A blood plasma aging clock

This 2019 Stanford human study developed an aging clock using blood plasma proteins:

“We measured 2,925 plasma proteins from 4,331 young adults to nonagenarians [18 – 95] and developed a novel bioinformatics approach which uncovered profound non-linear alterations in the human plasma proteome with age. Waves of changes in the proteome in the fourth, seventh, and eighth decades of life reflected distinct biological pathways, and revealed differential associations with the genome and proteome of age-related diseases and phenotypic traits.

To determine whether the plasma proteome can predict chronological age and serve as a “proteomic clock,” we used 2,858 randomly selected subjects to fine-tune a predictive model that was tested on the remaining 1,473 subjects. We identified a sex-independent plasma proteomic clock consisting of 373 proteins. Subjects that were predicted younger than their chronologic age based on their plasma proteome performed better on cognitive and physical tests.

The 3 age-related crests were comprised of different proteins. Few proteins, such as GDF15, were among the top 10 differentially expressed proteins in each crest, consistent with its strong increase across lifespan. Other proteins, like chordin-like protein 1 (CHRDL1) or pleiotrophin (PTN), were significantly changed only at the last two crests, reflecting their exponential increase with age.

We observed a prominent shift in multiple biological pathways with aging:

  • At young age (34 years), we observed a downregulation of proteins involved in structural pathways such as the extracellular matrix. These changes were reversed in middle and old ages (60 and 78 years, respectively).
  • At age 60, we found a predominant role of hormonal activity, binding functions and blood pathways.
  • At age 78, key processes still included blood pathways but also bone morphogenetic protein signaling, which is involved in numerous cellular functions, including inflammation.

These results suggest that aging is a dynamic, non-linear process characterized by waves of changes in plasma proteins that are reflective of a complex shift in the activity of biological processes.”

https://www.biorxiv.org/content/10.1101/751115v1.full “Undulating changes in human plasma proteome across lifespan are linked to disease”


A non-critical review of the study was published by the Life Extension Advocacy Foundation. Frequent qualifiers like “could,” “may,” and “possible” were consistent with the confirmation biases of their advocacy.

There were several misstatements of what the study did, including the innumerate:

  1. “used around half of the participant data to build a “proteomic clock”
  2. tested it on the other half of the participants
  3. a total of 3000 proteins”

Per the above study quotation, the numbers were actually:

  1. Closer to two thirds (2,858 ÷ 4,331), not “around half”;
  2. The other third (1,473 ÷ 4,331), not “the other half”; and
  3. 2,925 not 3000.

The final paragraph and other parts of the review bordered on woo. Did a review of the findings have to fit LEAF’s perspective?


In contrast, Josh Mitteldorf did his usual excellent job of providing contexts for the study with New Aging Clock based on Proteins in the Blood, emphasizing comparisons with epigenetic clock methodologies:

“For some of the proteins that feature prominently in the clock, we have a good understanding of their metabolic function, and for the most part they vindicate my belief that epigenetic changes are predominantly drivers of senescence rather than protective responses to damage.

Wyss-Coray compared the proteins in the new (human) proteome clock with the proteins that were altered in the (mouse) parabiosis experiments, and found a large overlap [46 proteins change in the same direction and define a conserved aging signature]. This may be the best evidence we have that the proteome changes are predominantly causal factors of senescence.

46 plasma proteins

Almost all the proteins identified as changing rapidly at age 78 are increasing. In contrast, a few of the fastest-changing proteins at age 60 are decreasing (though most are increasing). GDF15 deserves a story of its own.

The implication is that a more accurate clock can be constructed if it incorporates different information at different life stages. None of the Horvath clocks have been derived based on different CpG sites at different ages, and this suggests an opportunity for a potential improvement in accuracy.”

A commentator linked the below study:

https://www.sciencedirect.com/science/article/pii/S0092867419308323 “GDF15 Is an Inflammation-Induced Central Mediator of Tissue Tolerance” (not freely available)

which prompted his response:

“Thanks, Lee! This is just the kind of specific information that I was asking for. It would seem we should construct our clocks without GDF15, which otherwise might loom large.”

An epigenetic clock review by committee

This 2019 worldwide review of epigenetic clocks was a semi-anonymous mishmash of opinions, facts, hypotheses, unwarranted extrapolations, and beliefs. The diversity of viewpoints among the 21 coauthors wasn’t evident.

1. Citations of the coauthors’ works seemed excessive, and they apologized for omissions. However:

  • Challenge 5 was titled “Single-cell analysis of aging changes and disease” and
  • Table 1 “Major biological and analytic issues with epigenetic DNA methylation clocks” had single-cell analysis as the Proposed solution to five of the Significant issues.

Yet studies such as High-Resolution Single-Cell DNA Methylation Measurements Reveal Epigenetically Distinct Hematopoietic Stem Cell Subpopulations were unmentioned.

2. Some coauthors semi-anonymously expressed faith that using current flawed methodologies in the future – only more thoroughly, with newer equipment, etc. – would yield better results. If the 21 coauthors were asked their viewpoints of Proposed solutions to the top three Significant issues of epigenetic clocks, what would they emphasize when quoted?

3. Techniques were praised:

“Given the precision with which DNA methylation clock age can be estimated and evolving measures of biological, phenotype-, and disease-related age (e.g., PhenoAge, GrimAge)..”

Exactly why these techniques have at times produced inexplicable results wasn’t examined, though. Two examples:

  • In Reversal of aging and immunosenescent trends, the Levine PhenoAge methodology estimated that the 51-65 year old subjects’ biological ages at the beginning of the study averaged 17.5 years less than their chronological age. Comparing that to the Horvath average biological age of 3.95 years less raised the question: exactly why did PhenoAge show such a large difference?
  • The paper mentioned the GrimAge methodology findings about “smoking-related changes.” But it didn’t explain why the GrimAge methylation findings most closely associated with smoking history also accurately predicted future disease risk with non-smokers.

Eluding explanations for these types of findings didn’t help build confidence in the methodologies.

4. A more readable approach to review by committee could have coauthors – in at least one section – answer discussion questions, as Reversing epigenetic T cell exhaustion did with 18 experts.

https://genomebiology.biomedcentral.com/articles/10.1186/s13059-019-1824-y “DNA methylation aging clocks: challenges and recommendations”

A GWAS meta-analysis of two epigenetic clocks

This 2019 UK human study conducted a meta-analysis of genome-wide association studies of two epigenetic clocks using 13,493 European-ancestry individuals aged between ten and 98 years:

“Horvath-EAA, described in previous publications as ‘intrinsic’ epigenetic age acceleration (IEAA), can be interpreted as a measure of cell-intrinsic ageing that exhibits preservation across multiple tissues, appears unrelated to lifestyle factors, and probably indicates a fundamental cell ageing process that is largely conserved across cell types.

In contrast, Hannum-EAA, referred to in previous studies as ‘extrinsic’ epigenetic age acceleration (EEAA), can be considered a biomarker of immune system ageing, explicitly incorporating aspects of immune system decline such as age-related changes in blood cell counts, correlating with lifestyle and health-span related characteristics, and thus yielding a stronger predictor of all-cause mortality.

The meta-analysis of Horvath-EAA identified ten independent associated SNPs [single nucleotide polymorphisms], doubling the number reported to date, and highlighted 21 genes involved in Horvath-based epigenetic ageing. Four of the ten Horvath-EAA-associated SNPs are mQTL [methylation quantitative trait loci] for CpGs used in the Horvath/Hannum epigenetic clocks. A possible interpretation of this is that the functional mechanism by which these SNPs influence the rate of biological ageing is via altering methylation levels.

Father’s age at death, a rough proxy for lifespan, was nominally significantly correlated with both EAA measures, and parents’ age at death was additionally correlated with Hannum-EAA. Aside from these, genetic correlations with age-related traits were surprisingly few: it is possible that this could reflect an overly conservative correction for the multiple tests carried out, or low statistical power, rather than a genuine lack of correlations.

Genetic correlation analysis should be restricted to GWAS with a heritability Z-score of 4 or more, on the grounds of interpretability and power, so the Horvath-based results particularly should be interpreted with caution.”


A non-apologetic way to explain the above graphic is that NONE of these 218 “health and behavioral traits” were any more associated with the studied genetic measurements than would be expected by chance!

Fervent believers in the GWAS methodology’s capability to exactly predict individual phenotypes eventually become victims of the scientific method. These GWAS researchers griped about “overly conservative correction, or low statistical power” and other predictable shortfalls, and ended a long limitations statement with:

“While we have identified a number of SNPs and genes significantly associated with EAA, including genes already known to be related to ageing, the analyses presented here fall short of providing a mechanistic explanation for how these variants and genes act to influence biological age.”

Outside of beliefs, it’s hard to understand why research money keeps pouring into the GWAS dead end. If these researchers and their employing institution and sponsors want to make a difference in human lives, they need to get busy in other areas.

These researchers were employed by the same institution that couldn’t be bothered to scrape together six more weeks of funds to study the transgenerational damaging effects of acetaminophen – an analgesic available to billions of people – in Epigenetics research that was designed to fall one step short of wonderful.

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1008104 “A meta-analysis of genome-wide association studies of epigenetic age acceleration”

Restrict information in the name of science?

A Stanford researcher was annoyed that we live in the 21st century, and advocated we return to previous centuries’ information-flow check valves of wise old men. No doubt the publishers of and subscribers to the Journal of the American Medical Association applauded the same old tired prescription.

Ten instances of the word “should” in the final two paragraphs provided ample evidence of the paper’s intent. Mirroring the current political climate, accusations made of others were items the accusers were guilty of themselves, such as:

“When these scientists act as investigators in the hundreds of observational studies that they publish, or as editors and peer reviewers in evaluating submissions from others, would they tolerate publishing analyses and funding proposals that might contradict their belief system?”

https://jamanetwork.com/journals/jama/fullarticle/2753533 “Neglecting Major Health Problems and Broadcasting Minor, Uncertain Issues in Lifestyle Science”


One of the paper’s references included an informative graphic:

“A histogram of the total number of rumor cascades in our data across the seven most frequent topical categories.”

https://science.sciencemag.org/content/359/6380/1146 “The spread of true and false news online”


I’ll borrow from the curation of another Stanford paper Online dating cuts out the middlemen in conclusion:

“Are there examples where it wouldn’t potentially improve a person’s life to choose their information sources? Friends, family, and other social groups – and religious, educational, and other institutions – have had their middlemen/guarantor time, and have been found lacking.

Make your own choices for your one precious life.”

Perinatal stress and sex differences in circadian activity

This 2019 French/Italian rodent study used the PRS model to investigate its effects on circadian activity:

“The aim of this study was to explore the influence of PRS on the circadian oscillations of gene expression in the SCN [suprachiasmatic nucleus of the hypothalamus] and on circadian locomotor behavior, in a sex-dependent manner.

Research on transcriptional rhythms has shown that more than half of all genes in the human and rodent genome follow a circadian pattern. We focused on genes belonging to four functional classes, namely the circadian clock, HPA axis stress response regulation, signaling and glucose metabolism in male and female adult PRS rats.

Our findings provide evidence for a specific profile of dysmasculinization induced by PRS at the behavioral and molecular level, thus advocating the necessity to include sex as a biological variable to study the set-up of circadian system in animal models.”

“There was a clear-cut effect of sex on the effect of PRS on the levels of activity:

  • During the period of lower activity (light phase), both CONT and PRS females were more active than males. During the light phase, PRS increased activity in males, which reached levels of CONT females.
  • More interestingly, during the period of activity (dark phase), male PRS rats were more active than male CONT rats. In contrast, female PRS rats were less active than CONT females.
  • During the dark phase, CONT female rats were less active than CONT male rats.

The study presented evidence for sex differences in circadian activity of first generation offspring that was caused by stress experienced by the pregnant mother:

“Exposure to gestational stress and altered maternal behavior programs a life-long disruption in the reactive adaptation such as:

  •  A hyperactive response to stress and
  • A defective feedback of the hypothalamus-pituitary-adrenal (HPA) axis together with
  • Long-lasting modifications in stress/anti-stress gene expression balance in the hippocampus.”

It would advance science if these researchers carried out experiments to two more generations to investigate possible transgenerational epigenetic inheritance of effects caused by PRS. What intergenerational and transgenerational effects would they possibly find by taking a few more months and extending research efforts to F2 and F3 generations? Wouldn’t these findings likely help humans?


One aspect of the study was troubling. One of the marginally-involved coauthors was funded by the person described in How one person’s paradigms regarding stress and epigenetics impedes relevant research. Although no part of the current study was sponsored by that person, there were three gratuitous citations of their work.

All three citations were reviews. Unlike study researchers, reviewers aren’t bound to demonstrate evidence from tested hypotheses. Reviewers are free to:

  • Express their beliefs as facts;
  • Over/under emphasize study limitations; and
  • Disregard and misrepresent evidence as they see fit.

Fair or not, comparisons of reviews with Cochrane meta-analyses of the same subjects consistently show the extent of reviewers’ biases. Reviewers also aren’t obligated to make post-publication corrections for their errors and distortions.

As such, reviews can’t be cited for reliable evidence. Higher-quality studies that were more relevant and recent than a 1993 review could have elucidated points.

Sucking up to the boss and endorsing their paradigm was predictable. Since that coauthor couldn’t constrain themself to funder citations only in funder studies, it was the other coauthors’ responsibilities to edit out unnecessary citations.

https://www.frontiersin.org/articles/10.3389/fnmol.2019.00089/full “Perinatal Stress Programs Sex Differences in the Behavioral and Molecular Chronobiological Profile of Rats Maintained Under a 12-h Light-Dark Cycle”

Would you return a lost wallet?

The researchers in this 2019 Swiss/US study intentionally “lost” > 17,000 wallets under experimental conditions:

“We conducted field experiments in 40 countries to examine whether people act more dishonestly when they have a greater economic incentive to do so, and we found the opposite to be true. Citizens were more likely to return wallets that contained relatively larger amounts of money. Neither nonexperts nor professional economists were able to predict this result.

When people stand to heavily profit from engaging in dishonest behavior, the desire to cheat increases but so do the psychological costs of viewing oneself as a thief.”


The study did well in some aspects, including publicity. However:

1. The researchers admitted in the final paragraph:

“Using average reporting rates across countries, we find substantial variation in rates of civic honesty, ranging from 14 to 76%. This variation largely persists even when controlling for a country’s gross domestic product, suggesting that other factors besides a country’s wealth are also at play.”

Yet the paper’s first page contained the above graphic, which used each country’s GDP as a dependent variable! Wasn’t a behavioral economics study of honesty required to present their data honestly, and use factors that were experimentally significant?

2. “Other factors..at play” were relegated to the supplementary materials. The paper was only three-and-a-half pages long, so there was room for further explanations.

Here’s one comment on cultural differences from a Chinese PhD student:

“Biased design. In China (and Asian countries), people seldom use email, and our merit is to leave things untouched (“路不拾遗“:no one picks up lost articles in the street (idiom)).”

3. The study design had nothing to do with avoiding taxes, but three of the four sentences in the paper’s first paragraph did. This impressed as pointless.

https://science.sciencemag.org/content/365/6448/70 “Civic honesty around the globe” (not freely available)

Wikipedia is a poor source of information on advanced glycation end products (AGEs)

A link to Wikipedia is usually on the first page of search results. The Wikipedia post on AGEs lacks the evidence that a reader may infer from its text.

For example, the second paragraph of the AGEs post, Dietary Sources, contained the following text and references:

  1. “However, only low molecular weight AGEs are absorbed through diet, and vegetarians have been found to have higher concentrations of overall AGEs compared to non-vegetarians. [4]
  2. Therefore it is unclear whether dietary AGEs contribute to disease and aging, or whether only endogenous AGEs (those produced in the body) matter. [5]
  3. This does not free diet from potentially negatively influencing AGE, but implicates dietary AGE may be less important than other aspects of diet that lead to elevated blood sugar levels and formation of AGEs. [4] [5]”

[4] https://www.sciencedirect.com/science/article/pii/S0278691513004444 “Advanced glycation end products in food and their effects on health” (not freely available) 2013 Denmark.

Please note on this linked page that a German researcher took the time to correct one bias of the Danish reviewers, citing evidence from his studies that:

“The deleterious effects of food-derived AGEs in subjects with type 2 diabetes mellitus are proven.”

[5] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257625 “Dietary Advanced Glycation End Products and Aging” 2010 US.


Both of these references were reviews.

Unlike study researchers, reviewers aren’t bound to demonstrate evidence from tested hypotheses. Reviewers are free to:

  • Express their beliefs as facts;
  • Over/under emphasize study limitations; and
  • Disregard and misrepresent evidence as they see fit.

Reviewers also aren’t obligated to make post-publication corrections for their errors and distortions. For example, the Danes didn’t correct their review with any findings the German researcher presented.

As such, reviews can’t be cited for reliable evidence.


A sample of other problems with each of the Wikipedia sentences:

1. “However, only low molecular weight AGEs are absorbed through diet, and vegetarians have been found to have higher concentrations of overall AGEs compared to non-vegetarians. [4]”

The first part of sentence 1 came from the review’s abstract:

“Only LMW AGEs..may be absorbed from the gut and contribute to the body burden of AGEs.”

But the reviewers didn’t support their abstract’s statement with direct evidence from any study!

2. “Therefore it is unclear whether dietary AGEs contribute to disease and aging, or whether only endogenous AGEs (those produced in the body) matter. [5]”

The “therefore” of sentence 2 was misplaced. Sentence 1 didn’t attempt to explain whether “dietary AGEs contribute to disease and aging” or “only endogenous AGEs matter.”

Since sentence 2 wasn’t a consequence of sentence 1, the Wikipedia contributor(s) needed to support sentence 2 with evidence. Citing an “unclear” 2010 reference [5] ignored dozens of studies that provided better clarity.

3. “This does not free diet from potentially negatively influencing AGE, but implicates dietary AGE may be less important than other aspects of diet that lead to elevated blood sugar levels and formation of AGEs. [4] [5]”

Wikipedia contributors tend to cite irrelevant references rather than get flagged with “citation needed.” The value judgment of sentence 3 was an example of this intentionally misleading masquerade.

“Dietary AGE may be less important..” wasn’t unequivocally supported by studies referenced in either review, and didn’t represent an authoritative body of evidence. Contrast those weasel words with:

“The deleterious effects of food-derived AGEs in subjects with type 2 diabetes mellitus are proven.”

Good job, Wikipedia contributors! You used lower-quality reviews to promote misunderstandings that DETRACTED from science.


Wikipedia’s premise is that since the group knows more about any subject than does any individual, everyone is entitled to contribute. The results are usually incoherent narratives that often substitute opinions for evidence.

The second paragraph of the Exogenous section of the Wikipedia glycation post provided an example:

  • Assertions of the first and third sentences needed citations. Did the contributor(s) think these would be unexamined?
  • Someone contributed a cancer reference as the fourth sentence, although it had little to do with the preceding sentences.
  • The fifth sentence was informative on exogenous glycations and AGEs. An editor would have removed “recently” and “recent” though, because the cited source was dated 2005.