Blinded by their paradigm?

This 2022 human study investigated another type of aging clock:

“The glycan clock of age, based entirely on immunoglobulin G (IgG) N-glycans, can predict biological age with high accuracy. Unlike DNA methylation, glycosylation of IgG does not predict chronological age with high accuracy.

Heritability analysis of plasma glycans revealed that the majority of traits have high heritability estimates, indicating a tight genetic control of glycosylation. To better understand genetic and environmental factors influencing glycan clock variation, we performed a heritability analysis on data from two cohorts included in the TwinsUK registry.

Glycosylation is a series of enzymatic reactions in which carbohydrates are attached to other molecules (e.g., proteins or lipids) resulting in formation of complex carbohydrates and glycoconjugates commonly referred to as ‘glycans.’ Glycosylation of IgG antibody is especially interesting as it dramatically affects its function, and acts as a molecular switch between pro- and anti-inflammatory immune responses.

Heritability of the glycan clock was estimated to decompose observed phenotypic variance into three latent sources of variation:

  • A—additive genetic variance [red] represents cumulative impact of genes;
  • C—shared/common environment variance [purple] results from influences to which both members of a twin pair are exposed; and
  • E—unique environment variance [green] is events occurring to one twin but not the other, and includes measurement error.

fcell-10-982609-g002

Despite tight genetic control of the IgG glycome:

  • Heritability analysis of the glycan clock revealed only a moderate genetic contribution averaging around 39% [A, left side].
  • Including age of the individuals as a covariate in heritability analysis averaged 71% heritability estimates [B, right side].
  • Mean time difference was 7.5 years for points 1 and 2, and 6 years for points 2 and 3.

Observed increase in the genetic component could be a consequence of chronological age as a shared environmental variance characteristic for every individual and determined by their genetic makeup and epigenetic regulation.”

https://www.frontiersin.org/articles/10.3389/fcell.2022.982609/full “Heritability of the glycan clock of biological age”


Although A rejuvenation therapy and sulforaphane was cited, these researchers missed its central premise: Pro-aging epigenetic programming is directional and not purely random. Contrasting their above graphic’s heritability estimates of 39% with the age-regressed, right side’s average 71% could hardly have been more clear in illustrating this fact.

This study instead stated “Aging in general leads to epigenetic mediated deregulation of genes.” This weak sauce accompanied speculations such as “supports the notion that the glycan clock can be rejuvenated by simple lifestyle choices.”

Researchers almost always want to claim being first in finding x, y, or z. These researchers could have done that in this glycan clock study by highlighting an outstanding finding. So what happened?

An alternate explanation to their paradigm blinding them could be sponsor expectations, peer pressures, etc. I’ll ask them about it, and will update here with their response.


PXL_20221231_185407365

Do broccoli sprouts treat gout and kidney stones?

This 2022 rodent study investigated glucoraphanin’s effects on reducing uric acid:

“Hyperuricemia is a chronic disease characterized by abnormally elevated serum uric acid levels. Sulforaphane could lower uric acid by decreasing urate synthesis and increasing renal urate excretion in hyperuricemic rats.

A hyperuricemia model was established by administering feedstuffs with 4% potassium oxonate and 20% yeast. Forty male Sprague–Dawley rats were randomly divided into the normal control, hyperuricemia, allopurinol, and sulforaphane groups. Animals were treated by oral gavage for six consecutive weeks, and then phenotypic parameters, metabolomic profiling, and metagenomic sequencing were performed.

1-s2.0-S209012322200251X-ga1_lrg

We identified succinic acid and oxoglutaric acid as critical host-gut microbiome co-metabolites. Sulforaphane improved diversity of microbial ecosystems and functions, as well as metabolic control of the kidney. Sulforaphane exerted its renoprotective effect through epigenetic modification of Nrf2 and interaction between gut microbiota and epigenetic modification in hyperuricemic rats.

Limitations of this study include:

  1. We used glucoraphanin bioactivated with myrosinase for our experiments. Future experiments may directly involve sulforaphane.
  2. Bioinformatics analysis resulted in speculations that require further experimental testing.
  3. Further investigation of interactions between microbiota and the host epigenome is still needed.”

https://www.sciencedirect.com/science/article/pii/S209012322200251X “Sulforaphane-driven reprogramming of gut microbiome and metabolome ameliorates the progression of hyperuricemia”


It was a stretch to label treatment subjects as the “sulforaphane group” by claiming “Glucoraphanin (10 mg/kg) was metabolized to SFN by myrosinase as described in previous studies.” Both this and the referenced 2014 study “(RS)-glucoraphanin purified from Tuscan black kale and bioactivated with myrosinase enzyme protects against cerebral ischemia/reperfusion injury in rats” measured glucoraphanin and myrosinase, but not sulforaphane.

A human equivalent to this study’s daily glucoraphanin intake of 10 mg / kg weight would be (.162 x 10 mg) x 70 kg = 113 mg. Whether 10 mg was dry or wet weight wasn’t disclosed.

If 10 mg was wet, 113 mg is a little more than twice our model clinical trial’s average glucoraphanin intake of 51 mg fresh weight from eating 30 grams / day of super sprouts. In April 2020’s Understanding a clinical trial’s broccoli sprout amount, a study coauthor said:

“We considered 30 g and 60 g to be 1/2 and 1 portion per day, respectively, of broccoli sprouts. When we carried out tests with consumers, previous to the bioavailability studies, higher amounts per day were not easy to consume and to get eaten by participants.”

PXL_20221112_201430280

Eat broccoli sprouts to combat effects of BPA?

This 2022 rodent study investigated abilities of both glucoraphanin and sulforaphane to reduce bisphenol A’s effects:

“There are only a few studies on the anti-obesogenic activity of sulforaphane (SFN) in bisphenol A (BPA)-induced obese C57BL/6J mice and 3T3-L1 cells. BPA is one of the endocrine disrupting chemicals that mimics bioidentical hormones and acts as an active agonist of glucocorticoid receptors to promote adipogenesis.

We investigated anti-obesogenic effects of broccoli sprouts powder (BSP) with a high glucoraphanin (GRA) content, mustard (Sinapis alba L.) seed powder (MSP) that has a high myrosinase activity, and sulforaphane-rich MSP-BSP mixture powder (MBP).

  • GRA content in BSP was 131.11 ± 1.84 μmol/g, and SFN was not detected.
  • SFN content in MBP was 162.29 ± 1.24 μmol/g, and GRA was not detected.
  • GRA and SFN were not detected in MSP.

Mice were administered:

  • BPA (500 μg/kg/day);
  • BPA supplemented with 100 mg/kg/day Gar (BPA + Gar);
  • 15 mg/kg/day MSP (BPA + MSP);
  • 150 mg/kg/day BSP (BPA + BSP); or
  • 100 mg/kg/day MBP (BPA + MBP)

for 12 weeks. The BPA + Gar group served as the positive control group, since studies showed that Garcinia cambogia extract induces weight loss.

bpa weight gain

Mice in the BPA group showed a significantly high body weight and epididymal adipose tissue weight, compared to the ND group mice. MSP treatment had no significant effects. Gar, BSP, and MBP treatment significantly decreased body weight and epididymal adipose tissue weight in BPA-induced obese mice.

BSP and MBP exert anti-obesogenic effects by activating the AMPK signaling pathway. Our results suggest that BSP and MBP could be effective in the treatment and prevention of BPA-induced obesity.”

https://www.mdpi.com/2072-6643/14/18/3814/htm “Anti-Obesogenic Effects of Sulforaphane-Rich Broccoli (Brassica oleracea var. italica) Sprouts and Myrosinase-Rich Mustard (Sinapis alba L.) Seeds In Vitro and In Vivo”


Human daily equivalent doses:

  • Sulforaphane was (.081 * 100 mg) x 70 kg = 567 mg, or (.567 g * 162.29 μmol/g) = 92 μmol. The μmol amount is reasonable, but the mg weight would be intolerable. I’ve contacted these researchers for clarification, and will update with their response.
  • Glucoraphanin in broccoli sprout powder at (.081 * 150 mg) x 70 kg = 851 mg looks reasonable. Broccoli sprout powder vendors recommend 1 gram.

PXL_20221002_203139302_exported_8128

Broccoli sprout compounds at different growth stages

This 2022 study investigated 12 glucosinolate compounds in 9 broccoli cultivars across seeds, 3-, 11-, and 17-day-old sprouts:

“Broccoli is rich in glucosinolates (GLs) which makes it an excellent source of these nutraceuticals. Composition and concentration of GLs vary among broccoli cultivars and throughout developmental stages of the plant.

9 aliphatic GLs and 3 indole GLs were identified from 9 broccoli cultivars. Aliphatic GLs concentrations decreased with broccoli sprouts and seedling growth for most cultivars. Indole GLs amounts increased after germination and reached the highest level in Stage B 3-day sprouts or C 11-day seedlings, and fell back to a low level in D 17-day seedlings.

Stage B was a stage that sprouts grew with no lights in medium and were about to be transplanted into pots. Stage C was a period that seedlings began to grow a main leaf, while in Stage D they were growing the second main leaf.

stages

Relatively high accumulation of glucoraphanin and glucoerucin in Chunqiujiali seeds suggests that CQJL broccoli seeds could be used for extraction of beneficial aliphatic GLs in nutraceutical industry.”

https://www.mdpi.com/2223-7747/11/12/1563/htm “Variation in Glucosinolate Accumulation among Different Sprout and Seedling Stages of Broccoli (Brassica oleracea var. italica)”


These researchers are probably early in their careers. They may have otherwise measured broccoli sprout compounds like glucosinolate-hydrosolate isothiocyanates and others, as did the cited 3-day-old broccoli sprouts have the optimal yields. As those researchers said:

“From the perspective of comparison methods, broccoli varieties, and germination processes, there is still lack of a systematic comparison of SF yields and other bioactive compounds contents between broccoli seeds and sprouts.”

Glucoraphanin is not sulforaphane highlighted one pitfall in concluding “CQJL broccoli seeds could be used for extraction of beneficial aliphatic GLs in nutraceutical industry.” Selecting broccoli varieties highlighted another, and provided an example of how human-applicable broccoli sprout compound research across different varieties could be done:

“We found a clear difference in selecting functional broccoli by considering only the GSL content or hydrolysates.

  • Even if total GSL content and individual GSL content were high, ITC content could not be produced at a high level.
  • When GSL content is high, if nitrile formation rate was also high, more nitrile than ITC would be produced.”

Preteen practicing handstands 15 minutes before sunrise

PXL_20220924_104001656

Variable aging measurements

Two papers on aging measurements, starting with a 2022 human study:

“We collected longitudinally across the adult age range a comprehensive list of phenotypes within four domains (body composition, energetics, homeostatic mechanisms and neurodegeneration / neuroplasticity) and functional outcomes. We integrated individual deviations from population trajectories into a global longitudinal phenotypic metric of aging.

blsa participant ages

We demonstrate that accelerated longitudinal phenotypic aging is associated with faster physical and cognitive decline, faster accumulation of multimorbidity, and shorter survival.”

https://www.nature.com/articles/s43587-022-00243-7 “Longitudinal phenotypic aging metrics in the Baltimore Longitudinal Study of Aging”


I disagree with this study’s methodology.

1. Although it acknowledged individual variability, nothing was done to positively adjust to those facts. What could have been done per A review of biological variability was:

“Obtain a measurement of variability that is independent of the mean to ensure to not confound changes in variability with shifts in mean.”

2. A usual research practice is to take at least three measurements, and use their average as representative. That wasn’t done here, maybe because of time and expense considerations?

3. An important measurement for physical function was the time to finish a 400 meter walk. I walk more than ten times that almost every day. I use the first 400 meters as a warmup period while getting to the beach to walk eastward and enjoy the predawn light and water animal activity. I concentrate on gait speed during the last third while walking westward on a straightaway bike path.

This study would measure my gait speed as a sometimes old person during the first 400 meters, rather than a gait speed that usually approaches a young person’s during the last 400 meters. Even if I tried to walk my fastest right out of the gate, I wouldn’t be surprised to find a decade or two difference by this study’s measurements between a morning walk’s first and last 400 meter gait speeds.

4. An important cognitive function measurement was the Digital Symbol Substitution Test, apparently taken during subjects’ fasted state? Sometimes after exercising, I’m okay cognitively when starting work in a fasted state at 6:30 a.m., and other times I’m tired.

Two days ago during the last hour of work 1:30-2:30 p.m., I did outstanding work, four hours after eating whole oats for breakfast, and after drinking two coffees and three teas. I took time to put together pieces of puzzles into proper contexts for management’s attention. My bosses weren’t too pleased with the story it told, but it is what it is.

5. Are measurements of how you start what matters? Or is it how you finish, as is common in competitive sports?

This study would measure my cognitive function as a sometimes old person, rather than performance that approaches a young person’s later in the workday. For both physical and cognitive function, my abilities to ramp up and come close to young people’s capabilities are features that I work on, not random, inconvenient measurement variability.

6. Blood measurements were downgraded as having “limited coverage of the four phenotypic domains.” These were taken to fit into specific paradigms and epigenetic clocks. They predictably failed to show causality, as acknowledged with:

“Our analysis showed strong associations between global longitudinal phenotypic score and changes in physical and cognitive function. We did not have sufficient observations to fully separate these two dimensions over time, which would have strengthened the assumption of causality.”

Nowhere in this study was it hinted that all measurements were downstream effects of unmeasured causes. A follow-on study could reanalyze these subjects’ blood samples, MRI, and other measurements for originating upstream factors of signaling pathways and cascades per Signaling pathways and aging and An environmental signaling paradigm of aging.


Reference 35 of this first study was a 2021 human and rodent study that was tossed in as a limitation with:

“We might not have all of the relevant phenotypic measures (for example, more detailed immune profiles) for all participants.”

Its findings included:

“From the blood immunome of 1,001 individuals aged 8–96 years, we developed a deep-learning method based on patterns of systemic age-related inflammation. The resulting inflammatory clock of aging (iAge) tracked with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians.

Canonical markers of acute infection such as IL-6 and tumor necrosis factor-α were not major contributors to iAge, indicating that, except for IL-1β, infection-driven inflammatory markers of the acute inflammatory response do not contribute to age-related chronic inflammation.

We conducted a follow-up study in an independent cohort of 97 extremely healthy adults (aged 25–90 years) matched for cardiovascular risk factors (including conserved levels of high-sensitivity C-reactive protein), selected from a total of 151 recruited participants using strict selection criteria. In this healthy cohort, inflammation markers were measured using a 48-plex cytokine panel. Only 6 circulating immune proteins were significantly correlated with age, with CXCL9 again the largest contributor to age-related inflammation.

CXCL9 is a T-cell chemoattractant induced by IFN-γ and is mostly produced by neutrophils, macrophages and endothelial cells (ECs). We find that CXCL9 is mainly produced by aged endothelium and predicts subclinical levels of cardiovascular aging in nominally healthy individuals.

We did not find any significant correlation between known disease risk factors reported in the study (BMI, smoking, dyslipidemia) and levels of CXCL9 gene or protein expression. We hypothesize that one root cause of CXCL9 overproduction is cellular aging per se, which can trigger metabolic dysfunction.

As ECs but not cardiomyocytes expressed the CXCL9 receptor, CXCR3, we hypothesize that this chemokine acts both in a paracrine fashion (when it is produced by macrophages to attract T cells to the site of injury) and in an autocrine fashion (when it is produced by the endothelium) creating a positive feedback loop. In this model, increasing doses of CXCL9 and expression of its receptor in these cells leads to cumulative deterioration of endothelial function in aging.

IFN-γ did not increase in expression in our cellular aging RNA-seq experiment, suggesting that there are triggers of CXCL9 (other than IFN-γ) that play a role in cellular senescence in the endothelium that are currently unknown. However, in our 1KIP study, IFN-γ was in fact the second-most important negative contributor to iAge, which could be explained by the cell-priming effect of cytokines, where the effect of a first cytokine alters the response to a different one.

iAge derived from immunological cytokines gives us an insight into the salient cytokines that are related to aging and disease. A notable difference compared to other clocks is that iAge is clearly actionable as shown by our experiments in CXCL9 where we can reverse aging phenotypes. More practical approaches range from altering a person’s exposomes (lifestyle) and/or the use of interventions to target CXCL9 and other biomarkers described here.

Our immune metric for human health can identify within healthy older adults with no clinical or laboratory evidence of cardiovascular disease, those at risk for early cardiovascular aging. We demonstrate that CXCL9 is a master regulator of vascular function and cellular senescence, which indicates that therapies targeting CXCL9 could be used to prevent age-related deterioration of the vascular system and other physiological systems as well.”

https://www.nature.com/articles/s43587-021-00082-y “An inflammatory aging clock (iAge) based on deep learning tracks multimorbidity, immunosenescence, frailty and cardiovascular aging”


PXL_20220721_093128925.NIGHT

Blanching broccoli sprouts

Three 2022 papers of interest cited Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease. Let’s start with a fairly straightforward analysis of blanching broccoli sprouts to produce sulforaphane:

“We investigated the effect of blanching conditions to determine the optimal treatment that maximizes sulforaphane (SFN) content in broccoli sprouts. Broccoli seeds grown under controlled conditions were harvested after 11 days from germination and subjected to different blanching conditions based on a central composite design with temperature and time as experimental factors.

Optimum conditions were blanching at 61 °C for 4.8 min, resulting in 54.3 ± 0.20 µmol SFN/g dry weight, representing a 3.3-fold increase with respect to untreated sprouts. This is the highest SFN content reported for sprouts subjected to any treatment so far.

sfn heat response curve

Broccoli sprouts (20 g) were put in plastic bags, which were vacuum-sealed, and then subjected to time (3.4–11 min)–temperature (32–88 °C) combinations.

  • Blanching at 60 °C for less than 8 min resulted in the highest SFN content.
  • Above this temperature, SFN content decreases.
  • The exceptionally high values obtained in this work may be related to treatment, but also to broccoli cultivar and culture conditions.

Different broccoli tissues and developmental stages express different myrosinase isoforms, and catalytic properties of the enzyme may vary among different tissues. Myrosinase found in broccoli florets has an optimal temperature of around 40 °C, and considering myrosinases from other sources, this temperature may vary between 30 and 70 °C.”

https://www.mdpi.com/2304-8158/11/13/1906/htm “Maximization of Sulforaphane Content in Broccoli Sprouts by Blanching”

This first study used heat-only techniques similar to the uncited Enhancing sulforaphane content. It similarly found a 60°C (140°F) myrosinase cliff as have many other uncited studies.


A second paper was a rodent study:

“We investigated the role of sulforaphane, a well-known NRF2 activator, on age-related mitochondrial and kidney dysfunction. Young (2–4 month) and aged (20–24 month) male Fischer 344 rats were treated with sulforaphane (15 mg/kg body wt/day) in drinking water for four weeks.

Sulforaphane significantly improved mitochondrial function and ameliorated kidney injury by increasing cortical NRF2 expression and activity and decreasing protein expression of KEAP1, a NRF2 repressor. Sulforaphane treatment did not affect renal NRF2 expression or activity and mitochondrial function in young rats.”

https://www.mdpi.com/2076-3921/11/1/156/htm “Age-Related Mitochondrial Impairment and Renal Injury Is Ameliorated by Sulforaphane via Activation of Transcription Factor NRF2”

A human equivalent to this second study’s daily dose was intolerable at (.162 x 15 mg) x 70 kg = 170 mg. I curated this study anyway just to show an example of negligible treatment effects in young animals even when a dose is too high for humans.


A third paper was a review that focused on sulforaphane and its analogs’ chemistry:

“Analysis of the Web of Science database shows that, since 1992, about 3,890 articles have been published on SFN, and over 5,600 on isothiocyanates. Its natural analogs include iberin, alyssin, iberverin, erucin, berteroin, cheirolin, and erysolin.

SFN is a biologically active, natural isothiocyanate found in cruciferous vegetables, and is non-toxic. It has been selected for phase I and II clinical trials, where it is administered in the form of an extract or broccoli sprouts. There are no differences in biological activity between SFN and its natural analogs, such as erucin or alyssin.

No synthetic analogs of SFN described in this review qualified for clinical trials. This is likely due to the toxicity of these compounds in higher doses.”

https://www.mdpi.com/1420-3049/27/5/1750/htm “Sulforaphane and Its Bifunctional Analogs: Synthesis and Biological Activity”


PXL_20220712_100018566

Exercise substitutes?

Two papers, starting with a 2022 abstract of an ongoing in vitro study with rodent cells:

“Exercise mimetics may target and activate the same mechanisms that are upregulated with exercise administration alone. This is particularly useful under conditions where contractile activity is compromised due to muscle disuse, disease, or aging.

Sulforaphane and Urolithin A represent our preliminary candidates for antioxidation and mitophagy, respectively, for maintaining mitochondrial turnover and homeostasis. Preliminary results suggest that these agents may be suitable candidates as exercise mimetics, and set the stage for an examination of synergistic effects.”

https://faseb.onlinelibrary.wiley.com/doi/10.1096/fasebj.2022.36.S1.R3745 “Exercise mimicry: Characterization of nutraceutical agents that may contribute to mitochondrial homeostasis in skeletal muscle” (study not available)


A second 2022 paper reviewed what’s known todate regarding urolithins:

“Urolithins (Uros) are metabolites produced by gut microbiota from the polyphenols ellagitannins (ETs) and ellagic acid (EA). ETs are one of the main groups of hydrolyzable tannins. They can occur in different plant foods, including pomegranates, berries (strawberries, raspberries, blackberries, etc.), walnuts, many tropical fruits, medicinal plants, and herbal teas, including green and black teas.

Bioavailability of ETs and EA is very low. Absorption of these metabolites could be increased by co-ingestion with dietary fructooligosaccharides (FOS).

Effects of other experimental factors: post-intake time, duration of administration, diet type (standard and high-fat), and ET dosage (without, low, and high ET intake) in ETs metabolism were evaluated in blood serum and urine of rats consuming strawberry phenolics. Highest concentrations were obtained after 2–4 days of administration.

Various crucial issues need further research despite significant evolution of urolithin research. Overall, whether in vivo biological activity endorsed to Uros is due to each specific metabolite and(or) physiological circulating mixture of metabolites and(or) gut microbial ecology associated with their production is still poorly understood.

  • Ability of Uros to cross the blood-brain barrier and the nature of metabolites and concentrations reached in brain tissues need to be clarified.
  • Specific in vivo activity for each free and conjugated Uro metabolite is unknown. Studies on different Uro metabolites and their phase-II conjugates are needed to understand their role in human health.
  • Evidence on safety and impact of Uros on human health is still scarce and only partially available for Uro-A.
  • It is unknown whether there are potential common links between gut microbial ecologies of the two unambiguously described metabotypes so far, i.e., equol (isoflavones) and Uros (ellagitannins).
  • Gut microbes responsible for producing different Uros still need to be better identified and characterized, and biochemical pathways and enzymes involved.”

https://onlinelibrary.wiley.com/doi/10.1002/mnfr.202101019 “Urolithins: a Comprehensive Update on their Metabolism, Bioactivity, and Associated Gut Microbiota”


PXL_20220514_181229426

Vitamin D and pain

This 2022 human study investigated epigenetic clock associations:

“We assessed the potential relationship of Vitamin D’s effects on pain intensity and disability through associations in epigenetic aging in individuals with and without knee osteoarthritis (KOA). We hypothesized that associations between Vitamin D levels with pain intensity and interference in persons with KOA would be significantly mediated by epigenetic aging.

As a whole, the sample had a mean Vitamin D serum level of 26.7 ng/mL (± 12.8 ng/mL). The mean AgeAccelGrim was 2.4 years (± 5.6 years). There were no significant differences in Vitamin D levels between sex, race, and study site categories.

There was a significant difference in Vitamin D levels between the pain groups, with individuals in the High Impact Pain group showing significantly lower mean levels of Vitamin D (24.01 ng/mL) compared to the Low Impact Pain (28.30 ng/mL) and No Pain (27.30 ng/mL) groups.

vitamin d and pain

Data from this study highlight the important role that Vitamin D plays within the genomic environment, as well as in relation to health outcomes including pain intensity and disability.”

https://link.springer.com/article/10.1007/s12603-022-1758-z “Accelerated Epigenetic Aging Mediates the Association between Vitamin D Levels and Knee Pain in Community-Dwelling Individuals” (not freely available)


It’s good to see a study relating biological age to nutrition status. I didn’t see much discussion of other obvious factors involved in either pain or biological age in their limitations paragraph.

Subjects’ Vitamin D 26.7 ng/mL ± 12.8 ng/mL status indicated that most didn’t spend a few cents every day for their own one precious life. And Vitamin D supplementation wasn’t an exclusion criterion.

The local fire and rescue squad came last Friday to take away a younger neighbor’s body who died overnight. Last I talked with them, they were at least 50 pounds overweight and never exercised. Expressed condolences to their spouse, but wasn’t shocked.

I don’t live in a community-dwelling situation (old people who live on their own as opposed to those taken care of in nursing homes) like this study’s subjects. My youngest neighbors are in their twenties.

Nature hasn’t cared about our lives after our early teens, because we survived long enough to reproduce. What happens in our lives after puberty is largely up to each individual.

PXL_20220502_215338364

Gut microbiota knowledge through 2021

I’ll curate this 2022 review of what’s known and unknown about our trillions of gut microbiota through its topic headings:

“Most microbial taxa and species of the human microbiome are still unknown. Without revealing the identity of these microbes as a first step, we cannot appreciate their role in human health and diseases.

A. Understanding the Microbiome Composition and Factors That Shape Its Diversity
Effect of Diet Composition on the Microbiome Diversity

  • Macronutrients and Microbiome Diversity
  • Nutrient and Mineral Supplements and Microbiome Diversity

Stress

Drugs

Race and Host Genetics

Aging

Lifestyle

  • Exercise
  • Smoking
  • Urbanization

B. Understanding the Microbiome Function and Its Association With Onset and Progression of Many Diseases

Microbiome Association With Inflammatory and Metabolic Disorders

  • Chronic Inflammation in GIT and Beyond
  • Development of Malignant Tumors
  • Obesity
  • Coronary Artery Disease
  • Respiratory Diseases

Microbiome Role in Psychiatric, Behavioral, and Emotional Disorders

C. Understanding the Microbiome Function as Mediated by Secreted Molecules

D. Conclusion and Future Directions – A pioneering study aimed to computationally predict functions of microbes on earth estimates the presence of 35.5 million functions in bacteria of which only 0.02% are known. Our knowledge of its functions and how they mediate health and diseases is preliminary.”

https://www.frontiersin.org/articles/10.3389/fmicb.2022.825338 “Recent Advances in Understanding the Structure and Function of the Human Microbiome”


I took another test last month at the 14-month point of treating my gut microbiota better. Compared with the 7-month top level measurements, what stood out was an increase in relative abundance from 1% to 7% in the Verrucomicrophia phylum that pretty much exclusively comprises species Akkermansia muciniphilia in humans:

top 5 phylum 2-2022

This review termed Akkermansia muciniphilia relative increases as beneficial. Go with the Alzheimer’s Disease evidence didn’t.

Preventing human infections with dietary fibers inferred that insufficient dietary fiber may disproportionately increase abundance of this species. But I already eat much more fiber than our human ancestors’ estimated 100 grams of fiber every day, so lack of fiber definitely didn’t cause this relative increase.

Resistant starch therapy observed:

“Relative abundances of smaller keystone communities (e.g. primary degraders) may increase, but appear to decrease simply because cross-feeders increase in relative abundance to a greater extent.”

I’ll wait for further evidence while taking responsibility for my own one precious life.

Didn’t agree with this review’s statements regarding microbial associations with fear. These reviewers framed such associations as if gut microbiota in the present had stronger influences on an individual’s fear responses than did any of the individual’s earlier experiences. No way.

I came across this review by it citing The microbiome: An emerging key player in aging and longevity, which was Reference 25 of Dr. Paul Clayton’s blog post What are You Thinking?

Also didn’t agree with some of the doctor’s post:

  • Heterochronic parabiosis of young and old animals is wildly different from fecal transfer. Can’t really compare them to any level of detail.
  • Using a rodent young-to-old fecal microbiota transplant study to imply the same effects would happen in humans? Humans don’t live in controlled environments, so why would a young human individual’s gut microbiota necessarily have healthier effects than an old individual’s?
  • Another example was the penultimate paragraph: “By adding a mix of prebiotic fibers to your diet and maintaining a more youthful and less inflammatory microbiome you will have less inflammation, less endotoxaemia and less inflammageing. You will therefore live healthier and longer.” I’m okay with the first sentence. Equivalating the first sentence to both healthspan and lifespan increases in the second sentence wasn’t supported by any of the 45 cited references.

Vitamin K2 and hypertension

This 2021 rodent study investigated effects of Vitamin K2 on salt-sensitive hypertension:

“Mice were supplemented with VK2 and gut bacteria were detected by 16S rRNA. Common signaling pathway-related proteins were detected to further verify signaling pathways before validation of clinical samples.

Diets for 4 weeks were:

  • Normal group diet containing 0.5% NaCl;
  • High salt group (HS) diet containing 8% NaCl;
  • High salt diet plus VK2 supplementation group (HS_VK2) diet containing 8% NaCl and additional 0.025% VK2.

VK2 supplementation protected blood pressure and aortic vessels in salt-induced mice:

mk-4 salt

VK2 treated salt-sensitive hypertension by inhibiting the renin–angiotensin system.

fnut-08-639467-g009

Possible mechanisms of VK2 for salt-sensitive hypertension.”

https://www.frontiersin.org/articles/10.3389/fnut.2021.639467/full “Network and 16S rRNA Sequencing-Combined Approach Provides Insightal Evidence of Vitamin K2 for Salt-Sensitive Hypertension”


The form of Vitamin K2 was MK-4 per its C31H40O2 molecular formula. Data wasn’t provided to calculate a human-equivalent dose for 0.025% MK-4.

This study was part of a 2022 Gut Microbial Response to Host Metabolic Phenotypes collection which included animal studies investigating gut microbiota in contexts of β-carotene and β-sitosterol supplementation. I found this collection by it citing the 2018 rodent study GLP-1 release and vagal afferent activation mediate the beneficial metabolic and chronotherapeutic effects of D-allulose which was Reference 27 of Dr. Paul Clayton’s blog post Sweet Nothing.

Signaling pathways and disordered proteins

This 2022 review explored the title subject:

“Cell signaling imposes many demands on proteins that comprise these pathways, including abilities to form active and inactive states, and to engage in multiple protein interactions. Signaling often requires amplifying signals, regulating or tuning responses to signals, combining information sourced from multiple pathways, all while ensuring process fidelity.

Sensitivity, adaptability, and tunability are possible, in part, due to inclusion of intrinsically disordered regions in many proteins involved in cell signaling.  This review highlights the critical role of intrinsically disordered proteins for signaling:

  • In widely diverse organisms (animals, plants, bacteria, fungi);
  • In every category of cell signaling pathway (autocrine, juxtacrine, intracrine, paracrine, and endocrine); and
  • At each stage (ligand, receptor, transducer, effector, terminator) in the cell signaling process.

Function of the glucocorticoid receptor is regulated in part by its intrinsically disordered C-terminal tail. Prior to activation, the glucocorticoid receptor resides in cytosol:

glucocorticoid receptor

Intrinsic disorder in the glucocorticoid receptor not only enables multiple allosteric regulatory interactions to impact function, but also allows deployment of different surfaces of the protein to enable binding to many different sets of macromolecules, and regulation of these interactions via mRNA splicing and phosphorylation.

Combinations of alternative translation initiation and alternative mRNA splicing result in production of multiple glucocorticoid receptor isoforms from one gene. Various isoforms exhibit distinctive tissue distribution patterns and altered transcriptional regulatory profiles.

Greater than 90% of transcription factors either contain intrinsically disordered regions of proteins or are entirely intrinsically disordered. The many advantages conferred by disorder to cell signaling cascades means that:

  1. Understanding signaling required definition of roles disorder plays in each pathway;
  2. Many more examples of disordered proteins in cell signaling pathways are likely to be discovered; and
  3. More mechanisms by which disorder functions remain to be elucidated.”

https://biosignaling.biomedcentral.com/articles/10.1186/s12964-022-00821-7 “Intrinsically disordered proteins play diverse roles in cell signaling”


Cells in vivo seldom act on their own impetus. I would have liked discussion – or at least mention – of bidirectional signals between genes / cells / tissues / organs / organism / environment. This review’s topic of cell signaling pathways excluded “interactions of complex, interconnected systems spanning hierarchical levels” as explored in An environmental signaling paradigm of aging.

Every baby needs a sugar mama

This 2021 in vitro study examined butyrate producers:

“Butyrate produced by gut microbiota has multiple beneficial effects on host health. Oligosaccharides derived from host diets, and glycans originating from host mucus, are major sources of its production.

Butyrate is the major energy source for epithelial cells in the distal colon, induces differentiation of colonic regulatory T cells, and functions as an inhibitor of host histone deacetylase. These activities are essential for documented beneficial properties of butyrate, including anti-inflammation, gut immune homeostasis, inhibition of proliferation, and induction of apoptosis of colorectal cancer cells.

FOS-type oligosaccharides (kestose, nystose, fructooligosaccharide) were metabolized by only 6 of 14 butyrate-producing strains tested:

Growth of butyrate producers

Faecalibacterium prausnitzii, which is the most abundant butyrate producer in the healthy human gut, metabolized only FOS-type oligosaccharides among tested oligosaccharides. Anaerostipes spp. exhibited a similar pattern, except that A. caccae metabolized kestose but not nystose.

Glycoside hydrolase (GH)32 enzymes exhibiting FOS degradation activities were conserved in all six strains metabolizing FOS, and in three of the eight strains that did not metabolize FOS. This suggests that GH32 enzymes in those three strains are not actively used in metabolism.

The present study highlighted that even if functional genes are present in microbes, they are sometimes unable to metabolize substrates. This should be carefully considered in metagenomic studies to understand metabolic potential of gut microbiota.”

https://www.tandfonline.com/doi/full/10.1080/19490976.2020.1869503 “Characterization of fructooligosaccharide metabolism and fructooligosaccharide-degrading enzymes in human commensal butyrate producers”


These researchers had some work to do to show that selected strains’ characteristics were representative of their species. This post’s title was excerpted from Citation 37.

PXL_20220112_201642006

Resistant starch, β-glucan, and inulin

This 2021 paper reported results of two related human clinical trials:

“Lean and prediabetic overweight/obese men were included in two randomized crossover studies. In one study, participants received supplements of either long-chain inulin+resistant starch (INU+RS), INU or maltodextrin (placebo, PLA) the day prior to a clinical investigation day. The second trial studied beta glucan+RS (BG+RS) versus BG and PLA.

1. In lean men, INU+RS increased breath hydrogen and fasting plasma butyrate, which was accompanied by increased energy expenditure, carbohydrate oxidation, and peptide YY, and decreased postprandial glucose concentrations compared to PLA.

In prediabetic men, INU+RS increased plasma acetate compared to INU or PLA, but did not affect metabolic parameters.

The three supplements were:

  • INU: 12 g long-chain inulin in combination with 5.43 g maltodextrin to make it isocaloric.
  • INU+RS: 12 g of long-chain inulin in combination with 9.39 g 80% resistant starch RS2 granular potato starch.
  • PLA: 11.43 g maltodextrin.

2. BG+RS increased plasma butyrate compared to PLA in prediabetic individuals, but did not affect other fermentation/metabolic markers in both phenotypes.

The three supplements were:

  • BG: 35.25 g 34% yeast beta glucan with 5.43 g maltodextrin to make it isocaloric.
  • BG+RS: 35.25 g 34% yeast beta glucan in combination with 9.39 g 80% RS2 granular potato starch.
  • PLA: 11.43 g maltodextrin + 13.1 g protein and 4.58 g fat, same type and amounts as in the beta glucan product.

Effects of one-day consumption with a ‘slowly fermentable’ complex INU or BG alone, or INU or BG combined with a more ‘rapidly fermentable’ RS on substrate and energy metabolism were studied. These fiber mixtures were selected based on a high distal colonic acetate and total SCFA production in a validated in vitro model of the human colon.

Further research should study whether longer-term supplementation periods are required to elicit beneficial metabolic health in prediabetic individuals.”

https://www.tandfonline.com/doi/full/10.1080/19490976.2021.2009297 “Fiber mixture-specific effect on distal colonic fermentation and metabolic health in lean but not in prediabetic men”


The Discussion section related these findings to other research. Not sure how these researchers determined a trial time period. As Reviewing clinical trials of broccoli sprouts and their compounds pointed out:

“Biomarkers of effect need more time than biomarkers of exposure to be influenced by dietary treatment.”

PXL_20211226_114804495

Eat broccoli sprouts for your workouts

This 2021 human study investigated effects of pre- and post-workout glucoraphanin intake:

“The tablets used in this study contained 30 mg/3 tablets of sulforaphane glucosinolate [properly termed glucoraphanin], a precursor of sulforaphane (SFN), which is converted to SFN in the intestinal lumen by intestinal microflora. Subjects took one tablet of SFN supplement per meal, three times a day. Healthy men without exercise habits, smoking, or medication were included in the experiment:

eccentric exercise subjects

Pain on palpation reached its peak 1–2 days after exercise and recovered to baseline 5 days after exercise. Muscle soreness on palpation and range of motion were significantly lower 2 days after exercise in the sulforaphane group:

range of motion

Serum malondialdehyde, an indicator of exercise-induced oxidative stress, showed significantly lower levels 2 days after exercise in the sulforaphane group. SFN intake may protect the balance of antioxidant capacity and suppress excessive oxidative stress caused by exercise.

Continuation of SFN intake – from 2 weeks before and up to 4 days after eccentric exercise – suppressed exercise-induced oxidative stress and inhibited muscle soreness and muscle damage. To our knowledge, this study is the first to analyze these effects of SFN in humans.”

https://physoc.onlinelibrary.wiley.com/doi/10.14814/phy2.15130 “Effect of a sulforaphane supplement on muscle soreness and damage induced by eccentric exercise in young adults: A pilot study”


This study found that four days wasn’t enough time for 19-to-23-year-old men to fully recover from bicep eccentric exercise, regardless of glucoraphanin treatment or control group status. What’s an appropriate exercise recovery time? found a similar result using taurine as treatment with 20-to-33-year-old recreationally fit men who didn’t fully recover from bicep eccentric exercise after three days.

These researchers referenced Autism biomarkers and sulforaphane to acknowledge that this study’s daily 30 mg of glucoraphanin wasn’t sufficient to fully activate Nrf2 signaling pathways:

When SFN was added to PBMCs of healthy subjects in ex vivo experiments, NQO1 expression was increased, while HO-1 was not increased at a low SFN concentration (0.5 µM). However, when 2 or 5 µM of SFN was added to PBMCs, both NQO1 and HO-1 gene expression were increased. Concentration of the SFN supplement may be a reason why the amount of supplementation used in our protocol did not increase HO-1 expression.”


I create isothiocyanates by microwaving 3-day-old broccoli / red cabbage / mustard sprouts at 1000 W to 60°C (140°F) shortly before eating them. Unlike this study, I don’t depend on metabolism after the stomach to produce isothiocyanates from glucosinolates:

  • Less dependence on these subjects’ gut microbiota for sulforaphane production would have reduced a source of dose variability. Broccoli sprout compounds and gut microbiota first paper reviewed that subject.
  • Glucoraphanin intake with nothing else an hour before and after would have also reduced chances of sulforaphane loss by reacting with food. See Week 19 item 2 for two studies that found eating protein, fats, and fiber along with broccoli sprouts lowered isothiocyanates’ bioavailability.

Still, this was a step forward in research. Have fun with New Year’s resolutions.

PXL_20211226_123100412

Human agency vs. brain dysfunction

This 2021 human study used epigenetic clock technology to assess chronic inflammation as a driver of cognitive decline through its effects on brain structure:

“An epigenetic measure of C-reactive protein (DNAm CRP) was assembled for each participant. We found that higher inflammatory burden, indexed by DNAm CRP scores, associated with poor cognitive and neuroimaging brain health outcomes.

inflammation vs cognitive ability

DNAm CRP exhibited significantly larger associations with brain structural MRI metrics (including global grey and white matter atrophy, poorer white matter microstructure, and increased white matter hyperintensity burden) than serum CRP. Given that the 7 CpGs which make up DNAm CRP score reside in inflammation and vascular-related genes, these DNAm CRP-brain MRI associations may be capturing the impact of upstream inflammatory activity above and beyond that of serum CRP levels.

Our results indicate that some cognitive domains (processing speed) may be more mediated by brain structural consequences of chronic inflammation than others (verbal memory, visuospatial ability).

Our results add to the evidence base that DNAm-based predictors of inflammation may act as a quantifiable archive of longitudinal effects of these exposures – and other unaccounted for health and genetic profiles – that serum CRP levels fail to capture. By utilising an epigenetic inflammation measure, which integrates information from multiple immune-related CpG sites, we may provide a more reliable measure of chronic inflammation and thus a more comprehensive overview of consequences of chronic inflammation on brain structure and function.”

https://n.neurology.org/content/early/2021/11/17/WNL.0000000000012997.long “DNA Methylation and Protein Markers of Chronic Inflammation and Their Associations With Brain and Cognitive Aging”


These researchers essentially negated many of their findings by acknowledging:

“Although we endeavoured to remove participants with cognition-related pathology, these were screened via self-reported diagnoses, and we may be missing undiagnosed or subclinical incident neurodegenerative pathology.”

It wasn’t sufficient to claim in the Abstract section “Participants (N = 521) were cognitively normal, around 73 years of age” then include in the Discussion section a one-sentence limitation of relying on self-reports. Everyone defends themself against current and past realities and experiences.

Hard to imagine that objective measures such as the three comprising cognitive ability weren’t better screens. But then too many 73-year-old subjects may not have been “cognitively normal” and this study wouldn’t be adequately powered?

Can humans counteract inflammation? Non-communicable diseases? Smoking? Immune system degradation? Yes. No personal-agency actions were mentioned.

Also note this study’s social norming. The above-pictured 30-year-old female was busy at work, and subsequently hoisted a cat instead of a child in later years.

Take responsibility for your own one precious life.

PXL_20211118_114858264