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and C20:5 in platelets were found predominantly asesters in membrane phospholipids; only trace amountswere detected as free fatty acids13 (table n). The im-mediate effects of stimulation of platelet aggregation byphysiological aggregating agents such as collagen orthrombin are the activation of a platelet phospholipaseand the selective release of C20:4 from membrane phos-polipids;14 but with the mackerel diet C20:5, obviouslyincorporated into the platelet membranes while the sub-jects were on that regimen, was released as well. Theliberated C20:5 could competitively inhibit plateletcyclo-oxygenase,2,3 as has been shown in vitro.4 FreeC20:5 in plasma may also have a direct effect on plateletcyclo-oxygenase. Such a mechanism seems, however, tobe less likely: aggregation and TX synthesis after

exogenous arachidonic acid were not diminished duringthe mackerel diet, indicating that endogenous C20:5levels in plasma did not inhibit platelet cyclo-oxygenasedirectly. In addition, in studies of control platelets resus-pended in PPP obtained when patients were on themackerel diet, we found no inhibition of platelet aggre-gation. Further, the concentration of C20:5 necessaryfor inhibiting platelet aggregation in vitro has been

reported to be 5-10 times higher2,3 than the free C20:5plasma level observed in subjects on the mackerel diet.

Thus, the reduction in platelet aggregation inducedby low-dose collagen stimulation during the mackereldiet may result from an increase of C20:5 and a reduc-tion of C20:4 in platelet membranes, which lead todiminished formation of proaggregatory TXA2. This in-terpretation is supported by our observation that thedecrease in platelet aggregation correlated with bothdiminished TXB2 formation and with an increase in theC20:5/C20:4 ratio in platelet phospholipids (figs. 3 and4). The highest C20:5/C20:4 ratio in platelet phospho-lipids reached in 1 subject during this study was 0-52.In Eskimos who have an even higher C20:5/C20:4 ratio(approx. 1 - 0) platelet aggregation is reduced to a greaterextent. The latter, however, could be also partly due toa reduced platelet count.1

In addition to these suggestions of how platelet aggre-gation is reduced, one should keep in mind that otherfatty acids are also much changed by the mackerel diet(i.e., C20:1; C22:6; C24:1). An increased proportion ofunsaturated fatty acids in microsomes could influenceplatelet function by other means-e.g., by changingphysicochemical characteristics, such as viscosity andfluidity of the platelet membranes. IS Furthermore, areduction in plasma cholesterol by the mackerel diet(although not significant in this study) could contributeto the decrease in platelet aggregation by causingdiminished cholesterol incorporation into platelet mem-branes. The opposite effect, an increased plateletaggregability in cholesterol-loaded platelets, has beenshown.16

In conclusion, our findings, taken in conjunction withprevious epidemiological studies in Eskimos, suggestthat a change of fatty-acid consumption such that thediet contained fat with a high C20:5/C20:4 ratio couldreduce platelet aggregability and hence exert a beneficialinfluence on certain cardiovascular disorders.

This study was supported by the Deutsche ForschungsgemeinschaftWe 681. We thank S. Fischer, H. Witzgall, Th. Dorbic, and A. Wiehlfor cooperation.

Requests for reprints should be addressed to P. C. W., MedizinischeKlinik Innenstadt der Universitat, Ziemssenstrasse 1, 8000 Müncher.2, West Germany.

REFERENCES

1. Dyerberg J, Bang HO. Hæmostatic function and platelet polyunsaturatedfatty acids in Eskimos. Lancet 1979; ii: 433-35.

2. Dyerberg J, Bang HO. Dietary fat and thrombosis. Lancet 1978; i: 152.3. Dyerberg J, Bang HO, Stoffersen E, Moncada S, Vane JR. Eicosapentaenoic

acid and prevention of thrombosis and atherosclerosis? Lancet 1978, ii

117-19.4. Needleman P, Raz A, Minkes MS, Ferrendelli JA, Sprecher H Triene

prostaglandins: prostacyclin and thromboxane biosynthesis and uniquebiological properties. Proc Nat Acad Sci. 1979; 76: 944-48.

5. Von Lossonczy TO, Ruiter A, Bronsgeest-Schoute HC, van Gent OM.Hermus RJJ. The effect of a fish diet on serum lipids in healthy humansubjects. Am J Clin Nutr 1978; 31: 1340-46.

6. Born GVR. Aggregation of blood platelets by adenosine diphosphate and itsreversal. Nature 1962; 194: 927-29.

7. Bills TK, Smith JB, Silver MJ. Metabolism of (14C)arachidonic acid byhuman platelets. Biochim Biophys Acta 1976; 424: 303-14.

8. Lehmann J, Schoene NW, Church JP. Essential fatty acid deficiency andplatelet fatty acids of normotensive and genetically hypertensive rats

Prostaglandins 1977; 13: 583-86.9. Oelz O, Seyberth HW, Knapp, Jr HR, Sweetman BJ, Oates JA. Effects of

feeding ethyl-dihomo&ggr;-linolenate on prostaglandin biosynthesis and plate-let aggregation in the rabbit. Biochim Biophys Acta 1976; 431: 268-77

10. Lowry OH, Rosebrough HJ, Farr AL, Randall RJ. Protein measurementwith the Folin phenol reagent. J Biol Chem 1951; 193: 265-75.

11. Needleman P, Moncada S, Bunting S, Vane JR, Hamberg M, SamuelssonB. Identification of an enzyme in platelet microsomes which generatesthromboxane A2 from prostaglandin endoperoxides. Nature 1976; 261:558-60.

12. Cohen P, Derksen A, van den Bosch H. Pathways of fatty acid metabolismin human platelets. J Clin Invest 1970; 49: 128-39.

13. Marcus AJ, Ullman HL, Safier LB. Lipid composition of subcellular particlesof human blood platelets. Lipid Res 1969; 10: 108-14.

14. Bills TK, Smith JB, Silver MJ. Selective release of arachidonic acid from thephospholipids of human platelets in response to thrombin. J Clin Invest1977; 60: 1-6.

15. Schaeffer BE, Curtis ASG. Effects on cell adhesion and membrane fluidityof changes in plasmalemmal lipids in mouse L929 cells. J Cell Sci 1977.26: 47-55.

16. Shattil SJ, Anaya-Galindo R, Bennett J, Colman RW, Cooper RA. Platelethypersensitivity induced by cholesterol incorporation. J Clin Invest 1975,55: 636-43.

ENDOSCOPIC RETROGRADECHOLANGIOGRAPHY AND

PANCREATOGRAPHY IN INVESTIGATION OFPOST-CHOLECYSTECTOMY PATIENTS

W. S. J. RUDDELLM. G. ASHTON

D. J. LINTOTTA. T. R. AXON

Departments of Gastroenterology and Diagnostic Radiology,General Infirmary, Leeds

Summary 102 severely symptomatic post-chole-cystectomy patients were studied by

endoscopic retrograde cholangiography (ERCP), andsuccessful ampullary cannulation was achieved in 101.All 29 patients with jaundice were correctly classifiedinto intrahepatic and extrahepatic causes. 8% (73) pa-tients with abdominal pain but no history of jaundicehad retained biliary calculi, and 25% had an abnormalpancreatogram suggesting pancreatitis. The measure-ment of bileduct calibre alone did not reliably dis-

tinguish between the presence or absence of retainedstones. It is suggested that ERCP is the investigation ofchoice in the symptomatic post-cholecystectomy patient

Introduction

As many as 40% of patients continue to have

symptoms after cholecystectomy. Many have only milddyspepsia, abdominal discomfort, or diarrhoea which ca

445

be attributed to non-biliary causes such as peptic ulcer,œsophagitis, diverticular disease, or the irritable-bowelsyndrome. However, after exclusion of other gastrointes-tinal causes there remain about 5% of post-cholecystec-tomy patients who have severe recurrent or continuoussymptoms which are often difficult to diagnose. Manyhave retained biliary stones, and a proportion have pan-creatitis, which may be coincidental or related to past orpresent gallstones. The clinician confronted with thisproblem has a wide variety of tests at his disposal. Endo-scopic retrograde cholangiography and pancreatography(ERCP) accurately defines the anatomy of both biliarytract and pancreas in most patients. However, the tech-nique is difficult to learn, and initial enthusiasm hasbeen tempered by the realisation that simpler methodscan give similar diagnostic returns. Intravenous cholan-giography and percutaneous transhepatic cholangiogra-phy will often diagnose common bileduct stones, butthey give no direct information about the pancreas.Grey-scale ultrasonography will usually distinguish"medical" from "surgical" jaundice2 and may give addi-tional information about the pancreas.3 These proced-ures are technically less demanding and more widelyavailable than ERCP, and thus it would be helpful todefine a population in whom the regular use of ERCPis justified by a particularly high diagnostic return. Inthis study we have examined the use of ERCP in 102post-cholecystectomy patients presenting with undiag-nosed abdominal pain and/or jaundice.

Patients and Methods

All post-cholecystectomy patients presenting with jaundiceand/or abdominal pain severe and continuous enough to war-rant intensive investigation, and in whom no diagnosis hadbeen made, were studied by ERCP. Patients had been pre-viously examined by various combinations of the following in-vestigations : barium meal and enema; endoscopy; intravenouscholangiography, and abdominal ultrasound. Several patientswith abdominal pain had also undergone coeliac axis angiogra-phy. Only those patients in whom all these investigations gavenegative or inconclusive findings are included in this study. Pa-tients who had undergone any surgery on the pancreas or bili-ary system other than simple cholecystectomy with or withoutexploration of the common bileduct were excluded, as were pa-tients referred specifically for endoscopic papillotomy, inwhom the diagnosis of retained stones was assumed (notalways correctly).

102 patients (84 females 18 males; age range 18-83 years)who met these criteria were examined by ERCP. Time sinceoperation varied from 3 months to 30 years, and 13 patientswere studied more than 20 years postoperatively. ERCP wasperformed using an Olympus JFB2 or JFB3 duodenoscope underlight sedation with intravenous pethidine and diazepam. Themaximum extrahepatic biliary diameter was measured on allcholangiograms of sufficient quality to allow accurate measure-ment. This figure was corrected for magnification by using theendoscope as a known standard and assuming that bileductand endoscope were in the.same plane.

Results

Visualisation of the ampulla and cannulation withadequate opacification of at least one duct (pancreatic orbiliary) was achieved in 101 patients (99%). 29 patientspresented with jaundice with or without abdominal

pain, and 73 patients had abdominal pain without jaun-dice.

Abdominal Pain without Jaundice (Table I)Successful cannulation with demonstration of at least

one duct was achieved in 72 of the 73 patients. In theremaining patient the ampulla could not be located.Adequate demonstration of both ducts was achieved in48 patients (66%), and in 32 of these both pancreato-gram and cholangiogram were entirely normal. A pan-

TABLE I-FINDINGS AT ERCP IN 73 POST-CHOLECYSTECTOMYPATIENTS WITH ABDOMINAL PAIN AND NO HISTORY OF JAUNDICE

creatogram was obtained in 65 patients (89%) and wasnormal in 47. 18 pancreatograms were abnormal, andthe changes were gross in 8, and minor in 10. A satisfac-tory cholangiogram was obtained in 55 patients (75%);6 had retained biliary calculi and 49 had normal bile-ducts (no obstructing lesion). A cholangiogram wasobtained in 13 of the 18 patients with abnormal pan-creatograms ; 12 had normal cholangiograms and 1 hadretained biliary calculi. A cholangiogram was not

obtained in the remaining 5 patients with abnormal pan-creatograms, and in 2 cases this was due to prematuretermination of the examination when early filling of apancreatic cyst occurred in a grossly diseased pancreas.

Patients with Jaundice (Table II)29 patients had jaundice, with or without abdominal

pain. A satisfactory cholangiogram was obtained in 28patients and showed retained stones in 16; stones and amalignant stricture of the bileduct in 1; malignant bile-duct stricture alone in 2; and benign bileduct strictures

TABLE II-FINDINGS AT ERCP IN 29 POST-CHOLECYSTECTOMYPATIENTS WITH JAUNDICE

in 2. In 7 patients an entirely normal extrahepatic cho-langiogram excluded an extrahepatic ("surgical") causeof jaundice, and in 3 of these patients diffuse minorcalibre irregularities were apparent on the intrahepaticcholangiogram. Liver biopsies and clinical follow-upconfirmed parenchymal liver disease as the cause of

jaundice in all these 7 patients. In the only patient inwhom retrograde cholangiography was unsuccessful, anobvious diagnosis of pancreatic cancer was made on thepancreatogram, and malignant biliary obstruction waslater confirmed by percutaneous transhepatic cholangi-ography.

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Patients Studied More than 20 Years afterCholecystectomy

13 patients were studied more than 20 years aftercholecystectomy. 6 of these patients had jaundice and 7had abdominal pain without jaundice. 4 jaundiced pa-tients were shown to have retained biliary stones; 1 hadpancreatic cancer obstructing biliary drainage; and 1

had a normal extrahepatic biliary system, primary bili-ary cirrhosis being subsequently diagnosed on liver

biopsy. 2 patients with abdominal pain but withoutjaundice had retained biliary calculi; 1 had abnormali-ties of the pancreatogram suggesting pancreatitis; no ab-normality in either system was found in 3 patients; andcannulation was unsuccessful in the remaining patient.

Thus, biliary stones were found in 6 of 13 patientsmore than 20 years after cholecystectomy, and 2 of thesepatients had never been jaundiced.

Bileduct Measurements

The maximum extrahepatic biliary diameter was

measured on 75 retrograde cholangiograms which out-lined the whole extrahepatic biliary system sufficientlyclearly for a maximum diameter to be measured withconfidence. The mean maximum (corrected) biliary dia-meter in 21 patients with retained stones, 15.9&plusmn;0.8 mm(mean+SEM), was significantly greater than that in 54patients with no radiological evidence of obstruction,10.5&plusmn;0.4 mm (p<0.01). However, there was a consider-able overlap in corrected biliary diameters between pa-tients with stones and those without an obstructinglesion-more than half of the bileducts containingstones fell within the range (5-17 mm) encompassingthat of the non-obstructed ducts (see accompanyingfigure).

Discussion

The use of ERCP has allowed a high diagnostic rolein this difficult group of symptomatic post-cholecystec-tomy patients. Positive information which adequatelyexplained patients’ symptoms was obtained in 52 of 102patients (51%), and in a further 32 patients both biliarytract and pancreatogram were radiologically normal andthus unlikely to be the source of chronic symptoms. All29 patients with jaundice were correctly classified intointrahepatic (medical) and extrahepatic (surgical)cholestasis. The site and nature of the obstruction were

correctly identified in all 22 "surgical" cases. 18 patients(25%) with abdominal pain and no history of jaundicehad abnormal pancreatograms suggestive or diagnosticof pancreatitis. Confirmatory evidence of pancreatic dis-ease was subsequently found in 7 of the 8 patients witha grossly abnormal pancreatogram, the remaining pa-tient being unavailable for further tests. It is more diffi-cult to be certain of the significance of the diffuse butminor pancreatic ductal abnormalities found in 10 pa-tients, although such changes do correlate with findingson tests of pancreatic function,4 and probably representsignificant pancreatic disease. 4 of these patients had awell-documented history of one or more attacks of acutepancreatitis (as distinct from the chronic pain underinvestigation), but the remaining 6 had no other evi-dence of pancreatic disease. These findings suggest thatpancreatitis is a common cause of chronic pain aftercholecystectomy, and that other evidence suggesting this

Corrected maximum extrahepatic biliary diameters of 75 post-cholecystectomy patients, according to presence or absence ofan obstructing lesion.

Horizontal bars indicate mean and standard error.

diagnosis may be absent. The finding of common bile-duct stones is unexpected in the absence of past or pres-ent jaundice. In this series 6 patients (8%) with abdomi-nal pain but no history of jaundice had retained (orre-formed) biliary calculi, and 2 of these patients weremore than 20 years post cholecystectomy. 5 of these 6patients had negative intravenous cholangiograms andbileduct calibres well within the non-obstructed range.The clinical relevance of these retained stones was

shown in all cases by relief of symptoms after stoneremoval by surgery or endoscopic sphincterotomy. Theoverlap in bileduct calibres between obstructed and non- obstructed ducts implies that bileduct calibre per se will not distinguish between the presence or absence of &deg;

retained stones in the post-cholecystectomy patient. This &deg;

has important implications for techniques such as ultra-sound which depend for their accuracy on the detectionof changes in calibre rather than the direct visualisationof an obstructing lesion.5Adequate contrast cholangiography is essential in all

post-cholecystectomy patients with symptoms, irrespec-tive of a history of jaundice, if retained stones are notto be missed. Alternative diagnostic methods such asultrasound2,6-8 and intravenous cholangiography9 will

frequently miss small stones in a non-dilated commonbileduct. Despite its disadvantages, ERCP is the investugation of choice in this difficult group of patients. It wiiireliably detect both biliary and pancreatic pathologyand offers the additional advantage that definitive treat-ment in the form of endoscopic sphincterotomy an3

447

stone removal can be performed at the same sitting inappropriate circumstances. 10

Requests for reprints should be addressed to W.S.J.R., gastroenter-ology unit, General Infirmary, Leeds LSI 3EX.

REFERENCES

1 Bodvall B. The postcholecystectomy syndromes. Clins Gastroenterol 1973;1: 103-26.

2. Taylor KJW, Rosenfield AT, Spiro HM. Diagnostic accuracy of grey-scaleultrasonography for the jaundiced patient: A report of 275 cases. Arch In-tern Med 1979; 139: 60-63.

3 Berger LA, Agnew JE, Chudleigh PM, et al. Screening for pancreaticdisease: A comparison of grey-scale ultrasonography and isotope scanning.Lancet 1979; i: 633-35.

4. Ashton MG, Axon ATR, Lintott DJ. Lundh test and ERCP in pancreatic dis-ease. Gut 1978; 19: 910-15.

5. Sample WF, Sarti DA, Goldstein LI, Weiner M, Kadell BM. Grey-scaleultrasonography of the jaundiced patient. Radiology 1978; 128: 719-25.

6. Vallon AG, Lees WR, Cotton PB. Grey-scale ultrasonography in cholestaticjaundice. Gut 1979; 20: 51-54.

7. Wheeler PG, Theodossi A, Pickford R, Laws J, Knill-Jones RP, Williams R.Non-invasive techniques in the diagnosis of jaundice&mdash;ultrasound andcomputer. Gut 1979; 20: 196-99.

8. Editorial. Non-invasive methods for diagnosis of jaundice. Lancet 1979; ii:18-20.

9 Osnes M, Larsen S, Lowe P, Gronseth K, Lotveit T, Nordshus T. Compari-son of endoscopic retrograde and intravenous cholangiography in diag-nosis of biliary calculi. Lancet 1978; ii: 230.

10. Safrany L. Endoscopic treatment of biliary-tract diseases. Lancet 1978; ii:

983-85.

ANTI-RANA ANTIBODY: A MARKER FORSERONEGATIVE AND SEROPOSITIVE

RHEUMATOID ARTHRITIS

K. C. NG

J. D. PERRYK. A. BROWN

E. J. HOLBOROW

Bone and Joint Research Unit (Immunology), The LondonHospital Medical College, and Department of Rheumatology,

The London Hospital

Summary Without prior knowledge of the diag-nosis or clinical status, sera from pa-

tients with various arthritides were screened for anti-

body against rheumatoid-arthritis-associated nuclear

antigen (RANA) with indirect immunofluorescence oncytocentrifuged Raji cell preparations. 93% of 103 sero-positive rheumatoid patients had anti-RANA antibody,in contrast to 16% of 50 normal controls and a mean of19% (10-29%) of 122 patients with other arthritides.Anti-RANA antibody was also demonstrated in 95% of21 patients with seronegative rheumatoid arthritis char-acterised by symmetrical erosive peripheral polyarthritis.No correlation was found in rheumatoid arthritisbetween anti-RANA antibody and disease activity,duration of disease, drug therapy, or extra-articularmanifestations. RANA staining was absent in a non-Epstein-Barr-virus (EBV) cell line (Ramos). In 10 rheu-matoid patients, discordance between the presence ofantibodies to EBV antigens (viral capsid antigen [VCA]and EBNA) and RANA antibody was demonstrated. Anti-RANA antibody could be a useful marker for rheuma-toid arthritis.

Introduction

IN 1976 Alspaugh and Tan1 demonstrated by im-munodiffusion a precipitating antibody in rheumatoid-arthritis (RA) sera reactive against extracts of human Blymphocyte cell lines known to contain the Epstein-Barr

virus (EBV) genome. This antibody, which they termedrheumatoid arthritis precipitin (RAP), was demon-strated in the sera of two-thirds of seropositive rheuma-toid patients-a much higher incidence han they foundin normal subjects, seronegative rheumatoid patients,and patients with other arthritides. The reactive antigenin the EB-virus-transformed lymphoblastoid cell lineswas demonstrated as fine-speckled predominantly nuc-lear staining by indirect immunofluorescence and called"rheumatoid arthritis associated nuclear antigen"(RANA) by Alspaugh et al. Their failure to demon-strate RANA in lines carrying other herpes-like virusesraised the possibility of an aetiological role for EB virusin RA. However, Elson et awl. inferred that this was un-likely because they failed to demonstrate a significantdifference between RA patients and normal subjects inincidence of the common EB viral antibody, anti-VCA.We report here a much higher frequency of anti-RANA

antibody in carefully diagnosed seronegative and sero-positive rheumatoid patients than in normal subjectsand patients with other arthritides. The relationship ofanti-RANA antibody to clinical features of RA was in-vestigated. We have also compared the frequency of twocommon EBV antibodies (anti-VCA and anti-EBV-deter-mined-nuclear-antigen [anti-EBNA]) with that of anti-RANA antibody in the rheumatoid patients.

Materials and Methods

Sera

Sera from 103 seropositive patients with definite or classicalRA according to the ARA criteria4 and from 21 persistentlyseronegative RA patients with symmetrical erosive peripheralpolyarthritis were tested. Other arthritides also examined in-cluded the following: psoriatic arthritis (14); ankylosing spon-dylitis (30); systemic lupus erythematosus (31); Reiter’s syn-drome (10); osteoarthrosis (37); and healthy subjects (50).

Sera were stored at -20&deg;C and tested without prior knowl-edge of their clinical status.

B Lymphoblastoid Cell Lines

Established human B lymphoblastoid cell lines, Raji andRamos, were kindly donated by Prof. M. A. Epstein (Bristol).Raji is an EBV-transformed cell line, whereas Ramos is devoidof EBV genomes.

Tests for EB Viral Antibodies

Two common EB viral antibodies (anti-VCA and anti-

EBNA) were detected by immunofluorescent methods.5&mdash;7 Serawere screened for anti-VCA and anti-EBNA antibodies at dilu-tions of 1 in 8 and 1 in 10 respectively.

Test for Anti-RANA AntibodyThe indirect immunofluorescent method of Alspaugh et awl. 2

was used, but with Raji cells instead of Wil2 cells as substrate.A rheumatoid-arthritis serum positive for anti-RANA was

kindly supplied by Dr E. M. Tan (Scripps Clinic and ResearchFoundation, California). RANA staining was recognised as adense fine nuclear&plusmn;cytoplasmic fluorescent speckling seen inthe majority of the cultured lymphoblastoid cells. Sera werescreened at dilution of 1 in 16. Class-specific sheep fluorescein-isothiocyanate (FITC) labelled anti-human immunoglobulin(Ig) conjugates (Wellcome Laboratories) were used at dilutionsof 1 in 20. Slides were read on an incident-light Zeiss micro-scope with a 40 x objective.