Biomarker Profiling by Nuclear Magnetic Resonance Spectroscopy for the Prediction of All-Cause Mortality: An Observational Study of 17,345 Persons

Background Early identification of ambulatory persons at high short-term risk of death could benefit targeted prevention. To identify biomarkers for all-cause mortality and enhance risk prediction, we conducted high-throughput profiling of blood specimens in two large population-based cohorts. Methods and Findings 106 candidate biomarkers were quantified by nuclear magnetic resonance spectroscopy of non-fasting plasma samples from a random subset of the Estonian Biobank (n=9,842; age range 18-103 y; 508 deaths during a median of 5.4 y of follow-up). Biomarkers for all-cause mortality were examined using stepwise proportional hazards models. Significant biomarkers were validated and incremental predictive utility assessed in a population-based cohort from Finland (n=7,503; 176 deaths during 5 y of follow-up). Four circulating biomarkers predicted the risk of all-cause mortality among participants from the Estonian Biobank after adjusting for conventional risk factors: alpha-1-acid glycoprotein (hazard ratio [HR] 1.67 per 1-standard deviation increment, 95% CI 1.53-1.82, p=5x10(-31)), albumin (HR 0.70, 95% CI 0.65-0.76, p=2x10(-18)), very-low-density lipoprotein particle size (HR 0.69, 95% CI 0.62-0.77, p=3x10(-12)), and citrate (HR 1.33, 95% CI 1.21-1.45, p=5x10(-10)). All four biomarkers were predictive of cardiovascular mortality, as well as death from cancer and other nonvascular diseases. One in five participants in the Estonian Biobank cohort with a biomarker summary score within the highest percentile died during the first year of follow-up, indicating prominent systemic reflections of frailty. The biomarker associations all replicated in the Finnish validation cohort. Including the four biomarkers in a risk prediction score improved risk assessment for 5-y mortality (increase in C-statistics 0.031, p=0.01; continuous reclassification improvement 26.3%, p=0.001). Conclusions Biomarker associations with cardiovascular, nonvascular, and cancer mortality suggest novel systemic connectivities across seemingly disparate morbidities. The biomarker profiling improved prediction of the short-term risk of death from all causes above established risk factors. Further investigations are needed to clarify the biological mechanisms and the utility of these biomarkers for guiding screening and prevention.Please see later in the article for the Editors' Summary Editors' Summary Background A biomarker is a biological molecule found in blood, body fluids, or tissues that may signal an abnormal process, a condition, or a disease. The level of a particular biomarker may indicate a patient's risk of disease, or likely response to a treatment. For example, cholesterol levels are measured to assess the risk of heart disease. Most current biomarkers are used to test an individual's risk of developing a specific condition. There are none that accurately assess whether a person is at risk of ill health generally, or likely to die soon from a disease. Early and accurate identification of people who appear healthy but in fact have an underlying serious illness would provide valuable opportunities for preventative treatment. While most tests measure the levels of a specific biomarker, there are some technologies that allow blood samples to be screened for a wide range of biomarkers. These include nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These tools have the potential to be used to screen the general population for a range of different biomarkers. Why Was This Study Done? Identifying new biomarkers that provide insight into the risk of death from all causes could be an important step in linking different diseases and assessing patient risk. The authors in this study screened patient samples using NMR spectroscopy for biomarkers that accurately predict the risk of death particularly amongst the general population, rather than amongst people already known to be ill. What Did the Researchers Do and Find? The researchers studied two large groups of people, one in Estonia and one in Finland. Both countries have set up health registries that collect and store blood samples and health records over many years. The registries include large numbers of people who are representative of the wider population. The researchers first tested blood samples from a representative subset of the Estonian group, testing 9,842 samples in total. They looked at 106 different biomarkers in each sample using NMR spectroscopy. They also looked at the health records of this group and found that 508 people died during the follow-up period after the blood sample was taken, the majority from heart disease, cancer, and other diseases. Using statistical analysis, they looked for any links between the levels of different biomarkers in the blood and people's short-term risk of dying. They found that the levels of four biomarkersplasma albumin, alpha-1-acid glycoprotein, very-low-density lipoprotein (VLDL) particle size, and citrateappeared to accurately predict short-term risk of death. They repeated this study with the Finnish group, this time with 7,503 individuals (176 of whom died during the five-year follow-up period after giving a blood sample) and found similar results. The researchers carried out further statistical analyses to take into account other known factors that might have contributed to the risk of life-threatening illness. These included factors such as age, weight, tobacco and alcohol use, cholesterol levels, and pre-existing illness, such as diabetes and cancer. The association between the four biomarkers and short-term risk of death remained the same even when controlling for these other factors. The analysis also showed that combining the test results for all four biomarkers, to produce a biomarker score, provided a more accurate measure of risk than any of the biomarkers individually. This biomarker score also proved to be the strongest predictor of short-term risk of dying in the Estonian group. Individuals with a biomarker score in the top 20% had a risk of dying within five years that was 19 times greater than that of individuals with a score in the bottom 20% (288 versus 15 deaths). What Do These Findings Mean? This study suggests that there are four biomarkers in the bloodalpha-1-acid glycoprotein, albumin, VLDL particle size, and citratethat can be measured by NMR spectroscopy to assess whether otherwise healthy people are at short-term risk of dying from heart disease, cancer, and other illnesses. However, further validation of these findings is still required, and additional studies should examine the biomarker specificity and associations in settings closer to clinical practice. The combined biomarker score appears to be a more accurate predictor of risk than tests for more commonly known risk factors. Identifying individuals who are at high risk using these biomarkers might help to target preventative medical treatments to those with the greatest need. However, there are several limitations to this study. As an observational study, it provides evidence of only a correlation between a biomarker score and ill health. It does not identify any underlying causes. Other factors, not detectable by NMR spectroscopy, might be the true cause of serious health problems and would provide a more accurate assessment of risk. Nor does this study identify what kinds of treatment might prove successful in reducing the risks. Therefore, more research is needed to determine whether testing for these biomarkers would provide any clinical benefit. There were also some technical limitations to the study. NMR spectroscopy does not detect as many biomarkers as mass spectrometry, which might therefore identify further biomarkers for a more accurate risk assessment. In addition, because both study groups were northern European, it is not yet known whether the results would be the same in other ethnic groups or populations with different lifestyles. In spite of these limitations, the fact that the same four biomarkers are associated with a short-term risk of death from a variety of diseases does suggest that similar underlying mechanisms are taking place. This observation points to some potentially valuable areas of research to understand precisely what's contributing to the increased risk. Additional Information Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001606 The US National Institute of Environmental Health Sciences has information on biomarkers The US Food and Drug Administration has a Biomarker Qualification Program to help researchers in identifying and evaluating new biomarkers Further information on the Estonian Biobank is available The Computational Medicine Research Team of the University of Oulu and the University of Bristol have a webpage that provides further information on high-throughput biomarker profiling by NMR spectroscopy