Vol. LI, No. 4 (August, 1970) was issued 22.8.70.

THE BRITISH JOURNAL OF

EXPERIMENTAL PATHOLOGY

VOL. LI OCTOBER, 1970 NO. 5

SERUM PROTEIN CHANGES IN STILL'S DISEASE,RHEUMATOID ARTHRITIS AND GOUT

H. G. MINCHIN CLARKE, T. FREEMAN AND W. E. M. PRYSE-PHILLIPS

From the C.R.C. Laboratory, Whittington Ilo8pital, London N.19*

Received for publication April 22, 1970

SUMMARY.-Numerous workers have reported the electrophoretic changes ingout, rheumatoid arthritis and Still's disease. The recent introduction of ananalytical technique capable of measuring discrete proteins rather than groupsof proteins prompted a reassessment of the protein changes in these conditions.Eleven discrete oa- and f-globulins were studied in all 3 conditions. All 3showed statistically significant differences from normal values. The resultsobtained are discussed in relation to previous work and to the so called " acutephase reaction ".

ALTHOUGH there is a large amount of information concerning the serum proteinalterations in rheumatoid arthritis, similar studies in children with the lesscommon condition of Still's disease are hard to find. The condition may beconsidered a form of rheumatoid arthritis which occurs in children and is markedby the severity of the constitutional signs and an almost invariably positiveRose-Waaler test (inhibition technique) (McEwen, Ziff, Carmel, Ditata andTanner, 1958). According to Ansell and Bywaters (1963) the serum proteinsshow no specific clhange but only an increase in ac2 and y-globulins, as are usuallyreported in rheumatoid arthritis (see Sandor, 1966, for review), and regarded aspart of the general reaction of the body to sterile, perhaps immunologically-mediated, inflammation.

If the protein changes normally referred to as the " acute phase reaction"differ in various pathological states, as we believe to be the case (Clarke, Freemanand Pryse-Phillips, 1970a), then if Still's disease and rheumatoid arthritis aresimilar entities, then the same changes should be observed despite the disparityof age: these changes should differ from other pathological states: e.g. gout.

The raised serum uric acid in gout has not yet been explained. Overproduc-

* Present address: SeruLm Protein Section, Clinical Research Centre, Northwick Park Hospital,Harrow, Aliddx.

41

442 H. G. MINCHIN CLARKE, T. FREEMAN AND W. E. M. PRYSE-PHILLIPS

tion, abnormal carriage in the blood, failure of urinary excretion or excessiverenal reabsorption are all theoretically possible mechanisms. Copeman, in areview of current knowledge (1968) suggested that the fault is a failure of feedbackcontrol of inosinic acid synthesis with, possibly, an added renal tubular defect.

Alvsaker (1965, 1966) investigated the carriage of uric acid in the plasma,and reported that a low-density ,8-lipoprotein as well as albumin and an cXl-X2-globulin are capable of interacting reversibly with urate ions. He suggestedthat the al_2-globulin is a specific transport protein for uric acid in human bloodand found evidence that this protein is absent from the plasma of patients withprimary gout.

In view of the much greater specificity and quantitative ability ofa developmentof Laurell's immunoelectrophoretic method (Clarke and Freeman, 1968) it seemedworthwhile to compare the plasma proteins of men with and without primarygout. This paper reports the results of the study undertaken using this method,and also similar studies on children with Still's disease and adults of both sexeswith rheumatoid arthritis.

METHODSPatient selection

Still's disease. Children with a polyarthritis affecting more than 4 joints, present for atleast 3 months, and without any evidence of other relation diseases such as rheumatic fever,systemic lupus erythematosus, polyarteritis nodosa, or ulcerative colitis or psoriasis witharthritis, were studied. With such clinical features they all satisfied the criteria for diagnosisof the disease given by Ansell and Bywaters (1963). Any other serious system disease whichwas active either currently or in the past; the onset of puberty; a past history of jaundicethe presence of proteinuria, and treatment with steroid drugs at any time were taken asexcluding factors.

Rheumatoid arthritis.-Adult European out-patients aged between 18-62 yr were selectedfor study from a single out-patients department. Their symptoms and signs comprised someof the first 8 criteria for the diagnosis of rheumatoid arthritis listed in the Diagnostic Criteriafor Population Studies (Bull. Rheum. Dis., 1962). In no case had the illness been present forlonger than 10 yr. In no case had steroid treatment been given, and no patient had anyevidence of splenic or other reticuloendothelial system involvement. No other majorsystem disease was present. All patients were in good general health and in a normal stateof nutrition. None was incapable of work.

Gout. Only patients who fulfilled the diagnostic criteria for population studies mentionedabove, were selected for study. Thus, 2 or more of the following clinical features werepresent: (1) serum uric acid over 7*0 mg./100 ml., (2) presence of tophi, (3) urate crystals insynovial fluid or tissues; (4) a history of attacks of painful joint swellings with abrupt onsetof severe pain and eventual complete clinical remission.

Only males aged between 20-65 yr, who had no other current disease nor any past historyof chronic major system disease or jaundice, and who were still at work or capable of it, wereselected. They were all out-patients. In all cases, the last acute attack had begun over1 month before the blood sample was taken. None had been treated with steroid drugsin the previous 6 months. In all cases, the nutritional state was normal.

Protein estimationVenous blood was collected without stasis, separated within 3 hr and stored at - 20°

until the estimations were performed.

EXPLANATION OF PLATEFIG. 1.--A typical separation of (a) rheumatoid arthritis (b) Still's disease and (c) gout.

BRITISH JOURNAL OF EXPERIMENTAL PATHOLOGY.

Minchin Clarke, Freeman and Pryse-Phillips.

VOl. LI, NO. 5.

STILL S DISEASE, RHEUMATOID ARTHRITIS AND GOUT

RESULTS

Patients with Still's diseaseSix children were studied (2 males) aged between 6-13 yr (mean age, 10.0 yr).

The mean age at onset of illness was 8'2 yr and the mean duration of the illnesswas 19 months. In 2 cases, a parent or sibling had had a similar arthritic illness.All were British by birth. Two were out-patients when studied, but the other 4had been in hospital (not at rest) for periods of up to 2 months in 3 cases, and 6months in 1.

Their symptoms included: morning stiffness (5); pain on movement or jointtenderness (6); joint swelling apreading to other small joints within 3 months(6); subcutaneous nocules (2). The joint involvement was symmetrical in 4cases. The mean number of small joints affected was 4'3 and of larger joints(such as hips, knees and shoulders) was 1-3. Three children had axial (spinal)involvement.

The disease was graded clinically as mildly active (3); moderately active (2)and severe (1). The results of serological tests were as follows: Rose-Waaler orR.A. Latex-I positive, 5 negative; ANF or LE Cells-I positive, 5 negative;Mean ESR (Westergren)-35 mm/hr (range 10-85 mm./hr).

All the children were taking salicylates and one had in the past had a course ofgold injections i.m.

TABLE I. Clinical Characteristics of the Patients with Rheumratoid ArthritisNumberFemales

Symtptomns and SignsMorning stiffnessPain on movement or joint tenderniessJoint swellingSymmetrical involvementNodules detectedX-ray changes consistent with the diagnosisHistology consistent with the diagnosisMean number small joint sites affected (fingers, hands, wrists,

elbows, feet, ankles, jaws)Mean number larger joint sites affected (shoulders, hips, knees)Axial (spine, Sacro-Iliac joints) involvementOther systems involvedCurrent other system disease

InactiveMildly activeModerately activeSevere

Clinical rating of severity

TestsX-ray changes consistent with diagnosisHistology consistent with diagnosisRA Latex or Rose-Waaler + ve in last 3 monthsNot knownMean haemoglobin levelMean fall in ESR (mm./hr)Proteinuria

DrugsSalicylates given in the previous 3 monthsButazolidine given in the previous 3 monthsGold given in the previous 6 months

1813

1717181811175 (of 5)

5 (range 2-7)1 * 5 (range 0-3)

111 (Iritis)0

0594

175 (of 5)

132

84 per cent (range 65-110)40 (range 10-95)1 (trace)

17113

4143

444 H. G. MINCHIN CLARKE, T. FREEMAN AND W. E. M. PRYSE-PHILLIPS

Patients with rheumatoid arthritisEighteen patients (5 males) were studied. They had had the disease for a mean

of 4-9 yr (range: 6 months-10 yr) and their mean age was 45-7 yr (range 22-62 yr).In 6 cases there was a family history of rheumatoid disease. All were British.Sixteen were able to continue full or part-time employment, but 2 were notemployed for reasons unrelated to the severity of their illness. Only 3 patientshad a past history of other illness, usually respiratory. In no case was any otherillness considered to be active at the time of study. One patient had had a mildattack of jaundice as a child.

The patient's symptoms are listed in Table I with other clinical information.

Patients with goutEighteen males were selected randomly from the out-patient clinics of 2

London teaching hospitals. Their average age was 54-5 yr (range: 32-64 yr)and the mean duration of their disease was 6-4 yr (range 3 months-10 yr). Themean attack frequency over the previous year was 1-7 (range: 0-5 attacks),and the mean number of joints affected was 2-7 (range: 1-7 joints). A familyhistory of gout was elicited in 4 cases. Other background and diagnostic dataare given in Table II.

TABLE II. Clinical Characteristics of the Patients with GoutNumber . 18Social class 1 + 2 10Social class 3 + 4 6Social class 5 and unknown 2

Type of gout:Acute gouty arthritis 7Chronic gout . 10Irregular gout 1

Clinical ratings of severity:Mild activity . 11Moderate activity . 6Severe 1

Other gouty lesions:Renal 3Hypertension . 3

Past history of other diseases:Cardiovaseular (hypertension, myocardial infaretion) 5Renal 2Alimentary (ulcer, diverticulosis) . 5

Tests:Mean fall in ESR (mm./hr) 0-20 10

Unknown 8Mean haemoglobin level . 102 per centMean uric acid level (mg./100 ml. serum) . 8 4 (range 5 5-12)X-ray changes compatible with the diagnosis . 11

Sixteen patients were taking some form of drug treatment, which compriseduricosurics such as probenecid in 7 cases; salicylates, butazolidine or colchicinein 4 and allopurinol in 5. In no case was any other major system disease presentand judged clinically to be active, apart from those conditions regarded as complic-tions of the gout.

STILL S DISEASE, RHEUMATOID ARTHRITIS AND GOUT

Protein values in Still's diseaseThese are shown in Table III, with normal values given alongside. The

normals quoted represent the values found in 100 normal subjects aged between16-65 yr. Values significantly raised above the adult normal levels were foundin the case of ao-PGp (152 per cent reference serum), acx-ATP (217 per centreference serum) and ac2-macroglobulin (180 per cent reference serum). However,ac2-macroglobulin is known to be raised above adult levels in normal children(Abrams and Freeman, 1969), and similar elevated levels have been found forac1-PGp (Abrams, personal communication). High values, not actually attainingstatistical significance, were found in the case of haptoglobin, haemopexin andtransferrin.

TABLE III.-Serum protein concentrations in rheumatoid arthritis, Still's disease andgout, expressed as a percentage of a reference serum (Code No. 67/86) meansand (SD).

Normals

Protein Rheum. Still's Gout Mean SDcol-Lp 93 34t 109 31 120 31 127 20ci-PGp 137 37 . 152 24* . 130 20 115 19a:l-AT 159 63$ . 217 52$ . 153 53* 114 18ox-GC . 121 29 124 39 . 127 27 . 117 15oe2-M . 132 43 . 180 17* . 108 24 . 122 25Caer 156 45 . 172 29 . 148 30 . 137 25Hpt . 227 96$ . 224 110 . 157 98 . 103 44Hpx . 132 41* . 142 34 . 133 27* . 107 13Trf . 106 32 . 145 42 . 100 30 . 110 15fl-Lp 135 61 . 126 28 137 55 . 177 43P1A-C . 174 84t . 129 23 . 92 25 . 107 24n . 18 . 6 . 18 . 100

Significance of difference between rheumatoid arthritis, Still's disease and gout and normalP = < 0.05* P = < 0-01t P = < 0.001$

Protein values in rheumatoid arthritisThese are also shown in Table 111. The cl-lipoprotein level (93 per cent

reference serum) is probably low due to deterioration of the sample with time.High values for ai-ATp, haptoglobin, haemopexin and I1A-C were found.

Protein values in goutHaemopexin (133 per cent reference serum) and ax-antitrypsin (153 per cent

reference serum) were found to be above the normal range. It should be notedthat in 6 of the 18 cases xi-antitrypsin was too high to measure accurately; sincethese values were excluded from the mean and standard deviation calculationsthe true mean value should be higher. The other proteins were within normallimits.

Correlations were made, using a digital computer, between protein concentra-tions and a variety of clinical data. Some results are shown in Table IV. Despitethe significant probability values found, we feel that these findings should beinterpreted only as an indication that study with a larger number of patientsis needed.

445

446 H. G. MINCHIN CLARKE, T. FREEMAN AND W. E. M. PRYSE-PHILLIPS

TABLE IV.-Correlations Found in Protein and Clinical DataRheumatoid arthritis

al-lipoprotein: caeruloplasmin r=076 n= 16 P= <0*001oc,-PGp : U2-macroglobulin . r= 078 n= 16 P= < O 001(xl-antitrypsin: ESR r=0-63 n= 16 P= <0-01Axial involvement y-cxl-antitrypsin . r=0-60 n= 16 P= <0-02Axial involvement: haemopexin r=0-59 n= 15 P= <0-02Duration of illness: haptoglobin . r=0-55 n=16 P= <0-02Axial involvement: cxl-lipoprotein . r=0-54 n= 16 P= <0-02Haemopexin: GC r=0-38 n= 16 P= >0-01

GoutHaemopexin: GC r=0-83 n= 16 P= <0-001Haptoglobin :uric acid conc. r=0-69 n= 15 P=<0-01Duration of last attack: 41A-C r=0-62 n= 16 P= <0-01Uric acid conc.: ac-antitrypsin . r=064 n=11 P= <0 02al1-lipoprotein: caeruloplasmin . r=0-39 n= 16 P= >0 -1ail-PGpt: cs2-macroglobulin . r= 061 n= 16 P= <001

DISCUSSION

Apartfromthe studies ofimmunological function, little information concerningthe levels of serum proteins in Still's disease has been reported. Ansell andBywaters (1963) mention that the " . . . serum proteins show no specific change,but merely an increase in the a2- and y-globulin and some decrease in the albumin ".The alterations of serum proteins in rheumatoid arthritis have, on the otherhand, been studied extensively over the years, but the only finding on whichthere is general agreement is that the protein changes are those usually acceptedas constituting the " acute phase reaction ". This ill-defined term is usuallytaken to mean a group of proteins showing an increase in concentration withinflammation and tissue damage; the use of the term " acute phase " tacitlyimplies that the increase in concentration is due to a single stimulus. Sinceconcentration is the resultant of synthesis, pool distribution, and catabolism,and since remarkably little is known of the mechanisms that control these para-meters, it is equally possible that there are many stimuli involved in normalprotein homeostasis. If this were so, one would expect different pathologicalprocesses to stimulate the organism differently so that the changes observed inprotein concentrations might be characteristic of the stimulus and hence of thedisease process. Some evidence to support this hypothesis is provided by acomparison of protein changes after simple surgery and after acute viral infection(Clarke et al., 1970a).

The results obtained for rheumatoid arthritis (18 cases) and Still's disease(6 cases) are essentially the same except for (1) a high a2-M and a,PGp in childrenwith Still's disease. (2) A low cl-Lp in rheumatoid arthritis. (3) A normallA-C in Still's disease and high in rheumatoid. The first of these 3 (high ot2-Mand cl-PGp) are entirely explicable on the basis that these proteins are normallyat higher concentration in children than in adults. The second (a low xl-Lp inrheumatoid arthritis) is not a striking difference, and is probably invalid becauseof the deterioration of this protein which occurs with storage. Finally the high8ilA-C (C3) in rheumatoid arthritis but not in Still's disease is unexplained, thoughit is conceivable that this is related to the chronicity; such a finding has beenseen in multiple sclerosis (Clarke, Freeman and Pryse-Phillips, 1970b).

The differences shown between gout and rheumatoid arthritis are not impressive

STILL S DISEASE, RHEUMATOID ARTHRITIS AND GOUT 447

at first sight. However, a haptoglobin value within normal limits in gout,despite the presence of a significantly raised al antitrypsin and a lowered,81A-C (C'3) seems sufficient to justify our reluctance to dismiss such changes as" acute phase reaction ".

We would like to express our thanks to Professor Bywaters and Dr. BarbaraAnsell of the MRC Rheumatism Unit, Taplow; Dr. J. T. Scott of the West LondonHospital, and Dr. F. Dudley Hart of Westminster Hospital, for allowing us tostudy their patients.

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Lab. Invest., 18, 227.ANSELL, B. M. AND BYWATERS, E. G. L.-(1963) Pediat. Clins N. Am., 10. 921.CLARKE, H. G., MINCHIN AND FREEMAN, T.-(1968) Clin. Science, 35, 403.CLARKE, H. G., MINCHIN, FREEMAN, T. AND PRYSE-PHILLIPS, W. E. M.-(1970a) Clin.

Science, in the press.-(1970b) Clin. Chim. Acta, in the press.COPEMAN, W. S. C.-(1968) Editor, 'Textbook of the Rheumatic Diseases'. Edinburgh

(Livingstone). 4th Edition.DIAGNosTIC CRITERIA FOR POPULATION STUDIES.-(1962) Bull. Rheum. Dis., 13, 291.McEWEN, C., ZIFF, M., CARMEL, P., DITATA, D. AND TANNER, M.-(1958) Arthritis

Rheumat. 1, 481.SANDOR, G.-(1966)' Serum Proteins in Health and Disease'. London (Chapman and Hall).

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