Stress

Sleep

gettingwell4 2-3 stars Required further work, Acetylcholine, Amygdala, Beliefs, Brainstem, Cerebrum, Cortisol, Epigenetics, Hippocampus, Hypothalamus, Immune system, Limbic system, Lower brain, Memory, Neurochemicals, Stress, Testosterone, Vasopressin , , , , , , ,

If you can stand the woo of two Californians trying to outwoo each other, listen to these five podcasts with a sleep scientist.

https://peterattiamd.com/matthewwalker1/

“Ambien, sedation, hypnotives, are not sleep.

Sleep is a life support system. It’s the Swiss army knife of health.

Lack of sleep is like a broken water pipe in your home that leaks down into every nook and cranny of your physiology.

Sleep research is not being transmitted to clinical practice.”

I live on the US East Coast. Hyperbole in normal conversations outside of urban centers is an exception.

It’s different on the West Coast. For example:

  • Interviewer assertions regarding heart rate variability should be compared and contrasted with Dead physiological science zombified by psychological research evidence that:

    “A broad base of further evidence was amassed within human cardiac, circulatory, and autonomic physiology such that the hypotheses do not work as described.”

  • Interviewer favorable comments for MDMA (Ecstasy) “to deal with issues of underlying trauma, anxiety, and depression.”

Week 20 of Changing to a youthful phenotype with broccoli sprouts

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

Following up Week 19:

1. I went overboard this week with broccoli sprout measurements. Rinsing broccoli sprouts on a 12-6-6 schedule, each day’s WET and DRY a.m. weights were usually less than p.m. weights. But were they significant weight differences?

  1. Having no moisture for 12 hours vs. 6 hours produced a significant difference in DRY a.m. vs. p.m. weights (hypothesis A).
  2. Moisture was the equalizer, though, as WET a.m. vs. p.m. weights weren’t significantly different (hypothesis B).
  3. (hypothesis C) Were a.m. WET weight gain percentages the same as p.m.? No.
  4. Although most of Week 19’s measurements were taken on an 8-8-8 schedule, its 65.5 ± 4.4 g WET average was confirmed by both 61.8 ± 6.2 g a.m. and 66.4 ± 4.7 g p.m. distributions.

I’ll guess that laboratory automated conditions would change differences in DRY a.m. vs. p.m. weights (A) and WET weight gain percentages (C) to become insignificant. IAW, removing human causes of variability would improve results.

2. Per Week 19’s item 2, eating protein, fats, and fiber along with broccoli sprouts lowered compounds’ bioavailability. I’m looking for accompaniments that won’t have adverse effects.

Eating 60+ grams of broccoli sprouts twice a day is a lot. Mixing in sauerkraut tastes alright, but commercial brands have too much sodium.

3. It’s been 11 weeks since I posted A claim of improved cognitive function. I made many such connections after a transformational Week 9.

I had no idea that would happen. And I won’t come down no more.

4. Tomorrow will be Day 140 of eating broccoli sprouts every day. I’ve successfully addressed inflammation, and will maintain current practices.

Left ankle and knee twinges are among reminders of other aging phenotype aspects. The body clock reset described in An environmental signaling paradigm of aging apparently requires more than what I’ve been doing.

Eat broccoli sprouts for your skin!

gettingwell4 2-3 stars Required further work, Epigenetics, Immune system, Memory, Stress ,

This 2020 Swiss review subject was the interaction of Nrf2 activators and skin:

“The electrophile and Nrf2 activator dimethyl fumarate (DMF) is an established and efficient drug for patients suffering from the common inflammatory skin disease psoriasis. DMF is being tested for pharmacological activity in several other inflammatory skin conditions.

dmf

Psoriasis is a chronic inflammatory skin disease affecting 2–4% of the population and plaque psoriasis is the most common type, affecting about 90% of all patients. As about 30% of all patients suffer from moderate and severe psoriasis, there is a strong need for efficient systemic treatment options with few side effects.

SFN [sulforaphane] blocks NF-κB activity by several mechanisms. SFN oxidizes IκB, thereby inhibiting its phosphorylation and downstream NF-κB activation, but also targets specific cysteine residues of p50/p65, causing a reduction in DNA binding.

More indirect effects have also been suggested. SFN induces HO-1 expression via Nrf2, which in turn inhibits NF-κB. The isothiocyanate can also react with and oxidize components of cellular redox buffers, such as glutathione and thioredoxin, which are required to retain NF-κB’s DNA-binding capacity.

NLRP3 is believed to be critically involved in common diseases, whereas its role in immunity is rather minor. The mechanisms underlying NLRP3 inflammasome activation are of high medical interest.

Electrophiles can directly inhibit inflammasome activation. SFN inhibits activation of NLRP3 in the absence of NRF2 expression in a very fast manner, suggesting that transcriptional effects are not relevant for NLRP3 inhibition. SFN inhibits NLRP3 even in KEAP1 knockout cells.

All these results demonstrate that electrophiles can inhibit the inflammasome pathway in a direct manner, perhaps via the modification of reactive cysteine residues of inflammasome proteins or those which regulate activation of these complexes.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072181/ “Electrophiles against (Skin) Diseases: More Than Nrf2”

These reviewers focused on a pharmaceutical. Read this article for progress made on a generic:

“Biogen is expected to appeal this ruling against its Tecfidera patent protection, which is not due to expire until 2028. Its list price is reported to be about $2,026 for a two-week (14 day) supply at 120 mg.

‘The District Court decision clears the legal pathway for us to bring our dimethyl fumarate product to market, and we are working with the FDA to accelerate our regulatory approval target action date, which currently is November 16.'”

Broccoli or Sulforaphane: Is It the Source or Dose That Matters? listed 15 mouse studies and 4 human studies of sulforaphane treatments of skin diseases in Supplementary Material Table S3.

From Novel Nrf2 activators from microbial transformation products inhibit blood–retinal barrier permeability in rabbits:

“The cysteine residue of sulforaphane works as a weak electrophile and it interacts with cysteine residues of Keap1. Dimethyl fumarate is also a weak electrophile.

Nrf2 activity was evaluated by NQO1 induction activity in Hepa1c1c7 cells. RS9 was the most potent and the concentration needed to double (CD) the specific Nrf2 activity was 0.2 nM. The CD values for bardoxolone methyl, sulforaphane and dimethyl fumarate were 0.9 nM, 154.4 nM and 13.3 μM respectively.”

1. This review didn’t mention dimethyl fumarate’s NQO1 induction CD value because..? It’s one of Nrf2 signaling pathway’s main studied parameters, along with HO-1. For example, from Autism biomarkers and sulforaphane:

“This time point was chosen based on our earlier observations of kinetics of upregulation of Nrf2-dependent genes by SF, and was expected to capture increased mRNA production of both very fast (HO-1) and relatively slow (NQO1) responders.”

2. What about adverse effects? From Sulforaphane and RNAs:

“DMF is the most successful Nrf2 activator, FDA-approved in 2013 for the treatment of relapsing remitting multiple sclerosis. However, DMF causes leukopenia and other side-effects.

Bardoxolone cleared Phase II clinical trials for the treatment of advanced chronic kidney disease and type 2 diabetes mellitus, but was halted in Phase III trials due to cardiovascular concerns.”

3. What about prevention mechanisms for skin problems? Skin care isn’t just cancer prevention.

So – what can a person do to treat an inflammation problem in our largest organ, our skin?

  • Pay $2,026 every two weeks to take a daily 120 mg dose of a brand name pharmaceutical?
  • Wait around for some hypothetical future “development of new tailor-made molecules and drugs for the many inflammatory conditions which are associated with Nrf2, NF-κB and inflammasomes”?
  • Try other treatments that just address symptoms, not causes?

Or eat daily clinically-relevant broccoli sprout dosages for < $500 a year?

Week 19 of Changing to a youthful phenotype with broccoli sprouts

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

To follow up Week 18:

1. Continued attention to broccoli sprout gardening details was this week’s theme. The 12-6-6 schedule had an extra rinse during lunch time.

I stopped when the 8/3 evening batch smelled bad. Broccoli sprouts don’t do well with too much moisture.

I didn’t have this problem on a 12-12 schedule of two rinses. But I also didn’t have good yields.

I switched to an 8-8-8 schedule, and the problem didn’t recur. However, intervals were 5:00 a.m., 1:00 p.m., and 9:00 p.m. That led to eating broccoli sprouts too early and too late, and disrupting my sleep.

8-8-8 also didn’t produce optimal yields. The top two yields this week were both on 8/5. Those two batches started on 8/2, and apparently benefited from a 12-6-6 schedule during their initial germination stages.

I switched back to 12-6-6 on 8/7 with an extra step of rinsing my strainer and teaspoon between batch rinses. Not sure this step addresses whatever happened on 8/3, but it protects against one batch’s problems spreading to other batches. I’ll continue 12-6-6 unless I cause moisture problems, in which case I’ll return to a 12-12 routine.

The (65.5 gram x 2) = 131 g daily average of this week’s broccoli sprouts has been factored into Estimating daily consumption of broccoli sprout compounds numbers for broccoli seeds, sprouts and their compounds. Some worst-case scenarios change to evidenced estimates, such as consuming 52 mg sulforaphane daily by microwaving 131 g of 3-day-old broccoli sprouts.

I’ll update the many blog posts that reference these estimates. Most of them can be recognized from strikethrough typography.

2. During the 8-8-8 schedule I ate microwaved broccoli sprouts with supplements and sauerkraut instead of during a meal. I wondered if meal composition made any difference to broccoli sprout compounds. My meals are breakfast started with 1/2 cup (82 grams) of steel-cut oats, Boring Chicken Vegetable Soup for dinner, and leftover soup at lunch.

A 2018 Netherlands study Bioavailability of Isothiocyanates From Broccoli Sprouts in Protein, Lipid, and Fiber Gels found:

“Compared to the control broccoli sprout, incorporation of sprouts in gels led to lower bioavailability for preformed sulforaphane and iberin.”

IAW, eating protein, fats, and fiber along with microwaved broccoli sprouts wouldn’t help. So I’ll keep eating them with supplements for synergies* but not immediately before or after meals.

A 2018 review with some of the same researchers Isothiocyanates from Brassica Vegetables-Effects of Processing, Cooking, Mastication, and Digestion offered one possible explanation for protein acting to lower broccoli sprout compounds’ bioavailability:

“In vitro studies show that ITCs can potentially react with amino acids, peptides, and proteins, and this reactivity may reduce the ITC bioavailability in protein‐rich foods. More in vivo studies should be performed to confirm the outcome obtained in vitro.”

3. I mix in homemade sauerkraut when I eat microwaved broccoli sprouts. It helps ensure that I thoroughly chew sprouts. Wouldn’t expect anyone else to like unsalted sauerkraut.


Continued with Week 20 of Changing to a youthful phenotype with broccoli sprouts.

*See Broccoli sprout synergies for details.

Sulforaphane clinical trials and COVID-19

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A plethora of articles have been published this year on how researchers’ favorite topics can / may / should / could / will fix COVID-19. This one was different in that relevant clinical trials were both completed and already underway before a Madness of Crowds behavioral contagion infected us:

“It is crucial to understand the most appropriate context for introducing an anti-inflammatory therapy to complement an antiviral therapy. Such therapy must control inflammation without altering the ability of the host to mount an efficient adaptive immune response against the virus.

We propose that boosting endogenous cellular defenses by targeting the cytoprotective transcription factor Nrf2 (gene name NFE2L2) will promote the resolution of COVID-19 associated inflammation and also restore redox homeostasis and facilitate tissue repair.

The isothiocyanate sulforaphane (SFN) is the most potent naturally occurring NRF2 activator, with well-documented antioxidant and anti-inflammatory effects. The high bioavailability of SFN makes it an excellent candidate for alleviating excessive anti-inflammatory responses and protecting the lungs.

Even though Nrf2 is the primary mediator, additional factors contribute to the anti-inflammatory effects of SFN. SFN inhibits NF-κB, inhibitor of NF-κB kinase subunit β (IKKβ), and STAT3.

By regulating the endogenous cytoprotective systems, Nrf2 may have a more physiological role in achieving a balance between the beneficial and adverse effects of inflammation. Nrf2 inhibits IL-6 and IL-1β gene expression.

Antioxidant and cytoprotective effects of Nrf2 activation are long-lasting and persist for several days after inducer elimination. They are mediated by enzymes that, in contrast to small molecules, have long half-lives and are not consumed, and are instead regenerated during the reactions which they catalyze.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359808/ “Can Activation of Nfr2 Be a Strategy against COVID-19?”

The paper also documented in vitro, animal, and non-clinical human Nrf2 activator studies relevant to causes and effects.

Drying broccoli sprouts

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This 2020 Polish study investigated dried broccoli sprouts characteristics:

“The aim of this study was to quantify the air-drying and freeze-drying kinetics of broccoli sprouts. The Page model exhibited a very good fit to experimental data obtained by both air-drying and freeze-drying techniques. Time of germination had less effect on drying kinetics of broccoli sprouts.

Water activity (aw) of fresh broccoli sprouts was 0.999 ± 0.03 and moisture content 82.6% (w.b.). Drying reduced the value of aw (between 0.287 ± 0.04 (freeze-dried sprouts) and 0.293 ± 0.06 (air-dried sprouts at 40 °C)).

Highest total phenolics content and antioxidant activity were observed in air-dried sprouts (40 °C) and freeze-dried sprouts.

Drying curves of dried broccoli sprouts after 3 days of germination with experimental and predicted data based on the Page model: MR-moisture ratio, SPD40, SPD60 and SPD80-sprouts air-dried at 40, 60 and 80 °C, respectively, SPF-freeze-dried sprouts. [x axis in minutes]

Processes were continued until moisture of samples decreased to 10% (±0.5%) wet basis (w.b.).”

https://www.mdpi.com/2227-9717/8/1/97/htm “Drying Kinetics, Grinding Characteristics, and Physicochemical Properties of Broccoli Sprouts”

Repeating a relevant section from Are sulforaphane supplements better than microwaved broccoli sprouts?, I contacted a distributor of a dried broccoli sprout powder. In correspondence the company founder said:

“Each 700 mg capsules yields around 15mg sulforaphane.”

The company founder has written several reviews, one of which was the 2016 Sulforaphane and Other Nutrigenomic Nrf2 Activators: Can the Clinician’s Expectation Be Matched by the Reality? Section 6.5 Sulforaphane stated:

“By calculation, MYR [myrosinase]-active whole broccoli sprout supplement yielding 1% SFN could deliver 10 mg SFN per gram of powder, corresponding to ~12 grams of fresh broccoli sprouts (dried powder retains ~8% moisture).”

I asked the current study’s lead coauthor for actual figures because eyeballing the above kinetics chart, the 60°C end point looks closer to 6% than 8%. No reply yet. 6% moisture content would give a 16.7-to-1 ratio.

Per Week 18 of Changing to a youthful phenotype with broccoli sprouts, twice a day I start a new batch of broccoli sprouts with one tablespoon (10.7 grams) broccoli seeds of unspecified variety. Per Week 19, wet-basis (soaked five minutes then drained) weights of 3-day-old broccoli sprouts average 65.5 grams, consumed twice a day.

Let’s assume for calculation purposes:

  • The 2016 review’s 12-to-1 ratio of fresh broccoli sprouts weight-to-dried broccoli sprout weight is fairly representative; and
  • Recent 65.5 grams average of 3-day-old broccoli sprouts consumed twice a day is fairly representative.

Calculations based on personal correspondence:

  • Sulforaphane yield of one vendor’s dried broccoli sprouts is 15 mg / 700 mg capsule = 2.14%.
  • Using the review’s 12-to-1 ratio, a dried broccoli sprout equivalent of my daily consumption would be (65.5 g x 2) / 12 = 10.9 grams.
  • A sulforaphane equivalent would be 10.9 g x 2.14% = 233 mg.

If I use this study’s “82.6% (w.b.)” rather than the review’s 12-to-1 ratio, a sulforaphane equivalent would be more than twice as much:

  • A dried broccoli sprout equivalent of fresh broccoli sprouts would be (65.5 g x 2) x (1 – .826) = 22.8 grams.
  • A sulforaphane equivalent would be 22.8 g x 2.14% = 488 mg.

These are both much too high. What isn’t right?

Subsequent investigation of a distributor’s site found this table:

autism sprout powder

The study referenced for equivalence was Sulforaphane treatment of autism spectrum disorder (ASD). Calculations:

  • The 100 µmol sulforaphane amount for 90 kg participants weighed 17.73 mg per https://pubchem.ncbi.nlm.nih.gov/compound/sulforaphane.
  • The equivalent broccoli sprout powder sulforaphane yield is 0.01773 / 3.6 g = 0.4925%. That’s 5 mg of sulforaphane per gram of broccoli sprout powder.
  • 0.4925% / 2.14% = 0.23. Decrementing the above sulforaphane weights gives 233 mg x .23 = 54 mg, and 488 mg x .23 = 112 mg.

The answer to my question What isn’t right? I relied on private correspondence rather than what a vendor publicly disclosed.

I’m not particularly concerned about analytical uncertainties for myself. Whatever the numbers are, microwaving techniques for fresh broccoli sprouts increase them.

I immerse 3-day-old broccoli sprouts in 100 ml distilled water, then microwave them on 1000W full power for 35 seconds to ≤ 60°C (140°F) per Microwave broccoli to increase sulforaphane levels. After microwaving I transfer broccoli sprouts to a strainer, and wait five minutes to allow further myrosinase hydrolization of glucoraphanin and other glucosinolates into sulforaphane and other healthy compounds.

Broccoli sprout drying techniques don’t increase sulforaphane content as does microwaving. A broccoli sprout powder vendor has to take care that their drying process doesn’t hydrolyze glucosinolates, because sulforaphane degrades quickly unless it’s stabilized.

A study compared in Measuring sulforaphane plasma compounds used a stabilized product made from broccoli seeds. One of that study’s findings was:

“We evaluated stability of SF concentration in these tablets when maintained at -20 °C. Decline in SF content in 2 separate lots, shipped in boxes containing blisterpacks of tablet measured over 1.5 years, equates to about 17.8% per year.”

Those researchers stipulated a sulforaphane amount of 94.4 μmol in two tablets given to study subjects. The sulforaphane amount would have been (1 / 0.1773 mg) x 10 mg x 2 = 112.8 μmol if that study’s researchers had found the labelled 10 mg sulforaphane weight in each tablet.

The product’s sulforaphane stabilized for a short time, yes. But it measurably degraded over 1.5 years despite favorable storage conditions.

Wouldn’t it be better to create broccoli sprout hydrolysis compounds by microwaving them just before eating, rather than depending on vendor claims or individual metabolism?

Week 18 of Changing to a youthful phenotype with broccoli sprouts

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

1. After eating broccoli sprouts every day for 120 consecutive days, I finally got serious enough to spend $16 on a kitchen scale. 🙂 It’s really a jewelers scale, as it has carat, grain, and troy ounce units of measure in addition to gram and ounce.

Twice a day I start a new batch of broccoli sprouts with one tablespoon broccoli seeds of unspecified variety. I measured successive tablespoons at 10.2, 11.4, and 10.6 grams, and have since started each new batch with 10.7 grams of broccoli seeds.

Each new batch soaks for 12 hours. I rehydrate the other five batches for five minutes before draining, and clump together developing sprouts to look like this:

At room temperature and darkness, each drained batch is close to being completely dry every 12 hours. Laboratories provide water more frequently during germination.

The below weight measurements of 3-day-old broccoli sprouts are dry and wet (soaked five minutes then drained). Microwaving acts directly on a material’s water content, so I don’t microwave dry broccoli sprouts.

The trend reminds me of an initial step of quality programs – improvements start with measurements.

I’ve used Estimating daily consumption of broccoli sprout compounds worst-cases in estimates of broccoli seeds, sprouts and their compounds. I’ve factored these estimates with the above numbers. Compare them with strikethrough numbers – you may be surprised.

I won’t update posts where I’ve used these new estimates quite yet. A convenient action for my work-from-home-during-this-manufactured-crisis situation would be to rinse developing broccoli sprouts at lunch time, about 6 hours into their current 12-hour cycle. I’ll try that for a few days to see if it’s an improvement.

2. I reordered broccoli seeds from the same vendor. Their label is cropped so that its information will neither be mistaken for an endorsement, nor confused with originating from any basis in science:

The label’s Serving Size is new. I moved from one to two tablespoons after Week 5 of Changing an inflammatory phenotype with broccoli sprouts.

I contacted the company for further clarification:

“Just received broccoli seeds. I notice that Serving Size is two tablespoons. Is there any specific research you can point me to for understanding the two tablespoons?”

They responded:

“If I understand correctly you are using the broccoli seed for sprouting?

If so, the 2 Tablespoon size is a recommendation/guideline, depending on the size jar you are using and how much you would prefer to yield. 2 Tablespoons – 1/2 cup is a good range to stay within for sprouts to have room to grow and be rinsed properly ( for most sprouting jars).

Serving size for eating is subjective and you should sprout, sprout, sprout if they are a regular to your daily meals 😊

Please let us know more about your inquiry so we can best help, thanks. 🌼”

“Subjective” is probably the basis of other vendors’ recommendations as well. At least this vendor now uses the word “seeds” on their broccoli seeds package label.

Continued with Week 19 of Changing to a youthful phenotype with broccoli sprouts.

 

A cherry-picked DNA methylation study

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This 2020 US/Sweden/Denmark human study measured twins during their old age:

“We evaluate individual differences in DNA methylation at individual CpG sites across the methylome across 10 years in two Scandinavian samples of same‐sex aging twins. We test two competing hypotheses about the longitudinal stability and change in DNA methylation:

  1. The contribution of genetic influences changes with age, reflecting diminishing influence across time; and
  2. Nonshared factors accumulate in importance, signaling an increasing diversity of response to environmental exposures.

Understanding epigenetic changes over time in the elderly may identify pathways of decline or plasticity (e.g., maintenance or even boosts in functioning) during the aging process and help with elucidating the biology of aging and survival.

Across time, stability in methylation is primarily due to genetic contributions, while novel experiences and exposures contribute to methylation differences. Elevated genetic contributions at age‐related methylation sites suggest that adaptions to aging and senescence may be differentially impacted by genetic background.”

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13197 “A decade of epigenetic change in aging twins: Genetic and environmental contributions to longitudinal DNA methylation”

Swedish subject measurements were taken at ages 62 and 72. Danish subject measurements were taken at ages 76 and 86.

One epigenetic clock that used 2019 technology was favored over three others, including Horvath’s 2013 original clock. For some reason this study didn’t use his 2018 skin-and-blood clock that had vastly improved technology such as an 18-fold increase in genomic coverage with Illumina 450k/850k bead arrays.

These researchers’ intentions became evident with:

“The 353 Horvath clock sites were selected as best predictors of chronological age using multiple tissues. The 71 Hannum clock sites best predicted age (adjusted for sex, BMI) based on methylation observed in whole blood while the 514 sites from the Zhang prediction model relied on methylation observed in blood and saliva samples (Zhang et al., 2019).

The current findings of moderately higher heritabilities in the Zhang and Hannum sites versus the other clock sites may be in part due to our use of blood tissue.”

The 18-fold increase improved accuracy in blood for the 2018 Horvath clock. Could these researchers ignore it and claim they did their due diligence in 2019 and 2020?

A larger issue was this study’s duality paradigm of either heritability or environment being solely responsible for observed changes. Consider what A blood plasma aging clock found at ages 60 and 78 peaks:

The above changes were due to life stage. Josh Mitteldorf did his usual excellent job of providing contexts for that study with New Aging Clock based on Proteins in the Blood, including:

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

Weren’t changes in subjects’ life stages relevant to their epigenetic changes? Why wouldn’t their life stages have been among the causes of observed effects?

Sulforaphane and RNAs

gettingwell4 2-3 stars Required further work, Epigenetics, Hippocampus, Immune system, Limbic system, Memory, Stress , , ,

This 2020 Texas review subject was long non-coding RNAs:

“We review the emerging significance of long non-coding RNAs (lncRNA) as downstream targets and upstream regulators of the Nrf2 signaling pathway, a critical mediator of diverse cellular processes linked to increased cell survival.

It is believed that more than 3% of human genes are regulated by the Nrf2/Keap1 pathway. In addition to the classical cytoprotective and oxidative stress response genes transactivated by Nrf2, emerging evidence suggests a role for non-coding transcript regulation at the level of noncoding RNAs, [which] far outnumber protein-coding genes in the human genome.

One important distinction between miRNAs and lncRNAs is that the latter are often species-specific, meaning that a human lncRNA typically cannot be studied in the mouse or rat, and vice versa.

Sulforaphane (SFN) acts via multiple mechanisms to modulate gene expression, including the induction of Nrf2-dependent signaling. In addition to the established canonical targets of Nrf2, such as NQO1 and HMOX1, SFN altered the expression of multiple lncRNAs.

Given that SFN induces NMRAL2P [a lncRNA pseudogene] and several other lncRNAs in colon cancer cells, further studies are warranted on their respective roles as upstream regulators and/or downstream targets of Nrf2 signaling.

Pharmacological modulation of Nrf2 is considered a viable strategy against chronic conditions that are accompanied by oxidative stress and inflammation:

  • DMF [dimethyl fumurate] is the most successful Nrf2 activator, FDA-approved in 2013 for the treatment of relapsing remitting multiple sclerosis. However, DMF causes leukopenia and other side-effects.
  • Bardoxolone cleared Phase II clinical trials for the treatment of advanced chronic kidney disease and type 2 diabetes mellitus, but was halted in Phase III trials due to cardiovascular concerns.
  • SFN is relatively unstable at room temperature.

We used reported bioinformatics approaches to search for putative ARE [antioxidant response element] sequences among the entire set of 16,000+ annotated human lncRNAs. 13,285 promoter regions contained one or more potential binding sites for Nrf2.”

https://www.sciencedirect.com/science/article/pii/S0304383520303670 “Emerging crosstalk between long non-coding RNAs and Nrf2 signaling”

This study hyped lncRNAs in that only 7 have been validated as Nrf2 targets, and 8 validated as Nrf2 regulators. For regulators, “protein and/or miRNA interacting partners are yet to be fully corroborated” as well.

Also, there’s no need for a “SFN is relatively unstable at room temperature” problem. Just create sulforaphane right before consuming it.

Twice a day I microwave an average 65.5 grams of 3-day-old broccoli sprouts immersed in 100 ml water with a 1000W microwave on full power for 35 seconds to ≤ 60°C. After microwaving I transfer broccoli sprouts to a strainer, and wait five minutes to allow further myrosinase hydrolization of glucoraphanin and other glucosinolates into sulforaphane and other healthy compounds.

Reprogram inflammation with β-glucan

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

This 2020 French human cell study found:

“Exposure of mononuclear phagocytes to β-glucan contributes to the induction of innate immune memory, which is associated with long-term epigenetic, metabolic, and functional reprogramming. We investigated how preincubation of human monocytes with particulate β-glucan affects the biological response of macrophages following NLRP3 inflammasome activation.

Upon infection or cellular damage, NLRP3 assembles into a multiprotein inflammasome complex leading to the release of IL-1β. However, NLRP3 inflammasome activity can also be detrimental to the host, and its aberrant chronic activation is associated with severe pathologies.

β-Glucan is a safe molecule present in food products and already widely used in food supplementation. Although β-glucan–induced innate memory is associated with a nonspecific protective effect against infections, the role of this functional reprogramming in autoinflammatory disorders is unknown.

Because of the administration frequency and conservation needs, IL-1β–targeted therapy is invasive, complex, and also costly. In addition, IL-1β, an acute-phase protein, is crucial for effective immune responses to infection, and inhibitors targeting IL-1β may lead to unintended immunosuppressive effects in addition to preventing NLRP3 inflammasome activity in itself.

Targeting the origin of the disease, i.e., NLRP3, would represent the best therapeutic strategy. Most of these candidate drugs directly interact with NLRP3, but none seems to regulate the early activation events upstream of NLRP3 inflammasome assembly.

β-Glucan acted upstream of the NLRP3 inflammasome. β-glucan–induced innate immune memory represses IL-1β–mediated inflammation and support its potential clinical use in NLRP3-driven diseases.”

https://www.jci.org/articles/view/134778 “β-Glucan–induced reprogramming of human macrophages inhibits NLRP3 inflammasome activation in cryopyrinopathies”

This study came closer to addressing causes than others with:

“Targeting the origin of the disease would represent the best therapeutic strategy.”

It’s apparently too recent with a July 27th published date to make it onto https://www.betaglucan.org/i-p/, but earlier β-glucan inflammation studies may be found there.

Topical sulforaphane protects skin

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This 2020 Rutgers rodent study explored topical application of sulforaphane to prevent UVB-induced skin carcinogenesis:

“We investigated the transcriptomic and DNA methylomic changes during tumor initiation, promotion, and progression and its impact and reversal by sulforaphane (SFN). The production of ROS and inflammation are closely linked to UVB-induced carcinogenesis. SFN protects skin cells from UVB-induced damage mainly through promoting anti-inflammatory, antioxidative and anticancer pathways.

We observed the changes after 2, 15 and 25 weeks of UVB exposure, which would represent the three different stages of skin cancer development. After 2 weeks of UVB exposure, we did not observe any obvious tumors in the UVB group. But after 15 weeks of UVB exposure, some obvious tumors were observed in the skin.

After 15 weeks of UVB treatment in epidermal tissue, the difference between the UVB group and the control group was significantly more than that between the SFN group versus the UVB group. SFN appears to have better cancer-protective effects in earlier time points (weeks 14 and 20) than later time point (week 24). At weeks 20, SFN had significantly fewer tumors with decreased total tumor volume and tumor number.

SFN plays a highly regulatory role in various signaling pathways during UVB irradiation. SFN impacts UVB-induced alterations of DNA methylation profiles, and importantly, SFN treatment attenuates some of these DNA methylation changes. We found a subset of genes associated with SFN treatment, and the relevant changes in gene expression may be driven by promoter CpG methylation status.”

https://cancerpreventionresearch.aacrjournals.org/content/13/6/551 “Epigenome, Transcriptome, and Protection by Sulforaphane at Different Stages of UVB-Induced Skin Carcinogenesis” (not freely available)

We’re getting closer to using epigenetic clocks in sulforaphane studies. This study ignored the 2018 A multi-tissue full lifespan epigenetic clock for mice in favor of their homegrown DNA methylation measurements.

A search of ClinicalTrials.gov didn’t turn up directly relevant human studies.

Caution on broccoli seed erucic acid content?

gettingwell4 2-3 stars Required further work, Epigenetics, Stress

1. While looking through PubMed “broccoli skin” search results, I read a 2018 study Comparative Study of Predominant Phytochemical Compounds and Proapoptotic Potential of Broccoli Sprouts and Florets that cautioned about erucic acid content in broccoli seeds:

“Our results revealed significantly higher total UFAs [unsaturated fatty acids] content in the sprouts in comparison to the florets, with very low amounts of harmful erucic [27] acid in sprouts (0.5%) and florets (2%), in comparison to the broccoli seeds (38% – data not shown).”

But its cited reference [27] Various concentrations of erucic acid in mustard oil and mustard said nothing about broccoli seeds.

Values were on a dry weight basis. Broccoli sprout age was four days.

2. Another search found this 2017 Erucic acid in feed and food position paper which stated:

“When in this Scientific Opinion the erucic acid content is reported as a percentage, this value refers to the percentage erucic acid in the total fatty acids on a weight basis.

A tolerable daily intake of 7 mg/kg body weight per day for erucic acid was established.”

See Beneficial dietary erucic acid? for important evidence available at the time that was intentionally overlooked and misinterpreted:

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

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

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

3. It referenced a 2002 Determination and Health Implication of the Erucic Acid Content of Broccoli Florets, Sprouts, and Seeds which stated:

“The erucic acid content of broccoli florets, sprouts, and seeds was found to be about 0.8, 320, and 12100 mg/100 g, respectively.”

Respective erucic acid percentages of total lipids on a fresh weight basis were provided as 0.4%, 1.1%, and 26.9%.

Florets, sprouts, and seeds had no relationships among them as they were different broccoli cultivars. Broccoli sprouts’ age wasn’t disclosed.

4. The 2002 study was updated in a 2004 Glucoraphanin and 4-Hydroxyglucobrassicin Contents in Seeds of 59 Cultivars of Broccoli, Raab, Kohlrabi, Radish, Cauliflower, Brussels Sprouts, Kale, and Cabbage which stated:

“All seed accessions contained substantial amounts of hexane-extractable lipids ranging from 21.8 to 42.0% (mean of 32.8%; 21.8-37.0 and 30.9% range and mean, respectively, for broccoli cultivars only), which were composed of 27.0-56.7% (mean of 46.7%;39.4-56.7 and 50.2% range and mean, respectively, for broccoli cultivars only) erucic acid.”

Seeds of the 2002 broccoli sprouts commercial product were measured at 31.4% lipids, with erucic acid content 51.6% of total lipids.

5. The 2018 study cited a 2013 Biochemical composition of broccoli seeds and sprouts at different stages of seedling development whose broccoli seed and sprout composition dry weights are in the below graphic:

  • Broccoli seed lipid percentage of total carbohydrates plus crude fiber would be 9.36 g / (58.89 g + 15.47 g) = 12.6%.
  • 3-day-old broccoli sprouts lipid percentage of total carbohydrates plus crude fiber would be 8.67 g / (54.4 g + 8.97 g) = 13.7%.
  • No erucic acid contents were disclosed.

These four studies all required further work:

  • 2002 couldn’t be bothered to use just one broccoli cultivar for its three measurements, or disclose broccoli sprout age.
  • 2004 couldn’t resolve many of their findings with other studies.
  • 2013 used weights to equate measurements, instead of relating germination stages back to a beginning number of seeds and their measurements.
  • 2018 provided a bogus reference and an unsupported “broccoli seeds (38% – data not shown).” It claimed similarity with 2013, but a statistics package would say otherwise. It also didn’t comply with disclosing fatty acids weight as a percentage of broccoli sprouts weight.

Home sprouting has to deal with:

  • unknown cultivar,
  • unknown glucoraphanin and other glucosinolates contents,
  • unknown sulforaphane and other healthy compounds, and now
  • unknown erucic acid content.

Let’s reverse Microwave broccoli seeds to create sulforaphane calculations with 3-day-old broccoli sprouts have the optimal yields information to estimate an erucic acid content in one tablespoon of broccoli seeds. Measurements from Week 18 and Week 19 of Changing to a youthful phenotype with broccoli sprouts.

  • Broccoli seed weight of one tablespoon 10.7 g.
  • Lipids weight (10.7 g x 12.6% [2013 study]) = 1.35 g.
  • Erucic acid weight in one tablespoon of broccoli seeds (1.35 g x 26.9% [2002 study]) = 0.36 g.

This 0.36 g erucic acid content would be lower than 2017 guidelines for my 70 kg weight (7 mg x 70) = 0.49 g.

Let’s reverse Estimating daily consumption of broccoli sprout compounds techniques to estimate an erucic acid content in my daily consumption of 3-day-old broccoli sprouts grown from two tablespoons of seeds:

  • 131 g 3-day-old broccoli sprouts.
  • Maximum lipids weight (131 g x 13.7% [2013 study]) = 17.9 g.
  • Maximum erucic acid weight in 3-day-old broccoli sprouts (17.9 g x 1.1% [2002 study]) = 0.20 g.

Plug in your own numbers, but it looks like caution isn’t warranted for broccoli seed consumption. Consequences of a possible erucic acid content may be less than broccoli seeds’ healthy aspects.

One mitigation may be to start germination. Pick a point between broccoli seeds’ % of total fatty acids and ending 0.5% of 4-day-old sprouts [2018 study].

Not concerned with a daily estimate < .49 g erucic acid for broccoli seeds and sprouts. Back to a PubMed “broccoli skin” search.

See Politically correct about erucic acid and broccoli seeds for a follow up.

Eat sauerkraut today!

gettingwell4 2-3 stars Required further work, Epigenetics, Immune system, Stress , ,

This 2017 Spanish article reviewed health benefits of sauerkraut:

“During cabbage shredding and fermentation, a disruption of cabbage cells occurs, and GLS [glucosinolates] are hydrolyzed by myrosinase enzyme to a variety of GLS breakdown products. In particular, glucobrassicin is hydrolyzed into indol-3-carbinol (I3C) by myrosinase.

As the pH decreases during cabbage fermentation, I3C reacts nonenzymatically with ascorbic acid to yield ascorbigen (ABG). Studies have shown that ABG is the main GLS breakdown compound in sauerkraut, and it is present at levels between 3 and 18 μmol/100 g fw.

The antioxidant activity observed for sauerkraut in all studies was higher than that observed in raw cabbage.

It has been reported that doses between 53 and 150 μmol of ITCs [isothiocyanates] are enough to display anticarcinogenic effects. Taking into account that the content of ITCs in sauerkraut is in the range 22 μmol/100 g fw, it could be assumed that a weekly consumption of 200–250 g of sauerkraut would provide effective ITC doses to exert cancer chemopreventive effects.

Many studies reported that LAB [lactic acid bacteria] isolated from sauerkraut are potential probiotics.”

https://www.sciencedirect.com/science/article/pii/B9780128023099000248 “Sauerkraut: Production, Composition, and Health Benefits” (not freely available)

This introductory article presented interesting facts, but oversold sauerkraut. Dose and other conditional dependencies in order to achieve health and disease prevention benefits seemed to be beyond its scope.

A more considered view was offered in Fermented Food and Non-Communicable Chronic Diseases which referenced this article:

“Clinical data about the effects of sauerkraut on the human organism, health and disease are scarce. There is knowledge concerning particular compounds in sauerkraut and their impacts on diseases; however, a literature search revealed mostly cell line or rat experiments with very limited conclusions for humans.”

Earlier this month I started eating refrigerated sauerkraut twice a day with microwaved broccoli sprouts. I mix in three heaping teaspoons each time, and finish a 50 oz (1418 g) container in a week.

The mixture tastes better than just microwaved broccoli sprouts. It requires more chewing, which assists myrosinase hydrolization of broccoli sprout glucosinolates into sulforaphane and other healthy compounds.

Although sauerkraut isn’t a primary source, there may be beneficial amounts of probiotics etc. that increase what I get with broccoli sprouts and supplements.

I also started making my own sauerkraut using the commercial product’s juice as a starter. I add garlic but not salt. No results yet.

Transgenerational epigenetic inheritance of epimutations

gettingwell4 5+ stars Premium, Epigenetics, Memory, Stress , , , , ,

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.

Take responsibility for your one precious life – β glucan

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

From the main page of https://www.betaglucan.org/, a compilation for researchers:

“Beta Glucan extracted from yeast cell wall, can be a potent immune response potentiator and modulator. A common test to determine a glucan’s immune response potentiation effectiveness is the measure of the degree to which a glucan increases the nitric oxide burst, a pathogen killing agent.

Determinants of immune response activation and effectiveness are beta glucan source, processing, sizing and uniformity of beta glucan particles ingested. Particle size of 1-4 microns is optimum. Ingestion is optimized to prevent reaggregation.”

A sample of research:

“The tested (and suggested) daily dose remains in the range of 100–500 mg for stimulation of the immune system, whereas for a decrease in cholesterol levels a daily dose of 3 g is recommended.

Glucan supplementation prevents or even treats metabolic syndrome and decreases insulin resistance, dyslipidemia, and obesity. Glucan supplementation is a highly promising and inexpensive method of treatment for chronic respiratory problems.

Reactions known to be influenced by glucan are represented in white, reactions where glucan has no confirmed effects are shown in black.”

https://www.mdpi.com/1420-3049/24/7/1251/htm “Beta Glucan: Supplement or Drug? From Laboratory to Clinical Trials”

“Supplementation with glucan and vitamin D resulted in significant increase of vitamin D levels, improvements of HDL levels, and strong decrease of the total level of cholesterol.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5897984/ “Effects of β-glucan and Vitamin D Supplementation on Inflammatory Parameters in Patients with Diabetic Retinopathy”

“β-glucan inhibits tumor growth through induced systemic tumor-antigen specific T cell response, increased activity of T-cells in tumor, and decreased number of tumor-caused immunosuppressive cells. Sulforaphane inhibits CRC [colorectal cancer] carcinogenesis by modulating Nrf2 activity and inhibition of HDAC enzymes.

In a women’s health initiative prospective cohort during their 11.7-year follow up of dietary fiber and omega-3, -6 fatty acids, the results pointed out a reduced incidence of CRC for the association between a low dose of soluble fiber, a high dose of insoluble fiber, and a high dose of EPA and DHA.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321468/ “Chemoprevention of Colorectal Cancer by Dietary Compounds”

I first curated the above review and graphic in Train your immune system every day! 12 days into a self-quarantine after coming back from Milano, Italy, Monday, February 24, 2020. There’s a substantial probability that my traveling companion and I were exposed to COVID-19.

Yet neither of us had any symptoms then or since. My β-glucan, Vitamin D3, and zinc amounts were the same as described in that post, in Take responsibility for your one precious life – Vitamin D3, and in Take responsibility for your one precious life – Zinc.