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Harangi et al. HDL structure and function in dyslipidemia

endocrine or active liver disease including type 1 per minute.
and 2 diabetes mellitus, chronic renal disease and
malignancy. To calculate PON1 phenotype the dual substrate
method was used [17] . The genetic polymorphism at
Furthermore, thirty-two individuals were enrolled as a codon 192 Q→R (Arg/Gln at position 192) has the
control population confirmed to be healthy by clinical most significant impact on the enzyme activity as
and laboratory examinations. hydrolysis of paraoxon is faster by the R allele than
by the Q allele. The allozyme determined by the R
Biochemical assays allele was designated type B, while the allozyme
After an overnight fasting period venous blood identified by the Q allele was nominated type A. In
samples were taken into evacuated tubes and sera contrast, both R and Q alleles had similar arylesterase
were prepared immediately by centrifugation at activity. The ratio of the hydrolysis of paraoxon
1,500 × g for 10 min at 4 °C. Multiple aliquots of in the presence of 1 mol/L NaCl (salt-stimulated
each samples were separated and stored at -70 °C. paraoxonase) to the hydrolysis of phenylacetate was
Routine laboratory analyses: total cholesterol, used to assign individuals to one of the three possible
HDL-C, LDL-cholesterol (LDL-C), C-reactive protein PON1 phenotypes: AA (low activity), AB (intermediate
(CRP), triglyceride, ApoA1, apolipoprotein B (ApoB), activity) and BB (high activity). Cut-off values between
lipoprotein(a), hemoglobin A1c (HbA1c), and super- phenotypes were as follows: ratio below 3.0 for AA,
sensitive thyroid stimulating hormone were performed ratio between 3.0 and 7.0 for AB and ratio over 7.0 for
from fresh sera with Cobas c501 autoanalyzer (Roche BB phenotype.
Ltd., Mannheim, Germany) in the Department of
Laboratory Medicine of University of Debrecen. Tests Serum myeloperoxidase concentration
were performed according to the recommendation of measurement
the manufacturer. All the reagents were purchased Myeloperoxidase serum concentrations were
from the same vendor. measured by commercially available sandwich
enzyme-linked immunosorbent assay (ELISA) kits
Paraoxonase-1 activities and phenotype (R&D Systems Europe Ltd., Abington, England).
PON1 paraoxonase activity was measured by a The ELISA assay was performed according to the
kinetic, semi-automated method. Briefly, paraoxon manufacturer’s instructions. The intra- and inter-assay
(O,O-diethyl-O-p-nitrophenyl-phosphate, Sigma, coefficient of variations was 6.5-9.4%.
Hungary) was used as a substrate, and the generation
of 4-nitrophenol was measured on a microtiter plate HDL subfraction analysis
(Greiner Bio-One GmbH, Germany). The total of HDL subfractions were determined using an
15 µL serum was mixed with 285 µL Tris-HCl buffer electrophoretic method on polyacrylamide gel with
(100 mmol/L, pH = 8.0) containing 2 mmol/L CaCl 2 and the Lipoprint System (Quantimetrix Corp., CA,
5.5 mmol/L paraoxon. The absorbance was monitored USA) according to manufacturer’s instructions.
at 405 nm (25 °C), in every minute for 6 min by a This commercially available system separates
Beckman Coulter DTX880 Plate Reader (Beckman HDL subfractions from human serum on the basis
Coulter, California, USA) equipped with multimode of their size applying preloaded gel tubes for HDL
detector. Enzyme activity was calculated using the determinations.
-1
-1
molar extinction coefficient 17,600 M cm . PON1
paraoxonase activity is expressed as units per liter Concisely, 25 μL serum was added to the
of serum, where 1 unit equals 1 µmol of substrate polyacrylamide gel tubes along with 300 μL loading
hydrolyzed per minute. gel solution. The tubes contained Sudan Black as
a lipophilic dye and were photopolimerized at room
PON1 arylesterase activity was assayed by a standard temperature for 30 min. Electrophoresis with tubes
containing 1 mmol/L phenylacetate substrate (Sigma, containing sera samples and the manufacturer’s quality
Hungary) in 20 mmol/L Tris-HCl, pH = 8.0. The controls were performed at a constant of 3 mA/tube
reaction was started by adding the serum and the for 50 min. Subfraction bands were scanned with an
absorbance was monitored at 270 nm. Blanks were ArtixScan M1 digital scanner (Microtek International
included to correct for the spontaneous hydrolysis Inc., CA, USA) and were identified by their mobility (Rf)
of phenylacetate. We calculated the enzyme activity using very-LDL (VLDL) + LDL as the starting (Rf 0.0)
using the molar extinction coefficient 1,310 M cm . and albumin as the ending (Rf 1.0) reference point.
-1
-1
PON1 arylesterase activity is expressed in U/mL;
1 U is defined as 1 μmol phenylacetate hydrolyzed Ten HDL subfractions were differentiated between
168 Vessel Plus ¦ Volume 1 ¦ December 28, 2017