2002 by The International Union of Biochemistry and Molecular Biology Printed in U.S.A.

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MOLECULAR BIOLOGY EDUCATION Vol. 30, No. 2, pp. 86 89, 2002

Articles Vitamin Requirements

RELATIONSHIP TO BASAL METABOLIC NEED AND FUNCTIONS
Received for publication, December 14, 2001 Robert B. Rucker and Francene M. Steinberg From the Department of Nutrition, University of California, Davis, California 95616-8669

The dietary requirements for most water-soluble vitamins in homeothermic animals, particularly vitamins utilized in energy-related pathways, are related directly to metabolic rate. As a consequence, vitamin 3 requirements are similar when expressed relative to empirical functions of metabolic body size, e.g. (Wtkg) 4 or body surface area. The vitamin requirements for a range of animals are expressed relative to their corresponding rates of basal metabolism. Data for the rates of ascorbic acid production and turnover in animals that produce ascorbic acid are also compared with the ascorbic acid requirements and turnover in humans and guinea pigs, species that require ascorbic acid as a dietary essential. Factors that are most important in dictating the relative need for a given vitamin from a chemical perspective include chemical stability, the relative number of catalytic events that are involved in the process, the nature of the interactions with associated enzymes, and the presence or absence of pathways for partial synthesis or regeneration of the given vitamin. Vitamins that are required daily in millimolar amounts are usually less stable chemically, are involved in numerous reactions, and often exist in tissues as dissociable cofactors. In contrast, vitamins that are required daily in micromolar amounts are involved in fewer reactions and are often covalently bound to the proteins or enzymes for which they serve as cofactors. Development of the preceding concepts allows linkages of relative vitamin requirements to energy utilization and related biochemical and oxidative processes, which can aid in developing a better understanding of the integrative nature of nutritional and biochemical relationships. Keywords: Vitamins, nutrient requirements, metabolic rate, cofactor function. or 12.6 kJ (3 kcal) per h per kg of body weight, when expressed to the 34 power, e.g. (Wtkg)34. In a similar fashion, vitamin need and their utilization may be shown to be related to the energy costs for basal metabolism (Table I). It is also useful in such discussions to develop a currency that facilitates going from physical equivalents (joules) to chemical quantities (moles). Baldwin [3] has provided some elegant examples that utilize moles of ATP as a currency to describe how energy from given fuels (i.e. the heat of combustion or enthalpy) is partitioned through various metabolic pathways. This can be done whether ATP is generated from fat or carbohydrate oxidation, because the heat of combustion (enthalpy) needed to generate an ATP from fat or carbohydrate is about the same. Note this value is 76 84 kJ or 18 20 kcal per mol of ATP generated, about twice the value for the Gibbs free energy that is reported for ATP hydrolysis [3, 6]. In addition, selected intermediates evolving from major metabolic pathways can also be used. We have used acetyl-CoA for this purpose. A daily energy expenditure of 2000 kcal or 8.4 MJ is equivalent to generating 6 7 mol of acetyl-CoA, whether from carbohydrate, or fat and/or protein digestion and oxidation [6]. By using ATP or acetyl-CoA, heuristic estimates of the catalytic cycling needed to utilize an ATP or acetyl-CoA equivalent can be made (e.g. how many catalytic cycles occur before replacement). As an example, one may conclude that most water-soluble vitamins

In presentations dealing with vitamin-derived cofactor function and metabolism, interest is enhanced when the information is developed in ways that link vitamin function to nutritional need. Empirical relationships that have evolved from studies of animal energetics can be used to conceptualize similarities between vitamin requirements for common animal species, including humans. Moreover, explanations as to why requirements for individual watersoluble vitamins vary by several orders of magnitude can also be developed as a way of underscoring the importance of chemical stability, the specificity of cofactor protein interactions, relative metabolic needs, and other chemically related parameters. In homeothermic animals, a case may be made that water-soluble vitamin requirements are influenced by the same factors that dictate energy requirements. This perspective comes from the work of Kleiber [1] and Brody [2] and more recently work by Baldwin [3] and Heusner [4, 5]. That is, in homeothermic animals, the estimation of relative metabolic rate correlates with metabolic size, when expressed as a function of (Wtkg)34, even for animals whose body weights vary by orders of magnitude (Fig. 1). At ambient temperatures and at rest, most fasting homeothermic animals produce 294 kJ (70 kcal) of heat per day
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persist through 10 to 10 catalytic cycles before catabolism or elimination occurs, because micromolar to millimolar amounts of vitamins are needed on a daily basis to facilitate the oxidation of 6 7 mol of acetyl-CoA or produce 110 20 mol of ATP. Further, in typical redox reactions, wherein oxygen, H2O2, or superoxide anions are
3 5

substrates or products, destructive modification of enzymatic catalytic sites and associated components can be viewed as a relative constant occurrence related to oxidative metabolism, i.e. the process is not stochastic.
VITAMIN REQUIREMENTS AND INTERSPECIES METABOLIC NEEDS

FIG. 1. Logarithm of the basal metabolic rate, relative rate of ascorbic acid production, and body weight. Data for the basal metabolic rate are taken from Kleiber [1]. The relative ascorbic acid production per animal per day was estimated using values provided by Grollman and Lehninger [8] for the optimal ascorbic acid synthesis per gram of liver (mammals) or kidneys (birds), originally expressed as mol/g of kidney or liver per h. The relative daily production of ascorbic acid was obtained by multiplying these values by typical liver and kidney weights for the animals indicated in Table II. The hourly values were multiplied by 24. The method of least squares leads to a coefficient of nearly 34 to the power of body weight (Kg) for both interspecies comparisons of basal metabolic rates or ascorbic acid production.
TABLE I Requirements for selected water-soluble vitamins (expressed as mg per 1000 kcal or 4200 kJ) Vitamin Thiamin Riboflavin Niacina Pyridoxinea Cata 23 12 2030 24 Animal Ratb 2 1 8 2 Mouseb 2 1 8 2 Chickc 1 0.5 68 12 Humand 12 1 5 1

a Cats do not effectively convert tryptophan to niacin; thus, there is absolute need for niacin. In this regard, 10 mg of niacin is produced per 4200 MJ of typical diets containing high quality protein, when utilized by the rat, mouse, chick, or human. The higher pyridoxine need in the cat is because of higher protein requirements of carnivores and higher concentrations of enzymes dedicated to nitrogen metabolism [20]. b See Ref. 21. c See Ref. 22. d See Ref. 23.

The relationship between vitamin and energy requirements may also be developed by comparing the need for animals that require a dietary source to corresponding production in those animals capable of synthesis. In guinea pigs and humans, the absence of gulonolactone oxidase dictates that ascorbic acid be consumed as a dietary essential [7]. Thus, one can ask whether the amounts of ascorbic acid synthesized per day in animals that produce L-ascorbic acid correspond to the amounts needed in guinea pigs and humans. Data offered by Grollman and Lehninger [8] and Ginter [9] are used to make the comparison. These data describe the potential synthesis of ascorbic acid from D-glucuronic acid for which glucose and galactose serve as precursors [8]. When expressed relative to the metabolic body size or basal metabolic need, extrapolation from the available animal data yields values that are in keeping with the human requirement. For example, Grollman and Lehninger [8] used liver homogenates and gulonic acid as substrates to measure ascorbic acid synthesis (see Table II). They report that synthesis varies from 0.01 g of L-ascorbic acid per day per kg of body weight for the pig to 0.2 g per kg of body weight for the rat. Pauling [10] used similar data to infer that the ascorbic acid needs in humans were in the grams per day range. However, ascorbic acid production can be no more than the amount of glucose or galactose shunted through the direct oxidative pathway. In a 70-kg animal, this value ranges from 515 g per day (cf. Refs. 8, 11, and 12). Moreover, only about 1% of the gulonate flux is in the direction of ascorbate synthesis [8]. This amounts to 50 150 mg of ascorbate per day, which is in keeping with the current human requirements [7]. Note also that the production of ascorbic acid is relatively constant when expressed as a function of (WtKg)34 (see Table II and Fig. 1). Chatterjee [11] has also provided data on the synthesis of L-ascorbic acid by crude liver microsomes using gulonolactone as substrate. Use of their values also leads to the same relationship (cf. Refs. 12 and 13).

TABLE II Ascorbic acid synthesis in whole liver homogenates using L-gulonic acid as substrate Data were taken from Grollman and Lehninger [8]. Weights were chosen that are typical of adult animals. Ascorbic acid production is expressed as the total synthesized per day (mol/liver/day) or the total synthesized per day divided by weight expressed to the 34 power. Animal Body weight kg 0.03 0.35 2 10 125 500 0.3 1 Liver weight kg 2 18 80 420 3750 6000 10 30 Ascorbic acid production Ascorbic acid production Ascorbic acid production (WtKg)3/4 400 1010 891 825 401 449 723 461

Mouse Rat Rabbit Dog Pig Cow Pigeon Chicken

mol/g liver/h 0.6 1.12 0.78 0.46 0.15 0.33 1.22 0.64

mol/g liver/day 29 484 1498 4637 13500 47520 298 461

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A similar perspective is developed further using data provided by Ginter [9] on the rates of ascorbate turnover in the rat, mouse, guinea pig, rabbit, hamster, and human. Values for ascorbic acid half-lives are given in Table III. If the daily nutrient transfer rate (tr) is related to (WtKg)34, turnover (tu) will be a function of (WtKg)14. This is derived by assuming that the body pool size (ps) of the substance (total content) is directionally proportional to kps (WtKg)1. Dividing this function by the daily transfer rate (ktr(WtKg)34) approximates the relative turnover rate, represented in Equation 1. turnover kps(WtKg)1 ktu(WtKg)1/4 ktr(WtKg)3/4 (Eq. 1)

BAMBED, Vol. 30, No. 2, pp. 86 89, 2002

over in humans and guinea pigs are very similar to those determined in vivo [9].
CHEMICAL FACTORS INFLUENCING VITAMIN REQUIREMENTS

The function, ktu (WtKg)14, can then be used to estimate the hypothetical turnover for ascorbic acid in the guinea pig and human. Accordingly, the values for ascorbic acid turnTABLE III Estimates for ascorbate turnover in guinea pig and man derived from known values for the mouse, hamster, rat and rabbit Values under the columns guinea pig and man were computed by dividing the guinea pig adult body weight (taken as 1 kg) or human body weight (taken as 70 kg) expressed to the 14 power by the body weights for the mouse, hamster, rat, or rabbit expressed to the 14 power and then multiplying by the appropriate values of ascorbate half-life [9]. The body weights that were used to calculate the values for (WtKg)1/4 were 30, 125, and 200 300 g and 2 kg for the mouse, hamster, rat, or rabbit, respectively. Animal (WtKg)1/4 Half-life Days 12 2.53.0 2.32.6 45 3.5 1011 Guinea pig Days 3.5 4.7 3.63.7 2.8 3.5 3.5 3.7 Human Days 9.8 13.7 10.410.8 8.010 10 1011

Mouse Hamster Rat Rabbit Guinea pig Human

0.414 0.569 0.6690.775 1.41 1.0 2.89

Regarding the magnitude of individual vitamin requirements within a given animal species, the categories and examples given in Table IV can be developed as rationale for why the requirements of different vitamins and their corresponding cofactors vary by several orders of magnitude. In this regard, the extent and type of utilization, regardless of whether sequesterization occurs, or regardless of whether the vitamin-derived cofactor is covalently linked to an enzyme or protein, are probably the most important factors. As an example, about half of the niacin associated with NAD is utilized, because NAD is a substrate in mono- and polyribosylation reactions [14], in addition to its essential role as a dehydrogenase cofactor. Cellular compartmentalization and sequesterization also take on importance when one considers that the respective half-lives of many vitamins are best estimated in minutes to hours in simple solutions at physiological temperatures [1518] but are stabilized when bound to enzymes or specific binding proteins. The stability constants of most vitamins are usually first order and altered by changes in solvent and pH levels. Association with targeted enzymes and binding proteins increases stability. Moreover, the covalent association of biotin, riboflavin, and pyridoxal-5-phosphate with selected enzymes influences the amounts that are needed, because covalent binding to specific proteins lessens the probability for nonspecific interactions that occur when cofactors are dissociated and, as a consequence, are subject to nonspecific chemical and solvent interactions. As a final point, some of these same considerations can also be applied to fat-soluble vitamins. However, instead of catalytic cycling in an enzymatic context, the amounts

TABLE IV Factors important in defining vitamin requirements Values taken from the Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids, Food and Nutrition Board of the Institute of Medicine, National Academy Press, Washington, D.C., 2000, and the Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic acid, Biotin, and Choline, Food and Nutrition Board of the Institute of Medicine, National Academy Press, Washington, D.C., 2000. Vitamin Ascorbic acid Niacin Pantothenic acid Riboflavin Pyridoxine Thiamin Folic acid Biotin Vitamin B-12
a

RDA or AIa M 90 mg/day F 75 mg/day M 16 mg/day F 14 mg/day M 5 mg/day F 5 mg/day M 1.3 mg/day F 1.1 mg/day M 1.31.7 mg/day F 1.31.5 mg/day M 1.2 mg/day F 1.1 mg/day M 400 g/day F 400 g/day M 30 g/day F 30 g/day M 24 g/day F 24 g/day Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Factors Chemically modified or destroyed after 510 catalytic cycles or events NAD, NADH, NADP, or NADPH serves as a dissociable cofactor Serves as a substrate and co-substrate Used by numerous enzymes CoASH is a dissociable cofactor Serves as a substrate carrier and activator Used in numerous reactions As cofactors are less dissociable than ascorbic acid, NAD, NADP, or CoASH when associated with corresponding enzymes Often are covalently linked to corresponding enzymes As a cofactor is dissociable but involved in fewer metabolic steps than ascorbic acid, niacin (NAD), pantothenic acid, riboflavin, and pyridoxine Used in a limited number of specific reactions related to single carbon transfers Tightly bound to associated enzymes and transport proteins Covalently bound Used in a limited number of specific reactions (e.g., carboxylations and transcarboxylation steps) Tightly bound, used by limited number of enzymatic steps (e.g., methyl transfer reactions)

RDA, recommended dietary allowance; AI, adequate intake; M, male; F, female.

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needed to saturate specific receptors or participate in oxidative defense take on more importance. As an example, for vitamin E, for which utilization in chemical processes is the primary function (e.g. peroxidative protection), knowledge that 15% of the daily oxygen need (300 400 liters or 14 18 mol) is converted to a reactive oxidative species aids in conceptualizing the daily need for vitamin E. The daily need for vitamin E in humans is of the order of 0.1 0.2 mmol per day or less, i.e. close to the RDA for vitamin E, 40 mg per day in humans. This amount can be viewed as reasonable when one considers all of the other factors important to reactive oxidative species defense [19].
CONCLUDING COMMENTS
[7] A. C. Carr, B. Frei (1999) Toward a new recommended dictary allowance for vitamin C based on antioxidant and health effects in humans, Am. J. Clin Nutr. 69, 1086 1107. [8] A. P. Grollman, A. L. Lehninger (1957) Enzymatic synthesis of Lascorbic acid in different species, Arch. Biochem. Biophys. 69, 458 463. [9] E. Ginter (1981) Endogenous ascorbic acid synthesis and recommended dietary allowances for vitamin C, Am. J. Clin. Nutr. 34, 1448 1451. [10] L. Pauling (1974) Are recommended daily allowances for vitamin C adequate? Proc. Natl. Acad. Sci. U. S. A. 71, 4442 4446. [11] I. Chatterjee (1973) Evolution and the biosynthesis of ascorbic acid, Science 182, 12711274. [12] R. B. Rucker, M. Dubick, J. Robinson (1980) Hypothetical calculations of ascorbic acid synthesis based on estimates in vitro, Am. J. Clin. Nutr. 33, 961964. [13] R. B. Rucker, M. Dubick (1981) Reply to letter by Ginter, Am. J. Clin. Nutr. 34, 1450 1451. [14] J. B. Kirkland, J. M. Rawling, in R. B. Rucker, J. W. Suttie, D. B. McCormick, L. J. Machlin, Eds. (2001) Niacin. Handbook of Vitamins, Marcel Dekker Inc., New York, pp. 213255. [15] G. B. Dahl, R. I. Jeppsson, H. J. Tengborn (1986) Vitamin stability in a TPN mixture stored in an EVA plastic bag, J. Clin. Hosp. Pharm. 11, 271279. [16] S. Kurano, N. Jurano, C. Leist, A. Fiechter (1990) Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture, Cytotechnology 4, 243250. [17] J. L. Smith, J. E. Canham, W. D. Kirkland, P. A. Wells (1998) Effect of Intralipid, amino acids, container, temperature, and duration of storage on vitamin stability in total parenteral nutrition admixtures, J. Parenter. Enteral Nutr. 12, 478 483. [18] L. Rover, J. C. B. Fernandes, G. Neto, L. T. Kubota, E. Katekawa, S. H. P. Serrano (1998) Study of NADH stability using ultravioletvisible spectrophotometric analysis and factorial design, Anal. Biochem. 260, 50 55. [19] K. Beckman, B. N. Ames (1998) The free radical theory of aging matures, Physiol. Rev. 78, 547581. [20] Committee on Animal Nutrition (1986) Nutrient Requirements of Cats, Revised Ed., National Research Council, NAS, p. 88. [21] Committee on Animal Nutrition (1995) Nutrient Requirements of Laboratory Animals, 4th Ed., National Research Council, NAS, p. 192. [22] Committee on Animal Nutrition (1994) Nutrient Requirements of Poultry, 9th Ed., National Research Council, NAS, p. 176. [23] Subcommittee on the Tenth Edition of the Recommended Dietary Allowances (1989) Recommended Dietary Allowances, 10th Ed., Food and Nutrition Board, Commission on Life Sciences, National Research Council, NAS, p. 302.

Although it is intuitive that vitamin utilization is connected to metabolic processes, that vitamin requirements are similar in homeothermic animals if described as a function of oxygen utilization and catalytic cycling is seldom emphasized. In this context, requirements can be defined or described in terms of chemical stability, i.e. the number of catalytic cycles before chemical modification or destruction dictates replacement. Importantly, this view allows the development of concepts that link relative vitamin utilization to energy utilization and related oxidative processes.
REFERENCES
[1] M. Kleiber (1975) The Fire of Life. An Introduction to Animal Energetics, Revised Edition, John Wiley & Sons, Inc., New York. [2] S. Brody (1945) Bioenergetics and Growth, Reinhold, New York. [3] R. L. Baldwin (1995) Modeling Ruminant Digestion and Metabolism, Chapman & Hall, New York. [4] A. A. Heusner (1985) Body size and energy metabolism, Annu. Rev. Nutr. 5, 267293. [5] A. A. Heusner (1987) What does the power function reveal about structure and function in animals of different size? Annu. Rev Physiol. 49, 121146. [6] E. A. Newsholme, C. Start (1973) Regulation in Metabolism, John Wiley & Sons, Inc., New York.