Two reproducible and sensitive liquid chromatographic methods to quantify atenolol and propranolol in human plasma and determination of their associated analytical error functions

Two liquid chromatography (LC) methods with fluorimetric detection have been developed to measure atenolol and propranolol in human plasma. The same 5 μm Nucleosil RP-18 column, extraction procedure and mobile phase (containing acetonitrile, water, triethylamine and phosphoric acid, pH 3) were used. The linearity ranges were 25–800 ng/ml for atenolol and 3.13–100 ng/ml for propranolol. The coefficients of variation for validation assays were lower than 15% at the concentration assayed. The functions of the analytical error were linear: SD (ng/ml)=7.698+0.037C for atenolol and SD (ng/ml)=0.126+0.036C for propranolol.     

Adrenergic effects on plasma levels of glucagon, insulin, glucose and free fatty acids in rabbits

Adrenergic effects on plasma levels of glucagon, insulin, glucose and free fatty acids were studied in fasted rabbits by infusing epinephrine, norepinephrine, isoproterenol, phentolamine (an adrenergic alpha-receptor blocking drug) and propranolol (an adrenergic beta-receptor blocking drug). The adrenergic effects on the plasma levels of insulin, glucose and free fatty acids were similar to those found in other species. The plasma levels of insulin were increased by beta-receptor stimulation (isoproterenol, phentolamine + epinephrine) and decreased by alpha-receptor stimulation (epinephrine, norepinephrine, propranolol + epinephrine). The plasma levels of glucose were increased by both alpha- and beta-receptor stimulation, and the epinephrine-induced hyperglycaemia was only blocked by combined infusions with phentolamine and propranolol. The plasma levels of free fatty acids were increased by saline and further increased by beta-receptor stimulation (isoproterenol), while epinephrine and norepinephrine gave variable results. Alpha-receptor stimulation (propranolol + epinephrine) slightly decreased the plasma levels of free fatty acids. The plasma levels of glucagon, however, were mainly increased by alpha-receptor stimulation (epinephrine, norepinephrine, propranolol + epinephrine) and increased only to a minor extent by beta-receptor stimulation (isoproterenol, phentolamine + epinephrine) in rabbits. This is in contrast to results reported for humans, where beta-receptor stimulation seems to be most important in stimulating glucagon release.     

Immunoreactive somatostatin in the hypothalamus and other regions of the rat brain: effects of insulin, glucose, alpha- or beta-blocker and L-dopa.

Effects of various hormonal and pharmacological manipulations on somatostatin distribution were investigated to elucidate the physiological significance of somatostatin in the hypothalamus and the other regions of the rat brain. Immunoreactive somatostatin (IRS) was measured by radioimmunoassay newly developed. Insulin induced an increase of hypothalamic IRS and a decrease of plasma RGH, while glucose administration resulted in the opposite responses, which were not significant. Insulin also increased IRS in the thalamus and the brain stem. The insulin-induced increase of hypothalamic IRS was reduced by hyperglycemia. Glucagon reduced IRS initially and then increased it with an elevation plasma RGH. L-dopa did not affect hypothalamic IRS, although it decreased plasma RPRL. Phentolamine slightly increased plasma RGH and decreased IRS in most regions of the rat brain, while propranolol increased IRS in these regions. Pretreatment with propranolol significantly increased plasma RGH 120 min after insulin administration, and hypothalamic IRS decreased initially by pretreatment with propranolol, and then it increased significantly. When pretreated with propranolol, glucagon markedly increased plasma RGH and decreased IRS significantly. From these findings it is concluded that hypothalamic IRS may participate in the hormonal regulatory system in correlation to plasma RGH, as observed in studies on plasma GH and hypothalamic IRS following insulin, glucose, propranolol or phentolamine administration, but IRS in other regions of the brain may have some other actions as a neurotransmitter or a modulator, because of no significant correlation between plasma GH or PRL and IRS in these regions following various stimuli. In addition, glucose homeostasis and adrenergic mechanism may be important factors in regulating IRS in the rat brain.     

β-Blockers and Plasma Renin Activity in Hypertension

Long-term treatment with propranolol was shown to produce a sustained suppression of the renin-aldosterone system in hypertensive patients, despite concurrent diuretic treatment. However, the antihypertensive effect of this treatment correlated poorly with its effects on plasma renin activity and urinary aldosterone excretion. When prindolol, another β-adrenergic blocking drug, was substituted for propranolol, blood pressure control was retained, but there was a prompt rise in plasma renin activity, which was not attributable to changes in electrolyte balance. These observations suggest that the antihypertensive action of propranolol and other β-blockers does not result from their effects on plasma renin activity.     

Stereochemistry of tissue distribution of racemic propranolol in the dog

Only limited information is available on the stereochemistry of the in vivo distribution of beta-receptor-blocking drugs. In this study we determined the levels of the propranolol enantiomers in plasma, cerebrospinal fluid (CSF) and central nervous system (CNS), and peripheral tissues in the dog following an intravenous dose of a deuterium-labeled pseudoracemate. The appearance of the propranolol enantiomers in the CSF was rapid and nonstereoselective, with maximum concentrations reached at 15 min after dosing. The levels of the enantiomers in both CSF and plasma then declined in a parallel biphasic fashion, with a terminal t1/2 of about 125 min. Except for an early high CSF/plasma concentration ratio of 0.35, the CSF propranolol levels corresponded to the unbound concentration in plasma, CSF/plasma 0.20. All areas of the brain showed a similar uptake of propranolol, with a tissue concentration exceeding that in plasma about 10-fold during the terminal phase of elimination. The uptake of propranolol by peripheral tissues varied widely, ranging from a 50-fold accumulation by the lungs compared to plasma to no accumulation by adipose tissue. However, as for the CSF, there was no evidence of stereoselective uptake of propranolol by any CNS or peripheral tissue except for the liver. A significantly higher level of (+)-vs. (-)-propranolol in liver tissue presumably was a reflection of stereoselective hepatic metabolism of (-)-propranolol by this tissue. The slight stereoselectivity in plasma binding of propranolol known to exist in the dog had no significant influence on tissue or CSF distribution.     

Increased plasma protein binding of propranolol in rabbits with acute renal failure

Acute renal failure was produced in rabbits either by uranyl nitrate or by glycerol. Uremia ensued in all uranyl nitrate treated animals and in the majority of glycerol treated animals. In the uremic rabbits binding of phenytoin to plasma proteins decreased with respect to treated non-uremic animals and controls. On the other hand binding of propranolol markedly increased in uremia. The increased propranolol binding was not due to the elevated creatinine and urea levels in uremic plasma. Charcoal treatment of uremic plasma did not restore normal propranolol binding. The findings indicate that the influence of renal failure on binding of drugs to plasma proteins is more complex than hitherto assumed.     

Beta-adrenoreceptor blockade in the conscious rabbit: effects on plasma renin activity and blood pressure.

The administration of a single dose of dl-propranolol, 1 mg/kg i.v., in the conscious unstimulated rabbit produced effective beta-adrenoreceptor blockade (inhibition of isoprenaline tachycardia) for 150 min. During this period there was a positive correlation between plasma concentrations of propranolol and the degree of beta-blockade observed. In a further group of animals treated with propranolol, plasma renin activity (PRA) fell to 50% of control (P < 0.001) within 60 min, the rate of change of PRA also correlating with plasma propranolol levels. Similarly, there were reductions in mean blood pressure (P < 0.025) and heart rate (P < 0.025). Statistical relationships between the fall in blood pressure and either pre-treatment PRA or the change in PRA were consistent with the hypothesis that the hypotensive effect of propranolol was dependent upon its suppression of renin release. However, an alternative possibility that the fall in blood pressure was due to an acute reduction in cardiac output could not be excluded.     

High dose propranolol in the treatment of angina pectoris: relationship of dose to blood levels and hemodynamic consequences

Propranolol blood levels and the effect of these levels on hemodynamic parameters were evaluated in 25 patients with coronary artery disease undergoing cardiac catheterization and coronary angiography. Fifteen patients were receiving high doses of propranolol (320--1920 mg/day) while ten patients were receiving conventional doses (80--240 mg/day). The high dose propranolol group had significantly higher plasma propranolol levels than the conventional dose group (788 +/- 134 SD vs. 43 +/- 7.2 ng/ml SD), and there was a direct linear relationship between propranolol dose and plasma drug levels (r = 0.85, P < 0.001). There were no significant differences between high and conventional dose propranolol groups in terms of all hemodynamic parameters measured, namely ejection fraction, ventricular volume, cardiac index, or peripheral vascular resistance. Despite high drug dosage and blood levels, only mild side effects were seen.     

Stereoselective pharmacokinetics of oxprenolol,propranolol, and verapamil: Species differences and influence of endotoxin

The influence of endotoxin-induced inflammation was studied on the pharmacokinetics of the enantiomers of the racemic drugs oxprenolol, propranolol, and verapamil in rabbits and dogs. Enantioselective pharmacokinetics were seen for oxprenolol and propranolol in the rabbit and for propranolol and verapamil in the dog. In the dog, the enantioselective differences in plasma concentrations are due to differences in both protein binding and metabolism, whereas in the rabbit the differences are due solely to differences in metabolism. In both species endotoxin treatment increases the plasma concentrations of the enantiomers of the three drugs; both protein binding and metabolism are influenced. In rabbits and in dogs, the influence of endotoxin on the disposition of the three drugs is less enantioselective than was previously observed in the rat. © 1995 Wiley-Liss, Inc.     

Fluorimetrische bestimmung von furosemid und 4-chloro-5-sulfamoylanthranilsäure in plasma durch direkte auswertung von dünnschichtchromatogrammen

Fluorometric determination of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine by direct measurement of thin-layer chromatographic platesThe quantitative analysis of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine is described. The drugs are extracted into 2-pentanol-heptane, and the solvent is concentrated. The whole residue is chromatographed on silica gel plates. The compounds are determined directly on the thin-layer plates without derivatization. The recovery of propranolol from plasma is 70%, with a standard deviation of ± 4%.     

Fluorimetrische bestimmung von propranolol und seines metaboliten n-desisopropylpropranolol in plasma und urin durch direkte auswertung von dünnschichtchromatogrammen

Fluorometric determination of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine by direct measurement of thin-layer chromatographic platesThe quantitative analysis of propranolol and its metabolite N-desisopropylpropranolol in plasma and urine is described. The drugs are extracted into 2-pentanol-heptane, and the solvent is concentrated. The whole residue is chromatographed on silica gel plates. The compounds are determined directly on the thin-layer plates without derivatization. The recovery of propranolol from plasma is 70%, with a standard deviation of ± 4%.     

Plasma Propranolol Levels Associated with Suppression of Ventricular Ectopic Beats

The plasma propranolol levels associated with the abolition of chronic, stable ventricular ectopic beats have been studied. Racemic propranolol (DL) suppressed the ectopic foci at plasma levels of 40-85 ng/ml in eight patients, but levels of 70-200 ng/ml were unsuccessful in four patients. Dextro-(D) propranolol levels of 180-310 ng/ml were ineffective in four patients who had previously responded at levels of 60-75 ng/ml of racemic propranolol. The significance of the sympathetic nervous system in the genesis of ectopic beats is discussed. It is concluded that propranolol is clinically effective by virtue of its beta-adrenergic blocking activity.     

High-pressure liquid chromatographic method for the simultaneous quantitative analysis of propranolol and 4-hydroxypropranolol in plasma

A simple and rapid high-pressure liquid chromatographic procedure is reported for the simultaneous quantitative determination of propranolol and 4-hydroxypropranolol in plasma. Following an extraction the samples are chromatographed on a reversed-phase column and the components in the column effluent are detected by fluorescence monitoring. Using 1-ml plasma samples propranolol and 4-hydroxypropranolo concentrations at least as low as 1 ng/ml and 5 ng/ml, respectively, can be quantitated. The reproducibility of the method is satisfactory and no interference from endogenous plasma components or other drugs has been observed. A single plasma sample can be analyzed in approximately 20 min.     

Sodium restriction amplifies and propranolol loading inhibits circadian rhythm of plasma renin-angiotensin and aldosterone

Five male and 5 female clinically healthy volunteers, 17-37 years of age, gave systemic venous blood at 0600, 0800, 1200, 1800, 2000 and 0000 for RIA of (supine values) plasma renin-angiotensin (PRA) and aldosterone (PA) under 4 conditions: a. unrestricted sodium intake, no treatment; b. unrestricted sodium intake and 40 mg propranolol per os every 6 h; c. sodium restriction, no treatment; d. propranolol loading on sodium deprivation. Cosinor methods were used for data analysis. Sodium restriction amplifies the circadian rhythms of PRA and PA, whereas propranolol loading inhibits these same rhythms on a unrestricted sodium intake, but much less so under conditions of sodium deprivation. The propranolol-induced inhibition of the circadian rhythms investigated on a unrestricted sodium intake suggests that the beta-adrenergic system is an effective mechanism coordinating the circadian rhythmic functions investigated. The persistence of the rhythms in sodium-depleted subjects under pharmacological blockade of beta-adrenoreceptors is in keeping with the concept that a second mechanism of the circadian rhythms examined is located in the sodium-sensitive macula densa cells of the renal distal tubule.     

Immediate plasma renin response to propranolol: differentiation between essential and renal hypertension.

The immediate short-term effect on plasma renin activity of intravenous injection of propranolol was studied in 31 normal subjects and 166 hypertensive patients. In patients with essential hypertension and normal subjects plasma renin activity fell considerably within 15 minutes; the fall was directly proportional to initial plasma renin levels. In contrast, in patients with renal hypertension the fall was much less pronounced or totally absent. These differences in response to propranolol provide, though presently only on a group basis, a biochemical means of differentiating between patients with renal hypertension and those with essential hypertension. The observations also indicate that, unlike normal subjects and patients with essential hypertension, in patients with renal hypertension sympathetic activity plays no part in the control of basal plasma renin levels.     

Plasma Propranolol Levels in the Quantitative Assessment of β-adrenergic Blockade in Man

Plasma propranolol levels associated with reductions in endogenous and exogenous cardiac β-stimulation were determined in normal people. The levels associated with a given degree of blockade of exercise-induced tachycardia were about three times greater after intravenous administration than after oral administration. This shows that an active metabolite of propranolol is formed only after the drug is taken by mouth. The greatest reduction in the tachycardia of strenuous exercise was associated with plasma levels of 40 ng./ml. with oral administration and 100 ng./ml. with intravenously administered propranolol.The effect on isoprenaline-induced tachycardia following intravenously administered propranolol showed that the dose ratio for isoprenaline was about 30 with plasma levels of 100 ng./ml. and 10 with levels of 10-20 ng./ml. These plasma levels give 100% and 20-30% blockade of exercise-induced tachycardia. These findings suggest that some of the therapeutic effects of propranolol may be unrelated to β-adrenergic blockade.     

Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

A stereoselective reversed-phase HPLC assay to quantify S-(−) and R-(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid–liquid extraction for sample clean-up and employed 2,3,4,6-tetra-O-acetyl-β-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C₁₈ column using a mixture of acetonitrile–water–phosphoric acid–triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at λ_ex=280 nm and λ_em=325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.     

Effect of propranolol on glucose disposal rate in patients with hyperthyroidism

The effect of propranolol treatment (60 mg per day, three days) on glucose disposal rate (K-value) was investigated in nine patients with hyperthyroidism. K-value was improved in 4 cases and aggravated in 5 cases. The fasting levels of plasma free fatty acids (FFA) before the administration of propranolol in the improved cases were significantly higher than those in the aggravated cases. The propranolol treatment markedly reduced FFA levels only in the improved cases. These results suggest that the impaired glucose tolerance frequently seen in hyperthyroidism patients could be partly attributed to the increased level of plasma FFA.     

Simultaneous determination of d- and l-propranolol in human plasma by high-performance liquid chromatography

A method for the determination of d- and l-propranolol in human plasma is described. The method involves extraction of propranolol from plasma, and the formation of diastereomeric derivatives with the chiral reagent N-trifluoroacetyl-1-prolylchloride. Separation and quantitation of the diastereomeric propranolol derivatives are carried out by a reversed-phase high-performance liquid-chromatographic system with fluorimetric detection. The reproducibility in the determination of d- and l-propranolol in human plasma was 4.5% (relative standard deviation) at drug levels of 10 ng/ml.In two subjects who received a single 40-mg tablet of racemic propranolol the plasma levels of the d-isomer were lower than of the l-propranolol. The half-lives of d- and l-propranolol were similar.     

Factors contributing to increased muscle fatigue with beta-blockers.

beta-Adrenoceptor blockers are widely used clinically and can be classified as nonselective (beta 1 and beta 2) or selective (beta 1). Impairment of exercise performance is a well-known side effect of this group of drugs. This paper reviews mechanisms that could potentially be responsible for this impairment. In addition to cardiovascular and metabolic effects, beta-blockade inhibits Na(+)-K+ ATPase pumps controlling ion movement between muscle and plasma and thus may contribute to muscle fatigue through this mechanism. To investigate the relationship between the change in plasma [K+] and exercise performance, we studied healthy male subjects taking propranolol. Eight subjects performed maximal incremental cycle ergometer exercise tests during control (no drug), low dose (LD) (40 mg daily), and high dose (HD) (265 +/- 4.3 (SE) mg daily) of propranolol. The control plasma [K+] (5.8 +/- 0.12 mequiv./L) during exercise was significantly lower than either the LD (6.4 +/- 0.05 mequiv./L) or HD (6.1 +/- 0.16 mequiv./L) values. There was no significant difference between plasma [K+] for the LD and HD of propranolol. However, maximum oxygen uptake was reduced only while taking the HD of propranolol. Six of the subjects also performed three 30-s bouts of high intensity exercise on an isokinetic cycle ergometer while taking the LD and HD of propranolol. There was no significant difference between doses for the increase in plasma [K+] (LD, 7.8 +/- 0.35 mequiv./L vs. HD, 7.6 +/- 0.36 mequiv./L) during exercise. However, exercise performance was significantly reduced during HD compared with LD. These results suggest that the increases in plasma [K+] with propranolol did not play a direct significant role in the reduced performance observed during the HD.