Determination of catecholamines in a small volume (25 μl) of plasma from conscious mouse tail vein

Abstract

The determination of plasma catecholamine levels is commonly used as a measure of the sympathetic nervous system’s response to stress and is highly important for diagnosis, therapy and prognosis of cardiovascular diseases, catecholamine-secreting tumors arising from the chromaffin cells of the sympathoadrenal system, and affective disorders. Diseases in which catecholamines are significantly elevated include pheochromocytoma, Parkinson’s disease, Alzheimer’s disease, neuroblastoma, ganglioneuroblastoma, von Hippel Lindau disease, baroreflex failure, chemodectina (nonchromaffin paraganglioma) and multiple endocrine neoplasia. Plasma norepinephrine levels provide a guide to prognosis in patients with stable, chronic, and congestive heart diseases. The method described here for the determination of plasma catecholamines is based on the principle that plasma catecholamines are selectively adsorbed on acid-washed alumina at pH 8.7 and then eluted at a pH between 1.0 and 2.0. Upon injection, catecholamines in elutes were separated by a reversed phase C-18 column. After separation, the catecholamines present within the mobile phase enter the electrochemical detector. Electrochemical detection occurs because electroactive compounds oxidize at a certain potential and thereby liberate electrons which create measurable current. Catecholamines readily form quinones under these conditions, get oxidized, release 2 electrons and create current. The electrochemical detector detects this electrical current which linearly correlates to the catecholamine concentration loaded into the ultra-performance liquid chromatography instrument. A 15-min mixing time during the adsorption and desorption steps was found to be optimal. If the washing step was omitted, the catecholamines could not be eluted from the acid-washed alumina. To prevent dilution, the alumina had to be centrifuged and not aspirated to dryness after the washing step. We report here that by changing the range in the electrochemical detector plasma catecholamines were measured with only 12.5 μl plasma and more reliably with 25 μl plasma. The detection limit was 1 ng/ml. This assay method is very useful as blood can be collected from the tail vein in a conscious mouse and the same mouse can be used for time-dependent or age-dependent studies.

1. Introduction

Catecholamines are important natural molecules containing a catechol ring, which act as neurotransmitters or hormones at central and peripheral levels¹. The main endogenous catecholamines are norepinephrine (NE), epinephrine (EPI), and dopamine (DA). Liquid chromatography with electrochemical detection is a reliable technique for catecholamine assay². The electrochemical detection of NE and EPI in plasma after separation by liquid chromatography was first described by Watson in 1981³, based on previous work by Keller et al.⁴. This procedure is unsurpassed by any other method for the determination of catecholamines e.g., liquid chromatography with fluorometric or electrochemical detection^5–7, gas liquid chromatography with electron capture detection⁸ and mass fragmentography^9,10, or by radioenzymatic assays^11–13.

We have documented a reliable, reproducible, simple to perform, and adequately sensitive method for determination of plasma catecholamines in 25 μl plasma. This method can be used to detect plasma catecholamines in response to the following conditions: (i) determining stress-induced catecholamine secretion at different time points from the same mouse, (ii) determining age-dependent catecholamine secretion from the same mouse, (iii) finding a correlation between glucose-stimulated insulin and catecholamine secretion from the same mouse, (iv) correlating glycogenolysis with catecholamine secretion from the same mouse, (v) correlating age-associated development of Alzheimer’s disease (dementia), hypertension (high blood pressure), diabetes (high blood glucose), and sarcopenia (loss of muscle function).

The present study reveals comparable plasma catecholamine levels when blood is drawn from conscious mouse and from deeply anesthetized (with isoflurane) mouse. This contrasts with the findings reported earlier, where the authors reported much higher plasma catecholamine levels between blood drawn from catheterized mice, decapitated, and CO₂-inhaled mice¹⁶. Therefore, isoflurane-induced anesthesia is a better choice if blood must be drawn in anesthetized mice.

2. Materials

2.1. Standard curve:

DL-Norepinephrine hydrochloride, (±)-Epinephrine hydrochloride, 3,4-Dihydroxybenzylamine hydrochloride (DHBA), and dopamine hydrochloride) were used for making the standard curve.

2.2. Tris buffer:

Trizma^® base (2-Amino-2-(hydoxymethyl)-1,3-propanediol)) and ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate were used to make Tris buffer.

2.3. Phosphate-citrate buffer:

NaH₂PO₄, Na₂EDTA, sodium citrate, diethylamine hydrochloride, 1-Octanosulfonic acid, phosphoric acid, and N, N-dimethylacetamide was used to prepare the phosphate-citrate buffer.

2.4. Mobile phase:

Phosphate-citrate buffer and acetonitrile.

2.5. Adsorption and desorption of catecholamines:

Aluminum oxide (Al₂O₃), laboratory tube mixer, centrifuge tube filters (0.22 μm pore size) and hydrochloric acid (HCl).

2.6. Instrument:

Ultra Performance Liquid Chromatography (UPLC), reverse-phase dC18 column, polypropylene tube, and electrochemical detector.

2.7. Software:

Empower-3 (Waters Corporation, MA).

3. Methods

3.1. Mouse tail vein plasma collection

Mouse was held in a mouse holder and tail was cut about 1 mm. Blood was collected with EDTA-coated Micro-Hematocrit Capillary Tubes until it is 4/5th full, then transferred to a 500 μl Eppendorf tube with 2 μl 0.5 M EDTA and kept on ice. Blood samples were spun at 8400 x g, 4°C, for 5 min. 25 μl plasma was transferred to a 2 ml Eppendorf tube and kept on −80°C for future assay.

3.2. Preparation of plasma samples

1. 25 μl plasma was thawed and DHBA (1 ng/sample) in 200 μl Tris buffer (0.1 M tris, 0.05 M EDTA, pH 8.7) as internal control was added to thawed plasma sample (see Note 1).

2. 10–15 mg Al₂O₃ was added to each sample for adsorption of catecholamines at pH 8.7 and tubes were rotated on a laboratory tube mixer for 10–15 min at room temperature.

3. The aluminum beads were spun down after completion of rotation, the supernatant was removed, 500 μl water was added to the beads, and the mixture was transferred to a centrifuge tube filter (0.22 μm pore size). At this point, the filter cartridge was moved to a 2 ml collection tube. After first spin (8400 x g 1 min), another 500 μl water was added and spun again until the aluminum beads are dry.

4. The filter cartridge was moved back to its original filter tube and 50 μl of 0.1N HCl was added for desorption of catecholamines. The lid was closed and ‘vortexed’ the tube for 5 seconds (see Note 2). Sample was spun down and the eluted sample used for UPLC assay.

3.3. Electrochemical detection of catecholamines

1. Eluted samples were transferred to polypropylene tube and placed on the sample plate. The running speed was set at 0.3 ml/min, and column temperature was set at 30°C. Chromatographic separation was achieved on a universal, silica-based, reversed-phase C-18 column, which was equilibrated with the mobile phase at least 6 h before use (see Note 3).

2. The software Empower-3 was used to control the performance and data analysis.

3. Catecholamines were detected by an electrochemical detector in DC mode. The electrochemical detector was set up with the following parameters: Temperature = 28°C, EC = +0.50 V, Filter= 0.1 s, Range = 100 pA (see Note 4).

4. Phosphate-citrate buffer was used for the mobile phase^14,15. Simply, 3.45 g NaH₂PO₄ (final conc. 25 mM), 100 mg Na₂EDTA (final concentration 268 μM), 14.7 g sodium Citrate (final concentration 50 mM), 1.1 g diethylamine hydrochloride (final concentration 10 mM), and 0.72 g 1-octanosulfonic acid (final concentration 0.072%) were added in about 900 ml HPLC grade water in a beaker and the solution was mixed with a stir bar. After adjusting pH to 3.1 with phosphoric acid, water was added to make 1000 ml solution, which was filtered (0.22 μm pore size) in a 500 ml tissue culture bottle. 21 ml N,N-Dimethylacetamide (final concentration 2.2%) was added to the phosphate-citrate buffer followed by addition of 5% acetonitrile (V/V), then 5% (V/V) acetonitrile was added as isocratic mode. The concentration of the mobile phase was kept constant throughout the chromatographic process (see Note 5).

3.4. Standard curve

1. 5 mg of NE, EPI, DHBA and DA were dissolved in 2.5 ml of 0.1N HCl to make a stock solution of 2 mg/ml.

2. Stock solutions were thawed and diluted to different concentrations for different RANGE of electrochemical detector. The standard mixture that was used for 100pA RANGE included the following: NE (50 pg/μl), EPI and DHBA (75 pg/μl) and DA (12.5 pg/μl). The following ratios were used to make the standard curve: NE:EPI:DHBA:DA = 4:6:6:1. (see Note 6).

3. Serial dilution was made 4 times to obtain 5 different concentrations that gave 5 different points in the standard curve.

4. The following concentrations of NE were used to generate its standard curve: 31.25, 62.5, 125, 250, and 500 pg/μl (Fig. 1a&b). The standard curve went through every point and generated an R² value of 0.992 (Fig. 1c) (see Note 7).

5. The following concentrations of EPI were used to generate its standard curve: 46.875, 93.75, 187.5, 375, and 750 pg/μl (Fig. 1a&b). Like NE, the standard curve went through every point and produced an R² value of 0.995 (Fig. 1d).

6. The following concentrations of DA were used to generate its standard curve: 7.813, 15.625, 31.25, 62.5, and 125 pg/μl (Fig. 1a&b). Like NE and EPI, the standard curve went through every point and produced an R² value of 0.998 (Fig. 1e).

Fig. 1. Determination of standard curve of catecholamines at 100pA current.

(a) The following concentration of the standards was used to generate the chromatogram shown here: norepinephrine (NE: 250 pg/μl), epinephrine (EPI: 375 pg/μl), 3,4-didroxybenzylamine (DHBA: 375 pg/μl) and dopamine (DA: 62.5 pg/μl). (b) Peak results showing retention time (RT), area, height, amount, and the unit (pg/μl). (c) NE standard curve: The following concentrations of NE were used to generate its standard curve: 31.25, 62.5, 125, 250, and 500 pg/μl. (d) EPI standard curve: The following concentrations of EPI were used to generate its standard curve: 46.875, 93.75, 187.5, 375, and 750 pg/μl. (e) DA standard curve: The following concentrations of DA were used to generate its standard curve: 7.813, 15.625, 31.25, 62.5, and 125 pg/μl.

3.5. Detection of catecholamines in different plasma volumes

1. 12.5 μl plasma: The lowest volume of plasma that was used to measure catecholamines was 12.5 μl (Fig. 2a&b). We found 16.842 ng/ml of NE and 5.919 ng/ml of EPI when catecholamines in 12.5 μl plasma was adsorbed in 200 μl Tris-buffer with ~10 mg Al₂O₃, desorbed in 50 μl 0.1N HCl, and 40 μl was injected into the system.

2. 25 μl plasma: Although plasma catecholamines were detected in 12.5 μl plasma, the volume that gave consistent and reliable plasma catecholamines was 25 μl. We detected 29.439 ng/ml of NE and 14.523 ng/ml of EPI when catecholamines in 25 μl plasma was adsorbed in 200 μl Tris buffer with ~10 mg Al₂O₃, desorbed in 50 μl 0.1N HCl, and 40 μl was injected into the system (Fig. 2c&d).

3. 50 μl plasma: The following values were detected: 15.117 ng/ml of NE and 11.898 ng/ml of EPI when 50 μl plasma was eluted with 50 μl 0.1 N HCl and injected 20 μl of eluent (Fig. 3a&b).

4. 100 μl plasma: The following values were detected: 14.949 ng/ml of NE and 14.140 ng/ml of EPI when 100 μl plasma was eluted with 100 μl 0.1 N HCl and injected 20 μl of eluent (Fig. 3c&d).

5. 200 μl plasma: The following values were detected: 10.603 ng/ml of NE and 14.747 ng/ml of EPI when 200 μl plasma was eluted with 100 μl 0.1 N HCl and injected 10 μl of eluent (Fig. 4a&b) (see Note 8).

Fig. 2. Estimation of catecholamines in 12.5 and 25 μl of plasma.

(a) Chromatogram showing NE and EPI concentrations in 12.5 μl plasma. The catecholamines were adsorbed in activated aluminum oxide, extracted with 50 μl 0.1N HCl, and 40 μl eluted solution was injected to determine the catecholamine concentration. (b) Peak results showing retention time (RT), area, height, and amount (pg/μl). (c) Chromatogram showing NE and EPI concentrations in 25 μl plasma. Elution volume was 50 μl and injection volume was 40 μl. (d) Peak results showing retention time (RT), area, height, and amount (pg/μl).

Fig. 3. Assessment of catecholamines in 50 and 100 μl of plasma.

(a) Chromatogram showing NE and EPI concentrations in 50 μl plasma. The catecholamines were adsorbed in activated aluminum oxide, eluted with 50 μl 0.1N HCl, and 20 μl eluted solution was injected to determine the catecholamine concentration. (b) Peak results showing retention time (RT), area, height, and amount (pg/μl). (c) Chromatogram showing NE and EPI concentrations in 100 μl plasma. Elution volume was 100μl and injection volume was 20μl. (d) Peak results showing retention time (RT), area, height, and amount (pg/μl).

Fig. 4. Measurement of catecholamines in 200 μl of plasma.

(a) Chromatogram showing NE and EPI concentrations in 200 μl plasma. The catecholamines were adsorbed in activated aluminum oxide, eluted with 100 μl 0.1N HCl, and 10 μl eluted solution was injected to determine the catecholamine concentration. (b) Peak results showing retention time (RT), area, height, and amount (pg/μl).

3.6. Comparable plasma catecholamine levels when blood was collected from the tail vein in conscious mice and from the heart in deeply anesthetized mice.

1. Blood collected from the tail vein: To ascertain whether plasma catecholamines vary when blood is drawn from the tail vein in a conscious mouse versus from the heart in deeply anesthetized mouse, blood was collected from the tail vein and the heart and determined plasma catecholamines in 25 μl plasma. In tail vein plasma (25 μl), 14.063 ng/ml of NE and 26.487 ng/ml of EPI were found (Fig. 5a&b).

2. Blood collected from the heart: 13.491 ng/ml of NE and 30.215 ng/ml of EPI were detected in plasma extracted from the blood drawn from the heart (Fig. 5c&d).

Fig. 5. Evaluation of catecholamines in 25 μl of plasma drawn from tail vein and heart.

(a) Chromatogram showing NE and EPI concentrations in 25 μl plasma. Blood was collected from the tail vein of conscious mouse and plasma catecholamines were adsorbed in activated aluminum oxide, eluted with 50 μl 0.1N HCl, and 20 μl eluted solution was injected to determine the catecholamine concentration. (b) Peak results showing retention time (RT), area, height, and amount (pg/μl). (c) Blood was collected from the heart of deeply anesthetized (isoflurane) mouse. Chromatogram showing NE and EPI concentrations in 25 μl plasma. Elution volume was 50 μl and injection volume was 20 μl. (d) Peak results showing retention time (RT), area, height, and amount (pg/μl).

4. Notes

1. Changes in sensitivity of the assay on the electrochemical detector was essential to detect low (increased sensitivity by decreasing range) and high (decreased sensitivity by increasing range) levels of catecholamines in plasma samples. Changes in sensitivity mandated changes in DHBA concentration.

2. Vortexing was crucial for desorption of catecholamines from Al₂O₃.

3. Equilibration with the mobile phase was essential to obtain a stable baseline.

4. 5 nA to 100 pA RANGE were used to reliably detect catecholamines from different tissues including adrenal gland, PC12 cells, heart, kidney, spleen, white adipose tissue, brain (hypothalamus, thalamus, hippocampus, striatum, cortex, cerebellum, brainstem, spinal cord), gut regions (esophagus, fundus, body, pylorus, duodenum, jejunum, ileum, and colon) as well as plasma. Since plasma concentration of catecholamines are low 100 pA RANGE was used.

5. De-gassing of the mobile phase was not required and was kept in room temperature for a week.

6. Different concentrations of catecholamines for the standard curve were used to get the comparable maximal peaks in the same run.

7. Although 31.25 pg/μl NE was used as the lowest dose in the standard curve, 1 ng/ml of NE was detected in plasma when the original plasma volume (dilution in Empower 3) and the injection volume (weight in Empower 3) were adjusted in 100 pA RANGE. Less than 1 ng/ml was detected when sensitivity was increased by decreasing RANGE such as 50 pA. However, detection at 50 pA RANGE was coupled with unstable baseline and increased signal to noise ratio.

8. Plasma catecholamines were determined using 12.5, 25, 50, 100, and 200 μl plasma volumes where the elution and/or injection volumes were changed so that the detected catecholamine values from the different plasma volumes are similar.