Somebody tell my mother if I die


I’m fine, thank you.

I was making Boring Chicken Vegetable Soup for dinner, staring into the filling garbage, and thought of this song from fifty years ago. Followed by its Vietnam-era moan. Most of that band is dead now, BTW.

And for today’s young people:

“1984
Knockin’ on your door
Will you let it come?
Will you let it run your life?

It’s time you started thinking inside your head
That you should stand up and fight
Oh just where will you be when your freedom is dead
Won’t you listen tonight?”

Eat broccoli sprouts for your skin

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 the kinetics of upregulation of Nrf2-dependent genes by SF, and was expected to capture the 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 – 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

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.

Sulforaphane clinical trials and COVID-19

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

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 the experimental data obtained by both air-drying and freeze-drying techniques. The time of germination had less effect on the drying kinetics of the broccoli sprouts.

The 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)).

The 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 the moisture of the 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).”


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:

  • 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?

I asked this study’s lead coauthor for actual figures because eyeballing the above kinetics chart looks closer to 6% than 8%.


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 achieve up to but not exceeding 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 the SF concentration in these tablets when maintained at -20 °C. The 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 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 just before eating them, rather than depending on vendor claims or individual metabolism?

Week 18 of Changing to a youthful phenotype with broccoli sprouts

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

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?