It’s the fiber, not the fat

I came across this 2020 fiber-vs-fat rodent study from its citation in Gut microbiota and aging:

“Dietary intervention studies largely revolve around altering fat content. Little consideration has been given to amount of fiber and whether or not it is soluble.

We examined age- and sex-specific effects of a refined high-fat/low soluble fiber diet (rHFD) on body weight and gut microbiota composition relative to mice fed a refined low-fat diet (rLFD) that is nutritionally and compositionally matched to rHFD.

Chow diet supplied energy as 13.4% fat, 28% protein, 57.9% carbohydrates, and 15% dietary fiber (range of total dietary fiber between 15 and 25% with 15–20% insoluble and 2–5% soluble fiber).

Two refined diets were used: rLFD supplying energy as 12% fat, 21% protein, and 67% carbohydrates; and rHFD supplying energy as 45% fat, 20% protein, and 35% carbohydrates. [Both rLFD and rHFD contained] 5% fiber in the form of insoluble cellulose.

Young adult animals consumed chow diet for 17 weeks, and 1-year aged animals consumed chow diet for 60 weeks. We included a 1-week transition period wherein all mice were fed rLFD. For the following 4 weeks, half of the animals remained on rLFD while the other half consumed rHFD.

After 4 weeks, young adult female mice showed resistance to weight gain to rHFD, consistent with previous reports. Aged females fed rHFD showed rapid body weight gain relative to rLFD-fed aged females.

Young adult and 1-year aged males showed a significant gain in body weight that was independent of refined diet formulation, suggesting that other components of the refined diet contribute to body weight gain that is independent of dietary fat.

Transition from chow diet to rLFD resulted in changes to microbiota community structure and composition in all groups, regardless of sex and age. This dietary transition was characterized by a loss within phylum Bacteroidetes and a concomitant bloom of Clostridia and Proteobacteria in a sex- and age-specific manner.

No changes to gut microbiota community structure and composition were observed between mice consuming either rLFD or rHFD, suggesting that transition to rLFD that lacks soluble fiber is the primary driver of gut microbiota alterations, with limited additional impact of dietary fat on gut microbiota.”

https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-0791-6 “It’s the fiber, not the fat: significant effects of dietary challenge on the gut microbiome”


It’s alright for researchers in the Abstract and Introduction section to interpret how their rodent study may apply to humans. I appreciate when they confine their statements elsewhere to what they actually measured and found.

This study didn’t measure inflammation, behaviors, neurobiologics, metabolic parameters, immune biomarkers, or hormones. They can qualify statements with “may” all they want, but this study didn’t provide direct evidence for either:

“Age-specific vulnerability to diet-induced body weight gain in females may be related to aging-related changes to estrogens.”

or

“The lack of differences between rLFD- and rHFD-fed mice may indicate that gut microbiota structure and composition can be dissociated from body weight and systemic inflammation.”

Papers that cite this study can’t rely on its Abstract for “regulating metabolic, immune, behavioral, and neurobiological outcomes” because its experiments didn’t directly measure such outcomes.

Removing 2-5% soluble fiber from subjects’ diet had large effects. I look forward to reading human studies that are informed by this study.

Gut microbiota and aging

This 2020 review explored the title subject:

“The human body contains 1013 human cells and 1014 commensal microbiota. Gut microbiota play vital roles in human development, physiology, immunity, and nutrition.

Human lifespan was thought to be determined by the combined influence of genetic, epigenetic, and environmental factors including lifestyle-associated factors such as exercise or diet. The role of symbiotic microorganisms has been ignored.

Age-associated alterations in composition, diversity, and functional features of gut microbiota are closely correlated with an age-related decline in immune system functioning (immunosenescence) and low-grade chronic inflammation (inflammaging). Immunosenescence and inflammaging do not have a unidirectional relationship. They exist in a mutually maintained state where immunosenescence is induced by inflammaging and vice versa.

Immunosenescence changes result in both quantitative and qualitative modifications of specific cellular subpopulations such as T cells, macrophages and natural killer cells as opposed to a global deterioration of the immune system. Neutrophils and macrophages from aged hosts are less active with diminished phagocytosing capability.

Gut microbiota transform environmental signals and dietary molecules into signaling metabolites to communicate with different organs and tissues in the host, mediating inflammation. Gut microbiota modulations via dietary or probiotics are useful anti-inflammaging and immunosenescence interventions.

The presence of microbiomic clocks in the human body makes noninvasive, accurate lifespan prediction possible. Prior to occurrence of aging-related diseases [shown above], bidirectional interactions between the gut and extraenteric tissue will change.

Correction of accelerated aging-associated gut dysbiosis is beneficial, suggesting a link between aging and gut microbiota that provides a rationale for microbiota-targeted interventions against age-related diseases. However, it is still unclear whether gut microbiota alterations are the cause or consequence of aging, and when and how to modulate gut microbiota to have anti-aging effects remain to be determined.”

https://www.tandfonline.com/doi/abs/10.1080/10408398.2020.1867054 “Gut microbiota and aging” (not freely available; thanks to Dr. Zongxin Ling for providing a copy)


1. The “Stable phase” predecessor to this review’s subject deserved its own paper:

“After initial exposure and critical transitional windows within 3 years after birth, it is generally agreed that human gut microbiota develops into the typical adult structure and composition that is relatively stable in adults.

gut microbiota by age phenotype

However, the Human Microbiome Project revealed that various factors such as food modernization, vaccines, antibiotics, and taking extreme hygiene measures will reduce human exposure to microbial symbionts and led to shrinkage of the core microbiome, while the reduction in microbiome biodiversity can compromise the human immune system and predispose individuals to several modern diseases.”

2. I looked for the ten germ-free references in the “How germ-free animals help elucidate the mechanisms” section of The gut microbiome: its role in brain health in this review, but didn’t find them cited. Likewise, the five germ-free references in this review weren’t cited in that paper. Good to see a variety of relevant research.

There were a few overlapping research groups with this review’s “Gut-brain axis aging” section, although it covered only AD and PD research.

3. Inflammaging is well-documented, but is chronic inflammation a condition of chronological age?

A twenty-something today who ate highly-processed food all their life could have gut microbiota roughly equivalent to their great-great grandparents’ at advanced ages. Except their ancestors’ conditions may have been byproducts of “an unintended consequence of both developmental programmes and maintenance programmes.

Would gut microbiota be a measure of such a twenty-something’s biological age? Do we wait until they’re 60, and explain their conditions by demographics? What could they do to reset themself back to a chronological-age-appropriate phenotype?


A follow-on study to 3-day-old broccoli sprouts have the optimal yields

This 2020 follow-on study to 3-day-old broccoli sprouts have the optimal yields investigated myrosinase enzyme activity:

“Myrosinase (MYR) can hydrolyze glucosinolates to produce sulforaphane [and other healthy compounds]. In this study, MYR activity in broccoli seeds and sprouts of different varieties were firstly compared. Then, after optimization for the microencapsulation condition of MYR, characteristics of free and encapsulated MYR enzyme were evaluated and compared.

The difference in MYR activity among seven broccoli varieties’ seeds was significant. However, total MYR activity in seeds and sprouts was actually not comparable. In the same weight of seeds and sprouts, dry matter content of sprouts was lower than that of seeds because of their high moisture content.

  • MYR activity of sprouts did not change significantly during the first 2 days of germination.
  • From the fourth day, enzyme activity increased significantly.
  • By the sixth day, its activity increased to the maximum, then decreased.

Broccoli variety significantly affected MYR activity during germination. 6-day-old LW variety was selected for further research.

myrosinase activity temperatures

When temperature was higher than 55° C, free MYR activity decreased rapidly. At 65° C, the free enzyme activity was less than half of the maximum enzyme activity. Specific activity of encapsulated MYR declined slowly, with the values of 82.1% at 65° C.

Activity of free MYR was the highest at pH 5.0, and it decreased rapidly when pH was less or higher. Encapsulated MYR could retain its activity under wider pH range and higher temperature than free MYR. Encapsulated MYR also kept higher activity during storage at room temperature.

Supplement of encapsulated MYR was favorable for SF production in broccoli sprouts during storage.”

https://onlinelibrary.wiley.com/doi/10.1111/jfpe.13567 “Selection and microencapsulation of myrosinase enzyme from broccoli sprouts of different varieties and characteristics evaluation” (not freely available)


Human stomach pH is 1.5. This study didn’t measure myrosinase activity below pH 4, maybe because it’s inactivated? Don’t count on myrosinase hydrolyzing glucosinolates into sulforaphane and other isothiocyanates like I3C after swallowing broccoli sprouts or supplements.

These researchers’ previous study heated broccoli seed powder at 55°C for 5 min to inactivate the epithiospecifier protein. I’m adjusting microwave practices for 3-day-old broccoli sprouts from ≤ 60°C to 55°C (131°F) in consideration of both the ESP and the 55-to-65°C decline in myrosinase activity. Also ordered a pH meter.

Haven’t seen sulforaphane supplements with microencapsulated myrosinase. This study provided evidence to support that method. Less clear is whether microencapsulated myrosinase continues on intact past the stomach.

Even if there were such supplements, though, wouldn’t it be more efficient and effective to create broccoli sprout hydrolysis compounds just before eating them? Why depend on vendor claims, myrosinase stability, or our own individual metabolisms?

The future of your brain is in your gut right now

A 2020 paper by the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease:

“The gut and brain communicate bidirectionally via several pathways which include:

  1. Neural via the vagus nerve;
  2. Endocrine via the HPA axis;
  3. Neurotransmitters, some of which are synthesized by microbes;
  4. Immune via cytokines; and
  5. Metabolic via microbially generated short-chain fatty acids.

How does nature maintain the gut-microbiome-brain axis? Mechanisms to maintain homeostasis of intestinal epithelial cells and their underlying cells are a key consideration.

The symbiotic relationship that exists between microbiota and the human host is evident when considering nutrient requirements of each. The host provides food for microbes, which consume that food to produce metabolites necessary for health of the host.

Consider function of the human nervous system, not in isolation but in integration with the gastrointestinal ecosystem of the host, in expectation of a favorable impact on human health and behavior.”

https://www.sciencedirect.com/science/article/pii/B9780128205938000148 “Chapter 14 – The gut microbiome: its role in brain health” (not freely available)


Always more questions:

  • What did you put into your gut today?
  • What type of internal environment did it support?
  • What “favorable impact on human health and behavior” do you expect from today’s intake?
  • How will you feel?
  • Will you let evidence guide feeding your gut environment?

See Switch on your Nrf2 signaling pathway for an interview with the author.

Eat heat-killed bacteria for health?

Two human studies investigated health effects of heat-killed lactic acid bacteria. The first from 2019 found:

“One hundred healthy subjects with a body mass index from 23.0 to 29.9 (51 men and 49 women, mean age 41.4 years) were enrolled in this randomized, double-blind, placebo-controlled, parallel group study. Subjects were randomly assigned to daily administration of a tablet containing heat-killed Lactobacillus plantarum L-137 (HK L-137) (10 mg) or a placebo tablet for 12 weeks. This study was conducted at Higashi Koganei Sakura Clinic (Tokyo, Japan) from December 2017 to March 2018.

HK L-137 improved TC and LDL-C levels, especially in subjects with high serum CRP, an indicator of total inflammation. Seasonal increases in levels of TC and LDL-C were observed in the control group, but not in the HK L-137 group, resulting in significant differences between groups at 12 weeks.

HK L-137 decreased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) biomarkers of hepatic inflammation. Daily intake of HK L-137 enhanced T-cell responses and suppressed hepatic inflammation and serum cholesterol in overweight subjects.”

https://link.springer.com/article/10.1007%2Fs00394-019-02112-3 “Daily intake of heat-killed Lactobacillus plantarum L-137 improves inflammation and lipid metabolism in overweight healthy adults: a randomized-controlled trial”

Four individuals in both the control and treatment groups – 8% – came down with influenza during the 12-week trial period.


Researchers of a 2020 study cited their previous work in mouse models and in a preliminary clinical study. Let’s start with their comment on the first study:

“Reduction in high-sensitivity C-reactive protein (hsCRP) or pro-inflammatory cytokines, the most important biomarkers of systemic chronic inflammation, was not found.

We selected Lactobacillus plantarum OLL2712 as an optimal anti-inflammatory LAB strain among hundreds in our LAB library. Administration of heat-treated OLL2712 cells alleviated chronic inflammation by suppressing pro-inflammatory cytokine levels in visceral adipose tissue and the serum and improved hyperglycemia in mouse models with obesity and diabetes.

In the present study, we conducted a randomized, double-blind, placebo-controlled, parallel-group trial to examine whether the 12-week ingestion of a test yogurt containing heat-treated OLL2712 cells is effective in improving glucose metabolism-related parameters in human prediabetic participants. Prediabetic adults (n = 130, age range: 20–64 years) were randomly assigned to either the placebo or OLL2712 groups (n = 65 each) and were administered conventional yogurt or yogurt containing more than 5 × 109 heat-treated OLL2712 cells, respectively, daily for 12 weeks in Minato-ku, Tokyo, Japan between July and December 2018.

HbA1c levels were significantly reduced in both groups at week 12 compared to baseline. 12-week reduction of HbA1c levels was significantly greater in the OLL2712 group.

Fasting blood glucose (FBG) levels did not change significantly in both groups. Fasting insulin levels were significantly increased in both groups compared to baseline due to seasonal fluctuations from summer to winter. However, they continued to increase consistently throughout the study only in the placebo group.

Increased chronic inflammation marker levels and insulin-resistant index (HOMA-IR) levels were higher at week 12 than at baseline in the placebo group but not in the OLL2712 group. HOMA-IR = fasting glucose (mg/dL) × fasting insulin (μU/mL)/405.

Overall, the only significant difference between groups was found for HbA1c levels. Effect size was very small compared to that of clinical trials of antidiabetic medication that target patients with severe diabetes.

Placebo yogurt used in this study contained some effective ingredients including more than 1011 cells of Lactobacillus bulgaricus and Streptococcus thermophilus, which might provide glycemic improvement and might affect benefits of OLL2712 cells.

Postprandial glucose excursions contribute more to HbA1c in participants with lower FBG levels. Ingestion of OLL2712 cells might reduce HbA1c levels in participants with lower FBG levels by suppressing postprandial glucose excursions.”

https://www.mdpi.com/2072-6643/12/2/374/htm “Effects of 12-Week Ingestion of Yogurt Containing Lactobacillus plantarum OLL2712 on Glucose Metabolism and Chronic Inflammation in Prediabetic Adults: A Randomized Placebo-Controlled Trial”

The placebo group’s Day 0 fiber part of their diet was 3% (11.0 / (11.0 + 232 + 62.6 + 69)). The treatment group was also 3%.


People are eating highly-processed food if fiber is only 3% of their diet. Can effects from other gut microbiota interventions be expected when basic soluble fiber requirements aren’t met?

Also, humans have 1014 gut microbiota. They outnumber the second study’s treatment “5 × 109 heat-treated OLL2712 cells” by 20,000 to one, and its placebo group by 1,000 to one. Could either group reach effective levels?

I’m not overweight, prediabetic, or diabetic. Like Day 70 results from Changing to a youthful phenotype with broccoli sprouts, it’s hard to make personal comparisons to populations represented by these two trials.

I eat less than half the fat, and several times more than the fiber shown above. Would my adding heat-killed lactic acid bacteria have any measurable effects?

How will you feel?

Consider this a partial repost of Moral Fiber:

“We are all self-reproducing bioreactors. We provide an environment for trillions of microbes, most of which cannot survive for long without the food, shelter and a place to breed that we provide.

They inhabit us so thoroughly that not a single tissue in our body is sterile. Our microbiome affects our development, character, mood and health, and we affect it via our diet, medications and mood states.

The microbiome:

  • Affects our thinking and our mood;
  • Influences how we develop;
  • Molds our personalities;
  • Our sociability;
  • Our responses to fear and pain;
  • Our proneness to brain disease; and
  • May be as or more important in these respects than our genetic makeup.

Dysbiosis has become prevalent due to removal of prebiotic fibers from today’s ultra-processed foods. I believe that dietary shift has created a generation of humans less able to sustain or receive love.

They suffer from reduced motivation and lower impulse control. They are more anxious, more depressed, more selfish, more polarized, and therefore more susceptible to the corrosive politics of identity.


Other recent blog posts by Dr. Paul Clayton and team include Skin in The Game and Kenosha Kids.

Image from Thomas Cole : The Consummation, The Course of the Empire (1836) Canvas Gallery Wrapped Giclee Wall Art Print (D4060)

Problematic rodent sulforaphane studies

I was asked to give an example of Human relevance of rodent sulforaphane studies. I’ll use the 2020 Sulforaphane Diminishes the Formation of Mammary Tumors in Rats Exposed to 17β-Estradiol.

1. This study’s sulforaphane dose was “100 μmol/kg SFN..gavage regimen on Monday, Wednesday and Friday for 56 weeks.” From the October 2019 Broccoli or Sulforaphane: Is It the Source or Dose That Matters? “Allometric scaling uses the correction factor for rat doses 0.162” and this graphic:

Interpreting the human-relevant range:

Interpreting that 8% of the rodent studies were clinically relevant to human sulforaphane doses as a lower boundary (1.43 µmol / kg) and tolerable to humans as an upper boundary (4 µmol / kg):

A human equivalent of this study’s dose is (100 μmol/kg x .162) = 16.2 µmol / kg. See the original blog post for a study showing that a majority of both treatment and control group subjects will refuse and stop with sulforaphane doses less than half of this study’s human equivalent.

2. From Week 28, “The maximum lifespan for rats and humans were set to 3.8 years and 122.5 years, respectively.” A human-equivalent multiplication factor that can be applied to a rat post-development time period is 122.5 / 3.8 = 32.2.

Assuming this study’s subjects could achieve maximum lifespan, a human equivalent of 56 weeks is (56 x 32.2) ≈ 1,803 weeks, or ≈ 34.7 years.

3. Let’s assert that the main purpose of animal studies is to help humans.

Was it possible for this study to achieve this goal when it used intolerable human-equivalent sulforaphane doses for a period equivalent to over three decades of our lives?

Yet its Discussion section proposed that it’s useful for human guidance on:

  • Obesity;
  • Breast cancer in premenopausal and postmenopausal women;
  • Hormone status;
  • Phospholipids for cellular health and homeostasis;
  • Serum free fatty acids and triglycerides; and
  • Lipid metabolism and DNA damage.

4. These researchers definitely knew what this study was going to do. A coauthor of the above referenced 2019 paper was also a coauthor of this study, who “conceived the original study design and supervised the project.”

Why did they do it? The coauthor’s shared apology – published in the October 2019 paper – for these types of studies was:

“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.

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

This study was published in July 2020.

It wasn’t just a waste of resources. It detracted from science because people won’t recognize that its findings are inapplicable to humans.

Part 2 of Switch on your Nrf2 signaling pathway

To follow up topics of Part 1‘s interview:

1. “We each have a unique microbial signature in the gut. Metabolites that you produce might not be the same ones that I produce. This makes clinical studies very difficult because you don’t have a level playing field.”

This description of inter-individual variability could inform researchers’ investigations prior to receiving experimental results such as:

Post-experimental analysis with statistical packages of these types of results is apparently required. But it doesn’t produce meaningful explanations for such individual effects.

Analysis of individual differences in metabolism can better inform explanations, because it would investigate causes for widely-variable effects. Better predictive hypotheses could be a result.

2. Today I’m starting my 40th week of eating a clinically-relevant amount of microwaved 3-day-old broccoli sprouts every day. To encourage sulforaphane’s main effect of Nrf2 signaling pathway activation, I won’t combine broccoli sprouts with anything else either during or an hour before or after.

I had been taking supplements at the same time. This interview got me thinking about the 616,645 possible combinations of my 19 supplements and broccoli sprouts.

That’s way too many to be adequately investigated by humans. Especially because contexts for each combination’s synergistic, antagonistic, or additive activities may be influenced by other combinations’ results.

I’ll just eat food and take supplements outside of this sulforaphane window. Two that I’ve started to further research because of this interview are:

A. I’ve taken 750 mg fructo-oligosaccharides (FOS) twice a day for sixteen years. I’ve considered it as my only prebiotic. Hadn’t thought of either of these points:

  • “Polyphenols are now considered to be a prebiotic food for microflora in the gut. They tend to focus on producing additional amounts of lesser known species like Akkermansia muciniphila, and have a direct prebiotic effect. Microbiota break these big, bulky molecules down into smaller metabolites, which clearly are absorbed. Some beneficial effects that come from polyphenols are not from the original molecule itself, but from a variety of metabolites produced in the gut.
  • We use a prebiotic, actually called an immunobiotic, which is a dead lactobacillus plantarum cell optimised for its cell wall content of lipoteichoic acid. Lipoteichoic acid attaches to toll-like receptor 2, and that sets off a whole host of immune-modulating processes, which tend to enhance infection control and downregulate inflammation and downregulate allergenicity.”

B. Every day I take a 400 mg capsule of 1/3, 1/6 yeast β-glucan to train my innate immune system. β-glucan also works with toll-like receptor 2, but differently than does lipoteichoic acid. Have a dozen browser tabs open on the subject.

3. “Quinone reductase is critical because it is the final enzyme in the phase two detox pathway that stops DNA being mutated or prevents deformation of DNA adducts which are mutagenic. I want to look at genes that govern redox balance, inflammation, detoxification processes, cellular energetics, and methylation.”

Gene functional group classifications are apparently required in studies, to accompany meaningless statistics. When I’ve read papers attaching significance to gene functional groups, it often seemed like hypothesis-seeking efforts to overcome limited findings.

I’ll start looking closer when study findings include Nrf2 signaling pathway targets quinone reductase, DNA damage marker 8-hydroxydeoxyguanosine, and enzymes glutathione peroxidase and glutathione S-transferase.

4. I bolded “unregulated inflammation” in Part 1 because it’s a phrase I’d ask to be defined if that site enabled comments. Thinking on inflammation seems to come from:

“We focus on the intestinal epithelial cell as a key player because if you enhance function of that cell, and Nrf2 is part of that story, once you get those cells working as they should, they are modulating this whole underlying immune network.”

An environmental signaling paradigm of aging and Reevaluate findings in another paradigm have a different focus. That paradigm looks at inflammation in the context of aging:

“A link between inflammation and aging is the finding that inflammatory and stress responses activate NF-κB in the hypothalamus and induce a signaling pathway that reduces production of gonadotropin-releasing hormone (GnRH) by neurons.

The case is particularly interesting when we realize that the aging phenotype can only be maintained by continuous activation of NF-κB. So here we have a multi-level interaction:

  1. The activation of NF-κB leads to
  2. Cellular aging, leading to
  3. A diminished production of GnRH, which then
  4. Acts (through the cells with a receptor for it, or indirectly as a result of changes to GnRH-receptor-possessing cells) to decrease lifespan.

Cell energetics is not the solution, and will never lead to a solution because it makes the assumption that cells age. Cells take on the age-phenotype the body gives them.

Aging is not a defect – it’s a programmed progressive process, a continuation of development with the body doing more to kill itself with advancing years. Progressive life-states where each succeeding life-stage has a higher mortality (there are rare exceptions).

Cellular aging is externally controlled (cell non-autonomous). None of those remedies that slow ‘cell aging’ (basically all anti-aging medicines) can significantly extend anything but old age.

For change at the epigenomic/cellular level to travel up the biological hierarchy from cells to organ systems seems to take time. But the process can be repeated indefinitely (so far as we know).”


Switch on your Nrf2 signaling pathway

An informative interview to start this year with the author of Sulforaphane: Its “Coming of Age” as a Clinically Relevant Nutraceutical in the Prevention and Treatment of Chronic Disease:

The Antioxidant Dilemma with Dr. Christine Houghton

“The thing about science is, the more you know, the more you realise you don’t know. And I have this enormous respect now for signalling processes that are going on within the cell, and not just signalling. The way mother nature switches on, switches off, foot on brake, foot on accelerator, continuously all of the time.

Things have changed in understanding the function of Nrf2 for a start, in controlling in many ways those cellular defences. We could then switch on Nrf2. You switch on a whole host of protective molecules all at the same time.

We use NAC [N-acetyl-cysteine] in the lab all the time because it stops an Nrf2 activation. So, that weak pro-oxidant signal that you use to activate Nrf2, you switch it off by giving a dose of NAC. It’s a potent antioxidant in that right, but it’s blocking signalling. And that’s what I don’t like about its broad use.

The real advantage of sulforaphane is not only is it the most potent inducer of Nrf2, or activator, but it’s also highly bioavailable. It’s a very tiny, low-molecular weight, lipophilic molecule that just glides straight in through cell membranes. It’s about 80% bioavailable. Whereas big, bulky polyphenols are about 1% bioavailable just simply because of their chemical structure.

We focus on the intestinal epithelial cell as a key player because if you enhance function of that cell, and Nrf2 is part of that story, once you get those cells working as they should, they are modulating this whole underlying immune network.

I’m particularly interested in looking at core upstream factors that govern cellular defences. So, I want to look at genes that govern redox balance, inflammation, detoxification processes, cellular energetics, and methylation.

Intestinal epithelial, just like any other cell in the body, will respond to Nrf2 activation. It will respond to NF-κB downregulation. That’s going to enhance redox control. It’s going to reduce unregulated inflammation. It’s going to enhance detoxification processes. It’ll increase glutathione synthesis.

All of those core factors that any cell needs to work normally will be enhanced by activating Nrf2. And I use a high-yielding sulforaphane supplement of about 20 milligrams a day to do that. So, that’s the beginning.

Probiotics don’t typically colonise in an adult. That’s where we come back to this idea of restoring the gut ecosystem and using prebiotic foods.

In an ideal world, we’d be looking at 600 to 800 grams of non-starchy plant foods a day. In a real world, that isn’t always going to happen.

I never use the term leaky gut because it isn’t that. It’s a dynamic structure that becomes unresponsive.”


Hadn’t thought about weighing my daily AGE-less Chicken Vegetable Soup dinner (half) then tomorrow for lunch. Its total weight tonight was 2,575.5 grams.

  • Subtract 207.2 g wine, 985.6 g chicken broth, and 64.2 g noodles;
  • Add 131 g 3-day-old broccoli sprouts microwaved to ≤ 60°C (140°F) eaten earlier;
  • Subtract an estimated 170 g (6 oz.) chicken, didn’t measure juice squeezed from one lemon, didn’t estimate evaporation from 20 minutes cooking; and
  • Didn’t include either 81 g dry weight steel-cut oats which becomes 308 g for breakfast, or 103.8 g 3-day-old hulled oat sprouts.
  • Net 1,279.5 grams non-starchy plant foods

I’m doing alright by the “600 to 800 grams of non-starchy plant foods a day” guideline. Should exercise more, though, because I eat a lot.

Topics continued in Part 2.

Part 2 of Eat broccoli sprouts for DIM

Continuing Part 1 with three DIM studies, the first of which was a 2020 chemical analysis investigating:

“Anti-estrogenic, anti-androgenic, and aryl hydrocarbon receptor (AhR) agonistic activities of indole-3-carbinol (I3C) acid condensation products.

I3C is a breakdown product [isothiocyanate] of glucobrassicin. Most biological activities attributed to I3C are believed to result from its acid condensation products, as it is expected that after ingestion of cruciferous vegetables, I3C is completely converted in the stomach before it reaches the intestine.

The reaction mixture was prepared from I3C under acidic conditions. Based on the various HPLC peaks, 9 fractions were collected and tested.

DIM (3,3-diindolylmethane) displayed clear estrogenic activity, showing an additive effect when co-exposed with low concentrations of E2 [estradiol] (below EC50) [effective concentration that gives half-maximal-response of a biological pathway]. However, an anti-estrogenic activity was observed when DIM was co-exposed with higher concentrations of E2, i.e. above EC50. None of the nine fractions was able to inhibit response of E2.

I3C and DIM showed clear anti-androgenic activities when co-exposed with concentrations of T [testosterone] at EC50 or ECmax. DIM showed a relatively strong antagonistic activity, and was able to completely inhibit response of T.

All fractions displayed an AhR agonist activity. Poor activity of fraction 3 seems surprising, as it contains ICZ, which was shown to be a strong AhR agonist. This is a strong indication that ICZ is only present at a very low concentration.

Observed estrogenic and anti-androgenic effects of the reaction mixture are most likely due to DIM.

The present study is the first that demonstrates that DIM also possesses anti-estrogenic properties when co-administered with E2 concentrations above EC50. Rather than ICZ, LTr1 and several other compounds present in fractions 1 and 4 (CTr), and larger molecules present in fractions 7, 8 (LTe1) and 9 seem responsible for observed AhR activity of the reaction mixture.”

https://www.sciencedirect.com/science/article/pii/S1878535220302811 “Acid condensation products of indole-3-carbinol and their in-vitro (anti)estrogenic, (anti)androgenic and aryl hydrocarbon receptor activities”

I came across this study as a result of its citation in Brassica Bioactives Could Ameliorate the Chronic Inflammatory Condition of Endometriosis.


A second 2016 study was with humans:

“Forty-five subjects consumed vegetables, a mixture of brussels sprouts and/or cabbage, at one of seven discrete dose levels of glucobrassicin ranging from 25 to 500 μmol, once daily for 2 consecutive days.

‘Blue Dynasty’ cabbage contained 33.5 ± 4.0 μmol glucobrassicin per 100 grams food weight. ‘Jade Cross’ brussels sprouts contained 206.0 ± 12.9 μmol per 100 grams.

At 50 μmol, variability in 24-hour urinary DIM levels appears to stem from both within an individual and between individuals. At 200 and 500 μmol dose levels, most variability is coming from between individuals rather than within an individual.

Inter-individual DIM variability may reflect the relative benefit an individual derives from consuming glucobrassicin from vegetables, responsive not only to how much glucobrassicin was consumed but also to variations in I3C uptake and DIM metabolism, many of which are not characterized.

Dose curve between glucobrassicin dose (25–500 μmol) [25, 50, 100, 200, 300, 400, 500] and urinary DIM. Bars represent SD. Estimated parameters in the original scale (95% CI): Maximum DIM 421.5 pmol/mL (154.7–1,148.4), minimum DIM 5.4 pmol/mL (0.7–44.3), EC50 90.2 μmol (29.1–151.3).

We conclude that urinary DIM is a reliable biomarker of glucobrassicin exposure and I3C uptake and that feeding glucobrassicin beyond 200 μmol did not consistently lead to more urinary DIM. Our data support the notion that cancer-preventive properties that might be derived from cruciferous vegetable consumption may require neither a large quantity of vegetables nor high-dose supplements.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5220883/ “Harnessing the Power of Cruciferous Vegetables: Developing a Biomarker for Brassica Vegetable Consumption Using Urinary 3,3′-Diindolylmethane”


1. Most subjects had trouble eating 500 μmol / 242.72 grams of Jade Cross brussels sprouts:

“At the 500 μmol dose level, two subjects could not finish due to the taste of the raw Brussels sprouts and were reassigned to 50 μmol dose level.

Two of the remaining five subjects at the 500 μmol dose level “Did not eat all of the assigned vegetables.” 🙂 That amount of brussels sprouts may have made two more sick because one “Missed one void during 2–6 hour collection period” and another “Missed 2 voids during the 6–12 hour collection period.”

2. From its supplementary material, there were ten subjects who ate a 200 μmol glucobrassicin dose. That’s a lot of raw cabbage (179.10 g) and brussels sprouts (67.96 g).

  • On Day 1 at the 2-6 hour point, Subject 27’s urinary DIM measured 10.21 pmol/mL and Subject 20’s measured 991.88, > 9700% higher.
  • At that 2-6 hour point on Day 2, the same subjects measured 16.15 and 687.44 pmol/mL, > 4200% higher.
  • From Table 1, their respective Mean 24-h DIM ± SE, pmol/mL measurements were 20.7 ± 4.0 and 1105 ± 45, > 5300% higher.

The 100 μmol glucobrassicin dose was 149.25 g Blue Dynasty cabbage and 24.27 g Jade Cross brussels sprouts. Could you eat that every day?

3. There’s sufficient data to make individual DIM bioavailability calculations. Don’t know why this study didn’t do that, nor did any of its 18 citing papers.

One study came close for broccoli and radish sprouts, 2017’s Bioavailability and new biomarkers of cruciferous sprouts consumption (not freely available) by researchers in the same group as Our model clinical trial for Changing to a youthful phenotype with broccoli sprouts. They didn’t disclose and analyze individual DIM bioavailability evidence, though:

“Broccoli and radish sprouts content in GB [glucobrassicin] were ~11.4 and ~7.7 μmol/20 g F.W, respectively. After ingestion of broccoli sprouts, 49% of GB was suitably metabolised and excreted as hydrolysis metabolites, calculated as the sum of I3C and DIM (~5.57 μmol /24 h). Following radish ingestion, the percentage of GB hydrolysed and absorbed was 38% (~2.92 μmol /24 h).

These results of bioavailability contrast with the extremely low percentage (< 1%) of GB excreted as DIM after consumption of Brussels sprouts and cabbage in a previous study (Fujioka, et al., 2014). Further studies about conversion of other indole GLS [glucosinolates] to I3C and DIM are needed to know more about bioavailability of these compounds, as there is no information in literature.”


A ten-subject study in Microwave broccoli seeds to create sulforaphane found inter-individual variability of sulforaphane and its metabolites in blood plasma for the highest and lowest individuals was > 500% (2.032 / 0.359 μmol). The urinary % of dose excreted by the same subjects was > 400% higher (86.9% and 19.5%, respectively.)

These studies present an opportunity for further discovery:

  1. Which researchers will try to understand causal experiences in people’s lives that produced such effects?
  2. Which researchers will produce evidence for factors that make people responsively either alive or dead to external influences on their internal environment?
  3. Where are studies that show when an individual needs to change their responses – their phenotype – they can successfully do so?

Herding, the story of 2020

Part 2 of Sprouting hulled oats

In Sprouting hulled oats, seeds were sprouted at 21°C (70°F) for 3 days. That post ended with a question raised by Oat sprouts analysis regarding desirability of enzymes.

Here’s that study’s analysis of its hulled oat variety’s enzymes, excluding results not pertinent to this post. There was neither a 72-hour measurement period nor a 20°C 60-hour period analyzed. Interpolate measurements accordingly.

1. α-amylase enzyme was described as:

“Alpha-amylase plays a key role during germination since it catalyzes hydrolysis of α-1,4 glucosidic linkages of starch, yielding maltose and glucose necessary for seedling development. Activity of this enzyme increased considerably during oat sprouting [reference to Degree of oat sprouting] but it is also de novo synthesized during this process.

High glucose content in sprouted flour can increase its glycemic index (GI). Foods with low GI are beneficial due to low postprandial glucose response compared to foods with a high GI. Selection of germination conditions is crucial to modulate α-amylase activity in oat for obtaining healthier sprouted flours with lower GI.”

A. 3-day-old hulled oat sprouts probably don’t have “High glucose content.” Studies such as Optimization of Oat Amylase During Sprouting to Enhance Sugar Production found:

“Maltose was the primary sugar, though there was a detectable but smaller amount of glucose.”

B. I understand that researchers have adopted a glycemic index. Does that one dimension indexed on glucose at 100 adequately inform health-choice decisions about oat sprout α-amylase enzyme content?

What’s the point of indexing healthy choices like sprouted whole grains to unhealthy choices that healthy people aren’t going to make anyway?

2. Increased protease enzyme activity was analyzed as desirable, and used as an optimization parameter.

3. Lipase activity increased from 18°C 60-hour to 20°C 96-hour measurements in the above graphic. All sprouted oat lipase levels were below unsprouted control oat flour, however:

“Lipase activity decreased in sprouted var. Meeri flour during germination. Our results suggest that there must be an important lipase activity in oat hull.

Lipase hydrolyse triglicerides to free fatty acids that are prone to oxidation and cause rancidity of cereal flours. According to our results, use of dehulled oat grains is desirable to obtain sprouted oat flours with increased stability and longer shelf life.”


Don’t know which enzyme is responsible for mild throat burn after eating 3-day-old hulled oat sprouts. It isn’t unpleasant, just unexpected. Research so far indicates that people pay for catalytic enzymes that increase proteolytic and digestive activity.

What if we index health decisions on a standard at 100 of drinking a beer first thing in the morning? Would anything scaled by that one dimension inform fine tuning of health-choice decisions?

“Woke up this morning and I got myself a beer
The future’s uncertain and the end is always near”

A case for carnitine supplementation

This 2020 review subject was carnitine, acetyl-L-carnitine, and its other molecular forms:

“Carnitine is necessary to deliver long-chain fatty acids from cytosol into mitochondria. Carnitine homeostasis is maintained by diet and renal absorption, as only a small amount (about 25%) is obtained by endogenous biosynthesis.

Defective fatty acid oxidation occurs with reduced intracellular levels of carnitine, leading to glucose consumption instead of lipid consumption, resulting in hypoglycemia. Non-metabolized lipids accumulate in tissues such as heart, skeletal muscle, and liver, resulting in myopathy and hepatic steatosis.

2000 mg/day is unlikely to provoke unwanted side effects and is safe for humans. In-depth studies are needed to identify a unique method of analysis which can guarantee efficient monitoring of supplement active component amounts.”

https://www.mdpi.com/1420-3049/25/9/2127/htm “The Nutraceutical Value of Carnitine and Its Use in Dietary Supplements”


The review listed animal studies of L-carnitine alone and in combination with:

  • Vitamin D3;
  • Coenzyme Q10;
  • Nicotinamide riboside;
  • Selenium;
  • L-arginine;
  • Anti-histamine drugs cetirizine hydrochloride and chlorpheniramine maleate; and
  • Hypertension drug olmesartan.

Human studies of its effects included:

  • Muscle soreness, damage biomarkers, and cramps;
  • Osteoarthritis knee pain and inflammation markers;
  • Ischemic cerebrovascular injury;
  • Peripheral neuropathy;
  • Nonalcoholic fatty liver disease;
  • Insulin resistance and Type 2 diabetes;
  • Kidney diseases;
  • Inherited diseases phenylketonuria and maple syrup urine;
  • Stress, depression, and anxiety;
  • Male infertility; and
  • Hepatitis C.

Sprouting hulled oats

My Sprouting whole oats trial was a hassle with hulls and a poor germination rate. This week I used hulled oat seeds from a different vendor, and a different study, Degree of oat sprouting, as my model.

  • Oat variety of Avena sativa was a small seed, 7 mm x 2 mm. The model used “huskless oat ‘Gehl'” which is a different species (Avena nuda).
  • 100 seeds weighed 1.5 grams. There were over 1,300 seeds per 20 g batches.
  • Oat sprout batches were processed the same way I do broccoli sprout batches. A new batch started soaking to start germination every 12 hours, then was rinsed three times every 24 hours on a 6 hours – 6 hours – 12 hours cycle. I have an open question to the model’s corresponding coauthor to explain their “4.5‐hr wet steeping, 19‐hr air rest, and 4‐hr steeping, all at 20°C” procedures to start germination, since I didn’t have access to its cited study. The model grew oat sprouts for 1, 2, and 3 days.
  • Temperature in my kitchen was 21°C (70°F) because it’s winter outside. The model grew oat sprouts at 10, 14, 20, 25, and 30°C. Their findings included “Temperatures between 20° and 25°C yielded the most dramatic changes in properties of sprouted oats.”

I evaluated germination results per the model’s Degree of Sprouting finding:

“Length of the coleoptile [shoot] was selected as a criterion of categorization of degree of sprouting. Grains of degree 0 do not show any radicle [root] or coleoptile growth. Degree:

  1. Has visible embryos (small white point), while radicles and coleoptile are not visible;
  2. Shows a developed embryo emerging from the seed coat;
  3. Coleoptile lengths of at least half the oat grain length;
  4. Coleoptile lengths between half and a full grain length; and
  5. Coleoptile longer than a full grain length.”

Most of this trial wasn’t a big deal, adding just a few extra minutes onto what I do three times a day with broccoli sprouts. Here’s what this oat variety’s hulled seeds and 3-day-old sprouts looked like:

The tedious part was evaluating degrees of sprouting. I took as large a bottom-to-top sample as I could tolerate sorting (235 seeds / sprouts, about 17%), with these results:

A 97% germination rate. 🙂 Average weight of three 3-day-old batches was 51.9 grams, for a 260% weight gain. My 5-day-old whole oat sprouts trial had a 22% germination rate and a 221% weight gain.

The model’s Figure 3 Degree of Sprouting finding for 20°C and 25°C at 3 days was hard to read:

Don’t know how 0 degrees of sprouting at 20°C and 25°C > 1% reconciled with their statement “Germinability after 3 days was about 99% at all temperatures.” A numerical table wasn’t provided – yet another question for the corresponding author. Meanwhile, I’ll estimate:

Their hard-to-read Figure 3 also wasn’t completely congruent with their statement:

“Around 20% of grains sprouted at 20° and 25°C had a coleoptile longer than a full grain length (degree of sprouting 5).”


These oat sprouts taste milder than my previous trial’s. With more than a third at a degree-of-sprouting 5 measurement, they’re sweet, concurrent with the model’s findings that:

“Increased amounts of reducing sugars and ascorbic acid were found particularly in the radicles and coleoptile. Coleoptile and radicle growth (input parameters for the degree of sprouting) and reducing sugars and α‐amylase activity are interdependent.”

Corresponding increased enzyme concentrations produce an aftertaste, though. I eat them along with either food or drink.

Can eating three-day-old oat sprouts of this Montana cultivated variety help with what I’m already doing? Here’s what I expect, given the model was a different oat species, and the Sprouting oats and Oat sprouts analysis studies used different oat cultivars.

1. In order of magnitude: increased antioxidants, GABA, phenolic compounds, protein, amino acids, β-glucan, and polyunsaturated fatty acids. Don’t know about GABA and protein, but the others may help counter inflammation.

2. Increased enzyme intake. The model study used α-amylase as a marker for α-amylase enzymes (catalyze starches), protease enzymes (catalyze proteins), and lipase enzymes (catalyze fats).

Oat sprouts analysis characterized increased α-amylase and lipase activities as undesirable in a sprouted oat flour context. More on enzymes in Part 2 of Sprouting hulled oats.