eleven vitreous samples were taken from seven patients with CMV retinitis at 24 and 72 hours after a 2 mg intravitreal injection of ganciclovir and the concentration of ganciclovir was measured by high performance liquid chromatography. RESULTS: The mean concentration of ganciclovir at 24 hours was 143.8 mg/l (95% confidence interval 97.8-190) and at 72 hours was 23.4 mg/l (95% CI 0-49.7). The half life ranged from 11.9 to 26.3 (mean 18.8) hours in the four patients who had two samples taken. The mean half life calculated from the mean concentrations at 24 and 72 hours was 18.3 hours, so the calculated mean concentration at 7 days was 0.6 mg/l. CONCLUSIONS: This suggests that it takes about 7 days to eliminate the intravitreal ganciclovir, and that it is not likely to accumulate with weekly injections. The intravitreal concentrations achieved with high dose therapy remained above the ID50 for CMV (0.25-1.22 mg/l) for up to 7 days.

Postnatal changes in maternal and neonatal plasma antioxidant vitamins and the influence of smoking

Objective: To study the postnatal changes in the plasma concentrations of fat soluble antioxidant vitamins and malondialdehyde (MDA) in mothers and their newborns and their relation to smoking. Design: Prospective cohort study. Setting: Tertiary perinatal centre. Subjects: Eighteen non-smoking and 14 smoking mothers and 33 infants. Main outcome measures: Plasma concentrations of vitamins E, A, and β-carotene and MDA were measured in mothers and infants at delivery and on day 4 post partum. Results: Neonatal plasma levels of vitamins E, A, and β-carotene were significantly lower than maternal levels both at delivery and on day 4 in both groups. There was a significant postnatal increase in plasma vitamin E levels in smoking mothers and neonates of both groups. A significant postnatal increase in maternal, but not neonatal, plasma vitamin A was noted in both groups. Cord plasma vitamin E levels were significantly lower in infants of smoking mothers (mean 4.7 v 6.5 μmol/l, p = 0.041). Plasma MDA was paradoxically lower in smoking mothers at delivery (3.19 v 4.01 μmol/l, p = 0.03) and on day 4 (1.37 v 3.29 μmol/l , p = 0.005) and in infants of the smoking group on day 4 (2.18 v 3.12 μmol/l, p = 0.014). Also, there was a significant postnatal fall in plasma MDA levels on day 4 in mothers and infants in the smoking group. Conclusions: The postnatal changes in plasma vitamin E were more pronounced in the smoking group. The postnatal changes in plasma vitamins A and β-carotene were similar in both groups. The rapid decline in plasma MDA in smoking mothers and their infants suggests withdrawal of oxidative stress from smoking around delivery. This coincided with the increase in plasma vitamin E.

Associations between plasma antioxidants and hypertension in a community-based sample of 415 Australians aged 60–64

It has been suggested that increased oxidative stress may be both a cause as well as a consequence of hypertension. In vivo oxidation of low-density lipoproteins by oxygen-free radicals may increase hypertension-related atherogenesis, and antioxidants may be beneficial in this regard. Previous findings concerning associations between serum measures of antioxidants and hypertension have however been inconsistent. Plasma levels of β-carotene, Vitamin A, E, uric acid, homocysteine and total antioxidant capacity, as well as two markers of oxidative stress, malondialdehyde (MDA) and protein carbonyls, were measured in morning fasting blood samples provided by 415 Australians aged 60–64 years, selected randomly from the community. Participants also provided information on sociodemographic attributes, mental and physical health, and provided two measures of resting blood pressure, allowing a diagnosis of definite or borderline hypertension. Those with hypertension had lower levels of β-carotene and higher levels of uric acid and MDA compared to normotensive participants. The last two of these associations persisted when the analyses controlled for lifestyle and health factors. The findings from this study offer limited support for the proposition that lower antioxidant status and higher oxidative stress are associated with hypertension, and suggest the need for longitudinal studies to examine causality and intervention studies to determine the benefit of antioxidants in this group.

Antenatal supplementation of antioxidant vitamins to reduce the oxidative stress at delivery: a pilot study

BACKGROUND Preterm infants are at increased risk of oxidative stress and free-radical mediated diseases partly related to deficient antioxidant state. The purpose of this study was to investigate if maternal supplementation of antioxidant vitamins prior to delivery would reduce the oxidative stress in the mothers and their infants. STUDY DESIGN A pilot case-control study. PATIENTS AND METHODS Five mothers at risk of preterm delivery between 30 and 36 weeks were given a daily oral dose of betacarotene 20 mg, vitamin E 167.8 mg and vitamin C 1000 mg until delivery. Plasma levels of MDA and vitamins A, E and beta-carotene were measured prior to treatment in mothers and at delivery in both mothers and neonates. Seven mother-infant pairs comparable in gestation and birthweight acted as controls. RESULTS In the supplemented group, median maternal plasma MDA at delivery was significantly lower compared to the pretreatment level (1.9 vs. 3.2 micromol/l, p=0.04) and it was also lower than the control group (1.9 vs. 3.65 micromol/l, p<0.001). In the supplemented group, median maternal plasma vitamin E at delivery was significantly higher than the levels prior to treatment (46 vs. 31 micromol/l, p=0.007) in the same group and those at delivery in the control group (46 vs. 30 micromol/l, p=0.03). There was a trend of lower plasma MDA and higher vitamin E at birth in infants born to supplemented mothers, but it did not reach statistical significance. CONCLUSION A short supplementation of multiple antioxidant vitamins to a small sample of preterm pregnant women reduced the oxidative stress at delivery in mothers and probably in their neonates. Larger studies probably using larger doses are needed to evaluate the efficacy of this strategy.

Vitamin K in preterm breastmilk with maternal supplementation

Six healthy lactating mothers who gave birth to preterm infants at a median post conceptional age of 29.5 (range 26‐30) weeks were given 2.5 mg phylloquinone (vitamin K1) orally daily for 2 weeks beginning at a median postconceptional age of 31.5 (range 28–32) weeks. Phylloquinone was measured in the breastmilk daily for 14 d. Trough plasma phylloquinone concentrations were also determined on four occasions. Phylloquinone levels in the breastmilk increased from a baseline of 3 ± 2.3ngml‐1 to 22.6 ± 16.3 ng ml‐1 (mean ± SD) after the first dose (p < 0:05); a gradual increase was noted until phylloquinone levels reached a plateau of 64.2 ± 31.4ng ml‐1 after the sixth daily dose.

The effect of vitamin C and E supplementation on lipid and urate oxidation products in plasma

Abstract Plasma malondialdehyde (MDA) and allantoin concentrations were measured in fifteen volunteers before and after ingestion of either vitamin C, vitamin E or both. Increasing doses in the range of 250 – 1000mg for vitamin C and 200 – 800 I.U. for vitamin E were used over a period of eight weeks. Our study showed that an intake of 250mg vitamin C and 200 I.U. vitamin E produced a 25% reduction allantoin levels, doubling the dose resulted in a 75% reduction. Supplementation with both vitamins produced the earliest reduction in allantoin levels. Combined administration of both vitamins or vitamin C on its own, resulted in a reduction of MDA levels in plasma. Current recommended dietary allowances are discussed in relation to the supplementation doses used in this study.

Antioxidant levels in peripheral blood, disease activity and fibrotic stage in chronic hepatitis C.

Background: This study addressed the suggested association between levels of the antioxidants glutathione (GSH), vitamin C and vitamin E in peripheral blood and the histological activity and fibrosis stage in chronic hepatitis C (CHC). We then determined whether regular antioxidant supplementation influenced these antioxidant levels or disease severity.