Digestion of micellar casein in duodenum cannulated pigs. Correlation between in vitro simulated gastric digestion and in vivo data.

Correlation and validation of the results of simulated gastrointestinal digestion of food compounds towards in vivo data is essential. The objective of this work was to monitor the digestion of milk micellar casein in the porcine upper intestinal tract and to match the outcome with the gastric in vitro digestion following the Infogest harmonized protocol. In pig duodenum, small amounts of intact caseins were present in all samples, while caseins were observed up to 60 min of gastric in vitro digestion. The peptide profile generated after in vitro and in vivo digestion showed clear similarities with specific overrepresented regions rich in proline and other hydrophobic residues. The statistical comparison of the in vivo and in vitro peptidome resulted in satisfactory correlation coefficients, up to 0.8. Therefore, the in vitro protocol used was a robust and simple model that provides a similar peptide profile than that found in porcine duodenum.

Peptidomic data in porcine duodenal effluents after oral administration of micellar casein.

The data in this article are related to the research publication "Digestion of micellar casein in duodenum cannulated pigs. Correlation between in vitro simulated gastric digestion and in vivo data" (Miralles et al., Food Chemistry, 2021, 343, 128428). Pig duodenum effluents were collected with a T-shaped cannula 15 min before and during digestion over 150 min after casein intake. The casein degradation profile of individual pigs during digestion is presented. All identified peptide sequences at different digestion times for six subjects are provided. The peptide profile of digests in the form of heat maps is shown for αs1-, αs2-, ß- and κ-casein. The sum of amino acids belonging to peptides released from ß- and αs1-casein has been used to determine correlation coefficients and range the inter-individual variability. Finally, the global amino acid composition, isoelectric point and sequence length of all released peptides has been determined.

Beta-lactoglobulin as source of bioactive peptides.

Beta-lactoglobulin (beta-Lg) is currently an important source of biologically active peptides. These peptides are inactive within the sequence of the precursor protein, but they can be released by in vivo or in vitro enzymatic proteolysis. Once released, these peptides play important roles in the human health, including antihypertensive, antioxidant and antimicrobial activities as well as opioid-like features and ability to decrease the body-cholesterol levels. Bioactive peptides derived from beta-Lg are currently a point of intensive research. Their structure, biological significance and mechanism of action are briefly presented and discussed in this review.

Synergistic effect between different milk-derived peptides and proteins.

Antimicrobial peptides derived from food proteins constitute a new field in the combined use of antimicrobial agents in food. The best examples of milk-derived peptides are those constituted by bovine lactoferricin [lactoferrin f(17-41)] (LFcin-B) and bovine alpha(s2)-casein f(183-207). The aim of this work was to study if the antimicrobial activity of a natural compound employed in food preservation, nisin, could be enhanced by combination with the aforementioned milk-derived peptides. Furthermore, the possibility of a synergistic effect between these peptides and bovine lactoferrin (LF) against Escherichia coli and Staphylococcus epidermidis was also studied. Finally, the most active combinations were assayed against the foodborne pathogens Listeria monocytogenes and Salmonella choleraesuis. Results showed a synergistic effect when LFcin-B was combined with bovine LF against E. coli. In the same way, the combination of LFcin-B with bovine LF was synergistic against Staph. epidermidis. Bovine LF and nisin increased their antimicrobial activity when they were assayed together with bovine alpha(s2)-casein f(183-207). It is important to note the synergistic effect among LFcin-B and bovine LF, because both compounds might be simultaneously in the suckling gastrointestinal tract and could, therefore, have a protective effect on it. The other synergistic effect high-lighted is that between alpha(s2)-casein f(183-207) and nisin against L. monocytogenes because of the ability of L. monocytogenes to develop resistance to nisin.

Sensory and mass spectrometric analysis of the peptidic fraction lower than one thousand daltons in Manchego cheese.

A total of 107 different peptides, all derived from alphaS1-, alphaS2-, and beta-casein, were identified in different fractions of artisan or industrial Manchego cheese at 4 and 8 mo of ripening, and their sequences were examined. Most of these peptides are described for the first time in Manchego cheese. Taste characteristics (umami and bitter) were assigned based on their AA sequence and the position of these AA within the sequence. The umami taste was predominant in all fractions analyzed by the panelists, and the peptides EQEEL, QEEL, and EINEL, containing a high number of glutamic residues, were found within the fractions. However, in several fractions described as having umami characteristics, no peptides responsible for this taste were detected. Therefore, compounds other than peptides seem to be involved in the umami properties of water-soluble extracts lower than 1,000 Da of Manchego cheese.

Short communication: Identification of the aroma compounds responsible for the floral/rose flavor in water-soluble fractions of Manchego cheese.

Water-soluble fractions from Protected Denomination of Origin Manchego cheese, with molecular weight <1,000 Da, were fractionated using gel permeation chromatography and studied using both instrumental and sensorial analysis. In 2 of the fractions, panelists detected a floral, rose-like flavor. Analysis of these fractions by gas chromatography-mass spectrometry after simultaneous distillation extraction with dichloromethane identified 2-phenylethanol and phenylacetaldehyde as the compounds responsible for this flavor.

In vivo inactivation of the yeast plasma membrane ATPase in the absence of exogenous catabolism.

Yeast plasma membrane ATPase is inactivated up to 80% in the absence of catabolism of exogenous nutrients (exogenous catabolism). This inactivation, that is not accompanied by a decrease in the cellular content of ATPase, is due to an irreversible decrease of the Vmax and does not require protein synthesis. The inactivated enzyme maintains the ability to be regulated by fermentable sugars but shows important alterations in the characteristics of this regulation. Upon addition of glucose, the Vmax of the inactivated enzyme increases as well as its Ki for vanadate but, in contrast to the normal enzyme, its affinity for ATP or its pH optimum do not increase. It is concluded that in the absence of exogenous catabolism an irreversible modification of the yeast plasma membrane ATPase takes place that affects several of its kinetic properties.

Angiotensin-converting enzyme inhibitory activity of peptides derived from caprine kefir.

In this study, a potent angiotensin-converting enzyme (ACE)-inhibitory activity was found in a commercial kefir made from caprine milk. The low molecular mass peptides released from caseins during fermentation were mainly responsible for this activity. Sixteen peptides were identified by HPLC-tandem mass spectrometry. Two of these peptides, with sequences PYVRYL and LVYPFTGPIPN, showed potent ACE-inhibitory properties. The impact of gastrointestinal digestion on ACE-inhibitory activity of kefir peptides was also evaluated. Some of these peptides were resistant to the incubation with pepsin followed by hydrolysis with Corolase PP. The ACE-inhibitory activity after simulated digestion was similar to or slightly lower than unhydrolyzed peptides, except for peptide beta-casein f(47-52) (DKIHPF), which exhibited an activity 8 times greater after hydrolysis.

Caseinophosphopeptides released after tryptic hydrolysis versus simulated gastrointestinal digestion of a casein-derived by-product.

The production of caseinophosphopeptides from a casein-derived by-product generated during the manufacture of a functional ingredient based on antihypertensive peptides was attempted. The casein by-product was submitted to tryptic hydrolysis for 30, 60 and 120min and further precipitated with calcium chloride and ethanol at pH 4.0, 6.0 and 8.0. Identification and semi quantification of the derived products by tandem mass spectrometry revealed some qualitative and quantitative changes in the released caseinophosphopeptides over time at the different precipitation pHs. The by-product was also subjected to simulated gastrointestinal digestion. Comparison of the resulting peptides showed large sequence homology in the phosphopeptides released by tryptic hydrolysis and simulated gastrointestinal digestion. Some regions, specifically αS1-CN 43-59, αS1-CN 60-74, ß-CN 1-25 and ß-CN 30-50 showed resistance to both tryptic hydrolysis and simulated digestion. The results of the present study suggest that this casein-derived by-product can be used as a source of CPPs.

Isolation and partial characterization of cholesterol pronucleating hydrophobic glycoproteins associated to native biliary vesicles.

Cholesterol is transported both in unilamellar phosphatidylcholine vesicles and in bile salts-mixed micelles in native bile. The vesicular carrier of biliary lipids apparently has a well defined protein profile with a potent cholesterol crystallization-promoting activity. This study was conducted to identify and further characterize these vesicular proteins and to test the effect of isolated vesicular proteins on the cholesterol crystal formation in supersaturated model bile. The results confirmed that proteins are a constant component of highly purified biliary vesicles both in hepatic and gallbladder bile. Immunoglobulins (IgA, IgG and IgM) and albumin are associated to the purified hepatic biliary vesicles. Furthermore, four different hydrophobic glycoproteins with a molecular mass of 130, 114, 86, and 62-67 kDa were isolated. These glycoproteins showed no reactivity with anti-human whole serum or anti-immunoglobulin antibodies, suggesting that these proteins are biliary-specific. Isolated 130, 114 and 62-67 kDa vesicular glycoproteins significantly decreased the cholesterol nucleation time in artificial model bile. We concluded that some, but not all, vesicular-bound hydrophobic glycoproteins have cholesterol pronucleating activity and they may be involved in the pathogenesis of cholesterol gallstone disease.

Cholesterol crystallization-promoting activity of aminopeptidase-N isolated from the vesicular carrier of biliary lipids.

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle-associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase-N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase-N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 micrograms/ml. Aminopeptidase-N showed a concentration-dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase-N may play a critical role in the pathogenesis of cholesterol gallstone disease.

Uptake of dodecanedioic acid by isolated rat liver.

The uptake of dodecanedioic acid (C12); a dicarboxylic acid with 12 carbon atoms, was studied in the isolated perfused rat liver. Fifty mumol of C12 were injected as a bolus into the perfusing liver solution. The concentration of C12 in perfusate samples taken over 2 h from the beginning of the experiments were analyzed by high performance liquid chromatography. An in vitro experimental session was performed to determine the binding curve of C12 to defatted bovine serum albumin. These data were then used to compute the perfusate C12 free fraction. The number of binding sites on the albumin molecule was equal to 4.29 +/- 0.21 (S.E.), while the affinity constant was 6.33 +/- 0.87 x 10(3). M-1. Experimental values of perfusate C12 concentration versus time were individually plotted and fitted to a monoexponential decay for each liver perfused. The predicted C12 concentration at time zero averaged 0.354 +/- 0.0375 mumol/ml. Prom this value the apparent volume of distribution of C12 was obtained and corresponded to 153.02 +/- 14.56 ml. The disappearance rate constant from the perfusate was 0.0278 +/- 0.0030 min-1. The C12 half life was 26.6 +/- 2.3 min. The mean hepatic clearance from the perfusate was 4.08 +/- 0.38 ml/min. In conclusion, C12 is quickly taken up by the liver so that in about 100 min it was completely cleared from the perfusate.

Mass mapping analysis as a tool for the identification of genetic variants of bovine beta-casein.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometric analysis of the tryptic digest of beta-casein A2 and beta-casein B was performed before and after the separation of the peptides by LC. The overlapping of the chromatograms showed that all peaks were present in both samples, except for one only found in the tryptic digest of the A2 variant and two in the B variant. Experimental masses could be assigned to those peptides produced by tryptic digest of beta-casein variant. This peptide mapping strategy and current methodological improvements represent a promising tool for the identification of milk genetic variants with the difference of an amino acid substitution.

Capillary electrophoretic analysis of genetic variants of milk proteins from different species.

Polymorphism of bovine, ovine and caprine milk proteins was studied by CE. Identification of some rare bovine variants was carried out by isoelectric focussing (IEF) using PhastSystem. Genetic variants A and D of bovine alpha s2-casein, beta-casein variants A1, A2, A3, B and C and alpha s1-casein variants B and C were determined by CE. In addition, the different casein fractions including some genetic variants of ovine and caprine milk were identified by CE. In order to carry out this identification, collected fractions from a cation-exchange FPLC separation were injected by CE.

Improved method for the simultaneous determination of whey proteins, caseins and para-kappa-casein in milk and dairy products by capillary electrophoresis.

A capillary electrophoresis method for the simultaneous determination of whey proteins, caseins and their degradation products, such as para-kappa-casein, was proposed. The effect of several parameters (pH, ionic strength and concentration of urea in the electrophoresis buffer and applied voltage) on the analysis time and on the separation efficiency of the major milk proteins was studied. Using a hydrophilically coated capillary, in combination with electrophoresis buffer 0.48 M citric acid-13.6 mM citrate-4.8 M urea at pH 2.3, and a separation voltage of 25 kV, a complete separation of beta-lactoglobulin and para-kappa-casein was achieved, permitting the quantification of both components.

Pneumothorax in active pulmonary tuberculosis: resurgence of an old complication?

With the recent resurgence of tuberculosis (TB) in western countries, the incidence of complicating secondary pneumothorax has also increased. The work-up and management of this complication differs from that in other types of secondary spontaneous pneumothorax (SSP). Our objective was to assess clinical features and therapeutic modalities of SSP in patients with and without active pulmonary tuberculosis (APTB). All patients diagnosed with SSP seen at the Hospital Xeral of Vigo from January 1990 to June 1995 were candidates for this study. Full clinical, radiological and examinations were performed in all patients. Invasive procedures (thoracic catheter aspiration, thoracoscopy and thoracotomy) and mean hospital stay were compared in patients with and without APTB. Forty-eight patients with SSP were enrolled. Eleven patients (10 males and one female, mean age 30 +/- 11 years) had APTB; and 37 patients (31 males and six females, mean age 49 +/- 20 years) had conditions other than APTB. Chest pain, cough and fever were more frequent in patients with APTB (90% vs 59%; 45% vs 13.5%; 36% vs 5%, respectively). Catheter aspiration was successful in three of 10 (30%) of patients with APTB and in 15/23 (60.86%) of those without APTB. Catheter aspiration time was longer in the former group (25 +/- 22 days vs 13 +/- 11 days, P = 0.17). As initial treatment, thoracoscopy was performed in seven of 37 (18.91%) of those without APTB and in one of 10 (10%) patients with APTB. For patients with unsuccessful catheter aspiration, thoracoscopy was performed in eight of nine (89%) patients without APTB and in none of the patients with APTB. Thoracotomy was performed in only one of nine (11%) without APTB and in four of seven (57%) patients with APTB. Patients with APTB had a longer hospitalization (41 vs 18 days, P < 0.001). We concluded that SSP and APTB was a frequent association in our study. Patients with APTB showed a lesser and slower response to catheter aspiration. Despite severe clinical presentation and demand for more invasive procedures, patients with APTB showed a favourable response.

Detection of bovine milk proteins in soymilk by Western blotting.

A Western blotting method for the detection of whey milk proteins in commercial soymilks was applied to assess the food safety. Soy proteins and milk proteins were separated by SDS-PAGE in PhastSystem equipment. After the electrophoretic separation, immunodetection with anti-bovine alpha-lactalbumin and anti-bovine beta-lactoglobulin antisera was performed. Adulteration with bovine protein in percentages of 0.1% in soy protein can be detected. Western blotting of bovine alpha-lactalbumin and bovine beta-lactoglobulin was applied to detect adulteration by bovine milk proteins in different soymilks: powdered soymilk and soy infant formulas.

Risk factors and pathogenesis of cholesterol gallstones: state of the art.

The aim of this article is to present an update of selected aspects of the pathogenesis and risk factors of cholesterol gallstones, a highly prevalent Western disease. The etiology of cholesterol cholelithiasis is considered to be multifactorial, with interaction of genetic and environmental factors. Mechanisms of cholesterol lithogenesis include biliary cholesterol hypersecretion, supersaturation and crystallization, stone formation and growth, and bile stasis within the gallbladder. Each of these various steps could be under genetic control and/or be influenced through intermediate pathogenic steps linked to a variety of environmental factors.

Tunicamycin inhibits capillary endothelial cell proliferation by inducing apoptosis. Targeting dolichol-pathway for generation of new anti-angiogenic therapeutics.

Bovine adrenal medulla microvascular endothelial cells used in this study undergo cellular proliferation and differentiation upon culturing in vitro as observed both by light and scanning electron microscopy. Cells also respond to the growth promoting activity of serum and basic fibroblast growth factor (FGF2). Flow cytometric analysis of a synchronized culture established that cells take 68 hours to complete one cell cycle spending 36 hours in the G1 phase, 8 hours in the S phase, and 24 hours in the G2 + M phase when cultured in EMEM containing 2% heat-inactivated fetal bovine serum (FBS). At 10% serum, or in the presence of FGF2 (10 ng/ml-100 ng/ml) length of the cell cycle is reduced to 56 hours due to shortening of the G1 phase by 12 hours. Tunicamycin (a glucosamine-containing pyrimidine nucleotide), and an inhibitor of glucosaminyl-1-phosphate (GlcNAc 1-P) transferase, the first step of Glc3Man9GlcNAc2-PP-Dol (OSL) biosynthesis is found to inhibit the endothelial cells proliferation by inducing apoptosis as observed by flow cytometry and DNA laddering. Cell shrinkage, compaction of nuclei, membrane fragmentation, etc., typical of apoptotic response are frequently seen by light microscopy in the presence of tunicamycin. Scanning electron microscopy also exhibited a considerable amount of cell surface blebbing. Accumulation of an immunopositive cell specific asparagine-linked (N-linked) glycoprotein, Factor VIII:C in the absence of Glc3Man9GlcNAc2-PP-Dol in tunicamycin treated cells has been proposed as an apoptotic triggering mechanism under the current experimental conditions.

[Adenosine deaminase activity in the pleural effusion. A study of 64 cases]. / Actividad de adenosindesaminasa en líquido pleural. Estudio realizado en 64 casos.

Clinical and analytic data of 64 patients with firm etiologic diagnosis of pleural effusion with adenosine deaminase (ADA) present, were analyzed retrospectively. The patients had entered our hospital over a 40-month period. ADA activity in pleural fluid was analyzed by the Blake and Berman kinetic method. Mean ADA activity of the total sample was 32 U/l (SD:23.9). In patients with tuberculous pleural effusion ADA activity was higher than in the remaining patients (47.7, SD:21.4, versus 15.5 SD: 13.2; p < 0.0001). In the group of patients with tuberculous pleuritis diagnosed by pleural biopsy (22 cases) the presence of necrotizing granulomas was associated with slightly higher ADA activity although the difference was not statistically significant (49.2 SD 10.1 versus 41.3 SD 8.9; p = 0.07). Among only patients with tuberculous pleuritis or neoplasia with lymphocytic exudate, a cut off point greater than 23 U for ADA predicted a diagnosis of tuberculous pleuritis with a sensitivity of 0.96, specificity of 1, positive predictive value of 1, negative predictive value of 0.94, and a confidence limit of 0.97. In conclusion, ADA activity greater than 23 U determined by the kinetic method in pleural fluid with signs of lymphocytic exudate is strongly suggestive of pleural tuberculosis based on our sample of patients with pleural effusion.