Coupled-column liquid chromatographic analysis of catecholamines, serotonin, and metabolites in human urine.

A column-switching HPLC system was utilized for the simultaneous determination of epinephrine, norepinephrine, dopamine, serotonin, and their metabolites metanephrine, normetanephrine, 3, 4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid in human urine. The sample was injected directly onto a C18-alkyl-diol silica precolumn, which separated the analytes from matrix. The analytes were eluted from the precolumn onto the analytical column by the use of column-switching techniques and were then separated on the analytical column by means of ion-pair reversed-phase HPLC. The analytes were then oxidized to the corresponding quinones and converted into fluorescent derivatives by reaction with meso-1,2-diphenylethylenediamine.

Decreased sympatho-adrenal activity in diabetic patients with autonomic dysfunction following mental stress.

The sympathetic nervous system is of major importance in the regulation of numerous physiological functions. While it is clearly established that there is a decreased noradrenergic status in people with autonomic neuropathy, the epinephrine secretion is much more controversial. Basal and mental stress-stimulated plasma catecholamine levels were measured in 42 diabetic patients with and without pathological cardiovascular function tests and in 13 healthy, non-diabetic control subjects. In addition, the excretion of catecholamines was measured in a 24 h urine collection and compared with the stress stimulated plasma levels. During mental stress exposure a diminished epinephrine secretion was found in diabetic patients with autonomic neuropathy compared with diabetic patients without neuropathy and the healthy control group (p < 0.05: respectively). The decreased epinephrine response to mental stress was strongly correlated with a diminished urinary excretion of this "neurotransmitter' (r = 0.46; p < 0.01). Diabetic patients suffering from cardiovascular autonomic neuropathy exhibit a diminished sympatho-adrenal response following mental stress exposure. Both measurement of urinary and mental stress stimulated plasma epinephrine levels following mental stress provide reliable information regarding sympatho-adrenal activity in diabetic patients.

Determination of anti GAD65 autoantibodies with an ELISA before and after standardization with the new international reference serum.

The serum of a stiff-man syndrome patient was declared international GAD reference standard at the "1st GAD Antibody Workshop" held at the "12th International Immunology and Diabetes Workshop" in Orlando, Florida, USA 1993. A comparative study was performed with 123 diabetic and non-diabetic patients to evaluate whether standardization of this reference serum had changed the properties of a commercially available ELISA assay. All samples classified positive with the old test were confirmed with the new assay. Four additional samples with high "normal" values became positive with the new test. One of them was a control person having a family history of diabetes and genetic loci DR4/DR11. These findings might implicate a higher risk for the development of IDDM. The new standardization and adaptation of the ELISA seems to have influenced the sensitivity of the test positively.

The effect of transportation stress on circulating corticosteroids, enzyme activities and hematological values in laboratory dogs.

Stress during transportation was monitored in truck-transported Beagle dogs "IVTK HD". Cortisol (F), corticosterone (B), red and white blood cell parameters, and enzyme activities in plasma were estimated. Plasma levels of cortisol and corticosterone were increased significantly during transportation and returned to basal values overnight post arrival. Hematocrit, hemoglobin, erythrocytes and leukocytes increased moderately. No obvious differences in enzyme activities were observed. Only alpha-HBDH values dropped significantly at the end of the 9.5 h transport period. This study confirms, although individual reaction is remarkably different, transportation as a potent stressor for Beagle dogs.

ACTH increases noradrenaline release in pithed rabbits with electrically stimulated sympathetic outflow.

ACTH 0.03-1 microgram/kg per min i.v. increased the noradrenaline spillover rate (the rate at which endogenous noradrenaline enters into plasma) and the plasma noradrenaline concentration in pithed rabbits with electrically stimulated sympathetic outflow. ACTH 0.1 and 1 microgram/kg per min decreased the mean arterial pressure (MAP). The effects of ACTH persisted in animals treated with propranolol. Corticosterone 10 micrograms/kg per min had no effect on the neurochemical and circulatory parameters. ACTH 0.03 and 1 microgram/kg per min increased plasma corticosterone and cortisol concentrations; the two doses of ACTH had approximately the same effect. The plasma corticosterone concentration reached after infusion of corticosterone 10 micrograms/kg per min was about twice that obtained after ACTH 0.03 or 1 microgram/kg per min. In a second series of experiments, a pressor dose of noradrenaline (1 or 2 micrograms/kg per min) was infused i.v. into pithed rabbits. ACTH 0.03 and 1 microgram/kg per min decreased blood pressure and increased heart rate in these animals. The results suggest that high doses of ACTH increase noradrenaline release by an action on postganglionic sympathetic neurons. The effect is probably not mediated through adrenal steroids. In addition, ACTH seems to decrease MAP and to increase heart rate through postsynaptic vascular and myocardial effects.

Destruction of the preganglionic nerves by beta-bungarotoxin does not interfere with normal embryonic development of the rat adrenal medulla.

Using beta-bungarotoxin (beta-BTX) as a tool to eliminate the preganglionic cholinergic nerve supply to the embryonic rat adrenal gland, we have investigated whether or not these nerves affect the differentiation of embryonic chromaffin cells (pheochromoblasts). Rat fetuses received a single injection of 1 or 2 micrograms beta-BTX or an identical volume of saline at embryonic day (E) 17 and were taken for morphological and biochemical analyses at E 21. Administration of beta-BTX caused a 15 to 20% reduction in body weight, crown-rump-length and adrenal weight. Spinal cord development was reduced and acetylcholinesterase-positive cells in ventral and lateral columns were virtually absent in toxin-treated animals. In adrenal glands, a decrease of choline acetyltransferase activity to 13% of control levels and a concomitant decrease of ultrastructurally identifiable nerve fibers and axon terminals revealed that application of 2 micrograms beta-BTX effectively reduced the neuronal input to E 21 adrenal glands. Values for total adrenal catecholamines, relative amounts of adrenaline and noradrenaline, tyrosine hydroxylase and phenylethanolamine N-methyltransferase activities were unaltered. All ultrastructural features of pheochromoblasts (except the lack of synapse-like axon terminals) were inconspicuous. Corticosterone levels in adrenals and plasma were identical to controls. These data strongly suggest that normal embryonic development of adrenal chromaffin cells does not require an intact nerve supply.

HPLC isolation and GC-MS characterization of a compound strongly cross reacting with tetrahydroaldosterone antiserum.

Urines from patients with hypertension and elevated aldosterone levels, i.e. primary aldosteronism due to adrenal adenoma or hyperplasia or carcinoma were extracted, paper chromatographed and thereafter chromatographed repeatedly with normal phase and repeatedly with reversed phase HPLC systems in an attempt to find new metabolites of aldosterone. Specific 3 alpha-hydroxy-5 beta-tetrahydroaldosterone antiserum was used in a radioimmunoassay system to detect possible aldosterone metabolites in the HPLC fractions after each isolation step. The immunoactive HPLC fractions were derivatized and analysed by GC-MS. A relatively nonpolar compound, 11 beta:18(S),18:20 alpha-diepoxy-5 beta-pregnan-3 alpha-ol, was isolated and identified in this manner. This material was originally described by Kelly et al., in 1962 after loading human subjects with huge amounts (25-160 mg) of exogenous aldosterone. This material has not yet been described from endogenously produced aldosterone. Very small amounts, if any, were similarly isolated from the urine of a control subject. Therefore, this compound could prove to be a new marker for hypertension due to hyper-production of aldosterone.

Development of plasma 21-deoxycortisol radioimmunoassay and application to the diagnosis of patients with 21-hydroxylase deficiency.

Specific 21-deoxycortisol (21-DF) antiserum was raised in New Zealand white rabbits using a 21-DF-3,20-oxime-bovine serum albumin complex. Plasma radioimmunoassay of 21-DF was developed and used together with a radioimmunoassay of 17-hydroxyprogesterone (17-OH-P) for diagnosis of patients with 21-hydroxylase deficiency of congenital and postpubertal forms. The assays were performed in plasma extracts after isolation by paper chromatography. The response of plasma 21-DF and 17-OH-P to i.v. ACTH (25 IU) was studied in 15 adult controls and compared to 8 women with the late onset form of 21-hydroxylase deficiency and 23 women with idiopathic hirsutism. Normal 21-DF values for women were 6.9 +/- 3.6 ng/dl and for men 9.71 +/- 2.73 ng/dl. Newborn children (age: 3-10 days) had a value of 8.3 +/- 4.8 ng/dl. These values are definitely lower than the lowest value ever published. This is possibly due to the specificity of the antibody. During the menstrual cycle the 21-DF values did not change. The baseline and post-stimulated concentrations of hormone were similar in controls and women with hirsutism but were significantly higher in women with the late onset form of 21-hydroxylase deficiency. In the congenital form of 21-hydroxylase deficiency the 21-DF values (baseline) were high. In general, the 21-DF and 17-OH-P values have shown parallel changes. However, one case of 21-hydroxylase deficiency with elevated 21-DF but normal 17-OH-P was observed. The use of 21-DF for the diagnosis of 21-hydroxylase deficiency is suggested.

[Human acid secretion during daily administration of H2-blockers]. / Menschliche Säuresekretion unter täglich einmaliger Gabe von H2-Blockern.

The potency and duration of a single dose of ranitidine and famotidine were compared in placebo-controlled studies. In addition, the effect of a single night-time dose of famotidine (40 mg) on gastric acid secretion and basal hormone levels was assessed before, during and after a 28-day treatment. Basal acid secretion was depressed by about 73% and 76% 12 hours after 300 mg ranitidine and 40 mg famotidine respectively. Pentagastrin-stimulated acid output was reduced by about 26% and 29%. 20 hours after both drugs, basal secretion was still inhibited by about 60%, whereas no effect on stimulated acid secretion could be detected. Nocturnal gastric acidity (23.00-07.00) was inhibited from 35.8 +/- 4.6 mmol/l to 1.8 +/- 0.5 mmol/l (94% inhibition) by 40 mg bedtime famotidine , and to 1.7 +/- 0.5 mmol/l (95% inhibition) by 300 mg ranitidine. Both drugs significantly reduced H+ concentrations during the following day. On day 29, i.e. 12 hours after the last famotidine dose, basal and stimulated acid secretion was reduced by some 50% and 26% respectively. On day 35, gastric acid output had returned to pretreatment values. Basal levels of prolactin, testosterone etc. were unchanged by 28-day famotidine treatment. Rantidine and famotidine may therefore be used as a single night-time dose in the acute treatment of peptic ulcer disease.

[4 weeks' administration of omeprazole: effect on acid behavior and basal hormone levels]. / Vierwöchige Omeprazol-Gabe: Einfluss auf Säureverhalten und basale Hormonspiegel.

We have assessed the effect of omeprazole (30 mg daily) on gastric acid secretion as well as on basal hormone levels (fasting gastrin; TSH, T3, T4, TBG; insulin, glucagon, C-peptide; prolactin, testosterone, 17-beta-oestradiol, dihydroepiandrosterone, cortisol and PTH) in 8 healthy volunteers before and after a 28 day treatment. On day 29, i. e. one day after the last omeprazole dose, mean stimulated acid output was still reduced from 27.4 +/- 3.5 mmol H+/h (+/- SEM) to 7.8 +/- 1.4 mmol H+/h (72% inhibition). Fasting gastrin levels were raised from 55.5 +/- 6.8 pg/ml to 80.9 +/- 6.7 pg/ml (33% increase). On day 39, stimulated gastric acid secretion and fasting gastrin levels have been returned to pretreatment values. Basal levels of prolactin, testosterone, TSH, T3, T4, TBG, cortisol, PTH, 17-beta-oestradiol, insulin, glucagon, c-peptide, dihydroepiandrosterone remained unchanged by a 28-day omeprazole treatment. Omeprazole is a highly effective antisecretory compound without any effect on the basal hormone levels tested. Even after 28 days its effect on acid secretion and fasting gastrin levels was fully reversible.

Hypothalamo-pituitary-gonadal axis in children with chronic renal failure.

Delayed puberty in children with chronic renal failure (CRF) may be due to gonadal dysfunction, increased plasma binding of gonadal hormones, or changes of the hypothalamo-pituitary axis. Plasma androgens were studied in 17 prepubertal boys with preterminal CRF. In addition, the response of luteinizing and follicle-stimulating hormones (LH, FSH) to luteinizing-releasing hormone (LHRH) was followed in the plasma of these boys and of 12 prepubertal girls with CRF. Plasma testosterone (T) was significantly lower in the CRF boys than it was in the controls (mean, 9 vs. 22 ng/ml) and concerned also the free T fraction (2.5% in both groups). Dihydro-T was similarly reduced in CRF, resulting in a normal T/DHT ratio. Basal plasma LH levels were significantly elevated in boys (1.0 vs. 0.5 ng/ml) and in girls with CRF (1.4 vs. 0.4 ng/ml), whereas mean basal FSH values were similar to controls. After LHRH administration, peak levels of LH and FSH were not different in CRF and control children; however, the absolute differences from basal to peak values were lower in CRF. These findings may indicate that Leydig cell dysfunction in CRF already occurs before the onset of puberty. The blunted LH and FSH responses to LHRH suggest an additional disturbance at the hypothalamo-pituitary level.

Aldosterone metabolites and possible aldosterone precursors in hypertension.

The value of the urine tests: free aldosterone, aldosterone-18-glucuronide, tetrahydroaldosterone 18-hydroxycorticosterone and 18-hydroxydeoxycorticosterone in distinguishing primary aldosteronism from essential hypertension was studied in patients with typical and atypical primary aldosteronism and in patients with essential hypertension. The discriminating function of the tetrahydroaldosterone determination was the best, followed by 18-hydroxycorticosterone, free aldosterone and aldosterone-18-glucuronide. The measurement of 18-hydroxydeoxycorticosterone was without distinguishing value. Three cases with hypertension, adrenal adenoma, elevated 18-hydroxycorticosterone but normal aldosterone values were observed. In longitudinal studies the excretions of aldosterone, aldosterone metabolites and possible precursors periodically varied independently of each other. Determinations of urine aldosterone, aldosterone metabolites, 18-hydroxycorticosterone and 18-hydroxydeoxycorticosterone were not applicable for differential diagnosis of the adenoma and hyperplasia forms of primary aldosteronism.

Characterisation of aldosterone metabolites cross reacting with aldosterone and tetrahydroaldosterone antibodies.

Metabolites of aldosterone were extracted from human urine collected over three days following the intravenous injection of a tracer dose of tritium labelled hormone. After enzymic hydrolysis, steroids were separated by column, paper and thin-layer chromatography and the polarities of the labelled metabolites were compared with the chromatographic properties of known aldosterone products. The pattern of metabolites changed over the three days, from that associated with typical aldosterone metabolites, to less polar metabolites. Materials in the organic extract from a pH-1 hydrolysate of pooled pregnancy urine, were located by their ability to bind with aldosterone and tetrahydroaldosterone antisera and exhibited similar chromatographic properties to the radioactive metabolites. Using GC-MS, the identity of this immunoactive material could not be established in extracts after purification from 200 ml pregnancy urine but some synthetic derivatives of aldosterone as candidate compounds, were excluded.

Met-enkephalin inhibits mineralocorticoid production in isolated human aldosteronoma cells.

In vitro application of the morphinomimetic met-enkephalin resulted in inhibition of mineralocorticoid production by aldosterone-producing adenomas. Aldosterone, deoxycorticosterone, and corticosterone production by adrenocortical cells isolated from aldosteronomas has been studied under basal conditions and after stimulation with ACTH-(1-24). The blocking effect of met-enkephalin on the rate of aldosterone, deoxycorticosterone, and corticosterone release was significant at a concentration as low as 10(-11) M (P less than 0.001, P less than 0.01, and P less than 0.001, respectively). Dose-dependent inhibition of steroid biosynthesis became more apparent with increasing amounts of met-enkephalin in the incubation medium (10(-11)-10(-5) M); at a concentration of 10(-5) M, met-enkephalin decreased the production of aldosterone by 45%, that of deoxycorticosterone by 51%, and that of corticosterone by 44%. Increased steroid biosynthesis stimulated by ACTH-(1-24) was also significantly blocked by met-enkephalin. In a concentration of 10(-5) M, met-enkephalin produced significant decreases in aldosterone (P less than 0.001), deoxycorticosterone (P less than 0.05), and corticosterone (P less than 0.001) production compared to the peak values obtained after stimulation with 0.85 X 10(-10) M ACTH-(1-24). These data allow us to conclude that the inhibitory effect of met-enkephalin on mineralocorticoid production exerted at the level of the adrenals might be complementary to the factor(s) thought to be involved in the regulation of adrenal steroid production, playing a role similar to that of the biogenic amines originating in the adrenal medulla and regulating the adrenal cortex by a peripheral neurohumoral paracrine mechanism.

Direct radioimmunoassays for "aldosterone" and "18-hydroxycorticosterone" in unprocessed urine, and their use in screening to distinguish primary aldosteronism from hypertension.

For distinguishing primary aldosteronism from essential hypertension, we use simple direct radioimmunoassays for "aldosterone" (aldosterone and other materials that react with the antibody to aldosterone) and "18-hydroxycorticosterone" (similarly) in unprocessed urine. Patients with primary aldosteronism have high values for "aldosterone." This diagnosis can be validated by assays of further urine samples from the same person and by additional direct assays for "18-hydroxycorticosterone." In none of 65 urine samples from 26 patients with primary aldosteronism were both "aldosterone" and "18-hydroxycorticosterone" values within their reference intervals. However, a few "aldosterone" and "18-hydroxycorticosterone" values for patients with essential hypertension and normal aldosterone excretion were also (moderately) increased. Thus, when high values are found, true aldosterone values must be estimated by extraction and chromatography, to eliminate false positives. The "aldosterone" and "18-hydroxycorticosterone" values by our procedure are much higher than the corresponding values for urinary free aldosterone and 18-hydroxycorticosterone. Although not identified, the immunoactive materials are probably metabolites of aldosterone and 18-hydroxycorticosterone.

Increased excretion of 18-hydroxycorticosterone in patients with adrenal adenomas and hypertension.

Two female patients, 54 and 34 years old, each presented with an adrenal adenoma and hypertension. Blood pressure fell after removal of the tumors. The first patient had high urinary 18-hydroxycorticosterone and periodically elevated 18-hydroxy-deoxycorticosterone excretions. The second patient had elevated 18-hydroxycorticosterone and free cortisol excretions. Urinary aldosterone, aldosterone metabolites and plasma aldosterone were not increased. Plasma renin activity was suppressed and serum potassium levels were normal. After surgery, no elevated steroid values were found. Elevated 18-hydroxycorticosterone excretion may be an indicator of yet unknown hypertensinogenic mechanisms. The role of 18-hydroxycorticosterone in the etiology of hypertension is still unknown.

[Plasma concentration and systemic effect of betamethasone after intra-articular injection (author's transl)]. / Plasmakonzentration und systemische Wirkung von Betamethason nach intraartikulärer Injektion.

Plasma concentrations of betamethasone, cortisol and corticosterone were measured before and after intraarticular injection of a betamethasone-depot preparation (Celestan-Depot) by radioimmuno-assay in 31 patients. Plasma concentration of betamethasone reached its maximum of between of 10 and 17 microgram/dl 30 min after injection. It had fallen to half after 2 hours, and practically to nil from the eighth day onwards. Lowest plasma levels of cortisol and corticosterone occurred after 6--24 hours, returning to the normal range after four days. In nine patients with knee-joint effusion and synovitis the plasma concentration of betamethasone was significantly higher after 24 hours, and cortisol and corticosterone values after 48 hours significantly more suppressed, than in patients without joint effusions and signs of inflammation. The results indicate that plasma concentration of betamethasone and the suppressant effect on the adrenal cortex after intra-articular injection is similar to that after intramuscular applications. Correspondingly, systemic application of the cortisol derivatives can cause significant side effects and be contra-indicated also after intra-articular injection.

Dissociation in the excretion of different aldosterone metabolites and unmetabolized ('free') aldosterone in hypertension.

1. The determination of aldosterone-18-glucuronide (pH 1-labile aldosterone) was complemented by concomitant measurements of free urinary aldosterone and tetrahydroaldosterone in 307 patients, most of whom were hypertensive. In 38 cases (12.3%) the normal, aldosterone-18-glucuronide concentration was clinically misleading, but increased free aldosterone and/or tetrahydroaldosterone values suggested the presence of hyperaldosteronism, which in many of these cases was confirmed by elevated excretion of the possible major aldosterone precursor 18-hydroxycorticosterone (18-OH-B). 2. Of 224 patients with essential hypertension and normal or low plasma renin activity 18 had an elevated free aldosterone and/or tetrahydroaldosterone excretion without increased aldosterone-18-glucuronide. These cases may represent early or pre-symptomatic forms of primary hyperaldosteronism. In other cases, particularly when tetrahydroaldosterone was increased alone, abnormalities of aldosterone metabolism were suspected. 3. In two out of 15 patients with primary hyperaldosteronism, aldosterone-18-glucuronide values were frequently found to be normal, although elevations were noted in other variables. However, no relation to the morphological abnormality (adenoma versus hyperplasia) was seen.