Reduction of serum advanced glycation end-products with a low calorie Mediterranean diet.

UNLABELLED: Dietary intake of advanced glycation end-products (AGEs) increases circulating and tissue levels of these substances, contributing to a state of increased oxidative stress and inflammation. A low dietary AGE intervention has been shown to reduce body AGE content. Mediterranean diets (MD) are theoretically considered low in AGEs, but the specific effects of a MD on AGEs serum levels has not been tested. METHODOLOGY: Forty-seven overweight and obese premenopausal women underwent a three-month calorie restriction treatment (20 kcal/kg initial weight) with a Mediterranean-type diet that excluded wine intake. The adherence to the MD was assessed before and at the end of treatment using an on-line questionnaire, which scores from 0 to 14 (minimal to maximal adherence). Body composition, insulin resistance, lipoproteins and carboxymethyl-lisine (CML) serum levels were measured at both time periods. Serum CML was assessed through ELISA (enzyme-linked immunosorbent assay). Compliance to calorie restriction was assessed according to weight loss (< or > 5% initial weight). RESULTS: Mean body weight, body fat, waist circumference, total cholesterol, triglycerides and serum CML fell significantly, together with an increase in the Mediterranean score, although none of the patients reached the highest score. Significant changes in CML and insulin resistance were observed in 17 women classified as compliant to caloric restriction, but not in the 27 participants who were considered adherent to the MD (according to improvement of the Mediterranean Score). CONCLUSIONS: CML serum levels can be reduced through calorie restricted-Mediterranean-type diet. We could not reach a high enough MD score, so we cannot conclude whether the MD itself has an additive effect to caloric restriction.

La ingesta dietaria de productos finales de glicación avanzada (AGEs) aumenta los niveles séricos y tisulares de estas sustancias, lo que contribuye a un estado de mayor estrés oxidativo e inflamación. Una intervención dietaria con bajo contenido de AGEs ha demostrado reducir el contenido de AGEs en el cuerpo. La dieta mediterránea (DM) se considera teóricamente baja en AGEs, pero los efectos específicos de este tipo de intervención en los niveles séricos de AGEs no ha sido probado. Metodología: cuarenta y siete mujeres premenopáusicas con sobrepeso u obesidad se sometieron a tres meses de restricción calórica (20 kcal por kg de peso corporal inicial) con una dieta de tipo mediterráneo que excluía la ingesta de vino. La adherencia a la DM se evaluó al comienzo y al final del tratamiento utilizando una encuesta on-line, con puntuaciones de 0 a 14 (mínima a máxima adherencia a la DM). La composición corporal, la resistencia a la insulina, los niveles séricos de lipoproteínas y carboximetil-lisina (CML) se midieron en ambos períodos. El CML sérico se evaluó mediante ELISA (ensayo inmunoenzimático). La adherencia a la restricción calórica se evaluó de acuerdo con la pérdida de peso (< o > 5% del peso inicial). Resultados: la media de peso corporal, grasa corporal, circunferencia de la cintura, colesterol total, triglicéridos y CML sérica disminuyeron significativamente, junto con un aumento en el puntaje de adherencia a la DM, aunque ninguno de los pacientes alcanzó la máxima puntuación. Hubo cambios significativos en los niveles de CML y de resistencia a la insulina en 17 mujeres clasificadas como adherentes a la restricción calórica, pero no en las 27 participantes que fueron consideradas adherentes a la DM (de acuerdo con la mejoría en el puntaje de la encuesta). Conclusiones: los niveles séricos de CML disminuyeron tras la restricción calórica con una dieta tipo mediterránea. Dado que no se pudo alcanzar la puntuación máxima en la encuesta de DM, no podemos concluir si la propia DM tiene un efecto aditivo a la restricción calórica.

Reduction of serum advanced glycation end-products with a low calorie Mediterranean diet / Reducción sérica de productos finales de glicación avanzada séricos con una dieta baja en calorías tipo mediterráneo

Dietary intake of advanced glycation end-products (AGEs) increases circulating and tissue levels of these substances, contributing to a state of increased oxidative stress and inflammation. A low dietary AGE intervention has been shown to reduce body AGE content. Mediterranean diets (MD) are theoretically considered low in AGEs, but the specific effects of a MD on AGEs serum levels has not been tested. Methodology: forty-seven overweight and obese premenopausal women underwent a three-month calorie restriction treatment (20 kcal / kg initial weight) with a Mediterranean-type diet that excluded wine intake. The adherence to the MD was assessed before and at the end of treatment using an on-line questionnaire, which scores from 0 to 14 (minimal to maximal adherence). Body composition, insulin resistance, lipoproteins and carboxymethyl-lisine (CML) serum levels were measured at both time periods. Serum CML was assessed through ELISA (enzyme-linked immunosorbent assay). Compliance to calorie restriction was assessed according to weight loss (< or > 5 % initial weight). Results: mean body weight, body fat, waist circumference, total cholesterol, triglycerides and serum CML fell significantly, together with an increase in the Mediterranean score, although none of the patients reached the highest score. Significant changes in CML and insulin resistance were observed in 17 women classified as compliant to caloric restriction, but not in the 27 participants who were considered adherent to the MD (according to improvement of the Mediterranean Score). Conclusions: CML serum levels can be reduced through calorie restricted - Mediterranean-type diet. We could not reach a high enough MD score, so we cannot conclude whether the MD itself has an additive effect to caloric restriction (AU)

La ingesta dietaria de productos finales de glicación avanzada (AGEs) aumenta los niveles séricos y tisulares de estas sustancias, lo que contribuye a un estado de mayor estrés oxidativo e inflamación. Una intervención dietaria con bajo contenido de AGEs ha demostrado reducir el contenido de AGEs en el cuerpo. La dieta mediterrá- nea (DM) se considera teóricamente baja en AGEs, pero los efectos específicos de este tipo de intervención en los niveles séricos de AGEs no ha sido probado. Metodología: cuarenta y siete mujeres premenopáusicas con sobrepeso u obesidad se sometieron a tres meses de restricción calórica (20 kcal por kg de peso corporal inicial) con una dieta de tipo mediterráneo que excluía la ingesta de vino. La adherencia a la DM se evaluó al comienzo y al final del tratamiento utilizando una encuesta on-line, con puntuaciones de 0 a 14 (mínima a máxima adherencia a la DM). La composición corporal, la resistencia a la insulina, los niveles séricos de lipoproteínas y carboximetil-lisina (CML) se midieron en ambos períodos. El CML sérico se evaluó mediante ELISA (ensayo inmunoenzimático). La adherencia a la restricción calórica se evaluó de acuerdo con la pérdida de peso (< o > 5% del peso inicial). Resultados: la media de peso corporal, grasa corporal, circunferencia de la cintura, colesterol total, triglicéridos y CML sérica disminuyeron significativamente, junto con un aumento en el puntaje de adherencia a la DM, aunque ninguno de los pacientes alcanzó la máxima puntuación. Hubo cambios significativos en los niveles de CML y de resistencia a la insulina en 17 mujeres clasificadas como adherentes a la restricción calórica, pero no en las 27 participantes que fueron consideradas adherentes a la DM (de acuerdo con la mejoría en el puntaje de la encuesta). Conclusiones: los niveles séricos de CML disminuyeron tras la restricción calórica con una dieta tipo mediterránea. Dado que no se pudo alcanzar la puntuación máxima en la encuesta de DM, no podemos concluir si la propia DM tiene un efecto aditivo a la restricción calórica (AU)

Concordancia entre gasto energético y reposo medido y estimado por fórmulas predictivas en mujeres con obesidad severa y mórbida / Agreement between measured and calculated by predictive formulas resting energy expenditure in severe and morbid obese women

Objetivo: Comparar el gasto energético de reposo medido con el gasto energético de reposo estimado (GER) por fórmulas predictivas diseñadas a partir de población con peso normal u obesidad, en mujeres con obesidad severa y mórbida. Material y métodos: a 66 mujeres (índice de masa corporal 44,7 ± 4,9 kg/m2; edad 35,6 ± 10,3 años) se les realizó calorimetría indirecta con monitor metabólico Deltatrac (Datex Inst., Finlandia), antes de someterse a cirugía bariátrica. Se estimó el GER con las siguientes ecuaciones: Harris-Benedict con peso real y peso ajustado, Ireton-Jones, Estimación Rápida de Carrasco (16,2 kcal × kg peso real) y Mifflin. Resultados: (x ± de). El GER medido fue 1.797 ± 239 kcal/día. Todas las fórmulas, excepto Harris-Benedict con peso ajustado, sobreestimaron el gasto energético; la ecuación de Ireton-Jones fue la que sobreestimó en mayor cuantía el GER (689 ± 329 kcal/día), mientras que la ecuación de Mifflin sobreestimó el GER sólo en 6 ± 202 kcal/día. No se encontraron diferencias significativas entre el GER medido y el GER estimado por Mifflin y Estimación Rápida. La ecuación de Mifflin fue la más exacta: en 68% de los casos la diferencia entre el GER estimado y medido estuvo dentro de ± 10%, seguida por Harris-Benedict con peso real (64%) y la Estimación Rápida (61%). Según el análisis de Bland-Altman, hubo una correlación significativa entre la diferencia GER estimado- medido y el promedio de GER estimado y medido con todas las ecuaciones, excepto con la Estimación Rápida de Carrasco. Esto implica que, con la excepción de esta última, las fórmulas estudiadas subestiman o sobrestiman el GER dependiendo de la magnitud del GER medido. Conclusión: En la serie pacientes con obesidad severa y mórbida evaluadas, la ecuación de Mifflin y la Estimación Rápida otorgan el menor error de estimación del gasto energético de reposo en mujeres. Antes de recomendar una ecuación en particular es necesario realizar estudios de validación para determinar cuál es la ecuación de predicción más exacta para este grupo de pacientes

Objective: To compare measured resting energy expenditure (REE) with that predicted by formulas derived from populations with normal weight or obesity and from women with severe and morbid obesity. Material and methods: 66 women (aged 35.6 ± 10.3 y and BMI of 44.7 ± 4.9 kg/m2) were evaluated by indirect calorimetry with a metabolic monitor Deltatrac (Datex Inst., Finland), before undergoing gastric bypass. REE was calculated with the following equations: Harris-Benedict’s with both actual and adjusted weight, Ireton-Jones’, Mifflin’s, and Carrasco’s Fast Estimation, which corresponds to 16.2 kcal × kg actual weight. Results: (mean ± sd). Measured REE was 1797 ± 239 kcal/day. All formulas, except Harris-Benedict’s with adjusted weight, overestimated REE. The Ireton-Jones’ equation presented the greater overestimation (689 ± 329 kcal/day), whereas Mifflin’s equation overestimated REE only by 6 ± 202 kcal/day. No significant differences were detected between measured and calculated REE by Mifflin’s and Carrasco’s Fast Estimation. Accuracy (defined as difference between calculated and measured REE within ± 10%) was greater with Mifflin’s equation (68%), followed by Harris-Benedict’s with actual weight (64%) and Carrasco’s Fast Estimation (61%). By using the Bland-Altman analysis, significant correlations were observed between calculated-measured REE and mean REE (calculated + measured/2) with all equations except Carrasco’s Fast Estimation. This means that all but one formula underestimate or overestimate REE depending on the level of measured REE. Conclusion: In severe and morbid obese women, Mifflin’s and Carrasco’s Fast Estimation equations provided the best performance to estimate REE. Before recommending an equation in an a subset of individuals it is necessary to make previous validation studies to determine that equation with the best predictive power for this particular group of patients

[Agreement between measured and calculated by predictive formulas resting energy expenditure in severe and morbid obese women]. / Concordancia entre gasto energético y reposo medido y estimado por fórmulas predictivas en mujeres con obesidad severa y mórbida.

OBJECTIVE: To compare measured resting energy expenditure (REE) with that predicted by formulas derived from populations with normal weight or obesity and from women with severe and morbid obesity. MATERIAL AND METHODS: 66 women (aged 35.6 +/- 10.3 y and BMI of 44.7 +/- 4.9 kg/m2) were evaluated by indirect calorimetry with a metabolic monitor Deltatrac (Datex Inst., Finland), before undergoing gastric bypass. REE was calculated with the following equations: Harris-Benedict's with both actual and adjusted weight, Ireton-Jones', Mifflin's, and Carrasco's Fast Estimation, which corresponds to 16.2 kcal x kg actual weight. RESULTS: (mean +/- sd). Measured REE was 1797 +/- 239 kcal/day. All formulas, except Harris-Benedict's with adjusted weight, overestimated REE. The Ireton-Jones' equation presented the greater overestimation (689 +/- 329 kcal/day), whereas Mifflin's equation overestimated REE only by 6 +/- 202 kcal/day. No significant differences were detected between measured and calculated REE by Mifflin's and Carrasco's Fast Estimation. Accuracy (defined as difference between calculated and measured REE within +/- 10%) was greater with Mifflin's equation (68%), followed by Harris-Benedict's with actual weight (64%) and Carrasco's Fast Estimation (61%). By using the Bland-Altman analysis, significant correlations were observed between calculated-measured REE and mean REE (calculated + measured/2) with all equations except Carrasco's Fast Estimation. This means that all but one formula underestimate or overestimate REE depending on the level of measured REE. CONCLUSION: In severe and morbid obese women, Mifflin's and Carrasco's Fast Estimation equations provided the best performance to estimate REE. Before recommending an equation in an a subset of individuals it is necessary to make previous validation studies to determine that equation with the best predictive power for this particular group of patients.

Transición epidemiologica en Chile: lecciones aprendidas del Proyeto North Karelia / Epidemiological transition in Chile: lessons learned from the North Karelia Project

Chile ha vivido un acelerado proceso de transición epidemiológica y nutricional, con una prevalencia creciente de enfermedades crónicas. Esta tendencia se ha visto reafirmada con los alarmantes resultados de la Encuesta Nacional de Salud. Por ello describimos intervenciones poblacionales que han sido exitosas en controlar los factores de riesgo cardiovascular en el proyecto North Karelia de Finlandia. Entre las intervenciones descritas destacan: el compromiso de los servicios de salud, un programa de control de la hipertensión, entrenamiento del personal, distribución de material impreso, liderazgo, programas en el lugar de trabajo, programas de televisión, competencias para dejar de fumar, competencias entre comunidades para bajar el colesterol promedio, proyecto Berry, colaboración con productores de alimentos y supermercados, políticas y legislación anti tabaco, concepción de un nuevo modelo de cambios de conductas. Finalmente se hace referencia a los costos del proyecto y se discute su aplicación en nuestro país.

Complementary effects of Mediterranean diet and moderate red wine intake on haemostatic cardiovascular risk factors.

OBJECTIVES: To compare the effect of alcohol-free Mediterranean-type diet (MD) and high-fat diet (HFD) on plasma concentration of emergent haemostatic cardiovascular risk factors (HCVRF). Also, to test if red wine supplementation modifies HCVRF, independent of diet. DESIGN, SUBJECTS AND INTERVENTION: Controlled prospective intervention study. Two groups, each of 21 healthy male university students (22+/-3.4 y), received either MD or HFD for 90 days. Between days 30 and 60, both diets were supplemented with 240 ml/day of red wine. Baseline and T30, T60 and T90-day samples were drawn. No drop out from the study was observed. SETTING: University campus and outpatient nutrition clinic. RESULTS: Volunteers on HFD at T30 had increases in pro-coagulants fibrinogen (22%), factor VIIc (9%), and factor VIIIc (4%), and decreases in natural anticoagulants antithrombin III (3%), protein C (11%) and protein S (6%) and of 20% in plasminogen activator inhibitor-1. At the same time, individuals on MD had increases in fibrinogen (4%), antithrombin III (5%), protein C (3%), protein S (2.7%), and decreases in factor VIIIc (9%), and plasminogen activator inhibitor-1 (21%). After adjusting by baseline values, MD was associated with lower plasma fibrinogen (P=0.03), factor VIIc (P=0.034) and factor VIIIc (P=0.0057) and with higher levels of protein S (P=0.013). Red wine supplementation, in both diets, resulted in decreased plasma fibrinogen (P=0.001) and factor VIIc (P=0.05), and increased tissue plasminogen activator antigen (P=0.01) and plasminogen activator inhibitor-1 antigen (P=0.0003). Wine consumption was also associated with significantly (P=0.01) divergent effects on antithrombin III: it decreased by 10% in individuals on HFD but increased slightly in those on MD. No effects of diet or wine were detected in plasma protein C and C-reactive protein. CONCLUSION: MD and moderate consumption of red wine have complementary, mostly beneficial effects on HCVRF.

The chondroitin sulfate chain of bikunin-containing proteins in the inter-alpha-inhibitor family increases in size in inflammatory diseases.

Inter-alpha-inhibitor (IalphaI) and pre-alpha-inhibitor (PalphaI) are the main members of a set of multichain serine proteinase inhibitors. Present in human plasma, they may be involved in control of the inflammatory process. They are composed of homologous heavy chains (H1 and H2 for IalphaI; H3 for PalphaI) covalently linked by a protein-glycosaminoglycan-protein cross-link to bikunin, which is a chondroitin 4-sulfate proteoglycan. During the acute-phase response, biosynthesis of IalphaI and PalphaI is downregulated and upregulated, respectively. In this work, we provide evidence that, in inflammatory diseases, the chondroitin sulfate chain of bikunin increases in size proportionally to the severity of the inflammatory response. As a consequence, all IalphaI-related components that contain bikunin are structurally modified. Therefore, the changes in glycosylation of the acute-phase proteins are not restricted to N-linked glycans but also affect glycosaminoglycans. The implications of these findings are discussed with regard to biosynthesis and biological role, especially the anti-inflammatory effects of IalphaI-related proteinase inhibitors.

Unmasking a hyaluronan-binding site of the BX(7)B type in the H3 heavy chain of the inter-alpha-inhibitor family.

The inter-alpha-inhibitor (I alpha I) family gathers together several plasma protease inhibitors such as I alpha I and pre-alpha-inhibitor (P alpha I) that are variously assembled from a set of polypeptide chain precursors designated H1P to H3P. In addition to their protease inhibitory activity, a major physiological function of I alpha I family members is hyaluronan (HA) binding and HA-dependent stabilization of the extracellular matrix surrounding various cell types. Also, binding of HA to these molecules has been shown to be an important event in tumor cell proliferation and rheumatoid arthritis. However, how HA and I alpha I family members first recognize each other has so far remained elusive. The so-called BX7B domain found in some HA-binding proteins is an HA-binding site in which B represents a basic amino-acid residue and X represents any nonacidic residue. This domain has now been identified in the N-terminal end of H3P that is a precursor of P alpha I. A series of wild-type or mutant recombinant H3P chains produced with a mouse cDNA expressed in Escherichia coli allowed us to demonstrate that this domain binds HA in a noncovalent fashion. Furthermore, unmasking this HA-binding activity required most of H3P to be trimmed off at its C-terminal end. The latter observation was confirmed with a natural, mature H3 chain purified from human plasma. Indeed, a thermolysin-generated, N-terminal fragment of this H3 chain strongly bound HA whereas the intact H3 chain did not. Therefore, in vivo, the HA-binding activity of the mature H3 chain within P alpha I may vary with the folding and/or fragmentation of this protein.

A high-fat diet induces and red wine counteracts endothelial dysfunction in human volunteers.

Endothelial dysfunction is associated with atherogenesis and oxidative stress in humans. In rat and rabbit blood vessels, wine polyphenol antioxidants induce vascular relaxation in vitro through the NO-cGMP pathway. To assess the effect of a regular high-fat diet (HFD) and moderate red wine consumption on endothelial function (EF), a study was performed in healthy male volunteers. EF was measured as flow-mediated dilatation of the brachial artery, employing high-resolution ultrasound after an overnight fast. Other clinical and biochemical parameters related to EF were also measured. Six volunteers received a control diet, rich in fruits and vegetables (27% calories as fat) and five volunteers received an HFD (39.5% calories as fat). Measurements were done twice on each volunteer: after a period of 30 d with diet plus 240 mL of red wine/d, and after a period of 30 d with diet, without wine. In the absence of wine, there is a reduction of EF with HFD when compared to the control diet (P = 0.014). This loss of EF is not seen when both diets are supplemented with wine for 30 d (P = 0.001). Plasma levels of n-3 fatty acids (R2 = 0.232, P = 0.023) and lycopene (R2 = 0.223, P = 0.020) show a positive correlation with individual EF measurements, but they do not account for the significant differences observed among dietary groups or after wine supplementation. These results help elucidate the deleterious effect of a high-fat diet and the protective role of wine, n-3 fatty acids and dietary antioxidants in cardiovascular disease.

Inflammation-induced systemic proteolysis of inter-alpha-inhibitor in plasma from patients with sepsis.

Inter-alpha-inhibitor (IalphaI) is a human plasma serine proteinase inhibitor. It contains one light peptide chain called bikunin that exerts antiproteinase activity and other antiinflammatory functions. Bikunin is covalently linked to two heavy chains that, after tissular diffusion, stabilize the extracellular matrix. Owing to its negative acute-phase reactant character and its susceptibility to proteolysis, IalphaI has been implicated in the pathophysiology of sepsis. Moreover, IalphaI has been shown to exert a protective effect on a pig model of endotoxic shock. Twenty patients admitted to the intensive care unit (ICU) for a septic syndrome were included in the present study. IalphaI and antithrombin III (ATIII) levels were measured on admission. Sequential measurements of IalphaI could be done in 4 patients. We demonstrate that IalphaI levels are significantly decreased in plasma samples collected on admission from patients with sepsis (59 +/- 32 mg/L vs 241 +/- 70 mg/L; P < .0001). This decrease was greater in severe sepsis and septic shock than in sepsis. Death was not predictable from initiol IalphaI levels. In 2 patients with a favorable course, IalphaI values regularly increased during the ICU stay. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analysis and microsequencing, we characterized IalphaI-related components in plasma from several patients; they obviously arise from IalphaI through proteolytic cleavage. Thus, systemic proteolysis and decreased biosynthesis both contribute to the fall in the plasma level of IalphaI. Because IalphaI is very sensitive to proteolysis by polymorphonuclear granulocytes (PMNs) that are stimulated during sepsis, we suggest that IalphaI plasma level would be a useful marker for neutrophil proteinase activity. ATIII, as well as IalphaI, is considered a negative acute phase protein. Because in vitro ATIII is less susceptible than IalphaI to proteolysis by PMNs and because their relative levels weakly correlated, we suggest that an unspecific systemic proteolysis is not significantly involved in the ATIII deficiency occurring in sepsis.

Human pre-alpha-inhibitor is a positive acute-phase protein that is more susceptible than inter-alpha-inhibitor to proteolysis by stimulated neutrophils.

BACKGROUND: Pre-alpha-inhibitor (PalphaI) is a human plasma serine-proteinase inhibitor that is structurally related to inter-alpha-inhibitor (IalphaI). It is composed of a heavy chain named H3 covalently linked to bikunin by means of a glycosaminoglycan chain. We developed an ELISA procedure making it possible to measure PalphaI for the first time and we investigated its levels in sera from patients with inflammatory diseases. MATERIALS AND METHODS: We generated rabbit anti-H3 immunoglobulins, which were used on solid phase and biotinylated antibikunin immunoglobulins to detect trapped PalphaI. RESULTS: We demonstrate that PalphaI is more susceptible than IalphaI to in vitro proteolysis by stimulated neutrophils. However, the degradation products thus released as well as the other members of the IalphaI family present in serum do not affect the ELISA test. In a panel of control sera we observed PalphaI concentrations of 25.6 +/- 7.8 mg L-1 (mean +/- SD; n = 30). These values increased to 64.2 +/- 16.06 mg L-1 (mean +/- SD; n = 15) in patients with inflammatory diseases, concording with the positive acute-phase protein nature of PalphaI. However, for all these patients, the serum concentrations of PalphaI and C-reactive protein poorly correlated (r = 0.476; P = 0.076). Indeed, four patients had a relatively weaker increase in their PalphaI level than that of C-reactive protein. More often than not their plasma elastase content was then elevated. CONCLUSION: During inflammatory diseases plasma PalphaI levels may be dependent on increased synthesis in combination with enhanced catabolism, perhaps implicating neutrophil or other proteinases.

Inter-alpha-inhibitor (IalphaI) concentration in pig plasma is independent of acute phase protein response.

Human inter-alpha-inhibitor (IalphaI) has been shown to exert a beneficial therapeutic effect in a porcine model of endotoxin shock. It is therefore useful to have a better understanding of IalphaI metabolism during severe inflammatory syndromes. Experimental bacterial pneumonia was induced in pigs. The acute phase response was highlighted by an increase in pig major acute phase protein (pig-MAP) and haptoglobin concentrations in plasma collected daily over 4 days. In the same samples, the IalphaI levels remained unchanged. Moreover, crossed-immunoelectrophoretic and immunoblot analyses did not show any qualitative modification of IalphaI throughout the experiment. IalphaI has been reported to be a negative acute phase protein in both humans and rats. Here we demonstrated that IalphaI behavior clearly differs in humans and pigs and is definitively species specific.

Plasma polyphenols and antioxidants, oxidative DNA damage and endothelial function in a diet and wine intervention study in humans.

An intervention study was performed to evaluate the influence of a Mediterranean diet, a high fat diet, and their supplementation with red wine in moderate amounts, on biochemical, physiological, and clinical parameters related to atherosclerosis and other chronic diseases. For 3 months two groups of 21 male volunteers each, received either a Mediterranean diet or a high fat diet; during the second month, red wine was added isocalorically, 240 ml/day. Participants were kept under close medical and nutritional surveillance. At days 0, 30, 60 and 90, clinical, physiological and biochemical evaluations were made. Plasma vitamin C was significantly decreased in the high fat diet group compared to the Mediterranean diet group. After wine supplementation to the Mediterranean diet, a significant 13.5% increase in plasma vitamin C was observed. Furthermore, when wine was added vitamin E decreased significantly in plasma, 15% in the high fat diet and 26% in the Mediterranean diet. Total plasma antioxidant capacity (total antioxidant reactivity) increased 28% above basal levels in the Mediterranean diet group, but not in the high fat diet group. In both groups, wine induced a marked increase in total antioxidant reactivity above basal levels, 56% and 23%, respectively. Oxidative DNA damage, detected as 8-hydroxydeoxyguanosine (8-OHdG) levels in blood leukocyte DNA, was markedly increased by the high fat diet; however, it was strongly reduced, to approximately 50% basal values, after wine supplementation, both in the high fat diet and Mediterranean diet groups. Endothelial function, evaluated noninvasively as flow-mediated vascular reactivity of the brachial artery, was suppressed by the high fat diet, and was normal after wine supplementation. These effects are attributed to oxidative stress associated with a high fat diet, and to the elevated plasma antioxidant capacity associated with wine consumption and the Mediterranean diet. The results presented support the following conclusions: a high fat diet induces oxidative stress; a diet rich in fruits and vegetables enhances antioxidant defenses; wine supplementation to a high fat or a Mediterranean diet increases plasma antioxidant capacity, decreases oxidative DNA damage, and normalizes endothelial function.

Disulphide bonds assignment in the inter-alpha-inhibitor heavy chains--structural and functional implications.

Human inter-alpha-inhibitor (IalphaI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan: a light one named bikunin, carrying the antiproteinase activity and two heavy chains H1 and H2. The amino acid sequences of these heavy chains are highly similar; however when IalphaI is digested by neutrophil proteinases, their proteolytic susceptibility strongly differs [Balduyck, M., Piva, F., Mizon, C., Maes, P., Malki, N., Gressier, B., Michalski, C. & Mizon, J. (1993) Human leucocyte elastase (HLE) preferentially cleaves the heavy chain H2 of inter-alpha-trypsin inhibitor (ITI), Biol. Chem. Hoppe-Seyler 374, 895-901]. We mapped the disulphide topology of the IalphaI heavy chains in order to investigate whether or not disulphide bonds might be responsible for their differential susceptibility to proteolysis. Using amino acid sequencing and mass spectrometry analysis, we demonstrate that the H1 heavy chain contains one free thiol group and two disulphide bridges of which one links two largely spaced cysteine residues (Cys239 and Cys511). Thus H1 is clearly different from H2 which contains two disulphide bonds between closely located cysteine residues. However, using immunoprint analysis, we show that, when IalphaI is subjected to a limited digestion by Staphylococcus aureus V-8 proteinase, the two polypeptide chains are similarly susceptible to proteolysis. This enzyme preferentially cleaves the IalphaI heavy chains from their N-terminal extremity. These results are consistent with the circular dichroism (CD) analysis, suggesting that the conformation of the polypeptide backbone of H1 is not very different from that of H2, with calculated alpha-helicities of 24% and 28%, respectively. The CD measurements reveal that the aromatic amino acids of H1 and H2 are in a different asymmetrical environment. Inside the IalphaI molecule, the heavy chains are linked to the glycosaminoglycan chain via their C-terminal aspartic acid residue. Thus we suggest that the affinity of cationic neutrophil proteinases for the anionic glycosaminoglycan is responsible for the cleavage of the heavy chains (mainly H2) near their C-terminal end and the high susceptibility of IalphaI to these proteinases.

Effects of inter-alpha-inhibitor in experimental endotoxic shock and disseminated intravascular coagulation.

We investigated the effects of human inter-alpha-inhibitor (I alpha I) on hemodynamics, oxygenation, and coagulation parameters in a porcine model of endotoxic shock. Four groups of six animals were studied: (1) control, (2) I alpha I group receiving 30 mg/kg I alpha I over 30 min, (3) LPS group receiving 5 micrograms.kg/min Escherichia coli endotoxin over 30 min, and (4) LPS + I alpha I group receiving 30 min after endotoxin 30 mg/kg/30 min I alpha I. We measured hemodynamic and oxygenation parameters, usual coagulation markers and plasma levels of thrombin-antithrombin complexes, antithrombin III activity, plasminogen activator tissue type, plasminogen activator inhibitor type 1, von Willebrand factor, tumor necrosis factor-alpha, and I alpha I at baseline and at 30, 60, 90, 120, 180, 240, and 300 min. In the I alpha I group, plasma I alpha I levels reached 447 +/- 23 mg/L just after injection and 287 +/- 39 mg/L at 300 min. I alpha I half-life was 7.3 +/- 1.9 h. In the IPS + I alpha I group, I alpha I plasma levels decreased more rapidly, reaching 260 mg/L at 300 min. Compared with the LPS group, administration of I alpha I normalized the mean arterial pressure and cardiac index, improved the LPS-induced pulmonary hypertension, and resulted in the blunted increase in blood lactate and oxygen extraction ratio. A significant decrease in thrombin-antithrombin complexes and plasminogen activator inhibitor type 1 levels were observed. There was no significant difference in plasma tumor necrosis factor-alpha levels. We concluded that in this hypodynamic model of endotoxin shock, I alpha I administration resulted in a marked improvement in the hemodynamic, oxygenation, and coagulation parameters.

Inter-alpha-inhibitor as marker for neutrophil proteinase activity: an in vitro investigation.

Human neutrophil proteinases have been implicated in the pathogenesis of a wide variety of inflammatory diseases. The degradation of plasma proteins such as coagulation and fibrinolysis factors has been attributed to the excessive release of elastase in septicemia and in other conditions in which heightened proteolysis occurs. Inter-alpha-inhibitor (IalphaI) is particularly sensitive to cleavage by leukocyte proteinases. For this reason, the determination of IalphaI has been proposed as a method for evaluating plasma protein proteolysis by neutrophil enzymes. In this article we provide evidence that intact residual IalphaI can be accurately quantified by enzyme-linked immunosorbent assay (ELISA) determination without interference from fragments released from IalphaI by incubation with triggered neutrophils. We demonstrate that under these conditions IalphaI was quickly and steadily proteolyzed in a cell dose-dependent manner. Alpha-1 proteinase inhibitor (alpha1PI) partially protected IalphaI; however, the proteolysis persisted when IalphaI was incubated with stimulated neutrophils in the presence of a large relative excess of alpha1PI over the amount of elastase theoretically present in cells. For the same amount of alpha1PI, serum provided a better protection than alpha1PI alone but did not completely inhibit the IalphaI degradation. Therefore, ELISA determination of IalphaI might be useful for monitoring the in vivo activity of neutrophil proteinases in systemic proteolytic states.

Human pre-alpha-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain.

Pre-alpha-inhibitor (P alpha I) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing P alpha I. Then, taking advantage of the differential affinity of the members of the inter-alpha-inhibitor family (I alpha I) for heparin-Sepharose and blue-Sepharose, we isolated P alpha I. Its specific antitryptic activity was 580 IU/g, higher than that of I alpha I: 420 IU/g. Its M(r), determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130,000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from P alpha I by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the HI and H2 heavy chains of I alpha I. In reducing conditions, it was quite similar to that of H2 (M(r) 85,000) but clearly different from that of H1 (M[r] 78,000). Thus, the so-determined apparent M(r) of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74,100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69,454) are really occupied by two N-glycans, probably of biantennary type.

Fecal beta-D-galactosidase production and Bifidobacteria are decreased in Crohn's disease.

Digestive bacterial microflora play a major role in the pathogenesis of Crohn's disease (CD). Bacterial enzyme activities, especially beta-D-galactosidase, are decreased in fecal extracts from CD patients. We hypothesized that an alteration of the colonic flora might be responsible for this decrease. Indeed, we demonstrate that beta-D-galactosidase production in supernates of anaerobic cultures was significantly (P < 0.01) reduced in feces from patients with active Crohn's disease (N = 7), when compared to healthy controls (N = 8). Therefore using X-gal and selective media, we enumerated bacteria able to release beta-D-galactosidase in feces from patients with active (N = 16) or quiescent disease (N = 5) and healthy controls (N = 14). Bifidobacteria numbers were significantly reduced in patients (P < 0.01 for active; P < 0.02 for quiescent disease) whereas Bacteroides and Lactobacilli counts remained unchanged. beta-D-Galactosidase activity and Bifidobacteria counts were significantly correlated (P < 0.03). Bifidobacteria are regarded as beneficial for the host. The reduction in Bifidobacteria is responsible for decreased beta-D-galactosidase activity. Thus oral administration of prebiotics that promote their growth might have potential therapeutic interest.

Purification and characterization of pig inter-alpha-inhibitor and its constitutive heavy chains.

With the view of investigating the metabolism of inter-alpha-inhibitor, a plasma serine-proteinase inhibitor, in an animal model of inflammatory syndrome, we isolated inter-alpha-inhibitor from pig plasma. A high yield was obtained (140 mg/liter) with a two-step procedure: anion-exchange chromatography followed by affinity chromatography on heparin-Sepharose. In contrast to bovine inter-alpha-inhibitor was highly similar to human inter-alpha-inhibitor: its heavy chains are homologous to the human H1 and H2 heavy chains, as shown by chromatographic and electrophoretic properties, cross-immunoreactivity and N-terminal sequencing. Pig may therefore represent a good animal model to study inter-alpha-inhibitor metabolism and elucidate its physiological role.

Pig I alpha I appears unmodified in plasma in case of endotoxin-induced disseminated intravascular coagulation.

The unrestricted activity of leukocyte proteinases is thought to contribute to the degradation of plasma proteins and thus amplify the coagulation disorders occurring in septic shock. Inter-alpha-inhibitor (I alpha I) is a plasma protein particularly susceptible to their action. Therefore we investigated its behavior in a porcine model of endotoxin shock which reproduces the coagulation changes observed in human sepsis. We did not detect any qualitative or quantitative modification of porcine I alpha I in plasmas collected from pigs after endotoxin infusion. To explain these data, I alpha I was incubated with polymorphonuclear neutrophils (PMN) stimulated by FMLP in the presence of cytochalasin B. We found that, unlike human PMN, porcine cells were unable to proteolyze I alpha I. Moreover, in the incubation medium of pig PMN, triggered either by FMLP or PMA, no measurable elastase activity was evidenced. Therefore, we urge to better take into account species differences in functional responses of PMN, to explain the experimental results obtained in animal models of septic shock.