Infection after prostatic transrectal fiducial marker implantation for image guided radiation therapy.

PURPOSE: The aim of this retrospective study is to assess the risk of infection after transrectal ultrasound-guided fiducial marker insertion for image-guided radiotherapy of prostate cancer. MATERIAL AND METHODS: Between January 2016 and December 2020, 829 patients scheduled for intensity-modulated radiotherapy for prostate cancer had an intraprostatic fiducial marker transrectal implantation under ultrasound guidance by radiation-oncologists specialized in brachytherapy. Patients received standard oral prophylactic antibiotic with quinolone. If Gram negative bacteria resistant to quinolone were detected at the time of the prostate cancer biopsies, the antibioprophylaxis regimen was modified accordingly. The resistance to quinolone screening test was not repeated before fiducial marker insertion. Infectious complications were assessed with questionnaires at the time of CT-planning and medical record reviewed. Toxicity was evaluated according to CTCAE v5.0. RESULTS: The median time between fiducial marker implantation and evaluation was 10 days (range: 0-165 days). Four patients (0.48%) developed urinary tract infection related to the procedure, mostly with Gram-negative bacteria resistant to quinolone (75%). Three had a grade 2 infection, and one patient experienced a grade 3 urosepsis. The quinolone-resistance status was known for two patients (one positive and one negative) and was unknown for the other two patients prior to fiducial marker implantation. CONCLUSION: Intraprostatic transrectal fiducial marker implantation for image-guided radiotherapy is well tolerated with a low rate of infection. With such a low rate of infection, there is no need to repeat the search of Gram-negative bacteria resistant to quinolone before fiducial marker implantation if it was done at the time of prostate biopsies. Optimal antibioprophylaxis should be adapted to the known status of Gram-negative bacteria resistant to quinolone.

T-tubule localization of the inward-rectifier K(+) channel in mouse ventricular myocytes: a role in K(+) accumulation.

1. The properties of the slow inward 'tail currents' (I(tail)) that followed depolarizing steps in voltage-clamped, isolated mouse ventricular myocytes were examined. Depolarizing steps that produced large outward K(+) currents in these myocytes were followed by a slowly decaying inward I(tail) on repolarization to the holding potential. These currents were produced only by depolarizations: inwardly rectifying K(+) currents, I(K1), produced by steps to potentials negative to the holding potential, were not followed by I(tail). 2. For depolarizations of equal duration, the magnitude of I(tail) increased as the magnitude of outward current at the end of the depolarizing step increased. The apparent reversal potential of I(tail) was dependent upon the duration of the depolarizing step, and the reversal potential shifted to more depolarized potentials as the duration of the depolarization was increased. 3. Removal of external Na(+) and Ca(2+) had no significant effect on the magnitude or time course of I(tail). BaCl(2) (0.25 mM), which had no effect on the magnitude of outward currents, abolished I(tail) and I(K1) simultaneously. 4. Accordingly, I(tail) in mouse ventricular myocytes probably results from K(+) accumulation in a restricted extracellular space such as the transverse tubule system (t-tubules). The efflux of K(+) into the t-tubules during outward currents produced by depolarization shifts the K(+) Nernst potential (E(K)) from its 'resting' value (close to -80 mV) to more depolarized potentials. This suggests that I(tail) is produced by I(K1) in the t-tubules and is inward because of the transiently elevated K(+) concentration and depolarized value of E(K) in the t-tubules. 5. Additional evidence for the localization of I(K1) channels in the t-tubules was provided by confocal microscopy using a specific antibody against Kir2.1 in mouse ventricular myocytes.

Vascular endothelial growth factor production by isolated rat hepatocytes after cold ischemia-warm reoxygenation.

Inflammatory disturbances in the liver microcirculation have been associated with preservation injury of hepatic grafts. Vascular endothelial growth factor (VEGF), a proinflammatory growth factor released by hepatocytes, acts on sinusoidal endothelial cells, but its implication in graft injury is still unclear. We studied VEGF production by rat hepatocytes after cold ischemia and warm reoxygenation and compared the capacity of University of Wisconsin (UW) and sodium-lactobionate-sucrose (SLS) preservation solutions to maintain this hepatocellular function. Isolated hepatocytes were kept for 0, 24, and 48 hours at 4 degrees C in either solution (cold ischemia), then incubated for 1 to 24 hours at 37 degrees C (warm reoxygenation). We assessed cell viability and production of VEGF messenger RNA (mRNA) and protein. Cell viability decreased in a linear time-dependent fashion by 10% after 48 hours of cold preservation and by an additional 40% after 24 hours of warm culture. Very little VEGF mRNA could be detected after up to 48 hours of simple cold preservation in either solution. However, subsequent warm culture led to a robust and rapid increase in VEGF mRNA expression within the first hour, which declined to close to background levels within 8 to 12 hours in culture. This effect was more important in cells preserved in SLS than UW solution. Similarly, cold preservation alone did not trigger VEGF secretion. VEGF secretion was detected after culturing hepatocytes at 37 degrees C and reached a maximal secretion rate within 12 to 15 hours. However, VEGF production by preserved cells was reduced compared with unstored cells. In conclusion, cold ischemia and warm reoxygenation triggers VEGF mRNA expression by hepatocytes, but subsequent VEGF secretion is partially impaired, suggesting posttranslational defects.

Gender-based differences in cardiac repolarization in mouse ventricle.

The mouse heart has become a widely used model for genetic studies of heart diseases. Thus, understanding gender differences in mouse cardiac repolarization is crucial to the interpretation of such studies. The objective of this study was to evaluate whether there are gender differences in cardiac repolarization in mouse ventricle and to gain insights into the ionic and molecular mechanisms underlying these differences. Action potential durations (APDs) and K(+) currents in male and female ventricular myocytes were compared using a patch-clamp technique. APD(20), APD(50), and APD(90) were found to be significantly longer in females than males. Examination of the different K(+) currents revealed that a significantly lower current density exists in female ventricular myocytes compared with male myocytes for the ultrarapid delayed rectifier K(+) current, I(Kur) (at +30 mV, male, 33.2+/-2.9 pA/pF [n= 22]; female, 20.9+/-1.73 pA/pF [n= 19], P<0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding for I(Kur)) in female compared with male ventricle. The additional K(+) currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression levels for the K(+) channels underlying these currents were detected between both sexes. This study demonstrates that adult mice exhibit gender differences in cardiac repolarization. The expression of Kv1.5 and of its corresponding K(+) current, I(Kur), is significantly lower in female mouse ventricle, and as a result, the APD is lengthened.

A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

Mathematical model of an adult human atrial cell: the role of K+ currents in repolarization.

We have developed a mathematical model of the human atria myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley-type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action potential data that are representative of recordings from a majority of human atrial cells in our laboratory and therefore provides a biophysically based account of the underlying ionic currents. This work is based in part on a previous model of the rabbit atrial myocyte published by our group and was motivated by differences in some of the repolarizing currents between human and rabbit atrium. We have therefore given particular attention to the sustained outward K+ current (I[sus]), which putatively has a prominent role in determining the duration of the human atrial action potential. Our results demonstrate that the action potential shape during the peak and plateau phases is determined primarily by transient outward K+ current, I(sus) and L-type Ca2+ current (I[Ca,L]) and that the role of I(sus) in the human atrial action potential can be modulated by the baseline sizes of I(Ca,L), I(sus) and the rapid delayed rectifier K+ current. As a result, our simulations suggest that the functional role of I(sus) can depend on the physiological/disease state of the cell.

A rapidly activating sustained K+ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle.

1. The K+ currents which control repolarization in adult mouse ventricle, and the effects of changes in action potential duration on excitation-contraction coupling in this tissue, have been studied with electrophysiological methods using single cell preparations and by recording mechanical parameters from an in vitro working heart preparation. 2. Under conditions where Ca(2+)-dependent currents were eliminated by buffering intracellular Ca2+ with EGTA, depolarizing voltage steps elicited two rapidly activating outward K+ currents: (i) a transient outward current, and (ii) a slowly inactivating or 'sustained' delayed rectifier. 3. These two currents were separated pharmacologically by the K+ channel blocker 4-amino-pyridine (4-AP). 4-AP at concentrations between 3 and 200 microM resulted in (i) a marked increase in action potential duration and a large decrease in the sustained K+ current at plateau potentials, as well as (ii) a significant increase in left ventricular systolic pressure in the working heart preparation. 4. The current-voltage (I-V) relation, kinetics, and block by low concentrations of 4-AP strongly suggest that the rapid delayed rectifier in adult mouse ventricles is the same K+ current (Kv1.5) that has been characterized in detail in human and canine atria. 5. These results show that the 4-AP-sensitive rapid delayed rectifier is a very important repolarizing current in mouse ventricle. The enhanced contractility produced by 4-AP (50 microM) in the working heart preparation demonstrates that modulation of the action potential duration, by blocking a K+ current, is a very significant inotropic variable.

Block of IKs by the diuretic agent indapamide modulates cardiac electrophysiological effects of the class III antiarrhythmic drug dl-sotalol.

Indapamide is a diuretic agent with direct electrophysiological effects on ionic currents involved in cardiac repolarization. In particular, indapamide blocks the slow component of delayed rectifier potassium current. In contrast, most class III antiarrhythmic agents, such as dl-sotalol, block the rapid component of delayed rectifier potassium current. Computer simulations have suggested potentiation of drug effects on cardiac repolarization by the combined block of the rapid component of delayed rectifier potassium current and the slow component of delayed rectifier potassium current. Therefore, the objective of our study was to evaluate the modulation of cardiac electrophysiological effects of dl-sotalol by indapamide. Two indices of cardiac repolarization, monophasic action potential duration at 90% repolarization and effective refractory period, at two basic cycle lengths (800 and 400 msec) were determined in 24 anesthetized open-chest dogs. In two treatment groups (n = 6/group), data were obtained at base line and every 2 min during steadily increasing concentrations of dl-sotalol (0-40 microg/ml) either alone or in the presence of indapamide (500 ng/ml). Data were also obtained in dogs receiving either a low-dose (500 ng/ml) or a high-dose (up to 7.5 microg/ml) infusion regimen of indapamide alone. Administration of dl-sotalol was associated with concentration-dependent increases in monophasic action potential duration at 90% repolarization and effective refractory period, whereas repolarization was only slightly altered by the administration of indapamide alone. However, concentration-response curves of dl-sotalol were shifted to the left in dogs treated with the combination of dl-sotalol and indapamide, and the EC50 values of dl-sotalol estimated for the prolongation of monophasic action potential duration at 90% repolarization and effective refractory period were decreased 3-fold during the coadministration of both drugs (P < .05 vs. dl-sotalol alone). Thus, under conditions of normal K+ levels, clinically relevant concentrations of indapamide modulate dl-sotalol effects on cardiac repolarization. Additional block of cardiac K+ currents, especially the rapid component of delayed rectifier potassium current and the slow component of delayed rectifier potassium current could explain these observations.

Shal-type channels contribute to the Ca2+-independent transient outward K+ current in rat ventricle.

1. The hypothesis that Kv4.2 and Kv4.3 are two of the essential K+ channel isoforms underlying the Ca2+-independent transient outward K+ current (It) in rat ventricle has been tested using a combination of electrophysiological measurements and antisense technology in both native myocytes and a stably transfected mammalian cell line, mouse Ltk- cells (L-cells). 2. The transient outward currents generated by Kv4.2 channels in L-cells exhibit rapid activation and inactivation properties similar to those produced by It in rat ventricular cells. The current-voltage relationships and the voltage dependence of steady-state inactivation are also very similar in these two preparations. However, the recovery from inactivation of Kv4.2 is much slower (time constant, 378 ms) than that of It in rat ventricular cells (58 ms). 3. The K+ current due to Kv4.2 can be blocked by millimolar concentrations of 4-aminopyridine in L-cells; a similar pharmacological response has been observed in rat ventricular myocytes. 4. Quinidine inhibits Kv4.2 in L-cells and It in rat ventricular cells in a similar fashion. In L-cells quinidine reduced the amplitude of Kv4.2 and accelerated its time course of inactivation, suggesting that quinidine may act as an open channel blocker of Kv4.2, as has been described for It in rat ventricle. 5. To provide further independent evidence that Kv4.2 and Kv4.3 channel isoforms contribute to It in rat ventricular cells, the effects of 20-mer antisense phosphorothioate oligodeoxynucleotides directed against Kv4.2 and Kv4.3 mRNAs were examined in ventricular myocytes isolated from 14- and 20-day-old rats, and in L-cells. In both preparations, Kv4.2 antisense pretreatment significantly reduced the transient outward K+ current (by approximately 55-60%). Similar reduction of It was produced by the Kv4.3 antisense oligonucleotide on the 14-day-old rat myocytes. 6. In 14-day rat ventricular cells, combination of Kv4.2 and Kv4.3 antisense oligonucleotides did not produce a significantly larger reduction of It than that observed after pretreatment with either antisense oligonucleotide alone. 7. L-cells stably transfected with Kv4.2 were treated with Kv4.3 antisense oligonucleotide to evaluate the possibility of cross-reactivity between Kv4.3 antisense and Kv4.2 mRNA. This antisense treatment produced no change in It, verifying the lack of cross-reactivity. 8. These biophysical and pharmacological results together with the antisense data show that Kv4.2 and Kv4.3 are essential components of the Ca2+-independent transient outward K+ current, It, in rat ventricular myocytes.

Thyroid hormone regulates postnatal expression of transient K+ channel isoforms in rat ventricle.

1. The ability of thyroid hormone to regulate the postnatal changes of the Ca2+-independent transient outward K+ current (It) was studied in rat ventricular myocytes. 2. In rat ventricle, It is very small at birth and then increases markedly between postnatal days 8 and 20. The time course of this increase in current density is similar to that of a significant rise in plasma thyroid hormone (T3) levels. 3. During early development, the density of expression of It can be altered by changes in thyroid hormone levels. Eight days after birth the density of It measured at +50 mV in control animals is 2.2 +/- 0.4 pA pF(-1). This value is about 3-fold larger (6.5 +/- 0.8 pA pF(-1)) in myocytes from age-matched hyperthyroid animals. When the plasma T3 level in newborn rats is not allowed to increase, or is decreased by making animals hypothyroid, this age-dependent increase in It fails to occur. 4. Using RNase protection assays, Kv4.2 and Kv4.3 mRNA levels were measured in ventricular tissues obtained from age-matched 8-day-old control and hyperthyroid rats. In hyperthyroid animals, where an approximately 3-fold increase in It was identified, increases in the mRNA levels for Kv4.2 and Kv4.3 were 1.6-fold and 2.6-fold, respectively. 5. These results show that thyroid hormone can regulate the development of It in rat ventricle. Direct measurements of It density and mRNA levels as a function of development and thyroid hormone levels also strongly suggest that the Kv4.2 and Kv4.3 channels are essential components of It in rat ventricular cells.

[3H]dofetilide binding to cardiac myocytes: modulation by extracellular potassium.

UNLABELLED: The radioligand [3H]dofetilide binds specifically to the delayed rectifier potassium channel and provides a biochemical approach to study interactions of Class III drugs with this channel. However, previous studies have examined the binding of [3H]dofetilide to cardiac myocytes only at extracellular potassium of 135 mM. Because previous electrophysiological studies have shown that hyperkalemia could alter the pharmacological responses to I(Kr) channel blockers, the hypothesis tested in this study was that changing ionic conditions would alter characteristics of [3H]dofetilide binding. RESULTS: under physiological conditions (Na+ 135 mM, K+ 5 mM), [3H]dofetilide bound to two sites on guinea-pig ventricular myocytes (a high-affinity site, K(d) 26+/-8 nM, B(max) 81+/-12 fmol/10(6) cells: and a low-affinity site, K(d) 1.6+/-0.8 microM, B(max) 1003+/-173 fmol/10(6) cells, n=11). Binding properties were not altered by changes in osmolarity or extracellular sodium. However, when extracellular K+ was increased to 20 mM, a single binding site was observed with an affinity K(d) of 120+12 nM and a B(max) of 303+/-57 fmol/10(6) cells (P<0.05; n=6). To establish whether this effect was mediated at the high-affinity site we assessed the effects of elevated extracellular potassium on a biological model, neonatal mouse myocytes, that expressed solely the high-affinity binding sites. The K(d) values for binding to fetal mouse cardiac myocytes at an extracellular K+ of 5 mM and 20 mM were also significantly different, 29+/-10 and 230+/-46 nM, respectively. In conclusion, [3H]dofetilide binding to its high-affinity site is modulated by extracellular potassium.

Hypomagnesemia: characterization of a model of sudden cardiac death.

OBJECTIVES: We sought to compare the incidence of sudden death in rats treated with magnesium-deficient and control diets and to address the electrophysiologic characteristics associated with these end points. BACKGROUND: Although magnesium deficiency is associated with an increased incidence of sudden cardiac death in patients, there has been no clear cause and effect relation because of a number of covariables, including diuretic use, hypokalemia, digitalis use and left ventricular dysfunction. METHODS: Hypomagnesemic rats and their paired control rats underwent in vivo electrophysiologic studies and measurements of the total calcium and magnesium content of their cardiac ventricles RESULTS: Serum magnesium levels were 0.5 +/- 0.3 mEq/liter (mean +/- SD) in hypomagnesemic animals and 1.2 +/- 0.9 mEq/liter in control animals. A modest but significant prolongation of the repolarization time was seen at the apical epicardial site (83 +/- 8 ms in hypomagnesemic rats vs. 68 +/- 13 ms in control rats, p < 0.05), but not at the other sites studied. Bradyarrhythmias and tachyarrhythmias were observed in 82% of the hypomagnesemic rats during the in vivo electrophysiologic studies, compared with 0% in the control group. During these studies, sudden, unexpected asystolic deaths were observed in 4 of 11 hypomagnesemic rats and 0 of 8 control rats. Polymorphic nonsustained ventricular tachycardia was provoked by rapid pacing in 5 to 11 hypomagnesemic rats and 0 of 8 control rats. Three of six hypomagnesemic rats exposed to auditory stimuli developed seizures, followed immediately by sudden deaths-two due to asystole and one due to ventricular fibrillation-although no end points occurred in the control animals. CONCLUSIONS: In this model, magnesium deficiency results in sudden cardiac death. The presence of startle induction of sudden death preceded by seizures suggests that sudden cardiac death results from a neurologic trigger.

[3H]dofetilide binding: biological models that manifest solely the high or the low affinity binding site.

Dofetilide is a Class III antiarrhythmic agent known to selectively block the rapid component of the delayed rectifier K+ current (IKr). [3H]Dofetilide binds to a low and a high affinity sites on guinea-pig myocytes. The purposes of this study were: (1) to find biological models which express solely the high or the low [3H]dofetilide binding sites; (2) to characterize the single binding site models; and (3) to establish which of the high or the low affinity binding sites is associated with IKr. We compared and characterized the [3H]dofetilide binding on guinea-pig myocytes, neonatal mouse ventricular homogenate and untransfected CHO cells. These tissue preparations were selected since the neonatal mouse tissue expresses IKr while this current is absent from CHO cells. We compared the IC50 concentrations of dofetilide and two other known IKr blockers E-4031 and sotalol, on [3H]dofetilide binding to these three preparations. Using steady-state and kinetic binding techniques, we characterized the interaction of E-4031 and sotalol with the high and the low [3H]dofetilide binding sites. We found that neonatal mouse ventricle manifest solely the high affinity site (Kd 20 +/- 4 nmol/l, Bmax 18 +/- 4 fmol/mg) while CHO cells manifest solely the low affinity binding site (Kd 1.6 +/- 0.1 mumol/l, Bmax 5.8 +/- 0.8 pmol/mg). We demonstrated that the high and low affinity binding sites present on guinea-pig myocytes show characteristics similar to the single high affinity site expressed on neonatal mouse homogenate and to the single low affinity site expressed on CHO cells, respectively. Class III antiarrhythmic drugs inhibited binding to the high affinity site at concentrations similar to those required to inhibit 50% of IKr current in electrophysiologic studies. In contrast, dofetilide and E-4031 inhibited [3H]dofetilide binding to the low affinity site only at supra-pharmacologic concentrations. We next demonstrated that Class III drugs interact in a competitive manner with the high affinity site on neonatal mouse tissue while they interact with a site allosterically coupled to the low binding site on CHO cells. These data suggest that dofetilide interacts with the high and low affinity sites in a fundamentally different manner. We defined biological models which express solely the high or low [3H]dofetilide binding sites. Only the high affinity site is related to IKr.

Influence of indapamide and chlorthalidone on reperfusion-induced ventricular fibrillation in isolated guinea pig hearts.

The delayed rectifier potassium current (IK) is a major repolarizing current in guinea pig ventricular myocytes. Blockade of IK or other repolarizing currents is of increasing interest for development of antiarrhythmic drugs; however, these interventions may also be proarrhythmic. In the present study, we compared the potential antiarrhythmic properties of indapamide and chlorthalidone, two structurally related sulfonamide diuretics which differ in their ability to block the slow component of the delayed rectifier (IKs) in isolated, buffer-perfused guinea pig hearts. Hearts underwent 30-min global no-flow ischemia and 10-min reperfusion. Dose-response (10(-7)-10(-4) M) effects of indapamide or chlorthalidone on reperfusion-induced arrhythmias, coronary flow, and heart rate (HR) were evaluated in a randomized blinded fashion. There was no significant difference in the incidence of ventricular fibrillation (VF) for either compound as compared with untreated controls. However, VF duration was reduced to < 40 s in all hearts treated with indapamide 10(-4) M). Mean VF duration with indapamide 10(-4) M was 31 +/- 4 versus 70 +/- 40 s in controls (p < 0.05). Chlorthalidone did not protect against reperfusion-induced arrhythmias. HR was unchanged with either compound; coronary flow during the control perfusion period increased approximately 43% with indapamide 10(-4) M (p < 0.05 vs. all treatment groups). These results demonstrate that indapamide, but not chlorthalidone, confers significant protection against reperfusion-induced VF in this experimental preparation and suggest that selective block of IKs may be antiarrhythmic.

Role of polymorphic debrisoquin 4-hydroxylase activity in the stereoselective disposition of mexiletine in humans.

It was reported previously that mexiletine undergoes stereoselective disposition in humans and that formation of three of its major metabolites co-segregates with polymorphic debrisoquin 4-hydroxylase (CYP2D6) activity. In this study, the hypothesis was tested that the CYP2D6-mediated oxidation pathways of mexiletine are responsible for the stereoselective disposition of the racemate in humans. Fourteen healthy subjects (10 extensive metabolizers [EMs] and 4 poor metabolizers [PMs]) participated in this study. They received a single 200-mg oral dose of racemic mexiletine hydrochloride on two occasions: once alone and once during administration of low-dose quinidine (50 mg four times a day). Blood and urine samples were obtained over 48 hr after the administration of mexiletine and analyzed by a stereoselective high-performance liquid chromatography assay. As reported previously, RS-mexiletine disposition was altered by a genetically determined (PM) or drug-induced (quinidine) decrease in CYP2D6 activity. In contrast, R/S ratio of the apparent total and nonrenal clearances of mexiletine and the R/S ratio of the urinary recovery of both enantiomers were similar in EMs and PMs. Moreover, these ratios were unaltered by quinidine administration. Partial metabolic clearance of N-hydroxymexiletine glucuronide, a non-CYP2D6 dependent metabolite, was highly stereoselective; the R/S ratio was 11.3 +/- 3.4. This ratio was similar in subjects with either an EM or a PM phenotype and was not altered by quinidine administration. Thus, the results obtained in this study suggest that non-CYP2D6-dependent metabolic pathways are responsible for the stereoselective disposition of mexiletine in humans.

Stereoselective disposition of (+/-)-sotalol at steady-state conditions.

The objective of this study was to assess, under steady-state conditions, the stereoselective disposition of (+/-)-sotalol in man. In all patients studied (n = 7) values of oral clearance (137 +/- 51 ml min-1), renal clearance (96 +/- 42 ml min-1) and nonrenal clearance (41 +/- 25 ml min-1) of (-)-sotalol were greater than those for (+)-sotalol (123 +/- 45 ml min-1, 89 +/- 39 ml min-1 and 34 +/- 23 ml min-1, respectively; P < 0.05, Student's paired t-test). Binding to plasma proteins was greater for (+)-sotalol (38 +/- 9% vs 35 +/- 9% for the (-)-enantiomer; P < 0.05) such that unbound oral clearance (+)/(-) ratio (0.95 +/- 0.06) and unbound renal clearance (+)/(-) ratio (0.97 +/- 0.06) were not stereoselective. In contrast, estimated unbound nonrenal clearance, which represents approximately 25% of the total unbound clearance of the drug, was greater for the (-)-enantiomer (64 +/- 42 ml min-1) compared with (+)-sotalol (57 +/- 42 ml min-1; P < 0.05). The difference in the pharmacokinetics of sotalol enantiomers is mainly related to stereoselectivity in plasma protein binding.

Stereoselective high-performance liquid chromatographic assay for the determination of sotalol enantiomers in biological fluids.

A high-performance liquid chromatographic (HPLC) assay for determination of sotalol enantiomers in biological fluids was developed to assess the stereoselective disposition of the drug in man. Following extraction at pH 9.0 with a mixture of chloroform-isopropanol (3:1, v/v), the organic phase was evaporated to dryness and the residue derivatized with (-)-methyl chloroformate. Diastereoisomeric derivatives were resolved by HPLC (C8 column) with fluorescence detection (lambda ex = 235 nm and lambda em = 300 nm). Retention times of l- and d-sotalol derivatives were 13 and 15 min while that of the internal standard, S-(-)-atenolol, was 12.3 min. The detection limit of each enantiomer was 12.5 ng/ml using 1 ml of plasma or urine. Intra-day and inter-day coefficients of variation were less than 10% for each enantiomer in the range 0.125-2.5 micrograms/ml in plasma and 0.25-2.5 micrograms/ml in urine.

Influence of debrisoquine phenotype and of quinidine on mexiletine disposition in man.

Mexiletine is a low clearance drug which undergoes extensive metabolism in man. In vitro studies with human liver microsomes have suggested that major oxidation pathways of mexiletine are predominantly catalyzed by the genetically determined debrisoquine 4-hydroxylase (cytochrome P450IID6) activity. In this study, we investigated the role of debrisoquine polymorphism and the effects of low dose quinidine, a selective inhibitor of cytochrome P450IID6, on the disposition of mexiletine. Fourteen healthy volunteers, 10 with the extensive metabolizer (EM) and 4 with the poor metabolizer (PM) phenotype, received a single 200-mg dose of mexiletine hydrochloride orally on two occasions (1 week apart), once alone and once under steady-state conditions for quinidine (50 mg QID). During the phase mexiletine alone, total clearance, nonrenal clearance and partial metabolic clearance of mexiletine to hydroxymethylmexiletine, to m-hydroxymexiletine and to p-hydroxymexiletine were decreased in PM compared to EM (all P less than .05). In EM, quinidine decreased mexiletine total clearance from 621 +/- 298 to 471 +/- 214 ml/min (mean +/- S.D.; P less than .05) and mexiletine nonrenal clearance from 583 +/- 292 to 404 +/- 188 ml/min (P less than .05). Moreover, quinidine increased mexiletine elimination half-life in EM from 9 +/- 1 to 11 +/- 2 h (P less than .05). In these subjects, partial metabolic clearance to hydroxymethylmexiletine, m-hydroxymexiletine and p-hydroxymexiletine were decreased by quinidine coadministration 5-, 4- and 7-fold, respectively, whereas partial metabolic clearance to N-hydroxymexiletine was unaffected. Changes induced by quinidine in EM were correlated to their debrisoquine metabolic ratio. Thus, genetically determined or pharmacologically induced modulation of cytochrome P450IID6 activity represents a major determinant of mexiletine disposition.

Influence of the menstrual cycle on the absorption and elimination of D-xylose.

Thirteen healthy female volunteers with regular menstrual cycles (28 +/- 2 days) received 25 gm oral and 5 gm intravenous doses of D-xylose on 2 successive days during the follicular, ovulatory, and luteal phases of two consecutive menstrual cycles. The ovulation time was characterized by luteinizing hormone levels, body basal temperatures, and progesterone and estradiol serum levels. D-Xylose was assayed in plasma and urine with a phloroglucinol-based colorimetric method. The findings of this study indicated that menstrual cycle did not significantly affect D-xylose absorption. After oral administration, the total clearance was significantly increased in cycle 2 during the luteal phase (p = 0.004). After intravenous administration in both cycles, D-xylose total clearance was also significantly faster during the luteal phase (p = 0.038 and p = 0.041, respectively). After oral administration, the renal clearance tended to be higher during the luteal phase in both cycles studied. After intravenous administration, this parameter was increased during the luteal phase by 24% and 25% in cycle 1 and by 8% and 12% in cycle 2. These findings are consistent with those of others showing an increase in glomerular filtration rate (GFR) during the luteal phase of the menstrual cycle. The findings of this study seem to be explained by the hormonal changes occurring during the menstrual cycle. Further investigations are warranted with use of specific probes of renal processes (GFR, renal reabsorption and tubular secretion) to confirm our findings and to elucidate the underlying mechanisms.