Conceived and designed the experiments: JKP JYK OYK YJ JHL GS. Performed the experiments: JKP JYK YL. Analyzed the data: JKP OYK. Contributed reagents/materials/analysis tools: T-SJ. Wrote the paper: JHL.
The authors have declared that no competing interests exist.
This study aimed to determine the association of lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in circulation and peripheral blood mononuclear cells (PBMCs) with inflammatory and oxidative stress markers in nonobese women and according to menopausal status. Lp-PLA2 activity, a marker for cardiovascular risk is associated with inflammation and oxidative stress.
Eighty postmenopausal women (53.0±4.05 yr) and 96 premenopausal women (39.7±9.25 yr) participated in this study. Lp-PLA2 activities, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β in plasma as well as in PBMCs were measured. Plasma ox-LDL was also measured. Postmenopausal women demonstrated higher circulating levels of ox-LDL and IL-6, as well as IL-6, TNF-α, and IL-1β in PBMCs, than premenopausal women. In both groups, plasma Lp-PLA2 activity positively correlated with Lp-PLA2 activity in PBMCs and plasma ox-LDL. In premenopausal women, Lp-PLA2 activities in plasma and PBMCs positively correlated with IL-6, TNF-α, and IL-1β in PBMCs. In postmenopausal women, plasma ox-LDL positively correlated with PBMC cytokine production. In subgroup analysis of postmenopausal women according to plasma ox-LDL level (median level: 48.715 U/L), a significant increase in Lp-PLA2 activity in the plasma but not the PBMCs was found in the high ox-LDL subgroup. Plasma Lp-PLA2 activity positively correlated with unstimulated PBMC Lp-PLA2 activity in the low ox-LDL subgroup (r = 0.627, P<0.001), whereas in the high ox-LDL circulating Lp-PLA2 activity positively correlated with plasma ox-LDL (r = 0.390, P = 0.014) but not with Lp-PLA2 activity in PBMCs.
The lack of relation between circulating Lp-PLA2 activity and Lp-PLA2 activity in PBMCs was found in postmenopausal women with high ox-LDL. This may indicate other sources of circulating Lp-PLA2 activity except PBMC in postmenopausal women with high ox-LDL. We also demonstrated that circulating Lp-PLA2 and PBMC secreted Lp-PLA2 associate differently with markers of oxidative stress and sub clinical inflammation in nonobese women, particularly according to the menopausal states.
Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as plasma platelet activating factor acetylhydrolase (PAF-AH), is unique among members of the phospholipase A2 superfamily due to its origin, its association with circulating lipoproteins, and its substrate preference for polar phospholipids, including those generated during the oxidation of low-density lipoprotein (LDL)
Many studies found correlations between Lp-PLA2 and triglycerides, LDL-cholesterol, high-density lipoprotein (HDL)-cholesterol, body mass index (BMI), age, sex, use of postmenopausal hormones, and smoking
A total of 176 healthy, nonobese women aged 20–68 years were recruited during routine check-ups at a health promotion center at Yonsei University Hospital. Postmenopausal status (n = 80) was defined as an absence of menstruation for at least 12 months and the presence of estrogen deficiency symptoms, including hot flushes, increased sweating, nervousness, irritability, depression, palpitations, insomnia, headaches, dyspareunia, and joint pains. Premenopausal status (n = 96) was defined as the presence of regular menses. At the time of subject enrollment, subjects were interviewed about smoking status (non-/ex-smoker and current smoker), and frequency of alcohol intake. Alcohol consumption was calculated as the grams of ethanol ingested per day and cigarettes smoking data were reported as the number of cigarettes smoked per day. All participants were clinically healthy and were not taking any medications known to affect the immune system, such as oral contraceptives, lipid-lowering agents, anti-hypertensive drugs, functional foods, or vitamin and/or mineral supplements. The purpose of the study was carefully explained to all participants and their written consent was obtained prior to their participation. The study design was approved by the Institutional Review Board of Yonsei University.
Body weight and height were measured in the morning, lightly clothed without shoes and the BMI was calculated as body weight in kilograms divided by height in meters squared. Waist circumference was measured at the umbilical level with the subjects standing after normal expiration and the hip girth was measured at the widest part of the hip and, the waist and hip ratio (WHR) was calculated.
Blood pressure (BP) was measured in the left arm of seated patients with an automatic blood pressure monitor (TM-2654, A&D, Tokyo, Japan) after a 20-min rest. After a 12-hour fast, venous blood specimens were collected in EDTA-treated or untreated tubes. The blood specimens collected in the EDTA-treated tubes were used for the isolation of PBMCs or separated into plasma and stored at −70°C until further analysis. The blood samples collected in non-treated tubes were separated into serum and stored until further analysis.
Fasting total-cholesterol and triglyceride levels were measured using commercially available kits on a Hitachi 7150 Autoanalyzer (Hitachi Ltd., Tokyo, Japan). After precipitation of serum chylomicrons with dextran sulfate magnesium, the concentrations of LDL- and HDL-cholesterol in the supernatants were enzymatically measured. Fasting glucose levels were measured using a glucose oxidase method with a Beckman Glucose Analyzer (Beckman Instruments, Irvine, CA, USA). Free fatty acids were analyzed with a Hitachi 7150 autoanalyzer (Hitachi Ltd, Tokyo, Japan).White blood cell (WBC) count was determined using the HORIBA ABX diagnostic (HORIBA ABX SAS, Parc Euromedicine, France).
Whole blood was mixed with the same volume of RPMI 1640 (Gibco, Invitrogen, Carlsbad, CA, USA) and gently laid on a histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). The sample was then centrifuged at 2000 rpm for 20 min at 10°C. After the separation, a thin layer of PBMCs was isolated and washed twice with RPMI 1640. The pellet was resuspended in RPMI 1640 with streptomycin. Isolated PBMCs were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, seeded in 12-well plates (1×106 cells/mL; Nunc, Roskilde, Denmark), and incubated at 37°C with 5% CO2 for 24 hours. After a 24-hour incubation, supernatants were collected and stored at −80°C until interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and Lp-PLA2 activity levels were assayed
Levels of IL-1β, IL-6, and TNF-α in serum and PBMC supernatants were measured using the Bio-Plex™ Reagent Kit on the Bio-Plex™ (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions.
Lp-PLA2 activity in plasma and PBMC supernatants was measured by using a modification of a previously described high-throughput radiometric activity assay
Plasma ox-LDL was measured using an enzyme immunoassay (Mercodia, Uppsala, Sweden). The resulting color reaction was read at 450 nm with a Wallac Victor2 multilabel counter (Perkin Elmer Life Sciences, Turku, Finland). Serum hs-c-reactive protein (CRP) levels were measured with an Express Plus™ auto-analyzer (Chiron Diagnostics Co., Walpole, MA, USA) using a commercially available, high-sensitivity CRP-Latex(II) ×2 kit (Seiken Laboratories Ltd., Tokyo, Japan).
Urine was collected in polyethylene bottles containing 1% butylated hydroxytoluene after a 12-hour fast. The bottles were immediately covered with aluminum foil and stored at −70°C until further analysis. The compound 8-epi-PGF2α was measured using an enzyme immunoassay (BIOXYTECH urinary 8-epi-PGF2α™ Assay kit, OXIS International Inc., Portland, OR, USA). Urinary creatinine levels were determined using the alkaline picrate (Jaffe) reaction. Urinary 8-epi-PGF2α levels are expressed as pmol/mmol creatinine.
Serum levels of FSH and 17ß-estradiol were measured using commercially-available kits (ADIVIA Centaur FSH and ADIVIA Centaur Estradiol, respectively, Siemens, USA) on the ADIVIA Centaur (ADIVIA Centur, Siemens).
Statistical analyses were performed using SPSS version 12.0 for Windows (SPSS Inc., Chicago, IL, USA). The independent t-test was used to compare parameters between the two groups. One-way analysis of variance (ANOVA) with Bonferroni correction was used to test whether there were effects from menopausal state and plasma ox-LDL levels (below or above the median level) in postmenopausal women. General linear model (GLM) analysis was also performed with adjustment for age or BMI and alcohol consumption. Frequency was tested with the chi-square test. Pearson and partial correlation coefficients were used to examine relationships between variables. For descriptive purposes, mean values are presented using untransformed values.
In this study, postmenopausal women had a significantly higher BMI and included a lower percentage of alcohol drinkers (
Premenopausal women (n = 96) | Postmenopausal women (n = 80) | P0 | P1 | |||||
Age (yr) | 39.7 | ± | 9.25 | 53.0 | ± | 4.05 | <0.001 | - |
Years since menopause | - | 3.49 | ± | 3.87 | - | - | ||
Body Mass Index (kg/m2) | 21.9 | ± | 2.84 | 22.8 | ± | 2.27 | 0.024 | - |
Cigarette smoker, n (%) | 1 (1.0) | 2 (2.5) | 0.592 | - | ||||
Alcohol drinker, n (%) | 62 (64.6) | 37 (46.3) | 0.022 | - | ||||
Waist hip ratio | 0.84 | ± | 0.05 | 0.88 | ± | 0.05 | <0.001 | 0.042 |
Systolic BP (mmHg) | 109.3 | ± | 14.2 | 118.6 | ± | 12.0 | <0.001 | 0.614 |
Diastolic BP (mmHg) | 74.2 | ± | 10.6 | 76.0 | ± | 8.75 | 0.238 | 0.592 |
Triglyceride (mg/dL) |
90.7 | ± | 41.6 | 98.2 | ± | 45.4 | 0.489 | 0.340 |
Total-cholesterol (mg/dL) | 182.6 | ± | 26.4 | 210.3 | ± | 32.7 | <0.001 | 0.004 |
LDL-cholesterol (mg/dL) | 109.1 | ± | 23.4 | 132.5 | ± | 28.6 | <0.001 | 0.006 |
HDL-cholesterol (mg/dL) | 55.4 | ± | 12.8 | 58.1 | ± | 14.1 | 0.185 | 0.145 |
Glucose (mg/dL) |
85.5 | ± | 7.98 | 92.1 | ± | 12.3 | <0.001 | 0.016 |
Free fatty acid (uEq/L) |
407.1 | ± | 191.8 | 409.5 | ± | 158.2 | 0.488 | 0.548 |
hs-CRP (mg/dL) |
0.51 | ± | 0.75 | 0.36 | ± | 0.92 | <0.001 | <0.001 |
Serum FSH (IU/L) | 10.6 | ± | 17.9 | 75.2 | ± | 28.9 | <0.001 | <0.001 |
Serum 17ß-estradiol (pg/mL) |
134.6 | ± | 126.9 | 18.2 | ± | 24.8 | <0.001 | <0.001 |
White blood cells (×109/L) |
4.94 | ± | 1.01 | 4.98 | ± | 1.15 | 0.785 | 0.948 |
Means ± SD. Tested by independent t-test or general linear model with the adjustment.
tested by log-transformed P0: unadjusted, P1: adjusted for age, BMI, and alcohol consumption.
Premenopausal women (n = 96) | Postmenopausal women (n = 80) | P0 | P1 | |||||
Plasma oxidized LDL(U/L) |
40.9 | ± | 11.4 | 56.7 | ± | 23.8 | <0.001 | 0.031 |
Urinary 8-epi-PGF2α (pg/mg creatinine) |
1079.1 | ± | 281.6 | 1199.8 | ± | 461.5 | 0.179 | 0.400 |
Serum IL-6 (pg/mL) |
1.94 | ± | 1.28 | 4.53 | ± | 5.27 | <0.001 | 0.005 |
Serum TNF-α (pg/mL) |
5.88 | ± | 7.03 | 6.64 | ± | 10.6 | 0.795 | 0.213 |
Serum IL-1ß (pg/mL) |
0.70 | ± | 1.17 | 0.77 | ± | 1.53 | 0.929 | 0.961 |
Lp-PLA2 activity (nmol/mL/min) | 28.3 | ± | 9.42 | 30.6 | ± | 7.77 | 0.084 | 0.447 |
Nonstimulated PBMC | ||||||||
IL-6 (pg/mL) |
786.1 | ± | 3107.9 | 1409.0 | ± | 6504.8 | <0.001 | 0.027 |
TNF-α (pg/mL) |
169.3 | ± | 803.4 | 626.9 | ± | 2044.3 | <0.001 | 0.008 |
IL-1ß (pg/mL) |
14.4 | ± | 42.4 | 119.7 | ± | 351.2 | <0.001 | <0.001 |
Lp-PLA2 activity (nmol/mL/min) | 2.01 | ± | 0.55 | 2.11 | ± | 0.61 | 0.265 | 0.621 |
Mean ± SD. Tested by independent t-test or general linear model with the adjustment.
tested by log-transformed. P0: unadjusted, P1: adjusted for age, BMI and alcohol consumption.
In both premenopausal and postmenopausal women, plasma Lp-PLA2 activity positively correlated with plasma ox-LDL and supernatant Lp-PLA2 activity from nonstimulated PBMC cultures (
Premenopausal women (n = 96) | Postmenopausal women (n = 80) | |||||||||||
Plasmaox-LDL | Plasma Lp-PLA2 activity | Nonstimulated PBMC Lp-PLA2 | Plasma ox-LDL | Plasma Lp-PLA2 activity | Nonstimulated PBMC Lp-PLA2 | |||||||
r0 | r1 | r0 | r1 | r0 | r1 | r0 | r1 | r0 | r1 | r0 | r1 | |
Plasma ox- LDL(U/L) |
- | - | 0.244 |
0.247 |
0.022 | 0.006 | - | - | 0.390 |
0.394 |
0.215 | 0.154 |
Serum IL-6 (pg/mL) |
0.042 | 0.051 | −0.150 | −0.154 | −0.020 | −0.032 | 0.171 | 0.143 | 0.022 | 0.016 | 0.019 | −0.017 |
Serum TNF-α (pg/mL) |
−0.037 | −0.016 | −0.178 | −0.202 | 0.101 | 0.068 | 0.090 | 0.031 | −0.022 | −0.018 | 0.030 | −0.008 |
Serum IL-1ß (pg/mL) |
−0.018 | −0.020 | 0.042 | 0.033 | 0.073 | 0.042 | −0.061 | −0.062 | −0.050 | −0.046 | −0.022 | −0.048 |
Lp-PLA2 activity (nmol/mL/min) | 0.244 |
0.247 |
- | - | 0.427 |
0.422 |
0.390 |
0.394 |
- | - | 0.380 |
0.374 |
Nonstimulated PBMCs | ||||||||||||
IL-6 (pg/mL) |
0.058 | 0.042 | 0.326 |
0.318 |
0.363 |
0.353 |
0.230 |
0.222 | 0.051 | 0.073 | 0.127 | 0.121 |
TNF-α (pg/mL) |
0.106 | 0.072 | 0.209 |
0.202 | 0.205 |
0.198 | 0.308 |
0.307 |
0.171 | 0.208 | 0.032 | 0.022 |
IL-1ß (pg/mL) |
0.056 | 0.035 | 0.277 |
0.269 |
0.272 |
0.255 |
0.304 |
0.301 |
0.220 | 0.251 |
0.080 | 0.067 |
Lp-PLA2 activity (nmol/mL/min) | 0.022 | 0.006 | 0.427 |
0.422 |
- | - | 0.215 | 0.154 | 0.380 |
0.374 |
- | - |
*P<0.05,
**P<0.01,
***P<0.001,
tested by log-transformed.
Since Lp-PLA2 is known to hydrolyze the sn2 ester bond of oxidized phospholipids including ox-LDL
§tested by log-transformed. Tested by Pearson correlation (
Data are means ± SD. §tested by log-transformed. P0: unadjusted, tested by one-way ANOVA with Bonferroni method P1: adjusted for BMI and alcohol consumption, tested by general linear model (GLM) analysis. P2: adjusted for age, BMI, and alcohol consumption, tested by GLM analysis.
The major finding of this study is the lack of relation between circulating Lp-PLA2 activity and Lp-PLA2 activity in PBMCs in postmenopausal women with high ox-LDL (≥48.715 U/L, above median). A significant increase in Lp-PLA2 activity in the plasma but not the PBMCs of postmenopausal women with high ox-LDL may indicate other sources of Lp-PLA2 production except PBMC. The extent of the increase in plasma Lp-PLA2 may depend not only on the levels of lipoproteins carrying Lp-PLA2 in circulation but also on the cellular synthesis of this enzyme
Lp-PLA2 is thought to play an atherogenic role by hydrolyzing oxidized phospholipids in ox-LDL, resulting in the generation of two bioactive lipid mediators, lysophosphatidyl choline, and oxidized free fatty acids
Ox-LDL stimulates Lp-PLA2 expression in monocytes through the pathway of phosphatidylinositol 3-kinase and p38 mitogen-activating protein kinase
In addition, the prospective observation needs to be performed in the future to investigate if Lp-PLA2 is a physiological responder or an inducer of vascular inflammation. Actually, the Lp-PLA2 activity may be different according to the ethnicities, for example, rare homozygous mutation of Lp-PLA2 V279F polymorphism, the F/F genotype indicating the loss of function of Lp-PLA2 activity and the less atherogenic properties is found in Korean but not in Western people