Fibrinogen and albumin synthesis rates in major upper abdominal surgery

Brief name: Flooding dose technique with stable isotope (D5-phenylalanine) for measuring de novo synthesis of fibrinogen and albumin. We will study patients undergoing liver resections of variable size , i.e. hemihepatectomy and extended hemihepatectomy (n=9). Incorporation of D5-phenylalanine into fibrinogen and albumin will analysed by gas chromatography – mass spectrometry. Patients will be investigated 3 times in the postabsorptive state (not less than 6 h after a warm meal, and not less than 4 h after coffee or tea, tap water is allowed without restriction). Postoperatively iv crystalloid solutions may be administered. If 5% dextrose is given, it should be paused not less that 2h before measurement. The measurements will be preoperatively, not less than 2 days but not more than 5 days before the measurement on postoperative day 1. A 3rd measurement will then be performed on postoperative day 3-5. All procedures in conjunction with anesthesia, surgery and postoperative care will be in accord with the local routines at Karolinska University Hospital Huddinge, and at the discretion of the attending anesthesiologist and/or surgeon. At the preoperative measurement two venous accesses will be inserted. At the postoperative measurements existing vascular accesses will be utilized. The routine blood sampling for preoperative and postoperative surveillance will be adjusted time-wise to occur simultaneously with the measurements. Measurement procedures will be identical except for the isotopic enrichment of D5-phenylalanine. D5-phenylalanine will be administered intravenous during 10 minutes of a 2% phenylalanine solution (45mg/kg, 5, 10 and 30 MPE [mols percent excess]) on the 3 measurement occasions. Thereafter venous blood samples will be obtained just before and at 5, 10, 15, 30, 50, 60, 70 and 90 minutes after the start of the tracer phenylalanine infusion. The samples, collected into citrate vials and kept on ice and thereafter centrifuged at 2,500 r/minute at 4oC and the plasma fraction will thereafter be stored at -80oC pending analysis. All the measurement will be performed by specific trained research nurses and a PhD-student. For the calculation of the fractional synthesis rates the enrichment of D5-phenylalanine in the free phenylalanine pool in plasma and in circulating albumin and fibrinogen will be measured. For the free plasma enrichment, proteins are precipitated with sulfosalicylic acid and the supernatant was cleaned using ion exchange resin. For the enrichment measurement in albumin and fibrinogen, these two proteins are isolated from plasma samples. Albumin is isolated by ethanol extraction from trichloroacetic acid precipitated proteins. Albumin is then hydrolyzed using 6M HCL (110oC for 24 hours). Fibrinogen is isolated by several times dissolving in sodium citrate and precipitation with L-ammonium sulphate. Finally fibrinogen is precipitated using perchloric acid and hydrolyzed using 6M HCL (110oC for 24 hours). Following hydrolyzation, the enrichment of phenylalanine from both proteins is analyzed after conversion of phenylalanine to phenylethylamine using tyrosine decarboxylase and extraction with diethylether. Enrichments of D5, phenylalanine in the proteins are estimated from the measurements of mass ratios of m+5/m+2, of phenylethylamine by gas chromatography – mass spectrometry (Agilent 5975C, Agilent, Kista, Sweden) and comparison with standard curve of known phenylalanine enrichments. Fractional synthesis rates will be calculated by dividing the increment of the enrichment in the protein by the area under the curve for the precursor enrichment in plasma.