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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
NUCLEIC ACIDS, PURINE, AND PYRIMIDINE NUCLEOTIDES AND NUCLEOSIDES
Physiology, Toxicology, and Dietary Sources EA Carrey, UCL Institute of Child Health, London, UK D Perrett, Queen Mary University of London, London, UK HA Simmondsw r 2013 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by E. A. Carrey, H. A. Simmonds volume 2, pp 260–269, r 2005, Elsevier Ltd.
Introduction
Nucleic Acid Biosynthesis in Humans
Nucleic acids, vital components of all living cells, were isolated in 1869 from the nuclei of pus cells and the spermatozoa of Rhine salmon. Later, it was shown that the major constituents of nucleic acids are sugars, phosphate groups, and the characteristic purine and pyrimidine bases. The chemical structures of the purine bases, including uric acid – the end (waste) product of purine metabolism in humans – were established at the end of the 19th century. The role of the nucleic acids in storing and translating the genetic information in the cells was elucidated in the 20th century. Although their calorific contribution to the diet is trivial, nucleotides, nucleosides, and bases have essential roles in metabolism and signaling within the cell and organism. This article outlines the structure of nucleic acids and gives a brief overview of the physiological functions of nucleosides, nucleotides, and nucleic acids. It describes the toxicity that may arise from degradation of both endogenous and dietary (exogenous) nucleic acids in humans and contains a summary of the nucleic acid content of foods.
Physiology Structure The hereditary material in the nucleus of human cells is packed into 46 chromosomes and additional DNA is found in the mitochondria. The capacity of DNA to be copied into two complementary strands arises from the well-known double-helical structure and underlies the transfer of genetic information in all living organisms. Interactions between the DNA and transcription factors determine the time and place in the body where genes are transcribed, controlling development and metabolism. RNA molecules are synthesized initially on a DNA template by a DNA-dependent RNA polymerase in a process called transcription, where ribonucleotides complementary to the bases of one strand of DNA are joined by 30 –50 phosphodiester bonds (Figure 1(a)). w
Deceased.
Encyclopedia of Human Nutrition, Volume 3
The first step in nucleic acid synthesis involves the formation of the purine and pyrimidine ribonucleotides. There are two endogenous routes: either the energetically expensive de novo route from small molecules such as carbon dioxide, amino acids, and ribose sugars, or the energetically less expensive ‘salvage’ pathway. Purine bases and pyrimidine nucleosides from the breakdown of nucleic acids and nucleotide cofactors are salvaged within the cells, generating nucleotides that can be incorporated into nucleic acids. In most cells, salvage processes are more important, and the ribonucleotides recycled in this way exert feedback control on the de novo routes.
Metabolic Roles of Nucleotides The most abundant ribonucleotide in the body is adenosine 50 –triphosphate (ATP), which is the universal energy carrier in living organisms (Figure 1(b)). In addition, nucleotides are precursors of several coenzymes, used in many reactions including the conversion of food into energy. Within cells adenosine and guanosine nucleotides also have roles in the transduction of external signals into cellular responses, and in the translation and synthesis of proteins. Pyrimidine nucleotides are present at much lower concentrations in cells but also fulfill diverse functions. Uridine diphosphate (UDP)-glucose and Cytidine diphosphate (CDP)-lipids are active intermediates in the synthesis of glycogen and membranes, respectively, and sugars linked to UDP or GDP are used in the glycosylation of proteins. UDP-glucuronic acid is an essential component of the pathways that convert exogenous molecules and endogenous steroids into soluble forms for disposal from the body. The free deoxyribonucleotides are very scarce in the normal cell because they are used exclusively for the synthesis of DNA.
Synthesis of Nucleic Acids Synthesis of both DNA and RNA is prominent in cells and tissues with high turnover or metabolism (e.g., liver, gut epithelium, skin, dividing lymphocytes, bone marrow, and hair follicles). Different complements of enzymes
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources NH2 C N 7 C 5 6 1N Adenine CH 8 4 3 2CH 9 N C N
O− −
O P O
O
CH2
5′ 4′
H
H
O Deoxyribose
H
3′
2′
H O P
O
CH2
O
O H
N NH C Guanine CH C C N H2N N
O
H
H
H CH2
O
HC 5 4 3 N 2 HC6 1 C N
O
O−
8 CH
N
H2C O O 5′ Ribose H 4′ H
1′
H
2′
OH
P O
O−
O− O
P
O
O
P
O−
O
3′ H OH
Adenosine 5′-triphosphate (ATP)
Cytosine O
H
O HH C 3
C
O O P
9
(b)
H
H H −
N
N
7
C
O O
6 5C 4 3 C
NH2 H
O P
CH
C
2
C
H
−
N1
1′
O −
NH2
O
CH2
O
HC O
H
N
NH C
Thymine O
H
H (a)
C
H
H
Figure 1 (a) Schematic representation of part of a DNA strand showing the structural formulae of the four constituent bases, adenine, guanine, cytosine, and thymine, linked via the 30 -OH group of the deoxyribose moiety to the 50 -phosphate group of the next nucleotide. Also shown is the numbering of the atoms in the deoxyribose, as well as the pyrimidine and purine rings. The bases are adenine (A), guanine (G), cytosine (C), and thymine (T). Two strands are wound in opposing chemical directions to allow the well-known double-helix structure, with hydrogen-bonding between complementary nucleotides (i.e., A–T and G–C), to form. The deoxyribose and phosphate groups form the outer sides of the ‘ladder’. The RNA molecule is single-stranded, but double-helical regions arise when stretches of complementary sequences allow hairpin loops to form. In addition, the base uracil (U) is found instead of thymine, and the pentose is ribose. (b) Structural formula of ATP indicating that the ribose, as distinct from deoxyribose, has an OH group at the 2’ position on the pentose ring. When a nucleoside triphosphate (NTP) is linked through the 50 phosphate groups to the 30 position of the previous residue on the growing chain, the chemical energy for the polymerization is provided by the removal of the second and third phosphate groups.
are expressed in each cell type, and therefore tissues have characteristic profiles of internal metabolites, including nucleotides and nucleosides. For example, in cells that do not continuously divide, such as heart and muscle, nucleotide profiles are relatively simple, relating to the major requirement to sustain high levels of ATP and cofactors. Contrastingly, rapidly dividing cells in liver and intestine show a complex nucleotide pattern, identifying these organs as major sites of nucleic acid metabolism. The gut is particularly important in this respect. The rate of cell turnover in the luminal villi is high, and it has been calculated that in rats approximately 30 mg of endogenous nucleic acid derived from dead cells enters the gut lumen daily. This means that nucleic acid synthesis in liver and intestine is much higher than in tissues such as muscle.
Metabolism of Endogenous Nucleic Acids and Excretion of Metabolic End Products There is a considerable daily turnover of endogenous nucleic acids and ribonucleotides during muscle work, wound healing, erythrocyte senescence, mounting an immune response, etc. However, only a small fraction of these vital endogenous compounds is degraded and lost from the body. Because
de novo purine and pyrimidine synthesis is energetically expensive the contents of dead cells are normally used by other cells, and degraded RNA or cofactors are recycled within living cells using active ‘salvage’ routes. Breakdown products of DNA and RNA enter the salvage pathways in the form of the purine bases hypoxanthine (Hx) and guanine or the pyrimidine bases uracil and thymine (Figure 2). Any pyrimidine bases that are not salvaged are then further catabolized in a series of steps to b-amino acids, which are soluble and readily excreted. There is thus normally no measurable toxic pyrimidine end product from endogenous or dietary nucleic acids, except in the case of a small number of very rare metabolic disorders (Figure 3). The purine base Hx is converted in the liver to the insoluble metabolites xanthine and then uric acid by the enzyme xanthine oxidase/xanthine dehydrogenase (XDH) in man. Urate can normally be disposed of in the urine, but high concentrations can crystallize and form kidney stones or deposits in the joints and under the skin. Some rare genetic disorders can remove feedback regulation of purine biosynthesis, or excessive breakdown of cells may overload the salvage system, each resulting in very high endogenous levels of uric acid. However most other animal species (with the notable exception of some dog varieties) possess an additional catabolic enzyme, uricase,
Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
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Glutamine + HCO3− + ATP
PRPP + Glutamine
Aspartate + Carbamoyl phosphate
5-Phosphoribosylamine
Carbamoyl aspartate Purine nucleotides
9 further step to form the purine rings
PRPP + Orotate Nucleosides
IMP
AMP
GMP
ADP
GDP
ATP
GTP
Hx/G
Uridine
OMP +ATP
UMP
+PRPP
dADP
dGDP
dATP
dGTP
UDP UTP
dUDP
CTP
dUTP
CDP
dUMP
Thymidine dTMP
dCDP
dTDP
dCTP
dTTP
+ATP
DNA Figure 2 De novo synthesis of ribonucleotides uses small molecules and amino acids. The purine base of IMP is built up in several steps on a ribose phosphate molecule, and separate pathways generate ATP and GTP. In pyrimidine biosynthesis, the six-membered ring is formed before addition of the ribose phosphate to orotate, and decarboxylation to form UMP. Ribonucleotides (ATP, GTP, UTP, and CTP, shown in bold) are used in the synthesis of RNA, whereas DNA is synthesized after conversion of the ribose to deoxyribose, and of dUMP to dTMP. Salvage pathways (shown with open arrows) all follow two patterns: purine nucleotides and nucleosides, entering the cells or derived from hydrolysis of cofactors or nucleic acids, are converted to the free bases hypoxanthine or guanine and then converted by a specific phosphoribosyltransferase enzyme to the nucleoside; in contrast pyrimidines are salvaged from the nucleosides uridine or thymidine using specific kinase enzymes. It should be noted that PRPP (phosphoribosyl pyrophosphate) is essential for both de novo pathways and the purine salvage pathways.
which cleaves the purine ring of uric acid forming the readily soluble allantoin. This compound in turn may be degraded to ammonia in water-dwelling species such as fish.
Metabolism of Dietary Nucleic Acids in Humans The human diet is naturally rich in nucleic acids because food is derived from once-living organisms. Because, as already described, nucleotides and nucleosides can be synthesized de novo and nucleobases liberated during catabolism can be salvaged, they are often considered to be dispensable nutrients in food. The metabolism of these exogenous nucleic acids follows a similar pattern to the intracellular process described above, but the bacterial flora of the intestine are the first point of degradation. This digestion is rapid. Studies in both pigs and humans demonstrated that up to 50% of dietary purine was degraded to carbon dioxide within 30 min, 43% was recovered in the urine and 5% excreted in the feces. Less than 2% of dietary purines is incorporated into nucleic acids. Humans thus have no apparent essential requirement for purines from the diet and the intestinal mucosa provides an
effective barrier to their uptake through a battery of enzymes that can rapidly degrade purine nucleotides, nucleosides, and bases especially unusual purines found in plant materials. Because of this enzyme activity, and the rapid turnover of intestinal mucosa, approximately 200 mg of urate is excreted daily in the feces. This phenomenon ensures that levels of ATP do not fluctuate in concert with the dietary intake of purines, or may represent an important evolutionary development to protect the integrity of cellular DNA. On the other hand, pyrimidine ribonucleoside monophosphates (NMPs) and ribonucleosides are absorbed readily from the intestine and utilized for nucleic acid synthesis. This has been demonstrated by studies of humans with hereditary oroticaciduria, a rare defect in conversion of orotic acid to uridine monophosphate (UMP) in de novo pyrimidine synthesis. Such patients have severe megaloblastic anemia. They have been sustained on oral uridine, indicating that the dietary pyrimidine nucleoside can compensate totally for lack of de novo synthesis in humans. Studies using radiolabelled purines and pyrimidines in mice provided further evidence for the incorporation of dietary pyrimidine nucleosides, but not purine nucleosides, into hepatic RNA.
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
AMP
Adenosine
IMP
GMP
Cytidine
Inosine
Guanosine
Uridine
Thymidine
Guanine
Uracil
Thymine
Dihydrouracil
Dihydrothymine
-Ureidopropionate
-Ureidoisobutyrate
-Alanine
-Aminoisobutyrate
Hypoxanthine
Xanthine
Uric acid
NH3 + CO2
Urea Figure 3 Breakdown of DNA and RNA begins when the molecules are degraded by nuclease enzymes to liberate nucleotides. The next step, degradation by specific 50 –nucleotidases (removing the phosphate groups) to nucleosides or deoxynucleosides, is essentially irreversible. Nucleoside phophorylases generate (a) the purine bases hypoxanthine and xanthine, which are converted to uric acid or (b) the pyrimidine bases uracil and thymine, which are converted to b-amino acids, ammonia, and CO2, and thus to urea, a solute in urine.
Nucleic Acid Content of Foods The nucleic acid content of foodstuffs is expressed generally in terms of purine equivalents or ‘total potentially available nucleosides (TPAN)’ released from food by hydrolysis with sodium hydroxide or hydrochloric acid and enzymes. The data for purines are thus derived by analysis of the resultant constituent bases. Analysis by Robert McCance, Elsie Widdowson, and colleagues from the 1930s onward forms the basis of tables listing the composition of foodstuffs although, with a few exceptions, this demanding work has not been repeated using more modern analytical methods such as liquid chromatography–mass spectrometry (LC–MS). Foods can be classified into three groups; high, low, or essentially free of purines (and hence of pyrimidines too) (Tables 1 and 2). As a general rule growing organisms such as yeast, or rapidly metabolizing tissues such as liver, will be rich in both nucleic acids. Seeds and grain are good sources of the genetic material, DNA, as well as free nucleotides, which are stored for use in germination. Muscle tissue is an excellent source of the nucleotide ATP and the nucleic acids in mitochondria. Offal is also metabolically very active so is usually high in free nucleotides as well as nucleic acids. Fish and shellfish that are eaten ‘whole’ and fish eggs and roe are also high in nucleic acids. Extracts of meat and yeast e.g., Bovril, Marmite, Vegemite, have very high purine contents, as do supplements such as Spirulina for sale in ‘Health Food’ shops, but are usually eaten in small quantities.
Fats, white flour, sugar, and fruit juices have been separated from the ‘living’ part of the food and so they are poor sources of nucleic acids.
Effect of Cooking on Nucleotide Content of the Diet Nucleic acids are relatively resistant to hydrolysis at the moderate temperatures and short periods of time associated with cooking in water or frying in oil. On the other hand nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs) break down readily during boiling in water forming first their related NMP and then their base. The rate of hydrolysis is significantly increased in acidic solutions. The rate of degradation is enhanced if any enzyme activity is still present. The levels of nucleic acids and NTPs are well maintained during prolonged storage at 20 1C or below.
Nucleic Acid and Related Compounds in Beverages Tea, coffee, and cola drinks contain a number of unusual nucleobases based on xanthine (Figure 4). Caffeine (1,3,7trimethylxanthine) and theobromine are the best known. Caffeine is found in various quantities in the beans, leaves, and fruit of many plants. It is mainly consumed by humans in infusions extracted from the coffee bean and leaves of the tea bush. A cup of coffee can contain up to 175 mg of caffeine whereas a cup of tea contains approximately 40 mg.
Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
Table 1 A quick reference guide to the purine (nucleic acid) content of foods Foods and Beverages Rich in Nucleic Acids/Purines Offal: sweetbreads, liver, kidney, heart, and pate´ Wild or farmed game meats (venison, pheasant, rabbit, hare) Seafoods: sardines, sprats, herring, bloaters, anchovies, fish roe, caviar, taramasalata, trout or salmon; lobster, crab, prawns Vegetables: asparagus, avocado pears, peas, spinach, mushrooms, broad beans, cauliflower Pulses and grains: legumes, pulses and soya products such as bean curd, tofu, Quorn Cereals: all bran, oat, rye or wheat cereals and products; wholemeal, rye and brown breads Other: beer and yeast extracts/tablets (BarmeneTM, TastexTM). Meat or vegetable extracts (MarmiteTM, VegemiteTM, BovrilTM, OxoTM) Blue-green alga extracts (Spirulina) Foods that are Moderate or Low Sources of Purines Beef, lamb, pork (steak or chops), bacon, ham, sausages, some poultry, tongue (all should be eaten in moderation) Carrots, parsnip, other root vegetables, potatoes, lettuce, leeks, cabbage, sprouts, marrow, squash, courgettes Peanuts, cashew nuts Breakfast cereals Some fish (see Table 2) Foods and Beverages that are Essentially Purine-Free Milk, cheese, eggs, butter, margarine, cream, ice cream White bread or flour, cakes, scones, biscuits Sugar, jam, marmalade, honey, and sweets Cucumber, tomato, onions, pumpkin Fresh, cooked or tinned fruits, nuts Puddings, custards, yogurt Fruit juices, soft drinks
Table 2 Concentrations of purines in some common foods; results are recorded relative to 100 g of food for purine and for protein, although serving size for each ingredient may be larger or smaller than 100 g based on Diem and Lentner (1970). Food
Purine (mg per 100 g)
Protein (g per 100 g)
Meat Beef liver Beef kidney Beef heart Beef tongue Beef steak Calf liver Sweetbreads Veal cutlet Sheep kidney Lamb chop Pork liver Pork cutlet Bacon Ham Sausage (beef) Sausage (pork) Rabbit Venison
333 285 285 167 151 348 1212 152 312 196 289 164 85 136 79 66 118 156
19.7 15.4 16.8 16.4 19.5 19 19.6 19.2 16.8 14.9 22 16.4 9.1 19.5 13.8 11.5 20.4 20 (Continued )
Table 2
193
Continued
Food Vegetables Asparagus Cauliflower Celery Kohlrabi Mushrooms Peas Spinach
Purine (mg per 100 g)
32 32 20 44 72 72 96
Protein (g per 100 g)
2.1 2.1 1.1 2.1 3.5 6.7 2.2
Dried Legumes Split peas Red bean Lentils Haricot beans Lima bean
195 162 222 230 149
21 20 28 22 21
Other BovrilTM MarmiteTM OxoTM cubes Yeast extracts
340 356 236 2257
18 2 10 46
Poultry Chicken flesh Chicken liver Chicken heart Duck Goose Turkey
181 372 223 181 177 239
20.6 22.1 18 16 16.4 20.1
Fish, Seafoods Anchovies Bass Bloaters Bream Cod Crab Clams Eel Fish cakes Herring Kippers Lobster Lemon sole Mackerel Plaice Salmon Sardines Scallops Sprats Squid Trout
411 73 133 72 62 61 136 108 36 378 91 100 54 246 53 250 345 117 250 135 92
20 19.5 22.6 19.7 18 19.2 17 18.6 12.1 17 21.2 20 19.9 29 18.1 23 23 22.3 25.1 15 19.2
Canned Seafoods Anchovies Herring Mackerel Oysters Salmon Sardines Shrimp Tuna
321 378 246 116 88 399 231 142
30 17 26 6 26 24 22 29
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources O
H3C N
N
N
CH3
O N CH3
Caffeine O HN O
CH3 N
N N CH3
Theobromine
O H3C O
N N H
O
CH3 N N
Paraxanthine
H3C O
N
H N
N N CH3
Theophylline
Figure 4 Chemical structures of unusual nucleobases derived from xanthine, and found in plant tissues such as cocoa beans, coffee beans, and tea leaves.
Many popular soft drinks, e.g., cola, may contain up to 300 mg per can. Theobromine (dimethylxanthine) occurs naturally in cocoa beans (20 mg g1 of cocoa powder) and is therefore present in chocolate. Theophylline (1,3-dimethylxanthine) occurs at trace levels in tea leaves and is now chemically synthesized for use as a drug treatment for asthma. Caffeine is rapidly absorbed by the stomach and small intestine and is distributed throughout total body water. It is rapidly metabolized in the liver to paraxanthine (84%), theobromine (12%), and theophylline (4%). In man, caffeine acts as a central nervous system stimulant, temporarily warding off fatigue and restoring alertness: it readily enters the brain and acts as a nonselective antagonist of purinergic adenosine receptors. Caffeine is the most widely consumed psychoactive substance; in North America, 90% of adults consume caffeine beverages daily. ‘Energy drinks’ contain very high levels of caffeine and some also contain alcohol, so over-use can lead to a ‘wide-awake but drunken’ state. The US Food and Drug Administration (FDA) lists caffeine as a ‘multiple purpose drug generally recognized as safe food substance’. Because beers and related drinks are produced by fermentation of grains with yeasts, the process is associated with vast increases in cell numbers and this leads to drinks with a considerable nucleic acid and nucleotide content, even if the yeasts are removed by filtration. The economic importance of brewing, as well as the clinical relevance of beer and wine in gout, means that there is considerable literature on the composition of beers and wines including their purine levels. A traditional British beer (ale or bitter) can contain up to 25 mg purine per litre (250 mmol l1) but lager beers have up to 20 mg l1. Ciders contain o1 mg l1 of purine. Wine also contains significant amounts of purines. Some low alcohol beers may contain three times these levels of purines. Spirits contain very little in the way of purines because these compounds are removed in the distillation step.
Nucleotides in Human Breast Milk and Infant Formula Milks Human breast milk contains nucleic acids, nucleotides, and nucleosides, particularly cytidine and uridine, with a profile
that reflects the diet of the mother. Average TPAN concentrations in human milk are 172–222 mmol l1 (59–76 mg l1) at all stages of lactation. The content derived from cells is approximately 18% of TPAN during the first few days of lactation but drops to less than 10% later. Proportions of nucleosides derived from RNA (43–48%); free nucleotides (36–40%); free nucleosides (6.6–8%); and nucleotide adducts (9–10%) are similar in milk from women of several races. It is not known if all of these compounds in human milk are used by the breast-fed infant. However there is also a movement to supplement formula milks (based on cow’s milk) with ribonucleotides derived from hydrolyzed RNA. Cow’s milk contains lower levels of nucleotides, with a different profile from the human, but significant levels of the de novo pyrimidine intermediate orotic acid, which is low in humans (raised in milk from mothers who smoke). In the late 1990s the FDA agreed to the nucleotide supplementation of infant formula based on cow’s milk, but at lower concentrations than in human milk. The EU Food Committee recommended in 2003 (updated advice in 2007) that the content of nucleotides, if added to infant formulae and in follow-on formulae, should not exceed 5 mg per 100 kcal. If added the maximum nucleotide contents should be: cytidine monophosphate (CMP) 2.5 mg per 100 kcal, uridine monophosphate (UMP) 1.75 mg per 100 kcal, adenosine monophosoate (AMP) 1.5 mg per 100 kcal, guanosine monophosphate (GMP) 0.5 mg per 100 kcal, inosine monophosphate (IMP) 1 mg per 100 kcal. Several trials have evaluated the effects of nucleotide addition to formula milk in infants but only two of the trials have studied formulae with ‘human’ nucleotide levels of 72 mg l1. Thus, there is no adequate scientific basis at present to conclude that the addition of nucleotides in higher concentrations than presently permitted for infant formula would provide additional benefits. Formula milks based on soy protein have a high natural content of nucleotides and are therefore not supplemented.
Beneficial Effects of Dietary Nucleosides and Nucleotides In healthy adults, a normal varied diet is a good source of nucleic acids, nucleotides, and nucleosides, and supplementation is thought to be unnecessary. There is substantial evidence (principally from research in animal models) that the presence of nucleotides or nucleosides in the diet helps cellular proliferation in the gut, in postoperative trauma, and in the development of the immune response in infants. A medical food supplement containing arginine, glutamine, nucleotides, and omega-3 fatty acids, demonstrates a better clinical outcome for (adult) surgical patients, and a 30% reduction in risk of infection. It should be noted that glutamine is a precursor for de novo synthesis of nucleosides as well as a source of energy for proliferating cells. Dietary nucleotides have been shown to promote the incorporation of essential fatty acids into membrane lipids in healthy new-born babies, and to enhance the integrity and maturation of the intestine and of the immune system, and thus may contribute to the improved immunity seen in breastfed infants. Studies in lower socioeconomic groups have found that supplementation of formula with 14.2 mg free
Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
nucleotides per 100 g milk powder resulted in a significant reduction of first episodes of diarrhea. This may be linked to an alteration in the bowel flora, leading to a predominance of lactobacilli as seen in breast-fed babies. An extract from sugar cane (trade name NucleomaxX), containing 17% nucleosides, is used in the HIV-positive community to counteract the unpleasant side-effects of HIV drugs that inhibit the formation of mitochondrial DNA and hence energy-producing processes. The use of oral uridine in metabolic disorders is described later. Nucleotides based on both adenosine and uridine can activate the purinergic receptors on a wide range of cell types. Nucleotides influence the transcription of several genes in intestinal cells, and have been shown to improve growth and maturation of the gut in weanling rats. In lymphocytes and other cells, synthesis of nucleotides de novo is expanded dramatically when a signal for proliferation is received; the rate of pyrimidine biosynthesis increases more than purine biosynthesis. Thus nucleotides are now considered to be ‘conditionally essential’ because their provision in the diet may provide help through the salvage system where cells are dividing rapidly or where other nutrients, used as precursors, are scarce.
Purine Ribonucleotides as Flavor-Enhancing Additives The purine 50 -nucleoside monophosphates IMP and GMP, derived from degradation of RNA, have received much attention as the taste-active components in a variety of seafoods and meat. These purine 50 -nucleotides, but not the pyrimidine nucleotides CMP and UMP, enhance the savory flavor generated by monosodium glutamate (MSG), by interaction with receptors on the specific umami taste buds in the mouth. Because ATP is the major free nucleotide in muscle cells, its breakdown into the flavor-enhancing IMP provides a scientific rationale for the improved palatability of meat or game birds that have been hung for several days after slaughter. Similarly, the distinctive flavors of several cheeses are related to the metabolism, by bacteria, of the characteristic range of nucleotides present in the original milk.
Purine Ribonucleosides and Bases as Markers of Food Quality Related to the above topic is the role of hypoxanthine (Hx) in the determination of food quality. As described earlier, when an animal is killed the tissues become ischemic and the intracellular ATP starts to degrade, forming first AMP and then Hx. At room temperature the majority of ATP will have degraded within 24 h. The Hx level will be maximal at approximately 2 weeks in meat stored at 4 1C. The change in Hx content of the food alters the sensory perception of the food, with higher Hx levels causing a bitterness in the taste of meat. This aspect of purine catabolism has been particularly well documented in seafood, which is perhaps the most perishable of foods. Hx in fish and fish products such as fish fingers increases linearly with storage time, and measurement of the Hx levels has been recommended as a marker of fish spoilage.
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Toxicology Pharmacological Uses for Nucleosides and Nucleotides Rare genetic disorders highlight the sensitivity of lymphocytes to the efficient removal of waste from DNA catabolism, and in fact have provided the basis for development of novel immunosuppressant drugs which inhibit enzyme activities crucial to removal of purine nucleotides, and lead to misincorporation of nucleotides in DNA synthesis. On the other hand, the use of certain nucleotide analogs as drugs depends on their incorporation into DNA – for example, analogs used in HIV therapy are incorporated by the reverse transcriptase of the virus, and bring the reaction to a halt. Toxicity associated with several analogs is now known to arise from erroneous incorporation into the patient’s mitochondrial DNA, because of less stringent proof-reading by the mitochondrial DNA polymerase enzyme. Azidothymidine (AZT) remains one of the most effective and least toxic drugs for AIDS, albeit now usually taken in triple therapy. Nucleotide analogs have been used to inhibit the de novo pathways for the synthesis of the precursor nucleosides and nucleotides, leading to depletion of metabolites and imbalance of dNTPs, and hence to mis-incorporation of nucleotides in RNA or DNA, respectively. Malaria and other parasites rely exclusively on de novo pyrimidine biosynthesis, thus they may be susceptible at drug doses that do not affect the host, because the human body can obtain nucleotides from the salvage pathway. Similarly, because of the increased requirement for nucleotides in rapidly proliferating cells, almost all the enzyme reactions (Figure 2) have been investigated as potential targets for treatment of cancer, inflammation, or to prevent rejection of transplanted organs. Once again, combinations of drugs with different modes of action have often proved most effective. Oral uridine, as described earlier, can be used where de novo biosynthesis of pyrimidines is defective, and it may be useful in reversing some effects of mitochondrial dysfunction, and to minimize the toxic effect of the antitumor drug 5-fluorouracil. Oral administration of compounds such as benzylacyclouridine, or 20 30 50 tri-O-acetyl uridine (PN401), inhibits the degradative processes in the liver and delivers more uridine into the circulation than oral uridine alone. Uridine is also a precursor for UDP-glucose, essential for the deposition of glycogen in the liver, and UDP-glucose has been proposed as a dietary supplement. Oral CDP-choline is rapidly converted to its components, CDP (which can be recycled to uridine) and choline, an essential component of lipid membranes. Each molecule can then cross the blood–brain barrier where CDP-choline is used in regeneration of membranes within and around nerve cells, and its pharmacological effects may extend to protection against dementia, memory loss, visual degeneration, and to recovery from ischemic strokes.
Toxicity of Exogenous Nucleic Acids to Humans The potential toxicity of dietary nucleic acids to humans usually arises from their metabolic end products (principally uric acid). Many investigators have shown that when normal
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
subjects are fed RNA, the increase in the urate excretion is dramatic, but there is only a modest rise in plasma urate concentrations. The body pool of urate, and hence the plasma urate concentration, is the result of a balance between production, ingestion, and excretion. If kidney function is normal, the chief causes of high plasma uric acid concentrations are either a high intake of exogenous nucleic acid in the diet or overproduction of endogenous purine. A low-purine (low-nucleic acid) diet containing less meat, seafood, and other purine-rich foods and beverages (Tables 1 and 2) leads to a lower risk of gout symptoms. In contrast, subjects with genetic defects that remove the usual controls on purine biosynthesis may have overwhelmingly high endogenous levels of the waste product, uric acid. The contribution of the two sources can be assessed by placing the subject on a purine-free diet for a week, and measuring the total urinary uric acid. In this way fewer than 5% of patients with gout are found to excrete abnormally large amounts of urate (43 millimoles per day) derived from endogenous purines. In these cases, overproduction of purine nucleotides leading to excess uric acid can be traced to a genetic defect. Two such sex-linked disorders are hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency and phosphoribosyltransferase superactivity (PRPS). Boys presenting in infancy usually have severe and eventually fatal neurological deficits. In those presenting as adolescents, neurological problems are milder or absent, and only gout may be evident.
Urolithiasis and Other Kidney Stones Although modest overindulgence in purine-rich food does not precipitate gout in normal subjects, it can predispose to uric acid lithiasis. Uric acid stones are relatively common in countries where the consumption of nucleic acid-rich beverages and food is high, and in hot climates if insufficient fluids are consumed. A number of compounds, such as vitamin C, increase uric acid clearance and thus can precipitate urolithiasis. Perhaps not so well recognized is the uricosuric effect of a high-protein diet and the fact that purine-rich foods also predispose to renal calcium stones. This may be because many purine-rich foods such as spinach are equally rich in calcium oxalate. Some vegetables may provoke gout attacks by virtue of their oxalic acid content rather than of purines, but legumes, fastgrowing parts of brassicas and asparagus tips may also have significant nucleic acid content. Pulses and grains have a particularly high nucleic acid content. Approximately 25% of vitamin C intake is also excreted as oxalate, which can compound the problem. The solubility of uric acid is very sensitive to the pH of the urine, which in turn may be made more acidic by a highprotein diet. The solubility of uric acid in urine at pH 5.0 is low (approximately 1 mmol l1), but it can be increased
12-fold by alkalinizing regimens such as sodium bicarbonate or potassium citrate, which raise the pH to 8.0. Excess uric acid from dietary purines can also precipitate symptoms that may draw attention to endogenous uric acid accumulating in adults with milder forms of HPRT deficiency or PRPS superactivity, or to a defect leading to raised levels of a uric acid analog related to adenine. The ideal diet for subjects at risk of gout or of uric acid lithiasis is no more than one meat meal per day, using only the low-purine meat and vegetables indicated, and treatment with allopurinol. The most common and effective treatment for gout is the drug allopurinol, which prevents conversion of xanthine to uric acid by inhibiting the enzyme xanthine oxidase. Although the uricase gene appears to be present in human cells, the promoter is not activated, so no enzyme activity is detected in the liver. Biochemical drugs using recombinant uricase are effective in refractory gout.
See also: Caffeine. Choline and Phosphatidylcholine. Ascorbic Acid (Vitamin C): Physiology, Dietary Sources, and Requirements
Further Reading Becker MA (2001) Purines and pyrimidines. In: Scriver CR, Beaudet AL, Sly WS, and Valle D (eds.) The Metabolic and Molecular Basis of Inherited Disease, 8th edn., Chapter 106, pp. 2513–2537. New York: McGraw-Hill. Carver JD (2003) Advances in nutritional modifications of infant formulas. American Journal of Clinical Nutrition 77: 1550S–1554S. Christopherson RI, Lyons SD, and Wilson PK (2002) Inhibitors of de novo nucleotide biosynthesis as drugs. Accounts of Chemical Research 35: 961–971. Diem K and Lentner C (eds.) (1970) Scientific Tables - Chemical Composition of Foodstuffs, 7th edn., pp. 230–243. Geigy: Basle. Fuke S and Konosu S (1991) Taste-active components in some foods: A review of Japanese research. Physiology and Behaviour 49: 863–868. Grahame R, Simmonds HA, and Carrey EA (2003) Gout: The ‘At Your Fingertips’ Guide. London: Class Publishing. Gutierrez-Castrellon P, Mora-Magana I, Diaz-Garcia L, Jimenez-Gutierrez C, Ramirez-Mayans J, and Solomon-Santibanez A (2007) Immune response to nucleotide-supplemented infant formulae: Systematic review and meta-analysis. British Journal of Nutrition 98: S64–S67. Lee H, Hanes J, and Johnson KA (2003) Toxicity of nucleoside analogs used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry 42: 14711–14719. Richette P, Brie`re C, Hoenen-Clavert V, Loeuille D, and Bardin T (2007) Rasburicase for tophaceous gout not treatable with allopurinol: An exploratory study. The Journal of Rheumatology 34: 2093–2098. Rolls ET (2000) The representation of umami taste in the taste cortex. Journal of Nutrition 130: 960S–965S. Secades JJ and Frontera G (1995) CDP-choline: Pharmacological and clinical review. Methods and Findings in Experimental and Clinical Pharmacology 17(supplement B): 1–54. Tressler RL, Ramstack MB, White NR, et al. (2003) Determination of total potentially available nucleosides in human milk from Asian women. Nutrition 19: 16–20. Yu VYH (2002) Scientific rationale and benefits of nucleotide supplementation of infant formula. Journal of Paediatrics and Child Health 38: 543–559.
Physiology, Toxicology, and Dietary Sources EA Carrey, UCL Institute of Child Health, London, UK D Perrett, Queen Mary University of London, London, UK HA Simmondsw r 2013 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by E. A. Carrey, H. A. Simmonds volume 2, pp 260–269, r 2005, Elsevier Ltd.
Introduction
Nucleic Acid Biosynthesis in Humans
Nucleic acids, vital components of all living cells, were isolated in 1869 from the nuclei of pus cells and the spermatozoa of Rhine salmon. Later, it was shown that the major constituents of nucleic acids are sugars, phosphate groups, and the characteristic purine and pyrimidine bases. The chemical structures of the purine bases, including uric acid – the end (waste) product of purine metabolism in humans – were established at the end of the 19th century. The role of the nucleic acids in storing and translating the genetic information in the cells was elucidated in the 20th century. Although their calorific contribution to the diet is trivial, nucleotides, nucleosides, and bases have essential roles in metabolism and signaling within the cell and organism. This article outlines the structure of nucleic acids and gives a brief overview of the physiological functions of nucleosides, nucleotides, and nucleic acids. It describes the toxicity that may arise from degradation of both endogenous and dietary (exogenous) nucleic acids in humans and contains a summary of the nucleic acid content of foods.
Physiology Structure The hereditary material in the nucleus of human cells is packed into 46 chromosomes and additional DNA is found in the mitochondria. The capacity of DNA to be copied into two complementary strands arises from the well-known double-helical structure and underlies the transfer of genetic information in all living organisms. Interactions between the DNA and transcription factors determine the time and place in the body where genes are transcribed, controlling development and metabolism. RNA molecules are synthesized initially on a DNA template by a DNA-dependent RNA polymerase in a process called transcription, where ribonucleotides complementary to the bases of one strand of DNA are joined by 30 –50 phosphodiester bonds (Figure 1(a)). w
Deceased.
Encyclopedia of Human Nutrition, Volume 3
The first step in nucleic acid synthesis involves the formation of the purine and pyrimidine ribonucleotides. There are two endogenous routes: either the energetically expensive de novo route from small molecules such as carbon dioxide, amino acids, and ribose sugars, or the energetically less expensive ‘salvage’ pathway. Purine bases and pyrimidine nucleosides from the breakdown of nucleic acids and nucleotide cofactors are salvaged within the cells, generating nucleotides that can be incorporated into nucleic acids. In most cells, salvage processes are more important, and the ribonucleotides recycled in this way exert feedback control on the de novo routes.
Metabolic Roles of Nucleotides The most abundant ribonucleotide in the body is adenosine 50 –triphosphate (ATP), which is the universal energy carrier in living organisms (Figure 1(b)). In addition, nucleotides are precursors of several coenzymes, used in many reactions including the conversion of food into energy. Within cells adenosine and guanosine nucleotides also have roles in the transduction of external signals into cellular responses, and in the translation and synthesis of proteins. Pyrimidine nucleotides are present at much lower concentrations in cells but also fulfill diverse functions. Uridine diphosphate (UDP)-glucose and Cytidine diphosphate (CDP)-lipids are active intermediates in the synthesis of glycogen and membranes, respectively, and sugars linked to UDP or GDP are used in the glycosylation of proteins. UDP-glucuronic acid is an essential component of the pathways that convert exogenous molecules and endogenous steroids into soluble forms for disposal from the body. The free deoxyribonucleotides are very scarce in the normal cell because they are used exclusively for the synthesis of DNA.
Synthesis of Nucleic Acids Synthesis of both DNA and RNA is prominent in cells and tissues with high turnover or metabolism (e.g., liver, gut epithelium, skin, dividing lymphocytes, bone marrow, and hair follicles). Different complements of enzymes
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources NH2 C N 7 C 5 6 1N Adenine CH 8 4 3 2CH 9 N C N
O− −
O P O
O
CH2
5′ 4′
H
H
O Deoxyribose
H
3′
2′
H O P
O
CH2
O
O H
N NH C Guanine CH C C N H2N N
O
H
H
H CH2
O
HC 5 4 3 N 2 HC6 1 C N
O
O−
8 CH
N
H2C O O 5′ Ribose H 4′ H
1′
H
2′
OH
P O
O−
O− O
P
O
O
P
O−
O
3′ H OH
Adenosine 5′-triphosphate (ATP)
Cytosine O
H
O HH C 3
C
O O P
9
(b)
H
H H −
N
N
7
C
O O
6 5C 4 3 C
NH2 H
O P
CH
C
2
C
H
−
N1
1′
O −
NH2
O
CH2
O
HC O
H
N
NH C
Thymine O
H
H (a)
C
H
H
Figure 1 (a) Schematic representation of part of a DNA strand showing the structural formulae of the four constituent bases, adenine, guanine, cytosine, and thymine, linked via the 30 -OH group of the deoxyribose moiety to the 50 -phosphate group of the next nucleotide. Also shown is the numbering of the atoms in the deoxyribose, as well as the pyrimidine and purine rings. The bases are adenine (A), guanine (G), cytosine (C), and thymine (T). Two strands are wound in opposing chemical directions to allow the well-known double-helix structure, with hydrogen-bonding between complementary nucleotides (i.e., A–T and G–C), to form. The deoxyribose and phosphate groups form the outer sides of the ‘ladder’. The RNA molecule is single-stranded, but double-helical regions arise when stretches of complementary sequences allow hairpin loops to form. In addition, the base uracil (U) is found instead of thymine, and the pentose is ribose. (b) Structural formula of ATP indicating that the ribose, as distinct from deoxyribose, has an OH group at the 2’ position on the pentose ring. When a nucleoside triphosphate (NTP) is linked through the 50 phosphate groups to the 30 position of the previous residue on the growing chain, the chemical energy for the polymerization is provided by the removal of the second and third phosphate groups.
are expressed in each cell type, and therefore tissues have characteristic profiles of internal metabolites, including nucleotides and nucleosides. For example, in cells that do not continuously divide, such as heart and muscle, nucleotide profiles are relatively simple, relating to the major requirement to sustain high levels of ATP and cofactors. Contrastingly, rapidly dividing cells in liver and intestine show a complex nucleotide pattern, identifying these organs as major sites of nucleic acid metabolism. The gut is particularly important in this respect. The rate of cell turnover in the luminal villi is high, and it has been calculated that in rats approximately 30 mg of endogenous nucleic acid derived from dead cells enters the gut lumen daily. This means that nucleic acid synthesis in liver and intestine is much higher than in tissues such as muscle.
Metabolism of Endogenous Nucleic Acids and Excretion of Metabolic End Products There is a considerable daily turnover of endogenous nucleic acids and ribonucleotides during muscle work, wound healing, erythrocyte senescence, mounting an immune response, etc. However, only a small fraction of these vital endogenous compounds is degraded and lost from the body. Because
de novo purine and pyrimidine synthesis is energetically expensive the contents of dead cells are normally used by other cells, and degraded RNA or cofactors are recycled within living cells using active ‘salvage’ routes. Breakdown products of DNA and RNA enter the salvage pathways in the form of the purine bases hypoxanthine (Hx) and guanine or the pyrimidine bases uracil and thymine (Figure 2). Any pyrimidine bases that are not salvaged are then further catabolized in a series of steps to b-amino acids, which are soluble and readily excreted. There is thus normally no measurable toxic pyrimidine end product from endogenous or dietary nucleic acids, except in the case of a small number of very rare metabolic disorders (Figure 3). The purine base Hx is converted in the liver to the insoluble metabolites xanthine and then uric acid by the enzyme xanthine oxidase/xanthine dehydrogenase (XDH) in man. Urate can normally be disposed of in the urine, but high concentrations can crystallize and form kidney stones or deposits in the joints and under the skin. Some rare genetic disorders can remove feedback regulation of purine biosynthesis, or excessive breakdown of cells may overload the salvage system, each resulting in very high endogenous levels of uric acid. However most other animal species (with the notable exception of some dog varieties) possess an additional catabolic enzyme, uricase,
Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
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Glutamine + HCO3− + ATP
PRPP + Glutamine
Aspartate + Carbamoyl phosphate
5-Phosphoribosylamine
Carbamoyl aspartate Purine nucleotides
9 further step to form the purine rings
PRPP + Orotate Nucleosides
IMP
AMP
GMP
ADP
GDP
ATP
GTP
Hx/G
Uridine
OMP +ATP
UMP
+PRPP
dADP
dGDP
dATP
dGTP
UDP UTP
dUDP
CTP
dUTP
CDP
dUMP
Thymidine dTMP
dCDP
dTDP
dCTP
dTTP
+ATP
DNA Figure 2 De novo synthesis of ribonucleotides uses small molecules and amino acids. The purine base of IMP is built up in several steps on a ribose phosphate molecule, and separate pathways generate ATP and GTP. In pyrimidine biosynthesis, the six-membered ring is formed before addition of the ribose phosphate to orotate, and decarboxylation to form UMP. Ribonucleotides (ATP, GTP, UTP, and CTP, shown in bold) are used in the synthesis of RNA, whereas DNA is synthesized after conversion of the ribose to deoxyribose, and of dUMP to dTMP. Salvage pathways (shown with open arrows) all follow two patterns: purine nucleotides and nucleosides, entering the cells or derived from hydrolysis of cofactors or nucleic acids, are converted to the free bases hypoxanthine or guanine and then converted by a specific phosphoribosyltransferase enzyme to the nucleoside; in contrast pyrimidines are salvaged from the nucleosides uridine or thymidine using specific kinase enzymes. It should be noted that PRPP (phosphoribosyl pyrophosphate) is essential for both de novo pathways and the purine salvage pathways.
which cleaves the purine ring of uric acid forming the readily soluble allantoin. This compound in turn may be degraded to ammonia in water-dwelling species such as fish.
Metabolism of Dietary Nucleic Acids in Humans The human diet is naturally rich in nucleic acids because food is derived from once-living organisms. Because, as already described, nucleotides and nucleosides can be synthesized de novo and nucleobases liberated during catabolism can be salvaged, they are often considered to be dispensable nutrients in food. The metabolism of these exogenous nucleic acids follows a similar pattern to the intracellular process described above, but the bacterial flora of the intestine are the first point of degradation. This digestion is rapid. Studies in both pigs and humans demonstrated that up to 50% of dietary purine was degraded to carbon dioxide within 30 min, 43% was recovered in the urine and 5% excreted in the feces. Less than 2% of dietary purines is incorporated into nucleic acids. Humans thus have no apparent essential requirement for purines from the diet and the intestinal mucosa provides an
effective barrier to their uptake through a battery of enzymes that can rapidly degrade purine nucleotides, nucleosides, and bases especially unusual purines found in plant materials. Because of this enzyme activity, and the rapid turnover of intestinal mucosa, approximately 200 mg of urate is excreted daily in the feces. This phenomenon ensures that levels of ATP do not fluctuate in concert with the dietary intake of purines, or may represent an important evolutionary development to protect the integrity of cellular DNA. On the other hand, pyrimidine ribonucleoside monophosphates (NMPs) and ribonucleosides are absorbed readily from the intestine and utilized for nucleic acid synthesis. This has been demonstrated by studies of humans with hereditary oroticaciduria, a rare defect in conversion of orotic acid to uridine monophosphate (UMP) in de novo pyrimidine synthesis. Such patients have severe megaloblastic anemia. They have been sustained on oral uridine, indicating that the dietary pyrimidine nucleoside can compensate totally for lack of de novo synthesis in humans. Studies using radiolabelled purines and pyrimidines in mice provided further evidence for the incorporation of dietary pyrimidine nucleosides, but not purine nucleosides, into hepatic RNA.
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
AMP
Adenosine
IMP
GMP
Cytidine
Inosine
Guanosine
Uridine
Thymidine
Guanine
Uracil
Thymine
Dihydrouracil
Dihydrothymine
-Ureidopropionate
-Ureidoisobutyrate
-Alanine
-Aminoisobutyrate
Hypoxanthine
Xanthine
Uric acid
NH3 + CO2
Urea Figure 3 Breakdown of DNA and RNA begins when the molecules are degraded by nuclease enzymes to liberate nucleotides. The next step, degradation by specific 50 –nucleotidases (removing the phosphate groups) to nucleosides or deoxynucleosides, is essentially irreversible. Nucleoside phophorylases generate (a) the purine bases hypoxanthine and xanthine, which are converted to uric acid or (b) the pyrimidine bases uracil and thymine, which are converted to b-amino acids, ammonia, and CO2, and thus to urea, a solute in urine.
Nucleic Acid Content of Foods The nucleic acid content of foodstuffs is expressed generally in terms of purine equivalents or ‘total potentially available nucleosides (TPAN)’ released from food by hydrolysis with sodium hydroxide or hydrochloric acid and enzymes. The data for purines are thus derived by analysis of the resultant constituent bases. Analysis by Robert McCance, Elsie Widdowson, and colleagues from the 1930s onward forms the basis of tables listing the composition of foodstuffs although, with a few exceptions, this demanding work has not been repeated using more modern analytical methods such as liquid chromatography–mass spectrometry (LC–MS). Foods can be classified into three groups; high, low, or essentially free of purines (and hence of pyrimidines too) (Tables 1 and 2). As a general rule growing organisms such as yeast, or rapidly metabolizing tissues such as liver, will be rich in both nucleic acids. Seeds and grain are good sources of the genetic material, DNA, as well as free nucleotides, which are stored for use in germination. Muscle tissue is an excellent source of the nucleotide ATP and the nucleic acids in mitochondria. Offal is also metabolically very active so is usually high in free nucleotides as well as nucleic acids. Fish and shellfish that are eaten ‘whole’ and fish eggs and roe are also high in nucleic acids. Extracts of meat and yeast e.g., Bovril, Marmite, Vegemite, have very high purine contents, as do supplements such as Spirulina for sale in ‘Health Food’ shops, but are usually eaten in small quantities.
Fats, white flour, sugar, and fruit juices have been separated from the ‘living’ part of the food and so they are poor sources of nucleic acids.
Effect of Cooking on Nucleotide Content of the Diet Nucleic acids are relatively resistant to hydrolysis at the moderate temperatures and short periods of time associated with cooking in water or frying in oil. On the other hand nucleoside triphosphates (NTPs) and nucleoside diphosphates (NDPs) break down readily during boiling in water forming first their related NMP and then their base. The rate of hydrolysis is significantly increased in acidic solutions. The rate of degradation is enhanced if any enzyme activity is still present. The levels of nucleic acids and NTPs are well maintained during prolonged storage at 20 1C or below.
Nucleic Acid and Related Compounds in Beverages Tea, coffee, and cola drinks contain a number of unusual nucleobases based on xanthine (Figure 4). Caffeine (1,3,7trimethylxanthine) and theobromine are the best known. Caffeine is found in various quantities in the beans, leaves, and fruit of many plants. It is mainly consumed by humans in infusions extracted from the coffee bean and leaves of the tea bush. A cup of coffee can contain up to 175 mg of caffeine whereas a cup of tea contains approximately 40 mg.
Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
Table 1 A quick reference guide to the purine (nucleic acid) content of foods Foods and Beverages Rich in Nucleic Acids/Purines Offal: sweetbreads, liver, kidney, heart, and pate´ Wild or farmed game meats (venison, pheasant, rabbit, hare) Seafoods: sardines, sprats, herring, bloaters, anchovies, fish roe, caviar, taramasalata, trout or salmon; lobster, crab, prawns Vegetables: asparagus, avocado pears, peas, spinach, mushrooms, broad beans, cauliflower Pulses and grains: legumes, pulses and soya products such as bean curd, tofu, Quorn Cereals: all bran, oat, rye or wheat cereals and products; wholemeal, rye and brown breads Other: beer and yeast extracts/tablets (BarmeneTM, TastexTM). Meat or vegetable extracts (MarmiteTM, VegemiteTM, BovrilTM, OxoTM) Blue-green alga extracts (Spirulina) Foods that are Moderate or Low Sources of Purines Beef, lamb, pork (steak or chops), bacon, ham, sausages, some poultry, tongue (all should be eaten in moderation) Carrots, parsnip, other root vegetables, potatoes, lettuce, leeks, cabbage, sprouts, marrow, squash, courgettes Peanuts, cashew nuts Breakfast cereals Some fish (see Table 2) Foods and Beverages that are Essentially Purine-Free Milk, cheese, eggs, butter, margarine, cream, ice cream White bread or flour, cakes, scones, biscuits Sugar, jam, marmalade, honey, and sweets Cucumber, tomato, onions, pumpkin Fresh, cooked or tinned fruits, nuts Puddings, custards, yogurt Fruit juices, soft drinks
Table 2 Concentrations of purines in some common foods; results are recorded relative to 100 g of food for purine and for protein, although serving size for each ingredient may be larger or smaller than 100 g based on Diem and Lentner (1970). Food
Purine (mg per 100 g)
Protein (g per 100 g)
Meat Beef liver Beef kidney Beef heart Beef tongue Beef steak Calf liver Sweetbreads Veal cutlet Sheep kidney Lamb chop Pork liver Pork cutlet Bacon Ham Sausage (beef) Sausage (pork) Rabbit Venison
333 285 285 167 151 348 1212 152 312 196 289 164 85 136 79 66 118 156
19.7 15.4 16.8 16.4 19.5 19 19.6 19.2 16.8 14.9 22 16.4 9.1 19.5 13.8 11.5 20.4 20 (Continued )
Table 2
193
Continued
Food Vegetables Asparagus Cauliflower Celery Kohlrabi Mushrooms Peas Spinach
Purine (mg per 100 g)
32 32 20 44 72 72 96
Protein (g per 100 g)
2.1 2.1 1.1 2.1 3.5 6.7 2.2
Dried Legumes Split peas Red bean Lentils Haricot beans Lima bean
195 162 222 230 149
21 20 28 22 21
Other BovrilTM MarmiteTM OxoTM cubes Yeast extracts
340 356 236 2257
18 2 10 46
Poultry Chicken flesh Chicken liver Chicken heart Duck Goose Turkey
181 372 223 181 177 239
20.6 22.1 18 16 16.4 20.1
Fish, Seafoods Anchovies Bass Bloaters Bream Cod Crab Clams Eel Fish cakes Herring Kippers Lobster Lemon sole Mackerel Plaice Salmon Sardines Scallops Sprats Squid Trout
411 73 133 72 62 61 136 108 36 378 91 100 54 246 53 250 345 117 250 135 92
20 19.5 22.6 19.7 18 19.2 17 18.6 12.1 17 21.2 20 19.9 29 18.1 23 23 22.3 25.1 15 19.2
Canned Seafoods Anchovies Herring Mackerel Oysters Salmon Sardines Shrimp Tuna
321 378 246 116 88 399 231 142
30 17 26 6 26 24 22 29
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources O
H3C N
N
N
CH3
O N CH3
Caffeine O HN O
CH3 N
N N CH3
Theobromine
O H3C O
N N H
O
CH3 N N
Paraxanthine
H3C O
N
H N
N N CH3
Theophylline
Figure 4 Chemical structures of unusual nucleobases derived from xanthine, and found in plant tissues such as cocoa beans, coffee beans, and tea leaves.
Many popular soft drinks, e.g., cola, may contain up to 300 mg per can. Theobromine (dimethylxanthine) occurs naturally in cocoa beans (20 mg g1 of cocoa powder) and is therefore present in chocolate. Theophylline (1,3-dimethylxanthine) occurs at trace levels in tea leaves and is now chemically synthesized for use as a drug treatment for asthma. Caffeine is rapidly absorbed by the stomach and small intestine and is distributed throughout total body water. It is rapidly metabolized in the liver to paraxanthine (84%), theobromine (12%), and theophylline (4%). In man, caffeine acts as a central nervous system stimulant, temporarily warding off fatigue and restoring alertness: it readily enters the brain and acts as a nonselective antagonist of purinergic adenosine receptors. Caffeine is the most widely consumed psychoactive substance; in North America, 90% of adults consume caffeine beverages daily. ‘Energy drinks’ contain very high levels of caffeine and some also contain alcohol, so over-use can lead to a ‘wide-awake but drunken’ state. The US Food and Drug Administration (FDA) lists caffeine as a ‘multiple purpose drug generally recognized as safe food substance’. Because beers and related drinks are produced by fermentation of grains with yeasts, the process is associated with vast increases in cell numbers and this leads to drinks with a considerable nucleic acid and nucleotide content, even if the yeasts are removed by filtration. The economic importance of brewing, as well as the clinical relevance of beer and wine in gout, means that there is considerable literature on the composition of beers and wines including their purine levels. A traditional British beer (ale or bitter) can contain up to 25 mg purine per litre (250 mmol l1) but lager beers have up to 20 mg l1. Ciders contain o1 mg l1 of purine. Wine also contains significant amounts of purines. Some low alcohol beers may contain three times these levels of purines. Spirits contain very little in the way of purines because these compounds are removed in the distillation step.
Nucleotides in Human Breast Milk and Infant Formula Milks Human breast milk contains nucleic acids, nucleotides, and nucleosides, particularly cytidine and uridine, with a profile
that reflects the diet of the mother. Average TPAN concentrations in human milk are 172–222 mmol l1 (59–76 mg l1) at all stages of lactation. The content derived from cells is approximately 18% of TPAN during the first few days of lactation but drops to less than 10% later. Proportions of nucleosides derived from RNA (43–48%); free nucleotides (36–40%); free nucleosides (6.6–8%); and nucleotide adducts (9–10%) are similar in milk from women of several races. It is not known if all of these compounds in human milk are used by the breast-fed infant. However there is also a movement to supplement formula milks (based on cow’s milk) with ribonucleotides derived from hydrolyzed RNA. Cow’s milk contains lower levels of nucleotides, with a different profile from the human, but significant levels of the de novo pyrimidine intermediate orotic acid, which is low in humans (raised in milk from mothers who smoke). In the late 1990s the FDA agreed to the nucleotide supplementation of infant formula based on cow’s milk, but at lower concentrations than in human milk. The EU Food Committee recommended in 2003 (updated advice in 2007) that the content of nucleotides, if added to infant formulae and in follow-on formulae, should not exceed 5 mg per 100 kcal. If added the maximum nucleotide contents should be: cytidine monophosphate (CMP) 2.5 mg per 100 kcal, uridine monophosphate (UMP) 1.75 mg per 100 kcal, adenosine monophosoate (AMP) 1.5 mg per 100 kcal, guanosine monophosphate (GMP) 0.5 mg per 100 kcal, inosine monophosphate (IMP) 1 mg per 100 kcal. Several trials have evaluated the effects of nucleotide addition to formula milk in infants but only two of the trials have studied formulae with ‘human’ nucleotide levels of 72 mg l1. Thus, there is no adequate scientific basis at present to conclude that the addition of nucleotides in higher concentrations than presently permitted for infant formula would provide additional benefits. Formula milks based on soy protein have a high natural content of nucleotides and are therefore not supplemented.
Beneficial Effects of Dietary Nucleosides and Nucleotides In healthy adults, a normal varied diet is a good source of nucleic acids, nucleotides, and nucleosides, and supplementation is thought to be unnecessary. There is substantial evidence (principally from research in animal models) that the presence of nucleotides or nucleosides in the diet helps cellular proliferation in the gut, in postoperative trauma, and in the development of the immune response in infants. A medical food supplement containing arginine, glutamine, nucleotides, and omega-3 fatty acids, demonstrates a better clinical outcome for (adult) surgical patients, and a 30% reduction in risk of infection. It should be noted that glutamine is a precursor for de novo synthesis of nucleosides as well as a source of energy for proliferating cells. Dietary nucleotides have been shown to promote the incorporation of essential fatty acids into membrane lipids in healthy new-born babies, and to enhance the integrity and maturation of the intestine and of the immune system, and thus may contribute to the improved immunity seen in breastfed infants. Studies in lower socioeconomic groups have found that supplementation of formula with 14.2 mg free
Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
nucleotides per 100 g milk powder resulted in a significant reduction of first episodes of diarrhea. This may be linked to an alteration in the bowel flora, leading to a predominance of lactobacilli as seen in breast-fed babies. An extract from sugar cane (trade name NucleomaxX), containing 17% nucleosides, is used in the HIV-positive community to counteract the unpleasant side-effects of HIV drugs that inhibit the formation of mitochondrial DNA and hence energy-producing processes. The use of oral uridine in metabolic disorders is described later. Nucleotides based on both adenosine and uridine can activate the purinergic receptors on a wide range of cell types. Nucleotides influence the transcription of several genes in intestinal cells, and have been shown to improve growth and maturation of the gut in weanling rats. In lymphocytes and other cells, synthesis of nucleotides de novo is expanded dramatically when a signal for proliferation is received; the rate of pyrimidine biosynthesis increases more than purine biosynthesis. Thus nucleotides are now considered to be ‘conditionally essential’ because their provision in the diet may provide help through the salvage system where cells are dividing rapidly or where other nutrients, used as precursors, are scarce.
Purine Ribonucleotides as Flavor-Enhancing Additives The purine 50 -nucleoside monophosphates IMP and GMP, derived from degradation of RNA, have received much attention as the taste-active components in a variety of seafoods and meat. These purine 50 -nucleotides, but not the pyrimidine nucleotides CMP and UMP, enhance the savory flavor generated by monosodium glutamate (MSG), by interaction with receptors on the specific umami taste buds in the mouth. Because ATP is the major free nucleotide in muscle cells, its breakdown into the flavor-enhancing IMP provides a scientific rationale for the improved palatability of meat or game birds that have been hung for several days after slaughter. Similarly, the distinctive flavors of several cheeses are related to the metabolism, by bacteria, of the characteristic range of nucleotides present in the original milk.
Purine Ribonucleosides and Bases as Markers of Food Quality Related to the above topic is the role of hypoxanthine (Hx) in the determination of food quality. As described earlier, when an animal is killed the tissues become ischemic and the intracellular ATP starts to degrade, forming first AMP and then Hx. At room temperature the majority of ATP will have degraded within 24 h. The Hx level will be maximal at approximately 2 weeks in meat stored at 4 1C. The change in Hx content of the food alters the sensory perception of the food, with higher Hx levels causing a bitterness in the taste of meat. This aspect of purine catabolism has been particularly well documented in seafood, which is perhaps the most perishable of foods. Hx in fish and fish products such as fish fingers increases linearly with storage time, and measurement of the Hx levels has been recommended as a marker of fish spoilage.
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Toxicology Pharmacological Uses for Nucleosides and Nucleotides Rare genetic disorders highlight the sensitivity of lymphocytes to the efficient removal of waste from DNA catabolism, and in fact have provided the basis for development of novel immunosuppressant drugs which inhibit enzyme activities crucial to removal of purine nucleotides, and lead to misincorporation of nucleotides in DNA synthesis. On the other hand, the use of certain nucleotide analogs as drugs depends on their incorporation into DNA – for example, analogs used in HIV therapy are incorporated by the reverse transcriptase of the virus, and bring the reaction to a halt. Toxicity associated with several analogs is now known to arise from erroneous incorporation into the patient’s mitochondrial DNA, because of less stringent proof-reading by the mitochondrial DNA polymerase enzyme. Azidothymidine (AZT) remains one of the most effective and least toxic drugs for AIDS, albeit now usually taken in triple therapy. Nucleotide analogs have been used to inhibit the de novo pathways for the synthesis of the precursor nucleosides and nucleotides, leading to depletion of metabolites and imbalance of dNTPs, and hence to mis-incorporation of nucleotides in RNA or DNA, respectively. Malaria and other parasites rely exclusively on de novo pyrimidine biosynthesis, thus they may be susceptible at drug doses that do not affect the host, because the human body can obtain nucleotides from the salvage pathway. Similarly, because of the increased requirement for nucleotides in rapidly proliferating cells, almost all the enzyme reactions (Figure 2) have been investigated as potential targets for treatment of cancer, inflammation, or to prevent rejection of transplanted organs. Once again, combinations of drugs with different modes of action have often proved most effective. Oral uridine, as described earlier, can be used where de novo biosynthesis of pyrimidines is defective, and it may be useful in reversing some effects of mitochondrial dysfunction, and to minimize the toxic effect of the antitumor drug 5-fluorouracil. Oral administration of compounds such as benzylacyclouridine, or 20 30 50 tri-O-acetyl uridine (PN401), inhibits the degradative processes in the liver and delivers more uridine into the circulation than oral uridine alone. Uridine is also a precursor for UDP-glucose, essential for the deposition of glycogen in the liver, and UDP-glucose has been proposed as a dietary supplement. Oral CDP-choline is rapidly converted to its components, CDP (which can be recycled to uridine) and choline, an essential component of lipid membranes. Each molecule can then cross the blood–brain barrier where CDP-choline is used in regeneration of membranes within and around nerve cells, and its pharmacological effects may extend to protection against dementia, memory loss, visual degeneration, and to recovery from ischemic strokes.
Toxicity of Exogenous Nucleic Acids to Humans The potential toxicity of dietary nucleic acids to humans usually arises from their metabolic end products (principally uric acid). Many investigators have shown that when normal
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Nucleic Acids, Purine, and Pyrimidine Nucleotides and Nucleosides: Physiology, Toxicology, and Dietary Sources
subjects are fed RNA, the increase in the urate excretion is dramatic, but there is only a modest rise in plasma urate concentrations. The body pool of urate, and hence the plasma urate concentration, is the result of a balance between production, ingestion, and excretion. If kidney function is normal, the chief causes of high plasma uric acid concentrations are either a high intake of exogenous nucleic acid in the diet or overproduction of endogenous purine. A low-purine (low-nucleic acid) diet containing less meat, seafood, and other purine-rich foods and beverages (Tables 1 and 2) leads to a lower risk of gout symptoms. In contrast, subjects with genetic defects that remove the usual controls on purine biosynthesis may have overwhelmingly high endogenous levels of the waste product, uric acid. The contribution of the two sources can be assessed by placing the subject on a purine-free diet for a week, and measuring the total urinary uric acid. In this way fewer than 5% of patients with gout are found to excrete abnormally large amounts of urate (43 millimoles per day) derived from endogenous purines. In these cases, overproduction of purine nucleotides leading to excess uric acid can be traced to a genetic defect. Two such sex-linked disorders are hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency and phosphoribosyltransferase superactivity (PRPS). Boys presenting in infancy usually have severe and eventually fatal neurological deficits. In those presenting as adolescents, neurological problems are milder or absent, and only gout may be evident.
Urolithiasis and Other Kidney Stones Although modest overindulgence in purine-rich food does not precipitate gout in normal subjects, it can predispose to uric acid lithiasis. Uric acid stones are relatively common in countries where the consumption of nucleic acid-rich beverages and food is high, and in hot climates if insufficient fluids are consumed. A number of compounds, such as vitamin C, increase uric acid clearance and thus can precipitate urolithiasis. Perhaps not so well recognized is the uricosuric effect of a high-protein diet and the fact that purine-rich foods also predispose to renal calcium stones. This may be because many purine-rich foods such as spinach are equally rich in calcium oxalate. Some vegetables may provoke gout attacks by virtue of their oxalic acid content rather than of purines, but legumes, fastgrowing parts of brassicas and asparagus tips may also have significant nucleic acid content. Pulses and grains have a particularly high nucleic acid content. Approximately 25% of vitamin C intake is also excreted as oxalate, which can compound the problem. The solubility of uric acid is very sensitive to the pH of the urine, which in turn may be made more acidic by a highprotein diet. The solubility of uric acid in urine at pH 5.0 is low (approximately 1 mmol l1), but it can be increased
12-fold by alkalinizing regimens such as sodium bicarbonate or potassium citrate, which raise the pH to 8.0. Excess uric acid from dietary purines can also precipitate symptoms that may draw attention to endogenous uric acid accumulating in adults with milder forms of HPRT deficiency or PRPS superactivity, or to a defect leading to raised levels of a uric acid analog related to adenine. The ideal diet for subjects at risk of gout or of uric acid lithiasis is no more than one meat meal per day, using only the low-purine meat and vegetables indicated, and treatment with allopurinol. The most common and effective treatment for gout is the drug allopurinol, which prevents conversion of xanthine to uric acid by inhibiting the enzyme xanthine oxidase. Although the uricase gene appears to be present in human cells, the promoter is not activated, so no enzyme activity is detected in the liver. Biochemical drugs using recombinant uricase are effective in refractory gout.
See also: Caffeine. Choline and Phosphatidylcholine. Ascorbic Acid (Vitamin C): Physiology, Dietary Sources, and Requirements
Further Reading Becker MA (2001) Purines and pyrimidines. In: Scriver CR, Beaudet AL, Sly WS, and Valle D (eds.) The Metabolic and Molecular Basis of Inherited Disease, 8th edn., Chapter 106, pp. 2513–2537. New York: McGraw-Hill. Carver JD (2003) Advances in nutritional modifications of infant formulas. American Journal of Clinical Nutrition 77: 1550S–1554S. Christopherson RI, Lyons SD, and Wilson PK (2002) Inhibitors of de novo nucleotide biosynthesis as drugs. Accounts of Chemical Research 35: 961–971. Diem K and Lentner C (eds.) (1970) Scientific Tables - Chemical Composition of Foodstuffs, 7th edn., pp. 230–243. Geigy: Basle. Fuke S and Konosu S (1991) Taste-active components in some foods: A review of Japanese research. Physiology and Behaviour 49: 863–868. Grahame R, Simmonds HA, and Carrey EA (2003) Gout: The ‘At Your Fingertips’ Guide. London: Class Publishing. Gutierrez-Castrellon P, Mora-Magana I, Diaz-Garcia L, Jimenez-Gutierrez C, Ramirez-Mayans J, and Solomon-Santibanez A (2007) Immune response to nucleotide-supplemented infant formulae: Systematic review and meta-analysis. British Journal of Nutrition 98: S64–S67. Lee H, Hanes J, and Johnson KA (2003) Toxicity of nucleoside analogs used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry 42: 14711–14719. Richette P, Brie`re C, Hoenen-Clavert V, Loeuille D, and Bardin T (2007) Rasburicase for tophaceous gout not treatable with allopurinol: An exploratory study. The Journal of Rheumatology 34: 2093–2098. Rolls ET (2000) The representation of umami taste in the taste cortex. Journal of Nutrition 130: 960S–965S. Secades JJ and Frontera G (1995) CDP-choline: Pharmacological and clinical review. Methods and Findings in Experimental and Clinical Pharmacology 17(supplement B): 1–54. Tressler RL, Ramstack MB, White NR, et al. (2003) Determination of total potentially available nucleosides in human milk from Asian women. Nutrition 19: 16–20. Yu VYH (2002) Scientific rationale and benefits of nucleotide supplementation of infant formula. Journal of Paediatrics and Child Health 38: 543–559.