Comparing patterns of volatile organic compounds exhaled in breath after consumption of two infant formulae with a different lipid structure: a randomized trial.

Infant formulae have been used since decades as an alternative to or a complement to human milk. Human milk, the "gold standard" of infant nutrition, has been studied for its properties in order to create infant formulae that bring similar benefits to the infant. One of the characteristics of milk is the size of the lipid droplets which is known to affect the digestion, gastric emptying and triglyceride metabolism. In the current study a concept infant milk formula with large, phospholipid coating of lipid droplets (mode diameter 3-5 µm; NUTURIS, further described as "active"), was compared to a commercially available formula milk characterised by smaller lipid droplets, further described as "control" (both products derived from Nutricia). We investigated whether we could find an effect of lipid droplet size on volatile compounds in exhaled air upon ingestion of either product. For that purpose, exhaled breath was collected from a group of 29 healthy, non-smoking adult males before ingestion of a study product (baseline measurements, T0) and at the following time points after the test meal: 30, 60, 120, 180 and 240 min. Volatile organic compounds (VOCs) in breath were detected by gas chromatography-time-of-flight-mass spectrometry. Any differences in the time course of VOCs patterns upon intake of active and control products were investigated by regularised multivariate analysis of variance (rMANOVA). The rMANOVA analysis revealed statistically significant differences in the exhaled breath composition 240 min after ingestion of the active formula compared to control product (p-value < 0.0001), but did not show significant changes between active and control product at any earlier time points. A set of eight VOCs in exhaled breath had the highest contribution to the difference found at 240 minutes between the two formulas. A set of ten VOCs was different between baseline and the two formulae at T240 with p-value < 0.0001. To our knowledge this is the first study that shows the ability of VOCs in exhaled breath to monitor metabolic effects after ingestion of infant formulae with different lipid structure. The statistically significant differences in compound abundance found between active and control formula milk may be related to: (i) specific differences in the digestion, (ii) absorption of lipids and proteins and (iii) assimilation of the products in the gut.

Infant milk fat droplet size and coating affect postprandial responses in healthy adult men: a proof-of-concept study.

BACKGROUND/OBJECTIVES: Fat droplets in human milk (HM) are larger and surrounded by a phospholipid membrane compared with infant milk formulas (IMF). Since the physical structure of fat droplets might affect digestion and postprandial metabolism, an IMF was developed more mimicking HM lipid structure than current IMF. SUBJECTS/METHODS: A randomised, double-blind, crossover study was performed in 29 fasted healthy men (aged 18-25 years, BMI: 18-25 kg/m2) to compare 5-hour postprandial responses after consumption of an experimental IMF (Concept, Nuturis) with a current IMF (Control). RESULTS: Postprandial triacylglycerol (TAG) concentrations tended to increase faster after intake of Concept IMF (P=0.054), but peaked 3 h after intakes at similar concentrations. ApoB48 increased steadily and peaked 3 h after consumption. Increases in plasma glucose concentrations were comparable, but peak concentrations were reached faster after consumption of Concept IMF (P<0.05). Peak insulin concentrations were higher and reached earlier after intake of Concept IMF, causing a sharper decremental glucose rebound (P<0.05) and an earlier time to nadir in non-esterified fatty acid (NEFA) concentrations (P<0.01). Changes in plasma amino acids (AA), apoB100 and apoA1 were comparable. The incremental or decremental areas under-the-curve did not differ between Concept and Control IMF. Satiety scores and changes in the satiety hormones ghrelin and peptide YY were comparable, while cholecystokinin responses were earlier and higher after consumption of Control IMF (P<0.05). CONCLUSIONS: This proof-of-concept study suggests that fats and carbohydrates from the Concept IMF with larger and phospholipid-coated fat droplets are more rapidly absorbed than those from the current IMF.

Dietary long chain n-3 polyunsaturated fatty acids prevent allergic sensitization to cow's milk protein in mice.

BACKGROUND: Cow's milk allergy is one of the most common food allergies in children and no treatment is available. Dietary lipid composition may affect the susceptibility to develop allergic disease. OBJECTIVE: Assess whether dietary supplementation with long chain n-3 polyunsaturated fatty acids (n-3 LCPUFA) prevents the establishment of food allergy. METHODS: Mice were fed a control or fish oil diet before and during oral sensitization with whey. Acute allergic skin response, serum immunoglobulins as well as dendritic cell (DC) and T cell subsets in mesenteric lymph nodes (MLN), spleen and/or small intestine were assessed. RESULTS: The acute allergic skin response was reduced by more than 50% in sensitized mice fed the fish oil diet compared to the control diet. In addition, anti-whey-IgE and anti-whey-IgG1 levels were decreased in the fish oil group. Serum transfer confirmed that the Th2-type humoral response was suppressed since sera of fish oil fed sensitized mice had a diminished capacity to induce an allergic effector response in naïve recipient mice compared to control sera. Furthermore, the acute skin response was diminished upon passive sensitization in fish oil fed naïve recipient mice. In addition, the percentage of activated Th1 cells was reduced by fish oil in spleen and MLN of sham mice. The percentage of activated Th2 cells was reduced in both sham- and whey-sensitized mice. In contrast, whey-sensitized mice showed an increased percentage of CD11b+CD103+CD8α- DC in MLN in association with enhanced FoxP3+ regulatory T cells (Treg) in spleen and intestine of fish oil fed whey-sensitized mice compared to sham mice. CONCLUSIONS AND CLINICAL RELEVANCE: Dietary n-3 LCPUFA largely prevented allergic sensitization in a murine model for cow's milk allergy by suppressing the humoral response, enhancing local intestinal and systemic Treg and reducing acute allergic symptoms, suggesting future applications for the primary prevention of food allergy.

Perinatal programming of murine immune responses by polyunsaturated fatty acids.

Linoleic acid and α-linolenic acid are essential fatty acids (eFAs) and have to be acquired from the diet. eFAs are the precursors for long-chain polyunsaturated fatty acids (lcPUFAs), which are important immune-modulating compounds. lcPUFAs can be converted into eicosanoids and other mediators. They affect membrane structure and fluidity and can alter gene expression. There has been a marked change in dietary fatty acid intake over the last several decades. Since eFAs are acquired from the diet and immune development occurs mainly perinatally, the maternal diet may influence fetal and neonatal eFA levels, and thereby lcPUFA status, and thus immune development and function. To study whether early exposure to eFAs can program immune function, mice were fed diets varying in the ratio of ω-3 to ω-6-eFAs during pregnancy and/or lactation. After weaning, pups received a Western-style diet. At 11 weeks of age, the effects of maternal diet on the offspring's allergic and vaccination responses were examined using the T-helper 2 driven ovalbumin-induced allergy model and the T-helper 1 driven influenza-vaccination model, respectively. Offspring of dams fed a high α-linolenic acid diet during lactation showed an enhanced vaccination response. As diets with either low or high ω-3/ω-6-eFA ratio attenuated the T-helper 2 allergic response, the high α-linolenic acid diet fed during lactation had the most pronounced effect. These results indicate that there is a programming effect of maternal diet on the offspring's immune response and that in mice the window of greatest susceptibility to maternal dietary intervention is the lactation/suckling period.

Perinatal and chronic hypothyroidism impair behavioural development in male and female rats.

A lack of thyroid hormone, i.e. hypothyroidism, during early development results in multiple morphological and functional alterations in the developing brain. In the present study, behavioural effects of perinatal and chronic hypothyroidism were assessed during development in both male and female offspring of hypothyroid rats. To induce hypothyroidism, dams and offspring were fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate; dams starting 2 weeks prior to mating and pups either until the day of killing (chronic hypothyroidism) or only until weaning (perinatal hypothyroidism) to test for reversibility of the effects observed. Neuromotor competence, locomotor activity and cognitive function were monitored in the offspring until postnatal day 71 and were compared with age-matched control rats. Early neuromotor competence, as assessed in the grip test and balance beam test, was impaired by both chronic and perinatal hypothyroidism. The open field test, assessing locomotor activity, revealed hyperactive locomotor behavioural patterns in chronic hypothyroid animals only. The Morris water maze test, used to assess cognitive performance, showed that chronic hypothyroidism affected spatial memory in a negative manner. In contrast, perinatal hypothyroidism was found to impair spatial memory in female rats only. In general, the effects of chronic hypothyroidism on development were more pronounced than the effects of perinatal hypothyroidism, suggesting the early effects of hypothyroidism on functional alterations of the developing brain to be partly reversible and to depend on developmental timing of the deficiency.

A signaling role for leptin in puberty onset in female rats?

The brain might initiate puberty in response to adequate leptin signaling from the periphery. We studied the link between whole body fat, plasma leptin levels, and puberty onset, in both controls and food-restricted female Wistar rats from age 22 to 42 days. Body fat correlated positively with the prevailing plasma leptin levels (r = 0.776) and with the time of puberty onset, i.e. vaginal opening (VO) (r = 0.691). Blood samples collected every other day at ZT 2, 6, and 12, showed a diurnal rhythm in leptin levels with a nadir at ZT 6. Furthermore, leptin levels increased over the pubertal period. Food restriction (FR) delayed the time of VO considerably (median VO at 38 vs 28 d), and body fat and plasma leptin levels were lower in the FR group (p <0.01), although the positive correlation between body fat and leptin levels remained. Only the absolute, but not the relative amount of body fat increased with age. These data support the notion that leptin could indeed serve as the link between nutritional status and the reproductive axis, and in this way participate in the timing of puberty.

Central application of IGF-1 postpones time of vaginal opening in normally fed, but not in food-restricted rats.

BACKGROUND/AIMS: Central but also peripheral IGF-1 is suggested to play a role in the initiation of puberty as it directly affects GnRH synthesis and release. A possible intermediate in the effects of IGF-1 on puberty might be the adiposity-signaling hormone leptin, whose plasma levels are decreased in food-restricted (FR) rats. METHODS: IGF-1 was chronically centrally infused in 23-day-old prepubertal female rats which were either normally fed or 30% FR, and the effects on time of vaginal opening (VO) and plasma leptin levels were monitored. RESULTS: FR treatment postponed time of VO and decreased plasma leptin levels. In normally fed rats centrally infused with IGF-1, time of VO was found to be postponed to the same extent as FR treatment did. The IGF-1 infusion did not affect plasma leptin levels in normally fed animals but increased leptin levels in the FR group compared to controls. Daily food intake was equal between all groups but body weight course was lower in FR rats. IGF-1 treatment did not significantly affect food intake or body weight course. CONCLUSION: FR treatment delays the moment of vaginal opening to the same extent as observed in normally fed rats that were centrally infused with IGF-1.

Chronic leptin infusion advances, and immunoneutralization of leptin postpones puberty onset in normally fed and feed restricted female rats.

Does leptin play a vital role in initiating puberty in female rats and can it overrule a nutrionally imposed (i.e. a 30% feed restriction, FR) delay in puberty onset? Prepubertal female rats were chronically infused for 14 days with leptin (icv or sc) or leptin-antiserum (icv) while puberty onset was monitored by means of scoring the moment of vaginal opening (VO). Median VO age was higher (35 days versus 27 days) in FR animals but leptin levels at VO were significantly decreased (1.44 +/- 0.17 ng/ml versus 2.79 +/- 0.31 ng/ml). Centrally (icv) and peripherally (sc) infused leptin (1 microg/day) advanced VO age compared to FR controls (30 days versus 35 days and 31 days versus 41 days, respectively). Congruently, centrally (icv) administered leptin-antiserum (0.6 microg/day) delayed puberty onset. In normally fed rats median VO age was only marginally advanced (26 days versus 27 days) but only if leptin was applied centrally. The effects of FR on puberty onset are counteracted or even normalized by the infusion of leptin, whereas immunoneutralization of central leptin postpones puberty onset. We therefore conclude that central leptin is crucial for initiating puberty in female rats.

Determination of actin content in gallbladder tissue and its relevance to normalization of tensiometry data.

OBJECTIVES: To compare the common procedure in tensiometry of normalization of the force (in N) produced by a gallbladder tissue strip to units of stress, with normalization of force to the strip content of contractile protein. DESIGN: A comparison was made in both healthy and in diseased gallbladder tissue strips between two normalization procedures involving anatomical parameters. The contractile response expressed in terms of tissue stress (in N/m2), which entails a normalization to the strip cross-sectional area, was set against normalization to the tissue content of contractile protein (in N/mg actin/g strip wet weight). METHOD: Dose-response curves for acetylcholine (ACh) (10(-8) to 10(-3) M) and sulphated cholecystokinin octapeptide (CCK) (10(-12) to 10(-6) M) were assessed in healthy guinea pig (n = 8) and in diseased human gallbladder tissue strips (n = 28). Assuming a tissue density of 1.05 g/cm3, the strip cross-sectional area was calculated from its weight and length. Actin content in homogenized strips was determined by polyacrylamide gel electrophoresis followed by densitometry. RESULTS: Actin content in human tissue was 19.06 +/- 1.42 mg/g strip wet weight, and 12.84 +/- 0.76 mg/g strip wet weight in guinea pig tissue. No correlation was found between strip cross-sectional area and actin content. In the diseased human tissue, no correlation was found between the inflammation score and either strip cross-sectional area or strip actin content. Maximal force (in mN) exerted in response to either ACh or CCK correlated much more closely in healthy guinea pig gallbladder (r = 0.97) than in diseased human tissue (r = 0.59). Normalization of maximal force to strip cross-sectional area (i.e. to stress) showed considerable more variation (% coefficient of variance) than the normalization to strip actin content in healthy guinea pig tissue, although both strip cross-sectional area and actin content per se showed little variation. Normalization to either parameter did not result in an improved correlation or a decreased variation in the case of diseased human gallbladder tissue. CONCLUSION: Normalization of muscle strip force in diseased tissue is questionable, as the assumptions made for healthy tissue are not valid.

Effects of ursodeoxycholic acid therapy on in vitro gallbladder contractility in patients with cholesterol gallstones.

During treatment with ursodeoxycholic acid (UDCA), the fasting gallbladder volume increases by a yet unknown mechanism. The present study tests whether in vitro human gallbladder contractility in response to acetylcholine and cholecystokinin is affected by UDCA therapy. Gallbladder tissue was obtained from 15 patients treated with UDCA (10 mg/kg/day) during three weeks prior to surgery, and from 15 comparable patients not treated. Data were correlated with in vivo contractility, bile composition, and gallbladder wall inflammation. The inflammation score was lower in the treated patient group. UDCA treatment enhanced gallbladder contractility in vitro: Dose-response curves for acetylcholine and cholecystokinin were both shifted to the left, and the maximal contractile stress generated in response to cholecystokinin was higher in the treated group, whereas the maximal acetylcholine-induced stress was not increased. Maximal cholecystokinin-induced stress correlated positively with fasting gallbladder volume and negatively with the biliary cholesterol saturation index, but not with bile salt hydrophobicity or gallbladder wall inflammation score. In conclusion, UDCA treatment improves in vitro gallbladder contractility, possibly related to a reduced biliary cholesterol saturation. Increased fasting gallbladder volumes during UDCA treatment thus do not appear to result from decreased gallbladder muscle contractile strength.

Cholesterol crystallization in human gallbladder bile: relation to gallstone number, bile composition, and apolipoprotein E4 isoform.

Patients with multiple cholesterol gallstones are at increased risk of recurrence after nonsurgical therapy, possibly because of fast biliary cholesterol crystallization. Serum apolipoprotein E4 (apo E4) is a risk factor for primary cholesterol gallstone formation as well as recurrence. We examined potential effects of stone number and apolipoprotein E genotype on crystallization and on various crystallization-influencing factors in gallbladder biles of 36 cholesterol stone patients (25 multiple stones: 10 carrying the epsilon4 allele). Biliary cholesterol saturation, bile salt composition or concentrations of total protein, immunoglobulin (Ig)A, IgG, alpha1-acid glycoprotein, haptoglobin, or mucin--all crystallization promoters--did not differ between multiple and solitary stone patients, apparently not explaining different speed of crystallization (crystal observation time 3.5 +/- 0.6 days vs. 12.7 +/- 2.4 days, respectively; P = .0003). In contrast, biliary aminopeptidase-N activities (2,607 +/- 592 mU/mL vs. 947 +/- 185 mU/mL; P = .04) were higher and IgM levels (179 +/- 39 vs. 65 +/- 8 mg/L; P = .09) tended to be higher in the case of multiple stones. Although patients carrying the epsilon4 allele had similar stone numbers and crystallization as patients without the epsilon4 allele, their cholesterol saturation index (CSI) was lower (1.08 +/- 0.09 vs. 1.54 +/- 0.13; P = .01), whereas total protein and bile salt concentrations tended to be higher with preferential taurine-conjugation. In conclusion, fast cholesterol crystallization is associated with multiple stones but not with apolipoprotein E4. Whereas fast crystallization may contribute to high recurrence rates after nonsurgical therapy in case of multiple gallstones, the mechanism for increased risk of gallstone formation in patients carrying the epsilon4 allele remains unknown.

Quantitation of cholesterol-carrying particles in human gallbladder bile.

The inter-mixed micellar/vesicular (non-phospholipid-associated) bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, thus allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC (Donovan, J. M., and A. A. Jackson. 1993. J. Lipid Res. 34: 1121-1129). We adapted this method to the more complex human gallbladder bile and examined the relationship between cholesterol solubilization and crystallization in gallbladder biles from 10 cholesterol gallstone patients. The IMC (mean +/- SEM) was 9.67 +/- 1.97 (range 3.56-35.02) mM with significant enrichment with hydrophilic bile salt species. Upon gel filtration of these biles with an eluant buffer containing 10 major bile salts at concentrations according to their IMC, cholesterol was found to be solubilized mainly in mixed micelles. Vesicles were detected in all 10 biles after separation by KBr density gradient ultracentrifugation but in only 5 of these biles with the IMC method. Biles without vesicles had a lower CSI (1.15 +/- 0.12 vs. 1.90 +/- 0.28, P < 0.05), a higher total lipid concentration (11.9 +/- 2.3 vs. 5.9 +/- 1.1, P < 0.05), and a higher bile salt/ (bile salt + phospholipid) ratio (0.83 +/- 0.01 vs. 0.74 +/- 0.04, P = 0.07). For both IMC and ultracentrifugation methods, vesicular cholesterol concentration showed a negative correlation with crystal observation time and a positive correlation with cumulative crystal score during 21 days. Our data indicate that methods such as density gradient ultracentrifugation overestimate vesicular cholesterol solubilization in human biles.

Membrane cholesterol content of cholesterol/phospholipid vesicles determines the susceptibility to both damage and protection by bile salts: implications for bile physiology.

OBJECTIVES: To investigate the effect of membrane lipid composition on the susceptibility to bile salt damage and protection. DESIGN: Artificial model cholesterol/phospholipid (c/p) membranes (vesicles) with a varying cholesterol (0-15 mM) and phospholipid content (3-30 mM), and with a c/p ratio ranging up to 1.70, were prepared by sonication. We examined the effect of incubation with increasing concentrations of either tauroursodeoxycholate (TUDC), taurocholate (TC) or taurodeoxycholate (TDC) alone, or with proportionally varying mixtures of TUDC and TDC. METHOD: Vesicle integrity was assessed by the change in optical absorbance at 340 nm. RESULTS: Absorption of the bile salt-vesicle mixture decreased, with increasing bile salt concentration and hydrophobicity: TUDC less than TC less than TDC. Moreover, bile salt-induced damage also depended on membrane composition: vesicles containing more than 5 mM cholesterol and with a c/p ratio greater than 0.8 were less likely to be solubilized by 30 mM bile salt. Similarly, only in cholesterol-rich vesicles (c/p > 0.5) was a protective effect of TUDC against membrane disruption by TDC revealed upon incubation with various TUDC/TDC mixtures. CONCLUSION: Apart from the bile salt concentration and hydrophobicity, the cholesterol content of vesicles is pivotal, both in the bile salt-induced solubilization of cholesterol/phospholipid vesicles and in the potency of TUDC to prevent this.

Quantifying vesicle/mixed micelle partitioning of phosphatidylcholine in model bile by using radiolabeled phosphatidylcholine species.

Phospholipid in vesicles and mixed micelles of (model) bile has been traced or quantitated (or both) by adding radioactively labeled phosphatidylcholine species. The question is whether these labeled species mix homogeneously with the phosphatidylcholine species mixture present, such that the label distribution reflects the already established mass partitioning of species. In this study, model bile containing egg yolk phosphatidylcholine was incubated with radioactive phosphatidylcholine species. Vesicle and mixed micelle fractions were separated by gel filtration. Radiochemical analysis of the species distribution confirmed chemical analysis: 1,2-di(14C)palmitoyl-phosphatidylcholine was enriched in the vesicles, the 1-palmitoyl-2-(14C)oleoyl species evenly distributed, and the 1-palmitoyl-2-(14C)linoleoyl species more expressed in mixed micelles. This indicates that the distribution of an added radioactive phosphatidylcholine species represents the vesicle/mixed micelle distribution of that particular phosphatidylcholine species. Consequently, the label distribution of a particular added radioactive phosphatidylcholine species can be used to calculate the vesicle/mixed micelle partitioning of total phosphatidylcholine only after it has been established that the radioactive species reaches the same partitioning as total phosphatidylcholine.

Effect of central administration of the kappa-opiate receptor agonist U 69.593 on neurohypophyseal hormone levels in blood and cerebrospinal fluid.

Intracerebroventricular (i.c.v.) administration of the kappa-opiate receptor agonist U 69.593 induces a rapid and short lasting suppression of oxytocin (OXT) levels in plasma of water deprived rats, whereas only a tendency towards a suppression of vasopressin (AVP) levels in plasma is observed. No change in neurohypophyseal hormone levels in CSF occurs following i.c.v. administration of U 69.593 at the various times points studied. It is concluded that, upon i.c.v. administration, the suppressive influence of U 69.593 is much weaker than that of the dynorphins and that neurophypophyseal hormone levels in CSF behave differently from those in the peripheral circulation.

Effects of bile salt hydrophobicity on crystallization of cholesterol in model bile.

Precipitation of cholesterol crystals is an essential step in gallstone formation. In the present study we found much faster and more extensive precipitation of various cholesterol crystal shapes in whole model biles containing the hydrophobic bile salt taurodeoxycholate than in biles containing the relatively hydrophilic taurocholate. Addition of taurodeoxycholate to isolated cholesterol-phospholipid vesicles also induced more crystallization than taurocholate. Crystallization behaviour in whole model biles and in vesicles after addition of corresponding bile salts was very similar. The very hydrophilic bile salts tauroursodeoxycholate and taurohyodeoxycholate never induced crystallization from vesicles, and crystallization in corresponding whole model biles did not occur. These bile salts also reduced crystallization dose dependently after addition of taurodeoxycholate to vesicles. Ultracentrifugation experiments suggested a higher vesicular cholesterol-phospholipid bile salts. These findings indicate that bile salt hydrophobicity influences shape of cholesterol crystals and extent of crystallization, possibly by modulating the vesicular cholesterol-phospholipid ratio.

A novel vesicular assay to study factors affecting cholesterol crystallization in vitro.

We present an assay to study cholesterol crystallization that is fast, facile, and highly reproducible. Cholesterol crystallization from metastable vesicles was induced upon addition of bile salts and depended on the hydrophobicity of the bile salt used and the cholesterol-to-phospholipid ratio of the vesicles. Bile salt-induced crystallization was stimulated upon addition of Con A-binding glycoproteins (CABGs), comparable to the results of the same CABGs in a crystal growth assay. The onset time and total measuring time, however, were much shorter. This assay might, moreover, provide a tool to study the mechanism of cholesterol crystallization in more detail.

Effect of bile salts on in vitro gallbladder motility: preliminary study.

Impaired postprandial gallbladder emptying may be an important factor in cholesterol crystals precipitation and subsequent gallstone formation. We previously found strongly increased bile salt concentrations in gallbladder bile of gallstone patients with weak (< 50% fasting volume) postprandial gallbladder contraction compared to patients with strong (> 50%) postprandial contraction. Therefore, we studied potential effects of various conjugated and unconjugated bile salts with different relative hydrophobicity on in vitro contractility of gallbladder muscle strips obtained at cholecystectomy. Strips were incubated 5 min with bile salt at concentrations of 10(-8)-10(-4)M. The effect of 10(-3)M acetylcholine was measured and related to preincubation control value. Bile salts used were, in order of increasing hydrophobicity: tauroursodeoxy-, ursodeoxy-, tauro-, taurodeoxy- and deoxycholate. Ursodeoxy- and tauroursodeoxycholate did not significantly reduce gallbladder contractility. Taurocholate significantly reduced contractility at concentrations of 10(-6) M and higher, taurodeoxycholate at 10(-7) M and higher and deoxycholate at 10(-5) M and higher. Contractility induced by acetylcholine 10(-3) M at a bile salt concentration of 10(-4) M was 66.0 +/- 11.7% (taurocholate), 50.2 +/- 6.2% (deoxycholate) and 44.8 +/- 11.5% (taurodeoxycholate) of control. The effect of bile salts correlated with their relative hydrophobicity (r = -0.97; p < 0.01). Suppressing effects on gallbladder muscle strip contractility were long lasting and remained after rinsing. Results show that bile salts in the physiological dose range inhibit in vitro gallbladder contraction. If this mechanism exists in vivo, it may have important implications for gallbladder motility regulation.

Gallbladder emptying in vivo, bile composition, and nucleation of cholesterol crystals in patients with cholesterol gallstones.

BACKGROUND/AIMS: Impaired postprandial gallbladder emptying may provide time for progressive bile concentration with formation of instable cholesterol-rich vesicles and fast nucleation of cholesterol crystals. The aim of this study was to assess postprandial gallbladder emptying, bile composition, and nucleation of cholesterol crystals in the same patient. METHODS: In 30 patients with cholesterol gallstones, postprandial gallbladder emptying was measured ultrasonographically. In each patient, gallbladder bile composition (obtained at cholecystectomy) and nucleation of cholesterol crystals was determined. Patients were divided in 22 strong contractors (> 50% postprandial gallbladder emptying) and 8 weak contractors. RESULTS: In weak contractors, bile salt and phospholipid concentrations were much higher than in strong contractors (234.6 +/- 24.7 vs. 130.3 +/- 10.8 mmol/L [P < 0.001] and 44.5 +/- 3.5 vs. 30.2 +/- 3.1 mmol/L [P < 0.05], respectively). Cholesterol concentrations were comparable in strong and weak contractors. Consequently, total lipid concentration was significantly higher (15.5 +/- 1.4 and 9.2 +/- 0.7 g/dL; P < 0.001) and cholesterol saturation index significantly lower (0.90 +/- 0.08 and 1.61 +/- 0.17; P < 0.001) in weak contractors. Nucleation time, percentage of cholesterol in vesicles, bile salt species, and molecular species of phosphatidylcholine were not significantly different. CONCLUSION: Differences in bile composition can be linked to different patterns of postprandial gallbladder emptying and may point to two different pathways of gallstone formation.

Cholecystokinin evokes vasopressin release from perfused hypothalamic-neurohypophyseal explants.

Cholecystokinin (CCK) may have a transmitter/modulator role in the hypothalamic magnocellular neurosecretory system. In the rat, the supraoptic and paraventricular nuclei display high affinity binding for radiolabelled CCK. Exogenously applied CCK depolarizes supraoptic neurons, acting at postsynaptic CCK-B type receptors. The present study evaluated the ability for the sulfated octapeptide of CCK (CCK-8S), which is a predominate form of this peptide in brain, to evoke release of vasopressin from the neurohypophysis of intra-arterially perfused hypothalamic explants. 3 min applications of 1 microM CCK-8S through the intra-arterial perfusion medium prompted an elevation of vasopressin in samples taken from the neurointermediate lobe in 10 of 14 preparations. Vasopressin levels rose from undetectable baseline values to a peak of 29.5 +/- 6.7 pg/ml (mean +/- S.E.M). This response was dose-dependent and was abolished by pituitary stalk transection (5/5 explants). Locally applied CCK-8S (25-200 pmol) through bilateral infusions onto the ventral surface of the supraoptic nucleus also induced a dose-dependent release of vasopressin (5/7 explants). These observations suggest that CCK can act at receptors located on (or near) the somata of supraoptic nucleus neurons to induce neuronal discharges that are conducted to the neural lobe where they evoke release of vasopressin from neurohypophysial axon terminals.