Clinical Chemistry AACC Online Job Center
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 50: 2309-2315, 2004. First published September 30, 2004; 10.1373/clinchem.2004.034439
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2004.034439v1
50/12/2309    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergmeier, C.
Right arrow Articles by Gross, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergmeier, C.
Right arrow Articles by Gross, W.
Related Collections
Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2004;50:2309-2315.)
© 2004 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Distribution Spectrum of Paraoxonase Activity in HDL Fractions

Christoph Bergmeier1,a, Rüdiger Siekmeier2 and Werner Gross1

1 Labor für Angewandte Biochemie, Gustav-Embden-Zentrum für Biologische Chemie, Klinikum der J.W. Goethe-Universität, Frankfurt/Main, Germany.
2 Bundesinstitut für Arzneimittel und Medizinprodukte (BfArM), Bonn, Germany.

aAddress correspondence to this author at: Labor für Angewandte Biochemie, Gustav-Embden-Zentrum für Biologische Chemie, Klinikum der J.W. Goethe-Universität, Atzelbergstrasse 67, 60389 Frankfurt/Main, Germany. Fax 49-69-47874641; e-mail cbergmei{at}stud.uni-frankfurt.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Paraoxonase (PON1) associated with HDL can be regarded as a cardio- and vasoprotective enzyme. However, because HDL is not a homogeneous fraction, it is important to investigate in which subgroups of HDL active PON1 is located. It would also be useful to determine density profiles of the HDL apolipoproteins (Apo) E and J.

Methods: We investigated the density range of HDL ({rho} = 1.063–1.256 kg/L) in healthy individuals, using the ultracentrifugation reference method and a newly introduced automated fractionation method. Profiles of PON1 activity and ApoA-I, ApoA-II, ApoE, ApoJ, and cholesterol concentrations were obtained by use of various density gradients.

Results: PON1 activity was highest in the more dense HDL3 and VHDL fractions where PON1 was not dissociated from the particles during centrifugation. The fraction in density range 1.175–1.185 kg/L showed not only the highest PON1 activity, but also the highest specific activity (activity per HDL particle). This fraction was the least-dense fraction containing both ApoE and ApoJ. Only the Q192R polymorphism had an effect on the distribution profile of PON1 activity. In contrast, L55M and the T(–107)C polymorphisms (determined by a novel nonradioactive method) were without effect on the density distribution of PON1 activity.

Conclusion: The HDL3 fraction, which is important in reverse cholesterol transport, also carries the highest PON1 activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human serum paraoxonase (PON1;1 EC 3.1.1.2 and EC 3.1.1.8) hydrolyzes numerous organophosphates and lipid oxides (1), and calcium is an essential cofactor for its activity and stability (2). Currently, there are three known independent polymorphisms of the enzyme: Q192R, L55M (3), and T(–107)C (4).

The distribution of PON1 activity within HDL has been investigated by four separate research groups. PON1 was initially assumed to be located in HDL2 (arylesterase activity without eserin but with EDTA) (5)(6) and later in HDL3 (7), but these investigators provided no detailed information on the centrifugation procedure or on the distribution profile of PON1 in HDL subfractions. Other studies led to the postulate that PON1 activity measurement was suitable for quantification of HDL3, but this was confirmed only on the basis of polyethylene glycol precipitation (8)(9).

Despite these studies, opinion on the location of PON1 within the HDL density classes is still equivocal, and it is apparent that the VHDL fraction ({rho} = 1.216–1.256 kg/L) must also be taken into consideration. The objective of this study was therefore to determine those HDL subspecies [using the family concept of Alaupovic et al. (10)] that contain active paraoxonase and to analyze the apolipoprotein composition. An additional objective was to examine the possibility that the distribution of PON1 activity within the HDL subfractions is dependent on PON1 polymorphisms.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
samples
A pool of 245 individuals was available for study, and these were confirmed to be healthy by clinical examination. Serum for ultracentrifugation analysis was obtained from a subgroup of 49 individuals (20% women). The median age of this group was 36 years; median body mass index was 24.5 kg/m2; and median blood pressure was 126/70 mmHg. All individuals were normolipidemic and taking no medications. A total of 39% were smokers with 22% smoking ≥10 pack-years (maximum of 52 pack-years); 29% consumed ≥15 g/day alcohol (maximum, 40 g/day).

EDTA tubes were used for sample collection for the lipid and PCR measurements and serum tubes (both purchased from Sarstedt) for samples used in the ultracentrifugation analysis. All samples were centrifuged immediately (2880g for 10 min), and the components were deep-frozen (–80 °C). After thawing, all measurements were completed within 57 h because of the instability of apolipoprotein E (ApoE) in the presence of the density gradient solution used.

pon1 activity
PON1 activity was determined by use of phenylacetate as substrate (11). In contrast to the original procedure, buffer solution without substrate was used for dilution instead of water. The interassay CV over 2 months was 8.2% (n = 112; mean concentration, 0.6 U/mL based on measurement of a 1:101 dilution). The PON1 activity was not significantly influenced by the phosphotungstic acid (PTA)/MgCl2 precipitation (described in the section on ultracentrifugation).

kinetic nephelometry
ApoA-I and ApoB were quantified on a Beckmann protein array system according to the instructions of the manufacturer and using the reagents supplied by the manufacturer. ApoA-II was determined on the same system with N-antiserum against human ApoA-II from Dade-Behring (polyclonal; prod. no. OQBA) and suitable standards and controls (prod. nos. OUPG and OUPH; Dade-Behring).

elisa
ApoE and ApoJ were measured by noncompetitive sandwich ELISA with polyclonal antibodies from DPC Biermann. As capture antibody for ApoE, we used anti-h-ApoE (prod. no. BP275; rabbit) diluted 1:2001 in 0.2 mol/L carbonate buffer (pH 10), and as detector for ApoE, we used anti-h-ApoE conjugated with biotin (prod. no. 50BG-G1-3a; goat) diluted 1:10001 in Dulbecco phosphate-buffered saline containing 0.5 mL/L Tween 20 and 10 g/L bovine serum albumin (Roth). As capture antibody for ApoJ, we used anti-h-ApoJ (prod. no. R1037P; goat) diluted 1:2001 in 0.2 mol/L carbonate buffer (pH 10), and as detector for ApoJ, we used anti-h-ApoJ (prod. no. R1037P; goat), conjugated with biotin by means of B-TAG (Sigma), diluted 1:2001 in Dulbecco phosphate-buffered saline containing 0.5 mL/L Tween 20 and 10 g/L goat serum albumin (Sigma). The incubation times, with the exception of the binding of the capture antibodies at the plate surface (overnight), were 2 h at room temperature. The detection limit of the ApoE-ApoE ELISA was 60 pmol/L, and that of the ApoJ-ApoJ ELISA was 2 nmol/L. All measurements were carried out in duplicate. The interassay CVs were 5% and 11% for the ApoE-ApoE ELISA and the ApoJ-ApoJ ELISA, respectively (n = 62 for both mean concentrations, 13 nmol/L for the ApoE-ApoE and 81 nmol/L for ApoJ-ApoJ ELISA, respectively).

ultracentrifugation
Before centrifugation, the lipoproteins containing ApoB were precipitated by use of PTA/MgCl2 (12). The correctness of the PTA/MgCl2 precipitation in this study was verified by means of sequential flotation (data not shown). The density of the samples ({rho} = 1.0285 kg/L after precipitation, higher than that of native serum) was increased by the addition of degassed solutions (NaCl/NaBr) of defined density (accuracy, 10–4 kg/L; DMA 55; Anton Paar KG). Each solution contained 1.32 mmol/L CaCl2. Aliquots of PTA supernatant (2.22 mL) and density solution (1.58 mL; {rho} = 1.3425 kg/L) were mixed, giving a density of 1.1600 kg/L. Ultracentrifugation conditions were as follows: centrifuge, Kontron T-1080; rotor, Beckman Ti50.4; tubes, Beckman open-top thick-walled polycarbonate (cat. no. 355645); filling density, 1.1600 kg/L; rotor temperature, 15 °C; relative centrifugal force, 241 336g; time, 22 h; acceleration, maximum; deceleration, maximum down to 500 rpm.

computer-controlled fractionated suction
We developed a new procedure for emptying tubes after ultracentrifugation that provided high reproducibility. This involved placing a cannula (Hamilton needle) centrally at the fluid surface and removing discrete volumes from the contents of the tube by suction through the needle. The tip of the needle was continually lowered so that it remained in contact but not below the lowering fluid surface to minimize turbulence. The contact between needle and fluid was maintained only by forces of surface tension, i.e., by the wetability of the needle tip in the fluid. A new circular meniscus around the needle was formed where the tip of the needle was always at the highest point in the center of this meniscus. Exact control over this step was achieved by use of a programmable pipetting robot (Lissy; Zinsser Analytic; with WinLissy 4.5 software). The conditions for collecting the nine 420-µL fractions were as follows: volume flow, 70 µL/s for aspiration and 150 µL/s for output; needle speed, 23.8 mm/s during liquid detection and 47.7 mm/s during withdrawal; 20 µL of system air and 20 µL transport air; delay before aspiration, 0.2 s. Teflon-coated needles were used, and the system fluid was degassed doubly distilled water. A delay before aspiration was necessary so that the tip of the needle could be brought to the start position before the subsequent lowering process.

genotyping
PON1192 and PON155 genotypes were determined by PCR in an Eppendorf Thermal Cycler followed by restriction enzyme analysis. We added 0.1 µg of DNA to each PCR reaction mixture containing 60 pmol of oligonucleotide primers, 62.5 mM KCl, 12.5 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 µM each deoxynucleotide triphosphate, and 1.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech) in a final volume of 50 µL. Primer sequences and the use of the restriction enzymes have been described by Humbert et al. (3).

The T(–107)C polymorphism was determined by use of the amplification refractory mutation system (ARMS). We used two external primers, PONpromotorA (5'-GACGCAAGGACCGGGATGGCACAAAGTGAGTG-3') and PONpromotorB (5'-TGGGCGCAGACACCGACGGGCTAGGAGGCTCT-3'), and two 5' internal allele-specific primers, PONpromotor –107C (5'-attgTAGCTGCGGACCCGGCGGGGAGGAGC-3') and PONpromotor –107T (5'-attgTAGCTGCGGACCCGGCGGGGAGGAGT-3'), to distinguish between the T and C alleles at position –107. The internal primers included a deliberate mismatch three bases upstream of the 3' end (G->A; underlined) to reduce nonspecific binding of 3'-noncomplementary primers. We also synthesized both internal primers with a 5' overhang (attg) to omit reverse (3'->5') chain elongation. The amplification protocol consisted of the standard reaction mixture described above plus 100 mL/L dimethyl sulfoxide. The external 5' and 3' primers and one internal primer (30 pmol each) were added to each reaction mixture, and 30 cycles were performed at 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 1 min with a final extension at 72 °C for 6 min. PCR fragments were analyzed on 2% agarose gels.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
fractionation
There are two common procedures in use for fractionating the contents of tubes after centrifugation. These are the readily reproducible slicing technique, which provides only two fractions and is time-consuming, and the expulsion method, which depends on the use of heavy fluorohydrocarbons such as FC70. With the latter method, small fractions with acceptable reproducibility can be obtained only with considerable difficulty. In contrast, the procedure we developed enables the rapid (eight tubes in <30 min) isolation of small fractions (down to 250 µL) with high reproducibility (CV {approx}2%). The overall interassay CV (including PTA/MgCl2 precipitation, preparation of single and mixed density solutions, centrifugation, fractionation, and cholesterol determination) was 8.9%.

filling density 1.1377 KG/L
We attempted to obtain HDL2 as a uniform fraction within the spectrum (see Fig. 1 ). The scaling of the ordinate was based on relative values (%) because absolute values for PON1 activity and apolipoprotein concentrations within the density gradient were dependent on the initial quantities present. ApoA-I (Fig. 1 , {square}) and cholesterol (Fig. 1 , {circ}) showed the anticipated distribution patterns with a common lowest point at {rho} = 1.125 kg/L. The distribution of PON1 activity (Fig. 1 , •) differed because an increase was discernible only at densities >1.145 kg/L. The decrease in the last fraction was associated with ApoA-I, and active PON1 was not present in the serum protein fraction (sediment). We measured no significant PON1 activity in the solubilized sediment. The HDL3 fraction contained 25 times more PON1 activity than the HDL2 fraction. The ratio of PON1 activity to ApoA-I (Fig. 1 , {triangleup}), expressed as activity/amount of apolipoprotein (U/nmol), in the less dense fractions was proportional to PON1 activity, but in the last fraction, where both values decreased, the ratio increased.



View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Density distributions of cholesterol and ApoA-I concentration and PON1 activity after centrifugation for 22 h at 240 000g using a filling density of 1.1377 kg/L.

The arrow indicates the operational density between HDL2 and HDL3. The first fraction (mean density, 1.1109 kg/L) contains HDL2 (1.063 < {rho} < 1.1119 kg/L), and the last fraction (mean density, 1.1673 kg/L) contains the more dense HDL3 and the VHDL (1.1610 < {rho} < 1.256 kg/L).

flotation of hdl3 and vhdl
Centrifugations were carried out at high densities ({rho} = 1.216 and 1.256 kg/L) to check whether the distribution patterns obtained were attributable to separation of active PON1 from HDL. For this purpose, HDL2 was removed by ultracentrifugation using the slicing technique (12), and the bottom fraction was centrifuged at a density of either 1.216 or 1.256 kg/L. ApoA-I and cholesterol floated completely at {rho} = 1.216 kg/L, but the percentage of active PON1 was relatively constant (12%) over the density range used. In contrast, flotation experiments with VHDL ({rho} = 1.256 kg/L) showed that this fraction contained all of the PON1 activity as well as all of the ApoA-I and all of the cholesterol (Fig. 2 ). The mean recovery of PON1 after two centrifugations was 87.5% (n = 5).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Density distributions of cholesterol and ApoA-I concentrations and PON1 activity after centrifugation of the HDL3 after removal of the HDL2 at a filling density of 1.256 kg/L (240 000g at 30 h).

Fractions 1–8 correspond to the mean densities 1.23 and 1.28 kg/L, respectively.

filling density 1.16 KG/L
ApoA-I (Fig. 3 , {square}) was distributed evenly across the density range investigated with percentages between 6.5% and 16%. The highest percentage was observed in the density range 1.063–1.1366 kg/L. Because all of the HDL was centrifuged, this corresponds to the first fraction, with a mean density of 1.1335 kg/L, containing HDL2 as well as the less dense HDL3. It was notable that the lowest point (6.5%) in the ApoA-I distribution was at approximately {rho} = 1.1675 kg/L. In contrast, ApoA-II was unevenly distributed (Fig. 3 , {circ}). In fractions with a density >1.158 kg/L, the percentage of ApoA-II was much lower than that of ApoA-I. In the most dense HDL3 fraction ({rho} = 1.1907 kg/L), <5% of ApoA-II was present, whereas the highest percentage (>20%) was observed in the least dense fraction ({rho} = 1.1335 kg/L).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 3. Density distributions of ApoA-I, ApoA-II, and cholesterol concentrations (filling density, {rho} = 1.1600 kg/L; centrifugation for 22 h at 240 000g).

The error bars indicate the interindividual mean variation. The first fraction (mean density, 1.1335 kg/L) contains HDL2 and less dense HDL3 (1.063 < {rho} < 1.1366 kg/L), and the last fraction (mean density, 1.1907 kg/L) contains the more dense HDL3 and the VHDL (1.1854 < {rho} < 1.256 kg/L).

The distribution of the other HDL proteins (ApoE, ApoJ, and PON1) differed markedly from the apolipoproteins described above (Fig. 4 ). All three occurred only in those fractions in which ApoA-II was present in small amounts. The percentage of ApoE (Fig. 4 , {blacksquare}) increased only above a density of 1.1675 kg/L and then reached a plateau. Only traces were detectable in the less dense fractions ({rho} <1.1675 kg/L). More than 99% of the ApoJ in HDL (Fig. 4 , {diamond}) was found in the last two fractions.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Density distributions of ApoE and ApoJ concentrations and PON1 activity (filling density, {rho} = 1.1600 kg/L; centrifugation for 22 h at 240 000g).

The error bars indicate the interindividual mean variation. The density ranges of the first and the last fraction are the same as in Fig. 3Up .

The distribution of PON1 activity (Fig. 4Up , •) increased at {rho} = 1.16 kg/L, i.e., earlier than in the case of ApoE, and the activity reached its maximum at {rho} = 1.18 kg/L. Only 18% of the total PON1 activity was found in the less dense region, i.e., before commencement of the increase in activity. A notable feature of the various distribution patterns was an inverse relationship between PON1 activity and the concentration of ApoJ.

relationship of pon1 activity to genetic factors
The gene frequencies according to Hardy–Weinberg within the group under investigation were as follows: Q192, 72% (n = 245); L55, 66% (n = 158); and T(–107), 44% (n = 239). With regard to the distribution in the density gradient, neither L55M nor the nature of the promoter polymorphism had an effect on the distribution pattern. On the other hand, the proportion of activity in the homozygous R192 carriers (Fig. 5 , {diamondsuit}) was only 43% of that of the wild-type allele (Fig. 5 , {triangleup}) in the fractions with densities of 1.063–1.16 kg/L and was 25% higher than the wild-type allele in the fraction with the highest PON1 activity (P = 0.002, Student t-test).



View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Relationship between the density distributions of PON1 activity and Q192R polymorphism (filling density, {rho} = 1.1600 kg/L; centrifugation for 22 h at 240 000g).

The error bars indicate the interindividual mean variation. The density ranges of the first and the last fraction are the same as in Fig. 3Up .

distribution of the molar apolipoprotein ratios
The percentage of ApoA-IV amounts to only one-tenth of that of the ApoA-II (13) and can be neglected. Lipoprotein (LP) A-II:A-II particles present in the HDL range are relatively rare (14). It was therefore assumed that HDL contains mainly Lp A-I:A-I and Lp A-I:A-II particles. From the progression of the curve for the molar ratio of ApoA-I and ApoA-II (Fig. 6 , {diamondsuit}), conclusions could be drawn both with regard to the particle composition and the number of HDL particles. The ratio of the more complex Lp A-I:A-II particles to the simpler Lp A-I:A-I particles decreased with increasing density. In the range of HDL2 and the less dense HDL3 ({rho} <1.1515 kg/L), the ratio was still 2:1. The equimolar ratio was reached at {rho} = 1.160 kg/L, and in the fraction with the highest PON1 activity approximately three Lp A-I:A-I particles were present for every Lp A-I:A-II particle. In this section of the HDL3, the proportion of Lp A-I:A-I particles had increased by a factor of 6, whereas in the last fraction, that with the most dense HDL3 and VHDL, it returned to 2:3.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 6. Density distributions of the molar ApoA-I/ApoA-II ratio and the PON1 activity per nmol of ApoA-I and ApoE (filling density {rho} = 1.1600 kg/L; centrifugation for 22 h at 240 000g).

The error bars indicate the interindividual mean variation. The density ranges of the first and the last fraction are the same as in Fig. 3Up .

profile of the ratio of pon1 activity to apolipoprotein amount for APOA-I, APOA-II, APOE, and APOJ
PON1 activity reached a maximum at a density of 1.18 kg/L, and this coincided with the maximum value (38%) for the total PON1 activity per unit of ApoA-I (Fig. 6Up ). Compared with the PON1/ApoA-I distribution pattern, that of PON1/ApoA-II showed a narrower peak (data not shown). On the basis of our findings in this study, we could express PON1 activity as units of PON1 activity per HDL particle. In the HDL fraction with the highest PON1 activity, the ratio was 14 units/nmol of HDL particle, and in the last fraction, the activity was 7 units/particle. In the less dense HDL3 fractions ({rho} <1.158 kg/L), the activity per particle was <1 unit. The fraction with the highest PON1 activity thus showed the highest specific activity per particle.

The profile of PON1 activity per nmol of ApoE (Fig. 6Up , {blacktriangleup}) differed markedly from the profiles for PON1 activity and ApoE concentration. The highest point (20%) lay not at the end of the density spectrum, but shortly after the middle (at {rho} = 1.1675 kg/L). Values for PON1 activity per nmol of ApoJ are not shown because only the last two fractions had detectable concentrations of ApoJ. The fraction with the highest PON1 activity overall had a 10-fold higher PON1 activity than the fraction with the highest ApoJ content.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, HDL fractions were separated in the density range {rho} = 1.063–1.256 kg/L. Even when HDL2 was obtained as an isolated fraction, PON1 activity in HDL2 was only 1/25th of that in HDL3 and VHDL. These initial investigations confirmed the results reported by Graham et al. (7). There was, however, a fraction of HDL3 ({rho} = 1.125–1.145 kg/L) that, like HDL2, contained low PON1 activity, indicating that the higher the density of the fraction, the higher the PON1 activity.

The influence of storage conditions on the ratio of the PON1 activity in HDL2 and HDL3 was considerable. Repeated freezing increased the value from 1:25 to 1:200, but the overall activity did not change noticeably, as described previously by Brackley and Carro-Ciampi (15). The change was apparently caused by a decrease in PON1 activity in HDL2 during storage.

Investigations on the floating characteristics of HDL3 and VHDL in solutions of various densities demonstrated that active PON1 floated only when VHDL ({rho} = 1.256 kg/L) was brought to flotation and not with the main portion of ApoA-I of HDL3. In the density range for HDL3, there was no correlation between PON1 activity and the concentration of ApoA-I, although PON1 is generally associated with ApoA-I. The investigations also showed that PON1 activity is not dissociated from the particles. The loss in activity after two centrifugations averaged 12.5% (n = 5). Because the serum protein fraction is free of PON1 activity, we could not confirm the conclusion of Forte et al. (16) showing that ultracentrifugation separates considerable amounts of PON1 activity from HDL, and this difference may be attributable to the higher centrifugation forces. According to Sorenson et al. (17), binding to HDL is not essential for PON1 activity but, it must be kept in mind, that separation from the particles does occur when sufficiently high forces are applied over a prolonged period (e.g., 66 h of centrifugation using SW rotors). In combination with a PTA/MgCl2 precipitation, centrifugation at {rho} = 1.256 kg/L offers practical advantages because it is possible to concentrate PON1 by a factor of 8 and eliminate all plasma proteins except for the HDL proteins. In this case, removal of the fractions can be carried out by hand, using a syringe.

The search for the fraction with the highest PON1 activity involved centrifugation at {rho} = 1.1600 kg/L, where the high percentage of cholesterol and ApoA-I in the first fractions are attributable to compression of the less dense fractions (HDL2), and at this filling density, the fraction with a density of 1.18 kg/L can be isolated. The other HDL apolipoproteins in this fraction were also measured to evaluate the HDL particles. The distribution of the molar ApoA-I/ApoA-II quotients (Fig. 6Up ) resembles that for the distribution of PON1 activity (Fig. 4Up ), and we therefore concluded that PON1-active particles can characterized by their lower ApoA-II content. In the fraction with the highest PON1 activity, not only was the ApoA-II content at its lowest, but the occurrence of Lp A-I:A-II was at a minimum.

A "stripping effect" can lead to lipid-free ApoE, but in our studies all ApoE remained bound to HDL because no ApoE was detectable in the albumin fraction. Both ApoE and ApoJ can be considered as apolipoproteins associated with the most dense HDL3 and VHDL particles. Because of the drop in the curve of PON1 activity per ApoE (Fig. 6Up ) in the density range higher than {rho} = 1.175 kg/L, it can be concluded that the HDL fractions containing active PON1 float on those containing ApoE.

The ApoJ/PON1 (activity determined by use of paraoxon) ratio has been reported to be more suitable for predicting the occurrence of atherosclerosis than the cholesterol/HDL-cholesterol ratio (18). However, not all HDL particles containing paraoxonase activity contained ApoJ. The fractions with the highest PON1 activity contained only one tenth of the total HDL-ApoJ, and the fraction of HDL richest in ApoJ had only ~15% of the total PON1 activity. The ApoJ/PON1 activity ratio is based on the presence of the two proteins in the same particle or in different particles. The basis of this ratio is therefore empirical, but this does not impinge on its diagnostic accuracy.

The Q192 allele showed a wider distribution in the density profile. This was surprising because it had been suspected that only the L55M polymorphism could have an effect on the distribution of PON1 activity (4). The L55M polymorphism, however, had no effect on the distribution of PON1 activity, but it must be pointed out that the view that only the L55M polymorphism can influence stability is based on the observation of Leviev et al. (4), who found a relationship between PON1 activity and the L55M polymorphism. Therefore, the conclusion that this polymorphism has a positive effect on the stability of the enzyme appears incorrect. A change of 25% in the distribution in the density range indicated a different stability for the Q192R polymorphism, and this could imply that the "highly active" allele (R192) can be inhibited in vivo more readily. Moreover, the observation that the distribution of PON1 activity at various densities is not dependent on the promoter polymorphism was to be expected because the polymorphism determines only the amount of enzyme.

In summary:

The following facts could thus be established with regard to HDL in the {rho} = 1.18 kg/L fraction: (a) it contains the highest PON1 activity and highest specific activity per HDL particle; (b) it has the lowest concentration of ApoA-II and lowest number of Lp A-I:A-II particles; and (c) the fraction (already) contained ApoE and ApoJ, but not at the maximum concentrations achievable. In contrast to the other fractions, this density range appeared to contain an agglomeration of widely varying HDL particles, which although all demonstrating the same density, differed in the proteins they carried in addition to differing in the structural proteins. It is presumed that they also carry out different functions.

In conclusion, measurement of PON1 activity is not a simple alternative to determinations based on ultracentrifugation of HDL3, but it is a simple method for isolating and determining the part of HDL that has a primary task in the repair of other lipoproteins.


   Acknowledgments
 
We are very grateful to Dr. Eva Fisher for excellent help in PON1 mutation analysis.


   Footnotes
 
1 Nonstandard abbreviations: PON1, paraoxonase; Apo, apolipoprotein; PTA, phosphotungstic acid; and Lp, lipoprotein.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. La Du BN, Aviram M, Billecke S, Navab M, Primo-Parmo S, Sorenson RC, et al. On the physiological role(s) of the paraoxonases. Chem Biol Interact 1999;119–120:379-388.
  2. Mackness MI, Mackness B, Durrington PN, Connelly PW, Hegele RA. Paraoxonase: biochemistry, genetics and relationship to plasma lipoproteins [Review]. Curr Opin Lipidol 1996;7:69-76.[ISI][Medline] [Order article via Infotrieve]
  3. Humbert R, Adler DA, Disteche CM, Hassett C, Omiecinski CJ, Furlong CE. The molecular basis of the human serum paraoxonase activity polymorphism. Nat Genet 1993;3:73-76.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  4. Leviev I, James RW. Promoter polymorphisms of human paraoxonase PON1 gene and serum paraoxonase activities and concentrations. Arterioscler Thromb Vasc Biol 2000;20:516-521.[Abstract/Free Full Text]
  5. Kitchen BJ, Masters CJ, Winzor DJ. Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase. Biochem J 1973;135:93-99.[Medline] [Order article via Infotrieve]
  6. Don MM, Masters CJ, Winzor DJ. Further evidence for the concept of bovine plasma arylesterase as a lipoprotein. Biochem J 1975;151:625-630.[Medline] [Order article via Infotrieve]
  7. Graham A, Hassall DG, Rafique S, Owen JS. Evidence for a paraoxonase-independent inhibition of low-density lipoprotein oxidation by high-density lipoprotein. Atherosclerosis 1997;135:193-204.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  8. Chemnitius JM, Winkel H, Meyer I, Schirrmacher K, Armstrong VW, Kreuzer H, et al. Age related decrease of high density lipoproteins (HDL) in women after menopause. Quantification of HDL with genetically determined HDL arylesterase in women with healthy coronary vessels and in women with angiographically verified coronary heart disease. MedKlin 1998;93:137-145.
  9. Meyer I, Chemnitius JM, Kreuzer H, Zech R. A new method for quantitation of HDL-subclasses determining HDL2- and HDL3-arylesterase. Eur Heart J 1994;15(Suppl):612.
  10. Alaupovic P, Lee DM, McConathy WJ. Studies on the composition and structure of plasma lipoproteins. Distribution of lipoprotein families in major density classes of normal human plasma lipoproteins. Biochim Biophys Acta 1972;260:689-707.[Medline] [Order article via Infotrieve]
  11. Furlong CE, Richter RJ, Seidel SL, Motulsky AG. Role of genetic polymorphism of human plasma paraoxonase/arylesterase in hydrolysis of the insecticide metabolites chlorpyrifos oxon and paraoxon. Am J Hum Genet 1988;43:230-238.[ISI][Medline] [Order article via Infotrieve]
  12. März W, Gross W. Ultracentrifugal determinations of high-density lipoprotein subfractions HDL2 and HDL3 in a high capacity fixed angle Rotor. Arztl Lab 1988;34:265-270.
  13. Kostner GM, März W. Zusammensetzung und Stoffwechsel der Lipoproteine. Schwandt P Richter WO Parhofer KG eds. Handbuch der Fettstoffwechselstörungen, 2nd ed 2001:1-57 Schattauer Stuttgart. .
  14. März W, Gross W. Immunochemical evidence for the presence in human plasma of lipoproteins with apolipoprotein A-II as the major protein constituent. Biochim Biophys Acta 1988;962:155-158.[Medline] [Order article via Infotrieve]
  15. Brackley M, Carro-Ciampi G. Stability of the paraoxonase phenotyping ratio in collections of human sera with differing storage times. Res Commun Chem Pathol Pharmacol 1983;41:65-78.[Medline] [Order article via Infotrieve]
  16. Forte TM, Oda MN, Knoff L, Frei B, Suh J, Harmony JA, et al. Targeted disruption of the murine lecithin:cholesterol acyltransferase gene is associated with reductions in plasma paraoxonase and platelet- activating factor acetylhydrolase activities but not in apolipoprotein J concentration. J Lipid Res 1999;40:1276-1283.[Abstract/Free Full Text]
  17. Sorenson RC, Aviram M, Bisgaier CL, Billecke S, Hsu C, La Du BN. Properties of the retained N-terminal hydrophobic leader sequence in human serum paraoxonase:arylesterase. Chem Biol Interact 1999;119–120:243-249.
  18. Navab M, Hama-Levy S, Van Lenten BJ, Fonarow GC, Cardinez CJ, Castellani LW, et al. Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio. J Clin Invest 1997;99:2005-2019.[ISI][Medline] [Order article via Infotrieve]



The following articles in journals at HighWire Press have cited this article:


Home page
 J. Lipid Res.Home page
P. A. C. McPherson, I. S. Young, B. McKibben, and J. McEneny
High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation
J. Lipid Res., January 1, 2007; 48(1): 86 - 95.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
E. Thomas-Moya, M. Gianotti, A. M. Proenza, and I. Llado
The age-related paraoxonase 1 response is altered by long-term caloric restriction in male and female rats
J. Lipid Res., September 1, 2006; 47(9): 2042 - 2048.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
A. Kontush and M. J. Chapman
Functionally Defective High-Density Lipoprotein: A New Therapeutic Target at the Crossroads of Dyslipidemia, Inflammation, and Atherosclerosis
Pharmacol. Rev., September 1, 2006; 58(3): 342 - 374.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
C. Y. Han, T. Chiba, J. S. Campbell, N. Fausto, M. Chaisson, G. Orasanu, J. Plutzky, and A. Chait
Reciprocal and Coordinate Regulation of Serum Amyloid A Versus Apolipoprotein A-I and Paraoxonase-1 by Inflammation in Murine Hepatocytes
Arterioscler. Thromb. Vasc. Biol., August 1, 2006; 26(8): 1806 - 1813.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
L. S. Rozek, T. S. Hatsukami, R. J. Richter, J. Ranchalis, K. Nakayama, L. A. McKinstry, D. A. Gortner, E. Boyko, G. D. Schellenberg, C. E. Furlong, et al.
The correlation of paraoxonase (PON1) activity with lipid and lipoprotein levels differs with vascular disease status
J. Lipid Res., September 1, 2005; 46(9): 1888 - 1895.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
A. Chait, C. Y. Han, J. F. Oram, and J. W. Heinecke
Thematic review series: The Immune System and Atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease?
J. Lipid Res., March 1, 2005; 46(3): 389 - 403.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
clinchem.2004.034439v1
50/12/2309    most recent
Right arrow Submit an electronic Letter to
the Editor about this paper
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (12)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bergmeier, C.
Right arrow Articles by Gross, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bergmeier, C.
Right arrow Articles by Gross, W.
Related Collections
Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS