Mario Baraldini

Assessment of portal hypertension by endoscopic ultrasonography

Endoscopic ultrasonography (EUS) was performed in 40 patients with portal hypertension (PH) and in 48 control subjects. The azygous, splenic, mesenteric, and portal veins were displayed in both groups. However, esophageal and gastric varices, periesophageal and perigastric collateral veins, and submucosal gastric venules were displayed only in patients with portal hypertension. EUS was inferior to endoscopy in detecting and grading esophageal varices (p less than 0.0005), but EUS was superior in the detection of varices in the fundus of the stomach (p less than 0.0005). Detection of periesophageal veins by EUS increased with increasing diameter of esophageal varices at endoscopy (57% in grade 1, 89% in grade 2, and 100% in grade 3), and there was a direct correlation between endoscopic grade and the diameter of the periesophageal collateral veins at EUS. The diameter of the azygous vein by EUS at its distal and proximal margins was significantly greater in patients with PH (p less than 0.001); the EUS diameter of the azygous vein was significantly larger with variceal grade 2 compared with grade 1 (p less than 0.02 and p less than 0.01, respectively). In portal hypertensive gastropathy, endoscopic and EUS detection were coincident. No correlation was found between the presence of portal hypertensive gastropathy, endoscopic grade of esophageal varices, and detection of gastric varices at EUS.

Reduced cardiovascular responsiveness to exercise-induced sympathoadrenergic stimulation in patients with cirrhosis

Cardiovascular responsiveness to sympathoadrenergic activation obtained by muscle exercise in the supine position was evaluated in 22 patients with cirrhosis (11 alcoholic, 11 postnecrotic/cryptogenic; 14 with ascites) and 10 controls of comparable age. Plasma norepinephrine, heart rate, diastolic arterial pressure and cardiac function, as evaluated by systolic time intervals, were monitored. At rest, cirrhotics had higher norepinephrine (154 +/- 19 S.E.M. ng/l) and heart rate (79 +/- 2 beats per min) than controls (71 +/- 3 ng/l, p less than 0.01; 67 +/- 2 beats per min, p less than 0.001), whereas diastolic arterial pressure was similar. Among systolic time intervals, electromechanical systole, pre-ejection period, electromechanical delay and pre-ejection period to left ventricular ejection time ratios were prolonged (p less than 0.05 or less). Exercise led to significant increases in plasma norepinephrine, heart rate and diastolic arterial pressure in both controls and patients. In the latter, however, whereas the increase in norepinephrine was greater (p less than 0.001), those in heart rate and diastolic arterial pressure were less (p less than 0.005). As expected, most systolic time intervals shortened, but the decrease in pre-ejection period (p less than 0.05), isometric contraction time (p less than 0.02) and pre-ejection period to left ventricular ejection time ratio (p = 0.06) was less in patients than in controls. Direct correlations between exercise-induced changes in norepinephrine and both diastolic arterial pressure (r = 0.81; p less than 0.005) and heart rate (r = 0.85; p less than 0.002) were observed in controls, while inverse correlations (r = -0.67, p less than 0.001 and r = -0.44; p less than 0.05) were found in cirrhotics. These results suggest that cardiovascular reactivity to the sympathetic drive is impaired in cirrhotics. The impairment of cardiac contractility may be due to altered electromechanical coupling.

Hyperdynamic circulation of advanced cirrhosis: a re-appraisal based on posture-induced changes in hemodynamics

Little is known about the effect of posture on the circulatory abnormalities of advanced cirrhosis. We evaluated the systemic hemodynamics, measured by Doppler-echocardiography, atrial natriuretic factor, plasma renin activity and plasma norepinephrine, in 10 patients with cirrhosis and ascites and 10 healthy controls, after 2 h of standing and during lying down for a further 2 h. Standing hemodynamic patterns of controls and patients with cirrhosis did not differ significantly. The latter, however, showed higher plasma renin activity, norepinephrine and atrial natriuretic factor. The assumption of the supine position led to greater increases in cardiac index and atrial natriuretic factor, and reduction in systemic vascular resistance in patients with cirrhosis. Norepinephrine and plasma renin activity declined in both groups to a similar extent, while heart rate only slowed in controls. Thus, after 2 h in the supine position, patients with cirrhosis showed hyperdynamic circulation with increased cardiac index and heart rate and reduced systemic vascular resistance. Norepinephrine, plasma renin activity and atrial natriuretic factor were also elevated. The hyperdynamic circulation in advanced cirrhosis appears during or is enhanced by lying down. This finding suggests that this syndrome is, at least in part, attributable to excessive blood volume translocation towards the central area. However, the persistent activation of renin-angiotensin and sympathoadrenergic systems suggests that a concomitant reduced vascular sensitivity to vasoconstrictors concurs in its development.

Separation techniques for bile salts analysis

The analysis of bile salts in biological samples has remained a difficult task, due to the complex nature of the salts and also to their low concentration in common sample fluids such as plasma and urine. Given their importance, the development of accurate and sensitive methods of instrumental analysis has been the subject of intensive research, and recent advances have eliminated or lessened some of the difficulties. Currently available techniques are the following: thin-layer chromatography, gas chromatography, high-performance liquid chromatography, supercritical fluid chromatography, gas chromatography-mass spectrometry and capillary electrophoresis. Liquid chromatography coupled with mass spectrometry (thermospray, fast atom bombardment, electrospray and ionspray), a method undergoing continuous improvement, is also being applied to bile salts analysis. In this paper, these various techniques, which differ greatly in specificity, accuracy and simplicity, are reviewed and discussed, in terms of analytical performance, applicability to a given sample fluid, major limitations, ability to identify uncommon bile salts, including unsaturated oxo derivatives, glucuronides, sulfates, glycosides and bile alcohols.

Chemiluminescent Low-Light Imaging of Biospecific Reactions on Macro- and Microsamples Using a Videocamera-Based Luminograph

The analytical performance of a low-light imaging luminograph for quantitative luminescence analysis was evaluated in terms of sensitivity, spatial resolution, accuracy, precision, and sample geometry, at the macrolevel and in combination with optical microscopy. The system allows for the detection of 400 amol of enzymes such as alkaline phosphatase and horseradish peroxidase using 1,2-dioxetanes and luminol/p-iodophenol or acridancarboxylate esters, respectively, as chemiluminescent substrates. Enzymatic activity and spatial distribution of nylon net immobilized-alkaline phosphatase was studied; the system permits the quantification of the immobilized enzyme with a spatial resolution as low as 1 μm. Other applications, such as the alkaline phosphatase localization in 8 μm intestinal mucosa cryosections, quantitative immunocytochemistry, and dot blot DNA hybridization reactions, were studied and optimized. The system was also employed for in situ hybridization assay of cytomegalovirus DNA in infected human fibroblasts. The presence of a viral genome was revealed with digoxigenin-labeled probes and alkaline phosphatase-labeled anti-digoxigenin antibody, using chemiluminescent substrate for this enzyme. The luminescent signal was intense and stable, and the probe was imaged and quantified within single cells with higher intensity in the nuclei, with a spatial resolution as low as 1 μm and very low background. The results show that this technique is an ultrasensitive and potent analytical tool to localize and quantify biomolecules at microscopic level, and it is suitable for many bioanalytical applications.

Renal function impairment induced by change in posture in patients with cirrhosis and ascites.

The assumption of upright posture by patients with liver cirrhosis leads to striking activation of adrenergic and renin-angiotensin systems. The tilting-induced modifications in renal function of eight healthy controls and 14 untreated patients with liver cirrhosis and ascites were related to plasma concentrations of noradrenaline, renin activity and aldosterone. All patients had preserved renal blood perfusion. All parameters were evaluated during bed rest for two hours and in the sitting posture for one hour. Basal plasma renin activity (0.1 greater than p greater than 0.05), aldosterone and noradrenaline concentrations (p less than or equal to 0.01) were raised in cirrhotics. The renal function tests (creatinine clearance, filtered sodium, tubular rejection fraction, urinary sodium excretion) were significantly reduced in cirrhosis. Under basal conditions, in cirrhotic patients tubular rejection fraction and urinary sodium excretion were inversely related to both noradrenaline and aldosterone concentrations. After tilting, the noradrenaline and aldosterone integrated outputs (sigma delta) were significantly greater in cirrhosis. All renal function tests significantly decreased in cirrhotics, whereas creatinine clearance only significantly decreased in controls. Patients tubular rejection fraction of sodium and sodium excretion were related to sigma delta aldosteronaemia (r = -0.72; p less than 0.01), but no longer to sigma delta plasma noradrenaline.

High-performance liquid chromatographic-electrospray mass spectrometric analysis of bile acids in biological fluids

The present work describes the development of HPLC-mass spectrometric systems equipped with an electrospray interface for the quantitative analysis of bile acids. Good separation of free as well as glycine- and taurine-conjugated bile acids was achieved with a C18 reversed-phase column (3 microns particle size, 70 x 4.6 mm I.D.) employing methanol-15 mM ammonium acetate as the mobile phase for both isocratic and gradient mode, at a flow-rate of 0.3 ml/min. This system permits post-column splitting of the eluate for analysis by two different detectors: (1) electrospray-mass spectrometer with a flow-rate of 18 microliters/min; and (2) a complementary evaporative light scattering mass detector. When bile salts were ionized in the electrospray interface operating in the negative-ion mode, only [M-H]- molecular ions were generated; the detection limit was 15 pg injected for all bile acids studied. In the second system, a semi-micro pre-column splitting apparatus (Acurate, LC Packings) was utilized: with this device the flow-rate from the HPLC pump was reduced to 1.4 microliters/min and bile acids were separated with a micro-bore C18 column (3 microns particle size, 150 x 0.30 I.D.), using the same mobile phase as above. With this latter system, a head-column enrichment technique can be used: the amount injected can be increased from 60 to 200 nl, permitting an improvement in the detection limit to 5 pg injected. Application of the HPLC-electrospray-mass spectrometric method to bile and serum bile acid analysis is described; preliminary data on the ability of the first system to determine the 13C/12C isotope ratio in 13C-labeled bile acid enriched serum is also critically discussed.

Bed-rest-induced hypernatriuresis in cirrhotic patients without ascites: does it contribute to maintain 'compensation'?

Renal function, plasma renin activity, plasma aldosterone concentration and urine excretion of free norepinephrine were evaluated in 13 cirrhotics without previous or ongoing ascites and in 13 healthy subjects, after 6 days of controlled electrolyte intake (40 mmol of Na and 70 mmol of K per day) and during 24 h of recumbency. Plasma concentrations of the atrial natriuretic peptide (ANP) were also measured in 8 patients and 8 controls. Despite a low-normal filtered load of sodium (14.6 +/- 1.2 vs. 17.1 +/- 1.2 mmol/min), cirrhotic patients showed supernormal natriuresis (141.5 +/- 14.1 vs. 78.8 +/- 8.6 mmol/day; p < 0.001). Whereas the fractional excretion of sodium in these patients was twice that of controls (0.70 +/- 0.05 vs. 0.36 +/- 0.04%; p < 0.001), potassium excretion (42.5 +/- 2.7 vs. 43.1 +/- 2.7 mmol/day) and urine volume (1270 +/- 98 vs. 1452 +/- 148 ml/day) did not differ. In cirrhotics, plasma renin activity was reduced (0.50 +/- 0.12 vs. 1.39 +/- 0.33 ng/ml/h; p < 0.02), and plasma aldosterone concentration tended to be lower (66 +/- 10 vs. 86 +/- 9 pg/ml; p = 0.09), while urine norepinephrine excretion did not significantly differ from controls (961 +/- 120 vs. 782 +/- 43 ng/h). ANP was higher in patients than in controls (92 +/- 17 vs. 48 +/- 9 pg/ml; p < 0.05). Natriuresis was directly correlated with ANP (r = 0.69, p < 0.005) and ANP/plasma aldosterone ratio (r = 0.63; p < 0.01) in patients and healthy subjects taken together.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronobiological Evaluation of Sympathoadrenergic Function in Cirrhosis

Intraday activity of the adrenergic system was investigated in 7 healthy controls and in cirrhotic patients without ascites (group 1, 7 cases) and with ascites (group 2, 9 cases) by determining the urinary norepinephrine and vanillylmandelic acid excretions at 4-h intervals for 24 h. Mean arterial pressure and heart rate were also recorded. In controls, the statistical evaluation by the cosinor method showed a circadian rhythm of such variables, with zenith in the morning and nadir at night. Intraday changes of urinary excretion of norepinephrine were closely related to arterial pressure and heart rate in most subjects. The most important change in cirrhotic patients was the achronia [no detection of a statistically significant (p greater than 0.05) rhythm] in urinary excretion of norepinephrine and arterial pressure. This occurred not only in group 2 patients, who had lower arterial pressure and higher NE mesors than controls (p less than 0.05), but also in group 1 patients, whose mesors were comparable to controls. The statistical significance of heart rate rhythmicity was preserved in patients, but its zenith was progressively displaced toward evening (group 1) and night hours (group 2, whose mesor was increased). Changes in urinary excretion of vanillylmandelic acid roughly paralleled those of norepinephrine both in controls and patients, but they did not significantly increase even in the group with ascites. In both groups of cirrhotic patients, the correlation between urinary excretion of norepinephrine, arterial pressure, and heart rate within the same subject was lost in most cases. This chronobiological study showed that the sympathoadrenergic activity can be deranged also in the early stages of cirrhosis, and suggested that an altered control of cardiovascular homeostasis is present even under steady state conditions. This alteration might blunt adrenergic responses to stress conditions.

Bioavailability of a new ketoprofen formulation for once-daily oral administration

A new sustained-release formulation (sustained release Ibifen) that gradually releases ketoprofen within 24 h and ensures therapeutic plasma concentration for the entire period has been developed. It consists of tableted pH-dependent barrier film-coated ketoprofen granules and was administered at a single dose of 200 mg to 12 volunteers. Ketoprofen plasma profiles were compared with: (1) administration of Orudis retard 200 capsule (200 mg); (2) two 12-h doses of prompt release Ibifen capsules (100 mg). In vitro dissolution kinetics and ketoprofen plasma levels were measured by HPLC. Sustained release Ibifen dissolution rate was constant for 10 h, whereas Orudis retard 200 dissolution profile presented one higher slope (0-6 h) and a lower one (6-12 h). Both formulations showed a delayed kinetics with respect to prompt release Ibifen. After sustained release Ibifen administration, ketoprofen plasma peak, reached within 2 h, remained practically constant for at least 12 h (average 4 microg/ml), which is higher than therapeutic levels. Differently, Orudis retard 200 produced a delayed, higher C(max) (5.91+/-0.66 vs. 4.51+/-0.65 microg/ml; P<0.01) and disappeared more quickly. In conclusion, sustained release Ibifen can ensure therapeutic ketoprofen plasma levels for the entire 24 h period, avoiding plasma concentration spikes, with bioavailability similar to other ketoprofen preparations.