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Possible Role of Serotonin Transporter in Irritable Bowel Syndrome: Evidence From Platelet Investigation
GASTROENTEROLOGY 2011;141:e18 – e19
CORRESPONDENCE Readers may submit letters to the editor concerning articles that appeared in GASTROENTEROLOGY within one month of publication. Detailed guidelines regarding the content are included in the Instructions to Authors.
Possible Role of Serotonin Transporter in Irritable Bowel Syndrome: Evidence From Platelet Investigation Dear Sir:
In the May 2011 issue of GASTROENTEROLOGY, Foley et al1 report that platelet serotonin transporter (SERT) is significantly reduced in patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and associated with reduced duodenal levels of SERT mRNA and duodenal immune activation. Although these interesting results contribute to the current discussion of whether the SERT is implicated in the pathophysiology of functional gastrointestinal disorders and whether platelets are useful to investigate the SERT in this condition, caution must be exercised in interpreting the findings. The study contains some methodologic problems and the way in which the correlation analysis is performed generates a range of questions. First, the expression of platelet serotonin uptake (PSU) rate per mg platelet protein is unusual. The results are given in nmol or fmol 5HT/mg protein ⫻ min. Which is correct? Although the assay includes 5 different concentrations of [3H]-5HT/5HT, the authors do not calculate the maximum uptake rate (Vmax) and Km (Michaelis constant); they report the PSU at 800 nmol [3H]-5HT/5HT/L, and declare this to be the maximum uptake rate. Methodologic issues of PSU assays are well discussed in a recent study.2 To characterize the SERT the authors also use a singlepoint binding assay with [3H]-paroxetine (4 nmol/L). Again, given the potential methodical pitfalls in this assay protocol, errors can not be excluded.3 Second, we would like to point out existing literature data, which are not mentioned by the authors. Two previous studies on IBS patients apply standard methods for the assay of [3H]paroxetine saturation binding or PSU kinetics.4,5 Significantly lower mean Bmax is found for [3H]-paroxetine in 12 female IBS-D patients with a strong inverse correlation between Bmax and symptom severity score in these patients (r ⫽ ⫺0.964).4 Surprisingly, the study by Foley et al1 reports significantly higher median [3H]-paroxetine binding at 4 nmol/L in 18 patients with IBS-D than in healthy controls. Recently, we compared PSU kinetics in 149 patients with IBS and 163 healthy control subjects and found a high rate (55.5%) of very low and low Vmax values (below the 25th percentile of male controls) in male IBS patients.5 Third, the inverse correlation between PSU and [3H]-paroxetine binding in the mixed group of healthy subjects and patients with IBS-D and celiac disease, with r ⫽ ⫺0.44 (r is probably the Spearman’s referred to in Material and Methods) requires some comments. In general, correlation coefficients ⬍0.5 are thought to be of low strength. It is difficult to understand why this inverse relationship should mean
that measure of [3H]-paroxetine binding could be a useful surrogate marker of impaired SERT in patients with IBS. It is also surprising to read that the single-point binding assay of [3H]-paroxetine would be an indirect measure of SERT function. Previous studies in which PSU and [3H]-paroxetine binding were investigated in parallel report a lack of any relationship between Vmax and Bmax. Only 1 study has reported a positive correlation between Vmax and Bmax in a group of 20 female healthy subjects.6 Unfortunately, Foley et al have not mentioned or discussed these findings. The most recent study on females with fibromyalgia shows that both Vmax and Bmax are on average significantly lower in patients, but Vmax and Bmax are not correlated.7 Fourth, the significant but low correlation between logtransformed SERT mRNA expression in duodenal biopsy specimens and log PSU (R ⫽ 0.37) or log [3H]-paroxetine binding (R ⫽ ⫺0.46) in a mixed sample of healthy controls and IBS-D (R is probably the Pearson r and the sample size is now indicated) should be interpreted with caution. It can be assumed that patients with celiac disease were not included in this correlation analysis because this group showed the lowest SERT mRNA expression but the PSU and [3H]-paroxetine binding were not different from healthy controls. Thus, a general relationship between the SERT mRNA expression in duodenum and platelet SERT measures (functional or binding assays) can not be assumed. Finally, because proinflammatory cytokines (tumor necrosis factor-␣ and interferon-␥) are known to regulate SERT expression and function differently in different cell lines8 the finding of a significant negative correlation between log mRNA expressions of interleukin (IL)-10 and SERT in the IBS-D group with a Pearson r ⫽ ⫺0.8, is notable. Unfortunately, the sample size is not indicated; nor is a reason given for why a Bonferroni correction was applied only in this particular correlation analysis. It would be interesting to prove whether the relative expressions of IL-10 and SERT itself are also significantly and linearly associated (Spearman’s ) or show an exponential relationship. LEONORA FRANKE Department of Psychiatry and Psychotherapy Charitè-Universitättsmedizin Berlin Berlin, Germany MARTIN SCHMIDTMANN HUBERTHUS MÖNNIKES Department of Medicine and Institute of Neurogastroenterology Martin Luther Hospital Academic Teaching Institute of Charitè-Universitätstsmedizin Berlin, Germany
November 2011 1. 2. 3. 4. 5. 6. 7. 8.
CORRESPONDENCE
Foley S, et al. Gastroenterology 2011;140:1434 –1443.e1. Banovic´ M, et al. Platelets 2010;21:429 – 438. Jurado N, et al. Arch Med Res 2003;34:422– 427. Bellini M, et al. Am J Gastroenterol 2003;98:2705–2711. Franke L, et al. J Gastroenterol 2010;45:389 –398. Maguire K, et al. Biol Psychiatry 1993;34:356 –360. Bazzichi L, et al. Arthritis Res Ther 2006;8:R99. Zhu CB, et al. Neuropsychopharmacology 2006;31:2121–2131.
Conflicts of interest The authors disclose no conflicts. doi:10.1053/j.gastro.2011.08.042
Reply. Thank you for your interest in our study which
demonstrated immune activation in the gut of patients with the irritable bowel syndrome with diarrhea together with depression of mRNA of SERT in the mucosa. We were able to correlate this with mucosal lymphocyte numbers and also to impairment of platelet 5HT uptake. You raise some important points that we attempt to address. First, we apologize for the confusion about the units for expressing 5HT uptake, which are as correctly stated in Table 3 expressed in nmol/min/mg protein not pmol/min/mg protein as stated in Methods. We agree that the best way of expressing uptake is from calculation of Vmax, which we had intended to do but as stated in the text, we found “uptake at lower concentrations were highly variable but the uptake had reached a plateau at 800 nmol/L concentration and this was taken as the best estimate of maximum uptake.” We think this is fair and, although not ideal, it is unlikely to bias our results or alter our conclusions. We agree that there are 2 previous studies on IBS patients that also assessed paroxetine binding and came to rather different conclusions. The study by Bellini found paroxetine binding was lower in IBS-D compared with controls1 while we found it to be increased. Although they used a lower range of paroxetine concentrations 0.01–1 nmol/L, whereas we used 4 nmol/L to ensure we saturated the binding site these methodologic differences would not account for the opposite findings which we can only suggest are due to differences in patient selection given the small numbers in both studies, 12 in Bellini’s and 20 in ours. The other very detailed study performed by Franke et al2 is highly relevant because it has much larger numbers with a total of 149 IBS patients, 47% IBS-D, 29.5% alternating-type IBS, and 23.5% constipation-predominant IBS. This did show an overall 10% reduction in platelet 5HT uptake Vmax in IBS, which appeared largely due to the male patients. Female controls showed lower values than male controls, but these did not differ from female patients, the reasons for which are unclear. Given that we only had 7 males, we cannot comment on any gender difference in our study. We agree that there is no simple relationship between paroxetine binding and SERT function because paroxetine binds to a different site than serotonin. Our data showed an inverse correlation only in the IBS-D patient group. Our interpretation was that this was a compensatory response to
e19
decreased SERT function, but we agree that confirmatory studies are needed. Despite the large literature on platelet serotonin uptake, there are surprisingly few studies where both paroxetine binding and 5HT uptake have been assessed, mostly within the psychiatric and alcohol dependence field. Maguire et al3 found a positive correlation in healthy volunteers. A study in fibromyalgia found both Vmax and Bmax depressed compared with controls, but did not comment on any correlation.4 Most studies have not reported a correlation between 5HT uptake and paroxetine binding, which can be altered independently by disease5 or drugs.6 One study on major depression did find a decreased 5HT uptake associated with an elevated paroxetine binding in male patients, which parallels our findings.7 They also found paroxetine binding to correlate with anxiety, something we could not confirm. You are correct that the celiacs were not included in the correlation between mRNA for SERT and platelet uptake since as stated in the text the mRNA in the biopsies was measured on a different group of patients (group 2) whereas the platelets were measured only in group 1. We agree that the correlation between log IL-10 and log SERT mRNA in the IBS-D patients (n ⫽ 20) of ⫺0.8 is of interest. We took this to indicate a general effect of inflammation since although anti-inflammatory, IL-10 expression is increased in many inflammatory conditions and log IL-10 in fact correlated significantly (P ⬍ .001) with all the other cytokines (r ⫽ 0.4–0.8), although their individual correlations with log SERT were not significant. We log-transformed the data because the raw data were skewed. Untransformed data give an exponential curve, as would be predicted. We have just completed a larger study of 120 IBS-D patients in whom we will assess paroxetine binding and its relationship to response to 5HT3 antagonist. We believe that intervention studies such as these are needed to go beyond mere correlation and demonstrate the significance or otherwise of these fascinating but at times confusing changes in platelet function in psychiatric and gastrointestinal disorders. ROBIN C. SPILLER STEPHEN FOLEY, on behalf of the authors National Institute for Health Research Biomedical Research Unit Nottingham Digestive Diseases Centre School of Clinical Sciences Nottingham, England 1. 2. 3. 4. 5. 6. 7.
Bellini M, et al. Am J Gastroenterol 2003;98:2705–2711. Franke L, et al. J Gastroenterol 2010;45:389 –398. Maguire K, et al. Biol Psychiatry 1993;34:356 –360. Bazzichi L, et al. Arthritis Res Ther 2006;8:R99. Hrdina PD, et al. Psychiatry Res 1997;66:73– 85. Marazziti D, et al. Biol Psychiatry 1999;45:443– 447. Neuger J, et al. Psychiatry Res 1999;85:189 –198.
Conflicts of interest The authors disclose no conflicts. doi:10.1053/j.gastro.2011.09.038
CORRESPONDENCE Readers may submit letters to the editor concerning articles that appeared in GASTROENTEROLOGY within one month of publication. Detailed guidelines regarding the content are included in the Instructions to Authors.
Possible Role of Serotonin Transporter in Irritable Bowel Syndrome: Evidence From Platelet Investigation Dear Sir:
In the May 2011 issue of GASTROENTEROLOGY, Foley et al1 report that platelet serotonin transporter (SERT) is significantly reduced in patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and associated with reduced duodenal levels of SERT mRNA and duodenal immune activation. Although these interesting results contribute to the current discussion of whether the SERT is implicated in the pathophysiology of functional gastrointestinal disorders and whether platelets are useful to investigate the SERT in this condition, caution must be exercised in interpreting the findings. The study contains some methodologic problems and the way in which the correlation analysis is performed generates a range of questions. First, the expression of platelet serotonin uptake (PSU) rate per mg platelet protein is unusual. The results are given in nmol or fmol 5HT/mg protein ⫻ min. Which is correct? Although the assay includes 5 different concentrations of [3H]-5HT/5HT, the authors do not calculate the maximum uptake rate (Vmax) and Km (Michaelis constant); they report the PSU at 800 nmol [3H]-5HT/5HT/L, and declare this to be the maximum uptake rate. Methodologic issues of PSU assays are well discussed in a recent study.2 To characterize the SERT the authors also use a singlepoint binding assay with [3H]-paroxetine (4 nmol/L). Again, given the potential methodical pitfalls in this assay protocol, errors can not be excluded.3 Second, we would like to point out existing literature data, which are not mentioned by the authors. Two previous studies on IBS patients apply standard methods for the assay of [3H]paroxetine saturation binding or PSU kinetics.4,5 Significantly lower mean Bmax is found for [3H]-paroxetine in 12 female IBS-D patients with a strong inverse correlation between Bmax and symptom severity score in these patients (r ⫽ ⫺0.964).4 Surprisingly, the study by Foley et al1 reports significantly higher median [3H]-paroxetine binding at 4 nmol/L in 18 patients with IBS-D than in healthy controls. Recently, we compared PSU kinetics in 149 patients with IBS and 163 healthy control subjects and found a high rate (55.5%) of very low and low Vmax values (below the 25th percentile of male controls) in male IBS patients.5 Third, the inverse correlation between PSU and [3H]-paroxetine binding in the mixed group of healthy subjects and patients with IBS-D and celiac disease, with r ⫽ ⫺0.44 (r is probably the Spearman’s referred to in Material and Methods) requires some comments. In general, correlation coefficients ⬍0.5 are thought to be of low strength. It is difficult to understand why this inverse relationship should mean
that measure of [3H]-paroxetine binding could be a useful surrogate marker of impaired SERT in patients with IBS. It is also surprising to read that the single-point binding assay of [3H]-paroxetine would be an indirect measure of SERT function. Previous studies in which PSU and [3H]-paroxetine binding were investigated in parallel report a lack of any relationship between Vmax and Bmax. Only 1 study has reported a positive correlation between Vmax and Bmax in a group of 20 female healthy subjects.6 Unfortunately, Foley et al have not mentioned or discussed these findings. The most recent study on females with fibromyalgia shows that both Vmax and Bmax are on average significantly lower in patients, but Vmax and Bmax are not correlated.7 Fourth, the significant but low correlation between logtransformed SERT mRNA expression in duodenal biopsy specimens and log PSU (R ⫽ 0.37) or log [3H]-paroxetine binding (R ⫽ ⫺0.46) in a mixed sample of healthy controls and IBS-D (R is probably the Pearson r and the sample size is now indicated) should be interpreted with caution. It can be assumed that patients with celiac disease were not included in this correlation analysis because this group showed the lowest SERT mRNA expression but the PSU and [3H]-paroxetine binding were not different from healthy controls. Thus, a general relationship between the SERT mRNA expression in duodenum and platelet SERT measures (functional or binding assays) can not be assumed. Finally, because proinflammatory cytokines (tumor necrosis factor-␣ and interferon-␥) are known to regulate SERT expression and function differently in different cell lines8 the finding of a significant negative correlation between log mRNA expressions of interleukin (IL)-10 and SERT in the IBS-D group with a Pearson r ⫽ ⫺0.8, is notable. Unfortunately, the sample size is not indicated; nor is a reason given for why a Bonferroni correction was applied only in this particular correlation analysis. It would be interesting to prove whether the relative expressions of IL-10 and SERT itself are also significantly and linearly associated (Spearman’s ) or show an exponential relationship. LEONORA FRANKE Department of Psychiatry and Psychotherapy Charitè-Universitättsmedizin Berlin Berlin, Germany MARTIN SCHMIDTMANN HUBERTHUS MÖNNIKES Department of Medicine and Institute of Neurogastroenterology Martin Luther Hospital Academic Teaching Institute of Charitè-Universitätstsmedizin Berlin, Germany
November 2011 1. 2. 3. 4. 5. 6. 7. 8.
CORRESPONDENCE
Foley S, et al. Gastroenterology 2011;140:1434 –1443.e1. Banovic´ M, et al. Platelets 2010;21:429 – 438. Jurado N, et al. Arch Med Res 2003;34:422– 427. Bellini M, et al. Am J Gastroenterol 2003;98:2705–2711. Franke L, et al. J Gastroenterol 2010;45:389 –398. Maguire K, et al. Biol Psychiatry 1993;34:356 –360. Bazzichi L, et al. Arthritis Res Ther 2006;8:R99. Zhu CB, et al. Neuropsychopharmacology 2006;31:2121–2131.
Conflicts of interest The authors disclose no conflicts. doi:10.1053/j.gastro.2011.08.042
Reply. Thank you for your interest in our study which
demonstrated immune activation in the gut of patients with the irritable bowel syndrome with diarrhea together with depression of mRNA of SERT in the mucosa. We were able to correlate this with mucosal lymphocyte numbers and also to impairment of platelet 5HT uptake. You raise some important points that we attempt to address. First, we apologize for the confusion about the units for expressing 5HT uptake, which are as correctly stated in Table 3 expressed in nmol/min/mg protein not pmol/min/mg protein as stated in Methods. We agree that the best way of expressing uptake is from calculation of Vmax, which we had intended to do but as stated in the text, we found “uptake at lower concentrations were highly variable but the uptake had reached a plateau at 800 nmol/L concentration and this was taken as the best estimate of maximum uptake.” We think this is fair and, although not ideal, it is unlikely to bias our results or alter our conclusions. We agree that there are 2 previous studies on IBS patients that also assessed paroxetine binding and came to rather different conclusions. The study by Bellini found paroxetine binding was lower in IBS-D compared with controls1 while we found it to be increased. Although they used a lower range of paroxetine concentrations 0.01–1 nmol/L, whereas we used 4 nmol/L to ensure we saturated the binding site these methodologic differences would not account for the opposite findings which we can only suggest are due to differences in patient selection given the small numbers in both studies, 12 in Bellini’s and 20 in ours. The other very detailed study performed by Franke et al2 is highly relevant because it has much larger numbers with a total of 149 IBS patients, 47% IBS-D, 29.5% alternating-type IBS, and 23.5% constipation-predominant IBS. This did show an overall 10% reduction in platelet 5HT uptake Vmax in IBS, which appeared largely due to the male patients. Female controls showed lower values than male controls, but these did not differ from female patients, the reasons for which are unclear. Given that we only had 7 males, we cannot comment on any gender difference in our study. We agree that there is no simple relationship between paroxetine binding and SERT function because paroxetine binds to a different site than serotonin. Our data showed an inverse correlation only in the IBS-D patient group. Our interpretation was that this was a compensatory response to
e19
decreased SERT function, but we agree that confirmatory studies are needed. Despite the large literature on platelet serotonin uptake, there are surprisingly few studies where both paroxetine binding and 5HT uptake have been assessed, mostly within the psychiatric and alcohol dependence field. Maguire et al3 found a positive correlation in healthy volunteers. A study in fibromyalgia found both Vmax and Bmax depressed compared with controls, but did not comment on any correlation.4 Most studies have not reported a correlation between 5HT uptake and paroxetine binding, which can be altered independently by disease5 or drugs.6 One study on major depression did find a decreased 5HT uptake associated with an elevated paroxetine binding in male patients, which parallels our findings.7 They also found paroxetine binding to correlate with anxiety, something we could not confirm. You are correct that the celiacs were not included in the correlation between mRNA for SERT and platelet uptake since as stated in the text the mRNA in the biopsies was measured on a different group of patients (group 2) whereas the platelets were measured only in group 1. We agree that the correlation between log IL-10 and log SERT mRNA in the IBS-D patients (n ⫽ 20) of ⫺0.8 is of interest. We took this to indicate a general effect of inflammation since although anti-inflammatory, IL-10 expression is increased in many inflammatory conditions and log IL-10 in fact correlated significantly (P ⬍ .001) with all the other cytokines (r ⫽ 0.4–0.8), although their individual correlations with log SERT were not significant. We log-transformed the data because the raw data were skewed. Untransformed data give an exponential curve, as would be predicted. We have just completed a larger study of 120 IBS-D patients in whom we will assess paroxetine binding and its relationship to response to 5HT3 antagonist. We believe that intervention studies such as these are needed to go beyond mere correlation and demonstrate the significance or otherwise of these fascinating but at times confusing changes in platelet function in psychiatric and gastrointestinal disorders. ROBIN C. SPILLER STEPHEN FOLEY, on behalf of the authors National Institute for Health Research Biomedical Research Unit Nottingham Digestive Diseases Centre School of Clinical Sciences Nottingham, England 1. 2. 3. 4. 5. 6. 7.
Bellini M, et al. Am J Gastroenterol 2003;98:2705–2711. Franke L, et al. J Gastroenterol 2010;45:389 –398. Maguire K, et al. Biol Psychiatry 1993;34:356 –360. Bazzichi L, et al. Arthritis Res Ther 2006;8:R99. Hrdina PD, et al. Psychiatry Res 1997;66:73– 85. Marazziti D, et al. Biol Psychiatry 1999;45:443– 447. Neuger J, et al. Psychiatry Res 1999;85:189 –198.
Conflicts of interest The authors disclose no conflicts. doi:10.1053/j.gastro.2011.09.038