Harry R. Keiser

II. Validity and reliability of liquid chromatography with electrochemical detection for measuring plasma levels of norepinephrine and epinephrine in man

Abstract Liquid chromatography with electrochemical detection (LCEC) provides a rapid, sensitive, and specific technique for measuring human plasma norepinephrine (NE) and epinephrine (E) levels. We tested the reliability and validity of this technique against that of the catechol-O-methyl-transferase radioenzymatic (COMT-RE) assay. In healthy, resting humans, mean NE and E values were similar using the LCEC and COMT-RE techniques (311 vs. 300 pg/ml for NE; 57 vs. 52 pg/ml for E). In a series of 25 plasma samples obtained from a variety of sources, the correlation between the two methods was 0.99 for both NE and E. Coefficients of variation were similar for catecholamine levels above 100 pg/ml, but below this, the COMT-RE technique appeared to be more reliable. The advantages of the LCEC method are its speed, simplicity of sample preparation, low cost per assay, lack of use of radionuclides, and ease in trouble-shooting. The COMT-RE technique is preferable for small sample sizes or large numbers of samples. LCEC offers a reasonable alternative to the COMT-RE technique for measuring plasma norepineprhine and epinephrine.

Acetaminophen‐induced hepatic injury: Protective role of glutathione in man and rationale for therapy

Recent studies of acetaminophen‐induced liver damage in animals indicate that acetaminophen is converted in the liver to a chemically reactive arylating agent that normally is detoxified by conjugation with glutathione. When the dose of acetaminophen is large enough to deplete hepatic glutathione, however, there is extensive arylation of hepatic macromolecules and cell death. This paper presents evidence that administration of glutathione‐like nucleophiles, such as cysteamine, protects mice from arylation of hepatic macromolecules, hepatic necrosis, and death caused by the reactive acetaminophen metabolite. Additional studies indicate that glutathione may serve a similar protective function in man as in other animals. Thus, logical treatment of patients overdosed with acetaminophen might be based on cysteamine or other nucleophiles.

Bartter's syndrome: A disorder characterized by high urinary prostaglandins and a dependence of hyperreninemia on prostaglandin synthesis

Urinary prostaglandins E2 and F2alpha were measured by gas chromatography-mass spectrometry in three adult women and an adolescent girl with Bartters syndrome. On a constant metabolic diet prostaglandin E2 ranged from 293 to 1,221 ng/day (mean, 640 ng/day) and exceeded the normal range for adults of 76 to 281 ng/day in all patients. Prostaglandins F2alpha ranged from 291 to 1,061 ng/day (mean, 747 ng/day) in the adult women. Only in a young girl did prostaglandins F2alpha (1,677 ng/day) clearly exceed the normal range for adults of 422 to 871 ng/day. Treatment with indomethacin, which decreased urinary prostaglandin E-like material by 69 per cent or more, did not affect blood pressure. Plasma renin activity, which ranged from 5.2 to 22.2 ng/ml/hour (patients supine) and from 23.3 to 30.4 ng/ml/hour (patients upright), and urinary aldosterone, which ranged from 14.0 to 45.6 ng/day, decreased by 79, 65 and 52 per cent, respectively. The clearance of creatinine was lower for the eight or nine days of treatment, the balances of sodium and potassium were positive, and serum potassium was higher than in control. Ibuprofen, an inhibitor of prostaglandin synthetase which differs in structure from indomethacin, produced metabolic effects which were qualitatively similar to those of indomethacin. The results indicate that the renal synthesis of prostaglandins is increased in Bartters syndrome and that prostaglandins mediate the hyperreninemia and hyperaldosteronism which characterize the disorder. The over-production of prostaglandins by the kidney could be proximal cause of the syndrome, or secondary to intrarenal changes of an unknown nature. This study provides additional evidence for an important role for prostaglandins in the release of renin.

Malignant Pheochromocytoma: Effective Treatment with a Combination of Cyclophosphamide, Vincristine, and Dacarbazine

STUDY OBJECTIVE To determine the efficacy and toxicity of combination chemotherapy in patients with advanced, malignant pheochromocytoma. DESIGN Nonrandomized, single-arm trial. SETTING Governmental medical referral center. PATIENTS Fourteen patients with malignant pheochromocytoma confirmed by histologic tests. All patients had metastatic disease and elevated urinary catecholamine secretion. INTERVENTIONS After optimization of antihypertensive therapy, patients received cyclophosphamide, 750 mg/m2 body surface area on day 1; vincristine, 1.4 mg/m2 on day 1, and dacarbazine, 600 mg/m2 on days 1 and 2, every 21 days. MEASUREMENTS AND MAIN RESULTS Combination chemotherapy with cyclophosphamide, vincristine, and dacarbazine produced a complete and partial response rate of 57% (median duration, 21 months; range, 7 to more than 34). Complete and partial biochemical responses were seen in 79% of patients (median duration, more than 22 months; range, 6 to more than 35). All responding patients had objective improvement in performance status and blood pressure. Toxicity included expected hematologic, neurologic, and gastrointestinal effects of chemotherapy without serious sequelae. There were four minor hypotensive episodes and one minor hypertensive episode. CONCLUSIONS Combination chemotherapy with cyclophosphamide, vincristine, and dacarbazine is effective for advanced malignant pheochromocytoma. Urinary catecholamines are useful to ascertain biochemical response to therapy.

Prostaglandin biosynthesis by rabbit renomedullary interstitial cells in tissue culture. Stimulation by angiotensin II, bradykinin, and arginine vasopressin.

Rabbit renomedullary interstitial cells were isolated and grown in tissue culture. These cells synthesize 0.8 ng of prostaglandin E2 (PGE2) per microgram cellular protein per hour in monolayer tissue culture; prostaglandins A2 and F2alpha (PGA2 and PGF2alpha) biosynthesis was 10 and 5% of PGE2 biosynthesis, respectively. Arachidonic acid markedly stimulated the production of PGE2 and PGF2alpha, with conversion rates of 0.24 and 0.02%/h, respectively. Angiotensin II, hyperosmolality, bradykinin, and arginine vasopressin each stimulated PGE2 biosynthesis; maximum stimulation was 20, 3.7, 3.6, and 3.2 times basal production, respectively. PGE2 biosynthesis by the renomedullary interstitial cells was inhibited by isoproterenol, potassium, nonsteroidal anti-inflammatory agents (indomethacin, naproxen, ibuprofen, suprofen, meclofenamate, and acetylsalicylic acid), mepacrine (a phospholipase inhibitor), hydrocortisone, and cortisone. The rabbit renomedullary interstitial cell in tissue culture is a model system for the study of hormonal regulation of PGE2 biosynthesis.

Effects of drugs on human blood platelet and plasma amine oxidase activity in vitro and in vivo.

Abstract A method employing radioactive substrates has permitted sensitive assay of monoamine oxidase (MAO) activity in blood platelets and plasma. Kinetic studies indicate that while platelet and plasma MAO are distinct from that of liver mitochondria, the platelet and liver enzymes are quite similar, particularly in the characteristics of their response to inhibitors. Administration of six different MAO inhibitors including furazolidone to human subjects resulted in marked inhibition of platelet MAO, accompanied by expected changes in urinary tryptamine excretion. Effects of these drugs on plasma MAO were variable; an additional drug, isoniazid, in therapeutic dose was found to inhibit markedly plasma, but not platelet, MAO. Use of platelet MAO assay in the direct assessment of the MAO-inhibitory potency of drugs in man is suggested.

Plasma Normetanephrine and Metanephrine for Detecting Pheochromocytoma in von Hippel–Lindau Disease and Multiple Endocrine Neoplasia Type 2

BACKGROUND The detection of pheochromocytomas in patients at risk for these tumors, such as patients with von Hippel-Lindau disease or multiple endocrine neoplasia type 2 (MEN-2), is hindered by the inadequate sensitivity of commonly available biochemical tests. In this study we evaluated measurements of plasma normetanephrine and metanephrine for detecting pheochromocytomas in patients with von Hippel-Lindau disease or MEN-2. METHODS We studied 26 patients with von Hippel-Lindau disease and 9 patients with MEN-2 who had histologically verified pheochromocytomas and 50 patients with von Hippel-Lindau disease or MEN-2 who had no radiologic evidence of pheochromocytoma. Von Hippel-Lindau disease and MEN-2 were diagnosed on the basis of germ-line mutations of the appropriate genes. The plasma concentrations of normetanephrine and metanephrine were compared with the plasma concentrations of catecholamines (norepinephrine and epinephrine) and urinary excretion of catecholamines, metanephrines, and vanillylmandelic acid. RESULTS The sensitivity of measurements of plasma normetanephrine and metanephrine for the detection of tumors was 97 percent, whereas the other biochemical tests had a sensitivity of only 47 to 74 percent. All patients with MEN-2 had high plasma concentrations of metanephrine, whereas the patients with von Hippel-Lindau disease had almost exclusively high plasma concentrations of only normetanephrine. One patient with von Hippel-Lindau disease had a normal plasma normetanephrine concentration; this patient had a very small adrenal tumor (<1 cm). The high sensitivity of measurements of plasma normetanephrine and metanephrine was accompanied by a high level of specificity (96 percent). CONCLUSIONS Measurements of plasma normetanephrine and metanephrine are useful in screening for pheochromocytomas in patients with a familial predisposition to these tumors.

Effects of Dietary Sodium and of Acute Saline Infusion on the Interrelationship between Dopamine Excretion and Adrenergic Activity in Man

The effects of dietary sodium and of saline infusion on urinary dopamine and norepinephrine and on the relationship of these catecholamines to adrenergic activity were determined. In seven normal subjects on a 9-meq sodium intake, urinary dopamine and norepinephrine were 136+/-18 (SE) and 37.4+/-5.3 mug/day, respectively. When sodium intake was increased to 209 or 259 meq/day, urinary dopamine increased to 195+/-20 mug/day (P<0.01) whereas urinary norepinephrine decreased to 21.1+/-3.0 mug/day (P<0.01). Infusion of saline in seven subjects increased sodium excretion and urinary dopamine (from 2.18+/-0.22 to 2.79+/-0.19 mug/20 min, P<0.01), but decreased plasma dopamine-beta-hydroxylase by 33% and urinary norepinephrine insignificantly. The clearance of inulin and p-aminohippurate did not change significantly and filtration fraction was the same. The data indicate that an increase in dietary sodium or infusion of saline results in an apparent decrease in adrenergic activity and an increase in urinary dopamine. Dopamine excretion would thus appear to relate inversely to adrenergic activity and to parallel sodium excretion. These findings suggest a possible role for dopamine and norepinephrine in the regulation of renal sodium excretion.

Plasma dihydroxyphenylglycol and the intraneuronal disposition of norepinephrine in humans.

We examined plasma levels of the sympathetic neurotransmitter norepinephrine (NE) and its deaminated metabolite dihydroxyphenylglycol (DHPG) during supine rest in healthy human subjects and in sympathectomized patients, during physiological (tilt) or pharmacological (yohimbine, clonidine) manipulations known to affect sympathetically mediated NE release, during blockade of neuronal uptake of NE (uptake-1) using desipramine, and during intravenous infusion of NE. Healthy subjects had a mean arteriovenous increment in plasma DHPG in the arm (10%, P less than 0.05), whereas sympathectomized patients had a mean arteriovenous decrement in DHPG in the affected limb (mean decrease 21%, P less than 0.05 compared with healthy subjects). Tilt and yohimbine, which stimulate, and clonidine, which inhibits, release of endogenous NE, produced highly correlated changes in plasma NE and DHPG (r = 0.94). Pretreatment with desipramine abolished DHPG responses to yohimbine while enhancing NE responses. To attain a given increase in plasma DHPG, about a tenfold larger increment in arterial NE was required during NE infusion than during release of endogenous NE. When plasma NE was markedly suppressed after administration of clonidine, plasma DHPG decreased to a plateau level of 700-800 pg/ml. The results indicate that (i) plasma DHPG in humans is derived mainly from sympathetic nerves; (ii) increments in plasma DHPG during stimulation of NE release result from uptake of NE into sympathetic nerve endings and subsequent intraneuronal conversion to DHPG; (iii) plasma DHPG under basal conditions probably is determined mainly by net leakage of NE into the axonal cytoplasm from storage vesicles; and (iv) increments in NE concentrations at neuronal uptake sites can be estimated by simultaneous measurements of DHPG and NE during NE infusion and NE release. Measurement of NE and DHPG provides unique clinical information about sympathetic function.

Evidence for the Existence of Atrial Natriuretic Factor-Containing Neurons in the Rat Brain

Immunoreactive atrial natriuretic factor- (ANF-)positive nerve fibers and cell bodies were observed in the preoptic area, hypothalamus, mesencephalon, and pons of rats. In colchicine-treated animals a large number of immunoreactive ANF-positive cell bodies were seen in the organum vasculosum of the lamina terminalis, in several hypothalamic nuclei (e.g. periventricular, arcuate, and ventral premammillary nuclei), and in the dorsolateral tegmental nuclei of the pons. Varicose nerve fibers containing ANF were generally observed in the vicinity of the cells. These findings indicate that a widespread network of ANF-containing neurons is present in the brain.