Fasting plasma carotenoids concentrations in Crohn's and pancreatic cancer patients compared to control subjects.

Carotenoids are colored molecules that are widespread in the plant kingdom, but animals cannot synthesize them. Carotenes are long, apolar molecules which require fully functioning digestive processes to be absorbed properly. Hence they could be interesting markers of intestinal absorption and digestion. Indeed, only few tests are available to assess these processes and only the D-xylose tolerance test is routinely used. However D-xylose is a sugar that tests only the absorption of water-soluble compounds and it only tests duodenal absorption. In this study, we have evaluated carotenoids as markers of digestion and absorption. We compared fasting plasma carotenoids concentrations in 21 control subjects, 20 patients with Crohn's disease, and 18 patients with pancreatic cancer. Crohn's disease alters intestinal absorption while pancreatic cancer decreases pancreatic enzyme secretion thus impairing digestion. Results show that all carotenoids are significantly lower in Crohn's and cancer patients as compared to control subjects and the multifactorial analysis shows that this decrease is mostly independent of dietary intake. Interestingly, maldigestion as seen in pancreatic cancer more strongly influences plasma lutein and lycopene concentrations while malabsorption in Crohn's disease acts on other carotenoids. Thus carotenoids could be interesting alternatives for testing and following patients that are suspected of having malabsorption or maldigestion syndromes.

[Kinetics of Pseudomonas aeruginosa virulence gene expression during chronic lung infection in the murine model]. / Cinétique d'expression des gènes de virulence de Pseudomonas aeruginosa au cours d'une infection pulmonaire chronique dans le modèle murin.

UNLABELLED: Pseudomonas aeruginosa is a Gram-negative bacillus frequently encountered in human diseases. P. aeruginosa produces a large number of secreted and cell associated virulence factors. Their production is coordinated by various systems of gene regulation. The correlation and sequential intervention of regulation systems during a pulmonary infection have not been determined yet. OBJECTIVE: The aim of this study was to analyze the expression of three P. aeruginosa virulence genes (exoS, lasI, and algD) during the first seven days of chronic lung infection. To do so, mice were infected intratracheally with agarose beads containing P. aeruginosa. RESULTS: The results were a progressive decrease of exoS transcription and an increase of algD, and lasI transcription during infection. This dynamic evolution was consistent with the clinical observation, which demonstrated a progressive loss of type III secretion system function and an increase in the mucoid phenotype development in P. aeruginosa strains from cystic fibrosis patients. CONCLUSION: The development of a P. aeruginosa pulmonary chronic infection associates a decrease of gene expression related to a type III secretion system and an increase of alginate production.

The high lipid content of respiratory mucins in cystic fibrosis is related to infection.

Seven human bronchial mucins secreted by patients suffering from chronic bronchitis or from cystic fibrosis were prepared by exclusion from a Sepharose CL-2B column in 6 M guanidinium chloride. Their behaviour in CsBr density gradient centrifugation was compared. The mucin fractions were associated with peptides and lipids, the abundance of which decreases with the buoyant density. The lipid content of the mucin fractions appears to be related to the infectious character of the bronchial secretion and seems to be independent of cystic fibrosis. The content of fatty acids remaining after delipidation of mucin fractions from patients with chronic bronchitis or with cystic fibrosis also appears to be related more to the purulent character of the sputum than to cystic fibrosis or chronic bronchitis.

[Recent knowledge about intestinal absorption and cleavage of carotenoids]. / Données récentes sur l'absorption et le catabolisme des caroténoïdes.

Our knowledge about intestinal absorption and cleavage of carotenoids has rapidly grown during the last years. New facts about carotenoid absorption have emerged while some controversies about cleavage are close to end. The knowledge of the absorption and conversion processes is indispensable to understand and interpret the perturbations that can occur in the metabolism of carotenoids and vitamin A. Recently, it has been shown that the absorption of certain carotenoids is not passive - as believed for a long time - but is a facilitated process that requires, at least for lutein, the class B-type 1 scavenger receptor (SR-B1). Various epidemiological and clinical studies have shown wide variations in carotenoid absorption from one subject to another, such differences are now explained by the structure of the concerned carotenoid, by the nature of the food that is absorbed with the carotenoid, by diverse exogenous factors like the intake of medicines or interfering components, by diet factors, by genetic factors, and by the nutritional status of the subject. Recently, the precise mechanism of beta-carotene cleavage by betabeta-carotene 15,15' monooxygenase (EC 1.14.99.36) - formerly called beta-carotene 15,15' dioxygenase (ex EC 1.13.11.21) - has been discovered, and a second enzyme which cleaves asymmetrically the beta-carotene molecule has been found. beta-carotene 15,15' monooxygenase only acts on the 15,15' bond, thus forming two molecules of retinal from one molecule of beta-carotene by central cleavage. Even though the betabeta-carotene 15,15' monooxygenase is much more active on the beta-carotene molecule, a study has shown that it can act on all carotenoids. Searchers now agree that other enzymes that can catalyse an eccentric cleavage of carotenoids probably exist, but under physiological conditions the betabeta-carotene 15,15' monooxygenase is by far the most active, and it is mainly effective in the small bowel mucosa and in the liver. However the conversion of provitamin A carotenoids into vitamin A is only partial, and requires a satisfactory protein status.

Direct double antibody sandwich immunoassay for Pseudomonas aeruginosa elastase.

A direct sandwich enzyme immunoassay was developed in order to quantify Pseudomonas aeruginosa elastase. As a solid phase the wells of a microtitre plate were coated with specific IgG and horseradish peroxidase labelled IgG was used as the second antibody. The detection limit of the assay was 0.26 ng/ml and a good agreement was found with elastolytic activity determined using elastin-Congo red. This assay was simple, specific, sensitive and reproducible, and permits the determination of low levels of elastase.

Identification of phospholipids in secretory granules of human submucosal gland respiratory cells.

Although it has been shown that tracheal epithelial cells in culture synthesize and secrete phospholipids, no direct evidence for in situ phospholipid storage in human respiratory secretory epithelial cells has been demonstrated. We used a high-resolution cytochemical enzyme-gold technique to identify and precisely localize phospholipids in human submucosal gland secretory cells. In addition, lysozyme, a specific serous cell marker, was identified using the biotinstreptavidin gold technique with lysozyme antiserum. This double labeling of phospholipids and lysozyme was performed using gold particles of diameters 15 nm and 5 nm, respectively. Quantitation of phospholipid labeling was performed on an image analyzer. Phospholipids were identified in serous granules (8.87 +/- 2.21 gold particles/microns 2) in a significantly (p less than 0.05) higher density than in mucous granules (5.57 +/- 3.07 gold particles/microns 2). These results support the hypothesis that submucosal human airway serous and mucous secretory cells produce phospholipids which may be secreted in the airway lumen.

Role of phospholipid lining on respiratory mucus clearance by cough.

Phospholipid lining, present at the respiratory mucus-mucosa interface, may have an important role in the protective function of the airways by its abhesive properties and may also facilitate mucus transport. To mimic respiratory mucus-mucosa interface, monolayers of three different forms of phosphatidylglycerol (PG) have been deposited on glass slides by the Langmuir-Blodgett technique. Mucus adhesion and clearance by cough of mucus on these PG-coated or noncoated surfaces have been analyzed and compared, using frog respiratory mucus as "normal" mucus. Among the three PG types studied, the phosphatidylglycerol distearoyl, which is the phospholipid with the longest saturated fatty acid chain, was found to significantly improve the mucus cough clearance by decreasing the mucus work of adhesion compared with the noncoated surfaces. On the other hand, phosphatidylglycerol dipalmitoyl did not improve mucus cough clearance although it decreased mucus adhesion, and phosphatidylglycerol dioleyl did not improve either mucus cough clearance or mucus adhesion.

Assay of human pancreatic lipase in biological fluids using a non-competitive enzyme immunoassay.

A sandwich enzyme immunoassay has been developed for human pancreatic lipase using polystyrene balls coated with specific IgG as the first antibody and peroxidase-labelled IgG as the second antibody. The detection limit was 0.5 microgram/l. Good parallelism was observed with the curves obtained from standard lipase and lipase present in serum, pancreatic juice and duodenal contents, demonstrating that the assay may be used to measure the level of the protein in different biological fluids. Mean values of lipase in human sera were 12.3 +/- 6.8 micrograms/l in adults and 4.5 +/- 2.7 in newborns. In all cases a good correlation was found in serum between the catalytic activity and the enzyme immunoassay. Lipase is detectable in amniotic fluids at the 18th week of pregnancy but at a very low level (0.95 +/- 0.32 microgram/l). In pancreatic juices, lipase concentration was 14.6% of the total protein content. A study on cystic fibrosis patients showed a poor correlation between blood pancreatic lipase concentration and fat malabsorption underlying the difficulty in assessing pancreatic function by the measurement of serum pancreatic enzymes. The use of the lipase assay in duodenal contents would permit better assessment of pancreatic function in patients presenting a severe or borderline defect in fat digestion and absorption.

Correlation between the cystic fibrosis protein detected by isoelectric focusing and a protein of 12 kDa detected by SDS-polyacrylamide gel electrophoresis.

We have studied both by isoelectric focusing and polyacrylamide gel electrophoresis in the presence of SDS, sera of individuals homozygous (25) and heterozygous (26) for cystic fibrosis and compared them to controls (13). As in our first study [1], the protein with a pI value of 8.4 called 'cystic fibrosis protein' or CFP, was found in about 70% of homozygous and carriers and in 15% of controls. When the same sera were analysed by polyacrylamide gel electrophoresis in the presence of SDS and after staining with Coomassie Blue, an additional protein with a molecular weight close to 12,000 (P12) was present in most of the sera containing CFP, suggesting a close relationship between the two proteins. By increasing the sensitivity of staining by using silver nitrate, P12 was detected with variable intensity in almost all sera of homozygotes and heterozygotes and at the level of traces in all normal sera. These data suggest that P12, like CFP, would be a normal serum protein quantitatively increased in affected subjects.

Relationships between the lipid content and the rheological properties of airway secretions in cystic fibrosis.

The lipid composition and rheological properties of expectorated airway secretions have been analyzed in 16 patients with cystic fibrosis (CF), separated into two groups according to whether their secretions were superinfected or not. The total lipid content was higher in the superinfected CF secretions. The content in cholesterol and GL1, GL3 and GL4 glycosphingolipid fractions were significantly higher in the superinfected in comparison to the non-superinfected group. The viscosity was two-fold higher in the CF superinfected group. A significant correlation (r = 0.72, p less than 0.01) was observed between the apparent viscosity and the total lipid content of CF secretions. Cholesterol, glycosphingolipids and sphingomyelin were the lipid components which were the most closely and positively correlated to the viscosity. On the opposite, phosphoglycerol was negatively correlated (r = -0.72, p less than 0.05) to the viscoelastic properties of CF airway secretions.

Plasma and serum lactoferrin levels in cystic fibrosis. Relationship with the presence of cystic fibrosis protein.

Plasma lactoferrin concentrations were measured in blood of cystic fibrosis patients, heterozygotes and controls using a specific and sensitive enzyme immunoassay. 67 plasmas were studied (26 controls, 23 heterozygotes, 18 cystic fibrosis patients) and the results showed a statistically significant increase (p less than 0.05) of the level of plasma lactoferrin in cystic fibrosis patients (265 +/- 224 micrograms/l) compared to controls (168 +/- 100 micrograms/l) and heterozygotes (150 +/- 72 micrograms/l). Since it is well established that plasma lactoferrin level could be influenced by the number of neutrophils, a second set of experiments was performed on 20 cystic fibrosis patients on whom leukocyte counts were also made. When the 15 plasmas with normal neutrophils (in the range 2 to 6 giga/l) were considered, the mean lactoferrin level was 318 +/- 116 micrograms/l, still far above the normal values. For serum, a similar significant increase of lactoferrin concentration was observed in 33 cystic fibrosis patients (610 +/- 551 micrograms/l) compared to the values observed for 25 controls (237 +/- 155 micrograms/l) and 37 heterozygotes (272 +/- 231 micrograms/l). Cystic fibrosis protein (CFP) was identified in the same sera by isoelectric focusing and the intensity of the band was closely related to the increase of lactoferrin concentration in cystic fibrosis patients. In contrast, no difference in serum lactoferrin concentrations was observed between heterozygotes with or without CFP, indicating that the increased CFP concentration cannot be due only to altered granulocyte function.

Phospholipid composition and surface-active properties of tracheobronchial secretions from patients with cystic fibrosis and chronic obstructive pulmonary diseases.

Among the various components of tracheobronchial secretions, lipids and particularly phospholipids have been shown to influence rheological properties of airway secretions in patients with cystic fibrosis. We studied the phospholipid composition of tracheobronchial secretions, collected from patients suffering from cystic fibrosis (CF) and other chronic obstructive pulmonary diseases (COPD), and we analyzed the possible relationship between the phospholipid profile and the wettability of tracheobronchial secretions evaluated by the measurement of contact angle. Although total phospholipid content and contact angle of tracheobronchial secretions were significantly increased (P less than 0.01) in CF compared to COPD, no significant relationship existed between these two parameters. The concentrations of the different phospholipid subclasses were not homogeneously modified according to the origin of the secretions. Compared to COPD secretions, the CF secretions were characterized by a significant (P less than 0.001) increase in rigidifying fractions such as sphingomyelin and phosphatidylserine/phosphatidylinositol and a significant (P less than 0.001) decrease in surface-active fractions, such as phosphatidylcholine and phosphatidylglycerol (PG) (P less than 0.001). In the two groups, the surface-active phospholipid fraction, PG, was negatively correlated to the contact angle of tracheobronchial secretions. These results suggest that a decrease in PG content in CF secretions may be one factor responsible for an increase in their adhesivity to the respiratory mucosa, and, consequently, for mucus stasis and severity of bronchial obstruction in cystic fibrosis.

[Carotenoids: 2. Diseases and supplementation studies]. / Les caroténoïdes: 2. Pathologie et études de supplémentation.

Inverse correlations have been found in most studies on the relationship between dietary intake and plasma concentrations of carotenoids on one side and degenerative diseases such as cancer and cardiovascular diseases on the other side. Protective effects of carotenoids have been found for pathologies of the retina and the skin. Concentrations of these molecules in blood are lower in digestive pathologies and HIV. Short- and long-term toxicity of carotenoids was found to be low. In combination with the beneficial effects found for diets rich in carotenoids, this has initiated trials with relatively high doses of carotenoid supplements. In the study in Linxian (China) in a rural population with poor nutritional status, supplementation with beta-carotene, zinc, selenium and vitamin E lowered total mortality and mortality from stomach cancer. Other studies (ATBC, Caret.) on well-fed subjects did not show beneficial effects on mortality from cancer and cardiovascular diseases. On the contrary, higher mortality and lung cancer incidence was found in supplemented subjects that were also exposed to asbestos and cigarette smoke. In these studies, doses of supplemental beta-carotene were high and varied from 20 to 50 mg/day. One still ongoing study, called Suvimax, doses subjects for eight years with a cocktail of vitamins and minerals including 6 mg per day of beta-carotene. This supplementation with physiologically seen more "normal" doses might give clarity on the question if beta-carotene is the protective factor in fruits and vegetables.

[Measurement of carotenoids by high pressure liquid chromatography: from difficulties to solutions]. / Dosage des caroténoïdes par chromatographie liquide haute performance: des difficultés aux solutions.

The measurement of serum carotenoids by HPLC has been largely improved during the last 10 years. However these techniques still require much time and skills, and direct application of published methods is rarely satisfying. We report here the difficulties that we met to transfer some HPLC methods described in the literature to our laboratories. We propose some solution to overcome the problems that we have encountered, our experience will perhaps help out other biologists. We reported also some results obtained in healthy populations.

[Carotenoids: 1. Metabolism and physiology]. / Les caroténoïdes: I. Métabolisme et physiologie.

Carotenoids are a family of pigments with at least 600 members. They derive from lycopene after steps of cyclisation, dehydrogenation and oxidation. It is their chemical structure that determines their physiochemical properties and, in part, their biological activities. About 50 carotenoids can be found in human diet and about 20 of them have been found in plasma and tissues. There is no RDA (Recommended Daily Allowance) for carotenoids. Quantities of carotenoids in diet are difficult to estimate, partly because methods used for the establishment of food composition tables were not specific and sensitive enough. Also, given values do not always take into account variations due to season and region of culture. Absorption of beta-carotene in humans has been the subject of numerous studies but only very little is known about other carotenoids. In general, absorption depends on bioavailability from the food matrix and solubility in micelles. After absorption through passive diffusion, carotenoids follow the chylomicrons metabolism. They are taken up by the liver and released in the blood stream in lipoproteins (VLDL). Carotenoids with no-substituted beta-ionone cycles (alpha and beta-carotene and beta-cryptoxanthin) have provitamin A activity. Highest activity has been found for all-trans beta-carotene. Not all steps of vitamin A biosynthesis and metabolism of other carotenoids have been clarified yet. Besides their provitamin A activity, carotenoids have numerous biological functions. They are efficient scavengers of free radicals, particularly of 1O2. In vitro they have been shown to protect LDL. However, results in vivo are inconsistent. Other functions include enhancement of gap junctions, immunomodulation and regulation of enzyme activity involved in carcinogenesis.

Functions of proteins and lipids in airway secretions.

Proteins and lipids synthesized by airway secretory cells or transudated are active components in the protection of respiratory epithelium. Proteins and ions are involved in the control of mucus hydration. Secretory proteins, such as secretory immunoglobulin A (IgA), transferrin and lysozyme, participate in the airway antibacterial defence. Other biochemical components found in secretions, such as anti-inflammatory and antioxidant agents as well as antiproteases, contribute significantly to the protection of the underlying epithelium.