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CHAPTER 1
Human Vitamins: Discovery and Characterization
1.1 What Are Human Vitamins?
Vitamins have been known for just over 100 years as organic molecules essential for human health. They are defined as pure molecules that, while required for acute and chronic human health, cannot be biosynthesized by human cells and tissues (see Figures 1.1 and 1.2) (Combs and McClung, 2017). They must be obtained exogenously. Historically this meant from diet. Since the 1950s, the total synthesis of vitamins on an economic, industrial scale has meant an ever-increasing growth of vitamin supplements in a cascading consumer market.
The first inklings of vitamins as molecules required for human health go back to ancient times when Hippocrates is ascribed the suggestion of a vegetable cure for night blindness (a condition known as xerophthalmia) – from deficiency of one of the forms of vitamin A (Lindeboom, 1984). At least three hundred years ago, the observations that citrus fruits, and rations of lime juice in particular, could cure the symptoms of scurvy that had plagued British sailors among other seafarers on long voyages in the age of sail were a harbinger of what turned out to be vitamin C (Lamb, 2016). During the first four decades of the 20th century one vitamin after another, until a total of thirteen, were identified as micronutrients essential first in animal models and then in human health (Figure 1.1).
One can argue that the discovery of vitamins gradually brought into focus the concept of nutritional deficiencies as the basis of a subset of human diseases (Price, 2015). This concept complemented the recently established platform of infectious diseases that had coalesced from studies by Pasteur, Koch, and other microbiologists during the latter part of the 19th century. Subsequent understanding of all eight B vitamins as coenzymes for a broad range of cell chemistries helped to frame the principles of human metabolism.
1.1.1 Vitamins as Micronutrients and Recommended Daily Allowances (RDAs)
Definitionally, vitamins are essential organic molecules required from exogenous sources, via classical diet and in minute amounts as micronutrients. Figure 1.2 shows a gradation at the low end from 2–30 µg (vitamin B12 and vitamin D, and biotin B7) to 90 mg per day of vitamin C, almost four orders of magnitude range from low to higher dose (Combs and McClung, 2017). Vitamin C and vitamin E, (with the second highest recommended dietary allowance (RDA) at 15 mg per day), are thought to be effective but nonspecific antioxidants against water-soluble and lipid-soluble radical species, respectively. Vitamins C and E may need to reach higher intracellular concentrations than the active forms of other vitamins to fulfill their likely collisional encounters with potentially toxic and destructive organic and inorganic radicals.
We will note in Chapter 13 that vitamin D could as easily be classified as a hormone rather than a vitamin, an attribute also of the retinoic acid oxidation state but not the retinal form of vitamin A. Such hormones working with intracellular nuclear receptors may have binding affinities in the 10-9 M range for their single target receptor proteins in the sea of thousands of other proteins in cells.
The other vitamins cluster around a recommended daily allowance of about 1 mg per day. For an average molecular weight of ~300 daltons for those vitamins, 1 mg per day corresponds to ~30 nmoles, a small amount but still ~1015 to 1016 molecules. If all were fully absorbed at ingested RDA levels and distributed equally to the ~4×10 human cells per adult, about 100 to 1000 vitamin molecules per cell per day would be present before consideration of the many recyclings of almost all the vitamins in cellulo.
1.2 Vitamin Discovery: A Golden Age from 1910–1948
All 13 of the human vitamins were discovered in an intense set of efforts in the first four decades of the twentieth century between animal and human nutritionalists, medical practitioners and researchers involved in the diagnosis of human diseases, natural product isolation biochemists, and synthetic chemists in academic and industrial pharmaceutical laboratories. Three elements had come together to power this sustained set of discovery efforts (Combs and McClung, 2017).
The first element was the concept that some human diseases and their animal counterpart diseases could be caused by one or more micronutrient deficiencies (Figure 1.3). This required a new mode of thinking among physicians and public health researchers about the etiology of human diseases. The second element was the availability of animal models as assays for the deficiency of a particular essential molecule required in metabolism. Here, the animal physiology research community was critical. Indeed, while the discovery of thiamin as the first vitamin was largely a triumph of human epidemiologic research, most of the subsequent vitamin discoveries were based on animal models of human disease. Combs and McClung (2017) are probably the definitive source in summarizing the several paths that went forward.
The third element in the confluence that enabled vitamin discovery, purification, and characterization as pure low molecular weight molecules (B12 is, exceptionally, a medium molecular weight compound) was the availability of diets of defined composition. These became available at the end of the 19th and beginning of the 20th century as nutrition scientists worked to define the essential components for healthy human diets and worked out recipes for pure samples of protein, carbohydrates, lipids, and trace metals as starting points.
Combs and McClung recount the discovery of vitamin B (niacin), from observation of inmates with pellagra in an orphanage in Jackson, Mississippi in the 1920s (Combs and McClung, 2017). Pellagra (from the Italian word corresponding to roughened skin) had been known for two centuries (initially as mal de la rosa for the characteristic skin and mouth lesions on presentation). Goldberger noted that the orphanage staff did not contract pellagra although many of the patients (pellagrins) did (Goldberger and Tanner, 1922): the hypothesis was that health status might be determined by the milk and meat consumed by the staff but not the patients. A two-year dietary experiment with addition of milk and meat to the pellagrins' diet ameliorated the disease symptoms. Subsequently a dog model (black tongue) led to the assay needed for identification of niacin as the missing dietary factor.
The vitamin deficiency symptoms in animal models could be complex and reflect different endpoints of pathology from alteration of metabolite throughput in essential metabolic pathways. While vitamin B3 deficiency gives rise to the symptoms of human pellagra, animal models of pellagra-like symptoms led to detection and purification of two other B vitamins as dividends. Combs and McClung noted that a rat pellagra model was useful for isolation of vitamin B6 (pyridoxine). When a chick pellagra model was used, instead it was pantothenate (vitamin B5) deficiency that dominated and helped lead to B5 isolation and characterization (Combs and McClung, 2017). Animal pharmacology is always an inexact mimic of the human situation but also key metabolic pathway disruptions play out in complex ways at organ and organism levels.
The lack of a high throughput model was part of the reason the characterization of vitamin B12 was prolonged over two decades. While Minot and Murphy in 1926 (Minot and Murphy, 1926) had found that raw liver contained an active substance in treating the symptoms of fatal pernicious anemia, the lack of animal models and the scarcity of human patients made for a twenty-year slog from initial detection of vitamin B12 biological activity to its crystallization 22 years later in 1948 by teams led by K. Folkers at Merck and E. Smith at Glaxo (Rickes et al., 1948; Scott and Molloy, 2012;Smith and Parker, 1948). The discovery in 1947 that Lactobacillus lactis required the pernicious anemia extrinsic factor (B12) for growth enabled rapid purification of B12 to final homogeneity only one year later. This completed the set of all thirteen human vitamins. Figure 1.3 matches a given vitamin with characteristic deficiency syndrome that led to its isolation.
Each vitamin, with its distinct chemical and physical properties, posed different challenges. Folic acid with its variable glutamyl side chains (from one to up to twelve γ-glutamyl residues in some bacteria) had distinct activity in different assays, e.g. megaloblastic anemia preventions versus growth of lactobacillus bacteria. Thus, the mono-, di-, and heptaglutamyl forms of folate gave results in different assays that slowed recognition that there was an identical scaffold and a variable side chain with a range of biologic activities.
The differential stability of vitamins to heat and solubility in organic versus aqueous solvent helped in the isolation of these small molecules from natural sources (e.g. yeast extract) in which they might be present in trace quantities and as mixtures. Most of the vitamins, consistent with their small molecule, organic chemical structures, are heat stable. Thus, removal of proteins by heat was a common early step in isolation protocols. Vitamin A, however, is heat labile.
It had been known for some time that fish oil, including the paradigmatic cod liver oil products, contained lipid-soluble factors that could ameliorate certain disease symptoms. Among the first lipid-soluble vitamin activity in fish oil was vitamin A, activity destroyed by heating. McCollum and coworkers in 1922 utilized this heat lability to show that after removal of the heat-labile, antixerpthalmic (night blindness prevention, vitamin A) factor by prolonged heating, the oil still contained an antirachitic factor (vitamin D) that was heat stable (McCollum et al., 1922). This allowed proof that vitamin D existed as a separate entity and gave a strategy for its isolation. Ultimately, both retinal and retinol forms of vitamin A were crystallized from fish liver oil some two decades later (Combs and McClung, 2017).
Figure 1.4 depicts the time line for discovery of each human vitamin and food sources that are plentiful in each specific vitamin. Three of the four fat-soluble vitamins are found in high levels in vegetable oils, while the fourth, vitamin K, is present in leafy vegetables, consistent with its production in plant chloroplasts.
1.3V itamin Deficiencies: Primary and Secondary Symptoms
1.3.1 Primary Deficiencies
One can define vitamin deficiencies based on whether there is adequate intake or not of a given vitamin, and, if so, whether one or more of the essential targets is nonfunctional. Primary vitamin deficiency would be an inadequate supply of the vitamin from diet. Recommended daily allowances (RDAs) have been proposed for each of the thirteen vitamins based on "average daily intake level sufficient to meet the requirements of nearly all (97–98%) of healthy individuals in each age-sex specific demographic group" (see Figure 1.2) (Combs and McClung, 2017).
The RDA levels are set periodically and revised based on nutritional and disease-based reports. Levels are meant to prevent overt disease, cover other health risks from mechanisms of action of a given vitamin that may not yet be understood or appreciated in a molecular or metabolic level. The levels are also meant to avoid hypervitaminosis toxicity.
Essentially none of the water-soluble vitamins are stored in high levels in body tissues as they do not partition into lipid spaces. Thus, large doses tend to be excreted every day. One exception can be vitamin C. At large to megadoses of vitamin C it can undergo substantial metabolism to oxalate, calcium salts of oxalate are insoluble and can form kidney or urinary stones. This appears to be a rare but not negligible event.
Two of the lipid-soluble vitamins, vitamin A and vitamin D, can cause hypervitaminosis toxicity. At high dose they can accumulate in tissues, both the retinoic acid vitamer form of vitamin A and the 1,25-dihydroxy form of vitamin Dare hormones and can turn on hundreds of genes in target tissues (Chapters 12 and 13). Vitamin A in the form of retinol at levels above 3 micromolar can cause acute and chronic toxicity symptoms ((Combs and McClung, 2017), Table 5.8 for details) including craniofacial development abnormalities as well as CNS, cardiovascular and thymus organ system aberrations. Vitamin D at physiologic doses controls calcium phosphate uptake from the GI tract (Chapter 13). At very high doses of D hypercalcemia can occur and those high levels of blood calcium can lead to calcification of soft tissues, e.g. heart, a pathology known as calcinosis. There seem to be no serious hypervitaminoses with the other ten vitamins.
1.3.2 Secondary Deficiencies
Secondary deficiencies, persisting even in the face of otherwise adequate levels of an ingested vitamin, suggest one or more of the absorption, distribution, and execution of metabolic roles of the vitamin are defective. That could be uptake from the GI tract. A famous historical case was the finding that absorption of exogenous B12 (extrinsic factor) required secretion of a protein carrier, intrinsic factor, by gastric parietal cells (W. Castle's discovery in 1928 reviewed in (Kass, 1978)). Many of the B12 deficiency symptoms are due to autoantibodies against intrinsic factor. There are also mutations in/or failure to make and secrete functional full length intrinsic factor. Both conditions prevent subsequent intestinal uptake of the vitamin/intrinsic factor complex by enterocyctes. Vitamins, especially the water insoluble vitamins A, D, E, and K need to be transported bound to carrier proteins in the blood to end organs. If one of these is defective it will be a secondary vitamin deficiency. Loss of one of the enzymes required to convert any of the eight B vitamins to their coenzymatically active forms will also show up as a vitamin deficiency.
Combs and McClung do their usual exemplary job of exhaustive scholarship in characterizing the causes and effects of vitamin deficiencies on human organ systems (see their Chapter 5). Four (of thirteen) abstracted examples give a sense of how deficiency in specific targets can be exhibited as organismal pathology (Combs and McClung, 2017).
Three sites of secondary B12 deficiency are (a) lack of functional intrinsic factor, (b) lack of the carrier protein transcobalamin in blood, (c) and nonfunctional methylmalonyl-CoA mutase, one of the two B12 target enzymes in human metabolism (Chapter 10). Symptoms, respectively, can present as juvenile pernicious anemia, megaloblastic anemia, or methylmalonic acidemia (low blood pH). The last of these symptoms results from buildup of methylmalonyl-CoA, its hydrolysis to the free acid and secretion of high intracellular levels of methylmalonic acid into the blood. We discuss the molecular causes of the anemia symptoms in Chapter 10.
Secondary B2 (riboflavin) deficiency in humans might arise from lack of functional methemoglobin reductase (Fe3+ back to Fe2+) or nonfunctional electron transfer flavoprotein, which is the acceptor for electrons removed from short, medium, and long chain acyl-CoAs by acylCoA dehydrogenases (Chapter 4). The results can show up as methemoglobinemia or metabolic acidosis, respectively.
Three examples of defects in biotin (vitamin B7) metabolism are (a) defective biotinidase, the enzyme that attaches free biotin covalently to its target carboxylases, (b) nonfunctional propionyl-CoA carboxylase, and (c) nonfunctional pyruvate carboxylase (Chapter 8). The systemic pathologies that follow are alopecia (baldness), propionic acidemia, and Leigh disease, respectively. Leigh syndrome presents with elevated levels of both pyruvate and lactate (Baertling et al., 2014). Elevated pyruvate levels are dealt with in part intracellularly by reduction to L-lactate, which is then excreted into the blood.
The last brief example covers secondary defects in vitamin B1 (thiamin) physiology. With a defect in the function of pyruvate dehydrogenase (Chapter 3) the main bridging catalyst between glycolysis and the citrate (tricarboxylate) cycle, pyruvate builds up, is reduced to lactate and lactic acidemia occurs with neurologic symptoms (Kraut and Madias, 2014). When the branched chain keto acid dehydrogenase is the nonfunctional catalyst instead, the buildup of the branched chain keto acids in urine is accompanied by the odor reminiscent of maple syrup, leading to the terminology of maple syrup urine disease (Podebrad et al., 1999).
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Excerpted from "The Chemical Biology of Human Vitamins"
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Copyright © 2019 Christopher T. Walsh and Yi Tang.
Excerpted by permission of The Royal Society of Chemistry.
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