Ann Rheum Dis: first published as 10.1136/ard.44.6.390 on 1 June 1985. Downloaded from Annals of the Rheumatic Diseases, 1985, 44, 390-394 Demonstration of an abnormality of C apoprotein of very low density lipoprotein in patients with gout D G MACFARLANE, C A MIDWINTER, P A DIEPPE, C H BOLTON, AND M HARTOG From the University Department of Medicine, Bristol Royal Infirmary, Marlborough Street, Bristol BS2 8HU, Avon SUMMARY The very low density (VLDL) apolipoproteins of 12 male patients with gout have been studied by analytical isoelectric focusing. The relative percentage distribution of the C apolipoproteins was calculated and compared with that from 12 normolipidaemic and 12 'lipid-matched' controls. In the gout patients apolipoprotein CII (apo CII) represented 19-6% of the VLDL C proteins and the CII/CIII2 ratio was 0-57. In the normolipidaemic controls apo CII represented 28*8% and in the lipid-matched controls 33*1% of VLDL apo C, and the CII/C0112 ratio approached one in each control group. These differences were significant. This suggests that a reduction in VLDL apo CII may predispose to the hypertriglyceridaemia that is commonly found in patients with gout. copyright. Key words: gout, hyperlipidaemia, apolipoprotein CII. Many studies have shown a high incidence of had been approved by the local ethical committee. hyperlipidaemia in patients with gout, commonly All their drug treatment was stopped for a period of hypertnxglyceridaemia due to raised endogenous six weeks, and they were also requested to reduce http://ard.bmj.com/ VLDL. -3 As well as lipid, VLDL particles contain their alcohol intake as much as possible and avoid B, C, and E apoproteins, and recent discoveries self medication. At the end of the six-week period about the function of these have had considerable serum was obtained after a 12-hour overnight fast. impact on the understanding of disorders of lipid metabolism. Lipoprotein lipase (LPL) catalyses the CONTROLS conversion of VLDL to LDL at capillary endothelial Serum from fasting subjects was obtained from two sites.4 5 Apo CII is an activator6 of LPL, and apo control groups. One group consisted of 12 normoli- on September 27, 2021 by guest. Protected CIII has been reported to inhibit this enzyme in pidaemic, normouricaemic men (mean age 34-5 vitro.7 years) with a normal social alcohol intake drawn To date, studies of hypertriglyceridaemia in gout from the hospital laboratory and medical staff. The have concentrated on the lipid moiety. The purpose other consisted of 12 normouricaemic men (mean of this study was to look for an abnormality in the C age 46) without a history of gout, who were matched apoprotein component of VLDL in men with gout, in lipid status with the gout patients: type IV (five which might explain their predisposition to hyper- patients), type Ilb (two patients), and five normo- triglyceridaemia. lipidaemic patients. SUBJECTS ASSESSMENT OF LIPID STATUS Twelve men with gout (mean age of 48 years) were Cholesterol was measured by cholesterol oxidase selected at random from the rheumatology clinic. and triglyceride by enzymatic assay after enzymatic They all gave informed consent to the study which hydrolysis (Boehringer Mannheim GmbH Diagnos- Accepted for publication 20 December 1984. tica). Patients were classifed into Fredrickson hyper- Correspondence to Dr D G Macfarlane, Lewisham Hospital, lipidaemia types according to the appearance of Lewisham High Street, London SE13 6LH. serum before and after standing for 24 hours at 4°C, 390 Ann Rheum Dis: first published as 10.1136/ard.44.6.390 on 1 June 1985. Downloaded from Very low density apolipoproteins 391 the serum lipid levels, and, where indicated, acetic acid v/v for a further 30 minutes to remove lipoprotein electrophoresis.' ampholine from the gel, and finally stained with 0*25% Coomassie brilliant blue according to the PREPARATION OF VLDL APOPROTEINS method of Weber and Osborn." VLDL was isolated from serum of fasting patients by ultracentrifugation9 in an MSE Prepspin 50 and IDENTIFICATION OF THE PROTEIN BANDS was subjected to a further 18-hour period of Each protein band was identified by its isoelectric ultracentrifugation at 40 000 rpm to remove any point (pl) from a standard graph prepared by contaminating albumin. The washed VLDL (con- running 20 samples under identical conditions and taining 1-5-3-0 g/l protein) was delipidated with cutting the unfixed gels into 2 mm segments which tetramethylurea (TMU).'0 were soaked for 24 hours in 1 ml deionised water. 300 iil VLDL was added to 300 ,tl 4-2 M TMU in The pH of each segment was measured and a scatter a small glass tube. The contents were mixed and diagram prepared by plotting the pH of each incubated at 37°C for 30 minutes. The tube was then segment against the positions of that segment along centrifuged at 2500 rpm for 10 minutes, and the the gel. A linear least mean square analysis gener- supernatant containing TMU-soluble apoproteins ated a straight line in the pH region 4-2-6-2, and a pl was removed and subjected to analytical isoelectric was determined for each band by measuring the focusing in polyacrylamide gel rods. distance along the gel, making allowance for gel shrinkage that occurred during fixing and staining. ANALYTICAL ISOELECTRIC FOCUSING The four major anodal bands were assigned on the Gel preparation basis of their pl to each of the four major apo C Polyacrylamide gels were prepared by mixing the proteins by reference to the published literature12 following solutions: 2-4 ml of 13-75% acrylamide and previous work from this department.)3 w/v-0*8% N,N-methylene bis(acrylamide) w/v in 8 M urea; 7 5 ml 8 M urea; 1-2 ml glycerol; 0-5 ml copyright. N,N,N'N'-tetramethylethylenediamine; and 0-6 ml QUANTIFICATION OF APO C ampholine pH 4-6 (LKB Produckter). Chemical The stained gels were scanned at 560 nm in a Pye polymerisation was carried out by adding 0 15 ml of Unicam (SP800) spectrophotometer fitted with a gel a 1-07% solution of ammonium persulphate w/v in 8 scanning attachment. The peak area (heightxwidth M urea, and the gels were cast to a height of 10-5 cm at ½/2 peak height) was calculated for each apo C in 0-5 x 12 cm glass tubes. The final concentration of band, and the contribution of each individual band urea was 6-8 M and of acrylamide 7-5%. was expressed as a percentage of the total area of quantified apo C. The within-assay coefficient of http://ard.bmj.com/ ELECTROFOCUSING variation was 2%, and between-assay coefficient of A disc gel electrophoresis apparatus (Shandon variation ranged from 2-7 to 7%. Scientific Co. Ltd) was adapted for isoelectric Previous work in this department'3 has estab- focusing by fitting a Perspex extension collar to lished that this method of quantification is valid. accommodate the longer gels. The lower electrode The absorption of the bands produced by the C chamber was filled with 250 ml of 0-1 M phosphoric apoproteins is linear over a wide ran e. This has also acid and the upper with 240 ml 0*1 M NaOH. The been confirmed by other workers. on September 27, 2021 by guest. Protected gels were run at a constant voltage with a current of 1 mA per tube for one hour to remove the STATISTICAL ANALYSIS persulphate and partially establish the pH gradient. The differences observed in the distribution of C 50 RI of a solution consisting of 2-5% v/v ampholine apoproteins in the three groups were tested for pH 4-6 in 5% w/v sucrose solution was then carefully statistical significance by Wilcoxon's rank sum test placed on top of each gel, and 100 Rl of delipidated for unpaired data. VLDL solution layered underneath this. A constant voltage of 44 V per tube was then applied for 24 Results hours at room temperature. Each sample was run in duplicate. No overheating was experienced with up Relevant clinical findings in the gout patients are to six tubes run simultaneously. shown in Table 1 and their serum and VLDL cholesterol and triglyceride levels in Table 2. The GEL FIXING AND STAINING serum and VLDL cholesterol and triglyceride level Gels were first fixed in a 11*5% w/v trichloracetic of the lipid-matched controls are shown in Table 3. acid +3-46% w/v sulphosalicylic acid mixture for 30 Seven of the 12 gout patients (58%) had a minutes, then washed with 25% ethanol v/v +8% hyperlipidaemia: five type IV and two type lIb. Ann Rheum Dis: first published as 10.1136/ard.44.6.390 on 1 June 1985. Downloaded from 392 Macfarlane, Midwinter, Dieppe, Bolton, Hartog Table 1 Clinical and laboratory features of the gout Table 3 Serum and VLDL levels in normouricaemic patients lipid-matched controls Patient Age SUA* Obesityt Alcohol Typet Matched Triglyceride Cholesterol Type* (mmolNl) intake control (mmol/l) (mmol/l) 1 38 0-44 - + N Total VLDL Total VLDL 2 53 0-42 - + N 3 30 0-54 - + + N 1 1-68 0-84 6-42 0-46 N 4 56 0-54 - + N 2 1-48 1-09 6-84 0-53 N 5 73 0-63 + + N 3 1-36 1-06 7-23 0-71 N 6 42 0-5 + + IV 4 1-52 0-94 8-23 0-92 N 7 43 0-42 + + IV 5 2-24 0-76 6-92 1-10 N 8 61 0-62 + + IV 6 3-35 2-66 7-41 1-30 IV 9 40 0-44 - + IV 7 2-87 1-78 8-64 1-42 IV 10 52 0-55 - 0 IV 8 4-88 2-22 6-84 0-68 IV 11 56 0-5 - 0 lIb 9 6-40 4-34 8-23 1-45 IV 12 37 0-54 - 0 IIb 10 3-86 2-02 7-30 1-09 IV 11 3-42 1-87 8-90 2-30 lIb *Serum uric acid.