Relative quantification of plasma N-glycans in type II congenital disorder of glycosylation patients by mass spectrometry.

BACKGROUND: Type II Congenital Disorders of Glycosylation (CDG-II) are a group of diseases with challenging diagnostics characterized by defects in the processing of glycans in the Golgi apparatus. Mass Spectrometry (MS) has been a valuable tool in the definition of CDG-II subtypes. While some CDG-II subtypes are associated with specific N-glycan structures, others only produce changes in relative levels, reinforcing the demand for quantification methods. METHODS: Plasma samples from control individuals were pooled, derivatized with deuterated iodomethane (I-CD3), and used as internal standards for controls and patients whose glycans were derivatized with iodomethane (I-CH3), followed by MALDI MS, LC-MS and -MS/MS analyses. RESULTS: Total N-glycans from fifteen CDG-II patients were evaluated, and 4 cases with molecular diagnosis were considered in detail: 2ATP6V0A2-CDG siblings, and 2 MAN1B1-CDG patients, one of them carrying a previously undescribed p.Gly536Val mutation. CONCLUSIONS: Our methodology offers a feasible alternative to the current methods for CDG-II diagnosis by MS, which quantify glycan structures as fractions of the total summed signal across a mass spectrum, a strategy that lowers the variability of minor components. Moreover, given its sensitivity for less concentrated yet biologically relevant structures, it might assist the uncovering of novel diagnostic glycans in other CDG-II subtypes.

Action and localization of vasoactive intestinal peptide in the coronary circulation: evidence for nonadrenergic, noncholinergic coronary regulation.

Vasoactive intestinal polypeptide, a neurotransmitter peptide detected in animal and human hearts, has been found in nerves of coronary arteries. To determine the amount and distribution of vasoactive intestinal polypeptide in the large coronary vessels and its possible participation in coronary vasoregulation, two groups of animals were studied. In the first group, 11 anesthetized dogs were sacrificed to collect three (1 cm) segments along the circumflex and left anterior descending coronary arteries. These segments represented proximal (I), middle (II) and distal (III) portions of the two arteries. Concentrations (ng/g) of vasoactive intestinal polypeptide-like immunoreactive substance were determined by radioimmunoassay. Vasoactive intestinal polypeptide-like immunoreactivity was present in the left anterior descending (I = 7.28 +/- 1.65, II = 3.74 +/- 0.57, III = 2.29 +/- 0.53) and circumflex (I = 4.16 +/- 1.52, II = 4.58 +/- 1.13, III = 4.00 +/- 0.81) coronary arteries. The difference in vasoactive intestinal polypeptide-like immunoreactivity among epicardial segments of the anterior descending artery was significant, but there was no significant difference among segments of the circumflex coronary artery. In the second group (eight closed chest anesthetized dogs), the effects of vasoactive intestinal polypeptide intracoronary infusion on epicardial coronary constriction were examined at rest and with the artery constricted by serotonin. Left anterior descending (segments I, II and III) artery responses (% area change) to vasoactive intestinal polypeptide and vasoactive intestinal polypeptide plus serotonin were examined using quantitative coronary angiography. Vasoactive intestinal polypeptide infusion resulted in significant vasodilation in all the segments (I, II and III) of the left anterior descending artery.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple technique for producing supravalvular aortic stenosis in animals.

A safe and reproducible technique to create supravalvular aortic stenosis was developed, which avoids many of the difficulties encountered in the production of aortic stenosis. Dogs were anaesthetised and artificially ventilated. The chest was opened and the venae cavae were encircled with umbilical tapes. The ascending aorta was then encircled by a 1.5-2 cm wide, 6-7 cm long dacron patch, venous return was stopped by tightening the tapes, and a J-shaped clamp applied to the ascending aorta at the dacron patch. Two layers of continuous mattress suture were placed adjacent to the clamp, plicating the aortic diameter by about 50%. After releasing the clamp and restoring normal venous return, left ventricular (LV) and aortic (AO) pressures were measured. Subsequently, one or two deep mattress sutures were placed below the running mattress sutures to increase the stenosis and to obtain the desired gradient. The LV-AO systolic pressure gradients obtained immediately after the operation ranged from 40 to 75 mm Hg. Two to 6 months after the operation the pressure gradients ranged from 50 to 200 mm Hg. Left ventricular to body weight ratios were 6.41 (SEM 0.26) v 4.24(0.20) for the controls. Heart weight to body weight ratios were 8.37(0.35) v 5.65(0.33). LV end diastolic pressures were normal. This technique can be used either in puppies or adult animals. The problem of aortic rupture is eliminated. The pressure gradient can be easily controlled during the operation and reproducible LV hypertrophy can be obtained in a shorter time than with aortic banding of puppies.

Neurovascular mechanisms and sodium balance in the pathogenesis of hypertension.

Physiological studies have clarified the role that the brain has in the interplay between salt balance and hypertension. Neural mechanisms and endocrine secretions play a pivotal role in the adaptation of mammals to changes in the intake and excretion of sodium. Maneuvers that alter the concentration of sodium in the plasma modify the sensitivity of baroreceptor reflexes and alter vascular reactivity. These changes may be mediated in part by the release of vasopressin. The research also suggests that the brain indirectly modulates the ability of the vascular endothelium to release vasoactive factors. Collectively, these studies illustrate the multiple effects of the sodium ion on the peripheral neural and central endocrine mechanisms that participate in the regulation of arterial pressure.

Attenuated vascular reactivity in dogs with anteroventral third ventricle lesions.

Lesion of the anteroventral portion of the third cerebral ventricle causes hypernatremia, adipsia, and attenuation of the pressor response to intravenous administration of angiotensin II and norepinephrine. In addition, these lesions prevent the development of several experimental models of hypertension. In this study, a lesion of the third cerebral ventricle region was made in 14 dogs. In seven dogs in which hypernatremia developed the lesions included the organum vasculosum of the lamina terminalis; seven animals in which the circumventricular organ was spared by the lesion remained normonatremic. Vascular responsiveness of isolated right carotid artery rings to angiotensin II and phenylephrine was assessed 3 days after lesioning the anteroventral portion of the third cerebral ventricle. In endothelium-denuded ring vessels, vasoconstrictor responses to phenylephrine were significantly decreased in animals both with and without inclusion of the organum vasculosum of the lamina terminalis. A similar effect was observed in intact vessels of dogs in which the circumventricular organ was spared but not in those with lesions that included this area. In contrast, angiotensin II-induced vasoconstriction was significantly decreased in the arteries with intact endothelium of both groups of lesioned animals. These data show that lesion of the anteroventral third ventricle area alters alpha 1-adrenergic and angiotensin II vascular responsiveness in isolated carotid artery rings with the possible participation of the endothelium.

The renin-angiotensin system during acute myocardial ischemia in dogs.

We used the technique of high-performance liquid chromatography combined with radioimmunoassay to establish the profile of angiotensin peptides in the periphery and across the circulation of the dog's heart. Data were obtained before and after blockade of angiotensin converting enzyme, and after acute myocardial ischemia produced by occlusion of the left anterior descending coronary artery. Baseline values of plasma renin activity and immunoreactive angiotensin II were higher in the aortic root than in the coronary sinus but concentrations of angiotensin I and angiotensin-(1-7) were similar. In untreated animals, coronary occlusion produced significant increases in renin activity and arterial and venous levels of angiotensin I and angiotensin II. Inhibition of converting enzyme with benazeprilat (CGS-14,831) increased baseline circulating levels of angiotensin I, whereas angiotensin II and its carboxyl terminal fragments were reduced markedly. Baseline plasma levels of angiotensin-(1-7) and its fragments did not change. Myocardial ischemia in benazeprilat-treated dogs increased plasma renin activity and circulating levels of angiotensin I. Concentrations of angiotensin II and angiotensin-(1-7) did not change either in peripheral blood or across the coronary circulation. These results indicate that angiotensin peptides can be formed endogenously by enzymatic pathways alternate to converting enzyme. Furthermore, these data provide the basis for a further understanding of the role of the renin-angiotensin system after myocardial ischemia.

Exercise training and responsiveness of isolated coronary arteries.

Exercise is associated with release of catecholamines and vasoactive intestinal polypeptides. Recurrent exposure to catecholamines modifies the sensitivity of adrenoceptors. To test the hypothesis that exercise training may affect the sensitivity of the epicardial coronary arteries, we performed studies on isolated coronary arteries from male dogs capable of running on a treadmill. The animals were separated randomly into two groups: sedentary and exercise training. After 11 wk, rings of left circumflex and left anterior descending coronary arteries were studied in vitro. Contractions to alpha 1-adrenergic agonists (norepinephrine and phenylephrine) were not affected by exercise training. During contractions with prostaglandin F2 alpha, endothelium-dependent relaxations to alpha 2-adrenergic agonists (norepinephrine and UK 14304) were not reduced significantly by exercise training. The concentration-relaxation curves to beta-adrenergic agonists (norepinephrine, isoproterenol, and epinephrine) were shifted to the right after training. The concentration-response curves to vasoactive intestinal polypeptide, but not that to substance P, were shifted to the right in rings with endothelium from exercise-trained animals. These findings demonstrate a decrease in responsiveness of canine vascular smooth muscle to beta-adrenergic agonists and to vasoactive intestinal polypeptide after exercise training.

Substance P distribution and effects in the canine epicardial coronary arteries.

Substance P (SP), a vasoactive neuropeptide detected in animal and human hearts has been reported to increase coronary blood flow in animals. However, no data are available on SP effects on epicardial coronary arteries, the site of coronary disease. To determine the amount and distribution of SP and its action in the large coronary vessels, we studied two groups of dogs. One group was anesthetized for collecting three 1 cm segments of the circumflex coronary artery (CX) and left anterior descending artery (LAD) through a left thoracotomy. These segments represented proximal (I), middle (II), and distal (III) portions of the two arteries. Concentrations (ng/g) of SP-like immunoreactivity (SP-LI) were determined by radioimmunoassay. SP-LI was present in LAD (I: 1.17 +/- 0.20, II: 1.08 +/- 0.36, III: 1.14 +/- 0.25) and CX (I: 1.44 +/- 0.38, II: 1.51 +/- 0.47, III: 0.70 +/- 0.20). SP differences among segments of LAD and segments I and II of CX were not significant, but there was a significant difference between segment III of CX and the others. In the second group of closed chest anesthetized dogs, we examined the effects of intracoronary SP infusion before and during administration of serotonin (5HT). LAD and CX artery responses (% area change) to SP and to SP plus 5HT were examined using quantitative coronary angiography. Intracoronary 133Xe in saline provided coronary flow data. SP infusion produced significant vasodilation in segment II (15% area increase) and III (17%) during the highest dose (1 microgram/min). The three SP doses infused with 5HT (0.05 mg/min) did not produce vasodilation, although LAD segment III constriction from 5HT was abolished during the highest dose of SP infusion. The presence of SP, and its dilatory effect on the coronary arteries, suggests a role in maintaining vasodilator tone in the coronary arteries.

Vascular anastomosis by argon laser beam.

Twenty-four mongrel dogs, weighing 13 to 24 kg, were used to study the effectiveness of anastomosis by Argon Laser beam. After anesthesia, intubation and controlled ventilation, they were submitted to three types of vascular anastomoses: saphenous vein intercarotid artery bypass; left mammary artery/left anterior descending coronary artery bypass; and veno-venous anastomosis. In all groups, 0.8 to 1.5 watts of Argon Laser power were applied for a total time of 90 to 300 seconds. The lower power was for veno-venous anastomoses and the greater was applied for arterial anastomoses. The mean values of resistance of the laser anastomosis to pressure-induced repture were 730 mm Hg in the immediate postoperative study, but increased to 2,500 mm Hg 30 days after surgery. No signs of occlusion were demonstrated at the anastomotic sites by the angiographic and anatomopathological studies performed.

Heart rate, heart rate variability, and blood pressure during perioperative stressor events in abdominal surgery.

STUDY OBJECTIVE: To define the behavior of power spectral heart rate variability (PSHR) during potentially stressful events in the perioperative period, and relate it to changes in blood pressure (BP) and heart rate (HR). DESIGN: Longitudinal clinical study. SETTING: Operating room and recovery suites of a large tertiary care referral center. PATIENTS: 26 ASA physical status I, II, and III patients undergoing elective abdominal surgery. INTERVENTIONS: Anesthesia was induced with thiopental sodium and fentanyl, and maintained with isoflurane/nitrous oxide (N2O)/relaxant or enflurane/N2O/relaxant. The trachea was intubated and intraabdominal surgery was performed. MEASUREMENTS AND MAIN RESULTS: Observations consisted of HR, noninvasive blood pressure, and PSHR. They were made before and after induction of anesthesia, tracheal intubation, and surgical incision, and during maximal surgical stimulation and skin closure. HR and mean arterial pressure (MAP) maxima were also recorded for one hour before and after emergence from anesthesia. PSHR was obtained using a special algorithm and data acquisition system for real time spectral analysis of the instantaneous HRversus time function. The HR power spectrum parameters analyzed were low-frequency (LFA; powerband = 0.04 to 0.10 Hz), respiratory-induced frequency (RFA; powerband = respiratory frequency +/- 0.06 Hz), and the ratio of LFA to RFA. With induction of anesthesia, only RFA power decreased significantly. LFA power reduction became significant only after intubation and continued so until after incision. Immediately after induction, the decline in RFA power (vs. preinduction) was more pronounced when compared with the decline in LFA power (76% vs. 34%; p = 0.01). Hence, the ratio LFA/RFA increased significantly after induction of anesthesia. It was significantly higher than at postintubation, preincision, or skin closure. A significant elevation in LFA, LFA/RFA ratio, and BP occurred with maximal abdominal surgical stimulation. Only preinduction LFA, RFA, and LFA/ RFA ratio were predictive of MAP changes with induction of anesthesia (p = 0.006). In 8 of the 15 patients who had MAP changes of at least 10 mmHg with induction, PSHR indices correctly predicted a change of this magnitude. Late intraoperative HR maxima were positively correlated with the change in HR and incision (r2 = 0.58; p < 0.01). The change in BP with incision was positively correlated with early postoperative HR maxima (r2 = 0.60; p < 0.01). CONCLUSIONS: On anesthetic induction, preoperative, but not intraoperative, spectral indices were predictive of BP changes. Power spectral analysis of HR may provide information about the autonomic state that is not evident from BP or HR. The HR power spectrum, in particular, indicated a striking autonomic imbalance immediately after the induction of anesthesia despite stable HR and BP. LFA and LFA/RFA ratio appeared to track sympathetic autonomic activation during abdominal surgical stimulation, but not during other perioperative stressor events.

Energy production, O2 consumption, and blood flow reserve in experimental aortic valve disease.

Left ventricular energy production and its relation to myocardial O2 consumption and blood flow reserve were studied in 11 dogs with surgically produced valvular aortic stenosis (AS, 6 of 11) or combined stenosis and insufficiency (AS + AI, 5 of 11), and 7 dogs undergoing sham operation (S). Two months after operation the combined AS + AI group had the highest left ventricular mass (S, 4.85 +/- 0.53; AS, 6.38 +/- 0.90; AS + AI, 7.23 +/- 0.39 g/kg), left ventricular end-diastolic pressure (S, 2.1 +/- 1.6; AS, 6.3 +/- 1.7; AS + AI, 8.6 +/- 3.1 mmHg), left ventricular end-diastolic volume (S, 61.1 +/- 8.5; AS, 73.0 +/- 7.8; AS + AI, 95.8 +/- 20.9 ml), and stroke work. At rest, total left ventricular myocardial blood flow was increased in AS and AS + AI compared with sham (S, 89 +/- 8; AS, 135 +/- 19; AS + AI, 164 +/- 9 ml/min, P less than 0.05); and coronary resistance was lower in both AS and AS + AI groups. Peak-to-resting flow ratio determined by adenosine vasodilation was reduced in AS and AS + AI despite normal resting function (peak-to-resting flow ratio: S, 6.78 +/- 2.24; AS, 3.19 +/- 0.58, AS + AI, 3.99 +/- 0.84, P less than 0.02). Peak-to-resting flow ratio was inversely proportional to left ventricular mass. In turn the degree of hypertrophy correlated with the total power requirement of the left ventricle, regardless of the type of overload lesion.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasodilation and mechanism of action of propofol in porcine coronary artery.

BACKGROUND: A decrease in myocardial perfusion pressure may reduce myocardial blood flow. However, it may not significantly affect myocardial perfusion when in presence of a concurrent coronary artery vasodilation. However, the effects of propofol in coronary arteries are not well determined. In this study, the effects of propofol on porcine coronary artery responses to vasoactive agents that operate through voltage- and receptor-mediated calcium mechanisms were investigated. METHODS: Hearts of adult pigs (n = 103) were obtained from a slaughter house, and the left anterior descending coronary arteries were dissected. The arteries were cut into vessel rings and prepared with and without the endothelium organ chambers filled with buffered salt solution. The effect of propofol (10(-7), 10(-6), 10(-5), and 10(-4) M) on vascular smooth muscle contraction caused by intracellular Ca(2+)-influx through voltage- and receptor-mediated mechanism also was studied at a cellular level. RESULTS: Propofol relaxed coronary rings that were contracted by KCl, norepinephrine (NE), serotonin (5-HT), or carbachol (CCh). The minimal concentrations of propofol that produced significant vasorelaxation ranged from 3.16 x 10(-7) M to 3.16 x 10(-6) M. Vasodilation was more pronounced in rings contracted by NE, 5-HT, and CCh than by KCl. Propofol (10(-5) M) attenuated coronary vasoconstriction in response to cumulative concentrations of KCl, NE, 5-HT, and acetylcholine. Maximal contractions produced by NE and 5-HT were inhibited to a greater degree than contractions produced by KCl. Propofol at concentrations of 10(-5) M and higher attenuated a contraction in response to CaCl2 in vascular rings depolarized by KCl, but concentrations of 10-M did not attenuate contractions. Vasoconstriction in response to calcium entry in the presence of NE (and nifedipine 10(-6) M) was attenuated by propofol at concentrations of 10(-6) M and higher. Caffeine-induced contraction, caused by intracellular calcium release, was attenuated only at 10(-4) M of propofol. CONCLUSIONS: Propofol possesses vasodilator effect and attenuates the effects of vasoconstrictor agents in porcine coronary artery. Further, an antagonism of calcium channels may be responsible for these effects of propofol.

Increased vasoconstrictor activity of proximal coronary arteries with endothelial damage in intact dogs.

In this study we examined the hypothesis that endothelial damage increases proximal coronary arterial vasomotor tone and sensitivity to vasoconstrictor stimulation. The response of the left anterior descending coronary artery (LAD) (% area change) to serotonin and nitroglycerin were examined in eight anesthetized (Innovar + nitrous oxide), closed-chest dogs by means of quantitative coronary angiography. Dose-response curves of percent change in arterial cross-sectional area for three doses of intracoronary serotonin were examined before and after endothelial damage produced by a balloon catheter in the LAD. Endothelial damage was verified by postmortem scanning electron microscopic examination. Intracoronary injection of 133Xe provided coronary flow data. The damaged segment of LAD showed spontaneous vasoconstriction and further constriction in response to serotonin (33 +/- 5% before and 52 +/- 6% area reduction after damage; p less than .05). Nitroglycerin reversed serotonin-induced vasoconstriction in LAD segments without damage but not in the LAD segment with endothelial damage. No significant changes were observed in aortic pressure, and heart rate was kept constant by pacing. Blood flow in the LAD was not affected by endothelial damage itself (control, 2.44 +/- 0.09 ml/min/g; damage, 2.53 +/- 0.22 ml/min/g). Endothelial damage induced spontaneous proximal coronary constriction and diminished the relaxant response to nitroglycerin in the presence of serotonin. These results suggest that focal coronary narrowing that occurs in some patients after provocation with vasoconstrictor agents may be caused by local areas of damaged endothelium.