Determination of Enzyme Activity in Single Bovine Adrenal Medullary

Adrenal medullary cells (also known as chromaffin cells) have been widely used as a model in studies of endocrine function, neuronal function, and bas...
0 downloads 0 Views 320KB Size
Download PDF
Anal. Chem. 1997, 69, 3907-3914

Determination of Enzyme Activity in Single Bovine Adrenal Medullary Cells by Separation of Isotopically Labeled Catecholamines Showchien Hsieh and James W. Jorgenson*

Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290

A microcolumn liquid chromatography method for determining norepinephrine (NE), epinephrine (E), and phenylethanolamine N-methyltransferase (PNMT) enzyme activity in single bovine adrenal medullary cells is presented. Single cells were isolated and treated with excess deuterated substrate, D3-NE (0.05 mM) for enzyme reaction. After 6 h, the reaction was quenched and the product, D3-E, was quantified along with endogenous NE and E. Separation and detection of deuterated and protiated NE and E were achieved with microcolumns (110-125 cm long, 25 µm inner diameter) packed with 3 µm octadecylsilane-modified particles and operated with amperometric detection. Of the 33 cells reported, most cells containing predominantly E have enzyme activity while cells containing predominantly NE and cells containing a mixture of both NE and E show no enzyme activity. After incubation with 10 µM hydrocortisone, of the 17 cells reported, most cells containing predominantly E and cells containing a mixture of both NE and E have enzyme activity while cells containing predominantly NE have no enzyme activity. Detection limits for NE and E were 42 and 48 amol, respectively. Cellular heterogeneity has spurred interests in the analysis of single cells.1-7 To further understand the function of specific cells, it is often necessary to characterize them by their chemical components. To accomplish the task of analyzing the chemical contents in the small sample volume of a single cell, microanalytical techniques such as capillary zone electrophoresis (CZE) and microcolumn liquid chromatography (LC) have been applied. These methods require only picoliter to nanoliter injection volumes, which are ideal for injecting the small sample of a single cell without much dilution. By coupling these separation techniques with microelectrodes or laser-induced fluorescence, important biological compounds such as amino acids, proteins and catechols have been detected in single snail neurons,1-3 human erythrocytes,4,5 and bovine adrenal medullary cells.6,7 Adrenal medullary cells (also known as chromaffin cells) have been widely used as a model in studies of endocrine function, (1) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 436-441. (2) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science 1989, 246, 57-63. (3) Olefirowicz, T. M.; Ewing, A. G. J. Neurosci. Methods 1990, 34, 11-15. (4) Hogan, B. L.; Yeung, E. S. Anal. Chem. 1992, 64, 2841-2845. (5) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992, 64, 3045-3051. (6) Cooper, B. R.; Jankowski, J. A.; Leszczyszyn, D. J.; Wightman, R. M.; Jorgenson, J. W. Anal. Chem. 1992, 64, 691-694. (7) Chang, H. T.; Yeung, E. S. Anal Chem. 1995, 67, 1079-1083. S0003-2700(97)00221-7 CCC: $14.00

© 1997 American Chemical Society

neuronal function, and basic cellular secretion. The adrenal medullary cells of mammals are of two types: one that contain and secretes mostly norepinephrine (NE) and the other mostly epinephrine (E). The two principle cell types have been characterized by histochemical and electron microscope studies, where the smaller NE-storing cells, which contain high-electron density vesicles, stain darker than E-storing cells.8-10 Furthermore, there is evidence that NE and E are secreted by separate cell populations.11,12 Recent results from analysis of single bovine adrenal medullary cells by microcolumn LC with electrochemical detection6 and CZE with laser-induced fluorescence7 demonstrated there are varying amounts of NE and E contained in each cell. In testing this hypothesis of the two cell type theory, the results by Cooper et al.6 showed that approximately 20% of the cells in culture contain appreciable amounts of both NE and E. In light of these findings, it is of interest to further characterize the heterogeneity of adrenal medullary cells in culture and determine the reasons for the existence of cells containing both NE and E, also known as the indeterminant type.6 In situ immunocytochemical studies have also demonstrated the existence of two adrenal medullary cell populations based on the presence or absence of phenylethanolamine N-methyltransferase (PNMT), the cytosolic enzyme that transfers a methyl group from S-adenosylmethionine to NE, forming E.13-15 Since PNMT catalyzes the final step in E biosynthesis, it is believed to be unique in E-type cells where E, rather than NE, is the final product. Therefore, characterization of both PNMT activity and catecholamine content in individual cells would provide evidence of the origin of the indeterminate type of cell. We describe here a microcolumn separation method to measure the NE and E content and the PNMT activity in single bovine adrenal medullary cells. In the past, assays for PNMT have been done using radioassays. This was accomplished by incubating subpopulations of cells with either NE or a NE analog, such as octopamine, normetanephrine, or norfenephrine and radiolabeled S-adenosylmethionine, generating a radiolabeled product. The radioassay method is not specific because it cannot (8) Eranko, O. Nature 1995, 175, 88-89. (9) Hillarp, N. A.; Hokfelt, B. Acta Physiol. Scand. 1953, 30, 55-68. (10) Coupland, R. E.; Pyper, A. S.; Hopwood, D. Nature 1964, 201, 1240-1242. (11) Kilpatrick, D. L.; Ledbetter, F. H.; Carson, K. A.; Kirshner, A. G.; Slepetis, R.; Kirshner, N. J. Neurochem. 1980, 35, 679-692. (12) Marley, P. D.; Livett, B. G. Neurosci. Lett. 1987, 77, 81-86. (13) Goldstein, M.; Fixe, K.; Hokfelt, T.; Joh, T. H. Experentia 1971, 27, 951952. (14) Schultzberg, M.; Andersson, C.; Unden, A.; Troye-Bolmberg, M.; Svenson, S. B.; Bartfai, T. Neuroscience 1989, 30, 805-810. (15) Moro, M. A.; Garcia, A. G.; Langley, O. K. J. Neurochem. 1991, 57, 363369.

Analytical Chemistry, Vol. 69, No. 19, October 1, 1997 3907

distinguish among the different N-methylated products that are generated by PNMT. In addition, low levels of NE, which are present in adrenal medullary cells, will inhibit the activity of these NE analogs.16,17 Quantitation will be more difficult since the reaction rate will be slow, with low product formation. However, if deuterated NE were used as the substrate, then the rate at which NE and deuterated NE are converted to their respective methylated product would be virtually identical. If the deuterated substrate is in large excess of the amount of nondeuterated endogenous substrate, NE, which is present in the cell, then the rate of deuterated product formation will be enhanced, and the amount of endogeneous NE in the cell after the enzyme assay would not be altered significantly. At high substrate concentration, the enzyme reaction is zeroth order and thus the rate of product formation is directly related to the amount of enzyme and not the substrate. A technique referred to as EMMA18-20 has been described for performing enzyme assays in capillary electrophoresis columns. This is accomplished by electrophoretically mixing substrate with the enzyme and then separating generated products on the basis of mobility differences among enzyme, substrate, and product. Xue and Yeung adapted this approach to assay lactate dehydrogenase isoenzymes in single red blood cells.21 Although the technique is very sensitive when laser-induced fluorescence is used to monitor the product, it suffers from poor peak resolution. Typically, unless the enzyme has extremely fast turnover rate, a time scale on the order of minutes is allowed for reaction incubation, which can adversely broaden the peaks due to molecular diffusion. For small molecules such as isotopically labeled catecholamines, which have faster diffusion coefficients than larger biomolecules, peaks can broaden significantly and the separation would not be adequate. Since R,R,β-D3-NE is the only deuterated substrate available commercially, separation is more difficult than if all sites were deuterated. Therefore, a highly efficient chromatographic system is needed to separate D3-NE and NE. The large number of theoretical plates from open tubular liquid chromatography (OTLC) has been shown to achieve near-baseline separation of isotopically labeled compounds.22 However, OTLC columns do not have sufficient retention of polar catecholamines. Feasibility of separation of isotopically labeled low molecular weight molecules has been shown by HPLC. However, to achieve nearbaseline resolution, most or all possible sites had to be deuterated.23-27 Mass spectrometry can discriminate compounds of different masses and thus can be used to separate and detect D3-NE, NE, D3-E, and E. However, presently, mass spectrometry (16) Fuller, R. W.; Hunt, J. M. Biochem. Pharmacol. 1965, 14, 1896-1897. (17) Fuller, R. W.; Roush, B. W.; Molloy, B. B. Adv. Enzyme Regul. 1974, 12, 311-341. (18) Bao, J.; Regnier, F. E. J. Chromatogr. 1992, 608, 217-224. (19) Wu, D.; Regnier, F. E. Anal. Chem. 1993, 65, 2029-2035. (20) Miller, K. J.: Leesong, I: Bao, J.; Regnier, F. E.; Lytle, F. E. Anal. Chem. 1993, 65, 3267-3270. (21) Xue, Q.; Yeung, E. S. Anal. Chem. 1994, 66, 1175-1178. (22) Dluzneski, P. R.; Jorgenson, J. W. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1988, 11, 332-336. (23) Baweja, R. Anal. Chim. Acta 1987, 192, 345-348. (24) Cartoni, G. P.; Ferretti, I. J. Chromatogr. 1976, 122, 287. (25) Jinno, K.; Hirata, Y.; Hiyoshi, Y. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1982, 5, 102-103. (26) Lockley, W. J. S. J. Chromatogr. 1989, 483, 413-418. (27) Masters, C. F.; Markey, S. P.; Mefford, I. N.; Duncan, M. W. Anal. Chem. 1988, 60, 2131-2134.

3908

Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

lacks the sensitivity to detect low femtomole to attomole amounts present in a single cell. Here, we describe our strategy to achieve baseline separation of D3-NE, NE, D3-E, and E by microcolumn LC and its application for determining PNMT activity in individual bovine adrenal medullary cells. EXPERIMENTAL SECTION Chemical Reagents. Standards, reagents, and mobile phase constituents were purchased from Sigma Chemical Co. (St. Louis, MO). R,R,β-D3-NE was obtained from Cambridge Isotope Laboratories, Inc. (Andover, MA). R,R,β-D3-E was obtained from Merck Sharp & Dohme/Isotopes (St. Louis, MO). Hexanesulfonic acid was obtained from Regis Chemical Co. (Morton Grove, IL). All chemicals were used as received. A balanced salt solution was composed of 150 mM NaCl, 4.2 mM KCl, 1.0 mM NaH2PO4, 11.2 mM glucose, 0.7 mM MgCl2, 2.0 mM CaCl2, and 10.0 mM HEPES and adjusted to pH 7.4 with NaOH. The sodium phosphate-buffered mobile phase was prepared by diluting 85% phosphric acid to 75 mM. The 5 mM hexanesulfonic acid in phosphate buffer was prepared by diluting 0.5 M hexanesulfonic acid in 75 mM phosphate buffer and adjusting the pH to 4 with NaOH. All mobile phases were filtered through a 0.45-µm Nylon filter (Alltech, Deerfield, IL). All solutions were made up in deionized, 0.2-µm filtered water (Barnstead, Dubuque, IA). Chromatographic System. The chromatographic columns used for all single cell analyses were 25 µm inner diameter (i.d.) fused-silica capillaries, 110-125 cm long, and were slurry packed in our laboratory with 3-µm spherical silica particles chemically modified with octadecylsilane (ODS-AQ, YMC, Wilmington, NC). All other chromatographic columns were 25, 27, or 41 µm i.d. and 135, 100, or 260 cm in length, respectively, slurry packed with 5-µm spherical silica particles chemically modified with octadecylsilane. The packing procedure is described elsewhere28 and was followed with minor modifications. The frit for the column was formed by tapping the end of the capillary into a pile of 5-µm spherical silica particles. To make room for carbon fiber electrode placement, the particles were forced 1 mm into the column with a 25-µm tungsten wire. These were formed into a frit by sintering in the flame of a match. The open end of the capillary was placed in a high-pressure stainless steel reservoir containing a slurry of 1:60 (w/v) packing to hexane. An Altex Model 110A (Beckman, Fullerton, CA) pump forced isopropyl alcohol into the reservoir at 210 bar (3000 psi). The microcolumn LC setup was similar to that described previously.29 A commercial pump (Model LC-600, Shimadzu Corp., Kyoto, Japan) was used to deliver flow rates that ranged between 0.040 and 0.1 mL/min. Splitter capillaries with similar inner diameter and length were used so that the flow rate through the packed capillary column was about 10 nL/min. For temperature studies, the microcolumn and splitter were jacketed by water flowing through 1/4-in. Tygon tubing, and the water temperature was controlled by circulation through a constant-temperature bath. Detection. The detector used was a carbon fiber microelectrode similar to that described previously.30 The cylindrical carbon (28) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128-1135. (29) Jorgenson, J. W.; Guthrie, E. J. J. Chromatogr. 1983, 255, 335-348.

fiber (9-µm diameter, 1-mm length) was inserted into the end of the capillary column close to the frit. The catecholamines were detected at an oxidation potential of +0.6 V vs Ag/AgCl reference electrode with 0.1 N KCl supporting electrolyte. The signal current was amplified by a Model 427 current amplifier (Keithley Instrument, Inc., Cleveland, OH) and fed directly into a 16-bit analog to digital converter. The rise time of the current amplifier was set at 300 ms. Data acquisition and manipulation were performed with a Hewlett Packard Vectra 386/25 computer. The data collection rate on the computer was 0.4 point/s. The amplifier gain was 10-11 A/V. To achieve reproducibility from run to run, the carbon fiber was electrochemically cleaned before each run by applying a triangular wave form from 0 to +1.8 V for 30 s at a rate of 1 V/s.31 The area, retention time, standard deviation, and the number of theoretical plates of the chromatographic peaks were determined by a macro written in-house using software IGOR (Wavemetrids, Inc., Lake Oswego, OR). Using the retention time and standard deviation, the resolution between two peaks can be determined from the following equation:

R)

t2 - t1 2(σ1 + σ2)

(1)

where t1 and t2 are the retention time of the two analytes and σ1 and σ2 are their respective standard deviations in units of time. Diffusion coefficients (Dm) for NE in two different mobile phases were measured by determining its dispersion in an open tube.32 The Dm values (in cm2/s) ((SD) for NE are 5.80 ((0.08) × 10-6 (n ) 4) in 75 mM sodium phosphate buffer, pH 4 and 5.85 ((0.06) × 10-6 (n ) 4) in 75 mM sodium phosphate buffer containing 5 mM hexanesulfonic acid, pH 4. Sample Preparation. The bovine adrenal medullary cells were cultured according to the protocol of Wilson and Viveros.33,34 Only cells that had been in culture for three days were used. For studies on hormonal effect on cells, 50 µL of 2 mM hydrocortisone was added to each cell plate containing 2 mL of cell suspension. Prior to analysis, the cell growth medium was rinsed several times and replaced with a balanced salt solution. A single cell was removed from the plate and transferred to a 300-nL microvial with the aid of a hydraulic microinjector containing a tapered micropipet that had a 20-µm i.d. Exactly 10.0 nL of a 0.1 M phosphate buffer solution, pH 7.9, containing 5.00 × 10-5 M D3-NE, 2.52 × 10-4 M S-adenosylmethionine, and 5.00 × 10-2 M reduced glutathione was added to the microvial with the use of a specially constructed microdispenser pipet described previously.35 To prevent evaporation of the nanoliter sample volume, the microvial was capped with several layers of Parafilm (American Can Co., Greenwich, CT) laboratory sealing film. The microvial was then frozen and thawed four times and centrifuged at 12000g for 10 min before being placed in an incubator for 6 h at 37 °C. The enzyme reaction was quenched with 10.0 nL of 0.3 N perchloric (30) Knecht, L. A.; Guthrie, E. J.; Jorgenson, J. W. Anal. Chem. 1984, 56, 479482. (31) St. Claire, R. L.; Jorgenson, J. W. J. Chromatogr. Sci. 1985, 23, 186-191. (32) St. Claire, R. L. Ph.D. Thesis, University of North Carolina at Chapel Hill, 1986. (33) Wilson, S. P.; Viveros, O. H. Exp. Cell Res. 1981, 133, 159-169. (34) Leszczyszyn, D. J.; Jankowski, J. A.; Viveros, O. H.; Dilberto, E. J.; Near, J. A.; Wightman, R. M. J. Neurochem. 1991, 56, 1855-1863. (35) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1988, 60, 1521-1524.

Figure 1. Chromatographic separation of D3-NE and NE with 40 µm i.d. × 260 cm microcolumn packed with 5-µm particles. Pressure: 4800 psi (331 bar) R ) 1.1.

acid containing 1.00 × 10-5 M of an internal standard of 3,4-dihydroxybenzylamine (DHBA). To perform an injection of the contents of a single cell onto the column, a tapered micropipet, which has been cut at the tip, was used to extract the cell sample from the microvial. With the aid of a micromanipulator, the tip of the pipet was placed inside the capillary column, which was mounted on a x-y positioning stage. The pipet was inserted far enough into the inlet of the column so that a tight seal was formed between the pipet and the column. By applying low pressure (approximately 20 psi (1.4 bar)) from a specially constructed microdispenser,35 the sample was forced into the column. RESULTS AND DISCUSSION Strategies for Separation of Isotopically Labeled Compounds. We describe here our strategies to achieve baseline resolution of D3-NE, NE, D3-E, and E within a reasonably short analysis time. The chromatographic resolution of two components can be described by three experimental parameters in the following relationship (Purnell equation36):

R)

xN(R - 1)k′ 4R(k′ + 1)

(2)

where R is the resolution of the two components, N is the number of theoretical plates, R is the separation factor, and k′ is the capacity factor. These three parameters can be increased independently to achieve the desired resolution. From eq 2, R increases with the square root of N, which can be manipulated through the following relationship:

N ) L/H

(3)

where L is the length of column and H is the plate height. As is evident in eq 3, one can increase N by increasing L and/or decreasing H. Increasing column length can provide more theoretical plates; however, the pressure available from a commercial pump places a limit on the column length, thus, restricting the ability to obtain more theoretical plates by this approach. Additionally, a drawback of longer columns is longer retention times. This is seen in Figure 1, where separation of D3-NE and NE on a long column (260 cm) packed with 5-µm particles required almost 14 h to obtain a resolution of 1.1. (36) Purnell, J. H. Nature 1959, 183, 2009.

Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

3909

Figure 2. Chromatographic separation of D3-NE and NE with 25 µm i.d. × 135 cm microcolumn packed with 5-µm particles. Pressure, 2000 psi (138 bar). (A) D3-NE and NE, R ) 1.31; (B) D3-E and E, R ) 1.79.

Another way to increase N is to decrease H. This can achieved in three different ways. By obtaining plate height data for the compound of interest as a function of flow velocity as described by the van Deemter equation, one can determine the optimum flow velocity (uopt) where the greatest column efficiency occurs. The uopt was determined for NE in the phosphate buffer mobile phase for every column before use. Separation of isotopically labeled compounds ultimately requires addition of hexanesulfonic acid in the mobile phase. This addition, however, does not significantly change the value of uopt since the Dm of NE did not change when 5 mM hexanesulfonic acid was added to the phosphate buffer. The second way to increase N without increasing length is to decrease the inner diameter of the microcolumn. It has been reported that hmin, the reduced plate height at the optimum velocity, decreases with decreasing microcolumn inner diameter.28,37 By decreasing the inner diameter from 41 to 25 µm, the number of plates increased from 65 000 to 133 000 plates/m for NE in the phosphate buffer mobile phase. This effect is seen in Figure 2 where better resolution and shorter retention times were obtained with the 25 µm i.d. column since more theoretical plates were obtained in the shorter column used in Figure 2. The third way to decrease plate height is to utilize the relationship between plate height and particle size,

Hmin ) 2.25(dp)

(4)

where dp is the stationary phase particle size and the coefficient 2.25 is for a conventional-sized, well-packed column. An Hmin of 11.25 µm would be expected for 5-µm particles and 6.75 µm for 3-µm particles. However, as a result of a decrease in flow (37) Karlsson, K.-E.; Novotny, M. Anal. Chem. 1988, 60, 1662-1665.

3910 Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

Figure 3. Chromatographic separation of D3-NE, NE, D3-E, and E with 25 µm i.d. × 110 cm microcolumn packed with 3-µm particles. Pressure: 4500 psi (310 bar). (A) D3-NE and NE, R ) 1.46; (B) D3-E and E, R ) 1.83.

dispersion and in the resistance to mass transfer as column inner diameter decreases, the coefficient is smaller than 2.25 in microcolumns.28 For NE, this coefficient approaches 1.0 in 12 µm i.d. microcolumns packed with 5-µm YMC-ODS AQ particles.38 Thus, a much smaller Hmin can be obtained by using both smaller inner diameter microcolumns and smaller stationary phase particles. The effect of particle size on resolution can be seen in Figure 3 where greater resolution was obtained for the same inner diameter column packed with the smaller 3-µm particles. A maximum of 133 000 plates/m was obtained for the 5-µm packed column at uopt in the phosphate buffer mobile phase. In comparison, a maximum of 200 000 plates/m was obtained for the 3-µm packed in the same inner diameter (25 µm) and shorter (110 cm) column. This column provided 240 000 theoretical plates for NE in the phosphate buffer. The combined effects of higher theoretical plates, shorter column, and faster optimum velocity with smaller particles translate into better resolution and shorter retention times as shown in Figure 3. A drawback associated with columns packed with smaller particles is the higher pressure needed to obtain optimum velocity. For the 5-µm packed column used in Figure 2, a pressure of 150 bar (2200 psi) was needed to run at uopt, which is 0.030 cm/s for NE. In contrast to the column packed with the smaller 3-µm particles, used for the separation shown in Figure 3, a pressure of 300 bar (4500 psi) was required to run at a higher uopt, 0.040 cm/s as determined by the van Deemter plot (not shown here). Note that the pressure required to operate the column packed with 3-µm particles is still achievable with most commercially available pumps, with a pressure limit about 400 bar (6000 psi). The second parameter in eq 2 involves the separation factor, R, which is a ratio of the capacity factors of the two components to be resolved. The separation factor is always geater than or equal to 1 and approaches 1 for very similar compounds such as (38) Hsieh, S.; Jorgenson, J. W. Anal. Chem. 1996, 68, 1212-1217.

Figure 4. Chromatographic separation of D3-NE and NE with 27 mm i.d. × 100 cm microcolumn packed with 5-µm particles. (A) 50 °C, R ) 0.66; (B) 25 °C, R ) 0.87; (C) 1 °C, R ) 1.08. Table 1. Effect of Temperature on the Separation Factor, r, for D3-NE and NE R

temp (°C)

R

temp (°C)

1.014 1.015

50 40

1.016 1.020

25 1

the deuterated and protiated isotopes. The value of R can be affected by the type of stationary phase, mobile phase, and column temperature. The stationary and mobile phase were not changed because the YMC stationary phase and mobile phase chosen for the NE and E separation is one of the few to provide truly Gaussian peak profiles. However, column temperature can be considered to affect an increase in R. For D3-NE and NE, the separation factor is 1.016 at room temperature. We found that by thermostating the column at different temperatures, R increases from 1.014 to 1.020 (Table 1) with a subsequent increase in resolution from 0.66 to 1.08 from 50 to 1 °C as seen in Table 1. Figure 4 shows that the resolution of D3-NE and NE increases at lower temperatures as a result of increase in R. The third parameter in eq 2 is dependent on the capacity factor, k′. Increasing k′ will result in higher resolution as indicated in the eq 2. By increasing k′, this parameter increases and approaches an asymptotic value of 1. A significant benefit in increased resolution can be achieved when a k′ of 4 or 5 is used. However, beyond a k′ of 5, increases in resolution are relatively modest and are only achieved at a great expense of additional analysis time. In reversed-phase chromatography, ion pairing agents are typically used to increase k′ for charged analytes. It

Figure 5. Portions of chromatogram from a single bovine adrenal medullary cell after enzyme assay performed with 25 µm i.d. × 125 cm microcolumn packed with 3-µm particles. Table 2. Effect of Ion Pairing Reagent, Hexanesulfonic Acid, on Capacity Factor, k′, of NE with Reversed-Phase (YMC ODS-AQ) Packed Microcolumn [hexanesulfonic acid] (mM)

capacity factor, k′

0 2.5 5

0.4 4.6 8.4

was found that addition of hexanesulfonic acid in the mobile phase elicits an increase in k′ for NE as shown in Table 2. The k′ effect is seen between separations of NE and E isotopes, where E isotopes are better resolved (part B of Figures 2 and 3) than NE isotopes (part A of Figures 2 and 3) since E has a larger k′ than NE. The final chromatographic parameters used to separate D3NE from NE and D3-E from E were 25 µm i.d. microcolumns, length 110-125 cm, packed with 3-µm octadecylsilane-modified particles using a mobile phase containing 5 mM hexanesulfonic acid in sodium phosphate buffer, pH 4, at room temperature. Although we have shown that decreasing the temperature can elicit an increase in resolution, the setup for controlling the temperature of the microcolumn was very inconvenient for routine injections of single cell samples. By using smaller inner diameter microcolumns packed with smaller particles (25-µm i.d., 3-µm Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

3911

Figure 6. PNMT activity and catecholamine content in single bovine adrenal medullary cells.

particles), we can routinely separate D3-NE and NE with a resolution of 1.46 and D3-E and E with a resolution of 1.79, at room temperature. Single Cell Analysis. Figure 5 shows portions of a chromatogram from a single cell after the enzyme assay. This corresponds to cell 7 in Table 3. Since more D3-NE than NE is present in the cell, the D3-NE peak size is larger than the NE as seen in part A of Figure 5. The peaks are identified by calculating the ratio of a particular analyte’s retention time to the retention time of DHBA, an internal standard. These retention time ratios were matched with standards which were run before and after every two single cell runs. The D3-NE peak is most easily identified since it is always a large peak. We found that the retention time ratios were very stable among standards and single cell runs and, thus, were very reliable for identification purposes. Since the entire sample was not injected onto the microcolumn, DHBA was used to determine the transfer efficiency and also the slight difference in electrochemical responses of NE and E. Calibration runs were performed by calculating ratios of the peak areas of NE or E to peak areas of DHBA. The amounts of NE or E used in the calibration were 5, 50, and 250 fmol of both NE and E with 100 fmol of DHBA. The sample of these three different standards was transferred to a microvial and then microinjected onto the column. The number of moles of NE or E present in a single cell was determined by interpolating the peak ratios from the calibration curves. The accuracy and precision of microinjection for packed microcolumns has been discussed previously.6 It has been shown that microinjection of the same sample can vary at most by 3%. This issue was addressed in this study by making two independent injections from the same sample, and the results were similar to that described previously. Since cells in culture can secrete NE and E without stimulus, blank runs were performed. This was done by transferring into 3912

Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

Table 3. Catecholamine Content and PNMT Activity in Individual Bovine Adrenal Medullary Cells cell no.

NE (fmol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

nda nd 15 27 19 6 11 31 37 14 11 8 25 24

15 16 17 18 19 20 21 22 23

34 13 13 32 36 38 13 62 19

24 25 26 27 28 29 30 31 32 33

30 111 36 24 56 40 82 52 24 18

E (fmol)

PNMT activity (fmol/h)

E-Type 41 34 73 76 101 20 65 69 87 50 74 42 63 132

Indeterminant Type 53 25 27 18 43 15 26 125 40

1.1 0.65 0.57 0.2 0.2 2.2 1.2 1.1 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

NE-Type

a

5 30 nd nd nd nd nd nd nd nd

nd, none detected (less than 0.01 fmol/h activity).

nd nd nd nd nd nd nd nd nd nd

Figure 7. Effect of hydrocortisone on PNMT activity and catecholamine content in single bovine adrenal medullary cells. Table 4. Effect of Hydrocortisone on Catecholamine Content and PNMT Activity in Individual Bovine Adrenal Medullary Cells cell no.

NE (fmol)

1 2 3 4 5 6 7

2 3 2 15 15 12 5

8 9 10 11 12

55 25 15 15 12

13 14 15 16 17

25 47 55 100 120

E (fmol)

PNMT activity (fmol/h)

E-Type

a

67 48 38 30 65 33 25 Indeterminant Type 72 38 30 25 20 NE-Type nd 8 nd nd 30

5 0.6 0.4 0.4 nda nd nd 0.8 1.2 0.4 0.2 nd nd nd nd nd nd

nd, none detected (less than 0.01 fmol/h activity).

the microvial a few nanoliters of the balanced salt solution from the cell culture plate. This blank sample was manipulated in the same manner as the enzyme assay for a cell. Blank runs were performed to ensure that the signal peaks seen in the chromatogram of a single cell were due to the cell and not from the culture plate media. Subjecting the blank sample to enzyme assay ensures that the product D3-E seen in the chromatographic cell run comes from PNMT catalysis and not from other source. Blank runs from every cell culture plate show the D3-NE peak but no NE, E or D3-E peak.

Table 3 shows the catecholamine content and the PNMT activity of cells randomly picked from 10 different cell preparations. The table shows the E-type, indeterminate type, and NE type. Catecholamine content and PNMT activity data from Table 3 are graphically displayed in Figure 6. Note that E-type cells lie close to the axis labeled E while the NE-type cells lie close to the NElabeled axis. The indeterminate type cells can be found enclosed by the two dotted lines. The PNMT activity is represented by the height of the bar in the third dimension. The cells were categorized as NE or E type according to the ratio of amounts of one type of catecholamine over the other. Previous results by Chang and Yeung indicated that the ratios of E to NE were 2.7 and 5.6 for cells high in E content from two different glands.7 In this paper, we categorize E-type cells as containing two or more times the amount of E than NE. The reverse is true for NE-type cells. The indeterminate type has ratios of amounts of catecholamines less than 2. This confirms the findings of Cooper et al.6 regarding more than two cell types and contradicts the findings of two cell types described by earlier histochemical studies. Most of the E-type cells show PNMT activity, although no correlation is seen between the amount of E in the cell and the level of PNMT activity. However, no detectable PNMT activity was found in NEtype cells, indeterminate cells, and several E-type cells. The detection limit for this technique was determined to be 42 amol for NE and D3-NE and 48 amol for E and D3-E at a signal-to-rms noise (S/N) of 5. This translates into a detection limit for PNMT activity of 0.01 fmol/h. Most E-type cells show PNMT activity, which suggests that PNMT is responsible for generating the E found in each cell. For the indeterminate and NE-type cells, it is apparent that PNMT is either not active or is not present. For cells containing only NE, it is logical to assume that PNMT is insufficiently present to generate E. The indeterminate and six of the E-type cells, which contain some E but show no PNMT activity, might have had active Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

3913

PNMT at some time to produce the E observed. One hypothesis as to why PNMT activity is not evident in these cells could be lack of appropriate chemical signals. PNMT activity has been shown to be regulated through hormonal and neural biochemical pathways where glucocorticoid release or splanchnic nerve stimulation can increase PNMT activity.39,40 Therefore, removal of the adrenal cortex during the cell culture process may have caused some loss of PNMT regulation, since the cortex is physiologically responsible for releasing glucocorticoids into the medulla. To investigate the effect of hormones on adrenal medullary cells in culture, 10 µM hydrocortisone was added to cells in culture on the first day of culture. The cells were then analyzed after three days in culture. The data are presented in Table 4 and

Figure 7. Cells that are considered indeterminate now display PNMT activity. All NE-type cells have no PNMT activity. This set of data suggests that hormonal regulation could affect the PNMT activity in adrenal medullary cells and thus affect the amount of NE and E present in the cells.

(39) Ciaranello, R. D.; Black, I. B. Biochem. Pharmacol. 1971, 20, 3529-3532. (40) Ciaranello, R. D.; Wooten, G. F.; Axelrod, J. J. Biol. Chem. 1975, 250, 32043211.

AC970221W

3914

Analytical Chemistry, Vol. 69, No. 19, October 1, 1997

ACKNOWLEDGMENT This research was supported by The National Institutes of Health (Grant GM39515). We thank Hewlett-Packard for the donation of the computer and Glaxo-Wellcome for the donation of the Shimadzu pump. S.H. expresses her deep gratitude to Dr. Bruce R. Cooper for his initial guidance on this work. Received for review February 27, 1997. Accepted July 6, 1997.X

X

Abstract published in Advance ACS Abstracts, August 15, 1997.