Stability studies of ubiquinol in plasma.

BACKGROUND: Ubiquinol is a sensitive redox marker in the first line of the antioxidative defence mechanism and is increasingly being measured in oxidation studies. Because of its apparent instability during storage and processing, we compared various storage conditions. METHOD: Blood was collected from three volunteers into tubes containing EDTA; it was then separated at 4 degrees C and cryopreserved with saccharose (final concentration 6 g/L). Aliquots were stored with or without glutathione or butylated hydroxytoluene at -20 degrees C and -80 degrees C. RESULTS AND CONCLUSION: Ubiquinol in samples stored at -20 degrees C was not stable; however, it was stable when stored at -80 degrees C, even without addition of antioxidant. By contrast, alpha-tocopherol was stable under all conditions studied.

Chylomicron processing in familial dysbetalipoproteinemia and familial combined hyperlipidemia studied with vitamin A and E as markers: a new physiological concept.

In previous work we identified a transfer/diffusion process occurring in the postprandial state that more or less contributes to the accumulation of beta-VLDL in familial dysbetalipoproteinemia (FD). Here we present a new theoretical concept underlying chylomicron processing developed on the basis of extended quantitative analyses of fat loading experiments, with both vitamins A and E, performed in patients with familial combined hyperlipidemia (FCH) in comparison to patients with FD and control subjects. Recovery of triglycerides from the fat load in the plasma triglyceride pool was <4%, indicating a very effective lipolysis process with an active remnant generation. Vitamin A from the fat load was, over 48 h, quantitatively recovered in the plasma lipoprotein pool; vitamin E was recovered to 2241%. Nevertheless, transfer/diffusion of both vitamins showed similar patterns. At equilibrium, their contents correlated strongly with the lipoprotein concentrations, the slopes being similar for control subjects and both groups of patients. Only in those FD patients with the highest lipid values, did the vitamin A/lipoprotein mass ratio in the Sf>100 fraction deviate from the total group mean. In the Sf 15-100 fraction, most specific for 'remnants', vitamin A/cholesterol ratios for all subjects were uniform proving that beta-VLDL formation is a thermodynamic process regulated by concentration gradients and the lipophilicity of lipoprotein constituents, not a typical feature for patients with FD. In patients with FD, vitamin A in the plasma pool was recovered excessively (276%) in line with recognition in various pools as a result of the transfer/diffusion process in plasma.

Plasma thiol status in preeclampsia.

OBJECTIVE: To measure plasma thiol levels in women with normal pregnancies, women with preeclampsia, and nonpregnant controls to define plasma thiol's effect on glutathione homeostasis and pathophysiology of preeclampsia. METHODS: Total plasma cysteine, gamma-glutamylcysteine, homocysteine, cysteinylglycine, and glutathione levels were measured in ten nonpregnant women, ten women with normotensive pregnancies, and 20 women with preeclampsia at delivery. RESULTS: Median total plasma levels of all thiols in normotensive pregnant women were significantly lower than in nonpregnant women. Median total plasma cysteine and homocysteine levels in women with preeclampsia were significantly higher compared with pregnant controls (254 versus 190 micromol/L, P < .001; and 13.3 versus 8.4 micromol/L, P < .02, respectively), whereas glutathione levels were significantly lower in women with preeclampsia compared with those in pregnant controls (5.1 versus 6.3 micromol/L, P < .05). CONCLUSION: In women with preeclampsia, homocysteine and cysteine levels, which are lowered in normotensive pregnancy, were comparable to levels in nonpregnant women, whereas glutathione levels were lower. Those results suggest that in women with preeclampsia, glutathione use is higher or its synthesis is disturbed. Therefore, glutathione might affect pathophysiology of preeclampsia.

Beta-VLDL accumulation in familial dysbetalipoproteinemia is associated with increased exchange or diffusion of chylomicron lipids to apo B-100 containing triglyceride-rich lipoproteins.

To gain more insight into the accumulation of beta-very low density lipoprotein (beta-VLDL) in familial dysbetalipoproteinemia (FD), we followed the courses of the levels of retinyl palmitate (rp), alpha-tocopherol (alpha-T) and apolipoprotein (apo) B-48 in various lipoprotein fractions for up to 48 h in eight patients with FD and six normolipidemic control subjects after an oral fat load (50 g fat/m2 containing 150000 IU of rp and 5000 IU of alpha-T). Alpha-T was added because of its rapid transfer to other lipoproteins. Fasting apo B-48 concentration in FD was normal to strongly elevated, dependent on the fasting lipid concentrations. 3 h after fat loading, total apo B-48 content did not abnormally increase; while the apo B-100 content in the triglyceride-rich lipoprotein fraction remained stable. The levels of both vitamins increased considerably, especially in the remnant fraction (Sf 15-100), which in due course exclusively contained apo B-100 in most hyperlipidemic patients. This, together with the observation that peaks for rp and alpha-T were observed 3-6 h later than for apo B-48 strongly suggests that both vitamins transfer or diffuse rapidly towards the apo B-100 containing VLDL. RP is thus more a marker for this process, which also comprises chylomicron lipids, than a specific marker for chylomicrons. This process, first described here, appears decisive in the pathogenesis of FD.

Fat loading experiments with the vitamins A and E suggest that in postprandial lipemia transfer/diffusion of chylomicron lipids to VLDL contributes to beta-VLDL formation.

Vitamins A and E differ in hydrophobicity. When added to a fat load more or less specific labeling of chylomicrons and their remnants can be expected and this allows to approach the mechanism of postprandial lipemia from a new sight of view. Applied in a study in which 20 patients participated (eight patients with familial dysbetalipoproteinemia (FD), six patients with familial combined hyperlipidemia (FCH) and six controls) we found that vitamin A, no longer paralleled the apo B-48 concentrations from 9 h after a fat load, especially in the remnant fraction with Sf 15-100. Qualitatively, the distribution of vitamin A to the more dense fractions mirrored that of vitamin E, but the latter was more rapid. Both vitamins at the maximum of remnant-accumulation, at 14 h after the fat load, correlated with the cholesterol content of the remnant fraction. For vitamin E there was a similar concentration dependent distribution to all other lipoprotein fractions. The results confirm our view that the lipoprotein mechanism can be regarded as a dynamic system. During regular episodes following the meals, exogenous fat is, like the vitamins, distributed over all endogenously formed lipoproteins. This transfer process results in the formation of beta-VLDL and contributes to the pathogenesis of FCH and FD.

Pro- and anti-inflammatory cytokines in healthy volunteers fed various doses of fish oil for 1 year.

Dietary supplementation with n-3 fatty acids from fish oil alleviates inflammation in various chronic inflammatory disease states. Reductions in the production of pro-inflammatory cytokines interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), and interleukin 6 (IL-6) have been seen in humans after short-term n-3 fatty acid supplementation. We investigated long-term effects of dietary n-3 fatty acids on circulating cytokine concentrations and on ex vivo stimulated whole-blood production of IL-1 beta, TNF-alpha and interleukin 1 receptor antagonist (IL-1Ra), the naturally occurring antagonist of IL-1. A total of 58 monks with a mean age of 56 years were randomized into four groups and their diets were supplemented with 0, 3, 6, or 9 g of fish oil, providing 0, 1.06, 2.13 or 3.19 g of n-3 fatty acids per day. Subjects received equal amounts of saturated fatty acids, vitamin E and cholesterol. Compliance was excellent and erythrocyte fatty acid profiles closely reflected the amounts of n-3 fatty acids ingested. In the group receiving 9 g of fish oil per day, no influence of n-3 fatty acids on circulating cytokine concentrations was observed relative to placebo. Endotoxin-stimulated whole-blood cytokine production was measured at 26 and 52 weeks after the start and at 4, 8 and 26 weeks after cessation of supplementation. In all groups, the production of IL-1 beta and IL-1Ra was higher during supplementation than afterwards. However, no differences in cytokine production were noted between the placebo group and the various treatment groups at any point in time. Our results suggest that long-term supplementation of fish oil does not affect ex vivo cytokine production in man.

Vitamin E and fatty acid intervention does not attenuate the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

We investigated the effect of different interventions on aortic atherosclerosis in Watanabe rabbits. Four groups of rabbits were fed either an oleic acid-enriched diet (80% of total fat intake) with or without vitamin E supplementation (250 IU/kg) or a diet enriched in linoleic acid with or without vitamin E supplementation for 6 months. At the start of the study, plasma cholesterol concentration was 21.4 +/- 3.6 mmol/L (n = 32). The diets did not influence the mean plasma lipids and lipoprotein concentrations except for HDL cholesterol, which was increased more on the oleic acid-enriched diets than on the linoleic acid-enriched diets. Vitamin E levels in plasma and LDL were increased on the oleic acid diet and reduced on the linoleic acid diet. On the latter diet, supplementation of vitamin E was quantitatively less effective in raising plasma or LDL vitamin E levels. The susceptibility of LDL to oxidation was determined in vitro. Both oleic acid-enriched diets increased the lag time by 140% from baseline. The linoleic acid diet supplemented with vitamin E increased lag time by 59%. Linoleic acid alone, however, decreased the lag time by 30%. Similar but inverse effects were seen on LDL oxidation rate. Thus, intervention protected LDL to oxidation in the following order: oleic acid plus vitamin E > oleic acid > linoleic acid plus vitamin E > linoleic acid. Despite the differences in LDL oxidizability induced by the four experimental diets, assessment of aortic atherosclerosis at the end of the 6-month dietary study period revealed no differences among the four study groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of predigested fat on intestinal stimulation of plasma cholecystokinin and gall bladder motility in coeliac disease.

Cholecystokinin (CCK) release and gall bladder emptying in response to a fatty meal are completely abolished in coeliac disease. To determine the effect of lipid digestion on CCK release and gall bladder motility, six patients with untreated coeliac disease and a flat jejunal mucosa were studied on two separate days. After an overnight fast, the plasma CCK concentration and gall bladder volume were measured before and at regular intervals after the intraduodenal instillation of 60 ml corn oil (triglycerides) incubated with 40 ml saline or with 40 ml bile and pancreatic juice. The mean (SEM) concentration of free fatty acids in the aqueous phase of corn oil after incubation with bile and pancreatic juice (predigested corn oil) was 78 (35) mM compared with 0.1 (0.1) mM in the aqueous phase of corn oil incubated with saline (undigested corn oil). Integrated plasma CCK in response to predigested corn oil was significantly greater than that in response to undigested corn oil (101 (18) pM. 80 min v-2 (9) pM.80 min; p < 0.005). Similarly, integrated gall bladder contraction in response to predigested corn oil was significantly larger than that after undigested corn oil (817 (210) ml. 80 min v-225 (243) ml. 80 min; p < 0.05). In contrast to undigested corn oil, corn oil that has been predigested with bile and pancreatic juice induces plasma CCK secretion and gall bladder contraction in patients with untreated coeliac disease, presumably by generating and rendering soluble lipolytic products.

Adrenoceptors on blood cells in patients with primary hypertension: correlation with blood pressure and related variables.

OBJECTIVE: Alterations in platelet alpha 2-adrenoceptor and mononuclear leucocyte beta 2-adrenoceptor characteristics in primary hypertension have been extensively studied. The results of the reports have not been consistent, possibly because of the small number of subjects in most of the studies. We therefore studied the blood-cell adrenoceptor characteristics in a relatively large group of primary hypertensive and normotensive subjects. DESIGN: Platelet alpha 2-adrenoceptor characteristics were compared in 65 hypertensive and 51 normotensive subjects. Mononuclear leucocyte beta 2-adrenoceptor characteristics were compared in 72 hypertensives and 67 normotensives. Untreated hypertensive subjects were selected from the outpatient clinic and the normotensive controls were recruited by a newspaper announcement. METHODS: Platelets and mononuclear leucocytes were isolated from blood samples obtained after at least 10 min supine rest. The alpha 2- and beta 2-adrenoceptor characteristics were determined with [3H]-rauwolscine and [125I]-(-1)cyanopindolol, respectively. Correlations between the adrenoceptor characteristics and clinical parameters of the subjects were studied. RESULTS: No differences in alpha 2- or beta 2-adrenoceptor densities were observed between the two groups. However, a significantly lower equilibrium dissociation constant for [3H]-rauwolscine was observed in the hypertensive group. The correlations between the adrenoceptor characteristics and clinical parameters were weak and mostly not statistically significant. The results were compared with the most relevant studies in the literature. CONCLUSIONS: From our study and the literature, we conclude that blood-cell adrenoceptor characteristics are unchanged in primary hypertension.

The effect of a natural high-fibre diet on faecal and biliary bile acids, faecal pH and whole-gut transit time in man. A controlled study.

Dietary fibre possibly protects against colonic cancer by effects on bile acid metabolism. We investigated the effect of a natural high-fibre diet on secondary bile acid formation. Twelve healthy subjects on an habitual low-fibre diet (for 4 weeks) consumed a high-fibre menu for 10 weeks (experimental group). A control group of 10 subjects consumed their regular high-fibre diet during this period. Faecal and biliary acid composition, faecal weight, faecal pH and gut transit time were studied before and after 6 and 10 weeks of fibre addition. Changes in the experimental group were compared to changes in the control group. The concentration, but not the excretion, of the secondary faecal bile acids was reduced in the experimental group. Faecal weight increased, faecal pH dropped and gut transit time was not altered. The biliary deoxycholic acid content decreased and the cholic acid content increased after 6 weeks, but returned to baseline values after 10 weeks of fibre addition. This study shows that a natural high-fibre diet lowers secondary faecal bile acid concentration through an increase in stool weight. The 7 alpha-dehydroxylation of primary bile acids is probably not or only transiently inhibited.

Glutathione and glutathione S-transferases in Barrett's epithelium.

Glutathione content, enzyme activity and isoenzyme composition of glutathione S-transferases were assayed in normal and Barrett's esophageal epithelium of ten patients with Barrett's esophagus. In addition, gastric and duodenal specimens from the same patients were also investigated. Glutathione content, glutathione S-transferase enzyme activity as well as glutathione S-transferase pi content were all significantly lower in Barrett's epithelium as compared to normal esophageal mucosa. In contrast, glutathione S-transferase class alpha enzymes are markedly expressed in Barrett's epithelium, whereas only low amounts are present in normal esophageal epithelium. Glutathione and glutathione S-transferase composition in Barrett's epithelium show striking similarities with gastric epithelium, whereas duodenal epithelium is provided with considerable higher amounts of glutathione and glutathione S-transferases, except for levels of glutathione S-transferase class pi, which are lower. A significant negative correlation exists between glutathione S-transferase enzyme activity in the mucosa along the gastrointestinal tract, and the tumour incidence. Since glutathione and glutathione S-transferase are correlated with protection against cellular or cytogenetic damage, the low content of glutathione and glutathione S-transferases in the Barrett's esophagus may be a factor of relevance for the increased tumour risk in this tissue.

Does treatment with beta-adrenergic blocking agents cause a decrease in beta 2-adrenoceptor affinity?

The effect of beta-adrenoceptor antagonists (BAAs) differing in lipophilicity and partial agonist activity (PAA), and a full agonist, on the dissociation constant for [125I]-(-)- iodocyanopindolol binding to beta 2-adrenoceptors (KD) has been investigated. Twelve healthy, normotensive male volunteers (mean age 22.3 y) were treated with different BAAs according to a cross-over design. The drugs used were propranolol (highly lipophilic BAA, no PAA), pindolol (moderately lipophilic BAA, strong PAA), dilevalol (highly lipophilic BAA, weak PAA) and salbutamol (full agonist). Before and after a single dose and an 8 day course of one of the drugs, blood pressure and the beta 2-adrenoceptor characteristics of mononuclear leukocytes (MNL) were determined. Between the treatment periods, there was a washout interval of 14 days. All BAAs decreased the blood pressure, but only propranolol lowered heart rate. Treatment with salbutamol decreased the diastolic and increased the systolic blood pressure and heart rate. Three hours after the single dose of any of the BAAs, a more than 2-fold increase in KD was observed, and the increase became larger after 8 days of administration (up to 3.7-fold increase). In contrast, no effect on KD was observed after treatment with salbutamol. BAAs with PAA and salbutamol induced a 30% decrease in beta 2-adrenoceptor density. It is concluded that treatment with BAAs, irrespective their lipophilicity or PAA, induces a decrease in the affinity of MNL beta 2-adrenoceptors for antagonists. This phenomenon may help to explain the contradictory relationship between the kinetics and dynamics of BAAs.

Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects.

Oxidative modification of low density lipoprotein (LDL) has been implicated as a factor in the generation of macrophage-derived foam cells, the hallmark of atherosclerotic plaques. Because LDL consists of discrete subfractions with different physicochemical characteristics, the question arises as to whether these LDL subfractions differ in their susceptibility to oxidative modification. To answer this question, three LDL subfractions, LDL1, LDL2, and LDL3, were isolated from the plasmas of 11 healthy volunteers by density gradient ultracentrifugation. The LDL subfractions were oxidatively modified by incubation with copper ions. Differences in the subfractions' susceptibilities to lipid peroxidation were studied by measuring the formation of the 234-nm-absorbing oxidation products every 3 minutes on an ultraviolet spectrophotometer. A significant inverse linear relation was found between LDL subfractions and lag time (regression coefficient = -8.50, p less than 0.001), indicating that both the dense LDL3 and the light LDL2 were less well protected against oxidative modification than the very light LDL1. The LDL subfractions showed a positive linear relation with the rate of oxidation (regression coefficient = 0.46, p less than 0.001) and the amount of conjugated dienes formed in the LDL subfractions after 4 hours of oxidation (regression coefficient = 9.24, p less than 0.001). Thus, both LDL3 and LDL2 were more extensively modified in time than LDL1, which may be explained by the significantly higher concentration of polyunsaturated fatty acids in LDL3 (micromoles per gram LDL cholesterol) compared with LDL1 (Tukey's test, p less than 0.05). These results indicate that the more dense LDL subfractions, that is, LDL2 and LDL3, are more susceptible to oxidative modification and therefore may contribute more to foam cell formation than the less dense LDL subfraction LDL1.

The use of a rat-derived microflora for providing colonization resistance in SPF rats.

To obtain a suitable species-specific microflora for a new rat SPF-unit, germ-free WAG/Rij rats were associated with a flora derived originally from selectively decontaminated Cpb: WU (Wistar) rats. Caecal and ileal contents of these rats had been cultured anaerobically (37 degrees C) for 7 days and harvested. This cultured flora was given to germ-free Cpb: SE (Swiss) mice, which were kept in an isolator system and acted as a source of the flora to associate germ-free Wag/Rij rats. In these associated rats, several parameters indicative of the 'quality' of the intestinal microflora were investigated and compared to those in rats with a mouse derived anaerobic microflora. Parameters included relative caecal weight, colonization resistance and the concentration of faecal bile acids. The cultured rat-derived microflora normalized the observed intestinal parameters better than the mouse derived microflora, and provided better colonization resistance. We conclude that culturing of intestinal contents of selectively decontaminated animals can be a useful way to obtain a species-specific donor-microflora which can be used to start new SPF units.

The relationship between adrenaline and beta 2-adrenoceptors on human lymphocytes.

To study the relation between plasma adrenaline (AD) and noradrenaline (NA) and the beta 2-adrenoceptor number (Bmax) on lymphocytes these variables were measured in the following three groups: a) In 67 normotensive volunteers after a rest period of at least 15 min. b) In 10 essential hypertensive (HT) and 10 age and sex matched normotensive (NT) subjects before and after a standardized bicycle exercise test (BE). c) In 10 bilaterally adrenalectomized females (AF) and 10 healthy age matched females (NF) before and after a mental arithmetic test (MA). Significant correlations were found between AD and Bmax in NT (r = 0.30, P = 0.015) and between the proportional increase in AD and the proportional increase in Bmax after BE (r = 0.60, P less than 0.005). The MA induced elevation of Bmax was blunted after adrenalectomy. All these findings point to a relation between AD and beta 2-adrenoceptor regulation.

Beta 2-adrenoceptor up-regulation in relation to cAMP production in human lymphocytes after physical exercise.

The effect of isotonic bicycle exercise on beta 2-adrenoceptors and cAMP production in lymphocytes was studied. The origin of the newly exposed receptors was studied by measuring radioligand binding in the presence of hydrophylic or lipophylic 'cold' ligands. During exercise beta 2-adrenoceptor density increased from 1207 +/- 144 to 1776 +/- 152 sites/cell (56 +/- 14%). Isoprenaline induced an increase in cAMP synthesis, which was 68 +/- 15% higher than those values measured before the test. Forskolin did not induce a higher increase in cAMP production after exercise. The increase in receptor density seems not to be due to a transfer of internalized receptors to the membrane surface. We conclude that physical exercise induces an up-regulation of beta 2-adrenoceptors on lymphocytes and that these adrenoceptors are functional.

The influence of stress and cheese-whey on intestinal parameters in mice.

In a mouse model the effects of environmental stress on gastrointestinal parameters and the effects of cheese-whey on the changes induced by stress were studied. Mice were subjected to overcrowding, lack of bedding, overcrowding together with lack of bedding, continuous light, and housing at 30 degrees C. The influence of stress on relative caecal weight, faecal enterobacteriaceae, colonisation resistance (CR), filamentous segmented bacteria in the small intestine, fusiform bacteria in the faeces, and concentration of faecal bile acids was studied. Stress had no influence on relative caecal weight, faecal enterobacteriaceae, and faecal bile acids. Stress did decrease colonisation resistance, presence of segmented filamentous bacteria in the small intestine, and fusiform bacteria in the faeces. Cheese-whey had no positive effect on the microbiological disturbances caused by stress. The number of filamentous segmented bacteria in the small intestine and fusiforms in the faeces decreased by giving cheese-whey.

The 'normalization' of germ-free guineapigs with host-specific caecal microflora.

Hysterectomy-derived germ-free guineapigs were given colonization-resistant caecal flora from mice (mCRF) or microflora obtained from the caecum of an antibiotic-decontaminated conventional guineapig (gpCRF) and compared with guineapigs raised conventionally with the sow. Body weight and the following intestinal parameters were determined for the groups: colonization resistance (CR) to Escherichia coli, relative caecal weight (RCW), beta-aspartylglycine (faeces), volatile fatty acids (caecum) and bile acids (faeces). mCRF guineapigs showed values quite different from control animals for CR and RCW, indicating the unsuitability of mouse CRF for normalizing guineapigs. In gpCRF guineapigs CR and RCW values were comparable with controls, indicating the suitability of the guineapig flora for normalizing guineapigs. mCRF guineapigs housed with gpCRF guineapigs, showed an improvement in CR and RCW, yielding values found in control animals.

Intestinal "normalization" of germ-free rabbits with rabbit caecal microflora: effect of dosing regimens.

Hysterectomy-derived germ-free (GF) rabbits were given strictly anaerobic microflora obtained from the caecum of an antibiotic-decontaminated conventional rabbit. One group was given the caecal flora diluted in doe's milk. The second group received caecal flora without doe's milk and administration of the flora was repeated when the animals were given pelleted diet. Body weight and intestinal parameters determined in the two groups of rabbits were compared with values in rabbits conventionally raised with the doe. In GF rabbits given caecal flora, the values for most intestinal parameters were similar to values recorded in conventional animals. However colonization resistance to Escherichia coli was limited in GF rabbits given caecal flora diluted in doe's milk and severely impaired in GF rabbits given caecal flora without doe's milk, although relative caecal weight was normal. It is concluded that enteric microfloras should be preferably incorporated in doe's milk when dosed to GF rabbits to obtain acceptable gastrointestinal "normalization".

Elimination of murine viral pathogens from the caecal contents of mice by anaerobic preparation.

The aim of this study was to investigate methods to eliminate pathogenic viral agents while preserving the 'normalizing' properties of the gut microflora of mice. Mouse hepatitis virus A59 (MHV), Reo 3 virus and Theiler GD VII virus were added to the caecal contents of 'normal' mice and following dilution, with or without subsequent culturing, given to germ-free mice. Four weeks later antibody titres against these and other viruses were determined. MHV and Theiler CD VII virus survived dilution but were eliminated during culturing. Reo-virus survived the 10(-1) dilution-culture step. All dilutions and dilution-cultures of caecal contents resulted in 'normalization' in germ-free recipients of the relative caecal weight, percentage faecal fusiform-shaped bacteria, faecal bile acids and colonization of small intestine by segmented filamentous bacteria.