Book

Gibson RS1,      Principles of
Nutritional Assessment:
Niacin

3rd Edition    August 2024


Abstract

The term "niacin" refers to three compounds: nico­tin­amide, nico­tinic acid, and nico­tin­amide ribose. They are precursors of nico­tin­amide adenine dinucleo­tide (NAD) and nico­tin­amide adenine dinucleo­tide phosphate (NADP), both of which function in a variety of oxidation and reduction reactions and are associated with both cata­bolic and anabolic processes. Pellagra, a disease of the "4Ds" (diarrhea, dermatitis, dementia, and death) is the syndrome of niacin deficiency. Although rare, outbreaks of pellagra still occur when diets are based on maize (without nixtamalization), a cereal low in bioavailable niacin. Secondary niacin deficiency may also develop in diseases that inter­fere with absorp­tion of niacin or tryp­to­phan (a precursor of niacin) (e.g., chronic colitis, liver cirrhosis) or reduce the con­ver­sion of tryp­to­phan to niacin (e.g., chronic alcoholism, prolonged isoniazid treat­ment for tuberculosis). Rich food sources of niacin include meat, poultry, and fish, followed by dairy products and enriched or fortified cereals. Coffee is also a good source of niacin. Absorption of niacin from food ranges from about 23% to 70% and is lowest from cereals and highest from animal products. Certain food preparation and pro­ces­sing practices may influence the content and bio­avail­ability of niacin in food. Intakes and requirements for niacin are expressed as niacin equivalents (NEs) to consider the contribution of tryp­to­phan to niacin (i.e., 60mg tryp­to­phan yields 1mg niacin). Dietary reference values for niacin are set as NE per day, or alternatively expressed as mg NE/MJ or mg NE/1000 kcal based on the relationship between the requirements for niacin and energy. Niacin toxicity may occur when large pharmacological doses of niacin are taken to prevent or treat metabolic diseases such as dyslipidemia. Tolerable Upper Intake Levels (ULs) are based on dermal vasodilative flushing.

At present, the best biomarker of niacin status is the measure­ment of two urinary meta­bolites: N'‑methyl­nico­tin­amide and N'‑methyl-2-pyridine-5-carbox­amide (2‑pyridone) using HPLC. Sampling strategies employed include 24h urines with results expressed as concen­trations of individual meta­bolites (mg/d), or random spot urines when the ratio (2‑pyridone to N'‑methyl­nico­tin­amide), or concen­trations relative to creatinine (mg/g creatinine) are used. Interpretive criteria for adults are available for urinary excretion of these niacin meta­bolites expressed relative to creatinine and as the ratio (2‑pyridone / N'‑methyl­nico­tin­amide). WHO has developed provisional criteria for the severity of public health problem of niacin deficiency. Concentrations of the co-enzyme NAD in erythro­cytes (but not whole blood) or the ratio of erythro­cyte NAD to NADP (called niacin index) are said to be sensitive to short-term changes in niacin intake, even in the absence of clinical signs of deficiency. Hence, they may serve as a sensitive and reliable functional biomarker for risk of niacin deficiency. With the development of a rapid LC-MS/MS method for their analysis, further investigation of erythro­cyte NAD and NADP concen­trations as biomarkers of niacin status is warranted.

CITE AS: Gibson RS. Principles of Nutritional Assessment. Niacin https://nutritionalassessment.org/niacin/
Email: Rosalind.Gibson@Otago.AC.NZ
Licensed under CC-BY-4.0
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20c.1 Niacin - Introduction

Niacin (also known as "vitamin B3") is the term used to describe a group of compounds with the biological activity of the vitamin. They include nico­tinic acid (also known as niacin) composed of a pyrimidine ring bound to a carboxylic group (pyridine-3-carboxylic acid), nico­tin­amide (also known as niacinamide) composed of a pyrimidine ring bound to a carbox­amide group (pyridine-3-carbox­amide), and nico­tin­amide riboside. The third compound, nico­tin­amide riboside has been identified more recently and is a pyridine-nucleoside form of vitamin B3 consisting of nico­tin­amide with a beta-D-ribofuranosyl moiety at the 1‑position (Mehmel et al., 2020). All three compounds have similar but not identical properties and are precursors of nico­tin­amide adenine dinucleo­tide (NAD) and its phos­phory­lated derivative, nico­tin­amide adenine dinucleo­tide phosphate (NADP). The structures of nico­tinic acid, nico­tin­amide, and nico­tin­amide riboside are shown in Figure 20c.1.
Figure 24c.1
Figure 20c.1. Vitamin B3 molecules (nico­tinic acid, nico­tin­amide, and nico­tin­amide riboside), dietary precursors that support the form­ation of nicotinamide adenine dinucleotide. Redrawn from Kirkland & Meyer-Ficca (2018).

20c.1.1 Functions of niacin

Most of the niacin in food is present as NAD and NADP; very little exists as free forms of niacin (Chungchunlam & Moughan, 2023). Prior to absorp­tion, these two com­ponents must first be hydro­lyzed to free nico­tin­amide by phos­phatases and NAD glyco­hydro­lases in the small intestine, the major site of absorp­tion. At low intakes, absorp­tion into the intestinal cell is by sodium-dependent, carrier-mediated diffusion, but at higher intakes, passive diffusion predom­inates (Hrubša et al., 2022). Once absorbed, free nico­tin­amide is trans­ported to all tissues for the synthesis of the two coen­zymes NAD and NADP within the cells. In general, concentrations of intra­cellular NAD are higher than NADP. All tissues of the body can syn­the­size NAD and NADP, although concentrations are greatest in the liver where some storage may occur. Some niacin can be syn­the­sized by colonic bacteria in the large intestine (Yoshii et al., 2019). NAD exists in two forms: NAD+ (oxidized) and NADH (reduced), while NADP exists as NADP+ (oxidized) and NADPH (reduced).

NAD+ can be syn­the­sized within the cell from nico­tinic acid, nico­tin­amide, nico­tin­amide riboside, and tryp­to­phan by three path­ways, all of which require the enzyme nico­tin­amide mono­nucleotide phosphoribosyl­transferase (NMNAT). The three path­ways include the con­ver­sion of tryp­to­phan to NAD via the de novo NAD synthesis path­way, the Preiss-Handler path­way from nico­tinic acid, comprising three individual metabolic conversions, and the salvage path­way involving the NAD+ precursors nico­tin­amide or nico­tin­amide riboside. Of the three, the salvage path­way is the domi­nant one; for more details on these synthetic path­ways see Kirkland & Meyer-Ficca (2018).

NAD+ primarily participates in cata­bolic reactions and is an essential substrate for the activity of three classes of NAD+ -consuming enzymes: (i) sirtuins (SIRTs), (ii) poly(ADP-ribose) polymerases (PARPs), and (iii) cyclic ADP-ribose (cADPr) synthases. These NAD+-consuming enzymes cleave NAD+, resulting in an ongoing loss of NAD+ during which nico­tin­amide is produced as a reaction by-product. Because loss of NAD+ cannot be compen­sated by intake of NAD from the diet, nico­tin­amide is recycled into NAD+ via the salvage path­way. This path­way involves the activity of NAMPT and nico­tin­amide mono­nucleotide adenylyl­transferase (NMNAT 1‑3). Hence, activity of these enzymes has a larger effect on NAD concen­trations than nico­tin­amide or dietary intakes of niacin. For more details on the action of these enzymes see Xiao et al. (2018) and Penberthy & Kirkland (2020).

NAD+ can be converted to NAD+phosphate (NADP+) by the activity of NAD+ kinase. NAD(H) can also be utilized as a substrate during which NADPH is produced instead of NADP+, a process involving yet another NAD+-consuming process. During meta­bolism both NAD+ and the phos­phoryl­ated form NAD(P)+ undergo redox reactions and generate two redox couples NAD+ / NAD(H) and NAD(P)+ / NAD(P)H. The NAD+ / NAD(H) redox couple participates in about 400 reactions, predominately in cata­bolic meta­bolism, and regulates cellular energy meta­bolism, that is glycolysis and mitochondrial oxidative phos­phoryl­ation. By contrast, the NADP+ / NAD(P)H pool participates in about 30 reactions, mainly in anabolic meta­bolism, and supports the biosynthesis of fatty acids, nucleic acids and chol­esterol / steroids. It is also involved in maintaining redox balance. For a review of the cata­bolic processes and anabolic processes involving NAD+ and NAD(P)+, see Hrubsa et al. (2022) and Kirkland & Meyer-Ficca (2018) for more details.

Loss of cellular redox homeostasis of NAD+ / NAD(H) has been linked to a variety of patho­log­ical conditions, such as cardio­vascular diseases, neuro­generative diseases, cancer, and aging (Xiao et al., 2018). More research is required to better under­stand the cellular functions of NAD(H) and NADP(H) and how loss of redox homeostasis affects energy meta­bolism.

In addition to its redox roles, NAD+ is also an essential cofactor for important non-redox signaling path­ways, thus regulating biological functions, including gene expres­sion, cell cycle progres­sion, DNA repair and cell death. For more details on the role of NAD in the central nervous system, see Gasperi et al. (2019).

The major path­way of catabolism of nico­tinic acid and nico­tin­amide is by methyl­ation in the liver to N'‑methyl­nico­tin­amide (NMN) and subsequent oxidation to N‑methyl-2‑pyridone-carbox­amide (2‑pyridone) and N'‑methyl-4‑pyridone-5-carbox­amide (4‑pyridone). Under normal conditions, 20‑35% and 45‑60% of niacin is excreted as N'‑methyl­nico­tin­amide (NMN) and 2‑pyridone respectively, although the amount varies depending on niacin and tryp­to­phan intake (EFSA, 2014).

A small percentage of the essential amino acid tryp­to­phan is converted directly to nico­tin­amide adenine dinucleo­tide (NAD) via a two-step path­way (Figure 20c.2), primarily in the liver. The con­ver­sion rate is dependent on the status of tryp­to­phan rather than niacin, and is controlled by the activities of several enzymes; for more details see Fukowatari & Shibata (2013). Low intakes of tryp­to­phan, interference with tryp­to­phan trans­port, as occurs in Hartnup's disease, and conditions associated with increased tryp­to­phan meta­bolism (e.g., inflam­matory bowel diseases) (Nikolaus et al., 2017), all reduce the con­ver­sion of tryp­to­phan to niacin, resulting in an increased need for preformed niacin. Pyridoxal 5‑phosphate (a vitamin B6 derived coen­zyme), iron, riboflavin, ascorbic acid, and glutamine are involved as cofactors in the con­ver­sion of tryp­to­phan to NAD (Penberthy & Kirkland, 2020).

Physiological amounts of both nico­tinic acid and nico­tin­amide prevent the classical signs of niacin deficiency (see section below). However, certain niacin compounds also have pharmacological properties. At supra­physio­logical doses, nico­tinic acid is known to decrease total cholesterol, LDL cholesterol, and triglycerides and increase high-density lipoprotein cholesterol (HDL-C) (Minto et al., 2017). Chronic sup­plement­ation with nico­tin­amide ribose may also have some positive benefits such as reducing blood pressure and arterial stiffness, and warrants further study (Martens et al., 2018). Recent research has also implicated niacin as a therapeutic neuro­protective agent (Gasperi et al., 2019), although more studies are needed to confirm the role of niacin for alleviating neuro­logical impairment (Wuerch et al., 2023). In addition, decline of NAD+ levels during the aging process has led to investigations on the role of niacin in aging-related processes. Results suggest that niacin supple­ments, as the major nutritional precursor of NAD, may provide anti­aging properties (Imai et al., 2014).

20c.1.2 Deficiency of niacin in humans

Pellagra is the characteristic syndrome of severe niacin deficiency and its dietary precursor tryp­to­phan. Pellagra is known as the disease of the "4Ds" (diarrhea, dermatitis, and dementia, culmin­ating in death). Early symptoms include diarrhea resulting from intestinal inflam­mation, accom­panied by anorexia, nausea, and abdominal pain. Later, dermatitis develops in sun-exposed skin such as dorsal parts of hands, feet, cheeks, forehead, and sun-exposed regions of the neck (WHO, 2000). These signs arise from deficits in poly (ADP-ribose) polym­erase activity in response to UV radiation-induced DNA damage. The third D refers to dementia and presents with hallucinations and delusions, symptoms said to be caused by impaired form­ation of cADP-ribose and nico­tinic acid ADP, which result in alterations in neural calcium signaling (Meyer-Ficca & Kirkland, 2016).

Migraine, a neuro­logical condition related to brain energy deficiency, has recently been associated with intakes of dietary niacin in a population-based study of US adults (>20y) participating in NHANES 1999-2004 (Liu et al., 2022). An L-shaped relationship between dietary niacin intake and migraine was reported, with the risk decreasing with increasing dietary niacin consumption in those with a dietary niacin intake of <21.0mg/d. However, there was no association between dietary niacin consumption and migraine when the daily niacin intake was >21.0mg/d, indicating that the risk of migraine no longer decreases with increasing dietary niacin intake (Liu et al., 2022). A few case reports have also suggested that niacin may be effective as an adjuvant treat­ment for acute migraine, although the underlying mechanism is uncertain.

Pellagra is often a problem in countries where maize and millet jowar (Sorghum vulgare) are dietary staples. The bio­avail­ability of niacin in maize is poor (only about 30%) because most of the niacin is present in two forms: niacytin and niacino­genes. Niacytin consists of nicotinic acid esterified to poly­saccharides, whereas niacino­genes are bound to poly­peptides and glyco­peptides; both forms are unavailable for absorp­tion by intestinal enzymes. Maize is also particularly low in tryp­to­phan, a precursor of NAD (WHO, 2000). In contrast, millet jowar contains suitable amounts of tryp­to­phan, but excessive amounts of leucine which inter­fere with the con­ver­sion of tryp­to­phan to niacin. The mechanism is uncertain. Excess leucine may disrupt the availability or utilization of riboflavin and vitamin B6, micronutrients required in the synthesis of niacin from tryp­to­phan (Kirkland & Meyer-Ficca, 2018).

Niacin fortification of cereals, notably maize, has been associated with wide­spread reductions in the preval­ence of pellagra (Vilijoen et al., 2022). Never­the­less, out­breaks of pellagra still occur where cereals are not fortified and diets are restricted. Such outbreaks have been reported in Malawi among Mozambican refugees (Malfait et al., 1993), and more recently, among adults in the Kasese Catchment Area, Dowa, Malawi (Matapandeu et al., 2017).

Secondary pellagra can develop from certain diseases or conditions that inter­fere with the absorp­tion of niacin or tryp­to­phan or reduce the con­ver­sion of tryp­to­phan to niacin. Diseases that inter­fere with absorp­tion may include chronic diarrhea, chronic colitis, cirrhosis of the liver, and tuberculosis of the gastro­intestinal tract (Prabhu et al., 2021). Chronic alcoholism causes a reduction in the con­ver­sion of tryp­to­phan to niacin, arising from inhibition of tryp­to­phan 2,3-dioxygenase, a rate-limiting enzyme of the hepatic kynurenine path­way of tryp­to­phan degrad­ation and niacin synthesis (Prabhu et al., 2021). Even in countries with niacin fortification of wheat flour such as the United States, alcohol-associated pellagra cases have been reported. Other diseases in which the con­ver­sion of tryp­to­phan to niacin is reduced include the genetic disorder Hartnup disease when there is a deficit in tryp­to­phan trans­port (Kirkland & Meyer-Ficca, 2018) and carcinoid syndrome in which tryp­to­phan is preferentially oxidized to 5‑hydroxy­tryptophan and serotonin (Gade et al., 2020).

Prolonged treat­ment of tuberculosis with the drug isoniazid also reduces the con­ver­sion of tryp­to­phan to niacin by competing with pyridoxal 5'‑phosphate (a vitamin B6 derived coen­zyme required in the tryp­to­phan-to-niacin path­way). In Malawi, an increased risk of pellagra has been associated with mass scale-up of isoniazid use for the preventive treat­ment of tuberculosis for people living with HIV. Continuous isoniazid preventive treat­ment for tuber­culosis and the annual period of food scarcity were reported to increase the risk in this matched case-control study (Nabity et al., 2022). Such findings indicate that co‑administration of niacin-containing multi‑B vitamins with isoniazid as a pellagra prevention, warrants exploration.

20c.1.3 Food sources and dietary intakes

Most of the niacin in food is present as a component of NAD or NADP as noted earlier; very little exists as free forms of niacin, with the exception of liver and beans. Rich food sources of niacin include meat (especially liver), poultry, and fish followed by dairy products, oilseeds, some cereals (especially when enriched or fortified with niacin), legumes (including peanuts) and baker's yeast (Hrubša et al., 2022). In milk, 40% of the niacin is present as nico­tin­amide riboside and 60% as nico­tin­amide (Bieganowski & Brenner, 2004). Animal products release nico­tin­amide from its nucleotide forms during food pro­ces­sing and digestion, whereas plant products largely deliver nico­tinic acid. Tea, coffee, and cocoa beverages also contains an appreciable amount of niacin.

Absorption of niacin from food ranges from about 23% to 70% and is lowest from cereals and highest from animal products (EFSA, 2014). Certain food preparation and pro­ces­sing methods can influence the content and bio­avail­ability of niacin in food. Milling of cereals reduces their niacin content. Treat­ment of cereals with alkali (e.g., lime water), baking with alkaline baking powders, and roasting whole grain maize, all increase niacin bio­avail­ability by releasing the bound forms of niacin. The thermo-alkaline pro­ces­sing of maize kernels termed "nix­tamaliz­ation" and practiced in Latin America, is responsible, at least in part for the very low preval­ence of pellagra in the region (Hrubša et al., 2022). Roasting green coffee also increases its nico­tinic acid content, by removing the methyl group from trigonelline (N'‑methyl­nico­tinic acid) (Hrubša et al., 2022). The coffee cultivar, degree of roasting, and brewing techniques all influence the amount of nico­tinic acid in a cup of coffee. Although resistant to high tem­per­atures, significant amounts of water-soluble niacin can be lost in cooking water, if discarded. More details of the content and bio­avail­ability of niacin in both animal and plant-based foods are available in Chungchunlam & Maughan (2023).

In industrialized countries, meat and meat products often contribute as much as one third of the total intake of niacin of adults, closely followed by enriched or fortified cereals, whole-grain breads and bread products, and fortified ready-to-eat cereals; vegetables and milk + milk products contribute about one-tenth of the total niacin intake (Gregory et al., 1990; McLennan and Podger, 1995; IOM, 1998; EFSA, 2014).

Because niacin can be derived from the amino acid L-tryp­to­phan, a precursor of niacin, intakes are usually expressed in terms of niacin equivalents (NE). About 60mg of tryp­to­phan yields 1mg niacin following digestion and absorp­tion. However, the con­ver­sion rate varies widely (30%) between individuals and tends to increase with increased tryp­to­phan consumption. The most common sources of tryp­to­phan in U.S diets are high protein foods such as fish, chicken, and milk, as well as bananas, chocolate, and peanuts (Richard et al., 2009). Conversion of niacin from tryp­to­phan does not meet the need for niacin, of which about 50% is provided by dietary niacin sources. In Europe in 2014, the average daily intakes of total niacin from nine countries ranged from 27 to 53mg NE/day, with those for males slightly higher than females due mainly to the larger quantities of food consumed (EFSA, 2014). A summary table of the niacin intakes in these nine European countries is available in ESFA (2014).

In the United States, the Dietary Reference Intakes (DRIs) are set by the Institute of Medicine (IOM, 2000). The Estimated Average Requirements (EARs) are 12mg NE/day and 11mg NE/day, respec­tively for male and female adults. Corres­ponding levels for the US Recommended Dietary Allowances (RDAs) are 16mg NE/day for males and 14mg NE/day for females. Both male and female RDA intake levels for adults are similar to the Recommended Nutrient Intakes (RNIs) set by WHO/FAO (2004). In contrast, EFSA (2014) have set the Dietary Reference Values (DRVs) for niacin based on the relationship between niacin require­ment and energy require­ment. The Average Requirement (i.e., EAR) for adults (both men and women) is set at 1.3mg NE/MJ (about 5.5mg NE/1000kcal) and the Population Reference Intake (i.e. the RDA & RNI) corresponds to 1.3mg NE/MJ (about 6.6mg NE/1000kcal) assuming a coefficient of variation of 10%. These are the same levels set by the Scientific Committee for Food (SCF) in 1993. For details of the DRVs for other age and life-style groups set by these agencies, see relevant publications.

20c.1.4 Effects of high intakes of niacin

Niacin toxicity may occur when large pharmacological doses of different forms of niacin, especially nico­tinic acid, are taken to prevent or treat some metabolic diseases, as noted earlier. For example, nico­tinic acid is used in high doses (>1000mg/d) to treat hyper­lipidemia but is sometimes accom­panied by side effects. Of these, facial flushing caused by prostaglandin D2-mediated vaso­di­latation of small sub­cutaneous blood vessels, may occur, and has even been reported at doses of nico­tinic acid below 50mg/d (MacKay et al., 2012).

Most agencies use dermal vaso­dilative flushing as the basis of their Tolerable Upper Intake Levels (ULs) for niacin compounds. The U.S. Food and Nutrition Board (IOM, 2000) have set the Tolerable Upper Intake Level (UL) for adults at 35mg/d for all forms of "niacin", whereas both Europe and the United Kingdom have set different ULs for free nicotinic acid and nico­tin­amide because nico­tin­amide does not produce vaso­dilative flushing but has no beneficial effects on lipid profiles (Minto et al., 2017). For free nico­tinic acid and nico­tin­amide, the ULs for adults set by the European Food Safety Authority (ESFA) are 10mg/d and 900mg/d, respec­tively (EFSA, 2014), and 17mg/d and 500mg/d, respect­ively for the UK Safe Upper Levels (SULs) (Food Standard Agency, 2003). Differ­ences in the methods used by these agencies to set the ULs may account in part for these dis­crep­ancies (Minto et al., 2017). There is some concern about whether the flushing effect is an appro­priate basis for the UL for nico­tinic acid (MacKay et al., 2012), and whether different ULs should be set for healthy and unhealthy persons (Minto et al., 2017). Note: the UL does not apply to persons taking high-dose niacin for treat­ment of dyslipidemia.

20c.2 Indices of niacin status

More recent research has focused on the effects of supra­physio­logical doses of niacin compounds and their potential health benefits. Fort­ifi­cation of cereals with multiple micro­nutrients, including niacin, has been accom­panied by a reduction in the preval­ence of pellagra in low- and middle income countries and may account for the lack of research on biomarkers of status and function for niacin. The tests available today reflect only recent dietary intakes of niacin and there­fore may not identify all persons at risk to deficiency.

Measurement of the urinary meta­bolite of N'‑methyl­nico­tin­amide, in combin­ation with one or more of the urinary pyridone turnover products, remains the biochemical test recommended by WHO (2000) to survey at risk popu­lations to determine the extent and severity of deficiency of niacin and/or tryp­to­phan. However, as simpler, and more rapid methods for measuring NAD and NADP in erythro­cytes become available in the future, inves­tiga­tions of their use as indices of niacin status may increase. The current tests based on niacin meta­bolites in urine and erythro­cytes are described below.

20c.2.1 Urinary excretion of niacin meta­bolites

Urine is the main route of excretion for niacin, with N'‑methyl­nico­tin­amide and N'‑methyl-2-pyridone-5-carbox­amide (2‑pyridone) being the major end products of niacin meta­bolism. These products are derived from either preformed niacin or niacin obtained from dietary tryp­to­phan Figure 20c.2.
Figure 24c.1
Figure 20c.2 The tryp­to­phan - nico­tin­amide path­way. Note the path­way consists of the two parts: the first part is from tryp­to­phan to quinolinic acid and the second is from quinolinic acid to 2‑pyridone and 4‑pyridone that includes the NAD cycle and nico­tinamide catabolism. Abbreviations: NaMN: nico­tinic acid mononucleotide; NMN: nicotinamide mononucleotide; MNA: N'‑methyl­nico­tin­amide; 2‑pyr: N'‑methyl-2-pyridone-5-carbox­amide; 4‑pyr: N'‑methyl-4-pyridone-3-carbox­amide. From Fukuwatari and Shibata (2013).

Healthy adults generally excrete 20% to 35% of nicotinic acid as the N'‑methyl­nico­tin­amide (NMN) form and 45%‑60% as 2‑pyridone (de Lange and Joubert, 1964), although this pattern does vary with the amount and form of niacin ingested and the niacin status of the subject.

The excretion of both N'‑methyl­nico­tin­amide and N'‑methyl-2‑pyridone-5-carbox­amide (2‑pyridone) decreases in niacin deficiency, and their measure­ment in 24h urine samples is considered the most reliable and sensitive measures of the adequacy of intakes of niacin and tryp­to­phan, a precursor of nico­tinic acid. For example, in an experimental study of Jacob et al. (1989), during the depletion phase there was a significant fall in both 24h urinary N'‑methyl­nico­tin­amide and 2‑pyridone excretion after 35 days on a "low" intake of 6.1NE/d. Based on these findings, measure­ment of these two urinary meta­bolites appears reliable for assessing very low niacin intakes (i.e., 6.1NE/d). Of the two, however, N'‑methyl­nico­tin­amide excretion is the easiest to measure, and is more sensitive to marginal intakes (i.e., 10.1NE/d) than urinary 2‑pyridone (Jacob et al., 1989) (Table 20c.1).

Table 20c.1 Variation in the concentration of urinary meta­bolites during a niacin depletion / repletion study on healthy young men. The concen­trations are the mean ± SEM for the meta­bolites at the end of each metabolic period. NMN, (N'‑methyl­nico­tin­amide; 2‑pyr, 2‑pyridone; Data from Jacob et al., 1989.
Metabolic
period 		Niacin
intake
(NE/d) 		n Day NMN
(mg/d) 		2-pyr (mg/d)
Stabilization 19.6 7 11–13 2.90 ± 0.41 7.21 ± 1.86
Depletion 6.1 3 47–49 0.8 ± 0.13 1.00 ± 0.05
Depletion 10.1 4 47–49 0.81 ± 0.14 3.10 ± 0.71
Repletion 19.2 6 62–64 1.82 ± 0.08 6.25 ± 0.04
High intake 25 3 77–79 2.56 ± 0.0411.40 ± 1.92
High intake 32 3 77–79 4.57 ± 0.3819.36 ± 0.55

In a later study in Angola of patients with clinical pellagra, low urinary excretion of the two urinary meta­bolites were shown to be a sensitive and specific indicator of clinical pellagra (Creeke et al., 2007).

Results of more recent obser­vational studies are consistent with these earlier findings, and indicate that 24h levels of urinary niacin meta­bolites can be used as a bio­marker of dietary intake of niacin. Significant linear correlations were observed between 24h urinary excretion of the sum of the three niacin meta­bolites (N'‑methyl­nico­tin­amide, 2‑pyridone, and N'‑methyl-4‑pyridone-3‑carbox­amide) and usual dietary niacin intake (expressed as niacin equivalents) in both apparently healthy college students (Tsuji et al., 2010) and school children (aged 10‑12y) in Japan (Tsuji et al., 2011).

In the early experimental studies and the observational surveys conducted in Japan, the urinary meta­bolites were mea­sured in 2h (de Lange and Joubert, 1964), or 24h collections (Jacob et al., 1989; Tsuji et al., 2010, 2011), and expressed as a concentration (mg/d, Table 20c.1) for the meta­bolites. However, the collection of timed urine samples is difficult in field studies. Instead, collection of random spot urine samples is a more practical sampling alternative, although few studies have investigated this approach.

In a pilot study, Creeke and Seal (2005) analyzed both N'‑methyl­nico­tin­amide and 2‑pyridone levels simultaneously in spot urine samples collected from two healthy subjects over four consec­utive days. In the early morning of each day, an overnight fasting urine sample was collected. In addition, on day 4, non-fasting urine samples were collected at 3h intervals over 9h from each subject. Use of vitamin supplements by each subject was recorded. Their preliminary findings suggest that the use of fasting, early morning urine samples for assessing niacin status has potential: the impact of recent dietary intake is reduced and the samples are more indicative of longer-term niacin status. They also noted that expression of the meta­bolites relative to creatinine in the fasting spot urine samples yielded more stable results. Never­the­less, they caution use of this expression when applied to individuals with protein malnutrition and thus reduced creatinine output. In such circumstances, use of the urinary meta­bolite / creatinine ratio is inappro­priate because the resultant ratio will be suggestive of a higher niacin status (Dillon et al., 1992).

In a later study in Angola, Creeke and co-workers (2007) used fasting spot urine samples to measure and compare the urinary meta­bolites of niacin — N'‑methyl-2‑pyridone-5-carbox­amide (2‑pyridone) and N'‑methyl­nico­tin­amide — collected from both healthy individuals (n=2) and from patients diagnosed with pellagra (n=34). In this study, results were expressed as the concentration of the meta­bolites relative to creatinine to provide the most stable measure­ment of status. When expressed in this way, individuals with clinical pellagra had lower concen­trations of the two urinary meta­bolites, which rose markedly following treat­ment with a nico­tin­amide supplement. These findings indicate that the use of spot urine sample 2‑pyridone and N'‑methyl­nico­tin­amide concen­trations, relative to creatinine, are a sensitive and specific measure of severe deficiency.

Based on earlier research, a ratio of 2‑pyridone to N'‑methyl­nico­tin­amide has been proposed as a convenient alternative index of niacin status, independent of age and creatinine excretion, and applicable to casual urine samples (de Lange and Joubert, 1964). However, the use of this ratio, has been challenged. Although useful for detecting severe niacin deficiency accom­panied by pellagra, the ratio appears less useful in less severe deficiency states. For example, in the experimental depletion-repletion study in adult males (Jacob et al., 1989), the ratio of 2‑pyridone / N'‑methyl­nico­tin­amide in urine was not as good a measure of the low (6.1mg NE/d intake) as the individual meta­bolite excretions and was not sensitive to the intake of 10.1mg NE/d (see Table 20c.1). Hence, the ratio appears insensitive to marginal intakes of niacin. Moreover, some investigators suggest that excretion of both N'‑methyl­nico­tin­amide and 2‑pyridone in the urine is strongly dependent on the level of protein intake, so that the ratio may be a measure of protein adequacy and not niacin status (Shibata and Matuso, 1989a; Shibata and Matuso, 1989b). In view of these uncertainties, the use of this ratio is not recommended in marginal niacin deficiency states. Never­the­less, WHO (2000) include the ratio of 2‑pyridone to N'‑methyl­nico­tin­amide as one of the biomarkers for assessing niacin status, although they recognize that its interpretation is rather equivocal. Measurement of urinary N'‑methyl­nico­tin­amide is not appro­priate for pregnant women, in whom elevated excretion levels of N'‑methyl­nico­tin­amide occur as a result of alterations in pyridoxine meta­bolism (Ftukijwatari et al., 2004).

Some of the early studies used a nico­tin­amide load test involving the intra­muscular admin­istration of a 50mg test dose of nico­tin­amide, followed by measure­ment of N'‑methyl­nico­tin­amide in urine collected at the end of a 4-5h post-dose period. Gontzea et al. (1976) reported a 14% recovery of the test dose in well-nourished subjects over a 3h period, compared with 8% for a rural population whose basal excretion of N'‑methyl­nico­tin­amide was at the lower end of the normal range. With a lower oral niacin load (nico­tin­amide at 20mg/70kg body weight), post-dose urinary changes in 2‑pyridone were more responsive to niacin status than those changes apparent in N'‑methyl­nico­tin­amide. Loading tests are impractical, however, for field surveys, so they have had very little use.

Increasingly, with the development of new analytical methods, relationships between the other urinary pyridone turnover products including N'‑methyl-4‑pyridone-3-carbox­amide (4‑pyridone) and N'‑methyl-6-pyridone-3-carbox­amide (6‑pyridone) and niacin intakes are being examined. During an outbreak of pellagra in Mozambican refugees, for example, French researchers mea­sured 4‑pyridone and 6-pyridone as well as N'‑methyl­nico­tin­amide in 24h urine collections (Dillon et al., 1992). They noted that the ratio of 6-pyridone to N'‑methyl­nico­tin­amide in 24h urine specimens correlated well with the development of clinical symptoms of pellagra, mainly dermatitis, as shown in Table 20c.2.
Table 20c.2 Urinary excretion of niacin meta­bolites by Mozambican women. Data from Dillon et al., 1992.
Metabolite Control
(n=9) 		No signs of
pellegra
(n=9)		 With
pellegra
(n=10)
N'methyl-6-pyridone-3-
carbox­amide
(mmol/24hr urine) 		557 ± 199 152 ± 104 42 ± 9
N'methylnicotinamide
(mmol/24hr urine) 		273 ± 50 162 ± 17 213 ± 41
Molecular ratio of
N'methyl-6-pyri-
done-3-carbox­amide to
N'methylnicotinamide		1.950.900.18

Interpretive criteria

Interpretive criteria for adults and pregnant women for the urinary excretion of niacin meta­bolites expressed relative to creatinine for N'‑methyl­nico­tin­amide, and as the ratio (2‑pyridone / N'‑methyl­nico­tin­amide) are shown in Table 20c.3. These criteria are based on early studies and have been compiled by WHO for two of their publications: Pellagra and its prevention and control in major emergencies (WHO, 2000a) and Management of nutrition in major emergencies (WHO, 2000b). As noted earlier, WHO do indicate that the interpretation of these biomarkers is equivocal.

Table 20c.3 Guidelines for the interpretation of urinary excretion of N'methylnicotinamide as mg/g creatinine in a 24hr sample and the molecular ratio of N'methyl-6-pyridone-3-carbox­amide to N'methyl-nicotinamide. Data from WHO, 2000b.
Defic-
ient		LowAccept-
able 		 High 
N'methylnicotinamide
Men; women, non-preg-
nant or 1st trimester
2nd trimester
3rd trimester
(mg per g creatinine
in 24h urine sample)		 <0.5
<0.6
<0.8		 0.5-1.59
0.6-1.99
0.8-2.49		 1.6-4.29
2.0-4.99
2.5-6.49		 4.3
5.9
6.5
Molecular ratio of
N'methyl-6-pyri-
done-3-carbox­amide
(2-pyridone) to
N'methylnicotinamide		 <0.5 <1.0 1.0 - 4.0

Note Creeke et al. (2007) developed interpretive criteria to identify clinical pellagra based on two urinary meta­bolites expressed relative to creatinine from spot urine samples collected on both healthy and pellagra patients. These interpretive criteria were reported to achieve a sensitivity of 91% and a specificity of 72% for identifying clinical pellagra in their study, and are shown below:

2‑pyridone: <3.0 µmol/mmol creatinine (<4.0mg/g creatinine)

N'‑methyl­nico­tin­amide: >1.3 µmol/mmol creatinine (>1.6mg/g creatinine).

WHO (2000a) also provide provisional criteria for assessing the severity of public health problem of niacin deficiency based on two urinary meta­bolites of niacin (Table 20c.4). Note they include the ratio of 2‑pyridone to N'‑methyl­nico­tin­amide in Table 20c.4, even though this ratio may not be useful in popu­lations with marginal niacin status. (Table 20c.4).

Table 20c.4 Provisional criteria for the severity of the public health problem niacin deficiency. Data from WHO, 2000b.
IndicatorMild Moderate Severe
≥ 1 clin. case,
<1% of pop.
in age group
concerned 		1-4% of pop.
in age group
concerned		≥ 5% of pop.
in age group
concerned
Urinary N'‑methyl
­nico­tin­amide
<0.50 mg/g
creatinine		5-19%20-49%≥50%
Ratio 2-pyridone:
N'‑methyl­nico­tin-
amide <1.0		5-19%20-49%≥50%
Dietary intake of
niacin equivalents
<5mg/day		5-19%20-49%≥50%

20c.2.2 Measurement of urinary excretion of N'‑methyl­nico­tin­amide and 2‑pyridone

With the development of improved HPLC techniques, several urinary pyridones, including 2‑pyridone, 4‑pyridone, and 6‑pyridone together with N'‑methyl­nico­tin­amide can be readily separated and mea­sured, with improved accuracy and sensitivity. Separation of the meta­bolites in urine can be achieved using reversed-phase HPLC, hydrophilic liquid interaction chrom­atography, normal-phase HPLC, and supercritical fluid chro­matog­raphy. Detection is carried out by UV absorp­tion, MS, or MS/MS.

Creeke & Seal (2005) have developed an HPLC method in which both N'‑methyl­nico­tin­amide and 2‑pyridone in urine can be analyzed in the same run. They use a polymer-based mixed mode anion exchange reverse-phase cartridge that is commercially available. Analysis is performed on a reverse-phase C18 column using a methanol gradient elution system.

20c.2.3 Niacin and niacin meta­bolites in plasma and erythro­cytes

The concen­trations of niacin compounds and niacin meta­bolites in the plasma, erythro­cytes, and leukocytes have been studied as potential measures of niacin nutriture. Results have been inconsistent. Niacin circulates in the plasma as nico­tin­amide and nico­tinic acid, although nico­tin­amide is the major form. These levels in plasma are low and reflect dietary intake rather than body stores: they do not appear to be very useful measures of niacin status (Jacob et al., 1989). Likewise, concen­trations of the niacin meta­bolites, N'‑methyl­nico­tin­amide and N'‑methyl-2‑pyridone-5-carboxamide (2‑pyridone) in plasma do not appear to be as reliable as the same meta­bolites in urine for assessing individuals at risk to low niacin intakes (Jacob et al., 1989). With the development of new HPLC measure­ment techniques with improved accuracy and sensitivity, more research is needed to confirm these earlier findings.

The co-enzymes NAD and NADP are the active anabolic products of niacin and thus theoretically should provide a more direct measure of functional niacin status. However, in a study in Angola where niacin deficiency is endemic (Creeke et al., 2007), neither NAD and NADP concen­trations in whole blood nor the NAD:NADP ratio were significantly depressed in patients with clinical pellagra (n=34). It is possible that such unexpected results were associated with inadequacies in the processing and storage of the whole blood under field conditions. This may be a factor limiting the usefulness of the whole blood NAD and NADP assay in the field.

In contrast, experimental niacin depletion - repletion studies have shown that erythro­cyte concen­trations of NAD (but not NADP) are sensitive to short-term changes in niacin intake, even in the absence of clinical signs of deficiency. In the depletion-repletion study in young men (Figure 20c.3),
Figure 20c.3
Figure 20c.3. Mean concentration of erythro­cyte nicotinamide (NAD) and nicotinamde adenine dinucleotide phosphate (NADP), and the NAD/NADP ratio during four dietary periods: P1, stabilization; P2, depletion (6.1 or 10.1mg NE/d); P3, repletion; and P4, high intake (25 or 32mg NE/d). Data from Fu et al., 1989.
there was a continuous decrease in erythro­cyte NAD concen­trations during the depletion period, levels falling by 70% in men fed low-niacin diets containing either 6 or 10mg NE/d, increasing during the repletion phase when intakes of niacin (as NE) were adequate. In contrast, erythro­cyte NADP concen­trations remained unchanged. However, after five weeks of intakes of 25 and 32mg NE/d, no further significant increase in erythro­cyte NAD concen­trations was observed compared with the concentration during the repletion intake of 19.2mg NE/day (Fu et al., 1989). Erythro­cyte NAD concen­trations followed a similar trend in an experimental niacin depletion study of elderly individuals. These trends in NAD relative to NADP concen­trations parallel those in fibroblasts grown in niacin-restricted cultures (Jacobson EL, 1993). Based on these results, erythro­cyte NAD levels may serve as a sensitive and reliable biomarker for risk of niacin deficiency.

The ratio of erythro­cyte NAD to NADP (called niacin index) is sometimes used as a measure of niacin status, as NADP content in blood remains relatively unchanged even when low-niacin intakes cause a fall in NAD concen­trations, as shown in Figure 20c.3. The ratio of erythro­cyte NAD to NADP of <1.0 has been set as an interpretive criterion to identify individuals at risk of niacin deficiency (Fu et al., 1989). Clearly, in populations at risk to niacin deficiency, the use of NAD and NADP concentrations in erythrocytes as well as the determination of the niacin index in whole blood should be explored further (Jacobson & Jacobson, 1997).

20c.2.4 Measurement of niacin coen­zymes in erythro­cytes

Accurate measure­ment of NAD+ in erythro­cytes is technically challenging, with differences in sample pro­ces­sing and measure­ment strategies profoundly altering the results. Earlier methods were based on multiple liquid chro­matog­raphy tandem mass spectrometry (MS) but the sample preparation was complicated, the run times were long, and separation of the meta­bolites was not always appro­priate (Bustamante et al., 2017).

Demarest and co-workers (2019) have developed a rapid LC-MS/MS method for the determin­ation of NAD+ and its meta­bolites and have employed this method for erythro­cytes. The method has a simple, optimized sample handling procedure and can be used for erythro­cytes as well as for skeletal muscle and cerebrospinal fluid. The method utilizes an Accucore HILIC column with a linear gradient with a total run time of 14min, less than half the run time of most other available methods. In addition, the sample preparation does not require drying steps or speed va, increasing the stability of NAD+.

1H NMR Spectroscopy can also be used to detect and quantify NAD+ and NADP in erythro­cytes (and platelets) and is claimed to be superior to mass spectrometry methods (Shabalin et al., 2018).

Acknowledgements

The author is grateful to Dr Mirella Meyer-Ficca who kindly suggested minor revisions to this chapter. This assistance is appreciated.