0-1 star Wasted resources

Research that wasted resources

The TMAO meme

gettingwell4 0-1 star Wasted resources

A common dilemma for researchers is how to follow the herd enough to get a paper published, while simultaneously presenting replicable evidence of tested hypotheses. But unlike study researchers, reviewers are free to:

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

Reviewers also aren’t obligated to make post-publication corrections for their errors and distortions.

Here’s one of a dozen 2023 papers I read this week on TMAO. I picked this review because they attempted to come clean at the end of several sections.

I rated it as Wasted resources rather than Detracted from science as there might be a slight sniff of facts underneath the stench. Facts do not include “is associated with” or “is correlated with.”

The meme (repeated many times):

“Overall diet, lifestyle choices, genetic predisposition, and other underlying health conditions may contribute to higher trimethylamine N-oxide (TMAO) levels and increased cardiovascular risk. This review explores the potential therapeutic ability of resveratrol (RSV) to protect against cardiovascular diseases (CVD) and affect TMAO levels.”

Sections starting with premises that contained contradictions included:

2.2. TMAO and cardiovascular disease

Higher TMAO levels raise the risk of adverse cardiovascular events.

Overall, eating fish with high TMAO levels has relatively few adverse effects on CVD.

2.2.1 Relationship between TMAO and atherogenesis

Collectively, these findings provide a possible link between gut bacteria, platelet activation, and the risk of thrombosis.

More research is required to show the function of TMAO in the formation of CVD.

3. Application potential of phytochemicals such as polyphenols, RSV and its modified derivatives in regulating CVD

TMAO is a unique and independent risk factor for developing AS, partly through suppression of hepatic bile acid production.

Eating plants in general affects TMAO levels.

3.1. TMAO-lowering phytochemicals

Since the discovery of TMAO as a pro-atherogenic metabolite is relatively recent, only relatively small numbers of polyphenol-rich extracts and single phenolic compounds have been investigated as TMA/TMAO lowering agents.

Cardioprotective function of phytochemicals may arise from a combination of different mechanisms.

And so on.

https://febs.onlinelibrary.wiley.com/doi/10.1002/2211-5463.13762 “Cardiovascular risk of dietary trimethylamine oxide precursors and the therapeutic potential of resveratrol and its derivatives”

How many people would be healthy after they stopped eating fish, meat, and foods that had choline, carnitine, betaine, or ergothioneine? There are no clinical trials that omit all of these “TMAO precursors” from human diets because people would die.

Propagating the TMAO meme is dumb. It isn’t politically driven AFAIK, though, so doesn’t drop to the sub-basement evidence levels of politically correct memes.

2016 meme

A flawed broccoli microgreen study

gettingwell4 0-1 star Wasted resources

Sometimes I wonder why knowledgeable researchers design studies they know are wastes of time and resources, yet they perform them anyway. I’ll stop at three items this 2023 human study did that these researchers knew weren’t right.

1. Subjects’ bioavailable sulforaphane amounts from a single 16-gram serving of broccoli microgreens weren’t going to be anywhere near the lab-created sulforaphane amount. Human bioavailability doesn’t work like that, and a broccoli microgreens serving needed to be at least doubled in order to be within achievable range of other studies.

There wasn’t any reason in the Discussion section to waste the reader’s time with guesses about finding “Total mean urine SFN metabolite excretion over the 2-day study was 50.5 ± 2.7 μmol..total excreted SFN metabolites was less than the 100 µmols consumed.” They knew.

total urine sfn metabolites

2. If these researchers wanted to improve subjects’ broccoli microgreen bioavailability, consume a serving by itself, not with bagels and orange juice. Okay, a small amount of broccoli microgreens first thing in the morning probably doesn’t taste all that pleasant. But subjects couldn’t do this one time?

I’ve eaten cruciferous 3-day-old sprouts (after microwaving them to create sulforaphane and other isothiocyanates) by themselves to start my day every day for three and a half years now, eating nothing else before, then waiting an hour after. Like my healthspan depends on it.

3. They cited three studies that found eating broccoli for a minimum of fourteen consecutive days changed gut microbiota composition. It’s silliness to analyze why a single serving didn’t have similar effects. If this study’s design was of suitable length, they could have produced the same finding from Day 1 measurements.

So what’s this dumbing down of sulforaphane research all about?

  • Is it that researchers just aren’t serious about advancing their field?
  • Do they have to burn through funding regardless of flawed designs to keep people employed?
  • Some kind of academic equity requires the least knowledgeable person to be in charge?
  • Or do problems with wastes of research resources lie elsewhere?

https://www.mdpi.com/2304-8158/12/20/3784 “Sulforaphane Bioavailability in Healthy Subjects Fed a Single Serving of Fresh Broccoli Microgreens”

Glucoraphanin is not sulforaphane

gettingwell4 0-1 star Wasted resources, Memory, Neurochemicals, Serotonin, Stress ,

A poorly-conceived and intentionally-misrepresented human 2022 broccoli product study:

“We investigated whether a sulforaphane (SFN) [actually, sulforaphane precursor glucoraphanin] intake intervention improved cognitive performance and mood states in healthy older adults in a 12-week, double-blinded, randomized controlled trial.

The SFN group showed improvement in processing speed and a decrease in negative mood compared to the placebo group. However, there were no significant results in other biomarkers of oxidant stress, inflammation, or neural plasticity.

These results indicate that nutrition interventions using SFN can have positive effects on cognitive functioning and mood in healthy older adults.”

https://www.frontiersin.org/articles/10.3389/fnagi.2022.929628/full “Effects of sulforaphane intake on processing speed and negative moods in healthy older adults: Evidence from a randomized controlled trial”

Contrary to this study’s title, actual sulforaphane intake was not measured. The glucoraphanin product used in this study was the same item and daily dose as Eat broccoli sprouts for your workouts, which investigated effects with 19-to-23-year-old men. The treatment was taken all at once at an unspecified time of day rather than three times a day with young subjects.

These researchers knew from the 2012 study cited for dose that:

“Individual conversions of glucosinolates [like glucoraphanin] to isothiocyanates [like sulforaphane] varied enormously, from about 1% to more than 40% of dose. In contrast, administration of isothiocyanates (largely sulforaphane)-containing broccoli sprout extracts, resulted in uniformly high (70-90%) conversions to urinary dithiocarbamates.”

Young or old, a daily 30 mg glucoraphanin intake isn’t sufficient to fully activate human Nrf2 signaling pathways. A daily 17 mg sulforaphane intake could accomplish that.

PXL_20220819_101050766

Don’t bother eating broccoli sprouts if you’re old?

gettingwell4 0-1 star Wasted resources, Cerebrum, Epigenetics, Hippocampus, Limbic system, Memory, Neurochemicals, Oxytocin, Stress, Vasopressin ,

I try to not curate research that wastes resources. Couldn’t help but present this 2022 rodent study:

“We aimed to evaluate if sulforaphane (SFN) long-term treatment was able to prevent age-associated cognitive decline in adult (15-month-old) and old (21-month-old) female and male rats.

Our results showed that SFN restored redox homeostasis in brain cortex and hippocampus of adult rats, preventing cognitive decline in both sexes. However, redox responses were not the same in males and females.

Old rats were not able to recover their redox state as adults did, but they had a mild improvement. These results suggest that SFN mainly prevents rather than reverts neural damage; though, there might also be a range of opportunities to use hormetins like SFN, to improve redox modulation in old animals.”

https://link.springer.com/article/10.1007/s10522-022-09984-9 “Long-term sulforaphane-treatment restores redox homeostasis and prevents cognitive decline in middleaged female and male rats, but cannot revert previous damage in old animals” (not freely available)

These researchers cited Sulforaphane in the Goldilocks zone for hormetic effects of sulforaphane, so I asked:

“Did you develop any preliminary dose/response data for stating ‘there might also be a range of opportunities to use hormetins like SFN to improve redox modulation in old animals’?”

They cited Broccoli sprouts activate the AMPK pathway for long-term effects of a small sulforaphane dose, so I asked:

“Also, the three studies cited for ‘0.5 mg/Kg, i.e. 2.82 μmol/Kg BW for 3 months’ were all mouse studies. Since this was a rat study, wouldn’t there be increased dose and duration equivalencies?”

I’ll update this blog post in the event either of my questions to these researchers are answered.

PXL_20220819_101656448

If you lose mobility, you lose cognitive function

gettingwell4 0-1 star Wasted resources, Cerebrum, Epigenetics, Memory , , ,

This 2022 human study used four epigenetic clocks to assess aging:

“This cohort study was a secondary analysis of 3 Women’s Health Initiative (WHI) ancillary studies among 1813 women eligible to survive to age 90 years by end of study period. The study found that increased epigenetic age acceleration (EAA) as measured by 4 epigenetic clocks was associated with lower odds of survival to age 90 years with intact mobility; results were similar when including intact cognitive functioning.

This study benefited from a large, racially and ethnically diverse sample of women who were followed up to at least age 90 years with detailed longitudinal data on a host of lifestyle and health history factors. This study is generalizable to WHI women owing to use of IPW weights, and may be generalizable to a large range of women in the United States.

zoi220662t1_1658260078.05222

Among 1813 women, there were:

  • 464 women who survived to age 90 years with intact mobility and cognitive functioning;
  • 420 women who survived to age 90 years without intact mobility and cognitive functioning; and
  • 929 women who did not survive to age 90 years.

Only 29 women were reclassified from the healthy longevity group to surviving to age 90 years without intact mobility and cognitive functioning. Although it was of great interest to investigate the association between EAA and survival to age 90 years with intact cognitive function independently, this study population did not have sufficient numbers of women who experienced loss of cognitive function (without loss of mobility) to do so.”

https://jamanetwork.com/journals/jamanetworkopen/fullarticle/2794706 “Analysis of Epigenetic Age Acceleration and Healthy Longevity Among Older US Women”

Early humans who lost mobility in our African savanna ancestral environment during the Pleistocene Epoch (approximately 2.6M to 12K years ago) were prey. I highly doubt that immobile individuals successfully became our ancestors.

I downgraded this study because these researchers misguidedly soiled worthwhile findings with BMI and education level non-causal associations. They intentionally did this, as several of them were coauthors of the execrable Epigenome-wide meta-analysis of BMI in nine cohorts: examining the utility of epigenetic BMI in predicting metabolic health.

See Findings, or fun with numbers? and Does a societal mandate cause DNA methylation? for opposing research.

PXL_20220813_102515183

Broccoli sprouts and your brain

gettingwell4 0-1 star Wasted resources, Brain development, Cerebrum, Dopamine, Epigenetics, Glutamate, Hippocampus, Hypothalamus, Immune system, Limbic system, Memory, Neurochemicals, Oxytocin, Serotonin, Stress , , , , ,

A 2022 review of Nrf2 signaling hilariously avoided mentioning sulforaphane, although of ~4,000 sulforaphane published articles, two were cited. I’ll curate it anyway to highlight referenced brain effects.

“A good stability of NRF2 activity is crucial to maintain redox balance and therefore brain homeostasis. In this review, we have gathered recent data about the contribution of the NRF2 pathway in the healthy brain as well as during metabolic diseases, ageing, and ageing-related neurodegenerative diseases.

A functional NRF2 system is important to regulate both neuroinflammation, i.e., activation of microglia and astrocytes, and oxidative stress in the brain. NRF2 and NF-κB transcription factors regulate cellular responses to inflammation and oxidative stress in order to maintain brain homeostasis. Both pathways have been described to inhibit each other.

Nrf2 brain aging

Future challenges will be to establish novel therapies to:

  • Increase NRF2 activation in specific cell types and/or brain regions; and
  • Modulate NRF2 pathway in senescent cells.

Modulation of NRF2 signalling pathway by using specific food products [like unmentioned broccoli sprouts] and phytochemicals [like unmentioned sulforaphane], dietary supplements [like unmentioned Vitamin D3], drugs, and epigenetic modifiers, alone or in combination, will help to limit inflammatory diseases, ageing process, and subsequently ageing-related diseases.”

https://www.mdpi.com/2076-3921/11/8/1426/htm “Normal and Pathological NRF2 Signalling in the Central Nervous System”

PXL_20220808_095334058

Choosing your gut immune response

gettingwell4 0-1 star Wasted resources, Epigenetics, Immune system, Memory, Stress , , , ,

This 2021 paper reviewed evidence for immune system effects associated with specific gut areas:

“The intestinal immune system must not only contend with continuous exposure to food, commensal microbiota, and pathogens, but respond appropriately according to intestinal tissue differences. The entire intestine, inclusive of its lymph nodes, is considered a immunosuppressive organ overall compared to most other tissues, indicating that a state of tolerance to food and commensals – yet vigilance toward pathogens – was an evolutionarily stable strategy.

By operating in compartments, the immune system may generate multiple immune outcomes, even with simultaneous opposite goals e.g., tolerance or inflammation. Generation of unique immunologic niches within the intestine is influenced by a combination of tissue intrinsic properties, extrinsic environmental factors, and regionalized immune populations.

intestinal immune compartmentalization

Complexity of intrinsic and extrinsic driving forces shaping an intestinal niche makes it very challenging to determine causality in disease development and predicting effective therapeutic approaches. We really only stand at the beginning of understanding this interplay.”

https://www.nature.com/articles/s41385-021-00420-8 “Intestinal immune compartmentalization: implications of tissue specific determinants in health and disease”

I patterned this post after Choosing your future with β-glucan:

“So where do you choose to be? In an 80% survival group who were administered β-glucan before they encountered a serious infection? Or in a < 20% survival group who didn’t take β-glucan?”

and Long-lasting benefits of a common vaccine:

“As inferred by “induction of trained immunity by both Bacillus Calmette-Guerin tuberculosis vaccine and β-glucan” many of these findings also apply to yeast cell wall β-glucan treatments.”

This paper’s food allergy references were interesting. It’s an area that personally requires further work, although avoidance has historically been effective.

This paper briefly mentioned broccoli’s effects in the proximal small intestine. It wasn’t informative per gut compartment with this year’s focus on making my gut microbiota happy, such as what our colonic microbiota can do to reciprocate their host giving them what they want.

This review’s human studies referenced what could be done post-disease like surgery etc. in different gut compartments. Very little concerned an individual taking responsibility for their own one precious life to prevent such diseases in the first place. Its Conclusions section claim was a fallacy:

“..very challenging to determine causality in disease development and predicting effective therapeutic approaches.”

PXL_20210911_104042916

Changing your immune system / gut microbiota interactions with diet

gettingwell4 0-1 star Wasted resources, Epigenetics, Immune system, Memory, Stress ,

This 2021 human clinical trial investigated associations between gut microbiota and host adaptive immune system components:

“Diet modulates gut microbiome, and gut microbes impact the immune system. We used two gut microbiota-targeted dietary interventions – plant-based fiber or fermented foods – to determine how each influences microbiome and immune system in healthy adults. Using a 17-week randomized, prospective study design combined with -omics measurements of microbiome and host and extensive immune profiling, we found distinct effects of each diet:

  • Those in the high-fiber diet arm increased their fiber consumption from an average of 21.5±8.0 g per day at baseline to 45.1±10.7 g per day at the end of the maintenance phase.
  • Participants in the high-fermented food diet arm consumed an average of 0.4±0.6 servings per day of fermented food at baseline, which increased to an average of 6.3±2.9 servings per day at the end of the maintenance phase.
  • Participants in the high-fiber diet arm did not increase their consumption of fermented foods (Figure 1.C dashed line), nor did participants consuming the high-fermented food diet increase their fiber intake.

fiber vs fermented

Fiber-induced microbiota diversity increases may be a slower process requiring longer than the six weeks of sustained high consumption achieved in this study. High-fiber consumption increased stool microbial protein density, carbohydrate-degrading capacity, and altered SCFA production, indicating that microbiome remodeling was occurring within the study time frame, just not through an increase in total species.

Comparison of immune features from baseline to the end of the maintenance phase in high-fiber diet participants revealed three clusters of participants representing distinct immune response profiles. No differences in total fiber intake were observed between inflammation clusters. A previous study demonstrated that a dietary intervention, which included increasing soluble fiber, was less effective in improving inflammation markers in individuals with lower microbiome richness.

In both diets, an individual’s microbiota composition became more similar to that of other participants within the same arm over the intervention, despite retaining the strong signal of individuality.

Coupling dietary interventions to longitudinal immune and microbiome profiling can provide individualized and population-wide insight. Our results indicate that fermented foods may be valuable in countering decreased microbiome diversity and increased inflammation.”

https://www.cell.com/cell/fulltext/S0092-8674(21)00754-6 “Gut-microbiota-targeted diets modulate human immune status” (not freely available). See https://www.biorxiv.org/content/10.1101/2020.09.30.321448v2.full for the freely available preprint version.

Didn’t care for this study’s design that ignored our innate immune system components yet claimed “extensive immune profiling.” Not.

There was sufficient relevant evidence on innate immunity cells – neutrophils, monocytes, macrophages, natural killer cells, and dendrites – when the trial started five years ago. But maybe this didn’t satisfy study sponsors?

This study found significant individual differences in the high-fiber group. These individual differences failed to stratify into subgroup p-value significance.

I won’t start eating fermented dairy or fermented vegetable brines to “counter decreased microbiome diversity and increased inflammation.” I’m rolling the die with high-fiber intake (2+ times more grams than this clinical trial, over a 3+ times longer period so far).

Changing to a high-fiber diet this year to increase varieties and numbers of gut microbiota is working out alright. No worries about “increased inflammation” because twice-daily 3-day-old microwaved broccoli sprouts since Day 70 results from Changing to a youthful phenotype with broccoli sprouts have taken care of inflammation for 15 months now.

What effects have this year’s diet changes had on my adaptive and innate immune systems? 2021’s spring allergy season wasn’t pleasant. But late summer’s ragweed onslaught hasn’t kept me indoors – unlike other years – despite day after day of readings like today’s:

ragweed

Regarding an individual’s starting point and experiences, those weren’t the same as family, friends, significant other, identified group members, or strangers. Each of us has to find our own way to getting well.

Agenda-free evidence may provide good guidelines. So does how you feel.

Evaluating a company-sponsored β-glucan paper

gettingwell4 0-1 star Wasted resources, Epigenetics, Immune system, Stress

This 2020 review subject was yeast cell wall β-glucan effects in humans:

“The first aim of this review is to collate and interpret the existing pre‐clinical research on β‐1,3/1,6‐glucan with regard to immunity in order to clarify its molecular mechanism of immunomodulatory action.

mnfr3715-fig-0001-m

The second aim of this review is to collate and evaluate the literature in order to provide a comprehensive overview of human studies assessing the effect of supplementation with high quality, well‐characterized β‐1,3/1,6‐glucan from commercially available sources on immunity across multiple populations. Inclusion criteria consist of randomized, double‐blind, placebo‐controlled human studies that investigated efficacy of orally administered β‐glucan with a purity of over 75%.”

https://onlinelibrary.wiley.com/doi/10.1002/mnfr.201901071 “β‐1,3/1,6‐Glucans and Immunity: State of the Art and Future Directions”

I don’t usually curate company-sponsored research, aka puff pieces. I wondered why, after taking WellImmune β-glucan 500 mg daily for over two months, I didn’t have expected results.

There are always several possible explanations for experimental failures. I didn’t see applicable items in this paper.

There was much information regarding things their sponsor’s customers don’t need to know. Just like their sponsor’s product label, there was little about what customers need to know, such as:

What was each product’s content, in specific percentages, of 1,3/1,6 terminal-linked glucose molecules? That makes a difference.

The sponsor knows, but doesn’t disclose it on their product’s label. These researchers could have found out and presented that information on their sponsor’s and other companies’ products for each study reviewed.

Not doing so deprived readers of an important evaluation criteria that could possibly explain variable results and provide a better measure for comparability. Stopping at “a purity of over 75%” instead of investigating and disclosing exact information was evasive.

A broccoli sprouts study that lacked evidence for human applicability

gettingwell4 0-1 star Wasted resources, Brain development, Epigenetics, Telomeres , , , , ,

A 2020 study Combined Broccoli Sprouts and Green Tea Polyphenols Contribute to the Prevention of Estrogen Receptor–Negative Mammary Cancer via Cell Cycle Arrest and Inducing Apoptosis in HER2/neu Mice (not freely available) conclusion was:

“Lifelong BSp [broccoli sprouts] and GTP [green tea polyphenol] administration can prevent estrogen receptor–negative mammary tumorigenesis through cell cycle arrest and inducing apoptosis in HER2/neu mice.”

These researchers had unaddressed insufficiencies in this study that were also in their 2018 study as curated below. The largest item that required translation into human applicability was rodent diet content of 26% “broccoli sprout seeds.”

You may be surprised to read the below previous study’s unevidenced advice to eat double the weight of broccoli sprouts that I eat every day. You won’t be surprised that it’s not going to happen. Especially when no alternatives were presented because rodent diet details weren’t analyzed and published.

Sulforaphane is an evolved defense mechanism to ward off predators, and eating it is evolutionarily unpleasant. Will people in general and pregnant women in particular eat a diet equivalent to 26% “broccoli sprout seeds?”

Where were peer reviewer comments and researcher responses? Are these not public as they are by all Open Access journals hosted on https://www.mdpi.com/?

Sponsors and researchers become locked into paradigms that permit human-inapplicable animal research year after year. What keeps them from developing sufficient human-applicable evidence to support their hypotheses?

This 2018 Alabama rodent study investigated the epigenetic effects on developing breast cancer of timing a sulforaphane-based broccoli sprouts diet. Timing of the diet was as follows:

  1. Conception through weaning (postnatal day 28), named the Prenatal/maternal BSp (broccoli sprouts) treatment (what the mothers ate starting when they were adults at 12 weeks until their pups were weaned; the pups were never on a broccoli sprouts diet);
  2. Postnatal day 28 through the termination of the experiment, named the Postnatal early-life BSp treatment (what the offspring ate starting at 4 weeks; the mothers were never on a broccoli sprouts diet); and
  3. Postnatal day 56 through the termination of the experiment, named the Postnatal adult BSp treatment (what the offspring ate starting when they were adults at 8 weeks; the mothers were never on a broccoli sprouts diet).

“The experiment was terminated when the mean tumor diameter in the control mice exceeded 1.0 cm.

Our study indicates a prenatal/maternal BSp dietary treatment exhibited maximal preventive effects in inhibiting breast cancer development compared to postnatal early-life and adult BSp treatments in two transgenic mouse models that can develop breast cancer.

Postnatal early-life BSp treatment starting prior to puberty onset showed protective effects in prevention of breast cancer but was not as effective as the prenatal/maternal BSp treatment. However, adulthood-administered BSp diet did not reduce mammary tumorigenesis.

The prenatal/maternal BSp diet may:

  • Primarily influence histone modification processes rather than DNA methylation processes that may contribute to its early breast cancer prevention effects;
  • Exert its transplacental breast cancer chemoprevention effects through enhanced histone acetylation activator markers due to reduced HDAC1 expression and enzymatic activity.

This may be also due to the importance of a dietary intervention window that occurs during a critical oncogenic transition period, which is in early life for these two tested transgenic mouse models. Determination of a critical oncogenic transition period could be complicated in humans, which may partially explain the controversial findings of the adult BSp treatment on breast cancer development in the tested mouse models as compared the previous studies. Thus long-term consumption of BSp diet is recommended to prevent cancers in humans.”

“The dietary concentration for BSp used in the mouse studies was 26% BSp in formulated diet, which is equivalent to 266 g (~4 cups) BSp/per day for human consumption. The concentration of BSp in this diet is physiological available and represents a practical consumption level in the human diet.

Prior to the experiment, we tested the potential influences of this prenatal/maternal BSp regimen on maternal and offspring health as well as mammary gland development in the offspring. Our results showed there was no negative effect of this dietary regimen on the above mentioned factors (data not shown) suggesting this diet is safe to use during pregnancy.”

I didn’t see where the above-labelled “Broccoli Sprout Seeds” diet content was defined. It’s one thing to state:

“SFN as the most abundant and bioactive compound in the BSp diet has been identified as a potent HDAC inhibitor that preferably influences histone acetylation processes.”

and describe how sulforaphane may do this and may do that, and include it in the study’s title. It’s another thing to quantify an animal study into findings that can help humans.

The study’s food manufacturer offers dietary products to the public without quantifying all contents. Good for them if they can stay in business by serving customers who can’t be bothered with scientific evidence.

Any difference between the above-labelled “Broccoli Sprout Seeds” and broccoli seeds? Where was any evidence that “Broccoli Sprout Seeds” and SPROUTED “Broccoli Sprout Seeds” were equivalent per this claim:

“Equivalent to 266 g (~4 cups) BSp/per day for human consumption. The concentration of BSp in this diet is physiological available and represents a practical consumption level in the human diet.”

To help humans, this animal study had to have more details than the food manufacturer provided. These researchers should have either tasked the manufacturer to specify “Broccoli Sprout Seeds” content, or contracted out analysis if they weren’t going to do it themselves.

Regarding timing of a broccoli sprouts diet for humans, this study didn’t provide evidence for recommending:

“Long-term consumption of BSp diet is recommended to prevent cancers in humans.”

http://cancerpreventionresearch.aacrjournals.org/content/early/2018/05/15/1940-6207.CAPR-17-0423.full-text.pdf “Temporal efficacy of a sulforaphane-based broccoli sprout diet in prevention of breast cancer through modulation of epigenetic mechanisms”

Measuring one dimension of health

gettingwell4 0-1 star Wasted resources, Epigenetics, Immune system

This 2020 human study asserted:

“Our data provides the first epidemiological evidence supporting evidence obtained in preclinical models of metabolic syndrome and NAFLD that demonstrated hepatoprotective effects of phenolic acids.

  • High dietary intake of total phenolic acids is associated with a lower prevalence of non-alcoholic fatty liver disease and insulin resistance.
  • High intake of hydroxybenzoic acids, a class of phenolic acids, is associated with a lower prevalence of steatosis and clinically significant fibrosis.
  • High intake of hydroxycinnamic acids, another class of phenolic acids, is associated with a lower prevalence of insulin resistance.

Data on polyphenol content in foods was obtained from the Phenol-Explorer database (www.phenol-explorer.eu).”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078532/ “Higher phenolic acid intake independently associates with lower prevalence of insulin resistance and non-alcoholic fatty liver disease”

It’s a bad weather day, so I investigated the phenolics database and ran some numbers:

coffee and tea phenolics

Phenolic contents of all the other food I eat is 9% of my coffee-and-tea 1,975 mg total phenolics. Microwaved broccoli sprouts contribute half of that 9%.

Subjects were grouped according to whether their phenolics daily intake was over 221 mg or not. The over 221 mg group drank 5 cups of coffee a day, whereas the other group drank 1 cup.

According to a phenolics dimension of health, all these researchers needed to do was ask subjects about their daily coffee intake. But then the study would be over, with few “is associated with” findings.

Do humans avoid insulin resistance and non-alcoholic fatty liver disease by drinking more than one cup of coffee and tea? Is an answer available from real people, not just from a statistics package?

A 2020 study that primarily sourced a database last updated in June 2015 selected a fertile ground for later hypothesis-seeking.

fitting data

Ignoring subsequent research helped when staking a claim of first for whatever niche provided a publication opportunity.

I didn’t upload a screenshot of the Excel workbook with entries for pictured items I eat every day. That June 2015 database was incomplete with respect to current science here in December 2020.

See Eat oats today! for current examples of phenolic compounds in my daily 81 grams of steel-cut oats.

Does reprogramming signaling pathways create memories?

gettingwell4 0-1 star Wasted resources, Epigenetics, Immune system, Memory, Stress

This 2020 study investigated genes and signaling pathways for inflammatory memory:

“Fibroblast-like synoviocytes (FLS) play a critical role in pathogenesis of rheumatoid arthritis (RA). Chronic inflammation induces transcriptomic and epigenetic modifications that imparts a persistent catabolic phenotype to the FLS, despite their dissociation from the inflammatory environment.

Sustained activated genes established pro-inflammatory signaling components known to act at multiple levels of NF-κB, STAT and AP-1 signaling cascades. Sustained repressed genes included critical mediators and targets of the BMP [bone morphogenic protein] signaling pathway.

We identified sustained repression of BMP signaling as a unique constituent of the long-term inflammatory memory induced by chronic inflammation.

FLS are synovial tissue-resident and specialized mesenchymal cells critical for homeostasis. Key features of these cells during homeostasis include the production of extracellular matrix components and providing nutrients to the synovial fluid. Healthy synovium is composed of multiple layers of FLS, which forms the synovial lining and sublining through cell–cell contacts.

Inflammatory and pro-resolving mediators are tightly regulated to maintain normal synovium functioning. However, in inflammatory and autoimmune diseases such as rheumatoid arthritis, an imbalance between these signals causes homeostasis disruption leading to synovial tissue damage, cartilage destruction and bone degeneration.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679373/ “Chronic exposure to TNF reprograms cell signaling pathways in fibroblast-like synoviocytes by establishing long-term inflammatory memory”

These researchers described a positive feedback loop that kept rheumatoid arthritis in place. No feedback diagram or explanation of what sustained a disease condition other than to say:

“Gene expression changes induced by short-term tumor necrosis factor-alpha (TNF-α) treatment were largely sustained in the FLS exposed to chronic inflammation.”

Okay – then what upstream signals sustained TNF-α? What would it take to interrupt that feedback loop? What initiated it?

Studies usually substantiate effects by also developing evidence for causes of opposite effects and of no effects. This study investigated neither reversibility nor no effect, and instead stated:

“Multiple signaling networks are irreversibly modified due to TNF-α-mediated long-term epigenetic and transcriptomic reprogramming. We speculate that sustained repression of BMP signaling may be critically required to ensure the persistently transformed phenotype of RA FLS.”

No evidence was offered for “irreversibly modified.” Anyway, that didn’t fit with:

“We postulate that simultaneous targeting of these activated and repressed signaling pathways may be necessary to combat RA persistence.”

Enduring epigenetic memories? Or continuous toxic stimulation? provided another perspective: “Enduring epigenetic effects may be symptoms rather than causes when toxic conditions persist.”

Been on a Steely Dan kick lately. Probably due to this year’s Royal Scam:

A cherry-picked DNA methylation study

gettingwell4 0-1 star Wasted resources, Epigenetics, Immune system, Stress , , ,

This 2020 US/Sweden/Denmark human study measured twins during their old age:

“We evaluate individual differences in DNA methylation at individual CpG sites across the methylome across 10 years in two Scandinavian samples of same‐sex aging twins. We test two competing hypotheses about the longitudinal stability and change in DNA methylation:

  1. The contribution of genetic influences changes with age, reflecting diminishing influence across time; and
  2. Nonshared factors accumulate in importance, signaling an increasing diversity of response to environmental exposures.

Understanding epigenetic changes over time in the elderly may identify pathways of decline or plasticity (e.g., maintenance or even boosts in functioning) during the aging process and help with elucidating the biology of aging and survival.

Across time, stability in methylation is primarily due to genetic contributions, while novel experiences and exposures contribute to methylation differences. Elevated genetic contributions at age‐related methylation sites suggest that adaptions to aging and senescence may be differentially impacted by genetic background.”

https://onlinelibrary.wiley.com/doi/full/10.1111/acel.13197 “A decade of epigenetic change in aging twins: Genetic and environmental contributions to longitudinal DNA methylation”

Swedish subject measurements were taken at ages 62 and 72. Danish subject measurements were taken at ages 76 and 86.

One epigenetic clock that used 2019 technology was favored over three others, including Horvath’s 2013 original clock. For some reason this study didn’t use his 2018 skin-and-blood clock that had vastly improved technology such as an 18-fold increase in genomic coverage with Illumina 450k/850k bead arrays.

These researchers’ intentions became evident with:

“The 353 Horvath clock sites were selected as best predictors of chronological age using multiple tissues. The 71 Hannum clock sites best predicted age (adjusted for sex, BMI) based on methylation observed in whole blood while the 514 sites from the Zhang prediction model relied on methylation observed in blood and saliva samples (Zhang et al., 2019).

The current findings of moderately higher heritabilities in the Zhang and Hannum sites versus the other clock sites may be in part due to our use of blood tissue.”

The 18-fold increase improved accuracy in blood for the 2018 Horvath clock. Could these researchers ignore it and claim they did their due diligence in 2019 and 2020?

A larger issue was this study’s duality paradigm of either heritability or environment being solely responsible for observed changes. Consider what A blood plasma aging clock found at ages 60 and 78 peaks:

The above changes were due to life stage. Josh Mitteldorf did his usual excellent job of providing contexts for that study with New Aging Clock based on Proteins in the Blood, including:

“The implication is that a more accurate clock can be constructed if it incorporates different information at different life stages. None of the Horvath clocks have been derived based on different CpG sites at different ages, and this suggests an opportunity for a potential improvement in accuracy.”

Weren’t changes in subjects’ life stages relevant to their epigenetic changes? Why wouldn’t their life stages have been among the causes of observed effects?

Measuring sulforaphane plasma compounds

gettingwell4 0-1 star Wasted resources, Epigenetics ,

This 2020 Australian human study investigated methods of measuring sulforaphane plasma compounds:

“A simplified methodology to allow high-throughput LC–MS [Liquid Chromatography-Mass Spectrometry] analysis of plasma samples for measurement of sulforaphane and its metabolites is described. Analysis time is greatly reduced by employing fast chromatography and simple plasma extraction procedure.

Participants were observed consuming four Broccomax capsules, each containing 30 mg of broccoli seed extract and a dose of 8 mg of sulforaphane, as per manufacturer certificate of analysis, resulting in a total dose of 32 mg of sulforaphane (120 mg of broccoli seed extract).

Mean peak of combined metabolites from our study (0.9 and 1 μM) using 120 mg of broccoli seed extract (~32 mg of SFN) was similar to work by Fahey et al. who investigated pharmacokinetics of 350 mg of purified broccoli seed powder (mean 1.3 μM ± 0.5 μM), though our dose was almost three-times less. Pharmacokinetic profiles of our study mirrored those of Fahey et al. in that excretion was complete 8 hrs after consumption. Our intervention peaked slightly later (~2hrs), than that of Fahey (~1 hr), likely due to our use of a capsule rather than liquid.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070302/ “Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method”

This study was thin on comparing their n = 2 results to previous work. Here are comparables from Broccoli or Sulforaphane: Is It the Source or Dose That Matters?


These researchers set up a strawman by stating a false comparison:

“Our dose was almost three-times less.”

The compared study was the n = 10 subjects row above, which stated its dose as:

“200 μmol of SF was contained in about 350 mg of SF-αCD powder dissolved in 25 mL of distilled water, which subjects were given to drink upon arrival at the clinic.”

If the current study wanted a true comparison, they would measure and compare sulforaphane dose weights or amounts:

  • https://pubchem.ncbi.nlm.nih.gov/compound/sulforaphane lists sulforaphane’s molecular weight as 177.3 g / mol.
  • A 5.64 μmol sulforaphane amount (.001 / 177.3) equals a 1 mg weight of sulforaphane.
  • 200 μmol / 5.64 μmol = 35 mg sulforaphane used in the compared study.

But these researchers couldn’t even do that! They asserted a 32 mg sulforaphane dose “per manufacturer certificate of analysis” when they had the resources to do otherwise!

What kind of study design would go to all the trouble of measuring plasma sulforaphane metabolite outputs, but not measure their sulforaphane input dose???

Had they closely read the compared study, they may have also noticed that its commercial supplement, Prostaphane, was tested to verify stated dosage. These researchers could have done the same with Broccomax.

Don’t overcook broccoli

gettingwell4 0-1 star Wasted resources, Stress

This 2020 US / Korea study set a low bar and jumped over it by finding:

“The abundance of GSL [glucosinolate] hydrolysis products in cooked samples was lower compared to the raw samples.

Regardless of different cooking methods and durations, the total GSL amount in MeJA [methyl jasmonate]-treated broccoli was still higher than in the non-treated broccoli. This suggests that the increased GSL concentration in broccoli samples was solely affected by MeJA treatment, and the effect of MeJA was not affected by cooking methods.

Effect of cooking and 250 µM MeJA treatment on (A) total aliphatic glucosinolates, (B) total indole glucosinolates, and (C) total glucosinolates in ‘Green Magic’ broccoli. * = detected significant different by Student’s T-test (p ≤ 0.05, n = 3) with a significant interaction between MeJA treatment and cooking treatment.”

https://www.mdpi.com/2304-8158/9/6/758/htm “Methyl Jasmonate Treatment of Broccoli Enhanced Glucosinolate Concentration, Which Was Retained after Boiling, Steaming, or Microwaving”

Did it advance science to only replicate mistakes in consumer broccoli cooking methods with:

“The abundance of GSL hydrolysis products in cooked samples was lower compared to the raw samples.”

No.

Did the study design have tests to provide cooking method guidance for:

“To date, methods of delivering cooked broccoli without losing its nutritional benefits are still lacking in the literature, although consuming cooked broccoli is the most common practice for consumers.”

No.

Were there cooking method and temperature recommendations to avoid:

“Cooking also inactivates myrosinase, the enzyme converting GSL into hydrolysis products, and then hinders the formation of hydrolysis products.”

No.

Were there cooking method tests to further enhance either control samples or:

“Exogenous methyl jasmonate (MeJA) treatment was known to increase the levels of neoglucobrassicin and their bioactive hydrolysis products in broccoli.”

No.

Why omit temperature measurements since:

“The major research questions of this study were to evaluate how MeJA application to broccoli plants will affect GSL concentration, myrosinase activity, GSL hydrolysis product amounts..”

Maybe Microwave broccoli to increase sulforaphane levels wasn’t yet published when this study’s design decisions were made. Still, why would a study:

  • Test microwave half power without also testing full power?
  • Select microwaving time as the sole measurement without also measuring temperature?

Table S1 and Figure 3 of the Chinese / USDA study showed a two-minute microwaving time at 50% power wouldn’t be expected to have any sulforaphane content significantly different from uncooked broccoli. Also, temperatures of a five-minute microwaving time at 50% power were guaranteed to completely deactivate myrosinase.

Supplementary material confirmed that this study’s microwaving parameters didn’t show anything of value for how to use your microwave to increase broccoli compound levels. Did the study’s findings provide much more than what not to do?

Poor design decisions created a large gap between what could have been studied and what was studied. Let’s hope there will be better use of resources next time.