Upsala J Med Sci 81: 159-166, 1976 Detection and Characterization of Hyperlipoproteinaemia in Middle-aged Men HANS HEDSTRAND and BENGT VESSBY From the Departments of Internal Medicine and Geriatrics, University Hospital, Uppsala, Sweden ABSTRACT Serum lipoprotein (LP) concentrations were determined and LP patterns were classified in 261 middle-aged men, re- cruited from a health examination survey, with serum lipid values above the 80th percentile of the same population. Individuals with hyperlipoproteinaemia (HLP) and normolipidaemic controls were characterized also regarding family history of cardiovascular disease, do-economic factors and clinical and laboratory variables. Subjects with HLP type IV-V and I I B were overweight and showed hyperuricaemia and hyperinsnlinaemia compared with normolipidaemic controls and subjects with HLP type I I A . The latter showed elevated erythrocyte sedimentation rate. In spite of being overweight, subjects with HLP type III showed normal fasting values of insulin and uric acid in serum and normal early insulin response to intravenous glucose. The glucose tolerance did not differ significantly between the groups. Men with HLP types IV-V had predo- minantly sedentary occupations, in contrast to those with type IIA. There were significantly more smokers in the groups with HLP type IIB and IV-V than in the control group. Thus, individuals with different types of HLP tend to show different metabolic profiles hut also different socio- economic and clinical patterns, suggesting that exogenous factors are of importance in the expression of the LP abnormalities. INTRODUCTION In prospective studies, several risk factors for the development of atherosclerotic coronary heart dis- ease (CHD) have been identified. These factors include elevated concentrations of cholester- ol (21) and triglycerides (11) in serum. The hyperlipoproteinaemias (HLP) are, according to Fredrickson et al., divided into separate types with different lipoprotein (LP) patterns (16). Etiological- ly, HLP may be primary, due to genetically de- termined metabolic defects, or secondary to dis- orders such as diabetes mellitus and hypo- thyroidism. However, environmental factors, e.g. dietary habits, stress, physical activity, may also influence the serum lipid levels. This report concerns a study of the serum LP composition in middle-aged men with hyper- lipidaemia recruited from a health examina- tion survey. Subjects with different types of LP pattern were characterized regarding family history of cardiovascular disease, smoking habits, physical activity and socio-economic factors. The associa- tion between different types of HLP and some clini- cal and laboratory variables were also studied. MATERIALS A health examination was offered to all men living in the city of Uppsala and born in 1920-24 (19). A total of 2322 men were examined, giving a participation rate of 83.9%. The present study comprised all men analysed between Sept. 1971 and Sept. 1973 with hyperlipidaemia defined as the mean of two values of serum cholesterol and/or triglycerides (TG) above the 80th percentile in this popula- tion (291 mgll00 ml and 2.63 mmol, respectively. The hyperlipidaemic subjects were invited to a special lipid clinic for lipoprotein analysis. The following individuals were excluded: 23 men with hypertension, supine diastolic blood pressure (DBP)SlOS mmHg, 3 men with treated diabetes mellitus and one man with untreated hypothy- roidism. A total of 270 men were invited to the lipid clinic but 9 of them did not show up. Ten of the 261 subjects with hyperlipidaemia suffered from medical disorders. Two of them had angina pectoris and one had suffered from a myocardial infarction. One case each of intermittent claudication, operated aortic coarctation, Morbus Crohn, and Morbus Parkinson were included. Three men were treated with drugs: one hyperlipidaemic, one man with corticosteroid treated bronchial asthma and one with epilepsy. A control group consisting of 100 men, hypertensives excluded (supine DBP2105 mmHg), with serum lipid val- ues below the 80th percentile at the initial examination were randomly selected: 5 men consecutively examined on the 15th of each month throughout the investigation were chosen. The laboratory and clinical variables as well as socio-economic data of this group were compared with those of the subjects with different types of HLP. The LP values in the different types of HLP were also Upsala J Med Sci 81 160 H . Hedstrand and B . Vessby compared with the corresponding values in a randomly selected group of 92 apparently healthy men from the same population study (12). METHODS Lipid and lipoprotein analyses All serum lipid and LP analyses were performed at the Department of Geriatrics, University of Uppsala. Blood samples were drawn after an overnight fast and serum was collected by low speed centrifugation. EDTA was added to a final concentration of 0.05%. Separation of LP den- sity fractions by preparative ultracentrifugation accord- ing to Have1 et al. (18) was begun on the same day. After centrifugation at 15°C for 16 hours at 40000 rpm using a 40.3 rotor the top fraction corresponding to the very low density lipoprotein fraction (VLDL, dC1.006) was col- lected. From the bottom fraction at d = 1.006 the low den- sity lipoprotein (LDL, d=1.006-1.063) and high density lipoprotein (HDL, d>1.063) fractions were isolated either by a second spin at d = 1.063 in the preparative ultra- centrifuge or by precipitation of LDL by a heparin- manganese chloride solution (6). Cholesterol and TG analyses in serum and in the isolated density fractions were performed by semi-automatic techniques in a Tech- nicon Auto-Analyzer type I1 according to Rush et al. (27). The recovery of the LP analysis, i.e. the sum of cholesterol and TG in VLDL, LDL and HDL, was always within 100+10% of total serum cholesterol and TG, re- spectively. Immediately after the ultracentrifugation the top and bottom fractions recovered at d=1.006 as well as whole serum were subjected to agarose electrophoresis accord- ing to Noble (25). A 1 % agarose gel containing 0.25 % albumin was used and the chromatogram was stained in Sudan black. The LP patterns were classified according to Fredrikson et al. using the recommendations by Beaumont et al. (2). Limits for HLP corresponding to the 85th percentile in a random sample of healthy men from the same population study were 190 mgll00 ml and 1.50 mmoln for LDL cholesterol and VLDL respectively (12). The definition of type I11 HLP was based on the presence of a slow-moving band in VLDL on agarose gel electro- phoresis, with beta or close to beta mobility, in combina- tion with a high ratio cholesterol/TG in VLDL and a high “111-index” (29). In 6 individuals no complete ultracentrifuge analysis was obtained because of a low recovery of TG in the LP density fractions. However, the information gained from the analyses performed including serum TG and chol- esterol concentrations, cholesterol concentrations in the LP classes and the results of the agarose electrophore- sis was sufficient for an adequate classification of the LP pattern. Other laboratory investigations The analyses of serum uric acid (SUA), erythrocyte sedimentation rate (ESR) and haematocrit were performed with the methods used routinely at the De- partment of Clinical Chemistry, University Hospital, de- scribed elsewhere (19). The intravenous glucose tolerance test was performed as described before (19). The subjects were asked to be fasting after midnight the day before the examination. No other dietary prescriptions were given. Serum insulin was determined in duplicate with the Phadebas Insulin Test (Pharmacia AB, Uppsala, Sweden). This method is based upon the radioimmunosorbent tech- nique, described by Wide et al. (30). The early serum insulin response to intravenous glucose was expressed as the mean value of the serum insulin concentrations de- termined at 4 , 6 and 8 minutes after the start of the glucose injection. The late serum insulin response was expressed as the value at 60 minutes. The serum insulin index was defined as the ratio between early insulin response and basal serum insulin concentration (5,31). Clinical investigations The supine BP was measured with a mercury manometer after 10 min rest and read to the nearest 5 mmHg. The DBP was recorded when the sound disappeared entirely (Korotkoff phase 5 ) . The tables of Lindeberg et al. were used for calculation of relative body weight (22). Subscapular skinfold thick- ness was measured with a Harpenden caliper. A 12-lead electrocardiogram was recorded and clas- sified according to the Minnesota Code. Questionnaire The family history, as well as the information regarding physical activity, was obtained by a self-administered questionnaire a d rnodum Collen (13). The question and the classification of physical activity used have been pre- sented elsewhere (19). The coding of marital status and social group was based on interview reports. The three conventional social classes were used. The information of smoking habits was also recorded by interview. Statistical calculations Conventional methods were used for calculation of mean value and standard deviation (S.D.). Significances of dif- ferences between mean values were estimated with Stu- dent’s t-test (two-tailed test). Logarithm transformed val- ues were used when testing the means of serum insulin concentrations, K-values and ESR because these parameters had a skew distribution (19). For the same reason serum TG concentrations, VLDL and LDL TG and VLDL cholesterol concentrations were also tested after logarithmic transformation (12). The X2-test with Yates’ correction was used for comparison of frequencies. The accepted level of significance was p