Anal. Chem. 1982, 5 4 , 1452-1454
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cision is not better than 2% of the maximum concentration of each concentration range. This means that an error of 0.02 mg/L S in the 0-1 mg/L S range corresponds to an error of 1mg/L S in the 0-50 mg/L S range. For optimum precision, the response of the analyzer should be kept large by selecting the syringe size according to the sulfur content of the analyte and maintaining a reasonably rapid syringe drive speed. ACKNOWLEDGMENT The author thanks Ward R. Gibson for his skillful assistance in experimental work and D. H. Gehring and C. J. Hensler for their helpful comments.
LITERATURE CITED Granatelli, L. Anal. Chem. 1959 31, 434. Pitt, E. E. H.; Rupprecht, W. E. Fuel lg84, 43, 417. Drushel. H. V. Anal. Lett. 1970, 3, 353. Wallace, L. D. Anal. Chem. 1070, 4 2 , 387. Carter, J. M. Analyst (London) 1972, 97, 929. (6) Killer, F. C. A. In "Recent Analytical Developments in the Petroleum Industry"; Hodges, D. R., Ed., Halsted Press: Wiiey: New York, 1974. (7) Drushel, H. V. Anal. Chem. 1978, 50, 76.
(1) (2) (3) (4) (5)
RECEIVED for review January 13,1982. Accepted March 29, 1982. This is Research and Development Division Publication No. 588.
Electrochemical Assay for Brain Ascorbate with Ascorbate Oxldase James 0. Schenk, Ellen Miller, and Ralph N. Adams" Department of Chemistty, University of Kansas, Lawrence, Kansas 66045
A major research effort in our laboratory for some time has been the development of electrochemicaltechniques for in vivo monitoring of neurotransmitter systems in small animal brains. Typical experiments utilize very small glass-sheathed graphite-epoxy electrodes implanted in specific areas of brain tissue. Chronoamperometric experiments are conducted at potentials sufficient to oxidize and thus detect catecholamines in the extracellular fluid (ECF). Unfortunately, with most of the electrodes currently available, ascorbic acid (AA) is oxidized at potentials almost identical with that of the catecholamines norepinephrine (NE) and dopamine (DA). Differentiating chemical analysis of the ECF immediately surrounding the electrode tip would be ideal, but sampling technologies for this particular type of in situ measurement have yet to be developed. Due to the complexities of the in vivo experiment, we have turned to the brain slice as a simple model for investigating electrochemical responses in brain tissue. This preparation allows one complete access to the area surrounding the electrode tip. Both electrochemical experiments and subsequent chemical assays on the region immediately adjacent to the electrode tip can be accomplished readily, enabling one to sort out the electroactive species giving rise to the response. Since AA is present in very high concentrations (ca. millimolar) in all neuronal tissue, knowledge of its exact local ECF concentration is very important in interpreting results from in vivo or brain slice electrochemical measurements. It is in the above context that we summarize herein a rapid, specific assay for AA that can be used for any small brain tissue sample. Its use requires no devices not already available to experimenters working with in vivo electrochemistry. It obviates the need to constantly maintain a satisfactorily performing liquid chromatography system for AA analyses which, at best, has proven to be difficult and tedious. It requires only the purchase of the enzyme ascorbic acid oxidase (L-ascorbate:oxygen oxidoreductase, EC 1.10.3.31, which is stable under stored refrigerated conditions. Ascorbic acid oxidase (AAO) catalyzes the reaction
-
L-AA + 1/202 dehydroascorbic acid
+ H20
The reaction is extremely rapid and specific and, under the conditions used, can be shown to cause negligible oxidation of the catecholamines. Following conventional tissue sampling and homogenization, the assay consists of three simple steps: (1) A chronoamperometric measurement is made on the homogenized sample in buffer solution. This becomes a 0003-2700/82/0354-1452$01.25/0
measure of all electroactive species oxidized at that potential. (2) A standard addition of a few microliters of standard AA solution is made and a second chronoamperometric run calibrates the electrode under actual assay conditions. (3) A fixed amount of AAO is added and a third amperometric response detects the almost instantaneous removal of all AA. Since each of the electrochemical measurements requires only 1 s or less, the entire assay for an individual sample is completed in a few minutes and a simple calculation gives the AA content. Since air oxidation of AA samples is a serious source of error, the minimal handling and rapidity of the proposed assay are particularly advantageous. EXPERIMENTAL SECTION Reagents and Solutions. All chemicals for buffer solutions, etc. were reagent grade and were used as received. AAO was purchased from Boehringer-Mannheim(Indianapolis). Enzyme solutions having a concentration of 1 mg/100 pL were prepared in 0.05 M phosphate buffer, pH 5.6, fresh before each set of assays. When not in use they may be kept at 0 "C and are stable for at least 2 weeks (longest time tested) with no deterioration. The AA stock solutions were made in doubled distilled water and were 1.00-1.50 mM. These solutions, stored in a refrigerator, were stable for at least 2 h. Two buffer media were evaluated for the assays. For routine tissue samples 0.05 M phosphate/0.001 M EDTA (ethylenediaminetetraacetic acid), pH 7.0, can be used. If samples are to be evaluated from brain slices or used in pharmacological or physiological experiments, the bicarbonate buffer for maintaining the functioning tissue works equally well. This has the composition: 124 mM NaCl, 5 mM KC1, 1.24 mM KHZP04, 1.3 mM MgS04, 2.5 mM CaCl,, 26 mM NaHC03,and 10 mM glucose. This buffer is saturated with a 95% 02/5% C 0 2 gas mixture while in use. Apparatus. A small electrochemical cell of approximately 100 pL volume (Figure 1)was constructed by cutting off the end of an Eppendorf plastic pipet tip and force-fitting the piece over the end of a glassy carbon electrode (BioanalyticalSystems, Model GCE). This gives an easily removable but watertight seal. When the electrode is used in the inverted position, it forms an extremely convenient cell, A small platinum wire auxiliary electrode and a Ag/AgCl wire reference electrode are positioned with a micromanipulator (or any convenient holder) as seen in Figure l. This arrangementprovides complete access for solution additions, etc. Miniature electochemical cells of this general design have been reported ( I ) . The potentiostat for applying the potential in the chronoamperometric measurements was a PAR 174A (Princeton Applied Research Corp.) and the current-time response was displayed on 0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 8, JULY 1982
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Calculations. Each of the currents at 500 ms is directly proportional to the actual concentrations of electroactive species present during each of the three stepwise measurements. The first current, il, has three components: the unknown concentrations of endogenous AA (the quantity to be determined) plus the concentration of other endogenous electroactive species and any residual current the electrode may exhibit. The residual currents were negligible in all cases. The value of iz is proportional to the concentrations in il plus the concentration of AA standard added. Then, after the addition of AAO, the remaining i3 is only due to other electroactive species plus residual current. This current, obtained after two volume additions, is to be increased in value by the dilution factor Dz,before subtracting from il, and by D3 before subtracting from iz. The algebra of this type of standard addition analysis with two dilutions is cumbersome but straightforward, and the calculation €or the unknown endogenous concentration of AA, CM, is best given as
(4 - D~iJCstd
CA.4 = (ip
Flgure 1. Inverted cell arssembly used in the chronoamperometric
determination of ascorbic acid in tissue homogenates. Details of its construction appear in text EPT = Eppendorf pipet tip; GCE = glassy carbon electrode; WE = surface of working electrode; REF = reference electrode (Ag/AgCI);AUX = auxiliary electrode (Ag or Pt wire). a Tektronix oscilloscope with SA22N differential amplifier (Tektronix, Inc.) or a Houlston Model 5000 X-Y recorder (Houston Instruments) with time base. Any strip chart recorder with fast chart speed can be used. The potential can be applied manually or with any gating device. Due to the highly irreversible oxidation of AA, other electrochemical measuring techniques (potential sweep or differential pulse voltammetry) are far less quantitative than chronoamperometry. Tissue Sampling of Brain Slices. All samples used in this study consisted of nervous tissue from 275 to 325 g male albino rats. This tissue was obtained from coronal slices of a single hemisphere at the level of the caudate nucleus. Each slice was approximately 400-500 p,m thick and corresponded to the coordinates AP 8620-8380 of Konig and Klippel (2). Slices were cut by hand with a single-edged razor blade or on a McIlwain tissue chopper. Before the sample was weighed the caudate nucleus portion of the slice was iiaolated by peeling away the cortex and corpus callosum dorsally and making a cut diagonally across the ventral portion of the slice dorsal to the anterior commissure. This latter procedure ensures t,hat only caudate nucleus is analyzed, with no contamination by the nucleus accumbens. Any other tissue sample procedure giving sample sizes in the range 3-25 mg will fit the assay procedure as given. Larger samples can be aliquoted accordingly. Assay Procedure. The small tinsue or other sample is weighed and placed in a small conical tube with 100 pL of either of the previously described buffer solutions. If samples are not to be analyzed directly, the tube is immediately frozen in dry ice. Just prior to analysis the contents are thawed and immediately sonicated. (If a series of samples are sonicated, they are then refrozen and thawed individually just prior to actual assay.) The chronoamperometric i-t measurements are made by using the following parameters: E,, = +0.55 V vs. Ag/AgCl, 500 ms duration, and current is measured off the oscilloscope or recorder trace at 500 ms. Usually each measurement is repeated in triplicate with the cell tapped briefly between each run (to negate any depletion at the electrode surface from the previous i-t curve). The triplicate runs are averaged. This initial average i at 500 ms represents total electroactive species oxidizable at +0.55 V. Next, 10 pL of ascorbate standard is pipetted into the cell and it is tapped for mixing. Triplicate i-t curves are run as above and the mean i a t 500 ms provides a "standard addition" calibration of the electrode ffor AA. Now 10 pL of the stock AAO solution is added with stirring and triplicate i-t curves are again run. This final current represents the sum of the electrode residual current and any other electroactivespecies less all AA present. (The amount of enzyme added is purposely large to ensure rapid and total removal of AA.)
- D3i3) + (il - DZi3)(Dz/D3 - 2)
Since il, i2, i3, Cntd,Dz, and D3 are experimental values, C u is best calculated by direct substitution. The values of il, iz, and i3 are typically in microamperes. The initial homogenate volume, v h , is calculated in the following manner. The actual volume after homogenization is the 100 pL of buffer plus 70% of the wet tissue weight expressed in milligrams. This correction, for the approximately 70% aqueous volume equivalent of tissue, is widely used in assays of endogenous brain constituents. One may use this entire volume or an aliquot, V,, for the actual electrochemical measurements. The dilution factors for the standard additions then become
D1 =
10 p L
v,+ 10 pL
where V , is defined above and the 10 pL represents the addition of the AA standard solution v, + 20 pL
Dz =
Va where here the 20 pL is the sum of the additions of AA standard and the AAO addition v, + 10 pL D3 = va
the dilution factor used to increase i3 to its value, before the addition of AAO. (If the total homogenate is analyzed, V, = V, in all the above dilution factors.) Dl is only used to calculate the actual diluted concentration of standard AA, C S ~as, where Cstockis the concentration of standard AA in micromolar units. Note D , and D3 are inverted compared to Dl since they are used to increase the observed i3 due to the two dilutions. If the above units are used, the value of CM is in micromolar units. Multiplication of CM by v h and division by tissue weight in grams give the final results of AA content in micromoles per gram of wet tissue. For precise results all microliter syringes for standard additions should be calibrated and the actual calibrated values used for dilution factors, etc. Obviously, incremental additions other than 10 pL can be used.
RESULTS AND DISCUSSION Enzyme Specificity. Ascorbic acid oxidase was found to be specific for ascorbic acid even in the presence of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC), two catechol compounds known to coexist with ascorbic acid in brain tissue (see Figure 2). The experiment was conducted in physiological HC03- buffer thoroughly saturated with a 95% 02/5% C 0 2gas mixture. The starting concentration of each species tested separately was approximately 100 pM. The currents recorded were i a t 500 ms from chronoamperometric pulses conducted every minute. The currents were normalized
Anal. Chem. 1982, 5 4 , 1454-7455
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Table I. Electrochemical-AAO Assay of Caudate
Tissue Samples concn sample no.a
sample
AA,
wt, mg
wol/g
1 2 3 4 5 6
13.6 7.6
0.675 2.04
4.4
2.30 2.04 2.44
11.6 12.2 6.2
2.71
Each sample is taken from a caudata slice removed Mean = 2.03 i 0.29 SEM from a different rat brain. w o l /g. a
I
I
2
I
I
rlat(min)
6
1
I
10
Flgure 2. Demonstration of ascorbic acid oxidase specificity for ascorbic acid. Experimental details in text. AA = ascorbic acid: DA = 3,44ihydroxyphenylethylamine (dopamine):DOPAC = 3,rldihydroxyphenylacetic acid.
to the first value obtained for ascorbic acid. At minute 3 (after the current data were taken) 10 p L of the AAO solution was added. No significant current decrease for either DA or DOPAC occurred, while the AA current value was reduced by 95% within 1-2 min. No reduction of AA current values was observed over this time range in solutions not treated with AAO. Subsequent studies in volumes similar to those used in the tissue analysis demonstrated that the reduction of AA current was quantitative (Le., 99%). Similar specificity was confirmed in the phosphate buffer system. These results agree with those of Matsumoto et al. (3) and Gerwin et al. (4). Effect of Sample Work-up on Ascorbic Acid Concentrations. Since ascorbic acid is susceptible to air oxidation, it is necessary to determine what effect sample work-up has on concentrations of ascorbic acid likely to be found in tissue samples. To test this, 100 p L of 200 pM ascorbic acid in buffer was treated like a tissue sample. This involved freezing a t -70 OC, thawing, sonicating, refreezing, rethawing, and then assaying. Chronoamperometric measurements were made on 50 pL of this "sample". A standard addition of ascorbic acid was then added and chronoamperometric measurements were made again. If one assumes that no ascorbate is lost during these manipulations, one should be able to predict the value of the second current measurement. In all cases, the current was adequately predicted within 6%. The agreement was 5.6% (f0.84 standard deviation) N = 4. However, the experimental value was systematically too low by this amount. Consequently, this value can be assumed to be the amount of ascorbic acid lost during work-up. This relative error is slight and approaches the uncertainty of the current measurement. The observed precision for repeated assays of
standard ascorbate samples expressed as relative standard deviation is f2.6% ( N = 4). Brain Slice Tissue Results. The present results can be best compared to the liquid chromatographic analyses which are also compound-specific analyses. Table I gives the results of caudate slice assays from six rats using the present assay method. The mean value of 2.03 f 0.29 SEM in pmol/g is in excellent agreement with the caudate values (range of 1.40-2.50 pmol/g) analyzed by liquid chromatography according to Milby et al. (5). Since individual animals show this range of variation even in the same anatomical area, no absolute comparisons can be made. I t can be noted that the liquid chromatographic analyses have been shown to agree well with older data on mouse, rat, and guinea pig whole brains (6).Thus, there appears to be no question about the equivalency of the results obtained with the new assay compared to previous methods. This assay method is readily adaptable to all other small brain samples containing AA (synaptosomes, vesicles, etc.). The exact protocol described will take tissue samples up to ca. 25 mg. If larger samples are used, appropriate aliquots should be taken following homogenization. LITERATURE CITED (1) Karolczak, M.; Dreiiing, R.; Adarns, R. N.; Fellce, L. J.; Kissinger, P. T. Anal. Len. 1076,9 , 783-793. (2) Konig, J. F. R.; Klippel, R. A. "The Rat Brain, A Stereotaxic Atlas of the Forebrain and the Lower Parts of the Brain Stern"; Robert E. Krieger: New York, 1963. (3) Matsumoto, K.; Yamada, K.; Otsajina, Y. Anal. Chem. lQ81,53, 1974-1979. (4) . . Gerwin. 6.: Berstein, S. R.: Westley, J. J . Biol. Chem. 1074, 149, 2005-2008. (5) Mllby, K.: Oke, A,; Adams, R. N. Neurosci. Len. 1082, 28, 15-20. (6) Thrlvikraman, K. V.: Refshauge, C.; Adams, R. N. Life Sci. 1074, 15, 1335-1342.
RECEIVED for review March 1,1982. Accepted April 23,1982. The support of this work by the National Science Foundation, Neurobiology Section, via Grant BNS 7914226 is gratefully acknowledged.
High-Voltage Device for Clearing Clogged Glass Jet Separators Frederlck L. Cardinall" and Luis E. Lowe Department of the Interior, United States Geological Survey, National Water Qualiw Laboratory, 648 1-H Peachtree Industrlal Blvd., Doraville, Georgia 30340
A common problem in gas chromatography/mass spectrometry (GC/MS) is blocked glass jet separators. Current methods to remove blockages include the use of fine wires,
ultrasound, and a combination of solvents and vacuum (I, 2). These methods are time-consuming and may result in damage to the separator. The high-voltage discharge method described
This artlcle not subject to U.S. Copyrlght. Published 1982 by the American Chemlcal Society
