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Reactivity of serotonin in whole blood: Reply
Correspondence
Flachaire E, Beney C, Berthier A, Salandre J, Quincy C, Renaud B (1990): Determination of reference values for serotonin concentration in platelets. Clin Chem 36:2117–2120. Heyssel R (1961): Determination of human platelet survival utilizing C14-labeled serotonin. J Clin Invest 40:2134 –2142. Humble M, Bejerot S, Bergqvist PB, Bengtsson F (2001): Reactivity of serotonin in whole blood: Relationship with drug response in obsessive-compulsive disorder. Biol Psychiatry 49:360 –368. Kema IP, De Vries EG, Schellings AM, Postmus PE, Muskiet FA (1992): Improved diagnosis of carcinoid tumors by measurement of platelet serotonin. Clin Chem 38:534 –540.
Reply We are happy to read the comments by Dr. Mulder and colleagues. Because our findings were contrary to our expectations and we still regard the mechanisms behind them as unresolved, we find their contribution to further interpretation of our data useful. 1. We did not suggest that changes in metabolic processes might explain our findings, but rather that they may relate to innate or acquired differences concerning the elimination of platelet serotonin, due to, for example, genetic polymorphism of any of the mechanisms involved. We certainly agree that a significant difference of platelet turnover between responders and nonresponders could have explained our findings (because platelet death is a powerful mode of platelet serotonin elimination). To our knowledge, a link between platelet turnover and psychotropic drug response has not previously been described and may serve as an intriguing focus for future investigations; however, we stated in our paper that “Careful investigation of the temporal pattern of SRI-induced changes of 5HTrelated measures may disentangle this [divergent decrease in whole blood serotonin (WB-5HT)].” 2. Indeed, measures of platelet turnover could have elucidated the mechanisms behind our findings, but, unfortunately, this was not included in our investigation. On the other hand, we had the opportunity to check for divergent platelet monoamine oxidase (MAO) activity as one possible explanation. In our patients, this was estimated by Professor Lars Oreland, Uppsala University, according to the method described in Hallman et al (1987), using phenylethylamine as substrate. We found no correlation between WB-5HT decrease after 1 week of SRI treatment and platelet MAO-activity (n ⫽ 24, r ⫽ .060, p ⫽ .78), thus precluding this as a possible explanation. We still believe, however, that other ways of platelet serotonin elimination may possibly be involved. Other investigators have described a variable mechanism of serotonin efflux from platelets, related to gender and diagnosis (Hallman et al 1990; Peterson et al 1984), and individuals may differ in their patterns of stimulated serotonin release by exocytosis. Even if these mechanisms are of negligible importance compared with the continuous uptake of serotonin in the untreated condition, their relative importance may increase
BIOL PSYCHIATRY 2002;51:266 –268
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considerably when SRI treatment inhibits serotonin uptake. In one of the quoted articles (Aranda et al 1994), the author states that “A proportion of platelets that have undergone degranulation in vivo may recirculate preserving both their functional ability and their capacity for 5-HT uptake.” If this is the case, there must be numerous possibilities for variability in the decrease of platelet serotonin. In a later work by the same author (Aranda et al 1996), now working with humans instead of dogs, it appears that the significant correlation between platelet 5-HT content and platelet survival time is entirely dependent on the inclusion of 11 individuals with immunologic thrombocytopenic purpura. Among the 25 healthy volunteers, no such correlation was found, clearly implying that other mechanisms were responsible for the fourfold variability of platelet 5-HT content in this group. 3. It is true that the patients treated with paroxetine (PXT) contributed greatly to our correlation, in our view because they were twice as many as there were patients treated with clomipramine (CMI). The correlation was almost as significant if we only included PXT cases (n ⫽ 18, r ⫽ ⫺.71, p ⫽ .0009). The CMI cases by themselves did not attain a significant correlation (n ⫽ 9, r ⫽ ⫺.10, p ⫽ .79), but they did not distract from the composite correlation either. Clearly, our finding is not explained by differences between the two drugs. Ideally, however, in a replication, the mode of serotonin reuptake inhibition should be as uniform as possible. 4. We have also noted the outlier described by Mulder et al. However, when this case was omitted, the significant correlation remained (n ⫽ 26, r ⫽ ⫺.52, p ⫽ .0067). Because of this, we chose to keep this case in the material. This was a 41-year-old male patient with considerable comorbidity. He suffered from two autoimmune diseases: insulin-dependent diabetes mellitus and vitiligo. He also had hypertension and otosclerosis. However, no thrombocytopenia was recorded. Psychiatrically, he had a typical OCD with checking compulsions but was also diagnosed with high-functioning autism and dysthymia. His Y-BOCS was 19 at baseline and increased to 25 after 8 weeks of PXT treatment, when he chose to discontinue. His baseline WB-5HT was 872 nmol/L (slightly above average in our material) and decreased to 10 nmol/L after 1 week. We do not know why the serotonin of this patient decreased with this unusual rapidity, but it seems likely that short platelet life span as well as immunologically triggered release mechanisms could have been involved. We find it thought provoking that this was linked to an unusually poor clinical response, however. 5. Because ascorbic acid was added as antioxidant immediately at the time of sampling, the samples were frozen as quickly as possible and kept frozen until analysis, we believe that oxidative losses of 5-HT were negligible. When our mean values were compared with a previous study in which a similar analytical method was applied (Larsson et al 1988); we did not find significant differences. Also, if losses during preparation did appear, we
268
Correspondence
BIOL PSYCHIATRY 2002;51:266 –268
can not see any reason why this should appear at different rates related to clinical response in this randomized study. 6. We thank Dr. Mulder and his colleagues for making us aware of the quoted consensus report, which does not appear on MedLine searches and was not available to us when we wrote the article. In fact, we had similar findings (no association between autistic traits and rate of WB-5HT decrease) and stated that “autistic traits are unlikely to explain the link between WB-5HT decrease and clinical response.” Finally, we agree that this is a preliminary finding in need of replication. We agree that divergent platelet life span is one plausible mechanism and that this variable should be accounted for in future investigations in this field; however, we also believe that other mechanisms may well be operational in SRI-induced decrease of WB-5HT. Mats Humble Department of Neuroscience, Psychiatry VAS Uppsala University Psychiatry of Northern Dalecarlia Mora Hospital S-792 85 Mora Sweden Susanne Bejerot Department of Neuroscience, Psychiatry VAS Uppsala University Central Stockholm Psychiatric Clinic St. Go¨ ran Hospital S-112 81 Stockholm Sweden
Peter B. F. Bergqist AstraZeneca R&D, Lund S-221 87 Lund Sweden PII S0006-3223(01)01316-6
References Aranda E, Pereira J, Ajenjo C, Prieto C, Sepu´ lveda S, Mezzano D (1996): Human intraplatelet 5-hydroxytryptamine is correlated with mean platelet survival time. Thromb Res 84:67–72. Aranda E, Pizarro M, Pereira J, Mezzano D (1994): Accumulation of 5-hydroxytryptamine by aging platelets: Studies in a model of suppressed thrombopoiesis in dogs. Thromb Haemost 71:488 – 492. Hallman J, Oreland L, Edman G, Schalling D (1987): Thrombocyte monoamine oxidase activity and personality traits in woman with severe premenstrual syndrome. Acta Psychiatr Scand 76:225–234. Hallman J, Sakurai E, Oreland L (1990): Blood platelet monoamine oxidase activity, serotonin uptake and release rates in anorexia and bulimia patients and in healthy controls. Acta Psychiatr Scand 81:73–77. Larsson M, Forsman A, Hallgren J (1988): HPLC assays of 5-HIAA and tryptophan in cerebrospinal fluid and 5-HT and tryptophan in blood: A methodological study with clinical applications. Meth Find Exper Clin Pharmacol 10:453– 460. Peterson LL, Bartfai T, Ernster L, Wickstro¨ m G, Roos BE, ˚ hs U, A ˚ gren H (1984): Uncoupler-accelerated efWehlin-A flux of 5-hydroxytryptamine from platelets of healthy subjects and patients with unipolar and bipolar depression. Psychiatry Res 13:141–150.
Flachaire E, Beney C, Berthier A, Salandre J, Quincy C, Renaud B (1990): Determination of reference values for serotonin concentration in platelets. Clin Chem 36:2117–2120. Heyssel R (1961): Determination of human platelet survival utilizing C14-labeled serotonin. J Clin Invest 40:2134 –2142. Humble M, Bejerot S, Bergqvist PB, Bengtsson F (2001): Reactivity of serotonin in whole blood: Relationship with drug response in obsessive-compulsive disorder. Biol Psychiatry 49:360 –368. Kema IP, De Vries EG, Schellings AM, Postmus PE, Muskiet FA (1992): Improved diagnosis of carcinoid tumors by measurement of platelet serotonin. Clin Chem 38:534 –540.
Reply We are happy to read the comments by Dr. Mulder and colleagues. Because our findings were contrary to our expectations and we still regard the mechanisms behind them as unresolved, we find their contribution to further interpretation of our data useful. 1. We did not suggest that changes in metabolic processes might explain our findings, but rather that they may relate to innate or acquired differences concerning the elimination of platelet serotonin, due to, for example, genetic polymorphism of any of the mechanisms involved. We certainly agree that a significant difference of platelet turnover between responders and nonresponders could have explained our findings (because platelet death is a powerful mode of platelet serotonin elimination). To our knowledge, a link between platelet turnover and psychotropic drug response has not previously been described and may serve as an intriguing focus for future investigations; however, we stated in our paper that “Careful investigation of the temporal pattern of SRI-induced changes of 5HTrelated measures may disentangle this [divergent decrease in whole blood serotonin (WB-5HT)].” 2. Indeed, measures of platelet turnover could have elucidated the mechanisms behind our findings, but, unfortunately, this was not included in our investigation. On the other hand, we had the opportunity to check for divergent platelet monoamine oxidase (MAO) activity as one possible explanation. In our patients, this was estimated by Professor Lars Oreland, Uppsala University, according to the method described in Hallman et al (1987), using phenylethylamine as substrate. We found no correlation between WB-5HT decrease after 1 week of SRI treatment and platelet MAO-activity (n ⫽ 24, r ⫽ .060, p ⫽ .78), thus precluding this as a possible explanation. We still believe, however, that other ways of platelet serotonin elimination may possibly be involved. Other investigators have described a variable mechanism of serotonin efflux from platelets, related to gender and diagnosis (Hallman et al 1990; Peterson et al 1984), and individuals may differ in their patterns of stimulated serotonin release by exocytosis. Even if these mechanisms are of negligible importance compared with the continuous uptake of serotonin in the untreated condition, their relative importance may increase
BIOL PSYCHIATRY 2002;51:266 –268
267
considerably when SRI treatment inhibits serotonin uptake. In one of the quoted articles (Aranda et al 1994), the author states that “A proportion of platelets that have undergone degranulation in vivo may recirculate preserving both their functional ability and their capacity for 5-HT uptake.” If this is the case, there must be numerous possibilities for variability in the decrease of platelet serotonin. In a later work by the same author (Aranda et al 1996), now working with humans instead of dogs, it appears that the significant correlation between platelet 5-HT content and platelet survival time is entirely dependent on the inclusion of 11 individuals with immunologic thrombocytopenic purpura. Among the 25 healthy volunteers, no such correlation was found, clearly implying that other mechanisms were responsible for the fourfold variability of platelet 5-HT content in this group. 3. It is true that the patients treated with paroxetine (PXT) contributed greatly to our correlation, in our view because they were twice as many as there were patients treated with clomipramine (CMI). The correlation was almost as significant if we only included PXT cases (n ⫽ 18, r ⫽ ⫺.71, p ⫽ .0009). The CMI cases by themselves did not attain a significant correlation (n ⫽ 9, r ⫽ ⫺.10, p ⫽ .79), but they did not distract from the composite correlation either. Clearly, our finding is not explained by differences between the two drugs. Ideally, however, in a replication, the mode of serotonin reuptake inhibition should be as uniform as possible. 4. We have also noted the outlier described by Mulder et al. However, when this case was omitted, the significant correlation remained (n ⫽ 26, r ⫽ ⫺.52, p ⫽ .0067). Because of this, we chose to keep this case in the material. This was a 41-year-old male patient with considerable comorbidity. He suffered from two autoimmune diseases: insulin-dependent diabetes mellitus and vitiligo. He also had hypertension and otosclerosis. However, no thrombocytopenia was recorded. Psychiatrically, he had a typical OCD with checking compulsions but was also diagnosed with high-functioning autism and dysthymia. His Y-BOCS was 19 at baseline and increased to 25 after 8 weeks of PXT treatment, when he chose to discontinue. His baseline WB-5HT was 872 nmol/L (slightly above average in our material) and decreased to 10 nmol/L after 1 week. We do not know why the serotonin of this patient decreased with this unusual rapidity, but it seems likely that short platelet life span as well as immunologically triggered release mechanisms could have been involved. We find it thought provoking that this was linked to an unusually poor clinical response, however. 5. Because ascorbic acid was added as antioxidant immediately at the time of sampling, the samples were frozen as quickly as possible and kept frozen until analysis, we believe that oxidative losses of 5-HT were negligible. When our mean values were compared with a previous study in which a similar analytical method was applied (Larsson et al 1988); we did not find significant differences. Also, if losses during preparation did appear, we
268
Correspondence
BIOL PSYCHIATRY 2002;51:266 –268
can not see any reason why this should appear at different rates related to clinical response in this randomized study. 6. We thank Dr. Mulder and his colleagues for making us aware of the quoted consensus report, which does not appear on MedLine searches and was not available to us when we wrote the article. In fact, we had similar findings (no association between autistic traits and rate of WB-5HT decrease) and stated that “autistic traits are unlikely to explain the link between WB-5HT decrease and clinical response.” Finally, we agree that this is a preliminary finding in need of replication. We agree that divergent platelet life span is one plausible mechanism and that this variable should be accounted for in future investigations in this field; however, we also believe that other mechanisms may well be operational in SRI-induced decrease of WB-5HT. Mats Humble Department of Neuroscience, Psychiatry VAS Uppsala University Psychiatry of Northern Dalecarlia Mora Hospital S-792 85 Mora Sweden Susanne Bejerot Department of Neuroscience, Psychiatry VAS Uppsala University Central Stockholm Psychiatric Clinic St. Go¨ ran Hospital S-112 81 Stockholm Sweden
Peter B. F. Bergqist AstraZeneca R&D, Lund S-221 87 Lund Sweden PII S0006-3223(01)01316-6
References Aranda E, Pereira J, Ajenjo C, Prieto C, Sepu´ lveda S, Mezzano D (1996): Human intraplatelet 5-hydroxytryptamine is correlated with mean platelet survival time. Thromb Res 84:67–72. Aranda E, Pizarro M, Pereira J, Mezzano D (1994): Accumulation of 5-hydroxytryptamine by aging platelets: Studies in a model of suppressed thrombopoiesis in dogs. Thromb Haemost 71:488 – 492. Hallman J, Oreland L, Edman G, Schalling D (1987): Thrombocyte monoamine oxidase activity and personality traits in woman with severe premenstrual syndrome. Acta Psychiatr Scand 76:225–234. Hallman J, Sakurai E, Oreland L (1990): Blood platelet monoamine oxidase activity, serotonin uptake and release rates in anorexia and bulimia patients and in healthy controls. Acta Psychiatr Scand 81:73–77. Larsson M, Forsman A, Hallgren J (1988): HPLC assays of 5-HIAA and tryptophan in cerebrospinal fluid and 5-HT and tryptophan in blood: A methodological study with clinical applications. Meth Find Exper Clin Pharmacol 10:453– 460. Peterson LL, Bartfai T, Ernster L, Wickstro¨ m G, Roos BE, ˚ hs U, A ˚ gren H (1984): Uncoupler-accelerated efWehlin-A flux of 5-hydroxytryptamine from platelets of healthy subjects and patients with unipolar and bipolar depression. Psychiatry Res 13:141–150.