20.2: Indices of niacin status (20c.2)

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 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 ± 0.04	11.40 ± 1.92
High intake	32	3	77–79	4.57 ± 0.38	19.36 ± 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	1.95	0.90	0.18

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.

	Deficient	Low	Acceptable	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.

Indicator	Mild	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. 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).