Enhanced oxidative stress in Hashimoto's thyroiditis: inter-relationships to biomarkers of thyroid function.

OBJECTIVES: Oxidative stress has been implicated in the pathogenesis of several inflammatory and immune-mediated disorders including Hashimoto's thyroiditis (HT). The objectives of the present cross-sectional investigation were to estimate serum glutathione (GSH) status and the activities of its recycling enzymes in HT and to explore their interrelationships with biomarkers of autoimmunity and thyroid function. DESIGN AND METHODS: Newly diagnosed females with HT (n=44) and 58 matched control subjects were recruited. Thyroid hormone profile, anti-thyroperoxidase anti-body (TPO-AB), anti-thyroglublin antibody (Tg-AB), thyroid volume (Tvol), urinary iodine excretion (UIE), GSH and the activities of glutathione peroxidase (GPx), glutathione reductase and gamma-glutamyltransferase were assessed. RESULTS: Median UIE in HT was slightly but not significantly higher than that of controls. HT group exhibited higher levels of TSH, TPO-AB, Tg-AB and larger Tvol when compared with controls (P<0.001). The means of GSH and GPx in HT patients were significantly different from those of controls (P<0.001). In HT subjects, significant associations were seen between Tvol on TSH, GSH on TPO-AB, GSH on TSH and TPO-AB titers on TSH, respectively. CONCLUSIONS: This is the first study to demonstrate a substantial reduction in GSH status in HT subjects. Secondly, the interrelationship between the GSH contents and TPO-AB titers in HT provides a preliminary data to support the notion that GSH diminution is a hallmark of in the events leading to oxidative stress activation and the development of immunological intolerance in HT. Further studies are required to elucidate the role of GSH in the etiology of down-regulation of thyroid function.

Evaluation of Accessibility of Iodinated Salt and Nutritional Iodine Status during Pregnancy.

BACKGROUND: To assess accessibility of iodinated salt and urinary iodine concentrations (UIC) during pregnancy. This cross-sectional study was carried out between October and December, 2009 in Urmia County, West Azerbaijan (WA), Iran. METHODS: Data on demographic characteristics and iodinated salt accessibility were gathered through a questionnaire at 1st trimester. Household salt samples and urine samples (1st -and 3rd trimesters) were analyzed for iodine content. Pregnant women (n=490) at 1st trimester were interviewed. Of these, 490 subjects (12 prenatal care centers) were enrolled. RESULTS: All participants declared that they were exclusive users of iodinated salt. Segregation of the household salt samples according to iodine content (0, 8, 15 and 30 ppm) revealed that the respective distributions were 3.3%, 1.4%, 23.7% and 71.6%. Median UIC levels at 1st and 3rd trimesters were 73.5 µg/L and 114µg/L respectively. Accordingly, 86% and 70% of participants exhibited UIC < 150 µg/L. CONCLUSION: Median UIC during pregnancy in WA is markedly lower than those previously reported for regions with adequate iodine status in the country. Thus, extra iodine is needed to maintain adequate iodine store during gestation. In addition, this preliminary study reveals that a significant proportion (28%) of the household salt samples had low iodine content (≤ 15 ppm) although a level (>20 and <40 ppm) is mandatory in Iran. Further studies are deemed necessary to elucidate the cause(s) for manifestation iodine deficiency among pregnant women despite 20 years after iodine fortification strategy.

Urinary 8-epi-PGF2alpha and its endogenous beta-oxidation products (2,3-dinor and 2,3-dinor-5,6-dihydro) as biomarkers of total body oxidative stress.

Although measurements of plasma F2-isoprostanes are established markers of oxidative stress, their quantification only reflects acute non-enzymatic lipid peroxidation. In this study, a new approach is described for the rapid isolation and measurement of urinary 8-epi-PGF2alpha and its endogenous beta-oxidation metabolites (2,3-dinor-8-epi-PGF2alpha and 2,3-dinor-5,6-dihydro-PGF2alpha) for use as index of total body oxidative stress. Isoprostanes were partitioned with ethyl acetate and subsequently purified by chromatography on an aminopropyl (NH2) and silica (Si) cartridge. Final analysis of F2-isoprostanes as trimethylsilyl-ester/pentafluorobenzyl ester derivatives was carried out by stable isotope dilution mass spectrometry. Overall recovery of F2-isoprostanes was 80+/-4%. Inter- and intra-assay coefficients of variation were 5% and 7%, respectively. In a group of healthy humans, the mean excretion rates expressed as nmol/mmol creatinine for 2,3-dinor-8-epi-PGF2alpha, 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha, and 8-epi-PGF2alpha were 5.43+/-1.93, 2.16+/-0.71, and 0.36+/-0.16, respectively. Correlations were obtained between 8-epi-PGF2alpha and 2,3-dinor-8-epi-PGF2alpha or 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.998 and r=0.937, respectively). A strong relationship was also seen between 2,3-dinor-8-epi-PGF2 and 2,3-dinor-5,6-dihydro-8-epi-PGF2alpha (r=0.949). The new technique allows for high sample throughput and avoids the need for HPLC and/or other expensive equipment required for the initial sample preparation. Simultaneous analysis of urinary 8-epi-PGF2alpha and its metabolites should provide unique tool in clinical trials exploring the role of oxidant injury in human disease.

Antibodies to high-density lipoprotein and beta2-glycoprotein I are inversely correlated with paraoxonase activity in systemic lupus erythematosus and primary antiphospholipid syndrome.

OBJECTIVE: To determine the prevalence of anti-high-density lipoprotein (anti-HDL) antibodies and to establish a possible relationship between anti-HDL, anticardiolipin antibodies (aCL), anti-beta(2)-glycoprotein I (anti-beta(2)GPI), and paraoxonase (PON) activity in patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (APS). METHODS: Thirty-two patients with SLE and 36 with primary APS were enrolled in a cross-sectional study. Twenty age- and sex-matched healthy subjects were used as controls. Serum levels of IgG and IgM aCL, anti-beta(2)GPI, and antiprothrombin antibodies and IgG anti-HDL were measured by enzyme-linked immunosorbent assay. Total cholesterol, HDL cholesterol, HDL(2), and HDL(3) were determined by standard enzymatic techniques. PON activity was assessed by quantification of nitrophenol formation, and total antioxidant capacity (TAC) by chemiluminescence. RESULTS: Levels of total HDL, HDL(2), and HDL(3) were reduced in patients with SLE compared with controls (mean +/- SD 0.51 +/- 0.3, 0.37 +/- 0.3, and 0.14 +/- 0.1 mmoles/liter, respectively, versus 1.42 +/- 0.9, 1.01 +/- 0.7, and 0.40 +/- 0.2). Patients with SLE and primary APS had higher titers of anti-HDL antibodies and lower PON activity than controls. In the SLE population, PON activity was inversely correlated with IgG anti-HDL titers (r = -0.48, P = 0.005) whereas in the primary APS population, IgG anti-beta(2)GPI was the only independent predictor of PON activity (r = -0.483, P = 0.003). In the SLE group, anti-HDL was inversely correlated with TAC (r = -0.40, P < 0.02), and PON activity was positively correlated with TAC (r = 0.43, P < 0.02). CONCLUSION: IgG anti-HDL and IgG anti-beta(2)GPI antibodies are associated with reduced PON activity in patients with SLE and primary APS. Since the physiologic role of PON is to prevent low-density lipoprotein oxidation with its attendant atherogenic effects, the reported interactions may be relevant to the development of atherosclerosis in SLE and primary APS.

A diet containing rapeseed oil-based fats does not increase lipid peroxidation in humans when compared to a diet rich in saturated fatty acids.

OBJECTIVE: To compare the effects of a rapeseed oil-based diet containing an increased proportion of easily oxidised polyunsaturated fatty acids such as alpha-linolenic acid with a diet rich in saturated fatty acids on the degree of lipid peroxidation in the human body. DESIGN: A randomised cross-over study. SUBJECTS AND INTERVENTIONS: Nineteen healthy moderately hyperlipidemic subjects (six women and 13 men, age 50+/-8 y and body mass index (BMI) 24.5+/-2.6 kg/m(2)) were given a rapeseed oil-based diet (RO) and a control diet (SAT) rich in saturated fatty acids during two consecutive 4 week periods separated by a 4 week wash-out period. Biomarkers of lipid peroxidation and antioxidants were analysed in plasma and urine. RESULTS: No significant differences in plasma or urinary levels of free 8-iso-prostaglandin F(2alpha), plasma total 8-iso-prostaglandin F(2alpha) plasma hydroperoxides or plasma malondialdehyde were observed between the RO and SAT diets (P=0.14-0.95). A higher concentration of serum gamma-tocopherol was detected after the RO diet compared to the SAT diet (P<0.001), whereas the serum alpha-tocopherol concentration and plasma antioxidative capacity did not differ between the two test diets. The total cholesterol, LDL cholesterol and LDL/HDL ratio were lower after the RO diet compared to the SAT diet (P<0.001), while HDL cholesterol and total triglyceride levels were similar after the two diets. CONCLUSION: These results suggest that a rapeseed oil-based diet rich in alpha-linolenic acid does not seem to increase the degree of lipid peroxidation in plasma and urine compared to a diet rich in saturated fats. This is possibly due to a sufficient content of antioxidants in the rapeseed oil diet to increase circulating concentrations of antioxidants that may protect unsaturated fatty acids from oxidation. SPONSORSHIP: Swedish Council for Forestry and Agricultural Research and Foundation for Geriatric Research.

Measures of oxidative stress in heterozygous familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) may be associated with increased oxidative stress which may contribute to atherogenesis. Plasma lipid hydroperoxides (ROOHs), 8-epi PGF(2alpha) and alpha-tocopherol were measured in normal subjects and in newly referred heterozygous FH patients and used as indices of oxidative stress. ROOH levels were higher (+16%), albeit non-significantly, in FH patients than in controls subjects (4.4+/-0.3 vs. 3.8+/-0.3 micromol/l; n=51 and 40, respectively). 8-epi PGF(2alpha) levels were significantly greater (+56%) in the FH patients than in controls (0.43+/-0.06 vs. 0.27+/-0.05 nmol/l; P<0.05; n=14 and 16, respectively). FH patients with vascular disease had significantly higher (+32%) levels of ROOH compared with patients without vascular disease (4.9+/-0.40 vs. 3.7+/-0.33 micromol/l; P<0.05; n=27 and 24, respectively). Similarly, 8-epi PGF(2alpha) concentrations were higher (+100%) in the FH patients with vascular disease than in those without it (0.6+/-0.08 vs. 0.3+/-0.10 nmol/l; P<0.05; n=6 and 8, respectively). Absolute alpha-tocopherol levels in FH patients were similar to those in controls (21.0+/-0.70 vs. 23.8+/-1.30 micromol/l). When alpha-tocopherol levels were expressed relative to cholesterol, however, the concentrations were found to be significantly lower (-43%) in FH patients than in controls (2.9+/-0.10 vs. 5.1+/-0.40 micromol/mmol, P<0.0005). There were no differences in absolute or cholesterol standardised alpha-tocopherol levels in patients with and without vascular disease. These data suggest that oxidative stress is increased in FH-patients and is particularly pronounced in those patients with vascular disease. It is possible that increased oxidative stress may precede the development of vascular disease.

F(2) isoprostanes, potential specific markers of oxidative damage in human retina.

F(2) isoprostanes (F(2)-IPs) are a series of prostaglandin-F(2)(PGF(2))-like compounds produced during peroxidation of arachidonic acid (AA) by a mechanism independent of the cyclo-oxygenase. Of these, 8-epi-PGF(2alpha) has proved a reliable marker of oxidative stress in studies both in vitro and in vivo. Human retinas (n = 28) were obtained from healthy donors (age 15-59 years) and analysed for the content of free (non-esterified) as well as total (sum of free and esterified) F(2)-IPs. Retina tissue was homogenised, total lipids were partitioned with ethyl acetate and isoprostanes were isolated by chromatography on an NH(2) cartridge. After formation of pentafluorobenzyl ester and trimethylsilyl derivatives, F(2)-IPs were analysed by negative ion-chemical ionisation mass spectrometry using tetradeuterated PGF(2alpha) as the internal standard. Analysis of retina samples revealed the presence of measurable amounts of esterified 8-epi-PGF(2alpha) (mean: 81.71 microg/g AA; range: 3.29-187.89 microg/g AA) but not free F(2)-IPs. There was no correlation between age and content of 8-epi-PGF(2alpha). Detection of esterified F(2)-IPs in human retina provides the first direct evidence for free-radical-catalysed peroxidation of AA in retinal tissue. Analysis of F(2)-IPs should provide new opportunities for studying the implications of oxidative injury in various diseases of retina, including diabetic retinopathy and age-related macular degeneration.

The steady-state levels of oxidative DNA damage and of lipid peroxidation (F2-isoprostanes) are not correlated in healthy human subjects.

Oxidative damage to DNA in human tissues can be determined by measuring multiple products of oxidative damage to the purine and pyrimidine bases using gas chromatography-mass spectrometry (GC-MS). Oxidative damage to lipids (lipid peroxidation) can be quantitated by the mass spectrometry-based determination of F2-isoprostanes, specific end-products of the peroxidation of arachidonic acid residues in lipids. For both DNA base damage products and 8-epi prostaglandin F2alpha (PGF2alpha), there is a wide variation in levels between different healthy human subjects. We measured multiple products of oxidative damage to DNA bases in white cells, and 8-epi PGF2alpha in plasma, from blood samples obtained from healthy human subjects in the UK and in Portugal. No correlation of 8-epi PGF2alpha levels with levels of any modified DNA base (including 8-hydroxyguanine) was observed. We conclude that no single parameter can be measured as an index of "oxidative stress" or "oxidative damage" in vivo.

alpha-Lipoic acid decreases oxidative stress even in diabetic patients with poor glycemic control and albuminuria.

In the present cross-sectional study, the influence of alpha-lipoic acid on markers of oxidative stress, assessed by measurement of plasma lipid hydroperoxides (ROOHs), and on the balance between oxidative stress and antioxidant defence, determined by the ratio ROOH/(alpha-tocopherol/cholesterol), was examined in 107 patients with diabetes mellitus. Patients receiving alpha-lipoic acid (600 mg/day for > 3 months) had significant lower ROOHs and a lower ROOH/(alpha-tocopherol/cholesterol) ratio than those without alpha-lipoic acid treatment [ROOH: 4.76 +/- 2.49 vs. 7.16 +/- 3.22 mumol/l; p < .0001] and [ROOH/(alpha-tocopherol/cholesterol): 1.37 +/- 0.72 vs. 2.16 +/- 1.17; p < 0.0001]. In addition, the influence of glycemic control and albuminuria on ROOHs and on the ratio of ROOH/(alpha-tocopherol/cholesterol) was examined in the presence and absence of alpha-lipoic acid treatment. Patients were subdivided into three groups based on (1) their HbA1 levels (< 7.5, 7.5-9.5, and > 9.5%) and (2) their urinary albumin concentrations (< 20, 20-200, and > 200 mg/l). Neither poor glycemic control, nor the presence of micro- or macroalbuminuria prevented the antioxidant effect of alpha-lipoic acid. Using stepwise multiple regression analysis, alpha-lipoic acid was found to be the only factor significantly predicting low ROOHs and a low ratio of ROOH/(alpha-tocopherol/cholesterol). These data provide evidence that treatment with alpha-lipoic acid improves significantly the imbalance between increased oxidative stress and depleted antioxidant defence even in patients with poor glycemic control and albuminuria.

Oxidative stress in systemic lupus erythematosus and allied conditions with vascular involvement.

OBJECTIVE: To evaluate the occurrence and clinical significance of lipid peroxidation (oxidative stress) in rheumatic diseases characterized by vascular involvement. PATIENTS AND METHODS: Plasma 8-epi-PGF2alpha (oxidative stress marker) was measured by gas chromatography-mass spectrometry in 36 patients with systemic lupus erythematosus (SLE), 13 with systemic sclerosis (SSc), 13 with systemic vasculitis [Wegener's granulomatosis (WG), n = 4; Churg Strauss syndrome (CSS), n = 3; Behcet syndrome, n = 6], 12 with rheumatoid arthritis (RA) and in 23 healthy controls (n = 23). RESULTS: 8-epi-PGF2alpha levels were higher in patients with SLE (P = 0.007), SSc (P < 0.001) and vasculitis (P = 0.001) than in controls. In SLE, a positive Coombs' test and arterial hypertension independently predicted 8-epi-PGF2alpha concentrations (P = 0.004 and P = 0.001, respectively). SLE patients not taking prednisolone showed higher 8-epi-PGF2alpha concentrations than SLE patients on prednisolone (P = 0.02). In the latter group, a dose response relationship was noted between 8-epi-PGF2alpha and steroid dosage (r = 0.6, P = 0.0003). In WG and CSS, 8-epi-PGF2alpha concentrations correlated with disease activity (r = 0.8, P = 0.01) and were higher than in patients with Behcet disease (P = 0.003). CONCLUSIONS: Oxidative stress may be pathogenetically relevant in some autoimmune rheumatic diseases with vascular involvement. Amelioration of some clinical manifestations of these diseases may be envisaged by targeting lipid peroxidation with dietary or pharmacological antioxidants.

Age-related accumulation of free polyunsaturated fatty acids in human retina.

The present study reports composition of free (nonesterified) as well as total (sum of free and esterified) fatty acids (FAs) in human retina (n = 13). For free fatty acid (FFA) analysis, retina tissue was homogenized, total lipids were partitioned with ethyl acetate and subsequently applied onto a aminopropyl (NH2) cartridge to isolate FFAs from the bulk of other lipids. FFAs were converted to methyl ester derivatives and analysed by gas chromatography using flame ionization detector. Analysis of FFAs revealed that the mean percentage composition of the major components including palmitic acid (PA), stearic acid (SA), oleic acid (OA), arachidonic acid (AA) and docosahexaenoic acid (DHA) were 17.2, 36.7, 15.6, 8.8 and 14.2%, respectively. There were significant correlations between age of the donors' and the content of both free AA and DHA (rPearson = 0.69, p = 0.005, and rPearson = 0.64, p = 0.009). The mean percentage of total PA, SA, OA, AA and DHA were 22.6, 23.2, 17.7, 11.4 and 21.9%, respectively. There was no association between age and any of the major FAs. The present study provides the first evidence for the presence of FFAs in the human retina as well as an age-related accumulation of polyunsaturated fatty acids (PUFAs). The latter finding suggests an alteration in the metabolism of retinal PUFAs which can be due to an increase of oxidative stress and/or decrease of antioxidant defences during ageing.

Increased oxidative damage to all DNA bases in patients with type II diabetes mellitus.

Gas chromatography-mass spectrometry was used to measure the oxidative DNA damage in diabetic subjects and controls. Levels of multiple DNA base oxidation products, but not DNA base de-amination or chlorination products, were found to be elevated in white blood cell DNA from patients with type II diabetes as compared with age-matched controls. The chemical pattern of base damage is characteristic of that caused by an attack on DNA by hydroxyl radical. An increased formation of the highly reactive hydroxyl radical could account for many of the reports of oxidative stress in diabetic subjects. There was no evidence of an increased DNA damage by reactive nitrogen or chlorine species.

F4-isoprostanes as specific marker of docosahexaenoic acid peroxidation in Alzheimer's disease.

F2-isoprostanes are prostaglandin-like compounds derived from free radical-catalysed peroxidation of arachidonic acid. Peroxidation of eicosapentaenoic acid produces F3-isoprostanes, whereas peroxidation of docosahexaenoic acid would give F4-isoprostanes. This study demonstrates the presence of esterified F4-isoprostanes in human brain and shows that levels are elevated in certain brain cortex regions in Alzheimer's disease. Our data with Alzheimer's disease suggest that analysis of F4-isoprostanes will provide new opportunities to study lipid peroxidation in the neurodegenerative diseases.

Effect of dialysis on oxidative stress in uraemia.

It has been postulated that dialysis of patients with chronic renal failure (CRF) is associated with increased lipid peroxidation which may contribute to vascular and other complications of the syndrome. In the present study, a specific and precise technique [ferrous oxidation in xylenol orange (FOX) assay] was used to measure plasma lipid hydroperoxides (ROOHs) in three groups of uraemic patients. Patients were either studied before starting dialysis (n = 12) or on continuous ambulatory peritoneal dialysis (CAPD, n = 12) or haemodialysis (HD, n = 36) and compared to healthy controls (n = 20). Plasma ROOHs were markedly elevated in HD patients compared with the controls (7.01 +/- 2.9 microM versus 4.25 +/- 2.05 microM; P < 0.005, Mann-Whitney test). Plasma ROOH concentrations in the CAPD patients were increased but not significantly higher than controls (5.36 +/- 3.56 microM versus 4.25 +/- 2.05 microM). By contrast, no differences in ROOH levels were found between controls and predialysis patients. There was no difference in plasma thiobarbituric acid reactive substances (TBARS) between control and the three CRF groups. Absolute and cholesterol standardised plasma alpha-tocopherol levels were lower in the patients (whether they were on dialysis or not) than in the controls (18.62 +/- 6.88 microM versus 22.73 +/- 5.33 microM; P < 0.01 and 1.99 +/- 1.88 microM/mM versus 5.25 +/- 1.0 microM/mM; P < 0.0005, respectively). This study provides direct evidence that enhanced oxidative stress in CRF patients is related to the dialysis treatment rather than the disease itself. Further studies will be necessary to establish the relationships between plasma measures of oxidative stress and cardiovascular complications in CRF patients under dialysis and whether treatment with antioxidants may reduce oxidative stress or reverse adverse effects associated with dialysis.

The generation of the F(2)-isoprostane 8-epi-PGF(2alpha) by human platelets on collagen stimulation.

F(2)-isoprostanes are prostaglandin (PG) F(2)-like compounds formed via non-enzymatic peroxidation of arachidonic acid, although some F(2)-isoprostane production may be cyclo-oxygenase (COX)-mediated. Of these substances 8-epi-prostaglandin F(2)alpha (8-epi-PGF(2alpha)) has received the most attention as it induces vasoconstriction and mitogenesis, and influences pathophysiological mechanisms relevant to arterial disease. Using improved methods for F(2)-isoprostane determination we examined collagen-stimulated platelet production of F(2)-isoprostanes in platelet-rich plasma (PRP), distinguishing between the free and esterified forms of these substances. Collagen stimulation caused marked release to the plasma (platelet-poor; PPP) of free 8-epi-PGF(2alpha) (2 +/- 2 pg/mg platelet protein vs 174 +/- 53 pg/mg protein, control (i.e. non-stimulated) vs collagen-stimulated, P < 0.05) and of free 9alpha ,11alpha-PGF (37 +/- 19 pg/mg protein vs 1948 +/- 643 pg/mg protein, control vs stimulated, P < 0.05), a COX derived product. Neither free nor esterified 9alpha, 11beta-PGF and 9beta, 11alpha-PGF(2alpha) were detectable in control or collagen stimulated samples. Sample concentrations of the esters of 8-epi-PGF(2alpha) and 9alpha, 11alpha-PGF(2alpha) were unaltered by collagen stimulation. These data confirm a previous report that activated platelets release the F2-isoprostane 8-epi-PGF(2alpha), accompanying the release of a COX-derived product, 9alpha, 11alpha-PGF(2alpha).

Re-evaluation of the ferrous oxidation in xylenol orange assay for the measurement of plasma lipid hydroperoxides.

The ferrous oxidation in xylenol orange version 2 (FOX2) assay coupled with triphenylphosphine has recently been employed for the measurement of total plasma hydroperoxides (ROOHs). In this study, we have evaluated sample handling and the effect of storage conditions on ROOH levels in human plasma (n = 32). Mean level of ROOHs in fresh plasma was 8.35 +/- 3.09 mumol/l (range 4.03-19.5 mumol/l). Addition of butylated hydroxytoluene (BHT) immediately after sample collection had no effect on the concentration of ROOHs. Storage of samples at -70 degrees C for 6 weeks was associated with a variable degree of loss of detectable ROOHs. A mean ROOH level of 6.00 +/- 2.23 mumol/l (range 2.88-13.5 mumol/l) was recorded after 6 weeks of storage at -70 degrees C. There was no difference in the mean level of ROOHs between samples stored for 6 and 60 weeks at -70 degrees C. Inclusion of BHT had no effect on the stability of plasma ROOHs during prolonged storage. Intra-assay coefficients of variation were < 12%, with the lowest variation in fresh samples (7.6%). In conclusion, these results suggest that the FOX2 assay should be a useful tool for measurement of ROOHs in fresh plasma samples but not in stored samples.

Hydroperoxides in plasma are reduced by intensified insulin treatment. A randomized controlled study of IDDM patients with microalbuminuria.

OBJECTIVE: An association between reactive oxygen species and diabetic micro- and macrovascular complications has been proposed. In the present study, we have examined the effect of an improved blood glucose control on plasma levels of hydroperoxides in patients with IDDM. RESEARCH DESIGN AND METHODS: Subjects included 30 young IDDM patients with microalbuminuria who were randomized to receive either continuous subcutaneous insulin infusion (CSII) by a portable insulin pump (n = 15) or conventional insulin treatment (CIT) (n = 15) for 24 months. Plasma levels of hydroperoxides were measured by the ferrous oxidation with Xylenol Orange, version 2 (FOX2) assay. This method measures total lipid hydroperoxides and, unlike other methods, does not suffer from extraction losses. RESULTS: The mean HbA1c level was lower in the CSII group at the end of the study than in the CIT group: (mean [95% CI]) 8.6 (8.1-9.1) vs. 9.6 (9.0-10.3)%, respectively (P < 0.002). The level of plasma hydroperoxides was very similar at the start of the study but was significantly lower in the CSII group compared with the CIT group at the end of the study: 2.9 (2.1-3.7) vs. 4.3 (3.2-5.4) mumol/l, respectively (P < 0.02). In the CSII group, hydroperoxides were reduced by 31% from baseline (P < 0.001), whereas there was no change in levels of hydroperoxides in the CIT group. Mean hydroperoxide levels correlated with mean HbA1c during the study (r = 0.39, P < 0.04). Hydroperoxide levels were associated with the levels of microalbuminuria (r = 0.45, P < 0.02). CONCLUSIONS: This study provides support for the hypothesis that hyperglycemia is an important factor in the generation of hydroperoxides, and, thus, reactive oxygen species, in the circulation of IDDM patients.