“Unimportant” Molecules?—Part 1

Abstract

About a century ago when research into the nutritional components of food began, scientists were limited by the technology and physiological understanding of the time. Nonetheless, this pioneering research revealed the importance of many nutrients for the maintenance of life and prevention of overt deficiency diseases. Unfortunately, the necessary simplifications led to the unrecognized assumption that the constituents in food not required for life were not important. This justified growing food chemically rather than organically because essential nutrients were largely (but not entirely) conserved. However, as technology advanced—especially at the turn of this century—much was revealed. Nutrients considered single molecules when discovered were now realized to represent multiple variants and vitamers with significantly different physiological effects, and many of the molecules considered “unimportant” have huge impacts on health and resistance to disease.

History of Nutrition Research

About 100 years ago when the scientific community was diligently working to determine the components of foods necessary for life, they were limited by the technology of the time and the early stages of our understanding of physiology. Since so little was known, they had little to guide them. Nonetheless, a lot was discovered. The term “vitamine” was coined in 1912 by Polish scientist Casimir Funk to designate a group of compounds discovered to be vital for life.¹ Considering the limitations of the time, the researchers focused on food components required for life in the animals used or whose need was determined by serious human deficiency diseases. They also had to focus on the main biochemical pathways since these were the only ones measurable with the available technology. As the research evolved over the first few decades of the last century, a number of vitamins (Table 1), minerals, amino acids, and fatty acids were determined to be necessary for life. The total number of essential nutrients (depending upon criteria used) ended up at about 42 with a few more under consideration (Table 2). This pioneering research was a huge advance for the prevention of death and disease from serious deficiencies.

Table 1.

Year of Discovery of Vitamins²

Year of discovery	Vitamin
1910	Vitamin B1 (Thiamine)
1913	Vitamin A (Retinol)
1920	Vitamin C (Ascorbic acid)
1920	Vitamin D (Calciferol)
1920	Vitamin B2 (Riboflavin)
1922	Vitamin E (Tocopherol)
1929	Vitamin K1 (Phylloquinone)
1931	Vitamin B5 (Pantothenic acid)
1934	Vitamin B6 (Pyridoxine)
1936	Vitamin B7 (Biotin)
1936	Vitamin B3 (Niacin)
1941	Vitamin B9 (Folic acid)
1948	Vitamin B12 (Cobalamin)

Table 2.

Nutrients Required for Human Health

Vitamins:	12
Minerals:	10
Amino acids:	9
Conditional:	6
Fatty acids:	5
Total:	42

Unfortunately, most of the research was trial and error, and there was also the apparent default assumption that anything in food not found necessary for life or prevention of serious overt disease was “unimportant.” So, when food production methodologies evolved into chemically assisted growth and chemically assisted insect protection, as long as these “important” food constituents were maintained, the food was considered healthy. Big mistake.

The obvious questions then arise, “How many other molecules are in food?” and “Are they important for health?”

How Many Molecules Are in Food?

This important question has not yet been fully determined. Around the turn of this century, sophisticated technologies like chromatography, mass spectrometry, infrared spectrometry, and nuclear magnetic resonance were applied to plants, allowing quantitative and qualitative measurements of the molecules plants contain. According to one group of researchers, approximately 50 000 molecules have been found in plants, and they predicted that the final number will exceed 200 000.³ The function of most of these molecules has not yet been determined. Nonetheless, many of these molecules have been shown to have very significant impact on human physiology with substantial consequences for health and disease resistance. Terms like “phytonutrients” and “nutraceuticals” have been coined to describe the clinical benefits of these molecules, molecules which should be in food but have been lost due to modern agriculture.

Especially egregious has been the chemical modification of some of these molecules to make them patentable drugs to treat the diseases caused by their loss from the food supply.⁴

Variants and Vitamers

Another problem is that the vitamins were assumed to be specific single molecules. We now know that most of the vitamins have multiple variants. Unfortunately, the variant found most important in an animal species has often turned out to not be the variant most important for humans. These variants exhibit not only slight differences in chemical structure but also optical isomers that greatly impact binding to receptor sites. This issue will be addressed more fully in a future editorial.

For the purposes of this editorial, vitamin E is quite illustrative. This vital nutrient was discovered by Herbert Evans and Katherine Bishop in 1922 through rat studies. They labeled it the “fertility” vitamin since fetuses died when pregnant rats were fed a purified diet.ⁱ The animal’s fertility was reestablished with the addition of wheat germ oil to their diet. Vitamin E was later purified from wheat germ oil in 1936.

The way the fetuses died was through reabsorption. So they developed the “fetal reabsorption assay” to determine the amount of vitamin E in a food. The image on the left side of Figure 1 is the ovary of a normal pregnant rat while the image on the right shows fetal reabsorption due to vitamin E deficiency. Not surprisingly, they were very confident in this methodology since the results were so dramatic.

Figure 1.

Uteruses from Pregnant Rats with Standard Rat Diet Compared to Vitamin E Deficient Diet

This assay was then used to validate synthetic vitamin E, which was then sold as a dietary supplement and food additive. And this is where the problem starts. When better technology allowed the speciation of vitamin E, 8 isomers were discovered, as shown in Figure 2, and each has optical variants.

Figure 2.

Vitamin E Isomers

It turns out that the fetal reabsorption assay was most sensitive to alpha tocopherol (α-tocopherol). The problem is that not only did this assessment methodology not differentiate optically active isomers, but worse, gamma tocopherol (γ-tocopherol) is clinically far more important in humans than α-tocopherol. When one tocopherol variant is administered at high dosages, blood levels of the others decrease due to competitive absorption. This helps explain the inconsistency of vitamin E research that utilized α-tocopherol, much less important to humans, causing a reduction in γ-tocopherol with significant adverse effects.

It’s a more potent anti-inflammatory agent than α-tocopherol. Gamma tocopherol traps reactive nitrogen species more effectively than α-tocopherol. Supplementation with γ-tocopherol, but not α-tocopherol, significantly lowers C-reactive protein concentrations in hemodialysis patients (from 4.4 to 2.1 mg/L; P < .02). Treatment with γ-tocopherol tripled PPARγ mRNA levels within 24 hours in human colon cancer cells, an increase twice that produced by α-tocopherol. PPARγ is one of a family of transcription factors—the peroxisome proliferator-activated receptors—which act as anti-inflammatory mediators by interfering with inflammatory signaling cascades such as the nuclear factor-kappaB (NF-κB) pathway.⁵

Both α- and γ-tocopherol modulate eicosanoid synthesis, but γ-tocopherol’s influence is significantly stronger since it is a much more potent inhibitor of cyclooxygenase and lipoxygenase than α-tocopherol. Under most inflammatory conditions, cyclooxygenase-2 (COX-2) is upregulated and is the primary enzyme responsible for the formation of the pro-inflammatory prostaglandin E2 (PGE2); 5-lipoxygenase (5-LOX) is the rate-limiting enzyme involved in the formation of the pro-inflammatory eicosanoid, leukotriene B4 (LTB4).⁶

Not only is γ-tocopherol more effective in inhibiting inflammation-associated disease than α-tocopherol, but as noted above, supplementation with α-tocopherol alone significantly decreases γ-tocopherol levels, thus potentially increasing inflammation.⁷

The next issue is the difference between a supposed active constituent, such as a vitamin, being expected to have the same clinical impact as a food concentrate, such as wheat germ oil. In the 1940s, 2 MDs—the Shute brothers—discovered that wheat germ oil was effective in treating cardiovascular disease. (Sadly, they experienced surprisingly vicious opposition from the medical establishment and were professionally and personally ostracized.⁸) Nonetheless, they and subsequent researchers then decided that the benefits of wheat germ oil were due to the recently characterized and now extractable and synthesizable vitamin E, which led to the obvious clinical trials.

This is where the story becomes quite interesting and illustrative of the core theme of this editorial. I strongly encourage anyone interested in evolution of our understanding of nutrition to read Franklin Bicknell, MD, and Frederick Prescott, MD, The Vitamins in Medicine, 1948. The whole book is available online as a PDF. Reading through nutrient after nutrient, I noticed a consistent pattern: a food concentrate shown effective in early research—both animal and human—was replaced in later research with the vitamin thought to be the active constituent. While some still showed efficacy, most of the later studies were equivocal or negative. This led to the vitamins being discredited as clinically beneficial except in situations of overt deficiency.

Most of this early research long predated PubMed, therefore much of it is not found when doing an internet search. Table 3 shows the research on the clinical efficacy of wheat germ oil as compiled by Bricknell and Prescott.

Table 3.

Clinical Efficacy of Wheat Germ Oil⁹

Infertility	Cardiovascular disease
Recurrent abortions	Intermittent claudication
Toxemia of pregnancy	Clotting from surgery
Muscular dystrophy	Amyotrophic lateral sclerosis
Dupuytren’s and other contractures

Most of these were not reproduced when using synthetic DL-alpha tocopherol. Synthetic α-tocopherol differs significantly from natural α-tocopherol.

All natural vitamin E tocopherols are found in the RRR form (they have a saturated 16-carbon phytyl side chain with 3 chiral centers at carbons 2, 20 and 80, all of which are in the R configuration). Synthetic α-tocopherol is a mixture of 8 stereoisomers (molecules with the same molecular formula and same sequence of bonds but different spatial arrangements), only one of which has the same spatial arrangement as naturally occurring RRR α-tocopherol. Not only are the other 7 stereoisomers not found in any food (ie, they are new-to-nature, unnatural molecules), the racemic form (containing equal amounts of dextrorotatory and levorotatory molecules) in which they appear is potentially antagonistic to natural RRR α-tocopherol.

In light of this fact, it is worth noting that only the RRR stereoisomer is biologically active (ie, capable of affecting membrane-resident enzymes and cellular signaling). Synthetic α-tocopherol may function as an antioxidant but is composed of 7/8 (87.5%) otherwise non-biologically active compounds.

Of the approximately 120 primary research studies published from 1973-2007 assessing vitamin E’s (α-tocopherol’s) effects in lipid structures (eg, lipoproteins, cell membranes), 25% used natural RRR-α-tocopherol, 25% used all-racemic α-tocopherol, and the remaining 50% did not identify the form used. None used natural vitamin E containing the full complement of tocopherols and tocotrienols.⁵

A more recent study of wheat germ oil confirmed some of the early research. As shown in Table 4, 30 grams of wheat germ oil consumed for 30 days was shown to improve many of the key blood parameters related to cardiovascular disease, supporting the early clinical observations of the Shute brothers. Interestingly, these lipid benefits lasted for 30 days after discontinuation.

Table 4.

Cardiovascular Benefits of Wheat Germ Oil¹⁰

Blood Measure	Impact
HDL cholesterol	Increased 3%-24%
LDL cholesterol	Decreased 4%-21%
Triglycerides	Decreased 12%-24%
Atherogenic factor	Improved 10%-25%

When I read this study, I was struck by its similarity to a review article I had recently read. Rather than looking at the impact of vitamin E on blood lipids, this article summarized the impact of policosanol on measures of cardiovascular health. Policosanol is a mixture of long-chain alcohols extracted from plant waxes, such as those found in wheat germ and sugar cane. Whole wheat contains 3.0-56.0 mg/kg, which is lost when wheat is refined. Table 5 shows the impact of 5-20 mg/d for periods of 1-3 months.

Table 5.

Cardiovascular Benefits of Policosanol¹¹

Blood Measure	Impact
HDL cholesterol	Increased 8%-29%
LDL cholesterol	Decreased 19%-31%
Platelet aggregation	Reduced
Cholesterol oxidation	Reduced
Total cholesterol	Lowering effect comparable to statin drugs

Note the striking similarity of the impact of wheat germ oil and policosanol on lipid parameters. The implication is that not only was the wrong variant of vitamin E used to test the efficacy of wheat germ oil in cardiovascular disease, but they may have been looking at the wrong molecule entirely! Please be clear, I am not asserting vitamin E does not promote cardiovascular health. Rather the folly of believing that only 1 isolated component of a food or food concentrate is responsible for its benefits.

The next challenge is the conflating of vitamers. Vitamers are chemically similar substances that have a qualitatively similar vitamin activity. While in some situations there appears to be little clinical difference, in others the differences are vast. The folate story is a great example of how conflating vitamers leads to the wrong intervention—that unfortunately is still being recommended. As can be seen in Figure 3, folic acid and folates are similar—but clearly not the same.

Figure 3.

Folic Acid and Folates

All readers of IMCJ are very familiar with the potential health damaging effects of MTHFR (methylenetetrahydrofolate reductase) polymorphisms. Here is where it gets so interesting. Folic acid is a synthesized molecule not found in food and not physiologically active in humans. It must be converted into methylated folate (5-methyltetrahydrofolate), which is why MTHFR polymorphisms are so important. Unfortunately, MTHFR functions poorly in a hugeⁱⁱ portion of the population.¹³ As can be seen in Figure 4, folates in the diet do NOT need to be converted by MTHFR—they are already in the active form. However, folates are very fragile molecules and are easily lost through food processing. This loss resulted in neural tube defects, which were prevented by folic acid supplementation and food fortification—though with far less benefit in those with MTHFR polymorphisms. In other words, when eating a healthy diet rich in natural folates, the MTHFR polymorphisms have little impact. But when the diet is distorted by eating overly processed foods, then the polymorphisms become important.

Figure 4.

Folates in the Diet Bypass MTHFR¹²

Worse, for those with poorly functioning MTHFR, much if not most of the supplemental folic acid is not converted. This results in excessive levels of folic acid which in themselves become problematic. This shows up as increased risk of several cancers from folic acid—but not folates, which decrease cancer risk.¹⁴

The message seems clear: the early simplification of nutrition research inappropriately focused on just a few molecules and only one variant of these molecules, resulting in serious misunderstanding of food and contributed to the epidemic of chronic disease in every age group humanity is suffering.

Loss of “Unimportant” Molecules from the Food Supply

As I noted in my editorial, “Thoughts on a Unified Theory of Disease” (IMCJ 19.6), growing foods with chemicals instead of organically has resulted in the dramatic reduction—and even total loss—of many molecules in foods. Research is increasingly showing significant clinical consequences. Figure 5 is especially illustrative—it shows the difference in flavonoid levels in tomatoes according to fertilizer used. This is an especially strong study: the tomatoes were grown in a greenhouse, so all the variables were fully controlled, and they measured the levels of flavonoids monthly for a full year.

Figure 5.

Tomatoes Grown Chemically Have Dramatically Lower Levels of Flavonoids Compared to Those Grown Organically¹⁵

As can be seen, not only are many of these flavonoid levels lower in chemically grown foods, but some are totally lost. Modern agriculture maintains enough of the molecules for the food’s characteristic color and key flavor, but what remains is just a shadow of the full nutrient/molecular profile of foods grown organically from heirloom seeds.

An obvious question is, “Why do plants produce so many molecules that don’t appear necessary for life and growth?” The answer is clear, “The plants produce these molecules to make them stronger and healthier.” These molecules are anti-bacterial, anti-fungal, anti-viral, anti-insect, antioxidant, anti-cancer, etc. Not surprisingly, humans eating plants containing these molecules are healthier.

Phytonutrients

A lot of research and consumer publications now tout “phytonutrients” as important for health. This is indeed true. The problem is that many of these nutrients were already in the food supply before it was distorted. This loss of “unimportant” molecules is remarkably similar to the impact of processing foods resulting in the loss of many important nutrients, then adding a few back and lauding the food as “fortified.” The clinical impacts of these “unimportant” molecules are quite remarkable, and the research is growing rapidly. Figure 6 shows a few of the many beneficial physiological effects of one of these, limonene.

Figure 6.

Beneficial Physiological Effects of the Terpenoid Limonene ¹⁶

Clinical Significance

Many epidemiological surveys and interventional clinical studies have now documented the benefits of phytonutrients in total, as isolated supplements, and as food concentrates.

Researchers using data from the PREDIMED study, a large 5-year feeding trial aimed at assessing the effects of the Mediterranean Diet in primary prevention of cardiovascular disease in high-risk patients, found that a high polyphenol intake resulted in a reduced risk of overall mortality compared to those with lower intakes (Figure 7). Quite interesting to see that those with the highest intake of phytonutrients die at about half the rate of those with the lowest. In this study, low intake is < 600 mg/d, medium is 600 to 750 mg/d, and high > 750 mg/d.

Figure 7.

Correlation Between Polyphenol Intake and Survival¹⁷

Similar beneficial results are seen with many polyphenols and many diseases. But perhaps most important and timely is the impact of flavonoids on pervasive public health challenges such as COVID-19.

Quercetin

As noted previously, one of the reasons plants manufacture flavonoids is for viral protection. One of these flavonoids, quercetin, is especially important since it both binds to the SARS-CoV-2 spike protein and inhibits the enzyme needed for viral replication, as shown in Figure 8.¹⁸

Figure 8.

Quercetin Binds to Spike Protein and Inhibits 3CLpro and PLpro

Of course, while theoretical and cell cultures are useful, clinical studies are required. The following 2 studies are early research with multiple limitations. Nonetheless, the initial results are encouraging. The first published study prospectively followed 152 outpatients with confirmed COVID-19 for 30 days. All received standard of care, and half also received 500 mg of liposomal quercetin twice per day (the liposomal form is critical since standard quercetin is poorly absorbed). Since the untreated group had slightly more co-morbidities, the result reported here only compares those with no co-morbidities. As can be seen in Table 6, the results were impressive.¹⁹

Table 6.

Liposomal Quercetin Decreases Hospitalization, Length of Hospitalization, and Death

Measure	Control	Intervention	P Value
Hospitalized	22.4%	8.5%	.08
Days of hospitalization	5.14	1.25	.01
Needed O2	12.9%	0	.005
Admitted to ICU	6.5%	0	.05
Deaths	6.5%	0	.05

Shortly after this study was published, Di Pierro’s group had accepted for publication a second impressive prospective study.²⁰ In this one, over 21 days they measured viral load in 42 SARS-CoV-2 positive patients as well as clinical manifestations of the infection. As with the first study, all followed standard of care with half also receiving 500 mg liposomal quercetin 3 times per day the first week and twice each day the second. As can be seen in Table 7, quercetin significantly decreased viral load, symptomatology, and several laboratory measures of inflammation and damage.

Table 7.

Quercetin Decreases Viral Load and Clinical Manifestations of SARS-CoV-2²⁰

	Group SC	Group QP	P
RT-PCR (positive subjects    At enrollment    At day 7    At day 14    At day 21	21/21 (100%) 19/21 (90.5%) 4/21 (19%) 0/20 (0%)	21/21 (100%) 5/21 (24%) 0/21 (0%) 0/21 (0%)	0.0002
Symptoms variation*    Healed    Improved    Unchanged	4/21 (19%) 17/21 (81%) 0/21 (0%)	12/21 (57%) 8/21 (38%) 1/21 (5%)	0.0118
CRP* (mg/L)    At enrollment    Day 7	30.5± 27.9 18.1± 22.9	27.2 ± 27.0 12.3 ± 16.5	n. s.
LDH* (U/L)    At enrollment    Day 7	364.9± 139.9 327.6± 128.9	418.6 ± 192.9 270.0 ± 119.6	0.0001
Ferritin* (ng/mL)    At enrollment    Day 7	687.8± 879.1 557.5± 642.6	532.7 ± 264.9 319.7 ± 151.6	0.0029
D-dimer* (ng/mL)    At enrollment    Day 7	262.0 ± 240.0 183.6± 111.8	211.5 ± 65.7 186.3 ± 50.8	n. s.
Hospitalized patients	1/21 (4.8%)	0/21 (0%)	n. s.
Patients in ICU	1/21 (4.8%)	0/21 (0%)	n. s.
Deaths	1/21 (4.8%)	0/21 (0%)	n. s.

Notes “Regarding to symptoms, “healed are those patients who manifest on Day 1 one or more symptoms, but no symptoms on Day 7: “improved “ are those patients who show fewer symptoms on Day 7 than on Day 1; “unchanged” are those patients not affected, between the two periods, by variations in their symptoms’ frequency

*Values are expressed as mean ± standard deviation.

Abbreviations: SC, standard care; QP, formulated quercetin (+Standard Care); RT-PCR, real-time reverse-transcriptase polymerase chain reaction; LDH, lactate dehydrogenase: ICU, intensive care unit; n. s. not significant.

Could the increasing incidence of epidemics and pandemics be due to not only increased world travel and researching and modifying biological warfare agents, but also the decreasing body load of antiviral flavonoids that have been lost from the food supply?

Conclusion

The early focus of nutrition research on components in food required for life was very important for the maintenance of life and prevention of overt nutrient deficiency diseases. Unfortunately, the limited technology and physiological understanding of the time resulted in the apparent assumption that the other components of food were not important. We now know that while these “unimportant” molecules are not required to prevent overt disease, they are critically important for health.

The research is now very clear that there is far more to food than the few elements and molecules initially thought to be “important.” The good news is that not only are these molecules useful clinically, but we can now show strong research validation for guiding our patients to increase their consumption of organically grown plant foods.

In the next part of this editorial, I will dive into the intriguing clinical research evaluating the impact of “unimportant” molecule levels in humans.

Biography