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Haptoglobin (Hpt), primarily synthesized by hepatocytes, can be produced in central nervous system (CNS) under inflammatory conditions. In fact, increased Hpt was found in Cerebrospinal Fluids from patients with neurodegenerative diseases. We previously reported that Hpt binds Apolipoprotein E (ApoE), and influences ApoE key roles in cholesterol homeostasis. ApoE is the major constituent of lipoproteins into CNS, and modulates Amyloid-beta (A-beta) deposition and clearance. A-beta is known to trigger an inflammatory response in the brain, which is involved in the pathogenesis of Alzheimer’s disease. Our aim was to verify whether Hpt affects the ApoE function in the regulation of A-beta accumulation in the brain. We report here that the abilities of Hpt and ApoE to bind A-beta are similar. In addition, our data demonstrate that Hpt enhances the binding of ApoE to A-beta, thus suggesting that the two proteins and A-beta might interact with each other to form a trimeric complex. Further, a SDS-stable complex between the two proteins (ApoE and Hpt) and A-beta is formed following incubation at 37°C, and A-beta aggregation is reduced when it is incubated in presence of Hpt and ApoE. Finally, we found that Hpt can bind astrocytes. In conclusion Hpt, as forming a trimeric complex with ApoE and A-beta, and binding astrocytes, might play a critical role, participating in the binding and clearance of A-beta, thus alleviating its toxicity and promoting its removal from the brain.

Haptoglobin (Hpt) is known to capture circulating free Haemoglobin (Hb), and bind apolipoprotein (Apo) A-I or E. We report here that Hb can be tightly bound by most of Hpt molecules (TB-Hpt, 80%), whereas loosely bound by a minor part of them (LB-Hpt, 20%). LB-Hpt amount was significantly increased (over 60%) in patients with acute coronary syndrome. LB-Hpt bound ApoA-I and ApoE worse than TB-Hpt (8- and 4-fold less, respectively), and did not affect their activity of stimulating the enzyme LCAT. LB-Hpt and TB-Hpt displayed comparable levels of nitrotyrosine residues, but differences in glycan chains. Changes in the LB-Hpt level might be associated to changes in Hpt functions.

S-nitrosothiols act as carrier and reservoir of nitric oxide (NO), and release NO under stimulation of Ascorbate (Asc). Erythrocyte can regenerate Asc from its oxidised products, thus saving this powerful antioxidant. In this paper the effect of donors of NO, superoxide and peroxynitrite (SpNONOate, KO2, and SIN-1, respectively) on the erythrocyte production of Asc was investigated. We report here that NO stimulated, while superoxide and peroxynitrite decreased, the Asc recycling. The NO-stimulating effect on the erythrocyte production of Asc was confirmed by using GSNO, a natural occurring S-nitrosothiol, as NO donor. These data highlight a new property of NO, that is the stimulation of erythrocytes for their Asc recycling. Such a property might contribute to regenerate Asc from its oxidised forms, thus preventing its depletion in the circulation. Temperature and pH significantly affected, both in absence and presence of NO, the recycling of Asc by erythrocytes. We propose that a positive feedback, involving the reciprocal stimulation between Asc and S-nitrosothiols, might enhance productions of Asc by erythrocytes and NO release by circulating S-nitrosothiols.

INTRODUCTION: ApoA-1, the major apolipoprotein of the high density lipoprotein (HDL), plays a key role in the removal of cholesterol excess from peripheral cells in a recognized anti-atherosclerotic process called “reverse cholesterol transport (RCT)”. Among known ApoA-1 variants two, with one amino acid change (Zavalla, L159P; Zaragoza, L144R), are associated to poor RCT. Both mutations are located within the domain of ApoA1 (Leu141 – Ala164) binding Haptoglobin (Hpt) 1, a protein of the acute phase of inflammation. Hpt binding to ApoA1 was suggested to impair the role of HDL in RCT1. AIMS: Our aims were to study whether peptides containing the mutations of Zavalla (Pep Zav) or Zaragoza (Pep Zara) are able to bind Hpt or compete with native ApoA1, exposed on HDL, for binding Hpt. METHODS: The binding of biotinylated peptides to Hpt and the experiments of competition of acetylated peptides with HDL for Hpt binding were performed by ELISA, as previously published1. RESULTS: Both Pep-Zav and Pep-Zara bound to Hpt with efficiency similar to that of the peptide harbouring the native sequence of ApoA1 (pep 141–164). Nevertheless, when the same peptides were analysed for their ability to influence Hpt binding to HDL, only Pep Zav effectively displaced (50% at 15μM) Hpt from HDL (fig), as was the case for the pep. 141-164. CONCLUSIONS: We suggest that the L>P conversion, in the Zavalla variant, enhances hydrophilicity in ApoA1, improving the binding to Hpt. Therefore, as ApoA-1 binding to Hpt negatively affects RCT, individuals with Zavalla mutation might be exposed to higher risk of cardiovascular disease. 1.Spagnuolo M.S. et al (2005) J. Biol. Chem. 280, 1193-1198

E' stato studiato l'effetto della supplementazione della dieta con silimarina sullo stato redox plasmatico di capre in lattazione. Sono state evidenziate correlazioni positive tra i livelli di antiossidanti plasmatici e lo stato redox del latte.

Haptoglobin (Hp) can be purified by affinity chromatography using hemoglobin(Hb)-linked Sepharose. Elution with 8 M urea is generally performed, resulting in Hp heavy contamination by the apolipoprotein AI (ApoAI), and partial loss of Hb binding activity. Hp, separated from ApoAI, was recovered by elution with glycine-HCl at pH 3. Complexes of the isolated protein with Hb or ApoAI were detected by enzyme-linked immunosorbent assay (ELISA). Competition between the two ligands in their interaction with Hp was observed. Concanavalin A (ConA), which binds the Hp carbohydrate chains, did not influence the Hp binding to ApoAI. The results suggest that changes in the plasma level of ApoAI or Hb affect the Hp role in regulating the cholesterol reverse transport or preventing Hb-dependent oxidative damages.

During exercise reactive oxygen species are produced. The antioxidants prevent or limit tissue damages by these species in physiological conditions. In particular, ascorbate and urate scavenge peroxynitrite, which can alter the function of many molecules, including the lecithin-cholesterol acyltransferase (LCAT) enzyme involved in reverse cholesterol transport. The aims of the present study were to compare the plasma antioxidant response to an ergometric test (ET) in hypertensive and healthy subjects, evaluate the exercise dependent nitrosative stress in plasma, and assess whether the LCAT activity is altered by the exercise. Plasma samples, prepared before and after ET from hypertensive or healthy volunteers, were analysed for their levels of Ascorbate, Urate, -Tocopherol, Retinol, nitrotyrosine, and LCAT activity. The -Tocopherol and Retinol levels did not significantly change in both groups during exercise, while the Ascorbate level changed displaying higher increase in controls (+38.8%) than in hypertensives (+17.2%). In these patients, during ET, the Urate and nitrotyrosine levels changed more than in normotensives (+13.5% and +40.6% versus –3.1% and +25.2% respectively). The antioxidants effectively prevented loss or reduction of LCAT activity, as it was similar in hypertensives and normotensives, and did not change after ET. Conclusions: The results demonstrate that exercise is associated with enhanced protein nitrosation, and suggest that the Ascorbate or Urate levels increase to limit oxidative damage.

Technological processes represent the main source of protein and lipid oxidation in food. The oxidative status was determined in a soft Italian cheese, namely mozzarella, produced from water buffalo or bovine milk. The amount of protein-bound carbonyls, dityrosine and alpha-lactalbumin aggregates were measured to evaluate the extent of protein oxidation. The alpha-tocopherylquinone/alpha-tocopherol ratio and the trolox-equivalent antioxidant capacity were used as redox markers in the fat fraction. The levels of protein-bound carbonyls and alpha-lactalbumin aggregates were found significantly higher in mozzarella from bovine than in that from buffalo. On the contrary, higher amounts of redox markers were found in buffalo mozzarella. The levels of dityrosine aggregates were similar in the two groups of cheese. The data suggest that protein and fat are more protected against structure oxidative alterations in buffalo mozzarella than in bovine mozzarella

In the preovulatory follicle, the LH surge stimulates progesterone production, reduces estradiol synthesis, and scales up the permeability of the blood-follicle barrier. The purpose of this study was to investigate whether the extent of these changes is correlated with the levels of estradiol, estradiol esters, and cholesteryl esters in the follicular fluid. The follicular levels of progesterone, estradiol, estradiol linoleate, cholesterol, and cholesteryl linoleate were measured by HPLC. The estradiol linoleate/estradiol ratio, which reflects the efficiency of in vivo estradiol esterification, and the cholesteryl linoleate/cholesterol ratio were calculated and found negatively correlated. The estradiol level was positively correlated with the cholesteryl linoleate/cholesterol ratio while negatively correlated with the estradiol linoleate/estradiol ratio. The in vitro activity of lecithin-cholesterol acyltransferase, the enzyme esterifying both cholesterol and estradiol, was assayed by incubating the fluid with labeled substrates. This activity was not correlated with either the estradiol linoleate/estradiol or the cholesteryl linoleate/cholesterol ratio. The enzyme K m and V max values were lower with estradiol than with cholesterol. Higher estradiol linoleate/estradiol ratios and lower cholesteryl linoleate/cholesterol ratios were associated with higher level of Haptoglobin penetration into the follicle. This level, which was determined by ELISA, was found increased with increased progesterone concentration and, therefore, used as a marker of the LH-stimulated permeability of the blood-follicle barrier. Our data suggest that early preovulatory follicles contain more cholesteryl esters and less estradiol esters than follicles closer to ovulation.

Apolipoprotein A-I and Apolipoprotein E promote different steps of reverse cholesterol transport, including lecithin-cholesterol acyltransferase stimulation. Our aim was to study the changes in the levels of Apolipoprotein A-I, Apolipoprotein E and lecithin-cholesterol acyltransferase activity during atherosclerosis progression in rabbits. Quantitative echocardiographic parameters were analysed in order to evaluate, for the first time, whether atherosclerosis progression in rabbit is associated to apolipoproteins changes and alteration of indices of cardiac function, such as systolic strain and strain rate of the left ventricle. Atherosclerosis was induced by feeding rabbits for 8 weeks with 2% cholesterol diet. The HDL levels of cholesterol and cholesteryl esters were measured by HPLC. The lecithin-cholesterol acyltransferase activity was evaluated both ex vivo, as cholesteryl esters / cholesterol molar ratio, and in vitro. Apolipoproteins levels were analysed by ELISA. The HDL levels of cholesterol and cholesteryl esters increased, during treatment, up to 3.7 and 2.5 fold respectively compared to control animals. The lecithin-cholesterol acyltransferase activity in vitro was halved after 4 weeks. During cholesterol treatment, Apolipoprotein A-I level significantly decreased, whereas Apolipoprotein E concentration markedly increased. The molar ratio Apolipoprotein E/Apolipoprotein A-I was negatively correlated with the enzyme activity, and positively correlated with both increases in the intima-media thickness of common carotid wall and cardiac dysfunction signs, such as systolic strain and strain rate of the left ventricle. Further studies on humans or other animal models could be of help to confirm whether the ratio Apolipoprotein E/Apolipoprotein A-I can be considered as a biomarker of atherosclerosis progression.

Improved diagnosis of psoriasis, by new biomarkers, is required for evaluating the progression rate of the disease and the response to treatment. Haptoglobin (Hpt), a glycoprotein secreted by hepatocytes and other types of cells including keratinocytes, was found with glycan changes in psoriasis and other diseases. We previously reported that Hpt isolated from plasma of psoriatic patients is more fucosylated than Hpt of healthy subjects. The aim of this study was to compare the glycosylation pattern of Hpt isolated from skin scales or plasma of patients with psoriasis with that of Hpt from cornified epidermal layer or plasma of healthy subjects. High performance liquid chromatography analysis of the glycans isolated from the protein backbone revealed that glycan patterns from skin and plasma of patients were similar, and mostly displayed quantitative rather than qualitative differences from normal pattern. Biotin-labeled lectins were used to evaluate quantitative differences in the glycoforms of Hpt from plasma and psoriatic skin scales. Hpt from skin and plasma of patients showed more fucosylated and branched glycans than Hpt from plasma of healthy subjects. Tryptic glycopeptides of Hpt were also analyzed by mass spectrometry, and a decreased amount of sialylated glycan chains was found in glycopeptides of skin Hpt, as compared with Hpt from plasma. High levels of glycans with fucosylated and tetra-antennary chains were detected on the peptide NLFLNHSENATAK from Hpt of psoriatic patients. Our data demonstrate that specific changes in glycan structures of Hpt, such as enhanced glycan branching and fucose content, are associated with psoriasis, and that differences between circulating and skin Hpt do exist. A lower extent of glycan fucosylation and branching was found in Hpt from plasma of patients in disease remission. Altered glycoforms might reflect changes of Hpt function in the skin, and could be used as markers of the disease.

Haptoglobin (Hpt) binds the Apolipoprotein A-I (ApoA-I), and impairs its stimulation on lecithin:cholesterol acyl-transferase (LCAT), an enzyme playing a major role in the reverse cholesterol transport (RCT). The Apolipoprotein E (ApoE), like ApoA-I, promotes different steps of RCT, including LCAT stimulation. ApoE contains aminoacid sequences which are homologous to the ApoA-I region bound by Hpt and involved in the interaction with LCAT. Therefore Hpt was expected to bind also ApoE, and inhibit the ApoE stimulatory effect on LCAT. Western Blotting and enzyme-linked immunosorbent assay experiments demonstrated that the Hpt subunit  actually binds ApoE. The Hpt affinity for ApoE was higher than that for ApoA-I. High ratios of Hpt with either apolipoprotein, as those associated with the acute phase of inflammation, inhibited in vitro ApoE in stimulating the cholesterol esterification activity of LCAT. Hpt also impaired human hepatoblastoma-derived cells in uptaking 3H-cholesterol from proteoliposomes containing ApoE or ApoA-I. We suggest that the interaction between Hpt and ApoE represents a mechanism by which inflammation affects atherosclerosis progression. Hpt might influence ApoE function in processes different from RCT.

Growing evidence suggests that atherogenesis is associated with inflammation or defective removal of cholesterol excess from peripheral cells. Apolipoprotein A-I [ApoA-I] activates the enzyme Lecithin-Cholesterol Acyl-Transferase to esterify cell cholesterol for transport to liver. Haptoglobin [Hpt] was previously found able to bind ApoA-I, and suggested to reduce the enzyme activation. The aim of this study was to demonstrate that enhanced levels of Hpt, as present during inflammation, are associated with low enzyme activity and increased thickness of the arterial wall. Enzyme activity and Hpt concentration were analysed in patients with rheumatoid arthritis having the same plasma levels of antioxidants (ascorbate, urate, alpha-tocopherol, retinol) or oxidation markers (nitrotyrosine, lipoperoxide) of healthy subjects. Cholesterol esterification, determined as ratio of cholesteryl esters with cholesterol in high-density lipoproteins, was lower in patients than in controls, and negatively correlated with the intima-media wall thickness of the common carotid. The ratio of Hpt with ApoA-I was negatively correlated with the enzyme activity, while positively correlated with intima-media wall thickness. The results suggest that high Hpt levels might severely impair the enzyme activity, thus contributing to cholesterol accumulation in vascular cells, and lesion formation in the endothelium.

Haptoglobin (Hpt) was previously found to bind the high density lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I structure was analyzed to detect the site bound by Hpt. ApoA-I was treated by cyanogen bromide or hydroxylamine; the resulting fragments, separated by electrophoresis or gel filtration, were tested by Western blotting or enzyme-linked immunosorbent assay for their ability to bind Hpt. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was determined by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, containing the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, respectively. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with hemoglobin for Hpt binding in enzyme-linked immunosorbent assay experiments. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding hemoglobin, might be used for treatment of diseases associated with defective LCAT function.

Milk is the most important source of Retinol and alpha-Tocopherol for calves. These antioxidants save the food quality and prevent lipid oxidation in the mammary gland and the calf growing tissues. In Bubalus bubalis, seasonal changes for the plasma levels of both antioxidants were not found. The levels of Retinol and alpha-Tocopherol in the milk were 2 and 1.7 times higher in winter than in summer, respectively. These levels were correlated with the plasma level of triiodothyronine, and markedly increased in cows injected with triiodothyronine in summer. The cytosol from alveolar epithelial cells of mammary glands was incubated with alpha-Tocopherol and 3H-Retinol and, after gel filtration chromatography, both antioxidants were found associated with proteins migrating as a single peak of 33 kD. The amount of alpha-Tocopherol and Retinol binding proteins was 1.5 and 2.3 times higher in winter than in summer respectively. The Retinol binding proteins migrated as two bands (33 and 16 kD) by electrophoresis in denaturing and reducing conditions. Our data suggest that triiodothyronine enhances the transport of both liposoluble antioxidants through the blood-mammary barrier, and demonstrate that proteins of the mammary epithelial cells are involved in such a transport.

Haptoglobin is a hemoglobin-binding protein presenting in humans three distinct phenotypes (Hpt 1-1, Hpt 1-2, or Hpt 2-2). The Hpt 1-2 and Hpt 2-2 phenotypes are in turn represented by populations of isoforms. The relative amounts of the major isoforms of Hpt 1-2 and Hpt 2-2 were found to differ not only in different individuals, but also in the same individual before and after a physical effort. Exercise-dependent changes in the plasma concentrations of ascorbate, urate, α-tocopherol, retinol, and glutathione were also observed, but correlations between such changes and those of the amount for any isoform were not found. Samples of Hpt 1-2 or Hpt 2-2 were challenged with oxidants (H2O2 with ferrous ions, spermine–NO, KO2, and 3-morpholinosydnonimine), but the isoform levels were not altered. Hpt 2-2 isoforms were present in Hpt 1-2, as minor species. Furthermore, different isoforms exhibited different hemoglobin binding abilities. Thus, these parameters should also be taken into consideration in studies correlating Hpt phenotypes prevalence with pathologies or functional differences.

Thyroid hormones stimulate oxygen consumption and heat production, inducing the synthesis of reactive oxygen species, which in turn trigger several pathologies including mastitis, and infectious diseases in the newborn calf. The retinol and tocopherol seasonal levels were measured by HPLC in the milk of buffalo cows (winter: 1.60 and 1.20 ?g/ml respectively; summer: 0.71 and 0.76 respectively). In the plasma, seasonal changes for the levels of both antioxidants were not found. The retinol levels in the milk were positively correlated with the plasma levels of T3 (P

Leptin, a hormone mainly secreted by adipose tissue, is involved in the regulation of food intake, energy expenditure and whole-body energy balance. The aim of this study was to evaluate whether leptin concentration changes in growing buffaloes. The correlation between plasma leptin level and some haematic parameters as well as between leptin level and some growing indices was also analysed. Plasma level of glucagone, insulin, glucose, NEFA and 3-hydroxybutyrate were measured. Liveweight, rump area, compactness index, bodily index, tronco-thoracic index and average daily gain were chosen as somatic parameters. Blood samples were collected at 30 days intervals for ten months from 20 young growing buffaloes, starting from animals 130 days old. We found that plasma leptin level increased with weight gain. In particular, plasma leptin level decreased between the 7th and 9th month of age, anddramatically increased between the 10th and the 12th month of age. Plasma insulin and somatic parameters were positively correlated with leptin level while glucose and NEFA were negatively correlated with leptin plasma level. These results suggest that leptin might play a central role in the regulation of glycaemia and fat mobilization during growth in buffaloes.

The enzyme lecithin–cholesterol acyltransferase (LCAT) transfers an acyl chain from lecithin to cholesterol or oestradiol, thus playing a crucial role in reverse cholesterol transport and follicular synthesis of potent long-lived oestrogens. The mechanism of catalysis is biphasic, as it is based on a phospholipase and an esterifying activity. Sulfhydryl groups were previously reported to be required for the esterification step. Lecithin–cholesterol acyltransferase has previously been shown to be inhibited by thiol oxidants such as peroxynitrite. Peroxynitrite also converts tyrosine to nitrotyrosines. In the present study, high levels of nitrotyrosine associated with low LCAT activity, and vice versa, were found in human preovulatory follicular fluids. Follicular fluids were also analysed for oestradiol (E) and progesterone (P) concentrations. The E/P ratio, which decreases as ovulation approaches, was used to evaluate the maturation status of each follicle. Enzyme activity was negatively correlated with the E/P ratio. Ascorbate (Asc) and α-tocopherol (Toc) were titrated in follicular fluid and plasma to evaluate their accumulation or consumption in the follicle. High LCAT activity was found in follicular fluids where Asc and Toc had accumulated, whereas lower activity was associated with Asc and Toc consumption. The consumption of both antioxidants was positively correlated with the E/P ratio. The results suggest that as follicle maturation progresses, Toc and Asc concentrations increase in follicular fluid, thus protecting LCAT from oxidative damage and loss of activity.

Blood flow interruption is associated with oxygen depletion and loss of factors for function and survival in downstream tissues or cells. Hypoxia and absence of gonadotropins trigger apoptosis and atresia in the ovary. We studied the antioxidant response of follicular cells to plasma deprivation in ovaries dissected from water buffalo. Aliquots of follicular fluid were aspirated from each antral follicle, before and during incubation of the ovaries at 39 degrees C. Urate, ascorbate, retinol and alpha-tocopherol in the fluid were, titrated by High Performance Liquid Chromatography (HPLC) with spectrophotometric or spectrofluorimetric detection. The total antioxidant capacity of follicular fluid was determined as absorbance decrease, following addition of a source of radical chromophores. The more the incubation progressed, the higher levels of urate, ascorbate and total antioxidant capacity were found. Conversely, changes in concentration of the liposoluble antioxidants were not observed. Ascorbate synthesizing activity in the follicle was demonstrated by detecting the enzyme L-gulono-gamma-lactone oxidase in microsomes prepared from granulosa cells. These cells were also analyzed for the expression of the enzyme CPP32. The enzyme level, measured as DEVD-p-nitroanilide cleaving activity, was found related with the immunoreactivity to anti-CPP32 antibodies. Negative correlation between the enzyme activity (which is known to be induced by peroxynitrite) and the follicular level of urate (which scavenges peroxynitrite) was also observed. The amount of nitrotyrosine, a product of peroxynitrite attack on proteins, was measured in follicular fluids by Enzyme Linked ImmunoSorbent Assay (ELISA). This amount was found positively correlated with the CPP32 activity, and negatively correlated with the urate level in follicular fluid. Alterations in concentrations of ascorbate or urate may be associated with oxidative stress during follicular atresia.