Leukocyte telomere length and serum polyunsaturated fatty acids, dietary habits, cardiovascular risk factors and features of myocardial infarction in elderly patients

This paper presents a cross-sectional substudy of data obtained at time of inclusion in the ongoing OMega-3 fatty acids in Elderly patients with Myocardial Infarction (OMEMI) trial [23], in which elderly survivors of AMI are randomized to 2 years intervention with either 1.8 g marine n-3 fatty acids in the form of Pikasol capsules (Axellus AS, Oslo/Orkla Health, Norway) or corn oil placebo. The present investigation was performed in the first 299 consecutively enrolled patients.

The project was performed in accordance with the Declaration of Helsinki, approved by the Regional Ethical Committee South East with (Reference no.: 2012/1422), and registered at ClinicalTrials.gov (NCT01841944). All patients gave written informed consent to participate.

Patients were recruited at three hospitals in the Oslo region of Norway; the Oslo University Hospital, Ullevål, Oslo, Vestre Viken Hospital Trust, Bærum and Akershus University Hospital, Lørenskog. Patients were screened during their initial hospital admission. Inclusion criteria were patients who had undergone MI, age between 70 and 82 years, and ability to understand verbal and written patient information in Norwegian language and provide written consent. Exclusion criteria were documented intolerance for n-3 fatty acids, and additional disease states thought to influence survival and/or ability to adhere to study medication and follow up for the study period. Previous regular intake of n-3 supplements was not an exclusion criterion, as such intake is fairly common in the general Norwegian population, and exclusion would introduce a selection bias to the study. Patients were included two to 8 weeks after hospital discharge. The inclusion period for the 299 patients was November 2012 to November 2014. The study design has previously been described in detail [23].

Previous medical history and data regarding the index MI were retrieved from hospital records, including the classification of the MI as ST-segment elevation MI (STEMI) or non-ST-segment MI (NSTEMI), and peak Troponin T levels occurring from sequential measures during hospital stay. Echocardiography was performed as part of the clinical management in a total of 162 patients, and left ventricular ejection fraction (LVEF) was registered. Previous hypertension was defined as previous diagnosis of hypertension, and previous hyperlipidemia was defined as previous diagnosis or treatment with lipid lowering drugs. Current smoking was defined as current smoking or cessation no more than 3 months prior to inclusion.

The patients’ diet was assessed using the SmartDiet form which is a validated food frequency questionnaire, previously described [24]. The SmartDiet form was filled out once, and is based on the patients’ recollection of intake of different food groups, with a special emphasis on fats. Each of the items are scored, and summarized to a total score ranging from 15 to 45 points. The scores are further divided into three groups, where a score of 15–27 is considered an unhealthy diet (designated poor), a score of 28–35 a diet should be improved (designated intermediate), and a score of 36–45 points is considered a healthy diet (designated healthy). As the subdivision into the three arbitrary categories may limit the discovery of association, data from SmartDiet score were also explored as a continuous variable in the present study.

Venous blood samples were drawn in fasting state between 08:00 and 11:00 at inclusion and routine laboratory analyses were performed by conventional methods.

Serum was prepared within 1 h by centrifugation at 2000 x g for 10 min and kept frozen at − 80 °C until analysis of the fatty acid profile. EDTA whole blood was drawn and kept frozen at − 80 °C until DNA extraction for LTL analysis.

Fatty acids composition in serum phospholipids was analyzed by gas chromatography and expressed as % weight of total fatty acids, as previously described [25]. Briefly, serum lipids were extracted according to the Folch procedure [26]. The organic phase containing total lipids was collected and another extraction was performed. Separation of the phospholipid fatty acid fraction from total lipids was performed as described by Burdge et al. [27]. Methylation of the fatty acids was performed before being analyzed by gas chromatography using a Varian 3900 gas chromatograph equipped with a CP-8400 autosampler, a flame ionization detector and a high-polarity polyester CP-Sil 88 60 m × 0.25 mm capillary column (Varian, Middleburg, The Netherlands).

The content expressed as % weight of serum phospholipids of the LCPUFAs linoleic acid (LA) 18:2 n-6, arachidonic acid (AA) 20:3 n-3, alpha-linolenic acid (ALA) 18:3 n-3, eicosapentaenoic acid (EPA) 20:5 n-3 and docosahexaenoic acid (DHA) 22:6 n-3 and the ratio of the n-6 to n-3 LCPUFAs were used for the present study, as well as the ratio of the selected n-6 PUFAs to the selected n-3 PUFAs. These have all previously been extensively studied in relation to CVD [25].

Leukocytes are a commonly used source for analysis of telomere length in clinical studies, as they are easily obtained from the circulation, also used in our study. These have been shown to have acceptable correlation to telomere lengths in other tissues.

DNA was extracted from EDTA whole blood by the same lot of the QIAamp DNA Blood Mini Kit throughout the study (Qiagen Gmbh, Hilden, Ger) and according to the manufacturers instruction. DNA purity and quantity was tested on the NanoDrop, ND-1000 (Saveen Werner, Sweden) and samples were stored at − 80 °C until LTL analyses.

All DNA samples were diluted to a final concentration of 2 ng/μL and LTL was measured by singleplex quantitative real-time PCR [28]. PCR amplification was performed on the VIIa7 PCR Instrument with the QuantStudio Real-Time Software (Applied Biosystems by Life Technologies Foster City, CA, USA),using telomere-specific primers (Invitrogen by Thermo Fisher Scientific, Waltham, MA, US) (Additional file 1: Table S1) and GoTaq®qPCR Master Mix (Promega, Madison, WI, US). LTL was relatively quantified (RQ) to the single-copy-gene (SCG) 36B34 (Invitrogen by Thermo Fisher Scientific) (Additional file 1: Table S1) and to an internal reference sample. The primers for both targets were diluted to a final concentration of 4 pmol /μl, and PCR conditions were as follow; an initial step at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 sek and 60 °C for 1 min. All samples were run in triplicates [28].. Amplification efficacy for both targets were equal, and approximately 100%. Individual amplification curves for all samples of both assays (LTLs and SCG) were carefully validated. Technical replicates with a standard deviation (SD) exceeding 0.5 Ct or outliers, if diversely to the other replicates were excluded from the analysis. A template negative control was included in each run. The intra- and inter assay coefficient of variation (CV) for the LTL Ct values were; 0.37 and 2.1%, and for the SCG Ct values 0.20 and 0.40% (based on mean CV value between replicates on 1 plate and multiple plates, respectively). The inter-run CV for the T/S ratio (LTL Ct signal divided on the CT mean of the reference sample) was 2.09% (based on mean CV for triplicates run on different plates). The inter-run CV for the exponentially calculated T/S ratio was 13.2%, and when relatively calculated to the reference sample, the CV was 13.3% (Additional file 3: Methods).

Continuous variables are presented as mean ± SD or median (25th, 75th percentiles) as appropriate. Categorical data are presented as numbers and/or percentages. As most data were non-normally distributed non-parametric statistical tests were performed. Differences between groups were examined by Kruskal-Wallis or Mann-Whitney U test. For correlation analyses Spearman’s rho were used.

LTL in the total cohort ranged from 0.11 to 1.55 and was skewed towards shorter telomeres, with a median of 0.55 (0.42, 0.72).

Levels of the selected LCPUFAs presented as % weight of total serum phospholipids and the n-3/n-6 ratio in the total population and stratified by self-reported use of n-3 polyunsaturated fatty acid (PUFA) supplementation are presented in Table 2. Patients who reported regular intake of n-3 PUFA supplements (n = 135), had significantly higher levels of both EPA and DHA than those who did not, and also lower levels of AA (all p < 0.001). The n-6/n-3 ratio was also lower in patients taking n-3 PUFA supplements (p < 0.001).

Levels of LTL correlated weakly, but statistically significantly to the content of LA in serum phospholipids (r = 0.139, p = 0.017). Neither of the remaining investigated LCPUFAs, nor the n-6/n-3 ratio correlated significantly to LTL (r ranging from − 0.107 to 0.087 and p ranging from 0.067 to 0.958) (Table 3). Associations were also examined for differences in fatty acids levels across quartiles of LTL, with no significant differences (p > 0.1) (Fig. 1). When excluding patients using n-3 supplementation, the correlation between LTL and LA was borderline significant (r = 0.141, p = 0.074) (Additional file 2: Table S2), whereas the differences across quartiles of LTL did not change (all p > 0.1).

The mean SmartDiet score was 29.4 (range 15, 40), with 71 (30.7%) in Group 1 (poor), 142 (61.5%) in Group 2 (intermediate) and 18 (7.8%) in Group 3 (healthy). The respective median (25, 75%) levels of LTL were 0.55 (0.39, 0.69), 0.55 (0.43, 0.70) and 0.65 (0.45, 0.87) (Fig. 2). Despite a trend for longer LTLs in the healthy group, the difference did not reach statistical significance (p = 0.073). No statistically significant correlation between LTL and score obtained by SmartDiet as a continuous variable was observed (r = 0.043, p = 0.52).

To explore any association between LTLs and traditional risk factors, levels of LTL were dichotomized according to values below or above median level (0.55). As show in Table 4, no significant differences were observed according to values below or above median level in number of patients with the risk factor or not.

Patients with STEMI had numerically longer LTL compared to NSTEMI (0.57 (0.43, 0.75) vs 0.53 (0.42, 0.72), but not statistically significant (p = 0.89). LTLs dichotomized by median level showed no significant difference between values below or above the median in any marker of myocardial injury or myocardial function, as shown in Table 4. There was also no difference in LTL of patients presenting with heart failure at any time.

A significant limitation of the present study is the lack of a control population at the same age, but free from known CVD. Also, as mentioned, any medication used before the index infarction, might have influenced the results. Related to marine n-3 fatty acids, measure of telomerase activity might have added to the results. Measures of myocardial injury and function were limited to peak troponin T, NT-proBNP and LVEF by routine echocardiography. A significant strength with regard to the influence of LCPUFAs, however, is the individual measurement of serum fatty acids.