ANALYTICAL
BIOCHEMISTRY
88, 495-503
(1978)
A Microassay for the Determination Oxidase Activity Using Electron Chromatography’ F. F. FARRIS,? E. 0. MAGARIAN. College
oj’pharmacy.
North
Dakota
State
of Monoamine Capture Gas
AND F. 0. SLININGER
University.
Fargo.
North
Dakota
58102
Received August 9. 1977; accepted March 9. 1978 A sensitive and specific assay for determining monoamine oxidase (MAO) activity in serum and platelets is described. The procedure employs m-iodobenzylamine as substrate. The product, m-iodobenzaldehyde, is separated on an OV-17 column and measured by electron capture. Gas chromatography offers the advantage of analytical specificity without the need for additional procedural steps to eliminate potential interferences. Electron capture detection of the iodinated aldehyde is sufficiently sensitive to allow routine analysis on platelet samples of less than 15 pg of protein and serum aliquots of 50 ~1 or less. Analysis of approximately 80 samples per day may be accomplished by a single worker by employing an automatic sampling system for gas chromatographic injection. This fact, in addition to the small sample size, makes the method particularly suitable for the determination of large numbers of clinical samples.
In recent years, it has become increasingly clear that the monoamine oxidases (MAO) present in blood may fluctuate significantly from one physiological state to another. Changes in the serum enzyme have been detected in congestive heart disease (1,2), diabetes mellitus (2,3), and hepatic fibrosis (4S) as well as in patients receiving various types of drug therapy (6,7). Alterations in platelet MAO have been considered valuable in the study of mental depression (8,9) and schizophrenia (10) for several years. Such variations make the routine monitoring of these enzymes of potential clinical significance. Monoamine oxidase activity has commonly been measured by manometric (11) or spectrophotometric (12,13) methods. These techniques generally suffer from a lack of specificity and sensitivity. Increased sensitivity may be attained by using fluorometric (14,15) or radiochemical (16,17) assays, but these may have associated interferences or require laborious steps in order to eliminate the interfering substances. 1 Presented in part at the 1 lth Great Lakes Regional American Chemical Society Meeting, Steven’s Point, Wisconsin, June 6-8, 1977. 2 Author to whom correspondence should be addressed. 495
0003~2697/78/0882-0495$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction m any form reserved.
496
FARRIS,
MAGARIAN,
AND
SLININGER
We have developed an assay which utilizes m-iodobenzylamine as a substrate. The oxidation product, m-iodobenzaldehyde, is extracted into cyclohexane and analyzed by electron capture gas chromatography. The technique is simple and highly sensitive. Gas chromatographic analysis also offers a distinct advantage in analytical specificity. METHODS
Reagents. m-Iodobenzylamine hydrochloride was obtained from ICN Pharmaceuticals, Inc. (Plainview, New York) and recrystallized a minimum of three times from isopropyl alcohol (mp, 188190°C uncorrected). m-Iodobenzaldehyde and p-chlorobenzaldehyde were obtained from Eastman Organic Chemicals, Inc. (Rochester, New York) and were purified by repeated sublimation at room temperature (uncorrected mp, 55-56°C and 46-48°C respectively). Pesticide quality cyclohexane from Matheson Coleman and Bell (Norwood, Ohio) was used without further purification. All other chemicals were analytical reagent grade and used as received. Serum collection. Blood was collected by venipuncture using siliconecoated Vacutainers without anticoagulant (Becton, Dickinson, Co., Rutherford, New Jersey) or by finger prick. For finger prick, the blood was collected directly in a O&ml Reacti-Vial (Pierce Chemical Co., Rockford, Illinois). After collection the samples were allowed to clot by standing for 15 to 30 min at 4°C. Following centrifugation at 15OOg for 10 min, the serum was transferred with a Pasteur pipet to a Reacti-Vial equipped with a Teflon-lined screw cap. All sera not utilized immediately were stored at -50°C until analysis (normally less than 2 weeks). Platelet preparation. Platelets were prepared by a modification of the method developed by Youdim (18). Blood was collected in sodium citrate Vacutainers and centrifuged at 200g for 10 min. The platelet-rich plasma was removed by a Pasteur pipet, placed into a clear glass conical centrifuge tube, and centrifuged at 2000g for 10 min. The supernatant was discarded, and the platelets were washed three successive times by employing 1.0 ml of 0.3 M sucrose solution for every 5 to 6 ml of discarded plasma. Following each washing, the platelets were resedimented at 2000g for 10 min. The platelets were then suspended in a volume of 0.3 M sucrose that was equal to 1.5 times the volume of discarded plasma. The platelets were disrupted by sonication for 45 set using a Branson Model S75 sonicator set at low energy. Unless the platelets were used immediately, the suspension was stored at -50°C in glass tubes with Teflon-lined screw caps until analysis (normally within 1 week). The protein content of the platelet suspension was determined by Folin-Lowry (19) and Bradford’s Coomassie blue (20) techniques. Chromatographic equipment. Gas-liquid chromatography was performed on a Packard Model 420 biomedical gas chromatograph (Packard
GAS CHROMATOGRAPHIC
MAO ASSAY
497
Instruments, Co., Inc. Downers Grove, Illinois) equipped with a lo-mCi Ni-63 electron capture detector. The instrument was operated in the pulsed mode with a pulse width of 1 psec and a pulse period of 200 psec. Glass columns (2 m x 4 mm, i.d.) were packed with 3% OV-17 on 100/120 mesh Gas Chrom Q (Applied Science Laboratories, State College, Pennsylvania). Prior to use the columns were conditioned at 180°C for 24 hr with a carrier gas (95% argon-5% methane, Linde Gas, New York, New York) flow of 10 to 30 ml/min. After column conditioning, the detector was connected, and the chromatograph was adjusted to normal operating conditions. The inlet, column, and detector temperatures were set at 180, 135, and 21o”C, respectively. Attenuation settings were varied as required to obtain the desired response, but settings of 8 and 16 were found to be most useful. Argon-methane was set for a column flow rate of 30 ml/min and a bypass rate of 15 ml/min. Injections of 1 ~1 were made using a Packard Model 700 Automatic Sampling System (Packard Instrument Co., Inc.) with a delay time of 8 min. A Linear Model 255 lo-in. recorder (Linear Instruments Corp., Irvine, California) set at 1 mV was connected to the gas chromatograph through a Hewlett-Packard Model 3370 B electronic integrator (Hewlett-Packard, Cupertino, California) with a noise suppression setting of 3. Assay. To a 2-ml Reacti-Vial equipped with a Teflon-lined screw cap was added 250 ~1 of 7.2 x lop4 M m-iodobenzylamine hydrochloride in 0.2 M phosphate buffer of pH 7.2. The uncapped vial was preincubated in a 30°C water bath for 5 min, after which 50 ,ul of sample (serum. platelet-poor plasma, or platelet preparation which had been equilibrated at 30°C) was added. The vial was then capped tightly, and the mixture was incubated at 30°C for 30 min, followed by the addition of 30 ~1 of 60% perchloric acid and 1 ml of extraction solution (cyclohexane containing 100 pg of p-chlorobenzaldehyde/pl).3 The capped vial was shaken vigorously by hand for a few seconds, mixed for 10 min on a Coulter Blood Mixer (Coulter Electronics, Inc., Hialeah, Florida), and centrifuged for 10 min at 1500g. The cyclohexane layer was removed with a Pasteur pipet and placed into a Packard sampling vial (Packard Instruments, Inc.) in preparation for injection. A series of blanks employed in the procedure (usually one for every six samples) involved incubating the substrate in the absence of serum or platelets. The enzyme source was added immediately following perchloric acid treatment but prior to extraction. Each day a series of standards, ranging in concentration from 0 to 100 pg of m-iodobenzaldehyde per 1~1 of extraction solution, was injected onto the column. A standard curve was prepared from the obtained data by plotting 3 The vials must be capped securely or some loss of m-iodobenzaldehyde volatilization.
may result from
498
FARRIS, MAGARIAN,
AND SLININGER
peak height ratios of m-iodobenzaldehydelp-chlorobenzaldehyde against the concentration of m-iodobenzaldehyde. The concentration of aldehyde generated in an unknown was then determined directly from this graph. RESULTS AND DISCUSSION
The selection of m-iodobenzylamine as substrate for our study was based on three important considerations. First, the aldehyde formed from the oxidative deamination of the amine is quite resistant to further oxidation. This stability was mentioned earlier by Zeller et al. (21) and was one reason for their selection of this amine as substrate for the spectrophotometric assay of mitochondrial MAO. Second, m-iodobenzylamine is a very good substrate for B type MAO (22). Since both serum (7,23) and platelet (24,2.5) enzymes have been shown to preferentially attack B type substrates, selection of m-iodobenzylamine provides some reaction specificity, as well as a relatively rapid rate of oxidation. Finally, miodobenzyldehyde is highly sensitive to detection by electron capture. Under ideal conditions, we observed detection limits of less than 1 pg and a full scale recorder deflection was obtained with 25 pg. To determine the optimal substrate concentration for the incubation
)
3
b TIME
3
A
3
(MINUTES)
FIG. I. Chromatograms obtained from l-pi injections of (a) an 80 pg/pl m-iodobenzaldehyde standard, (b) the product of a 30-min incubation of a 50-~1 serum sample, and (c) the product of a 30-min incubation of substrate only. Peak 1 corresponds to 100 pg/pl of the internal standard,p-chlorobenzaldehyde. Peak 2 corresponds to m-iodobenzaldehyde.
GAS CHROMATOGRAPHIC
MAO ASSAY
499
mixture, assays were performed using different concentrations of miodobenzylamine. The maximum rate of aldehyde production with serum was obtained at substrate concentrations from 3 to 8 x 10m4 M. Marked substrate inhibition was observed at concentrations greater than IOpy M. McEwen (26) has reported a similar effect with serum MAO with both benzylamine and octylamine. Our platelet preparations were less sensitive to the effects of substrate concentration than was serum. At m-iodobenzylamine concentrations up to 3 x lo-” M, there was no indication of substrate inhibition. Instead, we found the expected saturation effect with only a small increase in the rate of aldehyde production occurring above 6 x lo-” M. Because of these substrate characteristics, 6 x lop4 M was chosen as the most appropriate concentration for the routine assay procedure. Figure 1 shows a set of three chromatograms obtained using the standard assay procedure. Approximate retention times for peaks of interest were 2 min for the internal standard (p-chlorobenzaldehyde, peak 1) and 5 min for m-iodobenzaldehyde (peak 2). Peak shape and resolution were excellent. allowing the use of either peak height ratios or peak area ratios as a means of quantification. Figure 2 illustrates a standard curve, obtained by plotting the peak height ratio ofm-iodobenzaldehydelp-chlorobenzaldehyde versus concentration of m-iodobenzaldehyde. The graph is linear over the normal working range of 0 to 100 pg of aldehyde/pl of cyclohexane. To investigate the effect of different amounts of enzyme on the rate of m-iodobenzaldehyde production, we performed a series of incubations
m- IODOBENZALDEHYDE
(pe/pl)
FIG. 2. Standard curve for the quantification ofm-iodobenzaldehyde. The peak height ratio of m-iodobenzaldehyde to p-chlorobenzaldehyde is plotted vs the concentrations in picograms per microliter of m-iodobenzaldehyde. Each point is the average of two injections. Vertical lines represent the range when these values are larger than the circle diameters.
500
FARRIS, MAGARIAN,
0
40
AND SLININGER
80 pl
150
160
SERUM
FIG. 3. Effect of serum MAO concentration on reaction rate. The total amount of miodobenzaldehyde generated is plotted vs the volume of serum in the 300-4 incubation mixture. Conditions are the same as those of the standard assay except that the serum volume was varied. Each point is the mean of three determinations. Vertical lines represent standard deviations when these values are larger than circle diameters.
containing various quantities of serum, or of the platelet preparation. The starting concentrations of substrate solutions were also changed to maintain a final substrate concentration of 6 x 10e4 M and a total incubation volume of 300 ~1. The results of the experiments for serum and platelets appear in Fig. 3 and 4, respectively. In each case, the rate of aldehyde production is proportional to the amount of serum or platelets incubated, and this proportionality extends well above and below the quantity of serum (50 ~1) and platelet protein (about 1.5 pug) normally analyzed. Under the standard assay conditions, oxidation of m-iodobenzylamine also proceeds proportionally with time (Fig. 5). Linearity appears to be maintained for at least 75 min. The precision of our method was determined by analyzing 26 aliquots of a single serum specimen over a period of 3 days. To prevent repeated freezing and thawing of the specimen, it was stored at -50°C in three separate vials, one for each day’s analysis. Eight samples were analyzed the first day and nine on each of the 2 following days. For the 26 aliquots analyzed we found a mean aldehyde production rate and standard deviation of 10.93 + 0.38 nmol/ml of serum/hr. The standard deviation corresponds to 3.5%. As described above, calibration curves were prepared by injecting cyclohexane solutions containing known quantities of m-iodobenzaldehyde and p-chlorobenzaldehyde into the gas chromatograph. Alternatively, aqueous standards of m-iodobenzaldehyde could be incubated and extracted, but this method is relatively inconvenient because of the low
GAS CHROMATOGRAPHIC
0
lb PLATELET
MAO ASSAY
2’0 PROTEIN
501
3b (p’~
)
FIG. 4. Effect of platelet MAO concentrations on reaction rate. The total amount of miodobenzaldehyde generated is plotted vs the platelet protein in micrograms used in the 300~1 incubation mixture. Conditions are the same as those of the standard assay except that the platelet preparation volume (1 I .2 pg of protein/50 ~1 of 0.2 M sucrose) was varied. Each point is the mean of three determinations. Vertical lines represent standard deviations when these values are larger than the circle diameters.
solubility of the aldehyde in water. Furthermore, in this paper has the advantage of minimizing vials that must be processed. On the other hand, complete extraction of m-iodobenzaldehyde and
the technique employed the number of sample our procedure assumes that no relative concen-
yr2.25x-2.79 rz0.996
160
E
P
0
2'0 INCUBATION
4'0 TIME
6'0
6.0
(MINUTES)
FIG. 5. Time course of the enzymatic oxidation. The total of m-iodobenzaldehyde generated is plotted vs the incubation time for (a) 50 ~1 of serum specimen and (b) 11.2 pg of platelet protein. Conditions are the same as those of the standard assay except that incubation time was varied. Each point is the mean of three determinations. Vertical lines represent standard deviations when these values are larger than the circle diameters.
502
FARRIS,
MAGARIAN,
AND
SLININGER
tration of solvent or aldehyde occurs during sample preparation. Our experimental observations support the validity of both assumptions. When 200+1 aqueous samples containing 15 ng of m-iodobenzaldehyde were acidified and extracted with 0.5 ml of extraction solution, the cyclohexane phase was found to contain an average of 29.0 pg of miodobenzaldehyde/pl (N = 3), a value corresponding to 97% recovery. No attempt was made to examine recoveries at other concentrations or at different incubation times, since the high precision obtained in our 3-day repetitive determination study and the excellent linearity of the incubation time and substrate concentration studies (Figs. 3, 4, and 5) strongly suggest that any variations in the procedure would necessarily be systematic. These observations indicate that the assumptions made concerning our calibration technique are valid and that the procedure as described in this paper gives an accurate estimate of the aldehyde generated. The suitability of m-iodobenzylamine as a substrate for mitochondrial MAO determinations has already been studied by Zeller et al. (2 1,22, 25). When used for platelet MAO analysis (25), this substrate was sensitive to differences in age as well as to changes in MAO activity of patients treated for parkinsonism. From our studies the substrate also appears to be sensitive to variations in serum MAO activity. We analyzed 23 sera and compared them with activities obtained using the spectrophotometric method of McEwen and Cohen (12). This gave a comparison of miodobenzylamine with one of the most commonly used MAO substrates, benzylamine. Benzylamine had an activity range of 6.2 to 22.0 nmol/ml of serum/hr as compared with 4.8 to 12.6 nmoYm1 of serum/hr for the iodinated substrate. The correlation between the two sets was good (r = 0.82), suggesting that the two substrates respond similarly to serum enzyme variations. The method as described applies to the analysis of serum and platelet samples of specific size. It appears, however, to be more broadly applicable. Successful analyses of serum samples ranging in volume from 5 to 200 ~1 have been accomplished, and it seems likely that the technique could be applied to tissue homogenates or mitochondrial preparations. The procedure involves a semiautomated system for determining MAO levels in large numbers of samples. The technique is simple and employs only small quantities of relatively inexpensive reagents. The high degree of sensitivity permits the analysis of serum samples obtained from finger prick quantities of blood or from extremely small platelet samples. This aspect may be of particular value in cases where routine patient monitoring is of interest. REFERENCES 1. McEwen, C. M., Jr., and Harrison, D. C. (1965) J. Lab. C/in. Med. 65, 546-59. 2. Nilsson, S. E., Tryding, N., and Tufvesson, G. (1%8)Acta Med. Stand. 184, 105- 112. 3. Tryding, N., Nilsson, S. E., Tufvesson, G., Berg, R., Carlstrom, S., Elmfors, B., and Nilsson, J. E. (1969) Stand. J. Clin. Lab. Invest. 23, 79-84.
GAS CHROMATOGRAPHIC
MAO ASSAY
503
4. Ito, K., Nakagawa, J.. Minakuchi. C., and Fukase, M. (1971) Digestion 4, 49-58. 5. Lin, A. W-S. M., and Castell, D. 0. (1974) B&hem. Med. 9, 373-85. 6. Klaiber, E. L.. Kobayashi. Y., and Broverman, D. M. (1971) J. C/in. Endocrinol. 33, 630-38. 7. Robinson, D. S.. Lovenberg. W.. Keiser. H.. and Sjoerdsma. A. (1968) Biochem. Pharmacol. 17, 109- 19. 8. Murphy, D. L., and Neiss. R. (1972) Amer. J. Psychiatv 128, 1351-57. 9. Landowski, J., Lysiak. W.. and Angielski. S. (1975) Biochem. Med. 14, 347-54. 10. Murphy, D. L.. and Wyatt, R. J. (1972) Nature (London) 238, 225-26. 11. Blaschko. H., Richter, D.. and Schlossmann. H. (1937) Biochem. J. 31, 2187-96. 12. McEwen, C. M., Jr., and Cohen. J. D. (1965) J. Lab. Clin. Med. 62, 766-76. 13. Tabor, C. W., Tabor, H., and Rosenthal. S. M. (1954) J. Biol. Chem. 208, 645-61. 14. Century, B., and Rupp, K. L. (1968) Biochem. Pharmacol. 17, 2012-13. 15. Takahashi. S.. and Karasawa. T. (197s) Clin. Chem. Acta 62, 393-400. 16. Wurtman. R. J., and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439-41. 17. Demisch, L., Bochnik, H. J., and Seiler. N. (1976) C/in. Chem. Acta 70, 357-69. 18. Youdim, M. B. H. (1976) in Monoamine Oxidase and its Inhibition, Ciba Foundation Symposium 39. pp. 405-06, ElsevieriExcerpta MedicaiNorth-Holland. New York. 19. Plummer. D. T. (1971) An Introduction to Practical Biochemistry, pp. 156-57, McGraw-Hill, New York. 20. Bradford, M. M. (1976) Anal. Biochem. 72, 248-54. 21. Zeller, V., Ramachander, G.. and Zeller. E. A. (1965) J. Med. Chem. 8, 440-43. 22. Bathina, H. B.. Huprikar. S. V., and Zeller. E. A. (1975) Fed. Proc. 34, 293. 23. McEwen, C. M.. Jr. (1%5) .I. Biol. Chem. 240, 2003- 10. 24. Belmaker. R. H., Ebstein. R.. Rimon, R., Wyatt, R. J., and Murphy, D. L. (1976) Acta
Psychiatr.
Stand.
54, 67-12.
25. Zeller. E. A.. Boshes. B., Arbit. J., Bieber, M., Blonsky. E. R.. Dolkart, M.. and Huprikar. S. V., (1976) .I. Neural Trans. 39, 63-77. 26. McEwen. C. M.. Jr. (1965)J. Biol. C/tern. 240, 2011-18.
BIOCHEMISTRY
88, 495-503
(1978)
A Microassay for the Determination Oxidase Activity Using Electron Chromatography’ F. F. FARRIS,? E. 0. MAGARIAN. College
oj’pharmacy.
North
Dakota
State
of Monoamine Capture Gas
AND F. 0. SLININGER
University.
Fargo.
North
Dakota
58102
Received August 9. 1977; accepted March 9. 1978 A sensitive and specific assay for determining monoamine oxidase (MAO) activity in serum and platelets is described. The procedure employs m-iodobenzylamine as substrate. The product, m-iodobenzaldehyde, is separated on an OV-17 column and measured by electron capture. Gas chromatography offers the advantage of analytical specificity without the need for additional procedural steps to eliminate potential interferences. Electron capture detection of the iodinated aldehyde is sufficiently sensitive to allow routine analysis on platelet samples of less than 15 pg of protein and serum aliquots of 50 ~1 or less. Analysis of approximately 80 samples per day may be accomplished by a single worker by employing an automatic sampling system for gas chromatographic injection. This fact, in addition to the small sample size, makes the method particularly suitable for the determination of large numbers of clinical samples.
In recent years, it has become increasingly clear that the monoamine oxidases (MAO) present in blood may fluctuate significantly from one physiological state to another. Changes in the serum enzyme have been detected in congestive heart disease (1,2), diabetes mellitus (2,3), and hepatic fibrosis (4S) as well as in patients receiving various types of drug therapy (6,7). Alterations in platelet MAO have been considered valuable in the study of mental depression (8,9) and schizophrenia (10) for several years. Such variations make the routine monitoring of these enzymes of potential clinical significance. Monoamine oxidase activity has commonly been measured by manometric (11) or spectrophotometric (12,13) methods. These techniques generally suffer from a lack of specificity and sensitivity. Increased sensitivity may be attained by using fluorometric (14,15) or radiochemical (16,17) assays, but these may have associated interferences or require laborious steps in order to eliminate the interfering substances. 1 Presented in part at the 1 lth Great Lakes Regional American Chemical Society Meeting, Steven’s Point, Wisconsin, June 6-8, 1977. 2 Author to whom correspondence should be addressed. 495
0003~2697/78/0882-0495$02.00/O Copyright 0 1978 by Academic Press. Inc. All rights of reproduction m any form reserved.
496
FARRIS,
MAGARIAN,
AND
SLININGER
We have developed an assay which utilizes m-iodobenzylamine as a substrate. The oxidation product, m-iodobenzaldehyde, is extracted into cyclohexane and analyzed by electron capture gas chromatography. The technique is simple and highly sensitive. Gas chromatographic analysis also offers a distinct advantage in analytical specificity. METHODS
Reagents. m-Iodobenzylamine hydrochloride was obtained from ICN Pharmaceuticals, Inc. (Plainview, New York) and recrystallized a minimum of three times from isopropyl alcohol (mp, 188190°C uncorrected). m-Iodobenzaldehyde and p-chlorobenzaldehyde were obtained from Eastman Organic Chemicals, Inc. (Rochester, New York) and were purified by repeated sublimation at room temperature (uncorrected mp, 55-56°C and 46-48°C respectively). Pesticide quality cyclohexane from Matheson Coleman and Bell (Norwood, Ohio) was used without further purification. All other chemicals were analytical reagent grade and used as received. Serum collection. Blood was collected by venipuncture using siliconecoated Vacutainers without anticoagulant (Becton, Dickinson, Co., Rutherford, New Jersey) or by finger prick. For finger prick, the blood was collected directly in a O&ml Reacti-Vial (Pierce Chemical Co., Rockford, Illinois). After collection the samples were allowed to clot by standing for 15 to 30 min at 4°C. Following centrifugation at 15OOg for 10 min, the serum was transferred with a Pasteur pipet to a Reacti-Vial equipped with a Teflon-lined screw cap. All sera not utilized immediately were stored at -50°C until analysis (normally less than 2 weeks). Platelet preparation. Platelets were prepared by a modification of the method developed by Youdim (18). Blood was collected in sodium citrate Vacutainers and centrifuged at 200g for 10 min. The platelet-rich plasma was removed by a Pasteur pipet, placed into a clear glass conical centrifuge tube, and centrifuged at 2000g for 10 min. The supernatant was discarded, and the platelets were washed three successive times by employing 1.0 ml of 0.3 M sucrose solution for every 5 to 6 ml of discarded plasma. Following each washing, the platelets were resedimented at 2000g for 10 min. The platelets were then suspended in a volume of 0.3 M sucrose that was equal to 1.5 times the volume of discarded plasma. The platelets were disrupted by sonication for 45 set using a Branson Model S75 sonicator set at low energy. Unless the platelets were used immediately, the suspension was stored at -50°C in glass tubes with Teflon-lined screw caps until analysis (normally within 1 week). The protein content of the platelet suspension was determined by Folin-Lowry (19) and Bradford’s Coomassie blue (20) techniques. Chromatographic equipment. Gas-liquid chromatography was performed on a Packard Model 420 biomedical gas chromatograph (Packard
GAS CHROMATOGRAPHIC
MAO ASSAY
497
Instruments, Co., Inc. Downers Grove, Illinois) equipped with a lo-mCi Ni-63 electron capture detector. The instrument was operated in the pulsed mode with a pulse width of 1 psec and a pulse period of 200 psec. Glass columns (2 m x 4 mm, i.d.) were packed with 3% OV-17 on 100/120 mesh Gas Chrom Q (Applied Science Laboratories, State College, Pennsylvania). Prior to use the columns were conditioned at 180°C for 24 hr with a carrier gas (95% argon-5% methane, Linde Gas, New York, New York) flow of 10 to 30 ml/min. After column conditioning, the detector was connected, and the chromatograph was adjusted to normal operating conditions. The inlet, column, and detector temperatures were set at 180, 135, and 21o”C, respectively. Attenuation settings were varied as required to obtain the desired response, but settings of 8 and 16 were found to be most useful. Argon-methane was set for a column flow rate of 30 ml/min and a bypass rate of 15 ml/min. Injections of 1 ~1 were made using a Packard Model 700 Automatic Sampling System (Packard Instrument Co., Inc.) with a delay time of 8 min. A Linear Model 255 lo-in. recorder (Linear Instruments Corp., Irvine, California) set at 1 mV was connected to the gas chromatograph through a Hewlett-Packard Model 3370 B electronic integrator (Hewlett-Packard, Cupertino, California) with a noise suppression setting of 3. Assay. To a 2-ml Reacti-Vial equipped with a Teflon-lined screw cap was added 250 ~1 of 7.2 x lop4 M m-iodobenzylamine hydrochloride in 0.2 M phosphate buffer of pH 7.2. The uncapped vial was preincubated in a 30°C water bath for 5 min, after which 50 ,ul of sample (serum. platelet-poor plasma, or platelet preparation which had been equilibrated at 30°C) was added. The vial was then capped tightly, and the mixture was incubated at 30°C for 30 min, followed by the addition of 30 ~1 of 60% perchloric acid and 1 ml of extraction solution (cyclohexane containing 100 pg of p-chlorobenzaldehyde/pl).3 The capped vial was shaken vigorously by hand for a few seconds, mixed for 10 min on a Coulter Blood Mixer (Coulter Electronics, Inc., Hialeah, Florida), and centrifuged for 10 min at 1500g. The cyclohexane layer was removed with a Pasteur pipet and placed into a Packard sampling vial (Packard Instruments, Inc.) in preparation for injection. A series of blanks employed in the procedure (usually one for every six samples) involved incubating the substrate in the absence of serum or platelets. The enzyme source was added immediately following perchloric acid treatment but prior to extraction. Each day a series of standards, ranging in concentration from 0 to 100 pg of m-iodobenzaldehyde per 1~1 of extraction solution, was injected onto the column. A standard curve was prepared from the obtained data by plotting 3 The vials must be capped securely or some loss of m-iodobenzaldehyde volatilization.
may result from
498
FARRIS, MAGARIAN,
AND SLININGER
peak height ratios of m-iodobenzaldehydelp-chlorobenzaldehyde against the concentration of m-iodobenzaldehyde. The concentration of aldehyde generated in an unknown was then determined directly from this graph. RESULTS AND DISCUSSION
The selection of m-iodobenzylamine as substrate for our study was based on three important considerations. First, the aldehyde formed from the oxidative deamination of the amine is quite resistant to further oxidation. This stability was mentioned earlier by Zeller et al. (21) and was one reason for their selection of this amine as substrate for the spectrophotometric assay of mitochondrial MAO. Second, m-iodobenzylamine is a very good substrate for B type MAO (22). Since both serum (7,23) and platelet (24,2.5) enzymes have been shown to preferentially attack B type substrates, selection of m-iodobenzylamine provides some reaction specificity, as well as a relatively rapid rate of oxidation. Finally, miodobenzyldehyde is highly sensitive to detection by electron capture. Under ideal conditions, we observed detection limits of less than 1 pg and a full scale recorder deflection was obtained with 25 pg. To determine the optimal substrate concentration for the incubation
)
3
b TIME
3
A
3
(MINUTES)
FIG. I. Chromatograms obtained from l-pi injections of (a) an 80 pg/pl m-iodobenzaldehyde standard, (b) the product of a 30-min incubation of a 50-~1 serum sample, and (c) the product of a 30-min incubation of substrate only. Peak 1 corresponds to 100 pg/pl of the internal standard,p-chlorobenzaldehyde. Peak 2 corresponds to m-iodobenzaldehyde.
GAS CHROMATOGRAPHIC
MAO ASSAY
499
mixture, assays were performed using different concentrations of miodobenzylamine. The maximum rate of aldehyde production with serum was obtained at substrate concentrations from 3 to 8 x 10m4 M. Marked substrate inhibition was observed at concentrations greater than IOpy M. McEwen (26) has reported a similar effect with serum MAO with both benzylamine and octylamine. Our platelet preparations were less sensitive to the effects of substrate concentration than was serum. At m-iodobenzylamine concentrations up to 3 x lo-” M, there was no indication of substrate inhibition. Instead, we found the expected saturation effect with only a small increase in the rate of aldehyde production occurring above 6 x lo-” M. Because of these substrate characteristics, 6 x lop4 M was chosen as the most appropriate concentration for the routine assay procedure. Figure 1 shows a set of three chromatograms obtained using the standard assay procedure. Approximate retention times for peaks of interest were 2 min for the internal standard (p-chlorobenzaldehyde, peak 1) and 5 min for m-iodobenzaldehyde (peak 2). Peak shape and resolution were excellent. allowing the use of either peak height ratios or peak area ratios as a means of quantification. Figure 2 illustrates a standard curve, obtained by plotting the peak height ratio ofm-iodobenzaldehydelp-chlorobenzaldehyde versus concentration of m-iodobenzaldehyde. The graph is linear over the normal working range of 0 to 100 pg of aldehyde/pl of cyclohexane. To investigate the effect of different amounts of enzyme on the rate of m-iodobenzaldehyde production, we performed a series of incubations
m- IODOBENZALDEHYDE
(pe/pl)
FIG. 2. Standard curve for the quantification ofm-iodobenzaldehyde. The peak height ratio of m-iodobenzaldehyde to p-chlorobenzaldehyde is plotted vs the concentrations in picograms per microliter of m-iodobenzaldehyde. Each point is the average of two injections. Vertical lines represent the range when these values are larger than the circle diameters.
500
FARRIS, MAGARIAN,
0
40
AND SLININGER
80 pl
150
160
SERUM
FIG. 3. Effect of serum MAO concentration on reaction rate. The total amount of miodobenzaldehyde generated is plotted vs the volume of serum in the 300-4 incubation mixture. Conditions are the same as those of the standard assay except that the serum volume was varied. Each point is the mean of three determinations. Vertical lines represent standard deviations when these values are larger than circle diameters.
containing various quantities of serum, or of the platelet preparation. The starting concentrations of substrate solutions were also changed to maintain a final substrate concentration of 6 x 10e4 M and a total incubation volume of 300 ~1. The results of the experiments for serum and platelets appear in Fig. 3 and 4, respectively. In each case, the rate of aldehyde production is proportional to the amount of serum or platelets incubated, and this proportionality extends well above and below the quantity of serum (50 ~1) and platelet protein (about 1.5 pug) normally analyzed. Under the standard assay conditions, oxidation of m-iodobenzylamine also proceeds proportionally with time (Fig. 5). Linearity appears to be maintained for at least 75 min. The precision of our method was determined by analyzing 26 aliquots of a single serum specimen over a period of 3 days. To prevent repeated freezing and thawing of the specimen, it was stored at -50°C in three separate vials, one for each day’s analysis. Eight samples were analyzed the first day and nine on each of the 2 following days. For the 26 aliquots analyzed we found a mean aldehyde production rate and standard deviation of 10.93 + 0.38 nmol/ml of serum/hr. The standard deviation corresponds to 3.5%. As described above, calibration curves were prepared by injecting cyclohexane solutions containing known quantities of m-iodobenzaldehyde and p-chlorobenzaldehyde into the gas chromatograph. Alternatively, aqueous standards of m-iodobenzaldehyde could be incubated and extracted, but this method is relatively inconvenient because of the low
GAS CHROMATOGRAPHIC
0
lb PLATELET
MAO ASSAY
2’0 PROTEIN
501
3b (p’~
)
FIG. 4. Effect of platelet MAO concentrations on reaction rate. The total amount of miodobenzaldehyde generated is plotted vs the platelet protein in micrograms used in the 300~1 incubation mixture. Conditions are the same as those of the standard assay except that the platelet preparation volume (1 I .2 pg of protein/50 ~1 of 0.2 M sucrose) was varied. Each point is the mean of three determinations. Vertical lines represent standard deviations when these values are larger than the circle diameters.
solubility of the aldehyde in water. Furthermore, in this paper has the advantage of minimizing vials that must be processed. On the other hand, complete extraction of m-iodobenzaldehyde and
the technique employed the number of sample our procedure assumes that no relative concen-
yr2.25x-2.79 rz0.996
160
E
P
0
2'0 INCUBATION
4'0 TIME
6'0
6.0
(MINUTES)
FIG. 5. Time course of the enzymatic oxidation. The total of m-iodobenzaldehyde generated is plotted vs the incubation time for (a) 50 ~1 of serum specimen and (b) 11.2 pg of platelet protein. Conditions are the same as those of the standard assay except that incubation time was varied. Each point is the mean of three determinations. Vertical lines represent standard deviations when these values are larger than the circle diameters.
502
FARRIS,
MAGARIAN,
AND
SLININGER
tration of solvent or aldehyde occurs during sample preparation. Our experimental observations support the validity of both assumptions. When 200+1 aqueous samples containing 15 ng of m-iodobenzaldehyde were acidified and extracted with 0.5 ml of extraction solution, the cyclohexane phase was found to contain an average of 29.0 pg of miodobenzaldehyde/pl (N = 3), a value corresponding to 97% recovery. No attempt was made to examine recoveries at other concentrations or at different incubation times, since the high precision obtained in our 3-day repetitive determination study and the excellent linearity of the incubation time and substrate concentration studies (Figs. 3, 4, and 5) strongly suggest that any variations in the procedure would necessarily be systematic. These observations indicate that the assumptions made concerning our calibration technique are valid and that the procedure as described in this paper gives an accurate estimate of the aldehyde generated. The suitability of m-iodobenzylamine as a substrate for mitochondrial MAO determinations has already been studied by Zeller et al. (2 1,22, 25). When used for platelet MAO analysis (25), this substrate was sensitive to differences in age as well as to changes in MAO activity of patients treated for parkinsonism. From our studies the substrate also appears to be sensitive to variations in serum MAO activity. We analyzed 23 sera and compared them with activities obtained using the spectrophotometric method of McEwen and Cohen (12). This gave a comparison of miodobenzylamine with one of the most commonly used MAO substrates, benzylamine. Benzylamine had an activity range of 6.2 to 22.0 nmol/ml of serum/hr as compared with 4.8 to 12.6 nmoYm1 of serum/hr for the iodinated substrate. The correlation between the two sets was good (r = 0.82), suggesting that the two substrates respond similarly to serum enzyme variations. The method as described applies to the analysis of serum and platelet samples of specific size. It appears, however, to be more broadly applicable. Successful analyses of serum samples ranging in volume from 5 to 200 ~1 have been accomplished, and it seems likely that the technique could be applied to tissue homogenates or mitochondrial preparations. The procedure involves a semiautomated system for determining MAO levels in large numbers of samples. The technique is simple and employs only small quantities of relatively inexpensive reagents. The high degree of sensitivity permits the analysis of serum samples obtained from finger prick quantities of blood or from extremely small platelet samples. This aspect may be of particular value in cases where routine patient monitoring is of interest. REFERENCES 1. McEwen, C. M., Jr., and Harrison, D. C. (1965) J. Lab. C/in. Med. 65, 546-59. 2. Nilsson, S. E., Tryding, N., and Tufvesson, G. (1%8)Acta Med. Stand. 184, 105- 112. 3. Tryding, N., Nilsson, S. E., Tufvesson, G., Berg, R., Carlstrom, S., Elmfors, B., and Nilsson, J. E. (1969) Stand. J. Clin. Lab. Invest. 23, 79-84.
GAS CHROMATOGRAPHIC
MAO ASSAY
503
4. Ito, K., Nakagawa, J.. Minakuchi. C., and Fukase, M. (1971) Digestion 4, 49-58. 5. Lin, A. W-S. M., and Castell, D. 0. (1974) B&hem. Med. 9, 373-85. 6. Klaiber, E. L.. Kobayashi. Y., and Broverman, D. M. (1971) J. C/in. Endocrinol. 33, 630-38. 7. Robinson, D. S.. Lovenberg. W.. Keiser. H.. and Sjoerdsma. A. (1968) Biochem. Pharmacol. 17, 109- 19. 8. Murphy, D. L., and Neiss. R. (1972) Amer. J. Psychiatv 128, 1351-57. 9. Landowski, J., Lysiak. W.. and Angielski. S. (1975) Biochem. Med. 14, 347-54. 10. Murphy, D. L.. and Wyatt, R. J. (1972) Nature (London) 238, 225-26. 11. Blaschko. H., Richter, D.. and Schlossmann. H. (1937) Biochem. J. 31, 2187-96. 12. McEwen, C. M., Jr., and Cohen. J. D. (1965) J. Lab. Clin. Med. 62, 766-76. 13. Tabor, C. W., Tabor, H., and Rosenthal. S. M. (1954) J. Biol. Chem. 208, 645-61. 14. Century, B., and Rupp, K. L. (1968) Biochem. Pharmacol. 17, 2012-13. 15. Takahashi. S.. and Karasawa. T. (197s) Clin. Chem. Acta 62, 393-400. 16. Wurtman. R. J., and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439-41. 17. Demisch, L., Bochnik, H. J., and Seiler. N. (1976) C/in. Chem. Acta 70, 357-69. 18. Youdim, M. B. H. (1976) in Monoamine Oxidase and its Inhibition, Ciba Foundation Symposium 39. pp. 405-06, ElsevieriExcerpta MedicaiNorth-Holland. New York. 19. Plummer. D. T. (1971) An Introduction to Practical Biochemistry, pp. 156-57, McGraw-Hill, New York. 20. Bradford, M. M. (1976) Anal. Biochem. 72, 248-54. 21. Zeller, V., Ramachander, G.. and Zeller. E. A. (1965) J. Med. Chem. 8, 440-43. 22. Bathina, H. B.. Huprikar. S. V., and Zeller. E. A. (1975) Fed. Proc. 34, 293. 23. McEwen, C. M.. Jr. (1%5) .I. Biol. Chem. 240, 2003- 10. 24. Belmaker. R. H., Ebstein. R.. Rimon, R., Wyatt, R. J., and Murphy, D. L. (1976) Acta
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25. Zeller. E. A.. Boshes. B., Arbit. J., Bieber, M., Blonsky. E. R.. Dolkart, M.. and Huprikar. S. V., (1976) .I. Neural Trans. 39, 63-77. 26. McEwen. C. M.. Jr. (1965)J. Biol. C/tern. 240, 2011-18.
