[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.

Plasma proteins immunologically related to inter-alpha-trypsin inhibitor.

SDS-polyacrylamide gel electrophoresis and immunoblot were applied to analysis of plasma proteins immunologically related to inter-alpha-trypsin inhibitor (ITI). In this system, anti-ITI sera were able to identify ITI and other components with an Mr near 120 kDa which would be degradation products of ITI by limited proteolysis. An anti-UTI (urinary trypsin-inhibitor) serum could detect, beside these derivatives, two minor components (Mr values near 90 and 60 kDa). Analysis of perchloric acid supernatants of plasma samples, using the same technic, induced visualization of a new component, similar to urinary trypsin inhibitor which could not be detected by direct analysis. This one was also characterized in a higher content in pathological samples (renal failure and infectious diseases).

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.

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.

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.

Development of an enzyme-linked immunosorbent assay for human plasma inter-alpha-trypsin inhibitor (ITI) using specific antibodies against each of the H1 and H2 heavy chains.

Inter-alpha-trypsin inhibitor (ITI) is a serine-proteinase inhibitor of human plasma enzymes. ITI is composed of three polypeptide chains covalently linked: bikunin, responsible for the antiprotease activity and two heavy chains H1 and H2. Human plasma also contains other components immunologically related to ITI such as pre-alpha-trypsin inhibitor (paI), inter-alpha-like inhibitor (IalphaLI) and free bikunin. The ELISA procedure we propose exclusively measures native ITI within the range 12.5-200 microgram/l. The intra- and interassay coefficients of variation were less than 5.6% and 8.7%, respectively. When ITI was added to plasma samples, full recovery was obtained. EDTA-plasma from 30 healthy individuals revealed a mean level of 241.5 mg/l (range 145.5-506). The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the specific measurement of native ITI in blood and thus might represent a useful tool for further physiopathological studies.

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.

A simple method for the measurement of different forms of alpha 1 proteinase inhibitor in the faeces of patients with Crohn's disease.

Alpha-1 proteinase inhibitor (alpha 1PI), formerly named alpha 1-antitrypsin, is excreted in the faeces of patients with Crohn's disease as isoforms clearly separated by SDS-PAGE and immunoblot analysis. Relapses in Crohn's disease are generally associated with the appearance in faeces of M(rs) 51,000 and 45,000 glycosylated forms of alpha 1PI, as compared with normal subjects and most of the patients in quiescent phases of their disease who excrete an M(r) 38,000 unglycosylated form of alpha 1PI. We used their differential Concanavalin-A reactivity to design a specific test. The proposed assay is potentially helpful for the follow-up of patients under therapy and for early recognition of attacks of Crohn's disease.

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.

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.

Urinary trypsin inhibitory capacity determination: study in patients with disseminated cancers.

We present a method for automated analysis of urinary trypsin inhibitory capacity. The validity of the method has been established. The mean value of urinary antitryptic activity is higher in patients with disseminated cancers (70 IU/1, n = 243) than in healthy donors (14,4 IU/1, n = 117). However, frequency distribution of urinary trypsin inhibitor values shows a great overlap, so that an increased level of urinary trypsin inhibitor cannot be considered as a marker of neoplastic diseases but seems to be a non specific indicator of inflammatory syndromes.

[Decrease of fecal beta-galactosidase activity in Crohn disease]. / L'activité bêta-galactosidasique fécale est diminuée au cours de la maladie de Crohn.

An increased degradation of colonic mucus by bacterial enzymes might participate in the development of mucosal lesions in inflammatory bowel disease. The biodisponibility of drugs used in the treatment of such disease relies upon the metabolic activity of colonic bacterial flora. This activity can be indirectly assessed by measuring fecal enzymatic activities. The aim of this study was to compare fecal beta-galactosidase (beta-gal) activity in controls, in patients suffering from extradigestive inflammatory disease and in patients with Crohn's disease (CD). Three groups were studied including 11 healthy volunteers (6 F, 5 M) mean age 29 years (21-37), 20 patients with rheumatoid arthritis (RA) (17 F, 3 M), mean age 61.5 years, and 34 patients with non operated CD (21 F, 13 M) mean age: 27 years (13-50). The Crohn disease activity index (CDAI) was > 150 in 24 and < 150 in 10. beta-gal activity was measured in fecal extracts by its ability to hydrolyze paranitrophenyl beta-D-galactopyranoside and expressed as units of enzymatic activity/gram of fecal proteins. beta-gal activity was significantly decreased in patients with CD (16 +/- 4.5 U/g) (m +/- sem) as compared with patients with RA (353 +/- 64 U/g) (P < 0.0001) and to controls (263 +/- 40 U/g) (P = 0.002). beta-gal activity was not significantly different in controls and in patients with RA. Patients with active CD had a significantly lower beta-gal activity than patients with quiescent CD (9.5 +/- 3.7 U/g vs 31.4 +/- 11.5 U/g) (P = 0.006).(ABSTRACT TRUNCATED AT 250 WORDS)

[Qualitative study of fecal alpha-1-antitrypsin in patients with inflammatory digestive disease and patients with ileostomy]. / Etude qualitative de l'alpha-1-antitrypsine fécale dans les maladies inflammatoires du tube digestif et chez des patients avec iléostomie.

Alpha-1-antitrypsin is a glycoprotein which is excreted in feces under three different forms of molecular weight 38, 45 and 51 kDa. The 45 and 38 kDa forms are the result of a partial or total removal of the carbohydrate moiety, respectively. We determined the molecular forms of fecal alpha-1-antitrypsin in 10 controls, 13 patients with protein-losing enteropathy other than inflammatory bowel disease, 70 patients with active (n = 55) (CDAI greater than 150) and inactive (n = 15) (CDAI less than 150) Crohn's disease, 14 patients with active (n = 12) and inactive (n = 2) ulcerative colitis, and 17 patients with ileostomy. Fecal 38 kDa alpha-1-antitrypsin was found in all controls, all patients with protein-losing enteropathy, in 82 percent of patients with inactive inflammatory bowel disease, and in 20 percent of patients with active inflammatory bowel disease. In contrast, the 51 kDa and 45 kDa forms were present in feces of 80 percent of patients with active inflammatory bowel disease, and in only 17 percent of patients with inactive inflammatory bowel disease. Patients with Crohn's disease and the 51 kDa form (n = 39) had significantly higher values of activity index (CDAI) and orosomucoid than patients with Crohn's disease and the 38 kDa form (n = 26) (P less than 0.01). Deglycosylated 38 kDa alpha-1-antitrypsin was never recovered in ileostomy samples. This suggests that deglycosylation of alpha-1-antitrypsin occurred in the colon and is impaired in patients with active inflammatory bowel disease.

[Application of polyacrylamide gel electrophoresis-SDS to the study of proteinuria (author's transl)]. / Application de l'électrophorèse en gel de polyacrylamide-SDS à l'étude des protéinuries.

The authors present a variant of the technique of sodium dodecylsulfate acrylamide gel electrophoresis (SDS-PAA) reported by Weber and Osborn, for the analysis of urinary proteins. SDS-PAA separates the proteins chiefly according to their molecular radius. SDS-PAA, as compared to acetate cellulose electrophoresis and immunoelectrophoresis, gives better resolution and may be recommended for the investigation of proteinuria.

[Antitrypsin activity of the urine and alpha 1 protease inhibitor]. / Activité antitrypsique de l'urine et alpha 1 protéase inhibiteur.

The urinary trypsin inhibitory capacity (TIC) is mainly due to the excretion of an inhibitor which is immunologically related to inter-alpha:trypsin inhibitor (ITI). However, alpha 1 protease inhibitor (alpha 1 PI) can be found in urine of patients with proteinuria. When this one is greater than 1 g/l, the measured TIC is more or less markedly related to the amount of alpha 1 PI present in the analyzed sample. The antitryptic activity of alpha 1 PI can be ruled out by incubating urine with specific anti-alpha 1 PI immunoglobulins. An identical result is obtained by acidification of the sample prior to TIC determination. Moreover, after freezing of urine, only the antitryptic activity of alpha 1 PI is strikingly decreased. Thus, in the presence of a significant proteinuria (greater than 1 g/l), a preliminary acidification of urine allows a suitable and specific measurement of TIC due to the inhibitor immunologically related to ITI. Thus, this one is a sensitive, useful and easy test for detecting and monitoring infections.

[Clinical value of the determination of urinary antitrypsin activity]. / Intérêt clinique de la détermination de l'activité antitrypsique urinaire.

Urinary trypsin inhibitory capacity is mainly due to the excretion of a glycoprotein which is immunologically related to the inter alpha-trypsin inhibitor and may be a proteolytic degradation product of that substance. It was tested in 133 subjects divided into 7 groups: 24 healthy controls (group A), 21 patients with bacterial infection (group B), 37 with bacterial infection under antibiotic therapy (group C), 25 with connective tissue disease (group D), 8 with infected connective tissue disease (group E), 14 with cancer (group F) and 4 with infected cancer (group G). Urinary trypsin inhibitory capacity level was very low in controls (3.32 +/- 0.8 U/g urinary creatinine), but it was dramatically increased when infection was present (149.67 +/- 23.6 U/g urinary creatinine). This test appeared to be more effective than serum C-protein measurement simultaneous carried out in the same patients. Urinary trypsin inhibitory capacity is not related to the degree of proteinuria in the urine sample, but it is increased in patients with chronic renal failure excluded from this study. Thus, its measurement is a sensitive, easy and useful test for detecting and monitoring infections. The return to its physiological value is a very good argument in favour of therapeutic effectiveness.

A proteoglycan related to the urinary trypsin inhibitor (UTI) links the two heavy chains of inter-alpha-trypsin inhibitor.

cDNA studies have suggested that inter-alpha-trypsin inhibitor (ITI) is a complex of several different peptide chains; the sequence of the inhibitory part of ITI is in excellent agreement with that of the urinary trypsin inhibitor (UTI). The present report demonstrates that a compound immunologically related to UTI is released by digestion with porcine pancreatic elastase or human leucocyte elastase. Since UTI has been shown to be a proteoglycan, ITI has been treated by chondroitinase. In these conditions, ITI is dissociated and gives rise to two heavy chains (78 and 85 kDa) and one light chain (26 kDa) immunologically related to UTI and which in PAGE moves close to UTIc (produced by chondroitinase treatment of UTI). We suggest that ITI is a non-covalent complex comprising two heavy chains and one light chain immunologically related to UTI and which is also a proteoglycan.