Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10–10 000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17°C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.     

Routine determination of plasma catecholamines using reversed-phase,ion-pair high-performance liquid chromatography with electrochemical detection

A procedure is described for the determination of plasma catecholamines using reversed-phase, ion-pair high-performance liquid chromatography coupled with electrochemical detection. Optimisation of chromatographic conditions with respect to detector performance and adherence to procedures and precautions described, render the method applicable to both neurochemical research and routine clinical analysis. The limit of quantitative detection of the method was found to be approximately 30 pg per injection for individual catecholamines. A single chromatographic run, providing adequate resolution of each component, could be completed in approximately 12 min.     

Determination of catecholamines in tissue and body fluids using microbore HPLC with amperometric detection

Performance of microbore reverse phase HPLC coupled with amperometric detection is detailed for the analysis of catecholamines in small tissue samples and human blood plasma and cerebrospinal fluid. Extraction procedures for pre-concentration and clean-up of these samples are described. Marked signal enhancement is observed due to the smaller column volume as well as the increased coulometric yield which results from the lower flow rates used with this technique. Detection limits of 0.2 to 0.5 picograms are obtained allowing analysis of catecholamines in extremely small tissue samples or small volumes of cerebrospinal fluid or plasma.     

Advances in Biochemical Screening for Phaeochromocytoma using Biogenic Amines

Biochemical testing for phaeochromocytoma is performed in diagnostic laboratories using a variety of tests with plasma, serum or 24-hour urine collections. These tests include catecholamines and their methylated metabolites - the metanephrines, either individually or in combination with their sulfated metabolites. High-performance liquid chromatography (HPLC) continues to be the dominant analytical method for biogenic amine quantitation. Chromatographic techniques are changing, with improvements in sample preparation procedures, column technology and more specific analyte detection using tandem mass spectrometry. Enrolments in quality assurance programs indicate that there are still many more laboratories in Australasia analysing urinary catecholamines than metanephrines. Nevertheless, clinical evidence and expert opinion favour metanephrines as the analytes with highest sensitivity for the detection of phaeochromocytoma. Practical issues such as better chemical stability and easier specimen collection also favour metanephrines over catecholamines. For these reasons, it is likely that laboratories increasingly will replace urine catecholamine testing with either plasma or urine metanephrines. However in interpreting positive results, the need remains to consider issues such as pre-test probability and use of potentially interfering medications.     

Determination of catecholamines in human plasma by high-performance liquid chromatography with electrochemical detection

In the present study, assays were improved for the determination of catecholamines in human plasma. High-performance liquid chromatography with electrochemical detection was employed for quantitative analysis. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase, and the detection potential, was investigated. An accurate solid-phase extraction procedure, after catecholamine complexation with diphenylborate, was developed. The efficiency yield for all catecholamines was in the range 92–98%. Relative standard deviation values for repeatability and for intermediate precision were less than 2% and 3%, respectively, for all three analytes.     

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 plasma and lysate. This method is useful in detecting total (free + sulfated + glucuronidated) catecholamines and the nature of conjugated catecholamines.     

High-pressure liquid chromatographic analysis of hemolymph plasma catecholamines in immune-reactive Aedes aegypti

Tyrosine and catecholamines have been implicated as substrates for the encapsulation reactions involved in the immune response of mosquitoes to microfilariae (mff). Identification and quantitation of tyrosine and catecholamines present in Aedes aegypti hemolymph plasma were accomplished by ion-pair high-pressure liquid chromatography with electrochemical detection at either +650 or +850 mV vs Ag/AgCl. Tyrosine, dopamine, and N-beta-alanyldopamine were detected in the hemolymph plasma of naive A. aegypti. Although no differences in these compounds were observed in hemolymph plasma from A. aegypti inoculated with Dirofilaria immitis mff, the chromatogram showed a single major peak (PI) (65 microM, expressed as dopamine equivalents) that was not present in naive hemolymph plasma. Saline-inoculated controls contained only 5% of the PI in immune reactive hemolymph plasma. A high concentration of PI (127 +/- 39 microM) was also detected after treatment of hemolymph plasma with mild alkaline conditions (pH 9.0), indicating that it is normally present as an electrochemically inert form in naive mosquitoes. High concentrations of PI were also detected in the naive hemolymph plasma from three other mosquito species, but no PI was found in A. trivittatus under any conditions. PI did not cochromatograph with any of the catecholamines commonly thought to be involved in immune responses of dipterans against metazoan parasites, suggesting that it may be a unique substrate for these reactions. The biological relevance of PI was evidenced by its appearance in the hemolymph plasma of two strains of D. immitis-inoculated A. aegypti.     

Free and conjugated plasma catecholamines, DOPA and 3-O-methyldopa in humans and in various animal species

The aim of the present study was to determine the extent to which plasma catecholamines are conjugated in different animals compared to man and how widespread is the presence of dihydroxyphenylalanine (DOPA) and 3-methoxy-4-hydroxyphenylalanine (3-OMD) in plasma among the different animal species. Free and conjugated norepinephrine, epinephrine, and dopamine were measured in plasma in humans and in several animal species (dog, rat, Gunn rat, cat, rabbit, guinea pig, African green monkey, young pig, calf, and one American black bear) using HPLC with electrochemical detection. The same technique was used to measure free and conjugated DOPA and 3-OMD in plasma of man, dog, rat, Gunn rat, calf, and American black bear. Human plasma contains the highest concentration of total (free and conjugated) catecholamines (46.1 pmole/ml), while low concentrations (below 15 pmole/ml) were observed in unstressed rats, calves, cats, and young pigs. In man, 95.3% of total plasma catecholamines were conjugated. The extent to which plasma catecholamines were conjugated varied greatly between animal species. The conjugated fraction expressed as percentages of the total catecholamines is lowest in the young pig (4.7%) and highest in the bear (100%). Conjugated dopamine was present in the plasma of all species, varying between 3% of the total catecholamine pool in young pig to 90% in dog. Conjugated norepinephrine was also present in plasma of all species except in unstressed rats with access to food. Conjugated epinephrine was detected only in cat and rat. Free DOPA and 3-OMD were present in plasma of all tested species with especially high levels of 3-OMD being present in dog. Conjugated DOPA and 3-OMD were not consistently found in any species. Our results indicate that man, dog, bear, and African green monkey are particularly good catecholamine conjugators and that young pig, guinea pig, rabbit, and calf are poor conjugators.     

Determination of plasma catecholamines by high performance liquid chromatography with electrochemical detection: comparison with a radioenzymatic method.

High performance liquid chromatography with electrochemical detection has been compared to a radioenzymatic method for the determination of plasma catecholamines. With the use of an internal standard highly accurate determinations of plasma noradrenaline, adrenaline and dopamine were performed on 0.2–2 ml plasma with the chromatographic method. The radioenzymatic method required only 3 × 50 μl plasma. A comparison of noradrenaline and adrenaline concentrations measured by the two methods in a set of nine plasma samples showed an excellent agreement between the methods (r=0.993 and 0.994, respectively). Advantages and disadvantages with the two methods are discussed.     

Use of plasma catecholamines in diagnosing pheochromocytoma and neuroblastoma

The diagnosis in two cases of neural crest tumours was confirmed by the measurement of concentration of plasma catecholamines. A histamine provocative test monitored by values of plasma catecholamines provided support for a diagnosis of pheochromocytoma in a patient with normal blood pressure and elevated values of urinary amines. In the second case the presence of a ganglioneuroblastoma secreting abnormal amounts of catecholamines was detected by plasma epinephrine and norepinephrine measurements when values of urinary free catecholamines were normal.     

Is the platelet phenolsulfotransferase involved in the sulfoconjugation of plasma catecholamines?

Catecholamines are predominantly present in the sulfoconjugated forms in human plasma. Phenolsulfotransferase (EC 2.8.2.1), which catalyses the sulfation of phenolic compounds, is widely distributed in human tissues. In blood, a phenolsulfotransferase, more specific for catecholamine sulfation is found exclusively in platelets. Free and sulfoconjugated catecholamines were measured in plasma and platelets of healthy volunteers and compared with those present in patients with uremia or pheochromocytoma to determine the ability of platelet phenolsulfotransferase to sulfurylate plasma catecholamines. In patients with pheochromocytoma, the rise in free and sulfoconjugated plasma catecholamines is accompanied by a simultaneous rise of these molecules in platelets. In uremia, where the level of plasma catecholamines is normal, the rise in the sulfoconjugates is not accompanied by a concomitant increase in either free or sulfoconjugated catecholamines in platelets. Platelet phenolsulfotransferase activity remains unchanged in pheochromocytoma and uremia. These data indicate that the platelet phenolsulfotransferase is involved in the sulfation of the catecholamines present in platelets, but its contribution, if any, to the high level of sulfoconjugated catecholamines found in plasma is negligible. This assertion is confirmed by our observations in thrombocytopenic patients. Indeed, despite the very low number of platelets and the absence of plasma phenolsulfotransferase activity, thrombocytopenic patients have normal plasma levels of free and sulfoconjugated catecholamines.     

Quantitation of 3,4-dihydroxyphenylacetaldehyde and 3, 4-dihydroxyphenylglycolaldehyde, the monoamine oxidase metabolites of dopamine and noradrenaline, in human tissues by microcolumn high-performance liquid chromatography.

We recently described the chemical synthesis of 3, 4-dihydroxyphenylacetaldehyde and 3,4-dihydroxyphenylglycolaldehyde, the monamine oxidase metabolites of dopamine and noradrenaline, respectively. We demonstrated the neurotoxicity of these compounds. Catecholamine nerve cells which synthesize these aldehydes die in degenerative brain diseases, such as Parkinson's and Alzheimer's. Here we describe a sensitive method for separating these catecholaldehydes from catecholamines and their other oxidative and methylated metabolites by microcolumn high-performance liquid chromatography with electrochemical detection. We then quantitate catecholamines and their major metabolites in human brain, plasma, and urine. The method can be used to determine the role of these catecholaldehydes in human disease.     

Modulation of insulin receptors and catecholamines in rat brain in hyperglycemia and hyperinsulinemia

Insulin receptor activity and its relationship with catecholamines and serotonin were investigated in rat whole brain membranes, synaptosomes and choroid plexus in alloxan induced short term and long term hyperglycemia and hyperinsulinemia. Insulin receptor activity was measured by [125I]insulin binding and catecholamines by high performance liquid chromatography with electrochemical detection. While choroid plexus insulin receptors modulate along with norepinephrine, dopamine and serotonin with the changes in insulin and/or plasma glucose levels, insulin receptor activity in synaptosomes and total membranes is not affected to a great extent except in long term hyperglycemia.     

Simultaneous analysis of human plasma catecholamines by high-performance liquid chromatography with a reversed-phase triacontylsilyl silica column

The clinical importance of simultaneous analysis of 3,4-dihydroxyphenylglycol with other human plasma catecholamines has been investigated to better understand the sympathetic nervous system. However, previous reports have had analytical difficulties with both resolution and extraction. The current study uses a reversed-phase triacontylsilyl silica (C30) column under the mobile phase condition without ion-pair reagents to separate catecholamines and their metabolites, with above 91% recoveries for intra-assay, above 85% for inter-assay, and less than 10% (n=5) coefficient of variation. Lower detection limits (S/N=4) and quantification limits (S/N=6) were 40 and 100 pg/mL for norepinephrine, 3,4-dihydroxyphenylglycol, and 3,4-dihydroxyphenylalanine, 10 and 20 pg/mL for epinephrine, 10 and 40 pg/mL for dopamine. Linear ranges were from 40 to 5000 pg/mL for norepinephrine and 3,4-dihydroxyphenylalanine, from 100 to 5000 pg/mL for 3,4-dihydroxyphenylglycol, and from 10 to 2000 pg/mL for epinephrine and dopamine. The C30 column may prove clinically useful, as it provides a convenient and simultaneous method of evaluation of human plasma catecholamines.     

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.     

Changes in plasma free and sulfoconjugated catecholamines before and after acute physical exercise: experimental and clinical studies.

To elucidate whether sulfoconjugated catecholamines in plasma, especially dopamine, serve as a source of free catecholamines, we examined the change in afterload on the deconjugating activity of catecholamines in isolated Langendorff perfused rat hearts. Dopamine-sulfate was administered under ordinary or high-work-load conditions. Free dopamine in the effluent was increased by the high-work-load of the hearts, whereas conjugated dopamine showed an apparent decrease. These results indicate the possibility that deconjugation of sulfoconjugated catecholamines is accelerated by a high-work-load. To obtain further evidence in humans, we also examined the changes in the plasma levels of free and sulfoconjugated catecholamines in healthy volunteers before and after marathon running. Free dopamine increased 1.99-fold from the baseline value after exercise, whereas conjugated dopamine decreased by 12%. Similarly, the plasma levels of free noradrenaline and adrenaline increased after exercise to 2.45- and 1.51-fold their respective baseline values, while conjugated noradrenaline and adrenaline both decreased. These clinical results, as well as those of the experimental studies, suggest that the increase in plasma free catecholamines after exercise is due not only to increased release from the sympathoadrenal system but also to accelerated conversion from sulfoconjugated catecholamines in the plasma.     

Measurement of catecholamines in biological fluids by high-performance liquid chromatography : A comparison of fluorimetric with electrochemical detection

An improved method for the determination of catecholamines in biological fluids, by reversed-phase high-performance liquid chromatography (HPLC) with fluorimetric detection is presented. The pH titration previously employed in the alumina extraction was abandoned in favour of the use of a molar excess of pH 8.5 Tris—HCl buffer. A novel lyophilisation step serves to concentrate the catechols and by reconstituting in mobile phase, chromatography disturbances are minimised. The addition of 2 mM octanesulphonic acid to a citrate—phosphate mobile phase at pH 6.0 gave optimal resolution and sensitivity.That HPLC separation can improve the specificity of the trihydroxyindole reaction, to the extent of providing a reliable analytical method, has been demonstrated and validated by the technique of HPLC with electrochemical detection. A correlation coefficient of 0.98 was obtained between the two techniques as applied to the measurement of urinary catecholamines. The HPLC—fluorimetric method was sensitive enough to measure 0.1 ng/ml of noradrenaline or adrenaline at a signal-to-noise ratio of 2.0. Application of the method to the quantitative determination of catecholamines in human urine, plasma and rat brain homogenates is demonstrated.     

Plasma catecholamines in resting rainbow trout,Salmo gairdneri Richardson,by high pressure liquid chromatography*

Plasma norepinephrine and epinephrine from cannulated trout were measured by high pressure liquid chromatography with electrochemical detection. The catecholamines were extracted with acid-washed alumina using a microfilter assembly which permitted analysis of small volumes of plasma. Mean (± S.D.) values for plasma norepinephrine and epinephrine were 1.83 (0.97) pmol ml^?1 and 8.95 (4.94) pmol ml^?1, respectively. These values are compared with catecholamine levels from other vertebrate species.     

Strain differences in rat adrenal biosynthetic enzymes and stress-induced increases in plasma catecholamines.

We have examined in two inbred rat strains basal and stress-induced increases in plasma levels of epinephrine (EPI) and norepinephrine (NE) and compared these with activities of the adrenal enzymes involved in the synthesis of catecholamines. There were no differences in basal levels of NE and EPI in plasma of adult male rats of the Wistar-Kyoto (WKY) and Brown-Norway (B-N) strains. However, following 5 min. of intermittent footshock, plasma levels of both catecholamines were twice as high in WKY rats as in B-N rats. In the adrenals of unstressed rats, activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were significantly higher in B-N rats. In addition, the adrenal weights and the contents of NE but not EPI were greater in B-N rats. Thus, in these two rat strains, the capacity of the adrenal gland to synthesize and store catecholamines appeared to be inversely related to plasma levels of NE and EPI after stress. The differences between the strains appeared to be due to differences in the rates of removal of catecholamines from the peripheral circulation as well as to differences in the rate of release of catecholamines from the sympatho-adrenal medullary system. Thus biosynthetic enzyme activities need not be related directly to the capacity to release and elevate plasma levels of catecholamines following stressful stimulation.