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Chapter 12
Chapter 12

Pre b1-HDL, a Native Lipid- poor HDL, and its Potential as a New Marker for HDL Metabolism

Takashi Miida and Satoshi Hirayama

Department of Laboratory Medicine, Juntendo University School of Medicine, Tokyo, Japan

INTRODUCTION

High density lipoprotein (HDL) has a density of 1.063 to 1.210, as determined by ultracentrifugation. Numerous epidemiological studies have shown that low HDL-cholesterol concentration is a strong predictor of coronary heart disease (CHD) (Miller et al., 1977; Gordon et al., 1989; Kitamura et al., 1994; Gordon, 1999; Foody et al., 2000 ). Although reducing the plasma concentration of low density lipoprotein-cholesterol (LDL-C) is the main therapeutic goal in the management of dyslipidemia (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults., 2001; De Backer et al., 2003; Teramoto et al., 2007 ), recent meta-analyses have clearly shown that statins (the strongest LDL-C-lowering agents) reduce the risk of cardiovascular events by only 20 e 30% ( Zhou et al., 2006 ). Moreover, the risk of CHD was shown to be negatively correlated with HDL-cholesterol concentration even in patients treated with statins (Mabuchi et al., 2002 ). Experimental studies strongly suggest that the anti-atherogenic activity of HDL centers mainly on the removal of excess cholesterol from atherosclerotic lesions. The removed cholesterol is then transported to the liver and excreted into the bile by a process known as reverse cholesterol transport ( Gomaraschi et al., 2006 ). In addition, HDL particles have anti-inflammatory, antioxidant, and anti-thrombotic effects. The compositions of lipids and apolipoproteins, particle size distribution, and electrophoretic mobility vary among HDL particles, and HDL particles exhibit different functions. Thus, HDL particles are classified into several

The HDL Handbook. ISBN: 978-0-12-382171-3 Copyright 2010 Elsevier Inc. All rights of reproduction in any form reserved.

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subfractions according to their physical and functional properties ( Barbaras et al., 1987; Lewis and Cabana, 1996; Miyazaki et al., 2000; Okazaki et al., 2005 ). Preb1-HDL is a unique lipid-poor HDL subfraction that is closely associated with cholesterol efflux from cellular membranes ( Miida et al., 1990, 1992; Fielding et al., 1991; Kawano et al., 1993 ). Over the past two decades, much has been learned about the physiological functions and clinical signifi- cance of pre b1-HDL (Miida et al., 1996, 1997, 1998, 2000a,b, 2003a, 2004a,b; Hirayama et al., 2007 ). In this chapter, we provide a brief overview of pre b1- HDL and discuss recent advances in the field.

CHARACTERISTICS OF HDL

HDL is a technical term referring to lipoproteins exhibiting a density of 1.063 e 1.210 with ultracentrifugation. Given that all other known lipoproteins, including chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL, contain apolipoprotein B (apoB: that is, apoB 48 or B 100 ), HDL may be defined as a lipoprotein that does not contain apoB. In patients with cholesteryl ester transfer protein (CETP) deficiency or cholestatic liver disease, large, apoE-rich lipoproteins without apoB accumu- late. They are called apoE-rich HDL, even though their density range overlaps those of LDL and apoE-poor HDL. In this chapter, we describe the nature of HDL in detail.

Lipid and protein constituents

As in the other classes of lipoproteins, HDL has a hydrophobic lipid core containing a cholesteryl ester (CE) and a triglyceride (TG) and is covered with a phospholipid monolayer. A small amount of free cholesterol also exists on the surface of HDL. Clinically, the cholesterol concentration associated with HDL particles is used as a marker for HDL and is referred to as HDL-cholesterol (HDL-C). However, cholesterol accounts for only 15 e 20% of the total weight of an HDL particle, while phospholipids and HDL-associated proteins account for 25 e 30% and 50%, respectively. Nascent HDL particles, which are secreted into the blood from the liver, consist mainly of phospholipids, apoA-I, and a little cholesterol, but no core lipids. Thus, the measurement of HDL-C concentration is unlikely to reflect the amount of lipid-poor HDL. HDL particles carry enzymes and transfer proteins as well as apolipopro- teins ( Table 12.1 ). The proteins associated with HDL particles reflect a heterogeneous distribution ( Francone et al., 1989 ). For example, some HDL particles are associated with lecithin-cholesterol acyltransferase (LCAT), and others are not ( Francone et al., 1989; Miida et al., 2004b ). Unlike apoB- containing particles such as LDL, some HDL particles contain weakly bound proteins that move to or from other lipoproteins during HDL metabolism. In

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TABLE 12.1 Lipid and protein constituents of HDL particles

HDL component Constituent

Function

Protein moiety

 

(a)

Apolipoproteins ApoA-I* ApoA-II*

Activation of LCAT Inhibition of LCAT? Activation of HL? Activation of LCAT? Activation of LCAT? Inhibition of CETP? Inhibition of HDL binding mediated by apoE-recognizing receptors? Inhibition of LCAT by dissociating apoA-I? Inhibition of LCAT by dissociating apoA-I? Binding to lipids and their transport? Binding to lipoprotein receptors? Binding to PON1 Preb1-HDL generation from b-HDL Dissociation of preb1-HDL

 

ApoA-IV

ApoC-I

ApoC-II

ApoC-III

ApoD

ApoE

ApoJ

ApoM

Serum amyloid A protein (SAA)

(b)

Enzymes

Lecithin-cholesterol

Conversion of FC to CE

 

acyltransferase (LCAT)

Paraoxonase-1 (PON1)

Inhibition of LDL and HDL oxidation Degradation of PAF and

Platelet activating

factor acetyl hydrolase (PAF-AH) oxidized PL

(c)

Transfer

Cholesteryl ester transfer protein (CETP)

Exchange of CE on HDL for TG on apoB-containing lipoproteins Transfer of PL on TG-rich lipoproteins Fusion of HDL particles and simultaneous dissociation of

proteins

Phospholipid transfer protein (PLTP)

 

preb1-HDL

Lipid moiety

 
 

Cholesterol (free and esterified forms) Triglyceride (TG) Phospholipids (PL)

HL, hepatic lipase; FC, free cholesterol; CE, cholesteryl ester * major apolipoproteins of HDL

 
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some cases, exogenous proteins dissociate the protein components from HDL particles (Lagocki and Scanu, 1980 ; Miida et al., 1999a, 2006).

Particle size

On average, HDL particles are 7 e 10 nm in diameter, which is nearly equal to the thickness of a cell membrane and is about 0.1 times the diameter of an influenza virus. As lipoproteins less than 70 nm in diameter can penetrate vascular walls (Mamo et al., 1998 ), HDL particles can easily access athero- sclerotic lesions in the subendothelial space. It is of great interest that HDL particles are detectable not only in blood but also in other body fluids such as lymph (Nanjee et al., 2001 ), follicular fluid (Jaspard et al., 1997 ), aqueous humor (Cenedella, 1984 ), and cerebrospinal fluid ( Roheim et al., 1979; Miida et al., 1999b, 2006; Kay et al., 2003 ). The HDL in cerebrospinal fluid has been extensively investigated by many researchers, and cerebrospinal HDL is believed to play a crucial role in cholesterol recycling ( Dietschy and Turley, 2001 ). Surprisingly, brain cholesterol has an extremely long half-life of about 5 years ( Bjo¨ rkhem et al., 1998 ).

HDL SUBFRACTIONS

HDL is composed of several different particles, known as HDL subfractions; however, the clinical significance of these subfractions as predictors of atherosclerotic disorders is controversial.

Analytical and separation methods

HDL particles vary in density, electrical charge, apolipoprotein composition, lipid composition, particle size, and physiological function. According to these differences, HDL particles can be separated into several subfractions using various analytical and separation methods, including ultracentrifugation ( Lewis and Cabana, 1996 ), gel filtration chromatography ( Kay et al., 2003 ), high performance liquid chromatography (Okazaki et al., 2005 ), agarose gel elec- trophoresis ( Peynet et al., 1986 ), gradient polyacrylamide gel electrophoresis, native two-dimensional gel (2-D gel) electrophoresis (Francone et al., 1989; Miida et al., 1990, 1992, 1996, 1997, 1998, 1999a, 2000a,b, 2003a, 2004b, 2006; Fielding et al., 1991; Kawano et al., 1993; Jaspard et al., 1997 ), capillary electrophoresis ( Inano et al., 2000; Zhang et al., 2006 ), immunoaffinity chro- matography ( Barbaras et al., 1987 ), enzyme-linked immunoassays (ELISAs) using monoclonal antibodies (Miyazaki et al., 2000; Miida et al., 2003a,b, 2004a, 2006; Hirayama et al., 2007 ), and crossed immunoelectrophoresis ( Neary et al., 1991 ). However, these methods present several problems. For example, ultracentrifugation exposes HDL subfractions to harsh conditions, including extreme gravity and salinity ( Lewis and Cabana, 1996 ). Furthermore,

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some HDL subfractions are rendered unstable by storage at 4 C or even 80 C ( Miida et al., 2003b ). We have analyzed the HDL subfractions in fresh plasma by native 2-D gel electrophoresis (Miida et al., 1990, 1992, 1996, 1997, 1998, 1999a ; 2000a,b, 2003a, 2004a,b, 2006; Fielding et al., 1991; Kawano et al., 1993 ). Although this method minimizes changes in the distribution of the subfractions during separation, it is time-consuming and costly, and considerable technical profi- ciency is required to obtain reproducible results. In addition, it is difficult to run several samples simultaneously. Our system uses a 0.75% agarose gel in the first dimension and a 2 e 15% gradient polyacrylamide gel in the second dimension. Unlike most 2-D gel systems, ours does not use sodium dodecyl sulfate in either the polyacrylamide gel or the electrophoresis buffer solution.

What is pre b1-HDL?

In agarose gel electrophoresis, plasma lipoproteins are separated by electric charge. Lipoproteins with greater negative charge will migrate further from the point of origin. LDL, VLDL, and HDL migrate with b-, pre b-, and a -mobility, respectively. In agarose gels stained with lipophilic dyes (e.g., Sudan Black B or Fat Red O), HDL is detectable as an a-lipoprotein. To detect lipid-poor HDL subfractions, the separated lipoproteins are transferred to a nitrocellulose membrane and visualized with anti-apoA-I antibodies. In addition to the a -migrating HDL, there is a minor apoA-I band with pre b-mobility, which is known as pre b-HDL ( Kunitake et al., 1985 ). Pre b-HDL is an LpA-I particle containing apoA-I but not apoA-II ( Castro and Fielding, 1988 ). Pre b-HDL may be separated further into three subspecies (pre b1-HDL, pre b2-HDL, and pre b3- HDL) by native 2-D gel electrophoresis (Figure 12.1 ) ( Francone et al., 1989 ). The estimated molecular weight of pre b1-HDL is 60 e 70 kDa ( Castro and Fielding, 1988; Miida et al., 2000a ). Pre b1-HDL consists mainly of apoAI and phospholipids (Castro and Fielding, 1988 ). The sphingomyelin/phosphatidyl choline ratio of pre b1-HDL is very similar to that of the outer leaflet of cellular membranes (Miida T and Fielding PE. unpublished data). Pre b1-HDL has almost no core lipids and little free cholesterol. The lipoproteins in fresh plasma were first separated on a 0.75% agarose gel (first dimension). Then, a longitudinal piece of the gel was run perpendicularly on a 2 e 15% gradient polyacrylamide gel without detergent (second dimen- sion). After electrical transfer to a nitrocellulose sheet, the HDL subfractions were visualized by Western blotting with 125 I-labeled anti-human apoAI antibody.

Physiological role and metabolism of pre b1-HDL

Studies have shown that pre b1-HDL is closely associated with the release of cellular cholesterol into plasma (i.e., the initial step of reverse cholesterol

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248 The HDL Handbook FIGURE 12.1 HDL subfractions separated by native two-dimensional gel electrophoresis. transport).

FIGURE 12.1 HDL subfractions separated by native two-dimensional gel electrophoresis.

transport). When plasma is incubated with 3 H-cholesterol-labeled cells, the radiolabel moves from the membranes to the plasma as a function of time. This is called cholesterol efflux and occurs via two independent pathways ( Kawano et al., 1993; Yokoyama, 2006): a specific lipid-free, or lipid-poor, apolipoprotein- mediated pathway; and a non-specific diffusion-mediated pathway. The former requires the specific interaction of free apoA-I or lipid-poor HDL with cellular membranes, and the latter involves various extracellular acceptors such as albumin and lipoproteins in the aqueous phase. In cultured fibroblasts, the magnitude of cholesterol efflux is proportional to the plasma pre b1-HDL concentration. Pre b1-HDL-dependent cholesterol efflux accounts for about 60% of the total cholesterol efflux (Kawano et al., 1993 ). Cholesterol efflux can be reduced to half the original value by the addition of monoclonal anti-pre b1-HDL antibodies or by the proteolytic treatment of cultured cells ( Kawano et al., 1993; Fielding et al., 1994 ). Among the many HDL subfractions, cellular cholesterol is first transferred preferentially to pre b1-HDL and then to pre b2-HDL (Castro and Fielding, 1988 ). Ultimately, cellular cholesterol is moved to pre b3-HDL, the largest pre b-HDL, where LCAT is located (Francone et al., 1989 ). LCAT converts free cholesterol to CE, a hydrophobic compound that enters the core of HDL particles. Finally, pre b-HDL is converted into large, CE-rich, spherical HDL particles with a -mobility upon agarose gel electrophoresis; this process is referred to as the maturation of preb1-HDL. Although CETP promotes the

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bidirectional transfer of CEs and TGs between lipoproteins (Moulin et al., 1992 ), CETP activity results in the net transfer of CEs from a -HDL to VLDL and LDL and of TGs in the reverse direction, owing to the CE and TG concentration gradients between lipoproteins. CEs carried by VLDL and LDL are transported to the liver where they are taken up via LDL receptors for further metabolism. Meanwhile, the TG-enriched a -migrating HDL is susceptible to hydrolysis by hepatic lipase, thereby regenerating pre b1-HDL.

Pre b1-HDL production

Recent studies have revealed that pre b1-HDL is generated by ATP-binding cassette transporter A-1 (ABCA1)-dependent and ABCA1-independent mechanisms.

ABCA1-dependent mechanism

The main source of plasma pre b1-HDL is probably direct secretion from the

liver. ABCA1 plays a crucial role in the lipidation of apoA-I with phospholipids and in the subsequent efflux of cholesterol from cellular membranes; however, there is considerable debate over its role in pre b1-HDL formation. Pre b1-HDL

is not free apoA-I but apoA-I coupled with phospholipids and a little free

cholesterol (Castro and Fielding, 1988 ). In vitro studies suggest that phos- pholipids are added to the apoA-I secreted from hepatocytes via ABCA1- dependent and -independent mechanisms (Kiss et al., 2003 ). This is consistent with the fact that patients with Tangier disease have pre b1-HDL despite a lack of ABCA1 activity ( Huang et al., 1995; Asztalos et al., 2001 ). Recent data strongly suggest that the production of biologically active pre b1-HDL involves at least two steps and that ABCA1 activity is closely related to pre b1-HDL synthesis. In J774 macrophages, ABCA1 activity creates two types of high-affinity apoA-I binding sites at the cell surface ( Vedhachalam et al., 2007 ). One is a low-capacity site formed by the direct interaction of apoA-I with ABCA1, and the other is a high-capacity site secondarily induced by the interaction of apoA-I with membrane lipids. In support of the multistep hypothesis, two C-terminal deletion mutants of apoA-I (D190-243 and D223- 243) can be cross-linked to ABCA1, although this significantly decreases their

binding affinity for the cell surface ( Vedhachalam et al., 2007 ). It has also been shown that C-terminal deletions in apoA-I (D185-243 and D220-243) do not prevent pre b1-HDL formation but greatly reduce ABCA1-mediated cholesterol efflux ( Chroni et al., 2007 ). A separate study examined the apoA-I secreted from HepG2 cells, Caco cells, and CHO cells expressing human apoA-I and found that apoA-I was first secreted as particles with pre b-mobility and

a Stokes radius of 2.6 nm ( Chau et al., 2006 ). With additional incubation,

however, 3.6-nm particles with pre b-mobility became predominant. The conversion from 2.6-nm to 3.6-nm particles required ABCA1 activity, and only

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the 3.6-nm particles were able to bind phospholipids. When the 3.6-nm parti- cles were incubated with smooth muscle cells, cholesterol efflux was increased to 28-fold that produced by phospholipids. These data strongly support the hypothesis that biologically active preb1-HDL is generated by ABCA1 activity and that pre b1-HDL promotes cholesterol efflux from cells.

ABCA1-independent mechanisms

In addition to the ABCA1-dependent mechanism described above, pre b1-HDL

is also generated from an a-migrating spherical HDL by ABCA1-independent

mechanisms. For example, a 2-h incubation of TG-enriched HDL 2 with rat hepatic lipase yielded pre b1-HDL, as determined by native 2-D gel analysis (58). Pre b1-HDL can be also formed from a -HDL during its hydrolysis by

bacterial TG lipase ( Miida et al., 2000a ). Lipase-dependent shedding of pre b1- HDL from a -HDL is likely to occur in vivo , because preb1-HDL was formed during recycling rat liver perfusion in the presence of heparin (Barrans et al.,

1994 ).

Other studies suggest that pre b1-HDL is also formed by lipoprotein lipase (LPL). Neary et al. (1991) incubated plasma samples, obtained before and after the injection of heparin, at 37 C and determined the total pre b-HDL concen- tration by crossed immunoelectrophoresis. When LCAT was inactivated by p-chloromercuriphenylsulfonic acid, total preb-HDL was increased by a much greater amount in the post-heparin plasma than in the pre-heparin plasma. This increase was especially marked in four hypertriglyceridemic subjects following

a fat load, suggesting that pre b1-HDL is generated from TG-rich lipoproteins

during lipolysis. Furthermore, Sviridov et al. (2003) examined pre b1-HDL formation in skeletal muscle during physical exercise by simultaneously measuring the concentration of preb1-HDL in the femoral artery and vein before and after 25 min of cycling. They found that the pre b1-HDL concentration was higher in venous blood than in arterial blood, independent of exercise, and that pre b1-HDL production (calculated as the difference in preb1-HDL concentra- tion between venous and arterial blood) increased 6.6-fold after exercise. Another report showed a 30% increase in the plasma preb1-HDL concentration after cardiopulmonary exercise followed by 4 km of jogging (Jafari et al., 2003). These results agree with those of another study showing that the plasma pre b1-HDL concentration was 46% greater in athletes than in control subjects (Olchawa et al., 2004). Although it is unclear whether preb1-HDL is generated from TG-rich lipoproteins or a -migrating HDL, LPL is likely to enhance pre b1-HDL formation during the passage of blood through skeletal muscle. Even without lipase activity, pre b1-HDL can be generated from a -HDL by exogenous apolipoproteins, which have greater affinity for HDL particles than does apoA-I. When serum amyloid A protein (SAA) was added to plasma, the pre b1-HDL concentration increased in a dose-dependent manner ( Miida et al., 1999a ). Dose-dependent pre b1-HDL formation was also observed when SAA

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was added to cerebrospinal fluid ( Miida et al., 2006 ). This phenomenon results from the displacement of apoA-I and phospholipids on HDL particles by SAA. In fact, pre b1-HDL production occurred immediately when SAA was added to ultracentrifugally isolated HDL coupled to agarose beads (Miida et al., 1999a ). Recent studies suggest that pre b1-HDL is also formed by a mechanism involving apolipoprotein M (apoM) ( Wolfrum et al., 2005 ). ApoM was recently identified in human plasma and is also found in the liver and kidney. ApoM is synthesized by hepatocytes and proximal tubule cells and is secreted with a hydrophobic signal peptide. Using this signal peptide as an anchor, apoM binds to the phospholipid bilayers of plasma lipoproteins such as HDL ( Christoffersen et al., 2006 ). In apoM knockout mice, large HDL particles accumulated, and no preb-HDL could be detected ( Wolfrum et al., 2005 ). ApoM promotes the formation of pre b1-HDL, resulting in increased choles- terol efflux in cultured cells.

PLASMA PRE b1-HDL CONCENTRATION IN VARIOUS DISORDERS

Several research groups have already reported the plasma pre b1-HDL concentration in healthy subjects based on native 2-D gel analysis followed by immunoblotting ( Miida et al., 1996, 1998, 2000a,b; Sasahara et al., 1997; Asztalos et al., 2000, 2001, 2004 ), ELISA ( Miyazaki et al., 2000; Miida et al., 2003a, 2004a; Sviridov et al., 2003, 2006; Olchawa et al., 2004; Hirayama et al., 2007 ), and an ultrafiltration-isotope dilution method ( O’Connor et al., 1998; Jafari et al., 2003 ). However, there is significant variation in the measured values between the different methods, and the same research group reported considerably diverse results ( Table 12.2 ). Such discrepancies may be the result of pre b1-HDL instability during storage ( Miida et al., 2003b ) or differences in the methods used to separate the HDL subfractions. To avoid these complications when analyzing clinical samples, we produced a monoclonal antibody specific for pre b1-HDL (Mab55201) and established an ELISA system for pre b1-HDL measurement ( Miyazaki et al., 2000 ). Mab55201 reacted with LpA-I but not LpA-II. The gel filtration analysis revealed that Mab55201 had strong reactivity against the lipoprotein fraction with a molecular weight of less than 67 kDa. Preincubation of human plasma with Mab55201 resulted in disappearance of pre b1-HDL spots in native 2D-gel analysis ( Miyazaki et al., 2000 ). When plasma was diluted 21-fold with 50% sucrose, the pre b1-HDL concentration did not change significantly for at least 5 days at 4 C or for 30 days at 80 C ( Miida et al., 2003b ). There was no significant difference in the plasma pre b1-HDL concentration between men and postmenopausal women ( Figure 12.2 ), although premenopausal women had less pre b1-HDL than either of the other two groups ( Miida et al., 2004a ). It is interesting that the plasma preb1-HDL concentration does not always parallel the HDL-C, apoA-I, and LpA-I concentrations (Miida et al., 1997,

252 The HDL Handbook TABLE 12.2 Reference ranges of plasma preb1-HDL concentration Number Relative conc.
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TABLE 12.2 Reference ranges of plasma preb1-HDL concentration
Number
Relative conc. Absolute conc.
Method
(M/F)
(% apoA-I)
(mg/L apoA-I) 1 Reference
Native
20
(11/9)
4.6
2.3
63
28
Miida et al. (1996)
2-D gels
12
(3/9)
4.2
1.6
59
23
Miida et al. (1998)
20
(18/2)
73
34
Miida et al. (2000a)
58
(22/36)
78
39 (M)
Miida et al. (2000b)
88
45 (F)
18
(10/8)
92
31
Sasahara et al. (1997)
79
(79/0)
79
49 (M)
Asztalos et al. (2000)
71
(30/41)
129
60 (M)
Asztalos et al. (2001)
120
85 (F)
1277
120
14 (M)
Asztalos et al. (2004)
(1277/0)
EIA
25 (15/10)
23
8
Miyazaki et al. (2000)
45
(24/21)
20
7
Miida et al. (2003a)
100 (47/53)
22
7 (M)
Miida et al. (2004a)
19
7(F)
7 (7/0)
150
33(M)
Sviridov et al. (2003)
33
(33/0)
37 3 2 (M)
Olchawa et al. (2004)
26
(21/6)
25
4 3
Sviridov et al. (2006)
30
(13/17)
1.5 0.4
20
6
Hirayama et al. (2007)
Ultrafiltration-
136 (46/90)
6.1 3.6
73
44 (Total)
O’Connor et al. (1997)
isotope
7.2
4.0 (M)
68
40 (M)
dilution
5.5
3.3 (F)
84
49 (F)
technique
19
(11/8)
7.9
3.8
100
60
Jafari et al. (2003)
1 Absolute concentration is presented as the amount of apoA-I associated with preb1-HDL.
2 Healthy subjects with TG < 150 mg/dL and HDL-C of 35 e 80 mg/dL.
3 Values are expressed as the mean SE.

2000a). Instead, the preb1-HDL concentration is positively correlated with the concentrations of LDL-cholesterol and TGs (Miida et al., 2000a,b). Previously, we compared the plasma preb1-HDL concentration between patients with angiographically defined coronary artery disease (CAD) and age- and sex- matched controls. We excluded those patients taking lipid-lowering agents. The absolute and relative preb1-HDL concentrations were 38% and 40% greater, respectively, in the CAD group than in the control group (P < 0.05 and P < 0.01, respectively) ( Miida et al., 1996). The increase was dominant among those CAD patients exhibiting low LCAT activity. Asztalos et al. (2000) also reported that CAD patients with low HDL-cholesterol levels had high preb1-HDL concentrations. They further examined the clinical significance of preb1-HDL using frozen samples collected for the Veterans Affairs HDL Intervention Trial (VA-HIT) (Asztalos et al., 2005). Those eligible for the VA-HIT were men below 74 years of age with a documented history of CHD, HDL-C 40 mg/dL,

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Chapter j 12 Pre b 1-HDL 253 FIGURE 12.2 Plasma pre b 1-HDL concentration according to

FIGURE 12.2 Plasma preb1-HDL concentration according to age and sex. Fasting plasma was obtained from 100 healthy Japanese subjects. The preb1-HDL concentration was measured by immunoassay using a specific monoclonal antibody.

LDL-C 140 mg/dL, and a TG of 300 mg/dL. The mean preb1-HDL concen- tration, measured by native 2-D gel electrophoresis, was significantly higher in the VA-HIT subjects than in those male controls from the Framingham Offspring Study who had a low HDL-C level ( < 40 mg/dL). Univariate analysis revealed that the preb1-HDL concentration is a significant risk factor for new cerebrovascular events, although the trend disappeared in a multivariate analysis. In patients undergoing hemodialysis (HD), the pre b1-HDL concentration markedly increased independent of the HDL-C concentration ( Miida et al., 2003a). In HD patients, the LCAT-dependent conversion of pre b1-HDL to a -HDL is severely delayed. When HD patients and control subjects were analyzed together, LCAT activity was negatively correlated not with the baseline pre b1-HDL concentration but with a decrease in the pre b1-HDL concentration during 120 min of incubation at 37 C ( Miida et al., 2003a ). When the HD patients were considered alone, however, LCAT activity was not correlated with the drop in pre b1-HDL. LCAT esterifies free cholesterol on LDL and HDL via its b- and a-LCAT activities, respectively ( Jonas, 1998 ). We previously examined pre b1-HDL metabolism in a patient with fish-eye disease (FED), which is characterized by a lack of a -LCAT activity as a result of a mutation in LCAT ( Miida et al., 2004b ). Although patients with FED usually show subnormal total plasma LCAT activity owing to the remaining b-LCAT activity, they have very low levels of HDL-C ( <10 mg/dL). In our patient, the plasma pre b1-HDL concentration was nearly normal, but little pre b1-HDL was converted to a -HDL at 37 C (Figure 12.3 ). These observations strongly suggest

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254 The HDL Handbook FIGURE 12.3 Impaired conversion of pre b 1-HDL into a -HDL in

FIGURE 12.3 Impaired conversion of preb1-HDL into a-HDL in fish-eye disease.

that plasma pre b1-HDL metabolism is closely related to a -LCAT activity but not to b-LCAT activity. Blood samples were obtained from a healthy normolipidemic volunteer and from a patient with (FED). The fresh plasma was incubated at 37 C for 90 min. The amount of pre b1-HDL in the normal plasma was decreased markedly, whereas the amount in the FED plasma was increased. Patients with poorly controlled type 2 diabetes, another group at high risk for CAD, have elevated fasting pre b1-HDL concentrations, which are associ- ated with the severity of carotid artery atherosclerosis ( Hirayama et al., 2007 ). Those patients with type 2 diabetes had mean absolute and relative pre b1-HDL concentrations that were 21 and 34% higher, respectively, than control levels. Stepwise regression analysis showed that the absolute and relative concentra- tions of pre b1-HDL were significant independent determinants for maximum intima-media thickness and plaque score. The mechanism underlying these findings should be clarified in future studies.

TURNOVER OF PREb1-HDL IN HUMAN PLASMA

When plasma is incubated at 37 C, the pre b1-HDL concentration is decreased exponentially as pre b1-HDL is converted to a -HDL by LCAT. Therefore, we

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Chapter j 12 Pre b 1-HDL 255 FIGURE 12.4 Determination of the conversion half-time of pre

FIGURE 12.4 Determination of the conversion half-time of preb1-HDL (CHT pre b1 ). Fresh plasma was incubated with or without DTNB, and paired samples were taken at baseline (C 0 ) and every 30 min. The pre b1-HDL concentration was measured by immunoassay. The difference in the pre b1-HDL concentration between the paired samples was assumed to reflect the conversion of pre b1-HDL to a -HDL by LCAT. The CHT pre b1 is defined as the time required for the pre b1-HDL concentration to reach 50% of C 0 .

determined the time required for a 50% reduction in the pre b1-HDL concen- tration from the baseline level and defined this as the conversion half-time (CHT pre b1 ) (Figure 12.4 ). The CHT pre b1 probably reflects the turnover rate of pre b1-HDL in vitro ( Miida et al., 2004a). In 100 healthy Japanese subjects, the mean CHT pre b1 was 47.4 13.0 min, with no significant difference according to sex or age. Therefore, CHT pre b1 may be used as a new marker for the turnover of HDL. At least in healthy subjects, CHT pre b1 is the strongest determinant of the plasma pre b1-HDL concentration (Miida et al., 2004a ).

CONCLUSIONS

It has been nearly 20 years since pre b1-HDL was first detected using native 2-D

gel electrophoresis; however, it is still controversial whether plasma pre b1- HDL is the initial acceptor of cellular cholesterol or is the first product of the interaction between free apoA-I and cellular lipids via the ABCA1-dependent pathway. Thus, the plasma pre b1-HDL concentration may be useful as

a marker for atherosclerosis or for the metabolic turnover of HDL. Additional studies are needed to distinguish between these possibilities.

ACKNOWLEDGMENTS

We thank Osamu Miyazaki (Sekisui Medical) and Ms Utako Seino (Niigata University) for their excellent technical assistance. We are most grateful to Ms Mayumi Okada for her invaluable support to our laboratory. This work was funded in part by Grants-in-Aid for Science Research from the Ministry of Education, Science and Culture of Japan (No. 12671102, 16590815, and 20590558).

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The HDL Handbook

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