Systematic review on the association between F 2 -isoprostanes and cardiovascular disease

Abstract

Background

Oxidative stress may play an aetiological role in the development and progression of cardiovascular disease (CVD). However, evidence on its biochemical markers has been controversial. This article aimed to assess the role of F₂-isoprostanes, a marker for measuring in vivo lipid oxidation, as a biomarker for CVD, including coronary artery disease, stroke and peripheral artery disease.

Methods

A literature search was performed using PubMed and EMBASE (from 1966 to February 2012). Studies that investigated the association between F₂-isoprostanes and CVD were eligible.

Results

Of the 22 eligible studies retrieved, 20 studies showed a significant association between F₂-isoprostanes and CVD. However, to date, there have been only four population-based studies, with one study reporting null association. Although data from prospective studies are ideal to examine a role of such biomarkers in predicting future CVD events, only two studies were prospective. In addition, differences in population characteristics, sample handling/storage and assays, coupled with a lack of confounding adjustment, may all contribute to the enormous variation in previous studies.

Conclusions

High levels of F₂-isoprostanes in urine or blood may be a non-specific indicator of CVD. However, further population-based studies are needed. In addition, multivariable analyses are required for future studies to control confounding and improve classification accuracy.

Introduction

First discovered in the early 1990s,^1,2 F₂-isoprostanes are a family of prostaglandin (PG)-like bioactive compounds that are formed in vivo independently of cyclooxygenases, principally via the free radical-mediated peroxidation of arachidonic acid. The radical attack occurs at carbon 7, 10 or 13 of arachidonic acid, resulting in four possible subfamilies of F₂-isoprostanes (series 5, 8, 12 and 15). F₂-isoprostanes are referred to as such because they contain F-type prostane rings, and are isomeric to PGF_2α.

Since its discovery, levels of F₂-isoprostanes have been recommended as a reliable biomarker for measuring in vivo lipid oxidation and oxidative stress.^3–5 As F₂-isoprostanes have been implicated in the pathogenesis of a number of diseases,⁶ their potential role in atherosclerosis and cardiovascular disease (CVD) has also been investigated.^6–8 Although clinical trials with supplemental antioxidants have generally failed to show benefits on CVD,^9–13 these studies do not necessarily exclude a role of oxidative stress in the pathogenesis of atherosclerosis. Little was known about the influence of these antioxidants on oxidative stress in humans and few trials have provided in vivo measurement of oxidative stress.¹⁴ Moreover, studies suggested F₂-isoprostanes might exert cellular effects, such as blood vessel contraction and smooth muscle proliferation in the vessels.¹⁵

Accumulating evidence has suggested an association between F₂-isoprostanes levels and CVD. This article provides a systematic review of the current evidence on the association between F₂-isoprostanes and CVD, including coronary artery disease (CAD), stroke and peripheral artery disease (PAD).

Methods

Study selection

A comprehensive literature search was performed using PubMed and EMBASE (1966 to February 2012). Keywords for searching included F₂-isoprostanes, F₂-IsoPs, F2IPs, 8-IP, 8-isoprostane, 8-epiPGF_2α, 15-F_2t-isoprostane, atherosclerosis, CVD, coronary heart disease, CAD, myocardial infarction, stroke and peripheral vessel disease. The search was restricted to studies published in English-language journals and conducted in human. References from recent review articles were also checked for relevant articles. The author determined the eligibility of studies by reading the title, abstract or full-text. As we did not attempt to evaluate the role of F₂-isoprostanes in risk classification or prognosis in this paper, only studies that investigated the association between F₂-isoprostanes and CVD were eligible. Studies were excluded if they met one of the following criteria: (1) an intervention agent/programme was involved; (2) the reference group was comprised of any other form of CVD; and (3) CVD occurred as a secondary disease. Figure 1 depicts the process for paper selection.

Figure 1

Flowchart of study selection process

Data extraction

Data on first author, publication year, study design, control populations, number of cases and controls, specimen, laboratory methods, biomarker and its measuring unit, main results, and confounding control were extracted and tabulated in a standard format. F₂-isoprostanes were presented as mean ± standard error in some studies. We converted the data to mean ± standard deviation using the following formula: standard deviation = standard error × √n. We summarized the evidence in relationships with the combined outcome of CVD, and with CAD, stroke and PAD, respectively.

Results

A total of 22 eligible studies were retrieved, including three on the combined outcome of CVD,^16–18 twelve on CAD,^19–30 four on stroke^31–34 and three on PAD.^35–37 The majority of papers were published between 2004 and 2008. All but two studies ^18,30 were retrospective in design. Four studies were based in a population, ^16,18,22,30 and the others were hospital-based. Only two studies did not find a significant association between F₂-isoprostanes and CVD.^24,30 The publication years and the significance level for the statistical tests of the association between F₂-isoprostanes and CVD (P values <0.01, <0.05 or >0.05) are shown in Figure 2.

Figure 2

The publication years of the included 22 studies and the P values of statistical tests for the association between F₂-isoprostanes and cardiovascular disease

Cardiovascular disease

All three included studies reported a statistically significant association between F₂-isoprostanes and the combined outcome of CVD or mortality.^16–18 A cross-sectional study with 2828 participants showed that the creatinine-indexed levels of urinary 8-epi-PGF_2α were significantly higher among those with a history of CVD (coronary heart disease, cerebrovascular disease, intermittent claudication or congestive heart failure) compared with those without.¹⁶ The results did not change meaningfully after adjustment with multiple covariates (linear regression β = 0.10, P = 0.002). Another recent study compared serum 8-iso-PGF_2α (a major F₂-isoprostanes isomer) levels between 54 patients with hypertrophic cardiomyopathy and 54 age- and sex-matched controls without CVD.¹⁷ The crude analysis without adjusting other covariates showed that the mean levels of 8-iso-PGF_2α were significantly higher in patients as compared with controls (35.4 ±10.2 versus 29.9 ± 9.9 pg/mL, P<0.001).

A nested case-cohort study showed a predictive role of urine 8-iso-PGF_2α in CVD (CHD or stroke) mortality in postmenopausal women.¹⁸ The concentration of 8-iso-PGF_2α was determined by liquid chromatographytandem mass spectrometry (LC-MS/MS) in overnight urine samples. After adjustment for systolic blood pressure, history of CVD, diabetes, smoking and body mass index (BMI), the highest quartile of urinary 8-iso-PGF_2α levels was associated with a higher risk of CVD mortality (odds ratio [OR] = 1.8, 95% confidence interval [CI]: 1.0-3.1, P = 0.05) compared with the lowest quartile.

Coronary artery disease

Of the 12 studies on CAD, only two studies did not find a statistically significant association between F₂-isoprostanes and CAD. The characteristics of design and main results for the 12 studies are shown in Table 1.

Table 1

Characteristics of the included studies investigating association between F₂-isoprostanes and coronary artery disease

First author, year	Study design	Controls	No. case/ control	Specimen	Laboratory methods	Biomarker, unit	Main results*	Confounding control
Woodward, 2009³⁰	Nested case-control	Healthy participants	227/420	Plasma	GC-MS	F₂-isoprostanes, pmol/L	Median 1146 versus 1250, P > 0.05; OR =1.08 (0.91–1.29) per 1SD	Age and gender
Basarici, 2008¹⁹	Case-control	Non-CAD patients	60/40	Urine	Enzyme immunoassay	8-isoprostane, pg/mL	68.75 (42.6) versus 38.27 (23.4), P < 0.001; OR = 7.19 (1.72–30.1), P = 0.007	Age, gender, hypertension, diabetes, hyperlipidaemia, smoking, obesity and hs-CRP
Noto, 2007²³	Case-control	Non-CAD patients	120/120	Plasma	ELISA	8-isoprostane, ng/L	Median 129, 152 and 184 for 1, 2 and 3 vessels with critical stenosis versus 88 in controls, P =0.0001; AUC = 0.873	-
Shishehbor, 2006²⁶	Case-control	Non-CAD patients	50/54	Plasma	LC-MS	F₂-isoprostanes, umol/mol arachidonate	9.4 (4.6) versus 6.2 (3.4), P < 0.001; OR = 9.7 (2.6–36.9) OR = 4.4 (1.3–15.1) OR = 2.7 (0.79–8.9) fourth, third, second versus first quartile	Framingham Risk Score and CRP
Ruef, 2006²⁴	Case-control	Non-CAD patients	108/46	Plasma	Enzyme immunoassay	8-isoprostane, unit not available	No difference (data not shown)	-
Wang, 2006²⁸	Case-control	Non-CAD	169/72	Plasma	Enzyme immunoassay	8-iso-PGF_2α, pg/mL	337.7 (80.2) versus 263.8 (74.2), P < 0.001; OR = 2.47 (1.44–4.26), P = 0.001, every 100 pg/mL increase	Age, sex, hypertension, diabetes, hyperlipidaemia, smoking, HDL-C and CRP
Wolfram, 2005²⁹	Case-control	Healthy volunteers	20/20	Urine Plasma Serum	Enzyme immunoassay	8-epi-PGF_2α, Pg/mg creatinine	297 (66) versus 236 (44), 25 (6) versus 20 (4), 262 (47) versus 217 (33), P < 0.05	Smoking
Gross, 2005²²	Cross-sectional	CAC-negative	NA	Plasma	GC-MS	F₂-isoprostanes, pmol/L	OR= 1.18 (1.02, 1.38) every 92.2 pmol/L increase	Sex, clinical site, age and race, BMI, smoking, serum lipids, CRP, antioxidant supplementation use, diabetes and blood pressure
Schwedhelm, 2004²⁵	Case-control	Participants without CHD	93/93	Urine	GC-MS	8-iso-PGF_2α, pmol/mmol	OR = 9.2 (2.8–30.0), P = 0.001; OR = 19.3 (5.4–68.8), P < 0.001	Diabetes, hypercholesterolaemia, hypertension and smoking
Vassalle, 2003²⁷	Case-control	Healthy control	27/13	Plasma	Enzyme immunoassay	8-epiPGF_2α, pg/mL	389.6(188.1) versus 123.2 (34.3), P < 0.001	-
Cipollone, 2000²⁰	Case-control	Healthy volunteers	20/40	Urine	Radioimmunoassay	8-iso-PGF_2α, Pg/mg creatinine	339 (122) versus 236 (83) versus 192 (71), P < 0.01	-
Delanty, 1997²¹	Case-control	Healthy volunteers	12/20	Urine	GC-MS	8-epiPGF_2α, pmol/mmol	265.8(141.3) versus 91.5 (52.8), P < 0.001	-

CHD, coronary heart disease; CAD, coronary artery disease; hs-CRP, high-sensitivity C-reactive protein; BMI, body mass index; CAC, coronary artery calcification; OR, odds ratio; SD, standard deviation; NA, not applicable; GC, gas chromatography; MS, mass spectrometry; ELISA, enzyme-linked immunosorbent assay, LC, liquid chromatography

The data are presented as mean (SD) or OR and 95% confidence interval

One early study of small sample size found that urinary levels of 8-epiPGF_2α were significantly higher in patients following acute myocardial infarction compared with healthy volunteers.²¹ Four studies found that plasma levels of 8-epi-PGF_2α were significantly higher in CAD patients compared with healthy volunteers. ^20,27,29 Note that none of these studies applied multivariable analysis to control confounding (Table 1).

Two studies applied multivariable analysis but showed inconsistent results.^25,30 One case-control study showed a significant association between urinary 8-iso-PGF_2α and coronary heart disease (OR = 19.3 for ≥131 mol/mmol versus ≤79 mol/mmol; OR = 9.2 for 60-130 versus ≤79 mol/mmol) after adjusting for multiple covariates.²⁵ In contrast, a larger case-control study nested in a cohort of a general population did not find a significant association between plasma F₂-isoprostanes and incident coronary heart disease (OR = 1.04, 95% CI: 0.93-1.15 for one interquartile increase in F₂-isoprostanes).³⁰

With the use of coronary angiography in clinical practice, the diagnosis of CAD is made based on the presence of angiographic stenosis at coronary artery. Four studies compared F₂-isoprostanes levels between consecutive patients with an angiographic diagnosis of CAD and those without evidence of significant stenosis during angiography, and showed significantly higher levels of F₂-isoprostanes in CAD patients compared with non-CAD patients.^19,23,26,28 In contrast, one study reported that there was no significant difference in plasma 8-isoprostane between CAD and non-CAD patients in a population of 154 consecutive patients undergoing angiography (data not shown in the original report).²⁴

Subclinical CVD that often precedes the manifestation of clinical CVD is typically associated with traditional coronary risk factors and strongly predicts future clinical CVD events. One study showed that plasma F₂-isoprostanes was associated with a higher risk of coronary artery calcification (OR = 1.18, 95% CI: 1.02-1.38, per 92.2 pmol/L increment).²²

Stroke

The characteristics of four included case-control studies are shown in Table 2. F₂-isoprostanes were all measured in a short time interval from the cerebrovascular events. Three studies reported higher levels of plasma F₂-isoprostanes in ischaemic stroke patients as compared with healthy participants.^31,33,34 In contrast, one study reported that patients with aneurysmal subarachnoid hemorrhage had higher levels of free form of F₂-isoprostanes after surgery in both plasma and cerebrospinal fluid as compared with controls.³² Note that only one of the four included studies performed multivariable analysis to control confounding.³⁴

Table 2

Characteristics of the included case-control studies investigating association between F₂-isoprostanes and stroke

First author, year	Cases	Controls	No. case/ control	Specimen	Laboratory methods	Biomarker, unit	Main results*	Controlled variables
Ward, 2011³⁴	Ischemic stroke	Healthy controls	44/44	Plasma	GC-MS	F₂-isoprostanes, pmol/l	3754 (538) vs. 1947(538), P < 0.02	Age, gender
Kelly, 2008³¹	Ischemic stroke	Controls without prior stroke	52/27	Plasma	GC-MS	F₂-isoprostanes, pg/mL	medians 0.041 vs. 0.0295, P = 0.012; AUC = 0.68 (0.55–0.8)	-
Lin, 2006³²	Hemorrhage stroke	Controls without stroke	15/11	Plasma Cerebrospinal fluid	GC-MS/NICI	F₂-isoprostanes, pg/ml	17.6(3.4) vs. 12.0(1.2) in plasma; 27.2 (5.7) vs. 8.7(1.2) in cerebrospinal fluid; P < 0.01	-
Sanchez-Moreno, 2004³³	Ischemic stroke	Controls	15/24	Plasma	Enzyme immunoassay	8-epiPGF_2α, pg/ml	198 (54.2) vs. 166(19.6), P = 0.02	-

GC, gas chromatography; MS, mass spectrometry; NICI, negative ion chemical ionisation

The data are presented as mean (SD), OR and 95% confidence interval, or area under receiver operating curve (AUC)

Peripheral artery disease

Peripheral arterial disease is an important manifestation of systemic atherosclerosis and thus shares some pathophysiological mechanisms including oxidative stress with CAD. However, epidemiological evidence linking F₂-isoprostanes to PAD has been limited (Table 3). All of the three included studies showed higher levels of urinary 8-iso-PGF_2α in patients with chronic lower limb ischaemia compared with healthy controls.^35–37 Only one study performed multi-variable adjustment. After adjustment with age, gender, diabetes, hypertension, BMI, creatinine, LDL, triglyceride, high-sensitivity C-reactive protein and homocysteine, an increment of every 10 pg/mL in serum 8-iso-PGF_2α was associated with an increment of 11% in the risk of lower limb ischaemia (OR = 1.11, 95% CI: 1.03-1.20).³⁶

Table 3

Characteristics of the included studies investigating association between F₂-isoprostanes and peripheral artery disease

First author, year	Study design	Controls	No. case/ control	Specimen	Laboratory methods	Biomarker, unit	Main results*	Controlled variables
Kals, 2008³⁵	Case-control	Healthy participants without PAD	39/34	Urine	Enzyme immunoassay	8-iso-PGF_2α, ng/mg creatinine	9.52 (5.6) versus 4.28 (2.51), P < 0.001	-
Mueller, 2004³⁶	Case-control	Participants without PAD	100/100	Serum	Enzyme immunoassay	8-iso-PGF_2α, pg/mL	Median 63 versus 42, P = 0.001; OR = 1.11 (1.03–1.20), for an increment of 10 pg/ml	Matching on age, gender, diabetes, adjusting with hypertension, BMI, creatinine, LDL, triglyceride, hs-CRP and homocysteine
Rossi, 2004³⁷	Case-control	Healthy volunteers	10/10	Urine	Radioimmunoassay	8-iso-PGF_2α, pg/umol creatinine	48.8 (8.9) versus 18.0(11.0), P < 0.001	-

PAD, peripheral artery disease; hs-CRP, high-sensitivity C-reactive protein; BMI, body mass index; OR, odds ratio

The data are presented as mean (SD) or OR and 95% confidence interval

Discussion

Accumulated evidence to date has indicated a positive association between F₂-isoprostanes and CVD, with the exception of only two out of the total 22 studies retrieved. Elevated F₂-isoprostanes may be indicative of CVD. However, these studies are heterogeneous in design, sample handling, assay, statistical analysis and data presentation. In particular, some of the following methodological problems need to be carefully addressed in future research.

First, more rigorous study design is need. Only two studies were prospective in design, and none adjusted for full Framingham Risk Score covariates.^18,30 In the absence of prospective studies, the temporality has not been established for F₂-isoprostanes and CVD, and the possibility of reverse causation is highly likely. Thus, prospective cohort studies are required in the field to provide effect estimates with better adjustment for confounding as well as establishing the temporal sequence between F₂-isoprostane elevation and CVD.

Second, the measurement of F₂-isoprostanes varied enormously from study to study. Besides different forms of F₂-isoprostanes (e.g. unmetabolized or metabolized form, Table 1) being measured across studies, the variation could also be due to specimen handling/storage. Not enough details were provided about the handling of biological samples, e.g. time length between collection and centrifugation, temperature of storage and freeze-thaw. Moreover, some studies investigated plasma F₂-isoprostanes, while others were based on urinary F₂-isoprostanes. Although plasma and urine are both non-invasive measures of F₂-isoprostanes, data are lacking regarding the correlation between the levels in plasma and that in urine. F₂-isoprostanes are cleared from the circulation in a rapid manner, approximately 16 min for 8-epi-PGF_2α.¹ Thus, the plasma F₂-isoprostanes would only represent the quantity for discrete short intervals in time. In addition, improper handling and/or prolonged storage of blood samples would lead to the generation of additional F₂-isoprostanes after the sample has been collected.³⁸ Furthermore, most of the previous studies did not provide details about which forms of F₂-isoprostanes, free, esterified or total, in plasma were measured. Note that in urine, only free F₂-isoprostanes are present.

Third, there are several approaches to final determination of F₂-isoprostanes in body fluids (mainly blood and urine), including gas chromatography/mass spectrometry (GC/MS), tandem mass spectrometry (GC/MS/MS), LC/MS/MS, radioimmunoassay and enzyme immunoassay.^39–43 Enzyme-linked immunosorbent assay (ELISA) methods measure one single isomer, i.e. 15-F_2t-isoprostane. GC-MS methods quantify some or all possible F₂-isoprostanes stereoisomers, while LC-MS methods separate and identify selected regio isomers and diastereomers. Indeed, the measurement from ELISA correlates very poorly with those from mass spectrometry.^44–47 Mass spectrametric methods are superior to radioimmunoassay and ELISA due to their sensitivity and specificity. Among the included studies, measurement of F₂-isoprostanes was conducted with different kits, and assays were constructed differently without validity data; thus, these measurements are not in complete agreement.

Finally, most studies did not apply multivariable analysis. The association observed could thus be due to, at least partly, other factors affecting both F₂-isoprostanes level and CVD risk. Multivariable analyses were carried out in several studies, but important covariates were not included in final models in some studies. The Framingham Risk Score needs to be adjusted. A list of additional factors to be considered for adjustment might include, but are not limited to, history of cancer, pulmonary disease, liver disease, kidney disease, tuberculosis, neurodegenerative diseases, thyroid disease, inflammatory diseases, diet, extensive exercise, pregnancy, obesity and hyperhomocysteinaemia. A biomarker should have added value beyond the predictive value of currently available markers. In addition, the presentation of data varies across studies. F₂-isoprostanes were presented as quartiles, tertiles, mean ± standard deviation or mean ± standard error. The units included ng (pg, pmol)/mmol with or without indexing to creatinine, and lg (ng/mmol). The reported effect estimates included difference in mean or median, OR and the area under the receiver operating characteristic curve. Further studies are needed to evaluate the net gain of predictive power after F₂-isoprostanes are incorporated.

In conclusion, evidence is gathering that high levels of F₂-isoprostanes in urine or blood may be a non-specific indicator for CVD. However, further population-based studies are needed. In addition, multivariable analyses are required for future studies to control confounding and improve classification accuracy.

Declarations

Competing interests: None.

Ethical approval: Not applicable.

Guarantor: Z-JZ.

Contributorship: Z-JZ conceived the study, researched data and wrote the manuscript.

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