Atherosclerosis develops gradually over a period of decades and its progress depends on several components such as hypercholesterolemia, lipid oxidation and inflammation [1]. Several inflammatory components take part in the pathogenesis of atherosclerosis [2, 3] and, of them, interleukin-6 (IL 6) and high sensitivity-C reactive protein (hs-CRP) are the most extensively studied. To date hs-CRP measurements are recommended for the assessment of cardiovascular risk in some asymptomatic adults, in spite of the fact that causality in ischaemic heart disease (IHD) has not been established [4].

Natriuretic peptides are released from the heart as a reaction to increased wall stress, pressure and hypertrophy and have been studied as prognostic biomarkers in IHD [5‐7]. Jørgesen et al. have previously shown that N-terminal pro b-type natriuretic peptide (NT- proBNP) in combination with changes in electrocardiography predicted all-cause mortality and cardiovascular events in patients without previous cardiac disease [8], but the predictive value of natriuretic peptides on the incidence of IHD has not been adequately studied.

Little is known about the significance and consequences of elevated troponin in the general population, despite the fact that troponin is commonly used to establish diagnosis and prognosis in myocardial infarction. Daniels et al. showed that a population of community-dwelling older adults (mean age 77 years) with elevated troponin T (TNT) (≥ 0.01 ng/ml) and NT-proBNP (≥ 450 pg/ml) ran a higher risk of both all-cause mortality and cardiovascular mortality during a median follow-up of 6.8 years [9]. A combination of biomarkers and traditional risk factors has previously been studied in terms of the prediction of death and major adverse cardiovascular events, showing different predictive abilities for increased risk [10‐12].

The objective of the present study was to investigate the additive value of a panel of biomarkers on top of clinical risk factors in the early prediction of the incidence of an IHD in a cohort of randomly selected male sample from the general population, investigated at 50 years of age and followed for 21 years. We hypothesised that while, clinical risk factors may be of limited predictive value for incident IHD in the general population, the addition of multiple biomarkers will resemble the complex and multifactorial nature of the atherosclerotic process and therefore facilitate the early identification of high-risk individuals in a general population.

Of 798 participants, 59 (7.5%) were excluded in total, of which 45 (5.6%) were excluded due to the lack of biomarker data and 14 (1.8%) due to the occurrence of an IHD event prior to baseline examination. A total of 739 (92.6%) participants of the original sample were therefore included in the current study.

During a median follow-up of 20.6 (range 0.6–21.6) years, 97 participants (13.1%) had an incident event of myocardial infarction (MI), unstable angina, PCI or CABG. The IHD events were divided as follows: MI with or without intervention with PCI or CABG (62.9%), PCI or CABG due to angina (27.8%) and hospitalised unstable angina (9.3%). The event rate was 7.0 [95% CI 5.8–8.6] per 1000 person-years. A total of 105 (14.2%) participants died, including all causes, before the end of follow-up on 30 Aug 2014. Of these, 33 (31.4%) died of cardiovascular causes and 72 (68.6%) died of other causes.

In this random population of middle-aged men without prior IHD followed for 21 years we found that, when biomarkers were studied separately, the number of high levels of biomarkers (hs-TNT, hs-CRP, IL-6 and NT-proBNP) is a significant predictor that provided highest predictive ability. In a multivariable prediction model, the addition of biomarker hs-TNT was an independent significant predictor of IHD and significantly improved prediction as tested by likelihood ratio test (p = 0.033). Furthermore, despite that addition of biomarker hs-TNT on top of clinical risk factors did not significantly enhance discriminative ability for IHD, it appeared to promote early prediction of incident IHD during 21-year long follow-up.

Previous risk scores are mostly based on clinical risk factors. The Framingham Risk Score (FRS) is a well-known, sex-specific multivariable risk factor algorithm [15]. This risk score was based on only 12 years of follow-up and only traditional risk factors, including age, total cholesterol, HDL, systolic BP, smoking and diabetes, but not including physical activity, BMI and biomarkers. Similarly, the Systematic Coronary Risk Evaluation (SCORE) is based on traditional risk factors and estimates only the 10-year risk of fatal cardiovascular diseases [16]. The QRISK (risk score using the QRESEQRCH database) incorporates family history and social deprivation and calibrates better to the UK population but their clinical CVD events were not adjudicated [17]. It can be argued that the above-mentioned risk prediction models might provide only a limited incremental risk of IHD in the general population in today´s clinical practice as they may not fully reflect the current observations of markedly decreasing population levels of smoking, and serum cholesterol, but instead an increase in overweight and obesity [18]. In our study we found that for risk estimation 20 years beyond FRS alone is a suboptimal clinical prediction model for IHD with an AUC about 0.69 in a population with fairly low cardiovascular risk profile. In the composition of FRS, HDL-C appears to be one of the most important factors, at least when FRS is applied in 50-years old men and their first event of IHD studied for approximately 20 years. Addition of further new biomarkers such as hs-TNT on top of clinical factors did improve the prediction, but did not significantly enhance discriminative ability. Together they are able to achieve AUC 0.72 which is more acceptable, as an acceptable c-statistics of at least 0.70 is recommended in clinical praxis. Furthermore, they appear to contribute to higher sensitivity for the similar levels of specificity throughout the follow-up, indicating a better prediction of IHD than FRS alone.

Atherosclerosis is a multifactorial process that involves many underlying components such as increased serum LDL-cholesterol, lipid oxidation and inflammation [1], and develops with preceding subclinical events. It is therefore reasonable to emulate this complex and multifactorial nature of the atherosclerotic process by combining different biomarkers representing different mechanisms. Indeed, when biomarkers were studied alone, the number of elevated levels of biomarkers (hs-TNT > 10 ng/L, hs-CRP > 1 mg/L, IL-6 > 8 ng/L and NT-proBNP > 100 pg/mL) is a significant predictor that provides highest predictive ability. Additionally, hs-TNT, hs-CRP and IL-6 were significant predictors for incidence of IHD. Hs-TNT has better prediction than smoking, BMI and glucose, and is an independent significant predictor when adjusted for HDL-C, cholesterol and hypertension. Also, the risk increases continuously with increasing units of the biomarker TnT indicating that this biomarker has a linear association with increased risk of IHD. Furthermore, HDL-C that is also a biomarker and widely used as a traditional risk factor, has the highest predictive ability compared with other known risk factors and the biomarkers investigated within this study. It is notable that this study involves a very long follow-up of 21 years which permit exploring long-term prediction of different biomarkers analyzed in a very early stage of disease process. A combination of different biomarkers made it possible to represent different mechanisms involved in the disease progression. It might be so that different underlying mechanisms are involved at different stages of the atherosclerotic process, and as a result the effect of biomarkers can differ along the way.

Previous studies have also studied similar biomarkers and often focused on a single biomarker for its prediction of the incidence of IHD. For instance, De Lemos et al. demonstrated that 25% of adults in the general population had detectable TnT levels (≥ 0.003 ng/mL) and that this was associated with structural heart disease and the risk of all-cause mortality during 6.4 years of follow-up [19], findings that were consistent in a subsequent meta-analysis [20]. Cesari et al. studied three inflammatory biomarkers, CRP, IL-6 and tumour necrosis factor-α, in 2225 individuals without previous cardiovascular disease. All the inflammatory biomarkers predicted cardiovascular events and the risk was highest among participants where all three inflammatory biomarkers were measured in the highest tertile [21]. In addition, other studies and meta-analysis show associations between inflammatory markers and ischaemic heart disease [2, 22, 23], but these findings have not been consistent [24] and the association can depend on conventional risk factors and additional inflammatory markers [25]. Furthermore, Daniels et al. studied the prediction of all-cause mortality and cardiovascular mortality by TNT and NT-proBNP in a cohort of 957 older adults and community residents in southern California. This study showed that participants with detectable TNT (≥ 0.01 ng/ml) and NT-proBNP (≥ 450 pg/ml) ran a higher risk of death [9]. However, to the best of our knowledge, whether these biomarkers are sensitive enough for the early prediction of IHD events at the age of 50 years in the general population has not been adequately studied previously.

Our study extends previous findings in two respects; firstly, we sought to determine the predictive value for incident IHD of combining different biomarkers representing different mechanisms such as inflammation (hs-CRP, ferritine, IL-6), cardiac stress (NTpro BNP) and cardiac damage (hs-TnT). In this study, a combination of different biomarkers provided the most powerful prediction of IHD in a univariable analysis. Secondly, we also sought to establish a risk factor model by adding biomarkers to clinical risk factors (hypertension, smoking, cholesterol, diabetes) and contemporary cardiovascular risk factors (BMI, physical inactivity). This strategy has previously been studied but without achieving consistent results. Zethelius et al. studied the prediction of cardiovascular death in elderly men (71 years) by a combination of biomarkers in addition to established risk factors and found that risk stratification was improved [12]. Wang et al. evaluated men and women (mean age 59 years) with a follow-up time of up to 10 years and found that a multimarker score only moderately increased the predictive value of major cardiovascular events when added to conventional risk factors [11]. Although not fully comparable, Kim et al. found similar results in a case–control study comprising only women [10]. In the present study, the addition of biomarker hs-TNT was an independent significant predictor of IHD and significantly improved prediction as tested by likelihood ratio test (p = 0.033). Furthermore, addition of biomarker hs-TNT on top of clinical risk factors appears to promote the early prediction of incident IHD during 21-year long follow-up. As compared with FRS, the AUC increased from 0.69 to 0.72 when adding biomarkers. This implies that prediction model consisting of FRS may be improved in a contemporary era of cardiovascular prevention with gradually declining incidence of IHD. Further studies with larger sample sizes are warranted. The reason why some biomarkers did not enhance further predictive power in addition to clinical risk factors in multivariable models, despite that they were significant in univarable analysis, might be lack of power in the present study. One other possible explanation is the fact that biomarkers such as hs-CRP represent mechanisms for inflammation, which might be overlapping with traditional risk factors such as HDL-C and hypertension in which inflammation is involved.

In this random, middle-aged male population sample, the addition of a biomarker hs-TNT was an independent significant predictor of IHD and adds to the early prediction of incident IHD in a multivariable model, in a general male population with lower risk than in previous generations. Further studies with long-term risk estimation are warranted giving consideration that the incidence rate of IHD is continuously declining.