Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Relative Efficiencies of Plasma Catechol Levels and Ratios for Neonatal Diagnosis of Menkes Disease

Background Menkes disease is an X-linked recessive neurodevelopmental disorder resulting from mutation in a copper-transporting ATPase gene. Menkes disease can be detected by relatively high concentrations of dopamine (DA) and its metabolites compared to norepinephrine (NE) and its metabolites, presumably because dopamine-beta-hydroxylase (DBH) requires copper as a co-factor. The relative diagnostic efficiencies of levels of catechol analytes, alone or in combination, in neonates at genetic risk of Menkes disease have been unknown. Methods Plasma from 44 at-risk neonates less than 30 days old were assayed for DA, NE, and other catechols. Of the 44, 19 were diagnosed subsequently with Menkes disease, and 25 were unaffected. Results Compared to unaffected at-risk infants, those with Menkes disease had high plasma DA (P < 10⁻⁶) and low NE (P < 10⁻⁶) levels. Considered alone, neither DA nor NE levels had perfect sensitivity, whereas the ratio of DA:NE was higher in all affected than in all unaffected subjects (P = 2 × 10⁻⁸). Analogously, levels of the DA metabolite, dihydroxyphenylacetic acid (DOPAC), and the NE metabolite, dihydroxyphenylglycol (DHPG), were imperfectly sensitive, whereas the DOPAC:DHPG ratio was higher in all affected than in all unaffected subjects (P = 2 × 10⁻⁴). Plasma dihydroxyphenylalanine (DOPA) and the ratio of epinephrine (EPI):NE levels were higher in affected than in unaffected neonates (P = 0.0015; P = 0.013). Conclusions Plasma DA:NE and DOPAC:DHPG ratios are remarkably sensitive and specific for diagnosing Menkes disease in at-risk newborns. Affected newborns also have elevated DOPA and EPI:NE ratios, which decreased DBH activity alone cannot explain.     

Plasma Catechols in Familial Dysautonomia: A Long-term Follow-up Study

This study tested whether familial dysautonomia (FD) involves progressive loss of noradrenergic nerves. Plasma levels of catechols, including dihydroxyphenylglycol (DHPG), norepinephrine (NE), dopamine (DA), and DOPA, were measured in 7 adult patients with FD and 50 healthy control subjects. FD patients were re-tested after a mean follow-up period of 13 years. Compared to controls, FD patients had low plasma levels of DHPG (P < 0.001), high DOPA and DA levels (P = 0.01, P = 0.0002), and high NE:DHPG (P < 0.0001), DA:NE (P = 0.0003), and DOPA:DHPG (P < 0.0001) ratios. At follow-up there were no changes in plasma levels of individual catechols; however, there were further increases in DOPA:DHPG ratios (mean 24 ± 7%, P = 0.01). In FD, plasma catechol profiles are sufficiently stable, at least over a decade, to be used as a biomarker of disease involvement. An increasing DOPA:DHPG ratio suggests slight but consistent, progressive loss of noradrenergic neurons.     

Prolonged changes in plasma concentrations of catecholamine metabolites following a single infusion of an MPTP analog

The magnitude and duration of effects of a single intravenous injection of 4'-amino MPTP, an analogue of the dopamine neurotoxin, MPTP, on plasma levels of catechols and normetanephrine were examined in conscious dogs. Plasma samples were collected prior to treatment with intravenous saline or 4'-amino MPTP.2HCl (22.5 mg/kg) and at weekly intervals for six weeks following treatment. Saline treatment had no effect on plasma levels of any of the measured compounds. Following 4'-amino MPTP, plasma DHPG fell to 14% of the pre-injection value and remained decreased for the full 6-week test period, with partial recovery by week 6 to 42% of the pre-injection value. Plasma DOPAC levels fell to 28% of pre-injection values 1 week after treatment with 4'-amino MPTP and showed no evidence of recovery during the 6-week test period. Plasma DOPA fell to 58% of the pre-injection level, while concentrations of the catecholamines NE, EPI, and DA were generally unaffected. The plasma concentration of the O-methylated NE metabolite, normetanephrine, was also unchanged by 4'-amino MPTP treatment. There were no differences in the concentrations of DA, NE or EPI within the adrenal medulla between saline and 4'-amino MPTP treated groups. This pattern of changes in plasma levels of catechols, which is consistent with presynaptic inhibition of MAO within sympathetic terminals, may be a useful indicator of exposure to MPTP-like neurotoxins.     

Chronic Blockade of Nitric Oxide Synthesis Elevates Plasma Levels of Catecholamines and Their Metabolites at Rest and During Stress in Rats

Formation of nitric oxide, an endothelium-derived relaxing factor, can be inhibited by administration of N-nitro-L-arginine methylesther (L-NAME). In the present study, the activity of the sympathoadrenal system in rats with blood pressure (BP) elevation induced by L-NAME was investigated. L-NAME was administered in a dose of 50 mg/kg, i.p. every 12 h for 4 days. Blood samples were collected via chronically inserted arterial catheters in conscious, freely moving rats at rest and during immobilization stress. Plasma epinephrine (EPI), norepinephrine (NE), and dopamine (DA), as well as catecholamine metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC) were measured by HPLC method. In L-NAME treated animals, which showed a significant increase in BP, plasma EPI levels were markedly elevated both before and during stress. Plasma NE levels were not significantly increased, however, DHPG levels, which indicate NE turnover and reuptake, were highly elevated. Plasma DA levels were not changed after L-NAME administration but DA metabolite DOPAC showed a significant elevation both under basal conditions and during stress. Thus, the present results indicate that the prolonged blockade of nitric oxide synthesis that causes arterial hypertension is associated with an activation of the sympathoadrenal system.     

Determination of DOPA,dopamine, DOPAC,epinephrine, norepinephrine, α-monofluoromethyldopa and α-difluoromethyldopa in various tissues of mice and rats using reversed-phase ion-pair liquid chromatography with electrochemical detection

A method for the determination of catecholic amino acids and amines by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection has been developed. By using octanesulfonic acid for ion pairing and by optimising ionic strength, pH and methanol concentration of the mobile phase, separation was achieved of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxypehnylacetic acid (DOPAC), norepinephrine (NE), epinephrine (EPI), and dopamine (DA). α-Difluoromethyldopa (DFMD) and α-monofluoromethyldopa (MFMD), two potent enzyme-activated irreversible inhibitors of aromatic amino acid decarboxylase were also separated from the natural catechols. Concentrations of catechols and inhibitors were measured in brains, hearts and kidneys of mice treated with small repeated doses of MFMD. The method has also been applied to the determination of catechols in other organs such as prostates and seminal vesicles of rats and in smaller tissues like mesenteric arteries. A semi-automated procedure making use of an automatic sample processor and a digital integrator permitted the analysis of as many as sixty samples per day.     

DOPA, dopamine, and DOPAC concentrations in the rat gastrointestinal tract decrease during fasting

The aim of the present study was to test the hypothesis that 3, 4-dihydroxyphenylalanine (DOPA) and dopamine (DA) in the gastrointestinal tract are to a large extent of exogenous origin and derived from food. Tissue concentrations of norepinephrine (NE), epinephrine (Epi), DA, DOPA, and 3,4-dihydroxyphenylacetic acid (DOPAC), as measured by reverse-phase HPLC with electrochemical detection, were studied in fed and 4-day-fasted Wistar rats as well as in sympathectomized and adrenodemedullated rats. Sympathectomy and adrenal demedullectomy decreased tissue concentrations of NE and Epi, respectively, but had no effect on the level of tissue DOPA. Large amounts of DOPA and DA were present in the gastrointestinal tract. Fasting decreased DOPA and DA in the stomach and DOPA concentrations in the quadriceps muscle but no concentrations in other organs. DOPAC in the heart decreased both in response to sympathectomy and to fasting, whereas DOPAC decreased in plasma after fasting and in skeletal muscle after sympathectomy. We conclude that the food content of DOPA and DA is of major importance for the metabolism of DA and, thus, for the dopamine-sulfate content in the gastrointestinal tract and in plasma. The decrease in muscle DOPA after fasting may be explained by less insulin being available during fasting for stimulation of DOPA uptake in the muscle depot. DOPAC in the organism seems to be of a dual origin, derived partly from DA in the food and partly from DA synthesized in sympathetic nerves.     

Differentiation of noradrenergic and dopaminergic neurons in the cardiovascular system

Dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and norepinephrine (NE) are present in the rat cardiovascular system. All of the catechols can be partially depleted by administering 6-hydroxydopamine (6-HODA). When animals are pretreated with desipramine before 6-HODA, there is a selective partial depletion of DA and DOPAC. NE can be partially depleted with minimal effects on DA and DOPAC by administering N-(2-chloroethyl)N-ethyl-2-bromobenzylamine (DSP-4). These results are consistent with the hypothesis that independent dopaminergic and noradrenergic elements are present in the rat cardiovascular system and that DA is not solely a precursor for NE. NE, DA and DOPAC were assayed in human vessels and the pattern of distribution of the catechols is consistent with the results reported for animals.     

A vesicular sequestration to oxidative deamination shift in myocardial sympathetic nerves in Parkinson's disease

In Parkinson's disease (PD), profound putamen dopamine (DA) depletion reflects denervation and a shift from vesicular sequestration to oxidative deamination of cytoplasmic DA in residual terminals. PD also involves cardiac sympathetic denervation. Whether PD entails myocardial norepinephrine (NE) depletion and a sequestration–deamination shift have been unknown. We measured apical myocardial tissue concentrations of NE, DA, and their neuronal metabolites 3,4‐dihydroxyphenylglycol (DHPG), and 3,4‐dihydroxyphenylacetic acid (DOPAC) from 23 PD patients and 23 controls and ascertained the extent of myocardial NE depletion in PD. We devised, validated in VMAT2‐Lo mice, and applied 5 neurochemical indices of the sequestration–deamination shift—concentration ratios of DOPAC:DA, DA:NE, DHPG:NE, DOPAC:NE, and DHPG:DOPAC—and used a kinetic model to estimate the extent of the vesicular storage defect. The PD group had decreased myocardial NE content (p < 0.0001). The majority of patients (70%) had severe NE depletion (mean 2% of control), and in this subgroup all five indices of a sequestration–deamination shift were increased compared to controls (p < 0.001 for each). Vesicular storage in residual nerves was estimated to be decreased by 84–91% in this subgroup. We conclude that most PD patients have severe myocardial NE depletion, because of both sympathetic denervation and decreased vesicular storage in residual nerves.

     

Simultaneous determination of catechols in thalamic slices with liquid chromatography/electrochemistry

A method was developed for the simultaneous determination of dopamine (DA), epinephrine (E), norepinephrine (NE), 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylglycol (MHPG), as well as L-3,4-dihydroxyphenylalanine (L-DOPA) with liquid chromatography (LC) using electrochemical (EC) detection. With a ODS column and a mobile phase consisting of a sodium acetate-citrate with heptasulfonic acid, this method was applied on simultaneous determination of catechols released from thalamic slices of ddY mouse. The pretreatment of the bathing medium required only centrifugation, and the supernatant was injected directly into the LCEC system. The high potassium stimulation of catecholaminergically innervated thalamic slices led to increase in the levels of DA, NE, DOPAC and MHPG, especially of NE, but not that of L-DOPA itself. In the present study, we designed to make simultaneous determination of catechols released from thalamic slices for estimation of the physiological status of catecholaminergic neuronal activity.     

Radioenzymatic assay of sulfate conjugates of catecholamines and DOPA in plasma

The addition of a commercially available sulfatase to the incubation mixture for the single isotope radioenzymatic assay of the catecholamines permits the simultaneous assay of the catecholamine sulfates in plasma. The sulfatase is compatible with the catechol-0-methyl transferase of the radioenzymatic assay and cleaves the sulfate to form the free catechol. With the multiple enzyme action it is possible to assay total (sulfated plus free) catecholamines and DOPA. A second series of assays performed in the absence of the sulfatase provides results for each of the free catecholamines and DOPA. In a study with ten normotensive male subjects supine free NE was 19% (range of 10–24%) of the total, E was 12% (range 6–16%), DA was 1% (range 0.5–2%) and DOPA was 45% (range 14–71%) of the total. Upon standing, free NE increased an average of 71% from 196±53 pg/ml (supine) to 336±69 pg/ml. Free NE (30%) and free E (18%) comprised a larger proportion of the total plasma NE and E in standing subjects than in the plasma of the supine subjects. Changes in the free levels and in the ratio of free to total levels of DA and DOPA were less than those noted for NE and E. This assay methodology may be useful in demonstrating individual variations in the ability to sulfate endogenous catechols.     

Asymmetric metabolism of hypothalamic catecholamines alternates with side of ovulation in a lizard (Anolis carolinensis).

We determined levels of monoamines and their metabolites in 2 hypothalami dissected from the right and left hemibrains of 15 females during the right-left alternating ovulatory cycle of Anolis carolinensis. Tissue contents of the following were measured using HPLC and electrochemical (coulometric) detection: dopamine (DA) and its metabolite 2,4-dihydroxyphenylacetic acid (DOPAC), norepinephrine (NE) and its metabolites 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxyphenylglycol (DHPG), and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). An asymmetry ratio (AR) was determined by subtracting hypothalamic content (pM/mg) on the larger ovary (LO) side from that on the smaller ovary (SO) side, divided by the sum of the 2 sides (AR = SO - LO/SO+LO). The Ar of MHPG and DHPG both decreased as the largest follicle in the LO grew during the cycle, from greater than 0 (content higher on the SO side) at the beginning of the cycle to less than 0 (content higher on the LO side). The average content of MHPG in the 2 sides significantly increased during the cycle. There were no significant asymmetric changes in hypothalamic DA or DOPAC. The average content of DA increased during the cycle, whereas the content of DOPAC, as well as DOPAC/DA, did not change. The average content of 5-HT increased, and the average metabolite ratio of 5-HIAA/5-HT decreased during the cycle without significant asymmetries. The metabolite ratios of NE and DA, but not 5-HT, were asymmetric on the same side in a given female.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transsynaptic Regulation of Olfactory Bulb Catecholamines in Mice and Rats

Norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylalanine (DOPA), and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured simultaneously by high performance liquid chromatography with electrochemical detection in extracts of olfactory bulbs at various intervals after chemical or surgical deafferentation. Chemical deafferentation of mice by intranasal irrigation with Triton X-100 or of rats by olfactory axotomy resulted in a rapid progressive decline of DA and DOPAC and an associated rise in NE in the olfactory bulb. However, after several weeks, these values returned to prelesion levels concomitant with reinnervation of the bulb by the afferent neurons. In contrast, deafferentation by procedures known to prevent reinnervation of the bulb by the afferent chemoreceptor neurons (i.e., a ZnSo4 solution in mice or a surgical procedure in rats) completely blocked the return to pre-lesion values of DA, DOPAC, and NE. The specificity of these effects was demonstrated by the inability of intranasal administration of the neurotoxin 6-hydroxydopamine to alter DA levels, resulting instead in a significant decline in olfactory bulb NE content. These data demonstrate that the DA content of the olfactory bulb can be influenced by either chemical or surgical modulation of the afferent pathway in two different species. This offers additional support for our hypothesis of transsynaptic regulation of intrinsic DA neurons of the bulb by the afferent olfactory chemoreceptor neurons.     

A method for very rapid determinations of catechols using ion-pairing reverse phase HPLC with electrochemical detection: effects of L-dopa treatment on the catechol content in various rat brain structures

A simple and rapid method for determination of 12 catechols (9 endogenous and 3 internal standards, i.s.) using ion-pairing reverse phase HPLC with electrochemical detection is presented. This study basically concentrates on the importance of optimizing the mobile phase composition in isocratic systems where ordinary 25 cm X 4.6 mm i.d. columns are used. Mobile phase compositions for three different purposes are reported: 1) separation of 9 endogenous catechols, possibly occurring in the samples, and 3 i.s. in a moderately short retention time (tR) (L-DOPA, DOPEG, alpha-Methyldopa (alpha-MeDOPA, i.s.), Noradrenaline (NA), DOPAC, Adrenaline (A), Dihydroxybensylamine (DHBA, i.s.), Norsalsolinol (NS), Dopamine (DA), Epinine (EPI), Salsolinol (S) and Isoprenaline (ISO, i.s.) within 11 min), 2) ultra rapid separation of detectable endogenous catechols except L-DOPA (NA, DOPAC, A, DHBA (i.s.), NS and DA within 5.2 min and with S within 5.7 min) and 3) moderately fast separation of detectable endogenous catechols (L-DOPA, NA, A, DHBA (i.s.), NS, DOPAC and DA within 7.6 min and with S within 10 min). By balancing the pH, concentration of organic modifier (2-propanol) and pairing ion (1-heptanesulphonic acid) as well as preconditioning new columns with more packing material (Nucleosil 5 micron C18) and to high pressures (5000 psi) for 7 days, very fast separations with good baseline resolution between the peaks are possible. The method was applied on L-DOPA treated rats (100 mg/kg), where the catechol content was analysed in 7 different brain structures during the time course of synthesis and degradation (4 hours) of catechols from L-DOPA.     

Determination of tyrosine, tryptophan and their metabolic derivatives by liquid chromatography-electrochemical detection: application to post mortem samples from patients with Parkinson's and Alzheimer's disease

A procedure is described for the rapid determination of the major indoles and catechols. Analysis with picogram detection limits was done by high-pressure liquid chromatography on a C18 reverse-phase column using electrochemical detection (LCEC). This method provides a comprehensive list of compounds which can be simultaneously determined in brain samples and for which there is no necessity of derivatization or pre-column purification. The regional distribution of 9 neurochemicals from rat brain and the levels of 10 neurochemicals from human brain are presented. DOPA, TYR, NE, MHPG, DOPAC, 5-HIAA, TRP, DA, HVA, 3-MT and 5-HT were detected in the caudate nucleus and putamen. The levels of neurochemicals from the caudate and putamen of a demented patient with Parkinson's disease were variably decreased; catechol and indole losses were greatest in the putamen. The levels of neurochemicals in the caudate and putamen of patients with Alzheimer's disease (SDAT) were also variably decreased; loss of NE was seen only in putamen and losses of DA, HVA and 5-HT were uniform across both caudate and putamen. The CSF of SDAT patients showed changes in NE only.     

Determinations of catechols in small volumes of plasma using ion-pair reversed phase liquid chromatography/electrochemistry

A method with improved sensitivity for detection of catechols (CA) in small volumes of plasma using an ion-pair reversed phase HPLC system with electromechemical detection is presented. Fast isocratic separations were obtained by using 7.5 cm x 4.6 mm (i.d.) reversed phase columns with 3C18 3 micron silica particles. The CA:s L-DOPA, Noradrenaline (NA), Adrenaline (A), Dihydroxybenzylamine (DHBA, i.s.), DOPAC and Dopamine (DA) were separated in less than 4 min. The performance of three different electrochemical cells was compared with respect to hydrodynamic voltammogram, band broadening effect, linearity and detection limit. The sample preparation procedure using alumina extraction of CA:s, was modified to improve recoveries and decrease dilution factors. A modified carbon paste cell (CP-O) gave a response 4-8 times higher than what is previously reported for GC cells. Detection limits were: L-DOPA 80, NA 1.25, A 1.25, DHBA 0.4, DOPAC 1.25 and DA 0.6 pg/injection. Application to plasma from rat and fish (cod) under rest, exercise and stress is reported. The method allows determination of CA:s in small volumes of plasma (less than 500 microliter) obtained several times a day from the same animal even if it is small (less than 1/2 kg), is under rest and parts of the plasma sample are to be used for analysis of other parameters than CA:s.     

Determination of Picomole Amounts of Dopamine, Noradrenaline, 3,4-Dihydroxyphenylalanine, 3,4-Dihydroxyphenylacetic Acid, Homovanillic Acid, and 5-Hydroxyindolacetic Acid in Nervous Tissue After One-Step Purification on Sephadex G-10, Using High-Performance Liquid Chromatography with a Novel Type of Electrochemical Detection

A determination of dopamine (DA), noradrenaline (NA), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindolacetic acid (5-HIAA) in nervous tissue is described. The method is based on a rapidly performed isolation of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA from one single nervous tissue sample on small columns of Sephadex G-10, followed by reverse-phase high-performance liquid chromatography with electrochemical detection. A new type of electrochemical detector based on a rotating disk electrode (RDE) was used. The rotating disc electrode was found to be a reliable and sensitive amperometric detector with several advantages over the currently used thin-layer cells. The detector appeared very useful for routine analysis. Practical details are given for the routine use of the RDE. Brain samples containing no more than 75-150 pg (DA, DOPA, DOPAC, HVA, and 5-HIAA) or 500 pg (NA) could be reproducibly assayed with high recovery (approx. 85%) and precision (approx. 5%), without the use of internal standards. Endogenous concentrations of DA, NA, DOPA, DOPAC, HVA, and 5-HIAA were determined in eight brain structures.     

Effect of cyclosporin A on rat kidney catecholamines

The immunosuppressive agent, Cyclosporin A, (CsA) has been associated with nephrotoxicity and hypertension. The mechanism for these effects are not known. We therefore determined the levels of the catecholamines; epinephrine (EPI), norepinephrine (NE) and dopamine (DA) and some of their metabolites; epinine, dihydroxyphenyl-acetic acid (DOPAC), homovanillic acid (HVA), metanephrine (ME) and 3-methoxy-4-hydroxy-phenylglycol (MHPG) in the kidneys of rats treated intraperitoneally with either CsA (120 micrograms/kg/body wt/day) or control vehicle (1 ml olive oil/kg body wt/day). Six control or CsA treated rats were sacrificed at 1 hour or 24 hours after a single treatment or after 7 days of daily treatment. Renal catecholamine levels were determined using HPLC-amperometric detector. Treatment with CsA increased renal NE and EPI levels by 59% and 70% respectively within 1 hour. In the rats sacrificed 24 hours after treatment, renal NE, EPI and DA levels were similar to or less than the control levels. Treatment with CsA for 7 days resulted in marginal increases in renal NE (22%) and EPI (30%). These changes were associated with a significant decrease in the levels of catecholamine metabolites in the CsA treated kidneys as compared to the controls. The above findings suggest that increases in renal catecholamines may be involved in the CsA-induced hypertension and nephrotoxicity, perhaps by increasing renovascular resistance.     

Stability of 3,4-dihydroxyphenylacetic acid in plasma extracts assayed by high-performance liquid chromatography with electrochemical detection

3,4-Dihydroxyphenylacetic acid (DOPAC) can be easily assayed by high-performance liquid chromatography (HPLC) with electrochemical detection at the same time as norepinephrine (NE), epinephrine (E), and dopamine (DA). The latter catecholamines are stable in perchloric acid extracts for over 6 h at 4°C in the dark whereas DOPAC levels drop rapidly by more than 50% in 6 h at 4°C in the dark. This study investigated the effects of reducing agents [ascorbic acid, dithiothreitol (DTT), reduced glutathione with or without a metal chelating agent (diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic acid)] on DOPAC. Extracted with alumina using 0.65 mmol/1 DTT prior to HPLC and electrochemical detection, DOPAC remained stable in the perchloric acid extract for 2 h at 4°C in the dark.     

Twenty-four-Hour Rhythms of Serum Acth,Prolactin, Growth Hormone,and Thyroid-Stimulating Hormone,and of Median-Eminence Norepinephrine,Dopamine, and Serotonin,in Rats Injected with Freund's Adjuvant

The effect of Freund's adjuvant injection on 24-h variation of circulating ACTH, prolactin, growth hormone (GH), and thyroid-stimulating hormone (TSH) levels, and of norepinephrine (NE) content, and dopamine (DA) and serotonin (5HT) turnover in median eminence, was examined in adult rats kept under light between 0800 and 2000 h daily. Groups of 6–10 animals Freund's complete adjuvant or its vehicle at 1 lOOh 3 days before sacrifice and were killed by decapitation at six different time intervals throughout a 24-h cycle. In rats injected with adjuvant's vehicle, serum ACTH and prolactin exhibited peak values around the light-dark transition (p < 0.0001 and < 0.04, respectively), while the maximum in TSH was found in the late afternoon (p < 0.0001, one-way ANOVA). GH levels did not vary on a 24-h basis. In Freund's adjuvant-injected rats, 24-h variations of TSH levels became blunted, while 24-h variations of prolactin and ACTH persisted. Freund's adjuvant augmented serum ACTH and prolactin levels, and decreased GH and TSH levels (p < 0.0007, factorial ANOVA). Median-eminence NE content, and turnover of DA, assessed by measuring dihydroxyphenylacetic acid, DOPAC/DA ratio, and of 5HT, assessed by measuring 5-hydroxyindoleacetic acid, HIAA/5HT ratio, varied on a 24-h basis in rats receiving adjuvant's vehicle (p < 0.02). Median-eminence NE content attained its maximum at 1600–2000 h, while maxima in DOPA/DA and HIAA/5HT ratios occurred at 0400 h. Injection with Freund's adjuvant reduced the amplitude of the daily variation of NE content, shifted the maximum of DOPAC/DA ratio toward the light-dark transition, and blunted the daily variation in HIAA/5HT ratio in median eminence. The administration at 1200 of the immunosuppressant drug cyclosporine (5 mg/kg, 5 days) restored the augmented ACTH and prolactin levels (p < 0.0001, factorial ANOVA) and depressed GH and TSH levels (p < 0.02) found in Freund's adjuvant-injected rats. Cyclosporine was also effective in restoring 24-h rhythmicity of serum ACTH and TSH, but not of prolactin, levels. Cyclosporine did not modify the effect of Freund's adjuvant on time-of-day changes of median-eminence NE content, but it was effective in counteracting the changes of DA and 5HT turnover found after immunization. The results are compatible with a significant effect of immune-mediated inflammatory response at an early phase after Freund's adjuvant injection on ACTH, GH, prolactin, and TSH release, which is partially sensitive to immunosuppression by cyclosporine. (Chronobiology International, 14(3), 253–265, 1997)