Selenium from selenium-rich Spirulina is less bioavailable than selenium from sodium selenite and selenomethionine in selenium-deficient rats.

The bioavailabilty of selenium (Se) from selenium-rich Spirulina (SeSp) was assessed in Se-deficient rats by measuring tissue Se accumulation and glutathione peroxidase (GSH-Px) activity. For 42 d, rats were subjected to dietary Se depletion by consumption of a Torula yeast (TY)-based diet with no Se; controls were fed the same diet supplemented with 75 microg Se/kg diet as sodium selenite. Se-deficient rats were then repleted with Se (75 microg/kg) by the addition of sodium selenite, selenomethionine (SeMet) or SeSp to the TY basal diet. Selenium speciation in SeSp emphasized the quasi-absence of selenite (2% of total Se); organic Se comprised SeMet (approximately 18%), with the majority present in the form of two selenoproteins (20-30 kDa and 80 kDa). Gross absorption of Se from SeSp was significantly lower than from free SeMet and sodium selenite. SeMet was less effective than sodium selenite in restoring Se concentration in the liver but not in kidney. SeSp was always much less effective. Similarly, Se from SeSp was less effective than the other forms of Se in restoring GSH-Px activity, except in plasma and red blood cells where no differences were noted among the three sources. This was confirmed by measuring the bioavailability of Se by slope-ratio analysis using selenite as the reference form of Se. Although Se from SeSp did not replenish Se concentration and GSH-Px activity in most tissues to the same degree as the other forms of Se, we conclude that it is biologically useful and differently metabolized due to its chemical form.

Iron availability from iron-fortified spirulina by an in vitro digestion/Caco-2 cell culture model.

Iron deficiency, one of the most important nutritional problems in the world, can be caused not only by foods deficient in iron but also by poor availability of dietary iron. Iron food fortification in combination with highly available iron from supplements could effectively reduce this deficiency. The aim of this study was to examine the iron availability from iron-fortified spirulina. We have used an in vitro digestion/Caco-2 cell culture system to measure iron spirulina availability and made a comparison with those of beef, yeast, wheat floor, and iron sulfate plus ascorbic acid as a reference. Iron availability was assessed by ferritin formation in Caco-2 cells exposed to digests containing the same amount of iron. Our results demonstrate a 27% higher ferritin formation from beef and spirulina digests than from digests of yeast and wheat flour. When iron availability was expressed per microgram of iron used in each digest, a 6.5-fold increase appeared using spirulina digest in comparison with meat. In view of this observed high iron availability from spirulina, we conclude that spirulina could represent an adequate source of iron.

Production of polyclonal antibodies towards the immunodetection of insecticide phosalone.

Hapten synthesis for the production of specific insecticide phosalone polyclonal antibodies was carried out starting from an intermediate of the phosalone synthesis, 6-chloro-2-benzoxazolone 1. Two haptens containing different spacers have been prepared: N-5-carboxypentyl-6-chloro-2-benzoxazolone 7 and N-(2-oxo-3-aza-5-carboxypentyl)-6-chloro-2-benzoxazolone 12. Each of these two haptens conjugated to bovine serum albumine (BSA) was used to immunize four rabbits. Immunoassays of phosalone were performed with ELISA using solid-phase bound hapten thyroglobulin conjugate and horseradish peroxidase labelled goat antirabbit IgG. The more sensitive response was observed when the antiserum obtained from the rabbit immunized with the hapten-BSA conjugate containing the N-2-oxo-3-aza-5-carboxypentyl spacer was in competition with the same hapten coupled to thyroglobulin. An identical response was obtained under the same conditions when using benzoxazolone instead of phosalone as competitor, showing a good recognition of the specific aromatic part of the organophosphate insecticide phosalone. Reduction of coating conjugate concentration (from 2 to 0.05 micrograms/well) and also the use of heterologous coating protein instead of homologous did improve the sensitivity, resulting in a concentration of phosalone required to inhibit binding by 50% of 2 mg/l and a detection limit of 0.02 mg/l.

Monospecificity of the antibodies to bovine alpha s1-casein fragment 140-149: application to the detection of bovine milk in caprine dairy products.

Comparison of the primary sequences of bovine, ovine and caprine alpha s1-casein shows a deletion of eight amino acid residues in the ovine casein region 141-148, which is identical in the bovine and caprine proteins except for a single difference in position 148 (Q or E). Polyclonal antibodies raised in rabbits against the bovine casein sequence 140-149 (QELAYFYPEL) appeared monospecific for bovine alpha s1-casein, since no antibody-antigen complex was formed with homologous ovine or caprine proteins. These antibodies remained unable to recognize the caprine sequence in the native protein even after extensive tryptic proteolysis. The lack of immunoreactivity of the antibodies against synthetic caprine alpha s1-casein peptide 138-149 (VNQELAYFYPQL) suggested that the glutamic acid residue in position 148 is essential for the antigenic character of the bovine peptide. From these observations, the use of these antibodies for the detection and quantitation of bovine milk present in ovine dairy products could be extended to caprine products.

Dietary grape seed tannins affect lipoproteins, lipoprotein lipases and tissue lipids in rats fed hypercholesterolemic diets.

The effects of monomeric and polymeric grape seed tannins on rat plasma lipoproteins, lipoprotein lipase, hepatic lipase and aortic and hepatic lipid concentration were studied. Male Sprague-Dawley rats received either a normal diet (with no added cholesterol and no tannins), a control diet (hypercholesterolemic diet) or hypercholesterolemic diets supplemented with 2% tannin monomers or 2% polymers 3 or 9 wk. Plasma total cholesterol, triacylglycerol, LDL cholesterol and VLDL concentrations were significantly higher and the HDL cholesterol concentration lower in controls and in rats fed the diet supplemented with monomers compared with rats fed polymeric tannins at both time points. Lipoprotein lipase and hepatic lipase activities were significantly higher in control and in monomer-fed groups than in the polymer-fed group. Hepatic and aortic cholesterol and triacylglycerol concentrations were significantly higher in control rats and those fed monomers than in the polymer-fed group. Moreover, plasma HDL cholesterol and hepatic lipase activity were closely associated with low aortic cholesterol and triacylglycerol in rats fed polymeric tannins. These rats also exhibited greater fecal excretion of cholesterol and especially bile acids than the control or monomer-fed groups. Thus dietary grape seed tannins have a pronounced anti-hypercholesterolemic effect by enhancing reverse cholesterol transport and also by reducing intestinal cholesterol absorption and increasing bile acid excretion.

Dietary grape seed tannins: effects of nutritional balance and on some enzymic activities along the crypt-villus axis of rat small intestine.

The aim of the study was to determine the nutritional and intestinal effects of grape seed tannins. For this purpose, tannins were incorporated in diets of rats at levels of 0.2 or 2.0% for 31 days in comparison to a control diet. The animals were pair-fed. Nutritional balances were not affected by feeding 0.2% tannins. At the highest dose (2%) grape seed tannins reduced growth as well as dry matter (DM) and nitrogen (N) digestibility. In rats fed protein-free diets, 2% tannins significantly increased endogenous fecal N. Starch and fat were well digested in all groups of rats. No changes in organ weights were observed. Duodenal alkaline phosphatase activity (AP) was never affected by tannins. On the other hand, in the jejunum, along the vilus-crypt unit, a reduction of AP and sucrase appeared at the tip villus which was balanced by an enhancement of 3H-thymidine incorporation in the middle of the crypt zone, giving evidence of endogenous N loss. This study did not reveal a major toxic effect of tannins except a reduced DM and N digestibility; nevertheless tannins directly interfere with mucosal proteins, thereby stimulating the cell renewal.

Effect of grape seed tannins on the activity of some rat intestinal enzyme activities.

The present study was designed to investigate the effects of grape seed tannins on rat intestinal alkaline phosphatase (AP), sucrase and dipeptidyl peptidase IV (DPP IV) activities. An experiment was performed in vivo by dietary supplementation with 2% tannins; this diet was tested on an experimental group of rats; a control group received a diet without tannins. After 31 days, tannins intake significantly decreased middle-jejunal AP from 123 to 45 mU/mg protein and sucrase activities from 310 to 195 mU/mg protein, while no significant difference appeared at the duodenal stage (p < 0.05). Ileal DPP IV activity was also significantly reduced (p < 0.05) from 190 to 110 mU/mg protein after tannin intake. Using in vitro experiments on purified brush border membranes, AP activity was found to be inhibited by grape tannins; this inhibition was prevented by the detergent Triton X-100. The addition of pancreatic-biliary (PB) juice to the incubation medium prevented or reversed the tannin-inhibited enzyme activity. The present data indicate that in the duodenal lumen, alkalinity and detergency from the PB secretion neutralized the ability of tannins to inactivate brush border hydrolase activities and suggest that enzyme inhibition took place once bile salts were reabsorbed while moving down the gut. This was confirmed by in vitro experiments where sucrase and DPP IV activities inhibited by grape seed tannins were largely recovered after the addition of PB juice to the incubation medium.

Comparative evaluation of the effects of two different forms of dietary fibre (rice bran vs. wheat bran) on rat colonic mucosa and faecal microflora.

The aim of the study was to compare the physiological consequences of two dietary fibre sources on the faecal microflora and colonic mucosal growth in rats. The studied sources, a moderately well-soluble fibre (rice bran, RB) and a less well-soluble fibre (wheat bran, WB), were included in diets of rats at a level of 10% for 3 weeks and compared with a totally fibre-deprived diet. RB significantly increased faecal water compared to the control diet (p < 0.05). Faecal nitrogen content and bacterial mass, as estimated from the 2,6-diaminopimelic acid (DAPA) output, were greatly and significantly increased by RB, and to a lesser extent by WB, compared to the control diet. Total bile acid excretion was significantly higher by rats fed RB than by those fed WB. Faecal bacterial enzyme activities tested (beta-glucuronidase, mucinase and nitroreductase) were significantly reduced by the two different fibre sources, but RB was more effective than WB, except for nitroreductase activity which was reduced at the same level for each fibre source. Although measurements of mucosal colonic weight and RNA content were significantly different between groups fed RB and WB (p < 0.05), DNA content and the ratio RNA/DNA did not significantly differ between these groups. Our results indicate that the differential changes observed in beta-glucuronidase and mucinase activities and DAPA and bile acid excretion may depend on the nature of the fibre consumed. They also suggest that RB, which had similar effects, sometimes more marked than WB, on the studied parameters, may be a new valuable fibre source.

Mathematical correlation between villus height and the nutritional state in Sprague-Dawley rats.

Three different experiments were carried out on growing male Sprague-Dawley rats. In the first experiment, dietary nitrogen was given in casein at four different protein values ranging from 5 to 16%. Rats were fed ad libitum for 21 days. In the second experiment, which lasted 17 days, animals were given three diets that differed in the molecular form of the nitrogen supply - that is, proteins or peptides. The protein value (N x 6.25) was 10% in each diet. In the third experiment, malnourished rats were refed diets with a protein value of 15% (N x 6.25) for eight days. The dietary nitrogen was either in the form of protein, peptide, or amino acid. Body weight was recorded daily. At the end of each experiment the intestinal villus height was measured by light microscopy. Data were statistically analysed by Exner's coefficient. The results assessed the validity of the correlation between villus height and gain in body weight.

Polyclonal antibodies with predetermined specificity against bovine alpha s1-casein: application to the detection of bovine milk in ovine milk and cheese.

Comparing the primary sequences of bovine and ovine milk proteins, some short peptide fragments are cow-specific, in particular the 141-148 fragment of bovine alpha s1-casein, which is deleted in its ovine counterpart. The 140-149 peptide was chemically synthesized on a solid phase matrix and directly used as an immunogen to produce polyclonal monospecific antibodies in rabbits. These antibodies recognized this fragment both on the peptidyl resin and in the native protein. They appeared to be monospecific, since no antigen-antibody complex was formed with homologous ovine or caprine proteins. Subsequently, a competitive enzyme-linked immunosorbent assay was successfully developed for the detection of defined amounts of cows' milk in sheep's milk from 0.125 to 64% (v/v) and in cheese from 0.5 to 25% (v/v) that was not influenced by heat treatment of milk or the degree of ripening of cheese.

Selective effects of PHA on rat brush border hydrolases along the crypt-villus axis.

The mechanism of the toxicity of lectin from Phaseolus vulgaris seeds has been investigated on rat enterocytes. Cell isolation procedures showed a selectivity in the loss of brush border hydrolases; this indicated that the microvilli blebbing was not the only mechanism of action of lectins on rat enterocytes.

Duodenal toxicity of dietary Phaseolus vulgaris lectins in the rat: an integrative assay.

It is now generally admitted that phytohemagglutinin (PHA) constitutes the main factor responsible for the dietary toxicity of raw kidney beans. In the growing rat, an impairment of growth is the unique expression of a malnutrition syndrome. The aim of this work was to precise to what extent the intestinal injuries may account for this malnutrition. PHA was administered for 9 days to growing rats at levels ranging from 0.0025 to 0.25% of food dry matter. One group of controls was fed ad libitum and other groups were restrained. In such conditions, PHA reduced the food intake when offered at a level higher than 0.04% as a linear function of the logarithm of lectin rate. Intestinal injuries were also dose-dependent: blebbing of microvilli and loss of alkaline phosphatase occurred at the smallest dose of PHA, cell loss occurred at higher doses. A compensatory hyperplasia was observed as a consequence of both intestinal injury and reduced food intake. Our main results are that, whatever may be the damages caused to the duodenal mucosa, the observed growth impairment was quasi-totally imputable to the reduction of food intake.

Effect of lectins from leguminous seeds on rat duodenal enterokinase activity.

Enterokinase activity from rat duodenal brush borders was assayed in vitro in the presence of purified lectins from 3 leguminous seeds. Noncompetitive inhibition of the enzyme was observed in each case. Phaseolus hemagglutinin was the most potent inhibitor among the 3 lectins tested.

[Formation of trypsin inhibitors during alkaline treatment of proteins]. / Mise en évidence de la formation d'inactivateurs de la trypsine au cours de traitements alcalins des protéines.

Casein is submitted to a severe alkaline treatment (NaOH 0,2 or 0,5 N, 1 hr., 80 degrees C). The hydrolysis by pancreatic enzymes (trypsin or chymotrypsin) is reduced in vitro and, in the case of the more severe treatment, stopped. After an extended (24 hrs.) trypsin and pronase hydrolysis, it is shown, by affinity chromatography, that peptides, which are not hydrolysable, can bind to trypsin and inactivate this enzyme in vitro.

[Effects of a phytohemagglutinin extract on growth, nitrogen digestibility and the activity of invertase and (Na+-K+)-ATPase in the intestinal mucosa of the rat]. / Effets d'un extrait de phytohémagglutinines sur la croissance, la digestibilité de l'azote et l'activité de l'invertase et de la (Na+-K+)-ATPase de la muqueuse intestinale, chez le rat.

A phytohemagglutinin extract is prepared from raw kidney beans (Phaseolus vulgaris) and incorporated at a level of 1% (dry matter) in the diet of young growing rats. Beside a decrease of feed intakes, the main effects of the experimental diet are the following : growth depression, decrease of dry matter and protein digestibility and hypoglycemia. Biological value, organs weight (liver, kidneys, spleen) did not change significantly. The hemagglutinin extract induces an inhibition of saccharase activity whereas (Na+-K+)-ATPase remains unchanged. Growth depressing effect may be due to an alteration of hydrolysis and absorption mechanisms at the level of brush border of enterocytes.

[Use of small peptides to study the loss of available methionine, lysine and tryptophan during technological processing]. / Utilisation de petits peptides pour l'étude des pertes de disponibilité de la méthionine, de la lysine et du tryptophane au cours de traitements technologiques.

Peptides such as glycyl-L-methionyl-glycine, glycyl-L-lysine, L-lysyl-glycine and glycine-L-tryptophyl-glycine are used to study side-chain reactivity of three essential amino acids during food processing. The treatment of glycyl-L-methionyl-glycine with sodium hypochlorite resulted in the two following types of reaction: 1. Methionyl residues are oxidised to the corresponding sulfoxide at sodium hypochlorite concentrations up to 0,1 p. 100 w/v; 2. Oxidation of methionine residues to methionine sulfone and deamination reactions also occur for sodium hypochlorite concentrations over 0,2 p. 100 w/v. Sodium hypochlorite treatment of glycyl-L-lysine and L-lysine-glycine causes, probably by deamination of the epsilon-NH2 groups, a loss of lysine of 20 and 30 p. 100, for sodium hypochlorite concentrations of 0,1 and 0,2 p. 100 w/v, respectively. Treatment of glycyl-L-tryptophyl-glycine with hydrogen peroxide (0,05 M) modifies tryptophan residues in such a way that it cannot be retrieved after hydrolysis of the tripeptide with methane sulfonic acid and subsequent chromatographic analysis; six new unidentified components appear on the chromatogram. Glycyl-L-methionyl-glycine was incubated with food constituents or additives such as reducing oses, acrolein, p-benzoquinone, methyl iodide, or dichloro I, I ethylene. Thin layer chromatography and RMN show that carbonyl compounds or quinones do not react with the thioether group. Alkylating agents sometimes used in food processing only gave traces of sulfonium compounds. Thus, it appears that the only reaction liable to render methionine residues unavailable in foods would be its oxidation to methionine sulfone.