Niacin, Riboflavin, and Thiamin



Niacin, Riboflavin, and Thiamin


W. Todd Penberthy, PhD∗



Common Abbreviations



Vitamins B3 (niacin), B2 (riboflavin), and B1 (thiamin or thiamine) are ultimately used by just over 400, 150, and 20 different proteins, respectively, and are thus intimately connected in essential bioenergetic, anabolic, and catabolic pathways. The thiamin and niacin deficiency diseases known as beriberi and pellagra, respectively, were the most devastating vitamin deficiency diseases in the history of the United States. These three vitamins are so important to human health that, together with folate, they are the only vitamins legally mandated for food enrichment by the government. Mandated enrichment is generally considered to have eliminated respective deficiency diseases; however, deficiency of these vitamins remains a primary concern in the modern developed world. This is due to poor food choices, adverse drug reactions, and infectious and autoimmune diseases, any of which can aggressively trigger pathways that actively deplete these vitamins. This chapter covers the history, physiological function, chemistry, and dietary requirements for each of these important vitamins.




Niacin History


Niacin deficiency was first described in Europe by Casal in 1762 as a condition of melancholia, crusty skin, and extreme weakness. Casal coined the disease “pellagra,” meaning angry skin. Pellagra became the most severe vitamin deficiency disease in U.S. history. Over 120,000 people died from pellagra epidemics during the first two decades of the twentieth century; pellagra was also the leading cause of death in mental hospitals in 1907.


By the 1950s medical doctors who witnessed pellagra noticed its similarities to schizophrenia. These doctors theorized that schizophrenics might have a genetic disorder that required far greater levels of dietary niacin. Experiments revealed that fixed administration of gram quantities of niacin to schizophrenic patients frequently resulted in therapeutic benefit with minimal or no adversity (Hoffer et al., 1957). However, this approach remains controversial and requires additional study. As a result of these initial experiments focused on schizophrenia, it was also determined that high-dose niacin (as nicotinic acid) is an effective treatment for dyslipidemia. Today high-dose niacin is a more effective elevator of high-density lipoprotein (“good” cholesterol) than any other pharmaceutical. This approach simultaneously lowers cholesterol, triacylglycerols, and very-low-density lipoprotein. Administration of high doses of various NAD precursors remains an active area of research, especially based on the many favorable results obtained in animal models of human diseases.



Niacin Nomenclature, Structure, and Biochemistry


Vitamin B3 is defined as the precursor to NAD and potentially includes three different molecular forms: nicotinic acid, niacinamide, and nicotinamide riboside. Nicotinic acid or pyridine-3-carboxylic acid is sometimes referred to as “flushing” niacin, whereas niacinamide or pyridine-3-carboxamide is referred to as “flush-free” niacin. The third form of vitamin B3, nicotinamide riboside, was recently discovered in 2004 and is not used clinically yet (Bieganowski and Brenner, 2004). Structures for the three molecules are shown in Figure 24-1.


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FIGURE 24-1 Three forms of vitamin B3. Vitamin B3 is a precursor to nicotinamide dinucleotide (NAD). The body converts the three forms of B3 to NAD through two enzyme reactions, or three in the case of nicotinic acid. We must obtain nicotinic acid, nicotinamide, nicotinamide riboside, or tryptophan from our diet to ultimately synthesize the essential molecule NAD.


Niacin Physiological Function


Molecular biology uses the elimination of gene expression to determine protein function. We can consider similar loss-of-function analysis for determining nutrient function. However, nutrient loss-of-function analysis is much more practically useful because we can readily address the loss-of-function symptoms by administering the vitamin molecule. Experiments involving vitamin deficiencies cannot usually be performed on humans; however, we can still learn from the history of pellagra in human populations and through experimentally controllable animal models.



Dietary Deficiencies of Niacin


The first symptoms of niacin deficiency (i.e., pellagra) include weakness, lassitude, anorexia, and indigestion. These are followed by the classic “three D’s”: dermatitis, diarrhea, and dementia. Additional symptoms include (1) functional changes in the gastrointestinal tract manifested as an absence of normal response to histamine, diminished secretion of hydrochloric acid in the gastric juice, and impaired absorption of vitamin B12, fat, glucose, and D-xylose; and (2) nonspecific lesions of the central nervous system. The dermatitis has a characteristic appearance on parts of the body exposed to sunlight, heat, or mild trauma, such as the face, neck, hands, feet, and elbows. These lesions are usually bilaterally symmetrical. Mental symptoms develop in untreated cases and include irritability, headaches, sleeplessness, loss of memory, and emotional instability. Pellagra-like symptoms are also seen in chronic alcoholism, in carcinoid syndrome, and in some patients who receive certain medications, such as the antituberculosis drug isoniazid or some chemotherapeutics.



Niacin-Responsive Genetic Disorders


Niacin-responsive genetic diseases such as aldehyde dehydrogenase and glucose-6-phosphate 1-dehydrogenase can be rescued with high doses of niacin (Table 24-1). High-dose vitamin therapies stimulate variant enzymes with decreased coenzyme binding affinity (increased Km), thereby improving the metabolic dysfunction (Ames et al., 2002). Given that the number of proteins that require NAD is greater than that for any other cofactor, scores of unmapped genetic polymorphisms are predicted to be responsive to high doses of NAD precursors, many of which have yet to be examined.




Proteins That Require Niacin


NAD participates in more reactions than any other known vitamin-derived molecule. Hundreds of redox reactions use NAD as a cofactor. Tens of reactions use NAD as a substrate and a few proteins use NAD as a ligand. At least 470 proteins use NAD or nicotinamide adenine dinucleotide phosphate (NADP[H]) in some capacity, and this number continually changes as more proteins are discovered. The function and importance of NAD concentrations remain active research areas.



Redox Reactions


NAD(P[H]) is used as a cofactor in a highly diverse set of reactions including the conversion of alcohols (often sugars and polyols) to aldehydes or ketones, hemiacetals to lactones,



image NUTRITION INSIGHT


Enrichment of Grain Products with B Vitamins


In the 1870s a technological development was made in mass grain milling. For the first time refined foods such as white flour and white rice became available to large segments of the population. Previously only wealthy individuals could have these. The milling of grains had the advantage of preventing spoilage; however, this processing depleted vitamins and minerals concentrated in the outer layers of the kernels (bran and aleurone) and germ. Wherever these refined foods were introduced, the diseases of pellagra and beriberi frequently appeared, depending on the variety of the diets.


Pellagra was particularly rampant in the southern United States, where diets frequently consisted simply of corn, molasses, and biscuits. For decades the search for the cause of pellagra centered on the association with corn-rich diets. Curiously, Native Americans did not suffer from pellagra, although corn was their main dietary staple. They treated corn with limewater, a process called nixtamalization, which released the NAD precursor tryptophan (and also inactivated mycotoxins), thus preventing pellagra.


Pellagra was finally understood in the 1930s thanks in large part to the initial work of the epidemiologist Dr. Joseph Goldberger. In 1926 he showed that a small amount of brewer’s yeast could prevent pellagra. Initially Goldberger’s hypothesis that pellagra was a deficiency disease was rejected in favor of hypotheses involving corn toxins or poor sanitation. In 1937 Dr. Conrad A. Elvjeham identified the pellagra-preventing factor as the molecule nicotinic acid. In these first experiments, dogs were fed deficient diets to cause tongue discoloration. The component of rice polishings that prevented tongue discoloration was subsequently isolated and named the pellagra-preventing factor.


Owing to the severity of the pellagra epidemics, the U.S. government established legally mandated standards of niacin enrichment of flour and other grain products. Beginning in the early 1940s these standards restored levels of thiamin, riboflavin, niacin, and iron to those found in whole-grain products. Enriched flour contains 2.9 mg thiamin, 1.8 mg riboflavin, 24.0 mg niacin, and 20 mg iron per pound of flour. Beginning as late as 1998, enriched-grain products have also been fortified with folate. Although products labeled “enriched” are responsible for eliminating pellagra epidemics, whole-grain products are still exceptional sources of nutrients because other vitamins and minerals lost in milling are not added as part of the enrichment program.


aldehydes to acids, and certain amino acids to keto acids. The common mechanism of operation for NAD(P[H]) cofactor redox reactions is generalized in Figure 24-2. Most dehydrogenases that use NAD or NADP function reversibly. Generally enzymes use NAD(H) in catabolic reactions, whereas NADP(H) is more commonly involved in anabolic (biosynthetic) reactions. For example, NADPH serves as an important reducing agent for the synthesis of fats and steroids (e.g., reactions catalyzed by 3-ketoacyl reductase, enoyl reductase, and 3-hydroxy-3-methylglutaryl-coenzyme A [HMG-CoA] reductase).


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FIGURE 24-2 Mechanism of substrate oxidation or reduction by pyridine nucleotide coenzymes. Typically, X is an electronegative atom (e.g., oxygen).

For pyridine nucleotide coenzymes to continue acting as catalysts, they must be recycled by coupling with other redox reactions. This may occur by coupling dehydrogenation reactions with hydrogenation reactions. For example, glyceraldehyde 3-phosphate dehydrogenase is coupled with lactate dehydrogenase in anaerobic glycolysis to produce NAD required for glycolysis. Similarly, NADPH produced by the pentose phosphate pathway is coupled to fatty acid synthesis. Alternatively dehydrogenation reactions are coupled with electron transport, as found in mitochondria.


Both NAD and NADP are part of the intracellular respiratory mechanism of all cells. They assist in the stepwise transfer of electrons or reducing equivalents from various energy substrates to the cytochromes. NADH usually donates its electrons to a flavin coenzyme in the mitochondrial electron transport chain responsible for ATP production. These reactions are outlined in Figure 24-3. A major source of NADH in the mitochondria is the β-oxidation of fatty acids. The main extramitochondrial source is NADH formed in glycolysis. Reducing equivalents are carried to the mitochondria by the malate–aspartate or the glycerol phosphate–dihydroxyacetone shuttles (see Chapter 12).


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FIGURE 24-3 Pyridine nucleotides and flavocoenzymes funnel reducing equivalents to the mitochondrial respiratory chain. The main extramitochondrial source is NADH formed in glycolysis. These reducing equivalents are carried into the mitochondria by the malate–aspartate or the glycerol phosphate–dihydroxyacetone shuttles. NADH yields 3 ATPs, whereas FADH2 yields 2 ATPs. Cyt, Cytochrome; ETF, electron transfer flavoprotein; FAD, flavin adenine dinucleotide; FeS, iron-sulfur protein domain; FMN, flavin mononucleotide; ThDP, thiamin diphosphate.

The approximately 57 different human cytochrome P450 monooxygenase systems use reducing equivalents of NADH and NADPH for a wide variety of functions, including synthesis and degradation of drugs, xenobiotics, prostaglandins, leukotrienes, retinoic acid, vitamin D, cholesterol, bile, and steroids (Nebert and Russell, 2002).



Nonredox Reactions and Control of Nad Levels


Four classes of enzymes play dominant roles in controlling NAD levels in response to DNA damage, immune activation, and other stimuli. These enzymes are poly(ADP-ribose) polymerase 1 (PARP1 through PARP18), the NAD-dependent deacetylases (sirtuin 1 through sirtuin 7), the ADP ribosyl-cyclases (CD38 and CD157), and indoleamine 2,3-dioxygenase (IDO). The first three of these enzymes produce nicotinamide as a side product that is recycled back to NAD via the salvage pathway as shown in Figure 24-4.


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FIGURE 24-4 Pathways proceeding from vitamins or essential amino acid tryptophan to NAD synthesis and fates are shown. NAD is used by ADP ribosyl-cyclase and sirtuin to generate potent activators of intracellular calcium release. Additional vitamins required for completion of the de novo NAD synthesis pathway starting from tryptophan include vitamin B2 (riboflavin), vitamin B6 (pyridoxyl phosphate), and vitamin C (ascorbate). Otherwise, vitamin B1 (thiamin) is required for synthesis of phosphoribosylpyrophosphate (PRPP) in the pentose phosphate pathway. Feedback inhibition loops are drawn with double lines. Nicotinic acid has distinguished capacity as a provider of NAD because nicotinate phosphoribosyltransferase (NAPRT) is not inhibited by NAD feedback inhibition (see text). By contrast when starting from nicotinamide, nicotinamide phosphoribosyltransferase (NAMPT) is inhibited by NAD. Nicotinic acid is converted to NAD via three reactions, whereas nicotinamide and nicotinamide riboside are converted to NAD via two reactions. The Preiss-Handler pathway begins with nicotinate combining with PRPP to make nicotinate mononucleotide (NaMN) in a reaction catalyzed by NAPRT. NaMN is then adenylated to form nicotinic acid adenine dinucleotide (NaAD) in an ATP-requiring reaction catalyzed by nicotinamide mononucleotide adenylyltransferase (NMNAT). NaAD is then converted to NAD by an ATP-requiring NAD synthetase (NADS) reaction, which transfers the amide from glutamine to NaMN to produce NAD. Nicotinamide and nicotinamide riboside are converted to NAD by just two reactions. Both are first converted to nicotinamide mononucleotide (NMN) followed by conversion to NAD in a reaction that requires ATP and is catalyzed by NMNAT. The NMNAT enzymes are used in NAD synthesis from all NAD precursors including tryptophan and nicotinic acid. Gln, Glutamine; Glu, glutamate; HAAO, 3-hydroxyanthranilate 3,4-dioxygenase; IDO, indoleamine 2,3-dioxygenase; KAT, kynurenine aminotransferase; KMO, kynurenine 3-monooxygenase; KYNU, kyureninase; NRK, nicotinamide riboside kinase; PAR, poly(ADP-ribose); PARP, PAR polymerase; PPAR, peroxisome proliferator-activated receptor, QPRT, quinolinate phosphoribosyltransferase; TDO, tryptophan 2,3-dioxygenase.

NAD-consuming activities are highly activated in response to any kind of DNA damage. PARP1 and PARP2 enzymes use NAD as a substrate to generate poly(ADP)ribose, an anionic polymer resembling DNA, to transfer ADP-ribose directly to histones and other proteins, including p53 and nuclear factor kappa B (NFκB). There are at least 18 different ADP-ribose transferring enzymes. PARP activation is directly proportional to the degree of DNA damage; it stops cell division and attempts DNA repair, a critical step in preventing cancer cells. More significantly, hyperactivation of PARP1 alone depletes intracellular NAD and ATP, which can lead to uncontrolled necrotic cell death.


Sirtuins can negatively regulate PARP activity by deacetylation. The sirtuins are NAD-dependent deacetylases that can target histones, p53, NFκB, and other important proteins. A tremendous amount of research has been devoted to sirtuins, owing to the general observation that increased NAD-mediated sirtuin activation affords health benefits. For example, sirtuin activators are useful for the treatment of diabetes and neurodegeneration in animal models.


We generally cannot change our genetics; however, we can change our epigenetics by altering NAD levels. Increased NAD activates sirtuin enzymes, which alters chromatin structure. For example, sirtuins are activated when calorie intake is reduced, and the resulting change in chromatin structure may be responsible for many of the therapeutically beneficial effects of calorie restriction. The other histone deacetylases are believed to possess constitutive activity that cannot be regulated. Thus NAD-dependent activation of sirtuin histone deacetylase activity is unique.


The ADP ribosyl-cyclase enzyme CD38 uses NAD as a substrate to generate the most potent known activators of intracellular calcium release, a mechanism required for chemotaxis of a variety of immune cells. The CD38-deficient mouse has persistent elevated NAD levels, increased energy expenditure, and does not become obese even when fed a high-fat diet. However, because CD38 is also required for chemotaxis of various immune cells, CD38-deficient mice are much more susceptible to infections. CD38 levels and activity are chiefly regulated at the transcriptional level, particularly by tumor necrosis factor-alpha (TNFα), which positively increases expression of CD38. CD157 is another NAD-dependent ADP ribosyl-cyclase that has important but less understood roles in neuron–glia cell interactions.


IDO plays major roles in controlling physiological NAD levels in humans by altering tryptophan concentrations within restricted cell types. Tryptophan is the essential substrate used for the de novo synthesis of NAD. IDO is highly activated within professional antigen-presenting immune cells (dendritic cells, macrophages, and B cells) during infections and in autoimmune disease. Unfortunately, persistent activation of IDO is pathogenic to neighboring cells because of this immune cell–specific consumption of tryptophan. The IDO pathways are complicated, but they are an area of intense research for both autoimmune diseases and cancer.



Niacin Sources, Chemical Stability, and Admet


Sources


Nicotinic acid, nicotinamide, and nicotinamide riboside are widely distributed in foods of both plant and animal origin. Good sources of preformed NAD precursors include milk, beef, poultry, fish, legumes, peanuts, and some cereals. Enriched grain products and flours are good sources as well. In uncooked foods of animal origin, the major forms of niacin are the cellular pyridine nucleotides, NAD(H) and NADP(H).




ADMET


The physiological effect of any ingested molecule depends on absorption, distribution, metabolism, excretion, and toxicity, together abbreviated as ADMET.




Distribution and Metabolism


Coenzyme forms of niacin in the gastrointestinal tract are first rapidly hydrolyzed to nicotinamide mononucleotide (NMN) by nonspecific pyrophosphatases in the intestinal lumen (Gross and Henderson, 1983). Alkaline phosphatase catalyzes further cleavage to nicotinamide riboside, which is converted to NAD by a two-step pathway starting with a reaction catalyzed by nicotinamide riboside kinase (see Figure 24-4) (Bieganowski and Brenner, 2004). NAD glycohydrolases (NADases) within mucosal cells may contribute to the breakdown of the coenzymes to nicotinamide. Once in the plasma, niacin enters cells by active or passive diffusion followed by metabolic trapping. Active diffusion of niacin into erythrocytes involves an anion transporter protein. Both erythrocytes and liver rapidly remove and convert niacin to NAD.


Tryptophan is an essential amino acid and also a precursor to NAD. Deficiencies of tryptophan transport (Hartnup disease) are primarily rescued by administering nicotinamide or nicotinic acid. Tryptophan is required for endogenous synthesis of NAD via the de novo pathway, which begins as the kynurenine pathway. This pathway requires



image CLINICAL CORRELATION


Niacin and Cancer


Cancer cells require more NAD and ATP to survive than nontransformed healthy cells. The rate of glycolysis in a cancer cell is at least 30 times greater than that of a nontransformed cell, making it more susceptible to cell death when deprived of essential molecules. Essentially all cells starve to death after some point of deprivation, whereas cancer cells will starve to death sooner than nontransformed cells when both are deprived of these essential molecules. Inhibition of NAD pathways kills cancer cells with greater sensitivity than healthy cells. New chemotherapeutics are being developed to target inhibition of the NAD-synthesizing enzymes NAMPT and IDO, essentially using a metabolic approach to killing cancer. PARP1 inhibitors are also being developed as cancer therapeutics.




image FOOD SOURCES OF PREFORMED NIACIN






Data from U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS). (2010). USDA National Nutrient Database for Standard Reference, Release 23. Retrieved from www.ars.usda.gov/ba/bhnrc/ndl


riboflavin, pyridoxyl phosphate (vitamin B6), and ascorbate (vitamin C) (see Figure 24-4). The de novo pathway is highly regulated by the immune system in specific cell types. Interferon gamma activates the rate-limiting enzyme, IDO, which is required for interferon gamma’s biological activities. Many intermediates in this NAD synthetic pathway such as kynurenine and kynurenate have significant physiological roles. Persistent activation of IDO is seen in many autoimmune diseases and in cancer, where decreased serum tryptophan is a common diagnostic indicator of poor prognosis. The complementary administration of high doses of alternative NAD precursors has been repeatedly shown to rescue these pathogenic processes in both animal models and some clinical cases (Penberthy, 2007).


The NAD salvage pathway recycles nicotinamide produced as a side product of NAD-degradation by PARP, sirtuin, and ADP ribosyl-cyclases back to NAD (see Figure 24-4). Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme controlling the rate of recycling of nicotinamide to NAD (Revollo et al., 2004). Importantly, NAMPT gene expression is strongly induced in response to a wide variety of stresses to provide an adequate level of NAD. Increased nicotinamide mononucleotide adenylyltransferse (NMNAT)-1, 2, or 3 also provides tremendous cell survival benefit in a wide range of stresses, particularly as seen in neurodegenerative models (Sasaki et al., 2006). Although NAMPT may be rate limiting for the salvage pathway under basal conditions, the NMNAT enzymes may limit NAD synthesis under stress-induced conditions where the levels of NAMPT have been dramatically increased. NAD inhibits NAMPT activity but not nicotinate phosphoribosyltransferase (NAPRT). This is partially why nicotinic acid provides greater levels of intracellular NAD than nicotinamide for many cell types.




Toxicity


Over the past several decades, dyslipidemia has been commonly treated with 3 to 5 g of nicotinic acid daily without serious adverse events (Carlson, 2005; Guyton and Bays, 2007). However, the flush response can be exceedingly unpleasant. The upper tolerable intake level (UL) for adults is designated at 35 mg/day of niacin, limited to niacin



image CLINICAL CORRELATION


Case Study of Pellagra in an Adult Woman


Oakley and Wallace (1994) reported a case involving a young woman in New Zealand who had a variety of neurological and dermal symptoms consistent with pellagra. However, she did not have diarrhea. Her symptoms were precipitated by prolonged lactation and increased activity. She had breast-fed her daughter for 3 years and had been particularly active while building her own home. Dietary intakes of niacin equivalents were within normal range. Her daily total excretion of urinary amino acids was abnormally high (77 mmol/L). Chromatography of urinary amino acids revealed elevated loss of tryptophan and other large neutral amino acids (alanine, serine, threonine, valine, methionine, isoleucine, tyrosine, and phenylalanine), whereas taurine, glycine, ornithine, lysine, arginine, and aspartate were present in normal amounts. Her symptoms were resolved after taking nicotinamide plus other B vitamins.



obtained from synthetic sources or fortified foods, based on the dose of nicotinic acid that can be associated with flushing effects. Although the flush can be uncomfortable, it is in fact linked to the beneficial effect of correcting dyslipidemia, because nicotinamide does not exert these therapeutically beneficial effects on lipid profiles.



Biochemical Assessment of Niacin Nutriture, DIETARY Requirements, and High-Dose Responses


Biochemical Assessment of Niacin Nutriture


Biochemical assessment of niacin nutritional status is usually based on whole blood measures of NAD/NADP (Jacobson and Jacobson, 1997). Restricting niacin/tryptophan intake to 50% of the Recommended Dietary Allowance (RDA) decreases NAD by 70% within 5 weeks while NADP levels remain constant. A decrease in NAD levels precedes pellagra symptoms and are frequently seen in carcinoid syndrome patients.



Dietary Requirements


The RDA was first developed during World War II to set minimal standards for food relief during wartime. These standards are updated regularly. The Estimated Average Requirement (EAR) is a measure of the amount needed to satisfy the needs of 50% of the population, while the RDA is designed to satisfy the needs of 97% of the population. Because the amino acid tryptophan may also serve as a precursor for NAD synthesis, the term niacin equivalent (NE) is used to quantify niacin intakes and requirements.


For niacin the EAR is 12 mg and 11 mg of NEs per day for men and women, respectively. RDAs are calculated as 30% greater than the EAR (1 coefficient of variation = 15%). The RDA is relatively easy to meet, with typical intakes in the United States of 25 to 40 mg of NEs per day, but it should be recognized that tryptophan rather than niacin is the major source of NEs in typical diets.



image DRIs Across the Life Cycle: Niacin


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Feb 26, 2017 | Posted by in PHARMACY | Comments Off on Niacin, Riboflavin, and Thiamin

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