PEEK tube-based online solid-phase microextraction-high-performance liquid chromatography for the determination of yohimbine in rat plasma and its application in pharmacokinetics study.

Yohimbine is a novel compound for the treatment of erectile dysfunction derived from natural products, and pharmacokinetic study is important for its further development as a new medicine. In this work, we developed a novel PEEK tube-based solid-phase microextraction (SPME)-HPLC method for analysis of yohimbine in plasma and further for pharmacokinetic study. Poly (AA-EGDMA) was synthesized inside a PEEK tube as the sorbent for microextraction of yohimbine, and parameters that could influence extraction efficiency were systematically investigated. Under optimum conditions, the PEEK tube-based SPME method exhibits excellent enrichment efficiency towards yohimbine. By using berberine as internal standard, an online SPME-HPLC method was developed for analysis of yohimbine in human plasma sample. The method has wide linear range (2-1000 ng/mL) with an R2 of 0.9962; the limit of detection was determined and was as low as 0.1 ng/mL using UV detection. Finally, a pharmacokinetic study of yohimbine was carried out by the online SPME-HPLC method and the results have been compared with those of reported methods.

A qualitative/quantitative approach for the detection of 37 tryptamine-derived designer drugs, 5 ß-carbolines, ibogaine, and yohimbine in human urine and plasma using standard urine screening and multi-analyte approaches.

The first synthetic tryptamines have entered the designer drug market in the late 1990s and were distributed as psychedelic recreational drugs. In the meantime, several analogs have been brought onto the market indicating a growing interest in this drug class. So far, only scarce analytical data were available on the detectability of tryptamines in human biosamples. Therefore, the aim of the presented study was the development and full validation of a method for their detection in human urine and plasma and their quantification in human plasma. The liquid chromatography-linear ion trap mass spectrometry method presented covered 37 tryptamines as well as five ß-carbolines, ibogaine, and yohimbine. Compounds were analyzed after protein precipitation of urine or fast liquid-liquid extraction of plasma using an LXQ linear ion trap coupled to an Accela ultra ultra high-performance liquid chromatography system. Data mining was performed via information-dependent acquisition or targeted product ion scan mode with positive electrospray ionization. The assay was selective for all tested substances with limits of detection in urine between 10 and 100 ng/mL and in plasma between 1 and 100 ng/mL. A validated quantification in plasma according to international recommendation could be demonstrated for 33 out of 44 analytes.

Effects of three antagonists on selected pharmacodynamic effects of sublingually administered detomidine in the horse.

OBJECTIVE: To describe the effects of alpha2 -adrenergic receptor antagonists on the pharmacodynamics of sublingual (SL) detomidine in the horse. STUDY DESIGN: Randomized crossover design. ANIMALS: Nine healthy adult horses with an average age of 7.6 ± 6.5 years. METHODS: Four treatment groups were studied: 1) 0.04 mg kg(-1) detomidine SL; 2) 0.04 mg kg(-1) detomidine SL followed 1 hour later by 0.075 mg kg(-1) yohimbine intravenously (IV); 3) 0.04 mg kg(-1) detomidine SL followed 1 hour later by 4 mg kg(-1) tolazoline IV; and 4) 0.04 mg kg(-1) detomidine SL followed 1 hour later by 0.12 mg kg(-1) atipamezole IV. Each horse received all treatments with a minimum of 1 week between treatments. Blood samples were obtained and plasma analyzed for yohimbine, atipamezole and tolazoline concentrations by liquid chromatography-mass spectrometry. Behavioral effects, heart rate and rhythm, glucose, packed cell volume (PCV) and plasma proteins were monitored. RESULTS: Chin-to-ground distance increased following administration of the antagonists, however, this effect was transient, with a return to pre-reversal values as early as 1 hour. Detomidine induced bradycardia and increased incidence of atrioventricular blocks were either transiently or incompletely antagonized by all antagonists. PCV and glucose concentrations increased with tolazoline administration, and atipamezole subjectively increased urination frequency but not volume. CONCLUSIONS AND CLINICAL RELEVANCE: At the doses administered in this study, the alpha2 -adrenergic antagonistic effects of tolazoline, yohimbine and atipamezole on cardiac and behavioral effects elicited by SL administration of detomidine are transient and incomplete.

The effects of yohimbine on the pharmacokinetic parameters of detomidine in the horse.

OBJECTIVE: To describe the pharmacokinetics of detomidine and yohimbine when administered in combination. STUDY DESIGN: Randomized crossover design. ANIMALS: Nine healthy adult horses aged 9 ± 4 years and weighing of 561 ± 56 kg. METHODS: Three dose regimens were employed in the current study. 1) 0.03 mg kg(-1) detomidine IV (D), 2) 0.2 mg kg(-1) yohimbine IV (Y) and 3) 0.03 mg kg(-1) detomidine IV followed 15 minutes later by 0.2 mg kg(-1) yohimbine IV (DY). Each horse received all three dose regimens with a minimum of 1 week in between subsequent regimens. Blood samples were obtained and plasma analyzed for detomidine and yohimbine concentrations by liquid chromatography-mass spectrometry. Data were analyzed using both non-compartmental and compartmental analysis. RESULTS: The maximum measured detomidine concentrations were 76.0 and 129.9 ng mL(-1) for the D and DY treatments, respectively. Systemic clearance and volume of distribution of detomidine were not significantly different for either treatment. There was a significant increase in the maximum measured yohimbine plasma concentrations from Y (173.9 ng mL(-1)) to DY (289.8 ng mL(-1)). Both the Cl and V(d) for yohimbine were significantly less (6.8 mL minute(-1) kg(-1) (Cl) and 1.7 L kg(-1) (V(d) )) for the DY as compared to the Y treatments (13.9 mL minute(-1) kg(-1) (Cl) and 2.7 L kg(-1) (V(d))). Plasma concentrations were below the limit of quantitation (0.05 and 0.5 ng mL(-1)) by 18 hours for both detomidine and yohimbine. CONCLUSION AND CLINICAL RELEVANCE: The Cl and V(d) of yohimbine were affected by prior administration of detomidine. The elimination half life of yohimbine remained unaffected when administered subsequent to detomidine. However, the increased plasma concentrations in the presence of detomidine has the potential to cause untoward effects and therefore further studies to assess the physiologic effects of this combination of drugs are warranted.

Pharmacokinetics of yohimbine following intravenous administration to horses.

Yohimbine is an alpha 2 adrenergic receptor antagonist used most commonly in veterinary medicine to reverse the effects of the alpha 2 receptor agonists, xylazine and detomidine. Most notably, yohimbine has been shown to counteract the CNS depressant effects of alpha 2 receptor agonists in a number of species. The recent identification of a yohimbine positive urine sample collected from a horse racing in California has led to the investigation of the pharmacokinetics of this compound. Eight healthy adult horses received a single intravenous dose of 0.12 mg/kg yohimbine. Blood samples were collected at time 0 (prior to drug administration) and at various times up to 72 h post drug administration. Plasma samples were analyzed using liquid chromatography-mass spectrometry (LC-MS) and data analyzed using both noncompartmental and compartmental analysis. Peak plasma concentration was 114.5 + 31.8 ng/mL and occurred at 0.09 + 0.03 h. Mean ± SD systemic clearance (Cls) and steady-state volume of distribution (Vdss) were 13.5 + 2.1 mL/min/kg and 3.3 + 1.3 L/kg following noncompartmental analysis. For compartmental analysis, plasma yohimbine vs. time data were best fitted to a two compartment model. Mean ± SD Cls and Vdss of yohimbine were 13.6 ± 2.0 mL/min/kg and 3.2 ± 1.1 L/kg, respectively. Mean ± SD terminal elimination half-life was 4.4 ± 0.9 h following noncompartmental analysis. Immediately following administration, two horses showed signs of sedation, while the other six appeared behaviorally unaffected. Gastrointestinal sounds were moderately increased compared to baseline while fecal consistency appeared normal.

Pharmacokinetics and pharmacodynamics of three intravenous doses of yohimbine in the horse.

Yohimbine is an alpha 2 adrenergic receptor antagonist, which has been shown to counteract the CNS depressant effects of alpha 2 receptor agonists in a number of species. Recently, our laboratory identified yohimbine in the absence of detectable concentrations of an alpha 2 agonist in a regulatory sample collected from a horse racing in California. This coupled with anecdotal reports of CNS stimulation and documented reports of cardiovascular changes when administered in conjunction with an agonist led us to investigate the pharmacokinetics and pharmacodynamics of yohimbine when administered alone. Nine healthy adult horses received a single intravenous dose of 0.1, 0.2, and 0.4 mg/kg yohimbine. Blood samples were collected at time 0 (prior to drug administration) and at various times up to 24 h postdrug administration. Plasma samples were analyzed using liquid chromatography-mass spectrometry (LC-MS), and resulting data analyzed using both noncompartmental and compartmental analysis. Peak plasma concentrations were 106.0 ± 28.9, 156.7 ± 34.3, and 223.0 ± 44.5 ng/mL for doses of 0.1, 0.2, and 0.4 mg/kg, respectively. Immediately following administration, two horses showed signs of sedation, one horse appeared excited, while the other six appeared behaviorally unaffected. Episodes of tachycardia were noted within minutes of administration for all horses at all doses; however, there was no correlation between behavioral responses and episodes of increased heart rate. Sixty-three percent of the horses (8, 6, and 4 of the 9 horses in the 0.1, 0.2, and 0.4 mg/kg dose groups, respectively) exhibited second-degree atrial-ventricular conduction blocks and bradycardia prior to drug administration that transiently improved or disappeared upon administration of yohimbine. Gastrointestinal sounds were transiently increased following all doses.

Severe acute intoxication with yohimbine: Four simultaneous poisoning cases.

Yohimbine is an indole alkaloid from the leaves and bark of the Pausinystalia johimbe tree that has acquired an enviable reputation in treating erectile dysfunction. This report presents four simultaneous severe poisoning/death cases caused by yohimbine. The test samples comprised the venous blood of four middle-aged men (aged 47-65) who were suspected of poisoning; one of the men died due to ineffective rescue. Ethanol concentration determination and toxicological routine screening were performed using gas chromatography with flame ionization detection (GC-FID) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A specific LC-MS/MS method was developed to quantify yohimbine, which showed concentrations of 459, 249, and 301ng/mL in three poisoned blood samples and concentrations as high as 5631ng/mL in the deceased. Moreover, the deceased's autopsy ruled out death from trauma and previous illness, and no other common toxic components were detected in his blood. Therefore, yohimbine poisoning appears to be the most likely cause of death. As a type of alkaloid that can be employed in the treatment of clinical diseases and additives for supplements, the danger of yohimbine should be of widespread concern in society.

Quantification of microdosed oral yohimbine and its major metabolite in human plasma in the picogram range.

Aim: Pharmacokinetics after oral microdosing of the anticipated CYP2D6 substrate yohimbine and its metabolite 11-OH-yohimbine is potentially useful for drug-drug interaction trials and profiling of CYP2D6 enzyme activity. Materials & methods: We developed an ultrasensitive ultra performance liquid chromatography coupled to tandem mass spectrometry assay for quantification of yohimbine and its main metabolite 11-OH-yohimbine in plasma with a linear calibration range of 5-2500 pg/ml and validated it according to US FDA's and EMA's guidelines. Sample preparation was performed using fast liquid-liquid extraction. The assay was applied for the determination of concentrations of yohimbine and 11-OH-yohimbine in plasma after oral administration of 50 µg yohimbine to two subjects. Conclusion: Ultrasensitive quantification of yohimbine and its metabolite enables the determination of their concentrations in plasma after microdosing which would be applicable to use in CYP2D6 phenotyping.

Variations in circulating catecholamines fail to alter human platelet alpha-2-adrenergic receptor number or affinity for [3H]yohimbine or [3H]dihydroergocryptine.

A series of studies were performed to determine the relationship between physiologic levels of circulating plasma norepinephrine and epinephrine and human platelet alpha-2 binding site number and the affinity (KD) of these sites for antagonist radioligands. In one study, alpha-2-adrenergic binding site number and affinity were compared using both [3H]yohimbine and [3H]dihydroergocryptine as radioligands. There was good absolute and relative comparison for binding site number, but only a relative relationship for KD. In 46 normal subjects, there was no significant relationship between site number or KD and age, plasma epinephrine, or plasma norepinephrine concentration. Even after plasma epinephrine was raised nearly 20-fold by means of an intravenous infusion for 4 h in seven normal subjects, neither sites (608 +/- 68 vs. 567 +/- 120 sites/platelet) nor KD (2.01 +/- 0.94 vs. 2.14 +/- 1.15 nM) were significantly changed. Similarly, neither sites (445 +/- 55 vs. 421 +/- 53 sites/platelet) nor KD (1.44 +/- 0.29 vs. 2.10 +/- 0.75 nM) were significantly changed in six normal subjects when plasma norepinephrine levels increased during oral administration of prazosin for 1 wk. Thus, in a cross-sectional analysis and after a change in plasma catecholamine concentrations, there was no relationship in normal subjects between platelet alpha-2 binding site number or affinity of these sites for antagonist radioligands and the circulating catecholamine levels to which the platelets were exposed. In a group (n = 7) of patients who lack epinephrine-induced platelet aggregation due to abnormal thrombopoiesis, binding site number was decreased (304 +/- 36 vs. 572 +/- 29 sites/platelet, P less than 0.001) and KD tended to be greater (8.69 +/- 2.44 vs. 5.40 +/- 0.31 nM, P = NS) than in normal subjects (n = 46), despite having similar plasma catecholamine levels. There was no difference in binding site number (491 +/- 116 sites/platelet) and KD (5.61 +/- 0.84 nM) in patients (n = 5) with autonomic insufficiency and low levels of upright plasma norepinephrine when compared with the normal subjects. Two patients were examined before and after the removal of a pheochromocytoma. Their binding site number and KD were normal before the operation and essentially unchanged after the tumor removal and fall of plasma catecholamines. Thus, this study demonstrates that within the physiologic and pathophysiologic range of plasma catecholamines (in men), there is no relationship between the circulating catecholamine concentration and either platelet alpha-2 adrenergic binding site number or the affinity of these sites for antagonist radioligands.

Acute reduction in human platelet alpha 2-adrenoreceptor affinity for agonist by endogenous and exogenous catecholamines.

The binding characteristics of l-epinephrine to intact human platelets were assessed under conditions of physiological and pharmacological variations in plasma catecholamine concentration. In competition with the alpha 2-adrenoreceptor antagonist yohimbine, mean platelet receptor affinity for l-epinephrine was decreased 3.4-fold after 2 h of upright posture and exercise. This change in agonist affinity correlated significantly with the increases in plasma epinephrine and norepinephrine that were stimulated by upright posture and exercise. Supine subjects infused with l-norepinephrine or l-epinephrine for 2 h also averaged a 3.3- and 2.7-fold decrease in platelet alpha 2-adrenoreceptor affinity for agonist with no change in receptor number or antagonist affinity. The alpha 2-adrenoreceptor agonist affinity changes were specific for alpha-agonists since they were blocked by phentolamine, and incubation with 10(-5) M isoproterenol produced no change in alpha 2-adrenoreceptor affinity for l-epinephrine. In vitro exposure of intact human platelets to 10(-6) to 10(-10) M l-epinephrine for 2 h produced a concentration-related decrease in alpha 2-adrenoreceptor affinity for agonist. In all three paradigms, average slope factors approached 1.0 as affinity decreased, which is consistent with a heterogeneous receptor population that becomes more homogeneous after agonist exposure. Incubation of platelet-rich plasma with 10(-6) to 10(-8) M l-epinephrine resulted in a dose- and time-related loss of aggregatory response to l-epinephrine; this demonstrates that agonist affinity changes are correlated with changes in receptor sensitivity. These observations demonstrate that physiological variations in plasma catecholamines acutely modulate the intact human platelet alpha 2-adrenoreceptor's affinity for agonist, and can thereby alter the sensitivity of platelets to alpha 2-adrenergic agonist.

Down regulation of alpha 2 adrenergic receptor numbers in platelets by insulin.

Incubation of platelets from normal volunteers, who had not taken any medication at least for 2 weeks, with insulin (200 mu units/ml), resulted in the inhibition of the potentiation of ADP-induced platelet aggregation in the presence of (-)-epinephrine by 50-60% when compared with the control. The inhibitory effect of insulin was not related to the increased cyclic AMP level or decreased thromboxane A2 synthesis in these cells. It was found that the treatment of platelets with insulin decreased alpha 2 adrenergic receptors number of these cells from 413 +/- 92/platelet to 206 +/- 84/platelet as determined by Scatchard analysis of [3H]yohimbine binding. The affinity of the receptors (1.05 +/- 0.02 nM) remained essentially unchanged due to the treatment of platelets with the hormone (1.40 +/- 0.60 nM).

Effects of Alzheimer's disease and normal aging on cerebrospinal fluid norepinephrine responses to yohimbine and clonidine.

BACKGROUND: The resting cerebrospinal fluid (CSF) norepinephrine concentration is unchanged or even increased in patients with Alzheimer's disease (AD). These in vivo findings appear to be inconsistent with the post-mortem locus ceruleus neuronal loss that is reported in patients with AD. METHODS: The effects of AD and advanced age on central nervous system noradrenergic status were estimated by comparing CSF norepinephrine concentrations following the administration of yohimbine hydrochloride, clonidine hydrochloride, and placebo in outpatients with AD and older and young normal subjects. Levels of yohimbine, its metabolite 11-hydroxy-yohimbine, and clonidine were measured in CSF and plasma samples. Behavioral responses were quantified by rating the Tension, Excitement, and Anxiety items on the Brief Psychiatric Rating Scale. RESULTS: Yohimbine-induced increases of CSF norepinephrine concentrations were greater in both patients with AD and normal older subjects than in normal young subjects. Clonidine-induced decreases of CSF norepinephrine concentrations did not differ among groups. Behavioral arousal following the administration of yohimbine was greater in patients with AD than in the other groups. CONCLUSIONS: Central nervous system noradrenergic responsiveness is enhanced in normal older subjects, and this age effect is retained in patients with AD. Behavioral sensitivity to increased central nervous system noradrenergic activity is enhanced in patients with AD.

Decreased alpha 2-adrenergic receptors on platelet membranes from diabetic patients with autonomic neuropathy and orthostatic hypotension.

Platelet adrenergic receptors were studied in normal subjects and diabetic patients with autonomic neuropathy to determine the relationship between adrenoreceptor status and orthostatic hypotension. The binding of [3H]clonidine and [3H]yohimbine to platelet membranes was measured in diabetic patients with autonomic neuropathy and orthostatic hypotension (n = 12) and without orthostatic hypotension (n = 11), diabetic patients without autonomic neuropathy (n = 12), and normal subjects (n = 9). Mean basal and standing plasma norepinephrine levels were not different in the four groups, and there was no relationship between orthostasis and norepinephrine responses. The diabetic patients with orthostatic hypotension had a significantly greater fall in mean blood pressure [31 +/- 2.8 (+/- SE) mm Hg] than any of the other three groups. Diabetic patients with diabetic autonomic neuropathy and orthostatic hypotension had a 30-40% decrease in number of platelet alpha 2-adrenergic receptors, as demonstrated by [3H]clonidine and [3H]yohimbine binding. The maximum number of binding sites for clonidine was 34 +/- 2.8 (+/- SE) fmol/mg protein in normal subjects, 27.4 +/- 3.4 in diabetic patients with neuropathy, 26 +/- 2.5 in diabetic patients with autonomic neuropathy without orthostatic hypotension, and 20.4 +/- 3.8 fmol/mg protein in diabetic patients with autonomic neuropathy with orthostatic hypotension (P less than 0.001). The maximum number of binding sites for yohimbine was 112 +/- 12.6 in normal subjects, 127 +/- 10 in diabetic patients without orthostatic hypotension, and 87 +/- 12.4 fmol/mg protein in patients with diabetic autonomic neuropathy with orthostatic hypotension (P less than 0.001). Reduced platelet alpha 2-receptors are associated with postural hypotension in diabetic autonomic neuropathy. If applicable to the postjunctional alpha 2-adrenergic receptor on sympathetic neurons, reduced vascular responses to changes in posture would be expected despite normal or enhanced norepinephrine secretion.

Platelet alpha-2-adrenergic dysfunction in negative symptom schizophrenia: a preliminary study.

The specific binding to platelet membranes (Bmax) of 3H-clonidine, an alpha-2 agonist, and 3H-yohimbine, an alpha-2 antagonist, was measured in nine drug-free male schizophrenic patients and repeated after 2 weeks of chlorpromazine (CPZ) treatment. Patients with a lower pretreatment Bmax for 3H-clonidine showed a significantly smaller change in Bmax after treatment, less improvement in their clinical state, as indicated by the change in the Global Assessment Scale (GAS), and a lower posttreatment GAS. Also, they had a significantly higher score for negative symptoms on the Affect Rating Scale both before and after treatment. These findings suggest that schizophrenic patients with relatively subsensitive platelet alpha-2-adrenergic receptors, as measured by 3H-clonidine binding, tend to have more negative symptoms and a diminished alpha receptor binding response and diminished clinical response to CPZ. There were no clinical correlations to 3H-yohimbine binding.

Low baseline and yohimbine-stimulated plasma neuropeptide Y (NPY) levels in combat-related PTSD.

BACKGROUND: Consistent with many studies demonstrating enhanced reactivity of the sympathetic nervous system in posttraumatic stress disorder (PTSD), the administration of yohimbine, a noradrenergic alpha(2)-antagonist, has been shown to increase core symptoms of PTSD and to induce greater increases in plasma 3-methyl-4-hydroxy-phenyl-glycol (MHPG) in subjects with PTSD compared with healthy control subjects. In turn, neuropeptide Y (NPY) has been shown to inhibit the release of norepinephrine from sympathetic noradrenergic neurons. METHODS: In the following study, plasma NPY responses to yohimbine and placebo were measured in a subgroup of 18 subjects with PTSD and 8 healthy control subjects who participated in the previous study of the effect of yohimbine on plasma MHPG. RESULTS: The PTSD subjects had lower baseline plasma NPY and blunted yohimbine-stimulated increases in plasma NPY compared with the healthy control subjects. Within the PTSD group, baseline plasma NPY levels correlated negatively with combat exposure scale scores, baseline PTSD and panic symptoms, and yohimbine-stimulated increases in MHPG and systolic blood pressure. CONCLUSIONS: This study suggests that combat stress-induced decreases in plasma NPY may mediate, in part, the noradrenergic system hyperreactivity observed in combat-related PTSD. The persistence of this decrease in plasma NPY may contribute to symptoms of hyperarousal and the expression of exaggerated alarm reactions, anxiety reactions, or both in combat veterans with PTSD long after war.

Patterns of cerebrospinal fluid catechols support increased central noradrenergic responsiveness in aging and Alzheimer's disease.

BACKGROUND: High cerebrospinal fluid (CSF) norepinephrine (NE) concentrations in aging and Alzheimer's disease (AD) could reflect decreased NE clearance from central nervous system (CNS) extracellular fluid or increased release of NE into CNS extracellular fluid. Measuring CSF concentrations of the intraneuronal NE metabolite dihydroxyphenylglycol (DHPG), an estimate of NE clearance, and the NE precursor dihydroxyphenylacetic acid (DOPA), an estimate of NE biosynthesis, can help differentiate these mechanisms. METHODS: NE, DHPG, and DOPA were determined by HPLC in CSF and plasma obtained following yohimbine, clonidine, and placebo. Ten AD, 10 older, and 11 young subjects were studied. RESULTS: CSF DOPA following yohimbine was higher in older and AD than in young subjects. CSF DHPG did not differ among groups. Plasma DOPA following yohimbine was higher in AD than in young subjects. CONCLUSIONS: During alpha-2 adrenoreceptor blockade in both aging and AD, there are increased responses of CNS NE biosynthesis and release with unchanged CNS NE clearance. This pattern is consistent with partial loss of CNS noradrenergic neurons with compensatory activation of remaining CNS noradrenergic neurons. Given the marked loss of locus coeruleus (LC) noradrenergic neurons in AD, achievement of high CSF NE suggests particularly prominent compensatory activation of remaining LC neurons in this disorder.

alpha 2-Adrenergic receptor binding in human platelets: alterations during the menstrual cycle.

We studied the effect of several clinically important variables on the characteristics of alpha 2-adrenergic receptors in human platelet membranes. The number and affinity of the receptor binding sites were determined from radioligand binding experiments, with [3H]yohimbine being the radioligand of choice. Platelets from female subjects had a cyclic variation in the number of alpha 2-adrenergic receptors that coincided with their menstrual cycles. The number of alpha 2-receptors was highest at the onset of menses and dropped to 74% to 79% of that value during the middle of the cycle. In concurrent experiments we did not observe comparable cyclic changes in receptor binding sites in platelets from male subjects. There was no age-dependent alteration in receptor number in a sample of 39 subjects ranging in age from 8 to 80 yr, but the number of alpha 2-receptors in platelets from male and female subjects differed. We also tested the possibility of a circadian rhythm in alpha 2-receptor number but found no cyclic changes as a function of time of day. There was no alteration in alpha 2-adrenergic receptor binding in the platelets from five subjects with Parkinson's disease. Finally, there was no change in receptor affinity as a function of any of the variables tested. These data should apply to the design of further studies on the clinical importance of platelet alpha 2-adrenergic receptors.

Human sympathetic activation by alpha2-adrenergic blockade with yohimbine: Bimodal, epistatic influence of cytochrome P450-mediated drug metabolism.

BACKGROUND: alpha2-Adrenergic blockade responses suggest adrenergic dysfunction in hypertension. alpha2-Blockade is also used to treat autonomic dysfunction. However, pharmacokinetic determinants of yohimbine disposition are not understood. METHODS: We evaluated alpha2-blockade with intravenous yohimbine in 172 individuals. Specific cytochrome P450 (CYP) isoform-mediated metabolism was investigated. Results were evaluated by ANOVA and by maximum likelihood analysis for bimodality of response distributions. RESULTS: Yohimbine metabolism to 11-hydroxy-yohimbine displayed greater than 1000-fold variability, with 17 individuals showing no metabolism. Nonmetabolizers differed from others in ethnicity but not in age, sex, body habitus, blood pressure, heart rate, or family history of hypertension. Bimodality of metabolism was suggested by frequency histogram, as well as maximum likelihood and cluster analysis. Among ethnic groups, subjects of European ancestry had the highest frequency of nonmetabolism. In vitro oxidation suggested that the major route of metabolism (lowest Michaelis-Menten constant and greatest intrinsic clearance) was likely via CYP2D6 to 11-hydroxy-yohimbine. In vivo genotypes at both CYP2D6 and CYP3A4 were necessary to predict metabolism (overall F = 3.03, P =.005); an interaction of alleles at these 2 loci (interaction F = 3.05, P =.033) suggested an epistatic effect on drug metabolism in vivo. Nonmetabolizers had greater activation of sympathetic nervous system activity. Yohimbine increased blood pressure, an effect mediated hemodynamically by elevation of cardiac output rather than systemic vascular resistance. Blood pressure and cardiac output responses did not differ by metabolizer group. CONCLUSIONS: We conclude that heterogeneous, bimodally distributed yohimbine metabolism depends on common genetic variation in both CYP2D6 and CYP3A4 and contributes to differences in sympathetic neuronal response to alpha2-blockade. These results have implications for both diagnostic and therapeutic uses of this alpha2-antagonist.

Peripheral markers of serotonergic and noradrenergic function in post-pubertal, caucasian males with autistic disorder.

Some studies have suggested that disorders in the peripheral and central metabolism of serotonin (5-HT) and noradrenaline may play a role in the pathophysiology of autistic disorder. This study examines serotonergic and noradrenergic markers in a study group of 13 male, post-pubertal, caucasian autistic patients (age 12-18 y; I.Q. > 55) and 13 matched volunteers. [3H]-paroxetine binding Kd values were significantly higher in patients with autism than in healthy volunteers. Plasma concentrations of tryptophan, the precursor of 5-HT, were significantly lower in autistic patients than in healthy volunteers. There were no significant differences between autistic and normal children in the serum concentrations of 5-HT, or the 24-hr urinary excretion of 5-hydroxy-indoleacetic acid (5-HIAA), adrenaline, noradrenaline, and dopamine. There were no significant differences in [3H]-rauwolscine binding Bmax or Kd values, or in the serum concentrations of tyrosine, the precursor of noradrenaline, between both study groups. There were highly significant positive correlations between age and 24-hr urinary excretion of 5-HIAA and serum tryptophan. The results suggest that: 1) serotonergic disturbances, such as defects in the 5-HT transporter system and lowered plasma tryptophan, may play a role in the pathophysiology of autism; 2) autism is not associated with alterations in the noradrenergic system; and 3) the metabolism of serotonin in humans undergoes significant changes between the ages of 12 and 18 years.

Serotonergic and noradrenergic markers of post-traumatic stress disorder with and without major depression.

Some studies have suggested that disorders in the peripheral and central metabolism of serotonin (5-HT) and noradrenaline (NE) may play roles in the pathophysiology of post-traumatic stress disorder (PTSD). This study examines (1) the availability of plasma total tryptophan, the precursor of 5-HT, and tyrosine, the precursor of NE; and (2) the platelet 5-HT transporter and alpha 2-adrenoceptor (alpha 2-AR) binding sites in patients with PTSD and healthy volunteers. High-performance liquid chromatography (HPLC) was employed to measure plasma tryptophan and tyrosine as well as amino acids known to compete with the same cerebral transport system; that is, valine, leucine, phenylalanine, and isoleucine. The maximum number of binding sites (Bmax) and their affinity (Kd) for binding to [3H]-paroxetine and [3H]-rauwolscine, a selective alpha 2-AR antagonist, were determined. [3H]-paroxetine and [3H]-rauwolscine binding Kd values were significantly higher in patients with PTSD than in healthy volunteers. [3H]-rauwolscine binding Kd values were significantly higher in patients with PTSD and concurrent major depression (MD) than in PTSD patients without MD and healthy volunteers. Plasma tyrosine concentrations and the ratio of tyrosine/valine + leucine + isoleucine + phenylalanine + tryptophan were significantly higher in PTSD patients with MD than in those without MD and healthy volunteers. The results show that PTSD is accompanied by lower affinity of paroxetine binding sites and that PTSD with concurrent MD is accompanied by lower affinity of alpha 2-ARs and increased plasma tyrosine availability to the brain. The results suggest that (1) serotonergic mechanisms, such as defects in the 5-HT transporter system, may play a role in the pathophysiology of PTSD; and (2) that catecholaminergic mechanisms, such as increased precursor availability and lowered affinity of alpha 2-ARs, may play a role in the pathophysiology of PTSD with concurrent MD.