Sensitive enantioseparation and determination of isoprenaline in human plasma and pharmaceutical formulations.

A simple, sensitive and fast RPHPLC method was developed and validated for the enantioselective determination of (RS)-isoprenaline (Ipn) in human plasma. The enantiomers were converted to diastereomeric derivatives using s-triazine (cyanuric chloride) based chiral derivatizing reagents. l-isoleucine and l-methionine were introduced as chiral auxiliary in s-triazine and two new monochloro-s-triazine reagents were synthesized. These reagents were characterized and used for synthesis of diastereomeric derivatives of (RS)-Ipn spiked in human plasma. (RS)-Ipn was isolated (purified and characterized) from a commercial pharmaceutical formulation and was used as the standard racemic sample. Structures of the two diastereomeric derivatives were optimized for lowest energy using the Gaussian 09 Rev A. 02 program and hybrid density functional B3LYP with 6-31G* basis set which showed the spatial orientation of hydrophobic groups on stereogenic centers in the diastereomeric derivatives. The results were correlated with the mechanism of separation and elution order. Limit of detection values were found to be 24.6 and 26.8 ng mL-1 for the first and second eluting diastereomeric derivatives, respectively.

Hydrogen bonding recognition and colorimetric detection of isoprenaline using 2-amino-5-mercapto-1,3,4-thiadiazol functionalized gold nanoparticles.

In this paper, we describe a rapid, low-cost and highly sensitive colorimetric method for the detection of isoprenaline, based on 2-amino-5-mercapto-1,3,4-thiadiazol (AMTD) functionalized gold nanoparticles (AMTD-AuNPs) as a sensing element. Hydrogen bonding interaction between isoprenaline and AMTD resulted in the aggregation of AuNPs and a consequent color change of AuNPs from red to blue. The concentration of isoprenaline could be detected with the naked eye or a UV-visible spectrometer. Results showed that the absorbance ratio (A650/A524) was linear with isoprenaline concentrations in the range of 0.2 to 2.6µM (R=0.997). The detection limit of this method was 0.08µM. The proposed method is simple, without using complicated instruments and adding salts for enhancing sensitivity. This probe could be successfully applied to the determination of isoprenaline in human serum samples and urine samples after deproteinization.

Isoproterenol induced hypertrophy and associated signaling pathways are modulated by somatostatin in H9c2 cells.

BACKGROUND: Somatostatin (SST), a growth hormone inhibitory peptide plays key role in regulation of cell proliferation via modulation of mitogen activated protein kinases (MAPKs) and cell survival pathway. In cardiac physiology, ß-Adrenergic receptors (ß-ARs) play crucial role in regulation of downstream signaling pathways in receptor specific manner. The aim of the current study was to delineate the mechanistic insight for the role of SST on ß-AR mediated signaling which promotes hypertrophy and apoptosis in rat fetal cardiomyocytes (H9c2 cells). Accordingly, SST dependent changes in signaling molecules including second messenger cAMP, PKA/CREB as well as MAPKs including ERK and p38 which are key mediators of hypertrophy and apoptosis were analyzed. METHODS AND RESULTS: In the present study, we determined receptor specific effects on intracellular cAMP levels, signaling by western blot analysis and apoptosis by using JC-1 and Hoechst-33258 staining. Here, we present the data which indicates that SST inhibits isoproterenol induced hypertrophy and apoptosis in H9c2 cells. Importantly, SST inhibits ß-ARs agonist induced cAMP activation and SST mediated inhibition of cAMP was enhanced in presence of ß-ARs antagonist. SST enhances ß2AR agonist formoterol mediated effects on PKA, CREB and ERK1/2 phosphorylations whereas it inhibits isoproterenol mediated ERK1/2 and p38 signaling in concentration dependent manner. CONCLUSIONS: Taken together, these results presented here provide a novel insight for the potential role of SST in regulation of ß-AR mediated effects on hypertrophy and modulation of hypertrophy promoting signaling in H9c2 cells.

Influence of plasma protein binding on pharmacodynamics: Estimation of in vivo receptor affinities of beta blockers using a new mechanism-based PK-PD modelling approach.

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B,vivo)). These values were compared with in vitro affinities (K(B,vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B,vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B,vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B,vivo)-K(B,vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B,vivo)/K(B,vitro) approximately 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics.

Interaction study of bioactive molecules with fibrinogen and human platelets determined by 1H NMR relaxation experiments.

In order to investigate the interaction processes between bioactive molecules and macromolecular receptors NMR methodology based on the analysis of selective and non-selective spin-lattice relaxation rate enhancements of ligand protons was used. The contribution from the bound ligand fraction to the observed relaxation rate in relation to macromolecular target concentration allowed the calculation of the normalized affinity index[A(I)(N)](L)(T) in which the effects of motional anisotropies and different proton densities have been removed. In this paper, we applied this methodology to investigate the affinity of epinephrine and isoproterenol towards two different systems: fibrinogen and platelets.

Primary sclerosing cholangitis in childhood is associated with abnormalities in cystic fibrosis-mediated chloride channel function.

OBJECTIVE: To determine whether primary sclerosing cholangitis (PSC) in childhood is associated with abnormalities in cystic fibrosis transmembrane conductance regulator (CFTR). STUDY DESIGN: Subjects with PSC diagnosed in childhood (n = 20) were recruited from Children's Hospital. Subjects had testing with sweat chloride concentration, nasal transmembrane potential difference, and extensive genetic analysis of the CFTR gene. Disease control subjects consisted of 14 patients with inflammatory bowel disease alone and no liver disease. t tests were performed to determine statistical significance. RESULTS: In the PSC group, CFTR chloride channel function (deltaChloride free + isoproterenol) was markedly diminished at -8.6 +/- 8.2 mV (reference range: -24.6 +/- 10.4 mV). In contrast, disease control subjects had normal function, at -17.8 +/- 9.7 mV (P = .008). Sweat chloride concentration in subjects with PSC was greater than in disease control subjects (20.8 +/- 3.4 mmol/L vs 12.0 +/- 1.6 mmol/L, P = .045). Comprehensive CFTR genotyping revealed that 5 of 19 (26.3%) subjects with PSC had a CFTR mutation or variant, compared with 6 of 14 (42.9%) disease control subjects. CONCLUSIONS: There is a high prevalence of CFTR-mediated ion transport dysfunction in subjects with childhood PSC.

beta-Adrenoceptor-mediated thermogenesis and lipolysis in patients with chronic obstructive pulmonary disease.

The present study investigated whether development or maintenance of a relatively increased fat mass in normal-weight patients with chronic obstructive pulmonary disease (COPD), despite periods of weight loss, may be related to impaired beta-adrenoceptor-mediated responses in lipid utilization and thermogenesis. Nine COPD patients and nine healthy controls (body mass index: 23.0 +/- 1.3 vs. 23.8 +/- 0.6 kg/m2, not significant; fat mass: 19.0 +/- 2.1 vs. 11.9 +/- 1.5 kg, P < 0.01) received consecutive 30-min infusions of 6, 12, and 24 ng x kg fat free mass(-1) x min(-1) isoproterenol. During beta-adrenergic stimulation, nonesterified fatty acid levels increased significantly less in COPD patients (P < 0.001). Respiratory exchange ratio decreased similarly in both groups, indicating a similar change in the rate of lipid to carbohydrate oxidation. Energy expenditure increased similarly in both groups during beta-adrenergic stimulation. However, because plasma isoproterenol concentrations were significantly higher in COPD patients, thermogenesis related to isoproterenol concentration was significantly reduced in this group (P < 0.05). In conclusion, beta-adrenoceptor-mediated lipolysis and thermogenesis are impaired in COPD patients. This may play a role in the development or maintenance of their relatively increased fat mass.

Evaluating hemodynamic and T wave criteria of simulated intravascular test doses using bupivacaine or isoproterenol in anesthetized children.

UNLABELLED: An increase in T wave amplitude > or =25% is a reliable indicator for detecting intravascular injection of lidocaine-epinephrine test dose in anesthetized children. We examined whether a simulated IV test dose containing bupivacaine instead of lidocaine, and isoproterenol instead of epinephrine, produces reliable changes in heart rate (HR) and T wave morphology. One hundred healthy infants and children (6-72 mo) were randomized to one of five groups (n = 20 each) during 1.0 minimum alveolar anesthetic concentration sevoflurane and 67% nitrous oxide in oxygen: atropine pretreatment (0.01 mg/kg IV) followed by 0.25% bupivacaine containing epinephrine 0.5 microg/kg IV, atropine followed by normal saline, atropine followed by 1% lidocaine containing isoproterenol 0.1 microg/kg, saline pretreatment followed by the lidocaine-isoproterenol test dose, and saline followed by saline. HR was recorded every 20 s and T wave amplitude of lead II was continuously recorded. All patients receiving the bupivacaine-epinephrine test dose and none receiving saline met the HR (positive if > or =10 bpm increase) and T wave criteria (positive if > or =25% increase in amplitude). The isoproterenol-containing test dose produced positive responses based only on the HR criterion with or without atropine pretreatment. Our results indicate that HR and T wave changes are useful if a bupivacaine-epinephrine test dose is used and that HR is the only useful indicator if an isoproterenol-containing test dose is used in sevoflurane-anesthetized children. IMPLICATIONS: To determine if an epidurally administered local anesthetic has been unintentionally injected into a blood vessel, a small dose of epinephrine or isoproterenol may be added to a local anesthetic. We found that an increase in heart rate > or =10 bpm and an increase in T wave amplitude of lead II >or =25% are useful indicators for detecting accidental intravascular injection of an epinephrine-containing test dose in sevoflurane-anesthetized children, whereas only a heart rate change is a reliable diagnostic tool if an isoproterenol-containing test dose is used.

Catecholamine handling in the porcine heart: a microdialysis approach.

Experimental findings suggest a pronounced concentration gradient of norepinephrine (NE) between the intravascular and interstitial compartments of the heart, compatible with an active neuronal reuptake (U1) and/or an endothelial barrier. Using the microdialysis technique in eight anesthetized pigs, we investigated this NE gradient, both under baseline conditions and during increments in either systemic or myocardial interstitial fluid (MIF) NE concentration. At steady state, baseline MIF NE (0.9 +/- 0.1 nmol/l) was higher than arterial NE (0.3 +/- 0.1 nmol/l) but was not different from coronary venous NE (1.5 +/- 0.3 nmol/l). Local U1 inhibition raised MIF NE concentration to 6.5 +/- 0.9 nmol/l. During intravenous NE infusions (0.6 and 1.8 nmol. kg(-1). min(-1)), the fractional removal of NE by the myocardium was 79 +/- 4% to 69 +/- 3%, depending on the infusion rate. Despite this extensive removal, the quotient of changes in MIF and arterial concentration (DeltaMIF/DeltaA ratio) for NE were only 0.10 +/- 0.02 for the lower infusion rate and 0.11 +/- 0.01 for the higher infusion rate, whereas U1 blockade caused the DeltaMIF/DeltaA ratio to rise to 0.21 +/- 0.03 and 0.36 +/- 0.05, respectively. From the differences in DeltaMIF/DeltaA ratios with and without U1 inhibition, we calculated that 67 +/- 5% of MIF NE is removed by U1. Intracoronary infusion of tyramine (154 nmol. kg(-1). min(-1)) caused a 15-fold increase in MIF NE concentration. This pronounced increase was paralleled by a comparable increase of NE in the coronary vein. We conclude that U1 and extraneuronal uptake, and not an endothelial barrier, are the principal mechanisms underlying the concentration gradient of NE between the interstitial and intravascular compartments in the porcine heart.

Study of human serum albumin structure by dynamic light scattering: two types of reactions under different pH and interaction with physiologically active compounds.

The effect of pH and binding of ten physiologically active compounds (isoproterenol, yohimbine, propranolol, clonidine, phenylephrine, carbachol, tripeptide fMLP, diphenhydramine, chlorpromazine and atropine) on the molecular structure of human serum albumin (HSA) has been studied using the dynamic light scattering. It was found that albumin globule has the most compact configuration (Stokes diameter 59-62 A) at physiological pH 7.4. The changes in pH, both increase to 8.0 and decrease to 5.4, result in the growth of globule size to 72-81 A. At acidic shift of pH an additional peak arises in the correlation spectra caused by the light scattering on the structures with the Stokes diameters of 29-37 A. Those conform to the sizes of the albumin subdomains. The indicated peak is not displayed at basic shift of pH. The interaction with propranolol, clonidine, phenylephrine, carbachol and tripeptide fMLP which hinder adenylate cyclase (AdC) and activate Ca-polyphosphoinositide (Ca-PPI) signaling system of a cell initiates structural rearrangements similar to acidic transitions. Isoproterenol, yohimbine diphenhydramine, chlorpromazine and atropine, which activate AdC and hinder Ca-PPI, cause conformational changes of HSA similar to basic transitions.

Simultaneous determination of the elimination profiles of the individual enantiomers of racemic isoproterenol using capillary electrophoresis and microdialysis sampling.

Microdialysis sampling and capillary electrophoresis with electrochemical detection (CE-EC) were used in combination to simultaneously define the elimination profile of each enantiomer of isoproterenol (ISP) administered as a racemic mixture to Sprague-Dawley rats. Resolution of the enantiomers of ISP was accomplished using a running buffer containing methyl-O-beta-cyclodextrin as a chiral recognition reagent. The CE-EC system provided a concentration limit of detection of 0.63 ng ml-1, allowing monitoring of the elimination of ISP for up to six half-lives. Microdialysis sampling was capable of continuously monitoring the concentration of ISP with 60 s resolution. The concentration versus time data for the elimination of (+) and (-) ISP were fit to a biphasic first order elimination model yielding average apparent distribution half-lives of 0.52 +/- 0.07 min and 0.55 +/- 0.08 min and average apparent elimination half-lives of 9.8 +/- 2.2 and 8.8 +/- 2.0 min for (-) and (+) ISP, respectively (n = 3 rats). No statistically significant difference in the average half-lives was found. However, because each enantiomer was simultaneously determined in each animal a paired two-sample t-Test could also be done. This statistical analysis demonstrated that a difference in the elimination half-lives of the enantiomers of ISP does exist.

Antibronchospasmic, tachycardiac, and hypokalaemic effects of L-isoproterenol in guinea-pigs.

The relationship between antibronchospasmic, tachycardiac, or hypokalaemic effects and plasma concentration of L-isoproterenol (ISP) hydrochloride was investigated in guinea-pigs in vivo. ISP was infused at the rate of 10, 30, 50, 100, and 300 ng kg-1 min-1. The antibronchospasmic effect was expressed as the attenuation of methacholine-induced bronchospasm. The EC50 values of ISP for antibronchospasmic and tachycardiac effects were 5.12 nM and 3.95 nM, respectively. Although they were comparable to the values reported in vitro (7.23-0.358 nM, 1.77 nM), the concentration response relationship of ISP for antibronchospasmic effect was quite steep with a slope factor of more than six. Moreover, a decrease in plasma potassium level was not clearly detected. The experimental procedure in our present study was useful for evaluating antibronchospasmic and tachycardiac effects of beta-agonists.

Highly sensitive and specific HPLC with fluorometric detection for determination of plasma epinephrine and norepinephrine applied to kinetic studies in humans.

An HPLC separation method combined with fluorometric detection was extended to enable simultaneous assessment of plasma 3H-labeled and endogenous epinephrine (E) and norepinephrine (NE). Forearm fractional extraction (FFE) of 3H-labeled E and NE and of endogenous E was measured in 40 healthy volunteers who were receiving a continuous infusion of 3H-labeled E and NE. Concentrations of arterial and venous E were 26.8 +/- 1.95 (mean +/- SE) and 6.8 +/- 0.75 ng/L, respectively. Arterial and venous NE and dopamine (DA) were also measured, with respective values of 140.7 +/- 8.5 and 192.1 +/- 15.1 for NE, and 13.1 +/- 0.78 and 11.3 +/- 0.70 ng/L for DA. The FFE of 3H-labeled E was slightly but significantly higher (0.790 +/- 0.016) than the that of either 3H-labeled NE or endogenous E (0.748 +/- 0.0146 and 0.745 +/- 0.0185, respectively; P < 0.001), the correlations being highly significant (r = 0.80, P < 0.001) in both cases. The small difference between the FFE of E and of 3H-labeled E allows the calculation of the apparent spillover of E. However, this spillover was negligible compared with forearm NE spillover (0.0112 +/- 0.0031 vs 1.369 +/- 0.128 ng/L per minute. The high sensitivity of this measurement of venous E widens the possibilities for studying E kinetics under physiological conditions.

Reduced sensitivity to beta-adrenoceptor stimulation and blockade in insulin dependent diabetic patients with hypoglycaemia unawareness.

Nine IDDM-patients with hypoglycaemia unawareness, seven IDDM-patients with hypoglycemia awareness and a control group of nine healthy persons were included in this study. The patients were recruited from the medical out-patients' department of the University Hospital of Tromsø. The pathophysiological changes which cause hypoglycaemia unawareness are today not clear. Reduced peripheral tissue sensitivity to catecholamines is suggested as one of several mechanisms which may contribute. For further investigation of beta-adrenergic sensitivity an isoprenaline/metoprolol sensitivity test was performed. Isoprenaline and metoprolol were administered intravenously, and the effects on heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and plasma levels of adrenaline (ADR) and noradrenaline (NA) were measured. All subjects were given the same doses of isoprenaline (0.25-8 micrograms) and metoprolol (0.5-8 mg). Metoprolol was given together with the dose of isoprenaline which increased heart rate by 25 beats min-1. The dose/response curves of both isoprenaline/HR and metoprolol/HR were significantly shifted to the right in IDDM-patients with hypoglycaemia unawareness compared with controls and IDDM-patients with hypoglycaemia awareness (P < 0.05). Reduced sensitivity of isoprenaline stimulation has also been shown before, whereas reduced sensitivity of a blocking agent has not earlier been shown. These findings support the hypothesis of reduced beta-adrenergic sensitivity as one pathophysiological component in hypoglycaemia unawareness.

The effect of age on the beta-adrenergic lipolytic response in healthy humans.

We evaluated the effect of age on the lipolytic response to intravenous infusion of isoproterenol in 12 elderly (age, > 60 years) and 12 young (age, 21 to 34 years) volunteers to examine if there is alteration in innervated beta-adrenergic responsiveness in tissues other than the heart. Lipolysis was evaluated by measuring the plasma concentrations of free fatty acids and glycerol. We also measured the plasma concentration of isoproterenol during infusion so that we could calculate comparable plasma isoproterenol concentrations to lipolytic responses in the two age groups. Our data show that, at equivalent infusion rates of isoproterenol, the two age groups achieved equivalent isoproterenol concentrations. The elderly had a higher concentration of free fatty acids but equivalent concentrations of glycerol as compared with the young subjects at equivalent isoproterenol plasma concentrations. However, our data were complicated by the fact that at the higher infusion rates of isoproterenol, the elderly showed a greater sympathetic stimulation than the young subjects as measured by plasma norepinephrine concentrations. Nonetheless, our data could not show that the elderly subjects were more resistant to beta-adrenergic receptor-mediated stimulated lipolysis. Thus innervated beta-adrenergic receptor hyporesponsiveness caused by aging may not necessarily extend to all organ systems.

Regulation of beta-adrenoceptors in the guinea-pig sinoatrial node.

This study examined the changes of beta-adrenoceptors in the guinea-pig sinoatrial nodal region following 7 day (-)-isoprenaline (400 micrograms/kg/h s.c.) infusion and the relationship between beta-adrenoceptor desensitization and receptor down-regulation. Changes in beta 1- and beta 2-adrenoceptor density were measured using quantitative autoradiography and function in organ bath studies. (-)-Isoprenaline treatment produced a marked decrease in total (from 57.5 to 33.9 fmol/mg protein), beta 1- (from 49.4 to 32.8 fmol/mg protein), and beta 2-adrenoceptor density (from 8.1 to 1.05 fmol/mg protein) in the sinoatrial node. In adjacent right atrium, treatment produced no change in total (39.5 and 36.7 fmol/mg protein) or beta 1-adrenoceptors (35.9 and 36.4 fmol/mg protein) but did decrease beta 2-adrenoceptors (from 3.7 to 0.3 fmol/mg protein). Chronotropic effects were measured in spontaneously beating right atrium. Procaterol, a selective beta 2-adrenoceptor agonist, caused a biphasic chronotropic response in control right atria, the first part of which was abolished in the tissue from treated animals. The maximum increase in right atrial rate to RO363, a beta 1-adrenoceptor selective partial agonist, was reduced from 114 bpm in control to 43 bpm in treated animals. In electrically driven right atrium with the sinoatrial node removed procaterol failed to produce a positive inotropic response via beta 2-adrenoceptors, but the maximum response to RO363 was reduced from 0.75 g in the control tissue to 0.12 g in the treated tissue. This study showed that changes in beta 2-adrenoceptor density following 7 day (-)-isoprenaline infusion are compatible with reduced functional responsiveness in the SA node. The reduction of beta 1-adrenoceptor number in the SA node was also compatible with the reduced chronotropic response in this tissue. However the lack of effect on beta 1-adrenoceptor density in the right atrium was not consistent with the decrease in beta 1-adrenoceptor mediated inotropic response in this tissue. This suggests that beta-adrenoceptor desensitization is not always associated with receptor down-regulation but depends also on the changes in the cell signalling system beyond the level of the receptor which differ according to the cardiac location.

Sensitive determination of (-)-isoproterenol and (-)-(R)-1-(3,4- dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol (T-0509), a cardiotonic agent, in rat plasma utilizing a fully automated catecholamine analyser.

(-)-Isoproterenol (ISO), used as bronchodilator, and (-)-(R)-1-(3,4-dihydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (T-0509), a new cardiotonic agent, were determined in rat plasma (only 25 microL) using a fully automated catecholamine (CA) analyser that includes in-line pre-extraction of CAs, coupled with an HPLC-ethylenediamine condensation reaction and a peroxyoxalate chemiluminescence detection system (HPLC-ED-PO-CL). The chromatographic conditions were: precolumn, SERUMOUT-CEX; buffer for delivering CAs in the precolumn, 10 mM potassium phosphate buffer (pH 7.5)/ethanol (92 + 8, v/v) (1 mL/min); precolumn clean-up solution, 4% phosphoric acid/acetonitrile (50 + 50, v/v) (1 mL/min); analytical column, TSKgel ODS-80Ts, 250 x 4.6 mm i.d.; eluent, 75 mM potassium acetate buffer (pH 3.2)/50 mM potassium phosphate buffer (pH 3.2)/acetonitrile (83.6 + 4.4 + 12, v/v/v for ISO and 76 + 4 + 20 v/v/v for T-0509) (1 mL/min); fluorogenic reagent solution, 105 mM ED and 175 mM imidazole in acetonitrile/ethanol (90 + 10, v/v) (0.25 mL/min); chemiluminogenic reaction solution, 0.25 mM TDPO, 150 mM hydrogen peroxide and 110 mM TFA in dioxane/ethyl acetate (50 + 50, v/v) (1.4 mL/min). Colterol, (+/-)-1-(3,4-dihydroxyphenyl)-2-[(1,1-dimethyl)ethylamino]ethanol, (+/-)-N-t-butylnorepinephrine was used as an internal standard (IS) for ISO, and (+/-)-1-(3,4-dihydroxyphenyl)-2-[(3,4,5- trimethoxyphenethyl)amino]ethanol, (+/-)-(3,4,5-trimethoxyphenethyl- aminomethyl)-3,4-dihydroxybenzylalcohol (T-1583) for T-0509. The detection limits for ISO and T-0509 were 1.3 and 0.9 fmol on injection, respectively.

Determination of metanephrines in plasma by liquid chromatography with electrochemical detection.

Metanephrines are O-methylated metabolites of catecholamines. We report the use of liquid chromatography with electrochemical detection to determine plasma concentrations of normetanephrine (NMN) and metanephrine (MN). Plasma NMN and MN in 32 normal volunteers and inpatients were compared with concentrations in 23 patients with pheochromocytoma. Metanephrines were adsorbed from plasma onto a cation-exchange column and eluted with ammoniacal methanol. The dried residue was dissolved in mobile phase and injected onto a reversed-phase column. Recoveries of NMN and MN from 1 mL of plasma averaged 50-70%, and results varied linearly with quantity injected over a range of 0.13-55 pmol. The detection limit was 25 fmol for NMN and 50 fmol for MN. Intra-assay CVs were < 5%. In normal volunteers and inpatients, plasma concentrations of NMN ranged between 0.12 and 0.73 nmol/L (mean 0.38 nmol/L), and MN between 0.06 and 0.63 nmol/L (mean 0.19 nmol/L). Plasma NMN concentrations were increased in all 23 patients with pheochromocytoma (range 1-172 nmol/L), whereas MN concentrations (range 0.10-382 nmol/L) were increased in only 9 patients. The assay method is reliable and sensitive and offers an approach to examine the extraneuronal metabolism of catecholamines. The method may also be useful in the diagnosis of pheochromocytoma.

The pharmacokinetics of isoproterenol in critically ill pediatric patients.

The pharmacokinetics of isoproterenol (ISO) in infants and children have never been reported. The authors studied ISO pharmacokinetics in two disparate groups of pediatric intensive care unit patients: postoperative cardiac patients (POC, n = 10), and reactive airway disease patients (RAD, n = 9). In all, 44 blood samples were taken at steady-state from the 19 patients, whereas from 15 patients samples were also taken just before and after discontinuation of ISO infusion. There were 12 male and 7 female patients in the study, and their ages ranged from 2 days to 14 years. The average ISO dosing rate was 0.30 micrograms/kg/minute for the whole study population, ranging from 0.01 to 5.5 micrograms/kg/minute. The POC patients received a significantly lower dosing rate than the RAD patients (0.029 +/- 0.002 vs 0.50 +/- 0.21 micrograms/kg/minute, P < .0001); the average steady-state plasma concentrations of ISO were also lower in the POC patients (1.3 +/- 0.3 versus 13.9 +/- 4.9 ng/mL, P < .0001). The steady-state plasma concentration, normalized to a dosing rate of .05 micrograms/kg/minute, was 1.9 +/- 0.3 ng/mL for all patients, and the clearance was 42.5 +/- 5.0 mg/kg/minute. Postoperative cardiac patients had a significant higher normalized steady-state plasma concentration and moderately significant lower clearance than did RAD patients (2.1 +/- 0.3 versus 1.7 +/- 0.4 ng/mL, P < .05 and 33.2 +/- 4.9 versus 48.4 +/- 7.3, P < .06, respectively). The average plasma half-life of ISO was 4.2 +/- 1.5 minutes, and the volume of distribution was 216 +/- 57 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma clearances and extractions of four catecholamines in the anesthetized rabbit: the role of amine removal by blood cells.

The purpose of the present study was to compare the plasma kinetics of norepinephrine (NA), epinephrine (A), isoprenaline (ISO), and dopamine (DA) in the anesthetized rabbit. To this end, a mixture of trace amounts of 3H-labeled NA, A, ISO, and DA was infused either into a femoral vein or into the ascending aorta, and the plasma amine clearances (Cl), the fractional amine extractions across the pulmonary (ERp) as well as systemic (ERs) circulation, and the cardiac output of plasma (COp) were determined at steady state of the amine infusion. The values of ERp, ERs, and COp were also used to calculate total-body fractional extractions [ERtot = ERp + ERs(1 - ERp)] and theoretical clearances (Clcalc = ERtot * COp). The four catecholamines differed as to their values of Cl and ERs and even more so with respect to ERp. The Cl was lowest for NA, intermediate for A and ISO, and highest for DA. Statistically significant pulmonary extractions were observed for NA (8.8%) and DA (24.0%), but not for A and ISO. The systemic extraction was lowest for ISO (63%) and highest for DA (75%). Cl values were higher than values of Clcalc and the ratio of Cl/Clcalc increased in the order of NA < A < ISO < DA. In vitro experiments, in which whole rabbit blood was incubated in the presence of added NA, A, ISO, and DA, showed a pronounced ability of blood cells to remove catecholamines from plasma. The four amines rapidly disappeared from plasma at rates increasing in the order of NA < A < ISO < DA.(ABSTRACT TRUNCATED AT 250 WORDS)