Whey proteins inhibit food intake and tend to improve oxidative balance in obese zucker rats.

BACKGROUND/AIMS: Whey proteins (WP), obtained from milk after casein precipitation, represent a heterogeneous group of proteins. WP are reported to inhibit food intake in diet-induced experimental obesity; WP have been proposed as adjuvant therapy in oxidative stress-correlated pathologies. This work evaluates the effects of WP in comparison with casein, as a source of alimentary proteins, on food intake, weight growth and some indexes of oxidative equilibrium in Zucker Rats, genetically prone to obesity. METHODS: We monitored food intake and weight of Zucker Rats during the experiment, and some markers of oxidative equilibrium. RESULTS: WP induced significant decrease of food intake in comparison to casein (WP 80.41 ± 1.069 ml/day; CAS: 88.95 ± 1.084 ml/day; p < 0.0005). Body weight growth was slightly reduced, and the difference was just significant (WP 128.2 ± 6.56 g/day; CAS 145.2 ± 3.29 g/day; p = 0.049), while plasma HNE level was significantly lower in WP than in CAS (WP 41.2 ± 6.3 vs CAS 69.61 ± 4.69 pmol/ml, p = 0.007). Mild amelioration of oxidative equilibrium was indicated by a slight increase of total glutathione both in the liver and in the blood and a significant decrease of plasma 4-hydroxynonenal in the group receiving WP. CONCLUSIONS: The effect of WP on food intake and weight growth in Zucker Rats is particularly noteworthy since the nature of their predisposition to obesity is genetic; the possible parallel amelioration of the oxidative balance may constitute a further advantage of WP since oxidative stress is believed to be interwoven to obesity, metabolic syndrome and their complications.

Vitamins D3 and K2 may partially counterbalance the detrimental effects of pentosidine in ex vivo human osteoblasts.

Osteoporosis is a metabolic multifaceted disorder, characterized by insufficient bone strength. It has been recently shown that advanced glycation end products (AGEs) play a role in senile osteoporosis, through bone cell impairment and altered biomechanical properties. Pentosidine (PENT), a wellcharacterized AGE, is also considered a biomarker of bone fracture. Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D3, are prerequisites for optimal osteoblasts functioning. Vitamin K2 is known to enhance in vitro and in vitro vitamin D-induced bone formation. The aim of the study was to assess the effects of Vitamins D3 and K2 and PENT on in vitro osteoblast activity, to convey a possible translational clinical message. Ex vivo human osteoblasts cultured, for 3 weeks, with vitamin D3 and vitamin K2 were exposed to PENT, a well-known advanced glycoxidation end product for the last 72 hours. Experiments with PENT alone were also carried out. Gene expression of specific markers of bone osteoblast maturation [alkaline phosphatase, ALP; collagen I, COL Iα1; and osteocalcin (bone-Gla-protein) BGP] was measured, together with the receptor activator of nuclear factor kappa-B ligand/osteoproteregin (RANKL/OPG) ratio to assess bone remodeling. Expression of RAGE, a well-characterized receptor of AGEs, was also assessed. PENT+vitamins slightly inhibited ALP secretion while not affecting gene expression, indicating hampered osteoblast functional activity. PENT+vitamins up-regulated collagen gene expression, while protein secretion was unchanged. Intracellular collagen levels were partially decreased, and a significant reduction in BGP gene expression and intracellular protein concentration were both reported after PENT exposure. The RANKL/OPG ratio was increased, favouring bone reabsorption. RAGE gene expression significantly decreased. These results were confirmed by a lower mineralization rate. We provided in vitro evidence that glycoxidation might interfere with the maturation of osteoblasts, leading to morphological modifications, cellular malfunctioning, and inhibition of the calcification process. However, these processes may be all partially counterbalanced by vitamins D3 and K2. Therefore, detrimental AGE accumulation in bone might be attenuated and/or reversed by the presence or supplementation of vitamins D3 and K2.

4-Hydroxyhexenal and 4-hydroxynonenal are mediators of the anti-cachectic effect of n-3 and n-6 polyunsaturated fatty acids on human lung cancer cells.

Cachexia, the most severe paraneoplastic syndrome, occurs in about 80% of patients with advanced cancer; it cannot be reverted by conventional, enteral, or parenteral nutrition. For this reason, nutritional interventions must be based on the use of substances possessing, alongside nutritional and energetic properties, the ability to modulate production of the pro-inflammatory factors responsible for the metabolic changes characterising cancer cachexia. In light of their nutritional and anti-inflammatory properties, polyunsaturated fatty acids (PUFAs), and in particular n-3, have been investigated for treating cachexia; however, the results have been contradictory. Since both n-3 and n-6 PUFAs can affect cell functions in several ways, this research investigated the possibility that the effects of both n-3 and n-6 PUFAs could be mediated by their major aldehydic products of lipid peroxidation, 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE), and by their anti-inflammatory properties. An "in vitro" cancer cachexia model, consisting of human lung cancer cells (A427) and murine myoblasts (C2C12), was used. The results showed that: 1) both n-3 and n-6 PUFAs reduced the growth of lung cancer cells without causing cell death, increased lipid peroxidation and Peroxisome Proliferator-Activated Receptor (PPAR)α, and decreased TNFα; 2) culture medium conditioned by A427 cells grown in the absence of PUFAs blocked myosin production and the differentiation of C2C12 muscle cells; conversely, muscle cells grown in culture medium conditioned by the same cells in the presence of PUFAs showed myosin expression and formed myotubes; 3) adding HHE or HNE directly to C2C12 cells maintained in culture medium conditioned by A427 cells in the absence of PUFAs stimulated myosin production and myotube formation; 4) putative consensus sequences for (PPARs) have been found in genes encoding fast isoforms of myosin heavy chain, by a bioinformatics approach. The overall results show, first, the ability of both n-3 and n-6 PUFAs and their lipid peroxidation products to prevent the blocking of myosin expression and myotube formation caused in C2C12 cells by medium conditioned by human lung tumour cells. The C2C12 cell differentiation can be due to direct effect of lipid peroxidation products, as evidenced by treating C2C12 cells with HHE and HNE, and to the decrease of pro-inflammatory TNFα in A427 cell culture medium. The presence of consensus sequences for PPARs in genes encoding the fast isoforms of myosin heavy chain suggests that the effects of PUFAs, HHE, and HNE are PPAR-mediated.

RAGE Expression and ROS Generation in Neurons: Differentiation versus Damage.

RAGE is a multiligand receptor able to bind advanced glycation end-products (AGEs), amphoterin, calgranulins, and amyloid-beta peptides, identified in many tissues and cells, including neurons. RAGE stimulation induces the generation of reactive oxygen species (ROS) mainly through the activity of NADPH oxidases. In neuronal cells, RAGE-induced ROS generation is able to favor cell survival and differentiation or to induce death through the imbalance of redox state. The dual nature of RAGE signaling in neurons depends not only on the intensity of RAGE activation but also on the ability of RAGE-bearing cells to adapt to ROS generation. In this review we highlight these aspects of RAGE signaling regulation in neuronal cells.

Role of Nrf2, HO-1 and GSH in Neuroblastoma Cell Resistance to Bortezomib.

The activation of Nrf2 has been demonstrated to play a crucial role in cancer cell resistance to different anticancer therapies. The inhibition of proteasome activity has been proposed as a chemosensitizing therapy but the activation of Nrf2 could reduce its efficacy. Using the highly chemoresistant neuroblastoma cells HTLA-230, here we show that the strong reduction in proteasome activity, obtained by using low concentration of bortezomib (BTZ, 2.5 nM), fails in reducing cell viability. BTZ treatment favours the binding of Nrf2 to the ARE sequences in the promoter regions of target genes such as heme oxygenase 1 (HO-1), the modulatory subunit of γ-glutamylcysteine ligase (GCLM) and the transporter for cysteine (x-CT), enabling their transcription. GSH level is also increased after BTZ treatment. The up-regulation of Nrf2 target genes is responsible for cell resistance since HO-1 silencing and GSH depletion synergistically decrease BTZ-treated cell viability. Moreover, cell exposure to all-trans-Retinoic acid (ATRA, 3 µM) reduces the binding of Nrf2 to the ARE sequences, decreases HO-1 induction and lowers GSH level increasing the efficacy of bortezomib. These data suggest the role of Nrf2, HO-1 and GSH as molecular targets to improve the efficacy of low doses of bortezomib in the treatment of malignant neuroblastoma.

The Nrf2/HO-1 Axis in Cancer Cell Growth and Chemoresistance.

The transcription factor, nuclear factor erythroid 2 p45-related factor 2 (Nrf2), acts as a sensor of oxidative or electrophilic stresses and plays a pivotal role in redox homeostasis. Oxidative or electrophilic agents cause a conformational change in the Nrf2 inhibitory protein Keap1 inducing the nuclear translocation of the transcription factor which, through its binding to the antioxidant/electrophilic response element (ARE/EpRE), regulates the expression of antioxidant and detoxifying genes such as heme oxygenase 1 (HO-1). Nrf2 and HO-1 are frequently upregulated in different types of tumours and correlate with tumour progression, aggressiveness, resistance to therapy, and poor prognosis. This review focuses on the Nrf2/HO-1 stress response mechanism as a promising target for anticancer treatment which is able to overcome resistance to therapies.

Effects of competition on acute phase proteins and lymphocyte subpopulations - oxidative stress markers in eventing horses.

The aim of the study was to evaluate markers of the acute phase response (APR) in eventing horses by measuring acute phase proteins (APP) (haptoglobin, Hp, and serum amyloid A, SAA), lysozyme, protein adducts such as pentosidine-like adducts (PENT), malondialdehyde adducts (MDA), hydroxynonenal adducts (HNE) and total advanced glycation/glycoxidation end products (AGEs), complete blood count and lymphocyte subpopulations (CD4+, CD8+ and CD21+) both at rest and at the end of an eventing competition. Blood samples were collected from eight Warmblood horses (medium age 10 ± 3) during an official national 2-day event competition at rest (R) and 10 min after the arrival of the cross-country test on the second day. Exercise caused a significant increase in red blood cell number, haemoglobin, packed cell volume, neutrophils, white blood cell and lymphocyte number; however, these values remained within the normal range. The CD4+ and CD8+ cells significantly increased, whereas the CD21+ lymphocytes decreased; a significant increase in serum SAA, lysozyme and protein carbonyl derivates was also observed. Two-day event causes significant changes in APR markers such as lysozyme, protein carbonyl derivates (HNE, AGEs, PENT) and lymphocyte subpopulations. The data support the hypothesis that 2-day event may alter significantly APR markers. Limitations of the study were the relatively small sample size and sampling time conditioned by the official regulations of the event. Therefore, further studies are needed to investigate the time required for recovery to basal values in order to define the possible effects on the immune function of the athlete horse.

p38MAPK inhibition: a new combined approach to reduce neuroblastoma resistance under etoposide treatment.

Neuroblastoma (NB) is the second most common solid pediatric tumor and is characterized by clinical and biological heterogeneity, and stage-IV of the disease represents 50% of all cases. Considering the limited success of present chemotherapy treatment, it has become necessary to find new and effective therapies. In this context, our approach consists of identifying and targeting key molecular pathways associated with NB chemoresistance. This study has been carried out on three stage-IV NB cell lines with different status of MYCN amplification. Cells were exposed to a standard chemotherapy agent, namely etoposide, either alone or in combination with particular drugs, which target intracellular signaling pathways. Etoposide alone induced a concentration-dependent reduction of cell viability and, at very high doses, totally counteracted cell tumorigenicity and neurosphere formation. In addition, etoposide activated p38 mitogen-activated protein kinase (MAPK), AKT and c-Jun N-terminal kinase. Pre-treatment with SB203580, a p38MAPK inhibitor, dramatically sensibilized NB cells to etoposide, strongly reducing the dosage needed to inhibit tumorigenicity and neurosphere formation. Importantly, SB203580-etoposide cotreatment also reduced cell migration and invasion by affecting cyclooxygenase-2, intercellular adhesion molecule-1, C-X-C chemokine receptor-4 and matrix metalloprotease-9. Collectively, our results suggest that p38MAPK inhibition, in combination with standard chemotherapy, could represent an effective strategy to counteract NB resistance in stage-IV patients.

Doloplus-2 pain assessment: an effective tool in patients over 85 years with advanced dementia and persistent pain.

Persistent pain in the elderly with dementia is neglected and effective pain diagnostic tools still lack. The aim of the study was to re-assess pain after 1 year in a group of elderly with dementia, resident in a Nursing Home. Doloplus-2 scale was adopted to detect pain and to evaluate its adequacy to address analgesia. Thirty-six out of 90 residents were affected by moderate-severe dementia and 23 patients with persistent pain were enrolled in the study. The results showed a significant reduction of Doloplus-2 score in the observational period (p <0.001) and of its mean score below the pain threshold (p <0.0001). This case analysis confirmed the clinical utility of Doloplus-2 to detect pain in patients with very advanced age and with severe dementia. The tool also confirmed its handiness and clinical applicability by nurses in order to support a systematic pain assessment in long term care.

Metalloproteinases and advanced glycation end products: coupled navigation in atherosclerotic plaque pathophysiology?

Matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and inflammatory cytokines, such as interleukin-1 (IL-1), are considered markers of evolution and/or instability of atherosclerotic plaques. Accumulation of Advanced Glycation Endproducts (AGE) is a well known phenomenon in diabetes and has also been considered in the pathogenesis of atherosclerosis. Aim of the present study was to analyse the levels of pentosidine, a fluorescent AGE, and to evaluate the expression of MMP-2, TIMP-3, and IL-1 in an ex vivo model of human advanced atherosclerotic plaques. We intended to test the possible correlation between pentosidine and markers of ECM remodelling and inflammation in the atherosclerotic process, and to investigate if classic risk factors, such as diabetes and hypertension, influenced these biochemical parameters. We found that diabetic plaques showed higher level of pentosidine, as expected, but much lower, or even undetectable, expression levels of MMP-2 and TIMP-3; IL-1 expression was not different between diabetic and non diabetic plaques. Hypertension did not influence any of these parameters. Although the statistical correlations between the expression of the considered genes and pentosidine did not reach significance, slight negative trends were noted between TIMP-3 and IL-1 expression vs. pentosidine content. We suggest that in mature diabetic plaques AGE accumulation can exert stabilizing effects on matrix proteins, while scanty cell presence leads to poor capacity of reactive responses, such as remodelling and inflammation.

Biochemical and morphologic effects after extended liver resection in rats: preliminary results.

After hepatic resection and transplantation with a partial graft, death and regeneration of the hepatocytes coexist in the liver. However, when the functional liver mass is inadequate to ensure a proper balance between regeneration vs functional and metabolic demands, small-for-size syndrome develops. We assessed the early effects of extended hepatic resection on liver function in a rat model. Six male Sprague-Dawley rats underwent 80% resection of the liver, and 6 rats served as a control group. At 6 hours after resection, blood samples were obtained from the hepatic vein for measurement of reduced glutathione (GSH), oxidized glutathione (GSSG), and hepatic venous oxygen saturation (Shvo(2)), and for standard liver function tests including determination of concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase, and total bilirubin. The remnant lobe was removed for GSH assay and histopathologic analysis. In the resection group, values were significantly higher for ALT (P = .002), AST (P = .002), and Shvo(2) (P = .01), whereas a significant decrease was observed for blood GSH (P = .009) but not liver GSH. Also in the resection group, we observed characteristic hepatocyte vacuolization with a gradient from periportal acinar zone 1 to the centrolobular area, the presence of hemorrhagic necrosis, and several leukocyte adhesions. The Shvo(2) and GSH data suggest early alteration of oxygen metabolism, as demonstrated by the reduction in oxygen uptake and decreased liver GSH secretion, with preservation of hepatic GSH. Mitochondrial dysfunction and oxidative injury seem to have a crucial role in early onset of liver damage.

Whey proteins influence hepatic glutathione after CCl4 intoxication.

Whey proteins (WP) are known to contain more cysteine than casein (CAS), so it is suggested that they should ameliorate the oxidative equilibrium in the organisms. To evaluate the influence of a WP-based diet on liver glutathione (GSH) content, male Sprague-Dawley rats were fed for 3 weeks a balanced liquid diet containing either WP or CAS as main source of protein. Liver GSH content was evaluated at the end of the treatment by high performance liquid chromatography (HPLC), both in basal conditions and after oxidative stress induced by CCl4 acute intoxication. In basal conditions, WP diet significantly increased hepatic GSH in comparison to CAS diet. After CCl4 intoxication, hepatic GSH was negligibly increased in CAS group, while its increase was much more marked in WP group, so that the difference between the two diets was significant; this suggests that WP provided rats with better ability to increase their GSH synthesis in case of need.

Induction of heme oxygenase 1 in liver of spontaneously diabetic rats.

It has been suggested that diabetes induces an increase in oxidative stress; the increased expression of heme-oxygenase 1 (HO-1) in liver is believed to be a sensitive marker of the stress response. The aim of this study was to examine whether diabetes is able to induce HO-1 expression in liver. The specific mRNA was amplified by RT/PCR and calibrated with amplified beta-actin mRNA. The mRNA HO-1 levels in the liver of spontaneously diabetic rats were increased by 1.8 fold compared with non diabetics; this supports the hypothesis of weak but significant oxidative damage due to chronic hyperglycaemia. This work represents the first in vivo study exploring the semi-quantitative expression of HO-1 in the liver of spontaneously diabetic rats.

Plasma protein oxidation and antioxidant defense during aging.

BACKGROUND: Oxidative stress is an important process that occurs in vivo during aging and is considered one of the main causes of molecular damage to cellular and tissue structures. These changes can accumulate in biological structures during aging. OBJECTIVE: The aim of this work is to evaluate plasma protein oxidative damage, measured as carbonyl groups content, and the concentration of some antioxidant molecules (vitamins and carotenoids) in 122 healthy volunteers (50 males and 72 females), 25 to 89 years old. RESULTS: Total plasma proteins slightly decreased with age, but the level of carbonyl groups was similar in the adult (< 65 years) and in the old, and was similar in both sexes. Plasma concentration of antioxidant molecules such as alpha-tocopherol, beta-carotene and other carotenoids, increased with age and correlated with the level of lipoproteins; plasma total cholesterol and triglycerides were significantly correlated with age as well. CONCLUSIONS: The surprisingly normal level of plasma protein carbonyl groups in our older subjects suggests two possibilities: a) the older people in our study are healthy and free from pathologies because of better protection against oxidative injury during their lifetimes, i.e., they maintained low-level oxidative damage on plasma proteins; or b) the level of carbonyl groups is normal because of the high turnover in plasma: the oxidized circulating proteins are preferentially and quickly removed; in this case oxidative damage is not discernible in plasma proteins but may proceed silently in other tissues.

Lipoperoxidation is selectively involved in progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by extensive neurofibrillary tangle (NFT) formation and neuronal loss in selective neuronal populations. Currently, no clues to the biological events underlying the pathological process have emerged. In Alzheimer disease (AD), which shares with PSP the occurrence of NFTs, advanced glycation end products (AGEs) as well as oxidation adducts have been found to be increased in association with neurofibrillary pathology. The presence and the amount of lipid and protein oxidation markers, as well as of pyrraline and pentosidine. 2 major AGEs, was assessed by biochemical, immunochemical, and immunocytochemical analysis in midbrain tissue from 5 PSP cases, 6 sporadic AD cases, and 6 age-matched control cases. The levels of 4-hydroxynonenal (HNE) and thiobarbituric acid reactive substances (TBARS), 2 major products of lipid peroxidation, were significantly increased by 1.6-fold (p < 0.04) and 3.9-fold (p < 0.01), respectively, in PSP compared with control tissues, whereas in AD only TBARS were significantly increased. In PSP tissue the intensity of neuronal HNE immunoreactivity was proportional to the extent of abnormal aggregated tau protein. The amount of protein oxidation products and AGEs was instead similar in PSP and control tissues. In AD, a higher but not significant level of pyrraline and pentosidine was measured, whereas the level of carbonyl groups was doubled. These findings indicate that in PSP, unlike in AD, lipid peroxidation is selectively associated with NFT formation. The intraneuronal accumulation of toxic aldehydes may contribute to hamper tau degradation, leading to its aggregation in the PSP specific abnormal filaments.

Scanning force microscopy reveals structural alterations in diabetic rat collagen fibrils: role of protein glycation.

BACKGROUND: The main functional property of collagen is to provide a supporting framework to almost all tissues: the effects of non-enzymatic glycation on this protein are deleterious and in diabetes mellitus contribute to the mechanism of late complications. The aim of this work is to provide evidence by scanning force microscopy of modifications in collagen structure caused by high glucose concentration, in vivo and in vitro, and to correlate the data with markers of non-enzymatic glycation. METHODS: Tendon fibrils were obtained from the tails of 8-month-old rats (BB/WOR/MOL¿BB) which developed diabetes spontaneously at least 12 weeks before they were killed, and from diabetes-resistant rats of the same strain (BB/WOR/MOL¿WB). A scanning force microscope (SFM; Nanoscope III) equipped with a Contact Mode Head was used for imaging. Band interval, diameter and depth of D-band gap were measured in non-diabetic and diabetic tail tendon fibrils and in fibrils incubated with glucose (0.5 M for 2 weeks). Fructosamine was determined in the tendon fibrils by a colorimetric method and pentosidine was evaluated in acid-hydrolyzed samples by coupled reverse phase-ionic exchange column HPLC. RESULTS: Incubated fibrils revealed modifications in radius (228+/-5 nm) and gap depth (3.65+/-0.10 nm) that closely reproduce diabetes-induced damage (236+/-3 and 3.20+/-0.04 nm respectively) and were significantly different from the pattern seen in non-diabetic fibrils (151+/-1 and 2.06+/-0.03 nm; p<0.001). Both fructosamine and pentosidine were higher in diabetic (3.82+/-1.43 nmol/mg and 2.23+/-0.24 pmol/mg collagen respectively) and in glucose-incubated fibrils (9.27+/-0.55 nmol/mg and 5.15+/-0.12 pmol/mg collagen respectively) vs non-diabetic tendons (1.29+/-0.08 nmol/mg and 0.88+/-0.11 pmol/mg collagen respectively; p<0.01); during the time course of incubation, an early increase in fructosamine was seen, whereas pentosidine increased later. The D-band parameter was similar in all three groups, indicating that axial organization is not modified by non-enzymatic glycation. CONCLUSION: This is the first description obtained with SFM of diabetes-induced ultrastructural changes in collagen fibrils. Moreover, the data presented are consistent with the concept that chronic exposure of collagen to glucose in vivo or in vitro leads to similar structural modifications in collagen fibrils, probably through crosslinks. The correlation between morphologic parameters and both markers of glycation provides strong evidence for a crucial role of this non-enzymatic modification.

Detection of circulating antibodies against malondialdehyde-acetaldehyde adducts in patients with alcohol-induced liver disease.

Acetaldehyde and malonildialdehyde can form hybrid protein adducts, named MAA adducts that have strong immunogenic properties. The formation of MAA adducts in the liver of chronic alcohol-fed rats is associated with the development of circulating antibodies that specifically recognized these adducts. The aim of this study was to examine whether MAA adducts might participate in the immune response associated with human alcohol-induced liver disease. Circulating antibodies against MAA adducts were evaluated in 50 patients with alcohol-induced hepatitis or cirrhosis, in 40 patients with non-alcohol-induced liver disease, in 15 heavy drinkers without liver damage and in 40 healthy controls by enzyme-linked immunosorbent assays (ELISA). Immunoglobulin G (IgG) reacting with MAA-modified proteins were significantly increased in the patients with alcohol-induced cirrhosis or hepatitis. The individual levels of anti-MAA IgG in those patients were associated with the severity of liver damage. Anti-MAA antibodies were also positively correlated with the levels of IgG recognizing epitopes generated by acetaldehyde and malonildialdehyde. However, competitive inhibition experiments indicated that the anti-MAA antibodies were unrelated to those against acetaldehyde- or malonildialdehyde-derived antigens and mainly recognized a specific, cyclic MAA epitope. Some degree of immune reactivity towards MAA adducts was also observed in patients with non-alcohol-induced liver injury. However, competitive ELISA showed that the antigens recognized by these sera were not the cyclic MAA adducts. Altogether, these results showed the formation of MAA antigens during alcohol-induced liver disease and suggest their possible contribution to the development of immunologic reactions associated with alcohol-related liver damage.

Lipoperoxidation in hepatic subcellular compartments of diabetic rats.

It is known that an accumulation of lipoperoxidative aldehydes malondialdehyde (MDA) and 4-hydroxynonenal (HNE) takes place in liver mitochondria during aging. The existence and role of an increased extra- and intra-cellular oxidative stress in diabetes, an aging-accelerating disease, is currently under discussion. This report offers evidence that lipoperoxidative aldehydes accumulate in liver microsomes and mitochondria at a higher rate in spontaneously diabetic BB/WOR rats than in control non-diabetic animals (HNE content, diabetes vs. control: microsomes 80.6+/-19.9 vs. 25.75+/-3.6 pmol/mg prot, p = .024; mitochondria 77.4+/-15.4 vs. 26.5+/-3.5 pmol/mg prot, p = .0103). Liver subcellular fractions from diabetic rats, when exposed to the peroxidative stimulus ADP/Fe, developed more lipoperoxidative aldehydes than those from non diabetic rats (HNE amount, diabetes vs. control: microsomes 3.60+/-0.37 vs. 2.33+/-0.22 nmol/mg prot, p = .014; mitochondria 3.62+/-0.26 vs. 2.30+/-0.17 nmol/mg prot, p = .0009). Liver subcellular fractions of diabetic rats developed more fluorescent chromolipids related to HNE-phospholipid adducts, either after in vitro peroxidation (microsomes: p = .0045; mitochondria: p = .0023) or by exposure to exogenous HNE (microsomes: p = .049; mitochondria: p = .0338). This higher susceptibility of diabetic liver membranes to the non-enzymatic attack of HNE may be due to an altered phospholipid composition. Moreover, a decreased activity of the HNE-metabolizing systems can be involved: diabetic liver mitochondria and microsomes were unable to consume exogenous HNE at the same rate as non-diabetic membranes; the difference was already significant after 5' incubation (microsomes p<.001; mitochondria p<.001). These data show an increased oxidative stress inside the hepatocytes of diabetic rats; the impairment of the HNE-metabolizing systems can play a key role in the maintenance and propagation of the damage.

Levels of carbonyl groups in plasma proteins of type 2 diabetes mellitus subjects.

Carbonyl groups result from protein oxidation and their level in tissues and plasma is a relatively stable marker of oxidative damage. Carbonyl content of plasma proteins in 43 type 2 diabetic subjects, 30-87 years of age (25 males and 18 females) and in 20 age-matched healthy controls (31-89 years of age, 12 males and 8 females) was evaluated with 2,4-dinitro-phenyl-hydrazine method. In both groups, lipids, tocopherols (HPLC) and glycated hemoglobin (HPLC) were studied. Fasting blood glucose, glycated hemoglobin and lipids were significantly higher in the diabetic group; carbonyl content and alpha-tocopherol were slightly, but not significantly higher in the diabetic group (1.06 +/- 0.03 vs. 0.97 +/- 0.04 nmol/mg protein, 27. 07 +/- 2.82 vs. 31.55 +/- 2.11 micromol/l, respectively). Significant relationships between age and lipids, alpha-tocopherol and proteins were found in controls, but not in diabetics. Alpha-tocopherol correlated with lipids in both groups; glycated hemoglobin, a marker of glycemic control, was related to lipids, alpha-tocopherol and protein carbonyl groups in diabetics, while only the correlation with carbonyls was found in controls. These results suggest that impaired glycemic control is connected to protein oxidation. Glycation cascade also releases free radicals, becoming responsible for further oxidative attacks. In conclusion, increased oxidative stress, if any, in the diabetic group, is doubtlessly induced by hyperglycemia, and the tocopherols are not seriously affected by a worsening of the metabolic control.