Red cabbage pigments and the brain

This 2020 sheep study measured red cabbage anthocyanin concentrations:

“Study aim was to determine whether strongly bioactive hydrophilic red cabbage anthocyanins cross the blood-cerebrospinal fluid barrier (blood-CSF barrier) and whether there is a selectivity of this barrier towards these compounds.

The blood-CSF barrier, apart from the vascular blood-brain barrier, is the second important barrier. Despite very tight connections between endothelial cells of blood vessels of the choroid plexus, blood-CSF barrier allows selective passing of substances from blood to CSF, which is considered as a medium actively involved in transport of information to nerve cells.

Uncharged, lipophilic, and small-sized substances (≤ 600 Da) can cross the brain barriers without major obstacles thanks to diffusion. The rate of these substances’ penetration into brain tissue is directly proportional to their lipid solubility, and inversely proportional to particle size. Hydrophilic substances require special carriers.

The average percentage level of native anthocyanins over the whole experiment was almost 39.5%, while their metabolites constituted just over 60.5%. However, the proportion of native forms vs. metabolites did not develop identically:

  1. Early term (0.5-4 hrs) was distinguished by native derivatives (> 76%).
  2. Second period (4.5 h) had a similar contribution of native anthocyanins (49.85%) and their metabolites (50.15%).
  3. Third interval (5.0-10 h) more than 87% of anthocyanins were metabolites.

For comparison, a human experiment showed only one period with maximum blood plasma anthocyanins concentration (2 h) after red cabbage consumption.

Only one of 17 native anthocyanins found in blood plasma was detected in CSF. Eleven of 17 metabolites found in blood were identified in CSF.

sheep csf cyanins

Due to their hydrophilic nature and considerable size (≥ 611 Da), there seems to be no possibility to use diffusion for permeation of red cabbage anthocyanins through the blood-CSF barrier. These pigments may pass through this barrier only by the use of special carriers. Other mechanisms of anthocyanins permeation through blood-CSF barrier cannot be eliminated.

Two maximal values of total anthocyanins concentration appeared in both blood and CSF. When the pool of cyanidin compounds available in blood became depleted, the decline of total anthocyanin concentration in CSF was also noted.

Nonacylated cyanidin derivatives penetrated the blood-CSF barrier, but acylated cyanidin derivatives did not. A significantly higher proportion of cyanidin sulfate forms in CSF (31%) compared to blood plasma (9%).

Further targeted studies are needed to determine which paths of permeation via blood-CSF barrier are actually responsible for anthocyanins passing, as well as what mechanisms are present during these processes. In addition, it is worth remembering that low molecular weight compounds formed mainly by colonic microbiota are very important metabolites of anthocyanins, and could be relevant in the context of permeation through brain barriers.”

https://pubs.acs.org/doi/10.1021/acs.jafc.0c03170 “The Blood–Cerebrospinal Fluid Barrier Is Selective for Red Cabbage Anthocyanins and Their Metabolites” (not freely available)


Don’t understand why this study hasn’t been cited even once. These researchers’ methods could be performed with broccoli and other red cabbage compounds.

Every hand’s a winner, and every hand’s a loser

Another great blog post Know When To Fold ‘Em by Dr. Paul Clayton:

“Newly formed proteins entering the endoplasmic reticulum must be correctly folded to achieve their final form and function. This is a complex procedure with a failure rate of over 80%.

When metabolism is sufficiently skewed, accuracy of protein folding in the endoplasmic reticulum falls below an already low baseline of 20%. Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum then triggers stress.

Integrated Stress Response (ISR) is something that cells do when they are affected by major stressors:

  • ISR turns down global protein synthesis, which is designed to kill virally infected or cancerous cells. If it kills the cancer cell or virally infected cell, that is the end of it.
  • If the stressor is in the heat / hypoxia / nutrient group, however, ISR effectively puts a cell into dark mode until hard times are over. Once the stressor has passed, a cell can then start to recover and return to homeostatic health.
  • But if the stressor is sustained, a low-grade ISR continues to smolder away, causing long-term impairment locally and ultimately systemically. Accumulation of misfolded or unfolded proteins activates ISR, leading to a down-regulation of protein synthesis, and increasing protein folding and degradation of unfolded proteins.

This is analogous to inflammation. Acute inflammatory responses to a pathogen or to tissue damage are entirely adaptive, and essential. Chronic inflammation, on the other hand, causes local and eventually systemic damage if left unchecked for long enough.”


A 2020 rodent study was cited for “reversing age-related cognitive decline”:

“This suggests that the aged brain has not permanently lost cognitive capacities. Rather, cognitive resources are still there, but have been somehow blocked, trapped by a vicious cycle of cellular stress.

Our work with ISR inhibition demonstrates a way to break that cycle, and restore cognitive abilities that had become walled off over time.

stress response inhibitor effects

If these findings in mice translate into human physiology, they offer hope and a tangible strategy to sustain cognitive ability as we age.”

https://elifesciences.org/articles/62048 “Small molecule cognitive enhancer reverses age-related memory decline in mice”


I’m curious as to why sulforaphane hasn’t been mentioned even once in Dr. Paul Clayton’s blog, which started three years ago. Do hundreds of sulforaphane studies performed in this century not contribute to his perspective? Polyphenols are mentioned a dozen times, yet they are 1% bioavailable compared with 80% “small molecule” sulforaphane.

Advice from the song depends on your definition of money:

“Know when to walk away
Know when to run
Never count your money
When you’re sitting at the table”

A bat epigenetic clock

This 2021 study subject was bats:

“Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity.

Hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that:

  • Age-related methylation change is influenced by developmental processes, while
  • Longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that
  • Bat longevity results from augmented immune response and cancer suppression.

Molossus molossus [a short-lived species] age genes are not enriched for immunity genes or genes that frequently mutated in cancer. However, M. molossus longevity genes exhibit significant overlap with genes involved in immunity and genes frequently mutated in human tumors.

Similar overlap patterns among immunity, longevity, and tumor-mutated genes also exist for long-lived bats.

Two species’ genetic adaptations for tumor suppression have been described to help explain their extreme longevity. Bats also have genetic mechanisms that enable strong antiviral immune responses without inducing damaging inflammatory reactions that may enable them to tolerate high levels of viral exposure.

Our results are consistent with an epigenetic clock theory of aging that connects beneficial developmental and cell maintenance processes to detrimental processes causing tissue dysfunction.”

https://www.nature.com/articles/s41467-021-21900-2 “DNA methylation predicts age and provides insight into exceptional longevity of bats”


The founder of the epigenetic clock has been busy, coauthoring more published studies than there have been weeks in this year! I’ve read five other 2021 studies he’s coauthored on dogs, horses, mammals (2), and humans in DNA methylation biomarker for cumulative lead exposure is associated with Parkinson’s disease. This one stood out for its “longevity results from augmented immune response and cancer suppression” findings.

If we’re interested in longevity, this clarity can direct efforts to both improve our immune systems and avoid problems like cancer. Symptoms may be subclinical, but that doesn’t provide adequate rationale to not address causes.

Peer review comments and responses were informative:

Reviewer #1 – “Developing an aging clock that works for a diverse set of bat species is a spectacular achievement.”

Reviewer #2 – “This is a tour de force study.”

Replies to Reviewer #3:

“Difference in recorded lifespans between three long-lived species and two short-lived species that we used to identify longevity DMPs [differentially methylated positions] is 20 years or more, even though they have similar body sizes (20-40 g). The three long-lived species [maximum ages 29.9, 30.5, and 37.1 years] also represent three different phylogenetic lineages.

CpG sites that undergo hypomethylation with age do so largely at random. In contrast, sites that undergo hypermethylation with age are highly nonrandom, and as has been noted before, are near genes associated with development. So yes, we believe there are predictable methylation changes with age.”

Choosing your future with β-glucan

This 2020 rodent study investigated yeast cell wall β-glucan effects on bacterial infections:

“β-glucan is a potent inducer of epigenetic and functional reprogramming of innate immune cells, a process called trained immunity, resulting in an enhanced host response against secondary infections. We investigate whether β-glucan exposure confers protection against pulmonary Mycobacterium tuberculosis (Mtb) infection.

  • β-glucan induces trained immunity via histone modifications. β-glucan-induced trained immunity confers protection against virulent Mtb via the IL-1 signaling pathway.
  • β-glucan-induced trained immunity enhances production of proinflammatory cytokines in human monocytes challenged with heat-killed Mtb. Increase in cytokine production capacity was the result of epigenetic reprogramming and mediated via the PI3K/Akt/mTOR pathway.

Most important, β-glucan-treated mice infected with Mtb demonstrated remarkably enhanced survival, which was dependent on IL-1 signaling.

survival Mtb

β-glucan epigenetically reprograms human monocytes, leading to a phenotype characterized by a unique IL-1 signature and anti-mycobacterial properties. β-glucan-treated mice were protected against pulmonary Mtb infection.

While both β-glucan and BCG [Bacillus Calmette-Guerin tuberculosis vaccine] reprogram HSCs to induce trained immunity, BCG reprogramming of HSCs was dependent on IFNγ signaling. β-glucan reprogramming of HSCs was mediated via IL-1 signaling, which was also required for protection against Mtb infection.

Considering safety of β-glucan in a human clinical trial, our results strongly suggest potential clinical implications of β-glucan for both prophylactic and therapeutic use in TB.”

https://www.cell.com/cell-reports/fulltext/S2211-1247(20)30587-8 “β-Glucan Induces Protective Trained Immunity against Mycobacterium tuberculosis Infection: A Key Role for IL-1″


My comment “many of these findings also apply to yeast cell wall β-glucan treatments” in Long-lasting benefits of a common vaccine lacked clarity. This post provides part of that evidence.

So where do you choose to be? In an 80% survival group who were administered β-glucan before they encountered a serious infection? Or in a < 20% survival group who didn’t take β-glucan?

Which is better for resolving a health situation before it becomes a problem?

  • Roll the dice, and hope for luck / providence?
  • Do nothing constructive, and depend on interventions after a problem occurs?
  • Take responsibility for your own one precious life?

PXL_20210327_111214712

Harnessing endogenous defenses with broccoli sprouts

This 2019 article was by the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease. It isn’t widely available, so I’ll quote liberally:

“Demand for solutions to digestive health issues is accelerating, especially since both scientific literature and popular press dedicate significant resources to promoting awareness of what has come to be known as ‘gut health’. In considering available therapies and the possibility that a somewhat different approach may more comprehensively optimise function of the gut ecosystem, a number of questions which do not yet have satisfactory answers are ponderable dilemmas:

  1. If diet alone can dramatically shift composition of the microbiome within 24 hours, what do we expect of a probiotic supplement?
  2. Even though probiotics as food or supplements demonstrate favourable clinical outcomes, they typically don’t colonise the gut. How do we expect them to restore diversity and lost species to the gut microbiome after antibiotics? If no trace of an administered probiotic organism can be found a few weeks later, is there any sustained benefit?
  3. Presence of obesity and other diseases is indirectly proportional to diversity of microbial organisms inhabiting the human gut. What can we expect of a few selected probiotic strains in helping to solve this problem?
  4. No antimicrobial approach selectively destroys a pathogen without impacting commensals to some degree. If we select a tool to eradicate gut pathogens, pathobionts or rogue commensals, how do we avoid damaging protective commensals with which we live symbiotically?
  5. The value of using a probiotic supplement after antibiotic therapy to recolonise the gut is uncertain. A 2018 multi-centre study showed that probiotic supplementation after antibiotics delayed gut microbiome reconstitution by around five months.
  6. If the gut can harbour around 1,000 different species, why do we expect a probiotic supplement harbouring just a few species to favourably modify a human microbiome?
  7. If Lactobacilli make up <0.1% of total microbes, why do we so readily choose them as probiotic supplements?
  8. If L-glutamine is a preferred energy source for the small intestine and not the colon, why is it used almost universally in gut repair programmes regardless of the affected region?

Removal of gluten and administration of probiotics have lesser impact than endogenous factors like elevated HbA1c:

Shift emphasis closer to optimising colonocyte metabolism as the primary driver of dysbiosis in the colon. Since these mechanisms within the human gut ecosystem already exist, intervene at this level, as distinct from using antimicrobials and exogenous probiotic strains to influence host cell function.

Phytonutrients that potently activate these core processes have been identified and are sufficiently bioavailable to achieve this end. Restoring homeostasis to the intestinal epithelial cells can be readily justified as a key initial step.

Sulforaphane is a potent inducer of hundreds of genes associated with cellular defences mechanisms. In this context, these genes include those that code for antioxidant and phase II detoxification enzymes, glutathione and metallothionein.

Sulforaphane exhibits other more specific gut and immune-related effects. As the most potent single food-derived activator of Nrf2, sulforaphane is capable of upregulating protective genes in colonocytes and other cells.

A growing body of work has identified the colonocyte as the driver of dysbiosis. Targeting colonocyte function provides an alternative to targeting microbes for remediation of dysbiosis.”

https://www.researchgate.net/publication/336578800_Restoring_Gut_Ecology_Harnessing_the_Inbuilt_Defence_Mechanisms_of_the_Gut_Epithelium “Restoring Gut Ecology: Harnessing the Inbuilt Defence Mechanisms of the Gut Epithelium” (registration required)


If you can’t access this paper, read The future of your brain is in your gut right now. If you can’t access that paper, listen to Switch on your Nrf2 signaling pathway.

The future of your brain is in your gut right now

A 2020 paper by the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease:

“The gut and brain communicate bidirectionally via several pathways which include:

  1. Neural via the vagus nerve;
  2. Endocrine via the HPA axis;
  3. Neurotransmitters, some of which are synthesized by microbes;
  4. Immune via cytokines; and
  5. Metabolic via microbially generated short-chain fatty acids.

How does nature maintain the gut-microbiome-brain axis? Mechanisms to maintain homeostasis of intestinal epithelial cells and their underlying cells are a key consideration.

The symbiotic relationship that exists between microbiota and the human host is evident when considering nutrient requirements of each. The host provides food for microbes, which consume that food to produce metabolites necessary for health of the host.

Consider function of the human nervous system, not in isolation but in integration with the gastrointestinal ecosystem of the host, in expectation of a favorable impact on human health and behavior.”

https://www.sciencedirect.com/science/article/pii/B9780128205938000148 “Chapter 14 – The gut microbiome: its role in brain health” (not freely available)


Always more questions:

  • What did you put into your gut today?
  • What type of internal environment did it support?
  • What “favorable impact on human health and behavior” do you expect from today’s intake?
  • How will you feel?
  • Will you let evidence guide feeding your gut environment?

See Switch on your Nrf2 signaling pathway for an interview with the author.

Part 2 of Switch on your Nrf2 signaling pathway

To follow up topics of Part 1‘s interview:

1. “We each have a unique microbial signature in the gut. Metabolites that you produce might not be the same ones that I produce. This makes clinical studies very difficult because you don’t have a level playing field.”

This description of inter-individual variability could inform researchers’ investigations prior to receiving experimental results such as:

Post-experimental analysis with statistical packages of these types of results is apparently required. But it doesn’t produce meaningful explanations for such individual effects.

Analysis of individual differences in metabolism can better inform explanations, because it would investigate causes for widely-variable effects. Better predictive hypotheses could be a result.

2. Today I’m starting my 40th week of eating a clinically-relevant amount of microwaved 3-day-old broccoli sprouts every day. To encourage sulforaphane’s main effect of Nrf2 signaling pathway activation, I won’t combine broccoli sprouts with anything else either during or an hour before or after.

I had been taking supplements at the same time. This interview got me thinking about the 616,645 possible combinations of my 19 supplements and broccoli sprouts.

That’s way too many to be adequately investigated by humans. Especially because contexts for each combination’s synergistic, antagonistic, or additive activities may be influenced by other combinations’ results.

I’ll just eat food and take supplements outside of this sulforaphane window.

I’ve taken 750 mg fructo-oligosaccharides (FOS) twice a day for sixteen years. I’ve considered it as my only prebiotic. Hadn’t thought of either of these points:

  • “Polyphenols are now considered to be a prebiotic food for microflora in the gut. They tend to focus on producing additional amounts of lesser known species like Akkermansia muciniphila, and have a direct prebiotic effect. Microbiota break these big, bulky molecules down into smaller metabolites, which clearly are absorbed. Some beneficial effects that come from polyphenols are not from the original molecule itself, but from a variety of metabolites produced in the gut.
  • We use a prebiotic, actually called an immunobiotic, which is a dead lactobacillus plantarum cell optimised for its cell wall content of lipoteichoic acid. Lipoteichoic acid attaches to toll-like receptor 2, and that sets off a whole host of immune-modulating processes, which tend to enhance infection control and downregulate inflammation and downregulate allergenicity.”

3. “Quinone reductase is critical because it is the final enzyme in the phase two detox pathway that stops DNA being mutated or prevents deformation of DNA adducts which are mutagenic. I want to look at genes that govern redox balance, inflammation, detoxification processes, cellular energetics, and methylation.”

Gene functional group classifications are apparently required in studies, to accompany meaningless statistics. When I’ve read papers attaching significance to gene functional groups, it often seemed like hypothesis-seeking efforts to overcome limited findings.

I’ll start looking closer when study findings include Nrf2 signaling pathway targets quinone reductase, DNA damage marker 8-hydroxydeoxyguanosine, and enzymes glutathione peroxidase and glutathione S-transferase.

4. I bolded “unregulated inflammation” in Part 1 because it’s a phrase I’d ask to be defined if that site enabled comments. Thinking on inflammation seems to come from:

“We focus on the intestinal epithelial cell as a key player because if you enhance function of that cell, and Nrf2 is part of that story, once you get those cells working as they should, they are modulating this whole underlying immune network.”

An environmental signaling paradigm of aging and Reevaluate findings in another paradigm have a different focus. That paradigm looks at inflammation in the context of aging:

“A link between inflammation and aging is the finding that inflammatory and stress responses activate NF-κB in the hypothalamus and induce a signaling pathway that reduces production of gonadotropin-releasing hormone (GnRH) by neurons.

The case is particularly interesting when we realize that the aging phenotype can only be maintained by continuous activation of NF-κB. So here we have a multi-level interaction:

  1. Activation of NF-κB leads to
  2. Cellular aging, leading to
  3. Diminished production of GnRH, which then
  4. Acts (through the cells with a receptor for it, or indirectly as a result of changes to GnRH-receptor-possessing cells) to decrease lifespan.

Cell energetics is not the solution, and will never lead to a solution because it makes the assumption that cells age. Cells take on the age-phenotype the body gives them.

Aging is not a defect – it’s a programmed progressive process, a continuation of development with the body doing more to kill itself with advancing years. Progressive life-states where each succeeding life-stage has a higher mortality (there are rare exceptions).

Cellular aging is externally controlled (cell non-autonomous). None of those remedies that slow ‘cell aging’ (basically all anti-aging medicines) can significantly extend anything but old age.

For change at the epigenomic/cellular level to travel up the biological hierarchy from cells to organ systems seems to take time. But the process can be repeated indefinitely (so far as we know).”


Switch on your Nrf2 signaling pathway

An informative interview to start this year with the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease:

The Antioxidant Dilemma with Dr. Christine Houghton

“The thing about science is, the more you know, the more you realise you don’t know. And I have this enormous respect now for signalling processes that are going on within the cell, and not just signalling. The way mother nature switches on, switches off, foot on brake, foot on accelerator, continuously all of the time.

Things have changed in understanding the function of Nrf2 for a start, in controlling in many ways those cellular defences. We could then switch on Nrf2. You switch on a whole host of protective molecules all at the same time.

We use NAC [N-acetyl-cysteine] in the lab all the time because it stops an Nrf2 activation. So, that weak pro-oxidant signal that you use to activate Nrf2, you switch it off by giving a dose of NAC. It’s a potent antioxidant in that right, but it’s blocking signalling. And that’s what I don’t like about its broad use.

The real advantage of sulforaphane is not only is it the most potent inducer of Nrf2, or activator, but it’s also highly bioavailable. It’s a very tiny, low-molecular weight, lipophilic molecule that just glides straight in through cell membranes. It’s about 80% bioavailable. Whereas big, bulky polyphenols are about 1% bioavailable just simply because of their chemical structure.

We focus on the intestinal epithelial cell as a key player because if you enhance function of that cell, and Nrf2 is part of that story, once you get those cells working as they should, they are modulating this whole underlying immune network.

I’m particularly interested in looking at core upstream factors that govern cellular defences. So, I want to look at genes that govern redox balance, inflammation, detoxification processes, cellular energetics, and methylation.

Intestinal epithelial, just like any other cell in the body, will respond to Nrf2 activation. It will respond to NF-κB downregulation. That’s going to enhance redox control. It’s going to reduce unregulated inflammation. It’s going to enhance detoxification processes. It’ll increase glutathione synthesis.

All of those core factors that any cell needs to work normally will be enhanced by activating Nrf2. And I use a high-yielding sulforaphane supplement of about 20 milligrams a day to do that. So, that’s the beginning.

Probiotics don’t typically colonise in an adult. That’s where we come back to this idea of restoring the gut ecosystem and using prebiotic foods.

In an ideal world, we’d be looking at 600 to 800 grams of non-starchy plant foods a day. In a real world, that isn’t always going to happen.

I never use the term leaky gut because it isn’t that. It’s a dynamic structure that becomes unresponsive.”


Hadn’t thought about weighing my daily AGE-less Chicken Vegetable Soup dinner (half) then tomorrow for lunch. Its total weight tonight was 2,575.5 grams.

  • Subtract 207.2 g wine, 985.6 g chicken broth, and 64.2 g noodles;
  • Add 131 g 3-day-old broccoli sprouts microwaved to ≤ 60°C (140°F) eaten earlier;
  • Subtract an estimated 170 g (6 oz.) chicken, didn’t measure juice squeezed from one lemon, didn’t estimate evaporation from 20 minutes cooking; and
  • Didn’t include either 81 g dry weight steel-cut oats which becomes 308 g for breakfast, or 103.8 g 3-day-old hulled oat sprouts.
  • Net 1,279.5 grams non-starchy plant foods

I’m doing alright by the “600 to 800 grams of non-starchy plant foods a day” guideline. Should exercise more, though, because I eat a lot.

Topics continued in Part 2.

Oat sprouts analysis

A research group published two 2020 studies on sprouting oat seeds. Their first study produced evidence over a range of germination parameters (hulled / dehulled seeds of two varieties, for 1-to-9 days, at 12-to-20°C):

“The aim was to investigate the influence of germination period and temperature on protein profile, bioactive potential (β-glucan and phenolic contents), antioxidant capacity, and on activity of enzymes (α-amylase, protease and lipase) from hulled and dehulled oat varieties. Multi-response optimization was used to identify optimal germination conditions that maximize sprouted oat flour quality.

  • Hulled (variety Barra) and dehulled (variety Meeri) germination was performed in dark at different temperatures (12, 14, 16, 18, and 20 ◦C) and duration (24, 60, 96, 156, and 216 h).
  • Germination at 16 ◦C for 216 h and 20 ◦C for 96 h produced the highest protein accumulation in varieties Barra and Meeri, respectively.
  • Germination for short periods (24–96 h) combined with medium temperatures (12–16 ◦C) retained β-glucan levels, but longer germination times (156–216 h) caused reductions of 47–64%. Endogenous β-glucanases increase activity during germination, causing hydrolysis of β-glucan.
  • Free phenolic compound content was between 1.6-fold and 2.8-fold higher when germination took place at high temperatures (16–18 ◦C) for longer times.
  • Antioxidant capacity was between 1.4 and 4.5-fold higher. High temperatures (16–18 ◦C) and longer germination times (156–216 h) positively influenced antioxidant capacity.

The effect of germination conditions strongly depended on genetic diversity and presence/absence of hull.

Optimal germination conditions maximize contents of β-glucan, free phenolic compounds, protease activity, and antioxidant capacity, and minimize activity of undesirable enzymes α-amylase and lipase. For variety Meeri, that corresponded to 18 ◦C and time 120 h.”

https://www.sciencedirect.com/science/article/abs/pii/S0023643820309440 “Changes in protein profile, bioactive potential and enzymatic activities of gluten-free flours obtained from hulled and dehulled oat varieties as affected by germination conditions” (not freely available)


Their second 2020 study analyzed properties of 4-day-old oat sprouts. Dehulled oat seeds (variety Meeri) were soaked at room temperature for 4 hours, then germinated in darkness at 18°C with humidity ≥ 90%.

“Sprouted oat powder was an excellent source of protein (10.7%), β-glucan (2.1%), thiamine, riboflavin, and minerals (P, K, Mg and Ca). It presented better amino acid and fatty acid compositions, and levels of γ-aminobutyric acid [GABA], free phenolics, and antioxidant capacity than control.

Protein content (g/100 g) and amino acid profile (g/100 g protein). Different letters within a row indicate p ≤ 0.05 statistical differences.

During germination, proteins are partially hydrolyzed increasing availability of free amino acids. Activity of glutamate decarboxylase enzyme is enhanced.

However, no significant reduction of glutamate content was observed. Glutamate is used for GABA and protein synthesis, but it is also produced by protein hydrolysis, glutamine synthetase-glutamate synthase cycle, and GABA transaminase reactions.

Sprouted oat powder exhibited 2.5-fold higher SPC [soluble (free) phenolic compounds] levels. De novo synthesis of phenolic compounds or liberation of phenolic compounds that are linked to macromolecules due to cell wall dismantling during germination could explain enhancement of SPC.

Sprouted oat powder displayed a 3-fold higher antioxidant capacity. Release of bound phenolic compounds and de novo synthesis of avenanthramides might be responsible.

Hydrolysis of β-glucan might also cause an increase in oxygen radical absorbance capacity. β-glucan oligosaccharides exhibit high radical scavenging activity and reducing power, and that could be related with exposure of their active hydroxyl groups and decrease of intermolecular hydrogen bonding during germination.”

https://www.sciencedirect.com/science/article/abs/pii/S0308814620318343 “Sprouted oat as a potential gluten-free ingredient with enhanced nutritional and bioactive properties” (not freely available)


Both studies started germination by:

“Twenty grams of oat seeds were used for germination. Soaking (1:6 ratio, w/v) was performed at room temperature (20 ◦C ±2 ◦C) for 4 h.”

Neither study included estimates of germination rates. I contacted the corresponding coauthor for that information, and they replied:

“The germination rate in hulled oat varieties was around 95% and in
dehulled one around 55-70% depending on the germination conditions.”


An oats β-glucan clinical trial

This 2020 human study investigated effects of processing β-glucan:

“Nutritional advantages of oats compared to many other grains include gluten-free nature, high content of polyunsaturated fatty acids, protein composition which complements that of pulses, and substantiated health effects of fibers, specifically oat β-glucan. Novel oat products, which are often semi-solid or liquid, generally need alterations of the physicochemical properties of oats.

The hypothesis in this study [Clinical trial NCT02764931] was that bioprocessing of oat bran with enzyme treatment, causing depolymerization of β-glucan, affects nutritional properties of bran and functional properties of β-glucan in human gastrointestinal tract.

The study meal consisted of oat bran concentrate treated with a commercial food-grade cell wall degrading enzyme preparation at 1 or 50 nkat β-glucanase g-1 dm-1. A control sample was prepared in the same way without added enzymes. Average MW [molecular weight] of β-glucan in:

  • Control oat bran concentrate was >1000 kDa [weight in kilodaltons] (High MW);
  • 1 nkat g-1 dm-1-treated 524 kDa (Medium MW); and
  • 50 nkat g-1 dm-1-treated 82 kDa (Low MW).

Results of this study supported the hypothesis that alteration of oat β-glucan MW with enzymatic treatment affects nutritional properties of oat bran and functional properties of β-glucan in the human gastrointestinal tract:

  • A High MW β-glucan meal resulted in the highest excretion of fecal bile acids, and the lowest excretion of phenolic compounds in urine.
  • A Low MW β-glucan meal resulted in the lowest excretion of fecal bile acids, but the highest excretion of phenolic compounds, especially ferulic acid, in urine.
  • Medium MW β-glucan was similar to High MW β-glucan in that it resulted in high excretion of fecal bile acids and low excretion of phenolic compounds to urine, but mean pressure in the duodenum was closer to Low MW than to High MW meal.

Perceived gut well-being after consumption of each meal did not differ between meals, but varied between genders, which should be further investigated.”

https://www.sciencedirect.com/science/article/abs/pii/S0308814620320811 “Effect of oat β-glucan of different molecular weights on fecal bile acids, urine metabolites and pressure in the digestive tract – A human cross over trial” (not freely available)


I eat 81 grams of steel-cut oats every morning, which is represented by this study’s high-molecular-weight control. Take responsibility for your one precious life.

No β-glucan for dolphins or seagulls

Zinc and broccoli sprouts – a winning combination

This 2019 study deserved better coverage than a one sentence mention in Reversal of aging and immunosenescent trends with sulforaphane:

“Obstructive sleep apnea syndrome is one of the most common breathing disorders in sleep, with a high prevalence of 3–7% and severe consequences. It is characterized by intermittent hypoxia (IH) due to recurrent episodes of partial or complete collapse of the upper airway during sleep, leading to blood hypoxemia, hypercapnia, sleep fragmentation, augmented respiratory efforts, and increased sympathetic activity.

Our study is the first investigation of the combination of BSE [broccoli sprout extract] and Zn [zinc] – Nrf2 and MT [metallothionein] inducers – to protect against IH-induced cardiomyopathy. By effectively activating Nrf2, its downstream targets, and MT, this combination can ameliorate defects associated with IH-induced cardiomyopathy more effectively than monotherapies.

Mice were administered with BSE (equivalent to SFN [sulforaphane] 2 mg/kg) and/or Zn sulfate heptahydrate (5 mg/kg) by gavage from 8 weeks of age at a frequency of once every other day for 8 weeks. Doses used in this study are safe to convert to human doses. [2 mg x .081 x 70 kg = 11 mg sulforaphane; 5 mg x .081 x 70 kg = 28 mg zinc]

  • Heart mass was significantly lower in the IH-BSE/Zn group than in IH and IH-BSE groups. Heart mass / tibia length ratio was significantly lower in the IH-BSE/Zn group than in IH and monotherapy groups.
  • Treatment with BSE and/or Zn can ameliorate myocardial fibrosis associated with IH, to a certain extent, and combination therapy has the best antifibrotic effect among treatments.
  • BSE or Zn can significantly ameliorate myocardial inflammation induced by IH, but the combination provides a better anti-inflammatory effect.
  • We used 3-NT as an indicator of the severity of oxidative stress. 3-NT protein levels were significantly reduced in IH mice for all treatment groups, and reduction was greater in the combination treatment group.
  • Combination is more effective than monotherapies to activate Nrf2-mediated antioxidant function.

  • In Zn-treated and combination treatment groups, MT protein expression was significantly higher than in the IH group, and there was only a slight increase in the IH-BSE group.”

Combination of Broccoli Sprout Extract and Zinc Provides Better Protection Against Intermittent Hypoxia-Induced Cardiomyopathy Than Monotherapy in Mice


One way to improve broccoli sprout compounds’ effects is to eat them with zinc. One way to improve zinc’s actions is to take it with broccoli sprouts.

Mild stress improves broccoli compound yields

This 2020 plant study by the same university as Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts investigated seasonal and stressful effects on broccoli compounds:

“In this study, three crop trials were carried out to evaluate effects of cultivation season, application of different dosages of methyl-jasmonate (MeJA) on overall quality and on total content of bioactive compounds of ‘Parthenon’ broccoli cultivated under field conditions of southeastern Spain.

Elicitation is the main tool used to increase content of secondary metabolites in vegetables, as it induces stress responses in plants. Several studies have involved application of elicitors to broccoli plants in order to improve their nutritional properties (although this application is more common for seeds and sprouts).

Content of total carotenoids, phenolic compounds and glucosinolates were higher in autumn compared with spring, showing increases of 2.8-fold, 2-fold and 1.2-fold, respectively. Moreover, a double application of MeJA increased contents of total carotenoids, phenolic compounds and glucosinolates by 22%, 32% and 39%, respectively, relative to untreated samples.

Controlled and timely (four days before harvest) application of 250 µM MeJA as an elicitor to aerial parts of plants, on two consecutive days, yielded florets of Parthenon broccoli with higher contents of bioactive compounds, without changing its overall quality.”

https://www.mdpi.com/2304-8158/9/10/1371/htm “Seasonal Variation of Health-Promoting Bioactives in Broccoli and Methyl-Jasmonate Pre-Harvest Treatments to Enhance Their Contents”


Findings by broccoli compound category were:

Glucosinolates

“Total content of GLSs was 2-fold higher in autumn than in spring. Total precipitation [2018] in spring was 361 mm compared with 185 mm in autumn. The water deficit in autumn could have contributed to the increase in total GLS content.

The main compound in samples of plants cultivated in spring (first and third assays) was glucoiberin (GIB), followed by glucoraphanin (GRA). The order was reversed in broccoli cultivated in autumn, with GRA being the main compound, followed by GIB, for all treatments.

Aliphatic GLSs were predominant in our Parthenon samples, representing on average 76%, 86% and 83%, of total GLSs in the first, second and third assays, respectively. In relation to the effect of MeJA on content of GLSs, neoglucobrassicin (NGB) was the only compound that showed a significant increase after application of MeJA in seasonal trials, since other GLSs decreased or did not differ with respect to the control group.

NGB increased significantly, from 0.3 mg/kg f.w., to 175 mg/kg f.w. in broccoli treated with two consecutive doses of 250 μM MeJA, and contents of GBSs, total indole GLSs and total GLSs also increased. In contrast, one single dose of 500 μM MeJA did not enhance contents of these compounds.”

Phenolics

“Contents of flavonols and chlorogenic acids were higher in autumn than in spring, whereas content of sinapic acid derivatives was higher in spring. Influence of light on individual phenolic compounds could explain the increase in flavonols and chlorogenic acid derivatives in autumn.

Although MeJA altered contents of phenolic compounds, this effect was not clearly associated solely with MeJA. We found a greater effect of the excipient and MeJA in autumn. When we added an extra stress factor – namely, MeJA – the impact was not as great as in autumn.”

Carotenoids

“In broccoli cultivated in spring, the order was β-carotene > lutein > violaxanthin > neoxanthin, while in autumn the order was β-carotene > violaxanthin > lutein > neoxanthin. Content of total carotenoids in broccoli cultivated in autumn (26 mg/kg) represented a 2.8-fold increase compared to broccoli grown in spring (9 mg/kg).

Treatment with MeJA significantly reduced total content of carotenoids in broccoli cultivated in autumn, whereas it did not show any effect on plants cultivated in spring, and in some cases even led to an increase in carotenoid content. Plants that received two applications of 250 µM MeJA content of carotenoids (34 mg/kg f.w.) increased in comparison with plants without this treatment (28 mg/kg f.w.) as well as those receiving one application of 500 µM MeJA (28 mg/kg f.w.).

Chlorophyll content was directly related to carotenoids content, with a strong correlation in autumn. Carotenoids absorb solar light in the spectral region not covered by chlorophylls and pass light energy to chlorophyll a, protecting it from harmful reactions that occur in conditions of excessive light, in the presence of oxygen. When high temperatures reduce content of carotenoids in spring, a reduction in total chlorophylls is also observed, possibly due to the photo-oxidation process.”


DEET and permethrin cause transgenerational diseases

This 2020 rodent study from the labs of Dr. Michael Skinner at Washington State University examined how great-grandmothers’ insect repellent exposures produced diseases in their great-grand offspring:

“Permethrin and DEET are the pesticides and insect repellent most commonly used by humans. These pesticides have been shown to promote the epigenetic transgenerational inheritance of disease in rats.

Direct exposure impacts an individual and their germ line. If germline epigenetics are modified, offspring generated with the affected germ cell can have epigenetic impacts on health and physiology.

Negative health effects of pesticides exposure do not stop with the individuals directly exposed. Epigenetic transgenerational inheritance occurs when future generations without exposure also exhibit alterations and disease. Epigenetic alterations are more common among individuals with disease than specific genetic alterations or mutations.

Pathologies examined are relevant to human populations including prostate, testis and kidney disease, as well as multiple disease incidence. No common DMR [differential DNA methylation region] among the different transgenerational disease DMR biomarkers was identified.

Observations suggest a common set of epimutations is not present between different diseases to alter general disease susceptibility. Although suggestions of such general molecular impacts for disease susceptibility may exist, the current study suggests predominately disease specific epimutations.

DMRs are present for each individual disease on all chromosomes, except the Y chromosome and mitochondrial DNA. The multiple disease signatures are present on the Y chromosome, as well as all other chromosomes. These results support the idea that transgenerational epigenetic effects of ancestral pesticides exposure are genome-wide.

The current study used an epigenome-wide association analysis to identify an epigenetic signature of transgenerational disease present in sperm. Biomarkers identified herein may potentially be used to assess paternal transmission of disease susceptibilities to future generations.”

https://ehjournal.biomedcentral.com/articles/10.1186/s12940-020-00666-y “Epigenome-wide association study for pesticide (Permethrin and DEET) induced DNA methylation epimutation biomarkers for specific transgenerational disease”


Don’t understand how studies on long-term effects of day-to-day human actions like applying insect repellent aren’t front page news. Everyone could benefit from this knowledge. When I explained this study to coworkers, they had a lot of questions and feedback.

An interesting side note was peer review exchanges. A human behavior indicator was pushback regarding repetition of key points among sections, which the researchers justified with:

“The reader does not have to skip back and forth between sections to understand the basic design and methods used.”

Behavioral aspects of epigenetic inheritance haven’t been investigated by this research group. Wouldn’t inherited conditions produce behavioral evidence of their consequences?


Anti-tumor effects of β-glucan

This comprehensive 2020 rodent study investigated dozens of scenarios for β-glucan in the context of anti-tumor immunity:

“Neutrophils and granulopoietic progenitors are major cellular effectors of β-glucan-induced trained immunity. The anti-tumor effect of β-glucan-induced trained immunity was mediated by qualitative changes in neutrophils.

A tumor-suppressive phenotype in neutrophils was associated with training of granulopoiesis mediated by type I IFN [interferon] signaling. Our analysis provided additional evidence for trained immunity-induced epigenetic rewiring of granulopoiesis toward an anti-tumor phenotype and corroborated the experimentally demonstrated IFN- and ROS-related mechanisms.

We observed inhibition of tumor growth by systemic transfer of trained neutrophils into already tumor-bearing mice. As granulocyte transfusion is currently considered as a therapy in humans with neutropenia, it is conceivable that cancer patients could receive as an adjuvant immunotherapy granulocytes from normal donors after induction of trained immunity in the latter.

Our study is the first to link the anti-tumor actions of β-glucan to trained immunity. We show here that the innate immune training and rewiring of granulopoiesis underlies the anti-tumor effect of β-glucan.”

https://www.cell.com/cell/fulltext/S0092-8674(20)31299-X “Innate Immune Training of Granulopoiesis Promotes Anti-tumor Activity”


Which do you prefer? The study’s graphical abstract:

or one of its volcano plots?

Here’s an overview of one investigated direction:

“To determine whether adaptive immunity is involved in the anti-tumor effect induced by β-glucan, mice that lack B and T cells were treated with β-glucan [1 mg] prior to the secondary tumor challenge. Pre-treatment with β-glucan decreased both B16-F10 [melanoma] and LLC [Lewis lung carcinoma] tumor burden also in [these] mice, showing that the anti-tumor effect of β-glucan-induced trained immunity does not require adaptive immunity.”


This study provided another example of what they called rewiring (but I term reprogramming) of the body’s environmental signaling pathways to achieve a desired phenotype, trained innate immunity. Whatever the terminology, almost every day over the past fifteen years I’ve eaten β-glucan in an oats breakfast and a 1/3, 1/6 yeast supplement at dinner as part of individually evolving.

Reprogramming other signaling pathways are in blog posts such as:

Take responsibility for your one precious life.

Flying over waves

Transgenerational epigenetic inheritance of epimutations

My 600th curation is a 2020 rodent study from Dr. Michael Skinner’s labs at Washington State University:

“Numerous environmental toxicants have been shown to induce the epigenetic transgenerational inheritance of disease and phenotypic variation. Alterations in the germline epigenome are necessary to transmit transgenerational phenotypes.

In previous studies, the pesticide DDT and the agricultural fungicide vinclozolin were shown to promote the transgenerational inheritance of sperm differential DNA methylation regions, non-coding RNAs and histone retention, which are termed epimutations. The current study was designed to investigate the developmental origins of the transgenerational differential histone retention sites (called DHRs) during gametogenesis of the sperm.

In addition to alterations in sperm DNA methylation and ncRNA expression previously identified, the induction of DHRs in the later stages of spermatogenesis also occurs. This novel component of epigenetic programming during spermatogenesis can be environmentally altered and transmitted to subsequent generations.

While the DHR may be consistent and present between the stages of development, the histone modifications may be altered. Several of the core histone retention sites absent in the DHRs had altered histone methylation. This adds a level of complexity to the potential role of histone retention in that it may be not only the retention, but also the alterations in histone epigenetic modifications.

The DHRs had positional associations with genes and the major functional categories were signaling, metabolism and transcription.

In the event the embryo stem cell population has a modified epigenetics and corresponding transcriptome, then all somatic cells derived from the stem cell population will have an altered cascade of epigenetic and gene expression programming to result in adult differentiated cells with altered epigenetics and transcriptomes. Previous observations have demonstrated in older adult human males alterations in histone retention develop and are associated with infertility.

Similar observations have also been provided for the development of differential DNA methylation regions (DMRs) induced by environmental toxicants such as DDT and vinclozolin. Since DHRs have a similar developmental programming, other epigenetic processes such as ncRNA are also anticipated to be similar.”

https://www.sciencedirect.com/science/article/pii/S0012160620301834 “Developmental origins of transgenerational sperm histone retention following ancestral exposures”


This study, like its dozens of predecessors performed year after year by this research facility, provided evidence for mechanisms of epigenetic transgenerational inheritance. The studied F3 generation members were great-grand-offspring, the first generation to have no direct exposure to DDT and vinclozolin.

As pointed out in A compelling review of epigenetic transgenerational inheritance:

“During the 1950s, the entire North American population was exposed to high levels of the pesticide DDT, when the obesity rate was < 5% of the population. Three generations later, the obesity frequency in North America is now ~45% of the population.”

There are varieties of mischaracterizations and hand-waving denials of epigenetically-inherited diseases. People don’t want to hear about and read proof that something we did or experienced disfavored our children, who unwittingly passed resultant problems on to their children, and which furthered on to their children’s children.