Clinical Science ( 1 989) 77,65 1-655
651
Anaesthetic agents and their effect on tissue protein synthesis
in the rat
S. D. KEYS'**,A. C. NORTON3, C. R. DUNDAS3, 0. EREMIN', K. F E R G U S O N 3 , ~ ~ ~
P. J. GARLICK2
Departments of 'Surgery and 'Anaesthetics, University of Aberdeen, Aberdeen and ZTheRowett Research Institute, Bucksburn,
Aberdeen, Scotland, U.K.
(Received 2 March/3 1 May 1989; accepted 14 June 1989)
SUMMARY
1. Rates of protein synthesis were measured, in vivo, in
lung, liver, heart and skeletal muscle of young male rats.
Groups of rats were exposed for 1 h duration to one of
the following anaesthetic regimens: 1.4% halothane, 2.2%
halothane, 1.4% halothane in 66% nitrous oxide, intravenous pentobarbitone (20 mg/kg) and intravenous
midazolam ( 18 mg/kg) combined with fentanyl(2 pg/kg).
F'ractional rates of protein synthesis were determined
by injecting [3H]phenylalanine( 150 pmo1/100 g body
weight).
2. Liver protein synthesis was depressed significantly
by all regimens, except midazolam/fentanyl, by up to
37.7% of control values. Lung protein synthesis was significantly reduced by all the anaesthetic agents by up to
30% of control rates.
3. The effects of the anaesthetic agents on skeletal
muscle and heart were small and not statistically significant.
4. There was no evidence of ventilatory depression as
manifested by changes in arterial blood gas partial
pressures of CO, and 0,,except in the group treated with
2.2% halothane.
Key words: anaesthetics, protein synthesis.
Abbreviation: ATP, adenosine 5'-triphosphate.
INTRODUCTION
Increased urinary nitrogen losses and a negative nitrogen
balance occurring after trauma were described in 1932 by
Cuthbertson [ 11. Subsequent studies have shown a negative nitrogen balance after surgery with an alteration of
the balance between protein synthesis and degradation
Correspondence: Mr S. D. Heys, The Rowett Research
Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB,
Scotland, U.K.
[2, 31. However, the role of anaesthetic agents in these
metabolic derangements is not clear and their effect on
protein synthesis in individual tissues in vivo has not been
fully evaluated.
Previous studies have investigated the volatile anaesthetic agent halothane and its effect on cell-culture
systems. These have demonstrated either a reduction in
protein synthesis in cultures derived from normal cells
[4-61 or no change in synthesis in tumour cell lines [7].In
addition, an inhibition of protein synthesis in isolated
intact perfused rat lungs has been shown by Rannels et al.
181.
The two studies in vivo carried out to date have shown
a reduction of whole body protein synthesis [9] and a
depression of hepatic albumin and transferrin synthesis
[lo], in response to halothane exposure. To obtain more
information on the effects of anaesthetics, we have
examined the effect of several general anaesthetic agents
and sedative drugs, used in clinical practice, on protein
synthesis in individual tissues.
MATERIALS AND METHODS
Animals
Male Hooded Lister rats of the Rowett strain (body
weight approximately 100 g) were housed in conditions of
controlled temperature and humidity with alternating
light and dark periods of 12 h duration. They were
allowed free access to water and food (Labsure, Manea,
Cambridgeshire, U.K.) at all times.
Anaesthetic agents
Halothane was obtained from ICI, Alderly Park,
Cheshire, U.K., fentanyl from Janssen Pharmaceutical
Ltd, Wantage, U.K., midazolam from Roche Ltd, Welwyn
Garden City, Herts, U.K., pentobarbitone from May and
Baker Ltd, Dagenham, Essex, U.K., and oxygen, nitrous
oxide and compressed air from BOC, Guildford, Surrey,
U.K.
652
S. D. Heys et al.
Anaesthetic procedures
removed, always in this order, and initially put into iced
water. They were then 'blotted' to remove excess-blood
and frozen in liquid N2 before being stored at -20°C
until analysis.
K, was determined from the specific radioactivities of
free and protein-bound phenylalanine using the formula
given by Garlick et al. [ 121:
Rats were prepared for anaesthesia by inserting a 26gauge needle plus cannula into a lateral tail vein, and a
continuous infusion o f 0.9n/o(w/v) NaCl (saline)was commenced at 1.3 ml/h. A temperature probe was positioned
into the rectum for continuous monitoring of core temperature and the rat was then restrained by wrapping in a
perforated cloth as previously described [ 1 I]. Groups of
six rats were then treated with the following anaesthetic
agents: (1) 1.4% halothane in 02-enriched air, F,o,= 0.33;
(2) 2.2% halothane in 0,-enriched air, F,o, = 0.33; (3)
1.4% halothane in 66% nitrous oxide, F,0,=0.33; (4)
pentobarbitone (20 mg/kg intravenous bolus), F,o, =
0.2 1; (5) midazolam ( 18 mg/kg) combined with fentanyl
(2 pg/kg) intravenously with a supplemental dose of 25%)
of the original dose of both agents, given after 30 min,
F,o, = 0.2 1; ( 6 ) saline infusion only (control group),
F,o, = 0.2 1.
Animals receiving inhalational anaesthetics were
placed in a 42-litre, gas-tight experimental chamber.
Chamber anaesthetic concentrations were monitored
using a Datex Capnomac Anaesthetic Multigas Analyzer
(Datex lnstrumentarium Corp.) that was calibrated with
air, Datex Quickcal calibrating gas, 100% 0, and 100°/n
nitrous oxide. Fresh anaesthetic gas flow was supplied at
6-8 litres/min and gas composition was adjusted to
ensure constant chamber concentrations and an F,o, of
0.33. Adequate CO, elimination was ensured by keeping
the chamber CO, concentration below 0.3%.
Radiant heat was supplied as necessary to all animals to
maintain their core temperature to within 1°C of the
measured temperature at the commencement of anaesthetic exposure.
K,
= S,/S,
x 100/t
where t is the incorporation time in days, and S, and S,
are the specific radioactivities of thc protein-bound and
free phenylalanine, respectively. The units of K, are
"/"/day, i.e. the percentage of tissue protein synthesized/
day.
Insulin and glucose
Plasma insulin was measured by radioimmunoassay
[ 131, and plasma glucose by a glucose oxidase technique
141.
Arterial blood gas partial pressures
Arterial blood partial pressures ( Po, and Pco,) and pH
were measured in groups of rats that were exposed to
these anaesthetic regimens by sampling arterial blood via
catheters inserted into the carotid artery. Samples were
immediately put on to ice and analysed using an ABL-4
blood-gas analyser (Radiometer, Copenhagen, Denmark).
Statistical analysis
Statistical significances between group means of the
control and treatment groups for free phenylalanine
specific radioactivities, K,, arterial blood gas partial
pressures, and plasma glucose and insulin concentrations
were assessed using two-tailed t-tests with a pooled
estimate of variance.
Measurement of protein synthesis rates
Fractional rates of protein synthesis (K,) were
measured during the final 10 min of a 1 h period of
exposure to the anaesthetic agent, using the 'flooding
dose' technique as described by Garlick et al. [12]. A
bolus of ~-[2,6-~H]phenylalanine
(150 pmol/l00 g body
weight; Amersham International, Amersham, Bucks,
U.K.) was given via the tail vein cannula and the saline
infusion was continued. Exactly 10 min after the bolus
injection, the rats were killed by decapitation and blood
was collected into a heparinized tube. The liver, right
lung, heart and gastrocnemius muscle were rapidly
RESULTS
ENect on tissue free phenylalanine specific activities
The values shown in Table 1 demonstrate that there
was no significant differences between means in each
tissue with different treatments. The precursor specific
activity had therefore not been altered by exposure to
anaesthetic agents.
Table 1. Free phenylalanine specific activities in individual tissues
Values are shown as means k SEM.
Free phenylalanine specific radioactivity (d.p.m./nmol)
Control
Heart
Lung
Liver
Skeletal muscle
291f12
298f 15
286f17
32 1 f 16
1.4%
2.2%
Halothane
Halothane
in N,O
Midazolaml
fentanyl
Pentobarbitone
Halothane
288f11
291 f 16
284-117
320 f 16
295f14
290f 17
284f16
329 f 18
303f14
290f 17
285f16
324 f 20
305f13
289f 16
285f16
346 f 25
307f 13
288 f 20
285f 15
321 5 2 2
653
Anaesthesia and tissue protein synthesis
Effect on protein synthesis
DISCUSSION
Liver protein synthesis rates were significantly reduced
by halothane alone, halothane in nitrous oxide and pentobarbitone, ranging from 62.8% to 72.7% of the control
values. Midazolam/fentanyl also caused a depression to
89.8% of control values but this was not significant (Table
2).
All anaesthetic regimens significantly depressed lung
protein synthesis, ranging from 67% to 86% of control
levels.
Reductions in skeletal muscle and heart protein synthesis also occurred but rates were always greater than
92.3% of controls and were not statistically significant.
This study, to the best of our knowledge, has demonstrated for the first time a fall in protein synthesis in
various tissues, in vivo, after exposure to anaesthetic
agents. The responses of individual tissues differed (Table
2), with no significant changes observed in heart and
skeletal muscle. Only in liver and lung were the changes
statistically significant, with depressions ranging from
40% to 16% of the control values. It is notable that these
tissues have the highest control protein synthesis rates
and, therefore, were probably the most susceptible to
agents depressing protein synthesis.
Many factors that are known to regulate protein
synthesis may have been instrumental in effecting the
changes observed in this study. In particular, food intake
and absorption can markedly alter K,, e.g. a 40% fall was
noted after 12 h of food deprivation in the rat [ 151. The
rats in the present study, however, were fed until the
experiment began. It is possible that lowered food absorption in response to anaesthetics could have influenced
protein synthesis, but this is unlikely because the pattern
of change in the various tissues is unlike that induced by
fasting, when muscle has been found to be the most sensitive [ 161.
A number of hormones may be expected to undergo
changes in concentration in response to anaesthesia.
Halothane treatment significantly increased plasma
insulin levels but there were no changes in response to
intravenous agents. However, as insulin has been shown
to bring about a rapid stimulation of protein synthesis
[ 151, these changes were not responsible for the decrease
in protein synthesis documented here. Stress induces the
release of corticosteroids and they must also be con-
Arterial blood gas analysis (Table 3)
Arterial blood gas analysis revealed no significant
changes between the control group and groups of rats
treated with 1.4% halothane, halothane in nitrous oxide,
pentobarbitone and midazolam/fentanyl. However,
animals treated with 2.2% halothane showed evidence of
respiratory depression with a significant reduction of Po2
and elevation of Pco,.
Plasma glucose and insulin concentrations
Insulin levels were significantly elevated in all groups
with halothane treatment (P<0.001), but there were no
significant differences with midazolam/fentanyl and
pentobarbitone. Glucose levels were elevated with 2.2%
halothane and also with halothane in nitrous oxide
( P < 0.00 1). However, pentobarbitone and fentanyl/
midazolam treatment did not significantly affect glucose
concentrations (Table 3).
Table 2. Effect of anaesthetic agents on tissue protein synthesis
Values are shown as means fSEM. Statistical significance: *P<0.05, **P< 0.001 compared with
control values.
K , ('/O/day)
Control
1.4%
Halothane
105.94f4.2 70.22f3.3**
Liver
53.70f0.8 46.25f1.4"
Lung
26.62f0.8 25.07f0.8
Heart
Skeletal muscle 20.25 f 0.6 19.49f 0.8
2.2%
Halothane
74.16f2.4'
36.05f1.8'
24.26f0.8
19.26 f 0.3
Halothane
in N,O
Midazolam/
fentanyl
65.99f4.8** 95.22i5.4
45.26f1.1*
39.47f1.7'
25.28f0.8
24.32f0.3
18.65 f 0.6
19.71 f 0.5
Pentobarbitone
77.06f2.1'
41.45f1.3'
24.27fl.O
20.32 f 0.5
Table 3. Effect of anaesthetic agents on arterial blood gas partial pressures and plasma insulin and glucose concentrations
Values are shown as means fSEM.Statistical significance: *P<0.05, **P< 0.001 compared with control values.
F, 0 2
PH
h,(kPa)
R o z (kPa)
Plasma insulin (p-i.u./ml)
Plasma glucose (mmol/l)
Control
1.4%
Halothane
2.2%
Halothane
Halothane
in N,O
Midazolam/
fentanyl
Pentobarbitone
0.2 1
7.3f0.15
14.92 1.8
4.6 f 0.3
7.0 f 1.3
7.3 f 0.3
0.33
7.2 f 0.03
12.6 f 1.3
6.11 f 0 . 5
42.7 f 5.7*
7.4 f 0.8
0.33
7.0+0.01**
9.3 f 0.6*
9.1 f 1.0'
51.2f7.3**
9.4 f 0.2*
0.33
7.2f0.09
11.9f2.0
5.9 f 0.7
66.9f 15.3**
10.3 f 0.3*
0.2 1
7.3 f 0.03
13.7 f 0.2
5.5 f 0.5
7.1 f0.8
7.7 f 0.5
0.21
7.3 f 0.05
12.3f 1.8
5.9 f 0.8
11.8 f 1.5
7.4 f 0.2
654
S. D. Heys et al.
sidered because high plasma corticosteroid levels are
known to suppress protein synthesis in certain tissues
[17]. This effect, however, takes 3-4 h to become
apparent and therefore is unlikely to have occurred in the
short ( 1 h) experimental period [ 181.
Previous studies have shown halothane to reduce
protein synthesis in human lymphocytes, mouse kidney
cells and rat hepatocytes [4-61, using clinically relevant
concentrations. Perfused organ studies also have demonstrated a reduction in protein synthesis (81, but only two
studies have considered the effects in vivo,demonstrating
a depression of whole body protein synthesis [9] and a
reduction in albumin and transferrin synthesis [lo].
The three inhalational regimens, 1.4% halothane, 2%
halothane and halothane in nitrous oxide, produced a
large and statistically significant fall in liver protein
synthesis (with smaller and non-significant reductions,
3.8-8.7%, in K , in heart and skeletal muscle). The
mechanism of action of halothane has not been fully elucidated, but Becker et al. [19], using isolated liver
mitochondria1 suspensions, showed that halothane inhibits electron transfer in the respiratory chain and reduces
the adenosine 5'-triphosphate (ATP)/adenosine 5'pyrophosphate ratios, thus limiting energy available for
use. Hypoxia might also limit ATP production but arterial
blood gas analysis from the present study revealed normal
Po,. However, the hepatic oxygen demand is met from
both arterial inflow and portal flow in varying proportions
depending on flow rates. Halothane and nitrous oxide can
cause a decrease in portal flow [20, 21) and although a
compensatory increase in hepatic arterial flow occurs, this
may be insufficient, resulting in a decrease in total hepatic
flow and oxygen availability 1221. Thus we cannot rule out
local hypoxia and low liver ATP levels in response to
halothane.
Lung protein synthesis also decreased significantly in
response to all treatments with halothane. However, in
contrast to the effects on high-energy phosphates demonstrated in liver mitochondria, Rannels et al. [8] were
unable to detect any changes in lung tissue ATP levels on
exposure to halothane. Total pulmonary blood flow may
be reduced due to a depression of cardiac output by halothane [23], and regional changes in alveolar Po, may
occur under halothane anaesthesia due to altered alveolar
ventilation and ventilation/perfusion ratios. In our study,
lung protein synthesis was measured using a sample taken
from a whole lung homogenate, and thus the measured
protein synthesis is an average of that in all lung regions.
Skeletal muscle and heart were least sensitive to the
effects of halothane; in heart halothane has been shown to
reduce coronary artery blood flow together with a parallel
reduction in myocardial oxygen consumption [24], and
Philbin & Lowenstein [25] have shown that halothane can
also reduce muscle blood flow. Preedy et al. [26] have
demonstrated an inhibition of cardiac and skeletal muscle
protein synthesis induced by hypoxia, but in view of the
small changes in protein synthesis observed in the present
study, however, a local hypoxia would not seem to have
occurred. Elevation of blood Pco, tensions has also been
suggested to depress protein synthesis in muscle but not
in heart or liver [ 111. Pco, was normal in all treatment
groups except that receiving 2.2% halothane, when an
increase occurred. However, those receiving 1.4% halothane had normal fa, values despite depressed protein
synthesis in liver and lung, but not in muscle. Taken
together, these data suggest that elevated fcoz is not
responsible for the changes in protein synthesis.
Pentobarbitone also significantly depressed protein
synthesis in the liver and lung, with smaller reductions in
the other tissues. Midazolam/fentanyl demonstrated a
similar trend, but its effect on liver was smaller and not
significant. It is known that pentobarbitone, like halothane, reduces portal blood flow with a reduction in total
hepatic flow [27], but midazolam increases portal flow
[22]. This is therefore consistent with a part of the
decrease in liver K , being due to a 'local' hypoxia brought
about by alteration in hepatic blood flow.
An inhibition of tissue protein synthesis by exposure to
anaesthetic agents is important because synthesis may
then fail to match continued breakdown, resulting in a
loss of tissue protein and failure to make secretory
protein. The reduction in liver protein synthesis may have
important clinical implications because of its role in the
synthesis and secretion of the acute-phase proteins. These
proteins, produced in response to stress, have been shown
to be necessary for both host resistance and tissue repair
mechanisms after trauma [28]. We d o not yet know how
long these effects of anaesthesia persist, but an attenuated
acute-phase response due to anaesthesia might, therefore,
be a negative factor in the recovery from surgery or injury.
The effect of midazolam/fentanyl on lung protein
synthesis is of particular interest and clinical relevance
because this is one of the sedative regimens used in
patients in intensive care units. It may then be given to
patients for prolonged periods of time to facilitate intermittent positive pressure ventilation. Lung proteins are
not only structural, but also comprise approximately 10%
of pulmonary surfactant and are essential for surfactant
structure and function [29]. The effect, therefore, of
anaesthetic agents on lung protein synthesis may be
important in pulmonary function and requires further
investigation.
ACKNOWLEDGMENTS
We thank the Wellcome Trust for financial support, and
Mr 1. Grant for his technical assistance. S.D.H. is a
Wellcome Research Training Fellow.
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