Anal. Chem. 1995,67, 1079-1083
Determination of Catecholamines in Single Adrenal Medullary Cells by Capillary Electrophoresis and Laser-Dnduced Native Fluorescence Huan=TsungChang and Edward S. Yeung* Ames Laboratory-USDOE and Department of Chemistty, Iowa State University, Ames, Iowa 5001 1
The present study demonstrates that native fluorescence detection combined with capilla~~ electrophoresis separation at low pH provides high sensitivity (down to nanomolar), high resolution, high speed, and low interference for the analysis of catecholamines. Further, this method has been employed successfully for the measurement of the amounts of epinephrine and norepinephrine in individual bovine adrenal medullary cells. Application of this method to the study of neurochemishy is promising. In the search for a better understanding of cellular differentiation, the physiological effects of external stimuli such as drugs, cell-cell communication, and neural transmission, single-cell analysis has generated much recent interest. Thus far, optical and electron microscopy,1 immunoassay? enzymatic radiolabeling3 fluorescence microscopy,4 and voltametric microelectrodes (ECD)5-7 have been most often employed for the study of single cells. However, there are still some limitations, such as inadequate sensitivity, poor quantitative reproducibility, the need to perform labeling with fluorescent dyes, and the inability to determine multiple components. Microcolumn liquid chromatography, with electrochemical or laser-induced fluorescencedetection, provides the advantages of separation of the components and high sensitivity because of the small sample volume needed for analysis.8-'3 Recently, capillary electrophoresis (CE) in narrow bore (2-75 pm4.d.) capillaries has become an important technique for bio~eparation.~~-'~ In addition, CE is ideally suited for single-cell (1) Tan, W.; Shi, Z.-Y.; Smith, S.; Bimbaum, D.; Kopelman, R Science 1992, 258,778-781. (2) Ishikawa, E.; Hashida, S.; Kohno, T.; Hirota, K Clin. Chim. Acta 1990, 194,51-72. (3) McCaman, R. M.; Weinrich, D.; Borys, H. J. Neurochem. 1973,21,473476. (4) Tank, D. W.; Sugimori, M.; Connor, J. A; Llinas, R R. Science 1988,242, 773-777. (5) Jankowski, J. A; Schroeder, T. J.; Holz, R. W.; Wightman, R M. J. Biol. Chem. 1992,267,18329-18335. (6) Lau, Y. Y.; Takayuki, A; Ewing, A G. Anal. Chem. 1992,64,1702-1705. (7) Leszczyzsyn, D. J.; Jankowski, J. A; Viveros, 0. H.; Diliberto, E. J.; Near, J. A; Wightman, R. M. J. Neurochem. 1991,56,1855-1863. (8) Kennedy, R T.; Oates, M. D.; Cooper, B. R; Nickerson, B.; Jorgenson, J. W. Science 1989,246,57-63. (9) Kennedy, R T.; St. Clair, R L.; White, J. G.; Jorgenson, J. W. Mikrochim. Acta 1987,2,37-45. (10) Kennedy, R T.; Jorgenson, J. W. Anal. Chem. 1989,61,436-441. (11) Moro, M. A; Lopez, M. G.; Gandia, L.; Michelena, P.; Garcia, A G. Anal. Biochem. 1990,185,243-248. (12) Oates, M. D.; Jorgenson, J. W. Anal. Chem. 1990,62,1577-1580. (13) Yeung, E. S. Acc. Chem. Res. 1994,27,409-414. (14) Landers, A P.; Oda, R P.; Madden, B. J.; Spelsberg, T. C. Anal. Biochem. 1992,205,115-124. (15) Stefansson, M.; Westerlund, D. J. Chromatogr. 1993,632, 195-200. 0003-2700/95/0367-1079$9.00/0 0 1995 American Chemical Society
analysis because on-line cell injection is easily implemented by either electroosmosisz0or hydrodynamic Laser-induced fluorescence (LIE),with its high sensitivity, is a suitable detection technique for CE, providing good mass limit of detection (LOD).na Hemoglobin and carbonic anhydrase in individual human erythrocytes have been determined by using native fluorescence with an Ar ion laser at 275 nm.24Derivatization and an indirect methodz1jz5have been used to overcome the limited applicability of LIF. Recently, indirect fluorescence detection has been employed for the determination of Na+ and K+ plus pyruvate and lactate in individual red blood ~ e l l s . 2 ~More 8 ~ ~ recently, we have taken advantage of enzyme amplification in an oncolumn reactionz8 to detect zeptomole levels of lactate dehydrogenase in individual red blood cells.29 Particle counting based on the oncolumn immunoassay between glucose &phosphate dehydrogenase (GGPDH) and an antibody was developed to detect zeptomole levels of G6PDH.3O Catecholamines are an important group of compound^.^^ Jorgenson and co-workers used 15-2@pm open-tubular liquid chromatography (LC) with ECD to determine the neurotransmitters and free amino acids in single neuron cells of the land snail, Helix a s p e ~ s i a In . ~addition, ~ ~ ~ ~ CEECD was demonstrated for the determination of catecholamine and serotonin in a neuronal ce11.19bz0 It is well known that chromaffin cells in the adrenal medulla synthesize and store the catecholamine hormones epinephrine (E) and norepinephrine (NE) and secrete these in response to stimulation by s e c r e t a g o g ~ e . ~Here, ~ . ~ ~we describe the use of ~
(16) Ong, C. P.; Pang, S. F.; Low, H. K; Li,S. F. Y.J. Chromatogr. 1991,559, 529-536. (17) Watson, E.; Yao, F. Anal. Biochem. 1993,210,389-393. (18)Nashabeh, W.; El Rassi, 2.J. Chromatogr. 1991,536,31-42. (19) Olefirowicz, T. M.; Ewing, A G. Anal. Chem. 1990,62,1872-1876. (20) Olefirowicz, T. M.; Ewing, A G. Chimia 1991,45,106-108. (21) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992,64,2841-2845. (22) Lee, T. T.; Lillard, S. J.; Yeung, E. S. Electrophoresis 1993,14,429-438. (23) Wu, S.; Dovichi, N. J. J. Chromatogr. 1989,480,141-155. (24) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992,64,3045-3051. (25) Yeung, E. S.; Kuhr, W. G. Anal. Chem. 1991,63,275A-282A (26) Li, Q.; Yeung, E. S. J. Capillay Electrophor. 1994,1, 55-61. (27) Xue, Q.; Yeung, E. S. J. Chromatogr, 1994,661,287-295. (28) Chang, H. T.; Yeung, E. S . Anal. Chem. 1993,65,2947-2951. (29) Xue, Q.; Yeung, E. S. Anal. Chem. 1994,66,1175-1178. (30) Rosenzweig, 2.; Yeung, E. S. Anal. Chem. 1994,66,1771-1776. (31) Frontiers in Catecholamine Research;Usdin, E., Snyder, S. H., Eds.; Pergarnon Press: New York, 1973. (32) Oates, M. D.; Cooper, B. R.; Jorgenson, J. W. Anal. Chem. 1990,62,15731577. (33) Cooper, B. R; Jankowski, J. A.; Leszczyszyn, D. J.; Wightman, R M.; Jorgenson. J. W. Anal. Chem. 1992,64,691-694. (34) Schroeder, T. J.; Jankowski, J. A; Kawagoe, K T.; Wightman. R M. Anal. Chem. 1992,64,3077-3083.
Analytical Chemistry, Vol. 67, No. 6,March 15, 1995 1079
CE-LIF with an Ar ion laser at 275 nm to determine quantitatively the amounts of E and NE in single bovine adrenal medullary cells. Also, the effects of pH and matrices on the fluorescence intensities and the separation of several important aromatic amine compounds are shown. EXPERIMENTAL SECTION
CE Instrumentation and Data Collection. The experimental setup is similar to that described in ref 29. Briefly, a highvoltage power supply (Glassman High Voltage, Whitehorse Station, NJ; EH series 0-40 kv) was used to drive the electrophoresis. Either 50- or Xpm-i.d. fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) were used for the separation. The total length was 60 cm, and the effective length was 40 cm. The entire electrophoresis and detection system was enclosed in a sheet-metal box with HV interlocks. The 275nm line from an Ar ion laser (Spectra Physics, Mountain View, CA; Model 2045) was isolated from other lines with an external prism and focused with a 1-cm focal length lens onto the detection window of the capillary tubing. Alternatively, the 284nm frequency-doubled output from a Kr ion laser (Coherent Radiation, Palo Alto, CA; Model FRED-Kr) was used. Two UG1 filters (Schott Glass Technologies, Duryea, PA) were used to prevent scattered light from reaching the photomultiplier tube. The fluorescence signal was either amplified by a current ampmer (Keithley, Taunton, MA; Model 427) or directly converted to voltage through a 10-kQ resistor. Data were collected via a 2 4 bit A/D interface at 5 Hz Uustice Innovation, Palo Alto, CA; Model DT 2802) and stored in a computer (IBM, Boca Raton, FL; Model PCIAT 286). Methods. Adrenal glands were obtained from a local butchery and used immediately. Chromafh cells were isolated from bovine adrenal medullae by the method of Livett and co-workers.36 After purification, the cells were stored at 4 "C before use. Before analysis, cells were washed five times with 10-mL portions of balanced salt solution. To prevent significant secretion of catecholamine, the cells need to be analyzed less than 30 min after they are washed. Just before injection of the cell into the capillary, 5 pL of cell suspension was added to 200 pL of water on a microscope slide. We found that unlike erythrocytes, the cells are not lysed by osmotic pressure under these conditions. To minimize the secretion of catecholamine from the cells and the adsorption of cells onto the slide surface, injection of a single cell into the capillary should be performed in less than 3 min. Therefore, the injection end of the capillary was etched with HF to form a narrow taper. The hydrodynamic injection method was similar to that described in ref 21. Excess matrix fluid is pushed out of the capillary by pressure once the cell adheres to the capillary wall. The cells lyse within seconds on contact with the running buffer. Separations were performed at 30 kV. Quantitation is achieved by measuring the peak areas and comparing them with those obtained for calibration runs with standard solutions of NE and E interposed between cell injections. Reagents. A balanced salt solution was composed of 150 mM NaCl, 4.2 mM KCl, 1.0 mM NaH2P04, 11.2 mM glucose, 0.7 mM MgC12, 2.0 mM CaC12, and 10.0 mM HEPES and adjusted to pH 7.4 with NaOH. The pH of the running buffer for CE separation (35) Ciolkowski, E. L.; Cooper, B. R.; Jankowski, J. A.; Jorgenson, J. M.; Wightman, R. M.I. Am. Chem. SOC. 1992,114, 2815-2821. (36) Livett, B. G.; Kozuousek, V.; Mizobe, F.; Dean, D. M. Nature 1979,278, 256-257.
1080 Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
PH Figure 1. Effect of pH on the fluorescence intensities of amines. Conditions are described in the text. 1, tryptamine; 2, serotonin; 3, E; 4, NE; and 5 , dopamine.
was adjusted with sodium hydroxide or phosphoric acid to the desired value. All standard amines, poly(ethy1ene glycol), and boric acid were purchased from Aldrich Chemical (Milwaukee, Wr) . Collagenase was obtained from Sigma Chemical (St. Louis, MO). Citric acid, sodium hydroxide, phosphoric acid, sodium phosphate, 2-propanol, and chemicals for preparation of the balanced salt solution were purchased from Fisher (Fair Lawn, NJ). RESULTS AND DISCUSSION
Detection. This is the frst demonstration, to our knowledge, of the use of native fluorescence to detect catecholamines in CE. Traditionally, ECD37-39and fluorescence detection with derivatization40 are more common. Although some amines with an aromatic or indole group have natural fluorescence, it is uncommon to use fluorescence for detection since conventional light sources are too weak.4l The use of an Ar ion laser at 275 nm has been shown to be suitable for the determination of native protein^.^^^^^^^ Recently, up to 50 mW of frequency-doubled output at 284 nm has become available in a commercial system. Since the excitation maxima of these amines is around 280 nm, except for serotonin at 298 nm, excitation at 275 or 284 nm is appropriate. Figure 1 shows the relative fluorescence efficiencies of the compounds in the 320-350-nm region from pH 2 to 9 when excited at 275 nm. In general, the fluorescence intensity is higher at low pH. Even though these amines are expected to be fully protonated below pH 7, solvent effects, presumably from the coions in the buffer solution, are probably responsible for the enhancement. Also, it is worth noting that at this condition amines are more stable. Generally, amines prepared at low pH and stored in the refrigerator can stay for at least 24 h without significant loss of fluorescence intensity. On the other hand, catecholamine is very unstable at high pH. For example, a yellow color can be found in E and NE solutions several hours after the solution has been prepared. Since biological samples such as cells generally are prepared in various organic matrices to preserve viability or to cause lysis, (37) Wallingford, R A; Ewing, A. G. Anal. Chem. 1989,61, 98-100. (38) Keating, J.; Dratcu, L.; Lader, M.; Shenvood, R. A.I. Chromatogr. 1993, 615, 237-242. (39) Olefirowicz, T. M.; Ewing, A. G. J. Neurosci. Methods 1993,34, 11-15. (40) Todoriki, H.; Hayashi, T.; " m e , H.; Hirakawa, A Y. J. Chromatogr, 1983, 276, 45-54. (41) Peat, M. A.; Gibb, J. W. Anal. Biochem. 1983,128, 275-280.
0
9
I ' 0 Matrix Figure 2. Fluorescence intensity of NE in various matrices normalized to that in 0.1 M citric acid at pH 2.8. Conditions are described in the text. Bar assignments: 1, 6% glucose; 2, 1% DMSO; 3,0.6% glucose; 4,balanced salt solution; 5, 0.1 M HCI; and 6, 0.8 M HC104. Table 1. Detection Limits of Amine Compounds for Laser-Induced Native Fluorescence after CE Separation
amine serotonin
concentration, nM pH 2.6* pH 5.7c
pH 2.8a
65 60 40
0.9 100 90 80
ndd
nd
80
nd nd nd nd nd nd
nd nd nd nd
25 5 200
nd
90
nd
8
0.5
epinephrine norepinephrine dopamine dopa
tryptophan metanephrine normetanephrine catechol homovanillic acid 3,4hydroxyphenylacetic acid
2.5
5 1 20 50 30
1.3
tryptamine
0.7
a Buffer, 3 mM citric acid; excitation, 275 nm. Buffer, 10 mM citric acid; excitation, 284 nm. Buffer, 15%2-propanol, 1%poly(ethy1ene glycol), and 0.1 M boric acid, pH adjusted by 0.1 M NaOH; excitation, 275 nm. Not determined.
it is important to know the effect of some common solvents on the fluorescence intensity. Figure 2 shows the effect of different matrices on the fluorescence intensity of NE. The fluorescence intensity of NE dissolved in organic solvents such as dimethyl sulfoxide OMSO) is higher. On the other hand, salts with high ionic strength or strong acids such as HClOd quench the fluorescence significantly. This may be due to the formation of ion-pair complexes between protonated amines with inorganic ions such as chloride or phosphate. Figure 2 indicates that HC104 is not a suitable agent for lysing adrenal medullary cells33in this detection method. Table 1shows the detection limit of representative amines at different pH conditions and excited at different wavelengths. While there is > loOx variation in the LOD among compounds, the LOD for each is quite constant as a function of experimental conditions. Since the injection volume of these analytes is in the nanoliter range, the absolute detectable amount is in the mid to low attomole level for most of these. There is little difference in performance between the two laser lines, which is also the case for the excitation of native fluorescence in proteins (data not shown). The results also show that native fluorescence detection of catecholaminesprovides better mass sensitivitythan traditional
2
4 Time (min)
6
8
Figure 3. Electrophoretic separation of amines at pH 5.4.Capillary, total length, 60 cm, effective length, 40 cm, and i.d., 50 pm; sample injection, 30 kV for 3 s; concentrations,6 pM for all analytes except serotonin, 0.3 pM, tryptophan, 10 nM, homovanillic acid, 3 pM, and 3,4-dihydroxyphenylaceticacid, 10 pM. Other conditions are described in the text. Peak assignments: 1, serotonin; 2, dopamine; 3,dopa; 4,NE; 5, E; 6, tryptophan; 7, catechol; 8, homovanillic acid; and 9, 3,4-dihydroxyphenylacetic acid.
fluorescence derivatization methods, which can only detect picomole to femtomole levels. Also, the concentration sensitivity is comparable to or better than that of ECD.37 The advantages of native fluorescence detection are (1)compatibility with separations at very low pH, (2) nonintrusive monitoring, (3) high selectivity and thus few interferencesfrom amino acids, and (4)simultaneous determination of various aromatic amines without prior chemical treatment. Separation. The separation of catechol and catecholamine is important since they play critical roles as neurotraxmitters in the central and peripheral autonomic system and as hormones exerting endocrine and exocrine effects. Figure 3 shows the separation of nine related amines at pH 5.4. High-speed separation in less than 8 min is achieved due to the additive effect of electroosmotic flow and electrophoretic motion of the analytes. The addition of 2-propanol and poly(ethy1ene glycol) into the running buffer partially decreases the electroosmotic flow and in turn provides sufficient resolution for all amines. The other reason to have poly(ethy1ene glycol) in the running buffer is to minimize the interaction between the amines and the capillary wall, since protonated amines can easily interact with the silanol groups of the capillary wall, which has net negative charge at pH higher than 4. One of the features of this separation scheme is that it is faster and more reproducible than that through the use of micellar electrokinetic chromatography (data not shown). A possible reason is that the protonated amines can form ion pairs with the anionic micelles, which results in decreased electrophoretic mobilities or increased interaction with the capillary wall. It is also possible to separate these amines merely on the basis of electrophoretic mobility at very low pH. Figure 4 shows the separation of four amines at pH 2.8. This is favorable for detection according to Figure 1. Separation under this condition provides an additional advantage in that there is less interference from acidic compounds and their metabolites, since only amines can migrate in this direction at such low pH values. Single-cellAnalysis. It is well known that adrenal medullary cells store large amounts, up to several hundred femtomoles, of Analytical Chemisfty, Vol. 67, No. 6, March 15, 1995
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1 3
I
i4
I
4 Time (mh)
2
0
0
2
4
8
6
Figure 4. Electrophoretic separation of amines at pH 2.8. Concentrations were 0.5 p M for NE and E, 10 nM for serotonin, and 5 nM for tryptamine; injection was performed at 30 kV for 4 s. Conditions are as in Figure 3. Peak assignments: 1, impurities; 2, tryptamine; 3, serotonin; 4, NE; and 5, E.
a
6
10
Time (min) Figure 6. Electrophoretic separation of lysed bovine adrenal medullary cells. Conditions are as in Figure 5. Peaks 1-3 are unidentified cellular components.
E
s -
E
3
0 0
2
4
6
Time (min) Figure 5. Electrophoretic separation of NE and E in a standard solution of 0.1 M citric acid at pH 2.3. Capillary, i.d., 16 pm, total length, 65 cm, effective length, 45 cm; concentration, 20 pM each. Other conditions are described in the text.
2
4
6
4
a
10
Time (min) Figure 7. Electrophoretic separation of intracellular components in a single bovine adrenal medullary cell. Conditions are as in Figure 5. Peaks 1-4 are unknown components. Table 2. Norepinephrine and Epinephrine Content in Individual Bovine Adrenal Medullary Cells
measured amount, fmol
catecholamines NE and E. Hence, it is a good test for our separation and detection scheme as applied to the analysis of biological samples. Figure 5 shows that NE and E from a standard sample are well separated at pH 2.3. Three different bovine glands were studied. Figure 6 shows the electropherogram of lysed bovine adrenal medullary cells (0.5 pL of cells/l50 pL of buffer solution) from gland 1. If the average size of a cell is 20 pm, the number of cells corresponding to 0.5pLis around 1.2 x 105.Based on this assumption and the volume of analytes injected into the capillary, the injected contents are derived from about 14 cells. The average amount of NE and E in individual cells can thus be estimated to be 15 and 39 fmol, respectively. Figure 7 shows the electropherogram of a single-cell analysis. ' h o major peaks corresponding to NE and E are depicted, with a good match of retention times with the standards. The signal-to-noise ratio is excellent, showing the power of native fluorescence detection. After several runs, the retention times changed due to the adsorption of cell membranes and other species onto the capillary wall. Fortunately, this can be easily overcome by flushing the capillary with running buffer and then re-equilibrating for 5 min. Since catecholamines are known to be secreted from cells even without any chemical or physical stimulation, a blank test after each run is necessary to guarantee that there is an insignifcant 1082 Analytical Chemisfry, Vol. 67, No. 6, March 15, 1995
cell no.
NE
E
gland 1 1 2 3 4 5 6 7 8 9 10 11 mean f SD
13 22 25 5.1 25 18 4.0 22 33 28 11 19i9
40 62 78 14 71 50 11 57 120 73 29 55 f 30
3.3 2.8 3.1 2.7 2.8 2.8 2.8 2.6 3.6 2.6 2.6 2.7 i 0.3
gland 2 1 2 3 4 5 6 mean f SD
107 81 91 86 48 17 72 f 30
524 514 373 48 1 279 112 380 i 150
4.9 6.3 4.1 5.6 5.8 6.6 5.6 f 0.8
E/NE
amount of catecholamine accumulated in the balanced salt solution. Results show that after the cells stay in the balanced salt solution for 30 min, the average amount of extracellular NE
and E which may be injected into capillary is less than 1.2 and 3.2 fmol/nL, respectively. In order to further minimize any interference from the balanced salt solution, we always pushed out the excess solution that was injected with a cell. This was possible because the cell is adsorbed onto the capillary wall once it is injected. The actual injected amount of extracellular NE and E is much less than the results from the blank test since excess matrix is pushed out after injection and since the cells are always analyzed within 30 min after transfer to the balanced salt solution. One of the most important features of this method is that the separation is much faster (less than 5 min) than that by using microcolumn LC (longer than 50 min).33 The lack of a chemical derivatization step further adds confidence in quantitation. Table 2 shows the amounts of NE and E in single adrenal medulla cells from two different glands. The amounts of catecholamines are signiiicantly different for each gland, which are from different cows. The values measured are quite comparable to those measured by using ECD.33 Cell-tocell differences in the amounts of NE and E are obvious. However, for each gland, the ratio is fairly constant. This shows that the variations for cells from the same gland are probably due to variations in cell size. The ratios (42) Kong, J. Y.; Thureson-Klein, A; Klein, R L. Neuroscience 1989,28,765775. (43) Muller, T. H.: Unsicker, IC J. Neurosci. Methods 1981,4,39-52. (44) Hochman, J.; Perlman, R L. Biochim. Biophys. Acta 1976,421,168-175. (45) Kilpatrick, D. L;Ledbetter, F. H.; Carson, K. A; Kirshner, A G.; Sleptis, R; Kinshner, N.J Neurochem. 1980,35, 679-692. (46) Greenberg, A; Zinder, 0. Cell Tissue Res. 1982,226,655-665.
of NE to E from the two glands are 2.7 and 5.6, which are close to the results found in the literat~re.42.4~Since we did not fractionate" or culture the cells before analysis, we did not observe both NErich and Erich cell types in the same gland among the limited number of runs. In a third gland (from a different cow), we did find two cens where the amounts of NE are larger than the amounts of E. Our results agree with Hochman's results,44 where the E content accounted for more than 95%of catecholamine. Also, the faster release of E compared to NE and the ease of NE conversion to E explain why, in general, the E content is higher than NE.45,46 ACKNOWLEDGMENT The authors wish to thank T.-H. Chen, W. H. Hsu, F. C. Minion, R F. Ross, and A S. Yeung for help in harvesting the adrenal medullary cells. We also thank Coherent Radiation for making available a FRED-Kr laser. The Ames Laboratory is operated for the US. Department of Energy by Iowa State University under Contract No. W-7405-Eng-82. This work was supported by the Director of Energy Research, Office of Basic Energy Sciences, Division of Chemical Sciences. Received for review September 27, 1994. Accepted January 12, 1995.@ AC940956Q @Abstractpublished in Advance ACS Abstracts, February 15, 1995.
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