June 2013 Blog with Durk and Sandy


There is a certain relief in change, even though it be from bad to worse; as I have found in traveling in a stage-coach, that it is often a comfort to shift one’s position and be bruised in a new place.
— Washington Irving

When you came to examine the American Constitution, you found that it was not really a constitution, but a Charter of Anarchism. It was not an instrument of government: it was a guarantee to the whole American nation that it never should be governed at all. And that is exactly what the Americans wanted.
— George Bernard Shaw, address in New York, 11 Apr. 1933

I will be brief. But not nearly as brief as Salvador Dali, who gave the world’s shortest speech. He said, “I will be so brief I am already finished,” and he sat down.
— E. O. Wilson

John Major relates a serious conversation with Boris Yeltsin on Russia’s economy: “Tell me, Boris, in one word, how is Russia doing? “Good.” “In two words, Boris, how is Russia doing?” “Not good.”
— from What Is Your One Sentence?
by Mimi Goss, a marketing book

The girl said she recognized me from the vegetarian club, but I’d never met herbivore.
— from the 3/29/13,
Casey Daily Dispatch

A fanatic is one who can’t change his mind and won’t change the subject.
— Winston Churchill



A new paper1 reports on highly protective effects of L-arginine (and also D-arginine) against endothelial dysfunction, increased AGE (advanced glycation endproduct) formation, and other features characteristic of type 2 diabetes by scavenging of methylglyoxal, a reactive dicarbonyl molecule produced during glucose, fatty acid, and amino acid metabolism that is a major precursor in the formation of AGEs.

The authors note that there is currently a lack of specific scavengers for methylglyoxal (MG), but that MG has been reported to have a high affinity for arginine. Hence, they tested the protective effects of arginine on the vascular smooth muscle cells and isolated aortic rings derived from rats and then incubated in high glucose (25mM) or methylglyoxal (MG) 100 mM) and tested for relaxation in response to acetylcholine.

Results showed that D-arginine and L-arginine prevented high glucose-induced elevation of MG levels in vascular smooth muscle cells and rat isolated aorta. MG and high glucose increased the expression and activity of the enzyme arginase (an enzyme in the urea cycle that catalyzes the formation of urea and ornithine) which competes with NOS (nitric oxide synthase) for L-arginine. Hence, increase in arginase can reduce the availability of arginine for conversion to nitric oxide by NOS, thus inducing endothelial dysfunction.

Another result of the study was the finding that D-arginine, L-arginine, and N-acetylcysteine prevented MG and high glucose-induced formation of the methylglyoxal-specific advanced glycation endproduct (AGE) N-carboxyethyl lysine (CEL). Coincubation of vascular smooth muscle cells with D-Arg (300 μM), L-Arg (300 μM) or NAC (N-acetylcysteine) (600 μM) attenuated the formation of CEL.

The authors note that, because L-Arg, but not D-Arg, is a precursor for NOS (nitric oxide synthase) and because D-Arg as well as L-Arg prevented MG- and high glucose-induced reduced relaxation, they conclude that “it strongly suggests that arginine, especially D-Arg, prevents MG- and high glucose-induced reduced relaxation by an eNOS-independent mechanism.” “The therapeutic potential of arginine against MG and high-glucose-induced pathology merits further investigation.”1


  1. Dhar et al. Arginine attenuates methylglyoxal- and high glucose-induced endothelial dysfunction and oxidative stress by an endothelial nitric-oxide synthase-independent mechanism. J Pharmacol Exp Ther. 342(1): 196-204 (2012).


Changes in DNA methylation (the methylome) with time are being studied for insights into aging and age-related diseases. A recent study reported some initial findings of a quantitative model of aging using measurements at more than 450,000 CpG markers (hot spots where DNA methylation takes place) from the whole blood of 656 people.1These subjects were aged 19 to 101 years.

The model was designed to “measure [ ] the rate at which an individual’s methylome ages.” The authors report that differences in aging rates were affected by gender and genetic variants and that, very interestingly, tumors reveal an accelerated aging rate.®

Changes in methylation are one reason that identical twins, which start out with the same DNA blueprint, become different with age (for example, becoming divergent in their susceptibility to various diseases), a phenomenon called “epigenetic drift.” Environmental factors, such as the dietary supply of methyl donating nutrients (e.g., choline and methionine) can also modulate gene expression via changes in DNA methylation.

The authors found that their model was able to predict the age of most individuals with high accuracy, with a correlation between age and predicted age of 96% and an error of 3.9 years. “The methylome of men appeared to age approximately 4% faster than that of women, even though the overall distributions of age were not significantly different between the men and women in the cohort (p > 0.05, KS test).”1 On the other hand, BMI did not contribute significantly to aging rate.

The authors report that “[n]early all markers in the model lay within or near genes with known functions in aging-related conditions …” However, “none of the genetic variants were significant predictors of age itself,” indicating that there was a pattern of changes in methylation that predicted age, but that methylation changes in a single gene did not do so. Examining all 70,387 markers of global methylation levels showed that 44% tend to increase and 56% tend to decrease with age. “[T]umors coincide with older values for 74% of the markers regardless of the trending direction.”1

“Interestingly, use of our aging model indicated that tumors appear to have aged 40% more than matched normal tissue from the same individual (Wilcox test, p<10-41).”1

One other interesting finding1 was that there was a loss of information (increase in entropy) in the methylome over time. “An increase in entropy of a CpG marker means that its methylation state becomes less predictable across the population of cells, i.e., its methylation fraction tends toward 50%. Indeed, over all markers associated with a change in methylation fraction in the sample cohort, 70% tended toward a methylation fraction of 50%.” “Furthermore, extreme methylome entropy for an individual was highly correlated with accelerated aging rate …”1

Another paper on DNA methylation changes with age reported on changes in development and aging of the human prefrontal cortex.2 “The human prefrontal cortex (PFC) plays a critical role in complex cognitive behaviors, personality, decision making, and orchestration of thoughts and actions and thus has been referred to as the CEO of the brain.”2

In this study, the researchers “investigated the genome-wide temporal dynamics of DNA methylation in a large cohort of well-characterized human PFC specimens from the second trimester of gestation until old age …” “DNA methylation at CpG dinucleotides has long been considered a key mechanism of transcriptional regulation and a critical factor in embryonic development and in cancer.”2

The researchers divided the PFC specimens by age into the fetal period, the childhood period, and those over ten years of age. The overall methylation changes were much greater during the fetal period, but involved fewer loci than in other life stages. During childhood and in later life, methylation changes occurred at a much slower rate (2–3 orders of magnitude slower).

One interesting finding was that many of the cancer-related genes began showing methylation changes during childhood and continuing into old age. Important tumor suppressor genes were found to have increased methylation levels during aging of adults. Hypermethylation silences the tumor suppressor genes, thereby increasing the risk of cancer.

We are beginning to see a deluge of papers on methylation changes with age. For example, another paper3 reported that age-dependent decreases in DNA methyltransferase and low transmethylation micronutrient levels (methyl donors) synergize to promote increased expression of genes implicated in autoimmunity and acute coronary syndromes. Generally, hypomethylation increases gene expression while hypermethylation silences genes by decreasing expression. Here, the researchers found that the age-associated decrease in DNA methyltransferase was synergistic with low folate, low methionine, or high homocysteine levels that demethylate and activate methylation-sensitive genes. Hypomethylation has also been shown to reactivate hypermethylated (silenced) tumor suppressor genes and that curcumin and EGCG are two natural materials that act as hypomethylating agents.5A,5B An additional paper4reported that axonal regeneration in the repair of the adult central nervous system in rodents was mediated at least in part through DNA methylation and that folic acid, as a methyl donor, promotes methylation.


  1. Hannum et al. Genome-wide methylation profiles reveal quantitative views of human aging rates.Mol Cell.49:359-67 (2013).
    2. Numata et al. DNA methylation signatures in development and aging of the human prefrontal cortex. Am J Hum Genet. 90:260-72 (2012).
    3. Li et al. Age-dependent decreases in DNA methyltransferase levels and low transmethylation micronutrient levels synergize to promote overexpression of genes implicated in autoimmunity and acute coronary syndromes. Exp Gerontol. 45:312-22 (2010).
    4. Iskandar et al. Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation. J Clin Invest. 120(5):1603-16 (2010).
    5A. Liu et al. Curcumin is a potent DNA hypomethylation agent. Bioorg Med Chem Lett. 19(3):706-9 (2009).
    5B. Fang et al. Tea polyphenol (-)-epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines. Cancer Res. 63(22):7563-70 (2003).


Here we continue our series on the use of hydrogen gas in the prevention and treatment of medical conditions, largely having to do with disorders induced by increased oxidative stress. As you may recall when we began this series of articles nearly a year ago (see “Hydrogen Therapy” in the June 2012 Life Enhancement magazine), we explained the special effectiveness of hydrogen as a selective antioxidant that scavenges the highly toxic hydroxyl radical, while having much less effect on radicals that play a role as signaling agents in normal physiology (such as superoxide and nitric oxide), and also scavenges the powerful oxidant peroxynitrite formed by the chemical reaction between superoxide and nitric oxide. We also pointed out that, unlike many other antioxidants, hydrogen is able to enter the mitochondria and scavenge free radicals there where most of the ROS (reactive oxygen species) are produced. Next, we explained the remarkable fact that hydrogen is produced in your lower intestine by gut bacteria, especially when they get larger quantities of fermentable dietary fiber called long chain fructooligosaccharides (LCFOS). From there, the hydrogen diffuses throughout your body, providing protection against hydroxyl radicals and peroxynitrite and in other ways, until leaving your body by exhalation from the lungs, diffusion from the skin, and from farts.

We have been following the development of this new field of medicine, hydrogen therapy, and writing up some of the studies in our newsletters. The one that follows shows how hydrogen was able to provide protection in Wistar rats against TIAs (transient ischemic events), where a section of the brain is briefly deprived of oxygen but not long enough for a complete stroke to take place.

In this study,1 researchers used a common model of these transient reductions of blood flow to the brain by occluding two blood vessels that enter the brain for ten minutes. The experimental animals received hydrogen gas for 3 hours (at a concentration of 2%) inhalation immediately after the operation that was done to occlude the vessels. As a result of this treatment, many neurons die in the targeted CA1 region of the brain. The cause is not entirely understood but oxidative stress is believed to be the main cause. Increases in oxidative stress create molecules that can be measured to follow this damage.

In this study, the transient ischemia in the rats that did not receive hydrogen resulted in increased lipid peroxidation (as indicated by increased levels of malondialdehyde) and the product of oxidation 8-iso-PGF2alpha, whereas the amounts of these ROS (reactive oxygen species) were significantly reduced by hydrogen in the rats treated with hydrogen. Moreover, the hydrogen significantly reduced neuronal death in the CA1 brain region. In a test of cognitive function, hydrogen-treated rats performed better than the rats subjected to ischemia but not receiving hydrogen in the Morris water maze, where animals have to find a submerged platform in a container of water in order to climb onto the platform to discontinue treading water. It took the hydrogen-treated rats a shorter length of time to find the hidden platform.

As the researchers of paper #1 report, there have been earlier studies finding that inhalation of hydrogen gas could decrease the infarct size (the volume of dead cells) in animal models of stroke, limit the size of heart infarction in animal models of heart attack, and protect against generalized inflammation and improve survival in animal models of poly-microbial sepsis.

While the animals in this study received hydrogen gas by inhalation, we have written earlier in this series (see, especially, our introductory article in the June 2012 issue of Life Enhancement magazine) on how hydrogen gas can be obtained conveniently (a gift of certain gut bacteria) in appropriate quantities and periods of time by consuming the LCFOS (long chain fructooligosaccharides) type of dietary fiber.


  1. Ge et al. Inhalation of hydrogen gas attenuates cognitive impairment in transient cerebral ischemia via inhibition of oxidative stress. Neurol Res. 34(2):187-194 (2012).


COMT—catechol-O-methyltransferase—if you haven’t heard of it before, then here is a bit of an introduction to an important part of your dopamine reward seeking system that is appearing in a considerable amount of recent research. It is important because it is the enzyme that degrades dopamine, thus having a major impact on the dopamine level in the brain, especially in the prefrontal cortex, a major site of higher cognitive processes.1 You make a lot of decisions in the prefrontal cortex, including assessing the tradeoffs between getting a reward now or waiting a while and possibly getting a larger reward later. Some researchers call COMT a factor in impatient choice (impulsivity).1

A specific variant (allele) of the COMT gene has been identified as associated with having a steep delay discounting, “the tendency to strongly discount future rewards as a function of their delay and thus choose tempting SSs [sooner-smaller rewards] over even substantially larger LLs [later-larger rewards].” Researchers in one paper1cite papers that show suboptimal life outcomes in financial, academic, and health domain and in different psychiatric or behavioral disorders such as substance abuse, overeating, relationship infidelity, and pathological gambling in people who have the steep delay discounting. The Val allele (compared to the Met Allele) of COMT is associated with a higher level of enzymatic activity and, as it degrades dopamine, lowers prefrontal cortex dopamine levels.1 COMT has a big effect on your life.

Using a sophisticated noninvasive EEG measurement system, the researchers exposed participants to a choice paradigm between SSs (sooner-smaller rewards) and LLs (later-larger rewards) and were able to calculate the intracortical electrical sources that generated the scalp-recorded activity of each of seven frequency bands. They were able, for example, to identify brain regions whose baseline activation correlates with DD (delay discounting), separately for each EEG frequency band.

The researchers concluded that: “Our research suggests that the Val allele predisposes individuals to a low level of baseline activation in the left DPFC [left dorsal prefrontal cortex] which then biases them toward impatient choice.”

There was another study, however, that found the Met allele to be associated with significantly steeper discounting rates, i.e., more impulsive choices, as compared with the Val allele (reported in #4). It was suggested that the discrepancy may be due to the fact that this study1 had adults as participants whereas the other study’s participants were adolescents. “Numerous aspects of PFC [prefrontal cortex] function, including the expression and activity of COMT, change over the course of adolescence.”4

But that isn’t the end of the story because there are natural products, such as EGCG, that can inhibit COMT, thus decreasing the degradation of dopamine, leading to higher levels of dopamine and, hence, potentially avoiding the disadvantage of impatient choice.

Inhibiting COMT To Decrease Dopamine Degradation in the Brain

EGCG ((-)-epigallocatechin-3-O-gallate, the major catechin found in green (or white) tea), has been recently identified as a high-potency inhibitor of the ubiquitous human enzyme catechol-O-methyltransferase (COMT).2 As of 2010 when another paper was published on EGCG’s effects on COMT,3 there were two drugs approved as COMT inhibitors: tolcapone and entacapone. “However, the use of tolcapone is only limited to fluctuating [Parkinson’s disease] patients who are refractory to other therapies, and requires heightened monitoring for the occurrence of hepatotoxicity [liver damage]. Although entacapone is relatively safer, it appears less efficacious than tolcapone.”3

The detailed molecular analysis of the mechanism by which EGCG inhibits COMT shows it to be a potent non-competitive inhibitor that, while able to interfere with the molecular mechanism by which COMT binds to dopamine, renders EGCG itself a poor substrate for COMT methylation.

In one of the papers,3 researchers studied the effect of EGCG both in vitro (human liver samples) and in vivo (modulation of L-Dopa methylation in rats). “When normal rats (treated with L-DOPA + carbidopa) were given an oral administration of EGCG (at 400 mg/kg), their 3-OMD levels [L-Dopa product after interaction with COMT] in circulation and striatum were reduced by approximately 30%, clearly reflecting an in vivo inhibition of L-DOPA methylation.” The breakdown of L-Dopa was reduced, but L-Dopa plasma concentration was increased only slightly (not statistically significant). This was similar to the results of treatment with L-Dopa + carbidopa plus tolcapone or entacapone in rats or humans.3

The authors3 concluded that: “The significant reduction of 3-OMD by EGCG may increase L-DOPA bioavailability in the central nervous system and particularly, reduce potential cytotoxicity associated with elevated levels of 3-OMD.”

Dopamine and Reward Processing

Another recent paper4 provided an overview of dopamine and reward processing that included COMT and a lot more, noting (for example) that reward processing takes place in multiple brain regions, including the VStr, midbrain, orbitofrontal cortex, anterior cingulate cortex, prefrontal cortex, ventral pallidum, and the medial dorsal nucleus of the thalamus. No wonder, then, that understanding the entire dopamine-reward system is far from complete and is very complex. One oddity, for example, is that “systemic administration of dopamine antagonists selectively reduces the motivation of animals to pursue rewards, without affecting their preferences for such rewards when they can be obtained without effort.”4 (Makes those who prefer getting rewards via the government’s freebies—rewards without effort—sound like they’re on dopamine antagonists.)

EGCG Increases Neurogenesis in the Sub-granular Zone of the Dentate Gyrus in Adult Mice

EGCG does a great deal more than just inhibit COMT, of course. In the area of cognition alone, EGCG also promotes neurogenesis, thus increasing the availability of new youthful adult-born neurons in those areas of the brain where neurogenesis takes place throughout life. One of those areas is the subgranular zone of the dentate gyrus in the hippocampus.

In one study of EGCG and neurogenesis, mice were divided into two groups of 7 mice each, one group of which received vehicle (controls) and one of which was treated with 25 mg/kg of EGCG for four weeks.5 Animals were treated with BrdU (5-bromo-2’-deoxyuridine) by injection and the neurons marked by Ki67 and DCX determined (these are markers of neuronal proliferation). “In the EGCG-treated groups, the number of Ki67+-positive cells were increased by 221.3% (p=0.01) compared to that in the vehicle-treated group (11.7 ± 0.17/section).”5

Hence, the authors conclude, EGCG enhances the survival of immature neuroblasts in the subgranular zone of the dentate gyrus in adult mice.

EGCG Increases the Number of Neural Stem Cells Around a Damaged Area After Rat Traumatic Brain Injury

EGCG is also potently neuroprotective in the injured brain. In one study,6 for example, male Wistar rats were subjected to acute brain trauma while under anesthesia after receiving either water or water with EGCG added to it at 0.1% w/v from 6 to 10 weeks of age. Excitotoxicity elicited under ischemia, such as that occurring during acute brain trauma, is known to cause excessive glutamate release from the injured neurons, with the subsequent influx of increased Ca2+ via glutamate receptors. The increased Ca2+ leads to a substantial increase in free radical generation, with cell degeneration and death following.

The researchers report that, following the initial mechanical insult, damage resulted from blood-brain barrier disruption, excitotoxic damage and free radical production. The scientists then looked for nestin (a marker of neural stem cells) in the cells around the damaged area. They found that the number of nestin-positive cells in the EGCG treatment group showed a significant increase when compared with the number in the water group. There was also a significant increase in the nestin-positive cells at days 3 and 7 following the brain trauma in the EGCG treatment group as compared with the number in the water group (73.0 ±25.8 and 12.4 ±3.3, respectively).6

“Therefore, we speculate that EGCG crosses the BBB [blood-brain barrier] and inhibits neuronal and NSC [neural stem cells] death by free radicals in the damaged area following TBI [traumatic brain injury].”6


  1. Gianotti et al. Why some people discount more than others; baseline activation in the dorsal PFC mediates the link between COMT genotype and impatient choice. Front Neurosci. 6:54 (May 2012).
  2. Zhu et al. Molecular modelling study of the mechanism of high-potency inhibition of human catechol-O-methyltransferase by (-)-epigallocatechin-3-O-gallate. Xenobiotica. 38(2):130-46 (2008).
  3. Kang et al. Dual beneficial effects of (-)-epigallocatechin-3-gallate on levodopa methylation and hippocampal neurodegeneration: in vitro and in vivo studies. PLoS One. 5(8):e11951 (Aug. 2010).
  4. Tunbridge et al. The role of catechol-O-methyltransferase in reward processing and addiction. CNS Neurol Disord Drug Targets. 11:306-23 (2012).
  5. Yoo et al. (-)-epigallocatechin-3-gallate increases cell proliferation and neuroblasts in the subgranular zone of the dentate gyrus in adult mice. Phytother Res. 24:1065-70 (2010).
  6. Itoh et al. (-)-epigallocatechin-3-gallate increases the number of neural stem cells around the damaged area after rat traumatic brain injury. J Neural Transm. 119:877-90 (2012).


Finding you have less time for cooking? Next time you have only a few minutes available and want to eat something tastier than scrambled eggs or (gasp) a frozen meal, try this:

The halfwich is basically a toasted piece of bread topped by meat, cheese, mustard, pickles, sauerkraut, a slice of avocado, and a slice or two of onion, then heated in a microwave until the cheese melts. The whole thing takes just a few minutes. The “secret” ingredient is: CORNED BEEF.

You can get lean corned beef in a convenient refrigerated serving size for 2 or 3 at most supermarkets.

You’ll need: 1 piece of bread, preferably high fiber, toasted corned beef, prepared in microwave, two slices of Swiss cheese, about ½ cup of sauerkraut, your favorite mustard, pickles, sliced so the pieces lie flat on the bread, a slice of avocado, and a slice or two of onion.

Cook corned beef as instructed (4–6 minutes in a microwave). Layer on a microwave-safe ceramic plate: toasted bread, slice of Swiss cheese topped by mustard, sauerkraut, sliced pickles, corned beef cut into thin slices, and slice or two of onion. Top the whole thing with another slice of Swiss cheese, then microwave for a minute or so until the cheese is melted.

That’s all there is to it for a great hot lunch!

Optional: Sprinkle ground caraway seed on sauerkraut.


At the start of a new paper,1 the authors note that patients with advanced atherosclerosis have severe deficiencies in the ability to generate new blood vessels to adapt to inadequate blood flow. Moreover, advanced age is a major risk factor for coronary and peripheral arterial diseases and is associated with impaired ability to generate new blood vessel formation to increase blood flow around areas of arterial occlusion. In addition, “the number and/or the functional activities of EPCs [endothelial progenitor cells] have been shown to be impaired by aging in both animals and in humans.”1

The researchers, searching for a mechanism to explain the defective neovascularization in the context of aging, have discovered that mice lacking NOX-2 (a subunit of the enzyme NADPH oxidase) were protected against age-associated impairment in reparative neovascularization following ischemia, as well as protecting EPCs against functional impairments associated with aging.

NADPH oxidase is a major source of ROS (reactive oxygen species) critical for neutrophil antimicrobial function.2

As part of the experimental protocol,1 the scientists studied the effect of aging on the expression of NADPH oxidase subunit NOX-2 in mouse ischemic hindlimb muscles. They found NOX-2 expression to be significantly increased in old as compared to young wild type animals. (NOX-2 knockout animals, of course, do not express the NOX-2 subunit in hindlimb muscles.) Hindlimb ischemia (impaired blood flow) was established in experimental animals with and without NOX-2 by surgery. In wild type mice, aging was associated with a significant impairment of blood flow recuperation [restoration by new blood vessel growth] at day 7, whereas, by contrast, NOX-2-/- [knockout animals] were protected against age-dependent impairment of blood flow recuperation. Likewise, wild type aging animals exhibited a significant increase in oxidative stress in ischemic muscles while NOX-2-deficient mice were protected against the age-associated increase in oxidative stress.

The scientists1 report that in previous studies (as cited), NOX-2-deficient mice were protected against the effects of ischemic strokes, some forms of hypertension, and aortic atherosclerosis induced by high blood cholesterol. Moreover, reduced neovascularization is associated with increased oxidative stress and some antioxidants (including red wine) have been reported to restore at least in part the ability to regenerate new blood vessels.

In one study,3 apocynin, thought to be an indirect NADPH oxidase inhibitor, increased collateral growth capacity, whether administered prior to, or 7 days following, arterial ligation. We were particularly interested, however, in the reported efficacy of annatto extract and beta-carotene in inhibiting NADPH oxidase and increasing expression/activity of antioxidant enzymes.4 Both annatto and beta carotene were able to increase the mRNA levels of SOD (superoxide dismutase) and CAT (catalase) in neutrophils from diabetic rats. As NOX-2 has been reported to be required for the microbiocidal function of neutrophils, it is possible that the annatto/beta-carotene treatments inhibited NOX-2.2 Previous papers published by the same group that published this paper3 were reported to show a reduction in ROS (reactive oxygen species) production in neutrophils of diabetic rats treated for 30 days with beta carotene and annatto extract. The researchers note that this study3 is the first (to their knowledge) to report an increased expression of CAT (catalase, an important antioxidant enzyme that protects against hydrogen peroxide) in neutrophils from diabetic rats treated with beta carotene and annatto extract.

The control group and diabetic group of rats3 received a modified standard diet, whereas experimental groups received the diet + annatto (control-annatto), and diet + annatto (diabetic-annatto) or diet + beta carotene (control-beta) and diet + beta carotene (diabetic-beta). The concentration of bixin (a potent carotenoid antioxidant) in the annatto extract was 2.17 g/100 g. As noted by the authors of the annatto/beta carotene study, “bixin, which is present in the extract of annatto, is one of the most effective biological singlet molecular-oxygen quenchers.” Moreover, they note that annatto, which is a natural yellow-red color, is widely used as a food colorant and has a relatively low cost of production and low toxicity, making it a very attractive pigment for the food industry.

The Terpenoid Bixin May Activate PPARgamma, Enhance Insulin Sensitivity and Increase Adiponectin Secretion from Adipocytes

A 2010 paper5 evaluated the effects of various terpenoids derived from herbal and dietary plants on the activity of PPARgamma, an important regulator of insulin sensitivity. (The synthetic glitazones that activate PPARgamma are drugs used in the treatment of diabetes). In a graph showing the effects of 24 hours of incubation of a number of terpenoids at 50 or 100 μM as compared to a vehicle control set at 100%, bixin showed the greatest activation of PPARgamma ligand at five-fold induction (compared to the vehicle control).

Another paper6 reported that, in mice, bixin also activated PPARalpha, a transcription factor that regulates the expression of genes involved in fatty acid oxidation. Activation of fatty acid oxidation in the liver by PPARalpha improved carbohydrate and lipid metabolism in the subject obese mice. Bixin treatment also improved obesity-induced dysfunctions including hyperglycemia, hyperinsulinemia, and adiponectin deficiency.

One of the effects of PPARgamma is to induce differentiation of preadipocytes to adipocytes. The new adipocytes, being small, have various properties that differ from mature adipocytes, such as being more insulin sensitive and secreting more adiponectin, an insulin-sensitizing adipokine.7


  1. Turgeon et al. Protection against vascular aging in Nox2-deficient mice: impact on endothelial progenitor cells and reparative neovascularization. Atherosclerosis. 223:122-9 (2012).
  2. Lamb et al. Endotoxin priming of neutrophils requires endocytosis and NADPH oxidase-dependent endosomal reactive oxygen species. J Biol Chem. 287(15):12395-404 (2012).
  3. Miller et al. Antioxidants reverse age-related collateral growth impairment. J Vasc Res. 47:108-14 (2010).
  4. Rossoni, Jr. et al. Annatto extract and beta carotene enhances antioxidant status and regulate gene expression in neutrophils of diabetic rats. Free Radic Res.46(3):329-338 (2012).
  5. Goto et al. Various terpenoids derived from herbal and dietary plants function as PPAR modulators and regulate carbohydrate and lipid metabolism. PPAR Res. Volume 2010, Article ID 483958, 9 pp. (this is an open access article).
  6. Goto et al. Bixin activates PPARalpha and improves obesity-induced abnormalities of carbohydrate and lipid metabolism in mice. J Agric Food Chem. 60:11952-8 (2012).
  7. Takahashi et al. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPARgamma activation. Biochem Biophys Res Commun. 390:1372-6 (2009).


For some time, there have been papers published on the anti-inflammatory and vasorelaxation activity of nitrated fatty acids.1–4 Now, a new paper5 reports that “conjugated linoleic acid (CLA) is the preferential unsaturated fatty acid substrate for nitration reactions during oxidative conditions and digestion.” It is reported that “multiple enzymatic and cellular mechanisms account for CLA nitration, including reactions catalyzed by mitochondria, activated macrophages, and gastric acidification.”5 It is reported here that CLA yields up to 105 greater extent of nitration products as compared to it’s natural competitor bis-allylic linoleic acid.

The nitrated fatty acids act as a circulating reservoir of nitrite for local conversion to nitric oxide. Hence, it is a way to increase nitric oxide availability throughout the body.

“Dietary CLA and nitrite supplementation [which can be derived from nitrate-rich foods such as spinach] in rodents elevates NO2–CLA levels in plasma, urine, and tissues, which in turn induces heme oxygenase-1 (HO-1) [an important antiinflammatory enzyme] expression in the colonic epithelium.” Incredibly, in human coronary artery endothelium, the paper notes, fatty acid nitroalkenes significantly influence the expression of ~400 metabolic and anti-inflammatory-related genes, including important inflammation regulators such as PPARgamma, Keap1/Nrf2, heat shock factor-1, and NF-kappaB. According to the authors, NO (nitric oxide) doesn’t directly nitrate protein or lipids, but must be oxidized to the proximal nitrating species NO2. “Importantly, NO2–[nitrite]-supplemented diets are associated with a variety of beneficial anti-inflammatory and metabolic actions, including the regulation of mitochondrial function, adipogenesis, oxygen delivery to tissues, and blood pressure. Although these events can in part be attributed to the generation of NO [nitric oxide], salutary responses to NO2–-derived oxides of nitrogen may also be transduced by the concomitant generation of electrophilic nitro-fatty acids (NO2–FA).”5

The Bottom Line

In this new paper5 the researchers show that CLA is the preferential endogenous fatty acid substrate for fatty acid nitration by NO and NO2– and provides tissue-protective and anti-inflammatory actions. Beneficial effects reported in the paper for nitrated fatty acids included: in mouse models of metabolic and inflammatory injury, fatty acid nitroalkene administration at nanomolar concentrations prevents restenosis after blood vessel injury, in mouse models of metabolic syndrome, nitrated fatty acids limit weight gain and loss of insulin sensitivity, nitrated fatty acids protect against ischemia-reperfusion injury, reduces plaque formation in a rodent model of atherosclerosis, and inhibits the onset of chemically induced inflammatory bowel disease. So, although the two of us do not use high-dose CLA for purported weight control, we do consider it’s use at lower, more physiological doses for the formation of nitrated-CLA as reported here5 to be potentially important.


  1. Baker et al. Fatty acid transduction of nitric oxide signaling. J Biol Chem. 280(51):42464-75 (2005).
  2. Lima et al. Nitrated lipids decompose to nitric oxide and lipid radicals and cause vasodilation. Free Radic Biol Med. 39:532-9 (2005).
  3. Trostchansky and Rubbo. Nitrated fatty acids: mechanisms of formation, chemical characterization, and biological properties. Free Radic Biol Med. 44:1887-96 (2008).
  4. Tsikas et al. Nitro-fatty acids occur in human plasma in the picomolar range: a targeted nitro-lipidomics GC-MS/MS study. Lipids. 44:855-65 (2009).
  5. Bonacci et al. Conjugated linoleic acid is a preferential substrate for fatty acid nitration. J Biol Chem. 287(53):44071-82 (2012).


A 2012 paper1 reported on the association between blood groups and the risk of coronary heart disease in two large cohorts: the Nurses’ Health Study (NHS, including 62,073 women) and the Health Professionals Follow-Up Study (HPFS, including 27,428 men).

The frequency for the blood types O, A, B, and AB was 42.9%, 36.0%, 13.3%, and 7.8% in women and 43.0%, 37.2%, 12.3%, and 7.5% in men. These frequencies were similar in both the NHS and HPFS across the four ABO blood groups. The authors determined that during up to 26 years of follow-up, there were 2055 confirmed coronary heart disease cases (1666 nonfatal MI and 389 fatal CHD) in the NHS, while during 20 years of follow-up, there were 2015 CHD cases (with 1420 nonfatal MI and 595 fatal CHD). The incidence of CHD among the blood groups per 100,000 person-years were 125, 128, 142, and 161 for those with blood type O, A, B, and AB in women and 373, 382, 387, and 524 for those with blood types O, A, B, and AB in men. The cumulative incidence of CHD was statistically significantly different among the 4 ABO blood groups in both cohorts (P=0.0048 in NGS and 0.0002 in HPFS, respectively.

“Compared with participants reporting blood group O, those with non-O blood type [A, B, and AB] had an age-adjusted hazard ratio of 1.09 (95% CI, 1.03–1.17).” “Compared with the O blood group, the non-O blood type (A, B, and AB) had a stronger relationship with CHD risk in overweight and obese women than those with BMI<25 kg/m squared. However, this interaction was not confirmed in men …”

The authors suggest that recent studies lend support to the relation between ABO blood type and cardiovascular risk. They had recently discovered that the ABO gene is located on chromosome 9q34 in association with the plasma soluble E-selectin levels in the NHS, as was found in another genome-wide association study. In addition, they say, the ABO locus was related to tumor necrosis factor alpha, a powerful proinflammatory cytokine that has been associated with increased CHD risk.1 Since most of the participants were white, however, the researchers suggest that the results may not necessarily apply to other races.


  1. He et al. ABO blood group and risk of coronary heart disease in two pro­spective cohort studies. Arterioscler Thromb Vasc Biol. 32:2314-20 (2012).


A new paper1 reports that in the Indian sub-continent and around the world, chewing fennel seeds (Foneiculum vulgare) is a common way to freshen one’s mouth after eating a meal. But surprisingly, there appears to be a lot more to it than that. In the new paper, the researchers explain that they found that fennel seeds contain significantly higher amounts of nitrites and nitrates when compared to other commonly chewed post-meal seeds. Nitrites and nitrates are “known to play crucial roles in maintaining vascular and digestive function”1 by being chemically reduced in the body to nitric oxide, an important vasoactive compound. In this new study, the researchers show that nitrites derived from fennel seeds can modulate vascular functions. Quite a nice benefit for a little bit of post-prandial chewing. (And, incidentally, if you haven’t tried them, they are tasty, with a sort of licorice flavor.)

The authors1 cite recent animal studies exploring different potential applications of what they call “nitrite therapy” that included pulmonary hypertension, acute tissue ischemic injuries, cerebral vasospasm, myocardial infarction, and stroke. “Studies have demonstrated that a high intake of nitrite containing fruits and vegetables in one’s diet has protective effects on the cardiovascular system.”1 The authors even suggest that chewing fennel seeds could be a useful way to maintain nitric oxide levels at high altitudes to help prevent the onset of acute hypoxia.2 Sounds like a good method for mountain climbers to use to avoid altitude sickness.

Cautionary note on increasing dietary intake of nitrates: A recent paper3 reports on the risk of esophageal and gastric cancer subtypes in relation to dietary N-nitroso compounds in 120,852 men and women aged 55-69 when they were recruited in the Netherlands Cohort Study in 1986. N-nitroso compounds are naturally occurring compounds containing a nitroso group attached to a nitrogen atom that can be converted in animals to carcinogenic forms in the acidic environment of the stomach or in the lower gastrointestinal tract by contact with heme-iron derived from meat. The Netherlands Cohort Study3 found that high dietary intakes of N-nitroso compounds (NOC) increased the risk of esophageal squamous cell carcinoma in men, though there was no increased risk in women. Importantly, adequate amounts of vitamin C, an inhibitor of endogenous nitrosation, can prevent this increased risk.3


  1. Swaminathan et al. Nitrites derived from Foneiculum vulgare (Fennel) seeds promotes vascular functions. J Food Sci. 77(12):H273-9 (2012).
  2. Erzurum et al. Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans. Proc Natl Acad Sci USA. 104(45):17593-8 (2007).
  3. Keszei et al. Dietary N-nitroso compounds, endogenous nitrosation, and the risk of esophageal and gastric cancer subtypes in the Netherlands Cohort Study. Am J Clin Nutr. 97:135-46 (2013).


A paper with this actual title was published very recently.1 Noting that large and increasing numbers of different foods and food constituents are being investigated for positive or negative associations with cancer, scientists investigated the appearance of 50 common ingredients from random recipes in a cookbook, evaluating the conclusions, statistical significance, and reproducibility of published associations with cancer appearing in papers in PubMed.

The authors found that forty ingredients (80% of those that they examined in the literature) had articles published on cancer risk. They found that “[a]ssociations with cancer risk or benefits have been claimed for most food ingredients. Many single studies highlight implausibly large effects, even though evidence is weak. Effect sizes shrink in meta-analyses.”

The authors also state that “[s]tudies may spuriously highlight results that barely achieve statistical significance or report effect estimates that either are overblown or cannot be replicated in other studies.” Overall, they found that 39% of studies concluded that the examined ingredient conferred an increased risk of malignancy, 33% concluded that the ingredient reduced the risk of malignancy, 5% concluded that there was a borderline statistically significant effect, while 23% could not discern a clearly increased or decreased risk. Studied ingredients included veal, salt, pepper spice, egg, bread, pork, butter, tomato, lemon, duck, onion, celery, carrot, parsley, mace, olive, mushroom, tripe, milk, cheese, coffee, bacon, sugar, lobster, potato, beef, lamb, mustard, nuts, wine, peas, corn, cayenne, orange, tea, and rum. You can imagine some of the difficulties in, say, examining the effects of lamb on the risk of cancer. How do you hold everything constant (including subject genetic diversity) while varying only the intake of lamb?

Some of the problems that likely contributed to the inconsistency included variation in definitions of “serving size,” “often,” “never” as well as there being no standardized, consistent selection of exposure contrasts for the reported risks. The authors claim that the vast majority of these claims (increased or decreased malignancy) were based on weak statistical evidence. We would also like to point out that the variation in human responses to the analyzed ingredients imposed a very difficult hurdle to the isolation of effects that can be generalized to large human populations. That is why we like to see considerable research done on mechanistic (cell free or cell culture) studies and animal studies considered together with human studies. The analysis of studies of a particular ingredient’s effects on cancer risk, considered as a whole, still is as much of an art as a science, depending upon the ability to see connections between, say, chemical structure and function of an ingredient. You don’t get definitive answers. You get leads.

See quotes on the next page, on the dangers of scientific bias, which appeared in the editorial commentary on this paper, published in the same issue of The American Journal of Clinical Nutrition.


Schoenfeld and Ioannidis. Is everything we eat associated with cancer? A systematic cookbook review. Am J Clin Nutr. 97:127-34 (2013).


Imagination, on the contrary, which is ever wandering beyond the bounds of truth, joined to self-love and that self-confidence we are so apt to indulge, prompt us to draw conclusions which are not immediately derived from facts; so that we become in some measure interested in deceiving ourselves.

— Antoine-Laurent Lavoisier1

White hat bias, defined by Cope and Allison as “bias leading to distortion of research-based information in the service of what may perceived as ‘righteous ends’” may be a factor in the overstatement of research find- ings. In addition, overstatement of results can be influenced by confirmation bias, by which the overstated results match preconceived views and hypotheses, leading to acceptance of the results even if the results are weak or nonsignificant.

— Bohan Brown, Brown, and Allison1

  1. Brown et al. Nutritional epidemiology in practice: learning from data or promulgating beliefs? Am J Clin Nutr. 97:5-6 (2013).



The Jan. 10, 2013 issue of The New England Journal of Medicine contained an article reviewing four recent Court decisions supporting the rights (under the First Amendment) of pharmaceutical companies to promote off-label uses of drugs approved by the FDA for a different purpose. Recognizing that the FDA’s authority does not extend to the practice of medicine, the article notes that the agency cannot prohibit physicians from prescribing approved drugs for non-approved uses.

It is widely recognized, though this was not discussed in the article, that a large percentage of drugs prescribed for the treatment of cancer (as an example) are for off-label uses (not the uses the FDA approved them for) and that this constitutes an important part of modern medicine, extending or saving large numbers of lives.

One of the main arguments made against the promotion of off-label uses of drugs approved by the FDA for a different purpose is that without being able to silence pharmaceutical companies from promoting off-label uses, manufacturers can seek approval only for certain limited uses of drugs, then promote that same drug for off-label uses, effectively circumventing FDA’s new drug requirements. In fact, the trial court in Caronia (a case discussed in the article) upheld the constitutionality of the FDA’s regulations prohibiting off-label uses of FDA approved drugs because it (the court) could not identify a less restrictive manner in which to prohibit pharmaceutical companies from circumventing the FDA approval process.

We note that just because the court could not think of any less restrictive manner (than an outright ban on First Amendment protected speech) to prevent the circumvention of the FDA’s approval process is hardly a legitimate basis for throwing out the First Amendment. (It certainly suggests that something is wrong, e.g., unconstitutional, with the FDA’s approval process if it cannot accommodate the First Amendment’s protections of speech and press.) The Constitution does have a procedure for amendment and the proper way to eliminate the First Amendment’s protection of some area of speech would be to pass an amendment to do that. Otherwise, it is just a court deciding on its own that it didn’t approve of the consequences of exercising one’s First Amendment’s rights and declaring the First Amendment doesn’t protect that speech.

The article noted that “[i]n overturning Caronia’s conviction, the three judge panel of the Second Circuit agreed that the FDA regulations were overly broad, specifically noting that nothing Caronia did constituted conspiracy to put a false or misleading or deficient label on a drug product. The court appeared particularly persuaded by the argument that the FDA regulations allow unfettered prescribing of approved drugs for off-label uses but then, through the off-label restrictions, refuse to allow the free flow of information that would result in a full vetting of the uses, limitations, and side effects of the drug. The Second Circuit held that such restrictions violate the principles of the First Amendment.”

The article concludes: “The question now is whether a host of other state and federal regulations can withstand such First Amendment scrutiny.” Thank goodness the First Amendment protection of speech and press still appears to have some teeth!

This case is a logical extension of our First Amendment arguments in Pearson v. Shalala.


  • Marcia M. Boumil, J.D., LL.M. Off-label marketing and the First Amendment.N Engl J Med. 368(2):103-5 (2013).


The model that has long been used to predict tissue damage from low dose radiation is based upon extrapolation from high dose radiation exposure. As has been noted in recent scientific studies,1–5 however, this model poorly predicts the effects in the low dose radiation range, whereas the hormetic model (a biphasic response) provides excellent predictions.

According to Edward J. Calabrese,1 the biphasic dose response model was originally proposed by Schulz in the 1880s based on his extensive testing of the effects of numerous disinfectants on the metabolism of yeast. Unfortunately, however, Schulz went on to claim that his dose-response findings provided the explanatory principle of homeopathy, leading to marginalization of both Schulz and his theory (due to the conflict between orthodox medicine and homeopathic practitioners). It would be another 60 years before the biphasic response (hormetic) explanation for low dose effects of radiation was to become a respected notion. However, government agencies that regulate toxic substances (such as the EPA) still use the linear threshold dose response model. By so doing, these agencies are costing huge sums of money using data that do not provide accurate estimates of toxicity at low doses, hence, not delivering greater safety in exchange for these immense costs.

We are beginning to see more scientific papers published in support of the hormesis explanation of why extrapolation of high dose radiation damage to tissues is not a proper way to estimate the damage, if any, to tissues derived from low dose radiation.

Dr. Calabrese1 reports his research and that of his colleagues during the past approximately ten years which, using three data sets, has found that “the threshold and the linear models poorly predicted the effects in the low dose zone whereas the hormetic model made uniformly accurate predictions.” The three data sets included1 publications in the peer-reviewed pharmacological and toxicological literature on a broad range of biological models, endpoints and chemicals, (2) an NCI series of 57,000 dose response involving over 2200 anti-tumor agents that had been tested on 13 strains of years considered models of human tumor cells, and (3) about 2100 agents in E. coli.

Dr. Calabrese notes that the threshold model was never validated and that during this long period during which a poor model was being used in toxicology and government agency regulations, it failed multiple, large objective validation tests. As Calabrese notes, getting the “dose response wrong was not simply an historical curiosity involving a conflict between homeopathy and traditional medicine, but one that has shaped the risk assessment process, profoundly affecting clinical medicine, public health, risk communication messages, personal health choices, the proper allocation of vast public/private resources as well as governmental legislation/regulatory programs.”

A second paper2 published in the latest FASEB J. examined the effects of low dose radiation exposure in a mouse model using mice that are very sensitive to low dose radiation, changing coat color in response to it. As the authors note in their introduction, “the human health risks of LDIR [low dose ionizing radiation] are still estimated by extrapolation from the biological effects observed at high doses, according to the linear no theshold (LNT) risk assessment model.” Pointing out that high doses of ionizing radiation can result in epigenetic modifications to DNA in adult mice, they wanted to study whether epigenetic alterations also occur in vivo in response to low dose ionizing radiation.

They used a type of mouse (the Avy mouse) that, in response to early developmental exposure to methyl donors (genistein, bisphenol A, ethanol) and in vitro culturing develop phenotype changes by altering the epigenome. In this study, the researchers show that low dose ionizing radiation causes both dose- and sex-dependent epigenetically induced adaptive changes at the Avy locus that in part depend on a cellular oxidative stress response.2

As the authors explain, this mouse is very sensitive to environmental effects on the fetal epigenome. Hypomethylation causes inappropriate Agouti gene expression in all tissues, leading to a yellow coat color but also other effects such as developing obesity, diabetes and cancer. When the promoter of this gene is hypermethylated, however, the animals have a much lower incidence of disease as well as having a pseudoagouti (brown) color. The experimental results showed that low dose ionizing radiation altered DNA methylation such that the animals ended up with a brown color and the reduced levels of disease as an adaptive response. This, the authors say, was due to exposure during pregnancy to doses of radiation equivalent to those received for a chest or head CT scan. “Hypomethylation of DNA at high doses of radiation and hypermethylation at low doses are indicative of a hormetic biphasic radiation dose response effect.”2 (The authors note that hypermethyl­ation was beneficial in this particular disorder but is not always beneficial. For example, tumor suppressor genes can become hypermethylated and, as a result, silenced, so that cancer risk is increased.)

The study2 “brings into question the assumption that every dose of radiation is harmful.”


  1. Calabrese. Hormesis: Toxicological foundations and its role in aging research. Exper Gerontol. 48:99-102 (2013).
  2. Bernal et al. Adaptive radiation-induced epigenetic alterations mitigated by antioxidants. FASEB J. 27:665-71 (2013).
  3. Son et al. Hormetic dietary phytochemicals. Neuromolecular Med. 10(4):236-46 (2008).
  4. Hayes. Nutritional hormesis and aging. Dose Response. 8:10-5 (2010).
  5. Wu et al. Multiple mild heat-shocks decrease the Gompertz component of mortality in Caenorhabditis elegans. Exper Gerontol. 44:607-12 (2009).


by Sandy Shaw & Durk Pearson
(originally appeared in Liberty)

A recent paper in Science1 on what caused the extinction of large North American canids (wolf-like carnivores) is startlingly parallel to what is happening to the government of the United States and its constituent states and leads us to a hypothesis concerning the death of democracies.

The paper reports that during the past 50 million years, successive branches of large carnivorous mammals have diversified and then gone into extinction. The authors1argue that “energetic constraints and pervasive selection for larger size (Cope’s rule) in carnivores lead to dietary specialization (hypercarnivory) and increased vulnerability to extinction.” They explain that Cope’s rule—the evolutionary trend toward larger size—is common in mammals because larger size makes it easier to evade predators and to capture prey. Moreover, larger size improves thermal efficiency, thus increasing the potential range of habitats into colder areas. As the size of carnivores increases beyond about 45 pounds, the amount of nutrition obtained from small prey becomes inadequate to cover the energy used in capturing them. Thus, the larger carnivores became what the authors call “hypercarnivores,” which hunt only large prey (as large as or larger than themselves). A plot of the index of hypercarnivory (PCI score) against estimated species duration shows that none of the hypercarnivorous species persisted for more than 6 million years, as compared to other, more omnivorous species that lasted as long as 11 million years. Hence, the authors propose, the hypercarnivores are more vulnerable to extinction. The researchers also note that hypercarnivores reverting to a more generalized diet and morphology was rare.

Reliance upon a smaller number of large prey increases the statistical variation in the nutritional intake. Moreover, the larger the carnivore, the lower their population density. Both of these are factors that increase the risk of extinction. It is interesting that the government of the United States and of its constituent states are moving rapidly in the direction of targeting large prey, with an increasing statistical variance in the yearly revenue from these relatively small number of prey. The federal government relies heavily on a steeply progressive income tax (with the well known result that the bottom 50% of income earners pay only 4% of income taxes). Relying upon the fat targets at the highest level of earnings has resulted in greater statistical variation in revenues, as well as increasing demand for government services from those paying little or nothing for them. Worse yet, today’s government is already preying upon the fat targets of the future by rapidly increasing government debt, something canids never had the option to do.

We suggest, therefore, that hypercarnivory may be one reason that democracies don’t last much longer than about 200 years. If we start counting from the passage of the 16th Amendment on Feb. 3, 1913 (which provided the means to target most of the large prey via unlimited progressive income taxation) rather than from 1787, the United States could theoretically last another century or so, though for a number of reasons (the immensely rapid expansion of government debt, for instance) we think it unlikely.

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