Silvia Lonati

Oxidative imbalance in patients with mild cognitive impairment and Alzheimer's disease

Increasing evidence supports a role of oxidative imbalance, characterized by impaired antioxidant enzymatic activity and increased reactive oxygen species (ROS) production, in mild cognitive impairment (MCI) and Alzheimers disease (AD) pathogenesis. Hyperhomocysteinemia, another risk factor for AD, also contributes to oxidative damage. Plasma total homocysteine (tHcy) and ROS levels, and total antioxidant capacity (TAC) were determined in 71 AD, 36 MCI and 28 vascular dementia (VaD) patients as well as in 44 age-matched controls. tHcy levels were significantly increased in patients with AD and VaD an a trend towards an increase in multiple domain MCI was observed. TAC was significantly decreased in AD as well as MCI, but not in VaD patients. In AD patients, a negative correlation was found between TAC and disease duration. ROS levels did not differ among groups, but were correlated with age. In conclusion, a pattern characterized by increased tHcy levels and decreased TAC is present in AD as well as MCI patients. While increased tHcy levels were also found in VaD, TAC modifications occur specifically in AD. ROS levels appear to be correlated with age rather than with a specific dementing disorder, thus leading to the hypothesis that oxidative imbalance observed in AD could be due to a decreased TAC.

Oxidative stress in kidney transplant patients.

Background. Little information is available about the role of oxidative stress in renal transplant patients. To evaluate the prevalence and severity of oxidative stress in renal transplantation, the authors conducted a cross-sectional study. Methods. In 112 cadaver or living-donor kidney transplant recipients with a follow-up of at least 6 months and with plasma creatinine less than or equal to 2.5 mg/dL, complete blood count, serum vitamin B12, serum folate (s-F), reactive oxygen species (ROS), thiol groups (−SH), total antioxidant activity (TAOC), serum homocysteine (Hcy), and intraerythrocyte folate (ery-F) were measured. Results. The mean levels of Hcy (21.1 &mgr;M vs. <10 &mgr;M), ROS (302.7 U. Carr (Carratelli units) vs. 250–300 U. Carr), and TAOC (410.6 &mgr;mol/HclO/mL vs. >350 &mgr;mol/HclO/mL), were higher than the reference interval, whereas −SH groups, vitamin B12, s-F, and ery-F were within the normal range. In the multivariate model, plasma creatinine (P =0.0062), vitamin B12 (P =0.0121), and TAOC (P =0.0007) were independently associated with oxidative stress. At multiple regression analysis, −SH groups and ROS were directly and inversely related to hematocrit (P =0.0007 and P =0.0073). There was also a negative correlation between −SH groups and blood pressure levels (P =0.0095). Conclusions. Renal transplant patients have a pattern of increased oxidant stress that is counterbalanced by an enhancement of the antioxidant mechanisms. Besides the well-known risk factors, the authors found that anemia is an independent risk factor for an increase of ROS. Further studies are needed to evaluate whether the correction of anemia might prevent or reduce the oxidative stress in renal transplant patients.

Free and total plasma malondialdehyde in chronic renal insufficiency and in dialysis patients

BACKGROUND Available data about oxidative status in patients with end-stage renal disease (ESRD) or on dialysis are contradictory. The present cross-sectional study aimed to investigate the role of renal insufficiency and dialysis on lipid peroxidation. To separate the effects of uraemia from dialysis-induced stress, we enrolled 26 patients with renal insufficiency on conservative treatment (ESRD), 23 on peritoneal dialysis (PD), 30 on haemodialysis (HD) and 30 controls. METHODS Plasma malondialdehyde (MDA) levels, both total (tMDA) and free (fMDA), were measured as indexes of oxidative stress by gas chromatography-mass spectrometry. Bound MDA (bMDA) levels were calculated as the difference between tMDA and fMDA. RESULTS Total and bMDA concentrations were significantly higher in patients than in controls (ESRD > HD > PD). In PD and HD patients, fMDA levels were similar and significantly higher than in ESRD. Multivariate analysis, with tMDA, fMDA and bMDA as dependent variables, showed similar and significant tMDA and bMDA relations with residual renal function (t = -2.160, P = 0.035) and albumin (t = -2.049, P = 0.045). Erythropoietin dose affected only fMDA values (t = -2.178, P = 0.034). CONCLUSIONS Free and bMDA concentrations identified different MDA patterns. Bound MDA, not excreted by kidneys, accounts alone for high tMDA concentrations in ESRD patients, while both fMDA and bMDA contribute to tMDA values in dialysis patients. These findings show that increased tMDA could be indicative not only of recent lipid peroxidation, and they also highlight the importance of evaluating free, bound and total MDA in patients with reduced renal function in order to assess their oxidative status.

Analytical performance and method comparison study of the total homocysteine fluorescence polarization immunoassay (FPIA) on the AxSYM analyzer

Abstract A fluorescence polarization immunoassay (FPIA) has been commercially released for routine large-scale testing of total homocysteine (tHcy) on the AxSYM analyzer. We evaluated the analytical performance of the AxSYM tHcy FPIA and compared it with the well established high-performance liquid chromatography (HPLC) and IMx tHcy FPIA methods. Homocysteine concentrations were measured by AxSYM and IMx tHcy FPIA and by a rapid isocratic HPLC method with fluorescence detection. Coefficient of variation (CV) of total imprecision for AxSYM tHcy was ≤5%, mean dilution recovery 102%, analytical sensitivity 0.70 μmol/l and linearity was good up to 1:8 dilution. Spearman rank correlations, rho, were 0.83 (p<0.0001) for AxSYM vs. HPLC, 0.97 (p<0.0001) for AxSYM vs. IMx and 0.83 (p <0.0001) for IMx vs. HPLC. Passing and Bablok regression Y-intercepts and slopes were: 2.944/0.937 (AxSYM vs. HPLC), −0.367/1.142 (AxSYM vs. IMx) and 2.632/0.805 (IMx vs. HPLC). Corresponding mean differences (AxSYM-Comparison Assay) recorded over a 5–50 μmol/l measured range were 1.80, −0.73 and 2.53 μmol/l. AxSYM tHcy FPIAs first rate precision, supported by the complete automation of the AxSYM analyzer, makes it fit for routine use and suitable for laboratories requiring homocysteine high-throughput testing capabilities.

Determination of serum holotranscobalamin concentrations with the AxSYM active B12 assay: cut-off point evaluation in the clinical laboratory

Abstract Background: A reliable early marker is required for diagnosis of cobalamin deficiency. We calculated an appropriate holotranscobalamin (HoloTC) cut-off point for identifying cobalamin deficiency using an immunoenzymatic assay. Methods: Determination of the cut-off threshold and correlation between HoloTC and the other diagnostic parameters routinely used for vitamin B12 deficiency [total vitamin B12 (tB12), folate, homocysteine] were measured in 250 routine blood specimens from 107 men (mean age 59.0±18.8 years) and 143 women (mean age 54.2±23.1 years). The inclusion criterion was serum tB12 concentration ≤221 pmol/L. Results: Analytical performance results agreed with those reported by others. A weak correlation (R=0.42) was found between HoloTC and tB12. A 40 pmol/L cut-off threshold was chosen for HoloTC and the associated sensitivity and specificity was 0.86 and 0.66, respectively. Out of 250 tested samples, 126 showed tB12 concentrations 139–221 pmol/L (gray zone, GZ) and 124 had tB12 concentrations <139 pmol/L (low, L). Values less than the cut-off for HoloTC were present in 68.2% and 37.9% of cases in the GZ and L group, respectively (p<0.01), and in 53.2% of subjects. Conclusions: Our results confirmed the analytical reliability of the AxSYM HoloTC assay. The method is adequate for routine use and a cut-off threshold of 40 pmol/L is appropriate for assessing cobalamin deficiency in populations with reduced tB12 values. Clin Chem Lab Med 2010;48:249–53.

Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate: a pilot study.

Abstract Background: Cigarette smoking, a cardiovascular risk factor leading to oxygen free radical formation, is involved in the development of serious pathological conditions. On the other hand, a healthy diet and adequate supplementation can help prevent many diseases. The aim of our study was to evaluate in healthy light smokers the effects of supplementation with mixed fruit and vegetable juice powder concentrate on homocysteine metabolism and oxidative status. Methods: In this pilot study, 32 healthy volunteers, 16 light smokers and 16 non-smokers, on twice daily supplementation were monitored at time zero and after 30days. Plasma homocysteine, and serum vitamin B12 and folate concentrations were measured by immunoenzymatic assays; reactive oxygen species, total antioxidant capacity and thiol groups by spectrophotometric methods; and total and free malondialdehyde concentrations by gas chromatography-mass spectrometry with isotopic dilution. Results: Baseline free malondialdehyde concentrations were significantly higher in smokers than in non-smokers and normalised after 30-day supplementation. Baseline results for all the other parameters remained unchanged after supplementation, with no significant differences between smokers and non-smokers. Conclusion: This is the first study showing a significant decrease in free malondialdehyde levels in light smokers after 1-month phytonutrient supplementation.

Circulating and progenitor endothelial cells are abnormal in patients with different types of von Willebrand disease and correlate with markers of angiogenesis.

von Willebrand disease (VWD) is the most common inherited bleeding disorder and is caused by quantitative or qualitative defects of von Willebrand factor (VWF). VWF, synthesized by endothelium and megakaryocytes (MK), circulates in plasma and is present in subendothelium and platelets. Circulating endothelial cells (CEC) and progenitor endothelial cells (EPC) have been recently proposed as markers of peripheral and bone marrow‐derived angiogenesis. To evaluate the association of CEC/EPC with known inherited defects of cellular and circulating VWF, we have measured the number of CEC/EPC together with cytokines involved in angiogenesis in different VWD types. A group of 74 patients was composed by the following VWD types: VWD1 (n = 22), VWD2A (n = 9), VWD2B (n = 19), VWD2M (n = 17), and VWD3 (n = 7). Healthy individuals (n = 20) were used as controls. CEC (CD146+, CD31+, and CD45−) and EPC (CD34+, CD133+, and CD45−) were evaluated by flow cytometry. Circulating serum levels of VEGF, E‐selectin, P‐selectin, EPO, and TPO were determined by ELISA. CEC, VEGF, E‐selectin, and EPO were higher and EPC lower in VWD patients than in controls (P < 0.01). Among the five groups of VWD patients and controls, a significant difference was found for CEC (one‐way ANOVA: P = 0.005), EPC (P = 0.001), E‐Selectin (P < 0.0001), EPO (P = 0.021), and TPO (P = 0.004): the latter was high in VWD3 patients. In VWD1, we found an inverse relationship between CEC and VWF:Ag levels (P = 0.048; R2 = 0.19). Based on these data, CEC are increased in VWD and are associated with the high levels of cytokines involved in angiogenesis (up‐regulation). EPC are decreased, suggesting down‐regulation of bone marrow‐derived angiogenesis in VWD. Am. J. Hematol. 2011.

Oxidative stress is increased in primary and post-polycythemia vera myelofibrosis

OBJECTIVE To determine if increased cell turnover in chronic myeloproliferative disorders can lead to hyperhomocysteinemia as a result of folate and/or cobalamin depletion, and contribute to oxidative stress. MATERIALS AND METHODS The clinical role of oxidative stress was investigated by measuring reactive oxygen species (ROS), total antioxidant capacity (TAC), and total homocysteine (tHcy), folate, cobalamin, and holotranscobalamin (HoloTC) levels in 51 chronic myeloproliferative disorders patients (male-to-female ratio: 1.1; median age: 64 years; range, 40-84 years), including 42 with primary myelofibrosis and 9 with post-polycythemia vera myelofibrosis. RESULTS Myelofibrotic patients had higher tHcy (p = 0.0201) and an unbalanced oxidative status (higher ROS and lower TAC levels; p < 0.0001) than controls. Presence of diabetes or another neoplasia was associated with higher ROS levels (p < 0.05), splenomegaly, hepatomegaly, and peripheral blasts with lower HoloTC levels (p < 0.005). The most severe forms of myelofibrosis (2-3) were associated with lower TAC (p = 0.045) and HoloTC levels (p = 0.017). Patients with Janus kinase-2 mutations had lower HoloTC levels (p = 0.0059). HoloTC deficiency was more frequently associated with Janus kinase-2 homozygosity (p < 0.0003). CONCLUSIONS Our findings suggest that the determination of HoloTC, tHcy, ROS concentrations, and TAC, can identify latent cobalamin deficiency and provide a rational basis for correcting the increased oxidation associated with disease progression.

Inhibitory activity of a synthetic pentapeptide on leukaemic myelopoiesis both in vitro and in vivo in rats

The synthetic pentapeptide pGlu‐Glu‐Asp‐Cys‐Lys has recently been proposed as the active component of a granulocyte‐derived inhibitor of normal haematopoiesis. We investigated its biological activity on leukaemic myelopoiesis both in vitro and in vivo in rats. Three different human permanent myeloid leukaemic cell lines (HL60, KG1, ML3) and a rat transplantable acute myeloid leukaemia (Shay leukaemia) were studied. Neither HL60 nor KG1 were sensitive to the peptide whereas a consistently reproducible inhibition of 3H‐TdR uptake was observed in ML3 cells. This effect was not due to a unspecific toxic action on target cells and was spontaneously reversible. When injected i.p. twice daily at an appropriate concentration in rats bearing Shay leukaemia, the peptide caused a significant increase in survival. Our results therefore indicate that the synthetic pentapeptide studied inhibits not only normal but also leukaemic myelopoiesis.

Erythrocyte ferritin concentration: Analytical performance of the immunoenzymatic IMx-Ferritin (Abbott) assay

Abstract Together with serum ferritin, erythrocyte ferritincan be a valuable diagnostic tool for evaluating the degree of impaired iron metabolism in different diseases. We collected peripheral blood samples from 64 subjects (22 healthy volunteers, 20 patients with hereditary hemochromatosis, and 22 patients on regular hemodialysis with secondary anemia) to evaluate whether an immunoenzymatic method generally used for serum ferritin can also be used to determine erythrocyte ferritin levels under various conditions of body iron status. Serum and erythrocyte ferritin levels were assayed in parallel using a microparticle enzyme immunoassay (MEIA) IMx-Ferritin kit and an IMx analyzer. The inter-assay imprecision of the serum and erythrocyte ferritin assays was 4.9% and 5.05%, the intra-assay imprecision was 2.2% and 2.3%, and the mean recovery was 102% (range 96–105%) and 101% (range 99–105%), respectively. Both serum and erythrocyte ferritin assays showed a detection limit of 1μg/L and good linearity (R 2=0.99) in the intervals 13.9–443 and 3.9–135.6μg/L, respectively. Our findings demonstrate that the IMx-Ferritin assay currently used to measure serum ferritin levels can also be adopted to measure erythrocyte ferritin insofar as it clearly discriminates high and low erythrocyte ferritin levels in cases of both iron overload and deficiency.