Gut: first published as 10.1136/gut.29.9.1167 on 1 September 1988. Downloaded from
Gut, 1988, 29, 1167-1172 Is ascites caused by impaired hepatic inactivation of blood borne endogenous opioid peptides?
J R THORNTON, H DEAN, AND M S LOSOWSKY From the Department ofMedicine, St James's University Hospital and Department of Pharmacology, University of Leeds, Leeds
SUMMARY Methionine enkephalin and catecholamines were measured in carefully collected plasma samples from 25 patients with cirrhosis and ascites, and 25 with cirrhosis without ascites, 15 disease and 15 healthy controls. Methionine enkephalin was invariably raised in the ascites group, the median value being 4.6-6.9 times that of the other three groups. Similarly, in the ascites group, median noradrenaline was increased 2.5-4.2 and median adrenaline 1X8-2X5 times that of the other groups. Plasma methionine enkephalin is considerably raised in patients with cirrhotic ascites and has actions which could enable it to be an initiating factor of ascites formation.
After the discovery of methionine and leucine the low peripheral vascular resistance, low arterial enkephalins in 1975,' more than 10 endogenous pressure and increased cutaneous blood flow often opioid peptides have been described.2 These peptides found in such patients.'7-2' http://gut.bmj.com/ have multiple actions, exerted via at least three We propose that, in cirrhosis, chronically- classes of receptors, mu, kappa, and delta.2 Many impaired, hepatic inactivation of methionine opioid peptides have been shown to circulate in the enkephalin and possibly other small opioid peptides, bloodstream- where they may act as hormones.57 may cause or contribute to vasodilatation and that We have suggested that the liver may play a major one important consequence may be the development role in the inactivation, largely by enzymatic of ascites, arising largely as a result of an imperfect
degradation, of blood borne opioid peptides com- homeostatic attempt to maintain systemic arterial on September 26, 2021 by guest. Protected copyright. prising eight or fewer amino acids, such as the pressure. Thus, the sodium and water retention of pentapeptide methionine enkephalin.8 If so, six ascites may be mediated largely by secondary activa- opioid peptides (methionine enkephalin, methionine tion of the sympathetic nervous system and adrenal enkephalin-arg-phe, methionine enkephalin-arg-gly- medullae. A similar mechanism may stimulate the leu, adrenorphin, leucine enkephalin and dynorphin non-osmotic release of vasopressin. Opioid peptide 1-8) may be found to be increased in the plasma of initiated lymphatic leakage may also contribute to patients with liver disease. ascites formation. This hypothesis is outlined in The very wide distribution of methionine Figure 1 and discussed in more detail below. enkephalin receptors includes blood vessel sites.9' ' Like opiates," opioid peptides are potent vaso- Methods dilators.'"'- Failure of the diseased liver to meta- bolise a vasodilator substance has long been SUBJECTS proposed.'7 Because cardiac output in patients with As an initial investigation of this hypothesis, we decompensated cirrhosis tends to be high,'7-'9 vaso- studied 50 patients with biopsy proven cirrhosis of the dilatation would seem the probable explanation for liver. Half of these patients had ascites, with or without peripheral oedema (group I). The ascites was Address for correspondence: Dr J R Thornton, Dept of Medicine, St James's unequivocally present on clinical examination, con- Hospital, Leeds LS9 7TF. firmed and shown to be free of infection by diagnostic Received for publication 7 April 1988. aspiration. The remaining 25 patients with cirrhosis 1167 Gut: first published as 10.1136/gut.29.9.1167 on 1 September 1988. Downloaded from
1168 Thornton, Dean, and Losowsky
,Opioid peptide elimination by disease liver cooled centrifuge at 40C and spun at 3000 rpm for 10 1 minutes. The plasma obtained was divided into *Plasma opioid peptide activity aliquots, acidified further to pH 3-0-4.0 with molar 01--/ hydrochloric acid and stored at -70°C. Part of the Vasodilation Histamine release blood sample obtained was sent for measurement of / \ creatinine, urea, electrolytes, and liver function aNordrenaline Lyrnphatic tests. vasopressin permeability As the pentapeptide enkephalins have a half life of only eight minutes in normal plasma,2"" preliminary Sodium /water Lymph investigations were done to ensure preservation of retention leakage methionine enkephalin. Immediate chilling of the Ascites bma blood sample, using heparin as the anticoagulant, Fig. 1 Proposed opioidpeptide pathogenesis ofthefluid was found to be inadequate. Aprotinin (Trasylol) has retention ofhepatic cirrhosis. Thick arrows indicate direction been suggested to preserve the peptide`4 but we, like ofchange. others,625 found it to be ineffective, even in associa- tion with chilling of the sample. Using it as a preservative gave methionine enkephalin values not did not have clinically apparent ascites and were not significantly different from those when heparin alone taking any diuretics (group II). These patients were was used. A combination of enzyme inhibitors may compared with 15 disease controls (three congestive be necessary to prevent methionine enkephalin cardiac failure, three chronic asthma, three chronic degradation in plasma7 and these inhibitors are pancreatitis, two untreated coeliac disease, two expensive. As an aminopeptidase which inactivates insulin-dependent diabetes mellitus, one chronic enkephalins is denatured by a pH below 5.5,`2 we intestinal pseudo-obstruction, one pneumococcal developed a technique using immediate chilling of pneumonia complicating carcinoma of the bronchus) the sample and its acidification to pH 5.0-5.5 using who had normal bilirubin and alanine aminotrans- citric acid. This procedure was shown to preserve ferase and no other evidence of liver disease (group more than 90% of methionine enkephalin for at least III) and with 15 healthy controls (group IV). In one hour. Addition of larger enkephalins containing addition, three further subjects with cirrhosis were the methionine enkephalin sequence (methionine studied before and after the development of ascites. enkephalin arg-phe, methionine enkephalin arg- http://gut.bmj.com/ All the subjects with ascites were hospital inpatients gly-leu, peptide E) to chilled, citric acid contain- and taking a sodium restricted diet of approximately ing plasma samples did not result in increased 40 mmol/day. None of the subjects were taking any methionine enkephalin values when measured by drugs which are known or likely to affect plasma either the radioimmunoassay or high performance methionine enkephalin or catecholamines. In par- liquid chromatography, indicating that acidification ticular, no subject was taking B-adrenergic blockers. does not promote and may prevent processing of
Subjects were excluded from the study for the larger enkephalins to methionine enkephalin. on September 26, 2021 by guest. Protected copyright. following reasons: (1) the presence of ascites was Further acidification of the plasma sample to pH equivocal. (2) They had had a previous episode of 3.0-4.0 and storage at -70°C was shown to maintain ascites and still required diuretics to maintain them methionine enkephalin for over a year. free frpm further fluid retention. (3) They had a plasma creatinine greater than the upper limit of Radioimmunoassay normal for our laboratory. (4) They had had a Methionine enkephalin was partially separated from gastrointestinal bleed within the previous five days. other plasma constituents using C18 Sep-Pak (5) They had been drinking alcohol within the columns primed with methanol and water. One previous three weeks. millilitre of the sample was added to the columns Pulse rate and arterial pressure were measured just which were then rinsed with 4% acetic acid and before blood sampling. eluted using methanol. The dried eluate was recon- stituted with bovine serum albumin-phosphate buffer Sample collection and 200 pl was assayed in duplicate. 100 pl of Venous blood was collected with the subjects at rest 1`25-methionine enkephalin was mixed with the in a sitting position, a heparinised cannula having sample, standard and non-specific binding tubes and been inserted 20 minutes earlier. The samples for incubated for 17 hours at 4°C. One hundred micro- methionine enkephalin measurement were collected lites of rabbit gamma globluin carrier was then using chilled syringes and chilled bottles containing added, followed by 500 R1 saturated ammonium citric acid. They were transferred rapidly to a pre- sulphate to separate bound from free tracer. After Gut: first published as 10.1136/gut.29.9.1167 on 1 September 1988. Downloaded from
Is ascites caused by impaired hepatic inactivation ofblood borne endogenous opioidpeptides? 1169
standing for 20 minutes, the tubes were centrifuged at be highly specific for methionine enkephalin. Its 1000 g for 10 minutes and the supernatant decanted. cross reactivity was: leucine enkephalin 5%, meth- The radioactivity of the precipitate was counted in a ionine enkephalin-arg-phe 3%, gly-gly-phe-met 2%, gamma scintillation counter for five minutes. The and methionine enkephalin-arg-gly-leu, dynorphin percentage of bound tracer divided by the tracer's 1-8, dynorphin 1-17, peptide E, f-endorphin all total counts was calculated for each sample and the <0.01%. These cross reactivity and reproducibility quantity of methionine enkephalin determined by findings are highly comparable with those of others reference to the standard curve. The antibody, using the same antibody.27 The specificity of the tracer, and reagents were purchased from Immuno antibody for pentapeptide methionine enkephalin Nuclear Corporation, Stillwater, Minnesota, USA. was confirmed further by reverse phase, high per- Our validation studies on the assay showed formance, liquid chromatography. A 6000 A solvent parallelism, when either pure methionine enkephalin delivery system with UGK universal injector, A460 or plasma samples containing high quantities of the electrochemical detector, 730 data module and mu peptide were serially diluted. We found a recovery Bonda Pak C18 column purchased from Waters rate of 86% and within- and between-assay coeffici- Associates were used. Plasma samples which ents of variation of 8% and 11% respectively. Non- the radioimmunoassay indicated to contain high specific binding ranged from 6*5-7.9%. Minimum amounts of methionine enkephalin, were injected sensitivity was 50 pmol/l (30 pg/ml). Using pure with a solvent system of 23% acetonitrile and 77% peptides purchased from either Bachem Inc or 0X1 M sodium dihydrogen phosphate buffer. A peak Peninsula Laboratories, the antibody was shown to corresponding in site to that of standard methionine enkephalin was observed and collected. This frac- tion, when measured by the radioimmunoassay, produced values for methionine enkephalin cornes- 01310 ponding well with that determined chromatographic- 900- ally. Compared with this fraction, immediately adjacent small peaks contained <2% methionine enkephalin immunoreactivity. Noradrenaline . and adrenaline were measured radioenzymatically.2930 The statistical significance of differences was determined by the Mann-Whitney U http://gut.bmj.com/ 700- test for the plasma measurements and by the paired t test for heart rate and arterial pressure.
0 Results
0 Plasma methionine enkephalin was invariably raised
in the patients with ascites (Fig. 2), with a range of 2.5 on September 26, 2021 by guest. Protected copyright. 500- a to 13.7 times the upper limit of normal. Its median pmol/l 0 value in the ascites patients was 4.6 to 6-9 times the median values in the other three groups (Table). There was no significant difference in methionine enkephalin between the 18 patients who were taking spironolactone, median 390 pmolIl, range 235-1315 300- a (223 pg/ml, 135-755) and the seven who were not, median 495 pmol/l, range 260-880 (284 pg/ml, 150- 0 505). In three further patients with cirrhosis who a developed ascites during the study, methionine enkephalin rose in all of them from 80, 95, and 175 pmolIl to 470, 255, and 660 pmol/l respectively (45, ire_inn-J 55, and 100 pg/ml to 270, 145, and 380 pg/ml). In the patients with ascites, the median value of noradrenaline was increased 2.5 to 4.2 times and O- Ascites No Ascites Disease Healthy median adrenaline concentration 1.8 to 2.5 times that controls controls of the remaining groups (Table). The patients with Fig. 2 Plasma methionine enkephalin in thefour groups of ascites also had a lower plasma albumin and higher subjects. Medians indicated by horizontal bars. plasma creatinine compared with the other groups. Gut: first published as 10.1136/gut.29.9.1167 on 1 September 1988. Downloaded from
1170 Thornton, Dean, and Losowsky
Table Plasma methionine enkephalin, noradrenaline, adrenaline, albumin, creatinine, heart rate, and mean arterial pressure in thefour groups ofsubjects. Data expressed as medians with range.
Ascites (I) No Ascites (II) Disease controls (III) Healthy controls (IV) Methionine enkephalin (pmol/l) 445 (235-1315)* 95 (50-285) 73 (50-110) 65 (50-95) Noradrenaline (pmol/l) 7120 (1790-20000)* 2820(1600-5940)t 1940 (710-6700) 1740(910-4680) Adrenaline (pmoUIl) 980 (300-2500)* 435 (110-1580) 330 (190-1030) 300(110-900) Albumin (g/l) 31 (23-43)* 42(33-48) 38(30-45): 42(39-48) Creatinine (gmolI) 86 (66-117)* 70(47-85) 73(52-87) 68(51-79) Heart rate (beats/min) 92 (84-105)t 76 (66-95) 81(68-121) 69(59-85) Mean arterial pressure (mmHg) 97 (93-104)t 102 (94-110) 104 (87-113) 109 (102-121)
Significant differences: *p<0.001; tp<001; tp<0 05. Conversion factors: pmol/l to pg/ml: methionine enkephalinx0-574, noradrenaline 0-170, adrenaline 0-184.
In the 50 patients with cirrhosis (groups I and II), enkephalin,27 28 rises in response to acute hypo- methionine enkephalin was directly correlated with volaemaemia,j'37 is not increased in patients with noradrenaline (r=0.359, p<0.01), adrenaline cirrhosis and ascites.' This suggests that the rise in (r=0.491, p<0.001), and creatinine (r=0.331, plasma methionine enkephalin is not a non-specific p<002). In addition, noradrenaline directly corre- response to vascular underfilling in these patients. lated with adrenaline (r=0.381, p<0.01) and The data clearly do not permit a definite conclusion, creatinine (r=0.323, p<002). Methionine however, as to whether the increase in plasma enkephalin (r= -0-434, p<0-002), noradrenaline methionine enkephalin in the patients with ascites is (r=-0-655, p<0-001), and adrenaline (r=-0*457, caused by increased secretion, diminished hepatic p
remainder, as most were chronically rather than Evidence that vasodilatation may initiate raised on September 26, 2021 by guest. Protected copyright. acutely unwell, and apparently no more so than those plasma catecholamines and ascites formation was patients in the disease control group. Furthermore, provided by a recent study in which patients with some of the relatively well subjects with cirrhosis refractory ascites had their ascitic fluid recirculated without ascites also had moderately raised plasma into their vascular compartment by peritoneovenous methionine enkephalin. Acute blood loss causes shunting.43 This procedure produced a marked release of this opioid peptide from the adrenal natriuresis and diuresis, and a return of raised plasma glands.2728 All the subjects with cirrhosis, however, catecholamines to normal. Despite these changes, were haemodynamically stable. the patients remained hypotensive with a low The source(s) of circulating methionine enke- peripheral vascular resistance. phalin is unclear. It can be released by the adrenal Raised plasma catecholamines in cirrhosis are a glands3 and is present in sympathetic nerves32 and the consequence of increased secretion rather than gut.33 Bilateral adrenalectomy has no effect on the diminished clearance."45 In decompensated normal concentration of methionine containing cirrhosis, increased renal artery resistance reduces enkephalins,34 however, and we have found renal blood flow, and intrarenal haemodynamics are markedly raised plasma methionine enkephalin in deranged.' Catecholamines are capable of causing the presence of normal sympathetic tone in patients such perturbations in renal perfusion47- and may, with acute liver disease.35 thereby, promote sodium and water retention in Beta-endorphin, a much larger opioid peptide decompensated cirrhosis.505' whose plasma concentration, like methionine Leakage of lymph from the hepatic lymphatics Gut: first published as 10.1136/gut.29.9.1167 on 1 September 1988. Downloaded from
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