Multi-detector row computed tomography angiography of peripheral arterial disease

With the introduction of multi-detector row computed tomography (MDCT), scan speed and image quality has improved considerably. Since the longitudinal coverage is no longer a limitation, multi-detector row computed tomography angiography (MDCTA) is increasingly used to depict the peripheral arterial runoff. Hence, it is important to know the advantages and limitations of this new non-invasive alternative for the reference test, digital subtraction angiography. Optimization of the acquisition parameters and the contrast delivery is important to achieve a reliable enhancement of the entire arterial runoff in patients with peripheral arterial disease (PAD) using fast CT scanners. The purpose of this review is to discuss the different scanning and injection protocols using 4-, 16-, and 64-detector row CT scanners, to propose effective methods to evaluate and to present large data sets, to discuss its clinical value and major limitations, and to review the literature on the validity, reliability, and cost-effectiveness of multi-detector row CT in the evaluation of PAD.
Introduction
Before multi-detector row CT (MDCT) technology was available, the evaluation of peripheral arterial disease (PAD) using CT was restricted to imaging only a portion of the peripheral arterial tree [1–8]. With the introduction of four-detector row CT (4D-CT) in 1998, this major limitation was overcome. A complete coverage of the lower extremity inflow and runoff arteries was possible with one acquisition using a single-contrast bolus. With the launch of the 16-detector row CT (16D-CT), the spatial resolution increased to near isotropic voxels and the contrast medium efficiency improved [9–11]. True isotropic high spatial resolution of the entire volume was possible using the 64-detector row CT (64D-CT) scanner. In addition, improved X-ray tube capacity and scan speed allow submillimeter acquisition of a large coverage without limitations. These developments made multi-detector row CT angiography (MDCTA) an accurate alternative for the assessment of the peripheral arteries [12–25]. Using standardized scanning and reviewing protocols, peripheral CT angiography is a robust non-invasive technique for evaluating chronic and acute disease of the peripheral arteries. We present a review concerning our experience with 4-, 16-, and 64-detector row CT scanners in patients with PAD.
Technique
Preparation
There are no specific prescanning preparations necessary for MDCTA of the peripheral arteries. The patient is placed comfortably to avoid movement, in the supine position with raised arms on the CT table. The legs are stabilized with cushions around the legs and slightly strapped with adhesive tape distally. It is important that the patient does not wear metal zippers or buttons on their clothing, since this can have a negative influence on the image quality, especially when using postprocessed images. Oral contrast should not be used, as this complicates postprocessing display (Table 1). Contrast material needs to be administered at body temperature to decrease the viscosity. The protocol can be completely programmed into the scanner.
Table 1Prescanning preparationParameterDescriptionClothingNo metal parts on clothingOral contrastNoneI.V. cannula antecubitalMinimally 22 G (0.6 mm inner diameter, blue valve)PositioningSupine, stabilized and lightly strapped, feet-first and arms elevatedRespiratory phaseInspiration during abdominal-pelvic range
Technical parameters
The main challenge for peripheral CT angiography is the great range of the vascular system that needs to be depicted. Using a scanogram of approximately 1,500-mm length, the coverage of the acquisition is planned from the celiac trunk (T12 vertebral body) to the level of the talus using 4D-CT, or to the level of the feet using 16D-CT or higher (Fig. 1).
Fig. 1(a) Scout image with three planned reconstruction batches of the abdomen, the upper legs, and the lower legs to preserve postprocessed image resolution. The frames 3-1, 3-2, and 3-3 depict the field of view of the three data sets, which need to be as narrow as possible to optimize pixel size. Whole-body volume maximum intensity projection (MIP) images after semiautomated bone removal of the abdominal data set (b), the femoral data set (c), and the crural data set (d)
Scan duration
The optimal scan duration for peripheral CT angiography varies between approximately 20 to 40 s, depending on the number of detector rows and the collimation (Table 2). The velocity of a contrast bolus to travel from the aorta to the popliteal arteries varies from 29 to 177 mm/s in patients with PAD [26]. This large variability is unpredictable and does not correspond to the severity of PAD. Based on these bolus travel times, it is recommended to limit the maximum table speed on faster scanners to 30 mm/s to avoid outrunning the bolus, leading to poor distal vessel opacification. This can be obtained, for example, by limiting the gantry rotation speed from 0.33 to 0.5 rotations per second or reducing the pitch (Table 2). Moreover, it is advised to program a second acquisition protocol into the scanner to start immediately if delayed distal enhancement is detected (Fig. 2). Because the time of the contrast bolus to travel from the aorta to the ankles varies from 7 to 40 s, a longer scan duration increases the risk of venous contamination, especially when there is critical ischemia and inflammation [26, 27]. Nevertheless, the discrimination of the arteries from the veins is often possible due to the stronger arterial enhancement and the anatomic 3D information [12].
Table 2Acquisition parameters for various multi-detector row computed tomography (MDCT) configurations for the angiography of peripheral arteriesType of scannerSection collimation width (mm)bRotation time (s)PitchcTable feed (mm/ rotation)Table speed (mm/s)Scan duration (s) dCharacteristics4D-CTa4 × 2.50.51.5153040Slow scan protocol, thick minimal slice width16D-CTa16 × 0.750.51.3153040Slow scan protocol, high resolution16 × 1.50.50.7173435Slow scan protocol, less resolution, better in obese patients16 × 1.50.51.0244825Fast scan protocol, less resolution, reduction of contrast media64D-CTa2 × 32 × 0.60.50.8153040Slow scan protocol, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible2 × 32 × 0.60.330.8164825Fast scan protocol, reduction of contrast media, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible2 × 32 × 0.60.331.019.86020Fast scan protocol, reduction of contrast media, high resolution, isotropic voxel, double z-sampling, scanning of obese patients possible, risk of outrunning the bolusaProtocols designed for Siemens CT scanners (Siemens Medical Systems, Erlangen, Germany) and should be modified appropriately for other models and manufacturersbValues are number of sections times section widthcPitch as the ratio of the table feed per rotation over the total width of the collimated beamdScan times representing a scanned range of 120 cmFig. 2Images from the first and second delayed acquisition of a 37-year old male with blue toe syndrome of the left hallux. (a and b) VRT images of the first acquisition show in the aneurysmatic abdominal aorta a short occlusion of the left femoral artery (white arrows) due to thrombo-embolism and an occlusion of the entire right superficial femoral artery. The anterior tibial arteries seem occluded in both legs. (c and d) VRT image of the feet with the first and the delayed second acquisition. The first acquisition (c) shows that the arteries of the feet are not enhanced yet due to slow flow (asterisk). The delayed acquisition (d) shows that both dorsal pedal arteries are patent and that the proximal arcuate artery and the first dorsal metatarsal artery (black arrows) of the left foot are occluded due to thrombo-embolism
Using recent MDCT scanners, fast scans (25 s or less) can be performed of the peripheral arteries to reduce the amount of contrast media. To allow fast scan speed using 16-detector row CT (16D-CT) scanners, a wider collimation must be used (Table 2). The 64-detector row CT (64D-CT) even allows to perform fast scans while maintaining submillimeter collimation. However, to ensure distal opacification, the scanning delay must be increased appropriately in fast scans. Another difficulty of a fast scan is that there is a greater risk of asymmetric enhancement in patients with severe unilateral vascular disease. Therefore, it is safer to choose a slower scan speed.
Collimation
To aim for maximal spatial resolution, a thin section collimation width allows a narrow effective slice width. Furthermore, the partial volume effect and blooming effect of calcium will be reduced (Fig. 3) [10]. The collimation should be chosen as narrow as possible but still allowing for a table speed of 30 mm/s and depends on the number of detector-rows and heat capacity. On a 4-detector row CT (4D-CT), the collimation is limited to 4 × 2.5 mm, whereas the 16D-CT and 64-detector row CT (64D-CT) allow a submillimeter collimation of 16 × 0.75 mm and 32 × 2 × 0.6 mm, respectively.
Fig. 3a, bImages of 16-detector row CT (16D-CT) acquired with a collimation of 0.75 mm showing the effect of slice width (SW) on the blooming of the arterial wall calcifications. (a) Reconstructed axial image of the right external and internal iliac artery with SW of 3.0 mm using a B46 reconstruction kernel shows more blooming of the calcifications than (b). (b) Reconstructed axial image of the right external and internal iliac artery with SW of 0.75 mm using a B46 reconstruction kernel with less blooming of calcifications
Using 16D-CT in obese patients, the thin collimation protocol leads to unacceptable noise levels in the abdomen and pelvis because the tube is unable to deliver the necessary dose in this submillimeter configuration. In order to enable the tube to deliver a higher dose, a wider collimation (16 × 1.5 mm) with a reduced pitch factor of 0.7 (Table 2) is used to improve the image quality in obese patients. For the 64D-CT scanner, there is no longer a tradeoff between resolution and scan speed, and it allows, even in obese patients, a fast submillimeter scan protocol.
Contrast injection
It is important in peripheral CT angiography to obtain a high and homogenous enhancement of the arterial tree and to synchronize the acquisition with the enhancement. The optimization of acquisition timing and contrast medium delivery is essential for vascular assessment and image postprocessing. Normally, attenuation values higher than 200 HU in the arteries is considered suitable in MDCTA [12, 13]. For the intravenous injection of contrast medium in the antecubital vein, 22- and 20-gauge intravenous cannulas are needed for the maximal flow rates of 3.5 and 5.0 mL/s, respectively.
Acquisition timing
Due to the interindividual hemodynamic variability in peripheral CT angiography, reliable timing techniques are preferred over using a fixed delay. The test-bolus technique relies on the dynamic monitoring of small contrast boluses to measure the contrast arrival and travel time at the proximal and distal arteries, respectively. The bolus-triggering technique is a commonly used timing technique that is based on repetitive low-dose sequential scans at the level of the abdominal aorta, to monitor the arrival time of the contrast media. The acquisition starts automatically when the preferred threshold is reached, approximately 100 to 150 HU above the baseline value. During a transition delay, which is the time needed for the table to move and start the scan, of approximately 4 s, breathing instructions can be given to the patient. During this delay, the enhancement of the aorta will further increase to an absolute value of more than 200 HU.
For a fast scan protocol, an extra delay must be added to the contrast arrival time to ensure distal arterial opacification [28]. This extra delay can be calculated as 35 s minus the scanning time. Thus, for a scan time of 25 s, a extra delay of 10 s. must be added. Another option is to monitor at the proximal level of the popliteal artery and to start the scan manually when enhancement is visualized. Consequently, the time of contrast arrival increases by approximately 8 s [26, 27] and the transition delay of the scanner increases to 11 s to travel from the knees to the diaphragm and then starting the acquisition.
Contrast injection
The volume of contrast material ranges from 120 to 160 ml for a typical scan duration of 40 s. The amount of contrast media depends on the scan duration and on the flow rate. Because the last volume of the bolus will not contribute to the enhancement when scanning below the knees, the injection duration can be shortened by 5 s, e.g., a 35-s injection time is used for an acquisition of 40 s. However, to ensure the enhancement of all arteries, the injection duration should not be shorter than 30 s and in fast scan protocols, a delay time needs to be added appropriately to prevent outrunning the contrast bolus. A flow rate of 3 to 4 ml/s is necessary for adequate arterial enhancement [12]. This corresponds to an iodine administration rate of 1.0 to 1.4 g/s using a contrast media concentration of approximately 320 to 350 mg I/mL. Based on the reported literature the average values of contrast media volume, concentration, injection rate, and administration rate are 134 ml, 341 mg I/mL, 3.5 ml/s, and 1.2 g/s, respectively [9–25, 29–36]. By increasing the iodine concentration to a concentration of 400 mg I /mL, the iodine administration rate can be increased to 1.6 g/s to increase the enhancement [37]. To optimize the enhancement, 20 to 60 mL of saline is injected immediately after the contrast media. A tighter bolus can be obtained to increase the attenuation.
Using a monophasic injection rate, the arterial enhancement increases over time to decrease at the end of the bolus. Consequently, the Hounsfield values of the enhanced arteries start lower at the level of the aorta and increase at the level of the popliteal artery to the highest attenuation value, and, subsequently, decrease distally in the runoff arteries, especially for longer scan durations [26]. A more homogenous enhancement can be achieved using a biphasic injection rate using a higher rate (5–6 ml/s) at the beginning (during the first 5 s) of the injection and a lower rate (3 ml/s) for the remaining volume. In clinical practice, a monophasic injection rate is often used because it is a simple method and has resulted in adequate image quality [37].
Patient dose in MDCT
A particular concern with MDCT scanners is delivering potentially higher radiation doses. To maintain the noise level in submillimeter slices, the dose needs to increase proportionally. On the other hand, with the increasing number of detector rows, the z-axis efficiency improves, since the overbeamed area decreases. Current MDCT scanners present an indication of patient dose on the scanner console for dose awareness and to help optimize the scan protocol. Useful in CT angiography is that, when reducing the X-ray energy, the contrast-to-noise ratio increases. Compared to a standard scan with 120 kVp, selecting 100 kVp, results in a dose saving of approximately 40% [38–40]. Furthermore, dose reduction can be achieved by decreasing the tube current using automatic tube current modulation. With angular tube current modulation, the tube current varies during the course of a rotation. The changing attenuation through different projections around the patient (e.g., at the level of the pelvis) can be used, to reduce unnecessary x-rays in the anterior–posterior projection without any substantial effect on image quality [9, 41, 42]. With longitudinal tube current modulation, the tube current varies along the z-axis based on the size, shape, and attenuation to maintain a predefined noise ratio. Compared with constant tube current, this technique results in acceptable image noise and a dose reduction of 20% or more without compromising diagnostic image quality [9, 41, 42].
The average patient dose reported in the literature in the assessment of PAD with CT angiography is 7.47 mSv [9, 12, 24, 31, 43]. The radiation risk from these doses is not a major concern in patients with PAD. Their life expectancy is shorter than the latency period of a radiation-induced fatal malignancy [44–46].
Display and evaluation
Image reconstruction
The raw data set is reconstructed using an increment with 50% to 70% overlap. Peripheral CT angiography generates more than 1,500 axial images, depending on slice width and reconstruction increment. It is recommended to reconstruct separate data sets. Routinely, we calculate three separate data sets of the peripheral runoff (Fig. 1). The first advantage is that it allows us to reconstruct thicker slices, e.g., of 1.5 mm for the abdominal and femoral data set, and thinner slices for the crural data set to optimize the resolution and to minimize the data load [9, 10, 16, 20, 23]. Secondly, longitudinal images that are calculated from the entire data set have a decreased resolution, due to the limited display matrix (e.g., 512 × 512) [32]. Images that are reconstructed from the separate data sets will preserve the initial longitudinal resolution.
A smooth kernel (B20 for Siemens CT scanners) is generally used in CT angiography and leads to an accurate depiction of the diameter of the vessels and is very appropriate for postprocessing. A sharp kernel (B46) is used when stents or severe vessel wall calcifications are present, as it minimizes the blooming effect at the cost of some increase in the noise level [47].
The field of view (FOV) is selected as small as possible to optimize pixel size. A FOV of 380 mm, 350 mm, and 300 mm for the abdominal, femoral, and crural data sets, results in pixel sizes of approximately 0.74 mm, 0.68 mm, and 0.58 mm, respectively. Also, the FOV can be further decreased to 200 mm by including only one leg, leading to a pixel size of 0.4 mm.
Advanced postprocessing and image evaluation
Additional two-dimensional (2D) and three-dimensional (3D) postprocessing techniques are required to facilitate interpretation and presentation. Reviewing exclusively the transverse images is inefficient and less accurate than reviewing a combination of reformatted images.
To preserve the study quality for clinical decision making, a standard set of postprocessed images needs to be included in the protocol. These include thin-slab maximum-intensity projections (MIPs) through visceral and renal arteries and the abdominal aorta, through femoropopliteal arteries, and through crural arteries (Fig. 4); whole-volume MIPs of the separate data sets after bone removal (Fig. 1) and when necessary, after removal of vessel wall calcifications (Fig. 5); and curved planar reformations (CPRs), e.g., through the iliac arteries. Volume-rendered (VR) images are fast and effortless created to present the pathology to clinicians, who normally do not have the possibility to review the data set interactively.
Fig. 4a–dStandard slab MIP images in the postprocessing protocol make MDCTA of peripheral arteries on a routine basis feasible. Slab MIPs are easy and fast to create and to evaluate. The images depict the vasculature without superimposing bones (a and b). From the abdominal data set, MIP images are created in coronal projection to depict the renal arteries (b) and in sagittal projection to depict the celiac trunk and mesenteric arteries (d). The aorta is also depicted for evaluation. (c) Standard coronal slab MIPs from the data set of the upper legs are created, which are parallel to the superficial femoral and popliteal artery. (d) Standard coronal slab MIPS from the data set of the lower legs display the crural arteriesFig. 5a–dVolume MIP images in anteroposterior projection show the result of three different threshold levels used for the segmentation of arterial wall calcifications. (a) Volume MIP before removal of the calcifications shows that the lumen is not visible. (b) Volume MIP after removal of the calcifications shows that, still, many voxels of calcification are present, hampering lumen assessment (arrows). (c) Volume MIP shows angiogram after calcium segmentation using a correct threshold level allowing lumen assessment. The rest of the voxels of the burden of calcifications are just visible as unesthetical noise, which is, however, preferable to introducing pseudo-stenoses (d) (arrows) by using a too low threshold level
The data sets are reviewed effectively by evaluating the standard set of postprocessed images and, interactively, exploring the data set using multiplanar reformations (MPRs) . The transverse images (or true cross-sectional images) need to be considered to verify diseased segments [4, 6, 19].
When extensive calcifications or stents are present, the vessel lumen visibility and the clinicians’ confidence in the CT images will decrease [25]. In whole-volume MIPs, superimposing calcifications can be selected to be removed digitally from the data set using thresholding and region-growing techniques (Fig. 6). However, the removal of the numerous arterial wall calcifications can be very time consuming. Another limitation of these segmentation techniques is that readers should be aware of artificial stenoses and occlusions. These can be introduced when voxels that represent lumen are inadvertently removed when in close contact with the bones (Figs. 7 and 8) or when a too low threshold value is used (Fig. 5). In addition, in VR images, the lumen is also obscured by vessel wall calcifications and, as a result, should not be used for the lumen assessment (Fig. 6). A more reliable technique for stenosis detection in extensive calcified arteries is CPR, which displays the lumen as a longitudinal cross-section (Fig. 6). When using an application that semi-automatically traces the vessel lumen, the risk of an inaccurately positioned central lumen line is minimized. The CPR projection should include at least two perpendicular longitudinal projections and true cross-sectional images can be viewed for lumen assessment [19]. Software tools are available for automatic quantitative evaluation of the traced lumen and to generate a graphical presentation of luminal diameter (Fig. 9). Multipath CPRs are under development and could enhance image evaluation.
Fig. 6a–dInfluence of vessel wall calcifications on postprocessed images and the ability for lumen assessment. (a) VRT image (medial view) of right femoropopliteal segment showing arterial wall calcification; does not allow luminal assessment. (b) CPR image (anteroposterior view) shows the interior of blood vessels as a longitudinal cross-section, even in the presence of the arterial wall calcifications. This is the preferred imaging technique when extensive calcifications of the vessel wall are present. Volume MIP (anteroposterior view) after bone removal using region-growing and threshold techniques (c) does not allow lumen evaluation. Volume MIP after additional calcification removal (d) removes superimposing calcification to enable lumen evaluationFig. 7a–cImages of segmentation artifacts due to bone removal in 16D-CTA. (a) Volume MIP after bone segmentation of the lower legs showing a pseudo-occlusion of both distal anterior crural arteries (arrows), which is caused by segmentation of the bones. (b) MIP of the lower legs showing the anterior tibial arteries in close proximity to the tibia (arrows), which is the cause of the false positive pseudo-occlusion. (c) Axial image of the lower legs just caudal from the pseudo-occlusion, showing the patency of both anterior tibial arteries of both legs in close proximity of the tibia (arrows)Fig. 8a–cApplying blue color to the voxels selected for removal helps to identify the sites of segmentation artifacts in VRT images. (a) VRT image before bone segmentation of the lower legs showing patent proximal anterior tibial arteries. (b) VRT with blue bones to indicate the voxels to be removed shows the voxels of the bone which are in contact with the proximal anterior tibial artery are not selected for removal and shows the voxels of the artery which are selected for removal. (c) Segmented VRT image showing the pseudo-occlusion of the anterior tibial arteryFig. 9a, bResults of semiautomated quantitative lumen assessment in aortoiliac arteries of a patient with in stent thrombosis. (a) Graph (upper section) displaying the maximum and minimum diameters of the lumen to quantify stenosis. CPR (lower section) through the aortoiliac arteries, which can be rotated around its longitudinal axis, depicts the luminal obstruction (asterisk) due to a thrombus inside an iliac stent. (b) Corresponding transverse image confirms the occlusion of the iliac stent
Wall calcification problem
The depiction of vessel wall calcifications using MDCT can be valuable, since severely calcified arteries may have consequences for bypass surgery. On the other hand, these wall calcifications are known to hamper the assessment of the lumen [2, 10, 14, 19]. Approximately 20% to 50% of the vascular segments contain wall calcifications, of which, 10% severely calcified [11, 19]. Patients with a history of diabetes mellitus, cardiac disease, or elderly age are very likely to have extensive calcifications [48]. Furthermore, we found that patients with Fontaine stage III/IV have more infrapopliteal arterial wall calcifications compared to stage IIb.
How can we deal with the vessel wall calcifications depicted with MDCTA? It is important to use a wider window width (WW) and higher window center (WC) level settings from the usual CT angiography level of around 150 WC ± 250 WW to 200 WC ± 1000 WW for a better differentiation of calcifications and stents from the enhanced lumen and to minimize the effect of blooming. A further minimization of blooming is reached by using a sharper reconstruction kernel and higher spatial resolution.
Especially in MIP images, the lumen is hidden by the circumferential calcifications. In these circumstances, transverse images, CPR images, and the digital removal of the calcifications help to depict the lumen, at least for the larger arteries. Despite all of the available tools, in particular in the smaller crural arteries, the concentric calcifications still hamper lumen assessment [11]. Recent publications showed that a subtraction technique using two acquisitions is feasible in some patients with PAD using MDCTA [24, 49]. In the near future, automated 3D applications could help to minimize the impediment of the calcifications [50]. Whether dual-energy CT angiography can improve this limitation of CT needs to be evaluated.
Clinical value
Because MDCT angiography for the imaging of the peripheral arteries is a rather new non-invasive technique, there are a small number of studies published on its performance and reproducibility (Table 3). The majority report on 4D-CT; two authors report on 16D-CT. There are no reports of the assessment of PAD using 64D-CTA. In our meta-analysis soon appearing in Radiology, which included 436 patients and 9,541 arterial segments, a pooled sensitivity and specificity for detecting a >50% stenosis of 92% and 93% was estimated, respectively.
Table 3Validity of CT angiography in peripheral arterial disease (PAD)AuthoraNo. of patientsNo. of analyzed segmentsNo. of detectorsReported sensitivity (%)eReported specificity (%)eAssessed segmentsStenosis category (%)hRichter et al. 199432ns184nsIliac>50Lawrence et al. 1995613419396Femorocrural>50Raptopoulos et al. 19963962419396Aortoiliac85–99Rieker et al. 199650400173–88b94–100bFemorocrural75–99Rieker et al. 19973021019399Aortoiliac75–99Kramer et al. 199810ns294nsIliocrural>90–99Ishikawa et al. 199949ns19795Bypass graftsnsBourlet et al. 20002231819590Aortoiliac>50Puls et al. 20013118648986Total tree50–99Willman et al. 20034676949199Aortoiliac graftsnsOfer et al. 20031841049192Total tree>50Heuschmid et al. 200318568491c92cTotal tree>50Martin et al. 2003411,31249297Total tree75–99Catalano et al. 2004501,14849693Total tree>50Mesurolle et al. 20041616829193Total tree>50Ota et al. 20042447049999Total tree>50Poletti et al. 2004d12144482/96gnsns>50Portugaller et al. 20045074049283Total treearea >70Romano et al. 2004423,40249395Total treensRomano et al. 2004221,78249294Total treensStueckle et al. 200452ns482100Total treensEdwards et al. 2005441,02447993Total tree50–99Fraioli et al. 2006751,425496–99h94–96hTotal tree50–99Schertler et al. 200517170169690Popliteocrural>50Willmann et al. 2005391,365169696Total tree>50Unpooled mean9194aBased on references [1–6, 8–10, 13–24, 31, 34, 35, 56, 57]bFor various anatomic levelscCalculated from the datadBased only on subtracted MDCTA images, the positive predictive value was 95%eSensitivity as published or calculated overall mean.fDiameter stenosis is mentioned unless specified (>50 means stenosis more than 50% including occlusion)gFor subtracted and nonsubtracted segments, respectivelyhDepending on the MDCTA protocol with varying mAs
Publications on the reproducibility of CT angiography reported a good intertest agreement between MDCTA and DSA (Table 4) and a good to excellent interobserver agreement for 4D-CTA [12, 21, 23, 35] and 16D-CTA (Table 5) [9, 11]. A few studies provide stratified data on the aortoiliac, femoropopliteal, and crural tract and show that the accuracy and reproducibility of the crural tract is lower than for the aortoiliac and femoropopliteal tracts [9, 11, 20, 22, 23].
Table 4Intertest agreement between CT angiography and digital subtraction angiography in PADAuthoraNo. of patientsNo. of assessed segmentsNo. of detectorsReported intertest agreementdAssessed segmentsRaptopoulos et al. 199639624190%AortoiliacBeregi et al. 199720521100%PoplitealTins et al. 200135219184%AortoiliacWalter et al. 2001224564κ = 0.68 (0.50–0.97)cTotal treeRubin et al. 2001183514100%Total treeHeuschmid et al. 2003231,136486%Total treeOfer et al. 200318444478%Total treeRomano et al. 2004423,4024κ = 0.68; 90%Total treeRomano et al. 2004221,7824κ = 0.68; 90%Total treeaBased on references [3, 7, 12, 14, 16–18, 35, 55]bBased on 97% of the segmentscAverage of the reported kappa values (ranges) of the individual anatomical segmentsdAn unweighted kappa statistic (κ) is reported for percentage agreementTable 5Interobserver agreement of CT angiography in PADAuthoraNo. of patientsNo. of analyzed segmentsNo. of detectorsReported interobserver agreementbAssessed segmentsRieker et al. 1997302101ρ=0.95AortofemoralWalter et al. 2001224564κ = 0.71–0.76cTotal treeTins et al. 200135219178%AortofemoralMartin et al. 2003411,3124κw = 0.84Total treeRomano et al. 2004423,4024κ = 0.84;0.86dTotal treeRomano et al. 2004421,7824κ = 0.85, 0.88, κ = 0.80eTotal treeCatalano et al. 2004501,1374κ = 0.80Total treeOta et al. 2004244704κ = 0.88IliacPortugaller et al. 2004507404κ = 0.81Total treeKock et al.f732,2684κw = 0.84Total treeOuwendijk et al. 2005792,41916κw = 0.85Total treeWillmann et al. 2005391,36516κ = 0.85–1Total treeaBased on references [4, 7, 9, 11, 15, 17–19, 21, 23, 35]bAn unweighted kappa statistic (κ) is reported, unless indicated (κw=weighted kappa statistic; ρ=intraclass agreement coefficients as a measure of agreement for ordinal or quantitative data). A linear weighting was used, except in one paper [15], where a quadratic weighting was usedcRange of kappa values of the individual anatomical segmentsdFor reader one and two, respectivelyeFor intraobserver (two readers) and interobserver agreement, respectivelyfBased on unpublished data
MDCTA leads to adequate decision making for treatment recommendations concerning both the anatomical level and the technique of revascularization [51]. A cost-effectiveness study showed that MDCTA is a cost-effective diagnostic strategy in the work-up of PAD [52, 53]. Randomized controlled trials confirmed that MDCTA in PAD is the optimal diagnostic imaging technique [25, 54] and reduces the diagnostic costs when compared to DSA and CEMRA with comparable clinical utility and patient outcomes. Besides these evidence-based results, local expertise and availability also define which modality to use in clinical practice (Table 6).
Table 6Advantages and limitations of multi-detector row CT angiography (MDCTA), contrast enhanced MR angiography (CEMRA), and digital subtraction angiography (DSA) MDCTACEMRADSAIntermittent claudication (Fontaine II)+++Chronic critical ischemia (Fontaine III or IV)−++Short examination time+−−Short postprocessing time−++Outpatient setting++−Availability+−+Non-invasive technique/patient comfortb++−Low diagnostic imaging costs+−−Contrast media tolerance−+−Three-dimensional imaging++−Non-interference of stentsc+−+Radiation risk+(−)d−+(−)dAcute clinical setting+−+Hemodynamic assessment−−a+Extraluminal pathology visualization+−a−aIs only possible when using additional sequencesbFrom [58]cFrom [59]dNegligible risk in population with chronic obstructive PAD
It is reported that arterial wall calcifications lead to false-positive interpretations and a decreased reproducibility in reading MDCTA [2, 14, 11]. We have to acknowledge this limitation with current technology. A preferential indication for MDCTA in patients with intermittent claudication (Fontaine stage IIb) is clearly justified. However, patients with critical limb ischemia (Fontaine stage III/IV), who are likely to have extensive calcifications of the smaller arteries, could be better off undergoing contrast-enhanced magnetic resonance angiography (CEMRA) or digital subtraction angiography (DSA).
Finally, MDCTA is an accurate technique to evaluate the patency after revascularization procedures [43]. The technique can be used in the evaluation of acute ischemia, e.g., after a revascularization procedure or in thrombo-embolic disease (Fig. 10). For aneurysmatic popliteal artery disease or entrapment syndromes of the popliteal artery, MDCTA is the preferred imaging modality (Fig. 11) [55].
Fig. 10a, bAcute thrombosis of the crural arteries in a 53-year-old woman with an acutely cold left leg after stopping anticoagulation therapy. The patient refused angiography. (a) VRT image (posteroanterior view) of MDCTA at the level of the crural arteries shows abrupt stoppage of arterial opacification in the left peroneal, anterior, and posterior tibial artery (arrows). The contralateral right crural arteries are patent. (b) Selective anterograde DSA image (posteroanterior view) confirms the occlusions of the three left crural arteries (arrows) due to thrombo-embolismsFig. 11a–dA 56-year old male patient who had a history of deep venous thrombosis with intermittent claudication of the right lower extremity. (a and b) Thin MIP image shows an aneurysmatic right popliteal artery with a tight stenosis distally. (c) VRT and volume MIP (d) confirm these findings and show patent proximal crural arteries
Conclusion
Multi-detector row CT angiography (MDCTA) is an outstanding non-invasive imaging test in the evaluation of patients with peripheral arterial disease (PAD) and is currently the modality of choice in patients with intermittent claudication. The technique can be used in the evaluation of patency after revascularization procedures and in acute ischemia. MDCTA has been shown to have high diagnostic performance and reproducibility in evaluating peripheral arterial disease (PAD). MDCTA reduces diagnostic costs and provides adequate information for decision making. The most important drawback is the limited lumen evaluation of extensive calcified arteries. MDCTA appears to be clinically less valuable in critical limb ischemia because of extensive crural artery calcifications.

tomography
sensitivity and specificity
human
peripheral vascular diseases
radiography
x-ray computed methods
reproducibility of results


Ophthalmologists, suicide bombings and getting it right in the emergency department

Background The number and extent of worldwide suicide attacks has risen sharply in recent years. The objectives of this retrospective study are: to determine the prevalence and outcome of the victims who sustained ocular injury, to describe the activities of ophthalmologists in the setting of an emergency department (ED) receiving mass casualties of a suicide bombing attack and to illustrate some of the treatment obstacles that they encountered and the protocol.
The number and extent of worldwide suicide attacks has risen sharply in recent years [1, 2]. The perpetrators typically mingle among crowds of civilians and detonate an explosive device that is usually strapped on their bodies with the intent of sacrificing their own lives in order to cause the death of as many others as possible. The injuries sustained by survivors of these well-planned attacks combine the lethal effects of penetrating trauma, blast injury, and burns [3]. Suicide attacks on civilians were historically confined to a limited number of countries, but the outrageous and devastating destruction of the Twin Towers in New York City on 11 September 2001 and the bombings in London and Madrid established the universality of such terrorism. With the rise in terror-related activities in urban settings, ophthalmologists worldwide may find themselves treating ocular trauma under conditions unlike any they had experienced before, and certainly remote from the relatively orderly setting of an emergency room in which traumatic ocular injuries can usually be counted on one hand at any given time.
Since the beginning of the latest Israeli-Palestinian Intifada on 29 September 2000, more than 4,600 people—mostly civilians—have been killed or injured by suicide bombings in that area [4]. The Tel Aviv Sourasky Medical Center (TASMC) is the largest hospital in the Tel Aviv metropolitan area. As such, most of the victims of the suicide bombing attacks in its catchment area are evacuated to TASMC, and a special treatment protocol has evolved for coping with the unusual logistic needs of such events. The objectives of this retrospective study are: to describe the activities of ophthalmologists in the setting of an ED receiving mass casualties of a suicide bombing attack, to illustrate some of the treatment obstacles that they encountered and the protocol that evolved for overcoming them, and to determine the prevalence and outcome of the victims who sustained ocular injury.
Methods
IRB approval was obtained for this retrospective interventional case series study. Clinical data on all casualties evacuated to the TASMC due to suicide bombing-related injuries were collected from the trauma registry records and reviewed. Their demographic data were obtained from the main admitting office records.
The senior surgeon stationed at the ED entrance is rapidly provided essential information on the type/location of injuries from the arriving ambulance’s paramedical personnel. He/she pages the designated on-site specialist according to prioritization for urgent management.
Ocular injuries are defined as any blunt, penetrating, or perforating trauma or blast-related damage to the eye, orbit, or ocular adnexa. The terms we used to describe ocular injuries conform to the recommendations the United States Eye Injury Registry and the International Society of Ocular Trauma [5]. Individuals with nonpenetrating or non-lacerating ocular injuries, non-penetrating debris in their eyelids, or superficial burns of the eyelids were excluded from this study, as were victims who died of their injuries before undergoing ophthalmologic treatment for whatever ocular damage had been suffered.
The ocular injuries for each eye were categorized according to type: it was possible to have multiple occurrences of the same type (i.e., multiple corneal lacerations) and of several types (i.e., corneal laceration and retinal detachment) in the same eye.
All records of ocular- and orbital-related trauma that were documented in the trauma registry were collected and analyzed together with hospital and outpatient clinic records. The analysis included age, gender, mechanism of injury, anatomic site of injury, Injury Severity Score (ISS) [6], length of stay, length of intensive care unit stay, and surgical procedures. Ophthalmic information included the initially diagnosed ocular condition, all surgeries performed during the ED stay and afterwards, and final ocular test findings. The data were entered using Excel spreadsheet (Microsoft Office) and a simple descriptive statistical analysis was performed.
Results
There were 13 suicide bombing attacks in the Tel Aviv metropolitan area between October 2000 and October 2004. A total of 352 patients were evacuated to the TASMC ED, and 198 of them were hospitalized. The other 154 patients suffered from minor injury or shock for which they were given appropriate treatment and instruction and sent home. The overall severity of suicide bomb-related trauma was very high: the mortality rate was 8.4% when the attack occurred in open spaces, 15.5% in closed spaces and 20.3% when the bomb exploded inside a bus. The ISS was 1–14 for 74% of the patients (non-hospitalized) and ≥16 for the remaining 26% of the patients (admitted to hospital). One of the prominent hallmarks of suicide bombing injuries is the extremely high prevalence of head injuries: among our patients, 49% suffered from head and neck injuries, 9% head and extremity injuries, 4% head and torso injuries, and 38% torso and other injuries (all data are taken from the experience with the 13 bombings in Tel Aviv).
Seventeen patients (4.8%) were listed in the trauma database as having any ocular or periocular trauma, and several had more than one type of injury. The types of recorded trauma were: open globe injuries (n=7), closed globe injury consisting of severe subconjunctival hemorrhage (n=2), partial thickness lamellar laceration of cornea (n=8) (lamellar flap) of which five were burn-related and three were due to small foreign bodies, and extraocular injuries (n=6), which included three orbital fractures due to primary blast injury and three eyelid lacerations. Primary repair of open globe was performed in six eyes that underwent primary closure of laceration. One patient who was diagnosed during the initial triage as suffering from open globe injury died during the initial trauma surgery, thus no ocular procedure was performed. Two other eyes underwent primary exploration of subconjunctival hemorrhage that was suspected as being open globe due to massive subconjunctival: no laceration was found intraoperatively, and contusion was diagnosed. The one eye that was found to be unsalvageable underwent primary enucleation.
After the initial eye surgery, two patients died from their other injuries within 24 h of the explosions.
Of the eight patients with partial thickness lamellar laceration of cornea, three were discharged and given instructions to return as outpatients on the following day. Five other patients who required and received medical treatment for non-ocular-related medical problems were hospitalized and continued eye treatment as inpatients. All patients in which superficial burns were found (n=5) were treated by manual removal of corneal foreign body and antimicrobial drops, and their recovery was uneventful.
Secondary surgical intervention was performed on four patients (all surviving patients who initially underwent primary closure of open globe) within 1 week of the initial trauma: large intravitreal foreign bodies were extracted in three of them, and an intra-lenticular foreign body was extracted from the fourth (Table 1). At final follow-up (≥2 years post-trauma), the visual acuity of the three patients who suffered from large intravitreal foreign bodies were finger counting (FM)-hand movement (HM). The silicone oil was removed in two of these patients and retained in the third; two of them are wearing large cosmetic contact lenses due to corneal opacities that were cosmetically disturbing. The patient who underwent surgery due to intra-lenticular foreign body and penetrating keratoplasty had final visual acuity of 6/12 (Table 1).
Table 1Relevant data of suicide bombing survivors who sustained open globe injuryNo.Age, years/sexPrimary surgical interventionSecondary surgical interventionForeign body extractedFurther surgeryFinal outcomeFinal visual acuity117/fClosure of open globePatient died within 24 h of trauma216/fNonePatient died within 24 h of trauma362/mEnucleation417/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionMetal shrapnell (5 mm)Silicone oil removalPreserved globe flat retinaFC518/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionMetal ball (2 mm)Preserved globe flat retinaHM619/fClosure of open globeLensectomy, vitrectomy, removal of foreign body, endolaser silicone oil injectionGlass fragment (3 mm)Silicone oil removalPreserved globe flat retinaHM727/mClosure of corneal perforationPenetrating keratoplasty, lensectomy, vitrectomy endolaser, intraocular lens implantationGlass fragment (1 mm)6/12FC, finger counting; HM, hand movement
Discussion
The dynamics of an emergency receiving center of victims of a suicide bombing attack are alien to most ophthalmologists. Today, the setting is characterized by large numbers of victims who sustain injuries that are more complex and more severe than those that had occurred during earlier periods of terror activity [7, 8]. Importantly, suicide bombings are more likely to occur in closed spaces, unlike other mass trauma scenarios such as car bombs, train wrecks, and other outdoor explosions: over 62% of injuries that occur in closed spaces are to the face, head, and neck, thus posing a far greater risk to the ocular structures.
In our experience, 17/352 (4.8%) of the survivors of suicide bombing attacks sustained eye injuries, and 9/17 (52%) required urgent attention. This rate is surprisingly lower when compared to previous reports, which documented that close to 10% of survivors of terrorist blasts have significant eye injuries [13]. We have no explanation for the low rate, but we can speculate that because of their close proximity to the hospital together with the efficiency of the Israeli Magen David (Red Cross), almost all survivors are speedily brought to the hospital for examination after terror-related episodes and are listed as admissions in the trauma records, even those with no complaints or only minor ones. Thus, the large number of admitted individuals artificially decreases the percentage of eye victims. Importantly, 41% of all of the reported ocular injuries [9–13] were severe in degree.
The final visual outcome of all the surgeries we performed was poor: globe preservation was successfully achieved in most cases (6/7), but only one patient with an intra-lenticular foreign body had useful vision postoperatively.
In order to provide the best treatment in such a complicated setting, special adaptations must be made to the treatment algorithm of the ophthalmology team. In terms of individual trauma cases, the victim of a suicide bombing attack is no different from any other eye trauma patient. The sudden presentation of large numbers of injured patients, however, presents two types of challenges: the logistical one of rapidly processing masses of casualties through the system and the medical one of providing the best possible trauma care to severely wounded patients [14]. According to our protocol, all patients who complain of eye symptoms and all unconscious patients who sustain head or face injuries must be checked by an ophthalmologist. This requires special disaster on-call lists of ophthalmologists who are able to arrive to the hospital on extremely short notice since, thanks to the highly efficient organization of our Red Shield ambulance facilities, blast victims usually arrive at the hospital within minutes and are hurried to either diagnostic tests or directly to operating theaters. Upon arrival to the ED, every victim of a terrorist attack is triaged by a senior surgeon who synchronizes the activities of the multifaceted operation. The ophthalmologists already present in the hospital and the ones on-call who arrive to the ED are in contact with that surgeon in order to expeditiously locate the victims with ocular injuries, examine them, and send them to the operation theaters, intensive care units, imaging studies or home. There is a directive in our department that all available staff members must contact the hospital immediately upon learning about any suicide bombing attack to check whether their services are required. In the event of large-scale attacks, they are prepared to be recruited to assist in triaging and in treating all the victims, not just those with ocular injuries. The triage procedure is the key to the successful management of large trauma events: the most important rule is that all patients must be checked by the ophthalmologist wherever they are located on the hospital premises. Trauma patients invariably require urgent treatment and some are sent directly from the ER to either imaging units or immediately to the trauma surgical unit. The senior ophthalmologist on the premises must contact trauma registration services, get a list of all admitted patients (usually assigned numbers upon admission) and make sure that each and every one of them is examined, even during emergency surgery or during imaging interventions for non-ophthalmological injuries. When an open globe is suspected, the eye is immediately patched, and the finding is reported to the surgeon in charge of the patient: the staff is instructed not to intervene in the treatment of the eye. Further evaluation is done only when it is certain that there is no danger of expulsive hemorrhage.
Only patients suspected as suffering from open globe injury undergo urgent primary closure of the wound. Since that patient invariably presents with multiple injuries and may not be fit for transfer to the ophthalmology operating theater, however, special alterations to the surgical protocol may be required. For instance, no ophthalmological microscope is available in our trauma center because space is limited in the trauma room due to the concomitant performance of many surgeries and given the cumbersome structure of an ophthalmic microscope. There is, however, a high-quality neurosurgical microscope that has a long arm that can be placed at sufficient distance from the patient and the life-support machines so that it can be used without disturbing the anesthesiologists and other trauma teams as they work, and this microscope is used with great success during primary closure. Other eye surgery procedures are postponed, either until the patient has been stabilized or they are scheduled for a later date. This highlights the first critical responsibility of the ophthalmologist in the mass trauma setting, that of identifying which surgical procedures must be carried out immediately. The order of surgical intervention deserves special attention: due to the characteristic complexity of the injuries, most of the patients required multiple procedures immediately following the trauma. The established protocol adopted among our surgeons is (in descending order): trauma surgery (for life-threatening conditions, performed by either trauma surgeons or neurosurgeons), ophthalmologic interventions (immediate surgery or instructions for palliative care), and orthopedic and plastic surgery interventions.
Finally, terrorist bombings present a danger to the ED staff members that is never associated with any other mass casualty situation: there is a very real chance of explosion by a second-hit, either by explosive material remaining on the perpetrator’s body, or, even more threatening, a second suicide bomber who infiltrates the ED disguised as one of the victims and detonates the bomb inside the crowded ED. Thus, a unique caveat in the ED protocol for terrorist bombing attacks is heightened vigilance, starting from the chaotic first minutes after the arrival of the victims.
Providing medical assistance in an ED to victims of suicide bombing attacks is a harrowing experience: physicians who work in an urban hospital are more and more likely to be exposed to such events [14]. In his excellent editorial, Hirshberg wrote "Urban terrorism, the scourge of the 21st century, is already at our doorstep and surgeons are called upon to play leadership roles in shaping the emergency response in their hospitals. Learning from the experience of those for whom the unthinkable has become a daily reality can help us develop and implement more effective answers to the threats in our own communities" [14].

terror
ocular trauma
vitrectomy
intra ocular foreign body


Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1

The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen, as well as the oral absorption of a number of drugs, including β-lactam antibiotics and angiotensin-converting enzyme inhibitors. Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains. Mutation of rabbit PepT1 arginine282 (R282, transmembrane domain 7) to a positive (R282K) or physiologically titratable residue (R282H), resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes. Neutral (R282A, R282Q) or negatively charged (R282D, R282E) substitutions gave a transporter that was not stimulated by external acidification (reducing pHout from 7.4 to 5.5) but transported at the same rate as the wild-type maximal rate (pHout 5.5); however, only the R282E mutation was unable to concentrate substrate above the extracellular level. All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type, except R282E-PepT1 which was faster. A conserved negatively charged residue, aspartate341 (D341) in transmembrane domain 8 was implicated in forming a charge pair with R282, as R282E/D341R- and R282D/D341R-PepT1 had wild-type transporter characteristics. Despite their differences in ability to accumulate substrate, both R282E- and R282D-PepT1 showed an increased charge:peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln, measured using two-electrode voltage clamp. This extra charge movement was linked to substrate transport, as 4-aminobenzoic acid, which binds but is not translocated, did not induce membrane potential depolarisation in R282E-expressing oocytes. A model is proposed for the substrate binding/translocation process in PepT1.
1
Introduction
The proton-coupled di- and tri-peptide transporter PepT1 (SLC15a1) is the major route of uptake of dietary nitrogen from the intestine, and is also important along with the higher affinity gene product PepT2 (SLC15a2) in the re-absorption of filtered peptides in the kidney (Daniel & Kottra, 2004; Meredith & Boyd, 2000). In addition, PepT1 is the route of entry of a wide class of orally bio-available pharmaceutically important compounds, including the β-lactam antibiotics, angiotensin-converting enzyme (ACE) inhibitors, antiviral and anticancer agents (Terada & Inui, 2004). Although these therapeutic compounds are not di- or tri-peptides, they are carried by virtue of their similar 3D shape to endogenous substrates, i.e. they are peptidomimetic, and modelling of the substrate-binding site from the features in common of this huge and diverse range of substrates has led to predictions concerning which parts of the PepT1 protein may be important. For example, a substrate template model has been developed by several groups (Bailey et al., 2000; Bailey et al., 2006; Biegel et al., 2005) which allows prediction of binding affinity for a potential substrate.
The rabbit PepT1 is a 707 amino acid protein, with twelve transmembrane spanning domains (TMDs) as confirmed by epitope mapping (Covitz, Amidon, & Sadee, 1998). In the absence of a crystal structure, attempts have been made to computer model the PepT1 transporter itself (Bolger et al., 1998), with site-directed mutagenesis used to test hypotheses generated by these models. One potential complication for these kind of studies is our recent report that PepT1 may form multimers in the plasma membrane (Panitsas, Boyd, & Meredith, 2006) although it is not clear how the subunits interact.
Site-directed mutagenesis has been a useful tool to identify functionally important residues in PepT1. One such residue is arginine282 in the rabbit PepT1 sequence. The mutation of arginine282 to a glutamate produced a peptide transporter (R282E-PepT1) that was no longer driven by a proton-gradient but behaved more like a facilitated peptide transporter, whilst simultaneously exhibiting peptide-gated currents that were proposed to be through a non-specific cation channel activity (Meredith, 2004). Residue 282 is located approximately halfway down the predicted transmembrane domain 7 (TMD7), and is either an arginine or a lysine in all cloned mammalian PepT1 sequences to date. The presence of a charged amino acid residue in a TMD, along with its conservation, suggested a functional role. Here, we have systematically investigated the role of arginine282 in rabbit PepT1 by making further mutations to determine the requirement for the charge and have identified an interacting residue, aspartate341, located in putative TMD8. Some of these data have been previously published in abstract form (Pieri, Boyd, & Meredith, 2004).
2
Materials and methods
2.1
Site-directed mutagenesis of the PepT1 gene
Oligonucleotides were custom synthesised (Sigma-Genosys, UK) for the following sequences (residues in bold are changed from wild-type PepT1):-R282-PepT1 mutants forward:where xxx was CAA for R282Q, AAG for R282K, GAT for R282D, CAT for R282H, and GCG for R282A.-D341-PepT1 mutants forward:where xxx was CGC for D341R.
Reverse primers for the PepT1 mutant PCR reactions were the reverse compliment of the forward primers. The site-directed PepT1 mutants were generated using the Quikchange protocol (Stratagene), and the resulting constructs confirmed by DNA sequencing (Department of Biochemistry, University of Oxford, UK).
2.2
cRNA synthesis and oocyte injection
PepT1 constructs were linearised with XbaI (New England Biolabs, UK) and cRNA generated by in vitro transcription (T7 mMessage mMachine, Ambion, Cambridgeshire, UK). X. laevis oocytes were obtained under MS222 anaesthesia (0.2%, w/v) in accordance with the UK Animals (Scientific Procedures) Act, 1986, and maintained at 18 °C in modified Barth's medium (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 2.4 mM NaHCO3, 0.42 mM CaCl2, 10 mM Hepes, 5 mM sodium pyruvate, 50 μg ml−1 gentamicin (Fluka, Poole, UK), adjusted to pH 7.6 with 1 M NaOH). Transport measurements were performed at least 72 h after micro-injection of oocytes with 27nl cRNA (1 μg/μl), with medium changed daily.
2.3
Transport experiments
Zero-trans uptake of [3H]-d-Phe-l-Gln (17.4 Ci/mmole, custom synthesised, Cambridge Research Biochemicals, Stockton-on-Tees, UK) was performed as previously described (Meredith, 2004). Briefly, 5 oocytes were incubated in 100 μl of uptake medium (95 mM NaCl, 2 mM KCl, 1 mM CaCl2, 0.42 mM MgCl2, 10 mM Tris/Hepes pH 7.4 or Tris/Mes pH 5.5) with tracer (0.4 μM) [3H]-d-Phe-l-Gln. After incubation, the oocytes were washed sequentially five times in 1 ml of ice-cold 120 mM NaCl solution, lysed individually with 100 μl 2% (w/v) SDS and liquid scintillation counted. As a control non-injected oocytes were also incubated in uptake medium with [3H]-d-Phe-l-Gln as above.
The affinity of wild-type and mutant PepT1 were assessed by competition studies with 0.4 μM [3H]-d-Phe-l-Gln and Gly-l-Gln present in the uptake medium in concentrations from 0 to 2 mM using the protocol above, and the Ki calculated using the method of Deves and Boyd (1989).
Efflux studies were performed as previously described (Meredith, 2004), with the exception that the extracellular trans-stimulant Gly-l-Gln was used at 5 mM, and an efflux time-course was performed.
Diethylpyrocarbonate (DEPC) inhibition of PepT1 was performed using a similar protocol to that of Terada, Saito, and Inui (1998). Briefly, PepT1 oocytes were preincubated with 1 mM DEPC for 15 min at pHout 5.5 in the absence or presence of the PepT1 substrates Gly-l-Gln, N-Acetyl-Phe (Meredith et al., 2000), and l-Ala-Tyramine (custom synthesised) and the non-substrate Tyr (all 5 mM). The oocytes were then washed in uptake medium before uptake assays were performed as detailed above.
2.4
Electrophysiology
Measurements of membrane potential were made by impaling oocytes with a single glass microelectrode (Intra 767 amplifier, WPI, Stevenage, Hertfordshire, UK) perfused in uptake medium (as above) with or without 0.4 μM d-Phe-l-Gln (synthesised in house), 0.6 mM Gly-l-Gln (Sigma, Poole, UK) or 10 mM 4-aminobenzoic acid (4-AMBA, Sigma). Two-electrode voltage clamp (TEVC) was performed by placing oocytes in a 0.1 ml recording chamber and perfusing with uptake solution (pH 5.5 or 7.4) at a rate of 15 ml/min. Oocytes were impaled by two agarose-cushioned microelectrodes filled with 3 M KCl (0.5–2.0 MΩ) and voltage-clamped at −60 mV using a Geneclamp 500B amplifier and PCLAMP 8.1 software (Axon Instruments, CA, USA). The holding potential was stepped from −60 mV over the range of −150 to +50 mV in 10 mV steps, each pulse lasting 100 ms, and returning to −60 mV in between test voltage pulses. Typically traces were filtered at 1 kHz during recording and digitized at 0.5–5 kHz using the DigiData 1200 interface (Axon Instruments, CA, USA). All experiments were carried out at room temperature.
2.5
Data analysis
All data are expressed as mean ± S.E.M., except for Fig. 6 where the error bars represent the maximum range of stoichiometry values when taking into account the errors for the uptake data and the currents.
In order to calculate the apparent charge:substrate stoichiometry, the peptide induced current that was measured by two-electrode voltage clamp at the oocyte resting membrane potential was divided by the radiolabelled dipeptide uptake in oocytes from the same preparation. This value was normalised to 1:1 for wild-type PepT1, the accepted stoichiometry for a neutral dipeptide (Fei et al., 1994; Steel et al., 1997).
2.6
Statistical analysis
Statistical analyses were performed using one-way ANOVA with differences considered significant if p < 0.05 when data were compared to the wild-type control, as detailed in the text and/or figure legend.
3
Results
3.1
pH dependence of d-Phe-l-Gln uptake into R282 mutants
Fig. 1 shows the pH dependence of d-Phe-l-Gln uptake into oocytes expressing mutant PepT1 transporters where R282 has been changed into number of amino acid residues. Of the residues tested, R282K- and R282H-PepT1 behaved like the wild-type PepT1, in that the initial rate of uptake (1 h incubation time) of dipeptide was significantly faster (p < 0.05, one-way ANOVA) at an external pH of 5.5 than at 7.4. The other amino acid substitutions tested, R282E- (as previously reported, Meredith, 2004), R282D-, R282A- and R282Q-PepT1 all gave the same initial rate of uptake at pH 5.5 and 7.4, indicating that transport by these mutants is not stimulated by an inwardly directed proton gradient. These changes cannot be ascribed to changes in the affinity of the mutant PepT1 proteins for their substrate, as the Ki of Gly-l-Gln inhibiting 0.4 μM [3H]-d-Phe-l-Gln was unchanged in the mutants (Fig. 2).
3.2
Can the R282 mutant PepT1 transporters concentrate substrates?
An earlier finding was that, unlike the wild-type, the R282E-PepT1 mutant was unable to concentrate substrate even in the presence of an inwardly directed proton gradient (Meredith, 2004). The ability to concentrate substrate was therefore tested for the other R282 mutants (Fig. 3a and b shows representative time-course experiments at pHout 5.5 and 7.4, respectively), and the mean accumulation levels are shown for 8 h uptakes in Fig. 3c for pHout 5.5. In contrast to R282E-PepT1, all were found to be able to concentrate peptide well above the equilibrium level when the external pH was 5.5 (an oocyte was assumed to have a volume of 1 μl, Petersen & Berridge, 1996; Yao & Tsien, 1997). In the absence of the proton driving force (pHout 7.4, Fig. 3d) a similar level of intracellular d-Phe-l-Gln concentration was reached by all the mutant PepT1 transporters, including R282E-PepT1 if the incubation time was increased to 24 h (accumulation of 2.3 ± 0.4-fold compared to 3.1 ± 0.5-fold for wild-type PepT1). Although at 24 h incubation times we observed that cell survival can be a limiting factor, there was no statistical increase in the accumulation for the wild-type PepT1 at pHout 5.5 or 7.4 between 8 and 24 h incubations, nor between 8 and 24 h for the wild-type PepT1 at pHout 7.4 (8.0 ± 1.4 vs. 8.3 ± 1.0, 2.5 ± 0.5 vs. 3.1 ± 0.5 and 1.0 ± 0.4 vs. 1.5 ± 0.3 respectively, all p > 0.05, one-way ANOVA).
3.3
Rates of d-Phe-l-Gln efflux from R282 mutants
Fig. 4 shows the rates of efflux of [3H]-d-Phe-l-Gln from oocytes expressing either wild-type PepT1, R282 PepT1 mutants or non-injected controls. All of the PepT1 constructs showed a significantly faster efflux than the non-injected oocytes (one-way ANOVA, p < 0.05), whilst R282E-PepT1 was significantly faster than the wild-type (one-way ANOVA, p < 0.05) as previously described (Meredith, 2004). Interestingly, R282E-PepT1 showed a faster efflux than all the other R282 mutants (one-way ANOVA, p < 0.05), which were not significantly different to the wild-type (p = 0.14).
3.4
Apparent transport stoichiometry (charge to substrate) using two-electrode voltage clamp
As well as uptake being pH independent and non-concentrative, the electrophysiological characteristics of R282E-PepT1 were strikingly different to the wild-type (Meredith, 2004). Since R282D-PepT1 is similarly pH independent but does accumulate substrate, the apparent proton to peptide stoichiometry was examined, where the uptake of 0.4 μM d-Phe-l-Gln was compared to the peptide-induced current in the same batch of oocytes. The current at the oocyte resting membrane potential (−27.0 ± 1.1 mV at pHout 5.5, and −36.8 ± 2.2 mV at pHout 7.4, n = 12) was taken to represent the membrane potential under uptake conditions, as the addition of 0.4 μM substrate does not produce a detectable change in membrane potential (Fig. 5). The ratio of proton to neutral dipeptide co-transported through wild-type PepT1 is 1:1 (Fei et al., 1994; Steel et al., 1997), yet in R282E-, R282D- and R282A-PepT1 the apparent stoichiometry is substantially higher (4, 5 and 5 respectively at pHout 5.5, Fig. 6).
3.5
Is the extra current measured dependent on substrate transport?
In an attempt to see if the extra current carried by R282E-PepT1 was dependent on substrate translocation rather than simply substrate binding, the non-translocated PepT1 substrate 4-aminobenzoic acid (4-AMBA, Darcel, Liou, Tome, & Raybould, 2005; Meredith et al., 1998) was used. As found for wild-type PepT1, 4-AMBA failed to induce a depolarisation in R282E-PepT1 oocytes at 10 mM, three times its Ki (Meredith et al., 2000), in contrast to the known transported substrate Gly-l-Gln, also at three times Ki (Fig. 5).
3.6
Identification of an interacting residue for R282
The conservation of a positively charged amino acid in a transmembrane domain (TMD7), and the results above, strongly suggested that the presence of a positive charged residue was necessary for wild-type-like PepT1 transport function. In TMD8, predicted to be at approximately the same level in the membrane, there is a conserved aspartate (D341), and it was an appealing hypothesis that the two oppositely charged side chains might be forming a charge pair. To test this, double mutants were made, R282E/D341R- and R282D/D341R-PepT1, to swap the charges over. As can be seen in Fig. 7, these double mutants showed the same pH dependence of influx as the wild-type transporter, providing strong evidence to support the hypothesis. Both R282E/D341R- and R282D/D341R-PepT1 were also able to concentrate substrate like the wild-type (data not shown).
3.7
Diethylpyrocarbonate inhibition of PepT1 function
Preincubation of wild-type PepT1-expressing oocytes with 1 mM diethylpyrocarbonate (DEPC) for 15 min completely inhibited the PepT1 mediated dipeptide uptake measured over 1 h, as shown in Fig. 8. This inhibition by DEPC was largely prevented by the presence of the known PepT1 substrates Gly-l-Gln and N-Acetyl-Phe (Meredith et al., 2000), but not by the amino acid non-substrate Tyr. Interestingly, despite being a good PepT1 substrate (Ki approximately 0.1 mM, data not shown), a modified peptide lacking a carboxyl terminus (l-Ala-Tyramine) only partially prevented DEPC inhibition (Fig. 8).
4
Discussion
The R282K mutation of rabbit PepT1 is not only the most conservative one regarding the charge, but in a number of species, including dog, rat and mouse, lysine is the naturally occurring residue at this position. Therefore it was not surprising to find that this mutant behaves like the wild-type rabbit PepT1 (pig, sheep, rhesus and crab-eating monkeys and human also have R282). The finding that R282H also behaved like the wild-type was interesting, as histidine has a side-chain that can be titrated over the range used in the experiments (pK of ∼6 in free solution). Our findings could be interpreted in several ways, including the possibility that only at pHout 5.5 is there the formation of a positive charge by side-chain titration that gives a stimulation of uptake over that seen at pHout 7.4. A second possibility is that the protein environment surrounding H282 is such that its side chain pK is shifted away from 6 and it is therefore always protonated, and thus behaves more like an arginine or lysine. This effect has been shown for example in the enzyme protein tyrosine kinase (Tishmack, Bashford, Harms, & Van Etten, 1997), where histidine residues had a pK as high as 9.2 when analysed by NMR.
In the original study on R282E-PepT1, it was concluded that the uptake of peptide by the mutant transporter was uncoupled from the movement of protons, and that in addition to acting as a facilitated peptide transporter, R282E-PepT1 also displayed a peptide-gated non-specific cation conductance (Meredith, 2004). However, it is possible that this conclusion needs updating in the light of the current findings that there are R282 mutants that, like R282E-PepT1, are not pH stimulated, yet are still able to accumulate substrate above equilibrium when an inwardly directed proton gradient is imposed. For a transporter to be able to accumulate substrate above equilibrium, an energy source must be involved, in this case the proton electrochemical gradient. Therefore, the mutant PepT1 proteins that can accumulate substrate but do not show pH stimulation (R282A and R282D) must still be coupled to the movement of protons down their electrochemical gradient. The lack of pH stimulation could be attributed to the fact that during the transport cycle these specific mutants have a different rate limiting step to the wild-type, and that for these mutants that step is not pH dependent. It has already been reported that R282E-PepT1 has a faster rate of efflux than wild-type, consistent with an uncoupling of peptide uptake from the proton driving force (Meredith, 2004); the finding here that the rates of efflux for the other R282 mutants are not different from that of the wild-type is in agreement with the hypothesis that they are still proton-coupled, as shown by their ability to accumulate substrate above the extracellular concentration at pHout 5.5 but not 7.4.
The simplest hypothesis was that in R282E-PepT1, the extra inward charge movement associated with peptide uptake collapsed the membrane potential, which is known to be the major driving force for proton-coupled peptide uptake (Temple & Boyd, 1998), hence the apparent lack of substrate accumulation. Therefore by extension one would expect R282D- and R282A-PepT1 to have the same charge coupling as the wild-type, as they too accumulate substrate, but this was not the case: both R282E-, R282D- and R282A-PepT1 showed the same increased charge:peptide apparent stoichiometry, i.e. substantially larger than the wild-type. This stoichiometry itself showed pH dependence, with a lower value of around 2 for the mutants at pH 7.4, suggesting that the current is either carried by protons or is a pH-sensitive phenomenon. There was no difference in the uptake in the absence or presence of sodium, either for R282E-PepT1 (Meredith, 2004) or R282D-PepT1 (data not shown).
The finding that the R282E/D341R- and R282D/D341R-PepT1 double mutants (the latter being a charge pair reversal of the naturally occurring residues in rabbit PepT1) had the same characteristics as the wild-type protein strongly suggests that these two residues do interact in the 3D protein (Pieri et al., 2004) as previously proposed (Meredith, 2004). During the preparation of this manuscript, Kulkarni et al. (2007) reported findings consistent with R282 and D341 forming a charge pair in human PepT1. The initial hypothesis that in R282E-PepT1 repulsion between E282 and D341 allowed the movement of extra ions through the protein when a peptide is transported was not supported however by the finding that the single D341R mutant also behaved like the wild-type, as one might have thought that R282 and R341 would repel in much the same way as E282 and D341. That D341R-PepT1 behaved like wild-type suggests that although R282 and D341 seem to form a charge pair, in rabbit PepT1 residue 341 being negatively charged is not crucial to PepT1 function; interestingly, in human PepT1 the D341R single mutant had reduced function (Kulkarni et al., 2007). The reason for this difference between rabbit and human PepT1 is not clear. The observation that the non-transported PepT1 substrate 4-AMBA did not induce a depolarisation in R282E-PepT1-expressing oocytes clearly shows that the charge movement is linked to substrate binding and translocation and not to binding alone. One explanation for the increase in proton–peptide stoichiometry is that in wild-type PepT1 the presence of a positively charged residue deep in the binding pocket at position 282 repels proton movement through the transporter protein during the translocation step. The data in Fig. 6 are consistent with this, as the apparent stoichiometry is lower at pHout 7.4 than it is at pHout 5.5, indicating that the proton electrochemical gradient is involved.
In the case of mutations where R282 was replaced with a non-positively charged amino acid, the rate-limiting step of the transport cycle must be insensitive to extracellular pH, hence the lack of stimulation when the pHout is dropped from 7.4 to 5.5. Kinetic analysis of peptide transport by PepT1 in rat renal brush border membrane vesicles (Temple & Boyd, 1998) showed that at pHout7.4/pHin7.4, it was the protonation of the carrier protein that was the rate-limiting step (Temple, Bailey, Bronk, & Boyd, 1996), whereas at pHout5.5/pHin7.4 it was the return of the unloaded carrier. The rate of peptide uptake by R282E-PepT1 (corrected for protein expression in the intact oocyte plasma membrane by luminometry, Panitsas et al., 2006) is the same as for the wild-type at pHout 5.5 (data not shown), but, unlike for the wild-type, is not slower at pHout 7.4. This indicates that the mutations to arginine282 that abolish pH sensitivity (R to E, D, A, or Q) are affecting the rate limiting step, so when pHout is 7.4 the rate limiting step has the same magnitude as that at pHout 5.5, hence the lack of sensitivity to changing pHout. The rate limiting protonation of the outward-facing carrier protein at pHout 7.4, the first step in the transport cycle (Temple et al., 1996), was proposed by us (Bailey et al., 2000; Meredith & Boyd, 1995) and others (e.g. Steel et al., 1997; Uchiyama, Kulkarni, Davies, & Lee, 2003) to be protonation of histidine57 (H57). In the R282-PepT1 mutants, except for R282K and R282H, the lack of a positively charged residue might result in a conformational change of the protein that changes the local environment and increases the pKa of H57, such that it is more easily protonated at a higher pHout. Thus at pHout 7.4 the rate limiting step is no longer the protonation of H57, but the return of the unloaded carrier, as it is at pHout 5.5, hence the similar transport rates at pHout 5.5 and 7.4.
A model for how proton–peptide transport might occur is shown in Fig. 9: the empty PepT1 is primed by protonation of H57, followed by the binding of a zwitterionic peptide, with the N-terminal co-ordinated at E595 and the C-terminal at H57-H+ (Meredith et al., 2000). Although the binding of the substrate C-terminal to His57-H+ is in disagreement with the conclusion of Terada et al. (1998), it is supported by the finding that N-Acetyl-Phe, a known PepT1 substrate which does not have a free amino terminal (Meredith et al., 2000), can protect PepT1 against inhibition by DEPC. Additionally, the finding that significant DEPC inhibition is still evident when oocytes were co-incubated with the PepT1 substrate l-Ala-Tyramine which lacks a carboxyl terminus further adds to the notion that H57-H+ binds the carboxyl terminus of the substrate (Fig. 8). H57-H+ then donates its proton to the C-terminal carboxyl group, and the transporter undergoes a conformational change that leads to the breaking of the salt bridge between R282 and D341, re-orientating the binding site to be inward facing as simultaneously the protonated N-terminal of the substrate binds to D341, neutralising the charge, and the R282 charge is stabilised by Y167 (the chemical properties of this tyrosine have been shown to be essential, Yeung et al., 1998). The peptide molecule is then released into the cytoplasm, whereby it returns to the zwitterionic state by releasing the proton from the carboxyl terminal. The transporter then undergoes the reverse conformational change to re-orientate the binding site to outward facing, and R282 reforms the salt bridge with D341.
As R282E-PepT1 cannot accumulate substrate, the implication must be that the movement of peptide is no longer coupled to the movement of the protons, whereas in all the other mutants coupling must be maintained. If for R282E-PepT1 the pKa of H57 was raised to the point that it was no longer favourable for it to donate its proton to the carboxyl terminus of the substrate, then peptide transport would no longer be proton-coupled, and this would explain the failure of R282E-PepT1 to accumulate substrate. Intriguingly, it can be seen in Fig. 6 that both R282D- and R282A-PepT1 appear to carry approximately one more charge per substrate peptide than R282E-PepT1, which is consistent with the hypothesis that one of the charges carried is coupled with the substrate.
In conclusion, the arginine at position 282 in rabbit PepT1 plays an intriguing role in the function of the transporter, with mutations to different residues revealing that a positive residue is required for pH dependence, whilst only R282E-PepT1 cannot concentrate substrate above equilibrium; this is despite other mutations, most notably R282D-PepT1, having a similarly increased charge:peptide stoichiometry. As previously proposed, R282 (TMD7) forms a charge pair with D341 (TMD8), with R282E/D341R-PepT1 showing normal transport characteristics. Further biological testing or a crystal structure of PepT1 will be required to establish the validity of the model proposed.

site-directed mutagenesis
slc15a1
epithelia
membrane transport
nutrient absorption
protein structure–function