Donor site morbidity of the fasciocutaneous radial forearm flap: what does the patient really bother?

The objective of this study was the evaluation of donor site morbidity in head and neck cancer patients after reconstruction using a free vascularized radial forearm flap with emphasis on subjective complaints. Fifty patients who underwent at least 6 months before a reconstruction using a free vascularized radial forearm flap were asked to fill out two questionnaires regarding cosmetics and sensibility and forearm disabilities. Furthermore, a function test including movement extensions (flexion–extension, ulnar–radial deviation and pronation–supination), strength (pinch and grip) and temperature (digiti I and V) of the donor and non-donor site were measured and compared. Thirty-five percent of the patients reported no complaints regarding cosmetics and sensibility and 75% mentioned no forearm disabilities. There was no difference in movement extensions, temperature and grip strength between donor and non-donor sites. The difference in pinch strength appeared to be significant (p < 0.001). The total score of the questionnaire on forearm disabilities correlated significantly with extension, pronation and grip strength of the donor arm. Donor site morbidity of the radial forearm flap measured by objective functional tests was limited but subjective self-ratings revealed complaints regarding cosmestics and sensibility and to a lesser extent regarding forearm disability. The present data may be used for solid patient counselling.
Introduction
The free radial forearm flap (FRFF), introduced by Yang et al. in 1981 [1], has established itself as a versatile, reliable and widely used method for reconstruction of defects in the head and neck region [2, 3]. The pliability and thinness of FRFF allow its use in complex reconstructions. The vascular pedicle of the FRFF provides adequate vessel diameter and length for microvascular anastomosis. A split skin graft is most frequently used for the forearm donor site reconstruction. Donor site morbidity includes functional and aesthetical outcome. Hand and wrist function is the most important issue in the assessment of morbidity of the FRFF. In a previous study we found that donor site morbidity of the radial forearm flap measured by objective functional tests is negligible but in the patient’s perception this is substantial [4]. Some retrospective studies describe a reduced forearm and/or wrist mobility and a reduced strength [5], stiffness of the wrist with movement disability, dysaesthesia, swelling of hand and/or wrist and pain [6], reduced sensibility of the radial nerve area and a less aesthetic result [7, 8], while others describe rare or no movement disabilities after using the FRFF [9, 10].
The subjective morbidity after harvest of the FRFF is one of the reasons that some reconstructive surgeons shift their attention to other fasciocutaneous flaps for reconstruction of defects in the head and neck [11, 12]. The goal of the present study is to investigate these subjective complaints in more detail and in relation to the objective function tests in a larger group of patients.
Methods
Patients
All patients who visited the outpatient clinic of the department of otolaryngology/head and neck surgery of the VU Medical Center during the period of March till July 2004 and had a reconstruction of the surgical defect by using a FRFF at least 6 months before were asked to participate in the study. Exclusion criteria were arthritis of the upper extremities or a recent trauma of the wrist and/or lower arm.
During this period 69 patients, who underwent such a operation, visited the outpatient clinic. Fifty patients (28 men and 22 women, aged between 26 and 77 years, mean 59.2 ± 11.1 years) met the inclusion criteria. Eight of these 50 patients had had a forearm or wrist injury before surgery (five patients had a wrist fracture, two had a Dupyutren’s contracture and one had surgery because of a ganglion on dorsal side of the wrist), of which four patients at the donor side. None of these patients had pre-existing complaints and/or range of motion disability of the hand or wrist. In 45 patients the left arm was used for the harvest of the FRFF and in five patients the right arm. In three patients the donor side was the side of the dominant hand.
Harvest of the flap was done simultaneously with the ablative procedure whenever possible. All patients underwent a preoperative and intra-operative Allen’s occlusion test to rule out inadequate blood supply from the ulnar artery. Fasciocutaneous flaps were raised under a tourniquet in a conventional subfascial or superfascial manner about 2 cm proximal to the wrist skin fold. The superficial radial nerve and branches of the lateral antecubital nerve were preserved. The cephalic vein was used as donor vein. The radial artery was not reconstructed in any patient. The donor defect was closed with a split skin graft (0.6 mm) taken from the upper thigh at the same site. In order to aid healing the arm a pressure dressing of a paraffin gauze and foam was placed over the skin and the arm was immobilized for 7 days in a dorsal hand-to-upper-arm splint.
Donor site morbidity questionnaires
Subjectively the donor site morbidity was measured by means of a questionnaire regarding cosmetics and sensibility (Table 1) and a questionnaire regarding forearm disabilities of the operated arm (Table 2). Response possibilities were yes, no, or not applicable. All themes relate to the last 24 h. A total score on each questionnaire was calculated by dividing all yes-scores by the number of questions answered yes or no. The total score ranged from 0 to 100 with 0 indicating no problems (no disability) and 100 indicating severe problems (severe forearm and/or wrist disabilities).
Table 1Questionnaire regarding sensibility and cosmetics of the donor arm (n = 50)QuestionsNumber of positive answer (%)No complaints17 (35)Complaints 1. Can you wear a wristwatch or bracelet?12 (24) 2. Does the hand feel numb?11 (22) 3. Does the scar itch?11 (22) 4. Do you experience problems in the cold?4 (8) 5. Does the appearance bother you?13 (27)In case of complaints, multiple answers were possibleTable 2Questionnaire regarding forearm disabilities in the last 24 h (n = 50)Questions Number of positive answer (%)No forearm disabilities38 (75)Disabilities 1. I wake up at night because of my forearm1 (2.0) 2. I have complaints lying on my forearm 4 (8.0) 3. I have complaints during daily life activities2 (4.0) 4. I have complaints during movements of my wrist3 (6.0) 5. I have complaints during leaning on my elbows or hands3 (6.0) 6. I have complaints with writing (or typing)1 (2.0) 7. I have complaints holding my wheel of my car or bike2 (4.0) 8. I have complaints during lifting an object5 (10.0) 9. I have complaints opening or closing a door2 (4.0) 10. I rub my wrist or forearm more than once a day9 (18.0) 11. I am irritable for people in my environment due to my forearm1 (2.0)
Donor site morbidity tests
Three tests were used for objective measurement of donor site morbidity. All measurements were performed at the operated and non-operated side. The first test covers movement extensions of the wrist. The angles of the maximal flexion and extension of the wrist, the ulnar and radial abduction and the lower arm pronation and supination were measured by the Mediclino Inclinometer (Bodybow-Holland, Nieuwegein, The Netherlands) with an accuracy of 2° and a range of 0–180° (Fig. 1).
Fig. 1Inclinometer used for measuring of movements
The second test measures the strength of the hand. The grip strength (strength measured in the hand) was measured in kg/m2 with a hydraulic dynamometer (Smith & Nephew Roylan, Germantown, WI, USA) with an accuracy of 2.0 kg and a range of 0–90.0 kg (Fig. 2). The pinch-strength (strength of digiti I and V) was measured with a pinch gauge (B&L Engineering, Santa Fe Springs, CA, USA) with an accuracy of 0.5 kg and a range of 0–12.5 kg (Fig. 3).
Fig. 2Hydraulic dynamometer used for measuring grip strengthFig. 3Pinch gauge used for measuring pinch strength
The third test measures the skin temperature of digiti I and V with a Tempcontrol MT 100 KC and probe (Tempcontrol Industrial Electronic Products, Voorburg, The Netherlands) on the skin surface with an accuracy of 0.1°C and a range of −200 to 1350°C.
Statistical analyses
Descriptive statistics were generated for the range of outcome variables in the study. Student’s t-tests were used to determine the differences in objective tests between the donor and non-donor site. Spearman correlation-coefficients were calculated to assess correlations between subjective and objective forearm disability.
For all tests, a two-sided p-value less than .05 was considered statistically significant.
Results
Regarding subjective evaluation of cosmetics and sensibility, 35% of the patients had no complaints. The other patients scored positively (thus indicating complaints) on several of the questions (Table 1).
Regarding forearm disabilities, 75% of the patients had no complaints (Table 2). The other patients showed a variety of complaints, especially on rubbing and lifting an object. The mean total score on the forearm disability questionnaire was 4.6 (SD 12.6) with a range from 0 to 82.
The results of the objective tests revealed that no differences were measured between the donor and non-donor arm regarding movement, grip and temperature. Pinch values appeared to be significantly different (Table 3).
Table 3Movement extensions, strength and temperature of donor and non-donor wrist and hand Donor sideNon-donor sideMeanSDMeanSDExtension57.311.759.811.0Flexion71.18.070.29.2Ulnar abduction57.78.258.98.0Radial abduction15.38.414.85.1Pronation85.410.885.311.0Supination79.612.280.412.9Grip28.710.429.99.7Pinch7.42.48.02.4*Temperature digit I29.33.329.43.6Temperature digit V29.03.929.14.1*Significant difference (p-value < .01))
The total score on the forearm disabilities questionnaire appeared to be significantly related to extension (r = 0.29, p < 0.05), pronation (r = 0.30, p < .05) and grip strength (r = 0.30, p < .05).
Discussion
The most important goal of reconstructive surgery in head and neck cancer patients is the optimal restoration of function. The FRFF is a very reliable flap which can restore function in the head and neck function very well [13, 14, 15]. However, the donor site morbidity after harvesting the flap is another important issue. While the main postoperative concern was the reconstruction site, during follow-up the donor site become more important to patients [16].
Hand and wrist functions are important in the assessment of morbidity of the FRFF. The only objectively observed difference in our study was pinch strength between operated and non-operated sides. It must be emphasized that in 94% of the patients the FRFF was harvested from non-dominant side. Therefore, the pinch strength on the operated arm may be already less than on the non-operated side before surgery. All other functional tests revealed no statistically significant differences between operated and non-operated sides. Also Ho et al. [17] did not find any significant difference between the operated and the non-operated arm for strength, range of motion and dexterity.
The questionnaires on the other hand revealed donor site complaints related to the FRFF. This difference between objective and subjective findings was also reported by others [4, 7, 8, 9]. The questionnaire on forearm disabilities showed that the vast majority (75%) of the patients had no functional complaints at all. The most frequent positive answer was about rubbing the forearm, which may be more a sensibility than a functional problem. In a study of Toschka et al. [18] postoperative hand function received a subjective rating of 80–100% of the preoperative function by 89% of their patients. Other studies report no subjective impairment of function in 63–100% of patients [7, 10]. Ho et al [17] found using a questionnaire consisting of a list of activities of daily living (ADL) that required use of wrist and forearm, that postoperative function appears to be quite satisfactory.
While the forearm disability questionnaire showed that functional problems are limited, the cosmetics and sensibility questionnaire revealed that only 39% of the patients had no cosmetic or sensible complaints. The most frequent (27%) complaint was on the appearance of the donor site. Other studies found complaints of poor aesthetic results in up to 28% of patients, particularly female patients [7, 9, 10, 16]. Bardsley et al. [10] examined the cosmetic result by a subjective assessment on a scale of 0–10. The cosmetic result was acceptable in men (mean score 1.5) but was less so in women (mean score 4). Ito et al. [16] scored the dissatisfaction about the FRFF donor site of 23 patients in five items: color (4%), scar width (0%), depression (30%), wrist mobility (0%) and sensation (4%). The majority of patients (61%) had no complaints at all. The mean number of items of which patients were dissatisfied was 0.39. On the other hand in the study of Toschka et al. [18] 94% of the patients rated the aesthetic outcome as fair or good. Lutz et al. [8] reported 98% of patients rating the aesthetic outcome as satisfactory.
Only extension, pronation and grip strength had a clear correlation with the score on the forearm disabilities questionnaire. For all other objective test no significant correlation with this score was found. Therefore, most objective test results may not be indicative for donor site complaints by the patient. This finding has been confirmed earlier by others [4, 7, 8, 9].
To diminish donor site morbidity several harvesting and donor site closure technique modifications have been proposed.
The technique of harvesting the flap may have some impact on donor site function. In the early days of this flap when radial bone was included for mandibular reconstruction, morbidity was substantial, even leading to wrist fractures [7]. Suprafascial dissection is claimed by some to lead to superior results from a standpoint of wound healing, but there are no comparative studies that confirm this [8, 18]. Wolff et al. [19] reported on a small series of prefabricated fascial-split-thickness skin flap: after a split-thickness skin graft is transplanted to the forearm fascia the flap can be raised with complete preservation of the forearm skin and microsurgically transplanted like a conventional radial flap. The prelaminated fasciomucosal radial forearm flap as described by Nehrer-Tairych et al. [20] claims to provide better cosmetic and functional donor site effects, although the series of patients they describe is rather small.
Avery et al. [5] reported good aesthetic results of repair of the FRFF donor site by full-thickness skin graft from the inner upper arm. Ho et al. [17] compared the functional and aesthetic outcomes of FRFF donor sites reconstructed with full-thickness skin graft, split skin graft alone and split skin graft overlying an acellular dermal matrix and found that all three methods of reconstruction have comparable low morbidity, postoperative satisfactory aesthetic and functional outcomes. Negative pressure wound dressing has been used for rapid healing and decreased donor site complications [21]. Bardsley et al. [10] used a ulnar artery-based transposition flap for primary closure to reduce wound healing and improve cosmetic results. Hsieh et al. [22] reported good results of primary closure of the FRFF donor site with a bilobed flap based on the fasciocutaneous perforator of the ulnar artery. Several types of tissue expansion have been reported to allow the use of local tissues for primary closure [23]. The preferred technique of closure is difficult to assess, because of limited comparative studies, limited number of patients and lack of significant differences.
The presented data can be used for solid counselling patients who are scheduled for FRFF reconstruction. Elaborate pre-surgical counselling may reduce the impact of functional and cosmetic impairment at the FRFF donor site [24]. Moreover, these data may serve as benchmark for future studies that use other free fasciocutaneous flaps.
Conclusion
Donor site morbidity of the radial forearm flap measured by objective functional tests is negligible but in the patient’s perception this is substantial. Subjective measurements show especially problems in cosmesis and sensibility. The present data may be used for solid patient counselling.

donor site morbidity
head and neck cancer
cosmetics
free radial forearm flap
functional results


Cellular pharmacokinetics of telavancin, a novel lipoglycopeptide antibiotic, and analysis of lysosomal changes in cultured eukaryotic cells (J774 mouse macrophages and rat embryonic fibroblasts)

Background Telavancin is a lipoglycopeptide with multiple mechanisms of action that include membrane-destabilizing effects towards bacterial cells. It shows bactericidal activity against forms of Staphylococcus aureus (phagolysosomal infection) with different resistance phenotypes [methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus]. We examine here the uptake, efflux and intracellular distribution of telavancin in eukaryotic cells as well as its potential to induce lysosomal changes (in comparison with vancomycin and oritavancin).
Introduction
Telavancin is a novel lipoglycopeptide derivative of vancomycin1 with marked bactericidal activity against vancomycin-susceptible and vancomycin-resistant organisms2 due to a multifunctional mechanism of action that combines inhibition of cell wall synthesis and disruption of bacterial cell membrane permability.3 It shows a high penetration in tissues,4 including human alveolar macrophages.5 In a recent study, we showed that telavancin exerts time- and concentration-dependent bactericidal activity against intraphagocytic Staphylococcus aureus, disregarding their resistance phenotypes (methicillin-resistant S. aureus, vancomycin-intermediate S. aureus or vancomycin-resistant S. aureus).6
The present investigation was initiated to further document and rationalize this observation by examining the uptake and subcellular disposition of telavancin in eukaryotic cells. Because previous studies had disclosed marked lysosomal alterations in eukaryotic cells exposed to another lipoglycopeptide, oritavancin,7 we also undertook to assess the impact of telavancin in this context, using vancomycin as a comparator. The study was performed with both phagocytic (J774 macrophages) and non-phagocytic (fibroblasts) cells because this allowed us, in the past, to obtain a comprehensive picture of the behaviour of other antibiotics accumulating in cells and causing specific lysosomal alterations.8–11
Materials and methods
Cells, cell cultures and assessment of membrane integrity
J774 mouse macrophages and rat embryo fibroblasts were cultivated, as previously described,7 in RPMI 1640 or in Dulbecco's modified Eagle's medium, respectively, both supplemented with 10% fetal calf serum (unless stated otherwise). The integrity of the pericellular membrane upon exposure to the antibiotics was assessed by measuring the release of lactate dehydrogenase in the culture medium, as described previously.6
Determination of cellular antibiotic concentration
Cells incubated with 14C-labelled telavancin were washed three times in ice-cold 0.9% NaCl, collected by scraping in distilled water and used for radioactivity determination (liquid scintillation counting) and protein assay (a detailed description of the methodology has been published previously12). In pilot experiments, cell-associated antibiotic was also measured by a microbiological technique (with Micrococcus luteus ATCC 9341 as test organism and antibiotic medium 11).12 A correlation coefficient (R2) of 0.95 was found between the two methods for cells incubated 24 h at concentrations spanning from 10 to 100 mg/L (n = 12).
Cell fractionation studies
Cells were collected by gentle scraping in 0.25 M sucrose/1 mM EGTA/3 mM imidazole pH 7.4, homogenized and fractionated, as described previously.12 In brief, homogenates were separated into an ‘unbroken cells/nuclei’ fraction by a low-speed centrifugation. The resulting cytoplasmic extract was separated into a ‘granules/membranes’ fraction and a final supernatant by high-speed centrifugation. The granule/membrane fraction was then further analysed by isopycnic centrifugation in a sucrose gradient. Protein and [14C]telavancin contents were determined in the fractions in parallel with the activity of marker enzymes of the main organelles, namely, inosine 5′-diphosphatase (E.C. 3.6.1.6.; plasma and endoplasmic reticulum membranes), cytochrome c oxidase (E.C. 1.9.3.1.; mitochondria) and N-acetyl-β-glucosaminidase (E.C. 3.2.1.30.; lysosomes). Results are expressed as the percentage of enzyme activity, protein or drug recovered in each fraction. For isopycnic centrifugation, distributions were standardized for equal density increments ranging from 1.08 to 1.21, as described previously.13
Biochemical studies
Cell sheets were washed three times in ice-cold 0.9% NaCl, collected by scraping in distilled water and lysed by sonication. Total phospholipids and cholesterol were extracted and assayed, as described previously.7 Proteins were assayed by the Folin–Ciocalteau/biuret method.14
Electron microscopy
Sample preparation was performed as described previously.15,16 Observations were made in a Zeiss electron microscope operated at 80 kV. Morphometric analysis was performed on pictures taken at random, using the Image J software available from the NIH web site (http://rsb.info.nih.gov/ij/) and examining a total cell surface of cell profiles of 1000–2000 µm2. For each cell profile, we manually selected the zones occupied by electron-dense material (for which a limiting membrane could not always be clearly recognized) or corresponding to large vesicles filled with heterogeneous material (see Figure 3 for examples). Results were expressed as percentage of the whole cell surface profile, which is numerically identical to the percentage of cell volume.17
Figure 3
Selected pictures illustrating typical changes observed in macrophages (a) and fibroblasts (b) after incubation with 90 mg/L telavancin for 24 or 72 h, respectively. Both cells show markedly enlarged and ill-shaped lysosome-like bodies with a pleiotropic material that is highly osmiophilic (often organized in a distorted concentric fashion). (c) Example of loose material and (d) example of highly osmiophilic material organized in multiple separate bodies. Bars are 1 µm. The dotted lines show examples of profiles selected for morphometric analysis: i, a large profile showing mainly a moderately osmiophilic and pleiomorphic material; ii, a small profile showing mainly osmiophilic material [possibly a polar section of a lysosome with mixed content (as in iii), but cut as shown by the arrow].
Materials
Telavancin hydrochloride (powder for microbiological evaluation and >90% purity) and [14C]telavancin trifluoroacetate (33.8 mCi/mmol and radiochemical purity >91%) were supplied by Theravance, Inc. (South San Francisco, CA, USA). The labelled drug was mixed with unlabelled telavancin to obtain a specific activity of 5 mCi/mmol. Stock solutions were prepared at a final concentration of 1–2 mg/L by vigorous vortexing in distilled water (the use of acidified DMSO recommended by the manufacturer was avoided because preliminary experiments disclosed a decrease in cell viability). Oritavancin (supplied as diphosphate salt fully hydrated) was obtained from Intermune (Brisbane, CA, USA). Vancomycin was procured as VANCOCIN® from GlaxoSmithKline, Belgium. Cell culture or microbiology media were from Invitrogen (Paisley, Scotland, UK) and Difco (Sparks, MD, USA). Other reagents were of analytic grade and purchased from E. Merck AG (Darmstadt, Germany) or Sigma-Aldrich-Fluka (St Louis, MO, USA).
Results
Influence of telavancin on pericellular membrane integrity
In preliminary experiments, we measured the release of lactate dehydrogenase, a cytosolic enzyme, from cells exposed to telavancin (90 mg/L, corresponding to the human Cmax18), in comparison with vancomycin (50 mg/L, corresponding to the human Cmax19) and oritavancin (25 mg/L, corresponding to the human Cmax20). No difference in controls (5.0 ± 0.2%) was seen with telavancin (4.9 ± 1.0%) or vancomycin (5.0 ± 1.5%) in J774 macrophages after 24 h of incubation, whereas oritavancin induced a small but significant increase in enzyme release (15.5 ± 1.5%; P < 0.001; n = 3 for all conditions). No significant effect was seen for fibroblasts after 72 h of incubation between controls (13.4 ± 3.7%) and cells incubated with telavancin (16.9 ± 2.5%), vancomycin (17.1 ± 1.3%) or oritavancin (18.2 ± 3.0%; n = 3 for all testing conditions).
Kinetics of uptake and release of telavancin
Figure 1(a and b) shows the kinetics of uptake of telavancin in J774 macrophages and fibroblasts incubated with an extracellular concentration of 90 mg/L. The uptake proceeded linearly over time at a rate that was similar in both cell types (see figure captions for details). Figure 1(c and d) examines the uptake of telavancin at increasing extracellular concentrations and at fixed time points (24 h for macrophages and 72 h for fibroblasts). The uptake was linearly related to the extracellular concentration in both cell types, allowing us to calculate a clearance rate for all conditions shown in Figure 1 of ~10 µL/mg of cell protein per h. For macrophages, we also examined the drug efflux (after an initial uptake of 12 h in the presence of 90 mg/L telavancin), which occurred at an apparent rate ∼5.7-fold lower than that of influx (Figure 1a).
Figure 1
(a and b) Kinetics of uptake of telavancin (filled symbols and continuous line) in J774 macrophages (a) or embryo fibroblasts (b) incubated for the indicated times with an extracellular concentration of 90 mg/L at 37°C in medium supplemented with 10% fetal calf serum. (a) Kinetics of efflux of the drug from J774 macrophages exposed to telavancin (90 mg/L) for 12 h and re-incubated in a drug-free medium for an additional 24 h (open symbols and broken line) are also shown. (c and d) Cellular concentration of telavancin in J774 macrophages (c) or embryo fibroblasts (d) incubated at 37°C for 24 h or 72 h, respectively, in the presence of telavancin at the extracellular concentrations indicated on the abscissa. Results are given as arithmetic means ± SD (n = 3) and analysed by linear regression to calculate the corresponding clearances (µL/mg of protein/h): J774 macrophages, 9.6 ± 0.6 (R2 = 0.98; a) and 10.0 ± 0.4 (R2 = 0.99; c); fibroblasts, 8.2 ± 0.4 (R2 = 0.97; b) and 9.0 ± 0.7 (R2 = 0.98; d).
The influence of serum on telavancin uptake was examined in J774 macrophages by performing experiments in culture medium not supplemented with calf serum. The incubation was limited to 5 h to ensure maintenance of cell viability. Telavancin uptake remained linear over the 10–90 mg/L range of extracellular concentrations, but proceeded at a rate ~1.7-fold faster than in the presence of serum (data not shown).
Subcellular distribution of telavancin
The subcellular distribution of cell-associated telavancin was examined in homogenates obtained from J774 macrophages incubated for 24 h with the drug at an extracellular concentration of 90 mg/L. The distribution of cell-associated telavancin and N-acetyl-β-glucosaminidase (lysosome marker) among the unbroken cells/nuclei, granules/membranes and supernatant fractions was similar (35% and 27%, 55% and 67% and 10% and 6%, respectively). The granule/membrane fraction was therefore subfractionated by isopycnic centrifugation. The density distribution of telavancin, in comparison with that of N-acetyl-β-glucosaminidase (lysosomes), cytochrome c oxidase (mitochondria) and inosine 5′-diphosphatase (plasma/endoplasmic reticulum membranes), is shown in Figure 2. The distribution pattern of telavancin was largely superimposable on that of the N-acetyl-β-glucosaminidase, clearly dissociated from that of cytochrome c oxidase and also distinct from that of inosine 5′-diphosphatase.
Figure 2
Density distribution of marker enzymes [N-acetyl-β-glucosaminidase (lysosomes), cytochrome c oxidase (mitochondria) and inosine 5′-diphosphatase (plasma and endoplasmic reticulum membranes)] and of telavancin after isopycnic centrifugation in a linear sucrose gradient of a granule fraction prepared from homogenates of J774 cells that were incubated for 24 h with 90 mg/L telavancin at 37°C in medium supplemented with 10% fetal calf serum. The ordinate shows the percentage of each constituent recovered in each fraction.
Ultrastructural alterations
Electron microscopy was used to examine whether telavancin induces morphological alterations in the subcellular organelles of cells incubated in its presence. Figure 3 shows selected pictures of alterations that could be observed in J774 macrophages and fibroblasts (90 mg/L; 24 and 72 h, respectively). There was an enlargement of lysosomes that were filled with a pleiotropic material, made of partly highly osmiophilic, concentric structures and partly of a more loose appearance material.
To gain quantitative information on these changes, a morphometric analysis was performed on pictures taken at random. Figure 4 shows the relative abundance of these abnormal lysosomal profiles in cells incubated with three different concentrations of telavancin, in comparison with control cells or cells exposed to 50 mg/L vancomycin or 20 mg/L oritavancin. In macrophages (24 h incubation) as well as in fibroblasts (72 h incubation), telavancin induced a concentration-dependent increase in the percentage of cell volume occupied by overloaded lysosomes. The morphometric analysis showed that: (i) changes induced in fibroblasts were more marked than those in macrophages (which may have been a consequence of the longer incubation time in fibroblasts); (ii) incubation of macrophages with 25 mg/L telavancin or of fibroblasts with 90 mg/L caused changes similar to those seen with 50 mg/L vancomycin in either cell types and (iii) alterations induced by telavancin at the highest concentration tested (90 mg/L) were considerably milder than in cells incubated with 20 mg/L oritavancin in both cell types.
Figure 4
Morphometric analysis of the material accumulated in macrophages (left-hand panel) or fibroblasts (right-hand panel) after 24 and 72 h of incubation, respectively, in control conditions or in the presence of 5, 25 or 90 mg/L telavancin (TLV), 50 mg/L vancomycin (VAN) or 20 mg/L oritavancin (ORI). Results are expressed as percentage of the cell surface occupied by the electron-dense material and/or large vesicles filled with a material of undetermined nature. Surface analysed was ~2000 µm2 for macrophages and 1000 µm2 for fibroblasts.
Influence of telavancin on cell phospholipid and cholesterol contents
In view of the ultrastructural changes observed, we looked for changes in phospholipids and cholesterol content of cells exposed to 90 mg/L telavancin in comparison with vancomycin (50 mg/L) and oritavancin (25 mg/L). Figure 5 illustrates the data obtained after 24 h in macrophages and 72 h in fibroblasts. Neither telavancin nor vancomycin caused detectable increase in the phospholipid content. In contrast, oritavancin induced significant increases in the phospholipid content in both cell types. Telavancin and vancomycin caused a slight but not significant increase in the cholesterol content, whereas oritavancin again induced a marked, statistically significant increase.
Figure 5
Total phospholipid content (upper panels) or total cholesterol content (lower panels) of J774 mouse macrophages (left-hand panels) or rat embryo fibroblasts (right-hand panels) exposed for 24 and 72 h, respectively, to glycopeptides at their human Cmax (VAN, vancomycin 50 mg/L; TLV, telavancin 90 mg/L and ORI, oritavancin 25 mg/L). Results are expressed as percentages of control values for total phospholipids and cholesterol. Values are arithmetic means ± SD (n = 6–8). Values for control macrophages and fibroblasts were, respectively, 204 ± 10 and 295 ± 8 nmol/mg of protein for total phospholipids and 72 ± 3 and 130 ± 16 nmol/mg of protein for total cholesterol. Statistical analysis (ANOVA): ***P < 0.001, **P < 0.01, *P < 0.05 when compared with the matching control. Other differences were not significant.
Discussion
The present study provides evidence that telavancin is taken up by cultured macrophages where it becomes associated with lysosomes, as assessed by cell fractionation studies. These results rationalize our previous data, showing that telavancin exerts a marked antibiotic activity against intraphagocytic S. aureus, which is known to develop in the phagolysosomes of infected macrophages.21,22
The accumulation level reached by telavancin in macrophages at 24 h (∼45-fold) is intermediate between that recorded previously in the same conditions for vancomycin (∼8-fold) and oritavancin (∼370-fold).12 Its uptake is not specific to phagocytic cells, as it is also observed with fibroblasts, both cell types taking up the drug at similar rates. The entry of drugs into lysosomes may occur through either diffusion/segregation or pinocytosis,23 as illustrated by the behaviour of macrolides8 and aminoglycosides,24 respectively. Diffusion–segregation seems unlikely in view of the slow efflux of telavancin, as this process is usually considered to be reversible (although binding of the drug to intracellular constituents could explain this slow efflux, as is observed for azithromycin in fibroblasts16). Pinocytosis, therefore, appears more plausible, especially if considering the size of the molecule that would prevent its fast diffusion through membranes. However, the clearance rates recorded here (∼10 µL/mg of protein/h) are ~15- to 30-fold higher than those reported for fluid-phase pinocytosis markers (∼0.7 µL/mg of protein/h in macrophages15 and ∼0.3 µL/mg of protein/h in fibroblasts24). Telavancin uptake, therefore, should involve a process of adsorptive pinocytosis (through binding to cell surface; see discussion and modelling in Silverstein et al.25) However, the lack of saturation of telavancin uptake upon increase of its extracellular concentration is intriguing as this (i) is an hallmark of adsorptive pinocytosis25 and (ii) was observed for oritavancin.12 The initial uptake clearance rate of oritavancin, however, is considerably larger (∼150 µL/mg of protein/h),12 which indicates that binding of telavancin should be much weaker and may not actually show saturation in the concentration range investigated here. A weaker membrane binding of telavancin is actually consistent with its lower lipophilicity when compared with oritavancin (reported logP values of 0.6 for telavancin versus 4.1 for oritavancin) [Advanced Chemistry Development Software Solaris V4.67, Sci Finder Scholar 2006, American Chemical Society, Washington, DC, USA].
Beyond its therapeutic interest, the lysosomal accumulation of telavancin may also be responsible for the morphological alterations observed in cells exposed to the drug. Yet, these changes appear quantitatively minor, as is also the case for vancomycin. Moreover, and in contrast to what is observed with oritavancin,7 telavancin did not significantly affect phospholipid or cholesterol cellular levels. This difference may be related to the lower uptake of telavancin, although we cannot exclude, at this stage, a true difference in the intrinsic capacity of the two molecules to interfere with lipid metabolism. Further studies are required to determine the exact nature of the accumulated material and to decipher the underlying molecular mechanisms. These may be more complex than those evidenced for aminoglycoside antibiotics, which mainly induce an accumulation of phospholipids.26 Interestingly, telavancin was without detectable effect on lactate dehydrogenase release. This suggests that the membrane-destabilizing properties exerted by telavancin towards bacterial membranes,3 which probably contribute to its marked bactericidal effect, involve constituents that are specific to or more abundant in prokaryotic cells.
Independent studies have shown that telavancin accumulates in human alveolar macrophages, reaching a cellular concentration of ~50 mg/L within 8–24 h after systemic administration of therapeutic doses.5,27 In our experiments, macrophages exposed for 24 h at 90 mg/L have an apparent cellular concentration of 4 mg/mL [based on an estimated mean cellular volume of 5 µL/mg of protein (see Van Bambeke et al.12 and references cited therein)]. The lower cellular uptake of telavancin reported in vivo may result from (i) its high protein binding (∼90% to 93%) and (ii) the fluctuation of its serum levels related to its once-daily administration and its half-life of 7.5 h.18 As extensively discussed in previous publications,6,7,12,22 our cellular model suffers from the limitation that it does not take into account these two important pharmacokinetic determinants. This may lead to an overestimation of the accumulation of telavancin versus that of vancomycin, for which the free fraction in vivo oscillates between 10% and 55%.19 Thus, assuming that only the free fraction of telavancin is available for the uptake by macrophages in vivo, its effective extracellular concentration will oscillate between ∼10 and 0.5 mg/L, which, in our model, would create a cellular concentration of ∼0.24 µg/mg of protein (i.e. ∼48 mg/L). At this concentration, which is of the same order of magnitude as that observed in vivo, telavancin does not cause any morphological change in our model.
Taken together, our data further document the tissue-directed pharmacokinetics of lipoglycopeptides.28 As telavancin is clearly superior to vancomycin with respect to bactericidal activity towards both extracellular and intraphagolysosomal S. aureus6 while showing a similar cellular safety profile (as far as lipid metabolism and subcellular morphology are concerned), the data support its potential interest for the treatment of infections where intracellular foci are present.29 Further studies using in vivo models are, however, required to confirm the improved intracellular efficacy and cellular inocuity of telavancin.
Funding
F. V. B. is Maître de Recherches of the Belgian Fonds National de la Recherche Scientifique. Funding: Belgian Fonds de la Recherche Scientifique (FRS–FNRS) and Belgian Fonds de la Recherche Scientifique Médicale (FRSM; grant nos 1.5.223.05 F, 3.4.549.00 F and 3.4.639.04 F); Belgian Federal Science Policy Office (Research project P5/33; research action P5); Fonds Spéciaux de Recherches and Actions de Recherches Concertées of the Université catholique de Louvain; Grant-in-Aid from Theravance, Inc. (for the pharmacokinetic studies with telavancin).
Transparency declarations
F. V. B. and P. M. T. are members of the European Advisory Board of Targanta Inc. (current owner of oritavancin). The remaining authors have none to declare.

cellular pharmacokinetics
glycopeptides
membrane
vancomycin
oritavancin
lipids


Cardiovascular molecular MR imaging

Introduction Cardiovascular molecular imaging is a rapidly evolving field of research, aiming to image and quantify molecular and cellular targets in vivo. MR imaging has some inherent properties that make it very suitable for cardiovascular molecular imaging. Until now, only a limited number of studies have been published on cardiovascular molecular imaging using MR imaging.
Introduction
Conventional imaging modalities, including magnetic resonance (MR), are primarily based on anatomical, functional or metabolic properties to study (patho)physiology. Molecular imaging is a rapidly evolving field of research, aiming to image and quantify molecular and cellular targets in vivo. Molecular imaging can be applied to a wide range of scientific and clinical fields of interest. One of the most promising applications of molecular imaging is in the field of cardiovascular imaging. Imaging of cardiac anatomy, dimensions and function has some limitations concerning, for example, prediction of therapy outcome. Addition of specific information on, for instance, plaque composition and total plaque burden can be very helpful in guiding therapy.
Imaging of molecular processes is desirable because cardiovascular disease may be detected earlier, risk stratification may be more accurate, monitoring of innovative therapies may be improved, or a more accurate prognosis may be provided [1].
MR imaging has some inherent properties that make it very suitable for cardiovascular molecular imaging. The interaction between inherent tissue properties and specific contrast agents may lead to more specific clinical conclusions and prediction of therapy outcome. Thereby, cardiovascular molecular MR imaging may help in diagnosing cardiovascular disease, and in deciding whether expected beneficial effects of (invasive) therapy counterbalance the risk of complications of therapy.
A conventional approach to molecular MR imaging concerns MR spectroscopy. Furthermore, there are two main innovative contrast agents that may be used clinically soon: (1) iron oxide MR contrast agents and (2) fibrin-targeted MR contrast agents.
MR spectroscopy
MR spectroscopy (MRS) allows non-invasive characterisation of myocardial metabolism. In principle, MRS is a pure form of molecular ‘imaging’ technique. Clinically, several nuclei allow noninvasive MRS of the heart. Initially, human MRS research was focussed on the 31P nucleus to study high-energy phosphate metabolism. An example concerning the effects of diabetes type 2 on myocardial high-energy phosphate metabolism is shown in Fig. 1 [2]. Another interesting new application of 31P-MRS was published by Smith et al. [3], who measured myocardial creatine kinase (CK) metabolite concentrations and adenosine triphosphate synthesis through CK, the primary energy reserve of the heart, to test the hypothesis that ATP flux through CK is impaired in patients with left ventricular hypertrophy (LVH) and chronic heart failure. It turned out that myocardial ATP levels were normal, but creatine phosphate levels were 35% lower in LVH patients than in normal subjects. Furthermore, the myocardial CK rate constant was normal in LVH, but halved in patients with LVH combined with chronic heart failure. Thereby, they could show that it is not the relative or absolute CK metabolite pool size but rather the kinetics of ATP turnover through CK that distinguishes failing from non-failing hypertrophic hearts. These observations support the hypothesis that a deficit in myofibrillar energy delivery contributes to chronic heart failure pathophysiology in human LVH. The same technique was applied by Weiss et al. to study ATP flux through CK in the normal, stressed and failing human heart [4]. The latter studies are elegant examples of the capability of MR to measure non-invasively the concentration of metabolites and even the rate constant of enzyme turnover.
Fig. 1Left panel: planscan of the volume of interest on transverse and sagittal spin-echo MR images. Right panel: phosphorus-31 magnetic resonance spectroscopy (31P-MRS) obtained at rest from the anterior left ventricular wall in a patient with diabetes type 2 (left) and a healthy subject (right). Note the decreased signal amplitude of phosphocreatine in the type 2 diabetes patient. Courtesy of Diamant et al. [2]
Another interesting application of metabolic imaging is 23Na MR imaging, as show by Jansen et al. They applied this innovative spectroscopic imaging technique as a diagnostic modality for early detection of myocardial ischaemia and viability in a rat model [5]. They tested whether 23Na MR imaging can be used to assess viability after low-flow ischaemia. 23Na MR chemical shift imaging was alternated with 23Na MR spectroscopy. Na image intensity increased with increasing severity of ischaemia. 23Na image intensity at end low-flow ischaemia was well correlated with CK release during reperfusion, as well as with infarct size. Therefore, their study indicates that 23Na MR imaging is a promising tool for the assessment of myocardial viability. Ouwerkerk et al. applied 23Na MR imaging to measure cardiac tissue sodium concentrations in the human myocardium [6]. They used a surface coil at 1.5 T MR to non-invasively quantify regional myocardial sodium concentrations in the left ventricular free wall, septum and adipose tissue. Their 23Na MR imaging results were consistent with prior invasive measurements on biopsy and autopsy specimens.
In the past, 1H-MRS of the myocardium was first applied to non-invasively study creatine depletion in non-viable infarcted myocardium [7]. Total creatine was measured in the posterior and anterior left ventricle and septum, and was significantly lower in regions of infarction than in non-infarcted regions of myocardium in patients or in the myocardium of healthy controls. Therefore, they showed for the first time that spatially localised 1H-MRS can be used to measure total creatine non-invasively throughout the human heart. The detection of regional creatine depletion may provide a metabolic means to distinguish healthy from infarcted non-viable myocardium. Szczepaniak et al. used 1H-MRS to measure myocardial lipid content [8]. Studies in rat tissue ex vivo and in healthy humans in vivo provided evidence that 1H-MRS constitutes a reproducible technique for the measurement of myocardial triglyceride. Increased myocardial triglyceride content was accompanied by elevated LV mass and suppressed septal wall thickening as measured by cardiac imaging. More recently, 1H-MRS of the human myocardium was improved by implementing the respiratory navigator technique to monitor diaphragmatic motion, and thereby correct data acquisition prospectively for breathing motion. First (unpublished) results from our institution show improved reproducibility of human cardiac 1H-MRS measurements when using the respiratory navigator technique, as compared with conventional continuous breathing. Respiratory navigator gated 1H-MRS was recently applied in an experimental setting in our institution to evaluate the effects of a very low calorie diet on myocardial triglyceride content. First (unpublished) results show that after a short very low calorie diet, there is an increase in intramyocardial triglyceride content (Fig. 2).
Fig. 2The surface coil was positioned just below the mitral valve level of the heart (a, b). Spectroscopic volume localisation in the interventricular septum on four-chamber (c) and short axis (d) views. Special care was taken to avoid contamination from epicardial fat. (e) Typical water-suppressed 1H-MR spectrum of myocardial tissue located in the interventricular septum. Peak heights are in arbitrary units
Gadolinium-based contrast agents
Gadolinium-based contrast agents can be applied to study regional myocardial perfusion. After a rapid intravenous contrast injection, there is marked signal enhancement first in the RV cavity, then in the LV cavity, and subsequently in the LV myocardium [9]. The peak signal intensity is related to the concentration of the contrast agent in the local tissue and is directly proportional to the coronary blood flow. Perfusion MR at rest and after infusion of pharmacological agents (adenosine and persantine) have been compared with standard methods (angiography or radionuclide scintigraphy) and demonstrated reasonable sensitivity (67–83%) and specificity (75–100%) [9].
Currently, multiple MR imaging techniques are available to assess myocardial viability. Cardiovascular MR imaging can be used to assess end-diastolic wall thickness and contractile function at rest [10]. Segments with an end-diastolic wall thickness <6 mm most likely represent transmural scar formation, and contractile function will not improve after myocardial revascularisation. Dobutamine MR imaging can be used to evaluate contractile reserve, in a similar manner to dobutamine echocardiography. Gadolinium contrast-enhanced MR imaging [11] allows for detection of the extent and transmurality of scar tissue (Fig. 3). Recently reported sensitivity and specificity are in the range of 74% and 82% respectively.
Fig. 3Left panel: short-axis MR images at rest and during dobutamine stress. Note the lack of improvement in myocardial wall motion in the anteroseptal region when dobutamine stress is applied. Right panel: delayed gadolinium contrast-enhanced MR images in two-chamber, four-chamber and short-axis views. Note the almost transmural delayed enhancement of the anteroseptal/apical region, corresponding to the region without contractile response due to dobutamine stress in the left panel. The anteroseptal region is considered as ‘non-viable’ myocardial tissue
Based on as yet unpublished scientific developments, it is expected that gadolinium-based delayed enhancement of the vessel wall may become reality. This MR imaging technique may allow fast total body screening for total plaque burden, an important predictor for morbidity and mortality.
In general, gadolinium-based contrast agents are not perfectly suited for molecular imaging because of the inherent high threshold of detectability. Therefore, new contrast agents are under development to potentiate the effect of distortion of the magnetic field.
Iron oxide MR contrast agents
Superparamagnetic iron oxide (SPIO) particles can be detected at micromolar concentrations of iron, and offer sufficient sensitivity for MR imaging [12]. SPIO-based cellular imaging has become an established technique to label and detect cells of interest. Imaging of macrophage activity was the initial and is still the most significant application, with several products either approved for or in clinical trials [12]. Another exciting application of SPIOs is labelling of myocardial stem cells. In a swine model for myocardial infarction, magnetically labelled stem cells were injected in the infarcted myocardial region (Fig. 4). Using delayed contrast-enhanced MR imaging, the infarcted area can be identified with high accuracy. New technical developments may even allow specific delivery of magnetically labelled therapeutics to the infarcted myocardial region [12]. Combined with measurements of myocardial function, MR imaging seems an excellent modality for planning, delivery and follow-up of myocardial stem cells as therapy for ischaemic heart disease.
Fig. 4Detection of delivery and migration of Feridex-labelled myocardial stem cells in a swine model. Hypointense lesions in spin-echo (SE),gradient-echo (GRE) and delayed-enhanced (Delayed) MR imaging (upper panel) of injection sites (arrows) within 24 h of intramyocardial injection. Cells were injected in the myocardial infarct (MI). Long-axis MR images (lower panel) show hypointense lesions (arrows) caused by labelled myocardial stem cells acquired within 24 h and 1 week. LV left ventricle, RV right ventricle. Courtesy of Bulte andKraitchman [12]
Another promising application of SPIO MR imaging is visualisation of vessel wall inflammation. SPIOs are ‘digested’ by macrophages, involved in inflammatory processes. Imaging of the SPIO-induced magnetic inhomogeneities allows for imaging of inflammation. Such an approach is currently only available in a research setting; it is, however, expected that these contrast agents will become available for clinical application soon.
Fibrin-targeted MR contrast agents
Exciting recent developments allow selective and non-invasive molecular MR imaging of thrombus [13]. The principle of the contrast agent is that it is targeted to fibrin. In an elegant study by Spuentrup et al., a swine model was used to test the innovative fibrin-targeted MR imaging contrast agent, which can be administered intravenously [14]. The imaging protocol consisted of coronary MR angiography to demonstrate the lumen of a coronary artery, combined with molecular thrombus MR imaging. Thereby, anatomical information could be linked to specific information of vessel wall components. In an area of focal coronary artery stenosis, intraluminal thrombus could be detected (Fig. 5).
Fig. 5Coronary thrombus visualisation with a fibrin-targeted molecular MR imaging contrast agent. Left panel: double-oblique white blood coronary MR angiography (multiplanar reconstruction) demonstrating the lumen of the left anterior descending artery with bright signal (arrowheads). Right panel: double-oblique MR images after administration of the fibrin-targeted contrast agent (multiplanar reconstruction, same orientation). Note the increased signal (arrow) in the left anterior descending coronary artery, corresponding to thrombus. Courtesy of Spuentrup et al. [14]
The same contrast agent can be applied to detect, for example, right atrial thrombus, a potential source of more distal emboli (Fig. 6). An atrial clot could be visualised easily with this molecular MR imaging technique, by intravenous administration of the fibrin-targeted contrast agent. Furthermore, MR clot imaging can be combined with functional imaging of the heart in the same imaging session. An even more exciting application of this fibrin-targeted contrast agent is detection of pulmonary emboli (Fig. 7).
Fig. 6Molecular MR imaging of atrial clot in a swine model. The left panel shows pre-contrast (upper) and post-contrast (lower) coronal images. Note the presence of high MR signal in the area of the right atrium, indicating an atrial clot. The right panel shows increased MR signal in the left atrium (LA), corresponding to a left atrial clot (arrow). These clots are potential sources for more distal emboli. LV left ventricle. Courtesy of Spuentrup et al. [14]Fig. 7Examples of molecular MR imaging of pulmonary embolus. Two examples are shown, each consisting of three adjacent coronal slices (horizontal). The MR imaging technique is such that signal from surrounding blood pool and lung parenchyma is suppressed. The upper row shows pulmonary embolus (arrow) in the right lower lobe. The lower panel shows bilateral pulmonary emboli (arrows) after intravenous administration of fibrin-targeted MR imaging contrast agent. Courtesy of Spuentrup et al. [14]
The above-described applications of molecular MR imaging may be especially suitable for fast screening for cardiovascular disease in an emergency setting. Patients presenting with chest pain in the emergency room can be studied by MR imaging to confirm or rule out ischaemic heart disease or pulmonary embolus. Molecular MR imaging using fibrin-targeted contrast agents allows selective visualisation of acute coronary, cardiac and pulmonary thrombi. Additional functional cardiac imaging can help determine the functional effects of detected thrombi.
Conclusion
Molecular MR imaging is an exciting and rapidly evolving new area of cardiovascular imaging. MR imaging seems very suitable for molecular imaging, although many technical difficulties have to be overcome. The main current limitation is the low sensitivity of MR imaging to detect small changes in magnet homogeneity. We expect that in the next decade, currently promising MR molecular imaging agents will be introduced into the clinical arena to guide diagnosis and therapy of cardiovascular disease.

cardiovascular molecular imaging
cardiovascular disease
contrast agents
mr spectroscopy