Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.
), National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:134-141.
ESTIMATION OF NEUROTRANSMITTERS IN THE BRAIN BY CHROMATOGRAPHIC METHODS
Deepti Nair, Ramkumar K, Srikumar BN, Raju TR and Shankaranarayana Rao BS Chromatography refers to a group of techniques used to separate complex mixtures on the basis of different physical interactions between the individual compounds and the stationary phase of the system. The basic components in any chromatography technique are the mobile phase (gas or liquid), which carries the complex mixture (sample); the stationary phase (solid or liquid), through which the mobile phase flows; the column holding the stationary phase; and the separated components (eluate). Modes of separation a. Adsorption chromatography (liquid-solid chromatography) It is based on the competition between the sample and the mobile phase for adsorptive sites on the solid stationary phase. There is equilibrium of solute molecules being adsorbed to the solid surface and desorbed and dissolved in the mobile phase. Those molecules, which are most soluble in the mobile phase, move fastest, whereas those, which are least soluble, move slowest. The stationary phase can be either acidic polar (eg, silica gel), basic polar (eg, alumina) or nonpolar (eg, charcoal). Commonly, the non-polar organic eluents for example hexane, dicholoromethane, and ethyl actetate are used. It can be used to separate compounds, which are highly soluble in organic solvents (eg., Fat-soluble vitamins). b. Partition chromatography Partition chromatography is also referred to as liquid-liquid chromatography. Separation of solute is based on the relative solubility in an organic (nonpolar) solvent and an aqueous (polar) solvent. Modern partition chromatography uses pseudoliquid stationary phases that are chemically bonded to the support or high-molecular-weight polymers that are insoluble in the mobile phase. Partition systems are called normal phase when the mobile phase is less polar than the stationary solvent and are termed reverse phase when the mobile solvent is more polar. When the elution strength of the mobile phase is constant throughout the separation, it is called isocratic elution and when varied is called as gradient elution. In liquid chromatography, the resolution is proportional to the column length (i.e., the number of theoretical end plates/unit length). Increasing the surface area leads to an increase in the number of theoretical plates. An increase in the surface area can be achieved by decreasing the particle size but comes with the disadvantage of increased resistance to flow, which impairs resolution because of backpressure. In the recent past, new stationary phases with small particle size that can withstand high pressures and offer better resolution have been developed. This has lead to the development of high performance liquid chromatography (HPLC). High Performance Liquid Chromatography (HPLC) The term High Performance Liquid Chromatography (HPLC) was coined to describe the separation of molecules under high pressure in a stainless steel column filled with a matrix and is used for the separation and determination of organic and inorganic solutes (mol.wt.< 1000) (Figure 1). Fundamentals of HPLC 1. Selectivity: It is the ability of a column to separate two components depending on its affinity and retention. 2. Capacity factor: It is the ability of a column to retain a particular compound. 3. Resolution factor: It is the resolving power of a column to separate two structurally closely related compounds.
Figure 1. Basic components of the High Performance Liquid Chromatography (Bender, 1972).
Reverse phase High Performance Liquid Chromatography Reverse phase High Performance Liquid Chromatography (rpHPLC) is a form of partition chromatography in which the chemically bonded phase is hydrophobic (nonpolar) and the starting mobile phase is more polar than the stationary phase. The HPLC consists of these basic components: pump, column, sample injector, detector and recorder. Pump: A pump forces the mobile phase through the column at a much greater velocity. The pump can be pneumatic syringe type, reciprocating or hydraulic amplifier. The most widely used pump is the mechanical reciprocating pump, which is now used as a multihead pump with two or more reciprocating pistons. During pumping, the pistons operate put of phase (180º for two heads, 120º for three) to provide constant flow. Column: The stationary phase is packed into long stainless steel columns. Usually, HPLC is run at ambient temperature, although columns can be placed in an oven and heated to enhance the rate of partition. Fine, uniform column packing results in much less band broadening but requires pressure to force the mobile phase through. The
packing can also can be pellicular (an inert core with a porous layer), inert and small particles or macroporous particles. The most common material used for column packing is silica gel. It is very stable and can be used as solid packing in liquid - solid chromatography or coated with a solvent, which serves as the stationary phase (liquid-liquid). Reversed phase HPLC is now very popular. The stationary phase is made up of nonpolar molecules (eg, octadecyl C-18 hydrocarbon) bonded to silica gel particles. For this type of column packing, the mobile phase commonly used is acetonitrile, methanol, water or any combination of solvents. Reverse phase columns are stable in the pH range of 2-7 and at elevated temperatures. Reversed phase column can be used to separate ionic, non-ionic and ionizable samples. A buffer is used to produce the desired ionic characteristics and pH for the separation of the analyte. Column packings vary in size (3-20mm), with the smaller particles used mostly for analytical separations and the larger ones for preparative separations. Sample injector: A small syringe can be used to introduce the sample into the path of the mobile phase that carries it into the column. The best and most widely used method is the loop injector. The sample is introduced into a fixed-volume loop.
When the loop is switched, the sample is placed into the path of the flowing mobile phase and is flushed into the column. Loop injectors have high reproducibility and are used at high pressures. Detectors: Modern HPLC detectors monitor the eluate as it leaves the column and ideally, produce an electronic signal proportional to the concentration of each separated component. Spectrophometers that detect absorbances of visible or ultraviolet light are commonly used. Fluorescence detectors are also used, because many biologic substances fluoresce strongly. Another common HPLC detector is the amperometric or electrochemical detector. These devices measure current produced when the analyte of interest is either oxidized or reduced at some fixed potential set between a pair of electrodes. Recorder: It is used to record detector signal versus the time taken for the mobile phase to pass through the instrument starting sample injection. The graph is called a chromatogram. The retention time is used to identify compounds when compared with standard retention times run under identical conditions. Peak area is proportional to the concentration of the compounds that produced the peaks. ESTIMATION OF AMINO ACIDS IN THE BRAIN There are several methods that were employed in the past to estimate the levels of different aminoacids in the brain. The commonly used two methods are described below. 1. Estimation of glutamate and GABA levels by multiple development paper chromatography Principle This method follows the principle of different partition coefficients that can be obtained from a stationary cellulose phase with a mobile solvent phase for different aminoacids, which aid in their separation. Extraction and quantification are done by reacting the aminoacids with ninhydrin (triketohydrindene hydrate). Ninhydrin reacts with an aminoacid to produce CO2, NH2 and its lower aldehyde and ultimately yields a chromophore known as Ruhemann’s purple with an absorbance maximum around 515nm.
Preparation of reagents 1. Solvent: butanol: acetic acid: water (12:3:5):‚ To 60 ml of butanol, 15 ml of acetic acid and 25 ml distilled water are added. 2. 0.25% Ninhydrin: 200 mg of Ninhydrin is dissolved in 99 ml of acetone. To this solution 1ml of pyridine is added. 3. 0.005% CuSO4 solution: 5 mg of cupric sulphate is dissolved in 10 ml 75% alcohol. Standards a. 2µM glutamate: 2.942 mg of glutamate is dissolved in 10 ml of distilled water. b. 2µM GABA: 2.062 mg of GABA is dissolved in 10 ml of distilled water. Assay procedure For the estimation of amino acids, the original method developed by Sadasivudu and Murthy (1978) is adapted with some modifications in our laboratory as described below (Nagaraja and Desiraju, 1993, Shailesh Kumar and Desiraju T, 1990, 1992; Shankaranarayana Rao et al. 1998; Sunanda et al 2000). After the dissection of different brain regions, each region is homogenized in 80% double distilled ethanol (for every 100mg of the brain tissue, 2ml of 80% alcohol is used). Homogenates are transferred to polypropylene tubes and centrifuged at 1200rpm for 10 min. 1ml of the supernatant is then transferred into small test tubes and evaporated to dryness at 70 oC in an oven. The residue is reconstituted in 100 ml distilled water and 10 ml is used for spotting on Whatman No.1 Chromatography paper. Standard solutions of glutamate and GABA at a concentration of 2 mM are also spotted using an Eppendorf micropipette; the spots are dried with a hair drier. The chromatograms are then stitched at the sides and placed in a chromatography chamber containing butanol: acetic acid: water (65: 15: 25, V/V) as solvent. When the solvent front reached the top of the papers, the papers are removed and dried. A second run is performed similarly, after which the papers are dried, sprayed with ninhydrin (0.25%
in acetone with 1% pyridine) and placed in an oven at 100 oC for 4 min. The portions which carry glutamate and GABA spots corresponding with the standard, are cut and eluted with 0.005% CuSO4 in 75% ethanol. Their absorbance is read against a blank in a LKB- 4050 spectrophotometer (Fig.M26) at 515nm and the levels are expressed as mmoles/ gram wet weight tissue. Calculations: The levels of glutamate and GABA are calculated by using the following formula;
Unknown OD A= Standard OD Standard in mg (3µg) X X Volume spotted (10µl) 100 x
3. Improving the sensitivity of detection of the compound The ability of OPA to react rapidly and completely at room temperature with primary amines led to its use as a pre-column derivatization reagent for amino acids. The resulting highly fluorescent 1-alkylthio-2-alkylisoindole derivatives are less polar than their respective amino acids and can be separated on C-18 reversed-phase HPLC Reverse phase separations are routinely carried out in the pH range of 6.0 to 7.5 for optimal fluorescence. Sensitivities in the femtomole to picomole range are achievable. Tissue Preparation and Estimation of Amino acid Neurotransmitters The following procedure has been used and standardized in our laboratory. The brain is rapidly removed and cooled in an ice-cold buffer. The different brain regions are micro dissected, weighed and dropped into chilled 2ml of sodium acetate buffer with 20% methanol in polycarbonate test tubes. The tissues are then homogenized in an icecold condition for 2 min. The homogenized tissue is centrifuged for 30 min at ~5000 rpm. The supernatant is filtered with 0.2-µm pore size cellulose acetate membrane and stored in a deep freezer (-800 C) till it is used for the analysis. Tissue samples are subjected for HPLC. The neurotransmitters are separated on a Supelco C18 column (15 X 0.46 ID and 3 µm). The chromatograms are analyzed using Winchrom data station. Preparation of the OPA reagent 5mg of o-pthalaldehyde is taken in a 1.5 ml eppendorf vial. 400µl of methanol, 100µl of borate buffer and 10ml of β- Mercaptoethanol is added and vertexed thoroughly. This reagent mixture has to be protected from light source and stored in the refrigerated condition. Preparation of the amino acid standards The standards for each amino acid are prepared individually by dissolving a known quantity of amino acid in warm distilled water to make 10mM
where, A = Aminoacid content in umoles/gram wet weight tissue 1000 = Conversion factor for gram wet weight tissue X = weight of the tissue in grams
2. Estimation of Amino Acids by HPLC Using Fluorescence Detector with Pre-Column Derivatization Principle Fluorescence detectors are probably the most sensitive among the existing modern HPLC detectors. When compounds having specific functional groups are excited by shorter wavelength energy and they emit light of higher wavelength radiation, called fluorescence. Usually, the emission is measured at right angles to the excitation. Roughly about 15% of all compounds have a natural fluorescence. Fluorescence intensity depends on both the excitation and emission wavelength, allowing selective detection of some components while suppressing the emission of others. Most of the amino acids, upon reacting with ophthaldehyde (OPA) at an alkaline pH give rise to fluorescent derivatives which could be separated on a C18 column by reverse phase high-pressure liquid chromatography. Chemical derivitization of a compound or a mixture of compounds prior to analysis is generally done for the following reasons: 1. Making a compound suitable for the analysis 2. Improving the analytical efficiency for the compound
concentration and stored in refrigerated condition. From this stock, working standards are prepared by diluting 100mL of stock solution to 4ml buffer (composition is same as the mobile phase). 20ml of the working standard is taken for the derivatization. Derivatization mixture for injection 20µl of supernatant of the tissue or standard 100µl of sodium tetra borate buffer (pH 9.5) 870µl of mobile phase 10µl of OPA reaction mixture The derivatization reaction are carried out in an amber colored vial and this reaction mixture incubated for 2 min at room temperature. After completion of the incubation period, 20ml is injected to HPLC for analysis, which gave the 100pico-mole concentration in the case of standard injection. The concentration of amino acids is estimated by isocratic separation. The column is saturated with the mobile phase before the analysis. The flow rate of mobile phase is maintained at 0.9 ml/min so as to generate a backpressure of around 120125 kg/cm 2 . The excitation wavelength and emission wavelength are set at 330 nm and 450 nm, respectively. The best response is obtained when the excitation and emission slit width is kept at 5nm. The chromatograms of amino acids is stored and analyzed after the completion of the experiment. The samples are analyzed using an external standard.
The rpHPLC chromatogram profile of the amino acid neurotransmitters viz. aspartate, glutamate, serine, glutamine, glycine, taurine and GABA are given in Figure 2. The concentrations of aminoacids is calculated based on the area under these respective peaks. ESTIMATION OF BIOGENIC AMINES AND THEIR METABOLITES BY HPLC The levels of biogenic amines and their metabolites are estimated using several chromatographic procedures. Two of the following methods are widely used in the recent past. 1. Estimation of Biogenic Amines by HPLC with Fluorimetric Detection The levels of noradrenaline (NA), dopamine (DA) and 5- hydroxy tryptamine (5-HT) are estimated by high performance liquid chromatography with Fluorimetric detection (HPLC - FD) as developed by Lakshmana and Raju (1997) and adopted with some modifications (Lakshmana et al. 1998; Shankaranarayana Rao et al 1998; Shelke et al. 1997; Sunanda et al 2000; Vijayakumar and Meti, 1998). Preparation of Reagents 1. 0.1M perchloric acid (PCA, mol. Wt = 100.46) is prepared by dissolving 10.8 ml of commercially available 60% PCA in 1 liter of quartz distilled water (QDW). 2. 0.02M Sodium acetate (Mol wt = 82.03) is prepared by dissolving 1.64g of sodium acetate in 1 litre QDW. 3. 0.1375% W/V Heptane sulfonic acid (HSA, Mol.wt = 202.24) is prepared by dissolving 1.375g of HSA in 1 litre QDW. 4. 16% V/V methanol is prepared by mixing 160 ml of methanol in 840ml of QDW.
Figure 2. The rpHPLC chromatogram profile of the aspartate, glutamate, serine, glutamine, glycine, taurine and GABA.
5. 0.1mM ethylene diamine tetra acetic acid (EDTA ; mol, wt = 372.2) is prepared by dissolving 37.2 mg of EDTA, disodium salt in 1 litre of QDW.
Chromatographic conditions The mobile phase consists of sodium acetate (0.02M), methanol (16%, V/V), heptane sulfonic acid (0.1375%), EDTA (0.2mM) and dibutylamine (0.01%, V/V). The pH is adjusted to 3.92 ± 0.01 with orthophosphoric acid and filtered through 0.45m membrane filter and degassed. The flow rate is set to 0.9ml per minute so as to yield a pressure of 125-130 kg/cm 2. The column is washed first with quartz-distilled water and then with 80% methanol. After- wards, it is equilibrated with the sodium acetate buffer, for atleast 24 hours before injecting standards/samples. Sample preparation Following decapitation the heads are collected into ice cold 0.1M PCA. Immediately the brains are removed and the desired brain regions are dissected. The tissues are weighed and homogenized in 1ml of PCA (0.1M). After centrifugation at 14,000rpm for 15 min at 4oC, the supernatant is filtered through 0.45 µm membranes and 100 ml of the filtrate is injected into the HPLC pump. After separation, NA, DA, Isoproterenol (IP) and 5-HT are detected at the excitation wavelength of 280 nm and an emission wavelength of 315nm, while keeping the slit width 10/10. All separations are isocratic and are carried out at room temperature. The peaks of the amines are plotted by a recorder on a chart paper running at a speed of 2.5 mm/minute. The biogenic amine peaks are identified by comparing the retention period of the peaks with that of external standards. The external standards are always injected before injecting the tissue samples. Standards Stock solutions of standards (1 mg/ml) are prepared in 0.1N Hydrochloric acid and are stored at -20 o C and used within two weeks of preparation. The working standard solution is prepared freshly in 0.1 M PCA for each experiment. The amount of standard is 3ng per 100 ml of injection volume for each NA, DA, IP and 5-HT. Monoamine peaks are identified by comparing their retention time in the sample (tissue extracts)
solution with that of standard solution and also by superimposing the chromatograms of the sample spikes with and without known amount of the standards. Calculations Monoamine contents in the tissues are quantified by comparing their peak heights with that of known standards after correcting for the recovery of internal standard. The fluorescence units obtained from brain tissue samples are converted into nanograms per gram wet tissue using the following formula (Lakshmana and Raju, 1997). IS X IT AS AT X 100 3 X X 1000
Where, IS = Fluorescence units for 3 ngs of isoproterenol standard. IT = Fluorescence units for 3 ngs of isoproterenol in tissue AT = Fluorescence units of amine in the tissue AS = Fluorescence units of 3 ngs of amine in standard 3 = ngs of amine standard injected. 100 = volume of sample injected in ml 1000 = Conversion factor for gram wet weight tissue X = weight of tissue in grams. Minimum detection limits The high efficiency of the HPLC separation for biogenic amines combined with the sensitivity of fluorometric monitoring enable the detection at picogram range. With these chromatographic conditions, it is possible to detect the levels of NA, DA, isoproterenol and 5-HT at 250 pg range, in less than 30 min in rat brain regions in a single chromatographic run. This sensitivity is found adequate for the analysis of these compounds in brain tissue extracts. A major advantage of the present procedure (isocratic HPLC-FD method) is the ease of sample preparation which reduces assay time and minimizes the chances for technical errors. The
separations are achieved at room temperature at 25±1oC and the use of special monitors for column temperature is not required. 2. Estimation of Biogenic Amines by HPLC with Electrochemical Detection Many compounds that are electrochemically active can be measured using HPLC-ECD due to the selectivity and sensitivity. This detection is based on the measurements of the current resulting from oxidation/reduction reaction of the analyte at a suitable electrode. Since the level of the current is directly proportional to the analyte concentration, this detector could be used for quantification. Principle At an applied positive potential, catecholamines and indoleamines are oxidized at ring hydroxyl groups with the release of two electrons. These electrons are transferred to the electrode and the resultant current is directly proportional to the number of molecules oxidized. Most of the analytes to be successfully detected require the pH adjustments. Mobile phase The mobile phase consists of sodium acetate (0.03 M), Methanol (7%), heptane sulfonic acid (50mM), EDTA (0.1 mM) and dibutyl amine (100µl). The solution is adjusted to pH 3.98 ± 0.01 with glacial acetic acid and filtered through 0.45µ pore size membrane. Preparations of the Standards Stock solution of biogenic amines and their metabolites is prepared at the concentration of 1mg/ml in the sodium acetate buffer (0.015M) with 20% methanol and is stored in 0º C and used within a week of preparation. The working standard solutions is prepared by diluting 10ml of stock standard solution to 10µl and from this 50µl is diluted to 5 ml, from this 20µl is injected to the HPLC injector port which gave the concentration of 200pM of the each standard.
The procedure for brain tissue preparation and processing is carried out as mentioned above for the amiinoacids. The monoamines and their metabolites peaks are identified by comparing their retention time in the sample (tissue extract) solution with that of the standard solution. Each monoamine in the tissue is quantified by comparing their peak area with that of known standards as described for aminoacids. References
1. Lakshmana, M.K. and Raju, T.R. (1997) An isocratic assay for neuroepinephrine, dopamine, and 5hydroxytryptamine using their native fluorescence by high performance liquid chromatography with fluorescence detection in discrete brain areas of rat. Anal Biochem 246: 166-170. 2. Lakshmana, M.K., Shankaranarayana Rao, B.S., Sudha, S., Dhingra, N.K., Ravikumar, R., Govindaiah and Raju, T.R. (1998). Role of monoamine oxidase type A and B on dopamine metabolism in discrete regions of primate brain. Neurochem Res 23: 1033-1039. 3. Nagaraja TN, Desiraju T (1993) Regional alterations in the levels of brain biogenic amines, glutamate, GABA, and GAD activity due to chronic consumption of inorganic arsenic in developing and adult rats. Bull Environ Contam Toxicol 50(1):100-107. 4. Ramesh, V, Lakshmana, MK, Shankaranarayana Rao BS, Raju, TR and Mohan Kumar V (1999) Alterations in monoamine neurotransmitters and dendritic spine densities in the medial preoptic area after sleep deprivation. Sleep Res Online 2: 49-55. 5. Sadasivudu, B and Murthy, Ch.R.K.(1978) Effects of ammonia on monoamine oxidase and enzymes of GABA metabolism in mouse brain. Arch Int de Phys etde Biochem 86:67-82. 6. Shailesh Kumar MV and Desiraju T (1990) Regional alterations of brain biogenic amines and GABA/ glutamate levels in rats following chronic lead exposure during neonatal development. Arch Toxicol 64(4):305-314. 7. Shailesh Kumar MV and Desiraju T (1992) Effect of chronic manganese exposure on rat brain biogenic amines and GABA/glutamate system. Biogenic amines 8(3-4):227-235. 8. Shankaranarayana Rao BS, Raju TR and Meti BL (1998) Selfstimulation of lateral hypothalamus and ventral tegmentum increases the levels of noradrenaline, dopamine, glutamate and AChE
activity, but not 5hydroxytryptamine and GABA levels in hippocampus and motor cortex. Neurochem Res 23 (8) : 10531059. 9. Shelke RJ, Lakshmana MK, Ramamohan Y and Raju TR (1997) Levels of dopamine and noradrenaline in the developing retina : Effect of light deprivation. Int J dev Neurosci 15: 139-143. 10. Sunanda, Shankaranarayana Rao BS and Raju TR (2000) Restraint stress- induced alterations in the levels of biogenic amines, aminoacids and AChE activity in the hippocampus. Neurochem Res 25 (12): 1547-1552. 11. Vijayakumar, M and Meti B. L (1998) Alterations in the levels of monoamines in discrete brain regions of clomipramine induced animal model of endogenous depression. Neurochem Res 27: 345-349. 12. Wu AHU (2000) Analytical techniques and instrumentation. In. Clinical chemistry; principles, procedures, correlations. IV Edn. Eds, M.L. Bishop, J.L. Duben-Engelkirk and E.P. Fody. LippincotWilliams and Wilkins.