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BIOAVAILABILITY R J Wood, Tufts University, Boston, MA, USA ª 2005 Elsevier Ltd. All rights reserved.
Introduction Bioavailability is an important consideration in a number of areas of nutrition, including the derivation of Dietary Reference Intakes, estimation of potential impact of changes in dietary pattern, the selection of specific food fortificants, and the formulation of whole meal products, such as infant formulas or meal replacements. In this article, the role of food processing in nutrient bioavailability, specific determinants of nutrient bioavailability, methods for measuring bioavailability, and bioavailability of food fortificants are discussed.
generally as growth stimulation under nutrient limiting conditions, etc. An exception to the ‘absorption equals bioavailability’ rule is selenium. One form of dietary selenium is selenomethionine. This selenium-containing compound is handled by the body exactly like the amino acid methionine and gets readily incorporated into methionine-containing proteins. However, the selenium found in selenium-dependent enzymes is in the form of a special amino acid called selenocysteine, which must be synthesized in the body during the process of incorporation of selenocysteine into these selenoproteins. Selenomethionine catabolism will result in the release of this selenium into an active endogenous selenium pool, which serves as the source of selenium for selenocysteine synthesis. Thus, selenium in selenomethionine is not immediately available to support selenium-dependent functions in the body.
Effects of Food Processing Definition Nutrient bioavailability is defined as the fraction of a nutrient in a food that is absorbed and utilized. In practice, however, measurements of bioavailability have focused on either direct measurement of absorption or determination of the change in some functional or biochemical endpoint reflecting absorption and utilization of the nutrient. In general, the bioavailability of all nutrients can be estimated by measuring absorption alone because, once absorbed, nutrients are freely available for biological utilization, irrespective of their original dietary source. For example, consider the case of iron bioavailability. Iron absorption can be measured directly from a food by a variety of methods (described in more detail later). In addition, absorbed iron enters the plasma iron pool carried on the protein transferrin. In turn, this absorbed iron will be used in large part (about 80%) immediately in the synthesis of hemoglobin by erythrocyte precursor cells in the bone marrow. The fraction of iron that is utilized for hemoglobin synthesis is not dependent in any way on the food source of that iron. Thus, food iron bioavailability can be conveniently measured and compared in relative terms among various sources by determining the change in blood hemoglobin after consumption of various forms of iron in iron-deficient subjects. Thus, nutrient bioavailability can be estimated by measuring these appropriate endpoints, such as hemoglobin incorporation for iron, hepatic tissue or mineral content of bone for various bone-seeking minerals, or more
Food processing can have positive or negative effects on nutrient bioavailability. For example, milling of grains removes all or part of the external covering of the grain (bran) that contains high amounts of phytic acid, an important inhibitor of bioavailability of divalent minerals, such as iron, zinc, and copper. One disadvantage of this form of food processing is that much of the mineral content of grains is in the bran fraction and is lost in the process of milling. To compensate for this loss of mineral (and some vitamins as well) grain products can be ‘enriched’ by fortifying the flour made from the milled grain with specific micronutrients. Simple food processing techniques, such as sprouting or fermentation, are also effective in lowering the phytate content and increasing mineral bioavailability of grains. Other techniques used in food manufacturing, such as browning, which produces the Maillard reaction, and extrusion, can have negative effects on bioavailability of certain nutrients. Processing practices that affect the polyphenol content of cereals and legumes can influence nutrient bioavailability. Polyphenols interact with plant proteins and form tannin–protein complexes that can inactivate enzymes or lead to protein insolubility adversely influencing amino acid and protein bioavailability. The antinutritional properties of polyphenols can be decreased by removing them from grain with chemical treatments (such as alkaline treatment and ammonia) or removing from grain the polyphenol-rich pericarp and testa by pearling.
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Importance of Nutrient Bioavailability to Human Nutrition Assessment of bioavailability of nutrients is an essential component of deriving dietary reference intakes (DRIs), guidelines for optimal intake of individual nutrients established for North American populations. Many DRIs are based on evaluation of available physiological data to determine the obligatory daily needs for a nutrient to replace losses, or the amount needed for optimal growth of tissues, and an estimate of overall dietary bioavailability of the nutrient in question. In many populations, the content of a nutrient in the diet (e.g., iron or zinc) may be sufficient to meet recommended intake, but bioavailability is suboptimal due to the presence of high levels of inhibitory substances (such as phytate) in the diet leading to a high risk of developing this nutrient deficiency.
Determinants of Nutrient Bioavailability Speciation
Speciation refers to the form of the nutrient found in food, which may in turn influence the absorption of the nutrient in the gastrointestinal tract. For example, this term could be applied to cis- or trans-forms of unsaturated fatty acids found in partially hydrogenated oils; individual coenzyme forms of vitamins, such as free thiamin, thiamin pyrophosphate (TPP), thiamin monophosphate (TMP) and thiamin triphosphate (TTP), or the various coenzyme forms of riboflavin – flavin mononucleotide (FMN) and adenine dinucleotide (FAD), or for vitamin B6 – pyridoxine, pyridoxamine, and pyridoxal; or minerals, such as ferrous or ferric forms of nonheme iron or heme iron; or the selenomethionine, selenite, or selenate form of selenium. Digestion and Metabolism
During the transit of digested food material through the gastrointestinal tract many changes occur in the intestinal lumen that could influence nutrient bioavailability. Digestion of food constituents is an important aspect of nutrient bioavailability. The secretion of acid into the stomach following food ingestion activates certain digestive enzymes, as well as creating an acidic environment that influences mineral solubility and extraction from food. In this regard, the choice of a mineral to use for supplementation purposes might be influenced by certain physiological conditions, such as achlorhydria. Aging is associated with a decrease in gastric acid secretion in many elderly persons leading to either hypochlorhydria or achlorhydria,
characterized by either low or complete absence of acid secretion, respectively, leading to a neutral or slightly alkaline gastric pH. This raised pH condition in the stomach can have detrimental effects on micronutrient bioavailability. For example, elderly persons with achlorhydria are at risk of vitamin B12 deficiency because of an inability to remove properly protein-bound vitamin B12 from food when it enters the stomach. In these subjects, the capacity to absorb vitamin B12 is normal, however, because they can readily absorb crystalline (nonproteinbound) vitamin B12 from a supplemental vitamin B12 dose. Lowering the gastric pH towards normal by administering hydrochloric acid restores vitamin B12 absorption from food. In achlorhydric elderly persons the absorption of calcium carbonate from a dietary supplement after an overnight fast is very poor, presumably because calcium carbonate is a relatively insoluble calcium salt and needs gastric acid to be solubilized. Again, these subjects have normal calcium absorption if the calcium is delivered in a more soluble form, such as calcium citrate. In addition to elderly persons who develop achlorhydria, a large number of people regularly use gastric acid-lowering medications, such as the gastric proton pump inhibitor omeprazole, for antiulcer therapy. These medications can reduce zinc absorption and presumably may affect the absorption of other divalent minerals. Acidification of the gastric contents and solubilization of minerals in the gastric juice is important because many mineral nutrients are preferentially absorbed in the duodenum (upper small intestine) and need to be available in free or low-molecular-weight complexes when they leave the stomach to facilitate contact with intestinal nutrient transporters on the apical (luminal) surface of the absorptive enterocytes. The lower gastrointestinal tract can also be a potentially important site affecting the bioavailability of bioactive substances found in food. In this regard, intestinal bacteria found in the large intestine can influence bioavailability. Bacteria are instrumental in the metabolic conversion of certain phytonutrients into forms that are more readily absorbed. In addition, bacteria likely play an important role in the enhancing effects of prebiotics on mineral bioavailability. Consumption of nonabsorbable carbohydrates, such as inulin, can have a positive effect on mineral absorption. A possible mechanism of this effect is that the nonabsorbable carbohydrates pass the small intestine and enter into the large intestine where they serve as a food substrate for bacteria. The metabolism of these prebiotic substances by intestinal bacteria lowers the pH of the lumen of the large intestine and may thereby
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serve to solubilize some insoluble mineral complexes that have passed through the small intestine. The freed mineral would then be available for absorption from the large intestine, thereby increasing overall mineral bioavailability. Other scenarios are possible as well, such as the release of short-chain fatty acids by the bacteria, that may facilitate mineral absorption. Chelation
Chelation is the process whereby an organic moiety acts as a ligand to bind a metal ion through two or more coordination bonds. Some low-molecularweight compounds that may be released during the digestion of food can act as metal chelators and increase metal solubility in the intestinal lumen. In some circumstances, chelated forms of metals are naturally present in food, such as heme iron (part of hemoglobin or myoglobin protein) found in meat. Heme is a stable protoporphyrin ring-containing compound that protects a central iron atom from interacting with other potentially deleterious compounds, such as phytic acid, that would reduce its availability and inhibit iron absorption. Nonheme iron bioavailability is affected by various enhancing and inhibitory substances found in food. In contrast, heme iron bioavailability is not. The heme moiety is absorbed intact by the enterocyte. Inside the enterocyte a cytosolic enzyme heme oxygenase breaks apart the protoporphyrin ring and releases the caged iron atom, which can then be transferred out into the blood. Bioavailability Enhancers and Inhibitors
Various food substances have been identified that act as enhancers or inhibitors of divalent mineral absorption. In general, these food factors influence nutrient bioavailability by either forming relatively insoluble complexes with nutrients or preventing them from interacting with their respective nutrient transporter, or by protecting the nutrient from such untoward interaction maintaining it in a state that can be absorbed or as an absorbable chelated complex (e.g., heme iron). A list of factors known to influence mineral bioavailability is given in Table 1.
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General Physiology of Nutrient Absorption
Nutrients enter the blood by passing through the intestinal mucosa. Intestinal nutrient transport can occur via two distinct pathways. One is termed the paracellular pathway and represents the movement of a nutrient between the absorptive enterocytes on the intestinal villi. This transport pathway is an energy-independent diffusional process and depends on the electrochemical gradient across the mucosa and its permeability characteristics to the nutrient in question. The characteristics of the diffusional pathway are not regulated in response to nutrient deficiency or excess. A second transport pathway represents the transcellular movement of a nutrient across the intestine. The transcellular transport rate of the nutrient is composed of both diffusional and carrier-mediated transport pathways. Often in response to changes in nutrient status the number of nutrient carriers will be changed to facilitate appropriate increases or decreases in intestinal absorption to help maintain nutrient homeostasis. Within a class of nutrients there can be substantial differences in absorption rates. For example, in the case of minerals, monovalent minerals (such as sodium, potassium, and iodine) are absorbed at very high efficiency approximating complete absorption, while multivalent elements (such as chromium, heavy metals, and iron) are relatively poorly absorbed (1–20%). In addition, because some nutrient transporters, e.g., the divalent metal transporter DMT-1 that is responsible for intestinal iron transport, can also transport more than one type of mineral, unintended alterations in the absorption rate of one mineral may occur by a process of co-adaptation in response to changes in the status or physiologic need for another mineral. For example, in iron deficiency, iron absorption is increased, but the absorption of cadmium and lead are also inadvertently increased. However, these effects (homeostatic regulation and co-adaptation) are responses to a change in physiological state and should not be confused with alterations in nutrient ‘bioavailability’ per se, a characteristic of an individual food, a complex mixed meal, or a longer term characteristic of a particular dietary pattern.
Table 1 Dietary enhancers and inhibitors of mineral bioavailability Enhancers
Inhibitors
Ascorbic acid Organic acids Meat factor Alcohol Inulin
Polyphenols (especially galloyl groups) Phytic acid Myricetin Chlorogenic acid (coffee) Insoluble dietary fiber
Methods for Measuring Nutrient Bioavailability In Vitro Bioavailability Technique
Nutrient bioavailability, estimated as absorbability alone, can be measured by various in vitro methods. In vitro methods have obvious distinct advantages
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in that they are less expensive, rapid, and amenable to high throughput analyses. Often, experimental in vitro methods involve an initial ‘digestion phase’ where the food is treated with acid and digestive enzymes to simulate the initial steps of food breakdown. The digestion phase is then followed by a second phase wherein the goal is to estimate the potential relative availability of a nutrient. This usually involves the measurement of the concentration of the soluble nutrient of interest in a supernatant of the digested food following centrifugation or after dialysis of the digested food products across a semi-permeable membrane designed to select only low-molecular-weight complexes. Variations on this theme include the addition of radioactive isotopes following the digestion phase and the in vitro measurement of cellular uptake of the nutrient in a cell culture preparation or some appropriate index of nutrient uptake. In the case of iron, for example, cellular synthesis of ferritin, an iron storage protein, has been used. Similar applications of this in vitro technique are now appearing in the scientific literature for the measurement of phytochemical bioavailability, such as beta-carotene, lycopene, lutein, etc. However, although promising at the moment, there is little confidence that these methods can adequately replace in vivo methods of measuring nutrient bioavailability. In Vivo Bioavailability Techniques
Various animal models and techniques have been used to estimate nutrient bioavailability. A primary concern that must be initially addressed in using animal models to estimate nutrient bioavailability is whether these various model systems accurately reflect nutrient bioavailability in humans. For example, usual experimental animal models, such as rats and mice, cannot be used to assess beta-carotene bioavailability because the absorptive mechanism in these rodents for this nutrient is quite different from that in humans. In contrast, the ferret appears to be a suitable animal model to at least mimic this carotenoid’s intestinal absorptive pathway. Similarly, poor correlation of iron bioavailability between chicks and humans for elemental iron powders has raised questions about the suitability of that species for estimating nutrient bioavailability of iron. Measuring Nutrient Bioavailability in Humans
The first consideration is often whether it is necessary to have an accurate quantitative estimate of nutrient bioavailability, or whether an estimate of relative bioavailability compared to a known
standard nutrient source will suffice. An accurate quantitative measure of bioavailability might be necessary when the intention is to provide data to derive a recommendation for dietary intake to meet a nutrient requirement. In this case, it is important to have a reasonably good estimate of the true fraction of a given dose of the ingested nutrient that could be absorbed and utilized, for example, to replace endogenous losses of the nutrient. A common application of bioavailability measurements is to compare relative bioavailability between two or more sources of a nutrient. For example, one might be concerned with determining the calcium bioavailability from milk compared to calcium derived from a vegetable source such as broccoli or calcium-fortified orange juice. There are many techniques available to measure relative nutrient bioavailability under in vivo conditions based on a comparison of the rise in plasma level (or urinary excretion) of the nutrient or rate of appearance in plasma of a radioactively labeled nutrient after an oral test dose. An important technical advance in measuring food mineral bioavailability in humans was the validation of an extrinsic tag method. Extrinsic tag studies were validated by measuring the extent of absorption of a mineral isotope mixed exogenously (the ‘extrinsic tag’) with a food compared to that of an intrinsic tag where the absorption of the isotope is determined from an intrinsically labeled food source. The intrinsic tag is often achieved by growing plants hydroponically in a solution enriched in a radioactive or stable mineral isotope to label the plant food of interest during growth, or by supplying the mineral isotope tag to a growing animal used for meat, or one that was used for milk production, for example. These studies have shown that in most cases the ratio of absorption of the extrinsic to the intrinsic isotope was approximately one, indicating that the extrinsically added isotope tag became homogenously incorporated into the pool of absorbed mineral found endogenously in the food of interest. The use of the extrinsic tag method has greatly facilitated the study of relative bioavailability of minerals from food in human subjects. A large and growing number of people are consuming dietary supplements. However, due to the relative difficulty of labeling these supplements, in most cases, little information on the bioavailability of the nutrients in the supplements is available. A study of vitamin and mineral bioavailability from a popular multinutrient supplement found good absorption of the water-soluble vitamins (B vitamins and vitamin C) from the tablet but relatively poor absorption of copper and zinc.
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Food Fortification
Bioavailability of Food Fortificants
In the summer of 1941 a National Nutrition Conference for Defense was held that led to the recommendation that there should be improvement of the nutritive value of certain low-cost stable food products (e.g., flour and bread) by nutrient enrichment to replace nutrients lost during the milling and refining process. This led to recommendations to fortify milk with vitamin D, margarine with vitamin A, and salt with iodine using the new recommended dietary allowances (RDAs) established by the Committee on Food and Nutrition of the National Research Council (currently the Food and Nutrition Board) as a yardstick to judge the appropriate levels of fortification. Standards of identity for ‘enriched’ flour’ were initially established that allowed for the addition of the ‘basic four’: iron, thiamin, niacin, and riboflavin (with optional calcium). In subsequent years, the standard of identity concept was expanded to include some other enriched foods. As of 1998, the Food and Drug Administration mandated that folate be added to the standards of identity for enriched breads, flours, corn meals, pastas, rice, and other grain products.
The bioavailability of nutrients added to food can be determined by a number of factors. For example, in some cases the reactivity of added nutrients can cause untoward reactions that adversely affect the organoleptic properties of food. In these cases, there must be a trade-off of some kind and it may be necessary to intentionally select a somewhat less bioavailable form of a nutrient to provide an acceptable consumer product or to provide an acceptable shelf life to the product under given field conditions. Moreover, once added to a food, the bioavailability of a fortificant can be altered by various food manufacturing processes, such as those that demand high heat and pressure. Normal home food preparation techniques can also affect nutrient bioavailability. In addition, plant breeding and horticultural practices can contribute to the development and use of superior plant varieties supplying additional or more bioavailable micronutrients. For example, genetic engineering of plants has led to the development of rice and other grain products that have lower phytate content and higher mineral bioavailability. The development of ‘Golden Rice,’ which is rich in -carotene, a dietary precursor of vitamin A, represents a well-known example of genetic plant engineering to enhance nutrient intakes. There is increasing interest in genetic manipulation of plant stocks to achieve higher content of potentially healthful phytonutrients, such as lycopene and lutein. Internationally, the traditional focus of fortification has been directed at the ‘Big 3’ – deficiencies of vitamin A, iodine, and iron – due to the widespread prevalence of deficiencies of these particular micronutrients and well-known adverse health effects of these nutrient deficiencies.
Micronutrient Fortification in the UK
In the UK, fortification of foods is subject to the Food Safety Act 1990. Fortification of certain micronutrients to margarine and most types of flour is mandatory. Calcium, iron, thiamin, and niacin are required to be added to both white and brown flours, but not to wholemeal flours. The level of required fortification is shown in Table 2. Margarine is required to be fortified with vitamin A and D to levels comparable with or exceeding those found in butter. Additional mandatory fortification requirements determine the nutrient content of infant formulas and follow-on formulas, weaning foods, and foods intended to be used in energyrestricted diets. In the UK, voluntary fortification is allowed for certain products, such as breakfast cereal, soft drinks, and milks. In most cases the level of fortification per serving is between 15% and 33% of the relevant RDA. Table 2 Nutrients required to be added to white and brown flours in the United Kingdom Nutrient
Amount of nutrient (mg) per 100 g flour
Calcium Iron Thiamin Niacin
235–390 Not less than 1.65 Not less than 0.24 Not less than 1.6
Bioavailability of Fortified Iron
A list of iron sources that are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) is given in Table 3. However, as shown in Table 4, the bioavailability of different iron sources varies widely. Moreover, even within a given source of iron, such as the elemental iron powders commonly used to fortify various ready-to-eat breakfast cereals and other products, a significant disparity (5–148%) in relative bioavailability (compared to ferrous sulfate) can be observed. To some extent, these differing bioavailability estimates reflect the influence of the characteristics of the fortified product in terms of its contribution of various enhancers or inhibitors (Table 1) on iron bioavailability. In addition, other factors also can affect the bioavailability of elemental iron powders, such as the particle size of the fortificant
200 BIOAVAILABILITY Table 3 Iron and zinc compounds listed as generally recognized as safe by the US Food and Drug Administration Iron compounds
Zinc compounds
Elemental iron Ferrous ascorbate Ferrous carbonate Ferrous citrate Ferrous fumarate Ferrous gluconate Ferrous lactate Ferrous sulfate Ferric ammonium citrate Ferric chloride Ferric citrate Ferric pyrophosphate Ferric sulfate
Zinc Zinc Zinc Zinc Zinc
sulfate chloride gluconate oxide stearate
compound – a finer particle size is associated with greater iron bioavailability. There is very limited information available concerning the bioavailability of calcium or other nutrients added as a fortificant to various products. One study in elderly women found that calcium citrate malate used to fortify orange juice had equivalent bioavailability to calcium from milk or calcium from a calcium carbonate supplement. In a study with adult subjects, the
Table 4 Average relative bioavailability in humans of various iron sources used as iron fortification compounds Average relative bioavailability
>90% group 106a 100 100 92 >60–<90% group 89 75 74 74 62 Variable (%) 21–74 25–32 13–148 5–20 a
Iron compound
Approximate iron content (%)
Ferrous Ferrous Ferrous Ferrous
lactate sulfate 7H2O fumarate succinate
19 20 33 35
Ferrous gluconate Electrolytic elemental Fe powders Ferric saccharate Ferrous citrate Ferrous tartrate
12 97
Ferric pyrophosphate Ferric orthophosphate H-reduced elemental Fe powder Carbonyl elemental Fe powder
25 28 97
10 24 22
99
Relative to absorption of iron from ferrous sulfate = 100% Adapted from Lynch S (2002) Food iron absorption and its importance for the design of food fortification strategies. Nutrition Reviews 60: S3–S15.
bioavailability of a single 25 000 IU dose of vitamin D2 was assessed from whole milk, skim milk, and vitamin D-fortified oil given with toast. No difference in peak serum vitamin D2 was found following these three treatments, suggesting that the fat content of whole milk does not influence vitamin D bioavailability. It has also been shown that consumption of vitamin D-fortified orange juice (1000 IU/240 ml) for 12 weeks significantly increased serum 25-hydroxyvitamin D concentrations. Fat content of a meal may have an important effect on carotenoid bioavailability. The absorption of carotenoids (-carotene, -carotene, and lycopene) from salad vegetables was found to be undetectable if a fat-free salad dressing was used, but substantially greater absorption occurred with a full-fat salad dressing. The amount of fat needed to promote optimal absorption of vitamin E and carotenoids may be rather limited. No difference in absorption of vitamin E and - or -carotene was observed when supplements were administered with either 3 g or 36 g of dietary fat. In contrast, lutein ester absorption was more than twice as great when consumed with the higher fat level. Bioavailability of food sources of folate are usually only about 50% of synthetic folic acid. This systematic difference may be due to the occurrence of polyglutamyl folic acid in foods that reduce folate absorption. See also: Calcium. Carotenoids: Chemistry, Sources and Physiology. Cobalamins. Copper. Food Fortification: Developed Countries; Developing Countries. Iodine: Physiology, Dietary Sources and Requirements. Microbiota of the Intestine: Prebiotics. Osteoporosis. Selenium. Vitamin A: Biochemistry and Physiological Role. Zinc: Physiology.
Further Reading Backstrand J (2002) The history and future of food fortification in the United States: a public health perspective. Nutrition Reviews 60: 15–26. Bouis HE (2003) Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost? Proceedings of the Nutrition Society 62(2): 403–411. Calvo MS and Whiting SJ (2003) Prevalence of vitamin D insufficiency in Canada and the United States: importance to health status and efficacy of current food fortification and dietary supplement use. Nutrition Reviews 61(3): 107–113. Chavasit V and Nopburabutr P (2003) Combating iodine and iron deficiencies through the double fortification of fish sauce, mixed fish sauce, and salt brine. Food and Nutrition Bulletin 24(2): 200–207.
BIOTIN Dary O and Mora JO (2002) Food fortification to reduce vitamin A deficiency: International Vitamin A Consultative Group recommendations. Journal of Nutrition 132(supplement 9): 2927S–2933S. Delange FM (2003) Control of iodine deficiency in Western and Central Europe. Central Europe Journal of Public Health 11(3): 120–123. Fairweather-Tait SJ and Teucher B (2002) Iron and calcium bioavailability of fortified foods and dietary supplements. Nutrition Review 60(11): 360–367. Johnson-Down L, L’Abbe MR, Lee NS, and Gray-Donald K (2003) Appropriate calcium fortification of the food supply presents a challenge. Journal of Nutrition 133(7): 2232–2238. Lutter CK and Dewey KG (2003) Proposed nutrient composition for fortified complementary foods. Journal of Nutrition 133(9): 3011S–3020S.
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Lynch S (2002) Food iron absorption and its importance for the design of food fortification strategies. Nutrition Reviews 60: S3–S6. Meltzer HM, Aro A, Andersen NL, Koch B, and Alexander J (2003) Risk analysis applied to food fortification. Public Health and Nutrition 6(3): 281–291. Penniston KL and Tanumihardjo SA (2003) Vitamin A in dietary supplements and fortified food: too much of a good thing? Journal of the American Dietetic Association 103(9): 1185–1187. Quinlivan EP and Gregory JK 3rd (2003) Effect of food fortification on folic acid intake in the United States. American Journal of Clinical Nutrition 77(1): 8–9. Rosado JL (2003) Zinc and copper: proposed fortification levels and recommended zinc compounds. Journal of Nutrition 133(9): 2985S–2989S.
BIOTIN D M Mock, University of Arkansas for Medical Sciences, Little Rock, AR, USA ª 2005 Elsevier Ltd. All rights reserved.
Biotin is a water-soluble vitamin that is generally classified in the B complex group. Biotin was discovered in nutritional experiments that demonstrated a factor in many foodstuffs capable of curing the scaly dermatitis, hair loss, and neurologic signs induced in rats fed dried egg white. Avidin, a glycoprotein found in egg white, binds biotin very specifically and tightly. From an evolutionary standpoint, avidin probably serves as a bacteriostat in egg white; consistent with this hypothesis is the observation that avidin is resistant to a broad range of bacterial proteases in both the free and biotinbound form. Because avidin is also resistant to pancreatic proteases, dietary avidin binds to dietary biotin (and probably any biotin from intestinal microbes) and prevents absorption, carrying the biotin through the gastrointestinal tract. Biotin is synthesized by many intestinal microbes; however, the contribution of microbial biotin to absorbed biotin, if any, remains unknown. Cooking denatures avidin, rendering this protein susceptible to digestion and unable to interfere with absorption of biotin.
Absorption and Transport Digestion of Protein-Bound Biotin
The content of free biotin and protein-bound biotin in foods is variable, but the majority of biotin in
meats and cereals appears to be protein-bound via an amide bond between biotin and lysine. Neither the mechanisms of intestinal hydrolysis of proteinbound biotin nor the determinants of bioavailability have been clearly delineated. Because this bond is not hydrolyzed by cellular proteases, release is likely mediated by a specific biotin—amide hydrolase (biotinidase, EC 3.5.1.12). Biotinidase mRNA is present in pancreas and, in lesser amounts, in intestinal mucosa. Biotinidase is also present in many other tissues, including heart, brain, liver, lung, skeletal muscle, kidney, plasma, and placenta. Biotinidase also likely plays a critical role in intracellular recycling of biotin by releasing biotin from intracellular proteins such as carboxylases during protein turnover. Intestinal Absorption and Transport into Somatic Cells
At physiologic pH, the carboxylate group of biotin is negatively charged. Thus, biotin is at least modestly water-soluble and requires a transporter to cross cell membranes such as enterocytes for intestinal absorption, somatic cells for utilization, and renal tubule cells for reclamation from the glomerular filtrate. In intact intestinal preparations such as loops and everted gut sacks, biotin transport exhibits two components. One component is saturable at a km of approximately 10 mM biotin; the other is not saturable even at very large concentrations of biotin. This observation is consistent with passive diffusion. Absorption of biocytin, the biotinyl-lysine product of intraluminal protein digestion, is inefficient relative to biotin, suggesting that biotinidase releases
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BIOAVAILABILITY R J Wood, Tufts University, Boston, MA, USA ª 2005 Elsevier Ltd. All rights reserved.
Introduction Bioavailability is an important consideration in a number of areas of nutrition, including the derivation of Dietary Reference Intakes, estimation of potential impact of changes in dietary pattern, the selection of specific food fortificants, and the formulation of whole meal products, such as infant formulas or meal replacements. In this article, the role of food processing in nutrient bioavailability, specific determinants of nutrient bioavailability, methods for measuring bioavailability, and bioavailability of food fortificants are discussed.
generally as growth stimulation under nutrient limiting conditions, etc. An exception to the ‘absorption equals bioavailability’ rule is selenium. One form of dietary selenium is selenomethionine. This selenium-containing compound is handled by the body exactly like the amino acid methionine and gets readily incorporated into methionine-containing proteins. However, the selenium found in selenium-dependent enzymes is in the form of a special amino acid called selenocysteine, which must be synthesized in the body during the process of incorporation of selenocysteine into these selenoproteins. Selenomethionine catabolism will result in the release of this selenium into an active endogenous selenium pool, which serves as the source of selenium for selenocysteine synthesis. Thus, selenium in selenomethionine is not immediately available to support selenium-dependent functions in the body.
Effects of Food Processing Definition Nutrient bioavailability is defined as the fraction of a nutrient in a food that is absorbed and utilized. In practice, however, measurements of bioavailability have focused on either direct measurement of absorption or determination of the change in some functional or biochemical endpoint reflecting absorption and utilization of the nutrient. In general, the bioavailability of all nutrients can be estimated by measuring absorption alone because, once absorbed, nutrients are freely available for biological utilization, irrespective of their original dietary source. For example, consider the case of iron bioavailability. Iron absorption can be measured directly from a food by a variety of methods (described in more detail later). In addition, absorbed iron enters the plasma iron pool carried on the protein transferrin. In turn, this absorbed iron will be used in large part (about 80%) immediately in the synthesis of hemoglobin by erythrocyte precursor cells in the bone marrow. The fraction of iron that is utilized for hemoglobin synthesis is not dependent in any way on the food source of that iron. Thus, food iron bioavailability can be conveniently measured and compared in relative terms among various sources by determining the change in blood hemoglobin after consumption of various forms of iron in iron-deficient subjects. Thus, nutrient bioavailability can be estimated by measuring these appropriate endpoints, such as hemoglobin incorporation for iron, hepatic tissue or mineral content of bone for various bone-seeking minerals, or more
Food processing can have positive or negative effects on nutrient bioavailability. For example, milling of grains removes all or part of the external covering of the grain (bran) that contains high amounts of phytic acid, an important inhibitor of bioavailability of divalent minerals, such as iron, zinc, and copper. One disadvantage of this form of food processing is that much of the mineral content of grains is in the bran fraction and is lost in the process of milling. To compensate for this loss of mineral (and some vitamins as well) grain products can be ‘enriched’ by fortifying the flour made from the milled grain with specific micronutrients. Simple food processing techniques, such as sprouting or fermentation, are also effective in lowering the phytate content and increasing mineral bioavailability of grains. Other techniques used in food manufacturing, such as browning, which produces the Maillard reaction, and extrusion, can have negative effects on bioavailability of certain nutrients. Processing practices that affect the polyphenol content of cereals and legumes can influence nutrient bioavailability. Polyphenols interact with plant proteins and form tannin–protein complexes that can inactivate enzymes or lead to protein insolubility adversely influencing amino acid and protein bioavailability. The antinutritional properties of polyphenols can be decreased by removing them from grain with chemical treatments (such as alkaline treatment and ammonia) or removing from grain the polyphenol-rich pericarp and testa by pearling.
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Importance of Nutrient Bioavailability to Human Nutrition Assessment of bioavailability of nutrients is an essential component of deriving dietary reference intakes (DRIs), guidelines for optimal intake of individual nutrients established for North American populations. Many DRIs are based on evaluation of available physiological data to determine the obligatory daily needs for a nutrient to replace losses, or the amount needed for optimal growth of tissues, and an estimate of overall dietary bioavailability of the nutrient in question. In many populations, the content of a nutrient in the diet (e.g., iron or zinc) may be sufficient to meet recommended intake, but bioavailability is suboptimal due to the presence of high levels of inhibitory substances (such as phytate) in the diet leading to a high risk of developing this nutrient deficiency.
Determinants of Nutrient Bioavailability Speciation
Speciation refers to the form of the nutrient found in food, which may in turn influence the absorption of the nutrient in the gastrointestinal tract. For example, this term could be applied to cis- or trans-forms of unsaturated fatty acids found in partially hydrogenated oils; individual coenzyme forms of vitamins, such as free thiamin, thiamin pyrophosphate (TPP), thiamin monophosphate (TMP) and thiamin triphosphate (TTP), or the various coenzyme forms of riboflavin – flavin mononucleotide (FMN) and adenine dinucleotide (FAD), or for vitamin B6 – pyridoxine, pyridoxamine, and pyridoxal; or minerals, such as ferrous or ferric forms of nonheme iron or heme iron; or the selenomethionine, selenite, or selenate form of selenium. Digestion and Metabolism
During the transit of digested food material through the gastrointestinal tract many changes occur in the intestinal lumen that could influence nutrient bioavailability. Digestion of food constituents is an important aspect of nutrient bioavailability. The secretion of acid into the stomach following food ingestion activates certain digestive enzymes, as well as creating an acidic environment that influences mineral solubility and extraction from food. In this regard, the choice of a mineral to use for supplementation purposes might be influenced by certain physiological conditions, such as achlorhydria. Aging is associated with a decrease in gastric acid secretion in many elderly persons leading to either hypochlorhydria or achlorhydria,
characterized by either low or complete absence of acid secretion, respectively, leading to a neutral or slightly alkaline gastric pH. This raised pH condition in the stomach can have detrimental effects on micronutrient bioavailability. For example, elderly persons with achlorhydria are at risk of vitamin B12 deficiency because of an inability to remove properly protein-bound vitamin B12 from food when it enters the stomach. In these subjects, the capacity to absorb vitamin B12 is normal, however, because they can readily absorb crystalline (nonproteinbound) vitamin B12 from a supplemental vitamin B12 dose. Lowering the gastric pH towards normal by administering hydrochloric acid restores vitamin B12 absorption from food. In achlorhydric elderly persons the absorption of calcium carbonate from a dietary supplement after an overnight fast is very poor, presumably because calcium carbonate is a relatively insoluble calcium salt and needs gastric acid to be solubilized. Again, these subjects have normal calcium absorption if the calcium is delivered in a more soluble form, such as calcium citrate. In addition to elderly persons who develop achlorhydria, a large number of people regularly use gastric acid-lowering medications, such as the gastric proton pump inhibitor omeprazole, for antiulcer therapy. These medications can reduce zinc absorption and presumably may affect the absorption of other divalent minerals. Acidification of the gastric contents and solubilization of minerals in the gastric juice is important because many mineral nutrients are preferentially absorbed in the duodenum (upper small intestine) and need to be available in free or low-molecular-weight complexes when they leave the stomach to facilitate contact with intestinal nutrient transporters on the apical (luminal) surface of the absorptive enterocytes. The lower gastrointestinal tract can also be a potentially important site affecting the bioavailability of bioactive substances found in food. In this regard, intestinal bacteria found in the large intestine can influence bioavailability. Bacteria are instrumental in the metabolic conversion of certain phytonutrients into forms that are more readily absorbed. In addition, bacteria likely play an important role in the enhancing effects of prebiotics on mineral bioavailability. Consumption of nonabsorbable carbohydrates, such as inulin, can have a positive effect on mineral absorption. A possible mechanism of this effect is that the nonabsorbable carbohydrates pass the small intestine and enter into the large intestine where they serve as a food substrate for bacteria. The metabolism of these prebiotic substances by intestinal bacteria lowers the pH of the lumen of the large intestine and may thereby
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serve to solubilize some insoluble mineral complexes that have passed through the small intestine. The freed mineral would then be available for absorption from the large intestine, thereby increasing overall mineral bioavailability. Other scenarios are possible as well, such as the release of short-chain fatty acids by the bacteria, that may facilitate mineral absorption. Chelation
Chelation is the process whereby an organic moiety acts as a ligand to bind a metal ion through two or more coordination bonds. Some low-molecularweight compounds that may be released during the digestion of food can act as metal chelators and increase metal solubility in the intestinal lumen. In some circumstances, chelated forms of metals are naturally present in food, such as heme iron (part of hemoglobin or myoglobin protein) found in meat. Heme is a stable protoporphyrin ring-containing compound that protects a central iron atom from interacting with other potentially deleterious compounds, such as phytic acid, that would reduce its availability and inhibit iron absorption. Nonheme iron bioavailability is affected by various enhancing and inhibitory substances found in food. In contrast, heme iron bioavailability is not. The heme moiety is absorbed intact by the enterocyte. Inside the enterocyte a cytosolic enzyme heme oxygenase breaks apart the protoporphyrin ring and releases the caged iron atom, which can then be transferred out into the blood. Bioavailability Enhancers and Inhibitors
Various food substances have been identified that act as enhancers or inhibitors of divalent mineral absorption. In general, these food factors influence nutrient bioavailability by either forming relatively insoluble complexes with nutrients or preventing them from interacting with their respective nutrient transporter, or by protecting the nutrient from such untoward interaction maintaining it in a state that can be absorbed or as an absorbable chelated complex (e.g., heme iron). A list of factors known to influence mineral bioavailability is given in Table 1.
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General Physiology of Nutrient Absorption
Nutrients enter the blood by passing through the intestinal mucosa. Intestinal nutrient transport can occur via two distinct pathways. One is termed the paracellular pathway and represents the movement of a nutrient between the absorptive enterocytes on the intestinal villi. This transport pathway is an energy-independent diffusional process and depends on the electrochemical gradient across the mucosa and its permeability characteristics to the nutrient in question. The characteristics of the diffusional pathway are not regulated in response to nutrient deficiency or excess. A second transport pathway represents the transcellular movement of a nutrient across the intestine. The transcellular transport rate of the nutrient is composed of both diffusional and carrier-mediated transport pathways. Often in response to changes in nutrient status the number of nutrient carriers will be changed to facilitate appropriate increases or decreases in intestinal absorption to help maintain nutrient homeostasis. Within a class of nutrients there can be substantial differences in absorption rates. For example, in the case of minerals, monovalent minerals (such as sodium, potassium, and iodine) are absorbed at very high efficiency approximating complete absorption, while multivalent elements (such as chromium, heavy metals, and iron) are relatively poorly absorbed (1–20%). In addition, because some nutrient transporters, e.g., the divalent metal transporter DMT-1 that is responsible for intestinal iron transport, can also transport more than one type of mineral, unintended alterations in the absorption rate of one mineral may occur by a process of co-adaptation in response to changes in the status or physiologic need for another mineral. For example, in iron deficiency, iron absorption is increased, but the absorption of cadmium and lead are also inadvertently increased. However, these effects (homeostatic regulation and co-adaptation) are responses to a change in physiological state and should not be confused with alterations in nutrient ‘bioavailability’ per se, a characteristic of an individual food, a complex mixed meal, or a longer term characteristic of a particular dietary pattern.
Table 1 Dietary enhancers and inhibitors of mineral bioavailability Enhancers
Inhibitors
Ascorbic acid Organic acids Meat factor Alcohol Inulin
Polyphenols (especially galloyl groups) Phytic acid Myricetin Chlorogenic acid (coffee) Insoluble dietary fiber
Methods for Measuring Nutrient Bioavailability In Vitro Bioavailability Technique
Nutrient bioavailability, estimated as absorbability alone, can be measured by various in vitro methods. In vitro methods have obvious distinct advantages
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in that they are less expensive, rapid, and amenable to high throughput analyses. Often, experimental in vitro methods involve an initial ‘digestion phase’ where the food is treated with acid and digestive enzymes to simulate the initial steps of food breakdown. The digestion phase is then followed by a second phase wherein the goal is to estimate the potential relative availability of a nutrient. This usually involves the measurement of the concentration of the soluble nutrient of interest in a supernatant of the digested food following centrifugation or after dialysis of the digested food products across a semi-permeable membrane designed to select only low-molecular-weight complexes. Variations on this theme include the addition of radioactive isotopes following the digestion phase and the in vitro measurement of cellular uptake of the nutrient in a cell culture preparation or some appropriate index of nutrient uptake. In the case of iron, for example, cellular synthesis of ferritin, an iron storage protein, has been used. Similar applications of this in vitro technique are now appearing in the scientific literature for the measurement of phytochemical bioavailability, such as beta-carotene, lycopene, lutein, etc. However, although promising at the moment, there is little confidence that these methods can adequately replace in vivo methods of measuring nutrient bioavailability. In Vivo Bioavailability Techniques
Various animal models and techniques have been used to estimate nutrient bioavailability. A primary concern that must be initially addressed in using animal models to estimate nutrient bioavailability is whether these various model systems accurately reflect nutrient bioavailability in humans. For example, usual experimental animal models, such as rats and mice, cannot be used to assess beta-carotene bioavailability because the absorptive mechanism in these rodents for this nutrient is quite different from that in humans. In contrast, the ferret appears to be a suitable animal model to at least mimic this carotenoid’s intestinal absorptive pathway. Similarly, poor correlation of iron bioavailability between chicks and humans for elemental iron powders has raised questions about the suitability of that species for estimating nutrient bioavailability of iron. Measuring Nutrient Bioavailability in Humans
The first consideration is often whether it is necessary to have an accurate quantitative estimate of nutrient bioavailability, or whether an estimate of relative bioavailability compared to a known
standard nutrient source will suffice. An accurate quantitative measure of bioavailability might be necessary when the intention is to provide data to derive a recommendation for dietary intake to meet a nutrient requirement. In this case, it is important to have a reasonably good estimate of the true fraction of a given dose of the ingested nutrient that could be absorbed and utilized, for example, to replace endogenous losses of the nutrient. A common application of bioavailability measurements is to compare relative bioavailability between two or more sources of a nutrient. For example, one might be concerned with determining the calcium bioavailability from milk compared to calcium derived from a vegetable source such as broccoli or calcium-fortified orange juice. There are many techniques available to measure relative nutrient bioavailability under in vivo conditions based on a comparison of the rise in plasma level (or urinary excretion) of the nutrient or rate of appearance in plasma of a radioactively labeled nutrient after an oral test dose. An important technical advance in measuring food mineral bioavailability in humans was the validation of an extrinsic tag method. Extrinsic tag studies were validated by measuring the extent of absorption of a mineral isotope mixed exogenously (the ‘extrinsic tag’) with a food compared to that of an intrinsic tag where the absorption of the isotope is determined from an intrinsically labeled food source. The intrinsic tag is often achieved by growing plants hydroponically in a solution enriched in a radioactive or stable mineral isotope to label the plant food of interest during growth, or by supplying the mineral isotope tag to a growing animal used for meat, or one that was used for milk production, for example. These studies have shown that in most cases the ratio of absorption of the extrinsic to the intrinsic isotope was approximately one, indicating that the extrinsically added isotope tag became homogenously incorporated into the pool of absorbed mineral found endogenously in the food of interest. The use of the extrinsic tag method has greatly facilitated the study of relative bioavailability of minerals from food in human subjects. A large and growing number of people are consuming dietary supplements. However, due to the relative difficulty of labeling these supplements, in most cases, little information on the bioavailability of the nutrients in the supplements is available. A study of vitamin and mineral bioavailability from a popular multinutrient supplement found good absorption of the water-soluble vitamins (B vitamins and vitamin C) from the tablet but relatively poor absorption of copper and zinc.
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Food Fortification
Bioavailability of Food Fortificants
In the summer of 1941 a National Nutrition Conference for Defense was held that led to the recommendation that there should be improvement of the nutritive value of certain low-cost stable food products (e.g., flour and bread) by nutrient enrichment to replace nutrients lost during the milling and refining process. This led to recommendations to fortify milk with vitamin D, margarine with vitamin A, and salt with iodine using the new recommended dietary allowances (RDAs) established by the Committee on Food and Nutrition of the National Research Council (currently the Food and Nutrition Board) as a yardstick to judge the appropriate levels of fortification. Standards of identity for ‘enriched’ flour’ were initially established that allowed for the addition of the ‘basic four’: iron, thiamin, niacin, and riboflavin (with optional calcium). In subsequent years, the standard of identity concept was expanded to include some other enriched foods. As of 1998, the Food and Drug Administration mandated that folate be added to the standards of identity for enriched breads, flours, corn meals, pastas, rice, and other grain products.
The bioavailability of nutrients added to food can be determined by a number of factors. For example, in some cases the reactivity of added nutrients can cause untoward reactions that adversely affect the organoleptic properties of food. In these cases, there must be a trade-off of some kind and it may be necessary to intentionally select a somewhat less bioavailable form of a nutrient to provide an acceptable consumer product or to provide an acceptable shelf life to the product under given field conditions. Moreover, once added to a food, the bioavailability of a fortificant can be altered by various food manufacturing processes, such as those that demand high heat and pressure. Normal home food preparation techniques can also affect nutrient bioavailability. In addition, plant breeding and horticultural practices can contribute to the development and use of superior plant varieties supplying additional or more bioavailable micronutrients. For example, genetic engineering of plants has led to the development of rice and other grain products that have lower phytate content and higher mineral bioavailability. The development of ‘Golden Rice,’ which is rich in -carotene, a dietary precursor of vitamin A, represents a well-known example of genetic plant engineering to enhance nutrient intakes. There is increasing interest in genetic manipulation of plant stocks to achieve higher content of potentially healthful phytonutrients, such as lycopene and lutein. Internationally, the traditional focus of fortification has been directed at the ‘Big 3’ – deficiencies of vitamin A, iodine, and iron – due to the widespread prevalence of deficiencies of these particular micronutrients and well-known adverse health effects of these nutrient deficiencies.
Micronutrient Fortification in the UK
In the UK, fortification of foods is subject to the Food Safety Act 1990. Fortification of certain micronutrients to margarine and most types of flour is mandatory. Calcium, iron, thiamin, and niacin are required to be added to both white and brown flours, but not to wholemeal flours. The level of required fortification is shown in Table 2. Margarine is required to be fortified with vitamin A and D to levels comparable with or exceeding those found in butter. Additional mandatory fortification requirements determine the nutrient content of infant formulas and follow-on formulas, weaning foods, and foods intended to be used in energyrestricted diets. In the UK, voluntary fortification is allowed for certain products, such as breakfast cereal, soft drinks, and milks. In most cases the level of fortification per serving is between 15% and 33% of the relevant RDA. Table 2 Nutrients required to be added to white and brown flours in the United Kingdom Nutrient
Amount of nutrient (mg) per 100 g flour
Calcium Iron Thiamin Niacin
235–390 Not less than 1.65 Not less than 0.24 Not less than 1.6
Bioavailability of Fortified Iron
A list of iron sources that are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA) is given in Table 3. However, as shown in Table 4, the bioavailability of different iron sources varies widely. Moreover, even within a given source of iron, such as the elemental iron powders commonly used to fortify various ready-to-eat breakfast cereals and other products, a significant disparity (5–148%) in relative bioavailability (compared to ferrous sulfate) can be observed. To some extent, these differing bioavailability estimates reflect the influence of the characteristics of the fortified product in terms of its contribution of various enhancers or inhibitors (Table 1) on iron bioavailability. In addition, other factors also can affect the bioavailability of elemental iron powders, such as the particle size of the fortificant
200 BIOAVAILABILITY Table 3 Iron and zinc compounds listed as generally recognized as safe by the US Food and Drug Administration Iron compounds
Zinc compounds
Elemental iron Ferrous ascorbate Ferrous carbonate Ferrous citrate Ferrous fumarate Ferrous gluconate Ferrous lactate Ferrous sulfate Ferric ammonium citrate Ferric chloride Ferric citrate Ferric pyrophosphate Ferric sulfate
Zinc Zinc Zinc Zinc Zinc
sulfate chloride gluconate oxide stearate
compound – a finer particle size is associated with greater iron bioavailability. There is very limited information available concerning the bioavailability of calcium or other nutrients added as a fortificant to various products. One study in elderly women found that calcium citrate malate used to fortify orange juice had equivalent bioavailability to calcium from milk or calcium from a calcium carbonate supplement. In a study with adult subjects, the
Table 4 Average relative bioavailability in humans of various iron sources used as iron fortification compounds Average relative bioavailability
>90% group 106a 100 100 92 >60–<90% group 89 75 74 74 62 Variable (%) 21–74 25–32 13–148 5–20 a
Iron compound
Approximate iron content (%)
Ferrous Ferrous Ferrous Ferrous
lactate sulfate 7H2O fumarate succinate
19 20 33 35
Ferrous gluconate Electrolytic elemental Fe powders Ferric saccharate Ferrous citrate Ferrous tartrate
12 97
Ferric pyrophosphate Ferric orthophosphate H-reduced elemental Fe powder Carbonyl elemental Fe powder
25 28 97
10 24 22
99
Relative to absorption of iron from ferrous sulfate = 100% Adapted from Lynch S (2002) Food iron absorption and its importance for the design of food fortification strategies. Nutrition Reviews 60: S3–S15.
bioavailability of a single 25 000 IU dose of vitamin D2 was assessed from whole milk, skim milk, and vitamin D-fortified oil given with toast. No difference in peak serum vitamin D2 was found following these three treatments, suggesting that the fat content of whole milk does not influence vitamin D bioavailability. It has also been shown that consumption of vitamin D-fortified orange juice (1000 IU/240 ml) for 12 weeks significantly increased serum 25-hydroxyvitamin D concentrations. Fat content of a meal may have an important effect on carotenoid bioavailability. The absorption of carotenoids (-carotene, -carotene, and lycopene) from salad vegetables was found to be undetectable if a fat-free salad dressing was used, but substantially greater absorption occurred with a full-fat salad dressing. The amount of fat needed to promote optimal absorption of vitamin E and carotenoids may be rather limited. No difference in absorption of vitamin E and - or -carotene was observed when supplements were administered with either 3 g or 36 g of dietary fat. In contrast, lutein ester absorption was more than twice as great when consumed with the higher fat level. Bioavailability of food sources of folate are usually only about 50% of synthetic folic acid. This systematic difference may be due to the occurrence of polyglutamyl folic acid in foods that reduce folate absorption. See also: Calcium. Carotenoids: Chemistry, Sources and Physiology. Cobalamins. Copper. Food Fortification: Developed Countries; Developing Countries. Iodine: Physiology, Dietary Sources and Requirements. Microbiota of the Intestine: Prebiotics. Osteoporosis. Selenium. Vitamin A: Biochemistry and Physiological Role. Zinc: Physiology.
Further Reading Backstrand J (2002) The history and future of food fortification in the United States: a public health perspective. Nutrition Reviews 60: 15–26. Bouis HE (2003) Micronutrient fortification of plants through plant breeding: can it improve nutrition in man at low cost? Proceedings of the Nutrition Society 62(2): 403–411. Calvo MS and Whiting SJ (2003) Prevalence of vitamin D insufficiency in Canada and the United States: importance to health status and efficacy of current food fortification and dietary supplement use. Nutrition Reviews 61(3): 107–113. Chavasit V and Nopburabutr P (2003) Combating iodine and iron deficiencies through the double fortification of fish sauce, mixed fish sauce, and salt brine. Food and Nutrition Bulletin 24(2): 200–207.
BIOTIN Dary O and Mora JO (2002) Food fortification to reduce vitamin A deficiency: International Vitamin A Consultative Group recommendations. Journal of Nutrition 132(supplement 9): 2927S–2933S. Delange FM (2003) Control of iodine deficiency in Western and Central Europe. Central Europe Journal of Public Health 11(3): 120–123. Fairweather-Tait SJ and Teucher B (2002) Iron and calcium bioavailability of fortified foods and dietary supplements. Nutrition Review 60(11): 360–367. Johnson-Down L, L’Abbe MR, Lee NS, and Gray-Donald K (2003) Appropriate calcium fortification of the food supply presents a challenge. Journal of Nutrition 133(7): 2232–2238. Lutter CK and Dewey KG (2003) Proposed nutrient composition for fortified complementary foods. Journal of Nutrition 133(9): 3011S–3020S.
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Lynch S (2002) Food iron absorption and its importance for the design of food fortification strategies. Nutrition Reviews 60: S3–S6. Meltzer HM, Aro A, Andersen NL, Koch B, and Alexander J (2003) Risk analysis applied to food fortification. Public Health and Nutrition 6(3): 281–291. Penniston KL and Tanumihardjo SA (2003) Vitamin A in dietary supplements and fortified food: too much of a good thing? Journal of the American Dietetic Association 103(9): 1185–1187. Quinlivan EP and Gregory JK 3rd (2003) Effect of food fortification on folic acid intake in the United States. American Journal of Clinical Nutrition 77(1): 8–9. Rosado JL (2003) Zinc and copper: proposed fortification levels and recommended zinc compounds. Journal of Nutrition 133(9): 2985S–2989S.
BIOTIN D M Mock, University of Arkansas for Medical Sciences, Little Rock, AR, USA ª 2005 Elsevier Ltd. All rights reserved.
Biotin is a water-soluble vitamin that is generally classified in the B complex group. Biotin was discovered in nutritional experiments that demonstrated a factor in many foodstuffs capable of curing the scaly dermatitis, hair loss, and neurologic signs induced in rats fed dried egg white. Avidin, a glycoprotein found in egg white, binds biotin very specifically and tightly. From an evolutionary standpoint, avidin probably serves as a bacteriostat in egg white; consistent with this hypothesis is the observation that avidin is resistant to a broad range of bacterial proteases in both the free and biotinbound form. Because avidin is also resistant to pancreatic proteases, dietary avidin binds to dietary biotin (and probably any biotin from intestinal microbes) and prevents absorption, carrying the biotin through the gastrointestinal tract. Biotin is synthesized by many intestinal microbes; however, the contribution of microbial biotin to absorbed biotin, if any, remains unknown. Cooking denatures avidin, rendering this protein susceptible to digestion and unable to interfere with absorption of biotin.
Absorption and Transport Digestion of Protein-Bound Biotin
The content of free biotin and protein-bound biotin in foods is variable, but the majority of biotin in
meats and cereals appears to be protein-bound via an amide bond between biotin and lysine. Neither the mechanisms of intestinal hydrolysis of proteinbound biotin nor the determinants of bioavailability have been clearly delineated. Because this bond is not hydrolyzed by cellular proteases, release is likely mediated by a specific biotin—amide hydrolase (biotinidase, EC 3.5.1.12). Biotinidase mRNA is present in pancreas and, in lesser amounts, in intestinal mucosa. Biotinidase is also present in many other tissues, including heart, brain, liver, lung, skeletal muscle, kidney, plasma, and placenta. Biotinidase also likely plays a critical role in intracellular recycling of biotin by releasing biotin from intracellular proteins such as carboxylases during protein turnover. Intestinal Absorption and Transport into Somatic Cells
At physiologic pH, the carboxylate group of biotin is negatively charged. Thus, biotin is at least modestly water-soluble and requires a transporter to cross cell membranes such as enterocytes for intestinal absorption, somatic cells for utilization, and renal tubule cells for reclamation from the glomerular filtrate. In intact intestinal preparations such as loops and everted gut sacks, biotin transport exhibits two components. One component is saturable at a km of approximately 10 mM biotin; the other is not saturable even at very large concentrations of biotin. This observation is consistent with passive diffusion. Absorption of biocytin, the biotinyl-lysine product of intraluminal protein digestion, is inefficient relative to biotin, suggesting that biotinidase releases