Epigenetic effects of plasma concentrate

“We use data from a safety study (n = 18, mean age 74) to investigate whether human umbilical cord plasma concentrate (hereinafter Plasma Concentrate) injected weekly (1 ml intramuscular) into elderly human subjects over a 10-week period affects different biomarkers, including epigenetic age measures, standard clinical biomarkers of organ dysfunction, mitochondrial DNA copy number (mtDNA-CN), and leukocyte telomere length.

More than 20 clinical biomarkers were significantly and beneficially altered. Telomere length and mtDNA-CN were not significantly affected by treatment.

An increase in entropy means that the methylome becomes noisier. We found that entropy was significantly decreased after treatment. Decreased entropy may implicate rejuvenation of the epigenetic landscape after plasma concentrate treatments.

changes in methylation entropy

Treatment reduced DNA methylation-based GrimAge by an average of 0.82 years, suggesting a reduction in morbidity and mortality risk. By contrast, no significant results could be observed for epigenetic clocks that estimate chronological age.

Our study lends credence to the notion that there are youth-promoting factors in the secretome of umbilical cord plasma. This conclusion has also been reached by other researchers that have provided treatment with stem cells, which do not work by plasma dilution but primarily by providing humoral factors and changing the microenvironment of cells and tissues. While there may be youth-promoting microvesicles or humoral factors that are at work, we do not want to rule out the possibility that it is ‘young and undamaged’ albumin that leads to the improvements noted, especially in light of recent evidence for such a mechanism.

This first human epigenetic clock study of plasma concentrate treatments revealed age-reversal effects according to a well-established DNA methylation-based estimator of morbidity and mortality risk. Future placebo-controlled replication studies are warranted with a larger number of participants over a longer study period, which our laboratory has undertaken to pursue.”

https://onlinelibrary.wiley.com/doi/10.1111/acel.13696 “Umbilical cord plasma concentrate has beneficial effects on DNA methylation GrimAge and human clinical biomarkers”


Garlic vs. broccoli

This 2022 human study compared effects of two supplements:

“We test the hypothesis that consuming glucoraphanin (from broccoli) or alliin (from garlic) results in the accumulation of sulforaphane and alliin and their associated metabolites in the human prostate gland in a randomised, double-blinded, 2 × 2-factorial, dietary supplement, four-week intervention study.

The predominant sulphur-containing metabolite in garlic is alliin, which is odourless and non-volatile. When the plant tissue is damaged, alliinase enzymes rapidly convert alliin to allysulfenates that condense to form allicin and other thiosulfinates, predominantly γ-glutamyl S-allyl-L cysteine (γ-SAC) and S-allyl-L cysteine (SAC).

The BroccoMax/GRN supplements (530 mg) contained 97.7 ± 6.70 µmol glucoraphanin. The Kwai/alliin supplements (715 mg) contained four garlic-derived metabolites: alliin (35.2 ± 0.52 µmol), γ-SAC (19.3 ± 1.91 µmol), SAC (1.8 ± 0.16 µmol), and allicin (21.4 ± 2.10 µmol).

Mean excretion of sulforaphane and its metabolites as a percentage of ingested glucoraphanin [aka bioavailability] was 56.21% (range 21–91%, SD ± 18.66).

sulforaphane bioavailability

Alliin was detected within the prostate of every participant. Estimation of dietary intake of alliaceous vegetables is challenging due to their widespread presence in processed foods, and it is likely that intake is often underestimated.

We provide evidence that sulforaphane can be detected in human prostate tissue following regular consumption of glucoraphanin supplements. In contrast, alliin and associated metabolites were not more abundant in prostates of men receiving the alliin garlic-derived supplement. It is conceivable that alliin does accumulate in human prostate tissue, but its turnover is much slower than that of sulforaphane so that a longer allium-free diet is required prior to an intervention to assess its accumulation.

Accumulation of sulforaphane and presence of alliin in prostate tissue, as demonstrated in this study, may result in local effects on healthy and cancerous cells through a variety of mechanisms. This may explain the reduced risk of prostate cancer incidence and progression following consumption of cruciferous and alliaceous vegetables.”

https://www.mdpi.com/2072-6643/14/16/3263/htm “Accumulation of Sulforaphane and Alliin in Human Prostate Tissue”


Epigenetic clocks so far in 2022

2022’s busiest researcher took time out this month to update progress on epigenetic clocks. If I curated every study he’s contributed to, it would require at least three blog posts a week. I’ll link to a few he’s posted in August 2022 that are more appreciated in the researcher community.

“In my lab, we are looking for clocks that apply to multiple species at the same time, for example, universal pan-mammalian clocks. It’s all about enhancing translation.

If you have an intervention that rejuvenates a mouse, a rat, a dog, and a cat according to the same clock, then chances are high that it will also work in humans. Naked Mole-Rat Hyaluronan Synthase 2 Promotes Longevity and Enhances Healthspan in Mice

Several groups, including mine, are working on single cell methylation clocks. Researchers are building clocks that respond to lifestyle interventions, such as exercise.

Moving away from methylation, it would be nice to build similar clocks for other ‘omics’ data. Many researchers build clocks on the basis of other omics data, such as for chromatin, proteomics, and gene expression.

There are different platforms, but they all attempt to measure the same thing: biological age. LINE-1 RNA causes heterochromatin erosion and is a target for amelioration of senescent phenotypes in progeroid syndromes

Epigenetic clocks are ‘life course clocks.’ I don’t know any other biomarkers of aging that applies to fetal tissues as well, because most other biomarkers measure organ dysfunction. Epigenetic profiling and incidence of disrupted development point to gastrulation as aging ground zero in Xenopus laevis

There’s this company called Intervene Immune, founded by Greg Fahy, and they are using GrimAge and other epigenetic clocks in clinical trials. They are doing a Phase II clinical trial. By the way, I’m one of the participants.

I could name several other groups who are using epigenetic clocks in clinical trials. It would be interesting if more people would measure epigenetic age in clinical trials in humans, at least as a secondary outcome, because there’s always an opportunity to make a discovery.

If you compare GrimAge to other biomarkers, such as cholesterol or glucose levels, you will see similar noise levels there. Epigenetic clocks are remarkably robust compared to what else is used in the clinic. I would say that the issue with technical noise in epigenetic clocks has been solved.

I’m really glad that different companies and researchers pursue different avenues, since it diversifies our risk. If one of these approaches works, it will change the world.”

https://www.lifespan.io/news/steve-horvath-on-the-present-and-future-of-epigenetic-clocks/ “Steve Horvath on the Present and Future of Epigenetic Clocks”


Non-patentable boron benefits

To follow up Is boron important to health? I’ll highlight a 2022 review of boron intake:

“Boron is essential for activity of several metabolic enzymes, hormones, and micronutrients. It is important for growth and maintenance of bone, reduction in inflammatory biomarkers, and increasing levels of antioxidant enzymes.

The average person’s daily diet contains 1.5 to 3 milligrams of boron. Boron intakes of 1–3 mg/day have been shown to improve bone and brain health in adults when compared to intakes of 0.25–0.50 mg/day.

One week of 10 mg/d boron supplementation resulted in a 20% reduction in inflammatory biomarkers TNF-α, as well as significant reductions (nearly 50%) in plasma concentrations of hs-CRP and IL-6. Calcium fructoborate, a naturally occurring, plant-based boron-carbohydrate complex, had beneficial effects on osteoarthritis (OA) symptoms. A double-blind study in middle-aged patients with primary OA found that all groups except the placebo group saw a reduction in inflammatory biomarkers after 15 days of food supplementation with calcium fructoborate.

Dietary boron intake significantly improves brain function and cognitive functioning in humans. Electroencephalograms showed that boron pharmacological intervention after boron deficiency improved functioning in older men and women, such as less drowsiness and mental alertness, better psychomotor skills (for example, motor speed and dexterity), and better cognitive processing (e.g., attention and short-term memory). Boron compounds can help with both impaired recognition and spatial memory problems.

We discussed the role of boron-based diet in memory, boron and microbiome relation, boron as anti-inflammatory agents, and boron in neurodegenerative diseases. Boron reagents will play a significant role to improve dysbiosis.”

https://www.mdpi.com/1420-3049/27/11/3402/htm “The Role of Microbiome in Brain Development and Neurodegenerative Diseases”


Findings, or fun with numbers?

This 2022 rodent study investigated bone mass phenotypes and sulforaphane:

“Mouse strains can have divergent basal bone mass, yet this phenotype is seldom reflected in the design of studies seeking to identify new modulators of bone resorption by osteoclasts. Sulforaphane exerts inhibitory effects on in vitro osteoclastogenesis in cells from C57BL/6 mice. We explore whether a divergent basal bone mass in different mouse strains is linked both to in vitro osteoclastogenic potential and to SFX-01 sensitivity.

osteoclasts in three mouse strains

Powerful antioxidants are an alternative to achieve beneficial bone effects and avoidance of osteoporotic bone loss. Sulforaphane (SFN) is a natural antioxidant found at high levels (as glucoraphanin) in cruciferous vegetables. SFN activates the NRF2 pathway and has anti-inflammatory effects, protecting against oxidative stress in many cell types.

These findings suggest that BM cells derived from animals with a high in vivo bone mass are less sensitive to M-CSF and RANKL in vitro leading to lower osteoclastogenesis. They also support the hypothesis that similar sensitivity extends to inhibitory effects of SFX-01 on osteoclast formation/function.

It is important to stress that osteoclasts generated in these strains may simply undergo multinucleation in a manner related to their underpinning genetics, and that by coincidence alone this is matched to their bone mass.”

https://onlinelibrary.wiley.com/doi/10.1002/cbf.3734 “High bone mass in mice can be linked to lower osteoclast formation, resorptive capacity, and restricted in vitro sensitivity to inhibition by stable sulforaphane”

I curated this study primarily for its honesty. I’ll link this post to future posts of studies where researchers lack similar honesty.


Broccoli sprouts and your brain

A 2022 review of Nrf2 signaling hilariously avoided mentioning sulforaphane, although of ~4,000 sulforaphane published articles, two were cited. I’ll curate it anyway to highlight referenced brain effects.

“A good stability of NRF2 activity is crucial to maintain redox balance and therefore brain homeostasis. In this review, we have gathered recent data about the contribution of the NRF2 pathway in the healthy brain as well as during metabolic diseases, ageing, and ageing-related neurodegenerative diseases.

A functional NRF2 system is important to regulate both neuroinflammation, i.e., activation of microglia and astrocytes, and oxidative stress in the brain. NRF2 and NF-κB transcription factors regulate cellular responses to inflammation and oxidative stress in order to maintain brain homeostasis. Both pathways have been described to inhibit each other.

Nrf2 brain aging

Future challenges will be to establish novel therapies to:

  • Increase NRF2 activation in specific cell types and/or brain regions; and
  • Modulate NRF2 pathway in senescent cells.

Modulation of NRF2 signalling pathway by using specific food products [like unmentioned broccoli sprouts] and phytochemicals [like unmentioned sulforaphane], dietary supplements [like unmentioned Vitamin D3], drugs, and epigenetic modifiers, alone or in combination, will help to limit inflammatory diseases, ageing process, and subsequently ageing-related diseases.”

https://www.mdpi.com/2076-3921/11/8/1426/htm “Normal and Pathological NRF2 Signalling in the Central Nervous System”


Eat broccoli sprouts for your offspring

This 2022 rodent study investigated effects of glucoraphanin supplementation during pregnancy and lactation:

“We investigated whether dietary intake of sulforaphane glucosinolate (SGS [properly termed glucoraphanin]) during pregnancy and lactation influenced composition of gut microbiota in offspring:

  • Dietary intake of SGS during pregnancy and lactation caused significant changes in diversity of gut microbiota in 3-week-old offspring (SGS-3W) and 10-week-old offspring (SGS-10W).
  • Plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in SGS-10W after injection of lipopolysaccharide were significantly lower than those of CON-10W group.
  • There were sex differences of gut microbiota composition in both SGS-3W and SGS-10W offspring.

glucoraphanin during pregnancy and lactation

This study has some limitations:

  1. We did not investigate mechanisms of how dietary intake of SGS during pregnancy and lactation modulated gut microbial communities in offspring.
  2. We found several signaling pathways in beneficial effects of SGS food pellet, and further study of the role of maternal intake of SGS food in these pathways is needed.
  3. We did not investigate mechanisms of relationships between maternal intake of SGS and long-term anti-inflammatory action in adult offspring, and further detailed study including epigenetic modification is needed.

These data suggest that dietary intake of SGS during pregnancy and lactation might produce long-lasting beneficial effects in adult offspring through persistent modulation of gut microbiota. It is likely that modulation of gut microbiota by maternal nutrition may confer resilience versus vulnerability to stress-related psychiatric disorders in offspring.”

https://www.sciencedirect.com/science/article/pii/S0955286322001681 “Long-lasting beneficial effects of maternal intake of sulforaphane glucosinolate on gut microbiota in adult offspring”

This study published results of a mother’s glucoraphanin intake where offspring never ate glucoraphanin, with beneficial effects at both 3 weeks (~prepubescent human) and 10 weeks (~young human adult). Maybe future studies will continue this paradigm on to a second or third generation to see whether there are also transgenerational epigenetic effects.

This study’s methods extracted glucoraphanin from 1-day-old broccoli sprouts into a powder containing 135 mg (0.31 mmol) glucoraphanin per gram. Each 1 kg of of treatment chow included pellets containing (2.3 mmol / 0.31 mmol) x 135 mg = 1 gram of broccoli sprout powder, 0.1% of food intake.

Per Drying broccoli sprouts, dried 3-day-old broccoli sprouts contain 10% moisture, and fresh 3-day-old broccoli sprouts contain 82.6% moisture. A gram of 1-day-old broccoli sprout powder may be an approximate equivalent of (.826 / .1) = 8 grams fresh 3-day-old broccoli sprouts for a mouse / kg of daily food intake. A human equivalent dose is (.826 / .1) x .081 x 70 kg = 47 grams of fresh 3-day-old broccoli sprouts / kg of daily food intake.

That’s about how much 3-day-old, microwaved, glucoraphanin-containing broccoli and red cabbage sprouts I eat every day, starting from 7.2 grams of seeds. I sprout another 3.5 grams of yellow mustard seeds into the mixture for taste.


Week 120 of Changing to a youthful phenotype with sprouts

It was time for an annual physical last Wednesday. My focus was to see whether reducing sulforaphane intake per Week 87 had the desired effect on thyroid measurements.

That and other adjustments did! Readings of TSH 2.91 (0.45 – 4.50 uIU/mL), free T4 1.22 (0.82 − 1.77 ng/dL), and free T3 2.4 (2.0 – 4.4 pg/mL) were all in-range. 🙂


I won’t repeat the Week 63 workbook calculations done after last year’s annual physical. To me, that’s another form of magical thinking.

Every one of those reference ranges, and optimal ranges built from all-cause mortality statistics, requires a suffix “of people who didn’t positively change their healthspan and lifespan.” What value is there in optimizing (pick a measurement) against those outcomes? Why compare my efforts, or results, or any other aspect of my life, to people who didn’t actionably care about their one precious life?

I’m not deflecting with poor measurements:

  • 3 of the 5 values in last year’s optimal ranges got better, and the other 2 stayed the same; and
  • 2 of the 4 values that weren’t in last year’s optimal ranges came into those ranges, and the other 2 got better but stayed outside an optimal range.

We each have a lot at stake. Bad things like diseases of old age happen on their own. If we want good things to happen, we have to make them happen.

Consider this from The impact of transgenerational epigenetic inheritance and early life experiences:

“Every disease is connected to the immune system.”

Are people making good choices every day for their immune systems?


Eat broccoli sprouts for metabolic syndrome

This 2022 rodent study investigated sulforaphane’s effects on insulin resistance:

“Insulin resistance is one of the defining clinical traits of metabolic syndrome, which represents a constellation of metabolic disorders, mainly comprising obesity, type 2 diabetes mellitus, atherogenic dyslipidemia, and hypertension. This study aimed to investigate therapeutic effects and potential mechanisms of sulforaphane (SFN) on high-fat diet (HFD)-induced insulin resistance. Control chow diet was 10 kcal% fat, and HFD was 60 kcal% fat.

  • SFN was found to effectively reduce body weight, fasting blood glucose, and hyperlipidemia, and improve liver function in HFD-fed mice.
  • SFN led to increased expression of antioxidant genes downstream of Nrf2, and decreased accumulation of lipid peroxides MDA and 4-HNE.
  • SFN significantly reduced glutathione peroxidase 4 (GPx4) inactivation-mediated oxidative stress by activating the AMPK and Nrf2 signaling pathways.

Data suggested that GPx4 could be a key target through which SFN might activate Nrf2/ARE signaling pathway to decrease the extent of insulin resistance induced in HFD-fed mice. Taken together, SFN ameliorated HFD-induced insulin resistance by activating the AMPK-Nrf2-GPx4 pathway, providing new insights into SFN as a therapeutic compound for alleviation of insulin resistance.”

https://www.sciencedirect.com/science/article/pii/S075333222200662X “Sulforaphane alleviates high fat diet-induced insulin resistance via AMPK/Nrf2/GPx4 axis”

This study’s sulforaphane dose was the same as Eat broccoli sprouts for your heart and Broccoli sprouts activate the AMPK pathway at 0.5 mg / kg. It was administered five times a week for 8 weeks to a subgroup of HFD-fed mice starting after 8 weeks of HFD.

A human equivalent oral dose to these three studies’ subcutaneous doses would be a low (0.5 mg x .081) x 70 kg = ~3 mg sulforaphane. Per Eat broccoli sprouts for your gut, my sulforaphane intake is six times that every day.


Is boron important to health?

Three papers on boron, starting with a 2022 review:

“Boron-containing compounds (BCC) have effects in the metabolism of living organisms. Information regarding effects and interaction of these compounds was compiled, and potential applications for treating human metabolic disorders was suggested.

Dietary boron supplementation affects metabolism of calcium, magnesium, triglycerides, glucose, amino acids, reactive oxygen, nitrogen species, and hormones such as 17β-estradiol, calcitonin, and 25-hydroxy-cholecalciferol. When food is boron-deprived, there are adverse effects like depressed growth, reduced serum steroid hormone concentrations, changes in plasma and organ calcium and magnesium concentrations, plasma alkaline phosphatase, and bone calcification on animal biological functions.

boron effects

Exploration of basic BCC as metabolism regulators is expanding. Although mechanisms of action are uncertain, limitation of damage induced by reactive species, inflammatory modulation, or activities on some enzymes and membrane transporters are often related to reported effects.

An increasing number of new BCC are emerging as potential tools for prevention, diagnosis, and therapy of metabolism maladies such as diabetes, metabolic syndrome, osteoporosis, cardiovascular, and liver diseases. For those innovative BCC, mechanisms of action are often clear.”

https://link.springer.com/article/10.1007/s12011-022-03346-9 “Boron‑Containing Compounds for Prevention, Diagnosis, and Treatment of Human Metabolic Disorders” (not freely available) Thanks to Dr. Marvin A Soriano-Ursúa for providing a link to a freely available document.

A second paper was a 2021 human study:

“In our elderly population-based sample, a boron-rich diet appeared to be characterized by high intakes of plant foods presumed to be healthy, low intakes of plant foods presumed to be less healthy, and low intakes of all kinds of animal foods.

Higher plasma boron concentrations were related to lower BMI and circulating concentrations of CRP. Plasma boron concentrations were associated with age, phosphate, and plasma lipid metabolism, and showed seasonal variations.

Human intervention studies are warranted to derive causal relationships of circulating and dietary boron with human health and metabolism. Robust databases on boron content of foods are needed to facilitate investigation of dietary boron intake in human studies.

Clarification of the non-/essentiality of trace element boron for human health will form the basis to derive recommendations for a dietary boron intake being sufficient to exert boron’s proposed beneficial physiological roles.”

https://link.springer.com/article/10.1007/s00394-021-02730-w “Plasma boron concentrations in the general population: a cross-sectional analysis of cardio-metabolic and dietary correlates”

As noted in this study, public agencies don’t consider dietary boron content important enough to include in public databases. My daily boron dietary intake estimated from published private databases is:

  • Walnuts, 1.63 mg x (28.3 g / 100 g) = .5 mg
  • Red kidney beans, 1.4 mg x (12 g / 100 g) = .2 mg
  • Chickpeas, 0.71 mg x (40 g / 100 g) = .3 mg
  • Celery, 0.5 mg x (72 g / 100 g) = .4 mg
  • Carrots, 0.3 mg x ( 76 g / 100 g) = .3 mg
  • Coffee .07 mg x 3 cups = .2 mg

2 mg boron daily dietary total

A third paper was a 2022 rodent study:

“Sodium pentaborate pentahydrate (NaB) 1 and 2 mg elemental B/kg supplementation induces the anagen phase in rats via Wnt-1, β-catenin, VEGF, PDGF, and TGF-β1 signaling pathways, which are important molecular mechanisms involved in hair growth.

NaB 4 mg B/kg suppresses these pathways and adversely affects hair growth.”

https://www.sciencedirect.com/science/article/abs/pii/S0946672X22000876 “Sodium pentaborate pentahydrate promotes hair growth through the Wnt/β-catenin pathway and growth factors” (not freely available)

A human equivalent of this study’s rat 1 mg elemental boron intake is (1 mg x .162) x 70 kg = 11 mg.


Trained immunity epigenetics

Two papers on trained immunity, starting with a 2022 review:

“Live attenuated vaccines such as the Bacillus Calmette–Guérin, measles-containing vaccines, and the oral polio vaccine have been shown to reduce overall mortality beyond their effects attributable to the targeted diseases.

After an encounter with a primary stimulus, epigenetic and metabolic reprogramming of bone marrow progenitor cells and functional changes of tissue immune cell populations result in augmented immune responses against a secondary challenge. This process has been termed trained immunity.

Main epigenetic events during induction of trained immunity are:

  1. Chromosomal reorganization on the level of topologically associated domains;
  2. Induction of long noncoding RNA activity;
  3. Histone modifications and chromatin accessibility; and
  4. DNA (de)methylation.

trained immunity mechanisms

An epigenetic enzyme belonging to the lysine methyltransferase family, Set7, possesses vital function in β-glucan training of monocytes. When inhibited, trained immunity phenotype is diminished, while Set7 deficient mice cannot establish innate immune memory.

β-glucan is recognized by Dectin-1, and has been known to lead to a shift from oxidative phosphorylation (OXPHOS) to glycolysis as an ATP source. However, a more recent study reported an increase in both glycolysis and oxygen consumption following training, which signals a higher rate of OXPHOS. This discrepancy is explained by the difference in concentration of β-glucan used in the experiments.

Stopping vaccination with measles and polio once the pathogens are eradicated, or replacing live attenuated polio with inactivated polio, should be done with caution, as it may have a substantial impact on childhood mortality. Trained immunity may also represent an important new approach to improve current vaccines, or to develop novel vaccines that combine induction of classical adaptive immune memory and innate immune memory.”

https://www.sciencedirect.com/science/article/pii/S0952791522000371 “Trained immunity: implications for vaccination”

Reference 34 was a 2020 study by two of the same coauthors that provided details on the above discrepancy:

“Findings presented by the current study suggest that the disparity in terms of the role of OXPHOS arises from the stimulatory dose of β-glucan [by intraperitoneal injection]. A β-glucan concentration of 1 μg/mL induces both glycolysis and OXPHOS, whereas a concentration of 10 μg/mL induces glycolysis but inhibits OXPHOS.”

https://www.cell.com/cell-reports/fulltext/S2211-1247(20)30458-7 “The Set7 Lysine Methyltransferase Regulates Plasticity in Oxidative Phosphorylation Necessary for Trained Immunity Induced by β-Glucan”


The goddess of rainbows

Two 2022 papers, starting with a review of irisin:

“This article is an overview of irisin generation, secretion, and tissue distribution. Its targeting of tissues or organs for prevention and treatment of chronic diseases is systematically summarized, with discussion of underlying molecular mechanisms.

Irisin is an exercise-induced myokine expressed as a bioactive peptide in multiple tissues and organs. Exercise and cold exposure are major inducers for its secretion.

Mechanistic studies confirm that irisin is closely correlated with lipid metabolism, insulin resistance, inflammation, ROS, endocrine, neurotrophic factors, cell regeneration and repairing, and central nervous system regulation. Irisin decreases with age, and is closely associated with a wide range of aging-related diseases.

A number of studies in elderly humans and animal models have shown that exercise can promote the body’s circulation and increase irisin levels in some tissues and organs. Resistance, aerobic, or combined exercise seem to play a positive role. However, exercise could not change serum irisin in some reported studies.

irisin human studies

There are large individual differences in exercise training in the elderly population. Since the half-life of irisin in the body is less than 1 h, it is necessary to pay attention to the time of blood sampling after a single exercise intervention. Some factors that impede detection of irisin levels in vivo include the half-life of irisin protein, sampling time, different tissues, and different health statuses before and after intervention.

It is worth noting that high-intensity exercise shows higher irisin levels even with the same energy expenditure during exercise. Precision studies of irisin in elderly subjects following exercise intervention need to be further clarified.”

https://www.sciencedirect.com/science/article/pii/S1568163722001222 “Irisin, An Exercise-induced Bioactive Peptide Beneficial for Health Promotion During Aging Process” (not freely available) Thanks to Dr. Ning Chen for providing a copy.

A second paper was a human study too recent to be cited by the first paper. I’ll highlight its irisin findings:

“We investigated the complex relationship among DNAm based biomarkers of aging, including DNAmFitAge, a variety of physiological functioning variables, blood serum measures including cholesterol, irisin level, and redox balance, and the microbiome on 303 healthy individuals aged between 33 and 88 years with a diverse level of physical fitness. Regular exercise was associated with younger biological age, better memory, and more protective blood serum levels.

Our research intends to show that regular physical exercise is related to microbiota and methylation differences which are both beneficial to aging and measurable. Our research provides the first investigation between microbiome derived metabolic pathways and DNAm based aging biomarkers.

Irisin levels decrease with age (0.23 average decrease for every 1 year older). We found age-related decreases in irisin levels were attenuated by exercise training. The link between irisin to GrimAge Acceleration and FitAge Acceleration is a novel observation.

HDL is positively associated with irisin. HDL and irisin have complex roles in physiology, and the positive relationship we observe between physical exercise and HDL and irisin align with protective effects seen between HDL and irisin with glucose homeostasis.

This work further supports the biological importance of irisin to the aging process. It is possible our research motivates interventions to boost irisin, like through physical exercise, as possible anti-aging therapies.”

https://www.medrxiv.org/content/10.1101/2022.07.22.22277842v1 “DNA methylation clock DNAmFitAge shows regular exercise is associated with slower aging and systemic adaptation


Variable aging measurements

Two papers on aging measurements, starting with a 2022 human study:

“We collected longitudinally across the adult age range a comprehensive list of phenotypes within four domains (body composition, energetics, homeostatic mechanisms and neurodegeneration / neuroplasticity) and functional outcomes. We integrated individual deviations from population trajectories into a global longitudinal phenotypic metric of aging.

blsa participant ages

We demonstrate that accelerated longitudinal phenotypic aging is associated with faster physical and cognitive decline, faster accumulation of multimorbidity, and shorter survival.”

https://www.nature.com/articles/s43587-022-00243-7 “Longitudinal phenotypic aging metrics in the Baltimore Longitudinal Study of Aging”

I disagree with this study’s methodology.

1. Although it acknowledged individual variability, nothing was done to positively adjust to those facts. What could have been done per A review of biological variability was:

“Obtain a measurement of variability that is independent of the mean to ensure to not confound changes in variability with shifts in mean.”

2. A usual research practice is to take at least three measurements, and use their average as representative. That wasn’t done here, maybe because of time and expense considerations?

3. An important measurement for physical function was the time to finish a 400 meter walk. I walk more than ten times that almost every day. I use the first 400 meters as a warmup period while getting to the beach to walk eastward and enjoy the predawn light and water animal activity. I concentrate on gait speed during the last third while walking westward on a straightaway bike path.

This study would measure my gait speed as a sometimes old person during the first 400 meters, rather than a gait speed that usually approaches a young person’s during the last 400 meters. Even if I tried to walk my fastest right out of the gate, I wouldn’t be surprised to find a decade or two difference by this study’s measurements between a morning walk’s first and last 400 meter gait speeds.

4. An important cognitive function measurement was the Digital Symbol Substitution Test, apparently taken during subjects’ fasted state? Sometimes after exercising, I’m okay cognitively when starting work in a fasted state at 6:30 a.m., and other times I’m tired.

Two days ago during the last hour of work 1:30-2:30 p.m., I did outstanding work, four hours after eating whole oats for breakfast, and after drinking two coffees and three teas. I took time to put together pieces of puzzles into proper contexts for management’s attention. My bosses weren’t too pleased with the story it told, but it is what it is.

5. Are measurements of how you start what matters? Or is it how you finish, as is common in competitive sports?

This study would measure my cognitive function as a sometimes old person, rather than performance that approaches a young person’s later in the workday. For both physical and cognitive function, my abilities to ramp up and come close to young people’s capabilities are features that I work on, not random, inconvenient measurement variability.

6. Blood measurements were downgraded as having “limited coverage of the four phenotypic domains.” These were taken to fit into specific paradigms and epigenetic clocks. They predictably failed to show causality, as acknowledged with:

“Our analysis showed strong associations between global longitudinal phenotypic score and changes in physical and cognitive function. We did not have sufficient observations to fully separate these two dimensions over time, which would have strengthened the assumption of causality.”

Nowhere in this study was it hinted that all measurements were downstream effects of unmeasured causes. A follow-on study could reanalyze these subjects’ blood samples, MRI, and other measurements for originating upstream factors of signaling pathways and cascades per Signaling pathways and aging and An environmental signaling paradigm of aging.

Reference 35 of this first study was a 2021 human and rodent study that was tossed in as a limitation with:

“We might not have all of the relevant phenotypic measures (for example, more detailed immune profiles) for all participants.”

Its findings included:

“From the blood immunome of 1,001 individuals aged 8–96 years, we developed a deep-learning method based on patterns of systemic age-related inflammation. The resulting inflammatory clock of aging (iAge) tracked with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians.

Canonical markers of acute infection such as IL-6 and tumor necrosis factor-α were not major contributors to iAge, indicating that, except for IL-1β, infection-driven inflammatory markers of the acute inflammatory response do not contribute to age-related chronic inflammation.

We conducted a follow-up study in an independent cohort of 97 extremely healthy adults (aged 25–90 years) matched for cardiovascular risk factors (including conserved levels of high-sensitivity C-reactive protein), selected from a total of 151 recruited participants using strict selection criteria. In this healthy cohort, inflammation markers were measured using a 48-plex cytokine panel. Only 6 circulating immune proteins were significantly correlated with age, with CXCL9 again the largest contributor to age-related inflammation.

CXCL9 is a T-cell chemoattractant induced by IFN-γ and is mostly produced by neutrophils, macrophages and endothelial cells (ECs). We find that CXCL9 is mainly produced by aged endothelium and predicts subclinical levels of cardiovascular aging in nominally healthy individuals.

We did not find any significant correlation between known disease risk factors reported in the study (BMI, smoking, dyslipidemia) and levels of CXCL9 gene or protein expression. We hypothesize that one root cause of CXCL9 overproduction is cellular aging per se, which can trigger metabolic dysfunction.

As ECs but not cardiomyocytes expressed the CXCL9 receptor, CXCR3, we hypothesize that this chemokine acts both in a paracrine fashion (when it is produced by macrophages to attract T cells to the site of injury) and in an autocrine fashion (when it is produced by the endothelium) creating a positive feedback loop. In this model, increasing doses of CXCL9 and expression of its receptor in these cells leads to cumulative deterioration of endothelial function in aging.

IFN-γ did not increase in expression in our cellular aging RNA-seq experiment, suggesting that there are triggers of CXCL9 (other than IFN-γ) that play a role in cellular senescence in the endothelium that are currently unknown. However, in our 1KIP study, IFN-γ was in fact the second-most important negative contributor to iAge, which could be explained by the cell-priming effect of cytokines, where the effect of a first cytokine alters the response to a different one.

iAge derived from immunological cytokines gives us an insight into the salient cytokines that are related to aging and disease. A notable difference compared to other clocks is that iAge is clearly actionable as shown by our experiments in CXCL9 where we can reverse aging phenotypes. More practical approaches range from altering a person’s exposomes (lifestyle) and/or the use of interventions to target CXCL9 and other biomarkers described here.

Our immune metric for human health can identify within healthy older adults with no clinical or laboratory evidence of cardiovascular disease, those at risk for early cardiovascular aging. We demonstrate that CXCL9 is a master regulator of vascular function and cellular senescence, which indicates that therapies targeting CXCL9 could be used to prevent age-related deterioration of the vascular system and other physiological systems as well.”

https://www.nature.com/articles/s43587-021-00082-y “An inflammatory aging clock (iAge) based on deep learning tracks multimorbidity, immunosenescence, frailty and cardiovascular aging”


Beneficial dietary erucic acid?

A 2022 review to follow up Caution on broccoli seed erucic acid content?:

“Erucic acid is found to cause cardiac lipidosis in young animals, yet direct evidence of cardiac injury does not exist for young humans. Concerns about erucic acid safety and cardiotoxicity have been published in the press which are based on scientific reports in the 1970s that erucic acid disrupted oxidative phosphorylation and lead to accumulation of lipids in rat cardiac tissue.

Spanish toxic oil syndrome was a major concern, leading to questions about erucic acid cardiotoxicity. Yet it was found that not rapeseed oil per se, rather its carcinogen anilin-dye refined derivative caused cardiotoxicity.

Later, it was understood that reduced ATP production with erucic acid treatment was due to unapt isolation of rat cardiac mitochondria and lipid accumulation that was unique to rats that inherently harbour a low β-oxidative peroxisomal activity and tissue-specific metabolism of erucic acid. Similar structural or metabolic perturbations and tissue injuries were not encountered in monkeys, humans, and pigs.

Potential mechanisms regarding antineoplastic effects of erucic acid in brain tumors:

erucic acid

In children (0 to 14 years), medulloblastomas accounted for less than 10% of brain neoplasias in China, African countries, and Ireland. The ratio was in the range 20%–29% in Brazil, Argentina, Thailand, Korea and Poland, the proportion was 30% in Ecuador, 31% in Taiwan and Jordan.

In adults, the ratio of brain neoplasias diagnosed as glioblastoma was:

  • Below 10% only in China;
  • In the range 10%–29% in India, Thailand, Malaysia, Nigeria, Algeria, Malta, Costa Rica, Ecuador, and the Russian Federation;
  • In the range 30%–49% in some South American countries, Singapore, Taiwan, Japan, Korea, Turkey, Denmark, Iceland, Italy, and Spain among others; and
  • In the range 50%–70% in North America, Puerto Rico, Martinique, Israel, Cyprus, Jordan, Kuwait, and in Oceania.

The low ratio of medulloblastomas in children and of glioblastomas in adult Chinese population cannot be easily attributed to a single genetic and nurture pattern. Very likely, many complex factors interact to explain this difference regarding the Chinese population.

Several hypotheses can be put forward to illuminate the cause of reduced ratios of high grade brain tumors in Chinese which would be of benefit for global reduction and prevention of brain tumors. Erucic acid is very highly consumed in the Chinese diet, and 8-fold higher erucic acid levels exist in Chinese women’s milk in comparison to many other countries.

We hypothesized that dietary erucic acid may be – at least among many factors – associated with reduced ratios of high grade brain tumors in Chinese. If epidemiological and animal studies would prove such an association, an effective, cheap, and relatively non-toxic dietary supplementary strategy may be employed to prevent brain tumors at erucic acid doses lower than those associated with any cardiotoxic effects.”

https://link.springer.com/article/10.1007/s11011-022-01022-4 “Could dietary erucic acid lower risk of brain tumors? An epidemiological look to Chinese population with implications for prevention and treatment” (not freely available) Thanks to Dr. Meric Altinoz for providing a copy.


Non-CpG methylation

Three 2022 papers on methylation epigenetic modifiers, starting with a human study focused on mitochondrial DNA non-CpG methylation involving nucleobases other than guanine (arginine, cytosine, or thymine):

“We collected brain tissue in the nucleus accumbens and prefrontal cortex from deceased individuals without (n = 39) and with (n = 14) drug use, and used whole-genome bisulfite sequencing to cover cytosine sites in the mitochondrial genome. Epigenetic clocks in illicit drug users, especially in ketamine users, were accelerated in both brain regions by comparison with nonusers.

Unlike the predominance of CpG over non-CpG methylation in the nuclear genome, the average CpG and non-CpG methylation levels in the mitochondrial genome were almost equal. The utility of non-CpG methylation was further illustrated by the three indices constructed in this study with non-CpG sites having better distinction between brain areas, age groups, and the presence or absence of drug use than indices consisting of CpG sites only. Results of previous studies on the mitochondrial genome that were solely based on CpG sites should be interpreted cautiously.

The epigenetic clock made up of age-related cytosine sites in mtDNA of the control group was consistently replicated in these two brain regions. One possibility for the correlation is the cycle theory that involves mitochondrial activity, mitochondrial DNA methylation, and alpha-ketoglutarate.

As mitochondrial activity fades with aging, mitochondria gradually lose the ability to eliminate methylation on cytosines through alpha-ketoglutarate. Further investigation of the underlying mechanisms is warranted.

To our knowledge, this is the first report that ketamine might change the mitochondrial epigenetic clock in human brain tissues. We believe this is the first report to elucidate comprehensively the importance of mitochondrial DNA methylation in human brain.”

https://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-022-01300-z “Mitochondrial DNA methylation profiling of the human prefrontal cortex and nucleus accumbens: correlations with aging and drug use”

A second rodent study focused on RNA methylation:

“We investigated the role of RNA N6-methyladenosine (m6A) in improved resilience against chronic restraint stress. A combination of molecular, behavioral, and in vivo recording data demonstrates exercise-mediated restoration of m6A in the mouse medial prefrontal cortex, whose activity is potentiated to exert anxiolytic effects. To provide molecular explanations, it is worth noting that epigenetic regulation, such as histone modification, microRNA, and DNA methylation all participate in mental and cognitive rehabilitation following exercise.

To generalize these rodent data to humans, we recruited a small group of patients with major depressive disorder with prominent anxiety disorders. Compared to age- and sex-matched healthy individuals, patients displayed decreased circulating methyl donor S-adenosyl methionine (SAM) levels. Serum SAM levels were found to be inversely correlated with the Hamilton Anxiety Scale, suggesting the potential value of SAM as a biomarker for depression or anxiety disorders.

Hepatic biosynthesis of methyl donors is necessary for exercise to improve brain RNA m6A to counteract environmental stress. The dependence on hepatic-brain axis suggests the ineffectiveness of exercise training on people with hepatic dysfunctions.

This novel liver-brain axis provides an explanation for brain network changes upon exercise training, and provides new insights into diagnosis and treatment of anxiety disorders. Exercise-induced anxiolysis might be potentiated by further replenishment of RNA methylation donors, providing a strategy of exercise plus diet supplement in preventing anxiety disorders.”

https://onlinelibrary.wiley.com/doi/10.1002/advs.202105731 “Physical Exercise Prevented Stress-Induced Anxiety via Improving Brain RNA Methylation”

A third paper was a review of mitochondrial-to-nuclear epigenetic regulation. I’ll highlight one mitochondrial metabolite, alpha-ketoglutarate (α-KG):

“Apart from established roles in bioenergetics and biosynthesis, mitochondria are signaling organelles that communicate their fitness to the nucleus, triggering transcriptional programs to adapt homeostasis stress that is essential for organismal health and aging. Emerging studies revealed that mitochondrial-to-nuclear communication via altered levels of mitochondrial metabolites or stress signals causes various epigenetic changes, facilitating efforts to maintain homeostasis and affect aging.

Metabolites generated by the tricarboxylic acid (TCA) cycle, the electron transport chain (ETC), or one-carbon cycle within mitochondria can act as substrates or cofactors to control epigenetic modification, especially histone acetylation and methylation and DNA methylation. α-KG produced in the TCA cycle serves as an essential cofactor for the chromatin-modifying Jumonji C (JmjC) domain-containing lysine demethylases (JMJDs) and ten-eleven translocation (TETs) DNA demethylases. Changes in α-KG levels are capable of driving nuclear gene expression by affecting DNA and histone methylation profiles.


α-KG deficiency in progenitor stem cells increases with age. For example, the level of α-KG is reduced in follicle fluids of aged humans, and supplementation with α-KG preserves ovarian function in mice.

α-KG extends lifespan in Drosophila by activating AMPK signaling and inhibiting the mTOR pathway. Supplementing α-KG in the form of a calcium salt promoted a longer and healthier life associated with decreased levels of inflammatory cytokines in old mice.

A human study showed a nearly 8-year reversal in DNA methylation clock biological ages of 42 individuals taking an α-KG based formulation for 4–10 months. α-KG supplementation leads to both demethylation and hypermethylation of some CpG sites in the genome, suggesting that α-KG may have a broader effect on methylation-based aging, such as metabolic functions.

Outstanding questions:

  1. How is production of mitochondrial metabolites regulated both spatially and temporally to elicit epigenetic changes in response to mitochondrial dysfunction?
  2. What are specific epigenetic factors involved in mitochondrial-to-nuclear communications, and how do they cooperate with transcription factors in response to various external and internal stimuli?
  3. Do various mitochondrial metabolites act alone or in concert on the epigenome to regulate the aging process?
  4. Are some organs or tissues more at risk than others in maintaining mitochondrial-to-nuclear communication during aging?
  5. Can intervention of mitochondrial-to-nuclear communications mimic beneficial epigenetic changes to delay aging or alleviate age-onset diseases?”

https://www.sciencedirect.com/science/article/pii/S0968000422000676 “Mitochondrial-to-nuclear communication in aging: an epigenetic perspective”