4-5 stars Advanced knowledge

Advanced knowledge

TMAO and heavy hydrogen

gettingwell4 4-5 stars Advanced knowledge, Acetylcholine, Epigenetics, Neurochemicals, Stress

A 2026 review subject was possible involvement of deuterium in TMAO levels, which contrasted with the usual TMAO meme. I’ll curate this paper through an outline of its sections:

“The human gut microbiome plays many essential roles, but an often-overlooked role is to maintain an abundant supply of deuterium depleted (deupleted) nutrients to fuel the host mitochondria. Excess deuterium (heavy hydrogen) damages mitochondrial ATP synthase nanomotors, leading to a decrease in matrix water production with increased reactive oxygen species (ROS) and inefficient ATP production. A microbial metabolite, trimethylamine N-oxide (TMAO) is a powerful signaling molecule whose plasma levels are high in association with many chronic diseases.

In this paper, we present a hypothesis that TMAO is a marker for deuterium overload in the methylation pathway, in addition to its role as an indicator of a disrupted gut microbiome. The original study that brought attention to TMAO involved feeding mice synthetic choline with fully deuterated methyl groups. Fully deuterated TMAO was subsequently detected in the plasma. By contrast, a diet rich in eggs, a natural source of choline (a precursor to TMAO), does not raise TMAO levels. Many of the pathologies that are linked to elevated TMAO can also be viewed as strategies to promote the supply of deupleted water to the mitochondria, systemically.

1. Introduction – DNA and histone methylation regulate epigenetic modifications and imprinting. Phosphatidylcholine is a precursor to acetylcholine, an excitatory neurotransmitter in the brain. Trimethylated lysine molecules, recovered during protein metabolism, are precursors to L-carnitine, which facilitates the transport of fatty acids into the mitochondria to be oxidized for fuel production. Dietary phosphatidylcholine and dietary L-carnitine, in addition to endogenous sources, are important nutrients that provide methyl groups to the methylation cycle. Choline and L-carnitine, as well as a close relative, betaine (trimethylglycine), are all precursors to trimethylamine (TMA), a small methylated amine produced through microbial enzymatic action. Obligate anaerobic hydrogen-dependent archaea called methylotrophs in the gut can reduce the three methyl groups in TMA to methane gas.

2. Evidence that deuterium disrupts mitochondrial function – Deuterium (2H) is a heavy isotope of protium (1H; hydrogen), and it is pervasive in nature, found in seawater at a concentration of 155 parts per million (ppm) relative to protium. Deuterium is highly damaging to the F1F0-ATP synthase (ATPase) nanomotors in the mitochondria that produce ATP, the primary fuel source of the cell. Deuterium loading suppresses the activity of many fundamental biologically important hydrolytic enzymes that depend on proton tunneling. It is likely that deuterium increases the frequency of unrepaired nuclear DNA mutations, by suppressing the activity of deuterium-sensitive repair enzymes.

The inherent collective proton tunneling (ICPT) process, which uses membrane-bound ATPase nanomotors in living organisms, is nature’s ultimate tool for discriminating hydrogen isotopes. This is because a deuteron (2H) cannot replace a proton (1H) in its tunnel protein during enzymatic transmembrane transport due to its doubled mass and twice larger atomic nuclear size. The result is large compartmental, inter- and intramolecular deuterium disequilibrium in 2H/1H ratios in all biomolecules, which readily distinguishes respiration from aerobic fermentation with adaptive significance. Deuterons irreversibly clog single proton tunneling ATP synthase nanomotors in mitochondria, resulting in the complete breakdown of ICPT.

This initiates many disease-causing molecular crowding mechanisms, which we review herein from the perspective of prokaryotic proton pumping and H2 gas formation in the organic molecular realm of mitochondrial proton-donating substrates. Understanding at the systems level how humans protect mitochondrial ICPT processes and ATP synthase is a fascinating journey reviewed herein. We hypothesize that the process uses TMAO as the microbial stepping stone, employing deuterium discrimination to become an active player in forming the biological reaction.

2.1 Quantum tunneling and proton-coupled electron transport – ICPT is a theoretical quantum mechanical phenomenon proposing that a single proton spontaneously passes through a potential energy barrier, typically within a hydrogen bond, in a manner that can be functionally irreversible. Unlike classical particles that must surmount energy barriers, protons can ‘tunnel’ through them due to their wave-like nature. Often, this single proton tunneling is part of a larger process where a proton and an electron are transferred simultaneously (or sequentially) as a single kinetic step, often in the presence of strong electric fields that stabilize the transferred state. This process is referred to as ‘proton-coupled electron transport’ (PCET).

Mitochondria exploit PCET to build the proton gradient that powers the nanomotors to produce ATP in the electron transport chain (ETC). Many enzymes, such as dehydrogenases and lipoxygenases, exploit proton tunneling to carry out their reactions. Deuterons are much less capable of tunneling, so this becomes a way to select for substrates containing protons rather than deuterons. The very large kinetic isotope effect (KIE) for soybean lipoxygenase is an example of this phenomenon.

NADH-ubiquinone oxidoreductase (Complex I) couples the transfer of two electrons between NADH and ubiquinone to the translocation of four protons across the membrane. This process provides the driving force for ATP synthase, which harnesses the gradient to produce ATP, but it also assures that few, or no, deuterons arrive on the other (intermembrane) side of the membrane, protecting the ATPase nanomotors.

SAMe, the universal methyl donor, plays a crucial role in regulating oxidative phosphorylation (OXPHOS). It is primarily synthesized in the cytoplasm and imported into the mitochondria via the import protein SAMC. SAMC is the only mitoSAM carrier and is required for OXPHOS and oxidative tricarboxylic acid (TCA) metabolism, showing a strong dependency of mitochondrial health on one-carbon (1C) metabolism. We hypothesize that the importance of SAMe to mitochondrial health is directly linked to the plausible theory that SAMe’s methyl groups are normally highly deupleted.

3. Are microbially synthesized methyl groups and butyrate deuterium depleted? – A careful tracing of multiple metabolic processes taking place in a human cell reveals that they are plausibly designed to greatly restrict the number of deuterons that are in the mitochondrial water. This strategy helps to minimize exposure of the ATPase nanomotors to deuterons. In part, this feat is accomplished through enzymes such as flavoproteins that greatly favor protium over deuterium in their reaction, i.e., that have a high deuterium KIE. The physics usually involves configuring the enzyme to support proton tunneling, since deuterons are much less capable of such tunneling.

Another way to support a reduced deuterium supply to the ATPase nanomotors is to select nutrients that are naturally low in deuterium to feed into the tricarboxylic acid (TCA) cycle. This is what makes the metabolites produced by the gut microbes via hydrogen recycling very significant. The enzyme expressed by anaerobic archaea that metabolize TMA, TMADH, is a flavoprotein with a high deuterium KIE (~ 8.6) due to vibrationally assisted hydrogen tunneling.

This means that the hydrogen recycling that takes place during its metabolism further scrubs deuterium from methylation pathways, while TMA that is left behind becomes enriched in deuterium. This unmetabolized TMA is converted to deuterium-enriched trimethylamine N-oxide (TMAO) in the liver and released into the circulation. Elevated TMAO levels in plasma are associated with increased risk to cardiovascular disease and a long list of other inflammatory diseases, as we will detail in the coming sections.

4. Natural and synthetic choline have different effects on TMAO levels: does deuterium play a role? – The original 2011 paper that first identified TMAO as a risk factor for heart disease, involved feeding mice phosphatidylcholine where all the protons in the methyl groups attached to the nitrogen atom were replaced with deuterium, so that the researchers could trace the products of the nutrient in the body. It turns out that they accidentally conducted an experiment testing what happens when phosphatidylcholine is extremely enriched in deuterium. 🙂 They also determined that supplementation of mice with deuterated choline, TMAO, or betaine resulted in upregulation of multiple macrophage scavenger receptors linked to atherosclerosis. TMAO was not produced if the mice were pretreated with antibiotics or in experiments with germ-free mice, confirming that microbial enzymatic action was a necessary precondition.

A paper in 2021 on human subjects compared choline intake from natural dietary sources with supplemental choline bitartrate and found that the latter but not the former raised blood TMAO levels. Notably, these authors wrote in the conclusion of the abstract: ‘Despite high choline content in egg yolks, healthy participants consuming four eggs daily showed no significant increase in TMAO or platelet reactivity.’ However, TMAO levels rose significantly following synthetic choline bitartrate supplementation. This occurred even though the subjects had normal kidney function, showing that elevated TMAO is not just a consequence of kidney disease.

The authors of the original 2011 study had published a follow-on study on human subjects in 2013, in which they supplemented the subjects with D9-PC, essentially repeating the mouse study but with humans as the subjects. They confirmed that D9-TMAO levels were sharply elevated in the plasma and urine following supplementation. Furthermore, an elevated TMAO level predicted an increased risk of major cardiovascular events, after adjustment for traditional risk factors. This study shows that it may not be phosphatidylcholine vs. choline bitartrate that matters, but rather whether the choline is deuterium depleted or deuterium enriched. By contrast, a survey involving over 14,000 participants found that dietary choline protects from both heart disease and stroke. L-carnitine is also a precursor to TMAO, and a mouse study in which the mice were fed deuterated L-carnitine also showed a sharp increase in plasma TMAO following supplementation, further supporting the idea that deuteration is the primary factor promoting TMAO accumulation.

5. TMAO directs metabolism towards deupleting peroxisomal-mitochondrial crosstalk – The synthesis of TMAO from TMA requires hydrogen peroxide. The source of hydrogen peroxide is via peroxisomal fatty acid chain modifications that utilize molecular oxygen dissolved in plasma to produce SCFAs, ketones, NADH and hydrogen peroxide (H2O2). The resulting metabolic water of the reaction is deupleted, as fatty acids are inherently deupleted molecules in biology. Although peroxisomes do not produce ATP directly, they reduce NAD+ for proton delivery to mitochondria via membrane-based intracellular proton transporters. Although the partial contribution of TMAO synthesis to intermediary metabolism is yet to be determined to efficiently deplete deuterium, it is certain that none of the above works well in the deuterium preserving glucogenic metabolic state. There is a strict dependence of peroxisomes on long chain saturated fatty acid substrates with particularly lower deuterium-related chemical mass.

Peroxisomal metabolism triggered by TMAO turnover utilizes very long and branched chain fatty acids (A) as well as dissolved molecular oxygen (B) carried in plasma. Peroxisomes produce SCFAs via β-carbon oxidation, ketones, NADH (C) and hydrogen peroxide (B). H2O2 is rapidly converted to metabolic water by catalase (CAT) that also yields molecular oxygen for the mitochondrial matrix (D) as well as for other cellular compartments. Peroxisomes can also reduce NAD+ for proton delivery to mitochondria via membrane-based intracellular proton transporters. oxidation of very long chain saturated fatty acid β carbons, purportedly of animal source, with the help of molecular oxygen, yields the most deupleted H2O2 by weight. CAT, one of the fastest enzymes in biology with that of isomerases, rapidly and irreversibly produces water from H2O2, while recycling oxygen. Metabolic hydrogen peroxide of fatty acid breakdown with low deuterium consequently provides ATP synthase nanomotor-sparing protons for energy production. High TMAO with hydrogen peroxide turnover can easily depend on CAT-mediated oxygen recycling.

6. Is TMAO an indicator of deuterium overload in the mitochondria?
6.1 TMAO inhibits S-adenosylhomocysteine hydrolase
6.2 TMAO induces reactive oxygen species
6.3 TMAO suppresses autophagy via PI3K/Akt/mTOR activation
7. TMAO and human diseases
8. Does oxidative stress lead to mitochondrial deupletion? – While oxidative stress is a major contributor to cellular damage, the processes involved in resolving ROS are an essential part of the mechanism by which the cell reduces deuterium levels in the mitochondria. Intracellular ROS are derived mainly from NOX, xanthine oxidase, and the mitochondrial electron-transport chain (mETC). Excess mitochondrial deuterium promotes increased ROS generated by the mETC. Superoxide dismutase (SOD) converts ROS to H2O2 , which can release the highly destructive hydroxyl radical in the presence of reduced iron (Fe2+). However, H2O2 is an excellent source of deuterium depleted water (DDW) in the mitochondria, as long as there is sufficient mitochondrial glutathione and both glutathione peroxidase and glutathione reductase are adequately expressed. H2O2 freely crosses the mitochondrial membrane, and, with adequate antioxidant support, it is rapidly converted to two molecules of DDW, catalyzed by glutathione peroxidase.

9. Do lipid-laden foam cells support mitochondrial deupletion?
10. Archaeobiotics
11. A crucial role for A. muciniphila
12. Strategies to lower TMAO levels – It is clear that elevated plasma TMAO is a risk factor for a broad range of chronic diseases, and therefore it is compelling that a strategy that reduces plasma TMAO should show health benefits. However, simply avoiding foods that provide precursors to TMAO is not likely to be productive. Choline, L-carnitine, and betaine are the primary sources that fuel the methylation pathway. Eggs and seafood, rich sources of these nutrients, also contain many valuable micronutrients and healthy fats that are also essential.

Deuterium depleted water (DDW) is commercially available, at dilution levels as low as 5 ppm. It can be mixed with tap water to simulate natural glacier water, typically containing around 100 ppm deuterium. Although the number of studies on the effects of therapeutic deuterium depletion on various health conditions is small, a review paper found that deuterium depletion has shown promise in preventing and treating cancer, improving long-term memory, enhancing sports performance, and reducing symptoms of depression.

It is apparent that the best way to reduce TMAO levels, while simultaneously boosting methylation supplies, is to promote an abundant colonization of anaerobic archaea in the gut, so that they can clear (fully metabolize) TMA before it has a chance to become TMAO.

13. Discussion – In this paper, we develop the argument that TMAO serves as a marker for excess deuterium in the methylation pathway, and, by extension, in the mitochondria, systemically. While methyl groups have powerful epigenetic effects, the ultimate fate of methyl groups is their metabolism to CO2 and water that is most likely deuterium depleted in the mitochondria. A microbial imbalance leading to reduced colonization by beneficial bacteria and an overgrowth of pathogenic species is the primary cause of overproduction of TMAO.

14. Conclusions – We have shown that TMAO, a causal factor for many diseases, may act as a marker for gut dysbiosis and for excess deuterium load in mitochondria, systemically. We traced through many of the biological pathways involving 1C metabolism and showed the integral role that gut bacteria play in stripping deuterium from methyl groups.”

https://link.springer.com/article/10.1007/s11306-026-02443-3 “The essential role of hydrogen gas recycling by gut microbes in reducing deuterium load in host mitochondria: is trimethylamine oxide a deuterium sensor?”

I take Now brand taurine, acetyl-L-carnitine, flush-free niacin, and betaine. I asked them whether these products are evaluated for their deuterium content. Will update with their response.

Self-reinforcing feedback loops of aging

gettingwell4 4-5 stars Advanced knowledge, Epigenetics, Memory, Stress, Telomeres , , , ,

A 2026 rodent study investigated age-associated queuine decline. Queuine is characterized as a “longevity vitamin” in the cited 2018 review Prolonging healthy aging: Longevity vitamins and proteins, along with ergothioneine, astaxanthin, and taurine.

“The contribution of transfer RNA (tRNA)-specific modifications to aging remains largely unexplored. We systematically profile tRNA modifications across multiple organs, species, and senescence models, and identify mannosyl-queuosine (manQ) as the first tRNA-specific modification that consistently declines with age.

Across species, queuine supplementation extends lifespan and enhances healthspan. In naturally aging mice, long-term oral administration beginning at 16-months-old (human equivalent 50 years) extends mean lifespan by 15.3%, reduces DNA methylation age, improves cognitive and motor performance, strengthens antioxidant defenses, remodels the gut microbiota, and alleviates inflammation and metabolic dysfunction without detectable toxicity.

These findings establish tRNA epitranscriptomic remodeling as a previously unrecognized layer of aging regulation, and identify restoration of manQ through queuine supplementation as a multi-system strategy to delay aging.

manQ hypomodification is selective rather than reflecting global tRNA depletion. Aging preferentially reduces the manQ-containing tRNAAsp fragment while leaving the corresponding unmodified tRNAAsp fragment, and other queuosine-modified tRNAs, relatively unchanged.

This pattern supports a regulated defect in modification homeostasis rather than a generalized change in transcript abundance. Such specificity argues that manQ loss is not merely a passive consequence of tissue degeneration, but instead represents a conserved, biologically meaningful aging-associated event with mechanistic impact.

Because proteostasis intersects with multiple canonical hallmarks (e.g. mitochondrial dysfunction, impaired stress resilience, and altered intercellular communication), translation-coupled proteome destabilization offers a unifying explanation for how a single tRNA modification defect can elicit multi-system consequences. In this view, manQ decline is not merely one of many molecular changes observed in aging, but rather a proximate determinant capable of amplifying downstream hallmarks through a common axis of proteome quality control.

Our findings further suggest that manQ depletion may engage self-reinforcing feedback loops that accelerate aging trajectories. This architecture offers a conceptual framework in which aging progressively erodes ‘epitranscriptomic integrity’ at the tRNA level, pushing translation toward an error-prone regime that accelerates proteostatic collapse and functional decline.

A distinctive implication of this work is that queuine introduces a microbiota-host epitranscriptomic axis into aging biology. Queuine is produced by gut microbiota and cannot be synthesized de novo by mammals. These findings expand the conceptual scope of geroscience by placing a microbiota-derived nutrient upstream of translational quality control.

Queuine supplementation offers a distinct therapeutic logic: rather than modulating a single signaling cascade, it restores a tRNA modification state that governs translational fidelity – an upstream determinant of proteome quality that can, in principle, influence multiple downstream hallmarks concurrently. These findings highlight an intervention paradigm centered on restoring molecular fidelity, rather than suppressing a single downstream phenotype, as a strategy to delay systemic aging.”

https://www.biorxiv.org/content/10.64898/2026.03.22.713446v1.full “Evolutionarily Conserved Decline of tRNA Mannosyl-Queuosine Links Translational Regulation to Aging and Is Reversed by Queuine”

Treat your gut microbiota well. Give them what they want, and expect reciprocity.

The hops compound xanthohumol

gettingwell4 4-5 stars Advanced knowledge, Stress

Two 2026 papers, with the first an in vitro study of over 2000 compounds to select those that best inhibit BACH1:

“BACH1 regulates the cellular oxidative stress responses by suppressing expression of cytoprotective genes. Dysregulated BACH1 activity has been implicated in a range of pathologies, including chronic inflammatory diseases, fibrosis, and cancer, making it a promising therapeutic target.

We identified four structurally distinct compounds that robustly inhibit BACH1 function. Notably, these compounds simultaneously activate transcription factor NRF2, suggesting the potential for a broader modulation of oxidative stress pathways.

However, while NRF2 induces expression of genes that protect against oxidative stress and inflammation and suppress ferroptosis, BACH1 represses them. While NRF2 broadly activates cytoprotective genes, BACH1 inhibition triggers a more restricted response but with a strong upregulation of HMOX1 (significantly stronger than the one obtained upon NRF2 activation).

As such, combined NRF2 activation and BACH1 inactivation is expected to produce a more potent antioxidant and anti-inflammatory effect than targeting either factor alone. Moreover, BACH1 regulates unique targets not shared with NRF2 and can dominantly repress genes even in the presence of active NRF2. This confers BACH1 inhibition distinct therapeutic value, particularly in contexts such as cancer cell invasion, where its suppression yields anti-metastatic effects.

  1. Auranofin is an FDA-approved gold salt used in rheumatoid arthritis whose primary mechanism of action is inhibition of thioredoxin reductases (TrxRs).
  2. Xanthohumol is a natural compound, prenylated chalcone, that belongs to the flavonoid family, with reported antimicrobial, anti-inflammatory, and antioxidant activities, and demonstrated safety in phase I and phase II trials.
  3. Alantolactone is a natural compound, member of the sesquiterpene lactone class with anti-inflammatory and antioxidant effects, and has been tested in several animal models without reported toxicity.
  4. CH55 is a synthetic retinoid with high affinity for RAR-α and RAR-β and antifibrotic activity and has not been yet tested in vivo.

In summary, this work establishes a robust screening platform for identification of functional BACH1 inhibitors, and provides new chemical scaffolds with potential for future therapeutic development.”

https://papers.ssrn.com/sol3/papers.cfm?abstract_id=6439073 “Development of a High-Throughput Screening Platform for Identification of Functional BACH1 Inhibitors Reveals Compounds with Anti-Invasive Potential”

Xanthohumol is ninth on the Nrf2 activator list. BACH1 interactions were also covered in Part 3 of Broccoli sprouts activate the AMPK pathway.

A 2026 paper that was too recent to be referenced in the above study described two xanthohumol clinical trials. The first trial was designed to assess bioavailability in healthy people (6 men and 6 women), and the second was designed to determine bioactivity in 16 healthy women:

“The aim of the present project was to systematically investigate bioavailability of native xanthohumol compared to micellar xanthohumol at two doses (86 mg vs. 172 mg) in a randomized crossover trial. We furthermore examined short-term effects of xanthohumol on resting energy expenditure (REE), blood pressure (BP), and heart rate (HR) in a randomized placebo-controlled crossover study.

Micellar solubilization significantly increased area under the curve (AUC), maximum plasma concentration of xanthohumol (Cmax), timepoint of maximum plasma concentration of xanthohumol (tmax), and apparent bioavailability compared to native xanthohumol. The dose also significantly influenced plasma kinetics, but apparent bioavailability and tmax were dose-independent in contrast to AUC and Cmax. In our subsequent study, xanthohumol did not affect REE, substrate oxidation, BP, or HR.

Two properties of xanthohumol impair its bioavailability. First, xanthohumol is relatively unstable in an acidic environment, and second, xanthohumol is highly lipophilic and hydrophobic, insoluble in the aqueous environment of the intestinal lumen, and poorly absorbed into enterocytes.

In addition to determining typical plasma kinetic parameters (e.g., Cmax, tmax, and AUC), we also calculated the amount of xanthohumol absorbed using maximum plasma xanthohumol concentration. These estimated amounts are minimum quantities of xanthohumol that had to be absorbed to achieve observed plasma xanthohumol concentrations.

Results of these calculations emphasized the relatively poor bioavailability of xanthohumol in humans. Only ∼0.1% and ∼1.2% of native and micellar xanthohumol were absorbed, respectively.

However, a limitation of this plasma estimation is that fractional absorption of xanthohumol was underestimated because distribution of xanthohumol in cells and tissues was not taken into account. It is likely that more xanthohumol was actually absorbed. Whether the bioavailability of xanthohumol is sufficient for physiological efficacy must be investigated further in humans.

In conclusion, oral bioavailability of micellar xanthohumol was higher than that of native xanthohumol. Systemic availability of xanthohumol did not differ between men and women. Our study provides no evidence that xanthohumol acutely affects REE, BP, and HR.”

https://onlinelibrary.wiley.com/doi/10.1002/mnfr.70413 “The Bioavailability of Xanthohumol in Humans and the Influence of Formulation and Dose: Randomized Controlled Trial Data”

2026 diet and supplement changes

gettingwell4 4-5 stars Advanced knowledge, Epigenetics, Immune system, Stress , ,

I switch things around pretty often, but I haven’t said much about diet and supplement changes since this time last year. Here’s what I’ve done in terms of changes that I’ve since abandoned or reduced, followed by additions or increases that I’ve kept.

Abandoned and reduced items

1. I stopped using Avena sativa oats to grow 3-day-old oats sprouts. I again ran into the same situation where I got < 10% yield.

The first time this happened in 2023, I related to the Montana farmer that degraded seed vitality was probably caused by the way that Amazon handled their oat products. I’m the customer, though, and I won’t make it my problem if the vendor can’t meet expectations.

I switched to sprouting Avena nuda oats based on Sprouting hulless oats. I’ll note that this Illinois farmer doesn’t let Amazon handle their organic Avena nuda oats, and they add on post office shipping costs. They don’t recommend sprouting, probably because of liability, although I’ve had a 91% germination rate over three days. I might have ordered Avena sativa oats directly from the Montana farmer bypassing Amazon if they were also organic.

2. I stopped taking alpha ketoglutarate. In my view, increasing tricarboxylic acid (TCA) cycle intermediate metabolites such as alpha ketoglutarate and CoQ10 should not be the primary way to improve mitochondrial electron transport chain function.

Instead of biochemical considerations, focus on photon modulation, which precedes biochemical reactions. Which means mitochondrial studies should be controlled for light exposures, and very few of them do that, although it’s the way nature works.

This past winter I increased indoor non-LED light exposure within a circadian rhythm framework. I’ll switch back to walking the beach at sunrise from being out in mid-day sun after it gets a little bit warmer.

3. I’ve taken creatine on and off during the past year. There’s a bit of literature on its use for improving methyltransferase system components like homocysteine.

Stopping creatine fits one of the overall patterns that studies demonstrate – people who are initially deficient in the studied item get a benefit, while people who are initially sufficient don’t benefit from treatment. I’ve always tested mid-range for homocysteine, which is desirable.

4. I had some cocoa powder lying around for a year or two, and I used it this past winter to improve the taste of coffee I bought on sale. Cocoa flavanols are supposed to improve various health measures. But I haven’t been provided access to the most recent human studies, so I won’t repeat their results without reading their details.

5. I saw this at Costco, and picked up a package:

A 2025 review covered pecan research, Pecans and Human Health: Distinctive Benefits of an American Nut. Eating pecans seems to have some health benefits, and they taste alright.

For me, though, the dryness of a chewed pecan bolus creates a swallowing problem that walnuts don’t have. YMMV.

6. I stopped taking 2 g magnesium L-threonate. I’ve always tested high for magnesium without using a specific supplement.

7. I reduced D3 by 25 mcg to a daily 2400 IU. Winter is over.

New and increased items

1. I curated five 2025 ergothioneine studies in Human studies of ergothioneine after stopping mushroom intake via AGE-less chicken soup. I wasn’t thrilled that none of them investigated long-term effects of persistent plasma ergothioneine levels.

This year I decided to start taking the higher 25 mg dose of the first study once a week. That should produce some benefits at a lower ergothioneine blood level than daily doses produce. I’ll check periodically for 2026 research.

2. The only paper I’ve curated on deuterium (heavy hydrogen) is Taurine and mitochondrial health. I started using Icelandic glacier water to make coffee and tea, and for just drinking.

It isn’t advertised as deuterium-depleted water, and it isn’t manufactured as such. But I think any glacier water contains less deuterium than local water. I use local filtered water for sprouting and cooking.

3. Per The return of the free radical theory of aging I started taking extra vitamin C separately from other supplements in the form of Now brand liposomal 1 gram twice daily this past winter. That study found vitamin C to be an anti-aging compound for primates.

Reference 72 of that 2026 paper is a freely available 2025 study https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(25)00339-X “Vitamin C conveys geroprotection on primate ovaries” that used the same vitamin C dose and duration in macaques to find:

“VC slowed aging in various ovarian cell types. Moreover, VC protected human ovarian endothelial and stromal cells (SCs) from aging partially via NRF2 activation. This study establishes a proof-of-concept for delaying primate ovarian aging with a single compound, and provides important insights into preventing and treating degenerative diseases related to ovarian aging.”

4. I restarted taking inulin last year, about 3 grams (a heaping teaspoon) daily after posting Inulin vs. FOS. My 2.5 year-old grandchild takes a level teaspoon daily, as inulin’s beneficial effects aren’t just for old people.

5. I started taking 12 mg astaxanthin twice in the morning. I use Nrf2 activators in the morning because Nrf2 is especially involved in the circadian cycle, as noted in papers such as Broccoli sprouts activate the AMPK pathway, Part 4.

6. I increased daily raw egg consumption from 3 eggs a day to 3 eggs twice daily.

7. This year, Ovega 3 algae oil DHA 420 mg/EPA 140 mg became no longer available AFAIK. I substituted Vegan Omega 3 algae oil DHA 300 mg/EPA 150 mg in the morning and Sports Research Omega 3 fish oil DHA 310 mg/EPA 690 mg in the afternoon.

8. I picked up this Korean seaweed in a 10-pack at Costco. The label doesn’t say what its iodine content is. I eat it as a snack whenever I get a salt craving, maybe once a week.

Eat broccoli sprouts for ALS?

gettingwell4 4-5 stars Advanced knowledge, Acetylcholine, Cerebellum, Cerebrum, Dopamine, Epigenetics, Glutamate, Hippocampus, Immune system, Limbic system, Lower brain, Memory, Neurochemicals, Serotonin, Stress , ,

A 2026 rodent study investigated sulforaphane’s ability to affect ALS-like symptoms:

“The objective of this study was to evaluate neuroprotective efficacy and safety of sulforaphane (SUFP) in a methylmercury (MMHg⁺)-induced preclinical rat model of amyotrophic lateral sclerosis (ALS). ALS is characterized by progressive motor neuron degeneration and muscle wasting, leading to impairments in gait, swallowing, salivation, and routine motor activities.

64 animals were classified into eight groups: 1st: normal control, 2nd: vehicle control; 3rd: SUFP perse (4 mg/kg, i.p.), 4th: MMHg + (5 mg/kg, p.o.), 5th: MMHg + 5 + SUFP (2 mg/kg, i.p.), 6th: MMHg+ 5 + SUFP (4 mg/kg, i.p.), 7th: MMHg+ 5 + omaveloxolone (OVX) (30 mg/kg, i.p.), and 8th: MMHg + 5 + dimethyl fumarate (DIMT) (50 mg/kg, i.p.). Neurotoxin MMHg + was orally administered at 5 mg/kg for the first 21 days. For the next 22 days, SUFP, OVX, and DIMT were administered intraperitoneally (i.p.).

SUFP modulates neurotransmitter levels such as acetylcholine (A), dopamine (B), GABA (C), glutamate (D), and serotonin (E).

SUFP4 exerted broad neuroprotective effects in ALS pathology by restoring antioxidant proteins (Nrf2, HO-1, SIRT1), suppressing apoptotic (Bax, caspase-3, Bcl-2) and inflammatory markers (TNF-α, IL-1β), and enhancing the anti-inflammatory cytokine IL-10. It also downregulated stress-related signaling pathways (PI3K/Akt, p75NTRECD, MAPKs) associated with neurodegeneration. These molecular effects translated into meaningful functional recovery, as evidenced by improvements in grip strength, locomotor performance, spatial memory, and depressive-like behavior.

Histopathological evaluation demonstrated attenuation of demyelination and preservation of neuronal architecture including the cerebral cortex, hippocampus, striatum, midbrain, and cerebellum. Beyond central neuroprotection, SUFP exerted systemic benefits by normalizing hepatic enzymes, improving skeletal muscle integrity, restoring redox balance, stabilizing neurofilament and myelin-associated proteins, and correcting hematological alterations.

Despite limitations related to study duration and animal sex, this work strongly positions SUFP as a promising, multi-target therapeutic candidate for ALS with both neural and systemic protective efficacy.”

https://link.springer.com/article/10.1007/s12035-026-05683-5 “Sulforaphane-Mediated Multitarget Therapeutic Effects in Methylmercury-Induced ALS-Like Pathology: Comparative Analysis and Multifaceted Approach to Neuroprotection and Systemic Recovery” (not freely available) Thanks to Dr. Sidharth Mehan for providing a copy.

Unlike A Nrf2 treatment for ALS?, this study didn’t present evidence that its treatment compound was effective for preventing ALS. For one thing, currently-known disease factors involving heat shock proteins and associated genes, some of which are Nrf2 targets, weren’t investigated.

Two Nrf2 activators were used in both studies as comparators of Nrf2 activation effects. Neither omaveloxolone nor dimethyl fumarate are ALS causal treatments, though, and have undesirable side effects.

A human equivalent of this study’s higher sulforaphane dose is ((4 mg x .162) x 70 kg) = 45 mg. 45 mg of sulforaphane might be too much to consistently take at one time because of unpalatability. But I documented taking an estimated 52 mg for a year during 2020-2021 by eating microwaved 3-day-old broccoli sprouts twice a day.

Nrf2 and stem cells

gettingwell4 4-5 stars Advanced knowledge, Cerebrum, Epigenetics, Glutamate, Hippocampus, Immune system, Limbic system, Memory, Neurochemicals, Stress, Testosterone , , ,

A 2026 review subject was mechanisms and therapeutic potential for Nrf2 activators in combination with mesenchymal stem cells:

“Mesenchymal stromal/stem cells (MSCs) are multipotent stem cells that can be isolated from various tissues – such as bone marrow (BM), umbilical cord (UC), adipose tissue (AD), dental pulp (DP), hair follicle (HF), and placenta – and differentiated into multiple lineages under appropriate conditions. Their functional repertoire includes immunomodulation, homing, and differentiation, which collectively help establish a balanced inflammatory and regenerative niche within damaged tissues during severe inflammation. MSCs-derived extracellular vesicles (MSCs-EVs) and conditioned medium (MSCs-CM) play remarkable roles, exhibiting potent anti-inflammatory and antioxidant properties that offer novel therapeutic alternatives for inflammatory diseases.

Therapeutic capacity of MSCs in inflammatory conditions is increasingly attributed to their potent paracrine activity rather than solely to their differentiation potential. A key mechanism underlying this paracrine effect is activation of the Nrf2 antioxidant pathway.

MSCs and their secreted products including exosomes (Exos), EVs, and CM, activate Nrf2 through multi-dimensional/target mechanisms, thereby enhancing cellular antioxidant defenses, modulating immune responses, and promoting tissue repair. It is noteworthy that therapeutic efficacy of MSCs and their derivatives can be enhanced through external modulation, including pretreatment with natural compounds.

Preconditioning refers to brief treatment of MSCs or their derivatives with physical, chemical, or biological factors prior to application, aiming to enhance their ability to counteract oxidative stress and improve their therapeutic efficacy. Flavonoids precondition and prime MSCs via the direct Keap1-Nrf2 pathway or indirect PI3K-Akt pathway, which enhances cellular resilience to adverse conditions by reducing apoptosis and promoting survival. Primed MSCs, in turn, remodel the microenvironment through an altered secretory profile, releasing bioactive factors that create more favorable conditions for their own persistence.

The core logic of these strategies lies in simulating or inducing adaptive stress, such as employing specific chemical molecules or drug stimuli, or utilizing physical / microenvironmental preconditioning to mimic specific physical conditions of the in vivo injury environment. The most straightforward strategy is overexpression of Nrf2 or its key downstream effector molecules.

The majority of existing studies remain at the level of observing correlations with Nrf2 upregulation, and there is still a lack of precise causal validation regarding key upstream signals – such as specific cytokines, miRNAs, or proteins – through which MSCs or derivatives initiate Nrf2 activation. Mechanistic insights are predominantly derived from in vivo or rodent (mouse/rat) model experiments, with a notable absence of clinical validation, insufficient long-term safety and pharmacokinetic data, and a lack of standardization in administration routes and dosages, all of which hinder clinical translation.

The essential role of the Nrf2 pathway has not been rigorously confirmed, as most studies have not employed reverse genetic validation using Nrf2-knockout animals or specific inhibitors. Consequently, it remains unclear whether therapeutic effects are necessarily and exclusively dependent on Nrf2, and potential synergistic contributions from other pathways may have been overlooked.

Most natural flavonoids face challenges such as low oral bioavailability, rapid metabolism, and poor targeting. Numerous challenges remain to be addressed in order to translate these promising preclinical findings into clinical practice. Future research should focus on the following aspects:

  1. Elucidating precise upstream molecular mechanisms by which MSCs activate Nrf2;
  2. Employing more clinically relevant chronic disorder models;
  3. Systematically evaluating long-term safety, optimal delivery strategies (including dosage and route of administration), and immunogenicity of MSCs-based therapies;
  4. Validating selection criteria (optimal source), quality control, batch-to-batch consistency of MSCs, and addressing regulatory and ethical barriers to clinical translation; and
  5. Integrating molecular docking, ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) prediction, and in vitro and in vivo validation to further elucidate regulatory effects of flavonoids and enhance understanding of their mechanisms of action.”

https://link.springer.com/article/10.1186/s13287-026-04925-6 “Activation of Nrf2 with natural flavonoids and mesenchymal stromal/stem cells: mechanisms and therapeutic potential for inflammatory diseases” (click pdf)

This paper was overly long at 127 pages, so I focused on the later sections. None of these treatments are currently ready for clinical trials.

I also didn’t mention specific flavonoids as Nrf2 activators. It’s beyond a reviewer’s task to rank Nrf2 activators, and a study’s researchers seldom address why they used a poorly-activating flavonoid instead of a higher-ranked natural plant compound such as sulforaphane.

Eat broccoli sprouts to prevent or treat obesity

gettingwell4 4-5 stars Advanced knowledge, Stress

This 2026 rodent study made mice obese with a high-glycemic-index diet, and intervened with different doses of sulforaphane during and after inducing obesity:

“To our knowledge, this is the first study investigating therapeutic effects of sulforaphane (SFN) on obesity resulting from feeding with high-glycemic-index diet (HGID). To evaluate the potential role of SFN on energy metabolism, obesity development, and insulin resistance, effects were tested by administering SFN at different doses [oral 5 mg and 20 mg] in addition to HGID and after animals were made obese with HGID.

Energy, macronutrient, and fiber contents of the HGID used in the experiment and the isocaloric control group feed were kept equal. The only difference between HGID and the control group feed was composition of the starch. While starch in the HGID was a waxy corn starch consisting of 100% amylopectin, it was natural starch (75% amylopectin, 25% amylose) in the control group.

This study is strengthened by its:

  • Experimental design, in which SFN was administered at multiple doses both during exposure to HGID and after development of HGID-induced obesity, allowing for a comprehensive evaluation of its effects on energy metabolism, obesity progression, and insulin resistance.
  • Focus on specific components of HGID. To be able to separate effects of the HGI diet pattern, one of the long-standing criticisms regarding GI, from individual components it contains, especially dietary fiber, we were able to evaluate glycemic index interaction by keeping energy, macronutrient, and fiber contents of feeds equal and the starch composition different.

Several limitations should be acknowledged.

  • Potential adverse effects of the 5 mg/kg/day and 20 mg/kg/day doses of sulforaphane in this study were evaluated in terms of clinical signs. However, systemic adverse effects, particularly those affecting the brain, cardiovascular system, or other organs, were not assessed.
  • The relatively short duration of the SFN intervention (five weeks following development of obesity induced by a HGID) may have limited the ability to fully capture all potential changes in the measured variables. It may be beneficial to observe for a longer period in future studies to provide evidence that SFN reverses HGID-induced obesity.
  • The ideal dose of SFN has not yet been determined. Dose and bioavailability are considered important parameters that need to be clarified for SFN to be considered as an anti-obesity agent.

Results indicate that SFN may provide potential benefits both as a protective agent in the obesity development process and as a therapeutic approach after obesity has developed.

  • While SFN suppresses obesity development by combating increased energy consumption, body weight, deteriorated lipid profile, and decreased insulin sensitivity upon exposure to HGID, it supports obesity treatment with its aspects of reducing food consumption and body weight gain and improving glycemic control.
  • SFN may reverse adverse effects of HGID in a time- and dose-dependent manner by regulating postprandial insulin, restoring IRS1/IRS2 function, inhibiting gluconeogenesis through coordinated activation of signaling between sirtuins and PGC-1α, and shifting liver metabolism from lipid synthesis toward mitochondrial oxidation.”

https://www.mdpi.com/2072-6643/18/4/574 “Sulforaphane Against the Metabolic Consequences of a High-Glycemic-Index Diet: Protective and Therapeutic Mechanisms Associated with Obesity and Insulin Resistance”

A human equivalent to this study’s daily oral low sulforaphane dose is (5 mg x .081) x 70 kg = 28 mg, which is achievable by eating broccoli sprouts every day. People won’t tolerate quadrupling 28 mg to a human equivalent of the study’s 20 mg daily oral sulforaphane dose, so I didn’t curate this study’s high-sulforaphane-dose-specific findings.

Human age equivalents to this study’s 8-week-old, 23-week-old, and 28-week-old mice are respectively 18-25 years, 25-35 years, and 28-38 years.

A 42-month human study of broccoli sprouts’ effects on cognitive function

gettingwell4 4-5 stars Advanced knowledge, Epigenetics, Memory ,

A 2026 paper provided details of a 2020-2023 human trial of broccoli sprouts:

“In a 42-month randomized, double-blind, placebo-controlled trial, 26 participants aged 63–90  years with memory impairment were randomly assigned to receive either 30 mg/day of glucoraphanin (GLR) or placebo. The primary outcome was the change in Memory Performance Index (MPI) scores from the mild cognitive impairment (MCI) screen. This study evaluated the long-term efficacy of GLR supplementation on cognitive function in older adults at an elevated risk for Alzheimer’s disease (AD), including those with MCI.

Participants were instructed to take three capsules of either the GLR or placebo supplements daily for 42 months. The GLR supplement contained 30 mg of GLR purified from broccoli sprouts, along with 120 mg of mustard powder per three capsules. Mustard powder was included as a source of exogenous active myrosinase to enhance the enzymatic conversion of GLR to sulforaphane. The placebo supplement contained 0 mg of GLR.

No significant group difference was observed in the initial 6 months. A marginal difference in favor of GLR appeared in the later phase (30 and 42 months), including the 42-month endpoint.

The GLR group demonstrated superior performance on immediate recall and delayed free recall tests. MCI participants showed a greater MPI improvement with GLR.

Long-term GLR supplementation may help preserve cognitive function in individuals at elevated risk for AD, particularly those with MCI. Larger trials are warranted to confirm efficacy and clarify underlying mechanisms.”

https://www.frontiersin.org/journals/nutrition/articles/10.3389/fnut.2026.1740494/full “Efficacy of 42-month oral administration of glucoraphanin in preventing cognitive decline in individuals at elevated risk of dementia, including those with mild cognitive impairment: a randomized, double-blind, placebo-controlled pilot study”

Again, 42 is the answer! 🙂

This study was funded by the supplement manufacturer. There was no explanation of what the supplement’s “purified from broccoli sprouts” entails. Also, I didn’t mention results of voluntary group exercise because there was a long gap in the participants’ data due to government response to covid.

  • For comparison of this study’s 30 mg glucoraphanin dose, Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts provided 30 grams of fresh broccoli sprouts that contained an estimated 51 mg of glucoraphanin for ten weeks. That study’s corresponding coauthor said of their 30 gram broccoli sprouts dose in Understanding a clinical trial’s broccoli sprout amount that “When we carried out tests with consumers, previous to the bioavailability studies, higher amounts per day were not easy to consume and to get eaten by participants.” There was no rationale provided for this study’s 30 mg dose other than citing two previous human studies that also used a 30 mg glucoraphanin dose.
  • For comparison of this study’s 120 mg mustard seed powder dose, my daily cruciferous food intake since five and a half years ago includes sprouted yellow mustard seeds started with 3.5 grams of seeds, along with sprouted broccoli and red cabbage started with 3.6 grams of each vegetable’s seeds, all sprouted for three days. I haven’t seen studies that show sprouting has effects on myrosinase enzyme activity.
  • This study cited Does sulforaphane reach the colon? which used 2% mustard seed powder to create sulforaphane from glucoraphanin. This study’s 120 mg mustard seed powder / 30 mg glucoraphanin is a lot more than 2%. My daily sprout intake started from 3.5 g mustard seeds / (3.6 g broccoli seeds +3.6 g red cabbage seeds) is also a lot more than 2%.

I’ve changed some items along the way, switching supplier from True Leaf to Johnny’s for organic broccoli seeds, and from non-organic Red Acre red cabbage seeds to True Leaf organic red cabbage seeds. I recently had to find another supplier of organic yellow mustard seeds when Naturevibe stopped carrying that product. I tried Food to Live, but their yellow mustard seeds when sprouted aren’t mild. I’ll next try Frontier Co-op to see if those are mild as advertised.

Eat broccoli sprouts for your heart, Part 2

gettingwell4 4-5 stars Advanced knowledge, Epigenetics , ,

A 2025 rodent study investigated synergistic effects of sulforaphane (SFN) and nicotinamide mononucleotide (NMN) on diabetic cardiomyopathy:

“Diabetic cardiomyopathy (DCM) as a significant diabetes complication remains a major human challenge. In this study, we provide evidence that the fat mass and obesity-associated protein (FTO) plays a pivotal role in DCM pathogenesis.

Downregulation of FTO in DCM acts as a critical inducer of ferroptosis by increasing expression of acyl-CoA synthetase long-chain family 4 (ACSL4), a key positive mediator of ferroptosis. FTO-mediated mitigation of ferroptosis occurs in an ACSL4-dependent manner which leads to increased methylation of Acsl4 transcripts.

  1. Ferroptosis plays an essential role in the pathogenesis of DCM.
  2. As the most widespread mRNA modification, N6-methyladenosine (m6A) is globally downregulated and implicated in diabetes and its complications.
  3. FTO, which is an m6A demethylase, was found to be downregulated in diabetes and its cardiovascular complications.
  4. NAD+ enhances the demethylase activity of FTO. Dietary supplementation with NMN, a critical intermediate in the NAD+ biosynthetic pathway, has been shown to efficiently elevate endogenous NAD+ levels.

Enhancing the demethylase activity of FTO with NMN combined with SFN targeting NRF2 could synergistically reduce the level of lipid peroxides to inhibit ferroptosis, providing an effective avenue for alleviating DCM.

We found that NMN could alleviate ferroptosis and improve heart function through enhancing FTO. SFN could prevent ferroptosis and partly rescue heart function via AMPK-mediated NRF2 activation.

We demonstrated that SFN combined with NMN treatment could significantly inhibit lipid peroxidation and rescue cardiac function in DCM compared to SFN or NMN treatment alone.

Although the combined regimen further suppressed ferroptosis and improved cardiac performance, it fell short of complete remission, underscoring that additional pathways also contribute substantially to the pathogenesis of DCM.”

https://link.springer.com/article/10.1007/s12012-025-10080-w “FTO-Mediated Mitigation of Ferroptosis Occurs in an ACSL4-Dependent Manner in Diabetic Cardiomyopathy”

The epigenetic mechanism involved with this study’s dietary dissolved-in-water 100mM NMN dose was Non-CpG methylation. This study used the same very low sulforaphane dose intraperitoneally injected as Eat broccoli sprouts for your heart. Discussion of that study provided an example that if a person waited until a diabetes-related disease condition became a problem, capabilities to adequately address causes and prevent the problem may be lost.

Notice in the last bar of the second graphic above taken from Figure 7 that the combined treatment was also provided to non-diabetic mice. These researchers provided over a dozen other measurements in Figure 7 to show similar short-term non-effects of the combined treatment, i.e. that it neither benefited nor harmed non-diabetic subjects. Grok interpreted this study’s 3-month-long intervention to be a 1-to-5 year human equivalent, depending on the measured effect (shorter for metabolic effects like MDA, longer for structural cardiac changes like reduced ferroptosis.)

The male subjects began at 2-months old, a human-equivalent 15-20 years old. These researchers gave them diabetes by feeding them a “high-fat diet for 3 months to induce insulin resistance, followed by a single intraperitoneal injection of streptozotocin (STZ) (in 0.1 mol/L of citrate acid buffer, 60 mg/kg) to induce partial insulin deficiency.” A 5-months old mouse is a 25-30 years old human equivalent.

Grok considered this study’s NMN human equivalent dose to be extremely high if provided in drinking water, but not if injected, depending on volume. However, the study didn’t state that its NMN dose was injected, and there was no dose volume indicated.

A Nrf2 / NAD+ connection?

gettingwell4 4-5 stars Advanced knowledge, Acetylcholine, Dopamine, Epigenetics, Glutamate, Hippocampus, Immune system, Limbic system, Neurochemicals, Serotonin, Stress , , ,

Here are two 2025 papers, starting with a rodent study that investigated interactions between the Nrf2 and kynurenine pathways:

“Exposure to the tryptophan metabolite kynurenine and its electrophilic derivative kynurenine-carboxyketoalkene (Kyn-CKA) leads to an increase in the abundance of transcription factor Nrf2 and induction of Nrf2-target genes. The Keap1/Nrf2 system is the main orchestrator of cellular defence against environmental stress, most notably oxidative and inflammatory stress.

Nrf2 can be activated pharmacologically by small molecules, the majority of which are electrophiles and oxidants that modify specific cysteine-based sensors in Keap1. C151 in Keap1 is the target of the isothiocyanate sulforaphane, a classical Nrf2 activator that has been employed in ∼90 clinical trials, as well as for the two Nrf2 activators that are clinically in use: dimethyl fumarate, for relapsing remitting multiple sclerosis, and omaveloxolone, for Friedreich’s ataxia.

Kynurenine is an endogenous metabolite derived from the essential amino acid tryptophan. Kynurenine and its metabolites, such as the electrophilic kynurenine-carboxyketoalkene (Kyn-CKA), have been demonstrated to activate Nrf2 in other pathologies, including sickle cell disease, attenuating inflammation. Moreover, identification of the gene encoding the kynurenine-metabolising enzyme kynureninase as a gene transcriptionally upregulated by Nrf2, provides a plausible negative feedback regulatory mechanism.

Because kynurenine is not electrophilic, whereas its metabolite Kyn-CKA is, we considered the possibility that Kyn-CKA is the actual Nrf2 activator. Using biochemical and cell-based assays, we found that Kyn-CKA reacts with C151 in the BTB domain of Keap1 and increases the thermostability of Keap1, indicating target engagement. Consequently, Nrf2 accumulates and induces transcription of antioxidant/electrophile-responsive element (ARE/EpRE)-driven genes.

These findings demonstrate that Kyn-CKA targets C151 in Keap1 to derepress Nrf2, and reveal that Nrf2 is a main contributor to the anti-inflammatory activity of Kyn-CKA in macrophages.”

https://www.sciencedirect.com/science/article/pii/S2213231726000078 “The electrophilic metabolite of kynurenine, kynurenine-CKA, targets C151 in Keap1 to derepress Nrf2”

A review subject was targeting nicotinamide adenine dinucleotide, oxidized form (NAD+) for clinical use:

“Mammalian NAD+ biosynthesis includes four known pathways, primarily occurring in cytoplasm:

  • (a) the NRH pathway;
  • (b) the salvage pathway;
  • (c) the Preiss–Handler pathway; and
  • (d) the kynurenine pathway.

The de novo kynurenine pathway metabolizes tryptophan (Trp) to NAD+, producing various intermediates that serve as biomarkers for different diseases. These intermediates show alterations in various pathological conditions.

While kynurenine and its metabolic derivatives are intermediates in the de novo NAD+ biosynthesis pathway, these are also produced independently in various physiological contexts, particularly in immune cells, where they act as immunomodulatory compounds.”

https://www.nature.com/articles/s43587-025-00947-6 “Emerging strategies, applications and challenges of targeting NAD+ in the clinic” (not freely available) Thanks to Dr. Jianying Zhang for providing a copy.

This second paper above showed a graphic of the Nrf2 and kynurenine pathways together in a diagram showing relationships between NAD+ augmentation and the hallmarks of aging, but didn’t elaborate other than labeling their box Dysbiosis. So how these two pathways interact is better outlined in the first paper above with explaining how a kynurenine-metabolizing enzyme is one of the hundreds of Nrf2 target genes, creating a natural feedback loop between Nrf2 activation and the kynurenine pathway.

These reviewers also lumped SIRT1 in their Dysbiosis box, and into several other boxes, probably due to the penultimate coauthor’s influence:

However, repeating something over and over doesn’t make it scientifically valid regardless of the number of citations. Or, as a 2022 review Sirtuins are not conserved longevity genes concluded:

“A global pursuit of longevity phenotypes was driven by a mixture of framing bias, confirmation bias, and hype. Review articles that propagate these biases are so rampant that few investigators have considered how weak the case ever was for sirtuins as longevity genes.

Acknowledging that a few positive associations between sirtuins and longevity have been identified after thousands of person-years and billions of dollars of effort, we review the data and suggest rejection of the notions that sirtuins (i) have any specific connection to lifespan in animals and (ii) are primary mediators of the beneficial effects of NAD repletion.”

Human studies of astaxanthin – Part 2

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Continuing Part 1, here are four more 2025 human studies of the Nrf2 activator astaxanthin, starting with a randomized, double-blind, placebo-controlled trial of its effects on reducing oxidative stress and inflammatory responses following eccentric exercise:

“This study investigated effects of astaxanthin supplementation on plasma MDA and HMGB1 levels following acute eccentric exercise in recreationally active male students. Fifty-four students were assigned to receive either 12 mg/day of natural astaxanthin (AST, n = 27) or placebo (PLA, n = 27) for 14 days.

A key consequence of eccentric-induced muscle damage is overproduction of reactive oxygen species (ROS). When ROS production exceeds the capacity of endogenous antioxidant systems, lipid peroxidation can occur. Malondialdehyde (MDA) is a stable end-product of lipid peroxidation and serves as a widely recognized biomarker for oxidative stress and cell membrane damage.

In parallel, muscle cell damage results in release of damage-associated molecular patterns (DAMPs) into the extracellular space. Among these, High Mobility Group Box-1 (HMGB1) plays a central role in inflammation when passively released from the nucleus. HMGB1 acts as a potent pro-inflammatory signal by activating innate immune receptors, recruiting immune cells, and upregulating cytokines such as IL-6 and TNF-α.

This heightened immune activity contributes to delayed-onset muscle soreness, which typically peaks 24–72 hours post-exercise, and is associated with impaired recovery. Sustained elevations in oxidative and inflammatory biomarkers, including MDA and HMGB1, may further impair recovery and contribute to long-term muscle pathology.

Astaxanthin’s antioxidant effects are mediated through both direct and indirect mechanisms. Structurally, astaxanthin is a xanthophyll carotenoid with a unique polar–nonpolar–polar configuration that enables it to span the phospholipid bilayer of cell membranes. This positioning allows it to neutralize ROS both at the membrane surface and within the lipid bilayer.

In addition, astaxanthin enhances endogenous antioxidant defenses by upregulating enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) through activation of the Nrf2–ARE signaling pathway. This dual mode of action provides both immediate and sustained protection against oxidative stress during and after exercise.

The placebo group showed substantial increases in MDA and HMGB1 after exercise, whereas the astaxanthin group experienced attenuated rises (~22% and ~27% smaller, respectively) and faster recovery toward baseline within 24 hours. These findings suggest that astaxanthin supplementation can be incorporated into recovery strategies for athletes and active individuals, especially during periods of heavy training or repeated bouts of intense eccentric exercise. By reducing oxidative damage and inflammation, astaxanthin may shorten recovery time, limit performance loss, and support overall training adaptations—benefits that are particularly valuable in sports requiring frequent high-intensity efforts.

Several limitations should be acknowledged in this study.

  1. Sample size was relatively small and limited to recreationally active young males, which may restrict generalizability of findings to other populations such as females, older adults, or elite athletes.
  2. Supplementation period was limited to 14 days; although this duration is sufficient to achieve plasma saturation of astaxanthin, longer interventions may produce different or more pronounced effects.
  3. Only two biomarkers were assessed (MDA and HMGB1), which provide important but incomplete insights into broader oxidative stress and inflammatory response. Including additional markers such as enzymatic antioxidants, cytokine profiles, and muscle damage indicators (e.g., creatine kinase) could yield a more comprehensive understanding.
  4. Dietary intake and physical activity outside the intervention were self-reported and not strictly controlled, which may have introduced variability in results.”

https://tmfv.com.ua/journal/article/view/3664/1922 “Taking Astaxanthin Supplementation Attenuates MDA and HMGB1 Following Eccentric Exercise: A Randomized Controlled Trial in Recreationally Active Students”

A clinical trial investigated astaxanthin’s effects with exercise in diabetic women:

“This study examined whether combined aerobic and resistance training (CT) and astaxanthin (AST) supplementation synergistically improve oxidant and inflammatory status as well as metabolic indices in T2DM, focusing on the mediatory role of Humanin (HN) and microRNAs (miRNA-122, miRNA-126-3p, and miRNA-146a).

Ninety women with T2DM were randomly assigned to six groups (n = 15 each):

  • Control (C), placebo (P), AST supplementation (S), combined training (CT), CT + placebo (CT + P), and CT + AST supplementation (CT + S).
  • CT, CT + P and CT + S groups underwent an 8-week training program (eight exercises, three sessions per week).
  • S and CT + S groups received 8 mg/day of AST.

This study only enrolled female participants age between 30 and 60 years old to minimize inter-individual biological variability arising from sex differences in hormone regulation, fat distribution, and gene expression related to inflammation and oxidative stress. Oxidative stress (OS) markers, inflammatory cytokines, HN levels, miRNAs expression, fasting blood glucose (FBG), insulin resistance (HOMA-IR), lipid profile, and hemoglobin A1c (HbA1c) were assessed.

HN is a member of a class of novel mitochondrial-derived peptides released during mitochondrial dysfunction. HN reduces ROS production, enhances antioxidant protein expression, maintains redox balance, and suppresses TNF-α, IL-1β, and IL-6 to inhibit inflammation. Furthermore, resistance and endurance training has shown to increase HN expression in patients with prediabetes. Exercise – aerobic and endurance – has been shown to increase circulating and skeletal muscle levels of HN, correlating with improved insulin sensitivity and mitochondrial function.

Our results showed:

  • CT and AST supplementation both improved antioxidant defense and reduced inflammation, and their combination was more effective than either intervention alone.
  • CT and AST supplementation increased blood concentration of HN, and their combination showed greater effects than AST supplementation, but not CT.
  • CT and AST supplementation increased blood levels of miRNAs-126-3p, and -146a and decreased miRNA-122, with their combination being slightly more effective in decreasing miRNA-122.
  • Both interventions improved lipid profile, with their combination being more effective in improving HDL and TG levels, although not total cholesterol.
  • FBG, HOMA-IR, and HbA1c were reduced by CT but not by AST supplementation.

Our data suggest that combining exercise with AST supplementation might improve oxidative status and inflammation through mechanisms involving HN and miRNAs 122, 126-3p, and 146a. Alleviating OS and inflammation could, in turn, lead to improvements in lipid profiles (e.g., TG, and HDL), IR, and reductions in HbA1c and FBG, as observed in our study. Furthermore, the combined approach seems to be more effective at improving cholesterol and TG levels.“

https://www.nature.com/articles/s41598-025-23914-y “Redox-sensitive miRNAs and Humanin could mediate effects of exercise and astaxanthin on oxidative stress and inflammation in type 2 diabetes”

A meta-analysis of randomized controlled trials reported until May 2025 assessed astaxanthin’s effects on lipid profiles. Neither of the two trials covered here nor the three trials covered in Part 1 were included in this meta-analysis.

“Astaxanthin, a xanthophyll carotenoid, has garnered significant interest due to its benefits with regard to dyslipidemia. This multifaceted functional food ingredient modulates several key enzymes associated with lipid regulation, including HMG-CoA reductase, CPT1, ACCβ, and acyl-CoA oxidase. It influences key antioxidant molecular pathways like Nrf2, limiting dyslipidemia occurrence and regulating liver cholesterol uptake through modulation of liver lipid receptors.

Astaxanthin daily doses and durations of analyzed studies: 12 mg for 8 weeks; 12 mg for 4 weeks; 20 mg for 12 weeks (two trials); 12 mg for 12 weeks; 8 mg for 8 weeks; 6 mg and 12 mg for 12 weeks; 6 mg, 12 mg, and 18 mg for 12 weeks.

This meta-analysis concludes positive effects of astaxanthin (6–20 mg/d) on HDL-C and triglyceride levels. Astaxanthin (6–20 mg/d) does not appear to significantly influence LDL-C and total cholesterol levels.

Regarding HDL-C, improvements were observed from 55 ± 8 mg/dL (pre-intervention) to 63 ± 8 mg/dL (post-intervention) (p < 0.01) in the 12 mg/d of astaxanthin groups. In triglyceride levels, results show a decrease from 151 ± 26 mg/dL (pre-intervention) to 112 ± 40 mg/dL (post-intervention) (p < 0.01) for 18 mg/d astaxanthin supplementation.

Further research is necessary to fully harness the potential of astaxanthin, which includes assessing astaxanthin in different subsets of patients, and in combination with other nutraceuticals to understand the compound’s effectiveness with regard to varying health conditions, genetic and epigenetic factors, and synergistic effects with other compounds.”

https://www.mdpi.com/1424-8247/18/8/1097 “Assessing the Effects of Moderate to High Dosage of Astaxanthin Supplementation on Lipid Profile Parameters—A Systematic Review and Meta-Analysis of Randomized Controlled Studies”

This same group of researchers assessed that in nine RCTs, astaxanthin had no effects on either body weight or BMI per https://www.mdpi.com/1424-8247/18/10/1482 “Therapeutic Potential of Astaxanthin for Body Weight Regulation: A Systematic Review and Meta-Analysis with Dose–Response Assessment”

Human studies of astaxanthin – Part 1

gettingwell4 4-5 stars Advanced knowledge, Epigenetics, Immune system, Stress , ,

Here are three 2025 clinical trials of the Nrf2 activator astaxanthin’s effects. Let’s start with a clinical trial of inflammation-related diabetic complications and insulin resistance:

“We investigated effects of 10 mg/day astaxanthin (ASX) supplementation for 12 weeks on microRNAs (miRNAs), lysophosphatidylcholine (LPC), and α-hydroxybutyrate (α-HB) as novel factors in development of a variety of diabetes-related complications.

  • LPC is believed to play a significant role in atherosclerosis and inflammatory diseases by modifying functions of multiple cell types, including smooth muscle cells, endothelial cells, monocytes, macrophages, and T cells. LPC can interfere with glucose-stimulated insulin secretion by impairing calcium homeostasis and other signaling pathways that are crucial for the proper functioning of beta cells. This impairment exacerbates hyperglycemia in diabetic patients. LPCs may impede insulin signaling pathways, thereby contributing to insulin resistance (IR).
  • α-HB is also an indicator of IR and impaired glucose regulation, both of which appear to result from excessive lipid oxidation and oxidative stress. The European population cohorts in 2016 identified α-HB as a selective biomarker for decreased glucose tolerance and prediabetes, which was independent of age, sex, BMI, and fasting glucose.
  • A number of studies have established a link between miR-21, miR-34a, and miR-155 and diabetic complications such as retinopathy and nephropathy.

In the ASX group, participants were divided into 2 subgroups according to the urinary albumin-to-creatinine ratio (ACR) (< 30 mg/g or ≥ 30 mg/g, an indicator of diabetic kidney disease).

  • The level of fasting plasma glucose before and after 12 weeks of treatment with ASX was 139.27 ± 21.18 vs. 126.43 ± 18.97 (p = 0.002), demonstrating a significant reduction compared to the placebo group.
  • In the ASX group, the mean HbA1c level at baseline was 7.89 ± 0.79 and declined to 7.05 ± 0.35 after the supplementation period, which was statistically significant.
  • Supplementation with ASX resulted in a statistically significant drop in HOMA-IR levels, whereas this parameter was not altered significantly in the placebo group.
  • The ASX group, in comparison with the placebo group, demonstrated marked changes in lipid profile factors such as TC, TG, and LDL (p = 0.011, p = 0.043, and p = 0.022, respectively).

Clinical studies indicate that rigorous diabetes management does not substantially diminish appearance of complications. Modifications in oxidative stress and IR markers, as well as miRNA expression, must be analyzed to identify biological markers with sufficient predictive power for development of complications in diabetic patients.

Supplementation with ASX substantially diminished the levels of α-HB, LPC, and inflammation-related miRNAs in diabetic patients with and without complications.”

https://onlinelibrary.wiley.com/doi/10.1155/ije/5878361 “Astaxanthin Modulates Inflammation in Type 2 Diabetes via Regulation of microRNAs, Lysophosphatidylcholine, and α-Hydroxybutyrate”

Another clinical trial investigated astaxanthin’s effects in heart failure patients:

“Chronic heart failure (HF) is often linked to increased oxidative stress and metabolic issues like high uric acid, which can worsen outcomes.This study aimed to investigate the effects of ASX supplementation on oxidative stress markers as the primary outcome and clinical symptoms in patients with HF.

80 patients with HF were enrolled and randomly assigned to receive either ASX (20 mg/day) or a placebo (20 mg/day of maltodextrin) for 8 weeks. Biomarkers including total antioxidant capacity (TAC), malondialdehyde (MDA), superoxide dismutase (SOD), serum uric acid (UA), and clinical symptoms (dyspnea, fatigue, appetite) were assessed pre-and post-intervention.

Daily supplementation with 20 mg of ASX for eight weeks in patients with HF resulted in significantly greater improvements in oxidative stress biomarkers compared to placebo group. This improvement included reductions in uric acid and MDA, along increases in TAC and SOD.

In our study, participants received the cis-isomer form of ASX. The cis-isomer of ASX demonstrates greater anti-inflammatory and antioxidant properties than the trans-isomer, along with enhanced bioavailability. Inconsistencies among studies may be attributed to differences in participants’ baseline antioxidant status, underlying medical conditions, dosage, isomeric form and formulation of ASX used, and the duration of intervention.

One of the strengths of this study is that it represents the first randomized clinical trial to evaluate the effects of ASX supplementation on oxidative stress markers, UA levels, and clinical symptoms in patients with HF. Additionally, potential confounding factors were controlled as much as possible. However, several limitations were identified, including the relatively short intervention duration, limited sample size, limited generalizability of the findings due to the single-center design, absence of blood ASX level measurements, and lack of long-term follow-up.”

https://link.springer.com/article/10.1186/s12872-025-05260-z “Impact of astaxanthin on oxidative markers, uric acid, and clinical symptoms in heart failure: a randomized clinical trial”

A third clinical trial evaluated astaxanthin’s effects as an adjunct to standard treatment of community-acquired pneumonia:

“Adult patients diagnosed with community-acquired pneumonia (CAP) were enrolled and assigned to receive either 12 mg/day ASX or a placebo in addition to standard antibiotic therapy for 7 days. Inflammatory markers, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-10 (IL-10), were measured at baseline and post-treatment. Secondary outcomes included Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation II (APACHE II) scores.

A total of 80 patients (40 per group) completed the study. Patients receiving ASX exhibited significant reductions in pro-inflammatory cytokines compared to the placebo group. IL-6 and TNF-α levels were significantly lower in the ASX group at the end of the study (P < 0.05). Additionally, SOFA and APACHE II scores showed greater improvements in ASX-treated patients, suggesting a potential role in mitigating disease severity.

These findings suggest that ASX may help preserve organ function, limit the progression of inflammatory injury, and reduce overall disease severity in hospitalized patients with CAP.

ASX is widely regarded as the most potent carotenoid, owing to its unique molecular structure. Its polar-nonpolar-polar configuration enables it to span lipid bilayers and neutralize ROS both within and outside cellular membranes—an advantage not shared by other carotenoids that tend to localize at the membrane surface.

Despite the positive findings of this study, some limitations should nevertheless be considered.

  • The relatively small sample size may have limited the statistical power to detect differences in some outcomes and affects the generalizability of the findings.
  • Microbiological data on CAP pathogens were not collected. As different microorganisms can trigger distinct inflammatory responses, this limits our ability to assess pathogen-specific variations in ASX efficacy.
  • A notable limitation of this study is the short follow-up duration, with outcomes assessed only over a 7-day period. While this timeframe offers insight into the acute effects of ASX on inflammatory and OS markers, it does not clarify whether these benefits are sustained beyond the immediate treatment window.
  • The fixed dose of 12 mg once daily may not have maintained optimal therapeutic levels throughout the day. Dose-ranging studies and evaluations of alternative regimens are needed to determine the most effective strategy.”

https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2025.1621308/full “The anti-inflammatory and antioxidant effects of astaxanthin as an adjunctive therapy in community-acquired pneumonia: a randomized controlled trial”

Part 2 continues with four more 2025 human studies of astaxanthin.

Plasmalogens Week #8 – Experience

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Wrapping up Plasmalogens Week with a summary of my plasmalogen-related experiences over the past two years since Plasmalogens, Part 3 in November 2023.

I took detailed plasmalogen measurements on July 24, 2025, with Dr. Goodenowe’s BioScan product. I’d guess that the populations against which BioScan personal Z-scores are derived are from Dr. Goodenowe’s research during this century, many frozen samples of which he’s kept. If so, I’d guess that these populations’ data probably don’t have bell-shaped curves, and that their data’s means and standard deviations may be skewed as they’re representing people who were diseased and/or old.

Here’s my peroxisomal function panel:

I wasn’t taking ProdromeNeuro or ProdromeGlia at the BioScan blood draw time. ProdromeNeuro and ProdromeGlia supplements contain plasmalogen precursors that bypass peroxisome organelles’ normal plasmalogen synthesis functions. I haven’t reordered these supplements in 2025, but took them until my supplies ran out in January 2025. Don’t know to what extent their effects may have continued for six months.

Every day for months before the BioScan, I took a fish oil capsule with 690 mg EPA and 310 mg DHA, and a flax seed oil capsule (700 mg alpha linolenic acid omega-3, 154 mg linoleic acid omega-6, and 168 mg oleic acid omega-9). I also ate 3 eggs a day.

These practices influenced the above peroxisomal function results. My Z-scores of DHA and EPA ethanolamine plasmalogens (DHA +1.3, EPA +1.7) are more than one standard deviation above their respective population means.

The next step of plasmalogen synthesis after peroxisomes takes place in endoplasmic reticulum organelles. Among other papers describing these steps in the ER link’s results, Improving peroxisomal function states:

“Proper functioning of peroxisomes in metabolism requires the concerted interaction with other subcellular organelles, including the endoplasmic reticulum (ER), mitochondria, lipid droplets, lysosomes, and the cytosol. A striking example of peroxisome-ER metabolic cooperation is de novo biosynthesis of ether phospholipids.”

ER stress involves the unfolded protein response, a protein homeostasis-maintaining system that monitors ER conditions by sensing inadequacy in ER protein folding capacity. ER stress is a very common occurrence for humans, in part because ER protein folding has an over 80% failure rate per Every hand’s a winner, and every hand’s a loser.

I haven’t read papers about ER stress directly influencing plasmalogen abundance. But I’ve curated papers, including several during this Plasmalogens Week, that demonstrate how oxidative stress reduces plasmalogens.

Here’s my BioScan inflammation / oxidative stress panel:

I don’t have a history of these measurements except for hsCRP, which has been below 1 for over five years since I started eating broccoli sprouts every day, along with taking taurine and betaine. That oxidative stress interventions may influence ER stress has been curated in papers such as Eat broccoli sprouts for stress, Part 2 of Eat broccoli sprouts for your eyes, Taurine week #7: Brain, Betaine and diabetes, and All about the betaine, Part 2.

Back to my peroxisomal function panel: I don’t consider my negative Z-scores (below the population mean) of Total PEs and Total PCs to be actionable. Both of them produced positive Z-scores (above the population mean) of their respective total plasmalogens (Total PLEs +1.3, Total PLCs +0.5). I view Total PEs and Total PCs as pools of raw materials for plasmalogen synthesis that are used when needed.

My July 2025 BioScan shows that my current practices provide adequate plasmalogens as compared with unknown populations. It indicates that to produce adequate plasmalogens, I don’t need ProdromeNeuro and ProdromeGlia plasmalogen precursor supplements that bypass normal peroxisomal function plasmalogen synthesis.

This year’s BioScan was a one-time event. I don’t agree with advocates for constantly tweaking health parameters, or obtaining frequent test results for ‘youthful’ targets, or competing with or conforming to other people’s measurements, or unfounded longevity beliefs. It’s every human’s choice whether or not we take responsibility for our own one precious life. Being overly obsessed about one’s health can be among the many symptoms of what’s ruining a person’s life.

I might use a future version of BioScan along with ProdromeNeuro and ProdromeGlia plasmalogen precursor supplements if I had to recover from an accident or some other health emergency that creates a substantial demand for plasmalogens’ antioxidant activities. But I’d first return to past practices I’ve found to be successful in combating oxidative stress, like increasing the frequency of Nrf2 activation by eating broccoli sprouts twice a day rather than once daily.

Plasmalogens Week #7 – Genes

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Continuing Plasmalogens Week with three 2025 papers, starting with a rodent study of genetically deleting a plasmalogen catabolizing enzyme:

“In this study, we investigated the impact of global and tissue-specific loss-of-function of a plasmalogen catabolizing enzyme, lysoplasmalogenase (TMEM86B), on circulatory and tissue lipidomes. Mice with homozygous global inactivation of Tmem86b (Tmem86b KO mice) were viable and did not display any marked phenotypic abnormalities.

Tmem86b KO mice demonstrated significantly elevated levels of plasmalogens alkenyl phosphatidylethanolamine (PE(P)) and alkenyl phosphatidylcholine (PC(P)), as well as lysoplasmalogens, in the plasma, liver, and natural killer cells compared to their wild-type counterparts. The endogenous alkenyl chain composition of plasmalogens remained unaltered in Tmem86b KO mice. Consistent with the global knockout findings, hepatocyte-specific Tmem86b knockout mice also exhibited increased plasmalogen levels in the plasma and liver compared to their floxed control counterparts.

Plasmalogens may be synthesized locally within various tissues, with each organ possessing the necessary enzymatic machinery to regulate its own plasmalogen levels. Plasmalogens are important structural constituents of the biological membranes of animals and certain anaerobic bacteria, and have several well-described functions, including regulating membrane dynamics and vesicular cholesterol transport and homeostasis.

  • One of the most interesting features of plasmalogens is their endogenous antioxidant activity, which is mostly due to the vinyl ether bond, which can scavenge reactive oxygen species and thereby protect other biomolecules from oxidative damage.
  • They increase the gene expression of multiple antioxidant enzymes to protect against chemically induced cytotoxicity and lipid peroxidation in cultured hepatocytes.
  • Plasmalogen derivatives such as polyunsaturated fatty acids (AA or DHA) and lysoplasmalogens can act as lipid mediators for multiple cellular signaling activities.
  • Plasmalogens are important for phagocytosis of macrophages, lipid droplet formation, and development and function of neuromuscular junctions.
  • They play vital roles in mediating immune responses, and mitochondrial fission to regulate adipose tissue thermogenesis, and protecting neuronal cells against cell death and inflammation.

All of these are suggestive of a critical role played by plasmalogens in maintaining cellular homeostasis.

While plasmalogen anabolism is well defined, its catabolism has been less studied. During catabolism, plasmalogens are deacylated by the action of a calcium-independent phospholipase A2 enzyme (iPLA2) to produce lysoplasmalogens. However, cytochrome C has also been shown to act as a plasmalogenase under certain circumstances.

The amount of lysoplasmalogens in cells is tightly regulated either by reacylation into plasmalogens through a coenzyme A-independent transacylase, or by degradation into fatty aldehydes and glycerophospholipids by an alkenyl ether hydrolase commonly known as lysoplasmalogenase. Lysoplasmalogenase is a microsomal transmembrane enzyme highly specific for lysoplasmalogens, and has no activity against plasmalogens.

While research on the distinct biological functions of lysoplasmalogens and plasmalogens is lacking, some reports indicate potential toxic effects of lysoplasmalogens. Degradation products of lysoplasmalogens, such as fatty aldehydes, are highly reactive electrophilic compounds that can form toxic adducts with cellular proteins and lipids. These interactions can lead to cellular dysfunction and contribute to various pathological conditions. Their accumulation in ischemic/reperfused tissues has been associated with cellular damage.

However, we observed that the amount of lysoplasmalogens as a proportion of total plasmalogens in the liver of Tmem86b KO mice was only ∼3.5%, indicating that elevated lysoplasmalogens are rapidly converted into plasmalogens within the liver. In adipose tissue-specific Tmem86a KO mice, which also exhibited higher lysoplasmalogens, no toxic effects were observed. Instead, these mice showed elevated mitochondrial oxidative metabolism and energy expenditure, offering protection from high-fat diet-induced metabolic dysfunction. These findings suggest that any potential toxic effects of lysoplasmalogens are largely mitigated by their rapid reacylation into plasmalogens.

This study enhances our understanding of regulatory mechanisms governing plasmalogen metabolism, and highlights the potential of targeting Tmem86b to therapeutically raise plasmalogen levels.”

https://www.jlr.org/article/S0022-2275(25)00068-9/fulltext “Modulation of endogenous plasmalogens by genetic ablation of lysoplasmalogenase (Tmem86b) in mice”

An independent researcher published a commentary on the above study:

“While the biosynthesis of this particular lipid subclass, starting in the peroxisomes and ending at the endoplasmic reticulum, has been the subject of extensive research, the degradation pathway of these compounds remains to be further elucidated. Plasmalogen breakdown is a complex process involving enzymatic hydrolysis, oxidative cleavage, and possibly also a recycling mechanism.

A fundamental unresolved question in the field of plasmalogen catabolism is which of the two possible reaction routes is actually the more important one. Either 1) directly via plasmalogenase or 2) via a deacylation step by a plasmalogen-specific phospholipase A2 (cPLA2, PLA2G4A), yielding a lysoplasmalogen as the first degradation product, and subsequent hydrolysis of the ether bond by a lysoplasmalogenase such as TMEM86A and TMEM86B. It is also unclear how these pathways interact or compensate for each other, how they are regulated, and whether they are tissue- or cell type–specific.

To make the story even more complex, a CoA-independent transacylase activity was described that reacylates lysoplasmalogen intermediates back to plasmalogens by transferring polyunsaturated fatty acids to the vacant sn-2 position of ether lysophospholipids. But no gene for this enzyme has so far been identified.

Why is plasmalogen breakdown so important? Disturbances in plasmalogen metabolism are associated with several human disorders. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis have been shown to be associated with reduced levels of plasmalogens.

Unfortunately, it is still too early to draw conclusions about the individual roles of TMEM86A and TMEM86B, as their cellular localisation and function are not sufficiently studied, and reliable antibodies for these proteins are not yet available. Localization of the two TMEM86 homologs overlaps to some extent, as shown, for example, by their gene expression in small intestine. However, whether one isoform is able to compensate for a deficiency in the other is uncertain, and was not found in small intestine of Tmem86b knockout mice [in the above study].

In contrast to the two proteins TMEM86A and TMEM86B, cytochrome c is much better studied. It is associated with the inner mitochondrial membrane, and can be released into the cytosol during apoptosis. It has a wide tissue distribution with most abundant gene expression levels in the digestive tract and heart.“

https://www.jlr.org/article/S0022-2275(25)00074-4/fulltext “Plasmalogen. Quo vadis?”

The statement “no gene for this enzyme has so far been identified” revealed a paradigm. But maybe what’s being observed evolved before genes?

One example of this principle is from the 1966 https://www.science.org/doi/10.1126/science.152.3720.363 “Evolution of the Structure of Ferredoxin Based on Living Relics of Primitive Amino Acid Sequences” which provided evidence pointing to heme protein evolution beginning before gene evolution. Its abstract included this statement:

“We explain the persistence of living relics of this primordial structure by invoking a conservative principle in evolutionary biochemistry: The processes of natural selection severely inhibit any change in a well-adapted system on which several other essential components depend.”

Maybe the process of reassembling plasmalogen breakdown products back into plasmalogens without involving a specific gene likewise became essential?

A role of plasmalogens in diabetic kidney disease was found in a third study that investigated a genetic rodent model of diabetes:

“Diabetic nephropathy (DN) represents a frequent cardiovascular complication of diabetes, affecting about 20–50% of individuals with the disease. Globally, it constitutes a primary etiology for end-stage kidney disease (ESKD) and chronic kidney disease (CKD), while also serving as a significant independent risk factor for cardiovascular morbidity and mortality.

Although intensive management strategies targeting blood pressure and glucose levels demonstrably attenuate the risk of DN development, they do not confer complete protection. This residual risk strongly implicates pathogenic factors beyond impaired glucose metabolism and hemodynamic alterations in DN pathogenesis.

In the present study, we employed the db/db mice as the DN model. When compared to other diabetes models, such as those induced by streptozotocin (STZ) or high-fat diet combined with STZ, the db/db model more accurately recapitulates the pathological features of human type 2 diabetes mellitus (T2DM). It also possesses a stable genetic background, making it particularly well-suited for the investigation of diabetes complications.

Transcriptomics revealed extensive dysregulation of metabolic and lipid regulatory pathways in db/db. Lipidomics uncovered pronounced abnormalities in cardiolipin species composition and plasmalogen profiles. Transcriptome-lipidome integration demonstrated impaired phosphatidylcholine (PC) biosynthesis, mechanistically linked to dysregulation of choline phosphotransferase 1 (chpt1), which correlated significantly with compromised tissue regeneration capacity.

Volcano plot analysis delineated specific lipid alterations, particularly in plasmalogen species in plasmalogen lipids. Plasmenylcholines (plas-PC) and plasmenylethanolamine (plas-PE) containing n-3 polyunsaturated fatty acids (PUFAs) were significantly decreased in the kidneys of db/db mice. Conversely, plas-PCs and plas-PEs esterified with n-6 PUFAs showed substantial accumulation in diabetic kidneys.

In conclusion, the highly sensitive and extensively targeted UHPLC-MS/MS methodology enabled a more in-depth characterization of renal metabolic and lipid perturbations in db/db mice. These alterations principally reflect the sustained inflammatory milieu and compromised antioxidant defenses characteristic of DN renal tissues.”

https://www.csbj.org/article/S2001-0370(25)00301-0/fulltext “Multi-omics characterization of diabetic nephropathy in the db/db mouse model of type 2 diabetes”

Plasmalogens Week #5 – Health and Diseases, Part 1

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Continuing Plasmalogens Week with three 2025 papers, starting with a human study that included plasmalogen biomarkers of non-communicable disease fatigue symptoms:

“This study explored the biological mechanisms underlying fatigue in patients with NCDs using a multi-omics approach. Our findings indicate that distinct metabolic pathways, salivary microbiota, and genetic factors may contribute to different dimensions of fatigue, including general, physical, and mental fatigue.

  • General fatigue is associated with unsaturated fatty acid biosynthesis, indicating its role in lipid metabolism.
  • Physical fatigue was associated with plasmalogen synthesis, mitochondrial beta-oxidation of long-chain fatty acids, and selenoamino acid metabolism, suggesting a potential contribution of impaired energy production.
  • Mental fatigue is associated with homocysteine degradation and catecholamine biosynthesis, which may influence cognitive fatigue.

This exploratory study suggests that fatigue in patients with NCDs may involve disruptions in lipid metabolism, neurotransmitter pathways, microbial composition, and genetic variations. Blood-based biomarkers showed better predictive potential for physical fatigue, whereas salivary-based models were more indicative of mental fatigue.

Although our findings support the role of lipid metabolism, the contribution of plasmalogen synthesis remains underexplored. Further studies are needed to validate these findings and understand their mechanisms of action.”

https://link.springer.com/article/10.1186/s12911-025-03034-3 “Visualizing fatigue mechanisms in non-communicable diseases: an integrative approach with multi-omics and machine learning”

A human study of metabolic dysfunction-associated steatotic liver disease (MASLD) included investigating plasmalogens:

“In this study, we applied untargeted metabolomic profiling to serum samples from individuals with and without MASLD, classified by the Fatty Liver Index, with the goal of identifying characteristic metabolic signatures and pathways that may underlie disease presence and progression. Individuals in the MASLD group displayed significantly higher levels of ALT, AST, ALP, and GGT, reflecting ongoing hepatic injury, cholestasis, and oxidative stress. However, albumin and bilirubin levels remained within normal limits, indicating early to intermediate disease stages rather than advanced fibrosis or cirrhosis.

A consistent and highly significant lipidomic pattern in the MASLD group is the depletion of plasmalogens and sphingomyelins. Depletion of these lipid classes was identified as a hallmark of insulin resistance as defined by the triglyceride-glucose index. In contrast, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol species were elevated in MASLD, pointing toward broader lipid remodeling events.

Reduced plasmalogen and sphingomyelin levels positions their depletion as a core feature of metabolic dysfunction. Plasmalogens are ether phospholipids with strong antioxidant capacity, and their reduction suggests a loss of protective buffering against oxidative stress, one of the main drivers of MASLD progression. Similarly, sphingomyelin depletion implicates altered membrane dynamics and signaling disturbances, further contributing to metabolic dysfunction.

Depletion of plasmalogens 1-(1-enyl-palmitoyl)-2-oleoyl-GPC (P-16:0/18:1), 1-(1-enyl-palmitoyl)-2-linoleoyl-GPC (P-16:0/18:2), 1-(1-enyl-palmitoyl)-2-palmitoyl-GPC (P-16:0/16:0), 1-(1-enyl-palmitoyl)-2-palmitoleoyl-GPC (P-16:0/16:1), 1-(1-enyl-palmitoyl)-2-oleoyl-GPE (P-16:0/18:1), 1-(1-enyl-palmitoyl)-2-linoleoyl-GPE (P-16:0/18:2), and disruption of the glutamate–gamma-glutamyl pathway stand out as central features of metabolic dysfunction in MASLD, with clear potential to inform biomarker discovery, disease classification, and the design of targeted therapeutic strategies.”

https://www.mdpi.com/2218-1989/15/11/687 “Metabolomic Signatures of MASLD Identified by the Fatty Liver Index Reveal Gamma-Glutamyl Cycle Disruption and Lipid Remodeling”

A rodent study investigated dietary sea squirt (AM) plasmalogen ethanolamine (PlsEtn) extract’s and dietary pig liver (PL) phosphatidyl ethanolamine (PtdEtn) extract’s effects on acetaminophen liver injury:

“We investigated dietary effects of PlsEtn from ascidian on chronic hepatic injury in acetaminophen (APAP)-treated mice. Five-week-old male mice were divided into four groups (n = 12), which were treated with experimental diets for two weeks and then the respective APAP-containing diet for five weeks.

Ingested PlsEtn is digested into lysoPlsEtn and free fatty acid in the small intestine. PlsEtn digests are absorbed and are subsequently resynthesized into PlsEtn preferentially with PUFA.

Acetaminophen is a frequently used analgesic and antipyretic. Approximately 90% of APAP is metabolized by UDP-glucuronosyltransferase and sulfotransferase into glucuronic acid and sulfate conjugates, respectively.

5–9% of APAP is metabolized into the highly reactive intermediate N-acetyl-p-benzoquinone imine (NAPQI). This metabolite is considered a pivotal molecule in APAP-induced hepatotoxicity and is conjugated by glutathione (GSH). Excessive NAPQI levels deplete GSH and covalently bind to cellular proteins, resulting in organelle dysfunction, such as mitochondria dysfunction. These impairments induce oxidative stress, cell malfunctions, and subsequently, cell death, such as ferroptosis and apoptosis.

Mice were treated with continuous APAP consumption to induce oxidative stress and impaired lipid metabolism in the liver. Effects of diets were evaluated based on levels of malondialdehyde (MDA), a marker of lipid oxidation, on fatty acid content, and on expression of apoptosis-related proteins in the liver.

The PlsEtn-rich diet effectively suppressed APAP-induced decrease in body and liver weights of mice. However, this suppressive effect was not observed in mice fed a PtdEtn-rich diet. APAP administration decreased the total fatty acid content in the liver, whereas a PlsEtn-rich diet alleviated this decrease and increased the hepatic content of docosahexaenoic acid (DHA).

Owing to the alkenyl linkage, which exhibits antioxidant properties, PlsEtn was expected to markedly suppress hepatic lipid oxidation. However, its suppressive effect was the same extent as that by PtdEtn. Both PlsEtn and PtdEtn contain an ethanolamine base in their structures, and free ethanolamine and its metabolite choline suppress lipid peroxidation. Dietary PlsEtn and PtdEtn may be metabolized into free ethanolamine and its further metabolites, which may alleviate APAP-induced hepatic lipid oxidation.

Dietary ethanolamine glycerophospholipids (EtnGpls) rich in PlsEtn or PtdEtn suppressed APAP-induced lipid oxidation in the liver. Protein expression results revealed that dietary EtnGpls reduced expression of certain apoptosis-related proteins compared to the APAP group. This reduction was more effective in mice fed the PlsEtn-rich diet than in those on the PtdEtn-rich diet.”

https://www.mdpi.com/2076-3417/15/11/5968 “Dietary Ethanolamine Plasmalogen from Ascidian Alleviates Chronic Hepatic Injury in Mice Treated with Continuous Acetaminophen”

This study neither demonstrated nor provided citations for its dietary plasmalogen recycling statements.

Three more plasmalogen health and disease papers are curated in Part 2.