Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors

Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P1 receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands.
Introduction
When chronically exposed to an agonist, G-protein coupled receptors (GPCRs) can undergo desensitisation, i.e. the response to a given level of agonist exposure declines, and/or more agonist is required to maintain the same response. Various mechanisms can be involved in desensitisation, some of which directly affect receptor responsiveness. One of these mechanisms involves phosphorylation of activated receptors by GPCR kinases (GRKs). This enables binding of arrestins to the receptor, leading to uncoupling from the G-protein and hence impaired signaling. Another, not necessarily mutually exclusive mechanism of desensitisation is internalisation of the receptor, which can be either homologous (because of activation of the receptor) or heterologous (because of activation of other receptors or to other stimuli). Receptor internalisation can be arrestin-dependent or independent (Bünemann and Hosey 1999) and involves translocation of the receptor from the cell membrane to the inside of the cell. There, it is entrapped inside clathrin-coated pits and can undergo either degradation or resensitisation.
Several approaches have been used in the past to detect and quantify GPCR internalisation. One classical method involves homogenisation of tissues or cells, differential centrifugation to yield a plasma membrane and a microsomal fraction followed by electrophoresis and autoradiographical analyses of the receptor of interest in both fractions (Simpson et al. 1984). Another classical method involves labeling of all receptors in intact cells with a lipophilic radioligand and competition by a hydrophilic ligand, which will replace the radioligand from sites at the cell surface but not from those entrapped intracellularly (Staehelin et al. 1983). More recently, genetic approaches have been used. An example is the use of a C-terminal green fluorescent protein (GFP; Barak et al. 1997; Kallal et al. 1998) to monitor receptor localisation after ligand stimulation sometimes combined with an algorithm to discriminate between internalised and membrane-bound receptors (Conway et al. 1999; Fukunaga et al. 2006; Schlag et al. 2004; Hirasawa et al. 2005). This latter method uses a cell-based fluorescence imaging system ArrayScanII, where the amount of internalised receptor/GFP spots are counted and normalised against the number of nuclei stained with Hoechst 33342. However, a GFP, or other fluorescent tag, is relatively large (200–300 amino acids) and can influence receptor function (McLean and Milligan 2000). Other methods use a combination of techniques, like quantitative flow cytometry analysis of the cell surface receptors and immunocytochemical confocal microscopy analysis to visualise the sub-cellular localisation of the receptor of interest (Hirasawa et al. 1998).
Detailed quantitative studies of internalisation are of interest as this process can be the mechanism of underlying physiological and pharmacological effects. For example, receptor internalisation is thought to underlie the modulation of immune function by sphingosine-1-phosphate (S1P; Matloubian et al. 2004). This also appears to be the mechanism of action of the immunosuppressive drug FTY720 (fingolimod; Chiba et al. 2006; LaMontagne et al. 2006), which is undergoing clinical trials for the treatment of multiple sclerosis and previously also for renal graft rejection (Budde et al. 2002; Kappos et al. 2006).
In our search to quantify internalisation of membrane receptors in a fast and easy way, we have setup a method, which uses an N-terminal HisG (6xHis+Gly)-tagged S1P1 receptor. Because the HisG-tag is N-terminal, it is extracellular. Therefore, when immunocytochemistry is performed without permeabilisation, only membrane-bound, tagged receptors will be detected. This way, internalisation is quantified as a difference in membrane-fluorescence between unstimulated and agonist-stimulated cells.
The use of small, N-terminal tags has been described before (Daunt et al. 1997; Vicentic et al. 2002; Wozniak and Limbird 1996; Von Zastrow et al. 1993; Miller 2004). Of these, Daunt et al. 1997 described a method where cells were plated onto 24 well tissue culture dishes. After stimulations with an agonist, incubations with first and second antibodies followed. Measurements were done after adding alkaline phosphatase substrate, resulting in a colorimetric reading. In this paper, we now describe the validation of a similar method by testing various antibody combinations and detection methods to quantify internalisation of N-terminal HisG-tagged receptors. This resulted in immunocytochemical method using a specific anti-HisG first antibody combined with a fluorescent second antibody (AlexaFluor® 488) as our preferred choice. The signal can either be visualised under the fluorescence microscope or measured in a fluorescence microplate reader. The latter results in quantitative measurement of the decrease in signal caused by receptor internalisation, in a direct, sensitive and fast way.
Materials and methods
Materials
pcDNA3.1 containing the entire coding region of the human S1P1 receptor was purchased from UMR cDNA Resource Center (Rolla, MO).
Cell culture media, hygromycine B, Lipofectamine™2000, pcDNA3.1/His C, pOG44, CHO-FlpIn cells, Alexa Fluor® 488 goat anti-mouse (IgG), anti-HisG (mouse monoclonal IgG2a), anti-HisG-horse radish peroxidase (HRP; mouse monoclonal IgG2a), pcDNA5/FRT/TO, NuPAGE® System, NuPAGE® Novex 4–12% Bis–Tris gel, MES running buffer, NuPAGE® transfer buffer and Invitrolon™ PVDF blotting membranes were obtained from Invitrogen (Breda, The Netherlands). BCA™ Protein Assay Kit was obtained from Pierce (Etten-Leur, The Netherlands). Formaldehyde solution was obtained from Sigma (Zwijndrecht, The Netherlands). Restriction enzymes (BamHI, XhoI, EcoRI and HindIII) were obtained from Fermentas Life Sciences (St. Leon-Rot, Germany). Black, clear bottom 96 well plates were obtained from Greiner Bio One (Alphen aan den Rijn, The Netherlands). S1P was obtained from Avanti-Polar Lipids (via Instruchemie B.V., Delfzijl, The Netherlands). Chamber slides were obtained from Nunc (Amsterdam, The Netherlands). BM Chemiluminescence Blotting Substrate [POD] and ABTS [2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] were obtained from Roche (Mannheim, Germany). Hyperfilm ECL was obtained from Amersham Biosciences (Diegem, Belgium). SEW2871 (5-(4-Phenyl-5-trifluoromethylthiophen-2-yl)-3-(3-trifluoromethylphenyl)-1,2,4-oxadiazole) was obtained from Calbiochem (via VWR, Amsterdam, The Netherlands). Goat anti-mouse-HRP was obtained from Jackson ImmunoResearch Europe (via Sanbio B.V., Uden, The Netherlands). RIPA buffer was obtained from Boston bio products (Worcester, UK). Halt protease inhibitor cocktail was obtained from Pierce (via Perbio Science Nederland B.V. Etten-Leur, The Netherlands).
Molecular cloning and transfection
An N-terminal HisG-tag was added to the S1P1 receptor via cloning into pcDNA3.1/HisC using BamHI and XhoI. A second cloning step was done using HindIII and XhoI to clone the HisG-tagged S1P1 receptor into the expression vector pcDNA5/FRT/TO. Transfection of plasmids into CHO-FlpIn cells and cell culture was done as described previously (Jongsma et al. 2006).
Western blot
Cells were washed and scraped in PBS, spun down and lysed in RIPA buffer with protease inhibitor. Protein concentration was determined using the BCA kit according to the manufacturer’s protocol. Ten micrograms of protein were loaded onto a 4–12% NuPAGE® Bis–Tris Gel. Electrophoresis was carried out at 200 V for 40 min in NuPAGE® MES running buffer. Protein was transferred to Invitrolon™ PVDF blotting membranes at 30 V for 1 h. The membrane was blocked for 1 h at room temperature in 50 mg/ml non-fat dry milk in PBS followed by overnight incubation at 4°C with a dilution of 1:5,000 for mouse anti-HisG. After washing with PBS-Tween (0.1%), the membrane was incubated with a 1:20,000 dilution of goat anti-mouse-HRP IgG at room temperature. Detection was done using BM Chemiluminescence Blotting Substrate and Hyperfilm ECL.
Immunocytochemistry
Cells (40,000 cells/well) were plated on day 1 in 96 wells black, clear bottom plates or eight well chamber slides. The next day, the medium was changed to serum-free medium. After incubation overnight, cells were stimulated with the indicated ligand in serum-free medium for 30 min at 37°C, unless otherwise indicated. The stimulation solutions were removed, and cells were fixed by applying 4% v/v formaldehyde in PBS for 15 min at room temperature. After washing three times with PBS, cells were incubated with the first antibody for 1 h at room temperature. Cells were washed again three times with PBS and, if necessary, incubated with the second antibody for 1 h at room temperature. Cells were washed three times with PBS followed by measurements on a Victor2 (Wallac, Perkin Elmer). The following antibody combinations were used: mouse anti-HisG 1:200 with AlexaFluor® 488 goat anti-mouse 1:500, mouse anti-HisG-HRP 1:200 and mouse anti-HisG 1:200 with goat anti-mouse-HRP 1:500. In case an HRP-bound antibody was used, either ABTS solution (50 μg/ml) or BM Chemiluminescence Blotting Substrate was added to generate an absorbance or luminescence signal, respectively. For the absorbance signal, a 405-nm filter was used; for the fluorescent signal, a 490-nm excitation and a 535-nm emission filter were used.
Data analysis
Concentration response curves were analysed by fitting sigmoidal functions to the experimental data using Prism 4 (Graphpad Software, San Diego, CA). Data are expressed as means ± SEM. Differences between groups were compared using a one way analysis of variance with a Dunnett’s correction or a Student’s t test where appropriate. P < 0.05 was considered significant.
Results
Characterisation of the HisG-tagged S1P1 receptor
The presence of HisG-S1P1 receptor in stably transfected CHO-FlpIn cells was confirmed in Western blot analysis as a band of approximately 50 kDa (Fig. 1). The HisG-S1P1 receptor was also visualised in the cell membrane by fluorescence microscopy using AlexaFluor® 488 goat anti-mouse as a secondary antibody (Fig. 2). CHO-FlpIn cells stably expressing the untagged S1P1 receptor were used as a negative control for both detection methods (Figs. 1 and 2). Addition of the N-terminal HisG-tag to the S1P1 receptor did not influence signaling via this receptor as it did not significantly alter the potency of S1P to inhibit forskolin-stimulated cAMP accumulation (pEC50 S1P1 9.0 ± 0.1 vs HisG-S1P1 8.9 ± 0.1; n = 6), measured as described previously (Jongsma et al. 2006).
Fig. 1Western blot of CHO-FlpIn cells stably expressing the S1P1 receptor (lane 1) and HisG-S1P1 receptor (lane 2) using a mouse anti-HisG first antibody and a goat anti-mouse HRP second antibody. HisG-S1P1 is detected at approximately 50 kDa as indicated by the arrow. The band at 80 kDa is nonspecific. The blot shown is from a typical experiment out of fourFig. 2Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 receptor (a) and the S1P1 receptor (b) using a mouse anti-HisG first antibody and AlexaFluor® 488 goat anti-mouse second antibody. Images shown are from one typical experiment out of nine
Internalisation of the S1P1 receptor
A 30-min incubation with 1 μM of S1P or 10 μM of the selective S1P1 agonist SEW2871 (Sanna et al. 2004) at 37°C decreased fluorescence (Fig. 3a, c, e). No such decrease in fluorescence was seen when cells were incubated with either agonist at 4°C (Fig. 3b, d, f). S1P concentration dependently internalised the HisG-S1P1 receptor as visualised with a fluorescence microscope (Fig. 4). Such decrease in fluorescence was also measured using a fluorescence microplate reader. The fluorescent signal of cells stably expressing the HisG-S1P1 receptor was significantly higher than for cells stably expressing the untagged S1P1 receptor. Stimulation with S1P (1 μM) for 30 min significantly decreased this signal (Fig. 5).
Fig. 3Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 using a mouse anti-HisG first antibody and AlexaFluor® 488 goat anti-mouse second antibody. Cells were stimulated for 30 min with 1 μM S1P or 10 μM SEW2871 at either 37°C (left panel) or 4°C (right panel). Images shown are from one typical experiment out of fourFig. 4Fluorescence microscope pictures of CHO-FlpIn cells stably expressing the HisG-S1P1 receptor using a mouse anti-HisG first antibody and AlexaFluor® 488 goat anti-mouse second antibody. Cells were stimulated with S1P at the indicated concentrations at 37°C for 30 min. Images shown are from one typical experiment out of four experimentsFig. 5Quantitative measurements of cells stably expressing HisG-S1P1 using a mouse anti-HisG first antibody and AlexaFluor® 488 goat anti-mouse second antibody. Cells were stimulated at 37°C for 30 min at the indicated S1P concentrations. The first bar represents cells stably expressing the S1P1 receptor without a HisG-tag. The second bar represents cells stably expressing HisG-S1P1 incubated without the first antibody. Statistical significance was shown between ‘vehicle’ and ‘1 μM S1P’ as well as between ‘no HisG’ and ‘vehicle’. Data are shown as relative fluorescent units (RFU) ± S.E.M. (n = 3)
Choice of detection method
Instead of the antibody combination mouse anti-HisG and AlexaFluor® 488 goat anti-mouse (Fig. 6a), other combinations were tested to optimise the method. The use of a first antibody mouse anti-HisG-HRP (Fig. 6b) or a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP (Fig. 6c) combined with the BM Chemiluminescence Blotting Substrate (POD) resulted in a luminescence signal, which proved to be very sensitive. However, the signal was very unstable over time, which required the addition of the substrate by the microplate reader. The combination of a first antibody mouse anti-HisG-HRP (Fig. 6d) or a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP (Fig. 6e) combined with ABTS resulted in an absorbance signal. This signal however, had less sensitivity compared to the other two detection methods. The use of a first antibody mouse anti-HisG-HRP resulted in a loss of sensitivity compared to the combination of a first antibody mouse anti-HisG with a second antibody goat anti-mouse-HRP. When the combination of a first antibody mouse anti-HisG and a second antibody AlexaFluor® 488 goat anti-mouse was used, we measured a concentration-dependent internalisation of HisG-S1P1 after stimulation with either S1P or SEW2871 (pEC50 S1P = 7.4 ± 0.1 [n = 8] and pEC50 SEW2871 = 5.7 ± 0.2 [n = 3], respectively; Fig. 7).
Fig. 6Quantitative measurements of HisG-S1P1. Ligand stimulations are carried out at 37°C for 30 min at the indicated concentrations (n = 3–4). Antibody and detection combinations used are mouse anti-HisG first antibody and AlexaFluor® 488 goat anti-mouse second antibody (a); mouse anti-HisG-HRP first antibody + POD (b); mouse anti-HisG first antibody and goat anti-mouse-HRP second antibody + POD (c); mouse anti-HisG-HRP first antibody + ABTS (d); mouse anti-HisG first antibody and goat anti-mouse-HRP second antibody + ABTS (e). Data are shown as percentage of unstimulated ± S.E.M. An asterisk indicates significant difference compared to unstimulated cells; a pound sign indicates significant difference compared to S1P (10 nM) stimulated cellsFig. 7Effect of increasing concentrations of S1P (closed squares) or SEW2871 (open squares) on CHO-FlpIn cells stably expressing the HisG-S1P1 receptor, using a mouse anti-HisG first antibody and AlexaFluor® 488 goat anti-mouse second antibody. Stimulations were carried out at 37°C for 30 min. The data shown represent the mean ± S.E.M. of six independent experiments performed in triplicate
Discussion
Previously reported methods to study the internalisation of GPCRs were very labor intensive, required specific equipment, added bulky tags and/or were poorly quantitative. We have developed and validated a method that uses stably transfected cells expressing a receptor with a minor modification only, i.e. an N-terminal HisG-tag, which did not influence receptor signaling. Several antibody combinations and detection methods were tested. The luminescent approach was very sensitive but highly dependent on strict adherence to a specific time interval between substrate addition and actual signal measurement. The absorbent signal obtained using another detection substrate resulted in a loss of sensitivity. The antibody combination of a first HisG specific antibody and a second AlexaFluor® 488 fluorescent antibody proved the most convenient, resulting in a sensitive and stable signal. Compared to previously published methods using a similar approach (Daunt et al. 1997; Vicentic et al. 2002; Wozniak and Limbird 1996; Von Zastrow et al. 1993), this immunocytochemical detection of the N-terminal HisG-tag is suitable to assess a large number of samples, e.g. to construct detailed concentration response curves and/or compare multiple ligands. In this regard, we showed that S1P was approximately 50 times more potent than SEW2871 in internalising the HisG-S1P1 receptor. This is in good agreement with the previously reported potency difference between the two agonists for activating the receptor (Sanna et al. 2004). As comparable methods have been described for adrenergic receptors as mentioned above (Daunt et al. 1997; Vicentic et al. 2002; Wozniak and Limbird 1996; Von Zastrow et al. 1993), it is likely that the method we described here is also suitable for receptors other than our model receptor, S1P1.
While our technique does not allow following internalisation in real time, the ability to process a large number of samples compensates for that by studying multiple time points. The only limitation to our method is the inability to measure internalisation of endogenous receptors. However, this would be possible when specific receptor antibodies are available, which is not yet the case for our model receptor.
In conclusion, we have validated a method to quantitatively measure receptor internalisation in which we use an N-terminal HisG-tag combined with immunocytochemistry. It proves to be sensitive enough to discriminate between different receptor ligands in a fast and non-radioactive way.

internalisation
g-protein coupled receptor
hisg-tag
sphingosine-1-phosphate receptors
quantification
cellular trafficking


Intestinal malrotation (IM) and cardiovascular defects (CCVD) are both common congenital defects. We investigated the prevalence and types of CCVD in a 25-year IM population, and its association with post-IM-operative morbidity and mortality. Data on the type of CCVD, other congenital defects, syndromes, associations, post-IM-operative morbidity and mortality were retrospectively reviewed from the records of IM patients born between 1980 and 2005. Data were analyzed on (significant) differences between CCVD subgroups, and risk factors for both morbidity and mortality were calculated. Seventy-seven of 284 IM patients (27.1%) were diagnosed with a major or minor CCVD (37 and 40 patients, respectively). Syndromes and associations were more frequently diagnosed in patients with major than with a minor CCVD (67.6 vs. 40%, respectively). Post-IM-operative complications, although frequently observed (61%), did not differ between patients with major and minor CCVD. Physical CCVD signs before IM surgery increased post-IM-operative morbidity significantly (OR 4.0, 95% CI 1.4–11.0). Fifteen patients died (19.5%), seven due to cardiovascular cause. Mortality risk was increased by intestinal ischemia and post-IM-operative complications and by major CCVD after correction for age at weight at the time of IM operation. Congenital cardiovascular defects in children with intestinal malrotation are common, with high morbidity and mortality rates after IM operation. Elective IM surgery in young patients with CCVD should be performed in a centre with adequate paediatric cardiac care. Benefits of laparoscopic intervention need further study.
Introduction
Intestinal malrotation (IM) is refers to all abnormalities of intestinal position and attachment and includes the concept of atypical malrotation or malrotation variant [1]. It is often accompanied by a narrow base of the small bowel mesentery. The incidence of IM is 1:500–1:3,500 living births [2, 3]. Volvulus, a rotation of the gut along its mesenteric stalk occurs in 60–70% of neonates diagnosed with IM, the ultimate strangulation in about 15% [4]. Eighty percent of the patients with symptomatic IM present in their first month of life [5] and require surgery. Delay in diagnosis may lead to intestinal ischemia, intestinal necrosis, septicaemia and short bowel syndrome. The surgical outcome is negatively influenced by the presence of intestinal necrosis [6], and the need for operation during the first 3 months of life [6–9]. To keep mortality and morbidity low, all asymptomatic children with intestinal malrotation are advised to undergo semi-elective surgery (Ladd’s procedure) in childhood and adolescence [10, 11].
Intestinal malrotation has been described in almost 100 syndromes and combinations. Associated serious abnormalities increase the mortality risk in patients with IM of about 22-fold according to Messineo [6]. Commonly associated congenital malformations are duodenal atresia, abdominal wall defects or diaphragmatic hernia [3, 12]. It is more frequently observed in patients with situs inversus [13] or heterotaxia, a syndrome associated by congenital cardiovascular defects [14].
Congenital cardiovascular defects (CCVD) are among the most common congenital birth defects occurring in about 1 in 100 living births [15]. Congenital malformations of the gastrointestinal, musculoskeletal and urinary systems are the most frequently seen extra cardiac malformations in patients with CCVD [16]. Intestinal malrotation has been observed in 2.8–4.1% of all the patients with CCVD, mainly in patients with single ventricle [17]. However, the incidence of CCVD in IM patients has not been precisely quantified in the recent literature and the full array of the associated cardiac anomalies has not been well described.
The purpose of our study was (1) to delineate and accurately describe the types and incidence of associated cardiac lesions in our most recent 25-year experience of patients with IM, and (2) to study the influence of a co-existing CCVDs on IM surgery morbidity and mortality.
Materials and methods
Population
A retrospective review at a single university centre was conducted on all the paediatric patients diagnosed with IM born and      between September 1980 and September 2005. Patients with CCVD were identified for further analysis. Patients demographics recorded included gestational age, weight and gender. Data on the type of CCVD, other congenital defects, known syndromes or associations, post-operative complications, morbidity and mortality were collected. The diagnosis of CCVD was based on clinical, ECG, echocardiography, heart catheterization and peri-operative findings. The IM diagnosis was based on radiological upper gastro-intestinal examination, or during acute surgery.
Data analysis
Collected data were analyzed by calculating prevalence percentages. Analysis of nominal variables was performed  using cross tables for calculating relative risk (RR) with the corresponding 95% confidence interval (95% CI). Fisher extact test was used for comparison of percentages of both CCVD groups, expressed as P values. For the comparison of continuous variables, Mann–Whitney U test was used to calculate P value. The P values are two-sided, and a value of P = 0.05 was considered statistical significant. For the calculation of risk factors for both post-operative morbidity and mortality, (adjusted) odds ratios (OR) with consequent 95% CI were calculated by uni- and multi-variate regression analysis. Statistical analysis was performed using SPSS software (version 12.0.1; SPSS Inc., Chicago, IL, USA).
Results
Demographics
During a period of 25 years, 284 patients were diagnosed in our centre with IM. Ninety-three of the 284 patients were identified with CCVD as well. Fifteen patients had persistent pulmonary hypertension (PPHT), all but one with congenital diaphragmatic hernia. One patient had a solitary pre-duodenal portal vein. The records of these 16 patients without structural CCVD were excluded, leaving 77 patients with both IM and CCVD (27.1% of the total IM population in the last 25 years) for further analysis. The gestational age range was 26–42 weeks with a median of 39 weeks. Median birth weight was 2.86 kg (range 0.72–4.53). A female predominance was seen with 47 girls (61%) and 30 boys (39%). Median follow-up period after IM diagnosis was 2.8 years (range 0–24 years). The overall survival at the end of follow-up was 80.5% (15 out of 77 patients died).
Congenital cardiovascular defects
The CCVDs were divided into the subgroups, major and minor CCVDs (see Table 1), varying from small ventricular septal defect (VSD) to common arterial trunk (CAT). Thirty-seven out of 77 patients (48.1%) were diagnosed with major CCVD and 40 patients (51.9%) with minor CCVD.
Table 1Types of major and minor congenital cardiovascular defectsMajor CCVDn = 37 (100%)Minor CCVDn = 40 (100%)Complex cardiovascular defectsn = 18 (48.6%)Ventricular septal defectn = 16 (40.0%)Tetralogy of Fallotn = 7 (18.9%)Atrium septal defectn = 7 (17.5%)Hypoplastic left heart syndromen = 3 (8.1%)Peripheral pulmonary stenosisn = 4 (10.0%)Atrioventricular septal defectn = 3 (8.1%)Persistent ductus arteriosusn = 3 (7.5%)Pulmonary valve atresian = 1 (2.7%)Pulmonary valve stenosisn = 3 (7.5%)Transposition of the great arteriesn = 1 (2.7%)Aortic valve stenosisn = 2 (5.0%)Tricuspid valve atresian = 1 (2.7%)Aortic coarctationn = 1 (2.5%)Common arterial trunkn = 1 (2.7%)Hypertrophic cardiomyopathyn = 1 (2.5%)Aortic arch hypoplasian = 1 (2.7%)Subvalvular aortic valve stenosisn = 1 (2.5%)Dextrocardian = 1 (2.7%)Left superior caval veinn = 1 (2.5%)Arteria lusorian = 1 (2.5%)CCVD Congenital cardiovascular defects
The characteristics of the whole group (n = 77) as well as that of the subgroups are described in Table 2. Forty-two patients underwent cardiac surgery, of which 20 patients had two or more cardiac surgical interventions. Patients with major CCVDs needed cardiac surgery more often. Minor CCVDs that needed surgical intervention most frequent were VSD, atrial septal defects (ASD) and aortic valve stenosis (AS). Six patients with severe CCVD did not undergo correction of their heart disease before IM operation: three patients died (n = 2 due to post IM surgery complications, n = 1 because of other congenital malformations). One patient with dextrocardia did not need cardiac correction. The two patients with Tetralogy of Fallot (TOF) underwent cardiac correction 6 months after IM surgery.
Table 2Characteristics of studied population and its subgroups of congenital cardiovascular defectsCharacteristicAll CCVDMajor CCVDMinor CCVDRelative risk (95% CI)/P value100% (n = 77)100% (n = 37)100% (n = 40)Boys39% (n = 30)35.1% (n = 13)42.5% (n = 17)0.85 (0.5–1.4)Syndromes or associations53.2% (n = 41)67.6% (n = 25)40% (n = 16)1.8 (1.1–3.1)Other congenital defects84.4% (n = 65)83.8% (n = 31)85% (n = 34)0.95 (0.5–1.8)Cardiac surgery54.4% (n = 42)83.8% (n = 31)27.5% (n = 11)4.3 (2.0–9.1)CCVD diagnosed before IM 57.1% (n = 44)78.4% (n = 29)37.5% (n = 15)2.7 (1.4–5.2)Age at CCVD diagnosis in days [median (range)]4.5 (0–210)4 (0–98)7 (0–210)P = 0.12Birth weight in kilograms [median (range)]2.86 (0.72–4.53)2.96 (0.96–4.53)2.84 (0.72–4.30)P = 0.4Age at first CCVD surgery in days [median (range)]102 (2–3,759)43 (2–3,759)117 (24–1,705)P = 0.15Values in bold represent statistical significant resultsCCVD Congenital cardiovascular defects, IM Intestinal malrotation
Congenital malformations, syndromes and associations
Congenital malformations other than IM and CCVD were observed in 65 of the 77 (84.4%) patients, see also Table 2. Most frequently observed congenital malformations included omphalocele (n = 7), congenital diaphragmatic hernia (n = 7), ano-rectal malformations (n = 6) and heterotaxia (n = 6).
Syndromes and associations were recognized in 41 (53.2%) patients, of which Down syndrome (n = 7), heterotaxia (n = 6), VACTERL (n = 5), Ivemark (n = 5) and lateralization abnormalities (n = 4) were most frequently recognized. Kartagener syndrome occurred in two patients. Several other syndromes all occurred once. Syndromes and associations were more frequent in patients with major CCVD.
Intestinal malrotation surgery
Characteristics of IM-operated patients and their major and minor CCVD subgroups are described in Table 3. Five patients did not undergo IM surgery yet; in three patients the surgery was about to take place in the near future, in one patient no surgical intervention was planned and one patient died of an unknown cause before IM surgery could take place. A Ladd procedure was performed for IM correction. Twenty-eight out of the 72 operated patients needed acute IM surgery, at a median age of 4.0 days (range 0–455). Only 4 out of these 28 patients had previously undergone surgical correction of the CCVD. Five patients had ischemic bowel disease at time of acute IM intervention; three of these patients died, one due to massive intestinal necrosis (one patient died due to cardiovascular cause, one patient died because of severe infection). At the time of IM surgery, signs and symptoms of CCVD, e.g., hepatomegaly, central cyanosis, cardiac murmurs and respiratory insufficiency, were more often observed twice in patients with a major CCVD (62.2 vs. 30.0%, respectively, P = 0.004). Most common complications were intestinal (Platzbauch, leakage of the bowel anastomosis resulting in sepsis, feeding difficulties with long-term necessity of nasogastrical tube feeding), infections and cardiovascular complications (e.g. bradycardia, thrombosis, arrhythmia) and they did not differ between both subgroups. Thirteen patients needed relaparotomy because of leakage of the bowel anastomosis (n = 4), Platzbauch (n = 2), suspected bowel obstruction with feeding difficulties (n = 1) and necrotising enterocolitis (n = 1). Five patients were reoperated for an adhesion ileus, all but one more than 1 month after surgery.
Table 3Characteristics of general population, major and minor congenital cardiovascular defects groups operated on intestinal malrotationCharacteristicAll CCVDMajor CCVDMinor CCVDP valueIM surgery100% (n = 72) 100%  (n = 34)100%   (n = 38)−Physical signs CCVD prior to IM surgery48.6%  (n = 35)67.6%  (n = 23)30.0%   (n = 12)0.004Post-IM-operative complications61.1%  (n = 44)67.6%  (n = 23)55.3%  (n = 21)0.3Post-IM-operative cardiovascular complications18.1%   (n = 13)20.6%    (n = 7)15.8%   (n = 6)0.8Relaparotomy18.1%     (n = 13)17.6%  (n = 6)18.4%  (n = 7)1.0Age at IM surgery in days [median (range)]17 (0–1,293)104 (0–1,196)6 (0–1,293)0.006Weight at IM surgery in kilograms [median (range)]3.21 (1.20–11.4)4.84 (1.20–11.4)2.88 (1.24–10.6)0.02Death in days after IM surgery [median (range)]28 (0–1,814)33 (0–1,814)3 (3–59)0.2Age at death in days [median (range)]40.5 (3–3,010)46 (22–3,010)8 (3–60)0.1Values in bold represent statistical significant resultsCCVD Congenital cardiovascular defects, IM Intestinal malrotation
Patients suffering from a post-operative IM surgery complication were younger (5.5 vs. 94.5 days, P = 0.031) and had a lower body weight (2.86 vs. 4.84 kg, P = 0.003) at time of IM surgery. Major CCVD, co-occurrence of a syndrome, or other congenital malformations did not significantly increase the risk on post-IM-operative morbidity. Correction for age and weight at the time of IM surgery in both CCVD groups showed, although not significant, a clear trend in post-IM-operative morbidity risk in patients diagnosed major CCVD (OR 2.9, 95% CI 0.9–9.6). Patients with pre-operative signs of CCVD had a four times increased risk on post-operative complications (95% CI 1.4–11.0). This risk factor was more prominent after correction for age and weight at the time of IM surgery (OR 8.2, 95% CI 2.1–30.7).
Mortality
Fifteen (19.5%) out of 77 patients died, 9 with major CCVDs. One patient with a minor CCVD died prior to IM surgery at the age of 1.4 years; cause of death remained unclear. Known causes of death after IM surgery were: cardiovascular complications (n = 7), massive intestinal necrosis (n = 2), sepsis (n = 2), respiratory failure (n = 2) and abstention of medical care because of unsuccessful surgical intervention on malformed trachea (n = 1). In both CCVD groups, death due to a cardiovascular cause remained the most frequent one. Median age at time of death was 40.5 days (range 3–3,010), with a median of 28.5 days after IM surgery (range 0–1,814). Of the 14 deceased patients, 9 (64.3%) had a major CCVD. Median age at time of death in the major CCVD group was 46 days (range 22–3,010) and in the minor CCVD group 8 days (range 3–60 days), although this difference was statistically not significant (P = 0.4). Twenty percent of the patients (7/35) with pre-operative physical signs of CCVD died, only one due to a cardiovascular cause. Three out of five patients with ischemic bowel at the time of IM surgery died post-operatively. The risk factors for mortality are shown in Table 4.
Table 4Risk factors (expressed as odds ratios and 95% confidence interval) for mortality in the whole study population (n = 77)CharacteristicDeceasedAliveOdds ratio (95% CI)Adjusting for weight and age at time of IM surgery OR (95% CI)Major CCVD9281.8 (0.6–5.7)5.0 (1.2–20.7)Minor CCVD634Syndrome/association7340.7 (0.2–2.2)1.0 (0.3–3.4) No syndrome or association828Other congenital defects13521.3 (0.2–6.4)1.1 (0.2–5.9)No other congenital defects210Physical CCVD signs prior to IM-surgery7281.1 (0.3–3.4)1.3 (0.4–4.4)No physical CCVD signs prior to IM-surgery730Intestinal ischemia327.6 (1.1–51.2)5.9 (0.9–42.4)No intestinal ischemia1156Post-IM-operative complications12711.3 (1.4–92.3)8.7 (1.0–75.7)No post-IM-operative complications1331Post-IM operative cardiovascular complications765.5 (1.5–20.5)4.5 (1.1–18.4)No post-IM-operative cardiovascular complications851Values in bold represent statistical significant resultsCCVD Congenital cardiovascular defects, IM Intestinal malrotation
Discussion
This study addresses the co-existence of malrotation with different types of congenital cardiovascular defects. We found 27.1% of all the patients registered in the last 25 years for IM to have a major or minor CCVD. Major CCVDs were seen in almost half of our population. Stewart et al. [2] found only 12% in a smaller IM population to have any CCVD. Complex CCVDs (23% of our population, 6.3% of all patients with IM in the last 25 years) and VSDs (21% of our population, 5.6% of all patients with IM in the last 25 years) were most frequently diagnosed. Pradat et al. [17] reported the co-occurrence of IM in patients with single ventricle to be as high as 8.5%. Further data on co-occurrence of CCVD in IM patients are missing.
The high prevalence of the CCVD in our IM cohort needs some thoughts on common aetiology. The gut is developed at a later stage in embryogenesis than the heart does. Abnormal abdominal development, e.g. by early development of an intra-abdominal cyst in embryogenesis, is known to enhance the chance of developing IM [18]. Changes in intra-abdominal space caused by a CCVD might also increase the risk on IM development during embryogenesis [4]. Another explanation might be a common genetic cause. The gut and the heart are both organs that show a characteristic left–right asymmetry, caused by a specific biochemical cascade during embryogenesis [19]. Two signalling molecules inevitable for this cardiac and gastro-intestinal asymmetry are nodal and pitx2c [20]. Reduced, diffuse or absent expression of pitx2c in the embryo cause abnormal looping from both heart and gut in animal studies [21–24], resulting mainly in abnormal outflow tract orientation (e.g. unseptated and misaligned great artery trunks, symmetrical outflow tract cushions, double outlet right ventricle, common atrioventricular trunk) [23, 24]. This disturbed expression of pitx2c could cause a spectrum of isomerism [25]. More research is needed to reveal a possible common genetic cause for both CCVD and IM.
Our study group explores a high percentage of syndromes or associations. Although IM has been recognized in many syndromes and associations, this high prevalence should make us aware of co-occurrence of CCVD in these patients, as well as the possibility of a syndrome or association in patients diagnosed with both IM and CCVD. The post-operative morbidity after IM surgery in the general paediatric population is between 0 and 16.7% [9, 26]. A higher morbidity rate (47%) has been reported by Murphy et al. [27], yet the surgical intervention was acute in 65% of their study population. Consistent with earlier reports [6–8], we found post-operative morbidity and mortality to be associated with younger age at the time of surgery. However, ischemic bowel (at the time of IM surgery) did not increase post-IM-operative morbidity as reported by others [2, 6, 8, 28]. Re-laparotomy in our population was consistent with earlier reports in the general population [26, 27]. The current study showed that patients with a CCVD had a relatively high morbidity rate after IM surgery (61.1%). Syndromes, associations or the presence of other congenital malformations were not found to be risk factors for post IM morbidity. Cardiovascular complications were seen in 18.1% of our population. There are neither data on prevalence of cardiovascular complications after IM surgery in the general paediatric population, nor on influence of physical condition prior to IM surgery on the morbidity after the operation. Although the type of CCVD was not significantly associated with an increased risk on morbidity, there was a clear trend (towards increased risk on post-operative morbidity) in patients with major CCVD. Furthermore, patients with pre-operative physical signs of CCVD had a high post-operative morbidity rate (77%).
The overall mortality rate in literature after IM surgery varies between 3 and 9% [6, 27–29]. Ilçe et al. [8] found a mortality rate varying between 0 and 36% in patients mainly younger than 1 year, having almost all emergency intervention. A recent study of Murphy et al. [27] reported only 1 post-operative death in 46 patients due to cardiovascular consequences in a patient with congenital cyanotic heart disease. In the current study, the mortality rate was higher: almost one-fifth of the patients died. Patients who died were significantly younger at the time of IM surgery. Other risk factors for death were major CCVD (after correction for age and weight at the time of IM surgery), ischemic bowel at the time of IM surgery, post-IM-operative complications in general and post-IM-operative cardiovascular complications.
Diagnosis and treatment of cardiac diseases have incredibly improved in the last two decades. Associated congenital abnormalities, including IM, have increased in significance. However, since mortality rates after surgical intervention for IM in patients with an associated CCVD are high, intervention should be well thought over. Recent studies discuss the advantages of laparoscopic inspection for IM, also in acute paediatric abdomen [30]. No data on laparoscopy in IM patients with CCVD are available. Possible advantages of laparoscopy for IM diagnosis, followed by operation, even in acute cases, might improve the outcome [30], even in patients with major CCVD. Referral to a centre equipped with paediatric cardioanesthesiological care is imperative.
Study limitations
This is a retrospective study of all consecutive live-born IM patients referred to a single university centre over a 25-year period. There are asymptomatic IM patients who never consult a doctor. Patients with IM and a very mild CCVD might have been missed. Screening for CCVD in IM patients could give us more information on the exact prevalence of CCVD in IM patients. Furthermore, since CCVD are very common congenital defects, a prospective study on the prevalence of IM in all patients with minor and major CCVD would also be of great interest.
Each retrospective study has its limitations, e.g. some variables were not (yet) available; e.g. syndromes and associations were common in our study population. Yet, the actual prevalence of syndromes might have been higher than reported. Certain syndromes and associations are quite new and nowadays there might be more attention on searching for a fitting syndrome or association diagnosis than 20 years ago.
As regards treatment, we perform 3 years laparoscopic inspection, followed by correction of the anomaly by laparoscopy or laparotomy in very young children [31].
Conclusion
The prevalence of CCVD in patients with IM is high (27.1%). Major and minor CCVDs are both frequently seen. Major CCVD is a risk factor for death after IM operation. Since morbidity and mortality rates in IM patients with CCVD are much higher than previously reported, a pre-operative screening for co-existing CCVD is indicated. Elective IM surgery in young patients with major CCVD should be conducted in a centre with adequate paediatric cardiac care. The benefit of laparoscopic intervention needs further follow-up.

congenital cardiovascular defect
intestinal malrotation
post-operative morbidity
post-operative mortality


Estrogen receptor α polymorphisms and postmenopausal breast cancer risk

Background The estrogen receptor alpha (ESR1) is a mediator of estrogen response in the breast. The most studied variants in this gene are the PvuII and XbaI polymorphisms, which have been associated to lower sensitivity to estrogen. We evaluated whether these polymorphisms were associated with breast cancer risk by means of an association study in a population of Caucasian postmenopausal women from the Rotterdam study and a meta-analysis of published data.
Introduction
Family history is one of the strongest risk factors for breast cancer [1]. It has been shown that the heritability of this disease is ∼30% [2]. The most important determinants of risk for breast cancer are related to endogenous hormone levels and major reproductive events [3], thus, suggesting that genes in the estrogen pathway may influence breast cancer risk.
The estrogen receptor alpha (ESR1) is one of the most important mediators of hormonal response in estrogen-sensitive tissues such as the breast [4] and plays a crucial role in breast growth and differentiation as well as in the development of cancer [5]. The human ESR1 gene is localized on chromosome 6q24-q27 [6], it extends more than 140 kb and includes eight exons [7]. The most studied variants in this gene are the PvuII (C/T) and XbaI (G/A) polymorphisms in intron 1, 397 and 351 bp upstream of exon 2 respectively [8, 9]. These variants have been implicated in gene expression by influencing transcription [10]. While some studies have found an increased risk for the A and T alleles of the XbaI and PvuII polymorphisms [4, 9, 10], others have found an increased risk only for the X (G) allele of XbaI [11, 12]. In addition, other studies found no effect at all for either of these polymorphisms [4, 13]. These alleles were correlated with high bone mineral density and height in other studies, including one performed in our study population, [14, 15], suggesting a stronger estrogenic effect in P(C) and X(G) allele carriers [14].
The aim of our study was to evaluate the effect of these polymorphisms on breast cancer risk by performing an association analysis in a population based study of Caucasian postmenopausal women. Further, we performed meta-analyses of all available published data on these polymorphisms and the risk of breast cancer.
Materials and methods
Study population and measurements
Our study population is part of the Rotterdam study [16]. Inhabitants of the suburb of Ommoord aged 55 or older were invited to participate and 7983 agreed to do so (response rate 78.1%). Study participants signed an informed consent and the Medical Ethics Committee of the Erasmus Medical Center approved the study. Our study group was composed of 4,878 postmenopausal women. Information on risk factors such as age at entry, age at menarche, age at menopause, parity, body mass index (BMI), waist hip ratio (WHR) and hormone replacement therapy use (HRT) was retrieved at baseline through a questionnaire. BMI was calculated by dividing the weight in kilograms by the height (in meters) squared [17].
Case identification and validation
Three different databases were used for patient identification. First, cases diagnosed by general practitioners in the research area (Ommoord) were collected (International Classification of Primary Care (code X76)). Second, the Dutch National Registry of all hospital admissions (LMR) was consulted to detect all malignancy related hospital admissions for study participants. Finally, regional pathology databases were linked to the Rotterdam Study to identify patients. Subsequently, breast cancer cases were validated by a physician on the basis of medical records of the general practitioner, discharge letters and pathology reports. Only pathologically confirmed cases were considered in the analysis. The index date was defined as the earliest date found in the pathology report.
Genotyping & data analysis
Out of the 4,878 women participating in our study, 3,893 (80 %) were successfully genotyped for the PvuII and XbaI polymorphisms. The genotyping procedures have been described previously [14]. Loss to follow up was assessed to verify it was independent of genotype. Categorical variables, such as parity and HRT, were compared between genotype groups using the chi-squared test. Continuous variables, (age at entry, age at menopause, BMI and WHR) were compared using the independent sample Mann–Whitney test. We used logistic regression to study the risk of breast cancer by ESR1 genotype. This analysis was performed using SPSS version 11, since there is no clear risk allele from the literature, we took the TT (PvuII) and AA (XbaI) genotypes as reference because they have been associated to lower sensitivity to estrogen in our population [14]. We also performed a trend test to evaluate if the number of risk alleles carried had an effect on disease risk. Hardy-Weinberg equilibrium (HWE) was assessed for both polymorphisms using Markov-Chain Monte-Carlo approximation of the exact test implemented in the GENEPOP package V 3.3 [18].
Meta-analysis
We searched PubMed until October 2006 for all case-control studies on the association of the PvuII and XbaI polymorphisms in the ESR1 gene and breast cancer. Our search strategy was based on the keyword “breast cancer” combined with “estrogen receptor” and “polymorphism”. To verify that all studies were retrieved, the reference lists of all publications were searched for additional studies. We excluded studies from our analyses if the genotype frequencies in the control population were out of Hardy-Weinberg or if their data had been previously used in another study. To quantify the strength of association, pooled odds ratios (ORs) and 95% confidence intervals (CI) were calculated using the random-effects model of the DerSimonian and Laird method [19]. The degree of heterogeneity between the study results was tested by the inconsistency statistic (I2). Funnel plots were used to evaluate publication bias [20]. Data were analysed using Review Manager, version 4.2 (Cochrane Collaboration, Oxford, UK).
Results
The total loss of follow-up for the genotyped participants was 8.4% and it was not dependent on ESR1 genotype (P = 0.51). The genotype frequencies of both polymorphisms were in HWE proportions (X2 P = 0.33 for PvuII and X2P = 0.31 for XbaI). In Table 1 we show the baseline characteristics of our study population. We found that all cases (incident and prevalent) were significantly younger at entry than controls (P < 0.001) and also died earlier during follow-up (P < 0.001), when using incident cases only we found the same significant differences (P for age at entry <0.0001, P for age at death <0.0001). We also found that cases had significantly fewer children than controls (P = 0.04). We did not find any significant differences in these baseline characteristics between genotype groups (data not shown).
Table 1General characteristics of the study populationCasesControlsTotalTotal studied (%)190 (4.7%)3513 (95.3)3703Mean age of entry (SD)*67.80 (7.7)70.36 (9.6)70.24 (9.6)Mean age at death (SD)*77.30 (8.6)84.46 (8.7)84.12 (8.8)Mean age at menarche (SD)13.57 (1.7)13.68 (1.8)13.67 (1.8)Mean age at menopause (SD)*49.51 (4.8)52.19 (13.6)52.07 (13.3)Mean number of children (SD)*1.77 (1.6)2.12 (1.7)2.10 (1.7)Parity (SD) (≥1 child)*121 (71.6)2640 (79.4)2761 (79)Hormone replacement therapy(%)27 (21.1)504 (19.5)531 (19.6)Mean BMI (SD)27.10 (3.9)26.67 (4.1)26.69 (4.1)Mean WHR (SD)0.87 (.09)0.87 (.09)0.87 (.09)* P-value < 0.05
There were 38 women with previously diagnosed postmenopausal breast cancer who entered the study. During follow-up, 152 were additionally diagnosed. There were no significant differences in the number of cases between the PvuII (P = 0.43 for overall cases) and XbaI genotypes (P = 0.33 for overall cases).
We carried out a logistic regression analysis adjusting for age at entry, age at menopause, BMI, WHR and HRT for both polymorphisms separately (Table 2). Since the T and A alleles of these polymorphisms have been correlated to lower estrogenic effects, we used the TT and AA genotypes as our reference categories in the analyses. There were no significant differences in risk for breast cancer among carriers of the different genotypes of the PvuII or XbaI polymorphisms in the ESR1 gene. There was a non-significant tendency of the C allele of PvuII (P-for trend = 0.22) and G allele of the XbaI (P-for trend 0.26) to be over represented in patients.
Table 2Odds ratios for breast cancer risk for PvuII and XbaI genotypesOverallIncidentPrevalentPvuIITTRefRefRefTC0.9 (0.6–1.4)1.0 (0.6–1.6)0.8 (0.3–2.1)CC1.4 (0.8–2.2)1.4 (0.8–2.5)1.2 (0.4–3.3)XbaIAARefRefRefGA1.2 (0.8–1.7)1.3 (0.8–2.0)0.8 (0.4–1.9)GG1.3 (0.7–2.2)1.5 (0.8–2.8)0.5 (0.2–2.4)
To evaluate our data together with those in the literature we performed meta-analyses. We identified nine articles studying the relation between XbaI and PvuII polymorphisms and the risk of breast cancer [4, 9–12, 21–24]. We excluded from our analyses one study [11], since the data was used in another study [4]. Furthermore, two studies were excluded since genotype frequencies of controls were out of HWE proportions [9, 10]. Using the random effects model we did not find any difference in risk among XbaI and PvuII genotypes (Figs. 1 and 2). High inter-study heterogeneity can render the interpretation of the results of a meta-analysis difficult and although we found high heterogeneity in the G/A versus GG comparison there was no significant heterogeneity in the other three comparisons. Additionally, the evaluation of the funnel plots did not suggest evidence for publication bias.
Fig. 1Meta-Analyses ESR1 XbaI polymorphism and breast cancer riskFig. 2Meta Analysis ESR1 PvuII polymorphism and breast cancer risk
Discussion
We performed an association study to evaluate the relationship of two well-studied polymorphisms in the ESR1 gene and the risk of breast cancer in Caucasian postmenopausal women from the Rotterdam Study. Using logistic regression analysis, we found no evidence of effect, with only a non-significant increase in breast cancer risk for AA carriers of the XbaI polymorphism (overall OR = 1.3, 95% CI = 0.7–2.2) and for TT carriers of the PvuII variant (overall OR = 1.4, 95% CI = 0.8–2.2). Additionally we performed meta-analyses of published data to examine the effect of both polymorphisms. These meta-analyses also suggest there are no differences in risk among genotype groups of these two ESR1 variants.
The XbaI and PvuII polymorphisms are situated in intron 1 and their functionality has not yet been demonstrated. Moreover, it has been suggested their effects could be the result of high linkage disequilibrium with functional variants that affect sensitivity to estrogen [13].
One of the limitations of our study is the limited number of breast cancer cases present in our population. Nevertheless, we have sufficient power (β = 0.8) to detect effects of 1.6 or higher. We further conducted meta-analyses off all studies conducted to date. Our data suggests that these two polymorphisms do not play a role in the susceptibility of breast cancer in elderly Caucasian women.

estrogen receptor
polymorphism
breast cancer


Active-site structure, binding and redox activity of the heme–thiolate enzyme CYP2D6 immobilized on coated Ag electrodes: a surface-enhanced resonance Raman scattering study

Surface-enhance resonance Raman scattering spectra of the heme–thiolate enzyme cytochrome P450 2D6 (CYP2D6) adsorbed on Ag electrodes coated with 11-mercaptoundecanoic acid (MUA) were obtained in various experimental conditions. An analysis of these spectra, and a comparison between them and the RR spectra of CYP2D6 in solution, indicated that the enzyme’s active site retained its nature of six-coordinated low-spin heme upon immobilization. Moreover, the spectral changes detected in the presence of dextromethorphan (a CYP2D6 substrate) and imidazole (an exogenous heme axial ligand) indicated that the immobilized enzyme also preserved its ability to reversibly bind a substrate and form a heme–imidazole complex. The reversibility of these processes could be easily verified by flowing alternately solutions of the various compounds and the buffer through a home-built spectroelectrochemical flow cell which contained a sample of immobilized protein, without the need to disassemble the cell between consecutive spectral data acquisitions. Despite immobilized CYP2D6 being effectively reduced by a sodium dithionite solution, electrochemical reduction via the Ag electrode was not able to completely reduce the enzyme, and led to its extensive inactivation. This behavior indicated that although the enzyme’s ability to exchange electrons is not altered by immobilization per se, MUA-coated electrodes are not suited to perform direct electrochemistry of CYP2D6.
Introduction
Heme proteins are a large class of proteins having a substituted iron porphyrin as a cofactor, with functions ranging from electron carriers to oxygen transport to catalysis. Heme enzymes such as cytochromes P450 (CYPs) and other oxygenases, being able to bind several substrates and to use electrons from their heme iron to drive a catalytic reaction, seem to be promising for nanobiotechnological applications in biosensing and biocatalysis [1, 2].
The immobilization of enzymes on solid supports or electrodes is a commonly used approach for constructing biosensors or biocatalytic devices [3, 4]. In principle, electrodes can be used as a source of electrons to drive a catalytic reaction, or as elements of biosensors which translate a molecular event such as substrate binding into a measurable electric signal.
The question arises whether immobilization preserves the protein function or rather induces losses of enzymatic activity, and what structural changes cause the dysfunction [1]. Therefore, techniques capable of probing the activity and the structure of enzymes immobilized on surfaces are valuable.
Surface-enhanced resonance Raman scattering (SERRS) has proven to be a sensitive and selective spectroscopic technique to study heme proteins immobilized on metal surfaces, and it has been used to characterize the active site of proteins such as cytochromes c, cytochrome c oxidase and CYPs [5]. This technique is based on the inelastic scattering of laser radiation whose wavelength is in resonance with an electronic transition of the molecule studied [6–8], leading to a signal enhancement denoted as the resonance Raman (RR) effect. In the case of proteins having a heme as a chromophore, the porphyrin vibrational modes are intensified over the other protein vibrations if the laser wavelength falls within an associated electronic absorption band. The heme moiety can thus be selectively investigated without a dominating interference due to the protein matrix. Additional sensitivity is gained through surface enhancement (or surface-enhanced Raman scattering effect) if the protein is close to a metal surface with particular characteristics. Depending on the molecule studied and on the experimental conditions applied, the synergy between the two signal-enhancing effects (i.e., SERRS) sometimes reaches single-molecule sensitivity [9–12], therefore allowing the study of enzymes immobilized on surfaces, typically consisting of very low amounts of sample (i.e., from a few layers to submonolayers of protein).
To fulfill the requirements to achieve both resonance enhancement and surface enhancement, silver is the surface-enhancing metal of choice [7]. Recently, heme proteins such as cytochromes c [5, 13–19] and CYP101 [20] have been investigated using SERRS on roughened Ag electrodes coated with self-assembled monolayers (SAMs) of several different alkanethiols and their derivatives. SAMs of functionalized alkanethiols are among the most versatile coatings for both electrostatic and covalent immobilization of proteins to metal surfaces, having the additional advantage of preventing possible negative consequences of the direct contact between the protein and the metal [5, 21]. Besides technological applications, SERRS on SAM-coated electrodes can become an important tool in fundamental studies about protein–membrane and protein–protein interactions. In fact, because of their structure and characteristics, SAMs can be thought of as a rough model for biological membranes [5, 13], or can be used to mimic the binding domain of a partner protein, since surface properties such as polarity and charge can be selected by choosing the appropriate alkanethiol derivative [13, 22]. Moreover, electrochemically roughened Ag electrodes coated with SAMs were recently shown to be excellent for studying immobilized proteins with voltammetric techniques, so both SERRS and voltammetry can be performed on the same sample [19].
In the work reported here, we exploited SERRS to study the behavior of the human enzyme CYP2D6 electrostatically immobilized on a Ag electrode coated with a SAM of 11-mercaptoundecanoic acid (MUA). CYPs are heme–thiolate monooxygenases capable of oxidizing and reducing a broad range of endogenous and exogenous substrates and can be found in all kinds of tissues in virtually all organisms [23]. CYP2D6 is a peripheral membrane-bound protein and is one of the most important CYPs in humans, being involved in the metabolism of about 30% of the currently marketed drugs, including β-blockers, neuroleptics, antidepressants and antiarythmics [24, 25]. Its characteristics make this enzyme not only attractive for technological applications as a biocatalyst or biosensor, but also from a toxicological and clinical point of view. In fact, a better understanding of its mechanisms of catalysis could help in developing more efficient drugs [26, 27].
Using a home-built SERRS spectroelectrochemical flow cell [28], we investigated the CYP2D6 active-site structure under diverse experimental conditions in order to check the functionality of the immobilized enzyme, its redox activity and its ability to bind reversibly a substrate or an exogenous heme ligand such as imidazole. Besides confirming the retention of the enzyme’s function upon immobilization, SERRS spectra yield information about structural changes in the active site associated with substrate binding and electron transfer, the initial two steps of a CYP catalytic cycle.
Materials and methods
Chemicals
The pSP19T7LT plasmid containing bicistronically human CYP2D6 with a C-terminal His6-tag and human NADPH-CYP reductase,was kindly provided by Magnus Ingelman-Sundberg. Dextromethorphan (DX), imidazole, MUA and sodium dithionite were purchased from Sigma-Aldrich (St Louis, USA). Emulgen 911 was obtained from KAO Chemicals (Tokyo, Japan). Nickel nitrilotriacetic acid (NTA) agarose was from Sigma. Phosphate buffer solutions (pH 7.4, 40 mM) were prepared from K2HPO4 and KH2PO4 (J.T. Baker, Deventer, The Netherlands). All other chemicals were of analytical grade and were obtained from standard suppliers.
Expression and purification of CYP2D6
The pSP19T7LT plasmids containing wild-type CYP2D6 were transformed in Escherichia coli, strain JM109. Expression, membrane isolation and purification were carried out as previously described [29]. In short, membranes were resuspended in 0.5% of the original culture volume of potassium phosphate–glycerol buffer (50 mM potassium phosphate buffer, pH 7.4, 10% glycerol) and CYP contents were determined by CO difference spectra [30]. The enzyme was solubilized by stirring in potassium phosphate–glycerol supplemented with 0.5% Emulgen 911 for 2 h at 4 °C. Insoluble parts were removed by centrifugation (60 min, 120,000g at 4 °C). The supernatant was mixed, while gently rocking it, with nickel nitrilotriacetic acid agarose for 60 min at 4 °C. The nickel nitrilotriacetic acid agarose was then applied to a polypropylene tube with a porous disk (Pierce, Perbio Science, Etten-Leur, The Netherlands), washed with potassium phosphate–glycerol buffer containing 2 mM histidine. Subsequently, bound CYP2D6 was eluted with 0.2 M histidine. After overnight dialysis in potassium phosphate–glycerol buffer the sample was concentrated on a Vivaspin 20 filtration tube (10,000 MWCO PES, Sartorius, Nieuwegein, The Netherlands) to a final concentration of 60 μM CYP2D6.
Spectroelectrochemical flow cell
SERRS measurements were performed using a modified version of a linearly moving low-volume spectroelectrochemical cell previously described [28]. The cell was originally designed to acquire potential-dependent SERRS spectra from proteins adsorbed on roughened Ag electrodes using a Raman microscope in a backscattering configuration. The modified version of this cell used in the present work has the possibility to flow a solution through the cell via two small holes (0.75-mm diameter each; Fig. 1). By using a valve, a liquid-chromatography pump (Gilson, Middleton, USA) and flexible PEEK tubing (VICI International, Schenkon, Switzerland), the thin layer (0.3 mm) of solution in contact with the Ag electrode surface can be changed by flowing different solutions though the spectroelectrochemical cell, without the need to disassemble the cell itself (Fig. 1). Moreover, by degassing the solutions with argon and by using oxygen-proof PEEK tubing, the presence of oxygen, which can interfere with electrochemical and spectroscopic measurements, can be minimized in the cell. It should be noted that small internal diameter of the liquid-chromatography tubing (0.75 mm) and the low internal volume of the cell (approximately 50 μL) allows the use of relatively small amounts of solutions. Fig. 1The surface-enhanced resonance Raman scattering (SERRS) spectroelectrochemical flow system. The degassed solutions are pumped though PEEK liquid-chromatography tubing into the cell and then to the waste. A switching valve is used to select which solution has to be pumped. A scheme of the cell is shown in detail, with the inlet and outlet for the flowing solution and the three electrodes: the Ag working electrode (WE), the Pt counter electrode (CE) and the connection though a porous frit to a saturated calomel electrode (SCE) reference electrode (RE). The laser for Raman excitation is focused by the microscope objective onto the Ag electrode through a glass cover and a layer of solution
Raman spectroscopy instrumentation
Spectroscopic measurements were conducted by placing the spectroelectrochemical cell under a home-built Raman microscope in a backscattering configuration: a Zeiss microscope (D-7082 with a ×40 objective, numerical aperture 0.60, working distance 2 mm) was coupled to an Andor Shamrock SR-303i-A single monochromator (Andor Technologies DV-420OE, Belfast, UK) with a mounted 2,400 g mm−1 holographic grating and an Andor Newton DU970N CCD camera. The 413.1 nm line of a continuous-wave Kr ion laser (Innova 300C, Coherent, Santa Clara, CA, USA) was used for excitation. The Rayleigh scattered light was removed using third millennium edge long pass filter. Laser powers of 5 mW at the sample and an accumulation time of 180 s were used throughout the experiments. To avoid sample photodegradation, the cell was put in a moving holder which ensured a constant linear movement of the metal electrode surface under the microscope objective, while keeping the focus approximately constant [28]. The monochromator slit was set to 120 μm, yielding a resolution of approximately 4 cm−1, with an increment of approximately 0.8 cm−1 per data point.
The sloping background of the spectra was subtracted using a baseline fitted to the experimental data with the Andor CCD camera software, while the fitting of the CYP2D6 spectrum at −1.0 V with Lorentzian functions was performed with PeakFit® version 4.12 (SeaSolve Software, Richmond, CA, USA).
Electrochemical instrumentation
Polycrystalline silver disk electrodes (IJ Cambria Scientific, Burry Port, UK) of 2-mm diameter (geometric area 0.0314 cm2) were used as working electrodes; a platinum ring and a saturated calomel electrode (AMEL Instruments, Milan, Italy), were used as the counter electrode and the reference electrode, respectively. The reference electrode was kept in a glass tube filled with a 40 mM potassium phosphate buffer solution and separated from the working solution by a PEEK porous frit. It was kept at constant room temperature (20 ± 0.1 °C) during all the experiments. The three-electrode system was controlled with a μAutolab potentiostat (Eco Chemie, Utrecht, The Netherlands). All potentials are referred to a saturated calomel electrode, previously calibrated against the methyl viologen (MV) MV2+/MV+ couple.
Sample preparation
SERRS measurements were performed on electrochemically roughened Ag electrodes coated with MUA, prepared as follows. Ag electrodes were treated as previously described [31] with water on aluminum oxide lapping film sheets (261× and 262×, 3M™) from 5- to 1-μm grain size until a mirrorlike appearance of the surfaces was obtained. After they had been polished, the electrodes were roughened ex situ with an oxidation–reduction cycle procedure similar to that described by Roth et al. [32] and then kept overnight in 2 mM ethanol solution of MUA. The oxidation–reduction cycles (approximately 0.67 C cm−2 of anodic charge passed per step) were performed in an ordinary glass electrochemical cell (5 mL) filled with a 0.1 M KCl solution using the three-electrode system described already.
The MUA-coated Ag electrodes were rinsed with ethanol to eliminate the excess of MUA, dried by gently blowing nitrogen on them and then immersed into 50 μL of a 3 μM solution of protein in potassium phosphate buffer (40 mM, pH 7.4, 10% glycerol) for at least 3 h at 4 °C, to induce adsorption of the protein on the electrode. Then, the coated electrodes with the adsorbed protein were rinsed with buffer to eliminate possible excess of loosely bound protein, and were put into the spectroelectrochemical cell described already.
Depending on the required experimental conditions, before every SERRS measurement the appropriate solution was flowed for 1–2 min into the cell: for measurements in the absence of DX or imidazole, a potassium phosphate buffer (40 mM, pH 7.4) was used, while for measurements in the presence of DX or imidazole, solutions of DX (5 mM, 0.02% of methanol) or imidazole (10 mM) in buffer were used. The solutions of DX were prepared by diluting with buffer a 2 M stock solution of DX in methanol. To chemically reduce the protein in the absence or presence of substrate, sodium dithionite solutions (5 mg mL−1) were prepared using buffer solutions or DX solutions (5 mM) in buffer, and were flowed for 1–2 min into the cell before every SERRS measurement. Prior to use, all the solutions flowed into the cell were deoxygenated for at least 3 h with argon.
RR measurements were performed using buffered CYP2D6 solutions in spinning glass capillaries as previously described [33]. To chemically reduce the enzyme in solution prior to RR measurements, solid sodium dithionite (to a final concentration of 5 mg mL−1) was added to a buffered 3–5 μM protein solution in presence of 5 mM DX, previously degassed with argon.
Results and discussion
The resting state
Intense SERRS spectra (Fig. 2, spectra a) were observed from CYP2D6 immobilized via electrostatic interaction [34] on MUA-coated Ag electrodes at open circuit (i.e.. no external potential is applied), in the presence of phosphate buffer. These experimental conditions should reproduce the “resting state” of the enzyme (i.e.. the starting point of its catalytic cycle) when no substrate is present in the active site and no reaction is taking place [35, 36]. A MUA-SAM was chosen as the coating because of its structural stability, its compatibility with CYP2D6 [34] and its use in recent SERRS studies on bacterial CYP101 [20] and on cytochrome c [5, 13]. Fig. 2A SERRS spectra of cytochrome P450 (CYP) 2D6 on an 11-mercaptoundecanoic acid (MUA)-coated Ag electrode obtained by consecutively flowing different solutions in the spectroelectrochemical cell (at open circuit), in the following order: a buffer, b dextromethorphan (DX) buffered solution, c buffer (rinse), d imidazole-buffered solution, e buffer (second rinse), f dithionite and DX solution in phosphate buffer. The 1,300–1,450-cm−1 regions of spectra b--e are identical to that of spectrum a and are omitted for visual clarity. B Detailed view of the 1,450–1,660-cm−1 region of the CYP2D6 SERRS spectra in presence of a buffer, b DX and d imidazole. Bands corresponding to the ν2 and ν3 vibrational modes for the six-coordinated low-spin and five-coordinated high-spin heme species are indicated. Experimental details are given in “Materials and methods”
The most intense band of the SERRS spectra is observed at 1,371 cm−1, and a group of bands are detected between 1,450 and 1,650 cm−1. The frequencies of the vibrational modes above 1,300 cm−1 are correlated with the oxidation, the spin and the coordination state of the iron atom and therefore they are referred to as “marker bands” for such properties [37]. The most relevant marker bands in CYP2D6 SERRS spectra are assigned to the corresponding vibrational modes on the basis of former RR studies of CYP2D6 [33] and other CYPs [37] in buffered solution (Table 1). According to the frequencies observed, the heme iron in the enzyme active site is oxidized (Fe3+), mainly six-coordinated low-spin (6cLS) state, as is usually expected from a CYP in its resting state [36, 37]. An extensive amount of experimental data from other CYPs as well as theoretical studies showed that in the 6cLS state the Fe3+ atom has the four pyrrolic nitrogens of the porphyrin macrocycle as equatorial ligands, and a cysteine and a water molecule (or hydroxyl ion) as axial ligands [35, 36]. Table 1Vibrational frequencies (cm−1) and relative intensities of most relevant oxidation- (4) and spin/coordination- (ν3, ν2) marker bands in SERR spectra of CYP2D6 adsorbed on MUA-coated electrodes, under different experimental conditionsModeFe3+Fe2+6cLS6cLS/5cHSWithout substrate/inhibitorWith inhibitor (imidazole)With substrate (dextromethorphan)5cHSaν21,582 (0.19)1,582 (0.14)1,582 (0.20)/1,567 (NA)b1,561 (0.91)ν31,501 (0.08)1,501 (0.10)1,501 (0.09)/1,486 (0.05)1,466 (0.65)ν41,371 (1.00)1,371 (1.00)1,371 (1.00)1,344c (1.00)Relative intensities are reported after the corresponding frequencies between parentheses; values are relative to the band with an intensity of 1.006cLS six-coordinated low spin, 5cHS five-coordinated high spin, NA not availableaReduced state is achieved by adding dithionite in presence of substratebBand not sufficiently resolved, frequency estimated by spectral difference [33]cTogether with this band, in spectra of CYP2D6 another one is observed at 1,360 cm−1, characteristic of reduced inactive cytochrome P420 species
The observation of an oxidized 6cLS state is in agreement with spectroscopic data on CYP2D6 previously reported. In particular, the SERRS spectrum of CYP2D6 under these conditions is identical to the RR spectrum of the same enzyme in buffer [33, 38] (Fig. 3 spectra b and b′). An analogous correspondence with RR data was recently reported for SERRS spectra of CYP2D6 adsorbed on Ag nanoparticles coated with MUA [34]. This striking similarity in the position and relative intensity of SERRS and RR spectra suggests that, differently from what was recently reported for CYP101 [20], CYP2D6 retains its active-site structure upon adsorption on the SAM-coated electrode, or at least that there are no structural changes inducing significant alterations in heme properties. Fig. 3Comparison between SERRS (a, b) and resonance Raman (a′, b′) spectra of reduced (a, a′) and oxidized (b, b′) CYP2D6. SERRS spectra were obtained from MUA-coated Ag electrodes in the presence of buffer alone (b) and a dithionite and DX solution in phosphate buffer (a), while resonance Raman spectra were obtained from CYP2D6 in solution under the same conditions. Marker bands for oxidation (ν4) and spin (ν3, ν2) states are indicated. Experimental details are given in “Materials and methods”
Reversible binding of a substrate
Although the active-site structure is likely to be unaffected by the protein–surface interaction, the functioning of the adsorbed enzyme still has to be verified. According to the accepted scheme of the catalytic cycle common to most CYPs, the heme iron may experience changes in its coordination sphere and its spin as a consequence of the presence of substrates in the active site [36]. RR spectra of CYP2D6 in the presence of DX, a substrate of this enzyme, showed the appearance of marker bands for a five-coordinated high-spin (5cHS) heme besides the 6cLS heme already present in the resting state [33]. In agreement with those RR data, SERRS spectra of CYP2D6 adsorbed on coated electrodes, upon flowing a DX solution into the cell (Fig. 2, spectra b), exhibit distinct differences from the spectra of the enzyme in the resting state, showing additional bands characteristic of a 5cHS heme (ν3 at 1,486 cm−1 and ν2 at 1,567 cm−1; Fig. 2, spectra b, Table 1).
This partial transition from 6cLS to 5cHS is explained by the displacement of a water molecule as the heme’s distal axial ligand by DX, which is accommodated in the active site on the distal side above the porphyrin macrocycle [33]. Water molecules, which may still be present in the active site, can then rebind to the heme iron, yielding an equilibrium between the 5cHS and 6cLS states [33, 37, 39].
The spectral changes taking place owing to the presence of DX therefore clearly indicate that the adsorbed enzyme retains its ability to accommodate a substrate into its active site, confirming similar results reported for CYP2D6 adsorbed on coated Ag nanoparticles [34]. Unfortunately, neither RR nor nanoparticle-based SERRS allowed the investigation of the reversibility of the substrate-binding process, since it is extremely difficult to rapidly eliminate the substrate from the enzyme-containing sample. Such problems are not encountered in the present spectroelectrochemical cell setup. The substrate is easily removed from the enzyme-coated electrode surface by flowing buffer through the cell, reestablishing the resting state conditions. Once the buffer had completely replaced the DX solution in the cell, 5cHS marker bands disappeared from SERRS spectra of CYP2D6, which recover the shape characteristic of the 6cLS heme in the resting state (Fig. 2, spectrum c). The exchange between the two solutions can be operated many times, showing every time the concomitant spectral changes, and indicating an intrinsic stability of the CYP2D6–SAM–electrode system. The similarity between the SERRS spectra of the same enzyme sample before the introduction of DX (Fig. 2, spectra a) and after its removal (Fig. 2, spectrum c) clearly demonstrates that the adsorbed CYP2D6 is able to reversibly bind a substrate, implying that the interaction with the coated electrode surface does not obstruct the substrate access routes to the enzyme active site.
Reversible binding of imidazole as an exogenous heme axial ligand
Imidazole and several imidazole derivatives are believed to form a relatively strong bond via their nitrogen with the heme iron of ferric CYPs, replacing the water molecule as a distal axial ligand, and thereby preventing the binding of oxygen to the heme in a subsequent step of the catalytic cycle [37, 40–43]. Since oxygen binding is essential for monooxygenase activity, often these compounds are effective inhibitors of CYPs [44].
The SERRS spectra of CYP2D6 collected upon flowing an imidazole solution over the enzyme-coated electrode are shown in Fig. 2, spectra d, displaying marker bands characteristic of a 6cLS state (Table 1), as expected from a CYP heme having an imidazole as the sixth axial ligand. Although the frequencies of the imidazole complex are identical to those reported for the resting state, the relative intensity ratio between the ν3 and ν2 bands is visibly increased with respect to the 6cLS resting state from 0.4 to 0.7 (Fig. 2spectra a and d, Table 1), in agreement with RR data of CYP2D6 in solution (see supplementary material) and with RR spectra previously reported for other CYP–imidazole complexes [41, 45–47]. This difference in relative intensities has been observed in all the RR spectra of CYP–imidazole complexes reported so far, and is generally regarded as the characteristic spectral feature which allows one to discriminate between the two 6cLS states having either imidazole or water as the heme axial ligand in CYPs. The explanation given for these characteristic relative intensity changes observed in the presence of imidazole in RR spectra of CYPs invokes the different resonance conditions due to redshift of the Soret absorption induced by imidazole in CYPs [37].
An additional but less pronounced feature observed in the SERRS spectrum of the CYP2D6–imidazole complex is the intensity decrease in the region around 1,630 cm−1, where three overlapping bands are expected: two stretching modes of the heme vinyl substituents around 1,620 and 1,630 cm−1, and the ν10 porphyrin mode around 1,635 cm−1 [37]. The presence of another vinyl stretching band, otherwise invisible because of its overlap with the more intense neighboring bands, can be substantiated by fitting the data with Lorentzian functions. A qualitative analysis of the fitting supports the hypothesis of a decrease in relative intensity of a vinyl band around 1,630 cm−1 for the imidazole complex (see supplementary material), suggesting a possible perturbation of the vinyls upon imidazole binding [48].
The observed change in relative intensity could be due to a variation in the rotational conformation of the vinyls [48]. Such variation might also cause a shift in the stretching frequencies of the vinyls [49, 50], although in SERRS spectra of CYP2D6 no significant shifts are detected. On the other hand, similar changes in relative intensities in the 1,610–1,640-cm−1 spectral region have been previously reported for the imidazole complexes of bacterial and microsomal CYPs [41, 45–47]. The fact that imidazole binding induces similar changes in vinyl rotational conformation in enzymes having rather different active-site structures is somewhat surprising. Since vinyl stretching modes in SERRS spectra are thought to be enhanced via an electronic coupling with the porphyrin [37], an alternative explanation of the changes detected upon imidazole binding could be the difference in resonance conditions due to the shift of the Soret band, similarly to what has been proposed for the ν3/ν2 intensity ratio. However, in absence of further experimental data from other techniques, the SERRS spectra presented cannot reliably give more detailed structural information about conformational changes of vinyls occurring upon imidazole binding.
Similarly to what was observed for DX binding, SERRS spectra obtained by flowing buffer through the cell in place of the imidazole solution (Fig. 2, spectrum e) have the ν3/ν2 relative intensity ratio restored to 0.4, and are identical to the spectra of the enzyme in the resting state (Fig. 2, spectra a), including the relative intensity pattern around 1,630 cm−1. Therefore, despite the imidazole being expected to form a relatively strong bond with the heme iron, the nonequilibrium situation due to the constant flow of buffer over the CYP–imidazole complex eventually causes the complete dissociation of the imidazole from the enzyme and the concomitant restoration of a water molecule as sixth ligand, demonstrating that the CYP–imidazole complex formation is reversible.
Redox activity
The catalytic activity of CYP2D6 relies on its ability to receive electrons from NADPH-dependent CYP reductase to drive the catalysis and to activate molecular oxygen to oxidize the substrate [36]. In particular, the first of the two electrons transferred during a catalytic cycle reduces the iron from Fe3+ to Fe2+, facilitating oxygen binding to the ferrous heme. In their resting state, CYPs usually have a formal reduction potential (E°′) lower than that of their electron-donor protein, so heme reduction does not occur [36]. However, the E°′ of some CYPs, including CYP2D6, is shifted to more positive values by the presence of a substrate in the active site, facilitating the reduction of the enzyme heme by the redox partner [1, 36, 51]. Therefore, the electron transfer reaction is believed to be triggered by substrate binding and is usually considered the third step of the catalytic cycle [36].
In absence of reductase, immobilized CYP2D6 might receive electrons from a reducing agent present in solution or directly from the electrode, by applying a proper voltage. The electron transfer process from the alternative electron source to the enzyme can be monitored by examining the relative presence of reduced heme species in SERRS spectra, using solutions previously deoxygenated with argon. This requirement is crucial since dioxygen rapidly binds to the ferrous heme to form a complex, which upon reaction with another electron quickly turns into an instable peroxo-ferric intermediate, continuing the catalytic cycle and hampering the observation of reduced species [36].
Upon flowing over the adsorbed enzyme a deoxygenated and buffered DX solution containing sodium dithionite as a reducing agent, the SERRS spectrum shown in Fig. 2, spectrum f is observed. It mainly presents bands with frequencies characteristic of a reduced 5cHS heme (Table 1), which are markedly different from all the spectra of oxidized CYP2D6 obtained so far (Fig. 2, spectra a–e). A noticeable feature is the coexistence of two ν4 oxidation marker bands at 1,344 and 1,360 cm−1, originating from the downshift and the splitting of the corresponding ν4 band observed at 1,371 cm−1 in the oxidized enzyme, and indicating the presence of two different heme species. While the ν4 band at 1,344 cm−1—consistent with the other bands observed—is typical of a ferrous CYP in its active form, the band at 1,360 cm−1 is characteristic of a ferrous inactive form called P420 [20, 37, 52, 53]. This strongly suggests that upon reduction a significant fraction of the immobilized enzyme undergoes structural changes leading to its inactivation. However, according to the RR spectra of the ferrous active and inactive forms previously reported for other CYPs, the active enzyme still contributes to a significant extent to the observed CYP2D6 SERRS spectrum, whereas no marker bands for the inactive species other than the one at 1,360 cm−1 can be detected. In fact, in all the RR or SERRS spectra of P420 species reported so far for various CYPs, the ν4 band is the strongest, with relative intensity much higher than for the other bands, whereas in spectra of active enzymes the intensity pattern is different, with many other bands having intensities comparable to that of the ν4 band [20, 37, 52–55]. In the spectra of reduced CYP2D6, the bands in the 1,400–1,650-cm−1 region have relative intensities comparable to that of the ν4 band of the active enzyme (at 1,344 cm−1), whereas the bands of the P420 species are likely to have relative intensities much smaller than that of the P420 ν4 band at 1,360 cm−1, and are therefore buried under the more intense bands due to the active enzyme.
Notably, this partial inactivation is not due to the immobilization as such, since every attempt to obtain reduced free CYP2D6 in solution by dithionite addition led to samples containing inactive enzyme besides the active one, yielding RR spectra which closely resemble the SERRS spectrum of the adsorbed enzyme (Fig. 4, spectra a). The inactivation might be due to laser-induced photodegradation, since laser illumination is common to both RR and SERRS. However, the use of laser powers lower than 5 mW yielded identical spectra (data not shown), and small amounts of inactive enzyme were previously detected in dithionite-reduced CYP2D6 samples using nonlaser techniques as well, such as UV–vis electronic absorption spectroscopy [38]. Fig. 4SERRS spectra of CYP2D6 on MUA-coated Ag electrodes in the presence of buffered solution with dithionite and DX (a) and without DX (b). The inset shows the oxidation marker band regions in detail, together with their spectral difference. Experimental details are given in “Materials and methods”
When reduction of CYP2D6 with dithionite is attempted in the absence of a substrate, the resulting SERRS spectrum presents some differences with respect to that of the substrate-bound enzyme (Fig. 4). In particular, the presence of DX in the dithionite solution increases the amount of reduced active enzyme at the expense of the oxidized and reduced inactive forms, as deduced from the increase in intensity of the band at 1,344 cm−1 and the concomitant decrease of the bands at 1,360 and 1,371 cm−1. Thus, the presence of DX in the heme pocket appears to affect the redox activity of the enzyme, possibly by causing a shift of its E°′ to more positive values, as previously observed for CYP2D6 on a polyaniline-doped glassy carbon electrode in the presence of fluoxetine [51].
On the other hand, in the electrochemical approach the control of the CYP2D6 oxidation state is attempted by applying a constant potential to the Ag working electrode, during which a SERRS spectrum is recorded to monitor the relative abundance of oxidized and reduced species characteristic for that voltage. Complete oxidation or reduction of the protein should in principle be obtained by applying a potential which is sufficiently more positive or more negative with respect to its E°′, respectively.
Besides the advantage of controlling the enzyme oxidation state and therefore, under adequate conditions, of supplying the electrons and driving the catalytic reaction, an additional benefit of this approach is the possibility to study the electron transfer process quantitatively. In stationary SERRS spectroelectrochemistry the analysis of a set of spectra acquired at various potentials can assess the Nernstian character the electron transfer process, yielding its E°′ value [5, 14, 19]. Moreover, a time-resolved approach based on potential-jump experiments can give information about the electron transfer kinetic parameters [5, 56].
However, despite the broad potential window investigated (from +0.15 V to −1.0 V) the electrochemical approach appeared to be unsuccessful in reducing the immobilized CYP2D6 completely, and failed to generate any reduced active enzyme at all. In fact, although there is some variability in the spectral data obtained from distinct electrodes, a common feature is the persistence of an intense marker band for the oxidized enzyme at 1,371 cm−1 even at potentials as negative as −1.0 V, together with the appearance of the marker band for a reduced inactive enzyme at 1,360 cm−1 (Fig. 5). An additional feature in the SERRS spectra of reduced CYP2D6 is the lack of any influence of DX (see supplementary material), contrary to what is observed with chemical reduction. Fig. 5Oxidation marker band (ν4) regions of SERRS spectra of CYP2D6 on MUA-coated Ag electrodes at −1.0 V (a), −0.8 V (b), −0.4 V (c) and −0.1 V (d) (vs. SCE), in the presence of buffer. The spectrum at −1.0 V is fitted with Lorentzian functions for the reduced and oxidized forms, represented as dotted lines and labeled with their Raman shift (1,371 cm−1 for the oxidized form and 1,360 cm−1 for the reduced inactive P420 form). Experimental details are given in “Materials and methods”
Although the E°′ of CYP2D6 on MUA-coated Ag electrodes might differ significantly from the E°′ reported for a polyaniline-doped glassy carbon electrode (−0.12 V) [1, 51], the persistence of a consistent amount of oxidized enzyme at −1.0 V suggests an incomplete electron transfer. Despite the large variability of the E°′ values found in the literature, depending on the CYP and on the method employed, no E°′ is reported below −0.6 V [1]. Consistent with this fact, CYP2D6 is effectively and completely reduced by a 5 mg mL−1 dithionite solution having a calculated redox potential of approximately −0.7 V [57]. Therefore, on a pure thermodynamical basis, the application of a potential more negative than −0.7 V to the Ag electrode should lead to a reduction of the adsorbed enzyme. The marked difference in the amount of reduced species obtained with chemical and electrochemical reduction (Fig. 6) might therefore be due to slower kinetics of the electron transfer process when using the second method. Fig. 6Comparison between SERRS spectra of CYP2D6 on MUA-coated Ag electrodes a chemically reduced with dithionite (open circuit) and b at −800 mV (vs.SCE). Both spectra were recorded in the presence of DX. Marker bands for oxidation (ν4) and spin (ν3, ν2) states are indicated. Experimental details are given in “Materials and methods”
The efficiency of the electrochemical reduction via the electrode is likely to be dependent on both the distance and the orientation of the adsorbed protein with respect to the electrode surface [1, 3], whereas these two factors probably do not influence the dithionite reduction process. In vivo, electron transfer between the electroactive heme center, deeply buried inside the protein structure, and the electron-donating reductase occurs via an interaction involving specific domains on the protein surfaces [1, 3], with a strictly defined orientation. Conversely, the electron transfer from dithionite in solution will not be sensitive to the protein orientation, considering the capacity of the reducing agent to approach the enzyme from more directions. Moreover, while the minimum distance between the enzyme active site and the electrode surface is equivalent to the thickness of the MUA coating (approximately 19 Å [22]) plus the heme–protein surface distance, dithionite is free to diffuse closer to the enzyme redox center. Therefore, an unfavorable enzyme orientation with respect to the coated metal surface or a too large heme–electrode distance would be consistent with experimental data, since it would slow down or suppress the electron transfer from the electrode, while dithionite would still be able to effectively reduce the CYP2D6 heme. Moreover, the inability of the electrochemical approach to generate any amount of reduced enzyme in its active form, which is otherwise observed in dithionite-reduced protein samples, suggests that CYP2D6 inactivation is triggered by the application of potentials between −0.4 and −1.0 V during the enzyme-reduction attempts.
The intense electric field present at the interface of the SAM-coated Ag electrodes, which has recently been indicated as the main reason for the extensive conversion of the immobilized bacterial CYP101 to its inactive P420 forms [20], might be the cause of this potential-induced inactivation. However, according to the theory originally proposed by Smith and White [58] and later adapted by Murgida and Hildebrandt [22] and Lecomte et al. [59] to explain the behavior of c-type cytochromes adsorbed on electrodes, the intensity of this interfacial electric field is expected to decrease as the applied potential approaches the potential of zero charge (−0.97 V for a polycrystalline Ag electrode) [13, 60–62]. Therefore, supposing that this theoretical model could be extended to CYPs (as it has been assumed in the case of CYP101 [20]), the interfacial electric field should be more intense at −0.1 V than at −1.0 V, whereas CYP2D6 inactivation occurs at more negative potentials, suggesting that in this case the interfacial electric field intensity as such is not responsible for the enzyme conversion to the P420 form upon electrochemical reduction. This hypothesis would be compatible with the electric-field-induced inactivation reported for CYP101 upon immobilization [20], considering that CYP101 is a soluble cytosolic enzyme, while CYP2D6 is a peripheral membrane protein, and the same interfacial electric field might have a very different effect on their respective active-site structures.
Besides tentatively excluding the interfacial electric field intensity as a cause, SERRS data alone cannot support any explanation for the CYP2D6 potential-induced inactivation. Additional detailed structural data on the secondary and tertiary structures of the adsorbed protein and on the nature and position of the amino acid residues interacting with the MUA monolayer would be highly valuable, and they might be retrieved in further studies using SERRS in combination with surface-enhanced infrared absorption spectroscopy [63] as well as other techniques [64–66].
Conclusions
Upon immobilization on MUA-coated Ag electrodes, the human enzyme CYP2D6 retains its active-site structure, its ability to reversibly bind a substrate (DX) and an exogenous heme ligand (imidazole), and its capacity to exchange electrons, demonstrating the efficiency of the coating to prevent major protein conformational changes or denaturation. However, despite its success in preserving an intact protein structure, the MUA coating appears to be unsuitable for performing direct electrochemistry experiments on immobilized CYP2D6, since its reduced state could not be adequately achieved by varying the electrode potential. The application of potentials from −0.4 to −1.0 V appears to induce enzyme inactivation instead. Among other factors influencing the redox activity, a more favorable enzyme orientation or distance with respect to the electrode surface might be achieved using other coatings, an approach currently under study in our group.
In general, SERRS spectroscopy on coated Ag surfaces was proven to be successful in monitoring the active-site structure of immobilized CYP2D6, enabling the detection of reversible substrate and ligand binding using only very low amounts of protein. Besides its technological relevance as an optical biosensing tool, this technique might have a significant potential for fundamental research on the mechanisms of catalysis for CYPs, as well as for other heme enzymes.
Electronic supplementary material
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surface-enhanced resonance raman scattering