Control group

Broccoli sprout compounds include sinapic acid derivatives

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

This 2020 study provided details about healthy sinapic acid broccoli sprout compounds:

“Anti-inflammatory effects of broccoli sprouts have been demonstrated in vivo, but the connection with composition is not yet fully explained. The aim of the present study was:

  • Provide a complex qualitative / quantitative insight into variability of SADs [sinapic acid derivatives] during germination of broccoli sprouts; and
  • Investigate their antioxidant and anti-inflammatory properties that might result from the presence of phenolics.

Sprouting in darkness results in overall decrease in total content of SADs with growth time, but promotes replacement of relatively low active constituents, such as sinapine, by stronger antioxidants. These structural changes are beneficial for total antioxidant capacity of broccoli sprouts, and are correlated with their increasing ability to scavenge free radicals, reduce transition metal ions, and inhibit lipid peroxidation.”

The graphic’s dotted line is sinapine.

“Anti-TNF-α activity of broccoli sprout extract and sinapic acid was less relevant than that of an anti-inflammatory drug DEX [dexamethasone]. However, contrary to DEX, they significantly stimulated release of IL-10, which is an anti-inflammatory and immunosuppressive factor. By downregulating secretion of pro-inflammatory cytokines, IL-10 may impede immunopathology by inhibiting activity of macrophages, natural killer cells and lymphocytes.

Most of the previous studies ascribed anti-inflammatory effects of broccoli sprouts to their sulfur compounds only. Research data from in vivo models confirmed that glucosinolates may stimulate IL-10 release while isothiocyanates do not influence significantly or even decrease its production.

The present study indicates that phenolic constituents may also be responsible for anti-inflammatory effects of broccoli sprouts, and stimulation of IL-10 might be most relevant in this context.”

https://pubs.rsc.org/en/content/articlelanding/2020/FO/D0FO01387K “Variability of sinapic acid derivatives during germination and their contribution to antioxidant and anti-inflammatory effects of broccoli sprouts on human plasma and human peripheral blood mononuclear cells” (not freely available)

Some aspects of this study:

1. Seed-2-4-6 day measurements complemented seed-3-5-7 day measurements of the superb 3-day-old broccoli sprouts have the optimal yields. Both studies used standard methodologies, provided convertible dry weight and fresh weight measurements in mg / g, and grew their sprouts in darkness, with the current study watering every 12 hours.

The current study measured composition changes of 31 compounds (18 sinapic acid derivatives, 8 glucosinolates, and 5 flavonoids). It was too complicated for me to sum up and convert these granular dry weight measurements to make them directly comparable with 3-day study total phenolics (TP below, and TF is total flavonoids). They decreased similarly to TP mg / g fresh weight measurements.

The 3-day study also compared each cultivated variety’s germination stage weights and measurements with its origin using a milligram-per-gram-of-seeds scale:

“To be comparable, the content of these bioactive compounds from 100 fresh sprouts was divided by the weight (g) of 100 seeds, and then this value was compared with their content from one gram seeds.

The seed weight of XM was nearly twice that of other varieties and its sprouts weight also had the highest increasing rate. LWW and LY had the lowest weight of seeds as well as sprouts.”

phenolics and flavonoids

Different lowercase letters meant significant differences in the same cultivar among sampling days, and uppercase in the same sampling day among cultivars.

A. That study’s mg / g fresh weight trend favored broccoli seeds, a subject explored in Microwave broccoli seeds to create sulforaphane. Seeds of all six cultivars were significantly higher than their sprouts in TP, TF, and sulforaphane in this paradigm.

B. The mg / g seed trend favored 3-day-old broccoli sprouts for TP (five of six cultivars), TF (all), but not sulforaphane (two of six). Both trends were scientifically informed.

Which paradigm suits you?

2. A 2005 study was cited for:

“Total glucosinolate content in fresh broccoli sprouts were reported to reach 4.02 μmol/g fw, which corresponds to 0.71 mg/g fw of active isothiocyanates. In our study, total content of SADs in 6-day-sprouts was 4.85 mg/g dw. Recalculating to fresh weight gives 0.37 mg/g fw, which suggests that SADs might well influence biological effects of glucosinolates.”

The current study measured eight glucosinolates but no isothiocyanates. Don’t think this 2005 study provided bases for close comparisons, since it had unknown broccoli cultivar, unknown sprout age, and unanalyzed isothiocyanates.

Comparisons to a two-decimal-point precision should use the same cultivar and contexts, to include growing conditions. For example:

  • The 3-day study showed wide variation in sulforaphane weights among six broccoli cultivars’ seeds, from 2.43 mg to 12.07 mg per gram of seeds. And:

    “Knowledge concerning the bioavailability of plant ITCs is essential to predict the potential level of exposure as GL determination alone may not accurately reflect how much of the final active ITC can be formed from sprouts.”

  • The three cultivars studied in Lab analyses of broccoli sprout compounds had broccoli sprout sulforaphane amounts vary from 0.3 to 1.2 μmol / g fw in one growing season, then from 0.2 to 0.6 μmol / g fw the next year.

But these researchers knew that:

“In a study, diminishing amounts of total phenolic acids in sprouts of three broccoli cultivars was observed only between 3rd and 7th day of germination under photoperiod conditions and only when expressed on fresh weight basis. After recalculating results to dry weight, amounts were increasing during the whole 14-day observation period.

Trends cannot be treated as universal, but are dependent on growth conditions and variety of the plant.”

“0.37 mg/g fw” of Day 6 sinapic acid derivatives would be lower than Day 7 of four 3-day study cultivars’ total phenolics, and between Days 5 and 7 of the other two. I’d guess that the current study’s cultivar, Cezar, would be neither the lowest nor the highest cultivar if comparably measured for total phenolics.

3. These researchers compared other studies to an extent that isn’t usually seen. The result was good information such as:

“The most significant difference is sinapine that was not detected in either of the two above mentioned works, but is a major component of extracts investigated in the present work.

In one study, 7-day-sprouts were germinated under photoperiod conditions and without monitoring the phenolic profile in preceding growing days. Dominating constituents identified were trisinapoyl-gentiobiose, that was also present in relevant quantities in 6-day-sprouts, and sinapoyl-malate, that was not detected in our study.

Taking into account the significant role of these compounds (especially sinapoyl-malate) in UV-protection, light availability might have caused much quicker sinapine degradation and synthesis of SADs more suitable for those conditions. The same study also demonstrated that additional UVA and UVB irradiation before harvest further induced production of some of the constituents.“

4. Flavonoids and hydroxycinnamic acids are subgroups of phenolic compounds. Sinapic acid derivatives are a category of hydroxycinnamic acids.

Would microwaving sinapic acid derivatives have similar effects to what was described in Microwave broccoli to increase flavonoid levels? That’s the subject of Microwaving broccoli sprouts may not affect phenolic levels.

Eat broccoli sprouts for pain?

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

This 2018 study investigated pain-relieving effects of two broccoli sprout compounds, sulforaphane and chlorogenic acid:

“Pharmacological evidence of the antinociceptive properties of broccoli aqueous extracts and bioactive metabolites were investigated in an experimental model of pain.

It was found that sprouts produced better antinociceptive response than seeds and inflorescence of broccoli, where SFN [sulforaphane] and CA [chlorogenic acid] were partial responsible. Opioid receptors were implicated in the antinociceptive effect of SFN, whereas calcium channels were involved in the concentration-dependent spasmolytic activity.

Our results give evidence of a dose-dependent antinociceptive effect of CA that might act in a synergic interaction with SFN and other metabolites to produce antinociceptive activity.”

https://www.sciencedirect.com/science/article/abs/pii/S0753332218333286 “Broccoli sprouts produce abdominal antinociception but not spasmolytic effects like its bioactive metabolite sulforaphane” (not freely available)

8-day-old broccoli sprouts were treated Days 5-8 with methyl jasmonate to increase glucosinolates as Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts did.

I hadn’t previously noticed papers on “Chlorogenic and Sinapic acid derivatives” that are part of my daily intake, but there’s much recent research. Consider these October 2020 chlorogenic acid papers for example:

I found If it stinks, it’s good for you as a result of it citing this study. See Broccoli sprout compounds include sinapic acid derivatives to follow on that subject.

I rated this study as Required further work. This is my 31st week of eating a clinically relevant amount of broccoli sprouts every day, and I still take acetaminophen.

If it stinks, it’s good for you

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

This 2019 review subject was hydrogen sulfide and broccoli sprout compounds:

“Release of H2S was identified as a hidden mechanism responsible for effects of natural compounds that were used for a long time for pharmacological, therapeutic or nutraceutical purposes. For instance, the release of H2S was recognized as the main mechanism accounting for the biological effects of garlic.

There is evidence of a close overlap between numerous physiological / biological effects attributed to natural ITCs [isothiocyanates] and H2S. They both behave as antioxidant and anti-inflammatory agents, are activators of potassium channels modulating a vasodilatory effect, and are chemopreventive agents. Moreover, an impressive overlap can be observed in the molecular mechanisms of action.

Vascular inflammation results from the persistence of oxidative and/or inflammatory stimuli on the endothelium and vascular smooth muscle. These types of stimuli can be a consequence of prolonged status of mild inflammation and are typical in certain metabolic / cardiovascular diseases, spreading to all organs and tissues.

Advanced glycation end products (AGEs) are responsible for an increase in LDL. Binding of AGEs to their receptors RAGE results in an increase in intracellular ROS generation and in activation of NF-kB. Oral consumption of SFN [sulforaphane] precursor-rich broccoli sprouts decreases the serum levels of AGEs in humans.

Inflammatory response plays a pivotal role in initiation and maintenance of chronic neuropathic pain. Inhaling low concentrations of H2S protects motor neurons from degeneration and delayed paraplegia in a mouse model of sciatic constriction injury. This effect has been ascribed to the activation of the Nrf2 pathway.

Dose-dependent rise of the pain threshold mediated by SFN was fully prevented by simultaneous administration of hemoglobin, confirming that H2S is likely to be the real player in ITC-induced analgesia. Kv7 channel activation can be considered one of the main mechanisms in the antinociceptive activity of H2S-releasing drugs.”

https://www.liebertpub.com/doi/10.1089/ars.2019.7888 “Organic Isothiocyanates as Hydrogen Sulfide Donors” (not freely available)

These reviewers were long on equivalencies and short on proofs. Unlike study researchers, reviewers aren’t bound to demonstrate evidence from tested hypotheses. Reviewers are free to:

  • Express their beliefs as facts;
  • Over/under emphasize study limitations; and
  • Disregard and misrepresent evidence as they see fit.

Study researchers are obligated to provide detailed analyses of why observed effects couldn’t have been produced from unobserved causes. That didn’t happen here.

Anti-tumor effects of β-glucan

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

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

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

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

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

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

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

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

or one of its volcano plots?

Here’s an overview of one investigated direction:

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

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

Reprogramming other signaling pathways are in blog posts such as:

Take responsibility for your one precious life.

Flying over waves

Nano-sulforaphane vs. barbecue chemicals

gettingwell4 4-5 stars Advanced knowledge, Brain development, Epigenetics, Stress , , ,

This 2020 chicken study investigated the capability of nano-sulforaphane to protect embryonic survival and neurogenesis from a barbecued meat chemical:

“Common teratogenic [of, relating to, or causing malformations of an embryo or a fetus] factors related to the development of the nervous system, such as alcohol consumption and smoking, have attracted wide attention. Teratogenic factors such as PhIP, the most abundant amine produced in common cooking procedures, can affect early embryonic development, leading to abnormal development of the nervous system.

Nano-sized medicine, in comparison with conventional medicine, leads to increased active concentrations and bioavailability. Both PhIP and nanoparticles can cross the placental barrier and enter the fetus from the external environment.

Chick embryos (100 per group) were incubated with 0.1% DMSO (Control); 20μM, 100μM, 200μM, or 300μM PhIP; or 200μM PhIP + 5μM Nano-SFN [sulforaphane] for 36 h:

  • Mortality rates were 0% for the Control, 8% with 20μM PhIP, 20% with 100μM PhIP, 53% with 200μM PhIP, 85% with 300μM PhIP, and 7% with 200μM PhIP + 5μM Nano-SFN.
  • Neural tube malformation rates [for the remaining live embryos] were 0% for the Control, 5% with 20μM PhIP, 14% with 100μM PhIP, 36% with 200μM PhIP, 14% with 300μM PhIP, and 6% with 200μM PhIP + 5μM Nano-SFN.

Women at the early stage of pregnancy should avoid barbecue. Instead, increase intake amount of cruciferous vegetables, which benefits fetal neural development.”

https://www.sciencedirect.com/science/article/abs/pii/S0940960220301618 “Nano-sulforaphane attenuates PhIP-induced early abnormal embryonic neuro-development” (not freely available)

PXL_20201022_225011002.NIGHT

Dietary contexts matter

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

Two papers illustrated how actions of food compounds are affected by their contexts. The first was a 2020 UCLA rodent study:

“Long-chain polyunsaturated fatty acids (PUFAs), particularly omega-3 (n-3) PUFAs, have been indicated to play important roles in various aspects of human health. Controversies are observed in epidemiological and experimental studies regarding the benefits or lack of benefits of n-3 PUFAs.

Dietary docosahexaenoic acid (DHA; 22:6 n-3) supplementation improved select metabolic traits and brain function, and induced transcriptomic and epigenetic alterations in hypothalamic and hippocampal tissues in both context-independent and context-specific manners:

  • In terms of serum triglyceride, glycemic phenotypes, insulin resistance index, and memory retention, DHA did not affect these phenotypes significantly when examined on the chow diet background, but significantly improved these phenotypes in fructose-treated animals.
  • Genes and pathways related with tissue structure were affected by DHA regardless of the dietary context, although the direction of changes are not necessarily the same between contexts. These pathways may represent the core functions of DHA in maintaining cell membrane function and cell signaling.
  • DHA affected the mTOR signaling pathway in hippocampus. In the hypothalamus, altered pathways were more related to innate immunity, such as cytokine-cytokine receptors, NF-κB signaling pathway, and Toll-like receptor signaling pathway.

DHA exhibits differential influence on epigenetic loci, genes, pathways, and metabolic and cognitive phenotypes under different dietary contexts.”

https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202000788 “Multi‐tissue Multi‐omics Nutrigenomics Indicates Context‐specific Effects of DHA on Rat Brain” (not freely available)

A human equivalent age period of the subjects was 12 to 20 years old. If these researchers want to make their study outstanding, they’ll contact their UCLA colleague Dr. Steven Horvath, and apply his new human-rat relative biological age epigenetic clock per A rejuvenation therapy and sulforaphane.

The second paper was a 2016 review Interactions between phytochemicals from fruits and vegetables: Effects on bioactivities and bioavailability (not freely available):

“The biological activities of food phytochemicals depend upon their bioaccessibility and bioavailability which can be affected by the presence of other food components including other bioactive constituents. For instance, α-tocopherol mixed with a flavonol (kaempferol or myricetin) is more effective in inhibiting lipid oxidation induced by free radicals than each component alone.

Interactions of phytochemicals may enhance or reduce the bioavailability of a given compound, depending on the facilitation/competition for cellular uptake and transportation. For example, β-carotene increases the bioavailability of lycopene in human plasma, and quercetin-3-glucoside reduces the absorption of anthocyanins.

Combinations of food extracts containing hydrophilic antioxidants and lipophilic antioxidants showed very high synergistic effects on free radical scavenging activities. A number of phytochemical mixtures and food combinations provide synergistic effects on inhibiting inflammation.

More research should be conducted to understand mechanisms of bioavailability interference considering physiological concentrations, food matrices, and food processing.”

Each of us can set appropriate contexts for our food consumption. Broccoli sprout synergies covered how I take supplements and broccoli sprouts together an hour or two before meals to keep meal contents from lowering sulforaphane bioavailability.

Combinations of my 19 supplements and broccoli sprouts are too many (616,645) for complete analyses. Just pairwise comparisons like the second paper’s example below would be 190 combinations.

binary isobologram

Contexts for each combination’s synergistic, antagonistic, or additive activities may also be influenced by other combinations’ results.

My consumption of flax oil (alpha linolenic acid C18:3) probably has effects similar to DHA since it’s an omega-3 PUFA and I take it with food. The first study’s human equivalent DHA dose was 100mg/kg, with its citation for clinical trials stating “1–9 g/day (0.45–4% of calories) n-3 PUFA.”

A 2020 review Functional Ingredients From Brassicaceae Species: Overview and Perspectives had perspectives such as:

“In many circumstances, the isolated bioactive is not as bioavailable or metabolically active as in the natural food matrix.”

It discussed categories but not combinations of phenolics, carotenoids, phytoalexins, terpenes, phytosteroids, and tocopherols, along with more well-known broccoli compounds.

Diving for breakfast

Rub some broccoli sprouts on it

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

This 2020 human/rodent study investigated treating and preventing skin photodamage with sulforaphane:

“Alterations in NRF2 signaling have been implicated in aging and stress-induced skin pigmentation disorders in the skin and hair follicles. NRF2 signaling regulates transcriptional programs involved in adaption and survival of cells in the setting of oxidative stress, and oxidative stress occurs in the setting of photodamage.

[1st human experiment with 14 subjects] Expression levels of NRF2 and its target heme-oxygenase-1 (HO-1) were evaluated by immunofluorescence (IF) in skin biopsies. Expression of NRF2 and HO-1 was significantly reduced in skin from individuals > 45 years old.

[2nd human experiment with 7 different subjects] The left arm was chosen for treatment with BSE [broccoli sprout extract], as there is typically more photodamage on the left arm due to chronic sun exposure through the car window while driving in the US. A photoprotected area of skin on an upper inner arm was also treated.

Expression of total NRF2 and phosphorylated NRF2 (NRF2-P) by IF microscopy was detected at low baseline levels in photoprotected skin, suggesting some activity of the pathway, whereas the expression of total NRF2 and NRF2-P was undetectable in untreated photoexposed skin (Un). There was significantly elevated IF expression and fold change of IF signal of NRF2 and especially NRF2-P in SF [sulforaphane]-treated skin compared with Un skin in most individuals.

There was no evidence of increased total NRF2 or NRF2-P expression in SF-treated photoexposed skin in 2 individuals. There was also no significant improvement in mottled hyperpigmentation or difference in melanin deposition following SF treatment.

[Six mouse confirmation/exploratory studies] SF is known to have several non-NRF2–mediated targets, such as NF-κB and AP-1. However, our findings suggest that negative regulation of UV-mediated hyperpigmentation observed following SF treatment is occurring in an NRF2-dependent fashion:

  • UVB+SF treatment resulted in more than a 50% decrease in skin pigmentation and melanin deposition, indicating that SF could prevent UVB-induced skin pigmentation.
  • The therapeutic effect of SF on reducing UVB-induced skin pigmentation was dependent on keratinocyte-intrinsic IL-6 receptor α (IL-6Rα) signaling that upregulated NRF2, which led to inhibition of melanogenesis.

Our results provide direct in vivo evidence of how NRF2 is involved in response to oxidative stress associated with photodamage and chronic UV exposure. Treatment of human or mouse skin hyperpigmentation with SF provided the proof of concept for targeting the NRF2 pathway as a therapeutic intervention.”

https://insight.jci.org/articles/view/139342 “Pathogenic and therapeutic role for NRF2 signaling in ultraviolet light–induced skin pigmentation”

Didn’t understand the 2nd experiment’s human dose of 5 nM sulforaphane. The lead author’s cited 2017 study Randomized, split-body, single-blinded clinical trial of topical broccoli sprout extract: Assessing the feasibility of its use in keratin-based disorders used “500 nmol of sulforaphane/mL.” Unless my math is off, the current study and previous study’s doses weren’t equivalent since 1 nM = 0.001 nmol/mL.

I’d like to know more about subjects who didn’t respond to topical sulforaphane treatment. What happened in their lives to make them dead to an evolutionarily-selected antioxidant and anti-inflammatory signaling pathway that influences many other internal environmental signals? Guess we’ll have to wait for:

“Further clinical studies with an increased number of human subjects, longer treatment regimens, and additional body sites are needed to further assess the long-term effects of NRF2 activation on photoaging.”

See Eat broccoli sprouts for your skin! and Eat broccoli sprouts for your hair! for similar studies.

Owl before sunrise

A research train wreck

gettingwell4 < 0 Detracted from science

I try to highlight research that advances science. Please humor a momentary break.

This 2020 study Effects of cooking methods on total isothiocyanate yield from cruciferous vegetables simulated microwaving broccoli to 210° C (410° F). These researchers knew or should have known that myrosinase inactivates at moderate temperatures, given studies published last century such as this with its “Thermal inactivation proceeded in the temperature range 30−60° C” finding. Without an active enzyme, no isothiocyanates are produced from cooking broccoli.

Incredibly, these researchers proved they knew their study detracted from science by citing Microwave broccoli to increase sulforaphane levels. That study found destructive effects on sulforaphane at temperatures above 60° C (140° F):

fsn31493-fig-0003-m

The sulforaphane (SFR) amount at HL70° on the right (broccoli heated to 70° C with a 950 W microwave operated on full power) was significantly less than uncooked broccoli (control) on the left!

“This work was supported by the National Cancer Institute.”

I haven’t found words to correspond with the coauthors. Egregious errors speak for themselves.

Eat broccoli sprouts to pivot your internal environment’s signals

gettingwell4 2-3 stars Required further work, Brain development, Cerebrum, Epigenetics, Glutamate, Immune system, Memory, Neurochemicals, Stress , , , , ,

Two 2020 reviews covered some aspects of a broccoli sprouts primary action – NRF2 signaling pathway activation:

“Full understanding of the properties of drug candidates rely partly on the identification, validation, and use of biomarkers to optimize clinical applications. This review focuses on results from clinical trials with four agents known to target NRF2 signaling in preclinical studies, and evaluates the successes and limitations of biomarkers focused on:

  • Expression of NRF2 target genes [AKR1, GCL, GST, HMOX1, NQO1] and others [HDAC, HSP];
  • Inflammation [COX-2, CRP, IL-1β, IL-6, IP-10, MCP-1, MIG, NF-κB, TNF-α] and oxidative stress [8-OHdG, Cys/CySS, GSH/GSSG] biomarkers;
  • Carcinogen metabolism and adduct biomarkers in unavoidably exposed populations; and
  • Targeted and untargeted metabolomics [HDL, LDL, TG].

No biomarkers excel at defining pharmacodynamic actions in this setting.

SFN [sulforaphane] seems to affect multiple downstream pathways associated with anti-inflammatory actions. NRF2 signaling may be but one pivotal pathway.

SFN is generally considered to be the most potent natural product inducer of Nrf2 signaling. Studies in which these actions are diminished or abrogated in parallel experiments in Nrf2-disrupted mice provide the strongest lines of evidence for a key role of this transcription factor in its actions.

It is equally evident that other modes of action contribute to the molecular responses to SFN in animals and humans. Such polypharmacy may well contribute to the efficacy of the agent in disease prevention and mitigation, but obfuscates the value of specific pharmacodynamic biomarkers in the clinical development and evaluation of SFN.”

https://www.mdpi.com/2076-3921/9/8/716/htm “Current Landscape of NRF2 Biomarkers in Clinical Trials”

Why do researchers still not use epigenetic clocks in sulforaphane clinical trials? Forty mentions of disease in this review, but no consideration of aging?

This was another example of how researchers – even when stuck in a paradigm they know doesn’t sufficiently explain their area (“No biomarkers excel”) – don’t investigate other associated research areas. Why not?

Here’s what Part 2 of Rejuvenation therapy and sulforaphane had to say to those stuck on biomarkers:

“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 epigenetic clocks capture aspects of biological age.”

The second review Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals also completely whiffed on epigenetic clocks. One mention of aging in this review, but it wasn’t of:

  • Citation 104 from Archives of Gerontology and Geriatrics; nor of
  • Citation 108 from the March 31, 2020, Aging journal; nor of
  • Citation 131 “Dietary epigenetics in cancer and aging.”

But epigenetic clock and aging associations were certainly in this review’s scope. For example, Citation 119 said:

“Nrf2 transcriptional activity declines with age, leading to age-related GSH loss among other losses associated with Nrf2-activated genes. This effect has implications, too, for decline in vascular function with age. Some of the age-related decline in function can be restored with Nrf2 activation by SFN.”

Why would people bother with phytochemicals (buzzword “compounds produced by plants”) unless to either ameliorate symptoms or address causes?

“Epigenetic Regulation of NRF2/KEAP1 by Phytochemicals” doesn’t occur in just laboratory situations. It’s also part of daily life.

These reviewers were straight-forward with side effects for two of the first review’s four items:

“The best known NRF2 activator that has obtained clinical approval is dimethyl fumarate for the treatment of multiple sclerosis. However, it has several side effects, including allergic reactions and gastrointestinal disturbance. There are a few related agents in clinical trials, such as Bardoxolone and SFX-01, a synthetic derivative of sulforaphane, which also exhibit less than desirable outcomes.”

Human relevance of rodent sulforaphane studies

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

After reading through findings of several dozen rodent studies this evening, I thought it would be worthwhile to revisit analysis of human relevance provided by one paper of How much sulforaphane is suitable for healthy people?

“Comparisons of published oral doses of sulforaphane administered to mice or rats and sulforaphane (tablets or sulforaphane-rich broccoli preparations) or glucoraphanin-rich broccoli preparations administered to humans.

The allometric scaling of the murine doses uses the correction factor of 0.081 and those for rat doses 0.162. Human doses were based on an estimate of 70 kg body weights in each study.”

A confession followed:

“Animal studies have not delivered all that might be expected of them. Pre-clinical experimentalists have not thought carefully about the selection of dose (or route) and its relevance to clinical utility.

Over two-thirds of the animal studies have used doses that exceed the highest (and bordering on intolerable) doses of sulforaphane used in humans.

Few studies have included a dose-response. The greater than 4-log spread of doses used in mice appears to be driven by needs for effect reporting in publications rather than optimization of translational science.

Authors of this review have contributed to this dose skewing.”

Let’s narrow this graphic to a human-relevant range:

human-rodent-relevant-dosages

48 of the 114 rodent study doses were in an allometric range applicable to humans.

Clinically relevant sulforaphane human doses start at a 100 µmol amount (17.73 mg). The graphic normalized human weights to 70 kg, so 100 µmol / 70 kg is 1.43 µmol / kg. Eyeballing the graphic, 43 of the 114 rodent study doses were in an allometric range applicable to human clinical doses.

But only three of the human sulforaphane study doses were above 4 µmol / kg. This indicator of the mentioned “intolerable doses” will limit clinically relevant oral doses to no more than 17.73 mg x (4 / 1.43) ≈ 50 mg in one serving.

Reviewing clinical trials of broccoli sprouts and their compounds described a sulforaphane study with doses above 4 µmol / kg:

“They proposed the intake of 15 capsules of broccoli sprouts at a time, giving 90 mg [508 µmol, 7.26 µmol / kg] of SFN and 180 mg of glucoraphanin, a never before tested dosage, which was established because of the poor life expectancy of the patients and the aggressive characteristics of this type of cancer.

Secondary effects of the chemotherapy, the lack of appetite, nausea, vomits, diarrhea, mouth sores, etc., were factors that made it very difficult for the patients to intake 15 pills at once, as the study initially planned.

Progression of the pancreatic cancer and the GI symptomatology led to a high rate of drop-off of:

  • 72% in the treatment group; and
  • 55% abandonment in the control group!

Therefore, the results were not significant.”

Our model clinical trial Effects of long-term consumption of broccoli sprouts on inflammatory markers in overweight subjects calculated subjects’ mean weight in Table 1 as “85.8 ± 16.7 kg.” Its average glucoraphanin dose per kg body weight was 117 μmol / 85.8 kg = 1.36 μmol / kg.

Per Estimating daily consumption of broccoli sprout compounds, my twice-a-day consumption of a total 131 grams microwaved broccoli sprouts represents a worst-case 52 mg sulforaphane daily intake. This is ≈ 3 µmol / kg per day, the graphic’s second-largest sulforaphane amount cluster. Half that per serving.

Only 8% of the rodent studies were in a sulforaphane range that was both:

  • Clinically relevant to humans as a lower boundary (1.43 µmol / kg); and
  • Tolerable to humans as an upper boundary (4 µmol / kg).

human-rodent clinically relevant tolerable dosages

The main purpose of animal studies is to help humans. Which researchers conducted sulforaphane studies that could actually help humans?

Natural sources of melatonin

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

This 2020 review subject was melatonin:

“The emergence of naturally occurring melatonin and its isomers in fermented foods has opened an exciting new research area. Melatonin is a hormone, an indolamine that predominantly appears in plants, microorganisms, and mammals.

The precursor of this molecule is solely the amino acid L‐tryptophan. Melatonin ensures a circadian and seasonal signal to vertebrate organisms; it is synthesized through a cascade of enzymatic reactions producing melatonin from serotonin in its final phases. The synthesis of melatonin is observed in almost all organs.

One melatonin molecule has the capacity to scavenge up to 10 ROS versus the other antioxidants that scavenge 1 or even less ROS. Melatonin antioxidant properties are accomplished with the indole ring that stimulates enzyme production (i.e., superoxide dismutase (SOD), glutathione‐peroxidase (Gpx), and catalase (CAT)), which mitigate free radicals to less toxic substances.

In addition to antioxidant properties, it plays a fundamental role in the modulation of various physiological functions, including circadian rhythmicity, bone integrity, and functionalization of the human reproductive system.

The presence of melatonin and its isomers is not exclusive for grapes and grape‐derived products. Other fruits such as sweet and sour cherries and fermented juices of orange and pomegranate may be also of interest.”

https://onlinelibrary.wiley.com/doi/full/10.1111/1541-4337.12639 “Naturally occurring melatonin: Sources and possible ways of its biosynthesis”

Treating psychopathological symptoms will somehow resolve causes?

gettingwell4 2-3 stars Required further work, Acetylcholine, Amygdala, Anterior cingulate cortex, Brain development, Cerebellum, Cerebrum, Cortisol, Dopamine, Epigenetics, Glutamate, Hippocampus, Hypothalamus, Immune system, Limbic system, Lower brain, Memory, Neurochemicals, Serotonin, Stress, Thalamus , , , , ,

This 2020 Swiss review subject was potential glutathione therapies for stress:

“We examine available data supporting a role for GSH levels and antioxidant function in the brain in relation to anxiety and stress-related psychopathologies. Several promising compounds could raise brain GSH levels by either increasing availability of its precursors, or expression of GSH-regulating enzymes through activation of Nrf2.

GSH is the main cellular antioxidant found in all mammalian tissues. In the brain, GSH homeostasis has an additional level of complexity in that expression of GSH and GSH-related enzymes are not evenly distributed across all cell types, requiring coordination between neurons and astrocytes to neutralize oxidative insults.

Increased energy demand in situations of chronic stress leads to mitochondrial ROS overproduction, oxidative damage, and exhaustion of brain GSH pools.

Several compounds can function as precursors of GSH by acting as cysteine (Cys) donors such as taurine or glutamate (Glu) donors such as glutamine (Gln). Other compounds stimulate synthesis and recycling of GSH through activation of the Nrf2 pathway including sulforaphane and melatonin. Compounds such as acetyl-L-carnitine can increase GSH levels.”

https://www.sciencedirect.com/science/article/abs/pii/S0149763419311133 “Therapeutic potential of glutathione-enhancers in stress-related psychopathologies” (not freely available)

Many animal studies of “stress-related psychopathologies” were cited without noting applicability to humans. These reviewers instead had curious none-of-this-means-anything disclaimers like:

“Comparisons between studies investigating brain disorders of such different nature such as psychiatric disorders or neurodegenerative diseases, or even between brain or non-brain related disorders, should be made with caution.”

Regardless, this paper had informative sections for my 27th week of eating broccoli sprouts every day.

1. I forgot to mention in Broccoli sprout synergies that I’ve taken 500 mg of trimethyl glycine (aka betaine) twice a day for over 15 years. Section 3.1.2 highlighted amino acid glycine:

“Endogenous synthesis is insufficient to meet metabolic demands for most mammals (including humans) and additional glycine must be obtained from diet. While most research has focused on increasing brain cysteine levels in order to drive GSH synthesis, glycine supplementation alone or in combination with cysteine-enhancing compounds are gaining attention for their ability to enhance GSH.”

2. Taurine dropped off my supplement regimen last year after taking 500 mg twice a day for years. It’s back on now after reading Section 3.1.3:

“Most studies that reported enhanced brain GSH following taurine treatment were performed under a chronic regimen, and used in age-related disease models.

Such positive effects of taurine on GSH levels may be explained by the fact that cysteine is the essential precursor to both metabolites, whereby taurine supplementation may drive metabolism of cysteine towards GSH synthesis.”

3. A study in Upgrade your brain’s switchboard with broccoli sprouts was cited for its potential:

“Thalamic GSH values significantly correlated with blood GSH levels, suggesting that peripheral GSH levels may be a marker of brain GSH content. Studies point to the capacity of sulforaphane to function both as a prophylactic against stress-induced behavioral changes, and as a positive modulator in healthy animals.”

Sunrise minus 5 minutes

Increasing carbon dioxide levels increases beneficial broccoli sprout compounds

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

This 2020 study used IPCC unscientific, politically-motivated, wild-ass guesses for year 2100 CO2 levels to find that broccoli sprouts – like most plants – benefit when CO2 is increased:

“Elevated CO2 (eCO2, 620 ppm, the expected IPCC-SRES B2-scenario prediction of eCO2 of the year 2100) was applied for 9 days to further improve nutritive and health-promoting values of three cultivars of broccoli sprouts.

  • eCO2 improved sprouts growth and induced GLs [glucosinolates] accumulation.
  • There were increases in myrosinase activity, which stimulated GLs hydrolysis to yield health-promoting sulforaphane.
  • Low levels of sulforaphane nitrile were detected and positively correlated with reduced epithiospecifier protein after eCO2 treatment.
  • High glucoraphanin and sulforaphane levels in eCO2 treated sprouts improved the anticarcinogenic and anti-inflammatory properties of their extracts.

In conclusion, eCO2 treatment enriches broccoli sprouts with health-promoting metabolites and bioactivities.”

https://www.sciencedirect.com/science/article/abs/pii/S030881462030964X “Elevated CO2 improves glucosinolate metabolism and stimulates anticancer and anti-inflammatory properties of broccoli sprouts” (not freely available)

This study was sponsored in Saudi Arabia. Would gathering such scientific evidence even be permitted in more “enlightened” countries?

Performing research on obvious lies and thinking for yourself isn’t allowed anymore in most “learning” institutions. You already knew that, didn’t you?

At the end of How much sulforaphane is suitable for healthy people? I applauded my high-school literature teachers for forcing their students to demonstrate that they could think for themselves. I didn’t initially mention that each monthly assignment to read two books, then compare-and-contrast them in a 3-page handwritten paper, was individualized so that students couldn’t undo the assignment’s purpose with parasitical collaboration.

This former practice remains a good measure of intentional dumbing-down of young people, the intent of which has become clearer.

Jet fuel exposure causes diseases in the great-grand offspring

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

This 2020 Washington State University rodent study examined how great-grandmothers’ JP-8 exposures produced diseases in their great-grand offspring:

“Ancestral exposure to environmental influences such as toxicants, abnormal nutrition, and traumatic stress can affect the germline epigenome and promote the epigenetic transgenerational inheritance of adult onset disease in various organisms from plants to humans. Biological mechanisms underlying transgenerational epigenetic inheritance induced by jet fuel exposure are further investigated in the current study.

Genome-wide association studies (GWAS) have found specific genetic mutations associated with human pathologies, however these genetic mutations generally appear in less than 1% of the disease population. In contrast, epimutations (DNA methylation, histone modifications, non-coding RNA, chromatin structure, and RNA methylation alterations) seem to have a higher frequency and appear in more individuals with the diseases. Determining epigenetic biomarkers for these diseases could become especially useful indicators of environmental exposures and disease susceptibility in the human population.

The number of differential methylated regions (DMRs) found in the transgenerational F3 males is between 100 and 500 for each individual pathology. Few DMRs overlap between the different pathologies which supports the possible use of epimutations as biomarkers of disease. Although further studies are required, the lack of a subpopulation of DMRs overlapping with all pathologies suggests that at a more stringent statistical threshold there are not common DMRs among specific diseases.

Although females develop transgenerational disease, insufficient numbers of oocytes can be obtained on individuals to allow epigenetic associations to be assessed. The study only examined male pathology and associated sperm epimutation associations.”

https://www.sciencedirect.com/science/article/pii/S0890623820301982 “Epigenome-wide association study for transgenerational disease sperm epimutation biomarkers following ancestral exposure to jet fuel hydrocarbons”

The only associations these study subjects had with JP-8 were their great-grandmothers’ jet fuel exposures while pregnant with their grandparents. Other environmental toxicants studied by this group that produced similar transgenerationally inherited diseases were DDT, atrazine, and vinclozolin.

Ever think about your great-grandchildren?

Unraveling oxytocin – is it nature’s medicine?

gettingwell4 2-3 stars Required further work, Acetylcholine, Amygdala, Brain development, Brainstem, Cerebrum, Cortisol, Dopamine, Epigenetics, Glutamate, Hippocampus, Hypothalamus, Immune system, Limbic system, Lower brain, Memory, Neurochemicals, Oxytocin, Serotonin, Stress, Vasopressin , , , , , , ,

This 2020 review attempted to consolidate thousands of research papers on oxytocin:

“Chemical properties of oxytocin make this molecule difficult to work with and to measure. Effects of oxytocin are context-dependent, sexually dimorphic, and altered by experience. Its relationship to a related hormone, vasopressin, have created challenges for its use as a therapeutic drug.

Widely used medical interventions i.e.:

  • Exogenous oxytocin, such as Pitocin given to facilitate labor;
  • Opioid medications that block the oxytocin system; or
  • Cesarean sections that alter exposure to endogenous oxytocin

have lasting consequences for the offspring and/or mother.

Such exposures hold the potential to have epigenetic effects on the oxytocin systems, including changes in DNA methylation. These changes in turn would have lasting effects on the expression of receptors for oxytocin, leaving individuals differentially able to respond to oxytocin and also possibly to the effects of vasopressin.

Regions with especially high levels of OXTR [oxytocin receptor gene] are:

  • Various parts of the amygdala;
  • Bed nucleus of the stria terminalis;
  • Nucleus accumbens;
  • Brainstem source nuclei for the autonomic nervous system;
  • Systems that regulate the HPA axis; as well as
  • Brainstem tissues involved in pain and social attention.

Oxytocin protects neural cells against hypoxic-ischemic conditions by:

  • Preserving mitochondrial function;
  • Reducing oxidative stress; and
  • Decreasing a chromatin protein that is released during inflammation

which can activate microglia through the receptor for advanced glycation end products (RAGE). RAGE acts as an oxytocin-binding protein facilitating the transport of oxytocin across the blood-brain barrier and through other tissues.

Directionality of this transport is 5–10 times higher from the blood to the brain, in comparison with brain to blood transport. Individual differences in RAGE could help to predict cellular access to oxytocin and might also facilitate access to oxytocin under conditions of stress or illness.

Oxytocin and vasopressin and their receptors are genetically variable, epigenetically regulated, and sensitive to stressors and diet across the lifespan. As one example, salt releases vasopressin and also oxytocin.

Nicotine is a potent regulator of vasopressin. Smoking, including prenatal exposure of a fetus, holds the potential to adjust this system with effects that likely differ between males and females and that may be transgenerational.

Relative concentrations of endogenous oxytocin and vasopressin in plasma were associated with:

These studies support the usefulness of measurements of both oxytocin and vasopressin but leave many empirical questions unresolved.

The vast majority of oxytocin in biosamples evades detection using conventional approaches to measurement.”

https://pharmrev.aspetjournals.org/content/pharmrev/72/4/829.full.pdf “Is Oxytocin Nature’s Medicine?”

I appreciated efforts to extract worthwhile oxytocin research from countless poorly performed studies, research that wasted resources, and research that actually detracted from science.

I was disappointed that at least one of the reviewers didn’t take this review as an opportunity to confess their previous wastes like three flimsy studies discussed in Using oxytocin receptor gene methylation to pursue an agenda.

Frank interpretations of one’s own study findings to acknowledge limitations is one way researchers can address items upfront that will be questioned anyway. Such analyses also indicate a goal to advance science.

Although these reviewers didn’t provide concrete answers to many questions, they highlighted promising research areas, such as:

  • Improved approaches to oxytocin measurements;
  • Prenatal epigenetic experience associations with oxytocin and OXTR; and
  • Possible transgenerational transmission of these prenatal epigenetic experiences.