Articles

Vitamin RequirementsRELATIONSHIP 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 vitaminsutilized in energy-related pathways, are related directly to metabolic rate. As a consequence, vitaminrequirements are similar when expressed relative to empirical functions of metabolic body size, e.g. (Wtkg)3⁄4

or body surface area. The vitamin requirements for a range of animals are expressed relative to theircorresponding rates of basal metabolism. Data for the rates of ascorbic acid production and turnover inanimals that produce ascorbic acid are also compared with the ascorbic acid requirements and turnover inhumans and guinea pigs, species that require ascorbic acid as a dietary essential. Factors that are mostimportant in dictating the relative need for a given vitamin from a chemical perspective include chemicalstability, the relative number of catalytic events that are involved in the process, the nature of theinteractions with associated enzymes, and the presence or absence of pathways for partial synthesis orregeneration of the given vitamin. Vitamins that are required daily in millimolar amounts are usually lessstable 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 areoften covalently bound to the proteins or enzymes for which they serve as cofactors. Development of thepreceding concepts allows linkages of relative vitamin requirements to energy utilization and relatedbiochemical and oxidative processes, which can aid in developing a better understanding of the integrativenature of nutritional and biochemical relationships.

Keywords: Vitamins, nutrient requirements, metabolic rate, cofactor function.

In presentations dealing with vitamin-derived cofactorfunction and metabolism, interest is enhanced when theinformation is developed in ways that link vitamin functionto nutritional need. Empirical relationships that haveevolved from studies of animal energetics can be used toconceptualize similarities between vitamin requirementsfor common animal species, including humans. Moreover,explanations as to why requirements for individual water-soluble vitamins vary by several orders of magnitude canalso be developed as a way of underscoring the impor-tance of chemical stability, the specificity of cofactor pro-tein interactions, relative metabolic needs, and otherchemically related parameters.

In homeothermic animals, a case may be made thatwater-soluble vitamin requirements are influenced by thesame factors that dictate energy requirements. This per-spective 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 relativemetabolic rate correlates with metabolic size, when ex-pressed as a function of (Wtkg)3⁄4, even for animals whosebody weights vary by orders of magnitude (Fig. 1). Atambient temperatures and at rest, most fasting homeo-thermic animals produce 294 kJ (�70 kcal) of heat per day

or 12.6 kJ (�3 kcal) per h per kg of body weight, whenexpressed to the 3⁄4 power, e.g. (Wtkg)3⁄4. In a similar fash-ion, vitamin need and their utilization may be shown to berelated to the energy costs for basal metabolism (Table I).

It is also useful in such discussions to develop a “cur-rency” that facilitates going from physical equivalents(joules) to chemical quantities (moles). Baldwin [3] hasprovided some elegant examples that utilize moles of ATPas a currency to describe how energy from given fuels (i.e.the heat of combustion or enthalpy) is partitioned throughvarious metabolic pathways. This can be done whetherATP is generated from fat or carbohydrate oxidation, be-cause the heat of combustion (enthalpy) needed to gen-erate an ATP from fat or carbohydrate is about the same.Note this value is 76–84 kJ or 18–20 kcal per mol of ATPgenerated, about twice the value for the Gibbs free energythat is reported for ATP hydrolysis [3, 6]. In addition, se-lected intermediates evolving from major metabolic path-ways can also be used. We have used acetyl-CoA for thispurpose. A daily energy expenditure of �2000 kcal or �8.4MJ is equivalent to generating �6–7 mol of acetyl-CoA,whether from carbohydrate, or fat and/or protein digestionand oxidation [6]. By using ATP or acetyl-CoA, heuristicestimates of the catalytic cycling needed to utilize an ATPor acetyl-CoA equivalent can be made (e.g. how manycatalytic cycles occur before replacement). As an exam-ple, one may conclude that most water-soluble vitamins

‡ To whom correspondence should be addressed. Tel.: 530-752-2089; Fax: 530-752-8966.

© 2002 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATIONPrinted in U.S.A. Vol. 30, No. 2, pp. 86–89, 2002

This paper is available on line at http://www.bambed.org86

persist through 103 to 105 catalytic cycles before catabo-lism or elimination occurs, because micromolar to millimo-lar amounts of vitamins are needed on a daily basis tofacilitate the oxidation of 6–7 mol of acetyl-CoA or pro-duce 110 � 20 mol of ATP. Further, in typical redoxreactions, wherein oxygen, H2O2, or superoxide anions are

substrates or products, destructive modification of enzy-matic catalytic sites and associated components can beviewed as a relative constant occurrence related to oxida-tive metabolism, i.e. the process is not stochastic.

VITAMIN REQUIREMENTS AND INTERSPECIESMETABOLIC NEEDS

The relationship between vitamin and energy require-ments may also be developed by comparing the need foranimals that require a dietary source to correspondingproduction in those animals capable of synthesis. Inguinea pigs and humans, the absence of gulonolactoneoxidase dictates that ascorbic acid be consumed as adietary essential [7]. Thus, one can ask whether theamounts of ascorbic acid synthesized per day in animalsthat produce L-ascorbic acid correspond to the amountsneeded in guinea pigs and humans. Data offered byGrollman and Lehninger [8] and Ginter [9] are used to makethe comparison. These data describe the potential synthe-sis of ascorbic acid from D-glucuronic acid for which glu-cose and galactose serve as precursors [8].

When expressed relative to the metabolic body size orbasal metabolic need, extrapolation from the available an-imal data yields values that are in keeping with the humanrequirement. For example, Grollman and Lehninger [8]used liver homogenates and gulonic acid as substrates tomeasure ascorbic acid synthesis (see Table II). They reportthat synthesis varies from �0.01 g of L-ascorbic acid perday per kg of body weight for the pig to 0.2 g per kg ofbody weight for the rat. Pauling [10] used similar data toinfer that the ascorbic acid needs in humans were in thegrams per day range. However, ascorbic acid productioncan be no more than the amount of glucose or galactoseshunted through the direct oxidative pathway. In a 70-kganimal, this value ranges from 5–15 g per day (cf. Refs. 8,11, and 12). Moreover, only about 1% of the gulonate fluxis in the direction of ascorbate synthesis [8]. This amountsto 50–150 mg of ascorbate per day, which is in keepingwith the current human requirements [7]. Note also that theproduction of ascorbic acid is relatively constant whenexpressed as a function of (WtKg)3⁄4 (see Table II and Fig. 1).Chatterjee [11] has also provided data on the synthesis ofL-ascorbic acid by crude liver microsomes using gulono-lactone as substrate. Use of their values also leads to thesame relationship (cf. Refs. 12 and 13).

FIG. 1. Logarithm of the basal metabolic rate, relative rate ofascorbic acid production, and body weight. Data for the basalmetabolic rate are taken from Kleiber [1]. The relative ascorbic acidproduction per animal per day was estimated using values providedby Grollman and Lehninger [8] for the optimal ascorbic acid syn-thesis per gram of liver (mammals) or kidneys (birds), originallyexpressed as �mol/g of kidney or liver per h. The relative dailyproduction of ascorbic acid was obtained by multiplying thesevalues by typical liver and kidney weights for the animals indicatedin Table II. The hourly values were multiplied by 24. The method ofleast squares leads to a coefficient of nearly 3⁄4 to the power of bodyweight (Kg) for both interspecies comparisons of basal metabolicrates or ascorbic acid production.

TABLE IRequirements for selected water-soluble vitamins (expressed as mg

per 1000 kcal or 4200 kJ)

Vitamin CataAnimal

Ratb Mouseb Chickc Humand

Thiamin 2–3 2 2 1 1–2Riboflavin 1–2 1 1 0.5 1Niacina 20–30 8 8 6–8 5Pyridoxinea 2–4 2 2 1–2 1

a Cats do not effectively convert tryptophan to niacin; thus, there isabsolute need for niacin. In this regard, �10 mg of niacin is producedper 4200 MJ of typical diets containing high quality protein, whenutilized by the rat, mouse, chick, or human. The higher pyridoxineneed in the cat is because of higher protein requirements of carni-vores and higher concentrations of enzymes dedicated to nitrogenmetabolism [20].

b See Ref. 21.c See Ref. 22.d See Ref. 23.

TABLE IIAscorbic 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 isexpressed as the total synthesized per day (�mol/liver/day) or the total synthesized per day divided by weight expressed to the 3⁄4 power.

Animal Bodyweight

Liverweight

Ascorbic acidproduction

Ascorbic acidproduction

Ascorbic acidproduction

kg kg �mol/g liver/h �mol/g liver/day (WtKg)3/4

Mouse 0.03 2 0.6 29 400Rat 0.35 18 1.12 484 1010Rabbit 2 80 0.78 1498 891Dog 10 420 0.46 4637 825Pig 125 3750 0.15 13500 401Cow 500 6000 0.33 47520 449Pigeon 0.3 10 1.22 298 723Chicken 1 30 0.64 461 461

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

turnover �kps(WtKg)1

ktr(WtKg)3/4 � ktu(WtKg)1/4� (Eq. 1)

The function, ktu (WtKg)1⁄4, can then be used to estimate thehypothetical turnover for ascorbic acid in the guinea pigand human. Accordingly, the values for ascorbic acid turn-

over in humans and guinea pigs are very similar to thosedetermined in vivo [9].

CHEMICAL FACTORS INFLUENCING VITAMIN REQUIREMENTS

Regarding the magnitude of individual vitamin require-ments within a given animal species, the categories andexamples given in Table IV can be developed as rationalefor why the requirements of different vitamins and theircorresponding cofactors vary by several orders of magni-tude. In this regard, the extent and type of utilization,regardless of whether sequesterization occurs, or regard-less of whether the vitamin-derived cofactor is covalentlylinked to an enzyme or protein, are probably the mostimportant factors. As an example, about half of the niacinassociated with NAD is utilized, because NAD is a sub-strate in mono- and polyribosylation reactions [14], in ad-dition to its essential role as a dehydrogenase cofactor.Cellular compartmentalization and sequesterization alsotake on importance when one considers that the respec-tive half-lives of many vitamins are best estimated in min-utes to hours in simple solutions at physiological temper-atures [15–18] but are stabilized when bound to enzymesor specific binding proteins. The stability constants ofmost vitamins are usually first order and altered bychanges in solvent and pH levels. Association with tar-geted enzymes and binding proteins increases stability.Moreover, the covalent association of biotin, riboflavin,and pyridoxal-5�-phosphate with selected enzymes influ-ences the amounts that are needed, because covalentbinding to specific proteins lessens the probability fornonspecific interactions that occur when cofactors aredissociated and, as a consequence, are subject to non-specific chemical and solvent interactions.

As a final point, some of these same considerations canalso be applied to fat-soluble vitamins. However, insteadof catalytic cycling in an enzymatic context, the amounts

TABLE IIIEstimates 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 bydividing the guinea pig adult body weight (taken as 1 kg) or humanbody weight (taken as 70 kg) expressed to the 1⁄4 power by the bodyweights for the mouse, hamster, rat, or rabbit expressed to the 1⁄4power and then multiplying by the appropriate values of ascorbatehalf-life [9]. The body weights that were used to calculate the valuesfor (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 Guinea pig Human

Days Days Days

Mouse 0.414 1–2 �3.5 9.8Hamster 0.569 2.5–3.0 4.7 13.7Rat 0.669–0.775 2.3–2.6 3.6–3.7 10.4–10.8Rabbit 1.41 4–5 2.8 –3.5 8.0–10Guinea pig 1.0 3.5 3.5 10Human 2.89 10–11 �3.7 10–11

TABLE IVFactors 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 theInstitute 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 AcademyPress, Washington, D.C., 2000.

Vitamin RDA or AIa Factors

Ascorbic acid M 90 mg/dayF 75 mg/day

Y Chemically modified or destroyed after 5–10 catalytic cycles or events

Niacin M 16 mg/day Y NAD�, NADH, NADP�, or NADPH serves as a dissociable cofactorF 14 mg/day Y Serves as a substrate and co-substrate

Y Used by numerous enzymesPantothenic acid M 5 mg/day Y CoASH is a dissociable cofactor

F 5 mg/day Y Serves as a substrate carrier and activatorY Used in numerous reactions

Riboflavin M 1.3 mg/dayF 1.1 mg/day

Y As cofactors are less dissociable than ascorbic acid, NAD, NADP, or CoASHwhen associated with corresponding enzymes

Pyridoxine M 1.3–1.7 mg/dayF 1.3–1.5 mg/day

Y Often are covalently linked to corresponding enzymes

Thiamin M 1.2 mg/dayF 1.1 mg/day

Y As a cofactor is dissociable but involved in fewer metabolic steps thanascorbic acid, niacin (NAD), pantothenic acid, riboflavin, and pyridoxine

Folic acid M 400 �g/day Y Used in a limited number of specific reactions related to single carbon transfersF 400 �g/day Y Tightly bound to associated enzymes and transport proteins

Biotin M 30 �g/day Y Covalently boundF 30 �g/day Y Used in a limited number of specific reactions (e.g., carboxylations and

transcarboxylation steps)Vitamin B-12 M 2–4 �g/day

F 2–4 �g/dayY Tightly bound, used by limited number of enzymatic steps (e.g., methyl transfer

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

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

needed to saturate specific receptors or participate inoxidative defense take on more importance. As an exam-ple, for vitamin E, for which utilization in chemical pro-cesses is the primary function (e.g. peroxidative protec-tion), knowledge that 1–5% of the daily oxygen need (300–400 liters or 14–18 mol) is converted to a reactive oxidativespecies aids in conceptualizing the daily need for vitaminE. The daily need for vitamin E in humans is of the order of0.1–0.2 mmol per day or less, i.e. close to the RDA forvitamin E, �40 mg per day in humans. This amount can beviewed as reasonable when one considers all of the otherfactors important to reactive oxidative species defense [19].

CONCLUDING COMMENTS

Although it is intuitive that vitamin utilization is con-nected to metabolic processes, that vitamin requirementsare similar in homeothermic animals if described as afunction of oxygen utilization and catalytic cycling is sel-dom emphasized. In this context, requirements can bedefined or described in terms of chemical stability, i.e. thenumber of catalytic cycles before chemical modification ordestruction dictates replacement. Importantly, this viewallows the development of concepts that link relative vita-min utilization to energy utilization and related oxidativeprocesses.

REFERENCES

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[10] L. Pauling (1974) Are recommended daily allowances for vitamin Cadequate? Proc. Natl. Acad. Sci. U. S. A. 71, 4442–4446.

[11] I. Chatterjee (1973) Evolution and the biosynthesis of ascorbic acid,Science 182, 1271–1274.

[12] R. B. Rucker, M. Dubick, J. Robinson (1980) Hypothetical calculationsof ascorbic acid synthesis based on estimates in vitro, Am. J. Clin.Nutr. 33, 961–964.

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[17] J. L. Smith, J. E. Canham, W. D. Kirkland, P. A. Wells (1998) Effect ofIntralipid, amino acids, container, temperature, and duration of stor-age 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 ultraviolet-visible spectrophotometric analysis and factorial design, Anal. Bio-chem. 260, 50–55.

[19] K. Beckman, B. N. Ames (1998) The free radical theory of agingmatures, Physiol. Rev. 78, 547–581.

[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 Lab-oratory Animals, 4th Ed., National Research Council, NAS, p. 192.

[22] Committee on Animal Nutrition (1994) Nutrient Requirements of Poul-try, 9th Ed., National Research Council, NAS, p. 176.

[23] Subcommittee on the Tenth Edition of the Recommended DietaryAllowances (1989) Recommended Dietary Allowances, 10th Ed.,Food and Nutrition Board, Commission on Life Sciences, NationalResearch Council, NAS, p. 302.

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