Psycho pharmacology
Psychopharmacology59, 193-197 (1978)
I bv Springer-Verlag 1978
Monoamine Oxidase Activity in Different Density Gradient Fractions of Human Platelets Dennis L. Murphy, Jonathan L. Costa, Brenda Shafer, and Laurence Corash Clinical NeuropharmacologyBranch, NIMH, and HematologyService,ClinicalPathologyDepartment, NIH, Bethesda, Maryland,U.S.A.
Abstract. Monoamine oxidase (MAO) activity measured in human platelets is reportedly altered by such drugs as epinephrine, lithium carbonate, and imipramine, and also reduced in a number of clinical disorders. To evaluate whether MAO activity might differ in platelet subpopulations, density gradient centrifugation with arabino-galactan was used to prepare four platelet fractions that differed in weight and volume. MAO activity in the lightest and smallest platelet subpopulation was approximately one-half that in the heaviest and largest subpopulation. Because platelet weight and size are thought to be related to platelet age, it is possible that some drug effects on platelet MAO activity might represent changes in platelet turnover. Factors other than platelet turnover rates may contribute to individual differences in platelet MAO activity, however, since one group of individuals with markedly reduced platelet MAO activity exhibited no shift in the proportion of lighter versus heavier platelets nor in the relative amount of MAO activity in each density gradient subfraction. Key words: Monoamine oxidase - Platelets - Density gradient
Human platelets provide an easily accessible tissue in which monoamine oxidase (MAO) activity can be evaluated for individual differences in response to MAO-inhibiting antidepressants (Robinson et al., 1973; Murphy et al., 1977b). Platelet MAO activity is also reportedly altered by such other drugs as lithium carbonate, tricyclic antidepressants, and epinephrine (Bockar et al., 1974; Sullivan et al., 1977; Gentil et al., 1975). In addition, over 40 papers have been published in the last several years reporting correlations between Address for offprint requests. Dennis L. Murphy, M. D., NIH
Clinical Center, 10-3S229, Bethesda, Md. 20014, U.S.A.
platelet MAO activity and psychiatric and medical diagnostic groups, personality characteristics in students, and behavioral characteristics and steroid hormone differences in the rhesus monkey (for review, see Wolstenholme and Knight, 1976). Several reviews have suggested that such factors as anticoagulant use, platelet preparation techniques, contamination by nonplatelet elements (plasma amine oxidase, leukocytes, or erythrocytes), and changes in platelet numbers or other platelet characteristics might influence measured values for platelet MAO activity (Murphy and Donnelly, 1974; Murphy and Wyatt, 1975). In addition, reduced platelet MAO activity has been reported in patients with iron deficiency anemia (Youdim et al., 1975). Nonetheless, direct studies of such factors have revealed that other patient groups with low platelet MAO activity do not have anemia or reduced serum iron levels, altered steroid hormone levels, differences in platelet numbers, or differences in such other platelet enzyme activities as succinate dehYdrogenase , lactate dehydrogenase, and cytochrome C reductase (Murphy et al., 1976a; 1977a). Some platelet enzymes, metabolites, and cell constituents vary with the density, size, and age of platelets (Karpatkin, 1972). In order to evaluate more critically the possibility that differences in platelet subpopulations studied contribute to differences in platelet MAO activities, we examined platelet MAO activity in four platelet fractions of differing buoyant densities and volumes prepared by a newly reported technique with significant advantage over previous procedures (Corash et al., 1977).
Materials and Methods
The techniques used in this study have been previouslydescribed (Corash et al., 1977; Murphy et al., 1976b) and are only briefly summarized here. Blood samples obtained by venipuncture were placed in plastictubes containing4 ~ sodiumcitrate dihydrateas an
0033-3158/78/0059/0193/$01.00
194 anticoagulant. A total platelet sample was obtained by pooling platelet-rich plasma (prepared by centrifugation of the whole blood at 600 g for 3 rain) together with platelets prepared from three successive washes of the erythrocyte pellet using an isosmolar, buffered saline-glucose solution. This procedure yielded more than 95 % of the available platelets free of all other blood-cell types. The total platelet preparation was then subfractionated into four populations using a discontinuous gradient of 15 %, 16 %, 17 %, and 20 % isosmofar arabino-galactan (Stractan). The buoyant densities (g/ml) for these 4 subfractions are (I) < 1.062, (II) < 1.066, ( I I I ) < 1.071, and (IV) < 1.084. Plasma proteins were also removed in this process. Platelet counts and platelet volume determinations were obtained using an electronic particle counter (Particle Data, Elmhurst, Illinois) equipped with logarithmic amplifier (Corash et al., 1977). Platelet monoamine oxidase activities were determined using 14C-benzylamine as substrate (Murphy et al., 1976b). Eleven subjects were studied. All were in good physical health with no hematologic abnormalities, and none had received any drugs with the preceding two weeks. Six subjects were staff personnel with MAO activities within the normal range; 5 other subjects were chosen from previously studied patients and other groups known to have extremely low platelet MAO activities (beyond two standard deviations of the normal mean (Murphy et al., 1976a; Buchsbaum et al., 1976)). Platelet MAO activities were redetermined using platelet rich plasma and our standard ant• and centrifugation procedures (Murphy et al., 1976b) for all subjects at the same time the samples for density gradient fractionation were obtained. The mean platelet-rich plasma M A O activity for the control subjects was 10.87 + 1.11 nmoles/108 platelets/h (range 8.52 to 14.20), and for the low M A O subjects it was 5.53 +_ 0.55 nmoles/108 platelets/h (range 4.66 - 6.17).
Resuits In the 4 platelet fractions obtained from the discontinuous Stractan gradient, MAO activity was proportionate to platelet density, with the lightest platelet fraction having a mean MAO activity 46 % that of the heaviest platelet fraction ( P < 0.05, Table 1). Most of the 11 subjects demonstrated a progressive increase of MAO activities from the lightest through the heaviest of the 4 platelet fractions. Two individuals (Nos. I and 8), however, had similar MAO activities in all fractions. In a similar fashion, mean platelet volumes progressively increased from the lightest to the heaviest fractions (Table 1). Platelets in Fraction I had a mean volume which was 80 % that of Fraction IV (P < 0.001). The two individuals with relatively little difference between MAO activities in Fraction I vs. IV had volume differences between Fraction I and IV of 1.40 and 1.56, values similar to the mean difference of 1.62 for the entire group. When the differences in volume and differences in MAO activity between Fractions I and IV were examined to determine if individuals with the greatest volume differences had the greatest differences in MAO activity, only a low, nonsignificant correlation was observed (r=0.12). The 3 heavier fractions each contained approximately equal proportions of the entire number of platelets collected; the
PsychopharmacoIogy 59 (1978) Table 1. Monoamine oxidase (MAO) activity in density gradient
subfractions of human platelets Subjects
MAO activity (nmols/108 platelets/h) in the four density fractions II
III
IV
2.40 5.06 7.50 6.18 8.50 7.34 11.10 11.86 13.72 18.82 20.80 t0.30 • 1.72
2.80 6.64 9.94 8.32 10.24 10.30 12.32 13.16 14.54 23.12 24.90 12.58 _+ 1.98
2.94 7.96 10.66 10.74 9.92 12.90 16.46 12.72 13.26 29.32 27.52 14.04 +_ 2.38
I
I 2 3 4 5 6 7 8 9 10 11 Mean • SEM
2.48 4.66 5.08 6.82 6.18 7.44 8.88 11.20 15.96 7.64 • 1.33
Platelet volume (g3) Platelets • (percent of total) _+
6.62 0.76
•
6.99 0.86
15.7 3.4
26.2 _+ 1.9
•
7.61 0.89
33.1 _+ 1.6
•
8.24 0.83
24.9 _+ 3.1
lightest fraction had somewhat fewer platelets (16 %, Table 1). When the subjects were divided into those with platelet MAO activities of less than 7 units (low MAO subjects) and those with MAO activities within a more normal range of 8 - 1 4 units, the low MAO subjects demonstrated a distribution of platelets across the 4 density gradient subfractions proportionate to that of the normal subjects (Fig. 1A). As indicated in the figure, the differences in mean MAO activity between the low and normal MAO groups were similar in all 4 subfractions, ranging from 52 % to 58 %. The differences between the 2 subject groups were statistically significant at the 0.05 level for all 4 density subfractions. When the MAO activities in each subfraction were expressed as a percent difference from the mean (Fig. 1B), the low MAO activity group closely resembled the normal subject group in the distribution of mean MAO activities across the 4 density gradient subfractions. In agreement with these density-based results, platelet volumes and platelet numbers were generally similar, and in no case varied more than 10 % (P = NS) in any of the subfractions when the low MAO and normal MAO groups were compared.Thus, an overrepresentation of lighter platelets in the individuals with low platelet MAO activity did not appear to contribute to the differences in MAO activities between these two subject groups.
D. L. Murphy et al. : Monoamine Oxidase Activity
195
Discussion 20
A
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Psychopharmacology59, 193-197 (1978)
I bv Springer-Verlag 1978
Monoamine Oxidase Activity in Different Density Gradient Fractions of Human Platelets Dennis L. Murphy, Jonathan L. Costa, Brenda Shafer, and Laurence Corash Clinical NeuropharmacologyBranch, NIMH, and HematologyService,ClinicalPathologyDepartment, NIH, Bethesda, Maryland,U.S.A.
Abstract. Monoamine oxidase (MAO) activity measured in human platelets is reportedly altered by such drugs as epinephrine, lithium carbonate, and imipramine, and also reduced in a number of clinical disorders. To evaluate whether MAO activity might differ in platelet subpopulations, density gradient centrifugation with arabino-galactan was used to prepare four platelet fractions that differed in weight and volume. MAO activity in the lightest and smallest platelet subpopulation was approximately one-half that in the heaviest and largest subpopulation. Because platelet weight and size are thought to be related to platelet age, it is possible that some drug effects on platelet MAO activity might represent changes in platelet turnover. Factors other than platelet turnover rates may contribute to individual differences in platelet MAO activity, however, since one group of individuals with markedly reduced platelet MAO activity exhibited no shift in the proportion of lighter versus heavier platelets nor in the relative amount of MAO activity in each density gradient subfraction. Key words: Monoamine oxidase - Platelets - Density gradient
Human platelets provide an easily accessible tissue in which monoamine oxidase (MAO) activity can be evaluated for individual differences in response to MAO-inhibiting antidepressants (Robinson et al., 1973; Murphy et al., 1977b). Platelet MAO activity is also reportedly altered by such other drugs as lithium carbonate, tricyclic antidepressants, and epinephrine (Bockar et al., 1974; Sullivan et al., 1977; Gentil et al., 1975). In addition, over 40 papers have been published in the last several years reporting correlations between Address for offprint requests. Dennis L. Murphy, M. D., NIH
Clinical Center, 10-3S229, Bethesda, Md. 20014, U.S.A.
platelet MAO activity and psychiatric and medical diagnostic groups, personality characteristics in students, and behavioral characteristics and steroid hormone differences in the rhesus monkey (for review, see Wolstenholme and Knight, 1976). Several reviews have suggested that such factors as anticoagulant use, platelet preparation techniques, contamination by nonplatelet elements (plasma amine oxidase, leukocytes, or erythrocytes), and changes in platelet numbers or other platelet characteristics might influence measured values for platelet MAO activity (Murphy and Donnelly, 1974; Murphy and Wyatt, 1975). In addition, reduced platelet MAO activity has been reported in patients with iron deficiency anemia (Youdim et al., 1975). Nonetheless, direct studies of such factors have revealed that other patient groups with low platelet MAO activity do not have anemia or reduced serum iron levels, altered steroid hormone levels, differences in platelet numbers, or differences in such other platelet enzyme activities as succinate dehYdrogenase , lactate dehydrogenase, and cytochrome C reductase (Murphy et al., 1976a; 1977a). Some platelet enzymes, metabolites, and cell constituents vary with the density, size, and age of platelets (Karpatkin, 1972). In order to evaluate more critically the possibility that differences in platelet subpopulations studied contribute to differences in platelet MAO activities, we examined platelet MAO activity in four platelet fractions of differing buoyant densities and volumes prepared by a newly reported technique with significant advantage over previous procedures (Corash et al., 1977).
Materials and Methods
The techniques used in this study have been previouslydescribed (Corash et al., 1977; Murphy et al., 1976b) and are only briefly summarized here. Blood samples obtained by venipuncture were placed in plastictubes containing4 ~ sodiumcitrate dihydrateas an
0033-3158/78/0059/0193/$01.00
194 anticoagulant. A total platelet sample was obtained by pooling platelet-rich plasma (prepared by centrifugation of the whole blood at 600 g for 3 rain) together with platelets prepared from three successive washes of the erythrocyte pellet using an isosmolar, buffered saline-glucose solution. This procedure yielded more than 95 % of the available platelets free of all other blood-cell types. The total platelet preparation was then subfractionated into four populations using a discontinuous gradient of 15 %, 16 %, 17 %, and 20 % isosmofar arabino-galactan (Stractan). The buoyant densities (g/ml) for these 4 subfractions are (I) < 1.062, (II) < 1.066, ( I I I ) < 1.071, and (IV) < 1.084. Plasma proteins were also removed in this process. Platelet counts and platelet volume determinations were obtained using an electronic particle counter (Particle Data, Elmhurst, Illinois) equipped with logarithmic amplifier (Corash et al., 1977). Platelet monoamine oxidase activities were determined using 14C-benzylamine as substrate (Murphy et al., 1976b). Eleven subjects were studied. All were in good physical health with no hematologic abnormalities, and none had received any drugs with the preceding two weeks. Six subjects were staff personnel with MAO activities within the normal range; 5 other subjects were chosen from previously studied patients and other groups known to have extremely low platelet MAO activities (beyond two standard deviations of the normal mean (Murphy et al., 1976a; Buchsbaum et al., 1976)). Platelet MAO activities were redetermined using platelet rich plasma and our standard ant• and centrifugation procedures (Murphy et al., 1976b) for all subjects at the same time the samples for density gradient fractionation were obtained. The mean platelet-rich plasma M A O activity for the control subjects was 10.87 + 1.11 nmoles/108 platelets/h (range 8.52 to 14.20), and for the low M A O subjects it was 5.53 +_ 0.55 nmoles/108 platelets/h (range 4.66 - 6.17).
Resuits In the 4 platelet fractions obtained from the discontinuous Stractan gradient, MAO activity was proportionate to platelet density, with the lightest platelet fraction having a mean MAO activity 46 % that of the heaviest platelet fraction ( P < 0.05, Table 1). Most of the 11 subjects demonstrated a progressive increase of MAO activities from the lightest through the heaviest of the 4 platelet fractions. Two individuals (Nos. I and 8), however, had similar MAO activities in all fractions. In a similar fashion, mean platelet volumes progressively increased from the lightest to the heaviest fractions (Table 1). Platelets in Fraction I had a mean volume which was 80 % that of Fraction IV (P < 0.001). The two individuals with relatively little difference between MAO activities in Fraction I vs. IV had volume differences between Fraction I and IV of 1.40 and 1.56, values similar to the mean difference of 1.62 for the entire group. When the differences in volume and differences in MAO activity between Fractions I and IV were examined to determine if individuals with the greatest volume differences had the greatest differences in MAO activity, only a low, nonsignificant correlation was observed (r=0.12). The 3 heavier fractions each contained approximately equal proportions of the entire number of platelets collected; the
PsychopharmacoIogy 59 (1978) Table 1. Monoamine oxidase (MAO) activity in density gradient
subfractions of human platelets Subjects
MAO activity (nmols/108 platelets/h) in the four density fractions II
III
IV
2.40 5.06 7.50 6.18 8.50 7.34 11.10 11.86 13.72 18.82 20.80 t0.30 • 1.72
2.80 6.64 9.94 8.32 10.24 10.30 12.32 13.16 14.54 23.12 24.90 12.58 _+ 1.98
2.94 7.96 10.66 10.74 9.92 12.90 16.46 12.72 13.26 29.32 27.52 14.04 +_ 2.38
I
I 2 3 4 5 6 7 8 9 10 11 Mean • SEM
2.48 4.66 5.08 6.82 6.18 7.44 8.88 11.20 15.96 7.64 • 1.33
Platelet volume (g3) Platelets • (percent of total) _+
6.62 0.76
•
6.99 0.86
15.7 3.4
26.2 _+ 1.9
•
7.61 0.89
33.1 _+ 1.6
•
8.24 0.83
24.9 _+ 3.1
lightest fraction had somewhat fewer platelets (16 %, Table 1). When the subjects were divided into those with platelet MAO activities of less than 7 units (low MAO subjects) and those with MAO activities within a more normal range of 8 - 1 4 units, the low MAO subjects demonstrated a distribution of platelets across the 4 density gradient subfractions proportionate to that of the normal subjects (Fig. 1A). As indicated in the figure, the differences in mean MAO activity between the low and normal MAO groups were similar in all 4 subfractions, ranging from 52 % to 58 %. The differences between the 2 subject groups were statistically significant at the 0.05 level for all 4 density subfractions. When the MAO activities in each subfraction were expressed as a percent difference from the mean (Fig. 1B), the low MAO activity group closely resembled the normal subject group in the distribution of mean MAO activities across the 4 density gradient subfractions. In agreement with these density-based results, platelet volumes and platelet numbers were generally similar, and in no case varied more than 10 % (P = NS) in any of the subfractions when the low MAO and normal MAO groups were compared.Thus, an overrepresentation of lighter platelets in the individuals with low platelet MAO activity did not appear to contribute to the differences in MAO activities between these two subject groups.
D. L. Murphy et al. : Monoamine Oxidase Activity
195
Discussion 20
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