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Research needing further work

Eat broccoli sprouts for DIM

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

This 2019 Spanish human study ran in parallel with Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts. I’ll focus on the aspect of diindolylmethane (DIM) from eating broccoli sprouts:

“The aim of this study is to evaluate the effect of gender or hormonal status (menopause) on the bioavailability of broccoli sprouts in different cohorts of overweight adult subjects: men, non-menopausal women and post-menopausal women.

3,3′-diindolylmethane (DIM) was detected and quantified in all volunteers. It increased significantly during broccoli [sprouts] ingestion in men. However, a steady decrease of its urinary concentration was observed in post-menopausal women that was significant at day 50. No significant changes were observed in premenopausal women. Albeit this different behaviour, no significant differences between the three groups were detected by the different statistical tests performed.

High increases observed in SFN-metabolites in the three cohorts confirm that the fresh product is a good source of bioactive compounds bioavailable in the organism. We detected high amounts of 3,3-DIM in urine samples, which can be related to the metabolism of glucobrassicin derivatives from our broccoli sprouts.

Post-menopausal women seem to metabolize isothiocyanates in a greater extension. Hormonal status and differences in gut microbiota may influence the bioavailability of isothiocyanates from broccoli sprouts but more studies are needed to support this statement.”

https://www.sciencedirect.com/science/article/abs/pii/S1756464619303147 “Bioavailability of broccoli sprouts in different human overweight populations” (not freely available)

“Post-menopausal women seem to metabolize isothiocyanates in a greater extension. A steady decrease of its [DIM] urinary concentration was observed in post-menopausal women that was significant at day 50.”

Subjects ate 30 grams of broccoli super sprouts every day through Day 35, then stopped, and were measured again at Day 50. The only example of measurements where Day 35 was less than Day 0 was postmenopausal women metabolizing more DIM.

That Day 35 data point didn’t have an asterisk next to it to indicate a statistically significant decrease. But the overweight postmenopausal women group’s next Day 50 significant “steady decrease” finding supported an interpretation that eating broccoli sprouts supplied them with DIM that they especially needed.

Regarding the huge percentage changes above, our model clinical trial found in a longer time frame:

The decrease in IL-6 levels was significantly related to the increase in 24 h urine SFN [sulforaphane] levels. In case of C-reactive protein, the decrease was significantly related to the increases in 24 h urine SFN-NAC [SFN-N-acetylcysteine] and SFN-CYS [SFN-cysteine].

I’ll guess that these parallel trial subjects also experienced similar benefits from eating broccoli  sprouts every day for five weeks. See Day 70 results from Changing to a youthful phenotype with broccoli sprouts for another guess that even shorter time frames would be effective.

Broccoli super sprout indolic compounds were as follows:

indolic glucosinolates

Assuming that only glucobrassicin is a precursor to DIM, subjects’ DIM bioavailability can be calculated as μmol DIM / 21.61 μmol. For example, overweight postmenopausal women Day 35 average of 0.5544 μmol DIM that ranged from 0.1771 to 0.8034 μmol DIM represented an average 2.57% DIM bioavailability with a range of 0.82% to 3.72% DIM bioavailability.

See Part 2 for DIM follow-up.

A claim of improved cognitive function

gettingwell4 2-3 stars Required further work, Cerebrum, Hypothalamus, Limbic system, Lower brain, Memory ,

I’ll describe evidence for claiming improved cognitive function in Week 9 of Changing to a youthful phenotype with broccoli sprouts.

I read parts of over a hundred research papers last week. That required substantial concentration to understand them, and stay on topic while learning new items, which started new searches. This wasn’t a new development, it was just to a much greater extent. I also worked forty hours for my job.

The main chain of blog posts began when I relooked at the presentation in Reversal of aging and immunosenescent trends after remembering it included before and after photos per A hair color anecdote. The presentation prompted last week’s most frequent self-question, Why didn’t I see this before?

One possible explanation is that people don’t usually see things outside their conditioned perceptions. (1) Reevaluate findings in another paradigm illustrated this with an example of how different frameworks viewed the same hypothalamus study differently.

I was interested to see what sulforaphane research had in common with the presentation topics, which produced (2) Reversal of aging and immunosenescent trends with sulforaphane. That required gaining a better understanding of PubMed search techniques, which led to (3) A pair of broccoli sprout studies.

Numerous presentation topics resulted in (4) Part 2 of Reversal of aging and immunosenescent trends with sulforaphane. I investigated one of its cited papers in (5) A review of sulforaphane and aging, which required further searches, some of which are still on tabs of my browser.

I was happy to oblige special requests with (6) Tailoring measurements for broccoli sprouts and (7) Uses of the lymphocytes to monocytes ratio.

Could I have done all of what I did last week without changing my internal environment? What exactly are the effects of eating a clinically relevant amount of broccoli sprouts every day for nine weeks?

A plausible explanation is in Upgrade your brain’s switchboard with broccoli sprouts.

A review of sulforaphane and aging

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

This 2019 Mexican review stated:

“We describe some of the molecular and physical characteristics of SFN, its mechanisms of action, and the effects that SFN treatment induces in order to discuss its relevance as a ‘miraculous’ drug to prevent aging and neurodegeneration. SFN has been shown to modulate several cellular pathways in order to activate diverse protective responses, which might allow avoiding cancer and neurodegeneration as well as improving cellular lifespan and health span.

NF-κB is in charge of inflammatory response regulation. Under basal conditions, NF-κB is sequestrated into the cytosol by IκB, but when pro-inflammatory ligands bind to its receptors, the IKK protein family phosphorylates IκB to degrade it via proteasome, so NF-κB is able to translocate into the nucleus and transcript several inflammatory mediators. Sulforaphane is capable to inhibit IκB phosphorylation and NF-κB nuclear translocation.

SFN upregulated Nrf2 expression by reducing DNA demethylation levels of the Nrf2 promoter. In another model using the triple-transgenic mouse model of Alzheimer’s disease (3 × Tg-AD), the use of SFN regulates the expression of the Brain-derived neurotrophic factor (BDNF) via HDAC inhibition, thus increasing H3 and H4 acetylation on the BDNF promoter. Enhancing BDNF expression as an effect of SFN treatment increased the neuronal content of several synaptic molecules like MAP 2, synaptophysin, and PSD-95 in primary cortical neurons of 3 × Tg-AD.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6885086/ “Sulforaphane – role in aging and neurodegeneration”

I came across this review while searching PubMed for sulforaphane commonalities with presentation topics in Part 2 of Reversal of aging and immunosenescent trends with sulforaphane. The review outlined some aging aspects and presented relevant sulforaphane studies. Others such as eye and muscle decline weren’t addressed.

Since sulforaphane’s “a ‘miraculous’ drug” in the Abstract, I expected but didn’t see corresponding excitement in the review body. Just phrases like “it is known” and non-specific “more research is needed.”

Other papers published after this review were found by a PubMed “sulforaphane signal aging” search:

A pair of broccoli sprout studies

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

This 2015 Oregon human study found:

  • “Plasma and urinary levels of total SFN [sulforaphane] metabolites were ~3–5 times higher in sprout consumers compared to BSE [broccoli sprout extract] consumers.
  • In sprout consumers, plasma concentrations were 2.4-fold higher after consuming the second dose than after the first dose.
  • Calculated SFN bioavailability from broccoli sprouts exceeded 100%.

a Following consumption of a single 200-µmol SFN dose. b Cumulative excretion of SFN metabolites from baseline collection through the 48-hr study period. c Bioavailability F calculated based on total micromoles excreted in urine. Cmax, maximum concentration observed; AUC, area under the curve; h, hour; L, liter; t1/2, half-life; Tmax, time at Cmax. Values represent mean ± SD, n = 10.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394840/ “Absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract”

Another way to state findings:

  1. Broccoli sprouts are better than supplements.
  2. Eating sprouts twice a day is better than eating them once a day.

No explanation was given for sprout weight variability, although one was needed, because 127.6 g / 2 = 63.8 g, not 46.8 g:

“In the divided-dose phase (two weeks later), subjects (fasting) consumed half the original dose (100 μmol SFN equivalents) at 8 AM from sprouts or the BSE and the other half (not fasting) 12 h later.”

A “SFN potential” process demonstrated sulforaphane amount equivalencies, but didn’t explain non-intuitive sprout weight measurements. Was it too difficult to control sprout variability? The difficulties were instead pushed onto other researchers trying to replicate the study, and consumers looking for practical guidance.

Regardless, I adjusted my practices to twice daily start a new broccoli sprout batch with one tablespoon of seeds rather than once a day with two tablespoons. I eat them with breakfast and dinner.

I microwave 3-day-old sprouts immersed in 100 ml distilled water on full 1000W power for 35 seconds to achieve 58°C. I immediately put them into a strainer to allow further myrosinase hydrolization of glucoraphanin and other glucosinolates into sulforaphane and other healthy compounds.

I’d overlooked the above study until I saw it referenced in its successor 2018 human study Untargeted metabolomic screen reveals changes in human plasma metabolite profiles following consumption of fresh broccoli sprouts and cited it in Reversal of aging and immunosenescent trends with sulforaphane for its DHEA findings. The clinical trial treatments included:

“Both dehydroepiandrosterone (DHEA) and metformin in an attempt to limit the “diabetogenic” effect of GH [growth hormone]. DHEA has many effects, in both men and women, that oppose deleterious effects of normal aging.”

A PubMed search on DHEA found Impact of Dehydroepianrosterone (DHEA) Supplementation on Serum Levels of Insulin-Like Growth Factor 1 (IGF-1): A Dose-Response Meta-Analysis of Randomized Controlled Trials which confirmed the clinical trial’s DHEA dose would increase IGF-1.

This study observed a significant decrease in DHEA after eating broccoli sprouts, but didn’t provide a plausible explanation for this finding, or cite relevant studies. Ten other significant decreases were related to antioxidants and fatty acids.

It isn’t clear that I needed to take DHEA anyway, since the clinical trial’s purpose for DHEA treatment was to oppose effects of growth hormone, which I’m not taking. But I’m getting good results, so I’ll just keep doing what I’ve been doing for a limited time.

The study said:

“While this study focuses largely on the potential effects of SFN, broccoli sprouts contain many other bioactive components that could be responsible for our observations as well as additional health benefits.”

Our model clinical trial Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects said much the same:

“The anti-inflammatory effects observed with broccoli sprouts intake are likely due to the combined effects of all the hydrolysis products of glucosinolates.”

The 3-day-old broccoli sprouts have the optimal yields study said:

“Although germination reduces SF [sulforaphane] yield to some extent, it is beneficial to the formation and accumulation of total phenol and flavonoids, ensuring the health properties of sprouts.”

Combining the pair of Oregon studies’ findings:

  1. Broccoli sprouts are better than supplements.
  2. Eating sprouts twice a day is better than eating them once a day.
  3. When in doubt, refer back to Item 1.

 

Do broccoli sprouts treat migraines?

gettingwell4 2-3 stars Required further work, Epigenetics, Limbic system, Lower brain, Memory , , ,

While rereading a review in Eat broccoli sprouts today, it occurred to me that I haven’t needed to take migraine medicine during the 9 weeks I’ve been eating broccoli sprouts every day. Since 14 weeks of lockdown overlap this period, it’s also possible that I’ve avoided triggering conditions. I look at brightly-lit screens all day, but don’t have cold air blowing on my head that’s the other half of my most common triggering condition.

I started having intermittent ~monthly episodes about ten years ago. I wouldn’t take sumatriptan unless I have a half-day-long headache that doesn’t respond to acetaminophen. It stops a headache from turning into a 3-day-long migraine.

I went over to PubMed and did a “sulforaphane migraine” search, which turned up exactly 1 (!!) result. A 2016 Chinese rodent study Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats found:

“Activation of the Nrf2/ARE pathway inhibited the activation of TGVS [trigeminovascular system] and prevented the induction of hyperalgesia. Sulforaphane might therefore be an effective agent for hyperalgesia.”

Plausible conclusion. Nitroglycerin definitely jolts a monster headache.

Two of the eleven papers citing this study were:

There wouldn’t be any potential payoff for a company to be interested in studying a sulforaphane-migraine connection. What sponsor would be interested enough to double the number of studies in this area?

See Part 2 of Do broccoli sprouts treat migraines? for a follow up.

A hair color anecdote

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

Will you excuse a poorly-evidenced observation that’s a positive development I left out of Week 8 of Changing to a youthful phenotype with broccoli sprouts?

I got a haircut last weekend after waiting for Governor Klan Robes Blackface to not arrest barbershop and hair salon owners for the crime of earning a living. A thirty-something tattooed barber wearing a face mask and face shield said my last haircut had been on February 1, 2020, so it had been 14 weeks. She used a #4 clipper to cut everything to about 1/2 inch.

I’d eaten broccoli sprouts every day for 7 weeks at that point. Post-haircut visible hair was all from that period, probably since Week 3, which was also when broccoli sprouts’ effects on inflammation became noticeable.

One evening as I brushed my teeth, I noticed overall hair appearance was mainly dark brown again, an unexpected phenomenon. Maybe white hair will show up as it gets longer?

Feedback on hair color from a back-of-the-head picture was mixed, ranging from “Yes. Definitely!” to Unsupported non-evidence since before and after pictures weren’t taken under the same lighting conditions. Even if validated, other factors could be in play, such as working from home without the stress of going into work.

While eating my usual steel cut oats for breakfast this morning, I remembered a super informative presentation by the lead researcher of clinical trial Reversal of aging and immunosenescent trends. I rewatched it, pausing after two minutes to reabsorb when he said:

“There’s a collapse that takes place somewhere between the ages of sixty to eighty in which you lose 98% of your ability to recognize foreign antigens.”

You will have forgotten why I drew your attention to this super interesting presentation by the 21:25 mark. But pause for the “Hair Rejuvenation?” slide with before and after photos:

“A couple of guys came to us and said they seemed to notice that their hair was growing in darker again. It’s an anecdote. It didn’t apply to most of the guys. But it’s a sign that maybe something interesting is going on.”

That’s followed by epigenetic clock findings using four different clocks. Note that no significant effects on biological age were found until the trial’s 9-month point, and those weren’t as strong as improvements by 12 months.

Improvements accelerated between 9 and 12 months, and at 12 months, subjects had increased their life expectancies by 2 years. The GrimAge clock showed subjects’ predicted lifespan and health span was unchanged 6 months after the trial ended.

I started and have continued four lifestyle “interventions” since last summer:

  1. In July I dramatically reduced my consumption of advanced glycation end products after reading Dr. Vlassara’s AGE-Less Diet: How a Chemical in the Foods We Eat Promotes Disease, Obesity, and Aging and the Steps We Can Take to Stop It.
  2. In September I started this trial’s non-prescription daily treatments of Vitamin D, zinc, and DHEA.
  3. Also in September, I started non-prescription intermittent quercetin treatments of Preliminary findings from a senolytics clinical trial.
  4. Eight weeks ago I started eating broccoli sprouts every day per clinical trial Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects.

In a month or so I should be able to say whether or not my hair really is growing in darker. One way to find out which “intervention” had the largest effect may be to stop one or more of them. That might happen anyway because:

  1. Consistently eating AGE-less food is boring.
  2. I’m leery of taking more than RDAs.
  3. Ehh.
  4. I still sadly hope against reality that we’re past the Madness of Crowds phase and can accelerate the “recover their senses slowly, one by one” phase. It would be harder to take care of my broccoli sprout farm if I have to go into work every day.

Or maybe An environmental signaling paradigm of aging is correct, and at a certain point, clocks are reset and none of these “interventions” will be needed? What do you think?

Lab analyses of broccoli sprout compounds

gettingwell4 2-3 stars Required further work, Epigenetics

To follow up Estimating daily consumption of broccoli sprout compounds, the main point of which was: how can a person guide their actions with evidence when a broccoli cultivated variety’s beneficial characteristics aren’t known?

I focus on broccoli sprout lab analyses of 3 cultivars made in a 2017 German study Isothiocyanates, Nitriles, and Epithionitriles From Glucosinolates Are Affected by Genotype and Developmental Stage in Brassica oleracea Varieties. Here are rounded numbers from the supplementary material:

1. What happened in 2015 to lessen all broccoli cultivars’ sprout compound values by 2015 / 2014 percentages?

I read and reread, and didn’t come up with a satisfactory answer. Maybe it’s a language difference, but this was an insufficient explanation:

“Decreased myrosinase activity probably was the reason for the low levels of hydrolysis products formed in broccoli cv. Sirtaki in 2015 compared to 2014, while 70% [67%] of the GLS concentration of 2014 was present.”

What caused “decreased myrosinase activity?” And why would that be explanatory for just one cultivar when two others also experienced “low levels of hydrolysis products?”

Greenhouse-grown broccoli sprouts clearly varied between batches. Temperatures and sun radiation were tossed in but not shown to have been explanatory variables.

2. Could a person infer from one cultivar’s 2014 lab report how much sulforaphane they would consume from that cultivar in 2015?

No.

3. Without knowing a broccoli sprouts’ cultivar, could a person infer from a glucoraphanin amount how much sulforaphane they would consume?

No. The 2014 Iron Man 5.2 μmol per g glucoraphanin wasn’t that different from the 2015 Sirtaki 5.4 amount. But Iron Man vs. Sirtaki differences of 0.3 vs. 0.6 μmol per g sulforaphane amounts and 5% vs. 11% hydrolyzed showed Sirtaki cultivar’s unrounded numbers had double or more those of Iron Man.

4. Could a person infer from a mature broccoli glucoraphanin amount anything about its broccoli sprout glucoraphanin amount, or vice versa?

No. The Sirtaki 8.0 μmol per g sprout glucoraphanin amount was the highest cultivar, but its fully developed head was lowest at 0.27.

5. Could differences be explained by the broccoli sprout batches’ one-day age difference?

6. Did both years use the same seed lot? If so, could a one year aging of seeds explain differences?

Here’s a graphic of the 2014 batch major broccoli sprout compounds. Glucoraphanin is red 4MSOB in A, and sulforaphane is red 4MSOB-ITC in C:

This study demonstrated that there are many unknowns even with lab analyses. It would seem that not much could be accurately inferred past knowing that broccoli sprouts of one cultivar produced more sulforaphane than another.

Would similar lab reports help current purchase decisions? I haven’t come across a broccoli seed bulk supplier who had lab reports for products they’re selling. When pressed on Amazon they at best say Calabrese, which has described hundreds of cultivars. Such as Iron Man and Marathon, which are also named Calabrese Iron Man F1 and Calabrese Marathon F1.

Obtaining information for each batch would probably cost them a lot of money, which would be passed on to their customers. Lab reports for a different batch than the broccoli seed offered may have limited utility to consumers.

It would assist broccoli seed consumers to at least know the cultivar. This study’s genotypes had a greater effect than did environmental influences.

See follow up Tailoring measurements for broccoli sprouts.

Enhancing sulforaphane content

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

This 2020 Chinese study experimented with enhancing sulforaphane content of broccoli florets in a range of conditions:

“For direct water blanching at 60°C, sulforaphane yield increased with treatment time from 1698.0 ± 121.9 μmol per kg DW (0 min) to 2833.3 ± 118.6 μmol per kg DW (1 min) and then steadily decreased to the lowest value of 2345.8 ± 57.7 μmol per kg DW for 5 min.

The sulforaphane yield of broccoli after 5 min thermal treatment at 65 °C was even lower than the value obtained for raw broccoli. The reason could be leaching of glucoraphanin into blanching water coupled with partial inactivation of myrosinase resulting in low yield of sulforaphane.

For direct water blanching, the best treatment temperature for maximizing sulforaphane yield was 60 °C.

Sulforaphane yield depends on the relative activity of myrosinase and ESP in the broccoli matrix and 3 min treatment at 65 °C during in-pack processing in this study was found to be the best condition that favours conversion into sulforaphane instead of sulforaphane nitrile. This indicates that the condition favours inactivation of ESP to a larger extent while maintaining sufficient myrosinase activity resulting in optimal conversion into sulforaphane.

Under this condition, it seems that all extractable glucoraphanin is converted to sulforaphane assuming 1 to 1 conversion, since glucoraphanin content of broccoli samples were determined to be 3141.2 μmol per kg DW whereas the sulforaphane yield was 3983 μmol per kg DW. The slightly higher sulforaphane yield than would be predicted from the level of glucoraphanin in raw broccoli requires further investigation.”

https://pubs.rsc.org/en/content/articlehtml/2020/fo/c9fo02089f “Mild heat combined with lactic acid fermentation: a novel approach for enhancing sulforaphane yield in broccoli puree”

1. The study presented evidence for kitchen practices:

  • Per the above graphic’s point a, I’ve changed to let broccoli sprout heating continue for 1 minute after microwaving to achieve up to but no more than 60°C (140°F). This allows further myrosinase hydrolization of glucoraphanin into sulforaphane. My practice had been to immediately cool them down, which was suboptimal point c on the 60°C line. I still transfer broccoli sprouts to a strainer immediately after microwaving.
  • The 60°C cliff finding of Microwave broccoli to increase sulforaphane levels was confirmed by this study’s 65°C direct blanching finding.

2. I didn’t view this study’s in-pack or lactic acid bacteria fermentation findings as having practical kitchen use. Maybe it’s a cultural difference?

3. Poor performance at 65°C after 5 minutes was partially attributed to “leaching of glucoraphanin into the blanching water.” But poor 65°C performance was evident at the 1 minute point compared with good 60°C performance.

“Partial inactivation of myrosinase” at 65°C was more likely to be the dominant factor.

4. Regarding:

“The slightly higher sulforaphane yield than would be predicted from the level of glucoraphanin in raw broccoli requires further investigation.”

The microwaving study author was on a productive investigation track with:

“Microwave irradiation might help to release more conjugated forms of glucosinolates and then get hydrolyzed by released myrosinase.”

That track developed in part from finding that broccoli florets microwaved on full power to 60°C increased glucoraphanin past control (raw) levels:

“The control GLR amount was 2.18 µmol/g DW, while the HL60 GLR amount was 2.78 µmol/g DW.”

Not to mention the coincident 1,114% increase in sulforaphane content of ordinary broccoli purchased at a grocery store!

I arrived at this study through it being referenced in the enjoyable 2020 Spanish review Functional Ingredients From Brassicaceae Species: Overview and Perspectives. Those reviewers noted that this study’s 2019 predecessor Fermentation for enhancing the bioconversion of glucoraphanin into sulforaphane and improve the functional attributes of broccoli puree (not freely available) found:

“Preferential formation of SFN-nitrile (less potential as inducer of phase II detoxification enzymes than SFN) instead of SFN.”

Part 3 of Rejuvenation therapy and sulforaphane

gettingwell4 2-3 stars Required further work, Brain development, Cerebellum, Cerebrum, Epigenetics, Hippocampus, Hypothalamus, Limbic system, Lower brain, Memory, Stress, Telomeres , , , , , ,

Part 1 focused on the study’s clinical biomarkers. Part 2 highlighted its epigenetic clocks. Now we’ll look at rejuvenation of cognitive function.

Charts for this study’s most relevant human aging applications – measured by the new human-rat relative biological age clock – were in supplementary data due to combining study untreated tissue samples into clock training data. Reanalyses showed:

“Using the final version of the epigenetic clocks, we find that treatment effects become even more significant especially for the hypothalamus.”

Human-rat relative clock percentages of rejuvenation were:

  • “Blood 70.6%
  • Liver 79.4%
  • Heart 61.6%
  • Hypothalamus 20.9%”

The Discussion section addressed hypothalamus rejuvenation:

“Why does plasma fraction treatment not reduce brain epigenetic age by the same magnitude as it does other organs? We can only begin to address this question after having first understood what epigenetic aging entails.

As it stands, our knowledge in this area remains limited, but it is nevertheless clear that:

  1. Epigenetic aging is distinct from the process of cellular senescence and telomere attrition,
  2. Several types of tissue stem cells are epigenetically younger than non-stem cells of the same tissue,
  3. A considerable number of age-related methylation sites, including some clock CpGs, are proximal to genes whose proteins are involved in the process of development,
  4. Epigenetic clocks are associated with developmental timing, and
  5. Relate to an epigenomic maintenance system.

Collectively, these features indicate that epigenetic aging is intimately associated with the process of development and homeostatic maintenance of the body post-maturity.

  • While most organs of the body turnover during the lifetime of the host, albeit at different rates, the brain appears at best to do this at a very much slower rate.
  • While most tissues harbor stem cells that are necessary for replenishment and turnover, stem cells in adult brain have only been detected in a defined and very limited area of the subventricular zone, olfactory bulb (in rats), hippocampus and hypothalamic proliferative region.

As such, if plasma fraction treatment’s rejuvenating effect is:

  • Mediated through the process of development and
  • Involves tissue stem cells

then its effect on epigenetic age of the brain would appear to be modest, which indeed it does.

It is to be noted however, that improving brain function does not depend on neurogenesis as much as it does on synapse formation and factors such as NMDA receptors, which decline in density with age.

Assessment of plasma fraction treatment on cognitive function (learning and memory). Rats were subjected to Barnes maze test – nine consecutive days of test where the time (in seconds) required by rats to find the escape hole (latency) was recorded and plotted. Error bars depict 2 standard errors.

Within a month of plasma fraction treatment, rats exhibited significantly reduced latency to escape, i.e., they learned and remembered better. After the second month, treated rats began with a slightly reduced latency period compared to untreated old rats, and once again, they learned much faster than the latter.

By the third month, it was clear that treated rats remembered the maze much better than untreated ones even from the first day of test as their latency period was significantly reduced. By the end of the test period, their latency was similar to that of young rats. This feature was sustained and repeated in the fourth month.”

Not sure why there’s a 62-day gap between “Second month” and “Third month.” Maybe it had something to do with “First month” starting 10 days after the first treatment and “Third month” similarly starting 13 days after the second treatment?

Many of us know older people who lived well past their time of good cognitive function:

  • We see how they’re helpless and dependent; and
  • We see how others take advantage of them in their morbidity phase, where healthspan stops increasing but lifespan continues.

We can make personal plans for that day, sure. But let’s also put some urgency into applying this study’s new human-rat relative biological age clock, and make:

“A step change in aging research. Although conservation of aging mechanism could be equally deduced from the existence of multiple individual clocks for other mammals (mouse, dog), the single formula of the human-rat clock that is equally applicable to both species effectively demonstrates this fact.”

Part 2 of Rejuvenation therapy and sulforaphane

gettingwell4 2-3 stars Required further work, Brain development, Cerebellum, Cerebrum, Epigenetics, Hippocampus, Hypothalamus, Limbic system, Lower brain, Memory, Stress, Telomeres , , , , , , ,

A rejuvenation therapy and sulforaphane focused on the study’s clinical biomarkers and not its biological age measurements. This Part 2 curation of the study highlights its epigenetic clocks because:

“While clinical biomarkers have obvious advantages (being indicative of organ dysfunction or disease), they are neither sufficiently mechanistic nor proximal to fundamental mechanisms of aging to serve as indicators of them. It has long been recognized that epigenetic changes are one of several primary hallmarks of aging.

DNA methylation (DNAm) epigenetic clocks capture aspects of biological age. The discrepancy between DNAm age and chronological age (term as ‘epigenetic age acceleration’) is predictive of all-cause mortality. Pathologies and conditions that are associated with epigenetic age acceleration includes, but are not limited to, cognitive and physical functioning, centenarian status, Down syndrome, HIV infection, obesity, and early menopause.

The [new] human-rat clocks apply to both species. The two human-rat pan-tissue clocks are distinct, by way of measurement parameters. One estimates absolute age (in units of years), while the other estimates relative age, which is the ratio of chronological age to maximum lifespan; with values between 0 and 1. This ratio allows alignment and biologically meaningful comparison between species with very different lifespan (rat and human), which is not afforded by mere measurement of absolute age.

Relative age estimation was made using the formula: Relative age = Age / maxLifespan where the maximum lifespan for rats and humans were set to 3.8 years and 122.5 years, respectively.”

From Supplementary Table 3, old control and old treatment subjects were males 109 weeks old, 55% of their maximum lifespan (109 / 197.6). Young control subjects were males 30 weeks old, 15% of their maximum lifespan.

The money charts for this study’s human aging applications – measured by the new human-rat relative biological age clock – were buried in Supplementary Figure 12, bar plots M through P:

“Human-rat clock measure of relative age defined as age/maximum species lifespan. Each bar-plot reports the mean value and one standard error.”

From Supplementary Table 8, the percentages of rejuvenation for the above bar plots, calculated as “(100 * (1 – Old Treated / Old Control)” were:

  • “Blood 70.6%
  • Liver 79.4%
  • Heart 61.6%
  • Hypothalamus 20.9%”

Let’s return to clinical biomarkers for comparison purposes. The current study measured pro-inflammatory cytokine IL-6 blood plasma levels at every time point, but didn’t publish numbers. Bar plots and narrative were:

“Inflammation is an important response that helps protect the body, but excess inflammation especially in terms of duration of this response can have very detrimental effects instead. This occurs when inflammation fails to subside and persists indefinitely; a condition referred to as chronic inflammation, which for reasons not well-understood, increases with age and is associated with a multitude of conditions and pathologies.

The levels of two of the most reliable and common biomarkers of chronic inflammation, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), are found to be considerably higher in old rats, and these were very rapidly diminished, within days by plasma fraction treatment, to comparable levels with those of young rats. This was especially stark with IL-6.

In time, the levels of these inflammatory factors began to rise gradually, but they were once again very effectively reduced following the second administration of the plasma fraction on the 95th day.”

Let’s compare the above IL-6 graphic with IL-6 concentration improvements of our 2018 model clinical trial, Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects, calculated as (100 * (1 – Day _ mean / Day 0 mean):

Mean pg/ml | % improvement | Period | Broccoli sprout consumption

  • 4.594 | 0% | Day 0 | “One week before the beginning of the intervention period, subjects were asked to avoid the consumption of Brassica vegetables (broccoli, radish, cauliflower, Brussel sprouts, mustards, among others) and their derived products.”
  • 1.748 | 62.0% | Day 0 to 70 | Subjects ate 30 g raw broccoli sprouts every day, and stopped eating them after Day 70.
  • 0.896 | 80.5% | Day 0 to 90 | “After the intervention period, a follow-up recovery period for all subjects continued for another 90 days with no ingestion of broccoli sprouts.”
  • 2.170 | 52.8% | Day 0 to 160 | Subjects had not eaten broccoli sprouts after Day 70.

Results between the studies were similar in that:

  1. IL-6 levels improved during early treatments through rat Day 8 and human Day 70, respectively.
  2. IL-6 levels continued decreasing shortly after treatments for 7 days (through rat Day 15) and 20 days (through human Day 90), respectively.
  3. IL-6 levels rose after rat Day 15 and human Day 90, respectively, but were still significantly below Day 0 values at rat Day 95 and human Day 160.

The current study measured Nrf2 but didn’t publish numbers. Bar plots and narrative were:

“The reduction of these inflammation markers is consistent with the profile of the nuclear factor erythroid 2-like 2 protein (Nrf2), which plays a major role in resolving inflammation, in part by inhibiting the expression of IL-6 and TNF-α. Nrf2 also induces the expression of antioxidants that neutralizes ROS [reactive oxygen species], which is also a significant feature in inflammation.”

A PubMed search on “nrf2 sulforaphane human” didn’t turn up relevant 2020 human in vivo studies. I disregarded reviews, cancer studies, disproven hypotheses, and other compounds listed in the below graphic.

I won’t repeat the entire Nrf2 section from the Part 1 curation, just one graphic and paragraph:

It [sulforaphane] is not only a potent Nrf2 inducer but also highly bioavailable [around 80%], so that modest practical doses can produce significant clinical responses. Other Nrf2 activators [shown in the above image] not only lack potency but also lack the bioavailability to be considered as significant intracellular Nrf2 activators.”

As noted in Reviewing clinical trials of broccoli sprouts and their compounds, there are no sulforaphane clinical trials that also use epigenetic clocks. Broccoli sprouts and their compounds’ effects on human aging is an area that hasn’t drawn attention and funding.

What effects may broccoli sprout compounds have on human aging? With this new human-rat relative biological age clock, researchers can get reliable answers from rat studies, with human clinical trials needed only to confirm those findings!

As rejuvenation research continues, what could people do easily, cheaply, and today for our long-term selves? Don’t know about the hypothalamus, but our blood, liver, and heart biological ages may decrease as we reduce inflammation and oxidative stress by eating broccoli sprouts.

I’m at a similar percentage of species maximum lifespan as were the study’s treated subjects. It’s my choice as to what my healthspan will be.

There isn’t evidence today to definitively say that changing my inflammatory phenotype with broccoli sprouts has had / will have rejuvenation effects on biological ages of my cells, organs, and body. But if eating broccoli sprouts every day not only reduces chronic inflammation and oxidative stress as expected, but also makes me younger, I could probably learn to live with that. 🙂

Continued with Part 3 of Rejuvenation therapy and sulforaphane.

A rejuvenation therapy and sulforaphane

gettingwell4 2-3 stars Required further work, Brain development, Cerebellum, Cerebrum, Epigenetics, Hippocampus, Hypothalamus, Limbic system, Lower brain, Memory, Stress, Telomeres , , , , , , ,

The founder of the epigenetic clock methodology with the coauthor of Aging as an unintended consequence released a 2020 rodent study “Reversing age: dual species measurement of epigenetic age with a single clock” at https://www.biorxiv.org/content/10.1101/2020.05.07.082917v1.full.pdf:

“We employed six clocks to investigate the rejuvenation effects of a plasma fraction treatment in different rat tissues. Two of these epigenetic clocks apply to both humans and rats.

The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus.

The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers and behavioral responses to assess cognitive functions. Cellular senescence, which is not associated with epigenetic aging, was also considerably reduced in vital organs.

Plasma fraction treatment consists of two series of intravenous injections of plasma fraction. Rats were injected four times on alternate days for 8 days. A second identical series of injections were administered 95 days later. In its entirety, the experiment lasted 155 days.

Overall, this study demonstrates that a plasma-derived treatment markedly reverses aging according to epigenetic clocks and benchmark biomarkers of aging.”

The study hasn’t been peer reviewed, so can’t be viewed yet as conclusive. Given that researchers’ single-most valuable asset is their reputations, though, will the findings have major revisions?

I was alerted to the study by Josh Mitteldorf’s blog post Age Reduction Breakthrough, who did his usual excellent curation:

“Most of the explosion in aging research (and virtually all the venture capital startups) are looking to treat aging at the cellular level. Their paradigm is that aging is an accumulation of molecular damage, and they see their job as engineering of appropriate repair mechanisms.

The truth, as Katcher [the lead lab researcher] understands it, is that, to a large extent, aging is coordinated system-wide via signal molecules in the blood. The problem is that there are thousands of constituents represented in tiny concentrations in blood plasma, but conveying messages that cells read. Which of these are responsible for aging?

The two-species clock[s] was [were] a significant innovation, a first bridge for translating results from an animal model into their probable equivalent in humans. Besides the methylation clock[s], the paper presents evidence of rejuvenation by many other measures. For example:

  • IL-6, a marker of inflammation, was restored to low youthful levels;
  • Glutathione (GSH), superoxide dismutase (SOD), and other antioxidants were restored to higher youthful levels;
  • In tests of cognitive function (Barnes maze), treated rats scored better than old rats, but not as well as young rats.;
  • Blood triglycerides were brought down to youthful levels;
  • HDL cholesterol rose to youthful levels; and
  • Blood glucose fell toward youthful levels.

These results bring together three threads that have been gaining credibility over the last decade. Mutually reinforcing, the three have a strength that none of them could offer separately.

  1. The root cause of aging is epigenetic progression = changes in gene expression over a lifetime.
  2. Methylation patterns in nuclear DNA are not merely a marker of aging, but its primary source. Thus aging can be reversed by reprogramming DNA methylation.
  3. Information about the body’s age state is transmitted system-wide via signal molecules in the blood. Locally, tissues respond to these signals and adopt a young or an old cellular phenotype as they are directed.”

Several of these aging measurements are also positively affected by sulforaphane. Using Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease as a reference:

1. “Chronic inflammation”

“Antioxidants in general and glutathione in particular can be depleted rapidly under conditions of oxidative stress, and this can signal inflammatory pathways associated with NF-κB. SFN [sulforaphane] has been shown to inhibit NF-κB in endothelial cells.

Two key inflammatory cytokines were measured at four time points in forty healthy overweight people [our model clinical trial, Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects]. The levels of both interleukin-6 (Il-6) and C-reactive protein (CRP) declined over the 70 days during which the sprouts were ingested. These biomarkers were measured again at day 90, wherein it was found that Il-6 continued to decline, whereas CRP climbed again. When the final measurement was taken at day 160, CRP, although climbing, had not returned to its baseline value. Il-6 remained significantly below the baseline level at day 160.”

OMCL2019-2716870.010

2. “Oxidative stress”

“As a mediator for amplification of the mammalian defence system against various stressors, Nrf2 [nuclear factor erythroid 2-related factor 2] sits at the interface between our prior understanding of oxidative stress and the endogenous mechanisms cells use to deal with it. Diseases known to be underpinned by oxidative stress are proving to be more responsive to amplification of cellular defences via Nrf2 activation than by administration of direct-acting antioxidant supplements.

SFN, with absolute bioavailability of around 80%, [is] capable of increasing several endogenous antioxidant compounds via the transcription factor, Nrf2.

Nrf2 is ubiquitously expressed with the highest concentrations (in descending order) in the kidney, muscle, lung, heart, liver, and brain. Nrf2 was shown to prevent endothelial cells from exhibiting a proinflammatory state. Nrf2 is required for protection against glucose-induced oxidative stress and cardiomyopathy in the heart.

Well in excess of 500 genes have been identified as being activated by SFN via the Nrf2/ARE [Antioxidant Response Element] pathway, and it is likely that this underestimates the number as others are being discovered. Of the available SFN clinical trials associated with genes induced via Nrf2 activation, many demonstrate a linear dose-response. More recently, it has become apparent that SFN can behave hormetically with different effects responsive to different doses.

It [sulforaphane] is not only a potent Nrf2 inducer but also highly bioavailable so that modest practical doses can produce significant clinical responses. Other Nrf2 activators [shown in the above image] not only lack potency but also lack the bioavailability to be considered as significant intracellular Nrf2 activators.”

The study’s most relentlessly questioned, scrutinized, and criticized findings may be the two new epigenetic clocks that apply to both humans and rats. The researchers invited other researchers to validate these clocks because:

“If validated, this would be a step change in aging research. Although conservation of aging mechanism could be equally deduced from the existence of multiple individual clocks for other mammals (mouse, dog), the single formula of the human-rat clock that is equally applicable to both species effectively demonstrates this fact.”

The commonalities of this study with efforts to change my inflammatory phenotype with broccoli sprouts were summarized in the Discussion section:

“Apart from rejuvenating the vital organs of the treated rats, plasma fraction also impacted two fundamental physiological processes that underlie a great number of pathologies, namely oxidative stress and inflammation. Within a week of treatment, the markers of chronic inflammation (IL-6 and TNF-α) were significantly reduced and remained low throughout the entire experiment.

Likewise, markers of oxidative stress in brain, heart, lung and liver, which were very much higher in control old rats, were at the end of the experimental period, indistinguishable between plasma fraction-treated old rats and young ones. Concomitant with this drastic reduction in oxidative stress was the augmented levels of antioxidants (GSH, Catalase and SOD) in these tissues, indicating that modulating the levels of ROS [reactive oxygen species] to that of youthful rats is at least one way by which plasma fraction suppresses oxidative stress. It remains to be ascertained whether the rate of ROS generation is also reduced.

The levels of Nrf2, a transcription factor that impacts on oxidative stress, as well as inflammation, were raised by plasma fraction treatment of old rats to those of the young ones, indicating yet another level by which this treatment modulates these two critical processes. Collectively, these results show that plasma fraction treatment impacts not only the overt performances of organs, but also the underlying physiological processes that are pivotal for optimal organ function and health.”

Great stuff, huh? Are you ready to change your phenotype?

Continued with Part 2 of Rejuvenation therapy and sulforaphane.

I’m kind of asleep right now

gettingwell4 2-3 stars Required further work, Beliefs

This should stay up on YouTube for a while since it uses satire. If it used informed opinions like:

YouTube would censor it.

“I’ll stay at home for the rest of my life if they tell me to. Who knows how to make the best choices for my health and my life? Definitely not me.

The last thing you want is for people to have the freedom to make their own choices and then experience the consequences of their choices.

This new scientific data is irrelevant to me. I already made up my mind when I was the most frightened, and I’m going to keep on believing what makes me the most frightened. Because it’s more congruent to my being that way.

The more scared I am, the more obedient I am.

Yes, I would like a mandatory vaccine. Sometimes the best medicine is like the best sex: non-consensual. They work on the same premise.

It’s pretty well proven that being proactive and taking care of your health won’t keep you healthy. Our only hope is in the pharmaceutical companies protecting us. They have a very good track record of never harming anyone.

I’d like two microchips please. Just to keep me extra safe. Or maybe we should just do a baby step, and use a mandatory tracking app on our phone to keep us safe and then go to the microchips.”

It was known to everybody that the lockdown would cause a catastrophe

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

To follow up If people don’t stand up for their rights, their rights will be forgotten which YouTube has taken down, here are excerpts from a subsequent interview which YouTube has also taken down:

“If you don’t present bad news, that’s not good news for the media.

On April 17, the Director of the CDC presented at the Presidential Briefing, this graph. Its a count of hospitals reporting some sort of symptom that might be influenza. If the number of people who show up at the hospital peaked around March 18th, that means the number of infections peaked around March 8th.

People don’t go to the hospital for their first symptoms. They give it three or four days, and if it doesn’t get better, then they go to the hospital.

If infections peaked around March 8th, then shutting down schools and the economy ten days later is totally absurd. Shutting down the economy ten days after the curve had already turned down is heartless.

New York hospitals were not overflowing. They were laying off people. 500 sick people is a drop in the bucket for the New York City hospital system.

It may have been unfortunate for the patients that there were so many respirators. That’s a different story.

Double-checking never happened with these models. You’re never off by orders of magnitude. You’re off by 10, 20, 30%. [The Imperial College model for UK deaths from COVID-19 changed from 510,000 to 20,000 IIRC] That was more than two orders of magnitude.

It was known to everybody that the lockdown would cause a catastrophe.

Isolating the nursing homes would have been the thing that would have prevented deaths, and would have prevented hospitals from becoming overloaded. Not letting children and young adults from becoming infected and developing immunity would not prevent the load on hospitals.

You don’t need to do anything to prevent a respiratory disease from running. What you should do – and what was not done in the United States – was to protect the elderly. From the experience in Italy, we already knew that the vast majority of people who died were people in their seventies, eighties, nineties, who had comorbidities.

We also had that in Seattle, people with comorbidities died in nursing homes. At that point in time, one should have isolated at least the nursing homes.

To isolate the children, who are not at risk, and put those at risk at risk, is a catastrophe. It’s a human catastrophe that should have never, ever, happened.

I don’t know where the government finds these so-called experts who don’t understand the very basics about epidemiology.

I have never heard of him and never read any publications on epidemiology by Bill Gates but maybe I overlooked some of his qualifications.

I don’t understand this mantra that ‘We will never go back to normal.’ Why not? The virus is gone. Let’s go back and have a life.

If people would be more active. If they would take part in political decisions. If they would be more awake. If they would fight for their democratic rights. This would never have happened.

It’s a failure of the people to take control of the government, and let the government take control of them.”

The Professor misunderstood the United States form of government. As a general principle, the federal government doesn’t order states to do such and such.

Florida, for example, did exactly what the Professor suggested, “protect the elderly.” Other states didn’t, like Washington, New Jersey, Virginia, Pennsylvania, and especially New York. Don’t know why those states’ residents don’t demand responsibility and accountability.

The Professor didn’t adequately present aspects of human behavior. For example, he cited a CDC chart of a drop in hospital reporting of influenza-like symptoms for his arguments without also citing the media frenzy to scare people away from hospitals for fear that they would catch COVID-19. So of course there were fewer instances of influenza-like symptoms reported by hospitals.

He also said “The virus is gone” but that statement had qualifications. Parts of this interview misplaced their relevant contexts.

Reanalysis of findings from a senolytics clinical trial

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

To follow up Preliminary findings from a senolytics clinical trial:

“The central hypothesis tested in our article is that a brief course of the senolytic drug combination, Dasatinib plus Quercetin (D+Q), can reduce senescent cell abundance in humans, specifically focusing on targeting adipose tissue in subjects with diabetes and kidney dysfunction, a condition in which adipose tissue senescent cell burden is known to be increased.

Although we reported a statistically significant decrease in skin senescent cells in the 9 subjects whose skin data were reported in the original article, that conclusion did not hold up upon reanalysis.

The overall conclusion of our article that D+Q can target senescent cells in humans holds upon reanalysis of the data, at least in adipose tissue and as reflected by a composite of blood SASP [senescence-associated secretory phenotype] factors, but we have not shown this in skin here.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994619/ “Corrigendum to ‘Senolytics decrease senescent cells in humans: Preliminary report from a clinical trial of Dasatinib plus Quercetin in individuals with diabetic kidney disease’”

Deaths in Italy attributed to COVID-19

gettingwell4 2-3 stars Required further work, Beliefs, Immune system

Why have so many coronavirus patients died in Italy? from the Telegraph today:

“According to Prof Walter Ricciardi, scientific adviser to Italy’s minister of health, the country’s mortality rate is far higher due to demographics – the nation has the second oldest population worldwide – and the manner in which hospitals record deaths.

‘The age of our patients in hospitals is substantially older – the median is 67, while in China it was 46,’ Prof Ricciardi says. ‘So essentially the age distribution of our patients is squeezed to an older age and this is substantial in increasing the lethality.

But Prof Ricciardi added that Italy’s death rate may also appear high because of how doctors record fatalities.

‘The way in which we code deaths in our country is very generous in the sense that all the people who die in hospitals with the coronavirus are deemed to be dying of the coronavirus.

On re-evaluation by the National Institute of Health, only 12 per cent of death certificates have shown a direct causality from coronavirus, while 88 per cent of patients who have died have at least one pre-morbidity – many had two or three,’ he says.”

Refactoring the current 4,825 deaths in Italy attributed to COVID-19 equals 579 (4,825 x .12). That number places Italy slightly above France’s 562 current total.

Evidence-based statements wouldn’t sufficiently frighten the herd, though. The article continued on to include now-obligatory, hyperbolic, unscientific WHO statements referencing a “miracle.”

Image from “Culture Audits: We Have Been Asking the Wrong Question”