Abnormalities of serum apo A1 containing lipoprotein particles in patients with primary biliary cirrhosis

Atherosclerosis 131 (1997) 203 – 210

Abnormalities of serum apo A1 containing lipoprotein particles in patients with primary biliary cirrhosis M.J. O’Kane a,*, P.L.M. Lynch a, M.E. Callender b, E.R. Trimble a,c a

Department of Clinical Biochemistry, Kel6in Building, The Royal Group of Hospitals, Gros6enor Road, Belfast BT12 6BA, UK b Department of Medicine, The Royal Group of Hospitals, Gros6enor Road, Belfast BT12 6BA, UK c Department of Clinical Biochemistry, The Queens Uni6ersity of Belfast, Institute of Clinical Science, Gros6enor Road, Belfast BT12 6BA, UK Received 6 September 1996; received in revised form 15 January 1997; accepted 19 January 1997

Abstract Patients with primary biliary cirrhosis (PBC) do not appear to have an increased risk of cardiovascular disease despite elevations in serum cholesterol. Recent evidence has pointed to LpA1 (an apo A1 containing particle which contains apo A1 but not apo A2) in protecting against atherosclerosis. The aim of this study was to investigate apo A1 containing particles in the serum of patients with PBC. Lipids and apolipoproteins were measured in 31 patients with PBC (30 females) and 27 control subjects (26 females). Patients were divided into 3 groups: group 1 with bilirubin B 18 mmol/l (n = 17); group 2 with bilirubin \18 mmol/l (n =11); and group 3 with end stage liver disease (ESLD, n= 3). As expected group 1 and 2 patients had higher total cholesterol, HDL cholesterol and phospholipids than control subjects. Apo B and apo A1 concentrations were similar to control subjects. However, LpA1 was greatly increased: 0.96 g/l (0.60 – 1.50), median (range) in group 1 and 1.09 g/l (0.75 – 1.33) in group 2 versus 0.62 g/l (0.45–0.93) for controls both PB 0.005 and the percentage of total apo A1 in the LpA1 fraction was increased: 54.8% (37.9–63.4) in group 1 and 55.7% (47.8–73.7) in group 2 versus 36.8% (25.1 – 49.1) for controls, both P B 0.005. Apo A2 concentration was reduced in group 1 0.38 g/l (0.30–0.51) and group 2 0.31 g/l (0.14 – 0.58) versus controls 0.43 g/l (0.36–0.57), PB0.05 and PB0.005 respectively. Patients with ESLD had reduced HDL cholesterol, apo A1, LpA1 and apo A2 compared to controls. These results suggest that PBC is associated with an altered distribution of apo A1 favouring an increased concentration of the protective LpA-I particles. Increased LpA1 concentration may be one of the factors contributing to the paradoxically low incidence of atherosclerosis in PBC patients. © 1997 Elsevier Science Ireland Ltd. Keywords: Primary biliary cirrhosis; Apolipoprotein A1; Apolipoprotein A2; Cholesterol

1. Introduction Primary biliary cirrhosis (PBC) is a chronic liver disease of autoimmune aetiology characterised by intrahepatic cholestasis. It primarily affects middle aged women and runs a chronic course of 10 years or more progressing to end stage liver failure [1]. Hyperlipidaemia together with xanthelasmata and xanthomata are recognised features of PBC [2]. Despite extensive investigation there has been much confusion concerning * Corresponding author. Tel.: +44 1232 894652; fax: + 44 1232 234029.

the changes in serum lipoproteins in PBC [3]. Jahn et al. [4] were among the first to demonstrate that the lipid abnormalities were variable and depended upon the stage of the PBC: in patients with early and intermediate histological disease (Scheuer stages 1 and 2 [5]) LDL cholesterol is raised with significant elevations in HDL cholesterol while in patients with advanced disease LDL cholesterol is increased with a decrease in HDL and the appearance of LpX. These abnormalities are thought to reflect changes in the synthetic rate of lipoproteins and alterations in the activities of enzymes involved in lipoprotein metabolism such as lecithin cholesterol acyl transferase (LCAT) and hepatic lipase (HL) [4,6].

0021-9150/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 2 1 - 9 1 5 0 ( 9 7 ) 0 6 1 0 8 - X

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Despite the increased serum concentration of atherogenic lipoproteins, patients with PBC do not appear to be at increased risk of atherosclerosis [7]. In one of the largest studies, reporting on 312 PBC patients, there was no increase in mortality from atherosclerotic related diseases compared to projected mortality rates for age and sex matched subjects from the general population [8]. The reasons for this anomaly remain unclear but may be related to reduced serum concentrations of apolipoprotein B (apoB) and lipoprotein (a) (Lpa) or to the marked increase in HDL cholesterol [9]. Although the inverse relationship between serum HDL cholesterol concentration and risk of cardiovascular disease is now well established the biological mechanisms underlying this relationship remain unclear [10]. Recent work postulating a direct role for HDL particles in reverse cholesterol transport has focused attention on HDL structure and protein composition [11]. Apolipoproteins (apo) A1 and A2 are the major proteins in HDL class particles with only minor amounts occurring in lipoproteins outside the HDL density range. Two major classes of apo A1 containing lipoproteins may be identified on the basis of apolipoprotein composition: LpA1 which contains apo A1 but not apo A2 and LpA1:A2 which contains both apo A1 and apo A2 [12,13]. Classification of particles in this manner, rather than by hydrated density is valuable in that it appears to describe metabolically distinct classes of lipoproteins. Three lines of evidence support the proposition that LpA1 and LpA1:A2 have different metabolic roles. Firstly, enzymes such as cholesterol ester transfer protein (CETP) and LCAT which play a key role in HDL metabolism are found mainly in LpA1 [14]. Secondly, cell culture studies have suggested that LpA1 promotes the efflux of cholesterol from adipocytes while the LpA1 promoted efflux is inhibited by LpA1:A2 particles [15]. Thirdly, there is evidence from case control studies that increased LpA1 levels protect against the development of coronary artery disease [16]. Thus the concept of LpA1 and LpA1:A2 particles is one that is clinically relevant. There is thus evidence from in vitro experiments and clinical studies supporting a direct role for LpA1 in the process of reverse cholesterol transport. Furthermore this evidence suggests that the serum concentration of LpA1 may be of value in assessing the risk of cardiovascular disease [16]. Although it is known that the serum concentrations of HDL cholesterol and total apo A1 are altered in cholestasis and PBC [9], there have been no studies examining the distribution of apo A1 between LpA1 and LpA1:A2 particles. This is an important point as changes in LpA1 concentration might be associated with an altered risk of cardiovascular disease. The aim of this study was to investigate the relative concentrations of LpA1 and LpA1:A2 particles in PBC patients together with other characteristics of apo A1

containing lipoproteins such as apo A1 isoform distribution and size.

2. Materials and methods

2.1. Patient selection Thirty one patients with PBC (1 male, 30 female; median age 62 years (range 22–81)) were recruited from the Liver Clinic of the Royal Victoria Hospital. In 27 patients the diagnosis was made on the basis of liver histology; in the remainder the diagnosis was inferred from the clinical presentation, elevated liver enzymes and the presence of high titres of anti-mitochondrial antibodies. None of the patients was on cholestyramine. Twenty seven normolipidaemic control subjects (1 male, 26 female; 57 years (range 23–73)) were recruited mainly from laboratory staff. A 30 ml sample of blood was collected from each subject after a 14 h overnight fast into both plain glass tubes and potassium EDTA tubes. Following centrifugation, the serum or plasma was separated and either analysed immediately, stored at 4°C or frozen at −70°C as appropriate.

2.2. Assay of LpA1 and apo A2 LpA1 was measured in terms of its apo A1 content by differential electroimmunoassay (DEIA) in a modified version of the method described by Atmeh and Shepherd [17]. In this technique flat bed agarose electrophoresis of serum is performed sequentially through agarose containing anti-apo A2 antiserum and then agarose containing anti-apo A1 antiserum. The concentration of anti-apo A2 antiserum in the gel is such that all apo A2 containing particles are retained in the proximal gel half allowing only the LpA1 particles to migrate into the anti-apo A1 containing gel. Thus the LpA1 concentration can be calculated by measuring the height of the rockets in the distal gel half. An immunogel (1.5× 107× 225mm) comprising 1% agarose and 3% polyethylene glycol 6000 in Tris barbital buffer (Tris 5.80 g, barbital 2.47 g, sodium barbital 9.76 g/l, pH 8.6) and containing 27 ml of anti-apo A2 antiserum (Immuno, Vienna, Austria) per ml of molten agarose was cast on one half of a sheet of Gelbond (Pharmacia LKB, Milton Keynes, UK). A second immunogel, of the same dimensions and composition, except that it contained 15 ml of anti-apo A1 antiserum (The Binding Site, Birmingham, UK) per ml in place of the anti-apo A2 antiserum was then cast on the remaining half of the Gelbond. Wells of 4 mm diameter and 7 mm apart were punched 7 mm from the long edge of the anti-apo A2 containing gel. The gel was placed in a Pharmacia LKB electrophoresis tank and 4 layers of Whatmann No. 1 filter

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paper, moistened with Tris barbital buffer were applied as wicks along the two long edges of the gel. Serum samples were diluted one part in 30 with Tris barbital buffer and 10 ml aliquots of these or standards were added to the wells. Electrophoresis was performed at 10.4 V/cm and at 4°C for 17 h. After electrophoresis the gel was processed by two cycles of washing and pressing followed by staining with Coomassie brilliant blue. The height of the LpA1 rockets was measured as the vertical distance from the tip of the rocket to the interface between the anti-apo A1 and anti-apo A2 containing gels. The assay was calibrated using serum from which the LpA1:A2 particles were removed by immunoprecipitation with anti-apo A2 antiserum: 100 ml of antiapo A2 antiserum was mixed with 20 ml of serum (collected from a healthy normolipidaemic subject) and incubated for 4°C for 48 h. The precipitate was removed by centrifugation and the completeness of precipitation was confirmed by the absence of further precipitation on the addition of a further 50 ml of anti-apo A2 antiserum. The concentration of apo A1 (and therefore LpA1) in the standard was assigned by electroimmunoassay using a commercially available apolipoprotein standard (Immuno, Vienna, Austria). The calibrator serum was uncontaminated by apo A2 containing particles as confirmed by DEIA. The between gel precision was 8.9% at an LpA1 concentration of 0.61 g/l (n =18) and 6.6% at an apo A2 concentration of 0.40 g/l.

2.3. The sizing of apo A1 containing particles The molecular size of apo A1 containing particles in serum was assessed by gel filtration chromatography. A 1 ml aliquot of serum was applied to a 1 × 90 cm column packed with Sephacryl S-300 (Pharmacia LKB) equilibrated with phosphate buffered saline (PBS; 0.15 mol/l NaCl in 0.1 mol/l phosphate buffer, pH 7.4) and eluted with PBS at 4°C. Fractions of 1.0 ml were collected; the protein content was analysed by measuring the absorbance at 280 nm and the apo A1 concentration of each fraction was measured by electroimmunoassay. The column was calibrated for molecular size using commercially available calibrants (MW-GF-1000, Sigma Chemical, Poole, Dorset, UK).

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(4:2:1 v/v/v) for 30 min at room temperature. The protein pellet was spun down and the supernatant discarded; the pellet was resuspended in 1 ml ether, shaken for a further 30 min and recentrifuged. The pellet was dried under air and dissolved overnight in 0.1 mol/l Tris, pH 7.4 containing 6 mol/l urea, 1 g/l sodium dodecyl sulphate and 25 mmol/l dithiothreitol. IEF was performed over a pH range 4.0–6.5 in polyacrylamide gel (5.6% acrylamide, 0.23% bisacrylamide containing 6.7 mol/l urea, 4.7% weight/volume glycerol and 7% by volume ampholine (Pharmalyte pI 4.0–6.5, Pharmacia LKB)) in a Hoefer Mighty Small SE–250 electrophoresis tank (Hoefer Scientific Instruments, CA). The anode reservoir contained 0.02 mol/l acetic acid and the cathode reservoir 0.025 mol/l NaOH. Ten microlitres of resolubilised delipidated samples were applied to the pre-cast wells in the gel, and electrophoresis was performed at 200 V for 2 h and 400 V for a further 2 h. Following electrophoresis protein was transferred from the gel by capillary blotting on to a polyvinylidenedifluoride (PVDF) membrane and the apo A1 in the membrane was detected as described previously by immunoblotting and chemiluminescent detection [18].

2.5. Other assays Total cholesterol, free cholesterol, triglycerides and phospholipids were measured on a Cobas Bio centrifugal analyser (Hoffman la Roche, Basle, Switzerland) by standard enzymatic techniques using reagents from Boehringer Mannheim (Sussex, UK). HDL cholesterol was measured following precipitation of VLDL and LDL by manganese–heparin (final manganese concentration 92 mmol/l). The presence of LpX was determined by agar electrophoresis followed by precipitation with heparin MgCl2 [19]. Apo A1 and apo B concentrations were measured on a Beckman Array protein analyser (Beckman Instruments, Brea, CA) with reagents supplied by the manufacturers. Measurement of liver profiles (AST, ALT, ALP, GGT, bilirubin and albumin) was perfomed on an Ortho Clinical Diagnostics Vitros 750 analyser (Ortho Clinical Diagnostics, Amersham, Buckinghamshire, UK).

2.6. Statistical analysis 2.4. Apo A1 isoform determination The apo A1 isoform distribution of whole plasma was determined by isoelectric focusing (IEF) followed by immunoblotting and chemiluminescent detection. EDTA plasma was delipidated by shaking 1ml of plasma with 1 ml of methanol – chloroform–ether

Statistical analysis was performed on SOLO software (BMPD Statistical Software, CA) using non parametric tests throughout. Lipid and lipoprotein concentrations were compared using the Mann-Whitney U test and apo A1 isoform distributions using the Kruskal-Wallis test.

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Table 1 Clinical and laboratory details of patients and controls

n (male/female) Age (years) Years since diagnosis Bilirubin (mmol/l) GGT (U/l) ALP (U/l) AST (U/l) albumin (g/l)

PBC group 1

PBC group 2

ESLD

Controls

0/17 62 5.7 11 265 243 59 42

1/10 61 7.7 28 488 492 123 40

0/3 61 9.7 101 73 315 158 21

1/26 57 (23 – 73)

(35–72) (0.6–9.8) (5–17) (40–688) (121–570) (32–133) (37–44)

(22 – 81) (0.2 – 12.0) (18 – 93) (397 – 817) (183 – 760) (53 – 212) (32 – 44)

(57 – 81) (4.0 – 16.0) (45 – 221) (50 – 486) (243 – 683) (134 – 168) (20 – 36)

10 30 78 28 41

(8 – 19) (15 – 68) (43 – 147) (12 – 42) (39 – 44)

Values are median (range).

3. Results

3.3. LpA1 and apolipoproteins

3.1. Characteristics of patients and controls

The total apo B concentrations were similar in PBC patients and controls (Table 2). The total apo A1 concentrations were similar for groups 1 and 2 but not for the ESLD group where they were significantly lower. The LpA1 concentration was increased by 60% in the combined groups 1 and 2 patients with a corresponding increase in the proportion of total serum apo A1 found in the LpA1 fraction. There was a tendency for the median LpA1 concentration to be higher in PBC group 2 than PBC group 1 patients although this was not statistically significant (P= 0.4). The ESLD patients had significantly lower LpA1 concentrations than either controls or the other PBC groups. Apo A2 concentrations were lower in patients (including ESLD) than controls with concentrations decreasing with disease severity in the order: PBC group 1 \ PBC group 2 \ ESLD.

The characteristics of the patient and control groups are given in Table 1. Three of the patients were on treatment with ursodeoxycholic acid, none was taking cholestyramine. The patients were divided into 3 groups of varying disease severity: group 1 with bilirubin B 18 mmol/l, (n =17); group 2 with bilirubin \ 18 mmol/l (n= 11) and group 3 with end stage liver disease (ESLD) as characterised by hepatic encephalopathy or portal hypertension (n = 3). Division of patients in this way was preferred over division by histological staging because of the high mean time elapsed since last biopsy (6.7 years, range: 0.2 – 16 years).

3.2. Lipids

3.4. apo A1 isoforms The results for lipids and apolipoproteins in PBC groups 1 and 2 were analysed both separately and in combination (Table 2). The combined PBC groups 1 and 2 had 13% higher total cholesterol, a 13% higher proportion of unesterified cholesterol, 25% higher HDL cholesterol and 19% higher phospholipids than the control subjects. The triglyceride levels were similar between patients and controls. There was a trend towards higher total and HDL cholesterol in PBC group 2 patients (bilirubin \ 18 mmol/l) as compared to PBC group 1 patients (bilirubin B18 mmol/l) although this was not statistically significant (P =0.4 and P= 0.1 respectively). The 3 patients with ESLD had cholesterol levels similar to those found in controls but lower HDL cholesterol than both controls and the PBC group 1 and 2 patients. 16 Patients had LpX present. In a separate experiment LpX was not detected in the supernatant following manganese – heparin precipitation for HDL cholesterol analysis for three subjects with LpX present in their serum.

The IEF/immunoblotting/chemiluminescent detection technique allowed ready identification of the five recognised apo A1 isoforms (Fig. 1). There was no difference in apo A1 isoform distribution between PBC patients and controls (Table 3).

3.5. Size of apo A1 particles Size of apo A1 containing particles was assessed by gel filtration chromatography of the serum from 4 control subjects and 4 PBC patients (2 patients with bilirubin B 18 mmol/l, 1 patient bilirubin \ 18 mmol/l and 1 patient with ESLD). In 2 of the 4 control subjects apo A1 eluted as a single broad peak of molecular weight circa 175 kDa, while in the other 2 control subjects 2 closely overlapping peaks were found (Fig. 2). In all 4 PBC patients the apo A1 elution profile was more clearly separated into 2 distinct peaks a higher molecular weight peak of circa 200 kDa and a lower molecular weight peak of circa 150 kDa (Fig. 3).

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Table 2 Serum concentrations of lipids and apolipoproteins in PBC patients and controls PBC group 1

Total chol (mmol/l) HDL (mmol/l) Triglycerides (mmol/l) Free cholesterol (mmol/l) Free:total cholesterol ratio (%) Phospholipid (mmol/l) Total apo A1 (g/l) LpA1 (g/l) % apo A1 in LpA1 Apo A2 (g/l) Apo B (g/l)

PBC group 2

Groups 1 and 2 combined

5.8 (4.7–8.4)* 6.2 (3.8 – 11.2)* 6.0 (3.8 – 11.2)* 1.7 (1.1–2.9)** 2.0 (1.4 – 3.5)** 1.7 (1.1 – 3.5)** 1.4 (0.8–2.8) 1.1 (0.5 – 1.5) 1.2 (0.5 – 2.8) 1.9 (1.2–2.5) 1.7 (1.2 – 3.6) 1.7 (1.2 – 3.6) 28.8 (25.0– 31.9 (27.1 – 34.3)** 29.6 (25.0 – 34.3)** 30.7)** 3.3 (2.3–4.5)* 3.0 (2.4 – 5.9)* 3.2 (2.3 – 5.8)* 1.73 (1.21–2.56) 1.66 (1.11 – 2.18) 1.71 (1.11 – 2.56) 0.96 (0.60– 1.09 (0.75 – 1.33)** 0.99 (0.60 – 1.50)** 1.50)** 54.8 (37.9– 55.7 (47.8 – 73.7)** 55.3 (37.9 – 73.7)** 63.4)** 0.38 (0.30– 0.31 (0.14 – 0.58)** 0.37 (0.14 – 0.58)* 0.51)* 1.08 (0.71–2.00) 1.06 (0.86 – 2.05) 1.07 (0.71 – 2.05)

ESLD

Controls

5.3 (3.5 – 6.6) 5.3 0.3 (0.2 – 0.5) 1.4 1.0 (0.7 – 1.5) 1.2 1.7 (1.3 – 2.0) 1.5 35.4 (34.4 – 36.4)** 26.3 2.2 (1.6 – 2.7) 0.35 (0.27 – 0.64)* 0.36 (0.23 – 0.40)**

(3.1–6.8) (0.6–1.9) (0.7–2.9) (1.1–2.0) (24.2–30.7)

2.7 (2.3–3.8) 1.71 (1.43–2.12) 0.62 (0.45–0.93)

87.0 (62.5 – 99.9)** 36.8 (25.1–49.1) 0.07 (0.05 – 0.15)

0.43 (0.36–0.57)

1.31 (0.86 – 1.87)

1.30 (0.46–2.00)

Values are median (range). * PB0.05 vs. control; ** PB0.005 vs. control.

4. Discussion This study has shown that although PBC patients in groups 1 and 2 have similar serum total apo A1 concentrations compared to control subjects there is a large increase both in the concentration of LpA1 particles and in the proportion of total apo A1 found in the LpA1 fraction. Although LpA1:A2 was not measured directly by its apo A1 concentration, the suggestion of an increase in LpA1 particles at the expense of LpA1:A2 is supported by the decrease in apo A2 concentration. To our knowledge this has not been demonstrated previously in patients with PBC. Other studies have demonstrated that the total apo A1 concentration is either normal [10] or decreased [9] in PBC patients and these conflicting results illustrate one of the difficulties in

Fig. 1. Apo A1 immunoblot following isoelectric focusing of serum. 6 Samples are shown, from left to right, 3 normolipidaemic controls and 3 PBC patient samples (Bilirubin \ 18 mmol/l). The five apo A1 isoforms in serum are clearly demonstrated: apo A1-1, -2, -3, -4, and -5.

investigating apolipoproteins–lipoprotein abnormalities in a chronic progressive disease such as PBC where the parameters under study will vary as the disease progresses. It is therefore essential that results in PBC patients are analysed with reference to disease severity. In this study the patients were divided into 3 groups (bilirubin B 18 mmol/l, bilirubin \ 18 mmol/l and ESLD). The ESLD group are clearly clinically and biochemically distinct and where many of the abnormalities found may be related to failing hepatic synthetic function. The use of a simple indicator of disease severity such as the serum bilirubin was preferred over histological staging because due to the long mean time since biopsy diagnosis (6.7 years) in a disease running a chronic progressive course of just over 10 years, an analysis based on the original histological stage was likely to underestimate the disease severity. Furthermore, histological staging itself can be inaccurate as the pathological changes may be patchy throughout the liver. The alterations in conventional serum lipid concentrations were similar to those reported previously for patients with PBC [4,9,10]: an increase in serum cholesterol, unesterified cholesterol:cholesterol ratio and phospholipid and HDL cholesterol. Within the PBC patients there was a trend towards higher total and HDL cholesterol in group 2 patients (bilirubin \ 18 mmol/l) as compared to group 1 patients (bilirubin B18 mmol/l) although this was not statistically significant. Patients with ESLD differed from the rest of the PBC patients in having HDL cholesterol concentrations lower than controls but similar total cholesterol concentrations. Since LpX does not remain in the supernatant following precipitation with manganese–heparin it makes no contribution towards HDL cholesterol concentration.

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Table 3 The distribution of apo A1 isoforms in patients and controls

Apo Apo Apo Apo Apo

A1-1 A1-2 A1-3 A1-4 A1-5

PBC group 1 (n = 10)

PBC group 2 (n = 8)

ESLD (n =1)

Controls (n = 10)

10.0 3.7 62.0 30.2 7.0

7.1 7.2 54.3 26.6 4.1

10.3 3.1 44.6 34.9 7.1

10.1 4.0 47.5 34.0 6.6

(5.1–2.8) (3.0–11.0) (32.0–45.0) (14.0–35.0) (1.2–23.5)

(2.4 – 10.2) (1.9 – 12.4) (40.7 – 63.0) (12.6 – 34.8) (2.6 – 12.7)

(8.3 – 12.0) (2.1 – 8.2) (32.0 – 63.0) (15.8 – 39.0) (3.0 – 13.8)

Results are expressed as a percentage of total apo A1 present as a given isoform. Median (range).

Fig. 2. Size distribution of apo A1 particles in serum as determined by gel filtration chromatography on Sephacryl S-300. The elution profiles for protein (absorbance 280 nm; shaded dots) and apo A1 concentration ( ) are shown for 4 control subjects; panels 1 – 4.

An explanation for the changes seen in LpA1 particle concentration was beyond the scope of the present study. In general terms the concentration of a lipoprotein may be affected by an alteration in its synthetic rate or alterations in its metabolism. There have been no studies addressing the synthetic rates of lipoproteins in early PBC although it is recognised that in ESLD (of whatever aetiology) synthetic function is decreased. Much of the work elucidating HDL metabolism in healthy subjects has focused on the metabolism of lipoproteins within specific hydrated density ranges, e.g., HDL2 and HDL3 rather than on particles of specific apolipoprotein composition such as LpA1 or LpA1:A2. It is clear that four enzymes, HL, lipoprotein

lipase (LPL), LCAT and CETP, play a key role in the interconversion of HDL species in blood and are important determinants of the concentrations of HDL and the HDL subclasses [19]. It seems probable that similar factors are at least in part responsible for determining LpA1 and LpA1:A2 concentrations and the distribution of apo A1 between these two particle types. Reductions in both LPL and HL activities have been shown in patients with both early and late PBC [4,20]. The reduction in HL was thought to be due to the presence of an unidentified plasma inhibiting factor [20]. A reduction in LCAT activity was also seen but only in patients with advanced PBC. Further work is needed to establish whether alterations in the activities

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Fig. 3. Size distribution of apo A1 particles in serum as determined by gel filtration chromatography on Sephacryl S-300. The elution profiles for protein (absorbance 280 nm; shaded dots) and apo A1 concentration ( ) are shown for 4 PBC patients: 2 patients with bilirubin B 18 mmol/l; 1 with bilirubin \ 18 mmol/l; and 1 with ESLD; panels 5 – 8 respectively.

of these enzymes are related to changes in LpA1 concentration. Experiments on a small number of PBC patients suggested that the disease is associated with an alteration in the size distribution of apo A1 particles as assessed by gel filtration chromatography. The sizing of apo A1 particles in serum is problematical with essentially only two techniques available: non-denaturing polyacrylamide gel electrophoresis and gel filtration chromatography. Non-denaturing gradient gel electrophoresis is commonly used but suffers from the disadvantage that the net charge of the lipoprotein may also affect electrophoretic mobility. In addition non spherical particles, e.g. discoid particles might also show aberrant migration. In contrast gel filtration chromatography is unaffected by particle charge and is likely to be affected less by deviations from the assumption of spherical particles and was thus chosen as the sizing method for this study. Gel filtration chromatography followed by immunoelectrophoresis proved to be a satisfactory method of assessing the size of serum apo A1. In control subjects the apo A1 particles eluted as one or two closely eluting peaks of molecular weight circa 175 kDa while in PBC patients two clearly defined

peaks of molecular weights circa 150 and 200 kDa are found. This pattern was not dependent upon the stage of the disease or LpA1 concentration with the ESLD disease patient having a similar pattern to the group 1 PBC patients. Thus PBC patients appear to have 2 distinct size populations of apo A-I particles. This may reflect alterations in the activities of the enzymes LPL, HL, LCAT and CETP which regulate HDL size in health. Apo A1 isoform analysis by IEF/immunoblotting/ chemiluminescent detection proved a robust and satisfactory technique. The five recognised isoforms of apo A1 were identified in plasma. apo A1-1 corresponds to pre-apo A1 which is converted to the mature apo A1 protein (apo A1-3 isoform) by the cleavage of a 6 amino acid N-terminal extension by a specific protease[21–26]. Apo A1-3 is the predominant plasma isoform and undergoes stepwise non enzymatic deamination to give the more acidic apo A1-4 and apo A1-5 isoforms. The apo A1-2 isoform which is generally present in small amounts only, may represent a degradation product of apo A1-1. In this study all five common isoforms were present in PBC patients with no additional forms noted. The distribution of apo A1

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among the different isoforms was similar in patients and controls. This suggests that both enzymatic and nonenzymatic processing of apo A1 is unimpaired in PBC. The results presented here have shown abnormalities in the concentrations of apo A1 particles in PBC. Case control studies have suggested that a high serum concentration of LpA1 is associated with a reduced risk of coronary heart disease [16] perhaps by a direct effect in promoting cholesterol efflux from peripheral tissues [15]. However, it must be noted that the relationship between LpA1 and coronary heart disease has not been rigorously tested in the setting of a long term prospective study. Extrapolating the significance of increased LpA1 concentrations to PBC patients, in whom the risk of heart disease does not appear to be elevated, is fraught with difficulties. The relative protection of PBC patients is probably multifactorial and further work is required to clarify the role of increased LpA1 concentrations

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