Epigenetic aging measurements can vary by the time of day at which they are taken, according to a study published in Aging Cell.
The circadian rhythm
Living on Earth, organisms have evolved to adapt to our planet’s day/night cycle. This adaptation is known as the circadian rhythm, and it influences many aspects of biology. As these researchers [1] and others have found, this even includes the number of white blood cells circulating in the bloodstream. Because different white blood cells often have different measured epigenetic ages, measuring the total epigenetic age of white blood cells can give different values over the course of a day.
Beginning with a one-person cohort
This research was done using white blood cells taken every three hours, over a period of 72 hours, from one 52-year-old man. Two sets of cells were measured: neutrophils and white blood cells that had been mostly depleted of neutrophils (WBC-Neu).
The researchers found 58,459 epigenetic sites in WBC-Neu that oscillated over 24 hours. These sites had significant overlap with the 2013 Horvath clock, which is touted to work on all tissues: that clock measured this person as being three years older at noon than at midnight. Similar oscillations were also found in the 2013 Hannum clock and the 2016 Lin clock: this particular clock had a variation of 5.5 years over the day.
In total, 8 of the 17 clocks were found to be significantly affected by the circadian rhythm when measuring WBC-Neu cells, and all of them followed the same pattern: older in the day and younger at night. Even GrimAge2, a 2022 clock, was significantly influenced in this way, as were clocks that are made with the principal components (PCs) of other epigenetic clocks.
To confirm their findings, then researchers then moved on to samples taken from a different group of people [2]. While that study had been originally geared to test stress rather than circadian rhythms, the researchers found the same thing: the participants were reported as having younger WBC-Neu values at 4:15 PM than at 12:45 PM.
Number and types of cells matter
Analyzing two other previously collected datasets [3, 4], the researchers found that natural killer (NK) cells were consistently found to be older than B cells and CD4+ T cells. Therefore, blood samples with more NK cells gave higher epigenetic ages. How much this affected the results varied by the particular clock.
While it is possible to partially offset these differences by adjusting for cell types, there are still variances within single cell types. Purified neutrophils taken from the 52-year-old man, along with another cohort of men aged 30 to 54, were reported as having statistically significant differences over the course of a day in 3 of the 17 clocks. Additionally, the number of different types of white blood cells is, itself, a biomarker of aging and disease.
Most critically, these oscillations may have confounded results from previous studies, as they can be stronger than the effect sizes found in those studies. For example, if researchers are testing lifestyle interventions and conclude that these interventions affect epigenetic age by roughly a year, this may be caused entirely by the time of day rather than the actual intervention. Significant work needs to be done to make sure that testing of interventions that affect epigenetic age are not being influenced by this or other confounders.
Literature
[1] Oh, G., Koncevičius, K., Ebrahimi, S., Carlucci, M., Groot, D. E., Nair, A., … & Petronis, A. (2019). Circadian oscillations of cytosine modification in humans contribute to epigenetic variability, aging, and complex disease. Genome biology, 20, 1-14.
[2] Apsley, A. T., Ye, Q., Etzel, L., Wolf, S., Hastings, W. J., Mattern, B. C., … & Shalev, I. (2023). Biological stability of DNA methylation measurements over varying intervals of time and in the presence of acute stress. Epigenetics, 18(1), 2230686.
[3] Reinius, L. E., Acevedo, N., Joerink, M., Pershagen, G., Dahlén, S. E., Greco, D., … & Kere, J. (2012). Differential DNA methylation in purified human blood cells: implications for cell lineage and studies on disease susceptibility. PloS one, 7(7), e41361.
[4] Wang, X., Campbell, M. R., Cho, H. Y., Pittman, G. S., Martos, S. N., & Bell, D. A. (2023). Epigenomic profiling of isolated blood cell types reveals highly specific B cell smoking signatures and links to disease risk. Clinical Epigenetics, 15(1), 90.
