Gluten and FODMAPs: The lady doth protest too much, methinks

This 2013 study in the journal of Gastroenterology

No Effects of Gluten in Patients With Self-Reported Non-Celiac Gluten Sensitivity After Dietary Reduction of Fermentable, Poorly Absorbed, Short-Chain Carbohydrates

has been heralded as definitive proof that avoiding gluten does not confer any health benefits, save for non-Celiacs (maybe). This is a tenuous claim at best and most likely results from:

  • a lack of attention to study design details
  • discounting past in vitro studies & in vivo studies in mice and humans
  • an annoyance towards those behaviors associated with ‘going gluten-free’

Before exploring why this study reframes the gluten question rather than dismisses it, delineating the specific hypothesis being tested is necessary. The authors ask:

How do people respond to different whey and gluten challenges in the context of a diet lowered in FODMAPs?

Uh, what are FODMAPs? They are fibers of the short chain Fermentable Oligo-Di-MonosAccharides & Polyols sort.

So who are we talking about? Entry criteria:

To find data pertaining to this question, they enrolled 37 non-Celiac subjects diagnosed with irritable bowel syndrome (IBS) and reporting stable improvements in their symptoms for at least 6 weeks when going on a gluten-free diet (GFD) prior to the study.

What did they feed to who and when? Study design:

The study was double-blind, randomized and with cross-over. This is reasonably rigorous. The authors settled on a sample size of 37 subjects as to attain 80% statistical power, which is “the probability that the test will give the right result when there is a real effect […] and [it] depends on the sample size, and on the size of the effect we hope to detect”. As far as nutritional studies go, this is good. Yet, in the words of David Colquhoun, “The minimum false discovery rate for p=0.05 is seen to be 0.289 [using Berger’s approach]. In other words, if you claim you have discovered something when you observe a p∼0.05, you will make a fool of yourself in about 30% of cases”. Let that settle in for a moment…Statistics aside, the study proceeded as follows.

  • 2 week run-in period: all subjects went on a gluten-free (GF) low FODMAP diet.
  • Randomization to 3 different diets for 7-days: High-gluten (16g/day), Low-gluten (2g/day) or a whey-control diet (16g/day).
  • 2 week wash-out period.
  • 3-day rechallenge on 1 of 3 different diets: High-gluten (16g/day), whey (16g/day) or a control diet (0g/day of whey or gluten).

Why diet for this length of time? Does it matter? Rationale for trial lengths:

The authors speak of the 7-day trial and the 3-day rechallenge as 2 separate trials. They give 2 reasons explaining why the rechallenge lasts 3 days. First of all, the original plan was for the participants to stay on each diet for 6 weeks (not 7 days) because “symptoms were uniformly induced within the first week of the original study”. Secondly, the 3 putative gluten responders in the 7-day trial had apparent symptoms within 3 days, thus negating the need for a longer rechallenge period. When discussing possible mechanisms surrounding negative and reproducible effects of gluten, this last point may act as a potential confounder when considering longer-term autoimmune reactions involving molecular mimicry with the thyroid gland or with the nervous system (e.g. cerebellar ataxia).

How do we tell if things got better or worse? Endpoints:

Their primary endpoint was “the change in overall symptom score” on the Visual Analog Scale (VAS) from the 2 week run-in period on GF low FODMAP diet to the treatment-period on 1 of 3 diets. Their secondary endpoints were that slice of participants with a change in overall and individual VAS symptom scores of >20mm as well as markers of protein metabolism byproducts, magnitude of gluten-specific T-cell receptors response, fatigue, activity levels and specifically reproducible GI symptom between the 7-day trial and the 3-day trial.

The easiest person to fool is yourself, so how did they mitigate that? Controls:

They tried to minimize the influence of added food chemicals, they (apparently) successfully reproduced the texture of gluten in gluten-free products and the whey-isolate product was lactose & FODMAP free.

How can we make sure the participants did what they were told to? Adherence:

How was the participants adherence monitored? Daily symptom cards were filled out, significant (>20mm) changes in VAS scores were recorded, notes were taken on fatigue using a daily fatigue scale (D-FIS) and an accelerometer checked activity levels. Furthermore, IgA and IgG specific T-cell responses to gliadin and deaminated gliadin were assayed along with IgE wheat antibodies. Lastly, poop was collected between days 5-7 so to monitor ammonia, β-defensin and calprotectin levels.

So what actually happened? Will gluten shoot your dicks off? Results:

A general trend emerged where overall VAS symptoms improved from baseline to week 2 of the low FODMAP GF run-in period. Only 8 participants (a mere 22% of the total IBS cohort) saw significant improvements of >20mm in abdominal symptoms. The 63% reduction in FODMAPs – from 19g at baseline to 12g during the run-in – may have been insufficient  to uncover subtler effects.

Baseline to run-in

Despite this general improvement, symptoms generally worsened on all 7-day trial diets compared to baseline assessments – irrespective of the quantity or absence of whey, gluten or FODMAPs. Considering that FODAMPs were lowered by 86.4-80.5% compared to baseline (down to 2.6-3.7g) across all 3 diets for 7 days, one could reasonably expects commensurate or bigger improvements showing up. Since this did not happen, could the nocebo effect be to blame? The authors suggest it might. This would be because diet content was not associated with degrees of symptomatic responses but diet order was – in both the 7-day trial (A) and 3-day rechallenge (B).

It was about diet order, not content

It was about diet order, not content

The possibility is reinforced when considering how, in both trials, 1st interventions had mean VAS score changes of 15.5mm whilst 2nd and 3rd ones only changed by 5.3mm and 4.0mm, respectively. In other words, transitioning from the run-in period to the diet-week has an effect that is independent of the kind of diet one transitions to. The fact that only 3 subjects showed gluten specific effects and 7 showed whey-specific ones also argues against an independent diet-content effect. Furthermore, fatigue D-FIS scores presented similarly, showing no noteworthy changes across the 7-Day diets but again, with worse fatigue symptoms when transitioning from the run-in period, irrespective of the diet transitioned to. The authors astutely raise the possibility of “more focused attention to anxiety and depression rather than fatigue might provide additional clues to why patients who follow a GFD feel better”, implying that the D-FIS scale is in fact less appropriate than measures of anxiety and depression. This is strongly supported by the enormity of clinical and anecdotal feedback.

Returning, again, to the order effect and baseline to run-in VAS improvements, these kind of results point to a relatively common sort of response, where gluten may be ‘necessary but not sufficient’ for inducing clinically observable negative effects that have also been reproduced elsewhere, by the very same authors and in other studies.

With the exception of 1 participant with a 3-fold Celiac-like T-cell specific response, that of all other participants was not noteworthy. Neither were the changes in biomarkers obtained from fecal samples. To their credit the authors recognized the discrepancy between their data set and that of others, explaining how “the serological pattern was mostly negative, but there were a lower proportion of cases with positive IgG AGA [emphasis mine] compared with recent data on gluten sensitivity[24]”.

Reference 24 is taken from the results of Umberto Volta et al.’s 2012 paper in the journal of Clinical Gastroenterology, informing us that “[…] IgG AGA were positive in 56.4% of GS [gluten sensitive] patients” out of a cohort of 78.

IgG antibody and Fc receptor guiding macrophage phagocytosis

IgG antibody and Fc receptor guiding macrophage phagocytosis

Interestingly, HLA-D status did not correlate to biomarker changes. I do not have a good enough explanation for this. It is all the more puzzling when one considers that 21 patients on the High-gluten diet, 13 on the Low and another 13 on the whey-control diet were all borderline positive for Whole gliadin IgA values, hovering around 19±3.5U/mol. A negative assay for Celiacs is at <20U/mol. So there is no gradient or dose-response relationship here. Yet, the double-blind placebo-controlled, larger sample study in journal of the American College of Gastroenterology by Carroccio et al. in 2012 demonstrated such a gradient amongst IBS sufferers, wheat sensitive patients and Celiacs. 10%, 40% and 72% respectively tested positive for serum Gliadin IgA. A longer time component, as previously alluded to and exemplified here by Carroccio et al., seems more appropriate for the type of effects being teased out: 4 weeks for the elimination diet, 1 week for between-diet wash-outs and 2 weeks for the single-item reintroduction diets (also with cross-over design).

We’re left with gluten showing no dose-dependent relationship with the severity of symptoms in IBS patients, yet symptoms get noticeably better across the board when FODMAPs are more than halved and gluten withdrawn. What’s more, although the 3-Day Rechallenge gluten & whey-free control diet ‘provoked’ lots of symptoms, all of the gluten containing diets also scored poorly in terms of symptoms.

Ultimately, this study in IBS patients adds to the notion that when gluten is eliminated, symptoms improve – but not just because of gluten. It’s as if it always hangs out in the wrong foods (neighbourhoods) where, when combined with FODMAPs and possibly other components, wreaks havoc. Here FODMAPs seem to have played the role of its context-dependent partner in crime. Knowing the laundry list of other possible offenders in industrialized processed diets, it would be foolish to assume other combinations with gluten won’t produce a vast and continuous (rather than discrete) presentation of symptoms.

All in all, gluten can both stimulate “zonulin, the only known physiologic modulator of intercellular TJs [Tight Junctions] described so far” and cause reproducible immunological reactions (here, here & here). It has both the key to the house and a weapon, its immunoreactive amino acid sequence. What’s more, a genetic model (Celiac disease) gets us lots of supporting information and a ‘most sensitive’ model to help disentangle other symptom presentations. This literal ‘textbook’ explanation of basic immunological mechanisms should help clarify why dismissing gluten as a fad is, actually quite brazen or down right silly.

“The clonal selection theory provides a useful conceptual framework for understanding the  cellular basis of immunological memory. In an adult animal, the peripheral lymphoid organs contain a mixture of lymphocytes in at least three stages of maturation: naïve cells, effector cells, and memory cells. When naïve cells encounter their antigen for the first time, the antigen stimulates some of them to proliferate and differentiate into effector cells, which then carry out an immune response (effector B cells secrete antibody, while effector T cells either kill infected cells or influence the response of other cells). Some of the antigen-stimulated naïve cells multiply and differentiate into memory cells, which do not themselves carry out immune responses but are more easily and more quickly induced to become effector cells by a later encounter with the same antigen. When they encounter their antigen, memory cells (like naïve cells), give rise to either effector cells or more memory cells (Figure 25–11).

Naive, Effector & Memory Cells

Naive, Effector & Memory Cells

Thus, the primary response generates immunological memory because of clonal expansion, whereby the proliferation of antigen-stimulated naïve cells creates many memory cells, as well as because these memory cells are able to respond more sensitively, rapidly, and effectively to the same antigen than do naïve cells. And, unlike most effector cells, which die within days or weeks, memory cells can persist for the lifetime of the animal, even in the absence of their specific antigen, thereby providing lifelong immunological memory “(p.1546, Chapter 25: The Adaptive Immune System, Molecular Biology of the Cell, 5th Edition, Alberts et al., 2008).

IgG belongs to the cell-surface proteins Ig superfamily. Amongst other tasks, IgG activates the complement system – “complement activation can also greatly increase the immune response to an antigen: the binding of an activated complement component to an antibody–antigen complex, for example, can increase the ability of the antigen to stimulate a B cell response more than a thousand fold” (Figure 25–74, p.1599, Chapter 25: The Adaptive Immune System, Molecular Biology of the Cell, 5th Edition, Alberts et al., 2008).

Some Ig superfamily cell-surface proteins

Some Ig superfamily cell-surface proteins

The Figure ‘Kinetics of Antibody Response’ depicts their 2-humped response pattern and the characteristically long time component of the mechanism within which they operate.

Kinetics of Antibody Response

Kinetics of Antibody Response

Lastly, consider the Carroccio et al. 2012 study highlights and points from “Non-celiac wheat sensitivity diagnosed by double-blind placebo-controlled challenge: exploring a new clinical entity

Study highlights: Now & Then

Study highlights: Now & Then

How gluten affects the brain is not clear. It doesn’t seem to need to cross the BBB itself for many of its neuropathological symptoms.

  • “In the present study, none of the patients had hypovitaminosis or malabsorption, and more than half of the patients did not even show any duodenal abnormalities. Neuropathologically, there is loss of Purkinje cells and/or degeneration of the dorsal columns (Bhatia et al., 1995; Hadjivassiliou et al., 1998) with facultative lymphocytic infiltration of the cerebellum, dorsal columns and peripheral nerves (Hadjivassiliou et al., 1998)”

Reproducing empirically observable effects with gluten cannot be ignored and is confirmed elsewhere.

  • ”Our results clearly showed that a relevant percentage — more than one-fourth — of the patients who underwent DBPC [double-blind placebo-controlled] wheat challenge were really suffering from WS [wheat sensitivity]”

The ‘necessary but not sufficient’ notion stated another way.

  • “[…] there was evidence that coexistent triggers, e.g., intestine-damaging drugs or dysbacteriosis, can lead to a more severe intestinal impairment (28 [mouse study – gasp!]). Clearly, wheat antigens may also act in a similar manner”

Whether it is gluten alone or gluten + whatever else is also in gluten-containing products that is not healthy for humans, the gluten-free recommendation stands. This does not include advice to sport a gas mask when walking past bakeries.

Myoclonic Epilepsy with Ragged-Red Fibers (MERRF)

1973 saw Tsairis et al. report on case studies within a family where myoclonus epilepsy was associated with ragged-red fibres (RRFs) from muscle tissue biopsies in addition to abnormal blood lactate and pyruvate levels(1). In 1980 Fukuhara N. et al. publish a case study(2) of 2 patients who presented in much the same way as described by Tsairis et al. 7 years ago. Fukuhara et al. entitled their study “Myoclonus Epilepsy associated with ragged-red fibres (mitochondrial abnormalities): Disease Entity or a Syndrome?” and in so doing, introduced a question highlighting difficulties that the classical ‘single disease’ medical model encounters when trying to account for overlapping constellations of symptoms. In November of the same year, Wallace et al. come to identify this particular constellation of symptoms by the acronym ‘MERRF’ following “pathophysiological and biochemical characterization of a mitochondrial DNA disease” as their study title reports(3). ‘Fukuhara’s Disease’ is another name for it. Soon thereafter in 1981, the human mitochondrial genomes’ sequence & organization is published in Nature(4), laying much of the ground work for answering the how’s & why’s behind Fukuhara et al.’s question. The molecular clue for MERRF was elucidated in 1990 by Shoffner, Wallace et al. who identified a mitochondrial tRNALys  (mt-tRNALys) point mutation responsible for much of the epilepsy, defective mitochondrial energy production and age-related genotype-phenotype associations observed in MERRF patients(5). The molecular mechanism boils down to defective oxidative phosphorylation (OXPHOS) in the mitochondrial electron transport chain (mETC), caused by faulty protein synthesis originating from a point mutation in the MTTK gene erroneously encoding the highly conserved TѰC loop of its mt-tRNALys gene product. This results in aberrant taurine modification, also known as a “wobble modification defect [which] is primarily responsible for dispossessing the mutant tRNALys of its cognate codon binding affinity, forcing the mutant tRNALysUUU to become translationally inactive”(6). Nowadays, disorders characterized by defective oxidative phosphorylation are termed ‘mitochondrial disorders’(7).

MERRF is diagnosed by meeting the following 4 criteria:

  1. myoclonus (spasmodic muscular contractions)
  2. generalized epilepsy
  3. ataxia (lack of voluntary coordinated muscle movements)
  4. red-stained ragged fibers from muscle biopsies (RRFs)

Shoffner & Wallace called the tRNALys point mutation in the (modifiable) highly conserved TѰC loop producing a CviJI restriction site “a simple molecular diagnostic test for the disease”(8). See a depiction of this in Figure 3A below, from Shoffner & Wallace’s seminal paper on MERRF.

TRNALys point mutation

Nowadays however, it is known that the mt-tRNALys point mutation exchanging an A base for a G base at nucleotide position 8344 in the mt-tRNA gene MTTK(9) is not solely responsible for all MERRF (and MERRF-like) conditions but is present in ≥80% of patients(1).

Other mutations in mitochondrial genes, including but not limited to: m.8356T>C, m.8361G>A and m.8363G>A account for another 10% and m.611G>A and m.15967G>A for the other 5%(11). This expanded range of mutations in addition to rare MERRF case studies presenting unique mutations (e.g. Mancuso et al. 2004(12)) speaks to the pleiotropic nature of mitochondrial diseases generally, in terms of origins & clinical phenotypes. Furthermore, the etiology of many diseases traditionally regarded as solidly Mendelian within the 1-gene-1-enzyme(13) paradigm is strongly challenged considering that a given clinical phenotype may originate from a staggering diversity of genotypes. A powerful visual depiction of this is seen in Figure 3 from DiMauro et Schon’s 2003 paper(14) which maps “Mutations in the Human Mitochondrial Genome That Are Known to Cause Disease”.

Mutations in the Human Mitochondrial Genome That Are Known to Cause Disease

Mutations in the Human Mitochondrial Genome That Are Known to Cause Disease

There are examples of multivariate influences affecting phenotypes of even classical Mendelian disease like cystic fibrosis. Although 70% of cystic fibrosis patients inherit a mutated CFTR gene (del508) in an autosomal recessive Mendelian manner, in 2005 it was found that in a cohort of 69 Italians carrying this mutation “those who also carried the R131 allele of the immunoglobulin Fc-gamma receptor II gene had a 4-fold increased risk of acquiring chronic Pseudomonas aeruginosa infection (p = 0.042) [which] suggested that FCGR2A locus variability contributes to this infection susceptibility in CF patients”(15). This suggests that nowadays, Mendelian labels are better used for qualifying inheritance patterns than meaningfully categorizing diseases.

MERRF reflects classical mitochondrial inheritance patterns where only an affected or unaffected mother can transmit the condition to her offspring. Fathers cannot transmit mitochondria to their children because sperm do not contribute mitochondria to the zygote which will, however, contain the mitochondria from the mother’s ovum. Interestingly, a few isolated cases of paternal mitochondrial inheritance have been observed but not replicated in such a way as to challenge the standard model of inheritance just mentioned”(16,17,18).

Barring a handful of exceptions, every human cell contains hundreds of mitochondrial organelles, each carrying about 2-10 little (~16.5kb) double-stranded closed-circles of mitochondrial DNA (mtDNA) that make up our mitochondrial genomes. We have inherited this genome from our most recent common female ancestor about 180,000(19) years ago (commonly referred to as ‘Mitochondrial Eve’). These polyploid mitochondria are normally mostly homoplasmic (identical or of the same kind). However, they do intrinsically mutate stochastically at higher rates than observed in our nuclear genome. Per this tendency, they become transiently heteroplasmic during cellular division (mitosis) and are then distributed into daughters cells – also in a random manner. Here heteroplasmy simply refers to the mixture of mutated and non-mutated (or mutated and wild-type) mtDNA molecules within a cell. MERRF is heteroplasmic condition. Affected patients demonstrate a ‘threshold’ effect, wherein the proportion of mutant mtDNA molecules within a certain tissue correlates positively with symptom severity and skeletal muscle anaerobic capacity. Older patients accumulate more deleterious mtDNA which also correlates positively with worse clinical phenotypes. Typical MERRF patients do not show overt symptoms during childhood. The age of onset is thus a strong indicator of disease severity and again adds to the evidence implicating the proportion of mutated to non-mutated mtDNA molecules as a powerful determinant of symptom severity.

Defective mitochondrial respiration can have various causes, originating from both inside and outside the mitochondrial organelle. Identifying the 8344A→G mt-tRNALys mutation as the most common cause of MERRF is based on the following lines of evidence proposed by Shoffner & Wallace in 1990:

  1. 2 mutations in the mtDNA of MERRF-positive patients were discovered in this experiment and 1 was in the MTTK gene encoding evolutionarily conserved gene product elements involved in adequate mitochondrial protein synthesis.
  2. There was a perfect correlation between the mutation and the disease: all 3 MERRF patients had the MTTK mutation whilst none of the 75 controls did.
  3. Reduced numbers of larger mitochondrial translation products were observed in MERRF patients which is expected because the gene products supposed to synthesize them are erroneously encoded due to mutated mtDNA tRNAs.
  4. An A (adenine) base is modified in tRNA’s highly conserved TѰC loop responsible for recognizing the ribosome and accommodating the tRNA-ribosome complex for synthesizing proteins.
  5. Biopsies from MERRF patients reveal mitochondrial heteroplasmy which is consistent with loss-of-function (LoF) mtDNA mutations of recent origin.
  6. The proportion of mutant mtDNA molecules correlates well with symptom severity as well as with skeletal muscle anaerobic threshold.

Interestingly, in Shoffner & Wallace’s observations emerged the case of a quasi entirely homoplasmic MERRF patient who started to manifest overt symptoms in her early teens, suggesting that a small amount of normal mtDNA can provide a disproportionately large protective effect on the phenotype for quite some time. This does not contradict the above statements relating the degree of heteroplasmy to the age of onset. Instead, it hints at just how little the proportion of mutated and wild-type mtDNA molecules need be shifted to spur major alterations in phenotype. This adds understanding to speculations regarding the emergence of phenotypic pleiotropy from putative genotypes.

Continuing along the lines of improving the resolution of genotype-phenotype linkage, starting out with a molecular marker of disease like the mt-tRNALys point mutation used for diagnosis is very useful. Beyond base assessment, quantifiable measures of what this disease marker is actually doing to a particular patient are crucial for subsequent prioritization of potential treatment avenues, discerning risk-reward ratios and also objectively assessing the situations’ general urgency. Shoffner & Wallace looked at oxidative phosphorylation activity levels in skeletal muscle as one such measure.

Fig.1 Shoubridge’s 2001 paper “Nuclear genetic defects of oxidative phosphorylation

Fig.1 Shoubridge’s 2001 paper “Nuclear genetic defects of oxidative phosphorylation

Oxidative phosphorylation occurs along 4 respiratory chain complexes and a final ATP synthase complex as depicted in Figure 1 of Shoubridge’s 2001 paper “Nuclear genetic defects of oxidative phosphorylation”. These complexes reside in the inner mitochondrial membrane which has a matrix side (bottom) and a cytosolic side (top). Electrons flow along the respiratory chain from NADH- and FADH-linked substrates to molecular oxygen as indicated by the narrow black  arrows. Protons are pumped to the cytosolic side of the inner membrane at locations indicated by the thick black arrows. This produces an electrochemical gradient for protons across the inner membrane. The gradient-directed route these protons follow along eventually leads them through the final ATP synthase complex (complex V) where they drive ATP synthesis, providing the universal cellular energy currency. Zeviani & Di Donato highlight how “the fundamental reaction of life, i.e. oxygen activation and the conservation of energy in cell respiration, is essentially a function of the integrity of the inner membrane respiratory chain”(20). Shoffner & Wallace found levels of oxidative phosphorylation obtained by skeletal muscle biopsy correlated best with the patients’ current clinical phenotype. For age-related prognoses, their second measure of mtDNA genotype correlated best. mtDNA genotype refers to the degree of hetero/homoplasmy discussed above. Previous case studies and the recognition that certain tissues have distinct energy demands predicts organ-specific energy thresholds affecting the age of onset in affected individuals. This expectation is confirmed experimentally“(21,22).

Many other variables can jumble the order in which symptoms are expected to manifest in tissues: those with the lowest aerobic thresholds succumbing first. “The prominent involvement of the nervous, cardiac and skeletal muscle systems supports the contention that tissues with high aerobic energy demands will be most affected in oxidative phosphorylation disorders, but this simple view cannot explain the selective vulnerability of different organs […]”(23). Part of the ever-growing list of candidate variables contributing to tissue-specific selective vulnerability includes: nuclear genes, environmental influences and especially the somatic replicative segregation of mitochondrial organelles. Our knowledge of these variables is substantially disparate when it comes to understanding how each might affect MERRF.

The influence of nuclear genes on the stability of mtDNA is obvious, yet complicated. This can be clarified by characterizing the 4 major ways they can exert their effect on mitochondrial processes. First of all, defects in nuclear genes can affect the stability of mtDNA. Secondly, they can also affect structural components & assembly factors involved in oxidative phosphorylation. Thirdly, even if the defects arise in nuclear genes encoding proteins only indirectly related to oxidative phosphorylation, pathology may arise nonetheless. Lastly, non-protein components encoded by nuclear genes are also important for maintaining a functional respiratory chain. With this in mind, we also know our nuclear genome encodes 1,700 mitochondrial proteins(24) and 69 of the 82 subunits composing complexes I to IV of the mETC. A host of partially characterized assembly and maintenance protein factors for mtDNA exert an influence that still needs figuring out(25). Despite their putative roles, statistical analyses of modern genetic issues suggest that “nucleotide changes in mtDNA that are not intrinsically pathogenic may predispose to, modulate the effects of, or reflect a propensity for the occurrence of deleterious mutations. In turn, deleterious mutations may promote the accumulation of somatic changes, through the generation of OXPHOS-related mutagens”(26). This lends explanatory power to the observation that MERRF phenotypes worsen with age. Nucleotide changes in nuclear or mtDNA are not necessarily the only level at which disease causing events can occur. Yasukawa et al. provided the “first evidence that a post-transcriptional modification deficiency causes a human disease”(27) by studying the wobble modification imparted by the 8344A→G tRNALys mutation.

Mitochondrial somatic replicative segregation was discussed above in terms of different tissues having different levels of heteroplasmy. Shoffner & Wallace emphasize the other aspect of heteroplasmic variation by making the point that the “same average mtDNA genotype could result in very different organ-specific genotypes and clinical phenotypes”(28). Their contention is that not only are different tissues segregated with different amounts of mutated mtDNA, but that the same amount of mutated mtDNA exerts differential effects on the phenotype of the tissue depending on that particular tissue’s threshold. Lertrit et al. believe different amounts of mutated mtDNA are distributed amongst different tissues during events originating in utero. They accounted for this by looking at 6 different tissues in a MERRF patient with the 8344A→G mt-tRNALys mutation and observed heteroplasmy in the cerebellum, cerebrum, pancreas, liver, muscle & heart. From this they concluded that “the mutated population of mitochondria must have existed before the formation of the 3 primary embryonic layers”(29). This phenomenon is termed according to the analogy it describes — a ‘mitochondrial bottleneck’ — where “a mother with a low degree of heteroplasmy in her mtDNA can transmit a higher level of heteroplasmy to her children”(30).

Environmental influences on MERRF are rife with speculation but are currently hard to formulate in a testable manner. It is worth mentioning, however, that anything with the potential to moderate inevitable effects of aging (such as practicing an evolutionarily concordant lifestyle) is at the very least worth exploring as an adjunct treatment option. Epigenetics is naturally taking on a more prominent role in the scientific study of ‘lifestyle factors’ and mitochondrial disorders. On a basic level, this is simply because it is a fascinating foray into understanding how environmental (external) information comes to sit ‘atop’ (‘epi’) our human databases (genomes) to change us in some way. The field of epigenetics carries a slew of tantalizing ‘known unknowns’ that are best set aside momentarily when disentangling the effects of improper aminoacylation in MERRF patients.

MTTK encodes mt-tRNALys which has a UUU anticodon that typically undergoes a post-transcriptional “τm5s2U modification … at the wobble position in the anticodon region”(31) if the ribosome is to decode it properly. The third position is the wobble one, containing a 2-thiouridine derivative, normally ready to be modified. Theory and experiment agree that MERRF 8344A→G mutant mt-tRNALys does not recognize AAA-programmed ribosomes and so the wobble base remains unmodified. A 1989 in vitro cybrid clone(32) experiment shows the consequence of this lack of anti-codon recognition by measuring the difference in respiration rate between MERRF 8344A→G mutant cybrid clone cells (ME1-4) and their wild-type control counterparts (Ft2-11). The respective respiration rates of ME1-4 and Ft2-11 was 1.7 and 5.3 fmol/min/cell(33). That same experiment also found the overall protein synthesis rate substantially decreased in the mutant cybrid clones. Could faulty aminoacylation be to blame? To answer this, the group then looked to compare charged (aminoacylated) or uncharged (non-aminoacylated) mt-tRNAs and found very similar aminoacylation levels between both mutant and wild-type control cells. Defective aminoacylation is not the problematic mechanism underlying MERRF. Kolesnikova et al. test this further. They find that yeast-derived “imported tRNALys [into human fibroblasts] is correctly amino-acylated and able to participate in mitochondrial translation, partially rescuing mitochondrial function(34). This view is strengthened by the fact that mt-tRNALys does not lose significant affinity for bovine-derived EF-TU elongation factor compared to wild-type cells, thus retaining its aminoacylation efficiency. Crystallographic analysis of the anticodon stem-loop belonging to mt-tRNALys shows it is important for recognizing the ribosome. It also strengthens the currently accepted molecular mechanism whereby decreased mETC respiration in MERRF stems from defective taurine modification in the wobble position of the anti-codon that results in the inhibition of proper base pairing at the mt-tRNALys-ribosome junction.

In vitro experimentation from the same group provides 2 plausible accounts for the observed increases in intracellular lysine. The ribosomes can stall facing an empty A-site and terminate translation prematurely. Alternatively, frameshifting at lysine codons may produce abortive proteins to be shuttled away from the mETC for degradation by ATP-dependent mitochondrial proteases. On principle at least, both explanations are not mutually exclusive and may conceivably occur together; sometimes the amino acid will not form because translation was prematurely terminated and other times the amino acid will form only to be subsequently degraded.

Oxidative phosphorylation is identified as the defective process in MERRF and the molecular interactions between ribosomes and tRNAs translating amino acids involved in the process have been described above. 2 logical questions might follow, such as; ‘which of the 5 complexes along the mETC cease to function normally as to impact respiration in this disorder?’; and ‘do nuclear genes contribute to mtDNA instability in MERRF (if so, how)? Of the 13 mitochondrially encoded polypeptides involved in oxidative phosphorylation, complexes I and IV have the most, with 7 and 3 subunits, respectively. Despite their numbers, in MERRF complex IV is in fact the most prominently affected whilst complex I only occasionally(35). Complex V just has 2 and complex III only 1 and they are not significantly affected. Defects in more than 1 respiratory complex are common in disorders with mtDNA mutations. As in MERRF, tRNA deletions or mutations predominate and tend to give rise to translation defects. MERRF is somewhat of an exception amongst mitochondrial disorders in that its 4 canonical features correlate very tightly to its predominant, heteroplasmic tRNALys 8344A→G point mutation. Until now, only LHON patients with ND gene mutations and those with exercise intolerances linked to the cytochrome b gene reflect this level of correlation(36).

In contrast to MERRF’s most affected complexes, complex II does not contain any subunits from mtDNA. However, its possible role as a cellular oxygen sensor has many interesting potential implications for the other 4 complexes in MERRF and other mitochondrial disorders(37). 3 genes encode subunits SDHB, SDHD & VHL in complex II that function as putative tumor suppressors by responding to hypoxic conditions via still unknown molecular mechanisms. It is curious that SDHA mutations can produce Leigh syndrome (LS) but mutations in the other 3 subunits do not. Instead, they are associated with cancer and not with nervous system disorders like LS which is puzzling. The mitochondrial disorder MNGIE (mitochondrial neuro-gastro-intestinal encephalomyopathy) is another example of non-mtDNA mutations affecting mitochondrial functions. It involves a defective nuclear gene encoding the TP enzyme (thymidine phosphorylase) impacting mtDNA replication rate and fidelity because of imbalanced dNTP pools containing excess dTTP(38). Thus, certain mitochondrial disorders are clearly affected by nuclear gene-encoded components and MERRF appears vulnerable to their exacerbations — at least in principle.

Table 1 Clinical and Genetic Heterogeneity of Disorders Related to Mutations in Mitochondrial DNA below, serves as a map of sorts highlighting where our predictions about which complexes will be affected in which disorders fall short. This is reflected in the fact that 50% of adult and 80-90% of paediatric patients suffering from a mitochondrial disorder remain unlinked to a defective gene. Why do abnormal mitochondria accumulate beneath the sarcolemmal membrane as to produce RRFs typical of the majority of mitochondrial disorders but not in Leigh syndrome? Thankfully the search field for factors capable of filling-in those explanatory gaps can be narrowed down by learning from those failed predictions and consequently also shed some light on how phenotypes emerge from genotypes. A more immediately answerable question is “whether discrete syndromes based on clinical features can be reliably identified or whether these disorders represent a continuum among the mitochondrial myopathies”?(39). Byrne et al. asked this in 1988 and since then, the evidence that has surfaced leans heavily towards a continuum. Nevertheless, because these diseases “include both Mendelian-inherited and cytoplasmic-inherited diseases”(40), some take this as a challenge to the idea of a continuum. However, this position is rapidly becoming less and less tenable with knowledge of epigenetic methylation and histone modification mechanisms. We also know 1 gene can often originate multiple gene products — and isoforms — because it is actually providing a template suitable for a vast repertoire of possible gene-products ready to be moulded by post-transcriptional and post-translation modifications. Lastly, in 1996 Poyton & McEwan successfully demonstrate the existence of a “bidirectional flow of information between the nuclear genome and the mitochondrial genome to adjust energy production in tissues to different energetic demands”(41), solidifying the idea of oxidative phosphorylation disorders sitting on a continuum. In light of this, it is important to note that diseases inherited in a Mendelian manner appear quite more vulnerable to therapeutic interventions if this ‘information stream’ can be successfully manipulated.

Table 1 Clinical & Genetic Heterogeneity of Disorders Related to Mutations in Mitochondrial DNA

Table 1 Clinical & Genetic Heterogeneity of Disorders Related to Mutations in Mitochondrial DNA

The idea of bidirectional genomic information flow underlies much of the discussion to follow where MERRF is compared and contrasted to other oxidative phosphorylation disorders. Consider MELAS (mitochondrial encephalomyopathy, lactic-acidosis & stroke-like episodes), a maternally inherited disease caused by a 3243A→G point mutation in the MTTL1 gene encoding tRNALeu. The point mutation is very similar to that in MERRF and there is significant symptom overlap (see Table 1 above). However, the proclivity of brain tissue to harbour different mutation concentrations is perplexing: in MERRF the small cerebral vessels contain high concentrations and in MELAS it is the dentate nucleus of the cerebellum(42). Complexes I and IV are also affected in MELAS but the situation is reversed: complex I is most affected and complex IV typically remains intact. Both MELAS and MERRF display COX-depleted RRFs via histochemical staining, although in the former it is not a canonical feature, unlike in MERRF. This fact harkens back to the exceptional phenotype-genotype correlation observed in MERRF that is unequaled in MELAS — even as regards COX-deficient tissues. To explore both disorders further, a timely case study was published this year by Liu et al. who described a 19 year old Chinese girl with overlapping MERRF and MELAS syndromes with a confirmed T3291C point mutation in the MTTL1 gene encoding tRNALeu(UUR). Biopsies from her left bicep revealed a mixed findings that may be somewhat expected with symptom overlap: there were about 10% of scattered RRFs, scattered COX-deficient fibers with 35% of them containing RRFs and scattered succinate dehydrogenase-reactive vessels (SSVs), 85% of which retained COX activity. Respiration assessments on fibroblasts revealed a heavy reliance on glycolysis instead of oxygen consumption, confirming defective oxidative phosphorylation. Western blot analysis of viable mitochondrial complexes in her fibroblasts revealed substantial decreases compared to controls: 24.9% for complex I and 14.8% for complex IV. The latter complex is typically nearly untouched in MELAS. In MERRF it is complex I that usually does not show as much dysfunction. The absence of dysfunction in complexes II, III and V is another clue as to the susceptibility of individual complexes to mutations in genes encoding faulty translation components. The authors offer an interesting observation regarding how heteroplasmic tissue segregates. They report that her muscle tissue retained a higher concentration of mutations compared to her lymphocytes and fibroblasts, convincing the authors that “negative selection for defective mitochondria is possible for rapid turnover cells, such as lymphocytes and fibroblasts, but difficult for highly differentiated, postmitotic syncytial muscle cells”(43). The authors hold progressive views regarding the pleiotropic nature of mitochondrial mutations, as they clearly state that there is a “clinical spectrum associated with the m.3291T>C mutation”(44).

NARP (neuropathy, ataxia, and retinitis pigmentosa) patients also evolve along a spectrum, all the way to (maternally inherited) Leigh syndrome when surpassing a 95% threshold of heteroplasmy. Strangely, NARP patients maternally inherit a  T—>G8993 or T—>C8993 point mutation in the complex V ATPase 6 subunit gene, despite Leigh syndrome associated mutations being present in complexes I to IV but not V! Furthemore, RRF-positive muscle biopsies are nearly unheard of in NARP patients. Why would they not have abnormal mitochondria accumulate beneath the sarcolemmal membrane like in other respiratory chain disorders? This mystery persists.

There are no mainstream cures for mitochondrial disorders, only treatments for the symptoms. Drugs are prescribed to affected patients but this unfortunately and mostly exchanges one symptom for another, or at best, abates symptoms without causing too much collateral damage. Much of this limitation stems from the 1-disease-1-drug paradigm under which conventional but not cutting-edge(45) medicine still functions. It simply does not fit with current knowledge of disease pleiotropy and plasticity. A fundamentally misplaced focus on treating symptoms rather than investigating root causes coupled to a failure to distinguish between these is largely to blame. After all, antibiotics treat infections, immune suppressants treat autoimmune flares, serotonin re-uptake inhibitors treat depression and anti-epileptics drugs (AEDs) treat epilepsy and so on and so forth. That being said, it must be recognized that treating disorders at their root is decidedly easier said than done and in the interim, symptoms must be somehow managed. The Hippocratic oath to ‘first do no harm’ is commonly jeopardized to that end. Unfortunately, the standard of care for MERRF patients conforms succumbs to that trend. MERRF patients are prescribed AEDs for seizure control and myoclonus(46) as well as standard pharmaceuticals for managing cardiac symptoms(47). Some L-carnitine and Coenzyme Q10 (CoQ10) is given; they help transport fatty acid derived components into mitochondria for aerobic respiration and aid the mETC to transfer electrons along the respiratory chain, respectively. Although CoQ10 is ineffective at significantly improving symptoms in MERRF patients it could still benefit those individuals also suffering with a primary CoQ10 deficiency. This benefit is apparent with its shorter chain analogue Idebenone that proves “effective in halting or even improving the hypertrophic cardiomyopathy in Friedreich’s ataxia”(48). CoQ10 is also known to be helpful with myopathies resulting from statin side-effects. It was initially supposed to go hand-in-hand with statin prescriptions, lending further support for trying it as an adjunct to AEDs. Standard physical therapy and aerobic exercise are encouraged as adjuncts to the aforementioned MERRF treatments along with minor surgery for ptosis correction (droopy eyelid). Creatine has been extensively studied; it is quite safe, cheap and supplementing with it is helpful in some mitochondrial disorders but not others. Lactic acidosis is very toxic to cells and is poorly managed, even with “the use of lactate-lowering agents including riboflavin, succinate and coefficient Q”(49).

What kind of futuristic treatments are proposed and what are their promises? Cytoplasmic transfer will enable dysfunctional mitochondrial components to be replaced by functional nuclear-encoded ones that exploit protein import machinery. It has been established as a proof of principle, both in vitro and in vivo(50,51)”. Tachibana et al.’s 2009 experiment provides a perspective on its imminence:

A recent noteworthy experiment featured the transfer of the nuclear genome from a primate oocyte to an enucleated oocyte of another primate containing only mitochondria (Tachibana et al., 2009). The oocytes generated contained the nuclear genome from two parents but mitochondria from the donor; when implanted in pseudopregnant mother they were able to successfully produce healthy rhesus macaque offspring(52)

This technique discusses the manipulation of prenatal events with the view of offering a cure or treatment. This begs the question: What about a prenatal MERRF diagnosis? Unfortunately, a prenatal diagnosis is not possible due to the tissue-partitioning of mitochondria that occurs in utero. Looking forward, gene therapy does not yet offer practical implementations of the exciting theories and principles developing under its purview. Stopping mutant mtDNA from being replicated is one theoretical approach on offer. It involves recognizing these mutant mtDNA sequences with sequence-specific antigenomic peptide-nucleic acids which should subsequently inhibit the replication of their target sequence. Although MERRF is a maternally inherited disease, there may be substantial methodological overlap with potential treatments for autosomal recessive oxidative phosphorylation disorders. For example, microcell-mediated chromosome transfer(53) could potentially help with extra-organellar components influencing MERRF’s severity even if the causal mutation is not resolved. Furthermore, direct cloning by complementation with retroviral cDNA expression libraries(54) is a tantalizing prospect with a meaningful track-record in vitro and in vivo. However, it needs to affect multiple tissues with particular heteroplasmic thresholds and precisely titrate expression in a timely manner. There appear to be many obstacles for every promising solution awaiting discovery.

The elimination of dysfunctional mitochondria by modulating mitophagy, apoptosis and other cellular quality control dynamics has been proposed. This approach is endorsed by DiMauro et Schon who argue that the “possibility of mitochondrial dysfunction needs to be taken into account by every medical subspecialty […] progress in this field has been striking enough to amply justify the term ‘mitochondrial medicine’”(55). Current pharmaceutical drugs targeting mitochondria are still imprecise, weak and replete with side-effects that are often times worse than the disorder itself. Fortunately, a lot of the scientific literature from disparate fields has uncovered epigenetic influences — also known as lifestyle factors — affecting numerous quality control mechanisms of the organelle, inside and outside of it. Despite these encouraging avenues, mainstream research remains committed to distilling these hypercomplex effects into drug form before exploring non-drug approaches. Thankfully, there are roads to explore in the mean time. Examples include  — but are not limited to — lifestyle factors guided by an evolutionarily concordant framework revolving around: nutrition, circadian rhythms entrainment, appropriate movement and activity and electrical stimulation of muscles and the brain(56,67). These attempts are both old and novel in many respects. They are often times significantly more reliable, reproducible, impactful and safe compared to conventional pharmaceutical drug treatments. Labelling such approaches as ‘holistic’ seems appropriate as the influences on mitochondria arise from many yet undiscovered pathways and feedback systems. What is being affected is not a single organ system, but organelles that allow the fundamentally life-sustaining process of respiration, carried out in nearly every cell in our body. Mitochondrial medicine is poised to modernize the medical paradigm.

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Will baby brains really be miswired if pregnant mom smokes pot?

Warning: my bias favors recreational & medicinal use of marijuana.

Which field of scientific research is of poorer quality: cannabis or nutrition?

It is honestly hard to say. They are neck and neck.

Why I am drawn to learning and writing about marijuana

At the most basic level it probably has to do with the immense opportunity to make fundamental pharmacological discoveries all the while exploding ancient walking-dead myths. Both opportunities are appealing.

I wanted to write about marijuana and health for some time now. I find it infinitely explorable and quite uncanny in its unremitting relevance to issues plaguing humanity, old and new. Just for a moment, imagine that for some peculiar reason you were to grant yourself a general education by focusing on a single ‘object’ or ‘thing’. The cannabis plant represents a stream of alluring questions splitting off into interminable rabbit holes. My confusing analogies aside, whether you are a history buff, a science nerd or more of a political junkie (you name it) there is something in it for everyone.

Super short introduction to the 3 Cannabis species

The genus cannabis contains 3 species; ruderalis, indica and sativa. Human intervention has mixed them up quite a bit and correct nomenclature would have us use terms like “indica-dominant” rather than just “indica”. I’m not sure if I’ll follow convention. But for simplicity’s sake, let us generalize. Cannabis sativa is more equatorial than not. It is a tall and lanky plant conferring a euphoric kind of high. In contrast, Cannabis indica does best at altitude, where it prefers to be shorter and quite sturdy. It produces more of a sedating high. Cannabis ruderalis is often shorter than your average sativa and is mainly used as a source of hemp because significant amounts of psychoactive substances are absent. The vast majority of cannabis consumed for medical or recreational purposes is an indica-sativa hybrid. This is true whether you are lighting up in Colorado, ingesting some in Amsterdam (The Netherlands) or vaporizing it in Australia. As far as I know, ‘pure’ strains of one or the other do not exist.

The science part

The study:

Miswiring the brain: D9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway

Sentence 1 of the abstract:

“Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments”

Before I deconstruct the study, I want to explain why I think certain terms and the 1st sentence of the abstract are inappropriate and incorrect, respectively.

First off, I agree that in utero cannabis exposure results in quantifiable changes to brain structures. However, I disagree with the authors’ implication that these changes are qualitatively understood (i.e. that their significance is known). In order to be credible, the terms “miswiring”, “permanent” and “impairments” should be backed-up by an unambiguous mix of epidemiological and randomized-controlled studies justifying their use. This is not the case. To see why, lets look at what the authors use to back-up their foregone conclusion, citing:

  • 1 prospective study “Prenatal marijuana and alcohol exposure and academic achievement at age 10”
  • 5 longitudinal studies “Effects of prenatal tobacco, alcohol and marijuana exposure on processing speed, visual–motor coordination, and interhemispheric transfer” & “The Effects of Prenatal Marijuana Exposure on Delinquent Behaviors are Mediated by Measures of Neurocognitive Functioning” & “Prenatal marijuana exposure contributes to the prediction of marijuana use at age 14” & “Prenatal Substance Exposure: Effects on Attention and Impulsivity of 6-Year-Olds” & “Intrauterine Cannabis Exposure Affects Fetal Growth Trajectories: The Generation R Study
  • 2 retrospective studies “Maternal smoking, drinking or cannabis use during pregnancy and neurobehavioral and cognitive functioning in human offspring” & “Prenatal marijuana exposure: Effect on child depressive symptoms at 10 years of age

Their list is thoroughly unimpressive. It represents a morass of statistical wand-waving, inappropriate sample sizes, inadequately controlled confounders, untested assumptions and blatant exclusions of contrary data. Even more troubling to me, is that many are solely or mainly funded by NIDA (National Institute of Drug ABUSE) whose grant criteria expects researchers to demonstrate drug harms, not drug effects (either good or bad). This is antithetical to sound methodology and a deal-breaker in terms of scientific credibility.

This compilation of literature refuting central claims made in the 7 above epidemiological studies is a good place to start [ is a great general resource for drugs, plants and experiences people have with them].

Alternatively, this 1994 ethnographic study of pregnant Jamaican women (funded by the March of Dimes Foundation) is interesting and well thought out. It concludes “The absence of any differences between the exposed or nonexposed groups in the early neonatal period suggest that the better scores of exposed neonates at 1 month are traceable to the cultural positioning and social and economic characteristics of mothers using marijuana that select for the use of marijuana but also promote neonatal development”. The authors explain 3 main limitations of their study:

  • recruitment “identification by fieldworkers, with assistance from local midwives, represented a contributive alternative to a random sampling strategy”
  • “although the sample size is small, it provided an opportunity to follow up drug-using women through pregnancy with the level of detail that often is lacking in retrospective studies of large numbers of women”
  • confounders “Although this study was successful in controlling for polydrug use and SES [Socio-Economic Status], other variables (financial independence, mothers education, and household child/adult ratio) emerged as meaningful during the course of this study”

Back to THC’s fascinating effect on cortical development in utero. This study used aborted foetuses, mice and in vitro as well as in vivo techniques. First of all, the authors describe an integrated signaling axis whereby (1) THC acts as the trigger binding to the (2) CB1 receptor (CB1R) which starts to transduce the signal by acting on (3) JNK that has SCG10 as its downstream target. SCG10 is a key neuron-specific protein. It is plentiful in the growth cones of developing neurons because it acts as a destabilizing factor. This means it promotes the disassembly of microtubules by binding to them as a dimer. Somewhat metaphorically, this grants neuronal structures “options”: they can branch out, make new connections, dissolve old ones and basically participate in the brains plasticity (i.e. the ability to change). You certainly want a degree of neuronal ‘instability’ if your are to learn or repair your brain.

After ensuring that SCG10’s downstream activity can in fact be mediated through CB1R transduction, the authors went on to argue that upon THC exposure, long-lasting CB1R signalling defects occur which would cause excessive SCG10 degradation, hence reducing destabilizing potential in neuronal structures. They based this on observations of increased & deregulated presynaptic activity. However, their own data (Fig.1F) does not support the view that these changes are long-term or permanent.

Fig.1F Protein & mRNA expression levels

Rather, the ”rewiring and reduced synaptic plasticity in the cortical circuitry were not associated [my emphasis] with long-lasting modifications of synaptic protein expression in the hippocampus of offspring prenatally exposed to THC”. They incorrectly extrapolated an observation of altered and increased presynaptic activity to mean that these changes were permanent and negative, despite their own data on protein and mRNA expression levels suggesting otherwise.

Furthermore, this other study explores the link between synaptic activity and morphological changes in synaptic constructs and cautions its readers about “the relationship between the number of synapses & their combined strength is likely to be highly complex & therefore one would not expect to find a linear relationship between structural plasticity & changes in synaptic transmission”. This is fancy way of saying: at this point in time, we should be cautious about labelling molecular changes we observe as good or bad – especially when a working theory of brain development is still under construction.

Regardless, you may rightfully ask, so what if the changes are not permanent? Maybe they are still bad for the baby! Fair question and fair point. The answer is that we do not know. However, we have a few points to consider in the mean time:

  1. As argued above, epidemiological studies do not currently suggest such an effect when adequately controlling for other factors. God knows they’ve tried.
  2. Cannabinoids are naturally found in a mother’s breast milk, shifting the burden of proof to those claiming their inherent danger.
  3. No clinical studies have reliably demonstrated children with neurocognitive or motor impairments that resulted from marijuana exposure (pre-natal or post-natal). God knows they’ve tried.
  4. There are tons of data demonstrating therapeutic effects of cannabis on neurocognitive markers* sans the unending list of side-effects constituting the rule rather than the exception for most classical pharmaceuticals.

*For a future post.

Interestingly, THC exposure was also shown to result in ectopic (abnormal) filopodia formation and altered axonal morphology (Fig.7E).

THC fuxxing with Filopodia & actin formation

Bad? Well, maybe. Good? Well, potentially. Lets assume it is bad. How do we then go about squaring that with population-wide data showing no link between marijuana exposure and neurocognitive impairments? Do we reconsider what is normal filopodial formation in utero compared to post-natal stages? Or do we question whether normal axonal morphology can in fact manifest in more varied forms that are context dependent (stage of development and access to nutrients for e.g.)?

This study is actually extremely valuable in terms advancing neuroscientific understanding of foetal brain development and the role played by our endocannabinoid system. In fact, the signaling cascade (THC—>CB1R—>JNK—>SCG10) shown to exist by this study is “the first signaling axis directly linking a GPCR to SCG10 as molecular effector”. GPCR = G-protein coupled receptor. It also demonstrated how “phosphorylation inhibits the microtubule destabilizing activity of SCG10 suggesting that this protein may link extracellular signals to the rearrangement of the neuronal cytoskeleton”. This has exciting implications about potential modes of action to explain successful treatment outcomes in patients using marijuana for depression and PTSD (and lots more).

Finally, maybe the gravest of errors made by the authors of this study was their lack of distinction between cannabis and THC. They certainly were not clear nor explicit about it. THC is not cannabis and cannabis is not THC. Cannabis is not just a ‘pharmacy’ but a polypharmacy. It has > 400 compounds amongst which +60 different cannabinoids and loads of terpenes.

It would have been nice to see the authors speculate about both negative AND positive effects (again, DUH!). From the scientific perspective, there is NO advantage to this kind of narrowed thinking. Politically, the opportunities are plentiful.

#EvoMed inspired: No 2 medicines

I contend that there is only medicine, not a variety of flavors of medicine. The medicine you should care for or call by that name is the kind that is testable and works. Keep in mind that clinical testing is one of many forms of testing that can validate medical interventions.

If I told you once, I’ll tell you again: definitions are important in science. Undoubtedly, the definitions below are imperfect and I’d appreciate alternatives making their way into the comments section. To my taste, they are fine for the purposes of this discussion.

Lets start by defining 3 major intersecting elements of the matter at hand:

These 3 concepts can be seen as feeding back and forth on one another.

  1. A belief arises, inexplicably or based on prior knowledge. It then needs to be broken down as succinctly as possible so that its essential principles can morph into a testable hypothesis with the minimal number of variables possible.
  2. This hypothesis does not become accepted because there is evidence to support it. It becomes accepted — progressively — by accumulating failed experimental attempts seeking to tear it down. Evidence supporting the hypothesis is often sought in parallel or may stem from the attempts to disprove it.
  3. If the hypothesis has enough supportive data of sufficient quality outweighing all evidence to the contrary then it can be termed medicine (in the context of beliefs relating health & disease).

How exactly a hypothesis is accepted as valid or invalid (point #3) is often messy business. This is because we are on the blurry edge of the space where ‘knowns’ & ‘unknowns’ collide.

There are a million different reasons why things that putatively ‘work’ cannot and should not be called medicine. This label is reserved for things with evidence stemming from sound scientific testing. Some things are simply unconfirmed medicines in the waiting. I think this is the case with some so-called ‘alternative medicines’ as well as with consistently poo-pooed lifestyle factors — both of which have roles to play in health. This is not an argument for only using tried-&-true treatments since there are many situations where novel experimentation and educated intuition are appropriate. It is a lot of what makes science exciting! However, until data is in for EVERYONE to question OPENLY, a good scientist should abstain from calling his or her intervention(s) medicine. Or simply making unwarranted claims of certainty or misrepresenting the limitations of their knowledge. A safe bet on whether or not a person is well aware of what they’re doing and saying is that most questions should typically be met with a flat “I don’t know”. Yes, N = 1’s are great and I am an avid self-experimenter finding lots of value in it. I am careful to not form too solid a world view based solely on my experiences. This is one of my pet-peeves with the Paleo blogoshpere (or any other health related blogospheres) which extrapolates the “everyone is different” (sound) meme to suggest that their own evaluation of themselves is — by default — more important than other lines of evidence. If only things were so simple…There is no clear road-map: you have to constantly adjust what you appear to experience with other information out there. It is a life-long lesson in humility to be a true scientist (at heart — not by title. Titles are meaningless).

Lets take 1 tangible example of each scenario. 1 scenario is an ‘alternative medicine’ intervention that is worth scrutinizing/testing. The other scenario is of an ‘alternative medicine’ intervention not deserving a 2nd glance.

Example of an ‘out there’ hypothesis fulfilling deserving further scientific scrutiny: acupuncture. More specifically, it is auricular acupuncture. It has been used for an immense variety of conditions, syndromes and symptoms. The only effect it appears to have is on pain management. The effect appears to be quite weak — at best. A recent review article entitled “Efficacy of Auricular Therapy for Pain Management: A Systematic Review and Meta-Analysis” blandly concludes that:

auricular therapy can be used as an adjunct therapy for pain management and, therefore, reduce analgesic use to minimize potential adverse effects and tolerance. Nonetheless, further studies—particularly large scale of RCTs—are needed to further confirm the efficacy of auricular therapy for pain and must take into consideration important features of methodological design, which include point specification, stimulation, treatment duration, placebo effects, and patient expectations of treatment outcomes

Acupuncture is certainly not quackery. It may just work very poorly. In the grand scheme of medical things however, it is not vying for a prominent role in medicine. Understanding the neurophysiology and placebo/nocebo effects relating to such interventions are interesting avenues to follow, maybe one day telling us more about the nature of acupuncture’s effect(s). Moving on…

Example of an ‘out there’ hypothesis undeserving of further scientific scrutiny: homeopathy

It fails simple logic. It fails right out of the gate, on many fronts. For one, it is based on the silly notion that the more diluted the supposed active component is in the solution, the higher its potency.  This contradicts the time-tested concept that has yet to receive a single piece of evidence disproving it: the greater the quantity of something, the larger the dose. U-curves are still dose-dependent U-curves, people. They simply describe non-linear relationships between 2 or more variables. A clear, logic fail (see below).

logic fail

Furthermore, the dilutions indices are so large that they cause uncontrollable laughing and scoffing amongst the scientifically inclined. See this table below for a good laugh:

The laughing table

The laughing table

This is overkill but deserved. Lets hammer the nail into the coffin of homeopathy by quoting the recent Optum “Overview Report on the Effectiveness of Homeopathy for Clinical Conditions: Evaluation of the Evidence”, which concludes:

There is a paucity of good-quality studies of sufficient size that examine the effectiveness of homeopathy as a treatment for any clinical condition in humans. The available evidence is not compelling and fails to demonstrate that homeopathy is an effective treatment for any of the reported clinical conditions in humans.

The fact that homeopathy got this level of scientific scrutiny when lower-school maths and high-school physics is enough to disprove its outrageous claims is a waste of time and energy. Hopefully they can no serve their intended purpose.

Being clear about what can be called medicine and what can’t or shouldn’t, does not mean one is close minded. On the contrary, it demonstrates an understanding of the general limits of knowledge generally and ones own knowledge. It’ll be hard to think outside of the box, so to speak, if you’re not willing to recognize you’re in a box in the first place. That box is an exciting place to be and it is vast, really vast. It should be cherished as a launching pad for expanding it. If done with a little intellectual integrity — the better!

Check-out the #EvoMed summit and make up your own mind.

[Edit/Mea Culpa: 23/11/2014]

I wrote “Acupuncture is certainly not quackery. It may just work very poorly.” I now realize that acupuncture can be  correctly labeled as quackery as per its definition on Wikipedia: “Quackery is the promotion of unproven or fraudulent medical practices”. I changed my mind, mainly due to 2 things:

1) the reassessment of the ‘significance’ of p-values in biomedical literature (see “An investigation of the false discovery rate & the misinterpretation of p-values” by David Coloquhon

2) the readjustment of my expectations regarding the statistical occurrence of actual ‘true’ results in biomedical literature (see “Why most published research findings & false” by John Ioannidis

I realized that I was applying a double-standard of ‘evidence required’ for acupuncture studies compared to my more rigorous approach with genetic, pharmacological or nutritional studies. My use of the word “weak” was somewhat euphemistic for “nothing to see here”.

Lastly, I would like to note that the definition of quackery can be applied problematically to viable medical treatments. For example, “ketogenic diets for epilepsy” or “marijuana as a treatment for pain” are recognized as unproven or ineffective medical treatments and the general consensus amongst medical institutions mirrors that position. This however, is incorrect. The science behind both of these unconventional modern medical approaches is sound.

This is me trying not to throw the baby out with the bath water.

Tip-toeing towards the question ‘What is consciousness?’

I do not know what ‘it’ is. No one does, quite yet.

Robin Carhart-Harris (PhD) and Professor David Nutt are both from Imperial College London and are very much interested in answering that question. You may have heard of Professor Nutt back in 2009 when he was sacked from his position as a drug advisor to the British government, for doing just that — communicating his understanding of drugs. So be it.

Both scientists describe their approach to the study of consciousness like this:

Perturbing a system and observing the consequences is a classic scientific strategy for understanding a phenomenon. Psychedelic drugs perturb consciousness in a marked and novel way and thus are powerful tools for studying its mechanisms [PMID: 24904346]

It’s straightforward, unassuming and an exciting way to study humans. Before we dive in, here is a quote (from their main paper discussed below) I can’t resist and a little information on one of their favorite tools for ‘disturbing the pond’ that is consciousness, namely Magic Mushrooms.

It does not seem to be an exaggeration to say that psychedelics, used responsibly and with proper caution, would be for psychiatry what the microscope is for biology and medicine or the telescope is for astronomy. These tools make it possible to study important processes that under normal circumstances are not available for direct observation (Grof, 1980)

A little information

2 ingredients in these shrooms can introduce you to dancing donuts. They are psilocybin and psilocin. Dry magic mushrooms contain anywhere from 0.2% to 1% of both compounds by weight. When psilocybin is fiddled with (aka dephosphorylated) it becomes the active metabolite, psilocin. In 1962 Wolback et al. said that “an equimolar dose to 1mol of psilocin is 1.4mol of psilocybin”. I’ll take their word. Funny how both compounds travel together. They have “predominant agonist activity on serotonin 5HT2A/C and 5HT1A receptors” [PMID: 24444771]. Why do you care? Because “5HT2A receptor agonism is considered necessary for hallucinogenic effects” [PMID: 24444771]. Damn straight it is! By the way – who better than Albert freakin’ Hofmann to isolate, identify and synthesize both compounds back in the early 60s? Thanks man! Magic mushroom use dates back about 3,000 years, in Mexico. They were and are still used far and wide for medicinal and non-medicinal purposes. They have a very low toxicity level and they are certainly not neurotoxic. Let’s explore that further. How does psilocybin compare to other molecules like natural vitamins, or over-the-counter drugs? We could look at it like this:

The lethal dose killing 50% of a population is called the LD50.The therapeutically effective dose for 50% of a population is called the ED50. The LD50/ED50 ratio is the therapeutic index of a drug. It tells you at what dosage a drug will exert its therapeutic effect on 50% of a population. So to put things into perspective, for shrooms “the LD50/ED50 ratio is 641 […] (compare this with 9637 for vitamin A, 4816 for LSD, 199 for aspirin and 21 for nicotine)”[PMID: 24444771]. Captain Obvious says it is very much safer than most over-the-counter drugs. Same goes for the many plant compounds found in your typical health foods store.

You want to know how shrooms compare to what your step-brother’s second cousin took back-packing across Asia? Well, psychonauts would be right to point out that “psilocybin is 45 times less potent than LSD and 66 times more potent than mescaline”[PMID: 24444771].

Onwards! Nutt and Carhart-Harris also employ MDMA (aka ecstasy) to probe consciousness. It is a drug belonging to the class of phenethylamines and amphetamines. Its empathogenic effects are of great interest in the treatment of various psychiatric disorders. However, it appears less suited to the study of consciousness when compared to the ‘classic psychedelics’ like LSD or magic mushrooms. The reason is simple. The shift it produces away form normal consciousness isn’t as dramatic. Don’t get me wrong, a lot of trippy-dippy stuff will still happen to you — just not quite in the same vein as a true psychedelic experience.

This is their paper that made me stand-up and take notice of neurosciences progress in the study of consciousness (clap clap). It should really be read in its entirety as it’s more of a scientific tour de force than any old paper:

The entropic brain: a theory of conscious states informed by neuroimaging research with psychedelic drugs [PMID: 24550805]

Let’s unpack this. Entropy, simplistically, is measure of disorder. In this brainy context it refers to the degree of randomness or uncertainty of a self-organized complex system.

Concept of Entropy

Our cerebral, self-organized complex networks are “functionally and structurally connected brain regions that show high spontaneous or ‘on-going’ metabolism”. Like a truck idling. These are described as DMNs (Default Mode Networks). Their resting-state functionality (RSFC) simply refers to the “temporal correlations between spatially distinct neurophysiological events” that characterize them during task-free states (like lying down with your eyes closed or sitting still). This is equivalent to describing the typical noise and pitch of an idling truck. When you focus on tasks or achieving goals, the DMN deactivates somewhat. In the metaphor, the truck changes what it’s doing, it is now in gear and driving.

Our brains have the kinds of systems that always teeter back and forth in a non-equilibrium state (i.e. it’s not sitting stably at the bottom of a metaphorical trough or valley but constantly adjusting, fidgeting). Typical of non-equilibrium systems, our brains display self-organized criticality (SOC). This means they naturally move towards a ‘critical’ point, situated at a weird intersection where states transition between order and disorder.

What does this mean when you eat a magic mushroom? Basically, you increase the entropy within those systems. There is “evidence that the brain exhibits more characteristics of criticality in the psychedelic state than are apparent during normal waking consciousness”. Remember, for our purposes criticality is where stuff gets weird and interesting. You brush shoulders with either extremes of the range granted by your non-equilibrium networks. Let’s put this into perspective with other life forms. I apologize in advance to any non-homo sapiens sapiens readers out there for the following passage:

the human brain exhibits greater entropy than other members of the animal kingdom, which is equivalent to saying that the human mind possesses a greater repertoire of potential mental states than lower animals

Booya! In your FACE pandas! [composes himself]

Carhart-Harris and Nutt proposes that we did not arrive at the consciousness we have by ‘taming’ it (entry suppression). Rather, we may have ‘challenged’ it (entropy expansion). This means the disorder of our brain systems increased compared to other animals. Only after that did we resort to entropy suppression (reorganizing, settling). This is the state we are currently purported to be in. This is the state that can be messed with.

To understand our consciousness subjectively, in experiential terms, we need to identify and define that which is being changed or played with: the ego. Freud’s ego, actually, which is “a sensation of possessing an identity or personality; most simply, the ego is our ‘sense of self’ […] Effectively, the terms ‘ego’ and ‘self’ are synonyms, except that ‘the ego’ has a background in Freudian metapsychology [where it] is not just a (high-level) sensation of self-hood; it is a fundamental system that works in competition and cooperation with other processes in the mind to determine the quality of consciousness”. Sorry for the long quote but definitions are important in science.

This paper proposes an Entropic Brain Hypothesis.

Primary consciousness = unconstrained cognition, less ordered (higher-entropy) neurodynamics.

Secondary consciousness = constrained cognition, more ordered neurodynamics, giving us an evolutionarily advantage balancing order and disorder, which might be more or less perfectly ‘critical’.

Driving a car = secondary consciousness

Thinking about the meaning of life = more in the wheelhouse of primary consciousness (but don’t discount the mix of both!)

Most importantly, “the relationship between normal waking consciousness and ‘primary consciousness’ is not perfectly continuous”.

Cognition spectrum

The seeming ‘expansion of mind’ and ‘collapse of ego’ can be reconciled with observations of decreasing rather than increasing measures of brain activity. This can be done by proposing a model characterizing primary consciousness and secondary consciousness integrating into an…”us”. It is the crux underlying the hypothesis put forth by the paper:

A distinction is being made between two fundamentally different styles of cognition, one that is associated with the consciousness of mature adult humans, and another that is a mode of thinking the mind regresses to under certain conditions, e.g., in response to severe stress, psychedelic drugs and in REM sleep. The style of cognition that is dominant in normal waking consciousness will henceforth be referred to as secondary consciousness and the (pre-ego) style of cognition that is associated with primitive states will be referred to as primary consciousness

So how did Nutt and Carhart-Harris start testing the effects of psychedelics on human consciousness? In a placebo-control design, they put people in an fMRI scanner to observe changes in CBF (cerebral blood flow) using a technique called ASL (arterial spin labelling). Strikingly, “results revealed decreased CBF after psilocybin and no increases. The decreases were localized to high-level association cortices, including key regions of the DMN […]”. Using another kind of measure, BOLD (blood-oxygen level dependent) they repeated the measurements and found signals consistent with CBF measurements just obtained.

So magic mushrooms don’t over activate certain areas, on the contrary, they seem to quiet them!

They confirmed these patterns using 2 other measures: functional connectivity (FC) between different networks and oscillatory power between different brain regions. FC and oscillatory power also decreased. They are careful to note that there was no direct measure of entropy, per se. What can be said with more confidence is that there was disorganizing of brain activity. Doesn’t that make you think of the artists’ butterfly mind, moved by whatever breeze comes along? And why do we care if the DMN has decreased activity? Because it is “relatively removed from sensory processing and is instead engaged during higher-level, metacognitive operations such as self-reflection, theory-of-mind and mental time-travel” (aka trippy stuff). Decreased activity of the DMN putatively reflects lesser ‘constraint’ (not lesser ‘ability’ overall).

They also found that the DMN and TPN (task-positive networks) had a reduced ‘anticorrelation’. All this means is that the DMN and the TPN become less dissimilar, there was higher entropy (they’re less organized and thus more similar). The analogy is the more untidy rooms get, the more they will tend to resemble each other. When you clean-up a room it is organized in such a way that if you moved things around just a bit, you would notice it. Not so much in very untidy rooms.

This “inverse coupling” between the DMN and TPN is characteristic during ‘dual awareness’ meditation which collapses the sense of duality we hold. This is the spiritual experience as can best be inferred currently. Where “you” and “the world” both start and end is not clearly distinct anymore. Hence the sense of “oneness”. This fits closely with anecdotal accounts and provides a mechanistic starting point for these subjective experiences. This is great science tip-toeing towards an expanse of knowledge usually regarded as outside its realm of investigation. It is not. This is prejudiced thinking. Nothing in principle excludes a scientific approach to understanding consciousness.

In the words of the authors:

mainstream psychology and psychiatry have underappreciated the depth of the human mind by neglecting schools of thought that posit the existence [of] an unconscious mind. Indeed, psychedelics’ greatest value may be as a remedy for ignorance of the unconscious mind

This developing theory of consciousness proposed by Nutt and Carhart-Harris reveals how the prediction of increased brain activity on a psychedelic, as a general pattern, is incorrect. Quite the opposite is observed. Primary and secondary consciousness have been redefined for the exploratory purposes of the paper and they benchmark 2 states the brain transitions between as part of the psychedelic experience. This is a continuum, not a binary situation. It also explores how the dynamics of coupling between different networks change as to become more similar, maybe explaining part of the ego dissolution phenomenon, the sense of “oneness”.


Again, this paper should be read in its entirety because it contains so much more than what I described. I did not do it justice.

Let me know about your experiences with psychedelics. Do you have a better theory for consciousness? Do you challenge the methodology in the paper? What does you astrologer think of all this?

Gut Microbial Metabolism Drives Transformation of Msh2-Deficient Colon Epithelial Cells

The great question posed in Belcheva et al.’s study “What is the nature of the interaction between our microbiota, colorectal cancer and inflammation?” has produced results that can help RECONCILE CONFLICTING EVIDENCE regarding the fiber-colorectal cancer question [CRC = colorectal cancer].

Generally speaking, epidemiological studies on the matter have only provided hints for generating hypotheses that are oftentimes more confused than the questions they’re trying to answer. In Good Calories, Bad Calories, Taubes’ position on fiber is echoed in a New England Journal of Medicine randomized controlled trial that he refers to. It concludes that “adopting a diet that is low in fat and high in fiber, fruits, and vegetables does not influence the risk of recurrence of colorectal adenomas […& that…] two previous trials [Toronto Polyp Prevention Trial & Australian Poly Prevention Project] also found that dietary changes had no effect on the overall risk of recurrence of colorectal adenomas”. This is damning evidence – damning evidence against adopting a modern day, grain-derived, sugar heavy & fat devoid ‘fibrous’ diet.

Ass cancer or Chipotle - that is the question.

Ass cancer or Chipotle – that is the question.

Buyer beware. Yes, yes, yes – the authors do unfortunately disenchant themselves by stating “Nor should we overlook the abundant data indicating that a diet low in saturated fats and rich in fruits, vegetables, and whole-grains has a favorable influence on the risk of chronic disease and mortality”. That statement is incorrect and frankly stupid in 2014. Let’s not waste time over it.

Now that we know consuming lots of fiber according to the ‘modern eating model’ is pretty useless – not only for preventing CRC – let’s do away with a whole load of variables and look at cellular mechanisms up-close to see what different kinds of murine (mouse) epithelial colon cells think of fibre. Let’s dive into Belcheva et al.’s study . Their message is essentially positive about butyrates’ effects on CRC, but cautions (at least in principle) against blind application.

Genes often affected in CRC

  • Adenomatous Polyposis Coli (APC) genes = tumor suppressing
  • DNA mismatch repair (MMR) genes (self-explanatory name)

The researchers approached their awesome question, keeping in mind the “role of inflammation in producing a niche for specific microbes to elicit their oncogenic effects” all the while recognizing that “the etiology of most CRCs does not have an inflammatory component”. Furthermore, “a comprehensive meta-analysis found a positive association of total carbohydrate intake with CRC”.

Bullshit aside - where's the CRC?

Bullshit aside – where’s the CRC?

To TEST their hypothesis, they make:

  • a mouse model [APCMin/+ (multiple intestinal neoplasia)] of human adenomatous polyposis
  • & cells with a “Mutation in or inactivation (via silencing) of MMR genes, such as MutS homolog 2 (MSH2)

They give an APCMin/+ mouse MSH-deficient cells and observe the growth of many more polyps.

Naturally, the question “Does the microbiota affect the #/growth of polyps?” arises. Well, lets see what happens when you take their microbiota away: “Polyp incidence is not reduced in germ-free APCMin/+ mice (Dove et al., 1997), indicating that gut microbes have little to no role in disease progression in this background.”

Ok – so what about antibiotics?

  • oral antibiotics dramatically reduced CRC specifically in APCMin/+MSH2-/- mice”

OK – since carbs “feed” bacteria and antibiotics “kill” ‘em, what’s up with carbs on the #/growth of polyps?

  • “a diet reduced in carbohydrates can phenocopy this effect and show that gut microbes stimulated CRC development through the production of carbohydrate-derived metabolites such as butyrate

INTERESTING. That which feeds our microscopic partners going back eons & eons is also implicated in feeding CRC. Hhhmm…if it’s not on already, tightly fasten your skeptic helmet.

The researchers elaborate further:

  • “The fact that antibiotic treatment led to a reduction in grade 1 polyps, which include aberrant crypt foci, argues that gut microbiota in APCMin/+MSH2-/- mice act at an early stage in the formation of CRC, perhaps even as a tumor initiator […with the caveat that…] not all members of the gut microbiota contribute equally to CRC development in this animal model”.

A hint as to possible mechanisms (or the elimination thereof):

  • “the mutation frequencies were similar between colon epithelial cells from untreated or antibiotic-treated MSH2-/- mice”

But how does the mice microbiota contribute to CRC formation?

  • “gut microbiota induce CRC through a mechanism that is independent of both inflammation and DNA damage.” So we know what mechanisms AREN’T responsible but still don’t know which one(s) IS/ARE.

Carbs as a function of microbial depletion – what happens in that paradigm?

They checked by setting up “Three-week-old APCMin/+MSH2-/- mice [who] were given a normal or low-carbohydrate diet (Table S1). Approximately 58% of the calories provided with the normal diet derived from carbohydrates, compared to 7%

2 things surfaced:

  • “Strikingly, the low-carbohydrate diet reduced polyp numbers in the small intestines and colons of APCMin/+MSH2-/- mice”
  • no additive effect on polyp number by combining the low-carbohydrate diet and the antibiotic treatment (Figures 3B & 3C) suggesting that both treatments function by the same mechanism

It is reasonable to think: maybe the decreased metabolite levels (i.e. poop from fermenting fiber/resistant starch) resulting from carb restriction is the deterministic factor in tipping the balance towards CRC? The authors think so:

  • “reducing either gut microbiota or dietary carbohydrates resulted in the reduction in both Ki-67 and β-catenin expression in APCMin/+MSH2-/- mice led us to hypothesize that bacterial metabolites might fuel the aberrant hyperproliferation of colon epithelial cells in these mice”.

So now we need a better mouse model to tease out the (potential) effect of metabolites (i.e. butyrate) on polyps (as a CRC proxy marker):

  • Previous mouse model = inadequate. It was MMR-deficient AND DDR-deficient (MMR also works via a ‘DNA Damage Response’ pathway)
  • New mouse model, succinctly named MSH2G674D/G674D = adequate ===because===> they have a functional DDR mechanism but CANNOT carry out other MMR functions (this is called controlling for variables).
    • ==> ‘what mechanisms do what’ can now be narrowed down via a process of elimination.

MSH2G674D/G674D mice revealed “the only SCFA that was statistically reduced by all antibiotic treatments and by the low-carbohydrate diet was butyrate”…Specifically, these treatments led to the reduction of three families within Firmicutes, namely Clostridiaceae, Lachnospiraceae, and Ruminococcaceae (Figure 6C), that are known to produce butyrate”.

From their controlled 2nd mouse model they remark on the plausible mechanism of butyrate’s **apparent** proliferative properties in murine epithelial colon cells

  • “butyrate modulates canonical Wnt signaling (Lazarova et al., 2004) and has been shown in some studies to promote CRC (Freeman, 1986; Lupton, 2004)”
  • Canonical Wnt signaling = regulates gene transcription, cell size and calcium levels inside the cell; all majorly important factors determining if cells live or die…so yes, of prime interest in cancer.

This is the CRUX of the study ===> “To TEST whether butyrate directly affects polyp formation in APCMin/+MSH2-/- mice, antibiotic-treated mice were fed a diet enriched in tributyrin, a stable form of butyrate that breaks down into three butyrate molecules in the gastrointestinal tract

  • 50 mM and 0.5mM of sodium butyrate, which represent concentrations of butyrate found in the distal part of the colon (Donohoe et al., 2012a), stimulated proliferation of colon epithelial cells in APCMin/+MSH2-/- mice but NOT in controls
  • AND “high concentrations of sodium butyrate (i.e., 10 and 100mM) did NOT increase colon epithelial cell proliferation in APCMin/+MSH2-/- mice

KEY POINT: I’m underlining “in APCMin/+MSH2-/- mice” like it’s going out of style because the study should not be extrapolated as saying

  • ‘butyrate = horrible for humans via mouse proxy’
  • but rather that ‘if certain cell repair mechanisms are non-functional, butyrate (through no fault of its own) can still fuel dastardly clever murine mutant epithelial colon cells
    • In technical speak: “because deregulated β-catenin signaling is a marker of early neoplastic changes of intestinal epithelium (Van der Flier et al., 2007), our results suggest that MSH2-deficient colonic epithelial cells are highly predisposed to transformation
  • And consequently, the authors say the “gut microbiota plays a key role in CRC by providing metabolites such as butyrate that ’fuel’ the transformation of murine APCMin/+MSH2-/- colonic epithelial cells” [into an “aberrant hyperproliferation phenotype”]

So, we’re left with:

  • Our study supports the carbohydrate-cancer link by showing that a diet reduced in carbohydrates resulted in reduced polyp formation in APCMin/+MSH2-/- mice
  • YET…”butyrate has been shown to modulate canonical Wnt signaling, and depending on the status of β-catenin activity, colon epithelial cells respond differently to butyrate (Lazarova et al., 2004)” ==> i.e. the butyrate paradox. It is not a paradox, just that the dose-response curve is not linear but rather U-shaped (as so often is the case) & dependent on the particular cellular metabolic milieu. OF COURSE this matters.

What to conclude? Let us think about these 2 points:

  • The authors’ conclusion of “a diet reduced in carbohydrates as well as alterations in the intestinal microbial community could be beneficial to those individuals that are genetically predisposed to CRC
  • A paper referenced by Belcheva et al.’s group states that “The Warburg effect dictates the mechanism of butyrate-mediated histone acetylation and cell proliferation”] and explains how butyrate metabolism is impaired in cancer cells. It remarks on the fact that “butyrate has opposing effects on cell growth: it inhibits cancer cell proliferation as an HDAC inhibitor but stimulates the proliferation of noncancerous cells (and cancerous cells when the Warburg effect is blocked) by being oxidized as an energy source
    • More details about 2 histone-base mechanism butyrate uses in its **seeming** flip-flops:
      • Colonocytes near the base of crypts receive tiny amounts (uMs) of available butyrate and so makes us of the acetyl-CoA/HAT mechanism for histone acetylation (inhibiting aberrant proliferation). The “acetyl-CoA/HAT mechanism involves metabolism of butyrate in the mitochondria followed by the subsequent ACL-catalyzed production of acetyl-CoA.”
      • Luminal colonocytes however receive more available butyrate (maybe in mM quantities) and thus uses another histone-based mechanism (for inhibiting aberrant proliferation), HDACinhibition, where higher levels of butyrate surpass the oxidative capacity of the cell, causing it to accumulate butyrate within.
  • NO FLIP-FLOP, just AWESOMENESS: “our transcriptome profiling results indicate that they upregulate different targets, with the former (acetyl-CoA/HAT) enriched for cell-proliferation genes and the latter (HDAC-inhibition) being enriched for apoptotic genes
  • THE GOLD: “These changes in gene expression are consistent with the lower doses of butyrate stimulating cell proliferation, while higher doses inhibit proliferation and increase apoptosis. These findings lead to a model whereby butyrate facilitates the normal turnover of the colonic epithelium by promoting colonocyte proliferation in the bottom half of each crypt while increasing apoptosis in those cells that exfoliate into the lumen

I conclude that our bodies really like fat as a fuel. Being the opportunists that we are, we do have long-term bacterial friends that can give us that oh-so-good fatty-fat-fat we love if we give them carbohydrate. Redundancy & mechanistic diversity are true evolutionary treats.

Taken together, it seems like a good idea to feed your colon cells plenty of butyrate and lower your total ‘sugar burden’. You can ingest sources of fiber that will not (usually) induce mutant cell genotypes. Not all sources are equal. Your colon cells like a high-fat diet and so do other cells in your human body. Fiber does and probably should play a role in a high-fat diet. How big a role? No idea. You probably can include quantities that satisfy your taste as a starting point.

Lastly, ketogenic diets are NOT synonymous with fiber poor diets. They fact that people implement them as such says more about their lack of understanding than what a ketogenic really is or can actually be when done properly.

TL;DR A varied and nutrient dense ketogenic diet combining evolutionarily concordant foods (for the most part), including vegetation feeding colon cells butyrate (& other metabolites) by bacterial proxy seems not only reasonable, safe and tasty but also a good strategy for avoiding god damn ass cancer.

Interview with Gary Taubes: Obesity & Calories [2012]

Around 2010-2011 my “Italian & Management Studies” course at University College London started boring me to the brink of death. Thankfully, I regained some child-like curiosity, pushing me to ask questions about our natural world. I also gained a newfound appreciation for just how good a companion the scientific method would be in this exploration.

At the same time, I started learning a lot about cannabis and soon thereafter nutrition grabbed my attention. Good Calories Bad Calories was the scientific ‘tour-de-force’ which laid the foundation for my voracious appetite concerning all things health, science (& more). I am currently in a part-time distance-learning MSc in Molecular Biology at Staffordshire University (UK).

 I emailed Gary Taubes asking him for an interview while making clear that I was essentially a fan, interested in pursuing a career in science/nutrition/something vague. He gladly accepted. I was and still am very grateful for the kindness he showed.


Caveat #1: my level of understanding about probiotics back then was essentially 0. Now it is significantly more informed. Yes – ‘the more you know, the less you know’ thing applies here: answers bring up more questions. Probiotics may be likened to the ‘other side’ of antibiotics, naturally granting them a fundamental role to play in our understanding of human health. A fascinating area of science!

 Caveat #2: This is the ‘full sentences’ transcript. I can provide the unedited one upon request. The write-up is mine alone and as far as I know Gary Taubes has not yet read it.


YouTube isn’t happy with me uploading the .MOV audio interview file so here it is provided as DropBox link:



 NuSIGary Taubes

Interview with Gary Taubes: Obesity & Calories [Transcript 11/06/2012]


Gary Taubes: […] Obesity is caused merely by taking in more calories than we expend, that’s the conventional wisdom. The argument that I’ve been making is that obesity is a hormonal regulatory defect just like any other growth disorder and the obvious trigger for it is the carbohydrate content of the diet working through the hormone insulin.

Raphael Sirtoli: Ok, so we can establish that first of all. What do you see as the major challenges to the food industry over the next 10 years?

GT: A large part of the food industry is selling processed carbohydrates and sugars, the bulk of which comes from supermarkets here with grains and cereals and products with sugars laced into them. So the question is what about fruit juices and sodas then?

R: We would effectively have to change the whole base [of food products] for the majority of the supermarket food stores.

GT: Let me rephrase this. I was thinking about this recently because it is not like we have stopped selling cigarettes because we have accepted that cigarettes cause lung cancer and probably heart disease and emphysema and a host of other ailments. We have just put warnings on them. It is hard to imagine warnings on cans of Coca-Cola and Pepsi and on bottles or cartons of apple juice. I’m not so optimistic that the medical community will ever get around to accepting that these things are kind of inherently deleterious.

R: It’s one thing to get these things accepted as empty calories, but another to get them to be accepted as something actually poisonous (with regards to fructose & sucrose).

GT: Exactly. It is something that literally causes chronic disease, so I do hope we do win that battle and it does eventually get accepted. However this challenges the existence of many food companies. These are not people who see themselves as peddling poison. They are people like you and me who basically think that they are doing good things with their lives and paying for their children’s college education. So I don’t know, I really don’t know. I hope the next 10 years will play out because the message is that you can think of them as a type of poison and you could argue that there is a dose beneath which they are harmless – which is probably true – but we don’t know what that dose is.

R: It would be hard to address that.

GT: Yes, and I would argue there is probably a dose at which cigarette smoking is effectively harmless, but the medical community disagrees on that too.

R: How do scientists typically investigate whether certain foods are good for us or not? Is there a ‘standard model’ of approach when studying the health aspects of food? Or does it mostly depend on what you’re looking for?

GT: That is a complicated question as well. Our Food and Drug Administration has mechanisms in place for determining whether or not a product is what they call generally recognized as safe. There are obligations that food companies have to fulfil if they have to add additives to foods so that the additives are considered safe enough to use. If you took naturally occurring foods like half a dozen apples and turned them into a glass of apple juice for instance, I don’t know if there is any precedent for establishing whether or not you have now turned that into something that in the long term will cause chronic disease. So how would you test it? Say you had a hypothesis that said that juice was toxic and so now you wanted to test that hypothesis. We are talking about diseases that develop over decades. The question is, what do you compare the juices to? If you just remove juice – say you take 20,000 people and randomize 10,000 of them to a juice-free diet – you are probably going to lower their caloric intake because you are going to remove the calories from the juice as well as the sugars in the juice that represent those calories. So, then you decide, what to replace those calories with and if I should give the other 10,000 people beverages that are sweetened only with glucose? In which case they might still drink less of them because they don’t stimulate their taste buds and their physiology the way beverages sweetened with sugar do. It is surprisingly difficult and complex.

I can tell you one thing we have done. We have actually started a not-for-profit organization, ‘We’ being my colleague Peter Attia and I. It is called the Nutrition Science Initiative and we now have backing from a very well endowed foundation from Houston in Texas. Our argument is that fundamentally the science of nutrition has been substandard over the years. Nutritionists and disease researchers have relied on very poorly controlled experiments or observational studies or animal models to come to their conclusions. None of these are then sufficiently rigorous or up to the task to determine what we really need to know about a healthy diet. So we are going to fund very well controlled experimental trials with humans and come up with innovative ways to do these studies. We can keep human subjects on the diet for 6 months or a year if necessary and will be able to establish if they did not eat or did eat and what was given to them. All of this would be a pipe dream if not for this Houston foundation, the ‘Lauren & John Arnold Foundation’ that is basically making it possible for us to do something we never imagined we could do before. We’re launching July 1st 2012.

R: That’s good news!

GT: Yes, that’s very exciting. We had our first meeting in Bethesda Maryland with members of our scientific advisory board and researchers from the NIH and elsewhere who want to do the first experiment that we want to fund.

The next couple of years are going to be learning experiences on how excruciatingly difficult this is – even when you have the money – but we are going to do it, and it is pretty fascinating.

R: There is a definite need for that. There is too much contrary evidence to the calories-in calories-out hypothesis to just have it be accepted as such.

GT: It is interesting. I already had a conversation with a physician researcher at Yale who very much believes the conventional wisdom. He effectively told me nothing we funded would make a difference in what he ultimately believes. This is because what he ultimately believes is determined by observational studies like the nurses’ health study.

The argument is we could do a 6 months study that could tell you that weight regulation is not determined by caloric intake and expenditure but by the nutrient content of the diet. This is the first study we want to do. It is the kind of study that should have been done rigorously in the 1960s but was not. Even if the counter argument will be, ‘well look, that only tells us what happens over 6 months, it does not tell us what happens over a lifetime’, well we just cannot do studies over a lifetime, so…

 R: It is a self-defeating argument: ‘we cannot do it because it is too difficult [& thus never try to]’. It completely ignores the fact that other studies encounter these barriers as well.

GT: In the mean while we use these observational studies which I believe are worthless and cannot be interpreted correctly. You know in the ‘Step 1’ of science they generate hypotheses, but that is all they do. However, [the over-interpretation of observational studies] is still rampant in medical research. They are everywhere in nutrition and chronic disease these days. One of our goals at the Nutrition Science Initiative (NuSI) is also to get people to understand how misleading these observational studies are and why they are not a substitute for hard experiment. If you don’t have the rigorous experimental trials you don’t know anything and it’s that simple.

R: We’ll definitely take note of that. Is a healthy diet the same for all populations or do certain populations require different diets? Like when considering geographic populations for instance.

GT:  That is an interesting question. I think the argument that I’m making in my book is that sugar and refined grains are sort of the fundamental problems, the primary nutritional evils in our diet. I would argue that that is true for every population. Although there might be populations that are more adapted to particular carbohydrate rich food. For instance, maybe South East Asians can tolerate rice better than other populations because the have been eating it for thousands of years. Although when white rice was introduced in the 20th century to populations it did cause Berri Berri I think. We will have to check this.

 R: Yes, it was a lack of Vitamin B12 I think.

GT: Yes so that is a possibility. The fundamental argument I’m making is that refined grains and sugars are the problem. You remove those from the diet and every population is going to be healthier. You minimize them in every population and they will be healthier. Are some populations more susceptible to these foods than others? I think that is pretty clear, because some populations like African-Americans in the U.S.A or Hispanics in the U.S.A have higher rates of obesity and diabetes than the Caucasian population which suggests that Caucasians had more time to adapt to these foods. Let’s say centuries or rather millennia instead of just a few centuries. So we can tolerate them a little bit easier. The argument that some people lose weight on say, low-fat diets and others lose weight on a low-carbohydrate diet and that that is a genetic issue or that it maybe depends on whether your insulin resistant is looking at it the wrong way I think. I think low-fat diets are just poorly constructed low-carbohydrate diets and some people can still benefit from them even though the carbohydrate restriction is not as great as it could be. If you look at low-fat diets they are just kind of restricted carbohydrate-diets in disguise.

R: […] because of the proportions.

GT: Yes exactly. Some people are metabolically healthy enough that their bodies will still respond to that level of carbohydrate restriction while others are so metabolically disturbed that they need much more carbohydrate restriction. You do that with a higher-fat diet. Not to say that the person who responds to the low-fat diet would not respond better to the low-carbohydrate diet, they just get enough benefit from a low-fat diet or at least from being told to go on a low-fat diet that that can be enough.

R: Should we then be confident in saying that a few thousand years – like in the case of Asians getting used to eating rice – is enough to see a small adaptation to a new introduction of food source?

GT: There is a lesson in evolution there. Have you ever read the book “Beak of the Finch”?

R: No I haven’t unfortunately (laughs).

GT:  Well it won the Pulitzer Prize here in the U.S.A a while back and I forget the author’s name at the moment. It is a terrific science book about evolution. The lesson from the research that the author was following was that 2 or 3 generations are enough to have a sufficient impact on a population’s ability to adapt to a new feature in the environment. So for instance you can imagine a situation in which sugar and refined grains are introduced into a population and the people are really susceptible – particularly the women – and they get obese and diabetic pretty quickly. This would have profound reproductive disadvantages and so would not take many generations before the most susceptible individuals to these aspects of the diet were weeded out because of the reproductive disadvantage. Maternal mortality during birth along with birth defects and the death of the infants are examples. All these things come at a pretty high frequency with uncontrolled diabetes.

R: The associated risks with diabetes will do the selecting.

GT: You can imagine this kind of situation where the women who are most susceptible to these carbohydrates might have been perceived as more or less attractive as mates. It is hard to say. One of the lines that keeps haunting me – I’m doing a new book now, very slowly but I’m working on it. It is on sugar and high-fructose corn syrup and the history and politics. I found an essay online by a physician writing around the 18th century. I think it might have been 1730s and he is actually defending sugar in the diet against claims that sugar is toxic, back then.

R: Back then!

GT: He is saying sugar isn’t toxic, it is completely harmless. But he then has an aside where he says ‘but of course it makes young girls fat’. Other than that…If that was true that it made young girls fat, those young girls in the 18th century would have been eating about 1/20th the amount of sugar our young girls are eating today. Is it possible that this was accurate? You would like a lot more evidence than one doctor’s word. However, it was interesting that it came in the context of an essay defending sugar and not in an essay attacking it.

R: Yes actually making that axiomatic point about sugar; that it will just do that to you [fatten you up].

GT: Yes, so the question is, ‘how much is necessary to do real harm when it is introduced into a population that is completely new to it?

I read a lot of memoirs of naturalists and explorers who interacted with aboriginal populations around the world. I read reports wherever I could find them; government reports, studies of aboriginal population and so on. There were these reports from Australia about the local Aborigines and they would describe them as almost decimated within a decade or one generation by the introduction of Western foods into their diets. The question is how, ‘how much of it was due to alcohol? Or sugar and flour? Or vegetable oil? Saturated fat? Lard?’ Who knows. These Western diets do, however, cause an enormous amount of harm. You would also have to worry about infectious diseases. However, those tended to be pretty obvious when they hit. The point is that the idea that these things did their harm at relatively low doses compared to those we take today and they did it quickly, is at least out there in the literature. Hopefully we will never have a chance to test that one.

R: Hopefully not! This leads me on to my next question. How does age affect one’s’ diet? Does it have an effect at all? If so, how?

GT: Many of us have this observation that we could eat anything in our youth. When I was in my early teens I played American Football in college and tried to get as large as humanly possible because that is what you want with people who play my position in football. Despite eating constantly and massive amount of foods I could not get over about 235 pounds. By the time I was 35 years old I could get to 235 pounds effortlessly and the battle was to not weigh 235 pounds. In fact, I got an email a couple days ago from a Green Beret (a Marine) who described his struggle with his weight and how despite being one of the most fit army/military men you can find he said that as he got older he kept getting bigger and bigger. This is a pretty common observation. The conventional wisdom is your metabolism slows down so you expend less energy and therefore the same amount of calories consumed comes in excess to the energy expenditure. The argument I make in my books and that I find more reasonable is that we change the way we partition our fuel. With the fuel we do consume, we go from burning to storing it. It is probably a result of a change in insulin signaling which is the primary hormone involved with this fuel partitioning. We thus go on to become more and more insulin resistant or our fat tissues become more and more insulin sensitive. Some combination of this and you could easily imagine a situation where the biggest proportion of the food you eat goes to being stored and a smaller proportion goes to energy expenditure. You perceive this as a decrease in metabolic rate and impulse to exercise, while in reality is it is simply a change; an effect of this change on your fuel partitioning.

R: Does the question then become more of a comparison argument where you are comparing, say, the natural metabolic derangements that accompany aging versus the chronically elevated levels of insulin which come with the Western Diet throughout a lifetime? Is it a mixture of both then?

GT: Well, again I would argue that it is the Western diet that is changing the insulin signaling and the carbohydrates in it changing the insulin signaling which is in turn changing the metabolic profile. It’s kind of interesting because you could imagine a decrease in energy expenditure in all people as we age. I doubt that 70 year-old hunter-gatherers (if they lived that long) were as active as 2 year-old hunter-gatherer’s or 4 year-old hunter-gathers. If you think of lions and predators, the adults sit around all day when they are not hunting and the children are playing and frolicking. This would all be a manifestation of how their bodies partition fuel and which energy is being used for expenditure. So when expenditure comes down, intake should come down to meet it. If somebody gets bed ridden there is no reason why they should continue to eat as much as they otherwise would. If a lumberjack had an accident and he is in bed for 6 months you would assume that his appetite is going to be less than when he was working as a lumberjack and cutting down trees all day long. That is kind of the issue where if you are gaining fat then there must have been some change in your regulatory system regulating fat accumulation. It’s not just enough to imagine you decrease expenditure because decreasing expenditure should go along with a decrease in intake.

R: Do you think that men and women require the same diets? Could you generalize or not?

GT: Would men and women both benefit from eating less refined grains and sugars? Yes absolutely. Is there such a thing as an ideal diet? That is I guess the question you could ask. What would that diet be? I guess it would maximize health and longevity and perhaps energy expenditure so that would mean that you are physically active. For women that would also mean fertility and the ability to nurse and so on. There are a lot of different things you would have to imagine this ideal diet could do. I would argue that if you remove the refined grains and sugars and easily digestible starches then you are pretty much along the way. You know you have done about most of what we know or what I think we know you could do to improve your diet. Although that does depend on what you replace it with. You could argue that women sort of evolved consuming a different diet if there were any evidence from hunter-gatherer populations or any evidence that they sort of naturally consumed more gathered foods and perhaps ate slightly less more hunted foods (laughs)…But I’m not a big fan of evolutionary arguments other than that it makes sense that we did not evolve to eat the foods we are eating now (laughs). One of the reasons that this makes sense is because we have all the chronic diseases that they seem to cause. So it is not just that a new food is inherently bad but if a new food goes along with new chronic diseases and those can be pretty easily explained by the presence of the new food, then there is a pretty good chance that the new food is bad. Whether certain combinations of old foods or foods that we did evolve to eat are more or less ideal than other combinations of these and whether this change is gender specific, who knows really.

R: Yes, the required studies have not been done yet.

GT: They have not been done. They probably never will (laughs).

R: (laughs) Ah, to hope! In your opinion what should concern us about genetically modified foods? Should we be concerned in the first place?

GT: I don’t really know in that sense. This is not one of my areas of expertise. My gut feeling is that they are relatively harmless. The foods we are modifying are foods that I’m arguing we probably should not be eating to begin with. So there you are.

R: Especially when you think about all the corn and grains.

GT: Yes. I will make one comment about this argument though. The primary problems come from genetically modified grain – wheat in particular and hence the wheat belly argument. The thing that I point out in the first chapter of ‘How We Get Fat’ is that all these populations that had high levels of obesity and diabetes were not eating genetically modified wheat. Usually it was diabetes researchers who were measuring weights in these populations that assessed this. The idea that you could still get significant levels of obesity and diabetes without any kind of genetically modified super wheat to do it is there. These were populations before the 1980s for the most part and some of them pre 1970s.

R: Historical basis.

GT: Yes, white flour and sugar were probably sufficient [to explain it]. Whether the way we make our wheat today makes it worse is possible but I would kind of doubt it on principle until I saw really compelling evidence to back it up.

 R: A simpler question I guess. What should the 3 most important points on a food label be?

GT: Contains sugar, contain HFCS, contains sucrose. Basically, CONTAINS SUGARS. I would not actually worry about 2 or 3.

R: The other people I interviewed are so quick to label as many as they can.

GT: I have these ongoing discussions with my wife. We have a 3 year old and a 6 year old and like with many kids it is hard to get them to eat and it is hard to get them to eat the foods you want them to eat. We live around Berkley California which is a kind of health mecca and the supermarkets are full of all kinds of supposedly healthy foods and healthy probiotic yoghurts that will improve my child’s gut biota. So the argument that I keep making to my wife in is that if it makes a health claim, look to see how much sugar it has in it.

R: I see, we should keep sugar as a point of reference.

GT: Yes. It is the same thing you see now with the gluten issue. It is very easy to make, or I assume it is relatively easy to make gluten-free products and then advertise them as gluten-free. However, if you then look for the thing that says that they are also sugars-free or sugar and HFCS-free, you are not going to find it because it is a lot harder to make a tasty food without sugar than it is without gluten. I feel like the gluten movement has been embraced by industry because it is a relatively easy way for them to look like they are producing a healthful food when in fact they are actually producing a food that still has sugar in it that their clientele and the public will buy now that we have become used to high levels of sugar in everything. It makes it a little bit more difficult.

R: It has skewed the norm.

GT: It has skewed the norm, yes.

R: Ok. What role do you think the probiotics have in healthy diet? Do they have any role at all?

GT: That is not an area of expertise for me…[sighs] I just have not really looked into it. I’m quite a bit of a one-trick pony. I spent all my effort learning enough to write Good Calories Bad Calories. By that time I had my second child and since then I have spent most of my effort and energy trying to get people to accept and understand these ideas. I want the medical research establishment to take them seriously and test them, but I literally don’t have time to or perhaps even the intellectual capacity anymore to take on a completely new subject. With the probiotics in general I could probably bet you that there is not enough randomized controlled evidence to suggest that they are actually doing something meaningful.

R: Yes.

GT: If you always asked yourself “what would it take to test this idea?” and this idea was for example that they are supposed to get rid of a symptom, then for instance you go on antibiotics, which tends to wipe out your gut biota. So you then replace it with the healthy bacteria in the probiotic. I can see the logic in that and I can imagine it would be pretty easy to test it either with an N=1 [type of experiment] or a relatively short experimental trial. Whether or not you are going to be healthy, live longer and have less chronic disease simply through a change in your probiotics is harder to accept. This is because you can imagine what it would take to test that. I can imagine that it has never happened [the experimental trials].

R: Yes. Another person I interviewed was telling me that out of the 800 health claims that probiotics seemed to have, all 800 had not yet been proven.

GT: [laughs]

R: I haven’t gone to check that for myself yet, although I wouldn’t be surprised if that were the case. [laughs]

GT: You get these huge efficacy effectiveness studies with pharmaceuticals because they can get patents out of them and there are people who stand to make a lot of money if they could come up with the drug that does something beneficial without an equal amount of harm. However, with naturally occurring bacteria there is nobody who is really motivated to spend the money necessary to find out about them and do the experimental trials to establish if they are really doing something. The fact that they might seem to make a difference if you were to take them yourself is interesting. But the history of science is a history of how easy it is to be deluded. I’m always amazed when people tell me – well first let me tell you, I have a friend who does a lot of, what is it called, ‘Self experimentation’. It is one thing when you are looking at the blood risk factors for disease and do something like measure your LDL particle number. However if your influences are subjective experiences like how much sleep you got, how smart you might feel that day, how energetic you are, even whether you have more or less gastrointestinal upset and so on, it then becomes extremely easy to be fooled. There could even be some kind of psychosomatic placebo effect, so you just never know.

R: Ok. Do you have time for a last question?

GT: Yeah sure.

R: I just had a cholesterol blood test recently and I was very curious to see my results after having significantly adapted my diet. What are the primary parameters that one should focus on when receiving a blood test/cholesterol analysis?

GT: I have almost been convinced that the most important number is the LDL particle number. There is a whole host of studies now that show that if you measure LDL particle number you can get a pretty good assessment by measuring Apo-B, the protein component of LDL particles. When we actually look at the number of LDL particles, the size and density issue kind of washes out. The idea that small dense LDL particles were bad – something that I wrote about in’ Good Calories Bad Calories’ – appears to be so because when you do have a lot of small dense LDL particles, you then also have a lot of LDL particles.

R: Ok, I see.

GT: So the total number of LDL particles is the factor that best tracks with heart disease and atherosclerosis. The hypothesis behind it is then driven by the triglycerides, which the state of the science today suggests is in turn driven by the carbohydrate content of the diet. It does not change the ultimate end point, which is if you want to improve that number as best you can, go on a low-carbohydrate high-fat diet. Although it does suggest that if there were one thing you wanted to measure that would be it, the LDL-P or Apo-B, both of which will give you the number. If you were to get the test for your particle size and density, you would probably see that [tracking] 199 times out of 200.

R: Ok, so are you saying that the LDL particle [composition] itself is less important than the actual number of the LDL particles (or specifically the Apo-B lipoprotein)?

GT: Yes. For Apo-B you will get LDL plus VLDL and IDL, but LDL makes up about 95% of it as I understand. Again, this is something where I have not spent a lot of time looking into so I’m relying on people who have and whose judgment I trust, including my colleague Peter Attia. What would the single best number be? Well, it is interesting when you talk to people who swear by LDL particle numbers. They will dismiss anything you could learn from any tests like these VAP tests which will give you size and density [of LDL particles] or simply getting triglycerides and HDL measured. You could say that in most cases you would learn enough from knowing if your triglycerides were high and your HDL was low and be pretty confident you are somewhat insulin resistant. I find it a bit odd to be dismissive about that. The way to deal with this [insulin resistance] is to get rid of the carbohydrates and replace them with fat. If your LDL particle number is very high, in most cases what that is telling you is that you are insulin resistant. The few times it is not, it is probably telling you that you have got what is called hypercholesterolemia which is a genetic defect. That is one of the reasons why you can argue that it is particle number and not size and density [that matters] because people with familial hypercholesterolemia have large fluffy LDL particles but just have so many of them and get heart disease at relatively young age. I think that if I were to pick a number circa 2012, that [LDL-P] would be it.

R: After following a relatively high-fat and high-protein diet I have to say how impressed I was by how closely my figures tracked to what the theory in ‘Good Calories Bad Calories’ suggested.

GT: Well thanks!

R: It was quite amazing. I had high total cholesterol, a pretty low Apo-B protein count that was within the ranges and good HDL. It is always nice to see that confirmed on an individual basis.

GT: I get emails everyday now from people discussing their LDL numbers or how their weight has changed. Some people like the Green Beret discussed how these books finally helped him understand what had been happening to him his whole life. It is one of the reasons why when I do get optimistic (I’m not a naturally optimistic person) it is because it seems so obviously right. It is hard to imagine that.

R: I think it really is paradigm shifting.

GT: Yes.

 R: You cannot really escape that conclusion.

GT: Yes.

R: Well it certainly was very interesting to speak with you.

GT: Thanks!

R: And thank you for the opportunity.