Arginine suppletion in free TRAM flap Based on literature at the initiation of the study, the most commonly used infusion dose of arginine was administered. Patients received intravenous supplementation of arginine or placebo (Bufa, Uitgeest, The Netherlands) prepared by the local pharmacist. Intravenous supplementation consisted of either a daily dose of 30 g of arginine, dissolved in 1000 ml of 0.9% sodium chloride and adjusted to pH 7.2 using 10% hydrochloric acid (net nitrogen intake, 45.7 mM), or a placebo treatment consisting of a daily dose of 25.2 g of alanine, dissolved in 1000 ml of 0.9% sodium chloride (net nitrogen intake, 44 mM). The amino acid alanine was chosen to control for nitrogen intake and because alanine does not participate in the arginine–nitric oxide pathway. The infusions were made isovolumetric and isonitrogenous. Although it was not possible to make the infusions isocaloric, the daily arginine infusion accounted for 120 kcal and the placebo infusion accounted for 100.8 kcal. Patients received an infusion of 1000 ml in two doses of 500 ml divided equally over 24 hours. A calibrated perfusion pump was used to ensure adequate administration. Blood samples were taken preoperatively, after completion of flap dissection, 1 hour after reperfusion, 24 hours after reperfusion, and on the fourth postoperative day. Blood samples were placed immediately on ice and processed as soon as possible (<5 minutes). Blood was centrifuged at 4°C at 4000 rpm at least within 1 hour after sampling. After centrifugation, 500 μl of plasma was deproteinized using 20 mg of dry sulfosalicylic acid, vortexed, and frozen in liquid nitrogen. Samples were stored at –80°C until analysis. A spectrum of 21 amino acids was determined using fully automated high‐performance liquid chromatography described in previous studies.22 Primary outcome was the incidence of partial flap loss, subdivided into minor partial flap loss and major partial flap loss. Photographs were taken daily with a ruler within the photograph to determine the area of partial flap loss. The partial flap loss was calculated by planimetry performed with the ImageJ (National Institutes of Health, Bethesda, Md.) program. Minor partial flap loss was managed conservatively and had no adverse effect on outcome (Figure 6.1). Major partial flap loss was débrided in the operating room and had a minor effect on aesthetic outcome (Figure 6.2). Definitions and abbreviations used for flap complications and their clinical implication are listed in Table 6.2. Laser Doppler flowmetry is an accurate method with which to assess localized microcirculation20,23 and was used to measure flap microcirculatory perfusion, which was measured using laser Doppler flowmetry with hooked probes with the Perimed (Jarfalla, Sweden) device in zones I and IV after flap dissection and hourly during the first 5 hours after reperfusion (Figure 6.3). Also taken into analysis were flap temperature, perioperative hemodynamics, and previously described risk factors.24 All data are presented as mean values ± SEM. The patient allocation was revealed to the researchers once recruitment, data collection, and laboratory analyses were complete and incorporated into the SPSS program (SPSS, Inc., Chicago, Ill.), which was then used for statistical analysis. Repeated measures analysis of variance was used for 89
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