Nicolas D. Vlachakis

Enzymatic deconjugation of catecholamines in human and rat plasma and red blood cell lysate

We have developed a method for enzymatic hydrolysis of both sulfated and glucuronidated catecholamines in plasma and red blood cell lysate. Hydrolysis occurs in the course of the radioenzymatic assay for catecholamines. In human plasma, catecholamines are conjugated almost entirely with sulfate while, in rat plasma, glucuronides are the main conjugates of epinephrine and dopamine but not norepinephrine. Rat plasma contains less percent conjugated catecholamine than human plasma. Human red blood cell lysate contains less conjugated catecholamine than plasma, whereas free E in lysate exceeds that of plasma and free NE has same level both in sulfated + glucuronidated) catecholamines and the nature of conjugated catecholamines.

Study of single and multiple dose pharmacokinetic/pharmacodynamic modeling of the antihypertensive effects of labetalol

This was an open-label, two-phase crossover study of labetalol in 11 patients with mild to moderate hypertension. A two- to four-week outpatient placebo phase was followed by a three-day inpatient placebo period. Patients were then randomly assigned to receive either labetalol, 200 mg, as a single dose and three times a day for three days and, on the final day, another single dose or a similar sequence with 300 mg as the single dose and multiple twice a day treatment. A two-week placebo outpatient period was followed by the second phase of the study in which the treatment regimen was reversed for the two groups. Blood samples for the determination of free and conjugated labetalol plasma levels were collected, and blood pressures and heart rate were recorded sequentially for 24 hours after the first and last dose of labetalol, and during the multiple dose treatment period before and two hours after each dose as well as four times daily with the patient supine and upright. Of the 11 patients analyzed, five were men and six were women, ranging in age from 33 to 62 years. Labetalol (200 mg and 300 mg) was rapidly absorbed with peak concentrations achieved in approximately one hour. The pharmacokinetic data best fit a two-compartment pharmacokinetic model with first order absorption. At steady state, the absorption, distribution, and elimination kinetics were similar for both dosage regimens with elimination half life of 7.65 and 7.92 hours for the 200 mg three times a day and 300 mg twice a day regimens, respectively. During the multiple dosing period average steady-state plasma drug concentrations were 0.149 mg/ml and 0.145 mg/ml for the 300 mg twice a day and 200 mg three times a day regimens, respectively. Approximately 12 percent of total plasma labetalol was free drug. The balance was conjugated. The first dose of 200 mg or 300 mg of labetalol significantly (p less than 0.01) lowered standing and supine mean blood pressure over a period of eight to 12 hours, respectively, with peak effects occurring at two (standing) and four (supine) hours. A significant reduction (p less than 0.01) in supine mean blood pressure was present 24 hours after the initial dose of 300 mg. At steady state the antihypertensive effects of the 200 mg three times a day and the 300 mg twice a day dosage regimens were similar.(ABSTRACT TRUNCATED AT 400 WORDS)

A radioenzymatic microassay for simultaneous measurement of catecholamines and their deaminated metabolites.

Abstract A simple and specific assay for measurement of norepinephrine, epinephrine, and their major deaminated metabolites 3,4-dihydroxyphenylglycol and 3,4-dihydroxymandelic acid in plasma has been developed. The assay is based on the conversion of these compounds to their O-methylated derivatives in the presence of catechol-O-methyltransferase and S-adenosyl-[methyl3H]methionine. The formed tritiated derivatives are selectively extracted in organic solvents and isolated by thin-layer chromatography. After oxidation to vanillin they are purified by solvent extraction and measured by liquid scintillation spectrophotometry. The assay is rapid and results can be obtained in less than 3 hr.

Catecholamines and their major metabolites in plasma and cerebrospinal fluid of man

In 36 patients undergoing elective surgery under spinal anesthesia, plasma and cerebrospinal fluid (CSF) concentrations of catecholamines and their major metabolic products were determined. The development of specific and sensitive radioenzymatic assays make these determinations possible. The levels of norepinephrine, epinephrine, and the O-methylated metabolite, normetanephrine, were greater in the plasma then the CSF, although the difference was significant for norepinephrine and epinephrine only (P less than 0.01 for both) On the other hand the levels of both deaminated metabolites dihydroxyphenylglycol (DOPEG) and dihydroxymandelic acid (DOMA) were greater in the CSF the in plasma, but the difference was significant for DOPEG only (P less than 0.01). Although there was a positive and significant correlation between the levels in plasma and CSF of all these compounds, their concentrations in CSF may reflect metabolism of catecholamines in the central nervous system.

A simple and specific radioenzymatic assay for measurement of urinary normetanephrine

Abstract A new, simple and, specific assay for measurement of urinary normetanephrine (NMN) has been developed. The assay is based on the conversion of NMN to its N-methylated, tritiated derivative, metanephrine ([3H]MN), utilizing phenylethanolamine-N-methyl-transferase and 3H-labeled S-adenosylmethionine. The [3H]MN formed is selectively isolated by the thin-layer chromatographic step and measured by liquid scintillation spectrophotometry. Ten microliters of a random urine from a normal subject gives at least three times the counts per minute of the blank. The assay is rapid and results can be obtained in less than 3 hr. Many drugs, including the antihypertensives, did not interfere with the assay.

Plasma levels of free and total catecholamines and two deaminated metabolites in man ― rapid deconjugation by heat in acid

We have described a procedure for deconjugation of plasma catecholamines, norepinephrine (NE), epinephrine (E) and dopamine (DA) and two catecholamine metabolites 3,4-dihydroxymandelic acid (DOMA) and 3,4-dihydroxyphenylglycol (DOPEG). Heat at 100 degrees C of the acidified specimen, pH 0.8, produced complete deconjugation of catecholamines in 7 minutes and of metabolites in 5-7 minutes. Subsequently all five products were simultaneously measured with a radioenzymatic assay. However, hydrolysis for 7 minutes produced approximately a loss of 5% in DA and E, 15% in NE and 50% in the metabolites. The percent of free compound in the plasma of 11 normotensive and healthy subjects was 23 +/- 16 for NE, 20 +/- 8 E, 0.8 +/- 1 DA, 20 +/- 7 DOMA and 42 +/- 12 for DOPEG. Similar results were obtained in a random specimen of six patients with primary hypertension. In a group of four patients with pheochromocytoma free levels of NE, DOPEG and DOMA were significantly greater than in the other two groups, whereas conjugates were not. The intravenous administration of NE or the activation of sympathetic nervous system by standing combined with exercise for 15 minutes did not produce a change in the levels of plasma conjugates. These findings suggest that short changes in plasma catecholamines are better reflected in the free than the conjugated part.

Increased plasma normetanephrine in spontaneously hypertensive rats.

Plasma catecholamine and total plasma normetanephrine concentrations were determined in 11 spontaneously hypertensive rats (SHR) and in 11 age and sex matched Wistar Kyoto control rats (WKy). Plasma norepinephrine concentration, obtained from tail vein blood of restrained rats, was 356 +/- 67 pg/ml in SHR and 297 +/- 61 in WKy (NS), whereas epinephrine was 1079 +/- 117 in SHR and 695 +/- 107 in WKy (p < 0.05). Total plasma normetanephrine concentration was 1825 +/- 472 pg/ml in SHR and 535 +/- 109 in WKy (p < 0.02). Since normetanephrine is mainly an extraneuronal metabolite of norepinephrine, its plasma concentration could reflect the amount of the neurotransmitter which reaches effector cell sites suggesting enhanced sympathetic function accounts for blood pressure elevation in SHR. A simple and specific radioenzymatic assay for measurement of total plasma normetanephrine in rat was described.

Red blood cells: in vivo site for transport and inactivation of biogenic amines in man and rats.

Abstract Red blood cell lysate contains catecholamines, octopamine and normetanephrine in various concentrations usually equal to or higher than respective plasma concentrations. During sympatho-adrenal activation an increase in plasma norepinephrine is associated with a simultaneous but smaller increase in lysate norepinephrine. The ratio of normetanephrine to norepinephrine is consistently higher in red cell lysate than in plasma. suggesting norepinephrine may be catabolized inside red cells. These data indicate a physiologically significant role for red blood cells in the disposition of peripheral amines and their metabolites.

Blood Pressure Response to Norepinephrine Infusion in Relationship to Plasma Catecholamines and Renin Activity in Man

B ECAUSE both norepinephrine infusion1 and sympathetic stimulation2 produce vasoconstriction and hypertension, there has been speculation about the role of norepinephrine in essential hypertension. In hypertensive patients urinary catecholamine metabolites have been reported as normal, elevated, or low,3 while recent reports failed to show uniformly elevated plasma catecholamines in essential hypertension when age-matched groups were studied.7’8 It has been shown for many years that neurovascular reactivity to various stimuli9’4 is increased in essential hypertension. Both the intravenous and intraarterial administrations of norepinephrine were associated with an increase in vascular reactivity with essential hypertension, but not with renal hypertension.’#{176} However, in these studies, although drug doses are calculated per kilogram of body weight, plasma concentrations of administered catecholamines were not measured. The present study was designed to examine vascular responsiveness to norepi-

Plasma normetanephrine measurements for detection of pheochromocytoma in patients with hypertension

We describe a simple and specific radioenzymatic assay for measurement of total plasma normetanephrine (NMN), which is an extension of a previously developed procedure for measuring of urinary NMN. Plasma NMN is deconjugated by acid hydrolysis at pH 1.0 and boiled for 20 min. The assay is based on the conversion of NMN to its N-methylated, tritiated derivative metanephrine (3H-MN), utilizing phenylethanolamine N-methyltransferase and S-adenosyl-[3H]methionine. The assay is rapid, sensitive and results can be obtained in less than 4 h. Many antihypertensive drugs tested did not interfere with the assay. This assay could be used for detection of pheochromocytomas in patients with hypertension.