Control group

An outstanding review of Vitamin K deficiency and disease

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

This 2019 review focused on one Vitamin K-deficiency biomarker. All parts I’ve quoted are outside the liver, so Vitamin K deficiency ≈ Vitamin K2 deficiency.

This is a hard read with many technical details, but sometimes that’s how researchers do it:

“Active MGP (matrix Gla protein), once released into extracellular space, acts as a local inhibitor of calcification. Widespread expression of MGP points to a role of MGP that by far exceeds its well-known function as local inhibitor of calcification.

Recent research confirmed this concept, usually by measuring plasma dp-ucMGP (desphospho-uncarboxylated MGP), a biomarker reflecting poor vitamin K status:

1160fig02

Vitamin K plays a pivotal role in maintaining bone health. Increasing evidence also implicates MGP in maintaining bone health.

In the Health, Aging and Body Composition study, 791 older community-dwelling adults underwent magnetic resonance imaging to measure bilateral knee structural features. The highest [25%] compared with the lowest fourth of the dp-ucMGP distribution had higher odds of having:

  • Meniscus damage;
  • Osteophytes;
  • Bone marrow lesions; and
  • Subarticular cysts.

Regarding Vitamin K supplementation:

  • Studies showed a dose-dependent decrease in circulating dp-ucMGP with an 86% decrease already observed after 4 weeks of substitution by 360 μg menaquinone-7 [in 50 hemodialysis patients];
  • In a randomized double-blind trial of 244 postmenopausal women followed up for 3 years, arterial stiffness as captured by aortic pulse wave velocity or stiffness index β, decreased in intervention compared with control group.

These results should be considered as hypothesis-generating in view of small sample size, and because there were no between-group differences in vitamin K–induced changes in elastic properties of the carotid artery.

Plasma dp-ucMGP levels ranging from 1.4 to 4.6 μg/L were optimal in terms of risk of mortality and macrovascular cardiovascular illness (4.6 μg/L threshold corresponding to the 65th percentile of dp-ucMGP distribution).

Vitamin K supplementation before irreversible organ damage sets in might find its application in prevention of a wide range of disabling diseases. Circulating dp-ucMGP levels might be measured over time to track risk of vascular complications.”

https://www.ahajournals.org/doi/10.1161/HYPERTENSIONAHA.119.12412 “Vitamin K–Dependent Matrix Gla Protein as Multifaceted Protector of Vascular and Tissue Integrity”

I usually don’t give 5+ stars to reviews. This one was different.

Yes, there could be factors other than this one Vitamin K deficiency biomarker involved in study findings. Sure, these coauthors cited their own studies. Its overall purpose, though, was to inform readers.

I’ll summarize this paper as providing evidence for a biomarker of Vitamin K2 deficiency being implicated in the development and progression of many diseases.

Part 2 of Vitamin K2 – What can it do?

gettingwell4 4-5 stars Advanced knowledge, Epigenetics, Stress

Two papers on Vitamin K2, and an online database to continue Part 1:

“Precise quantitative assessments of vitamin K bioavailability in humans is challenging due to unquantified tissue conversion of PK [phylloquinone, Vitamin K1] to MK [menaquinone, Vitamin K2]-4, and contributions of gut microbiota. Absorption of long-chain MKs (MK-7, MK-8 and MK-9) from natto, cheese and egg yolk is close to 100%.

Long-chain MKs have a longer half-life. Long half-life may not necessarily indicate increased bioavailability, but instead non-preferential utilisation by tissues compared to PK and MK-4. A long half-life may also indicate that long-chain MKs may be of particular importance for extrahepatic tissues.

12 databases list vitamin K content of individual food items, which is required to more accurately determine vitamin K intake. The Dutch database is the most comprehensive, and includes PK and several types of MKs, ranging from MK-4 to MK-10.”

https://pubs.rsc.org/en/content/articlelanding/2020/FO/C9FO02321F “Quantifying dietary vitamin K and its link to cardiovascular health: a narrative review” (not freely available)

One online database mentioned is at https://www.rivm.nl/en/dutch-food-composition-database:

“The Dutch Food Composition Database (NEVO) contains data on the composition of foods eaten frequently by a large part of the Dutch population. These foods contribute significantly to the intake of energy and nutrients. Foods of importance for specific groups of the Dutch population are also included.

The NEVO online website contains data on 133 nutrients of 2152 food items. The most recent version of NEVO online dates from November 2019.”

I downloaded a copy in Excel format, selected all, and sorted by column EE “VITK2_mug” in descending order. There were 263 food items with Vitamin K2 measurements.

Vitamin K2 food content

A second paper detailed a 2021 double-blind, placebo-controlled, feasibility study:

“INTRICATE will assess the influence of combined vitamin K2 and vitamin D3 supplementation on micro-calcification in carotid artery disease. Considering recent advancements in medical imaging, ultrasound, PET/MRI, and computed tomography can be used for selection and stratification of patients with atherosclerosis.

Subjects will be randomized (1:1) to a vitamin K2 (400 µg MK-7/day) and vitamin D3 (80 µg [3200 IU]/day) dose or to placebo. Primary endpoint is change in Na[18F]F PET/MRI (baseline vs. after 3 months) in treatment group as compared to placebo arm. Secondary endpoints are changes in plaque composition and in blood-biomarkers.

Studies suggest positive effects of vitamin D on vitamin K-dependent metabolism. The MGP-gene promoter contains a vitamin D response element, capable of a two to threefold enhanced MGP expression after vitamin D binding.

Upregulation of MGP due to vitamin D needs vitamin K to ensure full activation of MGP for optimal functioning. This implies that the combination of both vitamin K and vitamin D could provide enhanced protection against progressive vascular calcification, cardiovascular disease, and mortality.”

https://www.mdpi.com/2072-6643/13/3/994/htm “Effects of Combined Vitamin K2 and Vitamin D3 Supplementation on Na[18F]F PET/MRI in Patients with Carotid Artery Disease: The INTRICATE Rationale and Trial Design”

The second study was somewhat of a tell in that after two decades, researchers are still testing Vitamin K2 dose efficacy. Researchers don’t consider it proper science to not use a statistics package to lump subjects into groups.

Someday researchers will thoroughly analyze each individual, and relate measurements to each individual’s causal and symptomatic characteristics. Then we’ll find out whether what did or didn’t matter to each individual, will or won’t matter to a group.

Until then they’ll focus on one dimension of health like Vitamin K2 foods per their sponsor’s directions. Nevermind that Vitamin K2-rich foods like cheeses are full of advanced glycation end products (AGEs) that humans can’t adequately metabolize, to our detriment.

Vitamin K2 – What can it do?

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A trio of papers on Vitamin K2, the first being a 2021 review that emphasized dual effects:

“Osteoporosis (OP) is the most common bone disease that affects elderly men and women. It is a metabolic skeletal disorder caused by an imbalance between bone formation and resorption, leading to a loss of bone mass and quality, skeletal structure deterioration, and an increased risk of fractures.

Vascular calcification is defined as ectopic deposition of mineral matrix in vessel wall. It occurs prevalently in aging and primary chronic conditions (hypertension, diabetes mellitus, and chronic kidney disease), representing an important risk factor for cardiovascular morbidity and mortality.

Studies have provided support for a close link between bone and vascular health. Findings suggest that bone loss in OP may promote and increase the risk of cardiovascular events and vascular atherosclerosis.

Vitamin K2 is involved in a phenomenon in which a low calcium deposition in bone tends to be associated with a parallel increase of calcium deposition in vessel wall as a consequence of impaired calcium metabolism. Most production of Vitamin K2 in humans takes place in intestines. However, the amount derived from intestinal bacteria is poorly absorbed, and is not able to reach concentrations required to exert physiological functions.

Vitamin K2‘s ability to reduce loss of bone mineral density and fracture risk, as well as to improve bone quality, has been described by several clinical studies, which have confirmed that osteocalcin (OC) γ-carboxylation is the main mechanism of action through which this natural compound is able to improve bone health. Clinical evidence suggests an analogous protective role of Vitamin K2 at the vascular level, emphasizing a strict association between:

  • Vitamin serum level;
  • Matrix gla protein (MGP) γ-carboxylation levels;
  • Reduction of vascular smooth muscle cells osteogenic trans-differentiation; and
  • Possible risk of cardiovascular events.”

https://www.mdpi.com/2072-6643/13/4/1222/htm “The Dual Role of Vitamin K2 in ‘Bone-Vascular Crosstalk’: Opposite Effects on Bone Loss and Vascular Calcification”

A second 2021 review emphasized aging:

“Vitamin K can:

  • Carboxylate OC (a protein capable of transporting and fixing calcium in bone);
  • Activate MGP (an inhibitor of vascular calcification and cardiovascular events); and
  • Carboxylate Gas6 protein (involved in brain physiology and a cognitive decline and neurodegenerative disease inhibitor).

By improving insulin sensitivity, Vitamin K lowers diabetes risk. It also exerts antiproliferative, proapoptotic, autophagic effects, and has been associated with a reduced risk of cancer.

The most common [Vitamin K2] subtypes in humans are the short-chain MK[menaquinone]-4, which is the only MK produced by systemic conversion of phylloquinone [Vitamin K1] to menaquinone, and MK-7 through MK-10, which are synthesized by bacteria. The main sources of Vitamin K2 are fermented foods, cheeses, eggs, and meats.”

https://www.mdpi.com/2076-3921/10/4/566/htm “The Role of Vitamin K in Humans: Implication in Aging and Age-Associated Diseases”

The third paper – somehow not cited by these two reviews – was a 2006 human study that performed four experiments:

“The synthetic short-chain vitamin K1 is commonly used in food supplements, but recently the natural long-chain MK-7 has also become available as an over-the-counter supplement. The purpose of this paper was to compare in healthy volunteers absorption and efficacy of K1 and MK-7.

Serum vitamin K species were used as a marker for absorption and OC carboxylation as a marker for activity. Both K1 and MK-7 were absorbed well, with peak serum concentrations at 4 hours after intake.

A major difference was:

  • Very long half-life time of MK-7, resulting in much more stable serum levels; and
  • Accumulation of MK-7 to higher levels (7- to 8-fold) during prolonged intake.

MK-7 induced more complete carboxylation of OC.

Vitamin K2 vs K1

Accumulation and efficacy of K vitamins during long-term daily administration. Participants received in a crossover design either K1 (○) or MK-7 (•) or placebo; in the latter case only K1 (▴) could be detected.

  • (A) Circulating levels of vitamin K; baseline levels for K1 were subtracted; no MK-7 could be detected at baseline.
  • (B) Ratio between circulating carboxylated and undercarboxylated osteocalcin (cOC/ucOC); at baseline the ratio was 1.74 for MK-7, 1.8 for K1, and 1.7 for the placebo group.

MK-7 accumulated during the first 2 weeks until it reached a plateau level of about 10 nM (6 μg/L), whereas K1 remained slightly above placebo values during the entire study period. Efficacy of both K vitamins for OC carboxylation was monitored using the ratio between circulating cOC and ucOC, and it turned out that within 3 days both vitamins had induced increased cOC.

But only by taking MK-7 did the effect continue to increase during the entire study period.

Taken together, these data demonstrate considerable differences between MK-7 and K1:

  • Higher and more stable serum levels are reached with MK-7; and
  • MK-7 has a higher efficacy in both hepatic and extrahepatic protein carboxylation.”

https://ashpublications.org/blood/article/109/8/3279/23729/Vitamin-K-containing-dietary-supplements “Vitamin K–containing dietary supplements: comparison of synthetic vitamin K1 and natto-derived menaquinone-7″

I’ve tried various things over the years to address hypertension. I stopped high blood pressure medications briefly to see if each intervention worked. They all haven’t, presumably because I didn’t address causes.

More recently, I broke my left big toe on furniture while walking around in the dark last month, and haven’t recovered. No pictures from walking on the beach at sunrise because it still isn’t possible. 😦

A link between these two health conditions could be Vitamin K2. I don’t eat fermented foods because of their high sodium, or dairy products, and haven’t supplemented Vitamin K2.

Next week I’ll start a 300 μg MK-7 daily dose. Current Vitamin D3 dose is 3800 IU, compared to the second paper of Part 2 of Vitamin K2 – What can it do? which is 400 μg MK-7 and 3200 Vitamin D3.

Are overdosed rodent studies relevant to humans?

gettingwell4 < 0 Detracted from science, Stress

I partially read more than a dozen studies this week of overdosed rodents producing p < .05 significant results. Net effect was to thwart the purpose of rodent studies – to help humans.

The latest came from search term “SIRT1” “DHEA” 2021 after I read a 2021 study Dehydroepiandrosterone protects against acetaminophen-induced liver damage in rats by upregulation of Bcl-2 and activation of sirt signalling that found:

“The study examined protective effect of exogenous administration of dehydroepiandrosterone (DHEA) against acetaminophen (APAP) -induced liver damage in rats, and tested underlying mechanisms. DHEA prevents APAP-induced liver damage by concomitant upregulation of Bcl-2 and SIRT1-dependent effect.”

The daily DHEA dose was 50 mg/kg, which is a (.162 x 50 mg) x 70 kg = 567 mg human equivalent. Eleven times the most frequent human dose of Take responsibility for your one precious life – DHEA. Anyone who took this study’s DHEA amount would hurt themself.

The one-time acetaminophen dose was 800 mg/kg which is a (.162 x 800 mg) x 70 kg = 9 grams human equivalent. Someone who took 18 pills of the most frequent 500 mg acetaminophen dose would be attempting suicide.

How does this help humans?

apap dhea overdoses

See posts like Problematic rodent sulforaphane studies and Human relevance of rodent sulforaphane studies for further evidence and observations.

Eat oats and regain cognitive normalcy

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This 2020 rodent study investigated effects of different diets:

“The present study aimed to evaluate effects of β-glucan on the microbiota gut-brain axis and cognitive function in an obese mouse model induced by a high-fat and fiber-deficient diet (HFFD). After long-term supplementation for 15 weeks, β-glucan prevented HFFD-induced cognitive impairment, assessed behaviorally by object location, novel object recognition, and nesting building tests:

  • Long-term β-glucan supplementation suppressed microglia activation and inflammation in hippocampus of HFFD-fed mice;
  • β-glucan attenuated deleterious engulfment of synapses by activation of microglia seen in HFFD mice;
  • β-glucan significantly prevented upregulation of TNF-α, IL-1β, and IL-6 mRNA expression in hippocampus; and
  • A broad-spectrum antibiotic intervention abrogated β-glucan-induced improvement in cognitive function, highlighting the essential role of gut microbiota to mediate cognitive function and behavior.

We found that short-term β-glucan supplementation did not change cognitive behavior in HFFD fed mice. HFFD feeding for 7 days dramatically changed gut microbial profile, with β-glucan-fed mice clustered apart from HFFD-fed mice sample, suggesting:

  • Quick changes in gut microbiota are induced by short-term β-glucan consumption and
  • Possible causality of gut microbiota profile on cognition.

7% β-glucan 7% nondigestible fiber

β-glucan supplementation increased place discrimination ratio in object location test compared with HFFD mice; however, there was no significant difference in total exploration time with objects during test phases between the two groups. Higher place discrimination index in β-glucan supplementation group was not due to better general performance, but increased recognition memory.

Results provide consistent evidence linking increased β-glucan intake to improved:

  • Gut microbiota profile;
  • Intestinal barrier function;
  • Reduced endotoxemia; and
  • Enhanced cognitive function via more optimized synaptic and signaling pathways in critical brain areas.

It is speculative that β-glucan improvement of gut microbiota composition, but not necessarily diversity per se, may be most critical for improved cognition. Enhanced consumption of β-glucan-rich foods is an easily implementable nutritional strategy to attenuate diet-induced cognitive decline.”

https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-00920-y “β-glucan attenuates cognitive impairment via the gut-brain axis in diet-induced obese mice”

This study did well by elaborating It’s the fiber, not the fat and Eat oats to prevent diabetes related findings. How many humans eat themselves into essentially the same situation as this HFFD group with no gut-microbiota-friendly dietary fiber?

Experiments were with β-glucan 1,3/1,4 found in oats. β-glucan 1,3/1,6 has separate effects, especially on innate immunity.

It’s a coin toss on whether observed cognitive improvement was due to 7% β-glucan soluble fiber, 7% indigestible fiber, or both since they were part of the same HFBG diet. I eat both fibers, beginning with Avena nuda oats for breakfast.

Astaxanthin bioavailability

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By request, research on astaxanthin bioavailability. I used a “astaxanthin” “bioavailability” “quinone reductase” 2021 search term, and read citing papers.

“The bioaccessibility, bioavailability, and antioxidative activities of three astaxanthin geometric isomers were investigated using an in vitro digestion model.

  • 13Z-Astaxanthin showed higher bioaccessibility than 9Z- and all-E-astaxanthins during in vitro digestion, and
  • 9Z-astaxanthin exhibited higher transport efficiency than all-E- and 13Z-astaxanthins.

These might explain why 13Z- and 9Z-astaxanthins are found at higher concentrations in human plasma than all-E-astaxanthin.

9Z- and 13Z- astaxanthins exhibited a higher protective effect than all-E-astaxanthin against oxidative stress.”

https://pubs.acs.org/doi/10.1021/acs.jafc.7b04254 “Bioaccessibility, Cellular Uptake, and Transport of Astaxanthin Isomers and their Antioxidative Effects in Human Intestinal Epithelial Caco-2 Cells” (2017, not freely available)

jf1c00087_0005

“Astaxanthin with a high proportion of Z-isomer (especially rich in 9Z- and 13Z-isomers) was prepared from (all-E)-astaxanthin by thermal treatment and solid–liquid separation. Z-isomer-rich astaxanthin diet resulted in higher levels of astaxanthin in blood and many tissues (in particular, skin, lung, prostate, and eye) compared to all-E-isomer-rich diet.

Z-isomer-rich diet enhanced the level of 13Z-isomer in blood and tissues rather than that of 9Z-isomer. (13Z)-astaxanthin would have higher bioavailability and tissue accumulation than other isomers.”

https://pubs.acs.org/doi/10.1021/acs.jafc.1c00087 “Z-Isomers of Astaxanthin Exhibit Greater Bioavailability and Tissue Accumulation Efficiency than the All-E-Isomer” (2021, not freely available)

“Astaxanthin is highly susceptible to light, oxygen, and heat stress degradation. In addition, poor water solubility and bioavailability limit its efficacy in vivo. Investigating novel astaxanthin delivery systems is necessary in order to solve these drawbacks.”

https://www.mdpi.com/1420-3049/24/14/2640/htm “The Neuroprotective Effects of Astaxanthin: Therapeutic Targets and Clinical Perspective” (2019)

“Astaxanthin Z-isomers potentially have greater bioavailability and biological activity than (all-E)-astaxanthin. However, stability of Z-isomers is lower than all-E-isomer, which is a serious problem affecting its practical use.

In this study, we investigated impacts of different suspension media (oils and fats) and additives on astaxanthin isomer stability.

  • Z-isomers of astaxanthin isomerized to all-E-isomer during storage.
  • When soybean and sunflower oils were used as the suspension medium, astaxanthin isomers were hardly degraded. However the total Z-isomer ratio decreased from ~80% to ~50% during 6-week storage at 30 °C.
  • (9Z)-astaxanthin showed higher stability than 13Z- and 15Z-isomers.”

https://www.sciencedirect.com/science/article/abs/pii/S0308814621003770 “Evaluation and improvement of storage stability of astaxanthin isomers in oils and fats” (2021, not freely available)

I looked for but didn’t find a graph similar to this one that comparatively plotted astaxanthin:

OMCL2019-2716870.006

I also didn’t find recent human studies.

It seems that a special delivery system is required for taking astaxanthin as a supplement. It would require investigating manufacturers’ claims about isomer content and stability.

Eating colorful seafood is another way to get astaxanthin. Don’t know about eating raw or dried algae.

Are rodent models of human neurodegenerative diseases realistic?

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This 2020 stem cell review argued against rodent models of human neurodegenerative diseases:

“Neuronal loss is not caused solely by intrinsic degenerative processes but rather via impaired interactions with surrounding glia and other brain cells. Dysfunctional astrocytes do not provide sufficient nutrients and antioxidants to neurons, while dysfunctional microglia cannot efficiently clear pathogens and cell debris from extracellular space, resulting in chronic inflammatory processes in the brain.

Human glia, especially astrocytes, differ significantly in morphology and function from their mouse counterparts. Recent advances in stem cell technology make it possible to reprogram human patients’ somatic cells to induced pluripotent stem cells (iPSC) and differentiate them further into patient‐specific glia and neurons, thus providing a source of human brain cells.

stem3309-fig-0002-m

Astrocytes do not efficiently utilize energy resources and cannot provide adequate metabolic support to neurons. A coculture of healthy human neurons with diseased astrocytes impaired neuronal calcium responses to glutamate and γ‐aminobutyric acid (GABA) as compared to coculture with healthy human astrocytes.

Treatment with sulforaphane:

  • Normalized basal level glycolysis;
  • Decreased basal level Aβ42 secretion; as well as
  • Ameliorated inflammatory response to pro‐inflammatory cytokines TNF-α and IL1-β in PSEN1 mutant iPSC astrocytes.

It is essential to make sure that what we see in the dish is the real patient‐specific phenotype. Transplantation of human brain organoids containing microglia into mice could provide a novel tool for drug screening in vivo.”

https://stemcellsjournals.onlinelibrary.wiley.com/doi/full/10.1002/stem.3309 “Metabolic and immune dysfunction of glia in neurodegenerative disorders: Focus on iPSC models”

This review’s thesis seems plausible. However, one problem with in vitro stem cell studies is that they often don’t have a control group.

Giving children allergies with pets

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This 2021 human study investigated development and persistence of allergies:

“Allergic rhinitis (AR) is a common IgE-mediated disorder involving troublesome symptoms of nasal congestion, nasal itch, sneezing, and associated eye symptoms. Like many chronic health conditions, AR stems from complex gene–environment interactions.

130 subjects with AR were recruited. Control population included 154 healthy children who underwent a regular physical examination in the same ear, nose and throat clinic as AR patients. Individuals with history of asthma or atopic dermatitis were excluded.

AR analysis

Plenty of contradictory associations exist as whether furred pet exposure (cats and dogs) may be a risk or a protective factor for AR development. Discrepancies are likely due to the ubiquitous nature of pet allergens, while pet owners are more concerned about sanitation and many other hygiene-related reasons.

Interaction of early-life pet exposure with methylation level of ADAM33 increased the risk for AR onset 1.423 times more in children. This study provides evidence that:

  • Early-life pet exposure and low methylation level of ADAM33 increase AR risk in children; and
  • The interaction between pet exposure and methylation level of ADAM33 may play an important role in development of AR.”

https://aacijournal.biomedcentral.com/articles/10.1186/s13223-021-00526-5 “Interaction between early-life pet exposure and methylation pattern of ADAM33 on allergic rhinitis among children aged 3–6 years in China”

There’s nothing children can do about who their parents were. Exposing them to pet allergens, though, may be another example of early-life experiences causing lifelong effects.

Happy Mothers Day

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This 2021 rodent study investigated effects on offspring of maternal high-fat diet (HFD) during gestation and lactation, and offspring HFD during young adulthood:

“We found that gestation was the most sensitive period to induce obesity in late life, and there was no difference between sexes in chance of obesity. Furthermore, we found that lactation and administration of a HFD post‐weaning increased incidence of lipid metabolism disorders and obesity in offspring.

gestational hfd effects on offspring

There are different windows of opportunity for programming epigenetically labile genes. Some studies support the alteration of epigenetic status during development as an important cause induced adult obesity.

Gestation is considered as the most sensitive period because high DNA synthesis and DNA methylation patterns are established for normal tissue development during the embryonic period. These two programming events are the times when the epigenetic state changes most widely in the life cycle.”

https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16551 “Gestational high-fat diet impaired demethylation of Pparα and induced obesity of offspring”

Hey mothers! Do what you please. But don’t turn around and deny consequences of your behavior and choices on your descendants’ physiology and behavior, and possibly those of further descendants.

Gestation, birth, infancy, and early childhood are critical periods for humans. There’s no going back to correct errors and problems.

Part 2 of Broccoli sprouts activate the AMPK pathway

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This 2021 review subject was metformin’s role in autophagy:

“Metformin had been used as the first choice for treating diabetes for almost a century. Autophagy is responsible for recycling and degrading cellular components, which significantly affects cell functions in physiology and pathology.

Effects of metformin on autophagy mainly depend on corresponding signaling pathways in specific organs or tissues. Metformin can induce autophagy in cells of many organs and tissues via affirmed signaling pathways, such as AMPK-related signaling pathways.

1-s2.0-S0753332221000718-gr5_lrg

Different signaling pathways (alone or in combination) mediated the process of metformin affecting autophagy in different organs or tissues. It is necessary to combine effects of metformin on autophagy with pharmacological effects on pathologies in different organs or tissues, which would provide indications for future metformin applications.”

https://www.sciencedirect.com/science/article/pii/S0753332221000718 “The effects of metformin on autophagy”

I characterized this review as Part 2 of Broccoli sprouts activate the AMPK pathway because that study’s experimental evidence showed sulforaphane activation of the AMPK pathway was a predecessor to sulforaphane’s main effects of Nrf2 pathway activation. This review didn’t even mention Nrf2 activation.

Do all of metformin’s cited effects apply to daily intake of broccoli sprouts? Probably not, but most people who take metformin every day aren’t healthy.

See Part 3 for updates.

One aspect of research on short-chain fatty acids

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

To further understand An overlooked gut microbiota product, a 2018 rodent study found:

“Microbial metabolites short-chain fatty acids (SCFAs) have been implicated in gastrointestinal functional, neuroimmune regulation, and host metabolism, but their role in stress-induced behavioural and physiological alterations is poorly understood

SCFAs are primarily derived from fermentation of dietary fibres, and play a pivotal role in host gut, metabolic and immune function. All these factors have previously been demonstrated to be adversely affected by stress.

Administration of SCFAs to mice undergoing psychosocial stress alleviated enduring alterations in anhedonia and heightened stress-responsiveness, as well as stress-induced increases in intestinal permeability.

experimental design

SCFA treatment alleviated psychosocial stress-induced alterations in reward-seeking behaviour, and increased responsiveness to an acute stressor and in vivo intestinal permeability. In addition, SCFAs exhibited behavioural test-specific antidepressant and anxiolytic effects, which were not present when mice had also undergone psychosocial stress.”

https://physoc.onlinelibrary.wiley.com/doi/pdf/10.1113/JP276431 “Short-chain fatty acids: microbial metabolites that alleviate stress-induced brain–gut axis alterations”

One way researchers advance science is to relate aspects of their findings to previous studies. That approach works, but may miss items that weren’t covered in previous research.

This study fed specific quantities of three SCFAs – acetate, butyrate, and propionate – apparently due to previous research findings. If other SCFAs produced by gut microbiota were ignored – like crotonate (aka unsaturated butyrate) – how would that approach advance science?

I found this study from its citation in Harnessing endogenous defenses with broccoli sprouts.

An overlooked gut microbiota product

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This 2021 review subject was histone crotonylation:

“Histone crotonylation is a newly identified epigenetic modification that has a pronounced ability to regulate gene expression. It belongs to an expanding group of short chain lysine acylations that also includes the extensively studied mark histone acetylation.

Histone Kcr was first identified in 2011 where it was found to be mainly associated with active chromatin. Kcr occurs on the ε-amino group of the lysine side chain, where it neutralizes the positive charge of this residue. The loss in positive charge on histone Lys residues weakens DNA interaction, thus making chromatin less compact and accessible to DNA-binding factors.

Crotonate, like other short chain fatty acids (SCFAs), is mainly produced by gut microbiota during fermentation of partially and nondigestible carbohydrates. Circulating SCFAs (acetate, crotonate, butyrate, and propionate) can be taken up by tissues and converted into their cognate short-chain acyl-CoAs, the direct donors of histone Lys acylations.

fcell-09-624914-g001

Crotonyl-CoA is generated as a by-product of fatty acid and amino acid metabolism. Synthesis of crotonyl-CoA can occur in mitochondria or the cytoplasm. Evidence suggests that histone acylations are directly sensitive to changes in concentrations of their corresponding acyl-CoA metabolites, and therefore can act as indicators of cellular metabolic state.

Only a small number of Kcr sites in human histones have been identified so far. This is in part due to a lack of commercially available Kcr site-specific antibodies, which has meant much of the research in this field has focused on studying total histone crotonylation. This is likely to limit our understanding of the importance of histone Kcr, as functional impact of modification at specific sites cannot be readily assessed.”

https://www.frontiersin.org/articles/10.3389/fcell.2021.624914/full “The Regulation and Function of Histone Crotonylation”

At first I thought I had missed recent studies of gut microbiota producing crotonate. Searching again for “crotonate” “microbiota” 2020 2021, I didn’t find any that weren’t cited by this paper.

A lack of research could be due to factors mentioned above. It may also be that researchers just don’t look for evidence of the circulating SCFA crotonate.

Effects of another broccoli sprout compound

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This 2020 rodent study investigated effects of broccoli sprout hydrolysis compound indole-3-carbinol:

“I3C metabolites act as ligands of the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals. We investigated how dietary AhR ligand supplementation influences AhR target gene expression and intestinal microbiota composition.

Environmental signals, such as dietary, microbial, or xenobiotic factors, are sensed in intestinal tissue AhR, an important regulator of metabolism. It influences immune cell homeostasis and immune activation in the intestine.

AhR activation plays an important role in intestinal immunity, contributing to intestinal homeostasis, inflammation, and host defense:

  • AhR activation through high affinity AhR ligands has been shown to stimulate production of antimicrobial peptides.
  • AhR has been shown to be an important regulator of T cell immunity.

This indicates a major role of AhR in resolving intestinal inflammation.

High fat diet and control diet lead to reduced expression of Ahrr in intestinal immune cells.

High fat diet and control diet lead to reduced expression of Ahrr in intestinal immune cells.

Mucosal surface area of the gut represents an enormous area in direct contact with the environment. In addition to occasional pathogen encounters, the intestinal immune system is constantly exposed to antigens from diet or microbiota.

Gut-associated immune cells maintain a balance between protection against harmful infections and tolerating harmless food-derived antigens and commensals.

Our findings are in agreement with reports that dietary I3C supplementation restored AhR activation in intestinal mucosa under conditions of malnutrition and deprivation of natural AhR ligands. In humans, such malnutrition may result from a severely reduced consumption of vegetables and fruit in favor of a carbohydrate rich, high fat diet.”

https://www.mdpi.com/1422-0067/21/9/3189/htm “Dietary AhR Ligands Regulate AhRR Expression in Intestinal Immune Cells and Intestinal Microbiota Composition”

Our gut microbiota outnumber our human cells. Treat them well with broccoli sprout compounds, resistant starch, and fermentable fibers, and expect reciprocity.

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

gettingwell4 5+ stars Premium, Cerebrum, Epigenetics, Hippocampus, Immune system, Limbic system, Memory, Stress , , ,

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

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

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

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

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

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

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

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

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

stress response inhibitor effects

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

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

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

Advice from the song depends on your definition of money:

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

Improving healthy compounds of broccoli sprouts

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

This 2020 study investigated known and experimental effects on sprouted broccoli, white mustard, red radish, and red cabbage compounds:

“We planned development of cruciferous sprouts in hydroponics elicited with LED lighting and Methyl-Jasmonate (MeJA) to bio-stimulate production of glucosinolates, comparing effects of two types of LEDs designed for indoor food production systems.

We aimed to gain knowledge on response (germination rate, biomass yield) and phytochemical composition of fresh edible sprouts of cruciferous varieties (broccoli, radish, cabbage and mustard) under these conditions for future food production recommendations:

  • Use of LED lights to grow edible cruciferous sprouts was positive in terms of biomass production and phytochemical content (glucosinolates) without any negative effects.
  • Use of MeJA was positive, confirming previous results. Intensity of response for different species is useful to focus production of sprouts for specific purposes.

3-day old sprouts were placed in a growth chamber with controlled conditions (Photoperiod 18/6 h; temperature 24/18 °C; and relative humidity 60/80%), irrigated every other day to maintain enough humidity in substrate, using 1% bleach in distilled water, and collected on day 7. Trays of germinating seeds were evenly sprayed daily with 10 mL of solution for 4 days.

4 sprouts glusosinolates affected by LED and MeJa

Total Glucosinolates (mg/100 g fresh weight) of White Mustard, Broccoli, Red Cabbage and Red Radish sprouts, under two different LED lightings, and elicited with MeJA (250 μM).

Combining MeJA spraying with different LED light treatment showed clear increases in total glucosinolate contents for all studied sprouts when sprayed for 4 days with MeJA 250 μM.”

https://www.mdpi.com/2504-3900/70/1/67/htm “The Quality and Glucosinolate Composition of Cruciferous Sprouts under Elicitor Treatments Using MeJA and LED Lights”

The research group of Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts keep ramping it up. They’ve published studies of MeJA effects and LED effects on sprouts separately, but not combined like this one did.

I ordered a pound of red cabbage seeds to see how I like their 3-day-old sprouts. I started soaking mustard seeds purchased from a grocery store’s spice section last year to see if they’ll sprout.

Although effects in the above graphic are compelling, I don’t want to turn my kitchen into a laboratory with LED lights and MeJA treatments. I’ll first see if red cabbage and mustard sprouts are tolerable.

See Broccoli sprouts’ immune effects and Week 56 of Changing to a youthful phenotype with sprouts to follow on.