Inhibition of liver protein synthesis during laparoscopic surgery

Hans Barle1, Björn Nyberg2, Stig Ramel2, Pia Essén1, Margaret A. McNurlan3, Jan Wernerman1, and Peter J. Garlick3

1 Department of Anesthesiology and Intensive Care and 2 Department of Surgery, Huddinge University Hospital, Karolinska Institute, S-141 86 Huddinge, Sweden; and 3 Department of Surgery, State University of New York at Stony Brook, Stony Brook, New York 11794-8191


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies have indicated that laparoscopic surgery is associated with a decline in liver protein synthesis. In this study, the fractional synthesis rate (FSR) of total liver protein and albumin was measured in patients undergoing elective laparoscopic cholecystectomy at different times after commencing the procedure (n = 8 + 8). Liver biopsy specimens were taken after 15 min of surgery in an "early" group and after 49 min of surgery in a "late" group. The liver FSR was higher in the early group (24.1 ± 4.7%/day) compared with the late group (19.0 ± 2.8%/day, P < 0.02). The fractional and absolute synthesis rates of albumin were similar in the two groups, 6.4 ± 1.5 vs. 6.5 ± 1.0%/day and 97 ± 19 vs. 96 ± 18 mg · kg-1 · day-1 for the early and late groups, respectively. It is concluded that laparoscopic surgery was accompanied by a decrease in total liver protein synthesis rate, which developed rapidly during surgery. In contrast, no change in the synthesis rate of albumin was apparent during the course of surgery.

laparoscopy; stable isotope; surgical trauma


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRAUMA AND CRITICAL ILLNESS induce profound changes in protein metabolism, resulting in a negative nitrogen balance (15, 43). The loss of body protein is a consequence of a shift in the balance between protein synthesis and degradation. Whole body protein degradation generally increases in response to a catabolic insult (24, 34), but the effect on protein synthesis is variable, depending on the severity of the insult (9, 10, 13, 14). However, whole body rates of protein turnover reflect averages of all the individual tissues, and individual tissues are known to respond differently to states of protein catabolism, e.g., skeletal muscle and liver (11, 28, 32, 38). In humans, the dynamics of protein metabolism in individual tissues have generally been investigated with stable isotope techniques. The majority of studies have involved skeletal muscle or plasma proteins, samples of which are relatively easily obtained by percutaneous biopsy or blood sampling (3, 25), and only a few investigators have attempted to measure protein synthesis in human liver. These studies have employed both the constant infusion method (21, 41) and the flooding technique (30), followed by liver biopsy during open abdominal surgery. These studies have shown that active inflammation of the bowel is associated with increased liver protein synthesis, whereas colonic malignancy leads either to no change or to a decrease in synthesis, possibly depending on the stage of the disease (21, 30).

The development of laparoscopic surgical techniques has offered an opportunity to obtain human liver tissue for research purposes from healthy subjects. Use of this technique has permitted us to characterize the hepatic free amino acid concentrations (5) and the synthesis rates of total liver protein and albumin in humans (7). In two further studies, the effects of parenteral nutrition and preoperative growth hormone administration on these parameters were also investigated (6, 8). However, liver protein synthesis rates in the control groups of these two studies were lower than those in the original study by 28 and 35%, respectively (7). This appears to have resulted from differences in the experimental protocol in the different studies. Thus the liver protein synthesis rate was measured after 20 min of surgery in the first study, instead of after 45-50 min as in the other two studies. This would indicate lower liver protein synthesis rates with longer times of surgery. However, the two later studies also included an additional liver biopsy specimen before measurement of protein synthesis to permit for the analysis of the hepatic free amino acid concentrations in the first biopsy and liver protein synthesis rate in the second, which was taken from an adjacent site. It is possible therefore that local injury to the liver by the first biopsy might have influenced the rate of protein synthesis in the second.

The primary aim of the present study was to determine in a randomized, controlled fashion whether or not total liver protein synthesis remains unaffected during laparoscopic surgery and in addition to investigate the effect of a laparoscopic surgical trauma on albumin synthesis rates.


    PATIENTS AND METHODS
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. L-[ring-2H5]phenylalanine, 99 atom percent (Mass Trace, Somerville, MA), was dissolved in sterile water together with unlabeled phenylalanine (Ajinomoto, Tokyo, Japan) to a concentration of 20 g/l and an enrichment of 10 mole percent excess (MPE).

Subjects. Patients (n = 16) undergoing elective laparoscopic cholecystectomy due to cholecystolithiasis, but otherwise healthy, participated in the study. There was no biochemical evidence of liver disease (alanine and aspartate aminotransferases, alkaline phosphatase, gamma -glutamyltransferase, and bilirubin were all within normal ranges of values). The patients were randomized into two groups to be investigated early or late during surgery ("early" and "late" groups, respectively). The groups were comparable regarding age, weight, and body mass index (Table 1). The nature, purposes, and potential risks of the experimental procedures were explained to the patients before obtaining their voluntary consent. The study protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki and had been approved by the Ethical Committee of the Karolinska Institute, Stockholm, Sweden.

                              
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Table 1.   Characteristics of patients

Experimental protocol. The general protocol has been used previously (5, 6, 7), but modifications were made for the purpose of this study. Thus, preoperatively, antecubital venous lines were inserted bilaterally and the patients were given Ringer solution (Ringeracetat, Pharmacia Upjohn, Stockholm, Sweden) in the left arm (300-600 ml/h). The line in the right arm was used for blood sampling. The patients were anesthetized in a standardized manner with sodium thiopentone 5-7 mg/kg, fentanyl 2.5-5.0 µg/kg, and atracurium 0.5 mg/kg intravenously at the induction. The anesthesia was maintained by isoflurane in a mixture of oxygen/air, with intermittent doses of fentanyl and atracurium when needed. Carbon dioxide was insufflated into the abdomen, resulting in a pressure of 10-13 mmHg, which was maintained throughout the operation, and four trocars were inserted through the abdominal wall. Ventilation was adjusted to maintain a normal end-tidal CO2 level. The patients in the early group received an injection of [2H5]phenylalanine (45 mg/kg, 10% enriched) intravenously over 10 min in the left arm at the time of induction of anesthesia. In these patients, a total of three liver biopsy specimens was obtained. The first liver biopsy specimen was taken after ~15 min of surgery (i.e., after the insertion of the first trocar into the abdomen) and ~29 min after the injection of phenylalanine, from the edge of the right liver lobe with laparoscopic scissors (Fig. 1). In order to investigate if proximity to the first biopsy site affects the results of liver protein synthesis rate in forthcoming liver biopsies, two more specimens were obtained ~60 min after the injection of phenylalanine. One of these was taken close to the first biopsy site (within 1 cm) and the other further away (more than 4 cm from the first biopsy site). The patients in the late group received the injection of phenylalanine after 15-20 min of surgery, and the only liver biopsy specimen obtained in these patients was taken ~33 min later, after ~49 min of surgery (Fig. 1). Biopsy sites were coagulated diathermally to prevent bleeding. No complications due to bleeding were observed. Blood samples were also drawn at the following intervals: 0, 5, 10, 15, 30, 50, 70, and 90 min after the injection of phenylalanine for the determination of the isotopic enrichment of phenylalanine in plasma and in albumin. All the blood samples were centrifuged at 2,000 rpm for 20 min and stored in a -80°C freezer pending analysis.


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Fig. 1.   Study protocols for two groups of patients [early (A) and late (B)] investigating association between duration of laparoscopic surgery (cholecystectomy) and liver protein synthesis, as assessed by flooding technique. X, induction of anesthesia; Y, start of intravenous phenylalanine injection (for 10 min); LB, acquisition of liver biopsy specimens. Small arrows, blood samples for precursor pool and albumin enrichments. Thin lines, ongoing anesthesia; bold lines, anesthesia and surgery. Please note temporal relationships between these events in the 2 groups.

Measurements of plasma volume were made with 131I-labeled albumin (100 kBq, Institutt for Energiteknikk, Kjeller, Norway), beginning 30 min after the the injection of phenylalanine. Blood samples were taken at 0, 20, 30, 40, and 45 min to assess isotope dilution.

Synthesis rates of total liver protein and albumin. The details of the preparation and analysis of liver, plasma, and albumin samples for the enrichment of [2H5]phenylalanine have been described elsewhere (7, 12, 35). We have previously demonstrated that plasma phenylalanine enrichment closely approximates the enrichment within the liver when a flooding amount of [2H5]phenylalanine is given (6, 8). For preparation, the liver tissue specimens were homogenized in cold 3% perchloric acid (PCA) on ice and centrifuged for 5 min at 8,000 rpm at 4°C. After being repeatedly washed in order to remove traces of free phenylalanine, the protein precipitate was suspended in 0.3 M NaOH and reprecipitated in 5% PCA. After further washing, proteins were hydrolyzed in 6 M HCl for 24 h at 110°C. The HCl was removed by evaporation in vacuo, and the hydrolysate was 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 (33). The purity of the albumin fraction was checked with matrix-assisted laser desorption time of flight mass spectrometry (Finnigan Laser mat 2000, Finnigan, Hemmel Hempstead, England) showing that a single peak with a molecular mass 67,450 kDa had been isolated. Thereafter, the protein precipitate was suspended in 0.3 M NaOH and reprecipitated in 5% PCA. After being washed with 2% PCA, albumin was hydrolyzed in 6 M HCl for 24 h at 110°C. The HCl was removed by evaporation in vacuo, and the hydrolysate was used for the measurement of isotopic enrichment.

Samples of plasma for the determination of free phenylalanine enrichment were treated with 8% sulfosalicylic acid to precipitate protein. The supernatants were purified with cation-exchange columns [Dowex-50, Biorad AG, 50W-X8, (H+) form, 100-200 mesh], eluted with 4 M NH4OH, and then dried in vacuo.

The enrichment of [2H5]phenylalanine from liver protein and albumin hydrolysates was determined by measuring the mass-to-charge ratio (m/z) at 106 and 109 of the n-heptafluorobutyryl derivative of phenylethylamine on a Fisons MD 800 mass spectrometer (Fisons, Beverly, MA; Refs. 12, 35). The 2H5 enrichment of free phenylalanine in plasma was measured by monitoring the ions at m/z 336 and 341 of the N-tert-butyldimethylsilyl derivative on an HP 5972 mass spectrometer (Hewlett-Packard, Palo Alto, CA).

Other analytical procedures. The serum concentration of albumin was measured with the bromocresol purple method on a Hitachi 917 automated analyzer (Hitachi, Naka, Japan; Ref. 39).

Calculations and statistics. The fractional synthesis rate of liver protein (FSRliver), i.e., the the daily amount of protein synthesized by the liver, both endogenous and secreted proteins, expressed as a percentage of the total protein content of the liver, was calculated according to the previously described formula (25)
FSR<SUB>liver</SUB> = P × 100/AUC
where P is the isotopic enrichment of phenylalanine (MPE) in total liver protein at the time of the second liver biopsy specimen and AUC is the area under the curve for plasma free phenylalanine enrichment (MPE) vs. time (days).

The fractional synthesis rate for albumin (FSRalb), i.e., the percentage of the intravascular albumin pool that is synthesized every day, was calculated with the same formula as previously described (3)
FSR<SUB>alb</SUB> = (P<SUB>2</SUB> − P<SUB>1</SUB>) × 100/AUC
where P2 and P1 represent the enrichment of phenylalanine in albumin between 50 and 90 min, after the curve becomes linear (based on 50-, 70-, and 90-min samples). The AUC for the enrichment of plasma free phenylalanine is adjusted for the secretion time, i.e., the temporal lag period before the appearance of labeled 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 (3). Absolute synthesis rates for albumin were calculated as the fractional rate of albumin synthesis times the intravascular albumin mass, calculated from the concentration of plasma albumin and the measured plasma volume.

Data are presented as means ± SD. Student's t-test was used for comparison, and repeated-measures ANOVA was used for comparisons within the early group.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The fractional rate of liver protein synthesis was 24.1 ± 4.7%/day (n = 8) when measured early during surgery. This was higher than the value in the late group (n = 8), which was 19.0 ± 2.8%/day (P = 0.019; Table 2).

                              
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Table 2.   Effect of surgical trauma on FSR of liver protein measured early and late during surgery in 2 groups of patients undergoing elective laparoscopic cholecystectomy

The fractional rates of liver protein synthesis calculated for the second (close to the first biopsy site) and third (>4 cm from the first biopsy site) liver biopsy specimens in the early group were not significantly different from each other (17.6 ± 3.4 and 18.2 ± 4.6%/day, respectively; Table 2). However, a significant depression was observed when comparing these two to the value obtained in the first measurement 30 min earlier (P < 0.001).

The rate of liver protein synthesis calculated from biopsy 1 in the early group correlated significantly with rates calculated from biopsy 2 (r = 0.77, P = 0.043) and biopsy 3 (r = 0.95, P < 0.01). The rates from biopsies 2 and 3 were also correlated (r = 0.82, P = 0.024).

The FSR of albumin was similar in the two groups: 6.4 ± 1.5%/day in the early group and 6.5 ± 1.0%/day in the late group. The absolute synthesis rates were also similar (97 ± 19 vs. 96 ± 18 mg · kg-1 · day-1; Table 3). Furthermore, no differences between the groups regarding plasma albumin concentration (36.1 ± 2.3 vs. 35.1 ± 3.1 g/l) or plasma volume (3.4 ± 0.6 vs. 3.0 ± 0.5 l) were observed.

                              
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Table 3.   FSR and ASR of albumin measured early and late during surgery in 2 groups of patients undergoing elective laparoscopic cholecystectomy

The first liver biopsy specimen in the early group was taken 35.5 ± 1.9 min after the induction of anesthesia, 14.8 ± 4.5 min after the start of the operation, and 29.4 ± 2.2 min after the injection of phenylalanine. The second and third liver biopsy specimens in this group were taken after 47.2 ± 6.2 min of surgery. The liver biopsy specimen in the late group was taken 72.5 ± 8.4 min after the induction of anesthesia, 48.8 ± 3.6 min after surgery was started, and 32.8 ± 1.8 min after the phenylalanine injection (Fig. 1 and Table 2).


    DISCUSSION
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ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have demonstrated that laparoscopic cholecystectomy depresses liver protein synthesis. This is consistent with results from our previous studies showing discrepant rates of liver protein synthesis, which we hypothesized might result from variations in the time between the measurement and the start of surgery. The results from the present and previous studies are summarized in Fig. 2. Thus measurements beginning 45-50 min after starting surgery are lower than those after 20 min of surgery (6-8). This indicates that the surgical trauma or associated procedures such as anesthesia or CO2 insufflation are associated with a rapid decrease of liver protein synthesis. In the present study, liver protein synthesis was 24.1 ± 4.7%/day, when measured after ~15 min of surgery in the early group, compared with 19.0 ± 2.8% in the late group, measured after ~49 min of surgery, indicating a significant decrease, by 21%, within 50 min of the start of surgery (Table 2). However, it is not clear from this study if the decrease, which may have been initiated before completing the measurement in the early group, occurred suddenly or as a progressive decline.


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Fig. 2.   Relation between length of surgery and fractional synthesis rate of total liver protein. Values for early (black-triangle) and late () groups from this study are compared with control values from previous studies: , taken after 18 ± 6 min of surgery, n = 9 (Ref. 7); black-diamond , taken after 45 ± 4 min of surgery, n = 9 (Ref. 6); and , taken after 52 ± 10 min of surgery, n = 9 (Ref. 8).

By contrast, no differences in albumin synthesis between the two groups were observed (Table 3). This may indicate that albumin synthesis rates were actually preserved during surgery, despite the decrease in the synthesis rates of total liver protein. However, the time period over which albumin synthesis was measured differs in some respects from that for total liver protein synthesis. Albumin synthesis rates were determined from the increase in albumin enrichment in blood samples taken between 50 and 90 min after the injection of [2H5]phenylalanine (Fig. 1). As the secretion time of albumin is ~32 min in the early group (Table 3), this means that albumin synthesis rates were effectively measured between 18 and 58 min after the injection of isotope and between 4 and 44 min of surgery. Similarly, in the late group, the period of measurement was between 35 and 75 min of surgery. Thus albumin synthesis is measured later in the course of surgery than liver protein synthesis, so it is possible that the effect of the surgical trauma on albumin synthesis may already have been established in the early group by the time of measurement. If that is the case, the depressive effect on albumin synthesis does not appear to be progressive, because the value in the late group is not different. However, given the results from this study, it is not possible to distinguish between these two interpretations (i.e., preserved vs. rapidly decreased albumin synthesis). Moreover, the albumin FSR found in this study, although low compared with rates in healthy subjects, still falls within the range of mean values obtained previously when the same technique was employed (2, 31). Regarding the absolute synthesis rate of albumin, it needs to be pointed out that it is a calculated value based on the analysis of plasma albumin concentration and plasma volume, factors which may change rapidly during the surgical procedure. Although of interest for further comparison between the groups, these results should be interpreted with caution.

As those studies in which we previously obtained low rates of liver protein synthesis also included a liver biopsy before isotope injection (6, 8), an alternative explanation would be that the removal of the first liver biopsy specimen may have influenced the rate of protein synthesis measured in the second biopsy. However, the results from this study do not support this notion. First, the late group showed a low rate of liver protein synthesis, even though only one liver biopsy was taken. Second, in the early group two extra biopsy specimens were taken ~30 min after the first, one close to the first biopsy site (within 1 cm) and one some distance away (>4 cm). There was no difference between the liver protein synthesis rates calculated for these different locations of the liver (Table 2), showing that proximity to the injury caused by the first biopsy specimen had no effects. However, when comparing the results from either of these two measurements to the first measurement, a decrease in the liver protein synthesis rate was observed (P < 0.01). The liver FSR in these later biopsies was lower than that in the late group, but this was expected. The measurement period of 1 h is longer than the secretion time of many plasma proteins (e.g., albumin), so the label is lost from the liver, lowering the apparent liver FSR. The fact that the results from the two extra measurements of liver FSR in the early group correlated strongly with the first measurement and with each other indicates that the flooding technique is a reproducible method to measure the rate of liver protein synthesis under these circumstances.

Although a decrease in liver protein synthesis during laparoscopic surgery was observed in our previous study, as assessed by ribosome analysis in repeated liver biopsy specimens (8), this is the first study where the immediate effect of a surgical trauma per se on human liver protein synthesis has been investigated specifically with a stable isotope technique. In a former study, liver protein synthesis was measured during open abdominal surgery with the flooding technique (30), and the result was 20.7 ± 3.1%/day (n = 5), i.e., intermediate between the results of the two groups of this study. In the other two published studies on human liver protein synthesis, the continuous infusion technique was employed, which renders comparison with this study difficult, because export proteins were included to a lesser extent in the measurements, because much longer time periods of isotope infusion were employed (21, 41). Few studies of the metabolic effects of laparoscopic surgery are available, because the technique is fairly new in clinical practice. There is some evidence that laparoscopic cholecystectomy elicits less pronounced metabolic effects compared with open cholecystectomy, including relatively smaller increases of urea synthesis (26) and insulin resistance (42). However, the decreases in skeletal muscle protein synthesis and glutamine free concentration as well as the nitrogen losses are similar, irrespective of the surgical approach (20, 27). During laparoscopic cholecystectomy, the splanchnic circulation has been shown not to be compromised during pneumoperitoneum, implying that the liver is not hypoxic (37). The possibility that the anesthetic agents used (primarily isoflurane) exert the observed effect on liver protein synthesis must be also taken into consideration. However, even though previous studies in rats have shown that halothane depresses liver protein synthesis, such an effect has not been shown for the main anesthetic agent used in this study, isoflurane (22, 29). Furthermore, in human muscle, general anesthesia had no effect on protein synthesis, whereas 90 min of surgery resulted in an inhibition (19).

Previous work on liver protein synthesis has generally shown that stress or inflammation causes an increase, rather than the decrease observed here. In human subjects with ulcerative colitis, liver protein synthesis was elevated (30). Similarly in animals, liver protein synthesis is stimulated by a variety of stressful interventions, such as surgery (28) and cancer (38), as well as by inflammation due to endotoxemia (32) or injection of turpentine or interleukin-1beta (4). However, decreases in liver protein synthesis were observed in rats with malaria (23) and during hypoxia (40). With inflammation induced by turpentine or interleukin-1beta , the increase in total liver protein synthesis has been shown to be associated with a decline in albumin synthesis (1). This is consistent with reports of decreased albumin messenger RNA levels during inflammation (36). However, the decline in albumin synthesis as a fraction of total liver protein synthesis takes place after several hours, at which time it is largely offset by the increase in total liver protein synthesis. Such a suppressive effect on the albumin synthesis rate during inflammation has also been demonstrated in humans (36). However, in intensive care unit patients, an extreme variability as well as increased rates of albumin synthesis has been reported (16-18). The results from the present study indicate that the time factor (i.e., when in the course of surgery sampling is performed) must be taken into consideration, when effects on liver protein synthesis are studied in association with a surgical trauma. Furthermore, the effect of the degree of trauma and/or the severity of the systemic inflammatory insult needs to be further investigated in humans.

In conclusion, the FSR of liver protein decreased significantly during laparoscopic surgery. This effect developed rapidly after commencement of surgery but was not reflected by a detectable decline in the rate of albumin synthesis.


    ACKNOWLEDGEMENTS

We wish to thank Dr. Sven Bringman, Dept. of Surgery, Huddinge University Hospital, for assistance during the surgical procedures. H. Barle is the grateful recipient of the 1993 ESPEN-Clintec Scholarship and of the 1997 AGA Travel Scholarship. The skilled technical and nursing assistance of George Casella, Viveka Gustavsson, Josefina Price, and Eva Skoog is gratefully acknowledged.


    FOOTNOTES

This investigation was supported by grants from the Swedish Medical Research Council (Project 04210) and the County Council of Stockholm.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: H. Barle, Dept. of Anesthesiology and Intensive Care, K 32, Huddinge Univ. Hospital, S-141 86 Huddinge, Sweden (E-mail: barle{at}ebox.tninet.se).

Received 20 January 1999; accepted in final form 20 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
PATIENTS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 277(4):E591-E596
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