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The synthesis rates of total liver protein and plasma albumin determined simultaneously in vivo in humans
The Synthesis Rates of Total Liver Protein and Plasma Albumin Determined Simultaneously In Vivo in Humans HANS BARLE,1 BJO¨RN NYBERG,2 PIA ESSE´N,1 KERSTIN ANDERSSON,3 MARGARET A. MCNURLAN,4 JAN WERNERMAN,1 AND PETER J. GARLICK4
Although the metabolism of liver-derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin in vivo were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. L-[2H5 ]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 { 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC-MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 { 3.1 %/d (mean { SD), and that of albumin was 5.9 { 1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 { 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human in vivo model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein. (HEPATOLOGY 1997;25:154-158.) The liver plays a vital role in protein metabolism. Results from animal studies have shown profound changes in the rate of protein synthesis of the liver in response to trauma and critical illness, including enhanced synthesis of acute phase proteins.1-4 However, these results may not be immediately applicable to man. Until recently, techniques have not been available for the direct study of human liver protein metabolism. Stable isotope techniques have been extensively employed
Abbreviations: APE, atoms percent excess; FSR, fractional synthesis rate; ASR, absolute synthesis rate. From the 1Department of Anesthesiology and Intensive Care and 2Department of Surgery, Huddinge University Hospital, 3Anesthesiological Metabolism Unit, Clinical Research Center, Novum, Karolinska Institute, Stockholm, Sweden and 4Department of Surgery, University Medical Center, State University of New York at Stony Brook, NY. Supported by grants from the Swedish Medical Research Council (Project 04210) and the Maud and Birger Gustavsson Foundation, Stockholm, Sweden, and the County Council of Stockholm. Hans Barle is the grateful recipient of the 1993 European Society of Parenteral and Enteral Nutrition-Clintec fellowship. Received June 26, 1996; accepted September 9, 1996. Address reprint requests to: Hans Barle, M.D., Dept. of Anesthesiology and Intensive Care, K 32, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2501-0028$3.00/0
to examine protein metabolism in man, especially for whole body measurements. Whole body protein synthesis has been shown to change after surgery of moderate severity,5,6 and to increase after major trauma.7 On the other hand, in major trauma, whole body protein degradation increases even more than protein synthesis.7 However, it is preferrable that individual tissues are studied separately, because studies of the whole body do not differentiate responses at the organ level. Thus, stable isotope techniques have also been used to assess protein synthesis in human skeletal muscle.8 Esse´n et al. have demonstrated a decrease in muscle protein synthesis after surgery of moderate severity.9 Most of the knowledge of human liver protein synthesis originates from investigations of the synthesis of export proteins, such as albumin and fibrinogen.10-14 Total liver protein synthesis in man has been much less studied, because such investigations require access to liver tissue, which raises technical difficulties and potential risks. In the few studies undertaken so far, stable isotopes have been employed to assess total liver protein synthesis in liver biopsy specimens obtained during open abdominal surgery.15-17 Although providing the first information about human liver protein synthesis, the effect of the surgical trauma can not be neglected in these studies. However, current laparoscopic surgical techniques have provided an alternative procedure for obtaining liver tissue for scientific purposes, where the influence of the surgical procedure is minimized.18 The aim of this study was to further substantiate the laparoscopic cholecystectomy procedure as a human in vivo model for obtaining liver tissue, to characterize hepatic protein synthesis, in terms of total liver protein synthesis and relate it to the synthesis of exported albumin. PATIENTS AND METHODS 2
Materials. L-[ H5 ]phenylalanine, 99 atom percent (Mass Trace Inc., Woburn, MA) was dissolved in sterile water together with unlabelled phenylalanine (Ajinomoto Company, Tokyo, Japan) to a concentration of 20 g/L, 10 APE (atoms percent excess). The solutions were prepared, heat-sterilized, and stored in sterile containers. Subjects and Experimental Protocol. Nine patients with no known disease apart from cholecystolithiasis, scheduled for elective laparoscopic cholecystectomy, participated in the study (Table 1). The patients were studied after a 12- to 15-hour fast. Before the operation, the subjects were given an injection of morphine-hyoscine intramuscularly, as premedication. Antecubital venous lines were inserted bilaterally and the patients were given Ringer’s solution (Ringeracetat, Pharmacia, Stockholm, Sweden) in the left arm (100-400 mL/ hr). The line in the right arm was used for blood sampling. The patients were anesthetized in a standardized manner with sodium thiopentone, fentanyl, and atracurium at the induction, and a combination of oxygen, air, and isoflurane for maintenance. At the time of induction of anesthesia L-[2H5 ]phenylalanine 45 mg/kg, 10 APE was given intravenously for 10 minutes (Fig. 1). Immediately after the insufflation of carbon dioxide into the abdomen (resulting in an increased intraabdominal pressure of 10 to 13 mm Hg, which was maintained throughout the operation) and the insertion of four trocars through the abdominal wall, but before the cholecystectomy per se was started, a liver biopsy specimen was
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TABLE 1. Characteristics of Patients Undergoing Laparoscopic Cholecystectomy, Being Examined Regarding Liver Protein Synthesis Sex (F/M) Age (yr) Weight (kg) Length (m) BMI (kg/m2)
47.2 70.2 1.68 24.7
6/3 { 10.0 { 13.8 { 0.08 { 3.5
NOTE. Results are expressed as means and SD. Abbreviation: BMI, body mass index.
taken using laparoscopic scissors, at a standardized location: the most ventral and medial part of the right liver lobe. To stop bleeding, the biopsy site was coagulated diathermally. No complications because of bleeding were observed. Blood Samples. Venous blood samples were drawn at the following intervals: 0, 5, 10, 15, 30, 50, 70, and 90 minutes after the injection of phenylalanine for the determination of isotopic enrichment in plasma and albumin, respectively (Fig. 1). Samples at 0 minutes (which were taken before any parenteral fluid had been provided), were also used to determine plasma albumin concentrations and serum concentrations of insulin and glucose. The samples were stored at 0807C before analysis. Measurements of plasma volume were made beginning 30 minutes after the injection of phenylalanine, with 131I-labelled albumin (Institutt for Energiteknikk, Kjeller, Norway) corresponding to 100 kBq. Blood samples were taken at 20, 30, 40, and 45 minutes to assess isotope dilution, according to the routine of the Department of Clinical Physiology, Huddinge University Hospital. The values of the measured plasma volumes were compared with the values obtained from the normogram using weight and age as described by Dagher et al.19 Ethics. The nature, purposes, and potential risks of the experimental procedures were explained to the patients before obtaining their voluntary consent. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and had received an a priori approval by the Ethical Committee as well as the Isotope Committee (blood volume measurements) of the Karolinska Institute, Stockholm, Sweden. Sample Preparation. The details of the preparation and analysis of samples of skeletal muscle protein, plasma albumin, and plasma for the enrichment of L-[2H5 ]phenylalanine have been extensively described elsewhere.20-22 Briefly, the liver tissue specimens were homogenized in cold 3% perchloric acid on ice in Eppendorff microfuge tubes (Sarstedt, Inc., Newton, NC) and centrifuged (Eppendorff 5402 centrifuge, Eppendorff-Netheler-Hinz GmbH, Hamburg, Germany) for 5 minutes at 8,000 rpm at 47C. The protein precipitate was washed repeatedly to remove traces of free phenylalanine, followed by suspension in 0.3 mol/L NaOH and reprecipitation in 5% perchloric acid. Following further washing, proteins were hydrolyzed in 6 mol/L hydrochloric acid (HCl) for 24 hours at 1107C. The hydrochloric acid was removed by evaporation in vacuo, and the hydrolysate used for the measurement of isotopic enrichment. Albumin in plasma samples was extracted from 9% trichloroacetic acid–precipitated protein fraction by differential solubility in absolute ethanol.23 The purity of the albumin fraction was checked with matrix-assisted laser desorption time of flight mass spectrometry (Finnigan Laser mat 2000, Finnigan Corporation, Hemmel Hempstead, England) showing that a single peak with a molecular weight of 67,450 kd had been isolated. Thereafter, plasma albumin samples were prepared in the same way as samples of total liver protein. Samples of plasma for the determination of free phenylalanine enrichment were treated with 8% sulphosalicylic acid to precipitate protein. The amino acid containing supernatants were purified with cation exchange columns (Dowex-50, Biorad AG50, 50W-X8, (H/) form, 100-200 mesh [Biorad, Inc., Hercules, CA]) and eluted from columns with 4 mol/L NH4OH. The samples were then dried in vacuo. Masspectrometry. The determination of enrichment of L-[2H5 ]phenylalanine in skeletal muscle protein, plasma albumin, and plasma has been described previously.20,24 In short, the enrichment of L-[2H5 ]phenylalanine from liver tissue protein and albumin hydrolysates was determined by measuring the mass-to-charge ratio at 106 and 109 of the n-heptafluorobutyryl derivative of phenylethylamine on a Fisons MD 800 mass spectrometer (Fisons, Beverly, MA). With this instrument, there was no change in the enrichment with sample loading and the previously employed calibration curves were
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not necessary.24 The L-[2H5 ] enrichment of free phenylalanine in plasma was measured by monitoring the ions at mass-to-charge ratio, 336 and 341 of the t-butyldimethylhylsilyl derivative on an HP 5972 mass spectrometer (Hewlett Packard, Palo Alto, CA). Other Analytical Procedures. Serum glucose was determined by a glucose dehydrogenase method.25 Radioimmunoassay was used to determine the serum concentration of insulin.26 Plasma albumin concentration was measured using the bromocresol green method.27 Calculation and Statistics. The fractional synthesis rate (FSR) of total liver protein, i.e., the fraction of the total pool of liver protein that is synthesized every day, was calculated according to the previously described formula:28 ks Å P 1 100/A
where ks is the FSRTLP (%/day) of total liver protein and P is the isotopic enrichment of phenylalanine in total liver protein at the time of the liver biopsy specimen (APE), and A is the area under the curve of plasma free phenylalanine enrichment (APE vs. time). The fractional synthesis rate of albumin, i.e., the fraction of the intravascular albumin pool that is synthesized every day, was calculated using the same formula in a modified version as previously described:12 ks Å (P(2) 0 P(1) ) 1 100/A
where ks is the FSRALB (%/day) for albumin, and P(2) and P(1) represent enrichment of phenylalanine in albumin at two time points, after the curve of enrichment becomes linear (usually 50 and 90 samples). A is also modified to the area under the curve for enrichment of plasma-free phenylalanine between time points adjusted for the secretion time (Ts ), i.e., the temporal lag period before the appearance of labelled albumin in plasma. The secretion time was assessed by plotting the individual regression line for the linear part of the albumin enrichment curve and extrapolating to the baseline enrichment.12 Calculation of absolute synthesis rates of albumin (ASR) was based on the fractional rate of albumin synthesis and the intravascular albumin mass calculated from the concentration of plasma albumin concentrations and the measured plasma volume. Data are presented as means and the SD. Difference between values of measured plasma volumes and values obtained from the normogram was assessed by Student’s t test for paired samples. The correlation between FSR of total liver protein and the ASR of albumin, as well as the correlation between age and the FSR of albumin were calculated using ‘‘simple regression’’ (r Å Pearson’s coefficient of correlation). RESULTS
The fractional synthesis rate of total liver protein was 24.7 { 3.1%/day as assessed from the liver biopsy specimens taken at 35 { 7 minutes after induction of anesthesia and injection
FIG. 1. Experimental protocol for measuring fractional synthesis rates of total liver protein and albumin, using the flooding dose method, in nine patients undergoing laparoscopic cholecystectomy. L-[2H5 ]phenylalanine (45 mg/ kg, 10 atoms percent excess [ÅAPE]) was administered for 10 minutes. A liver biopsy specimen (one per patient) was obtained at 35 { 7 minutes (mean and SD), and blood samples were taken regularly for 90 minutes. Isotopic enrichment in total liver protein (from liver biopsy specimens), plasma albumin, and plasma were then measured.
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TABLE 2. The FSRs of Total Liver Protein in Patients (n Å 9) Undergoing Laparoscopic Cholecystectomy, Investigated With the Flooding Technique Patient
Liver Biopsy Time (min)
FSR, Total Liver Protein (%/d)
1 2 3 4 5 6 7 8 9
38 50 40 30 33 30 30 30 30 34.6 { 7.0
27.4 24.2 26.0 27.6 25.4 27.5 18.6 21.4 23.7 24.7 { 3.1
NOTE. The individual time point for taking the liver biopsy specimen in minutes after the injection of isotope, is also presented. Results are expressed as means and SD.
of phenylalanine (Table 2). Biopsy specimens were taken 18 { 6 minutes into the operation. The fractional synthesis rate of albumin was 5.9 { 1.2%/ day and the absolute synthesis rate of albumin was 109 { 21 mg/kg/d (Table 3). There was no statistically significant correlation between the FSR of total liver protein and the ASR of albumin (r Å .41). Furthermore, there was no correlation between age and the FSR of albumin (r Å .37). The albumin secretion time (i.e., the time elapsed before isotopically labelled albumin started to appear in peripheral blood) was 39.4 { 4.8 minutes, after initiation of the injection of the stable isotope (Table 3). Plasma volumes were 3.3 { 0.6 L as measured with 131I-labelled albumin, compared with 3.0 { 0.5 L according to the normogram by Dagher et al. (Table 3).19 The mean intraindividual difference between the two values was 11.6 { 4.2% (P õ .05). Hence, for seven of the nine subjects, plasma volume measurement gave a higher value than the normogram. The serum concentration of glucose was 5.7 { 0.6 mmol/L (reference interval, 3.4-6.4 mmol/L) and of insulin was 13.4 { 5.3 mU/L (reference interval for fasted subjects, 3-35 mU/ L). DISCUSSION
In the present study, the laparoscopic cholecystectomy procedure was further characterized as a human in vivo model to obtain liver tissue specimens. The fractional synthesis rate of total liver proteins was estimated by the incorporation of 2 L-[ H5 ]phenylalanine. In addition, the fractional synthesis rate as well as the absolute synthesis rate of exported albumin were measured. The technique for obtaining liver biopsy specimens in a standardized and safe manner during laparoscopic upper abdominal surgery, as a human in vivo model, has recently been characterized.18 From a scientific point of view, alternative liver biopsy techniques have disadvantages, which, at least in part, have been overcome by this model. Percutaneous and transvenous biopsy techniques provide relatively small specimens, and are used primarily for diagnostic purposes.28 Furthermore, these techniques are potentially riskful, and may not be considered ethically acceptable in healthy volunteers. Perioperative (i.e., via a laparotomy) techniques also carry potential risks, but the surgical procedure allows inspection of the biopsy site. However, after the introduction of laparoscopic surgical techniques, laparotomy is performed more seldom, except in patients with underlying malignancy and in acute situations. Furthermore, the conventional laparotomy exposes the organism for a trauma which may not be negligible, possibly affecting the results. In the few studies performed, the laparoscopic cholecystectomy has been shown
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to have less pronounced metabolic effects compared with conventional cholecystectomy, including smaller increase of urea synthesis and less reduction of insulin sensitivity.29,30 In this study, the laparoscopic model for aquiring liver tissue was combined with the flooding technique to study liver protein synthesis. The flooding technique is suitable during non–steady-state conditions, like an operation, because the periods of measurement are short. Furthermore, the problem of recycling of amino acids into the precursor pool of amino acids is minimized.8,9 This was the first study where total liver protein synthesis was assessed by gas chromatography mass spectrometry using L-[2H5 ]phenylalanine as the isotopically labelled amino acid. The sensitivity of gas chromatography mass spectrometry (GC-MS) for the determination of protein enrichment facilitates the use of smaller biopsies than was possible with L-[13C]leucine and gas isotope ratio mass spectrometry.17,20,24 The liver biopsy specimens were taken as soon as possible after the start of the operation, i.e., immediately after the intraabdominal insufflation of carbon dioxide. This was done to minimize the influence of the anesthetic agents or the operative trauma on liver protein synthesis. The immediate effects of trauma as well as opioids, barbiturate induction, and volatile anesthetic agents on liver function in general and liver protein synthesis in particular are largely unknown in man.31 However, even though previous studies in rats have shown that halothane may depress liver protein synthesis, especially after prolonged exposure, such an effect has not been shown for either isoflurane (at least not after exposure for 1 hour) or for a combination of midazolam-fentanyl.32,33 Furthermore, because isoflurane is metabolized to a very small extent,31 it was chosen as the main anesthetic agent. The mean FSR of total liver protein for this group of nine patients was 24.7%/d and was quite consistent between subjects, with a range between 18.6-27.6%/d (Table 2). These values agree well with those obtained previously with L-[13C]leucine.17 The FSR of total liver protein is about 10-fold higher than that of skeletal muscle in man.9 A similar relationship between these two tissues has been observed in fasting young rats, although the absolute values are three- to fourfold higher in the rat.34,35 The results for FSR of albumin in this investigation were in accord with values previously reported in healthy individuals, even though values in young males are somewhat higher (Table 3).11,12,21,22 In the present investigation the FSR of albumin showed a comparatively larger scatter among the subjects, when compared to the scatter of FSR of total liver protein in the same subjects (Tables 2 and 3). There was no correlation between age and FSR of albumin. TABLE 3. The FSRs and ASRs of Albumin in Patients Undergoing Elective Laparoscopic Cholecystectomy (n Å 9)
Patient
1 2 3 4 5 6 7 8 9
Secretion Time (min)
FSR Albumin (%/d)
Plasma Albumin Concentration (g/L)
Plasma Volume (L)
ASR Albumin (mg/kg/d)
36.0 32.4 45.1 47.3 38.6 35.5 40.2 42.3 36.8 39.4 { 4.8
5.8 6.5 8.1 6.2 6.6 4.4 4.5 4.5 6.4 5.9 { 1.2
39 39 39 43 40 40 41 37 41 39.9 { 1.7
2.3 2.7 3.2 3.0 4.2 4.3 3.3 3.0 3.4 3.3 { 0.6
113 131 134 106 128 89 87 76 118 109 { 21
NOTE. The time from the injection of isotope until the appearance of labelled albumin in plasma is also shown (‘‘secretion time’’), as well as plasma volume measurements and plasma albumin concentrations. Results are expressed as means and SD.
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The ASRs of albumin showed somewhat lower values than previously reported in healthy subjects (Table 3).11,12,36 The reasons for this are not clear, but all the parameters used in the calculation (FSR of albumin, plasma concentration of albumin, plasma volume, and body weight) as well as the category of patients that is investigated, are of importance when the results are interpreted. Above all, the possible effects of anesthesia and surgery must be taken into consideration. Regarding plasma volumes, there was a statistically significant difference when the measured values were compared with the values obtained from the normogram of Dagher et al.19 For seven of the patients, the measured values were actually higher, possibly because of the parenteral fluid that was provided perioperatively. This emphasizes the need to measure the plasma volume when investigating the absolute synthesis rates of export proteins. We calculated whether or not there was a statistically significant correlation between total liver protein synthesis (FSR) and ASR. However, no such correlation was found within the range of values of this study. Still, total liver protein synthesis and albumin synthesis can be related to one another in absolute terms if some basic assumptions concerning the protein content of the liver are made. Assuming that the liver accounts for 2% of body weight37 in the adult (weighing 70 kg) and that the total protein concentration is 150 g/kg fresh liver,37 the liver contains approximately 210 g of protein. If the FSR of total liver protein is 25%/d, this means a de novo protein synthesis of about 52 g/d, or 750 mg/kg body weight per day. Because the ASR of albumin is approximately 110 mg/kg/d as found in this study, the contribution of albumin synthesis to total liver protein synthesis, would be about 15%, as reported previously in animals (Table 3).22,38,39 In general, when estimating the fractional synthesis rate of total liver protein, employing a constant infusion of an isotopically labelled amino acid, lower values are produced15,16 than with the flooding technique.17 However, when estimating the FSR of albumin, the two methods give more uniform values.11,22 The most likely explanation for the difference in liver FSR involves whether or not the synthesis of exported proteins is included in the measurement. With the flooding technique, the synthesis of exported protein is included because the time of measurement is shorter than the secretion time of the exported proteins. With the constant infusion technique, labelled export proteins have escaped from the liver because of longer periods of measurement, and are therefore not included in the measured FSR. In this study, the intention was to take the liver biopsy specimen at 30 to 40 minutes after the injection of phenylalanine, to enable maximal labelling before label was lost from the liver into export proteins. Albumin has been shown to have a secretion time of 30 to 40 minutes.12 In one patient, however, the liver biopsy was taken at 50 minutes, because of technical reasons (Table 2). In conclusion, in this study, a human in vivo model for obtaining liver tissue specimens during laparoscopic cholecystectomy was applied to measure the synthesis rate of total liver proteins and relate it to that of albumin isolated from plasma. The flooding technique was employed, using L-[2H5]phenylalanine as the isotopically labelled amino acid. In the future, this study model may be used to study the effects of different hormones and nutrition upon hepatic protein metabolism. Acknowledgment: The skilled technical and nursing assistance of Mrs. Viveka Gustavsson, Ms. Eva Skoog, and Mr. George Casella is gratefully acknowledged. REFERENCES 1. Jepson MM, Pell JM, Bates PC, Millward DJ. The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochem J 1986;235:329-336.
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35. Garlick PJ, Wernerman J, McNurlan M, Heys SD. Organ-specific measurements of protein turnover in man. Proc Nutr Soc 1991;50:217-225. 36. Rotschild MA, Oratz M, Screiber SS. Serum albumin. HEPATOLOGY 1988; 8:385-401. 37. Widdowson EM, Dickerson JW: Composition of the body. In: Lentner C, ed. Geigy Scientific Tables. Volume 1. Basel: Medical Education Division, Ciba-Geigy Corporation, 1981:220. 38. Ballmer PE, McNurlan MA, Grant I, Garlick PJ. Down regulation of albumin synthesis in the rat by human recombinant interleukin-1 or turpentine and the response to nutrients. J Parent Ent Nutr 1995;19:266271. 39. Pain VM, Clemens MJ, Garlick PJ. The effect of dietary protein deficiency on albumin synthesis and the concentration of active albumin messenger RNA in rat liver. Biochem J 1978;172:129-135.
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Although the metabolism of liver-derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin in vivo were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. L-[2H5 ]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 { 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC-MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 { 3.1 %/d (mean { SD), and that of albumin was 5.9 { 1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 { 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human in vivo model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein. (HEPATOLOGY 1997;25:154-158.) The liver plays a vital role in protein metabolism. Results from animal studies have shown profound changes in the rate of protein synthesis of the liver in response to trauma and critical illness, including enhanced synthesis of acute phase proteins.1-4 However, these results may not be immediately applicable to man. Until recently, techniques have not been available for the direct study of human liver protein metabolism. Stable isotope techniques have been extensively employed
Abbreviations: APE, atoms percent excess; FSR, fractional synthesis rate; ASR, absolute synthesis rate. From the 1Department of Anesthesiology and Intensive Care and 2Department of Surgery, Huddinge University Hospital, 3Anesthesiological Metabolism Unit, Clinical Research Center, Novum, Karolinska Institute, Stockholm, Sweden and 4Department of Surgery, University Medical Center, State University of New York at Stony Brook, NY. Supported by grants from the Swedish Medical Research Council (Project 04210) and the Maud and Birger Gustavsson Foundation, Stockholm, Sweden, and the County Council of Stockholm. Hans Barle is the grateful recipient of the 1993 European Society of Parenteral and Enteral Nutrition-Clintec fellowship. Received June 26, 1996; accepted September 9, 1996. Address reprint requests to: Hans Barle, M.D., Dept. of Anesthesiology and Intensive Care, K 32, Huddinge University Hospital, S-141 86 Huddinge, Sweden. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2501-0028$3.00/0
to examine protein metabolism in man, especially for whole body measurements. Whole body protein synthesis has been shown to change after surgery of moderate severity,5,6 and to increase after major trauma.7 On the other hand, in major trauma, whole body protein degradation increases even more than protein synthesis.7 However, it is preferrable that individual tissues are studied separately, because studies of the whole body do not differentiate responses at the organ level. Thus, stable isotope techniques have also been used to assess protein synthesis in human skeletal muscle.8 Esse´n et al. have demonstrated a decrease in muscle protein synthesis after surgery of moderate severity.9 Most of the knowledge of human liver protein synthesis originates from investigations of the synthesis of export proteins, such as albumin and fibrinogen.10-14 Total liver protein synthesis in man has been much less studied, because such investigations require access to liver tissue, which raises technical difficulties and potential risks. In the few studies undertaken so far, stable isotopes have been employed to assess total liver protein synthesis in liver biopsy specimens obtained during open abdominal surgery.15-17 Although providing the first information about human liver protein synthesis, the effect of the surgical trauma can not be neglected in these studies. However, current laparoscopic surgical techniques have provided an alternative procedure for obtaining liver tissue for scientific purposes, where the influence of the surgical procedure is minimized.18 The aim of this study was to further substantiate the laparoscopic cholecystectomy procedure as a human in vivo model for obtaining liver tissue, to characterize hepatic protein synthesis, in terms of total liver protein synthesis and relate it to the synthesis of exported albumin. PATIENTS AND METHODS 2
Materials. L-[ H5 ]phenylalanine, 99 atom percent (Mass Trace Inc., Woburn, MA) was dissolved in sterile water together with unlabelled phenylalanine (Ajinomoto Company, Tokyo, Japan) to a concentration of 20 g/L, 10 APE (atoms percent excess). The solutions were prepared, heat-sterilized, and stored in sterile containers. Subjects and Experimental Protocol. Nine patients with no known disease apart from cholecystolithiasis, scheduled for elective laparoscopic cholecystectomy, participated in the study (Table 1). The patients were studied after a 12- to 15-hour fast. Before the operation, the subjects were given an injection of morphine-hyoscine intramuscularly, as premedication. Antecubital venous lines were inserted bilaterally and the patients were given Ringer’s solution (Ringeracetat, Pharmacia, Stockholm, Sweden) in the left arm (100-400 mL/ hr). The line in the right arm was used for blood sampling. The patients were anesthetized in a standardized manner with sodium thiopentone, fentanyl, and atracurium at the induction, and a combination of oxygen, air, and isoflurane for maintenance. At the time of induction of anesthesia L-[2H5 ]phenylalanine 45 mg/kg, 10 APE was given intravenously for 10 minutes (Fig. 1). Immediately after the insufflation of carbon dioxide into the abdomen (resulting in an increased intraabdominal pressure of 10 to 13 mm Hg, which was maintained throughout the operation) and the insertion of four trocars through the abdominal wall, but before the cholecystectomy per se was started, a liver biopsy specimen was
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TABLE 1. Characteristics of Patients Undergoing Laparoscopic Cholecystectomy, Being Examined Regarding Liver Protein Synthesis Sex (F/M) Age (yr) Weight (kg) Length (m) BMI (kg/m2)
47.2 70.2 1.68 24.7
6/3 { 10.0 { 13.8 { 0.08 { 3.5
NOTE. Results are expressed as means and SD. Abbreviation: BMI, body mass index.
taken using laparoscopic scissors, at a standardized location: the most ventral and medial part of the right liver lobe. To stop bleeding, the biopsy site was coagulated diathermally. No complications because of bleeding were observed. Blood Samples. Venous blood samples were drawn at the following intervals: 0, 5, 10, 15, 30, 50, 70, and 90 minutes after the injection of phenylalanine for the determination of isotopic enrichment in plasma and albumin, respectively (Fig. 1). Samples at 0 minutes (which were taken before any parenteral fluid had been provided), were also used to determine plasma albumin concentrations and serum concentrations of insulin and glucose. The samples were stored at 0807C before analysis. Measurements of plasma volume were made beginning 30 minutes after the injection of phenylalanine, with 131I-labelled albumin (Institutt for Energiteknikk, Kjeller, Norway) corresponding to 100 kBq. Blood samples were taken at 20, 30, 40, and 45 minutes to assess isotope dilution, according to the routine of the Department of Clinical Physiology, Huddinge University Hospital. The values of the measured plasma volumes were compared with the values obtained from the normogram using weight and age as described by Dagher et al.19 Ethics. The nature, purposes, and potential risks of the experimental procedures were explained to the patients before obtaining their voluntary consent. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and had received an a priori approval by the Ethical Committee as well as the Isotope Committee (blood volume measurements) of the Karolinska Institute, Stockholm, Sweden. Sample Preparation. The details of the preparation and analysis of samples of skeletal muscle protein, plasma albumin, and plasma for the enrichment of L-[2H5 ]phenylalanine have been extensively described elsewhere.20-22 Briefly, the liver tissue specimens were homogenized in cold 3% perchloric acid on ice in Eppendorff microfuge tubes (Sarstedt, Inc., Newton, NC) and centrifuged (Eppendorff 5402 centrifuge, Eppendorff-Netheler-Hinz GmbH, Hamburg, Germany) for 5 minutes at 8,000 rpm at 47C. The protein precipitate was washed repeatedly to remove traces of free phenylalanine, followed by suspension in 0.3 mol/L NaOH and reprecipitation in 5% perchloric acid. Following further washing, proteins were hydrolyzed in 6 mol/L hydrochloric acid (HCl) for 24 hours at 1107C. The hydrochloric acid was removed by evaporation in vacuo, and the hydrolysate used for the measurement of isotopic enrichment. Albumin in plasma samples was extracted from 9% trichloroacetic acid–precipitated protein fraction by differential solubility in absolute ethanol.23 The purity of the albumin fraction was checked with matrix-assisted laser desorption time of flight mass spectrometry (Finnigan Laser mat 2000, Finnigan Corporation, Hemmel Hempstead, England) showing that a single peak with a molecular weight of 67,450 kd had been isolated. Thereafter, plasma albumin samples were prepared in the same way as samples of total liver protein. Samples of plasma for the determination of free phenylalanine enrichment were treated with 8% sulphosalicylic acid to precipitate protein. The amino acid containing supernatants were purified with cation exchange columns (Dowex-50, Biorad AG50, 50W-X8, (H/) form, 100-200 mesh [Biorad, Inc., Hercules, CA]) and eluted from columns with 4 mol/L NH4OH. The samples were then dried in vacuo. Masspectrometry. The determination of enrichment of L-[2H5 ]phenylalanine in skeletal muscle protein, plasma albumin, and plasma has been described previously.20,24 In short, the enrichment of L-[2H5 ]phenylalanine from liver tissue protein and albumin hydrolysates was determined by measuring the mass-to-charge ratio at 106 and 109 of the n-heptafluorobutyryl derivative of phenylethylamine on a Fisons MD 800 mass spectrometer (Fisons, Beverly, MA). With this instrument, there was no change in the enrichment with sample loading and the previously employed calibration curves were
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not necessary.24 The L-[2H5 ] enrichment of free phenylalanine in plasma was measured by monitoring the ions at mass-to-charge ratio, 336 and 341 of the t-butyldimethylhylsilyl derivative on an HP 5972 mass spectrometer (Hewlett Packard, Palo Alto, CA). Other Analytical Procedures. Serum glucose was determined by a glucose dehydrogenase method.25 Radioimmunoassay was used to determine the serum concentration of insulin.26 Plasma albumin concentration was measured using the bromocresol green method.27 Calculation and Statistics. The fractional synthesis rate (FSR) of total liver protein, i.e., the fraction of the total pool of liver protein that is synthesized every day, was calculated according to the previously described formula:28 ks Å P 1 100/A
where ks is the FSRTLP (%/day) of total liver protein and P is the isotopic enrichment of phenylalanine in total liver protein at the time of the liver biopsy specimen (APE), and A is the area under the curve of plasma free phenylalanine enrichment (APE vs. time). The fractional synthesis rate of albumin, i.e., the fraction of the intravascular albumin pool that is synthesized every day, was calculated using the same formula in a modified version as previously described:12 ks Å (P(2) 0 P(1) ) 1 100/A
where ks is the FSRALB (%/day) for albumin, and P(2) and P(1) represent enrichment of phenylalanine in albumin at two time points, after the curve of enrichment becomes linear (usually 50 and 90 samples). A is also modified to the area under the curve for enrichment of plasma-free phenylalanine between time points adjusted for the secretion time (Ts ), i.e., the temporal lag period before the appearance of labelled albumin in plasma. The secretion time was assessed by plotting the individual regression line for the linear part of the albumin enrichment curve and extrapolating to the baseline enrichment.12 Calculation of absolute synthesis rates of albumin (ASR) was based on the fractional rate of albumin synthesis and the intravascular albumin mass calculated from the concentration of plasma albumin concentrations and the measured plasma volume. Data are presented as means and the SD. Difference between values of measured plasma volumes and values obtained from the normogram was assessed by Student’s t test for paired samples. The correlation between FSR of total liver protein and the ASR of albumin, as well as the correlation between age and the FSR of albumin were calculated using ‘‘simple regression’’ (r Å Pearson’s coefficient of correlation). RESULTS
The fractional synthesis rate of total liver protein was 24.7 { 3.1%/day as assessed from the liver biopsy specimens taken at 35 { 7 minutes after induction of anesthesia and injection
FIG. 1. Experimental protocol for measuring fractional synthesis rates of total liver protein and albumin, using the flooding dose method, in nine patients undergoing laparoscopic cholecystectomy. L-[2H5 ]phenylalanine (45 mg/ kg, 10 atoms percent excess [ÅAPE]) was administered for 10 minutes. A liver biopsy specimen (one per patient) was obtained at 35 { 7 minutes (mean and SD), and blood samples were taken regularly for 90 minutes. Isotopic enrichment in total liver protein (from liver biopsy specimens), plasma albumin, and plasma were then measured.
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TABLE 2. The FSRs of Total Liver Protein in Patients (n Å 9) Undergoing Laparoscopic Cholecystectomy, Investigated With the Flooding Technique Patient
Liver Biopsy Time (min)
FSR, Total Liver Protein (%/d)
1 2 3 4 5 6 7 8 9
38 50 40 30 33 30 30 30 30 34.6 { 7.0
27.4 24.2 26.0 27.6 25.4 27.5 18.6 21.4 23.7 24.7 { 3.1
NOTE. The individual time point for taking the liver biopsy specimen in minutes after the injection of isotope, is also presented. Results are expressed as means and SD.
of phenylalanine (Table 2). Biopsy specimens were taken 18 { 6 minutes into the operation. The fractional synthesis rate of albumin was 5.9 { 1.2%/ day and the absolute synthesis rate of albumin was 109 { 21 mg/kg/d (Table 3). There was no statistically significant correlation between the FSR of total liver protein and the ASR of albumin (r Å .41). Furthermore, there was no correlation between age and the FSR of albumin (r Å .37). The albumin secretion time (i.e., the time elapsed before isotopically labelled albumin started to appear in peripheral blood) was 39.4 { 4.8 minutes, after initiation of the injection of the stable isotope (Table 3). Plasma volumes were 3.3 { 0.6 L as measured with 131I-labelled albumin, compared with 3.0 { 0.5 L according to the normogram by Dagher et al. (Table 3).19 The mean intraindividual difference between the two values was 11.6 { 4.2% (P õ .05). Hence, for seven of the nine subjects, plasma volume measurement gave a higher value than the normogram. The serum concentration of glucose was 5.7 { 0.6 mmol/L (reference interval, 3.4-6.4 mmol/L) and of insulin was 13.4 { 5.3 mU/L (reference interval for fasted subjects, 3-35 mU/ L). DISCUSSION
In the present study, the laparoscopic cholecystectomy procedure was further characterized as a human in vivo model to obtain liver tissue specimens. The fractional synthesis rate of total liver proteins was estimated by the incorporation of 2 L-[ H5 ]phenylalanine. In addition, the fractional synthesis rate as well as the absolute synthesis rate of exported albumin were measured. The technique for obtaining liver biopsy specimens in a standardized and safe manner during laparoscopic upper abdominal surgery, as a human in vivo model, has recently been characterized.18 From a scientific point of view, alternative liver biopsy techniques have disadvantages, which, at least in part, have been overcome by this model. Percutaneous and transvenous biopsy techniques provide relatively small specimens, and are used primarily for diagnostic purposes.28 Furthermore, these techniques are potentially riskful, and may not be considered ethically acceptable in healthy volunteers. Perioperative (i.e., via a laparotomy) techniques also carry potential risks, but the surgical procedure allows inspection of the biopsy site. However, after the introduction of laparoscopic surgical techniques, laparotomy is performed more seldom, except in patients with underlying malignancy and in acute situations. Furthermore, the conventional laparotomy exposes the organism for a trauma which may not be negligible, possibly affecting the results. In the few studies performed, the laparoscopic cholecystectomy has been shown
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to have less pronounced metabolic effects compared with conventional cholecystectomy, including smaller increase of urea synthesis and less reduction of insulin sensitivity.29,30 In this study, the laparoscopic model for aquiring liver tissue was combined with the flooding technique to study liver protein synthesis. The flooding technique is suitable during non–steady-state conditions, like an operation, because the periods of measurement are short. Furthermore, the problem of recycling of amino acids into the precursor pool of amino acids is minimized.8,9 This was the first study where total liver protein synthesis was assessed by gas chromatography mass spectrometry using L-[2H5 ]phenylalanine as the isotopically labelled amino acid. The sensitivity of gas chromatography mass spectrometry (GC-MS) for the determination of protein enrichment facilitates the use of smaller biopsies than was possible with L-[13C]leucine and gas isotope ratio mass spectrometry.17,20,24 The liver biopsy specimens were taken as soon as possible after the start of the operation, i.e., immediately after the intraabdominal insufflation of carbon dioxide. This was done to minimize the influence of the anesthetic agents or the operative trauma on liver protein synthesis. The immediate effects of trauma as well as opioids, barbiturate induction, and volatile anesthetic agents on liver function in general and liver protein synthesis in particular are largely unknown in man.31 However, even though previous studies in rats have shown that halothane may depress liver protein synthesis, especially after prolonged exposure, such an effect has not been shown for either isoflurane (at least not after exposure for 1 hour) or for a combination of midazolam-fentanyl.32,33 Furthermore, because isoflurane is metabolized to a very small extent,31 it was chosen as the main anesthetic agent. The mean FSR of total liver protein for this group of nine patients was 24.7%/d and was quite consistent between subjects, with a range between 18.6-27.6%/d (Table 2). These values agree well with those obtained previously with L-[13C]leucine.17 The FSR of total liver protein is about 10-fold higher than that of skeletal muscle in man.9 A similar relationship between these two tissues has been observed in fasting young rats, although the absolute values are three- to fourfold higher in the rat.34,35 The results for FSR of albumin in this investigation were in accord with values previously reported in healthy individuals, even though values in young males are somewhat higher (Table 3).11,12,21,22 In the present investigation the FSR of albumin showed a comparatively larger scatter among the subjects, when compared to the scatter of FSR of total liver protein in the same subjects (Tables 2 and 3). There was no correlation between age and FSR of albumin. TABLE 3. The FSRs and ASRs of Albumin in Patients Undergoing Elective Laparoscopic Cholecystectomy (n Å 9)
Patient
1 2 3 4 5 6 7 8 9
Secretion Time (min)
FSR Albumin (%/d)
Plasma Albumin Concentration (g/L)
Plasma Volume (L)
ASR Albumin (mg/kg/d)
36.0 32.4 45.1 47.3 38.6 35.5 40.2 42.3 36.8 39.4 { 4.8
5.8 6.5 8.1 6.2 6.6 4.4 4.5 4.5 6.4 5.9 { 1.2
39 39 39 43 40 40 41 37 41 39.9 { 1.7
2.3 2.7 3.2 3.0 4.2 4.3 3.3 3.0 3.4 3.3 { 0.6
113 131 134 106 128 89 87 76 118 109 { 21
NOTE. The time from the injection of isotope until the appearance of labelled albumin in plasma is also shown (‘‘secretion time’’), as well as plasma volume measurements and plasma albumin concentrations. Results are expressed as means and SD.
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The ASRs of albumin showed somewhat lower values than previously reported in healthy subjects (Table 3).11,12,36 The reasons for this are not clear, but all the parameters used in the calculation (FSR of albumin, plasma concentration of albumin, plasma volume, and body weight) as well as the category of patients that is investigated, are of importance when the results are interpreted. Above all, the possible effects of anesthesia and surgery must be taken into consideration. Regarding plasma volumes, there was a statistically significant difference when the measured values were compared with the values obtained from the normogram of Dagher et al.19 For seven of the patients, the measured values were actually higher, possibly because of the parenteral fluid that was provided perioperatively. This emphasizes the need to measure the plasma volume when investigating the absolute synthesis rates of export proteins. We calculated whether or not there was a statistically significant correlation between total liver protein synthesis (FSR) and ASR. However, no such correlation was found within the range of values of this study. Still, total liver protein synthesis and albumin synthesis can be related to one another in absolute terms if some basic assumptions concerning the protein content of the liver are made. Assuming that the liver accounts for 2% of body weight37 in the adult (weighing 70 kg) and that the total protein concentration is 150 g/kg fresh liver,37 the liver contains approximately 210 g of protein. If the FSR of total liver protein is 25%/d, this means a de novo protein synthesis of about 52 g/d, or 750 mg/kg body weight per day. Because the ASR of albumin is approximately 110 mg/kg/d as found in this study, the contribution of albumin synthesis to total liver protein synthesis, would be about 15%, as reported previously in animals (Table 3).22,38,39 In general, when estimating the fractional synthesis rate of total liver protein, employing a constant infusion of an isotopically labelled amino acid, lower values are produced15,16 than with the flooding technique.17 However, when estimating the FSR of albumin, the two methods give more uniform values.11,22 The most likely explanation for the difference in liver FSR involves whether or not the synthesis of exported proteins is included in the measurement. With the flooding technique, the synthesis of exported protein is included because the time of measurement is shorter than the secretion time of the exported proteins. With the constant infusion technique, labelled export proteins have escaped from the liver because of longer periods of measurement, and are therefore not included in the measured FSR. In this study, the intention was to take the liver biopsy specimen at 30 to 40 minutes after the injection of phenylalanine, to enable maximal labelling before label was lost from the liver into export proteins. Albumin has been shown to have a secretion time of 30 to 40 minutes.12 In one patient, however, the liver biopsy was taken at 50 minutes, because of technical reasons (Table 2). In conclusion, in this study, a human in vivo model for obtaining liver tissue specimens during laparoscopic cholecystectomy was applied to measure the synthesis rate of total liver proteins and relate it to that of albumin isolated from plasma. The flooding technique was employed, using L-[2H5]phenylalanine as the isotopically labelled amino acid. In the future, this study model may be used to study the effects of different hormones and nutrition upon hepatic protein metabolism. Acknowledgment: The skilled technical and nursing assistance of Mrs. Viveka Gustavsson, Ms. Eva Skoog, and Mr. George Casella is gratefully acknowledged. REFERENCES 1. Jepson MM, Pell JM, Bates PC, Millward DJ. The effects of endotoxaemia on protein metabolism in skeletal muscle and liver of fed and fasted rats. Biochem J 1986;235:329-336.
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2. Hasselgren P-O, Jagenburg R, Karlstro¨m L, Pedersen P, Seeman T. Changes of protein metabolism in liver and skeletal muscle following trauma complicated by sepsis. J Trauma 1984;24:224-228. 3. Wolfe RR, Jahoor F, Hartl WH. Protein and amino acid metabolism after injury. Diab Met Rev 1989;5(2):149-164. 4. Neuhaus OW, Balegno HF, Chandler AM. Induction of plasma protein synthesis in response to trauma. Am J Physiol 1966;211:151-156. 5. Crane CW, Picou D, Smith R, Waterlow JC. Protein turnover in patients before and after elective orthopaedic operations. Br J Surg 1977;64:129133. 6. Harrison RA, Lewin MR, Halliday D, Clark CG. Leucine kinetics in surgical patients; a study of the effect of surgical ‘stress.’ Br J Surg 1989;76: 505-508. 7. Birkhahn RH, Long CL, Fitkin D, Geriger JW, Blakemore WS. Effects of major skeletal trauma on whole body turnover in man measured by L(1,14C)-leucine. Surgery 1980;88:294-300. 8. Garlick PJ, Wernerman J, McNurlan MA, Esse´n P, Lobley GE, Milne E, Calder GA, et al. Measurement of the rate of synthesis in muscle of postabsorptive young men by injection of a flooding dose of (1-13C) leucine. Clin Sci 1989;77:329-336. 9. Esse´n P, McNurlan MA, Wernerman J, Vinnars E, Garlick PJ. Uncomplicated surgery, but not general anesthesia, decreases muscle protein synthesis. Am J Physiol 1992;262:E253-E260. 10. McFarlane AS. Measurement of synthesis rates of liver-produced plasma proteins. Biochem J 1963;89:277-290. 11. Olufemi OS, Humes P, Whittaker PG, Read MA, Lind T, Halliday D. Albumin synthetic rate: a comparison of arginine and alpha-ketoisocaproate precursor methods using stable isotope techniques. Eur J Clin Nutr 1990;44:351-361. 12. Ballmer PE, McNurlan MA, Milne E, Heys SD, Buchan V, Calder AG, Garlick PJ. Measurement of albumin synthesis in humans: a new approach employing stable isotopes. Am J Physiol 1990;259:E797-E803. 13. de Feo P, Horber FF, Haymond MW. Meal stimulation of albumin synthesis: a significant contributor to whole body protein synthesis in humans. Am J Physiol 1992;263:E794-E799. 14. Ballmer PE, Walshe D, McNurlan MA, Watson H, Brunt PW, Garlick PJ. Albumin synthesis rates in cirrhosis: correlation with Child-Turcotte classification. HEPATOLOGY 1993;18:292-297. 15. Stein TP, Mullen JL, Oram-Smith JC, Rosato EF, Wallace HW, Hargrove WC III. Relative rates of tumor, normal gut, liver and fibrinogen protein synthesis in man. Am J Physiol 1978;234(6):E648-E652. 16. Fearon KCH, McMillan DC, Preston T, Windstanley FP, Cruickshank AM, Shenkin A. Elevated circulating interleukin-6 is associated with an acutephase response but reduced fixed hepatic protein synthesis in patients with cancer. Ann Surg 1991;213:26-31. 17. Heys SD, Park KGM, McNurlan MA, Keenan RA, Miller JDB, Eremin O, Garlick PJ. Protein synthesis rates in colon and liver: stimulation by gastrointestinal pathologies. Gut 1992;33:976-981. 18. Barle H, Ahlman B, Nyberg B, Andersson K, Esse´n P, Wenerman J. The concentrations of free amino acids in human liver tissue obtained during laparoscopic surgery. Clin Physiol 1996;16:217-227. 19. Dagher FJ, Lyons JM, Finlayson DC, Shamsai J, Moore FD. Blood volume measurement: a critical study. Prediction of normal values: controlled measurement of sequential changes: choice of a bedside method. Adv Surg 1965;1:69-109. 20. McNurlan MA, Esse´n P, Thorell A, Calder AG, Anderson SE, Ljungqvist O, Sandgren A, et al. Response of protein synthesis in human skeletal muscle to insulin: an investigation with L-[2H5]phenylalanine. Am J Physiol 1994;267:E102-E108. 21. Ballmer PE, McNurlan MA, Esse´n P, Anderson SE, Garlick PJ. Albumin synthesis rates measured with (2H5ring)phenylalanine are not responsive to short-term intravenous nutrients in healthy humans. J Nutr 1995;125: 512-519. 22. Hunter KA, Ballmer PE, Anderson SE, Broom J, Garlick PJ, McNurlan MA. Acute stimulation of albumin synthesis rate with oral meal feeding in healthy subjects measured with [ring-2H5]phenylalanine. Clin Sci 1995; 88:235-242. 23. Korner AD, Debro JA. Solubility of albumin in alcohol after precipitation by trichloroacetic acid: a simplified procedure for separation of albumin. Nature 1956;178:1067. 24. Calder AG, Anderson SE, Grant I, McNurlan MA, Garlick PJ. The determination of low d5-phenylalanine enrichment (0.002-0.09 atom percent excess), after conversion to phenylethylamine, in relation to protein turnover studies by gas chromatography/mass spectrometry. Rapid Comm Mass Spec 1992;6:421-424. 25. Banauch D, Bru¨mmer W, Ebeling W, Metz H, Rindfrey H, Lang H. Eine Glucose-Dehydrogenase fu¨r die Glucose-Bestimmung in Ko¨rperflu¨ssigkeiten. J Clin Chem Clin Biochem 1975;13:101-107. 26. Midgley AR, Rebau RW, Niswender GD. Radioimmunoassays employing double antibody techniques. Acta Endocrin Suppl 1969;142:247-254. 27. Doumas BT, Watson W, Biggs HG. Albumin standards and the measurement of serum albumin with bromocresol green. Clin Chim Acta 1971;31: 87-96. 28. Lebrec D. Various approaches to obtaining liver tissue—choosing the biopsy technique. J Hepatol 1996;25:20-24. 29. Glerup H, Heindorff H, Flyvbjerg A, Jensen SL, Vilstrup H. Elective lapa-
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roscopic cholecystectomy nearly abolishes the postoperative hepatic catabolic stress response. Ann Surg 1995;221:3:214-219. Thorell A, Nygren J, Esse´n P, Gutniak M, Loftenius A, Andersson B, Ljungqvist O. The metabolic response to cholecystectomy; insulin resistance after open vs. laparoscopic surgery. Eur J Surg 1996;162:187-191. Miller RD, ed. Anesthesia. New York; Churchill-Livingstone, 1990:135-655. Heys SD, Norton AC, Dundas CR, Eremin O, Ferguson K, Garlick PJ. Anaesthetic agents and their effect on tissue protein synthesis in the rat. Clin Sci 1989;77:651-655. Ferguson K, Heys SD, Norton AC, Dundas CR, Garlick PJ. Effect of volatile anaesthetic agents on liver protein synthesis [Abstract]. Proc Nutr Soc 1990;49:182A. McNurlan MA, Tomkins AM, Garlick PJ. The effect of starvation on the rate of protein synthesis in rat liver and small intestine. Biochem J 1979; 178:373-379.
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35. Garlick PJ, Wernerman J, McNurlan M, Heys SD. Organ-specific measurements of protein turnover in man. Proc Nutr Soc 1991;50:217-225. 36. Rotschild MA, Oratz M, Screiber SS. Serum albumin. HEPATOLOGY 1988; 8:385-401. 37. Widdowson EM, Dickerson JW: Composition of the body. In: Lentner C, ed. Geigy Scientific Tables. Volume 1. Basel: Medical Education Division, Ciba-Geigy Corporation, 1981:220. 38. Ballmer PE, McNurlan MA, Grant I, Garlick PJ. Down regulation of albumin synthesis in the rat by human recombinant interleukin-1 or turpentine and the response to nutrients. J Parent Ent Nutr 1995;19:266271. 39. Pain VM, Clemens MJ, Garlick PJ. The effect of dietary protein deficiency on albumin synthesis and the concentration of active albumin messenger RNA in rat liver. Biochem J 1978;172:129-135.
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