Metabolic Effects of a Hydrophobic Alginate Derivative and Tetrahydrolipstatin in Rats Fed a Diet Supplemented with Palm Fat and Cholesterol.

The effects of octadecylamide of alginic acid (amidated alginate) and tetrahydrolipstatin on serum and hepatic cholesterol, and the faecal output of fat and sterols, were investigated in rats. Amidated alginate is a sorbent of lipids, tetrahydrolipstatin is an inhibitor of pancreatic lipase. Rats were fed diets containing cholesterol and palm fat at 10 and 70 g/kg, respectively. Palm fat was provided by coconut meal. Amidated alginate at 40 g/kg diet significantly decreased serum total cholesterol, low-density lipoprotein and hepatic cholesterol, and hepatic lipids and increased the faecal output of fat and coprostanol. Tetrahydrolipstatin at 300 mg/kg diet significantly decreased low-density lipoprotein cholesterol and hepatic lipids and increased the faecal output of fat. The intake of feed was not significantly influenced; however, the weight gains in rats fed amidated alginate were lower than in rats of the control group. Both amidated alginate and tetrahydrolipstatin modified the fatty acid profile in excreta lipids. Concentrations of saturated fatty acids were decreased and those of unsaturated fatty acids increased. Despite different modes of action, amidated alginate and tetrahydrolipstatin were equally efficient in removing the dietary fat from the body.

Beneficial effects of enrichment of chicken meat with n-3 polyunsaturated fatty acids, vitamin E and selenium on health parameters: a study on male rats.

Consumption of chicken meat enriched with bioactive compounds such as n-3 polyunsaturated fatty acids (PUFAn-3), vitamin E (vE) and selenium (Se) can help prevent many diseases and can be used to deliver those substances to humans. This might be of importance as chicken meat consumption is increasing worldwide. The effects of enriching chicken meat with PUFAn-3, vE and Se through dietary interventions were studied in rats. Four groups of Ross 308 female broilers from day 22 to day 35 of age were fed control diet (L) that contained lard and 80 mg vE and 0.3 mg Se/kg, or diets that contained rape seeds and fish oil with the same level of Se and vE as in the control diet, the same level of Se as in the control and 150 mg vE/kg, or 150 mg of vE and 0.7 mg Se/kg. Broiler carcasses were boiled, deboned, lyophilized and pooled by group. Boiled edible components of chicken carcass (BECC) were included (240 g/kg) in the diets fed to four groups of ten 10-week-old Wistar male rats for 8 weeks. Inclusion of BECCs modulated dietary fatty acid profile in the rat diets. Feeding these diets did not influence parameters related to growth or relative weights of internal organs in the rats. Feeding BECCs with lower PUFAn-6/n-3 decreased the n-6/n-3 ratio in the rat brain and liver, and increased the proportion of docosahexaenoic acid in the brain lipids. Liver cholesterol level was similar among the experimental groups, whereas the concentration of vE in the liver of rats fed BECC with increased vE levels was higher than that in the rats fed BECC with the basal vE level. Haematological and biochemical parameters in blood were within the normal range for rats, but a few rats showed a tendency towards increased levels because of the higher vE and Se level. The health-promoting effect of feeding rats PUFAn-3 enriched BECC was more pronounced when an increased dietary level of vE was used, but the increased level of Se did not provide the rats with additional benefits. Thus, the findings indicate that BECC enriched with PUFAn-3 and vE by a dietary intervention is a functional food with great potential of implementation.

Gonadoliberin (GnRH) and its copper complex (Cu-GnRH) enzymatic degradation in hypothalamic and pituitary tissue in vitro.

The amount of decapeptide decapeptide gonadoliberin (GnRH) that reaches pituitary gland depends not only on transcriptional, translational and posttranslatonal processes but also on the extent of degradation exerted by specific proteolytic enzymes. The copper-gonadoliberin (Cu-GnRH) complex preserves the native GnRH amino acid sequence but contains Cu(2+) ion bound to the nitrogen atom at the imidazole ring of the His(2). The aim of this study was to determine whether GnRH and Cu-GnRH molecules differ in their susceptibility to proteolysis in male rat hypothalamic and pituitary tissue in vitro. RIA was applied for a time-dependent study based on 0-90 min incubations at 30°C of exogenous peptide (2.5 µg GnRH or Cu-GnRH) in respective hypothalamic/pituitary supernatant and pellet fractions. To compare the protective effect of bacitracin, a competitive PEP inhibitor, incubations were made with (125 µg/sample) or without an inhibitor. In the second experiment 100 µg of GnRH or Cu-GnRH were incubated for 5 h at 37°C in hypothalamic and pituitary tissue in vitro and then HPLC analysis was applied both to characterize the elution pattern of GnRH and Cu-GnRH degradation products as well as to determine their AA composition. In both tissues, Cu-GnRH remained more resistant to enzymatic degradation and fully protected in the presence of bacitracin. In conclusion, the obtained data suggest that copper ion changed GnRH conformation and significantly modified its physiological properties due to a hindered endopeptidases access to specific AA bonds. Therefore, the Cu-GnRH complex might be considered as GnRH analog potentially able to prolong the occupation of a GnRH receptor at the gonadotrope cells.

Alkaline saponification results in decomposition of tocopherols in milk and ovine blood plasma.

Alkaline saponification of entire sample matrixes for quantification of alpha-, gamma-, delta-tocopherols (alpha-T, gamma-T, delta-T) and alpha-tocopherol acetate (alpha-TAc) was examined. High-performance liquid chromatography was used to measure alpha-T, gamma-T, delta-T and alpha-TAc in tocopherol standard solutions, milk and ovine blood plasma. Saponification in the presence of vitamin C decreases the concentration of tocopherols, especially alpha-T and gamma-T. The poor recovery of tocopherols is due to the decomposition of tocopherols in saponified standard solutions, milk or plasma. Saponification of samples in the presence of 2,[6]-ditertbutyl-p-cresol or flushed only with a stream of Ar resulted in a major decrease in the concentrations of alpha-T, gamma-T, delta-T and alpha-TAc in comparison with saponification in the presence of vitamin C.

Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids.

Dietary cis-9, trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect, in vitro incubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. Linoleic acid (LA) or alpha-linolenic acid (LNA) was incubated (1.67 g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay-concentrate ration. Adding fish oil (up to 4.17 g/l) tended to decrease the initial rate of LA (P=0.025) and LNA (P=0.137) disappearance, decreased (P<0.05) the transient accumulation of conjugated isomers of both fatty acids, and increased (P<0.05) the accumulation of trans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100 mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50 mg/l) to pure cultures inhibited the growth and isomerase activity of B. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised by B. fibrisolvens in pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects on B. fibrisolvens.

Effects of exclusive formula or breast milk feeding on oxidative stress in healthy preterm infants.

BACKGROUND: Compared to formula, breast milk is considered to have superior antioxidant properties and consequently may reduce the occurrence of a number of diseases of prematurity associated with oxidative stress. AIMS: To test whether the antioxidant properties of breast milk in healthy premature infants are different to those of formula milk by comparing vitamin E levels in milk and determining the excretion of malondialdehyde (MDA) in urine. METHODS: Vitamin E was measured in the breast milk of 20 mothers who had given birth prematurely. Urinary MDA was measured in 10 exclusively breast milk fed and 10 exclusively formula fed healthy preterm infants receiving no vitamin supplements. MDA was measured after derivatisation with 2,4-dinitrophenylhydrazine and consecutive HPLC with UV detection. RESULTS: Urinary MDA concentrations were consistently very low (0.074+/-0.033 microM/mM Cr and 0.078+/-0.026 microM/mM Cr in breast and formula fed infants respectively) and not significantly different between healthy breast milk and formula fed infants. Both breast and formula milk contained satisfactory levels (0.3-3.0 mg/100 ml) of vitamin E. CONCLUSION: Antioxidant properties of both breast milk and formulae are sufficient to prevent significant lipid peroxidation in healthy premature infants.

Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection.

Positional and geometric isomers of mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms were separated on commercially available reversed-phase columns in gradient systems composed of acetonitrile and water, utilizing photodiode array detection. The biological samples were hydrolyzed with 2 M NaOH for 35-40 min at 85-90 degrees C. After cooling, the hydrolysates were acidified with 4 M HCl and the free fatty acids were extracted with dichloromethane. The organic solvent was removed in a gentle stream of argon. The fatty acids were determined after pre-column derivatization with dibromacetophenone in the presence of triethylamine. The reaction components were mixed and reacted for 2 h at 50 degrees C. Separations of derivatized fatty acids were performed on two C18 columns (Nova Pak C18, 4 microm, 250x4.6 mm, Waters) by binary or ternate gradient programs and UV detection at 254 and 235 nm. The geometric and positional isomers of some unsaturated fatty acids were substantially retained on the C18 columns and were distinct from some saturated fatty acids, endogenous substances in biological samples or background interference. Only slight separation of critical pairs of cis-9 C18:1/cis-11 C18:1 and cis-6 C18:1/trans-11 C18:1 was obtained. A ternate gradient program can be used for complete fractionation of a mixture of conjugated linoleic acid isomers (CLA) from cis-9, cis-12 and trans-9, trans-12 isomers of C18:2. The CLA isomers in the effluent were monitored at 235 nm. The CLA isomers were differentiated from saturated and unsaturated fatty acids using a photodiode array detector. The utility of the method was demonstrated by evaluating the fatty acid composition of duodenal digesta, rapeseed and maize oils.

Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine urine by high-performance liquid chromatography and photodiode array detection.

A HPLC method for the determination of allantoin, uric acid, hypoxanthine and xanthine (purine metabolites) in ovine urine without the disadvantages inherent in derivatization is described. After dilution 1:6 with water, urine samples were injected onto the column. Separation and quantification of purine metabolites was achieved using two Nova-Pak C18 columns (4 microm, 250x4.6 mm, Waters). A binary gradient program and UV detection were used for purine metabolites analysis. Clear resolution of purine metabolites was obtained in less than 15 min. Allantoin, uric acid, hypoxanthine and xanthine in the effluent were monitored at 225, 284, 250 and 267 nm, respectively. The average recoveries of purine metabolite standards added to urine samples were satisfactory (100.2-102.9%). The low coefficients of variation (0.29-0.73%) as well as the low detection (0.16-0.70 nmol) and quantification (0.52-2.32 nmol) limits indicate satisfactory precision, reproducibility and sensitivity of the proposed method. This procedure is suitable for routine quantification of purine metabolites in a large number of urine samples.

Simultaneous measurement of allantoin, uric acid, xanthine and hypoxanthine in blood by high-performance liquid chromatography.

A high-performance liquid chromatographic method for determining catabolism products of nucleic acids and purines, such as oxypurines (i.e. uric acid, xanthine and hypoxanthine) and allantoin in the blood plasma of ruminants was developed. The plasma was deproteinized with 10% trichloroacetic acid. The method enabled determination of oxypurines without derivatization. Allantoin was determined after conversion with 2,4-dinitrophenylhydrazine to a hydrazone (GLX-DNPH). Separation of converted allantoin, uric acid, xanthine and hypoxanthine derivatives was carried out using two reversed-phase C18 columns. The combination of pre-column derivatization and gradient elution with monitoring of the effluent at 205, 254 and 360 nm provides a simple and selective analytical tool for studying oxypurines and allantoin in plasma. The total run time of the HPLC analysis was 60 min. The recovery of the purine derivatives (i.e. oxypurines and allantoin) added to the plasma was between 95 and 106%. Purine derivatives were stable when the processed samples were stored for 7 days at -10 degrees C. The low values of the intra-assay coefficient of variations (2.5-4.6%) and the low values of the detection limits (0.187-0.004 nmol) point to the satisfactory precision and sensitivity of the method.

Use of INAA to study the interaction between Se and Te in cells of Saccharomyces cerevisiae.

The differences in the effects of inorganic Se (IV and VI) compounds and seleno-cystine [(CySe)2] on the Te (as Na2TeO3) uptake by the yeast, Saccharomyces cerevisiae, has been studied. Se, Te, Ag, Zn, Fe and Co contents of the cells were measured by instrumental neutron activation analysis. For the determination of the Ag content, the monostandard method was applied as the analytical method. The contents of other elements were determined by comparison with standards having similar amounts of the determined element as the sample. Results obtained show that an antagonist interaction occurs between SeO2 and Te. There was a significant increase in the concentrations of Se and Te when the yeast was incubated in the medium containing (CySe)2 and Te. (CySe)2 markedly increased the Ag content of cells, especially within the first 8 h of incubation. The low level of SeO2 in the medium are the exterior factor which produce an observable increase of the Ag concentration in the cells. The higher level of SeO2 in the medium causes a long-term marked increase in the Ag content of the cells. The uptake yield of Ag also increased in the presence of (NH4)2SeO4 in the medium. The Te supply produced a significant enhancement in the Ag content of the cells during the initial 8 h of incubation. The presence of Se and/or Te in the medium causes change in the intracellular Zn, Fe and Co levels.

Use of INAA to study the determination of Se, Th, Zn, Co and Fe levels of yeast cells.

Differences in the effects of seleno-cystine (CySe)2, glutathione (GSH), Se(IV) [as SeO2] and Se(VI) [as (NH4)2SeO4] on Th(IV) [as Th(CO3)2] uptake by the cells, Saccharomyces cerevisiae, have been studied. The Th, Se, Zn, Co and Fe levels of the yeast cells were measured by instrumental neutron activation analysis. Results obtained show that the addition of Th alone to the culture medium resulting in the Th content of the cells and the Th level of the yeast slightly decreased during the incubation. The addition of Th in combination with GSH produced a higher decrease of the Th content in comparison with the single Th dosage. During the initial 48 h of the incubation the presence of Th and Se(VI) in the medium produced a decrease of the Th level of the cells in comparison with the addition of Th alone. (CySe)2 or SeO2 does not produce a regular change of the Th level of the cells. Th uptake by the yeast influenced the retention of Se in the cells. In fact, the Se levels of the cells were always significantly higher when the yeast was incubated in the medium containing Th and SeO2 or Se(VI). The enhance in the Se level of the cells rises with increasing concentrations of SeO2 in the culture medium. Th decreased the Se content of the yeast when the cells were incubated in the medium containing (CySe)2 and Th. GSH supply in combination with Th and SeO2 produced a very significant enhancement of the Se abundance in the cells in comparison with the single addition of SeO2. Se-compounds and/or Th dosages affected the Zn, Co and Fe contents of the cells. The Fe level of the yeast is below the quantitative detection limit of Fe when the cells were incubated in the medium containing Th.

[Biodistribution studies on selenium and mercury in mice using instrumental neutron activation analysis]. / Badanie biodystrybucji selenu i rteci u myszy metoda instrumentalnej neutronowej analizy aktywacyjnej.

An attempt was made to compare the Se and Hg abundance's in liver, kidneys and blood after simultaneous intraperitoneal injections of inorganic mercury (as HgCl2) and SeO2 or organic Se-compound (i.e. seleno-cystine, seleno-methionine or selenodiglutathione). Instrumental neutron activation analysis was applied as the analytical method due to the advantages of both its sensitivity and chemically non-destructive procedure. No neurological or other lesion symptoms of Hg and Se intoxication were found. Especially high concentrations of Hg and Se in liver and blood were found after simultaneous i.p. injections of HgCl2 and Se-compounds. Moreover, significantly high abundance's of Se and Hg in liver and blood were found after simultaneous injections of seleno-methionine and HgCl2. On the other hand, only for kidneys the Hg content after the single injections of HgCl2 was considerably higher in comparison with the simultaneous injections of Hg and Se. We suggest that Se-compounds protects against renal lesions by decreasing the concentration of Hg in kidneys. Hg2+ ions are bounded by selenohydryl groups of the metabolites of injected Se-compounds. Moreover, the binding yield of Hg2+ ions with the metabolites of Se-compounds depends upon the chemical form of injected Se-compounds.

Use of INAA to study Se, Sb, Zn and Co levels of yeast cells.

Yeast cells, Saccharomyces cerevisiae, were exposed to Sb(V)(10-5M) and SeO2(10-4M) or seleno-cystine (CySe)2(5 x 10-5M). Se, Sb, Zn and Co levels of the yeast were measured by instrumental neutron activation analysis. The results obtained show that in the absence of Se, Sb is taken up by the cells and the highest concentration of Sb in the yeast was observed during the initial 2.5 h of incubation. Both Se-compounds resulted, in general, in a minute decrease of uptake yield of Sb by the cells. This effect can be particularly observed in the presence of SeO2. The presence of Sb in the yeast medium slightly increased the Se level only after long incubation times. Se uptake by the yeast was higher (regardless of Sb dosage) when the yeast was incubated in the medium containing (CySe)2 (in comparison with SeO2). The presence of Se-compounds and/or Sb caused decrease in the levels of Zn found in the cells. While SeO2 presence resulted in minor changes of the Co level of the yeast, the combined presence of Sb and Se-compounds produced the significant enhancement of Co abundance. The similar effect was noted in the yeast incubated in a medium containing only (CySe)2 or Sb.

Effect of Cd on tissue levels of Se, Zn, Fe, Co and Rb.

The interaction of Cd (as CdCl2) with organic Se-compounds and SeO2 was investigated. Instrumental neutron activation analysis was applied as the analytical method. Concentrations of Se in liver and kidneys were always significantly higher after simultaneous i.p. injections with Cd and Se-compounds in comparison with single Se-injection. Cd or Se-injections also affect the content of Zn, Fe, Co and Rb in all the investigated organs. We suggested that the formation of Cd-Se-protein complexes allow for an orderly transfer of Cd to metallothionein (MT). The results showed that Cd was a very effective inducer of MT, and the maximum formation of MT was observed in the liver.

Studies on the interaction between SeO2 and sulfur compounds and distribution of Rb, Zn, Co, Fe and Hg in mice by instrumental neutron activation analysis.

Content of Se, Rb, Zn, Co, Fe and Hg in liver, kidneys, spleen, brain and blood of SAS/4 mice were determined after i.p. injection with SeO2, gluthathione, cysteine, cysteamine or methionine. Instrumental neutron activation analysis (INAA) was applied as the analytical method. Se was incorporated in all the examined organs, the the efficiency of the incorporation depended upon the sulfur compounds injected. Injection with above compounds affects the contents of the other elements in all mice organs.

Variation of Se, Zn, Co, Fe and Rb distribution in rats upon sequence of injection with SeO2 and glutathione.

The contents of Se, Zn, Co, Fe and Rb in several organs of Wistar rats were determined by instrumental neutron activation analysis (INAA) after injections of SeO2 and glutathione (GSH). Se was incorporated in all the examined organs, and the efficiency of incorporation does not depend upon the sequence of injection with SeO2 and GSH. The sequence of these injections affects the contents of the other elements in all the examined organs.

Variation of Se, Zn, Co, Fe and Rb distribution in rats after injection with selenium or sulfur compounds.

Contents of Se, Zn, Co, Fe and Rb in liver, kidney, heart, spleen, brain, pancreas and testicle of Wistar rats were determined 2 h after i.p. injection with selenodiglutathione, selenomethionine, selenocystine, selenocystamine, methionine, cystine and cystamine. Instrumental neutron activation analysis (INAA) was applied as the analytical method. It was found that Se was incorporated to all examined rat organs. High Se incorporation was observed after injection with selenomethionine and selenocystine. Variations in the Zn, Co, Fe and Rb contents were observed in all the investigated organs after the injections. These variations depended upon injected compounds.

The incorporation of Yb and Se into the brains of rats and their effect on the Zn level.

The concentrations of Yb, Se and Zn in the brains of rats were determined after the injection of ytterbium chloride and selenodiglutathione--single or together--by neutron activation analysis. Both Yb and Se were incorporated in all the investigated brain tissues and they also caused changes in the Zn concentration.

The incorporation of selenium and ytterbium into the eyes of mice.

The incorporation of Se and Yb into the eyes of mice has been studied. Selenodiglutathione, (GS)2Se, or ytterbium chloride, YbCl3, were injected intraperitoneally into mice: either alone, combined, or after various time intervals. Instrumental neutron activation analysis was applied as the analytical method for the determination of the levels of Se and Yb. The concentrations of both investigated elements were highest in the retinal tissue of the eye. YbCl3 influenced the distribution of Se in the eye.

Simultaneous determination of some trace elements in biological materials by neutron activation analysis.

Neutron activation analysis (NAA) for the determination of some trace elements in biological samples is described. This method permits the simple and rapid determination of Se, Hg, Fe, Co, Zn, Ag, Sn, Cr, Yb, Sb, Au and As after a radiochemical separation from Na, Cs, Rb and, partially, Br. For this purpose, precipitation by adding sulphide and hydroxyl ions was used. An attempt was made to simplify the radiochemical procedure. The present (destructive analysis) was tested in radiotracer experiments. Further tests were made using standard biological materials. The method should find applications in neutron activation analysis, particularly of biological materials.