Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Preservation of Foods with

Natamycin

Types of Food Suitable for Natamycin Use

0016 The principal use of natamycin is on the surface of

cheese and dry sausages. Due to its low solubility,

natamycin remains effective on the surface for

extended periods. When first applied, only 30–50

p.p.m. will be present in solution on the surface –

the remainder is present in the more stable crystal

formation. The preservative then gradually dissolves,

insuring a slow release and prolonged effectiveness.

An additional advantage of surface treatment is that

natamycin does not penetrate far into the food; a limit

of penetration of 1– 4 mm has been reported in cheese

rind. This is particularly useful for the preservation of

blue cheese, especially in comparison to sorbate. Sor-

bate migrates into the cheese matrix where it inhibits

the desired blue mold development inside the cheese.

Natamycin remains on the surface, acting only where

it is needed to prevent the growth of surface spoilage

molds.

0017 Much of the early work on natamycin investigated

its ability to inhibit fungal growth on fruit. Straw-

berries, raspberries, and cranberries sprayed with 50

p.p.m. natamycin in the field prior to harvesting

showed reduced spoiling during storage. Treatment

by immersion in 10–100 p.p.m. proved more effect-

ive. A lecithin coating containing natamycin can be

used to treat harder fruit, such as apples and pears.

An addition level of 1–10 p.p.m. natamycin may be

effective for a variety of beverages, including juices,

beer, wine, and beverages containing tea or milk

solids. Natamycin (at 20 p.p.m.) has proved effect-

ive against S. cerevisiae spoilage of orange juice.

Further potential applications include ready-to-eat

frostings, salad dressings, and mayonnaise susceptible

to yeast spoilage. Natamycin control of Aspergillus

niger and S. cerevisiae in cottage cheese has been

investigated. Levels of 20–100 p.p.m. added to wash

water or 1–5 p.p.m. added to the cheese dressing

inhibited fungal growth. A level of 5–10 p.p.m. can

be effective in controlling yeast and mold growth in

yogurt. Natamycin has also been shown to be effect-

ive against molds isolated from bakery products.

A level of 100 p.p.m. has been shown to inhibit

the growth of yeasts and molds in fillings and icings.

(See Yeasts.)

0018 Natamycin can also be used to treat the surface of

cured-meat products such as raw hams and investi-

gations have been conducted into its ability to inhibit

the spoilage microflora on raw cut chicken. Dipping

in a solution of 10 p.p.m. inhibited yeast counts for

12–15 days at refrigeration temperature.

Mode of Application

0020To treat the surface of cheese and sausages, the food

can be immersed, sprayed, or coated in an aqueous

suspension or the suspension can be used in a plastic

coating. Penetration into the food then depends on

the food type, being greater in soft cheese compared

to hard cheese.

0021Aqueous dipping solutions for cheese usually con-

tain 1250 p.p.m. natamycin, but concentrations vary

depending on cheese type. For example, dips for blue

cheese may require dip concentrations as high as

2500 p.p.m. The cheese should be dipped into the

natamycin slurry for a few seconds, whilst the slurry

is kept constantly agitated to maintain the natamycin

in suspension. The concentration of natamycin in the

dipping solution becomes reduced after each dipping,

at a rate dependent on the ratio of surface area to

weight of each piece of cheese.

0022A suspension of 1250 p.p.m. is recommended for

spraying cheese, applied at a rate of 6 l t

1

to achieve

7–15 p.p.m. natamycin on the surface. It is important

that the natamycin is evently applied as it binds rap-

idly and tightly on contact with the cheese surface, so

that subsequent mixing after initial application will

not achieve better distribution of the preservative. A

tumbler of at least 1 m in length is recommended with

a space of at least 0.3 m between the natamycin and

flow agent application.

0023Lower levels of natamycin (100–750 p.p.m.) can

also be added to an emulsion of a polymer in water,

mostly polyvinyl acetate, which can be applied as a

cheese coating. Alternatively, 100–20 000 p.p.m. of

natamycin can be mixed with 0.5–50 g l

1

of a suit-

able thickener and approximately 20–250 g l

1

salt

and used on the surface of cheese or sausages. This

method of application overcomes the problem of

uneven distribution of the fungicide caused by the

heterogeneity of the fat content of the product. Coat-

ings can be applied by ‘painting’ multiple layers on to

the surface with a sponge or soft cloth, or by a com-

mercially available coating machine.

0024Natamycin can also be used in natural and fibrous

casings of dry fermented sausage products, prevent-

ing mold growth during the ripening and storage

process. Commonly, casings can be prepared by im-

mersion for 2 h in a 1000-p.p.m. suspension. Alterna-

tively the sausages can be dipped or sprayed in a

1000–5000-p.p.m. natamycin suspension.

Fermentation and Production

0025Natamycin is produced by fermentation of Strepto-

cocous natalensis in an aqueous nutrient medium

containing a carbon source (e.g., starch, molasses,

4114 NATAMYCIN

glycerol, glucose, lactose, maltose, sucrose, alcohols,

organic acids), a fermentable organic and/or inor-

ganic nitrogen source (e.g., corn steep liquor, casein,

zein, lactalbumin, soya bean meal). The carbon

source usually comprises 0.5–5% of the medium.

Inorganic cations (potassium, sodium, or calcium)

and anions (sulfate, phosphate, or chloride) may be

needed as well as trace elements such as boron, mo-

lybdenum, or copper. Fermentation is aerobic and

mechanical agitation and use of antifoaming agents

can aid the process. The temperature is usually 26–



C and the pH range pH 6–8. The period of fer-

mentation, varying with the medium, can be between

48 and 120 h.

0026 Due to its low solubility in water, natamycin will

accumulate mainly as crystals in the broth. Recovery

commonly involves dissolving the natamycin using

polar solvents with limited water miscibility such as

butanol, methanol, and acetone and adjusting the

medium to approximately pH 10. The fermentation

broth is filtered to remove the mycelial biomass and

impurities, and adjusted to a lower pH (c.pH7)in

order to crystallize the natamycin, which is then re-

covered and finally dried. An alternative process that

does not use organic solvents involves the disintegra-

tion of the fermentation biomass by homogenization,

high shear mixing, or ultrasonic techniques or treat-

ment with heat, alkali, or enzymes.

See also: Legislation: History; Mycotoxins:

Classifications; Preservatives: Classifications and

Properties; Food Uses; Analysis; Spoilage: Molds in

Spoilage; Yeasts in Spoilage; Yeasts

Further Reading

Anonymous (1992) Cheese and cheese rind determination

of natamycin content method by molecular absorption

spectrometry and by high-performance liquid chroma-

tography. IDF Standard 140A. Brussels: International

Dairy Federation.

Davidson PM and Doan CH (1993) Natamycin. In: David-

son PM and Branen AL (eds) Antimicrobials in Foods,

pp. 395–407. New York: Marcel Dekker.

Hamilton-Miller JMT (1973) Chemistry and biology of the

polyene macrolide antibiotics. Bacteriological Reviews

37: 166–196.

Raab WP (1972) Natamycin (Pimaricin). Its Properties and

Possibilities in Medicine. Stuttgart: Georg Thieme.

Raghoenath D and Webbers JJP (1997) Natamycin

recovery. AU patent application no. 1999717229 B2.

Stark J (1999) Permitted preservatives – natamycin. In:

Robinson RK, Batt CA and Patel PD (eds) Encyclopedia

of Food Microbiology, pp. 1776–1781. London:

Academic Press.

Struyk AP and Waisvisz JM (1975) Pimaricin and Process

of Producing Same. US patent 3892850.

Struyk AP, Hoette I, Drost G et al. (1957) Pimaricin, a new

antifungal antibiotic. Antibiotics Annual 878–885.

Nectarines See Peaches and Nectarines

Neural Tube Defects See Pregnancy: Maternal Diet, Vitamins, and Neural Tube Defects; Folic Acid:

Properties and Determination; Physiology

NATAMYCIN 4115

NIACIN

Contents

Properties and Determination

Physiology

Properties and Determination

K J Scott, Formerly of Institute of Food Research,

Norwich, UK

P M Finglas, Institute of Food Research, Norwich, UK

Introduction

0001 Niacin (nicotinic acid and nicotinamide) occurs

widely in nature. Chemically, nicotinic acid is 3-pyri-

dine carboxylic acid, and nicotinamide is 3-pyridine

carboxylic acid amide (Figure 1). It should also be

noted that in the USA niacin is used as a specific name

for nicotinic acid, and niacinamide for nicotinamide.

0002 Nicotinic acid (mol wt 123.11) occurs as color-

less (white) nonhygroscopic needles or crystalline

powder. It has a melting point of 235.5–236.5



and sublimes. It is soluble in water (1.67 g per

100 ml at 25



C). It is also soluble in ethanol, acids,

alkalis, and propylene glycol, but insoluble in ether.

It has an absorption maximum at 261 nm, but the

intensity is pH-dependent.

0003 Nicotinamide (mol wt 122.11) also occurs as col-

orless (white) needles or crystalline powder but is

slightly hygroscopic. It has a melting point of 128–

131



C and a distilling point of 150–160



Cat

5 10

4

mmHg. It is very soluble in water (100 g

per 100 ml at c.25



C), ethanol (67 g per 100 ml)

and glycerol (10 g per 100 ml) but is almost insoluble

in ether. Like nicotinic acid, it has an absorption

maximum at 261 nm. Both nicotinic acid and the

amide are stable in the dry form and in neutral

aqueous solutions. Nicotinamide can be converted

to nicotinic acid by treatment with acids or alkalis.

Nicotinamide adenine dinucleotide (NAD) and nico-

tinamide adenine dinucleotide phosphate (NADP) are

coenzyme forms. They combine with a wide variety

of proteins and catalyze a large number of oxidation

and reduction reactions and substrates in living

organisms. (See Coenzymes.)

0004The human requirement of nicotinic acid or

nicotinamide is dependent on the tryptophan intake

(60 mg of tryptophan is equivalent to 1 mg of nico-

tinic acid). Tryptophan is converted via a complex

pathway. This pathway from tryptophan to NAD

does not directly produce nicotinic acid or nicotin-

amide; NAD

is transformed to nicotinamide, which

is further N-methylated, and reenters the pyridine

nucleotide cycle or may be deaminated to nicotinic

acid, which is further converted to nicotinic acid

mononucleotide.

Occurrence

0005Nicotinic acid and nicotinamide are found in a wide

variety of foods: yeast, liver, heart, and muscle meats

are good sources (Table 1). Because of the tryptophan

contribution, milk, milk products, and eggs are also

considered to be sources of niacin activity, although

their actual content of niacin itself is relatively low. In

natural products, niacin is usually in the bound form.

Thus, in order to measure the total content it is neces-

sary to include a hydrolytic stage. It is important to

realize that this measure of total niacin must not

necessarily be equated with what is biologically avail-

able. Most nicotinic acid in maize is in the form of

nicotinyl esters, which are thought not to be hydro-

lyzed in the gut. In wheat bran it is bound to polysac-

charides, peptides, and glycopeptides. Around 50%

of niacin in unenriched wheat and wheat products has

been reported to be in a bound form. The treatment of

maize with lime water has been reported to liberate

bound niacin, making it biologically available to pigs.

It has also been claimed that, in areas where corn is

routinely steeped in lime water, there is an absence of

pellagra compared to areas with an equal consump-

tion of unsteeped corn where pellagra was endemic.

However, the various claims for bound forms of

Nicotinic acid Nicotinamide

fig0001 Figure 1 Structural formulae for nicotinic acid and nicotin-

amide. Reproduced from Niacin: Properties and Determination,

Encyclopaedia of Food Science, Food Technology and Nutrition,

Macrae R, Robinson RK and Salder MJ (eds), 1993, Academic

Press.

4116 NIACIN/Properties and Determination

niacin require thorough reinvestigation. (See Bioa-

vailability of Nutrients.)

Biological Fluids (Blood) and Tissues

0006 Nicotinamide is the primary circulating form of the

vitamin. All tissues can incorporate nicotinamide into

NAD. In the liver, nicotinic acid is incorporated

into NAD, which is broken down into nicotinamide.

The primary regulatory substance of the homeostasis

in whole animals is nicotinamide itself. The levels in

blood are buffered by the liver by being converted

into a storage form of NAD.

Determination

Biological Assays

0007 Historically, biological assays with animals such as

the chick or the rat have been used as a baseline for

other methods and to provide an evaluation of vita-

min potency and availability of vitamins. However,

biological assays are expensive to carry out and have

an inherent lack of precision but, most importantly,

are directly relevant only to the species used and

conditions of the assay procedure. Thus, ‘animal’

assays for the determination of niacin are generally

no larger used.

Other Assays

0008Unlike a biological system, which responds to the

bioavailable vitamin in a food material, nonbiologi-

cal assays are usually geared to measuring the total

vitamin content. However, it is possible, using differ-

ential hydrolysis in conjunction with the micro-

biological assay, to make some assessment of the

distinction between the ‘free’ and ‘bound’ vitamins,

although this information must be treated with cau-

tion. Hydrolysis to release bound forms is normally

carried out with sulfuric acid, although in some

methods the use of alkali has been recommended,

particularly for cereals.

0009Chemical assays This assay is based on the forma-

tion of colored complexes formed as a result of

the reaction between niacin and cyanogen bromide.

The reaction is as follows: an a-unsubstituted pyri-

dine forms a pyridinium salt with cyanogen bromide,

which reacts with an amine under ring opening to

form a derivative of glutaconaldehyde. The color

tbl0001 Table 1 Nicotinic acid, potential nicotinic acid from tryptophan, and niacin equivalents in various foods (mg 100 g

1

)

Food Nicotinic acid Tryptophan/60 Niacin equivalents

Marmite 58 9 67

Liver

Lamb (raw) 14.2 4.3 18.5

Lamb (fried) 19.9

4.9

24.8

Ox (raw) 13.4 4.5 17.9

Ox (stewed) 10.3

5.5

15.8

Heart

Lamb (raw) 6.9 3.6 10.5

Ox (raw) 6.3 4.0 10.3

Ox (stewed) 4.7 6.7 11.4

Kidney

Lamb (raw) 8.3 3.5 11.8

Lamb (fried) 9.6 5.3 14.9

Ox (raw) 6.0 3.4 9.4

Ox (stewed) 6.2

5.5

11.7

Meats (lean, raw)

Beef 5.0

4.7

9.7

Lamb 5.4

3.9

9.3

Pork 6.9

4.5

11.4

Chicken 7.8 4.3

12.1

Milk (whole, average) 0.16

0.6

0.8

Eggs

Chicken (whole, raw) 0.1 3.7 3.8

Chicken (boiled) 0.1 3.7 3.8

Cheese

Cheddar 0.1 6.8

6.9

Danish blue 0.63

5.5

6.1

From:

McCance RA and Widdowson EM (1991) The Composition of Foods. In: Holland B, Welch AA and Unwin ID, Buss DH, Paul AA, Southgate DAT (eds)

The Composition of Foods, 5th edn. Cambridge: Royal Society of Chemistry and Ministry of Agricultural, Fisheries and Food;

Food Standards Agency

(2002) McCance and Widdowson’s The Composition of Foods, Sixth Summary Edition. Cambridge; Royal Society of Chemistry.

Estimated.

NIACIN/Properties and Determination 4117

produced can be measured spectrophotometrically. It

has been reported that the colored products from the

amide are not as stable or intense as from the acid.

The amide is usually hydrolyzed to the acid. The

Association of Official Analytical Chemists (AOAC)

method employs sulfanilic acid as the chromogenic

base in the manual and automated methods for nico-

tinic acid and nicotinamide in drugs, foods, feed, and

cereal products, and barbituric acid in the analysis

of the amide in multivitamin preparations. Foods

and feeds are extracted with acid, and cereal products

with calcium hydroxide. Factors which must be

controlled for the method to produce reliable results

include reaction temperature, pH, and preparation of

blank corrections. However, this method lacks speci-

ficity, since all 3-pyridoxine derivatives react, and it

requires the use of the highly toxic reagent cyanogen

bromide. Thus, this method has been largely replaced

by microbiological and high-performance liquid

chromatography (HPLC) methods. (See Spectroscopy:

Visible Spectroscopy and Colorimetry.)

0010 Microbiological methods Although probably not

suited for occasional use because of the specialist

facilities and expertise required, the microbiological

assay has been widely used for the determination of

nicotinic acid in a variety of materials. It is more

sensitive and specific than the chemical assay. The

microbiological assay is based on the specific nutri-

tional requirements in a defined medium for a

particular vitamin(s) by a microorganism. The most

widely used organism is Lactobacillus plantarum

(ATCC 8014). In order to present the vitamin in a

biologically active form to the organism the material

to be assayed is normally subjected to acid hydrolysis.

This procedure also converts any amide into nicotinic

acid. This organism responds to nicotinic acid, nico-

tinamide, and nicotinuric acid (an inactive metabol-

ite), and NAD, but not tryptophan, and it is also able

to utilize bound nicotinic acid, present in cereals, to a

considerable extent. It is specified in official methods

for the determination of total niacin activity in food.

The AOAC and AACC extraction procedures involve

autoclaving the sample at 121–123



C for 30 min

with 1 mol l

1

sulfuric acid.

0011 High-performance liquid chromatography HPLC

offers an alternative to the chemical or micro-

biological assay, although the initial equipment cost

and subsequent recurrent costs are relatively high.

Analysis is commonly carried out after alkali, acid,

or acid/enzyme hydrolysis, by separation on a

reversed-phase column and ion pair reagents in the

mobile phase and ultraviolet detection. A particular

problem in the HPLC analysis of niacin in food

materials is that because of its relatively low ultravio-

let absorption, interference from other compounds

can make peak identification and quantification diffi-

cult. The application of this technique to food prod-

ucts often requires clean-up procedures, like cartridge

extractions and column switching. The use of fluores-

cence detection increases specificity and sensitivity,

but requires postcolumn derivatization, because

niacin is not natively fluorescent. (See Chromatog-

raphy: High-performance Liquid Chromatography.)

Dietary Requirements

0012As stated earlier, the evaluation of data obtained from

the analysis of foods is complicated by the uncertain-

ties surrounding the bioavailability of bound forms

of the vitamin and the contribution of tryptophan.

These factors must be considered when considering

the nutritional implications of niacin intake. (See

Dietary Requirements of Adults.)

0013The presently accepted conversion rate of trypto-

phan to niacin is 60:1. Using this conversion rate and

the tryptophan requirement to maintain nitrogen bal-

ance, it is possible to establish the niacin requirement.

0014Recommended daily amounts or reference nutrient

intake values are based on niacin equivalents. The UK

figures are calculated in terms of resting metabolism,

that is, 2.7 mg of niacin equivalents per MJ or

11.3 mg per 1000 kcal. The daily level for men is

about 17 mg, and about 13 mg for women, and is

independent of activity. The recommended level

does not fall with increasing age. An additional 2 mg

is recommended during lactation. Recommended

levels for infants and children are dependent on age,

ranging from 3 to 5 mg for infants under 1 year, about

11 mg at 5 years and about 12 mg at 10 years of age.

US recommendations are very similar.

See also: Bioavailability of Nutrients; Chromatography:

Thin-layer Chromatography; High-performance Liquid

Chromatography; Gas Chromatography; Coenzymes;

Dietary Reference Values; Dietary Requirements of

Adults; Spectroscopy: Visible Spectroscopy and

Colorimetry; Vitamins: Overview; Determination

Further Reading

AACC (2000) Niacin-microbiological method. AACC

methods 86-51 and 86-50A. In: Grami B (ed.) American

Association of Cereal Chemists Methods, 10th edn.

St. Paul, MN, USA: American Association of Cereal

Chemists.

AOAC (2002) Niacin and niacinamide. Methods 985.34,

944.13 and 975.41. In: Horwitz W (ed.) Official

Methods of Analysis, 17th edn. Gaithersburg, MD:

AOAC International.

4118 NIACIN/Properties and Determination

Ball GFM (ed.) (1998) Niacin and tryptophan. In: Bioavail-

ability and Analysis of Vitamins in Foods, pp. 319–

359.London: Chapman & Hall.

Eitenmiller RR and DeSouza S (1985) Niacin. In:

Augustin J, Klein B, Becker D and Venugopal P (eds)

Methods of Vitamin Assay, 4th edn, pp. 385–398. New

York: Wiley.

Lahely S, Bergaentzle M and Hasselmann C (1999)

Fluorimetric determination of niacin in foods by high-

performance liquid chromatography with post-column

derivatization. Food Chemistry 65: 129–133.

Report of the Committee on Medical Aspects of Food

Policy (1991) Dietary Reference Values for Food Energy

and Nutrients for the UK. DHS Report on Health and

Social Subjects, no. 41. Report of the Committee on

Medical Aspects of Food Policy. London: Her Majesty’s

Stationery Office.

Shibata K and Taguchi H (2000) Nicotinic acid and nicotin-

amide. In: De Leenheer A, Lambert WE and Van

Bocxlaer JF (eds), Modern Chromatographic Analysis

of Vitamins, 3rd edn. pp. 325–364. New York: Marcel

Dekker.

Physiology

D A Bender, University College London, London, UK

Introduction

0001 The vitamin niacin (nicotinic acid and nicotinamide)

forms the functional moiety of the nicotinamide nu-

cleotide coenzymes, nicotinamide adenine dinucleo-

tide (NAD) and nicotinamide adenine dinucleotide

phosphate (NADP). These act as electron carriers in

a wide variety of oxidation and reduction reactions.

NAD is also the source of adenosine diphosphate

(ADP)-ribose for the ADP-ribosylation of proteins, a

mechanism of enzyme regulation, and the poly(ADP-

ribosylation) of nucleoproteins controlling the deoxy-

ribonucleic acid (DNA) repair mechanism. Nicotinic

acid adenine dinucleotide and cyclic ADP-ribose both

have a role in regulation of intracellular calcium

concentration in response to hormone action.

0002 Although niacin is generally regarded as a vitamin,

it is not strictly a dietary essential, since the nicotin-

amide moiety of the coenzymes can be synthesized

in vivo from the essential amino acid tryptophan.

Under normal circumstances, the intake of trypto-

phan is probably adequate to meet niacin require-

ments without the need for any preformed niacin in

the diet.

Metabolism of Niacin

Dietary Forms and Sources

0003Niacin is present in tissues, and therefore in foods,

largely as NAD and NADP. The postmortem hydroly-

sis of NAD(P) is extremely rapid in animal tissues, so

it is likely that much of the niacin of meat (a major

dietary source of the vitamin) is free nicotinamide.

0004Coffee may provide significant amounts of nico-

tinic acid, formed as a result of the pyrolysis of trigo-

nelline (N-methyl nicotinic acid) during roasting.

0005In the calculation of niacin intakes, the niacin con-

tent of cereals is normally ignored. Although chemical

analysis reveals niacin in cereals (largely in the bran),

this is mostly biologically unavailable, since it is

bound as niacytin – nicotinoyl esters to a variety of

polysaccharides and glycopeptides with molecular

weights ranging between 1500 and 17 000. Although

niacytin is not hydrolyzed by digestive enzymes, a

small amount is hydrolyzed nonenzymically by

gastric acid, and up to 10% of the niacytin in cereals

may be biologically available.

0006Treatment of cereals with alkali (e.g., soaking over-

night in calcium hydroxide solution, the traditional

method for the preparation of tortillas in Mexico) or

baking with alkaline baking powders releases much

of the bound nicotinic acid. Roasting of whole-grain

maize has a similar effect, since there is enough

ammonia released from glutamine to form free

nicotinamide by ammonolysis.

Digestion and Absorption

0007Nicotinamide nucleotides in the intestinal lumen are

not absorbed as such, but undergo hydrolysis to free

nicotinamide. A number of intestinal bacteria have

nicotinamide deamidase activity, and a proportion

of dietary nicotinamide may be deamidated in the

intestinal lumen.

0008Both nicotinic acid and nicotinamide are absorbed

from the small intestine by a sodium-dependent

saturable process of active transport, although at

unphysiologically high concentrations there is also

passive diffusion across the intestinal mucosa.

Synthesis of the Nicotinamide Nucleotide

Coenzymes

0009As shown in Figure 1 NAD(P) can be synthesized from

either of the niacin vitamers, or from quinolinic acid,

which is a metabolite of the amino acid tryptophan.

0010In liver there is little utilization of preformed

niacin for nucleotide synthesis. The enzymes for

nicotinic acid and nicotinamide utilization are more

or less saturated with their substrates at normal

concentrations in the liver, and hence are unlikely to

NIACIN/Physiology 4119

be able to use additional niacin for nucleotide synthe-

sis. The liver synthesizes relatively large amounts

of NAD(P) from tryptophan, followed by hydrolysis

to release nicotinic acid and nicotinamide into the

circulation for use by other tissues.

0011 In most extrahepatic tissues, nicotinic acid is a

better precursor of nucleotides than is nicotinamide.

However, muscle and brain are able to take up

nicotinamide from the blood stream effectively, and

apparently utilize it without prior deamidation.

Synthesis of Nicotinamide Nucleotides from

Tryptophan

0012The oxidative pathway of tryptophan metabolism is

shown in Figure 2. Under normal conditions, almost

all of the dietary intake of tryptophan, apart from the

COOH

PRPP

Nicotinic acid

PPi

ATP

Nicotinic acid mononucleotide

Nicotinic acid adenine dinucleotide

Nicotinamide adenine dinucleotide (NAD)

Glutamine

PPi

ATP

PRPP

Glutamate

CONH

OH OH

CONH

OP P

OH OH

Quinolinic acid

Quinolinic acid phosphoribosyltransferase

Nicotinic acid

phosphoribosyltransferase

Nicotinamide

deamidase

Nicotinamide

phosphoribosyltransferase

NAD glycohydrolase,

ADP-ribosyltransferase,

poly(ADP-ribose) polymerase

Nicotinamide

PRPP

PPi

fig0001 Figure 1 Synthesis of nicotinamide adenine dinucleotide (NAD) from nicotinamide, nicotinic acid, and quinolinic acid. Quinolinate

phosphoribosyltransferase EC 2.4.2.19, nicotinic acid phosphoribosyltransferase EC 2.4.2.11, nicotinamide phosphoribosyltransferase

EC 2.4.2.12, nicotinamide deamidase EC 3.5.1.19, NAD glycohydrolase EC 3.2.2.5, NAD pyrophosphatase EC 3.6.1.22, ADP-

ribosyltransferases EC 2.4.2.31 and 2.4.2.36, poly(ADP-ribose) polymerase EC 2.4.2.30.

4120 NIACIN/Physiology

small amount that is used for net new protein synthe-

sis, is metabolized by this pathway, and hence is

potentially available for NAD synthesis. About 1% of

tryptophan metabolism is by way of 5-hydroxylation

and decarboxylation to form the neurotransmitter

5-hydroxytryptamine (serotonin).

0013 The equivalence of dietary tryptophan and pre-

formed niacin as precursors of the nicotinamide

nucleotides has been assessed by determining the

excretion of N

-methylnicotinamide and methyl-

pyridone carboxamide in response to test doses of

the precursors, in subjects maintained on controlled

diets. There is a considerable variation between sub-

jects. It is generally accepted that in order to allow for

individual variation it should be assumed that 60 mg

of tryptophan is equivalent to 1 mg of preformed

Tryptophan

Kynurenine

C CH COOH

Kynurenic acid

Xanthurenic acid

COOH

CCH

3-Hydroxykynurenine

3-Hydroxyanthranilic acid

Aminocarboxymuconic

semialdehyde

Alanine

COOH

CHO

COOH

Acetyl CoA

NAD

COOH

Quinolinic acid

Tryptophan dioxygenase

and formylkynurenine formamidase

Kynurenine hydroxylase

Kynureninase

3-Hydroxyanthranilic

acid oxidase

Picolinate carboxylase

Nonenzymic cyclization

COOH

fig0002 Figure 2 Tryptophan metabolism and the formation of quinolinic acid: tryptophan dioxygenase EC 1.13.11.11, formylkynurenine

formamidase EC 3.5.1.9, kynurenine hydroxylase EC 1.14.13.9, kynureninase EC 3.7.1.3, 3-hydroxyanthranilate oxidase EC 1.10.3.5,

picolinate carboxylase EC 4.1.1.45.

NIACIN/Physiology 4121

niacin – an overestimate of the average requirement

of tryptophan for NAD synthesis. This is the basis for

expressing niacin requirements and intake in terms of

niacin equivalents – the sum of preformed niacin and

1/60 of the tryptophan.

0014 The synthesis of NAD from tryptophan involves

the nonenzymic cyclization of aminocarboxymuconic

semialdehyde to quinolinic acid. The alternative

metabolic fate of aminocarboxymuconic semialde-

hyde is decarboxylation, catalyzed by picolinate car-

boxylase, leading to the formation of acetyl coenzyme

A. There is thus competition between an enzyme-

catalyzed reaction, which has hyperbolic, saturable

kinetics, and a nonenzymic reaction which has linear,

first-order kinetics.

0015 At low rates of tryptophan metabolism, most will

be by way of the enzyme-catalyzed pathway, leading

to oxidation, and there will be little accumulation

of aminocarboxymuconic semialdehyde to undergo

nonenzymic cyclization. As the rate of formation of

aminocarboxymuconic semialdehyde increases, and

picolinate carboxylase becomes saturated, there will

be an increasing amount available to undergo cycliza-

tion to quinolinic acid, and hence onward metabol-

ism to NAD. There is thus not a simple stoichiometric

relationship between tryptophan and niacin, and the

equivalence of the two coenzyme precursors will vary

as the amount of tryptophan to be metabolized and

the rate of metabolism vary.

0016 The activities of three enzymes, tryptophan dioxy-

genase, kynurenine hydroxylase, and kynureninase,

may all affect the rate of formation of amino-

carboxymuconic semialdehyde, as may the rate of

uptake of tryptophan into the liver.

0017 Tryptophan dioxygenase Tryptophan dioxygenase

(also known as tryptophan oxygenase or tryptophan

pyrrolase) has a short half-life in vivo (of the order of

2 h) and is subject to regulation by three mechanisms:

stabilization by its heme cofactor, hormonal induc-

tion, and feedback inhibition and repression by high

concentrations of NADP.

0018 The holoenzyme is more stable than the apo-

enzyme, and in the presence of relatively large

amounts of heme both the activity and the total

amount of enzyme protein in the liver are increased.

Induction of heme synthesis thus results in increased

oxidative metabolism of tryptophan. This is not

induction of tryptophan dioxygenase apoenzyme,

but the result of reduced catabolism of the enzyme

protein.

0019 Tryptophan and a number of tryptophan analogs

have a similar effect by promoting conjugation of the

apoenzyme with hematin, and thus stabilizing the

holoenzyme.

0020Tryptophan dioxygenase is induced by glucocorti-

coid hormones and glucagon; the mechanisms

involved are different, and the effects are at least

partially additive.

0021Glucocorticoid hormones cause induction of the

new messenger ribonucleic acid (mRNA) and protein

synthesis, unlike the increase in activity observed

in the presence of higher than normal amounts of

tryptophan or heme. In response to the administra-

tion of the synthetic glucocorticoid, dexamethasone,

there is increased transcription of the rat liver

tryptophan dioxygenase gene, resulting in a 10-fold

increase in tryptophan dioxygenase mRNA in the

liver.

0022Glucagon, mediated by cyclic adenosine monopho-

sphate (cAMP), increases the synthesis of tryptophan

dioxygenase following the administration of gluco-

corticoids, although it has little effect in unstimulated

animals. The effect of glucagon appears to be the

result of an increase in the rate of translation of

mRNA rather than an increase in transcription, and

is antagonized by insulin.

0023Kynurenine hydroxylase and kynureninase The

activities of kynurenine hydroxylase and kynureni-

nase are only slightly higher than that of tryptophan

dioxygenase under basal conditions. Impairment of

the activity of either enzyme may reduce the rate

of tryptophan metabolism, and so reduce the

accumulation of aminocarboxymuconic semialde-

hyde, and the synthesis of NAD.

0024Kynurenine hydroxylase is a flavoprotein, and in

riboflavin-deficient rats its activity is only 30–50% of

that in control animals. Riboflavin deficiency may

thus be a contributory factor in the etiology of

pellagra when intakes of tryptophan and niacin are

marginal.

0025In a number of studies, sexually mature women

show a higher ratio of urinary kynurenine to

hydroxykynurenine than do children, postmeno-

pausal women, or men, suggesting impairment of

kynurenine hydroxylase by endogenous estrogens or

their metabolites. In experimental animals the admin-

istration of estrogens results in a very considerable

reduction in kynurenine hydroxylase activity. The

mechanism of this effect is unclear, but it is physiolo-

gically important; in most areas where pellagra was

common, about twice as many women as men were

affected.

0026Kynureninase is a pyridoxal phosphate-dependent

enzyme; impairment of its activity in vitamin B

deficiency leads to accumulation of kynurenine and

hydroxykynurenine, and their transamination prod-

ucts, kynurenic and xanthurenic acids. This is the basis

of the tryptophan load test for assessing vitamin B

4122 NIACIN/Physiology

nutritional status. It is also inhibited by estrogen me-

tabolites, and both vitamin B

deficiency and inhib-

ition reduce the rate of metabolic flux through the

oxidative pathway, hence reducing the formation of

quinolinic acid and NAD from tryptophan.

Catabolism of the Nicotinamide Nucleotide

Coenzymes

0027 There is no evidence of any specific storage of niacin

or the nicotinamide nucleotide coenzymes in the

body. Free NAD(P), which is not associated with

enzymes, is rapidly hydrolyzed, and the resultant

nicotinamide is either used for resynthesis of nucleo-

tides or is methylated and excreted. The catabolism

of NAD is catalyzed by four enzymes:

0028 NAD glycohydrolase, which catalyzes the hy-

drolysis of NAD(P)

at the N-glycoside linkage

to yield nicotinamide and either ADP-ribose or

ADP-ribose phosphate. This enzyme catalyzes

hydrolysis of both NAD

and NADP

. It is also

important in the formation of nicotinic acid

adenine dinucleotide and cyclic ADP-ribose

0029 NAD pyrophosphatase, which releases nico-

tinamide mononucleotide. This can either be

hydrolyzed by NAD glycohydrolase to release

nicotinamide, or be a substrate for nicotinamide

mononucleotide pyrophosphorylase, to form NAD

0030 ADP-ribosyltransferase(s)

0031 Poly(ADP-ribose) polymerase

003 2 ADP-ribosyltransferases and poly(ADP-ribose)

polymerase normally transfer ADP-ribose on to ac-

ceptor proteins (see below), although both are also

able to catalyze simple hydrolysis of NAD

in the

absence of an acceptor protein.

Urinary Excretion of Niacin and Metabolites

0033 There is normally little or no urinary excretion of

either nicotinamide or nicotinic acid, because both

vitamers are actively resorbed from the glomerular

filtrate. It is only when the plasma concentration is

so high that the resorption mechanism is saturated

that significant excretion occurs.

0034 The metabolites of niacin are shown in Figure 3,

and the use of their urinary excretion as an index of

niacin nutritional status in Table 1. The principal

metabolite of nicotinamide is N

-methylnicotina-

mide, which is actively secreted into the urine by the

proximal renal tubule. N

-Methylnicotinamide can

also be metabolized further, to yield methylpyridone

carboxamide. The extent to which this oxidation

occurs, and the relative proportions of the 2-pyridone

and 4-pyridone, varies from one species to another

and also shows considerable variation between differ-

ent strains of the same species. Aldehyde oxidase

catalyzes the formation of both pyridones, and some

additional 2-pyridone arises from the activity of

xanthine oxidase.

0035Nicotinamide can also undergo oxidation to nico-

tinamide N-oxide. This is normally a minor metabol-

ite in humans, unless large amounts of nicotinamide

are ingested. At high levels of nicotinamide intake,

some 6-hydroxynicotinamide may also be excreted.

0036Nicotinic acid can be conjugated with glycine to

form nicotinuric acid (nicotinoyl glycine), or may be

methylated to trigonelline (N

-methylnicotinic acid).

Small amounts of 6-hydroxynicotinic acid may also

be formed.

0037It is not clear to what extent urinary excretion of

trigonelline reflects endogenous methylation of nico-

tinic acid. There are significant amounts of trigonel-

line in foods, and some is formed by the intestinal

bacterial metabolism of niacytin. Trigonelline is

absorbed, but cannot be utilized as a source of niacin,

and is excreted unchanged.

0038Nicotinic acid can give rise to N

-methylnicotina-

mide and methyl pyridone carboxamide as a result of

its incorporation into NAD(P) and subsequent hy-

drolysis to release nicotinamide. Similarly, nicotina-

mide can give rise to nicotinic acid, and hence

nicotinuric acid and trigonelline, as a result of deami-

dation. However, test doses of nicotinamide are ex-

creted mainly as N

-methylnicotinamide, pyridones,

and nicotinamide N-oxide, while doses of nicotinic

acid are excreted mainly as nicotinuric acid.

Metabolic Functions of Niacin

Redox Function of NAD(P)

0039The nicotinamide nucleotide coenzymes are involved

as proton and electron carriers in a wide variety of

oxidation and reduction reactions. Before their chem-

ical structures were known, NAD and NADP were

known as coenzymes I and II respectively. Later,

when the chemical nature of the pyridine ring of

nicotinamide was discovered, they were called

diphosphopyridine nucleotide, or DPN (NAD), and

triphosphopyridine nucleotide, or TPN (NADP).

These names will still be found in the literature, and

the nicotinamide nucleotide coenzymes are sometimes

referred to as the pyridine nucleotide coenzymes.

0040As shown in Figure 4, the oxidized coenzymes have

a formal positive charge, and are represented as

NAD

and NADP

, while the reduced forms, carry-

ing two electrons and one proton (and associated

with an additional proton) are represented as

NADH and NADPH. The two-electron reduction of

NAD(P)

proceeds by way of a hydride (H



) ion

transfer to carbon-4 of the nicotinamide ring.

NIACIN/Physiology 4123