Surgical and pathological outcomes of laparoscopic surgery for transverse colon cancer

Purpose Several multi-institutional prospective randomized trials have demonstrated short-term benefits using laparoscopy. Now the laparoscopic approach is accepted as an alternative to open surgery for colon cancer. However, in prior trials, the transverse colon was excluded. Therefore, it has not been determined whether laparoscopy can be used in the setting of transverse colon cancer. This study evaluated the peri-operative clinical outcomes and oncological quality by pathologic outcomes of laparoscopic surgery for transverse colon cancer.
Introduction
Since its first report [1], laparoscopic colon surgery has been controversial with regard to its use for colorectal cancer. Several prospective randomized trials including the COLOR and CLASSIC studies have demonstrated that laparoscopic-assisted surgery for colorectal cancer resulted in a shorter hospital stay, reduced analgesic use, and earlier recovery to bowel movement [2–6]. Moreover, the COST study established the long-term oncological safety of laparoscopic-assisted surgery for colon cancer, and currently the laparoscopic approach is accepted as an alternative to open surgery for colon cancer [7]. However, transverse colon cancer was excluded from prior randomized controlled trials. The reasons for exclusion of transverse colon cancer include the following: difficulty in deciding the appropriate operative procedure and extent of lymph node dissection, as well as technical difficulties with the laparoscopic identification, ligation, and lymph node dissection around the middle colic vessels. Therefore, there continues to be debate on whether to use laparoscopic surgery for transverse colon cancer.
Materials and methods
Between August 2004 and November 2007, the medical records of all patients who underwent laparoscopic surgery for colorectal cancer were reviewed. Laparoscopic colorectal cancer resection was started August 2004 in our clinic. Pathologic confirmation, colonoscopy, barium enema, computed tomography (CT), ultrasonography, and chest PA were performed for diagnosis in all patients preoperatively. All patients with colorectal adenocarcinoma admitted to our clinic were considered for laparoscopic surgery. Exclusion criteria for laparoscopic surgery were as follows: (1) patients with colorectal cancer obstruction and failure of stent insertion, (2) patients with colorectal cancer perforation requiring emergency surgery, (3) patients with T4 colorectal cancer lesion that could not be resected laparoscopically, and (4) patients with compromised cardio-pulmonary function in whom pneumoperitoneum under general anesthesia was contraindicated. In this study, transverse colon cancer was defined as lesions between the hepatic flexure and splenic flexure in the colon, requiring ligation of the middle colic vessels at their origin. CT or barium enema was performed in all patients with colon cancer preoperatively for localization of the tumor. If radiological localization was unclear, preoperative colonoscopic Indian ink tattooing or endoscopic clipping was performed.
The procedure used for the transverse colon cancer was chosen based on the location of the tumor. A tumor located at the hepatic flexure or within 10 cm distal to hepatic flexure was treated by an extended right hemicolectomy, and a tumor located at the splenic flexure or within 10 cm proximal to the splenic flexure was treated by an extended left hemicolectomy. A tumor located between the above two lesions was treated by a transverse colectomy. An extended right hemicolectomy was defined as lymphadenectomy simultaneously with ligation of ileocolic, right colic, and middle colic vessels at their origins; an extended left hemicolectomy was defined as lymphadenectomy simultaneously with ligation of left colic and middle colic vessels at their origins. A transverse colectomy was defined as lymphadenectomy simultaneously with ligation of middle colic vessels at their origins. Extra-corporeal anastomosis was performed in all cases of laparoscopic surgery for transverse colon cancer. All patients started a diet after passing flatus. To evaluate the postoperative surgical outcomes and oncological quality of laparoscopic surgery for transverse colon cancer, we compare the age, gender, body mass index (BMI), operating time, blood loss, time to passing flatus, time to start of diet, hospital stay, surgical morbidity, surgical mortality, conversion to open surgery, tumor size, distal resection margin, proximal resection margin, radial margin, and number of harvested lymph nodes between the transverse colon cancer group (TCC) and other site colon cancer group (OSCC). In this study, a single surgeon (YS Lee) performed all operations. Comparisons between the two groups were made by applying the independent samples t test and χ2 test. Differences were considered to be significant for P value < 0.05.
Results
Postoperative clinical outcomes
Three hundred and twelve patients underwent colorectal cancer resection in our clinic during these periods. Among them, 44 patients underwent conventional open surgery according to exclusion criteria (five cases of stent failure or colon obstruction, five cases of emergency operation, 12 cases of far advanced tumor, 12 cases of recurrent cancer operation, seven cases of patient’s refusal, and three cases for old age with high risk for pneumoperitoneum). There was no transverse colon cancer in the conventional open surgery group. A total of 268 patients underwent laparoscopic resection for colorectal cancer. Of the 268 patients, 140 patients who underwent laparoscopic resection for rectal cancer were excluded in this study. Finally, 128 patients who underwent laparoscopic resection for colon cancer were enrolled in this study. Of the 128 patients, 34 patients had transverse colon cancer, counting for 10.8% of total colorectal cancers, and 94 patients had other sites of colon cancer. In the TCC group, extended right hemicolectomy was performed in 18 cases, transverse colectomy was performed in eight cases, and extended left hemicolectomy was performed in eight cases. In the OSCC group, right hemicolectomy was performed in 38 cases, left hemicolectomy was performed in five cases, and anterior resection was performed in 51 cases. There were no statistical differences in age, gender distribution, BMI, operating time, intraoperative blood loss, time to flatus, time to start of diet, and hospital stay between patients with TCC and OSCC (Table 1). Four patients in the OSCC group had a major complication, one had colon injury, and two had anastomosis leak. A simple laparoscopic closure was performed in the case of colon injury, and two patients with anastomosis leak underwent re-operation. Minor complications that occurred in four cases in the OSCC group (one case of ileus, two cases of port site minor infection, and one case of urinary retention) and two cases in the TCC group (one case of ileus and one case of atelectasis) were treated successfully conservatively. One case among the TCC group and three cases in the OSCC group were converted to open surgery; all of these converted cases were due to tumor-related factors, T4 lesion, or huge tumor. There was no surgical mortality in this study.
Table 1Clinical characteristics of patients TCC (N = 34)OSCC (N = 94)P valueAge (years)64.1 ± 11.362.5 ± 12.1NSSex (M:F)15:1946:48NSBody mass index (kg/m2)23.5 ± 3.024.0 ± 3.1NSOperating time (min)211.1 ± 52.2 (140–360)a220.4 ± 94.3 (60–620)aNSBlood loss (ml)100.0 ± 83.8 (0–400)a114.2 ± 145.2 (0–700)aNSTime to pass flatus (days)2.8 ± 0.82.6 ± 1.0NSDiet start (days)4.2 ± 1.84.2 ± 2.8NSHospital stay (days)11.4 ± 4.111.2 ± 6.0NSTCC Transverse colon cancer, OSCC other site colon cancer, NS not significantaValues are ranges.
Oncological quality by pathologic outcomes
There were no statistical differences in the tumor size, proximal resection margin, distal resection margin, radial margin, and number of harvested lymph nodes between patients with TCC and OSCC (Table 2).
Table 2Pathological outcomes of patients TCC (N = 34)OSCC (N = 94)P valueTumor size (cm)5.2 ± 2.5 (0.7–11)a4.6 ± 2.4 (0.1–14)aNSPRM (cm)19.5 ± 10.2 (10.0–25.0)a15.3 ± 11.0 (4.2–25.0)aNSDRM (cm)13.9 ± 6.9 (6.0–25.0)a11.6 ± 5.3 (3.0–32.0)aNSNo. of lymph nodes24.4 ± 11.7 (3–59)a21.1 ± 8.4 (3–59)aNSRadial margin (cm)0.9 ± 0.8 (0–3.0)a1.0 ± 0.9 (0–3.5)aNSTCC Transverse colon cancer, OSCC other site colon cancer, DRM distal resection margin, PRM proximal resection margin, NS not significantaValues are ranges.
Discussion
Since laparoscopic colon resection was first reported in 1991 [1], laparoscopic surgery has been widely employed for various benign colorectal disease such as benign mass, diverticular disease, inflammatory bowel disease, rectal prolapse, and now increasingly for colorectal malignant disease. Evidences of the safety and efficacy of the laparoscopic surgery for colorectal cancer had been reported from several prospective randomized controlled studies and meta-analysis of several trials, which favored the laparoscopic surgery for colorectal cancer over conventional open surgery due to its many short-term benefits such as shorter hospital stay, reduced use of analgesics, and earlier recovery of bowel movements [2–6, 8–13]. Eventually, long-term oncological safety of laparoscopic colon cancer resection was established, and the laparoscopic approach was accepted as an alternative to open surgery for colon cancer by the COST study [7].
Transverse colon cancer occurs in about 10% of cases of colorectal cancer, and it often presents a challenge for the choice of the best surgical procedure based on the location of the tumor and extent of lymph node dissection. There could also be technical difficulties with laparoscopic identification, ligation, and lymph node dissection around the middle colic vessels depending on the surgeon’s experience. Because of these reasons, transverse colon cancer was excluded from almost every prior prospective randomized trial. Therefore, there is continued debate over the validity of laparoscopic surgery for transverse colon cancer. The major controversy about laparoscopic surgery for transverse colon cancer lies on whether or not it is feasible to perform sufficient extent of lymph node dissection around the middle colic artery laparoscopically. As experiences of laparoscopic surgery are accumulating and surgical techniques and instruments are developing, we consider that the extent of laparoscopic lymph node dissection for transverse colon cancer is not less than the extent of conventional lymph node dissection for transverse colon cancer.
Some of the difficulties with laparoscopic surgery for transverse colon cancer that need to be resolved include the following: First, precise localization of carcinoma of the transverse colon is important; this is because the extent of resection and lymph node dissection depends on the location of the tumor in the transverse colon. Diminished tactile guidance can make localization of the tumor in the transverse colon more difficult in small tumor. There are a number of methods used to localize the tumor. Preoperative barium enema is useful for localization of large and advanced tumor, but radiological localization is inconclusive and difficult in early cancer. In such cases, colonoscopic tattooing with Indian ink or placing endoscopic clips is very effective. Using endoscopic clip at the time of preoperative colonoscopy and using fluoroscopy for localization, the clip migration could be the main problem, and using fluoroscopy in the operating theater is troublesome and time consuming. However, in the case of hepatic flexure or splenic flexure colon cancer, by checking X-ray just after placing the endoscopic clip, we could precisely localize both flexure tumors easily. Intraoperative colonoscopy can be used for localization of the tumor, but it is also time consuming, and moreover, it can cause colonic insufflation that makes laparoscopic surgery difficult [14]. Properly placed tattoos are long lasting and can be placed at the time of diagnostic colonoscopy [15]. In the event of tattoo failure, one can use intraoperaitve colonoscopy for localization easily. In this study, we performed preoperative colonoscopic tattoo in three cases to localize the mid transverse colon cancer, colonoscopic clipping in three cases of hepatic flexure colon cancer, and barium enema in the other cases. There was no case with non-localization. We think that colonoscopic tattooing is effective in small mid transverse colon cancer, and endoscopic clipping is effective in small hepatic or splenic flexure colon cancer. Second is the laparoscopic identification, ligation, and lymph node dissection around the middle colic vessels. Fujita et al. reported laparoscopic techniques that can be used for identification of middle colic vessel. They suggested that the ventral aspect of the caudal portion of superior mesenteric vein be exposed, and the exposed vessel brought cephalad toward the caudal portion of the pancreas, with identification of the middle colic vessels [16]. Baca et al. introduced the ‘window technique’ for lymphadenectomy with simultaneous resection of the vascular stem [17]. Ichihara et al. introduced the technique of rotation of mesocolon to identify middle colic artery [18]. The authors have identified the middle colic vessels using surgical techniques introduced by Fujita in most of the cases, and the in case of slender patients we could directly identify pulsating middle colic artery by stretching the transverse mesocolon by traction of both ends of the transverse colon by the first assistant. Identifying middle colic vessels, excessive traction of the transverse mesocolon by the first assistant may cause tearing of the vein and bleeding. Moreover, the length of the gastrocolic trunk of Henle is relatively short, and attempt to control bleeding from the gastrocolic trunk of Henle can injure the superior mesenteric vein [19]. It is especially important for a laparoscopic colorectal surgeon to recognize the variable drainage of the right colic and middle colic vein to the gastrocolic trunk of Henle and to have a precise knowledge of superior right colic vein anatomy. Third is the low incidence of transverse colon cancer. Consecutive cases are needed for overcoming the learning curve. Therefore, it might take surgeons longer time to become experienced in the techniques used for laparoscopic transverse colon cancer resection.
In this study, there were no significant differences between patients with TCC and OSCC in terms of operating time, blood loss, resection margin, and number of lymph nodes. Schlachta et al. reported that the operating time was longer in the TCC group compared to the OSCC group, and the number of harvested lymph nodes in the TCC group was greater than in the OSCC group because of the extended resection and larger specimens, as well as the need to attend to the middle colic vessels in patients with TCC [20]. However, in this study, there was no statistical difference in operating time and number of harvested lymph nodes, as well as the surgical outcomes between the two groups. We think that operating time could be similar in the TCC and OSCC groups in experienced hands, and actually, the extent of lymph node dissection in transverse colon cancer is not too wide than that of other site colon cancer, so there were no statistical differences in pathological outcomes between the two groups in this study.
There was one case of colon injury proximal to anastomosis caused by electrocautery during the operation in the OSCC group; this was treated by a laparoscopic simple closure on postoperative day 3. Three cases of anastomosis leak, one case of right hemicolectomy, and two cases of anterior resection occurred in the OSCC group. A patient with anastomosis leak was diagnosed by clinical symptoms or sign, fecal or purulent discharge from drain, fever, leukocytosis, and peritoneal irritation sign. Radiologic study using a water-soluble dye was not performed in this study. Of the three cases, one patient with a leak of anterior resection was treated conservatively successfully, and two patients with leak cases underwent laparoscopic re-operation. All other minor complications were successfully treated conservatively.
Conversion to open surgery occurred in one case in the TCC group and in three cases in the OSCC group; these differences were not significant. Factors related to the tumor were the cause of conversion to open surgery. One case in the TCC group was converted to open surgery due to T4 lesion in the transverse colon cancer that invaded the anterior wall of the stomach. In the OSCC group, one case with a larger tumor that was hard to handle by laparoscopy, and two cases of sigmoid colon cancer that invaded the uterus were converted to open surgery. However, T4 colon cancer is not a contraindication for laparoscopy if en bloc resection could be performed with the laparoscopy. In those cases converted to open surgery, laparoscopic en bloc resection was impossible.
This study has some weak points. First, the number of patients with transverse colon cancer is too small, making up 10% of the total colorectal cancer resection and 12% of the total laparoscopic colorectal resection in this study. The second is that although the data in this study were collected prospectively, the data were not derived from a larger case series and this study was not randomized controlled. The third is that the mean follow-up period was too short to evaluate the oncological outcomes (15.9 months; range 1–40 months). We think that large-scale prospective controlled trials and long-term analysis are mandatory to overcome these limitations and confirm the oncological safety of laparoscopic transverse colon cancer surgery. We are going to report a long-term analysis after a long-term follow-up involving more cases prospectively.
Conclusions
The results of this study show no significant differences with regard to surgical outcomes and oncological quality by pathologic outcomes between patients in the OSCC and TCC groups. Further investigations with large-scale prospective studies and long-term analysis of laparoscopic surgery for transverse colon cancer are mandatory to establish the oncological safety of laparoscopic surgery for transverse colon cancer.

transverse colon
colon cancer
laparoscopy


An outwardly rectifying anionic background current in atrial myocytes from the human heart

This report describes a hitherto unreported anionic background current from human atrial cardiomyocytes. Under whole-cell patch-clamp with anion-selective conditions, an outwardly rectifying anion current (IANION) was observed, which was larger with iodide than nitrate, and with nitrate than chloride as charge carrier. In contrast with a previously identified background anionic current from small mammal cardiomyocytes, IANION was not augmented by the pyrethroid tefluthrin (10 μM); neither was it inhibited by hyperosmolar external solution nor by DIDS (200 μM); thus IANION was not due to basal activity of volume-sensitive anion channels. IANION was partially inhibited by the Cl− channel blockers NPPB (50 μM) and Gly H-101 (30 μM). Incorporation of IANION into a human atrial action potential (AP) simulation led to depression of the AP plateau, accompanied by alterations to plateau inward calcium current, and to AP shortening at 50% but not 90% of complete repolarization, demonstrating that IANION can influence the human atrial AP profile.
The electrophysiological behaviour of cardiac myocytes from mammalian hearts is determined by the combined activity of a range of different cation and anion channel types. The reversal potential for chloride (Cl−) ions in the heart (ECl) lies between ∼−60 and −40 mV [1]. Negative to ECl outward Cl− movement generates depolarizing ionic current, whilst positive to ECl inward Cl− movement generates repolarizing ionic current. Therefore, the activation of Cl− channels can influence both the resting membrane potential and the duration of cardiac action potentials (APs) ([1–3] for reviews). Several different anion channel types have been identified that may contribute to cardiac physiology and pathophysiology [1–3]. Of the cardiac anion channel currents thus far identified, the three major types are: (i) a cystic fibrosis transmembrane conductance regulator (CFTR) current-activated through cAMP-dependent phosphorylation (ICl,cAMP; e.g. [4–6]); (ii) a stretch- or swelling-activated Cl− current (ICl,Swell; e.g. [7–9]) and (iii) a Ca2+-activated Cl− current (ICl,Ca; e.g. [10–12]).
Recently, an outwardly rectifying anionic background current (IAB) has been identified in cardiac myocytes from two commonly studied model species (rat and guinea-pig) using whole-cell patch-clamp measurements [13,14]. IAB is distinct from previously identified Cl− currents as it has a distinct permeability sequence  and is insensitive to the stilbene diphosphonate Cl− channel inhibitor DIDS, to cell swelling and to intracellular Ca2+ and cAMP [13,14]. IAB can also be differentiated from other major cardiac anion currents as it can be activated by the pyrethroid agent tefluthrin [14]. Anion substitution experiments have provided evidence that IAB can influence AP duration (APD) [13]. There is some disagreement as to whether or not a basally active anionic current exists in human atrium [15,16] and there is no information as to whether humans exhibit an IAB with the characteristics of that seen in small mammal hearts. Therefore, the present study was undertaken to determine whether or not IAB exists in adult human cardiac myocytes. The resulting findings indicate the presence in human atrial myocytes of an outwardly rectifying anionic background current (IANION). Notably, the IANION observed in this study has the potential to contribute to human atrial electrophysiology, but is distinct from both the IAB recorded previously from myocytes from small mammal hearts [13,14] and from outwardly rectifying stilbene diphosphonate-sensitive anionic currents recorded previously from human atrium [17].
Methods
Atrial myocyte isolation
The study was approved by the local Central and South Bristol Research Ethics Committee and was conducted in accordance with the principles of the Declaration of Helsinki. Human right atrial appendages were obtained, with consent, from 32 patients (27 males, 5 females, average age 69.7 ± 1.7 years) undergoing coronary artery bypass surgery. Single human atrial myocytes were isolated from right atrial appendages by mechanical and enzymatic dispersion. Tissue samples were quickly immersed in cardioplegic solution (see Table 1; solution G, 100% O2, ice cold). The samples were chopped into small chunks and washed with an EGTA-containing solution (see Table 1; solution H) gassed with 100% O2 for 15 min at 37 °C. The chunks were then incubated in the same solution from which EGTA was excluded and protease type XXIV (3 U/ml, Sigma) and collagenase type V (250 U/ml, Sigma) were added. The medium was continuously gassed with 100% O2 at 37 °C. After 15 min, the incubation medium was substituted for the same solution containing collagenase only. Myocytes were progressively released from the chunks into the supernatant and their yield monitored under a microscope. The suspension was washed in enzyme-free solution and the myocytes were stored at room temperature until use (within ∼8 h of cell isolation).
Electrophysiology
Solutions used. Experimental solutions for the investigation of anionic current were similar to those used previously to study IAB
[14]; the composition of all solutions used is given in Table 1. Osmolarity values given for each of the solutions listed in Table 1 were measured using a micro-osmometer employing a freezing-point method (Advanced Instruments, Norwood, MA, USA). Myocytes used in whole-cell voltage-clamp experiments were superfused (20–25 °C) with a standard Hepes-buffered Tyrode’s solution (see Table 1; solution A) until the whole-cell recording configuration had been obtained. For isolation of background anion current, sodium-free Tyrode’s solutions were used (solutions B–E) in which Na was replaced by N-methyl-d-glucamine (NMDG), with one of several possible dominant anions: solution B, chloride; solution C, aspartate; solution D, iodide; solution E, nitrate. All drugs used were added to solution E from stock solutions made in dimethyl sulfoxide (DMSO) with an exception of N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (Gly H-101), which was solved in distilled water. The hyperosmotic external solution (solution F) was prepared by adding 70 mM sucrose to solution E. A Cs-based pipette solution (solution I) was used for all experiments. Solution I was sodium-free to prevent contamination of chloride currents by the sodium–calcium exchanger current.
Drugs. Diisothiocyanostilbene-2,2′-disulfonic acid (DIDS, final concentration 200 μM), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, final concentration 50 μM) and tefluthrin (TEF, final concentration 10 μM) were purchased from Sigma Chemical Co. (Poole, UK). N-(2-naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (Gly H-101, final concentrations of 10 and 30 μM) was purchased from Merck (Frankfurt, Germany). All the drug-containing solutions were protected from light throughout.
Electrophysiological recording
In electrophysiological experiments, junction potential changes were minimized by immersing the reference Ag/AgCl electrode in a 3 M KCl solution with a continuous agar bridge (4% agar in 3 M KCl). Borosilicate glass pipettes (Harvard Apparatus, UK) were pulled using a vertical two-step Narishige PP-830 microelectrode puller (Narishige, Japan) and had a tip resistance of 5–7 MΩ when filled with the pipette solution. During anion substitution experiments, background anion current was elicited from voltage-clamped myocytes (superfused with solutions B, C, D and E in the whole-cell configuration) by depolarizing ramps from −90 to +70 mV from a holding potential of −50 mV (ramp rate of 0.32 V s−1; sweep duration 1.03 s). A holding potential of −50 mV was used to inactivate the Na+-current and T-type Ca2+-current.
Recordings were made using an Axopatch 200A amplifier, and data were recorded on computer using pClamp v. 9.0 software (Axon Instruments, Forster City, CA). Data were analyzed using the Clampfit program of pClamp v. 9.0.
Mean values of averaged original signals over five command pulses were used for statistical analysis. Hyperpolarizing voltage steps of −20 mV and 5 ms duration were applied at 20 Hz to record the capacitance transients required for direct integration and the calculation of cell capacitance. The statistical significance between control and the drug periods or  and other anions were determined by the Paired Student’s t-test using either Microsoft Excel or GraphPad Prism v. 4.0. The statistical significance between the normal and hyperosmotic  solutions was calculated with two-way ANOVA test using GraphPad Prism v. 4.0. Statistical significance was considered to refer to the 95% level of confidence (p < 0.05).
Human atrial action potential simulations
The Courtemanche et al. human atrial action potential (AP) model [18] was modified to incorporate a formulation for IANION based on the experimental data obtained with  and Cl− in Figs. 1 and 2. Readers are referred to [18] for the general equations required to set up the model. The following equation was used to simulate anionic background current (IANION)where EANION represents the current reversal potential and gANION is the conductance of IANION. By fitting Eq. (1) to experimental data shown in Fig. 1B and scaled to the mean data shown in Fig. 2A, we obtained gANION = 0.37 pS/pF, EANION = −45.64 mV, c = 0.87, d = 8.4 × 10−4 mV−1 for the -sensitive IANION, and gANION = 0.19 pS/pF, c = 0.94, d = 2.5 × 10−4 mV−1 for the Cl−-sensitive IANION.
Results and discussion
The voltage protocol used for these experiments was similar to that used previously to study IAB present in cardiomyocytes from small mammal hearts [14] and is shown as an inset to Fig. 1A. From a holding potential of −50 mV, ascending voltage ramps were applied between −90 and +70 mV. This protocol was applied to cells superfused first with aspartate (Asp)-containing solution (solution C) and then with different superfusates containing more permeant anions. Fig. 1A shows an example of the net current traces obtained from a cell superfused serially with solutions containing Asp−, Cl−,  and I−. Both inward and, particularly, outward current components were greater with Cl−,  and I− than with Asp− in the external superfusate. Fig. 1B shows current traces for the same cell, obtained by subtracting the current in Asp− from that with each of the more permeant anions. With each of Cl−,  and I−, the Asp−-sensitive difference current showed marked outward rectification. Fig. 1C compares the mean outward current amplitude at +60 mV (normalized to membrane capacitance) for the three anions. Compared to , the observed current was significantly greater with I− and smaller with Cl− as charge carrier. These observations support the presence in human atrial cells of a basally active, anionic current (IANION); however, the relative current amplitudes with the three permeant anions differ from those observed previously with for the IAB observed in myocytes from guinea-pig and rat hearts, where IAB was largest with  (permeability sequence ; [13,14]).
Fig. 2A shows the mean IANION–voltage relation for 19 atrial cells, with  as the major external anion (with IANION measured as the  − Asp− difference current). The mean current–voltage relation for the resulting current showed clear outward rectification, with an observed reversal potential (Erev) for IANION in these experiments of −45.7 ± 2.2 mV (obtained by pooling Erev values from individual experiments). Previous studies provide evidence that human atrial cells exhibit ICl, Swell (e.g. [15,17,19–21]). Therefore, in order to determine whether or not IANION could be attributed to basal activity of channels mediating ICl,Swell, Asp− to  substitutions were also made using hyperosmolar external solution [14]. The mean data from eight such experiments are shown in Fig. 2B. There was no statistically significant difference between the plotted densities of IANION from the I–V relation obtained in hyperosmolar solution and that shown in Fig. 2A, suggesting that IANION is distinct from ICl,Swell.
In order to characterize further IANION from human atrial myocytes, the sensitivity of the current to a range of pharmacological interventions was tested. Fig. 2C summarises the effects of the various interventions (expressed as % changes in -sensitive current at +60 mV). The stilbene disulphonate DIDS failed to inhibit IANION at a concentration (200 μM) that would be anticipated to inhibit ICl,Swell
[19,21]. On the other hand, tefluthrin (10 μM), which we have previously reported to activate the IAB seen in myocytes from guinea-pig hearts [14], failed to alter significantly the amplitude of IANION from human atrial myocytes. Together with the relative IANION amplitudes in I−, Cl− and , the lack of effect of tefluthrin indicates that IANION is distinct from the previously reported rat/guinea-pig IAB
[13,14]. Moreover, the lack of significant inhibition of the current by DIDS or hyperosmolar solution makes the IANION observed in the present study distinct from ICl,Swell
[1] and from an osmolarity- and stilbene-sensitive outwardly rectifying chloride current recently reported by Demion and colleagues [17]. NPPB (50 μM) produced a partial, statistically significant (p < 0.05) inhibition of IANION. The glycine hydrazide Cl− channel inhibitor Gly H-101 failed to produce a significant blockade of IANION at 10 μM (∼7-fold greater than the reported IC50 for CFTR channel inhibition at +60 mV [22]); but produced partial attenuation of the current at 30 μM (∼20-fold the reported IC50 for CFTR [22]). Evidence for the presence of CFTR (ICa,cAMP) in human atrial cells is mixed [1,15,19–21,23], with a number of studies failing to observe the current in response to β-adrenergic stimulation, forskolin or cAMP (e.g. [15,19–21]). Previous work has failed to find evidence for ICl,Ca in human atrial myocytes [24] and, moreover, the presence of EGTA in the pipette dialysate (Table 1, solution I) and external [Ca2+] replacement in our experiments would have inhibited any [Ca2+]i-activated conductances on membrane depolarization. Therefore, the IANION seen here appears to differ not only from guinea-pig and rat IAB
[13,14] but also from the three major reported cardiac anion conductances in: (i) being basally active and (ii) its overall pharmacological profile and sensitivity to anion substitution.
In order to gain insight into the physiological role of IANION, the current was incorporated into human atrial AP simulations as outlined in the ‘Methods’. Fig. 3A shows the simulated APs (at an AP frequency of 1 Hz) from the Courtemanche et al. model [18] both without (Control) and with inclusion of IANION, whilst Fig. 3B shows the corresponding current profiles during the time-course of the AP. With either  and Cl− as charge carrier, incorporation of IANION into the model produced shortening of AP duration at 50% repolarization (APD50; the measured APD50 values were 184, 165 and 159 ms for Control, Cl-sensitive IANION and NO3-sensitive IANION, respectively). In contrast, APD90 was comparatively unaffected (the measured APD90 was ∼305 ms under each condition) and resting potential also changed relatively little (with resting potential values of −81, −79 and −78 mV, respectively, for Control, IANION with Cl− and IANION with ). The more marked effect of IANION inclusion at less negative potentials during AP repolarization is concordant with the outwardly rectifying nature of the current. An additional observation made from the AP simulations is that incorporation of IANION also influenced the profile of L-type calcium current (ICa,L) during the AP plateau: the initial rapid component of ICa,L was unaffected by IANION incorporation, but the sustained component during the AP plateau showed a modest reduction. Thus, both an increase in repolarizing current carried by IANION and the consequent decrease in the sustained component of ICa,L combined to lead to AP plateau depression and abbreviation of APD50. The results shown in Fig. 3 demonstrate clearly that IANION is able to influence human atrial AP repolarization. Further work is now warranted to determine both the extent to which the incorporation/omission of IANION influences the susceptibility of human atrial cells and tissue to arrhythmia and to pursue the underlying identity and regulation of this novel background conductance.

anion
anion
background current
atrial myocyte
heart
patch-clamp
action potential
cardiac
atrium
anionic
computer modelling


Acute neuropsychological effects of MDMA and ethanol (co-)administration in healthy volunteers

Rationale In Western societies, a considerable percentage of young people expose themselves to 3,4-methylenedioxymethamphetamine (MDMA or “ecstasy”). Commonly, ecstasy is used in combination with other substances, in particular alcohol (ethanol). MDMA induces both arousing as well as hallucinogenic effects, whereas ethanol is a general central nervous system depressant.
Introduction
In Western societies, a considerable proportion of young people expose themselves to 3,4-methylenedioxymethamphetamine (MDMA or ‘ecstasy’; Gross 2002; Parrott 2001; Tancer and Johanson 2007). Ecstasy has gained widespread use in the ‘club’ scene, typically all-night parties with loud music and intense lights (Winstock et al. 2001). The average dose of ecstasy used recreationally is reported to be around 80–90 mg of MDMA with considerable individual variation (Tanner-Smith 2006). Ecstasy users are generally multidrug users who have experience with various recreational drugs and use these in combination with ecstasy (Gouzoulis-Mayfrank and Daumann 2006b). Probably due to its availability, alcohol remains one of the most co-used substances (Barrett et al. 2005). As the use of alcohol is known to induce impairment of cognitive function and decrease the awareness of this impairment, this can lead to dangerous behaviour like driving under influence (Lamers and Ramaekers 2001; Riley et al. 2001).
MDMA acts primarily by releasing serotonin (5-hydroxytryptamine (HT)) from pre-synaptic 5-HT terminals. It reverses the direction of the reuptake transporter and increases 5-HT levels at the post-synaptic receptors (Liechti and Vollenweider 2000; Mlinar and Corradetti 2003; Pifl et al. 1995). MDMA is also a potent releaser of dopamine and (nor)adrenaline (Colado et al. 2004; Liechti and Vollenweider 2001).
MDMA is rapidly absorbed following oral administration. Within 30 min, MDMA is detectable in the blood. Plasma levels peak at 1–2 h after drug administration, and maximum behavioural and subjective effects occur around 1–2 h and have declined by 4 h in spite of persisting plasma levels (de la Torre et al. 2004; Green et al. 2003). Increasing the dose does not result in a proportional rise in plasma concentrations, which is indicative of non-linear pharmacokinetics (de la Torre et al. 2000).
The behavioural effects of MDMA resemble but are not restricted to effects of psychostimulants (e.g. amphetamines or ‘speed’) as well as hallucinogenics (e.g. lysergic acid or ‘lysergic acid diethylamide’), although MDMA’s most characteristic effects are described as an increase in empathy and friendliness (Vollenweider et al. 2002). This led to MDMA being categorized as an ‘entactogen,’ as coined by Nichols and Oberlender (1990).
Most research into the cognitive effects of MDMA in humans has focused on the long-term effects, where only memory was consistently found to be impaired (Verbaten 2003; Verkes et al. 2001). Our review of the acute effects of MDMA in humans showed that cognitive effects were assessed only in a limited number of studies, using diverse tests and generally addressing only certain aspects of neuropsychological function. As such, no consensus on MDMA’s cognitive effects could be reached (Dumont and Verkes 2006). Since then, reports on the effects of MDMA generally confirmed previous findings (Kuypers et al. 2006; Kuypers et al. 2007; Ramaekers et al. 2006; Tancer and Johanson 2007). Interestingly, two studies reported effects of MDMA on memory, which had not been assessed previously. These reports showed acute impairment of immediate and delayed recall of words as well as spatial memory by MDMA (Kuypers and Ramaekers 2005, 2007).
Drinks containing ethanol, commonly referred to as alcohol, are widely available and regularly used in Western society. Ethanol is chiefly a central nervous system (CNS) depressant. It inhibits both excitatory and inhibitory post-synaptic potentials by potentiating the action of gamma-aminobutyric acid at its receptor (Suzdak et al. 1988). Reports of the cognitive effects of combined use of MDMA and ethanol in humans have been sparse in the literature. Studies that were performed assessed psychomotor function, attentional performance and subjective effects (Hernandez-Lopez et al. 2002; Kuypers et al. 2006; Ramaekers et al. 2006). In general, MDMA and ethanol had no or opposite effects on effect measures, and as such co-administration did not exacerbate single-drug effects.
In the current study, we employed a series of tests sensitive to changes in all common neuropsychological domains induced by several pharmacological compounds, including amphetamines (Wezenberg et al. 2004).
It is generally acknowledged that the combined use of alcohol with other CNS-depressant drugs may enhance the effects of ethanol or of the other drugs. MDMA, however, has stimulant effects while ethanol is a sedative agent, suggesting that the effects of co-administration are diminished rather than augmented compared to the effects following single administration. This hypothesis was investigated during acute co-administration of MDMA and ethanol in healthy volunteers.
Materials and methods
Study design
This study utilised a four-way, double-blind, randomised, crossover, placebo-controlled design. Sixteen volunteers were randomly assigned to one of four treatment sequences. Each volunteer received a capsule containing either 100-mg MDMA or placebo and an ethanol–placebo infusion (target blood alcohol concentration (BAC) of 0.6‰) with a washout of 7 days between each treatment.
Study outline
Subjects arrived in the morning and were admitted to the study after a negative urine drug screen (opiates, cocaine, benzodiazepines, amphetamines, methamphetamines and delta-9-tetrahydrocannabinol), as well as a negative alcohol breath test and recording of signs and symptoms of possible health problems. A light breakfast was offered. Drug administration was scheduled at 1030 hours and the alcohol infusion was started at 1100 hours for a duration of 3 h. At 1130 hours, subjects performed the psychological test battery as described below. Specific test times are reported in Table 1. Subjects received lunch at 1400 hours and were sent home at 1700 hours after a medical check. Adverse events where recorded throughout the study day. Vital signs were monitored using a Datascope® Accutorr Plus™ cardiovascular monitor and Braun® type 6021 ThermoScan during the study day. The data presented in this report are a subset of a larger data set, which will be reported elsewhere.
Table 1TimelineNeuropsychological testsDescriptionTime (h:m)Drug administration0:0018-word list immediate recallImmediate recall of 18-word list1:00SDSTTranslate symbols to digits with key present in 90 s1:05SDRTTranslate symbols to digits from memory1:08Pursuit taskKeep dot within moving circle1:10Tangles taskTangled line leads to which target?1:13Switch taskFollow, possibly conflicting, instructions (choice between left or right)1:1718-word list delayed recallDelayed recall of 18-word list1:2218-word list delayed recognitionRecognise words of 18-word list memorised earlier among 18 distracters1:23Point taskKeep pen steady in air, measures tremor1:25Visual analogue scales16 100-mm scales for subjective experiences1:30Times are relative to drug administration.
Subjects
Sixteen healthy volunteers (nine male, seven female), regular users of ecstasy and alcohol, aged 18–29 years and within 80–130% of their ideal bodyweight, were recruited through advertisement on the internet and at local drug testing services. They were all in good physical and mental health as determined by assessment of medical history, a medical, electrocardiogramme and clinical, haematological and chemical blood examination. Previous drug use was assessed using a structured interview. Fifteen volunteers were right handed and one was left handed. The study was approved by the local Medical Ethics Committee. All subjects gave their written informed consent before participating in the study and were compensated for their participation. Subject demographics and drug history are reported in Table 2.
Table 2Volunteer demographics–drug history MeanSDMinMaxAge (years)22.12.918.029.0Education (years)16.51.61218Height (cm)174.712.3147.0189.1Weight (kg)67.512.445.788.4Opiates0.10.301LSD2.56.6025Amphetamines37.381.10250Ecstasy94.6138.414431Cannabis1,174.31,665.5205,840Cocaine33.7105.70400Alcohol2,367.91,981.6505,200Solvents3.613.3050Barbiturates0000Benzodiazepines18.657.30216Psilocybin6.910.4030Drug quantities mentioned are lifetime drug exposures, not further specified.
One subject had a mild adverse reaction (local vascular reaction) to the alcohol infusion and one subject did not refrain from drug use; both (one male, one female) were excluded from further participation and results obtained were not included in the data analysis.
Drugs and dosages
MDMA (or matched placebo) was given as a capsule in a single dose of 100 mg via oral administration (dose range; 1.1–2.2 mg/kg). MDMA was obtained from Lipomed AG, Arlesheim, Switzerland and encapsuled according to Good Manufacturing Practise by the Department of Clinical Pharmacy, UMC St Radboud, Nijmegen, the Netherlands. MDMA 100 mg orally is a relevant dose in the range of normal single recreational dosages. Previous experiments in humans used doses up to 150 mg without serious adverse events. Ethanol (or matched placebo) was administered continuously by intravenous infusion of 10% ethanol in glucose solution resulting in an ethanol blood concentration of 0.6‰ with a duration of 3 h as described below.
Alcohol clamping
To standardise alcohol delivery and maintain a constant alcohol blood concentration over time, an intravenous ethanol clamp was used. Ethanol was administered by infusion of a 10% ethanol in glucose solution for a duration of 3 h. The infusion rate was calculated using frequent breath alcohol concentrations measurements, according to a previously designed algorithm (Amatsaleh et al. 2006a). Breath alcohol concentration was assessed using a HONAC AlcoSensor IV® Intoximetre.
An intravenous administration route was chosen, ensuring standardisation of the rate and bioequivalence of ethanol administration. This is an important pre-requisite for predictable pharmacokinetics of ethanol. The process was semi-automated using a computer spreadsheet programme, which uses measured breath alcohol concentrations to calculate the infusion rate needed to maintain the ethanol level at 0.6 mg/mL. This is a relevant dose equivalent to peak levels of approximately two to three units of alcoholic beverages. In many European countries driving is prohibited at BAC above 0.5‰. This limit has been confirmed by a report that shows that at an average BAC of 0.6‰ psychomotor performance is significantly impaired (Amatsaleh et al. 2006b). A BAC of 0.6‰ is equivalent to approximately two to three alcoholic beverages commonly used in social settings in Western society, which is considered to be a safe and relatively moderate dose, despite its significant CNS effects.
MDMA blood analysis
For the assessment of serum levels of MDMA, blood samples were collected 90 min after drug administration from each subject on each study day. Venous blood samples (10 ml) were collected into heparinised tubes, centrifuged immediately at 4°C for 15 min. Plasma was split into aliquots of 2 mL (to prevent over-freezing–thawing), frozen rapidly using liquid nitrogen and stored at −80°C. Samples were analysed for MDMA and MDA concentration by the Toxicology unit of the Leyenburg hospital, The Hague, the Netherlands.
Neuropsychological tests, apparatus and procedure
The performance on all neuropsychological tests was recorded by means of a digitising tablet (WACOM UD-1218-RE), a laptop computer, a pressure-sensitive pen (which could also be used as a cursor) and test forms. The x and y coordinates of the pen tip on and up to 5 mm above the digitiser were sampled with a frequency of 200 Hz and a spatial accuracy of 0.2 mm. The time schedule of the tests is summarised in Table 1.
To familiarise the subjects with the tests and procedures, they were invited to the hospital to perform a practise session within 1 week before the actual study days. All tests had five equivalent versions for four test days and one practise day, test versions were counterbalanced over test days.
Executive function
Switch task This test is a reaction time task measuring simple as well as complex reaction time, assessing executive performance (Baker and Letz 1986). After a random period of 0.75 to 1.75 s, two rectangular fields appeared on both sides of a circle in the centre of the screen. Only one of the two fields provided the subjects with information, either a colour, an arrow or both. The other non-informative field always had a neutral grey colour. Five conditions were subsequently presented to subjects. If only green fields appeared, subjects had to move as fast as possible into the green field. If green and red fields appeared, subjects had to move into the green field and away from the red field as soon as they appeared.
If green fields with a left or right arrow were presented, subjects were to move into the direction of the arrow. Green and red fields with a left or right arrow indicated that subjects were to follow the direction of the arrows in the green field, but the opposite direction of the arrows in the red field. Finally, the first condition was repeated. All conditions contained 20 trials except condition four in which there were 40 trials (total = 120 trials). The outcome measures were the mean reaction times per condition. The last condition is a repetition of the first to check for possible changes in attention.
Memory
Eighteen-word list A verbal memory test based on the classic Auditory Verbal Learning Test (Vakil and Blachstein 1993) was used. A variant was made consisting of a list of 18 words. The classic test uses 15 words. A longer wordlist was chosen, however, to prevent ceiling effects. The list was presented verbally three times. Under normal circumstances, subjects are supposed to remember an increasing number of words after each trial. Directly after each presentation, and after an interval of 20 min, subjects were asked to recall as many words as possible. After the delayed recall trial, a list of 36 words was presented from which they were asked to recognise the 18 words previously presented. The incorrect words were distracters and resembled the correct words in a semantic or phonologic manner. Responses were either correct positive (when a word that was recognised was indeed part of the list presented during immediate recall) or false positive (when a word was recognised but was not part of the list presented during immediate recall, e.g. the word was a distracter). The outcome measure was the number of correctly recalled or recognised words for the average of the three immediate recall trials, the delayed recall trial and the delayed recognition trial.
Symbol digit recall test The symbol digit recall test (SDRT) followed directly after the Symbol Digit Substitution test (SDST), which is discussed in the last paragraph of this section. After subjects had finished the SDST, they were shown the symbols of the SDST without the translation key, one at a time, and asked to produce the corresponding numbers. This test is based on an extended procedure of the SDST to measure incidental learning (Kaplan et al. 1991). The outcome measure was the number of correctly translated symbols.
Psychomotor function
Pursuit task To measure implicit procedural learning, a computerised version of the rotor pursuit task was used. This test is based on the classic rotary pursuit task (Ammons 1951). It is a continuous motor task. Subjects had to follow the movement of a large target stimulus on the computer screen with a cursor by moving the pen over the XY tablet. The speed of the target gradually increased when the cursor was contained within the target but decreased considerably when it was not. The target followed a spatially predictable circular path over the screen. The outcome measure for this test was the total number of rotations within 2 min.
Point task The point task, a measure for tremor, required subjects to try to keep the cursor inside a very small circle for 1 min, while avoiding contact between the pen and the test form. The outcome measure for this test was the deviation from the target.
Visuospatial and visuomotor function
Tangle task The tangle task required the subject to visually track a particular line winding through two to four other lines. On subsequent trials, the tangles increased in complexity; they got longer and made more 90° turns. The paper form had a start area and five target areas, numbered 1 to 5, which reflect the maximum target areas on the screen, starting with only three target areas.
This test is modelled after the visualisation test from the ‘kit for factor referenced cognitive tests.’ It was selected by the US NAVY to study environmental and other time-course effects and has good task stability and reliability (Bittner et al. 1986). The outcome measures are the reaction time per trial and the number of correct trials in 2 min.
Attention
Symbol Digit Substitution test This test is a version of the subtest from the Wechsler Adult Intelligence Scale (Wechsler 1981). Subjects had to substitute the nine symbols for the digits 1–9 on the basis of a given translation key. The outcome measure was the total number of digits completed in 90 s.
According to Hege et al. (1997) this task measures many cognitive components, e.g. visuospatial scanning, intermediate memory, perceptual motor speed and speed of cognitive processing. Therefore, subsequent analyses were performed in order to attempt and disentangle these cognitive processes. Based on pen pressure, movement trajectories were defined as either pen-up periods or pen-down periods. This allowed for subsequent analysis of matching times and movement (writing) times in the Symbol Digit Substitution test. For the motor component, the mean writing times were computed. For the more cognitive component, the mean matching times were computed. These analyses have been previously performed (Sabbe et al. 1999; Wezenberg et al. 2005).
Subjective
Subjective effects were recorded using the Bond and Lader (Visual Analogue) Mood Rating Scale (BLMRS). This inventory was completed at the end of each neuropsychological test battery on each study day.
The BLMRS scale consisted of 16 lines, each 10 cm in length, with opposite terms at each end of the line (alert–drowsy, calm–excited, strong–feeble, muzzy–clear-headed, well coordinated–clumsy, lethargic–energetic, contented–discontented, troubled–tranquil, mentally slow–quick witted, tense–relaxed, attentive–dreamy, incompetent–proficient, happy–sad, antagonistic–amicable, interested–bored, withdrawn–gregarious). Subjects were asked to indicate which item was more appropriate by marking the line. The outcome measure was the distance to the marker on each scale. These scale scores were then aggregated to scores for ‘calmness,’ ‘alertness’ and ‘contentedness’ as described by Bond et al. (1974).
Statistical analyses
Statistical evaluation (using SPSS 11.5 for Windows) was performed with general linear model repeated-measures analysis of variance. Main and interaction effects were tested using a two-factor (‘ethanol’ and ‘MDMA’), two-level (absent versus present) multivariate model.
The analysis of the data was based on Maxwell and Delaney (2004) and Kirk (1995). First the presence of interaction (non-additivity) was tested with alfa = 0.05. When the interaction was not statistically significant we proceeded by testing the main effects, each at alfa = 0.05. In the case of a significant interaction, we proceeded by testing simple main effects of each drug, i.e. MDMA vs. placebo and ethanol vs. placebo.
Results
Subject demographics are summarised in Table 2. Out of 16 subjects, 14 completed the study procedure. One subject had a mild adverse reaction (local vascular reaction which subsided with infusion stop) to the alcohol infusion and one subject did not refrain from drug use; both were discontinued from study participance and data already obtained were not included in statistical analysis. Only significant results are mentioned in this section, unless stated otherwise.
MDMA blood concentration 90 min after administration did not differ for MDMA single vs. MDMA and ethanol co-administration and was on average 196 μg/L (SD = 83 μg/L). Blood alcohol concentration was maintained at an average of 0.54‰ (SD = 0.07‰).
Executive function
Executive function (switch task) did not show any significant main or interaction effects.
Memory function
Memory function was assessed by the 18-word list (outcome measures were ‘immediate recall,’ ‘delayed recall’ and ‘recognition,’ see Fig. 1) as well as the SDRT. Immediate recall was impaired only by ethanol (F(1, 12) = 8.71, p = 0.011).
Fig. 1Memory effects (18-word list), Immediate: immediate recall, average score of three trials of correctly recalled verbally presented words, Delayed: correctly recalled verbally presented words 20 min after presentation, Recognition: correctly recognised verbally presented words containing 18 distracters, 20 min after presentation (mean and SEM). Immediate recall was impaired only by ethanol (F(1, 12) = 8.71, p = 0.011). Delayed recall was impaired by MDMA (F(1, 12) = 10.447, p = 0.007) as well as by ethanol (F(1, 12) = 16.031, p = 0.002); recognition was not affected by any drug condition
Delayed recall as assessed by the 18-word list was impaired by MDMA (F(1, 12) = 10.447, p = 0.007) as well as by ethanol (F(1, 12) = 16.031, p = 0.002). The SDRT, also a test for delayed recall, showed a similar pattern of impairment by MDMA (F(1, 12) = 5.300, p = 0.038) as well as by ethanol (F(1, 12) = 7.654, p = 0.016).
Psychomotor function
Psychomotor function was assessed with tests for tremor (point task), accuracy (pursuit task) and speed (SDST motor time, see Fig. 2); other SDST results are reported in the section “Attention.” Ethanol impaired psychomotor speed as reflected in the increase in SDST motor time (F(1, 12) = 9.295, p = 0.009).
Fig. 2Psychomotor effects: SDST writing time (mean, SEM). Ethanol increased writing times (F(1, 12) = 9.295, p = 0.009)
Visuospatial and visuomotor function
Visuospatial and visuomotor function were measured with the tangle task, subdivided into ‘total number correctly solved’ and ‘reaction time,’ and did not show any significant effects, although a trend of impairment by MDMA (F(1, 12) = 3.966, p = 0.068) was observed.
Attention
Attention was assessed with the SDST task; the outcome measures were ‘motor time’ (see “Psychomotor function”), ‘matching time’ (Fig. 3) and ‘total number correctly substituted.’ The time required to match symbols to the corresponding numbers showed a significant MDMA and ethanol interaction (F(1, 12) = 6.214, p = 0.027). Tests for simple main effects revealed that both single-drug conditions reduced attention compared to placebo (ethanol F(1, 13) = 6.248, p = 0.027; MDMA F(1, 13) = 6.822, p = 0.022; see Fig. 3).
Fig. 3Attention effects: SDST matching time, i.e. time needed for translation (mean and SEM). A significant MDMA by ethanol interaction was found (F(1, 12) = 6.214, p = 0.027)
Subjective effects
Subjective effects are depicted in Fig. 4. Feelings of ‘contentedness’ where increased significantly by MDMA only (F(1, 12) = 4.710, p = 0.049).
Fig. 4Subjective effects (aggregated Bond and Lader scores, mean and SEM). Feelings of ‘Contentedness’ where increased significantly by MDMA only (F(1, 12) = 4.710, p = 0.049) A significant interaction effect (F(1, 12) = 7.358, p = 0.018) was found for feelings of ‘Alertness.’ Feelings of ‘Calmness’ were reduced only by MDMA (F(1, 12) = 20.259, p = 0.001)
A significant interaction effect (F(1, 12) = 7.358, p = 0.018) was found for feelings of ‘alertness.’ Tests for simple main effects revealed that ethanol but not MDMA significantly decreased feelings of alertness compared to placebo (F(1, 13) = 50.613, p < 0.001). Feelings of ‘calmness’ were reduced only by MDMA (F(1, 12) = 20.259, p = 0.001).
Discussion
This study demonstrates that the effects of 100-mg MDMA, commonly known as ecstasy, on cognitive function are no greater than the effects of a relatively low dose of ethanol. This is remarkable as these results suggest that the effects of 100-mg MDMA are comparable to the peak effects of two to three alcoholic beverages. Co-administration of these compounds did not result in any significant cognitive impairments beyond those observed after administration of only ethanol. The use of moderate amounts of alcohol is common in Western societies and, although impairing cognitive function, socially accepted, while ecstasy use remains very controversial. Of course, our findings only relate to the acute neuropsychological implications of ecstasy use and not to the physiological and long-term effects, which rightfully remain topics of discussion (Gouzoulis-Mayfrank and Daumann 2006a; Nutt 2006; Parrott 2007).
Drug effects observed in this placebo-controlled crossover study were moderate. Co-administration was well tolerated as indicated by the subjective scores, which were comparable to those found after single administration of MDMA. An interaction of MDMA and ethanol was found for subjective alertness scores. Ethanol, as expected, reduced subjective alertness, while MDMA co-administration reversed the reduction of subjective alertness by ethanol. In the present study, MDMA by itself did not significantly affect subjective alertness, although this effect has been consistently reported in other studies and is a well-known effect of amphetamines. However, MDMA did significantly reduce subjective calmness, i.e. subjects felt more excited after MDMA use. Probably, the Bond and Lader Mood Rating Scale is not well suited for the assessment of subjective effects of psychoactive drug effects and future studies should employ more appropriate subjective drug effect measures such as the Profile Of Mood States (de Wit et al. 2002).
When considering the results for each neuropsychological domain, executive function was not affected by any drug condition. A previous study showed impairment of executive function by ethanol but not MDMA, although ethanol impaired performance in only one out of three tests of executive function (Lamers et al. 2003). The BAC in this study was 0.3‰ at the time of testing compared to 0.56‰ in our current study, suggesting a lack of sensitivity of the test employed in the current study.
The above-mentioned previous study also reported visuospatial and visuomotor impairment by MDMA but not by ethanol. Although not significant, our current results show a similar pattern where MDMA showed a trend of impairment of visuospatial and visuomotor function, whereas ethanol did not.
Psychomotor function was impaired only after ethanol administration (SDST motor time, see Fig. 2). The majority of studies addressed in our review of acute effects of MDMA in humans (Dumont and Verkes 2006) did not report any change in psychomotor function after MDMA either. However, increased psychomotor function after MDMA has also been found (Lamers et al. 2003; Ramaekers et al. 2006). These studies administered 75 mg instead of 100 mg. Possibly, the effects of MDMA are biphasic, with a low dose of MDMA exhibiting more amphetamine-like effects, e.g. arousal, increasing performance, whereas higher doses may elicit more hallucinogenic effects and impair performance (Liechti et al. 2001; Solowij et al. 1992).
As mentioned above, MDMA co-administration reversed the ethanol-induced feelings of sedation, although MDMA was unable to reverse the psychomotor impairment induced by ethanol. This dissociation between subjective and objective sedation confirms previous findings by Hernandez-Lopez et al. (2002).
Several studies assessed MDMA’s effect on attention using the Digit Symbol Substitution Task (DSST), although no significant effects were found (Cami et al. 2000; Farre et al. 2004; Kuypers and Ramaekers 2005). One study reported decreased DSST performance after ethanol as well as after ethanol and MDMA co-administration but no effect of MDMA (Hernandez-Lopez et al. 2002). Our findings confirm these findings to a large extent. We found no main effects of MDMA or ethanol on attention, although an interaction of ethanol and MDMA for ‘matching time’ (time required to match the number to the corresponding symbol) was found. Co-administration of MDMA and ethanol increased ‘matching time’ comparable to the increase observed after both MDMA and ethanol single administration, compliant with our hypothesis of competitive mechanisms of action of both drugs (see Fig. 3).
Studies investigating the long-term effects of MDMA consistently found memory to be affected (Verbaten 2003). In the present report, almost all memory measures showed quantitatively comparable impairment for each drug condition (see Fig. 1), although the effect of MDMA on immediate recall did not reach statistical significance. Only delayed recognition was not impaired in any drug condition. These findings suggest a deficit in the retrieval of verbal information encoded in memory, rather than impairment in the storage of information. Our findings are similar to the results of a previous study on MDMA-induced effects on memory (Kuypers and Ramaekers 2005). In this previous study, no memory impairment was observed after methylphenidate administration, a pronounced dopamine and norepinephrine releaser, suggesting the involvement of serotonin in memory impairment. Several other studies also have shown serotonin-mediated modulation of memory function through interaction with the cholinergic neurotransmitter system, although the details of this complicated interaction remain elusive (Cassel and Jeltsch 1995; Garcia-Alloza et al. 2006; Meneses 2007). Generally, subjects stated that they were well aware of their impaired memory after MDMA.
BAC was on average 0.56‰. At this level, driving is prohibited by law in many European countries because of its interference with normal functioning. Although the effects were moderate, ethanol impaired cognitive performance in various tests. Similar moderate effects were observed with MDMA 100 mg, considered to be slightly above the average recreational dose (Tanner-Smith 2006). This might be considered surprising for a drug with reported robust subjective stimulating and hallucinogenic properties. However, because the effects caused by a single dose of 100-mg MDMA were comparable to the effects of a BAC of 0.56‰, this dose should by inference be considered unacceptable in motorised traffic.
Arguably, the moderate drug effects as found in this study could be explained by ‘missing’ the time of the maximal drug effects. Although the average MDMA blood concentration reported here (196 μg/L) is comparable to Cmax of 100-mg MDMA (199.8 μg/L) as reported by de la Torre et al. (2000), MDMA concentration was assessed at the end of the testing procedure. However, Hernandez-Lopez et al. (2002) found significant effects at 60 min as well as 90 min after drug administration, arguing against the suggestion of ‘missing’ peak drug effects.
The circumstances in which these substances are normally used cannot be fully recreated in the laboratory and this may have suppressed the effects of both substances. It is not unlikely that these substances show enhanced effects when tested under typical circumstances and surroundings. Recently, Parrott et al. (2006) concluded that the increase in physical activity and body temperature typically experienced when using MDMA enhance MDMA effects. Ball et al. (2006) demonstrated that a familiar surrounding increased MDMA-induced locomotor response as well as single-neuron activity in rats, compared to unfamiliar surroundings. Therefore, the psychosocial context in which MDMA is used, along with the different expectations and behaviour, probably influences its effects (Sumnall et al. 2006). It is unlikely, however, that this affects the quality of the interactions of MDMA and ethanol.
In conclusion, co-administration of MDMA and ethanol did not impair cognitive function significantly more than MDMA or ethanol administration alone. The most prominent effect of (co-)administration of MDMA and ethanol was an impairment of memory. Ethanol also impaired psychomotor function. Although the impairment of performance by each drug condition was relatively moderate, this significant impairment of cognitive function should be considered intolerable in motorised traffic and other cognitively demanding situations as confirmed by previous research and as defined by law. However, the effects of these drugs in the concentrations used in the present study on established neuropsychological tests appear to be smaller than one would assume based on their reputation.

acute
neuropsychologic
effects
mdma
ethanol
healthy volunteers
ecstasy
alcohol
interaction