EDITOR-IN-CHIEF Benjamin Caballero Johns Hopkins University Center for Human Nutrition School of Hygiene and Public Health 615 North Wolfe Street Baltimore, Maryland 21205-2179 USA EDITORIAL ADVISORY BOARD EDITORS Luiz C Trugo Laboratory of Food and Nutrition Biochemistry Department of Biochemistry, Institute of Chemistry Federal University of Rio de Janeiro CT Bloco A Lab 528-A Ilha do Fundao, 21949-900 Rio de Janeiro Brazil Paul M Finglas Institute of Food Research Norwich Laboratory Colney Lane Norwich, NR4 7UA UK Peter Belton AFRC Institute of Food Research Norwich Laboratory Colney Lane Norwich NR4 7UA UK Peter Berry Ottaway Berry Ottaway Associates Ltd 1A Fields Yard Plough Lane Hereford HR4 0EL UK vi EDITORIAL ADVISORY BOARD Ricardo Bressani Universidad del Valle de Guatemala Institute de Investigaciones Aparto 82 Guatemala 01901 Barbara Burlingame Food and Agriculture Organization of the United Nations Viale delle Terme di Caracalla Rome 00100 Italy Jerry Cash Michigan State University Department of Food Science and Human Nutrition East Lansing MI 48824 USA Colin Dennis Campden & Chorleywood Rood Research Association Chipping Campden Gloucestershire GL55 6LD UK Johanna T Dwyer Tufts University USDA Human Nutrition Research Center 711 Washington Street USA Tee E-Siong Institute of Medical Research Division of Human Nutrition Jalan Pahang Kuala Lumpur 50588 Malaysia Patrick F Fox University College Department of Food Chemistry Cork Republic of Ireland Jesse Gregory University of Florida Food Science and Human Nutrition Department PO Box 110370 Newell Drive Gainesville FL 32611-0370 USA RJ Hamilton 10 Norris Way Formby Merseyside L37 8DB UK George D Hill Lincoln University Plant Sciences Group Field Service Centre Soil, Plant and Ecological Sciences Division PO Box 84 Canterbury New Zealand Harvey E Indyk Anchor Products Limited PO Box 7 Waitoa New Zealand Anura Kurpad St John’s Medical School Department of Nutrition Bangalore India Jim F Lawrence Sir FG Banting Research Centre, Tunney’s Pasture Health and Welfare Canada, Health Protection Branch Ottawa Ontario K1A 0L2 Canada F Xavier Malcata Universidade Catolica Portugesa Escola Superior de Biotecnologia Rua Dr Antonio Bernardino de Almeida Porto 4200 Portugal Keshavan Niranjan University of Reading Department of Food Science and Technology Whiteknights PO Box 226 Reading Berkshire RG6 2AP UK John R Piggott University of Strathclyde Department of Bioscience and Biotechnology 204 George Street Glasgow Scotland G1 1XW UK Vieno Piironen University of Helsinki Department of Applied Chemistry & Microbiology PO Box 27 Helsinki FIN-00014 Finland EDITORIAL ADVISORY BOARD Jan Pokorny Prague Institute of Chemical Technology Department of Food Science Technicka Street 5 CZ-16628 Prague 6 Czech Republic Terry A Roberts 59 Edenham Crescent Reading Berkshire RG2 6HU UK Délia B Rodriguez-Amaya University of Campinas Department of Food Science Faculty of Food Engineering PO Box 6121 Campinas SP 13081-970 Brazil Jacques P Roozen Wageningen University Agrotechnology and Food Sciences Laboratory of Food Chemistry PO Box 8129 6700 EV Wageningen The Netherlands Steve L Taylor University of Nebraska Lincoln Department of Food Science and Technology 143 H C Filley Hall East Campus Lincoln NE 68583-0919 USA Jean Woo Chinese University of Hong Kong Department of Medicine Prince of Wales Hospital Shatin N.T Hong Kong David C Woollard AgriQuality NZ Ltd Lynfield Food Services Centre 131 Boundary Road PO Box 41 Auckland 1 New Zealand Steven Zeisel University of North Carolina at Chapel Hill Department of Nutrition 2212 McGavran-Greenberg Hall Chapel Hill NC 27599-7400 USA vii FOREWORD There are no disciplines so all-encompassing in human endeavours as food science and nutrition. Whether it be biological, chemical, clinical, environmental, agricultural, physical – every science has a role and an impact. However, the disciplines of food science and nutrition do not begin or end with science. Politics and ethics, business and trade, humanitarian efforts, law and order, and basic human rights and morality all have something to do with it too. As disciplines, food science and nutrition answer questions and solve problems. The questions and problems are diverse, and cover the full spectrum of every issue. Life span is one such issue, covered from the nutritional basis for fetal and infant development, to optimal nutrition for the elderly. Another such issue is the time span of the ancient and wild agro-biodiversity that we are working to preserve, to the designer cultivars from biotechnology that we are trying to develop. Still another is the age-old food preparation methods now honoured by the ‘eco-gastronomes’ of the world, to the high tech food product development advances of recent years. As with most endeavours, our scientific and technological solutions can and do create new, unforeseen problems. The technologies that gave us an affordable and abundant food supply led to obesity and chronic diseases. The ‘‘green revolution’’ led to loss of some important agro-biodiversity. The technological innovation that gave us stable fats through hydrogenation, flooded the food supply with trans fatty acids. All these problems were identified through a multidisciplinary scientific approach and solutions are known. When technology created the problem and technology has found the solution, implementation is usually more successful. Reducing trans fatty acids in the food supply is case in point. Beyond the technologies, the solutions are more difficult to implement. We know how obesity can be reduced, but the solution is not directly technological. Hence, we show no success in the endeavour. Of all the problems still confounding us in food science and nutrition, none is so compelling as reducing the number of hungry people in the world. FAO estimates that there are 800 million people who do not have enough to eat. The World Food Summit Plan of Action, the Millennium Development Goals and other international efforts look to food science and nutrition to provide the solution. Yet we only have part of the solution—the science part. The wider world of effort in food science and nutrition needs to be more conscientiously addressed by scientists. This is the world of advocacy and action: advocacy for food and nutrition as basic human rights, coupled with action to get food where it is needed. But all those efforts would be futile if they are not based on sound scientific information. That is why works such as this Encyclopedia are so important. They provide to a wide readership, scientists and non-scientists alike, the opportunity to quickly gain understanding on specific topics, to clarify questions, and to orient to further reading. It is a pleasure to be involved in such an endeavour, where experts are willing to impart their knowledge and insights on scientific consensus and on exploration of current controversies. All the while, this gives us optimism for a brighter food and nutrition future. Barbara Burlingame 25 February 2003 INTRODUCTION There is no factor more vital to human survival than food. The only source of metabolic energy that humans can process is from nutrients and bioactive compounds with putative health benefits, and these come from the food that we eat. While infectious diseases and natural toxins may or may not affect people, everyone is inevitably affected by the type of food they consume. In evolutionary terms, humans have increased the complexity of their food chain to an astounding level in a relatively short time. From the few staples of some thousand years ago, we have moved to an extraordinarily rich food chain, with many food items that would have been unrecognizable just some hundred years ago. In this evolution, scientific discovery and technical developments have always gone hand in hand. The identification of vitamins and other essential nutrients last century, and the development of appropriate technologies, led to food fortification, and thus for the first time humans were able to modify foods to better fulfill their specific needs. As a result, nutritional deficiencies have been reduced dramatically or even eradicated in many parts of the world. This evolution is also yielding some undesirable consequences. The abundance of high-density, cheap calorie sources, and the market competition has facilitated overconsumption and promoted obesity, a problem of global proportions. As the food chain grows in complexity, so does the scientific information related to it. Thus, providing accurate and integral scientific information on all aspects of the food chain, from agriculture and plant physiology to dietetics, clinical nutrition, epidemiology, and policy is obviously a major challenge. The editors of the first edition of this encyclopedia took that challenge with, we believe, a great deal of success. This second edition builds on that success while updating and expanding in several areas. A large number of entries have been revised, and new entries added, amounting to two additional volumes. These new entries include new developments and technologies in food science, emerging issues in nutrition, and additional coverage of key areas. As always, we have made efforts to present the information in a concise and easy to read format, while maintaining rigorous scientific quality. We trust that a wide range of scientists and health professionals will find this work useful. From food scientists in search of a methodological detail, to policymakers seeking update on a nutrition issue, we hope that you will find useful material for your work in this book. We also hope that, in however small way, the Encyclopedia will be a valuable resource for our shared efforts to improve food quality, availability, access, and ultimately, the health of populations around the world. Benjamin Caballero Luiz Trugo Paul Finglas A Acceptability of Food See Food Acceptability: Affective Methods; Market Research Methods ACESULFAME/ACESULPHAME J F Lawrence, Health and Welfare Canada, Ontario, Canada Production and Physical and Chemical Properties Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Acesulfame K (Figure 1) is structurally related to saccharin. It also has many of the same physical and chemical properties. Acesulfame was one of a series of sweet-tasting substances synthesized by Hoechst AG in the late 1960s. All of these had in common the oxathiazinone dioxide ring structure. The synthesis involved reaction of fluorosulfonyl isocyanate with either acetylene derivatives or with active methylene compounds such as a-diketones, a-keto acids, or esters. The latter reaction is used for the commercial production of acesulfame K. A generalized reaction scheme for synthesis of the oxathiazinone dioxide ring structure is shown in Figure 2. Many analoges have been prepared and evaluated for taste properties. The potassium salt of the 6-methyl derivative, acesulfame K, displayed the best sensory and physical properties and thus it has received extensive testing aimed at obtaining approval for its use in diet foods. Acesulfame K is a white crystalline material which is stable up to 250
C, at which temperature it decomposes. The free acid form of the sweetener has a distinct melting point of 123.5
C. Acesulfame K has a specific density of 1.83. When dissolved in water it produces a nearly neutral solution while the free acid is strongly acidic (pH of a 0.1 mol l1 aqueous solution being 1.15). The sweetener is very soluble in water; a 27% solution can be prepared at 20
C. The solubility of acesulfame K increases significantly with temperature. At 80
C, 50% solutions can be prepared; because of this, greater than 99% purity can be obtained by crystallization. It is substantially less soluble in common solvents such as ethanol, methanol, or acetone. Background 0001 Acesulfame K (potassium salt of 6-methyl-1,2,3oxathiazine-4(3H)-one-2,2-dioxide; Figure 1) is a high-intensity artificial sweetener which is about 200 times as sweet as sucrose (compared to a 3% aqueous sucrose solution). It was accidentally discovered in 1967 by Dr. Karl Clauss, a researcher with Hoechst AG in Frankfurt, FRG, during his experiments on new materials research. The sweetener is not metabolized by the human body and thus contributes no energy to the diet. It is now approved for use in more than 20 countries. Sweetness 0002 The sweetness properties of acesulfame K are similar to saccharin. It has a clean, sharp, sweet taste with a rapid onset of sweetness and no lingering aftertaste at normal use levels. However, at high concentrations, equivalent to 5% or 6% sucrose solutions, acesulfame K does possess a bitter, chemical aftertaste. The intensity of sweetness of acesulfame K, in common with other artificial sweeteners, varies depending upon its concentration and the type of food application. For example, it is 90 times sweeter than a 6% sucrose solution, 160 times sweeter than a 4% sucrose solution and 250 times sweeter than a 2% sucrose solution. Mixtures of acesulfame K with other intense sweeteners, such as aspartame or cyclamate, result in some synergistic increases in sweetness. Mixtures with saccharin are somewhat less synergistic. 0003 0004 0005 0006 2 ACESULFAME/ACESULPHAME Table 1 Typical use levels of acesulfame K in diet foods CH3 O C O N− SO2 K+ fig0001 Figure 1 Structure of acesulfame K. Reproduced from Acesulphame/Acesulfame, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press. O C N O O O SO2F N H O Fluorosulfonylisocyanate O N H 0007 0008 O NaOH SO2 NH SO2F O Figure 2 Synthesis of the acesulfame ring structure using fluorosulfonyl isocyanate and tert-butylacetoacetate as starting materials. Reproduced from Acesulphame/Acesulfame, Encyclopaedia of Food Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds), 1993, Academic Press. The stability of acesulfame K in the solid state is very good. It can be stored at ambient temperature for 10 years without decomposition. Aqueous solutions at pH 3 or greater may also be stored for extended periods without detectable decomposition or loss of sweetness. However, below pH 3, significant hydrolysis may occur at elevated temperatures. For example, at pH 2.5 an aqueous buffered solution of acesulfame K would decompose by about 30% after 4 months of storage at 40
C, whereas no decomposition occurs under the same conditions within the pH range of 3–8. At 20
C, less than 10% decomposition of acesulfame K occurs after 4 months’ storage at pH 2.5, indicating that under normal storage conditions aqueous solutions of the sweetener are very stable. Acesulfame K is stable under most food-processing conditions, including the elevated temperature treatments encountered in pasteurization and baking. Food Uses 0009 Concentration (mg kg ) Soft drinks Coffee and tea Jams and marmalades Ready-to-eat desserts Chewing gum 1000 267 3000 1000 2000 Safety and Regulatory Status O SO2F H3C O fig0002 ∆ O Food products desserts, breakfast cereals, and chewing gum. Table 1 lists approximate concentration levels of acesulfame K typically used in several types of foods. O + tbl0001 1 Because of its stability, acesulfame K has been evaluated in a wide variety of diet food products, including table-top sweeteners, soft drinks, fruit preparations, Acesulfame K has been subjected to extensive feeding studies in mice, rats, and dogs. The substance is not considered to be carcinogenic, mutagenic, or teratogenic. It is excreted unmetabolized in test animals or humans. The current maximum acceptable daily intake (ADI: the maximum amount that can be consumed daily for a lifetime without appreciable risk) established by the Food and Agriculture Organization/World Health Organization (FAO/WHO) Joint Expert Committee on Food Additives in 1990 is 5 mg per kg body weight. This value is based on the highest amount fed to animals for which there was no effect. The first regulatory approval for acesulfame K was by the UK in 1983. Since then it has received approval for specific uses in more than 20 countries. 0010 0011 Analysis Thin-layer chromatography, isotachorphoresis, and high-performance liquid chromatography (HPLC) have been evaluated for the determination of acesulfame K in a variety of matrices, including liquid and solid food products, animal feed, and biological fluids. Of the three, HPLC is perhaps the most useful since the efficiency of the chromatography coupled with selective detection (ultraviolet absorbance) enable quantitative measurements to be made in rather complex food samples. In addition, the sample preparation is minimal, usually involving a water extraction for solid samples or a filtration and dilution of liquid samples before direct HPLC analysis. Acesulfame K has been incorporated into a multisweetener analytical method employing HPLC. See also: Carbohydrates: Sensory Properties; Chromatography: High-performance Liquid Chromatography; Gas Chromatography; Legislation: Contaminants and Adulterants; Saccharin; Sweeteners: Intensive 0012 ACIDOPHILUS MILK 3 Further Reading Franta R and Beck B (1986) Alternatives to cane and beet sugar. Food Technology 40: 116–128. Kretchmer N and Hollenbeck CB (1991) Sugars and Sweeteners. Boca Raton: CRC Press. Lawrence JF and Charbonneau CF (1988) Determination of seven artificial sweeteners in diet food preparations by reverse-phase liquid chromatography with absorbance detection. Journal of the Association of Official Analytical Chemists 71: 934–937. O’Brien-Nabors L and Gelardi RC (1991) Alternative Sweeteners. New York: M. Dekker. ACIDOPHILUS MILK W Kneifel and C Bonaparte, University of Agricultural Sciences, Vienna, Austria Copyright 2003, Elsevier Science Ltd. All Rights Reserved. derived from acido (acid) and philus (loving) and this designation reflects the acidotolerant potential of this species. In 1959, Rogosa and Sharpe presented a detailed description of this bacterium. Background and History 0001 0002 0003 Since the first documentation of the beneficial role of Lactobacillus acidophilus in correcting disorders of the human digestive tract in 1922, products containing L. acidophilus, especially various types of Acidophilus milk, have become increasingly popular. Today, a multitude of such products are commercially available, many of them being assigned to the category of probiotic foods. Most of these probiotics possess a bacterial microflora of well-documented and scientifically proven bacterial strains with several benefical properties. Besides other categories of foods containing special ingredients, these products have also recently been subclassified under the umbrella of functional foods. In general, the human body is inhabitated by more than 500 different bacterial species; among them, the lactobacilli play an important ecological role. Besides their important gut-associated function, lactobacilli are also part of various other human-specific microbial ecosystems, e.g., skin, vagina, mouth, nasal, and conjunctival secretions. L. acidophilus is the best known of the health-promoting lactobacilli of mammals and a naturally resident species of the human gastrointestinal tract. It colonizes segments of the lower small intestine and parts of the large intestine, together with other lactobacilli species, such as L. salivarius, L. leichmanii, and L. fermentum. It is interesting to note that these resident Lactobacillus species should be distinguished from the spectrum of so-called transient Lactobacillus species, which are represented by L. casei. Historically, in 1900, Australian researchers isolated L. acidophilus from fecal samples of bottle-fed infants for the first time and named it ‘Bacillus acidophilus.’ The actual nomenclature L. acidophilus is Fundamental Characteristics of Lactobacillus acidophilus Together with 43 other species, L. acidophilus is listed as a member of the genus Lactobacillus which belongs to the heterogeneous category of lactic acid bacteria. Lactobacilli are Gram-positive, nonmotile, catalase-negative, nonspore-forming rods with varying shapes, ranging from slender, long rods to coccobacillary forms. They are considered as (facultative) anaerobes with microaerophilic properties. L. acidophilus usually appears as rods with rounded ends, with a size of 0.6–0.9  1.5–6 mm, mainly organized singly or in pairs or short chains (Figure 1). The cell wall peptidoglycan is of the Lys-d-Asp type; the mean proportion of guanine and cytosine in the DNA ranges between 34 and 37%. With rare exceptions, this bacterium shows good growth at 45
C but not below 15
C, having an optimum growth temperature in the range of 35–38
C. Substrates with pH values of 5.5–6.0 are preferred. Metabolically, it is a typical obligately homofermentative bacterium and produces racemic lactic acid (both the lþ and the d enantiomeric forms) from lactose, glucose, maltose, sucrose, and other carbohydrates. Usually, it follows the Embden–Meyerhof–Parnas pathway for glucose metabolism. Important growth factor requirements are acetic or mevalonic acid, riboflavin, pantothenic acid, niacin, folic acid and calcium, but not cobalamin, pyridoxine, and thymidine. Starch and cellobiose are fermented by most strains. Another differential key criterion for the distinction from other lactobacilli (e.g., L. delbrueckii subsp. bulgaricus) is its capability of cleaving esculin. Further differential criteria are the utilization of trehalose, melibiose, raffinose, ribose, and lactose. While 0004 4 ACIDOPHILUS MILK 10µm fig0001 Figure 1 Microphotograph of a Lactobacillus acidophilus culture (deep-frozen culture concentrate cultured in MRS broth; for details see Table 1). lactic acid bacteria. Because of their beneficial L. acidophilus-related properties, products containing this bacterium have been used in the treatment of gastrointestinal disorders and to reestablish the function of the intestine after treatment with antibiotics. Other features of these products are the provision of b-galactosidase to humans having an enzymatic deficiency for lactose digestion or, particularly when used in conjunction with fructooligosaccharides (oligofructose), the reduction of fecal enzymes (glucuronidase, nitroreductase, azoreductase) which obviously play some role in some stages of precancerogenesis. Since L. acidophilus produces equimolar amounts of l(þ) and d() lactic acid, products fermented with this bacterium offer the advantage of a reduced d() lactate content, compared to classical yogurt. However, the acidification potential of this bacterium is often low and varies considerably among strains. Products with Lactobacillus acidophilus physiological parameters allow some distinction from other food-relevant lactobacilli, it is not possible to use a phenotypical basis to discriminate sufficiently among L. acidophilus, L. johnsonii, L. gasseri, L. crispatus, and L. amylovorus. All five species are usually assigned to the L. acidophilus cluster. A distinction of these species can be facilitated by applying genotypical techniques and methods based on DNA homology, the molar amounts of guanine plus cytosine in the DNA, or by the analysis of certain cell wall components. Physiological Actions of Lactobacillus acidophilus 0005 Because of the properties described above and its pronounced bile salt resistance, L. acidophilus is well adapted to the environmental conditions of the gastrointestinal tract. Proteins in the cell wall may be important in attaching the bacterium to the mucosal cells of the intestine. With strain-dependent variations, L. acidophilus contributes to the inhibition of the multiplication of pathogenic and putrefactive bacteria in the intestine due to the production of organic acid and trace amounts of H2O2. Furthermore, strain-specific inhibitory substances can be excreted by certain strains. In this context, numerous antagonistic peptides (bacteriocins) have been isolated from certain strains of L. acidophilus. For example, some of them were described as lactocidin, acidophilin, acidolin, lactosin B, and lactacin B and possess some ‘antibiotic’ potential against salmonellae, staphylococci, Escherichia coli, and clostridia, and partly also against other species of At present, a broad variety of products containing L. acidophilus is on the market. This bacterium has been incorporated into fermented as well as nonfermented milks of different levels of dry matter and fat (Figure 2). Cows’ milk is the main substrate which is processed using the same basal technology as applied for the manufacture of yogurt or other cultured dairy products. Hence, continuous production lines with conventional or aseptic filling systems are used. Some of the products also contain added fruits and flavoring agents. Fermented dairy products containing L. acidophilus as a single bacterial culture are primarily of local importance in Russia, Eastern European countries, and Scandinavia. In contrast, in other European regions, L. acidophilus is usually used in combination with other microorganisms (e.g., Bifidobacterium spp., Streptococcus thermophilus, L. delbrueckii subsp. bulgaricus, L. casei). Milk cultured with such multicomponent starter cultures (including L. acidophilus) is produced in increasing numbers and varieties and consumed frequently by many people. Among these dairy products, a distinction can be made between the so-called ‘mild’ yogurt products (yogurt-related fermented milks, with or without fruits) which are based on a fermentation with various thermophilic bacteria (many of them are assigned to the area of probiotics), and so-called ‘Acidophilus milk’ products which are usually fermented by means of mesophilic lactic acid bacteria (e.g., strains of Lactococcus lactis or Leuconostoc cremoris or combinations of both), in addition to L. acidophilus. A general flow diagram for the production of such an Acidophilus milk fermented under mesophilic 0006 0007 ACIDOPHILUS MILK 5 Acidophilus products Single-culture products Multiple-culture products Soymilk-based Acidophilus milk 'Sweet' Acidophilus milk Fermented Acidophilus milk (liquid, dried) fig0002 0008 0009 Other products Acidophilus milk cofermented with mesophilic lactic acid bacteria Fermented milk manufactured with L. acidophilus and other thermophilic lactic acid bacteria and/or bifidobacteria Fermented milk manufactured with L. acidophilus and yeasts, facultatively plus meso- or thermophilic lactic acid bacteria Fermented Acidophilus paste or cubes (enriched with sugar), texturized Figure 2 Survey of the diversity of food products containing Lactobacillus acidophilus. conditions is presented in Figure 3. Deep-frozen culture concentrates or freeze-dried bacteria or, very rarely, liquid cultures are inoculated into the milk base. The fermentation is usually performed overnight for 15–20 h. Stirred products, with a liquid character, are usually made, but set-style fermented Acidophilus milk products with increased levels of solid-nonfat are also available. Other categories of products include specially fermented drinks (e.g., L. acidophilus plus yeasts, with or without other lactic acid bacteria, resembling kefir and named acidophilin), texturized products with a reduced water content, which are offered in a pasty form or cut in cubes, or powdered milk which has been fermented with L. acidophilus before drying. Many of these product types have a local significance as dietary adjuncts. In Russia, these products even play some role as therapeutic agents and have been well recognized with regard to their medical relevance. Nonfermented milk containing L. acidophilus is also offered by some dairies. Such products are usually produced from standardized milk which is supplemented with a culture concentrate (deep-frozen pellets or lyophylisate) of L. acidophilus under cooled conditions, followed by stirring before filling into cartons or beakers. Some of these products are also fortified with fat-soluble vitamins (A, D, E), watersoluble vitamins (thiamin), and trace elements (iron). While a pronounced metabolic activity of the L. acidophilus strains is desired for all those products which are produced by fermentation, storage-resistant but not fast-growing cultures (strains) are needed for the manufacture of ‘sweet’ (nonfermented) Acidophilus milk in order not to alter the sensory properties during storage. The sensory characteristics of nonfermented ‘sweet’ Acidophilus milk are comparable with regular milk; those of fermented Acidophilus milk (mesophilic varieties) are similar to those of regular cultured or sour milks which are manufactured using a butter flavor-producing mesophilic culture, since almost no acetaldehyde, which is typical for yogurt, but some diacetyl-based butter aroma is generated during fermentation caused by citrate-fermenting mesophilic lactic acid bacteria. Since L. acidophilus possesses alcohol dehydrogenase activity, which is capable of reducing acetaldehyde, only low levels of this compound are found in the corresponding products. Thus, yogurt-related dairy products (thermophilic varieties) containing L. acidophilus often exhibit a milder and less acidic taste than classical yogurt, i.e., that manufactured by a cofermention of S. thermophilus and L. delbrueckii subsp. bulgaricus. Sensorically, this classical yogurt is dominated by acetaldehyde which introduces some kind of astringent characteristic and typical sharpness. Moreover, many classical yogurt cultures, in particular owing to the Lactobacillus component of the culture, exhibit a continued acidification activity even under cooled conditions on the shelves of retail shops. Besides the sensory changes, this ‘overacidification’ can also lead to textural problems (syneresis, whey separation). 0010 6 ACIDOPHILUS MILK Process milk with variable dry matter and fat contents Dual-step homogenization with 15 000-20 000 kPa at 65-70 8C Heat treatment for 5-10 min at 90-95 8C Cooling to fermentation temperature (varies from 22 to 30 8C) Inoculation with L. acidophilus and a mesophilic starter culture Fermentation period at defined temperature (varies from 22 to 30 8C) Stirring, cooling to 10-12 8C and filling into packaging units (beakers, cartons, etc.) fig0003 Figure 3 General production steps of the manufacture of fermented Acidophilus milk using a combined fermentation with Lactobacillus acidophilus and mesophilic lactic acid starter culture. Data compiled after various manufacturers’ recommendations. Obviously because of these effects, preferences of consumers for the milder yogurts with L. acidophilus have been observed in many countries. A completely different group of Acidophilus products are the pharmaceutical preparations containing L. acidophilus. Capsules, suppositories, and soluble powders packaged in sachets are enriched with bacterial lyophylisates and used for therapeutic purposes. They are marketed in pharmacies and health stores. 0011 Bacterial Viable Count and Bacterial Stability of Acidophilus milk Although milk is a substrate containing almost a universal array of nutrients, it does not fully meet the growth requirements of L. acidophilus. For this purpose, additives and growth promoters consisting of a mixture of natural compounds which support and enhance bacterial growth are recommended for supplementation of the fermentation milk by most culture suppliers. Usually, they are added to the milk base in small amounts, before inoculation. In addition, the use of multicomponent cultures offers the advantage of inducing synergistic effects among the bacterial microflora which may also positively influence the propagation rate and the stability of the bacteria. According to legal aspects and to consumer expectations, products labeled as Acidophilus milk or as containing L. acidophilus necessarily have to contain a significant number of these microorganisms. In this context, a group of experts of the International Dairy Federation has recommended that L. acidophilus shall be detected in such products at a level of at least 1 million CFU ml1 or g, at their sell-by dates. Recently, studies performed in several countries have shown that many commercially available products can meet this limit, but with a considerable number of products a decrease in the L. acidophilus counts has been observed during a storage period of approximately 3–5 weeks. Due to the fact that the expression of beneficial effects is based on a high number of active bacteria, a high viable count and pronounced bacterial stability have become important goals in product development and optimization. Viable counts of L. acidophilus-containing dairy products are usually enumerated by culture methods based on plate count techniques with media designed for culturing lactic acid bacteria (e.g., MRS, Rogosa agar, TGV agar; for details see Table 1). To enhance the discriminatory power of these media (this is of particular relevance in the examination of products which contain a mixed microflora), media are modified by slight acidification and/or by supplementation 0012 0013 0014 0015 ACIDS/Properties and Determination tbl0001 Table 1 Media used for culturing Lactobacillus acidophilus Media References Lactobacillus agar according to De Man JD, Rogosa M and Sharpe ME (1960) Journal of Applied Bacteriology 23: 130–135. Rogosa agar Lactobacillus selective agar according to Rogosa M Mitchell and JA, Wiseman RF (1951) Journal of Bacteriology 62: 132–133. TGV agar Agar medium according to Galesloot T, Hassing F and Stadhouders J (1961) Netherlands Milk and Dairy Journal 15: 127–150. 7 Yogurt: The Product and its Manufacture; Yogurt-based Products; Dietary Importance MRS agar with antibiotics (e.g., vancomycin at different levels) or with other selective agents (cellobiose, conjugates with chromogenic indicator dyes, esculin, etc.). In many cases, the parallel use of different media selective for each of the bacterial components is necessary to allow the reliable microbiological monitoring of these Acidophilus products. Moreover, microscopical verification of isolates harvested from the different media usually completes their routine assessment. Although a number of media and methodologies have been described in the literature, no official standard method is available yet. See also: Fermented Milks: Types of Fermented Milks; Functional Foods; Lactic Acid Bacteria; Probiotics; Further Reading Fondén R, Mogensen G, Tanaka R and Salminen S (2000) Effect of culture-containing dairy products on intestinal microflora, human nutrition and health – current knowledge and future perspectives. In: IDF Bulletin, no. 352, pp. 5–30, Brussels: International Dairy Federation. Hammes WP (1995) The genus Lactobacillus. In: Wood JB and Holzapfel WH (eds) The Genera of Lactic Acid Bacteria, pp. 19–54. London: Blackie Academic & Professional. Kanbe M (1992) Uses of intestinal lactic acid bacteria and health. In: Nakazawa Y amd Hosono A, (eds) Functions of Fermented Milk. Challenges for the Health Sciences, pp. 289–304. London: Elsevier Applied Science. Kneifel W and Pacher B (1993) An X-Glu based agar medium for the selective enumeration of Lactobacillus acidophilus in yogurt-related milk products. International Dairy Journal 3: 277–291. Lee YK, Nomoto K, Salminen S and Gorbach SL (1999) Handbook of Probiotics. New York: John Wiley. Mital BK and Garg SK (1992) Acidophilus milk products: manufacture and therapeutics. Food Reviews International 8: 347–389. Tamime AY and Robinson RK (1999) Yoghurt Science and Technology. Cambridge: CRC, Woodhead Publishing. ACIDS Contents Properties and Determination Natural Acids and Acidulants Properties and Determination J D Dziezak, Dziezak & Associates, Ltd., Hoffman Estates, IL, USA Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Background 0001 In very general terms, an acid is a compound that contains or produces hydrogen ions in aqueous solutions, has a sour taste, and turns blue litmus paper red. A more comprehensive definition, given by the US chemist G.N. Lewis, states that acids are substances that can accept an electron pair or pairs, and bases are substances that can donate an electron pair or pairs. This definition, applicable to both nonaqueous and aqueous systems, requires that an acid be either a positive ion or a molecule with one or more electron-deficient sites with respect to a corresponding base. The definition most widely used to describe acid– base reactions in dilute solution is one that was proposed independently by two scientists in 1923 – the Danish chemist J.N. BrØnsted and the US chemist T.M. Lowry. The BrØnsted–Lowry theory defines an acid as a proton donor, that is, any substance (charged or uncharged) that can release a hydrogen ion or proton. A base is defined as a proton acceptor or any substance that can accept a hydrogen ion or proton. 0002 tbl0001 Table 1 Structure, ionization constant, pKa, and key physical and chemical properties of acidulantsa Acid Structure Ionization constant(s) pKa Physical form Melting point (
C) Solubility (g per100 ml of water) Hygroscopicity Taste characteristics Acetic acid CH3COOH 1.76  105 at 25
C 4.76 Clear, colorless liquid 8.5 Soluble na Tart and sour Adipic acid COOH K1 ¼ 3.71  105 4.43 Crystalline powder 152 1.9 g at 20
C Low level of hygroscopicity Smooth lingering tartness; complements grape flavors CH2 K2 ¼ 3.87  106 at 25
C 5.41 COOH K1 ¼ 7.10  104 3.14 Moderately hygroscopic Tart; delivers a ‘burst’ of flavor CH2 K2 ¼ 1.68  105 K3 ¼ 6.4  107 at 25
C 4.77 6.39 Nonhygroscopic Tart; has an affinity for grape flavors 83 g at 90
C CH2 CH2 CH2 COOH Citric acid HO C COOH Crystalline powder CH2 COOH Anhydrous Hydrous Fumaric acid COOH K1 ¼ 9.30  104 3.03 K2 ¼ 3.62  105 at 18
C 4.44 CH HC COOH White granules or crystalline powder 153 135–153 286 181 g at 25
C 208 g at 25
C 0.5 g at 20
C 9.8 g at 100
C 1.99  104 (for gluconic acid) 3.7 White crystalline powder 153 59 g at 25
C Nonhygroscopic Neutral taste with acidic aftertaste, when hydrolyzed 1.37  104 at 25
C 3.86 Liquid; also available in dry form 16.8 Very soluble na Acrid K1 ¼ 3.9  104 3.40 Crystalline powder 132 62 g at 25
C Nonhygroscopic Smooth tartness K2 ¼ 7.8  106 at 25
C 5.11 Phosphoric acid K1 ¼ 7.52  103 2.12 Very soluble in hot water na Acrid 7.21 12.67 Tartaric acid K2 ¼ 6.23  108 K3 ¼ 2.2  1013 K1 and K2 at 25
C; K3 at 18
C K1 ¼ 1.04  103 147 g at 25
C Nonhygroscopic Extremely tart; augments fruit flavors, especially grape and lime K2 ¼ 4.55  105 at 25
C 4.34 Glucono-d-lactone O C HC OH HO CH O HC OH HC CH2OH Lactic acid CH3 HC OH COOH Malic acid COOH HO CH CH2 COOH COOH 2.98 Liquid Crystalline powder 168–170 HO CH HC OH COOH na, not applicable. a Adapted with permission from Food Technology, Acidulants: Ingredients that do more than meet the acid test. 44(1): 76–83. Institute of Food Technologists, Chicago, Illinois, USA. 10 ACIDS/Properties and Determination 0003 This article discusses the physicochemical properties of acids and describes several methods for their analysis. Strong Versus Weak Acids 0004 The strength of a BrØnsted–Lowry acid depends on how easily it releases a proton or protons. In strong acids, owing to their weaker internal hydrogen bonds, the protons are loosely held. As a result, in aqueous solutions, almost all of the acid reacts with water, leaving only a few unionized acid molecules in the equilibrium mixture. The reaction takes place according to eqn (1): HA þ H2 O Ð H3 Oþ þ A 0005 In this equation, HA represents the undissociated acid, H3Oþ the hydronium ion formed when a proton combines with one molecule of water, and A the conjugate base of HA. Unlike strong acids, weak acids exist largely in the undissociated state when mixed with water, since only a small percentage of their molecules interact with water and dissociate. Most acids found in foods, including acetic, adipic, citric, fumaric, malic, phosphoric and tartaric acids, and glucono-d-lactone, are classified as weak or medium strong acids. Physicochemical properties, including the ionization constant, pH, the apparent dissociation constant (pKa) and buffering capacity, are discussed below and are listed in Table 1. Ionization Constant 0007 The tendency for an acid or acid group to dissociate is defined by its ionization constant, also denoted as pKa. The ionization constant, given at a specified temperature, is expressed as: Ka ¼ 0008 ½H3 Oþ ½A , ½HA pH Measurement of acidity is an important aspect of ascertaining the safety and quality of foods. Such measurements are given in terms of pH, which is defined as the negative logarithm of the hydronium ion concentration (strictly, activity): ð1Þ Physicochemical Properties 0006 acid. Acids such as citric acid and phosphoric acid, which have three transferable hydrogens, are called triprotic or tribasic acids. Ionization of polyprotic acids occurs in a stepwise manner with the transfer of one hydrogen ion at a time. Each step is characterized by a different ionization constant. ð2Þ where the brackets designate the concentration in moles per liter. The ionization constant is a measure of acid strength: the higher the Ka value, the greater the number of hydrogen ions liberated per mole of acid in solution and the stronger the acid. Acids with more than one transferable hydrogen ion per molecule are termed ‘polyprotic’ acids. Monoprotic or monobasic acids are those that can liberate one hydrogen ion, such as acetic acid and lactic acid. Those containing two transferable hydrogen ions are called diprotic or dibasic acids and include, for example, adipic acid and fumaric pH ¼ log10 1 ¼  log10 ½H3 Oþ : ½H3 Oþ 0009 ð3Þ The lower the pH value, the higher the hydrogen ion concentration associated with it. A pH value of less than 7 indicates a hydrogen ion concentration greater than 107 M and an acidic solution; a pH value of more than 7 indicates a hydrogen ion concentration of less than 107 M and a basic solution. When the hydronium and hydroxide ions are equal in concentration, the solution is described as neutral. (See pH – Principles and Measurement.) It is also important to note that, because the pH scale is logarithmic, a difference of one pH unit represents a 10-fold difference in hydrogen ion concentration. 0010 0011 pKa The term pKa is defined as the negative logarithm of the dissociation constant: pKa ¼ log10 1 ¼  log10 Ka : Ka 0012 ð4Þ The pKa corresponds to the pH value at the midpoint of a titration curve developed when one equivalent of weak acid is titrated with base, and the pH resulting from each incremental addition of base is plotted against the equivalents of hydroxide ions added. The pH of a system is at the pKa when the concentrations of acid (HA) and conjugate base (A) are equal. At the pKa and, to a lesser extent, in the area extending to within one pH unit on either side of the pKa, the system resists changes in pH resulting from addition of small increments of acid or base. In other words, at the pKa, acids and their salts function as buffers. The number of pKas that an acid has depends on the number of hydrogen ions it can liberate. Monoprotic acids have a single pKa, whereas di- and triprotic acid have two and three pKas, respectively. 0013 0014 0015 ACIDS/Properties and Determination 0016 Strong acids have low pKa values, and strong bases have high pKa values. Buffering Capacity 0017 0018 0019 0020 A solution of a weak acid (or a weak base) and its corresponding salt is called a buffer solution. In these systems, the hydronium ion content is not significantly changed when a small amount of acid or base is added to that solution. The reason that buffer solutions resist appreciable changes in pH can be best illustrated by an example. If a small amount of hydrochloric acid is added to a buffer solution composed of acetic acid and sodium acetate, the protons from the hydrochloric acid would associate with the acetate ions to form unionized molecules of acetic acid. As the newly formed acid molecules ionize, the equilibrium would shift towards forming more hydronium ions (eqn (1)). This would result in only a very slight increase in pH. Similarly, the addition of a small amount of sodium hydroxide to the same buffer solution would have little effect on pH. Hydroxide ions from the sodium hydroxide would combine with hydronium ions in the equilibrium mixture, forming undissociated molecules of sodium hydroxide. More of the acid molecules would then dissociate to replace the hydronium ions lost; though a new equilibrium system would be created, it would produce only a minimal effect on pH. The quantity of acid or base that a buffer solution is capable of consuming before a change in pH is realized is termed the ‘buffering capacity.’ The buffering capacity is defined as the number of moles of strong acid or base required to increase the pH by one unit in 1 l of buffer solution. The buffering capacity of a solution is greatest at its pKa value where the concentrations of acid and conjugate base are equal. adding a suitable indicator to a solution and matching the color of the solution to a standard solution containing the same indicator. This method can estimate pH to the nearest 0.1 pH unit. A more accurate technique and the one most frequently employed, the potentiometric method, uses a pH meter to determine hydrogen ion concentration. The two electrodes of the meter – a calomel reference electrode and a glass indicator electrode – are immersed in the solution, of known temperature, whose pH is to be measured. The electrode potential of the indicator electrode is linearly related to changes in hydrogen ion concentration and therefore pH. The total concentration of acid in a solution can be determined by titration. The titration process is performed by placing in a flask a known volume of acid solution whose concentration is unknown. To the flask, a few drops of indicator, e.g., phenolphthalein, which is colorless in acid solutions and pink in basic solutions, is introduced. A base solution of known concentration is then gradually added until the acid is completely neutralized. This point is indicated when the solution permanently changes color. The concentration of acid can then be calculated from the volume of base solution used. The value obtained, called titratable acidity, is an estimate of the total acid in the solution. It accounts for both the free hydronium ions present in the equilibrium mixture and the hydrogen ions released from undissociated acid molecules. For weak acids, the titratable acidity is different from the actual acidity (hydrogen ion concentration), since these compounds exist largely in the undissociated state in solution. For strong acids, however, titratable acidity and actual acidity are virtually the same, since strong acids and their salts are completely ionized in solution. Chromatographic Methods Quantitative determinations of acidity play an important role in ensuring food product quality and stability. Information obtained on acid levels can help in detecting cases of food adulteration, monitoring fermentation processes, and evaluating the organoleptic properties of fermented foods. pH determination, titratable acidity, chromatographic methods, and capillary electrophoresis are procedures commonly employed by the food industry to determine food acids. (See Adulteration of Foods: Detection.) Gas chromatography (GC) and high-performance liquid chromatography (HPLC) have almost entirely replaced paper and thin-layer chromatography as methods for identifying and quantifying food acids. pH can be measured by two techniques: colorimetric and potentiometric. The colorimetric method involves 0022 Titratable Acidity Analytical Methods pH Determination 0021 11 Gas Chromatography GC has been used to analyze organic acids in fruit and fruit juice. Analysis involves preparing volatile derivatives such as methyl esters of the organic acids, prior to their injection into the gas chromatograph. Derivatives are chromatographed on a nonpolar stationary phase column and detected by a flame ionization detector. By use of GC, malic acid has been shown to be a major constituent of many fruits, including apples, pears, grapes, peaches, and nectarines, and significant 0023 0024 0025 0026 0027 12 ACIDS/Natural Acids and Acidulants levels of citric acid have been found in citrus fruits such as orange, lemon, and grapefruit, and in noncitrus fruits, including pears, nectarines, cherries, and strawberries. (See Chromatography: Gas Chromatography.) phenols, respectively. Acids are detected by a UV detector, and the signal is sent to a data collector. The resulting separation is graphically represented as an electrophoregram. Enzymatic Analysis 0028 0029 0030 High-performance Liquid Chromatography HPLC is used more extensively than GC to determine organic acids because the technique requires little or no chemical modification to separate these nonvolatile compounds. Separation is usually done on either a reversed-phase C8 or C18 column or a cationexchange resin column operated in the hydrogen mode. Acids are detected by either refractive index (RI) or ultraviolet (UV) detectors. RI detection requires prior removal of any sugars present that potentially can interfere with quantification; sugar removal is not required for UV detection at 220–230 nm. Adulteration of a commercial cranberry juice drink was detected using HPLC when the test yielded different results for organic acids, sugars, and anthocyanin pigments than those obtained for a standard juice drink. Atypical citric and/or malic acid contents and presence of a natural colorant, probably grape skin extract, confirmed that the drink was adulterated. In wine-making, HPLC is used to monitor concentrations of tartaric, malic, succinic, citric, lactic, and acetic acids, which contribute tartness and stability to the finished product. A common approach involves using a column containing a strong cation exchange resin and eluting the sample with dilute sulfuric acid; the eluant is then analyzed for acids by RI detection. This column has the additional advantage of permitting the simultaneous detection and quantification of ethanol and the monitoring of wine for adulteration with methanol. Organic acids in wine can also be separated using ion chromatography with a conductivity detector. (See Chromatography: Highperformance Liquid Chromatography.) Enzyme assays provide another means of analyzing acids. For example, an enzymatic assay of l-malic acid uses an NAD(P)-linked malic enzyme and involves spectrophotometrically measuring the absorbance of NADPH, a reaction product, at 340 nm. 0032 See also: Adulteration of Foods: Detection; Chromatography: High-performance Liquid Chromatography; Gas Chromatography; pH – Principles and Measurement Further Reading Fennema OR (ed.) (1979) Food Chemistry. Principles of Food Science, Part 1. New York: Marcel Dekker. Lehninger AL (1975) Biochemistry, 2nd edn. New York: Worth. Macrae R (1988) HPLC in Food Analysis. London: Academic Press. Pomeranz Y and Meloan CE (1978) Food Analysis: Theory and Practice. Westport: AVI. Suye S, Yoshihara N and Shusei I (1992) Spectrophotometric determination of l-malic acid with a malic enzyme. Bioscience, Biotechnology, and Biochemistry 56(9): 1488–1489. Natural Acids and Acidulants J D Dziezak, Dziezak & Associates, Ltd., Hoffman Estates, IL, USA Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Capillary Electrophoresis 0031 A relatively new technique, capillary electrophoresis, is also useful for separating and quantifying organic acids in food systems. This technique utilizes an electrical field to separate molecules on the basis of their charge and size. Small volumes of sample, usually a few nanoliters, are injected on to a fused silica capillary tube, which is usually less than 1 m in length and 50 mm in internal diameter. The ends of the tube are placed in electrolyte reservoirs containing electrodes. A voltage in the range of 20–30 kV is delivered to the electrodes by a power supply and causes the charged molecules to move. Because organic acids are negatively charged, they migrate away from more neutral or positively charged molecules, such as sugars and Background Acids, or acidulants as they are also called, are commonly used in food processing as flavor intensifiers, preservatives, buffers, meat-curing agents, viscosity modifiers, and leavening agents. This article discusses the functions that acidulants have in food systems and reviews the more commonly used food acidulants. 0001 Functions of Acidulants The reasons for using acidulants in foods are numerous and depend on what the food processor hopes to accomplish. As outlined above, the principal reasons 0002 ACIDS/Natural Acids and Acidulants for incorporating an acidulant into a food system are flavor modification, microbial inhibition, and chelation. Flavor Modification 0003 0004 Sourness or tartness is one of the five major taste sensations: sour, salty, sweet, bitter, and umami (the most recently determined). Unlike the sensations of sweetness and bitterness, which can be developed by a variety of molecular structures, sourness is evoked only by the hydronium ion of acidic compounds. Each acid has a particular set of taste characteristics, which include the time of perceived onset of sourness, the intensity of sourness, and any lingering of aftertaste. Some acids impart a stronger sour note than others at the same pH. As a general rule, weak acids have a stronger sour taste than strong acids at the same pH because they exist primarily in the undissociated state. As the small amount of hydronium ions is neutralized in the mouth, more undissociated acid (HA) molecules ionize to replace the hydronium ions lost from equilibrium (eqn (1)). The newly released hydronium ions are then neutralized until no acid remains. Taste characteristics of the acid are an important factor in the development of flavor systems. HA þ H2 O ! H3 Oþ þ A 0005 0006 ð1Þ As pH decreases, the acid becomes more undissociated and imparts more of a sour taste. For example, the intense sour notes of lactic acid at pH 3.5 may be explained by the fact that 70% of the acid is undissociated at this pH, compared with 30% for citric acid. In addition to sourness, acids have nonsour characteristics such as bitterness and astringency, though these are less perceptible. At pH values between 3.5 and 4.5, lactic acid is the most astringent. Acids also have the ability to modify or intensify the taste sensations of other flavor compounds, to blend unrelated taste characteristics, and to mask undesirable aftertastes by prolonging a tartness sensation. For example, in fruit drinks formulated with low-caloric sweeteners, acids mask the aftertaste of the sweetener and impart the tartness that is characteristic of the natural juice. In another example, in substitutes for table salt, acids remove the bitterness from potassium chloride and provide the salty taste of sodium chloride. Other acids, such as glutamic and succinic acids, possess flavor-enhancement properties. (See Flavor (Flavour) Compounds: Structures and Characteristics; Sensory Evaluation: Taste.) Because acids are rarely found in nature as a single acid, the combined use of acids simulates a more natural flavor. Two acids that are frequently blended together are lactic and acetic. 13 Microbial Inhibition Acidulants act as preservatives by retarding the growth of microorganisms and the germination of microbial spores which lead to food spoilage. The effect is attributed to both the pH and the concentration of the acid in its undissociated state. It is primarily the undissociated form of the acid which carries the antimicrobial activity: as the pH is lowered, this helps shift the equilibrium in favor of the undissociated form of the acid, thereby leading to more effective antimicrobial activity. The nature of the acid is also an important factor in microbial inhibition: weak acids are more effective at the same pH in controlling microbial growth. Acids affect primarily bacteria because many of these organisms do not grow well below about pH 5; yeasts and molds, in comparison, are usually acid-tolerant. (See Spoilage: Bacterial Spoilage; Molds in Spoilage; Yeasts in Spoilage.) In fruit- and vegetable-canning operations, the combined use of heat and acidity permits sterilization and spore inactivation to be achieved at lower temperatures; this minimizes the degradation of flavor and structure that generally results from processing. (See Canning: Principles.) Acidification also improves the effectiveness of antimicrobial agents such as benzoates, sorbates, and propionates. For example, sodium benzoate – an effective inhibitor of bacteria and yeasts – does not exert its antimicrobial activity until the pH is reduced to about 4.5. (See Preservation of Food.) Blends of acids act synergistically to inhibit microbial growth. For example, lactic and acetic acids have been found to inhibit the outgrowth of heterofermentative lactobacilli. 0007 0008 0009 Chelation Oxidative reactions occur naturally in foods. They are responsible for many undesirable effects in the product, including discoloration, rancidity, turbidity, and degradation of flavor and nutrients. As catalysts to these reactions, metal ions such as copper, iron, manganese, nickel, tin, and zinc need to be present in only trace quantities in the product or on the processing machinery. (See Oxidation of Food Components.) Many acids chelate the metal ions so as to render them unavailable; the unshared pair of electrons in the molecular structure of acids promotes the complexing action. When used in combination with antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, or tertiary butylhydroquinone, acids have a synergistic effect on product stability. Citric acid and its salts are the most widely used chelating agents. (See Antioxidants: Natural Antioxidants; Synthetic Antioxidants.) 0010 0011 14 ACIDS/Natural Acids and Acidulants 0012 0013 0014 0015 Other Functions Commonly Used Acidulants One of the most common reasons for adding acids is to control pH. This is usually done as a means to retard enzymatic reactions, to control the gelation of certain hydrocolloids and proteins, and to standardize pH in fermentation processes. In the first example, the lowering of pH inactivates many natural enzymes which promote product discoloration and development of off-flavors. Polyphenol oxidase, for example, oxidizes phenols to quinones, which subsequently polymerize, forming brown melanin pigments that discolor the cut surfaces of fruits and vegetables. The enzyme is active between pH 5 and 7 and is irreversibly inactivated at a pH of 3 or lower. In the second example, acidification to 2.5–3 is required for high-methoxyl pectins to form gels. Because pH influences the gel-setting properties and the gel strength obtained, proper pH control is critical in the production of pectin- and gelatin-based desserts, jams, jellies, preserves, and other products. In the final example, standardization of pH is done routinely in fermentation processes, such as wine-making, to ensure optimum microbial activity and to discourage growth of undesirable microbes. Acids are also added postfermentation to stabilize the finished wine. (See Beers: Biochemistry of Fermentation; Colloids and Emulsions; Enzymes: Functions and Characteristics; Phenolic Compounds.) Acid salts function as buffers in various systems. (See Acids: Properties and Determination.) For example, in confectionery products, acid salts are used to control the inversion of sucrose into its constituents, glucose and fructose, the latter being hygroscopic. The resulting lower concentration of fructose yields a less hygroscopic food system and a longer shelf-life. Acids are a major component of chemical leavening systems, where they remain nonreactive until the proper temperature and moisture conditions are attained. The gas evolved by reaction of the acid with bicarbonate produces the aerated texture that is characteristic of baked products such as cakes, biscuits, doughnuts, pancakes, and waffles. The onset and the rate of reaction of these compounds are controlled by such factors as the solubility of the acid, the mixing conditions for preparing the batter, and the temperature and moisture of the batter. Many chemical leavening systems are based on salts of phosphoric and tartaric acids. (See Leavening Agents.) Acids have also been used for other purposes. For example, they are added to chewing gum to stabilize aspartame and to cheese to impart favorable textural properties and sensory attributes. Among the most widely used acids are acetic, adipic, citric, fumaric, lactic, malic, phosphoric, and tartaric acids. Glucono-d-lactone, though not itself an acid, is regarded as an acidulant because it converts to gluconic acid under high temperatures. 0016 Acetic Acid Acetic acid is the major characterizing component of vinegar. Its concentration determines the strength of the vinegar, a value termed ‘grain strength,’ which is equal to 10 times the acetic acid concentration. Vinegar containing, for example, 6% acetic acid has a grain strength of 60 and is called 60-grain. Distillation can be used to concentrate vinegar to the desired strength. (See Vinegar.) Fermentation conducted under controlled conditions is the commercial method for vinegar production. Bacterial strains of the genera Acetobacter and Acetomonas produce acetic acid from alcohol which has been obtained from a previous fermentation involving a variety of substrates such as grain and apples. Vinegar functions in pH reduction, control of microbial growth, and enhancement of flavor. It has found use in a variety of products, including condiments such as ketchup, mustard, mayonnaise, and relish, salad dressings, marinades for meat, poultry, and fish, bakery products, soups, and cheeses. Pure (100%) acetic acid is called glacial acetic acid because it freezes to an ice-like solid at 16.6
C. Though not widely used in food, glacial acetic acid provides acidification and flavoring in sliced, canned fruits and vegetables, sausage, and salad dressings. 0017 0018 Adipic Acid Adipic acid, a white, crystalline powder, is characterized by low hygroscopicity and a lingering, high tartness that complements grape-flavored products and those with delicate flavors. The acid is slightly more tart than citric acid at any pH. Aqueous solutions of the acid are the least acidic of all food acidulants, and have a strong buffering capacity in the pH range 2.5–3.0. Adipic acid functions primarily as an acidifier, buffer, gelling aid, and sequestrant. It is used in confectionery, cheese analogs, fats, and flavoring extracts. Because of its low rate of moisture absorption, it is especially useful in dry products such as powdered fruit-flavored beverage mixes, leavening systems of cake mixes, gelatin desserts, evaporated milk, and instant puddings. 0019 0020 ACIDS/Natural Acids and Acidulants Citric Acid 0021 0022 0023 0024 0025 0026 The most widely used organic acid in the food industry, citric acid, accounts for more than 60% of all acidulants consumed. It is the standard for evaluating the effects of other acidulants. Its major advantages include its high solubility in water; appealing effects on flavor, particularly its ability to deliver a ‘burst’ of tartness; strong metal chelation properties; and the widest buffer range of the food acids (2.5–6.5). Citric acid is naturally present in animal and plant tissues and is most abundantly found in citrus fruits including the lemon (4–8%), grapefruit (1.2–2.1%), tangerine (0.9–1.2%) and orange (0.6–1.0%). (See Citrus Fruits: Composition and Characterization.) The principal method for commercial production of the acid is fermentation of corn. Formerly, the acid had been obtained by extraction from citrus and pineapple juices. Citric acid is available in a liquid form, which solves processing problems related to incorporating the acid into a food system, such as predissolving citric acid crystals and caking or crystallate deposits on processing equipment. Also available are granulated forms which allow the particle size to be customized to meet the particular need. Citric acid has numerous applications. It is commonly added to nonalcoholic beverages where it complements fruit flavors, contributes tartness, chelates metal ions, acts as a preservative, and controls pH so that the desired sweetness characteristics can be achieved. Sodium citrate subdues the sharp acid notes in highly acidified carbonated beverages; in club soda, it imparts a cool, saline taste and helps retain carbonation. The acid is also used in wine production both prior to and after fermentation for adjustment of pH; in addition, because of its metalchelating action, the acid prevents haze or turbidity caused by the binding of metals with tannin or phosphate. The calcium salt of citric acid is used as an anticaking agent in fructose-sweetened, powdered soft drinks, where it neutralizes the alkalinity of other ingredients that support browning, such as magnesium oxide and tricalcium phosphate. Citric acid has also found use in confectionery and desserts. In hard confectionery, buffered citric acid imparts a pleasant tart taste; it is added to the molten mass after cooking, as this prevents sucrose inversion and browning. Citric acid is used in gelatin desserts because it imparts tartness, acts as a buffering agent, and increases the pH for optimum gel strength. Low levels of the acid, ranging from 0.001 to 0.01%, work with antioxidants to retard oxidative rancidity in dry sausage, fresh pork sausage, and dried meats. Citric acid is also used in the production of frankfurters: 3–5% solutions are sprayed on the 15 casings after stuffing and prior to smoking to aid in their removal from the finished product. Used at 0.2% in livestock blood, sodium citrate and citric acid act as anticoagulants, sequestering the calcium required for clot formation so that the blood may be used as a binder in pet foods. In seafood processing, citric acid inactivates endogenous enzymes and promotes the action of antioxidants, resulting in an increased shelf-life. Citric acid also chelates copper and iron ions that catalyze the oxidative formation of off-flavors and fishy odors associated with dimethylamine. In processed cheese and cheese foods, citric acid and sodium citrate function in emulsification, buffering, flavor enhancement, and texture development. Sodium citrate is also combined with sodium phosphate as a customized emulsification salt for processed cheese. Cogranulation of citric acid with malic and fumaric acids yields new tart flavor profiles. 0027 Fumaric Acid The extremely low rate of moisture absorption of this acid makes it an important ingredient for extending the shelf-life of powdered food products such as gelatin desserts and pie fillings. Fumaric acid can be used in smaller quantities than citric, malic, and lactic acids to achieve similar taste effects. Fermentation of glucose or molasses by certain Rhizopus spp. is the method used to produce fumaric acid commercially. The acid is also made by isomerization of maleic acid with heat or a catalyst, and is a byproduct of the production of phthalic and maleic anhydrides. Fumaric acid is also made in particulate form, where the acid makes up about 5–95% of the particulate, with the remainder being other acids such as malic, tartaric, citric, lactic, ascorbic, and related mixtures. Applications of fumaric acid include rye bread, jellies, jams, juice drinks, candy, water-in-oil emulsifying agents, reconstituted fats, and dough conditioners. In refrigerated biscuit doughs, the acid eliminates crystal formations that may occur in all-purpose leavening systems. In wine, it functions as both an acidulant and a clarifying aid, although it does not chelate copper or iron. 0028 0029 0030 Glucono-d-Iactone (GDL) A natural constituent of fruits and honey, GDL is an inner ester of d-gluconic acid. Unlike other acidulants, it is neutral and gives a slow rate of acidification. When added to water, it hydrolyzes to form an equilibrium mixture of gluconic acid and its d- and g-lactones. The acid formation takes place slowly when cold and accelerates when heated. As 0031 16 ACIDS/Natural Acids and Acidulants 0032 0033 GDL converts to gluconic acid, its taste characteristics change from sweet to neutral with a slight acidic afteraste. GDL is produced commercially from glucose by a fermentation process that uses enzymes or pure cultures of microorganisms such as Aspergillus niger or Acetobacter suboxydans to oxidize glucose to gluconic acid. GDL is extracted by crystallization from the fermentation product, an aqueous solution of gluconic acid and GDL. Because of its gradual acidification, bland taste, and metal-chelating action, GDL has found application in mild-flavored products such as chocolate products, tofu, milk puddings, and creamy salad dressings. In cottage cheese prepared by the direct-set method, GDL ensures development of a finer-textured finished product, void of localized denaturation. It also shortens production time and increases yields. In cured-meat products, GDL reduces cure time, inhibits growth of undesirable microorganisms, promotes color development, and reduces nitrate and nitrite requirements. (See Curing.) Lactic Acid 0034 0035 Lactic acid is one of the earliest acids to be used in foods. It was first commercially produced about 60 years ago, and only within the past two decades has it become an important ingredient. The mild taste characteristics of the acid do not mask weaker aromatic flavors. Lactic acid functions in pH reduction, flavor enhancement, and microbial inhibition. Two methods are used commercially to produce the acid: fermentation and chemical synthesis. Most manufacturers using fermentation are in Europe. Confectionery, bakery products, beer, wine, beverages, dairy products, dried egg whites, and meat products are examples of the types of products in which lactic acid is used. The acid is used in packaged Spanish olives where it inhibits spoilage and further fermentation. In cheese production, it is added to adjust pH and as a flavoring agent. Malic Acid 0036 This general-purpose acidulant imparts a smooth, tart taste which lingers in the mouth, helping to mask the aftertastes of low- or noncaloric sweeteners. It has taste-blending and flavor-fixative characteristics and a relatively low melting point with respect to other solid acidulants. The low melting point allows it be homogeneously distributed into food systems. Compared with citric acid, malic acid has a much stronger apparent acidic taste. As dl-malic acid is the most hygroscopic of the acids, resulting in lumping and browning in dry mixes, the encapsulated form of this acid is preferred for dry mixes. Malic acid occurs naturally in many fruits and vegetables, and is the second most predominant acid in citrus fruits, many berries, and figs. Unlike the natural acid, which is levorotatory, the commercial product is a racemic mixture of d- and l-isomers. It is manufactured during catalytic hydration of maleic and fumaric acids, and is recovered from the equilibrium product mixture. The acid has been used in carbonated beverages, powdered juice drinks, jams, jellies, canned fruits and vegetables, and confectionery. Its lingering profile enhances fruit flavors such as strawberry and cherry. In aspartame-sweetened beverages, malic acid acts synergistically with aspartame so that the combined use of malic and citric acids permits a 10% reduction in the level of aspartame. In frozen pizza, malic acid is used to lower the pH of the tomato paste without chelating the calcium in the cheese, as would citric and fumaric acids. This application improves the texture of the frozen pizza. 0037 0038 Phosphoric Acid The second most widely used acidulant in food, phosphoric acid, is the only inorganic acid to be used extensively for food purposes. It produces the lowest pH of all food acidulants. Phosphoric acid is produced from elemental phosphorus recovered from phosphate rock. The primary use of the acid is in cola, root beer, and other similar-flavored carbonated beverages. The acid and its salts are also used during production of natural cheese for adjustment of pH; phosphates chelate the calcium required by bacteriophages, which can destroy bacteria responsible for ripening. As chemical leavening agents, phosphates release gas upon neutralizing alkaline sodium bicarbonate; this creates a porous, cellular structure in baked products. The main reason for incorporating phosphates into cured meats such as hams and corned beef is to increase retention of natural juices; the salts are dissolved in the brine and incorporated into the meat by injection of brine, massaging, or tumbling. When used in jams and jellies, phosphoric acid acts as a buffering agent to ensure a strong gel strength; it also prevents dulling of the gel color by sequestering prooxidative metal ions. 0039 0040 Tartaric Acid Tartaric acid is the most water-soluble of the solid acidulants. It contributes a strong tart taste which enhances fruit flavors, particularly grape and lime. This dibasic acid is produced from potassium acid tartrate which has been recovered from various byproducts of the wine industry, including press cakes from fermented and partially fermented grape 0041 ACIDS/Natural Acids and Acidulants 0042 juice, less (the dried, slimy sediments in wine fermentation vats), and argols (the crystalline crusts formed in vats during the second fermentation step of wine-making). The major European wine-producing countries, Spain, Germany, Italy, and France, use more of the acid than the USA. Tartaric acid is often used as an acidulant in grapeand lime-flavored beverages, gelatin desserts, jams, jellies, and hard sour confectionery. The acidic monopotassium salt, more commonly known as ‘cream of tartar,’ is used in baking powders and leavening systems. Because it has limited solubility at lower temperatures, cream of tartar does not react with bicarbonate until the baking temperatures are reached; this ensures maximum development of volume in the finished product. See also: Acids: Properties and Determination; Antioxidants: Natural Antioxidants; Synthetic Antioxidants; Canning: Principles; Citrus Fruits: Composition and Characterization; Colloids and Emulsions; Curing; Flavor (Flavour) Compounds: Structures and Characteristics; Leavening Agents; Oxidation of Food Components; Phenolic Compounds; Preservation of Food; Sensory Evaluation: Taste; Spoilage: Bacterial Spoilage; Vinegar Further Reading Anon. (1995) Spotlight on ingredients for confectionery and ice cream: Pointing and Favex point the way. Confectionery Production May: 350–351. Anon. (1995–1996) Citric acid is no lemon. Food Review Dec./Jan.: 51–52. Arnold MHM (1975) Acidulants for Foods and Beverages. London: Food Trade Press. Bigelis R and Tsai SP (1995) Microorganisms for organic acid production. In: Hui YH and Khachatourians GG (eds) Food Biotechnology: Microorganisms, pp. 239– 280. New York: Wiley-VCH. Bouchard EF and Merritt EG (1979) Citric acid. In: Grayson M (ed.) Kirk–Othmer Encyclopedia of Chemical Technology, 3rd edn, vol. 6, p. 150. New York: Wiley. Brennan M, Port GL and Gormley R (2000) Post-harvest treatment with citric acid or hydrogen peroxide to extend the shelf life of fresh sliced mushrooms. Lebensmittel-Wissenschaft & Technologie 33: 285–289. 17 Dziezak JD (1990) Acidulants: ingredients that do more than meet the acid test. Food Technology 44(1): 76–83. Farkye NY, Prasad B, Rossi R and Noyes QR (1995) Sensory and textural properties of Queso Blanco-type cheese influenced by acid type. Journal of Dairy Science 78: 1649–1656. Fowlds R and Walter R (1998) The Production of a Food Acid Mixture Containing Fumaric Acid, PCT Patent application WO 98/53705. Gardner WH (1972) Acidulants in food processing. In: Furia TE (ed.) CRC Handbook of Food Additives, 2nd edn, vol. 1, p. 225. Cleveland, OH: CRC Press. Garrote GL, Abraham AG and DeAntoni GL (2000) Inhibitory power of kefir: the role of organic acids. Journal of Food Protection 63(3): 364–369. Goldberg I, Peleg Y and Rokem IS (1991) Citric, fumaric, and malic acids. In: Goldberg I and Williams R (eds) Biotechnology and Food Ingredients, pp. 349–374. New York: Van Nostrand Reinhold. Hartwig P and McDaniel MR (1995) Flavor characteristics of lactic, malic, citric, and acetic acids at various pH levels. Journal of Food Science 60(2): 384–388. International Commission of Microbiological Specifications for Foods (1980) Microbial Ecology of Foods, vol. 1. New York: Academic Press. Kummel KIF (2000) Acidulants use in sour confections. The Manufacturing Confectioner Dec.: 91–93. Miller Al and Call JE (1994) Inhibitory potential of fourcarbon dicarboxylic acids on Clostridium botulinum spores in an uncured turkey product. Journal of Food Protection 57(8): 679–683. Oman YJ (1992) Process for Removing the Bitterness from Potassium Chloride, US Patent No. 5,173,323. Phillips CA (1999) The effect of citric acid, lactic acid, sodium citrate and sodium lactate, alone and in combination with nisin, on the growth of Arcobacter butzleni. Letters in Applied Microbiology 29: 424–428. Sun Y and Oliver JD (1994) Antimicrobial action of some GRAS compounds against Vibrio vulnificus. Food Additives and Contaminants 11(5): 549–558. Suye S, Yoshihana N and Shusei I (1992) Spectrophotometric determination of l-malic acid with a malic enzyme. Bioscience, Biotechnology, and Biochemistry 56(9): 1488–1489. Synosky S, Orfan SP and Foster JW (1992) Stabilized Chewing Gum Containing Acidified Humectant. US Patent No. 5,175,009. Vidal S and Saleeb FZ (1992) Calcium Citrate Anticaking Agent. US Patent No. 5,149,552. 18 ADAPTATION – NUTRITIONAL ASPECTS ADAPTATION – NUTRITIONAL ASPECTS P S Shetty, Food and Agriculture Organization, Rome, Italy J C Waterlow, London School of Hygiene & Tropical Medicine, London, UK Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Introduction 0001 0002 The word ‘adaptation’ is used in many different contexts: biological or Darwinian; physiological or metabolic; behavioral or social. In nutrition, we are concerned with the last two. The difference between ‘adaptation’ and ‘homeostasis’ is that the latter represents the maintenance of a set point for some physiological characteristic such as body temperature or pH – this is Claude Bernard’s ‘fixité du milieu intérieur.’ Adaptation involves a change in the set point, for example, the increase in hemoglobin concentration found in people living at high altitude or the decrease in sodium concentration in the sweat in people exposed to high environmental temperatures. Such adaptations take time; one speaks of people ‘becoming adapted,’ whereas homeostasis is a rapid and continuous process. For adaptation to be more than just a response, it must represent a new steady state, capable of being maintained, and we think of it as beneficial to the organism, preserving, within limits, normal function. It is here that the real difficulty arises. For most bodily characteristics or functions, there are no clear definitions of a ‘normal’ range, within which physiological adaptations can operate. Basal metabolic rate (BMR) is an exception, but for most functions that are important for the quality of life, such as work capacity or resistance to infection, there are no such defined limits, so it is difficult to decide whether an adaptation is ‘successful.’ We shall return to this point later. In nutrition, it is convenient to look separately at adaptation to inadequate intakes of energy and protein before going on to the more realistic situation of overall deficiency of food and deficiency or excess of micronutrients. Adaptation to Low Energy Intakes 0003 The human body responds to an inadequate intake of food energy by a whole series of physiological and behavioral responses. Experimental studies of semistarvation in normal adults have helped in understanding the physiological changes that characterize this adaptive response to a lowered energy intake in humans. The metabolic responses that occur during acute energy restriction and the physiological mechanisms that are involved may, however, be different from the changes observed in individuals who are chronically undernourished as a result of longstanding marginal energy intakes. In previously well nourished adults, a reduction in BMR is a constant finding during experimentally or therapeutically induced energy restriction. This finding has been explained on the basis of a loss of active tissue mass, as a result of the loss of body weight, together with a decrease in the metabolic activity of the active tissues. The latter would indicate a greater efficiency or metabolic adaptation, on the assumption that the same amount of work is being done at lower cost. Recalculating the data from the two separate semistarvation studies, one short term and the other longer term, it has been shown that the early fall in BMR seen during energy restriction is mainly accounted for by enhanced metabolic efficiency (Table 1). This reduction in BMR per kilogram of active body tissues seen in the first 2 weeks of energy restriction remained essentially unchanged over the subsequent period of semi-starvation. The greater contribution to the fall in BMR during prolonged energy restriction, however, was the result of a slow decrease in the total mass of active tissues. It seems reasonable, therefore, to suggest that the reduction in BMR during energy restriction occurs in two different phases. In the initial phase, there is a marked decrease in the BMR, which is not attributable to the changes in body weight or body composition. This decrease in BMR per unit active tissue is a measure of increase in Table 1 Changes in body weight, active tissue mass (ATM), and basal metabolic rate (BMR) following short- and long-term semistarvation in humans Semistarvation Short-terma Body weight (kg) ATM (kg) BMR: MJ day1 kcal day1 BMR decrease: kJ day1 kJ kg1 ATM a Long-termb Baseline Day14 Baseline Day168 71.6 44.9 7.3 1742 65.4 42.2 5.7 1370 67.5 38.8 6.6 1575 51.7 28.7 4.2 1004 21.4% 16.3% 36.3% 13.8% Data from experiments conducted on 12 human subjects by Grande et al. (1958). b Data from experiments conducted on 12 human subjects by Keys et al. (1950). 0004 tbl0001 ADAPTATION – NUTRITIONAL ASPECTS 0005 ‘metabolic efficiency’ in well-nourished individuals who are energy-restricted and is often cited as evidence of ‘metabolic adaptation.’ With continued energy restriction, the lowered level of cellular metabolic rate remains nearly constant, and any further decrease in BMR is accounted for by the loss of body weight. Thus, the longer the duration of energy restriction, the more important the contribution of decreased body tissues becomes to the reduction in BMR. This reduction in lean body tissue with prolonged energy restriction is considered to be a passive process and a consequence of body tissues being used as substrates and metabolic fuel to compensate for the lack of food energy. The biochemical and physiological mechanisms involved in reducing the cellular metabolic rate are poorly understood. It has recently been estimated that *90% of BMR is contributed by mitochondrial oxygen consumption, of which only *20% is uncoupled by mitochondrial proton leak and the rest coupled to ATP synthesis. It is not known how much changes in mitochondrial function contribute to the increasing efficiency of tissue metabolism. Several physiological changes in hormonal and substrate function may operate to influence the changes in metabolic efficiency seen during the early part of energy restriction. Several hormones are now known to be sensitive to changes in the levels of energy intake, dietary composition, and energy balance status of the individual. Changes in sympathetic nervous system (SNS) activity and catecholamines, alterations in thyroid hormone metabolism, and changes in insulin and glucagon play an important role in this response. The reduction in SNS activity and catecholaminergic drive that we observed was counter to traditional views on the control of substrate mobilization during starvation. Traditionally, the increase in lipolysis, maintenance of glucose homeostasis, and increase in glucagon output on fasting have been considered as being the result of an enhanced sympathetic drive during energy restriction. It now appears that the lipolytic activity associated with energy restriction appears to be under the dominant control of declining plasma insulin levels. Insulin is the primary hormonal signal that allows for an orderly transition from the fed to the fasted state without the development of hypoglycemia. While the SNS activity is toned down, signaled by the decrease in energy flux, the energy deficit lowers insulin secretion and initiates changes in peripheral thyroid metabolism. The reduction in the activities of these three thermogenic hormones acts in a concerted manner to lower cellular metabolic rate. Changes in other hormones such as glucagon, growth hormone, and glucocorticoids may also participate and, in association with insulin 19 deficiency, help promote endogenous substrate mobilization leading to an increase in circulating free fatty acids (FFA) and ketone bodies. Contribution may also be made by the reduction in Naþ–Kþ pumping across the cell membrane and futile substrate cycling, although how much they contribute to the reduced energy output is not known. The elevated FFA levels, alterations in substrate recyling, and protein catabolism will also influence the resting energy expenditure. These changes are thus not only aimed at lowering the metabolic activity of the active cell mass but also essential for the orderly mobilization of endogenous substrates and fuels during a period of restricted availability of exogenous calories. These hormonal and metabolic changes aid the survival of the organism and may be considered as being ‘adaptive’ in nature. Adaptation to lowered energy intake in chronically undernourished adults on subsistence food intakes in the developing world appears, however, to be different. Ferro-Luzzi summarized the adaptive responses in individuals who were maintaining energy balance in spite of life-long exposure to low energy intakes – the state of so-called ‘chronic energy deficiency.’ Adaptation was represented as a series of complex integrations of several different processes that occurred during energy deficiency and resulted in a new level of equilibrium being achieved at a lower level of energy intake. People who have gone through the adaptive process may be expected to exhibit more or less permanent sequelae (or costs of adaptation), which include smaller stature and body size, altered body composition and a lower BMR, with the likelihood of enhanced metabolic efficiency of energy handling. However, this has been difficult to prove, largely because marked changes in the body composition (in particular in the fat and lean compartments) make interpretation of changes in the metabolic rate per unit of active tissue mass highly unreliable as indicators of metabolic efficiency. Changes in body composition as well as in body size and dimensions may play a dominant role in adaptation to long-term inadequacy of energy intake from childhood, however undesirable they may be. These physiological adaptations are not beneficial changes, as they influence employability and economic productivity, although they may help in furthering survival of the individual. Adaptations to a reduction in food energy intake may also be manifest as physiological and behavioral changes in physical activity, aimed at reducing the energy expended by the individual every day to make up for the energy deficit. Reductions in either intensity or duration of physical activity can save much energy and hence may be a crucial response to 0006 0007 20 ADAPTATION – NUTRITIONAL ASPECTS 0008 0009 energy restriction and an important feature of the adaptive response. Studies on semistarvation of previously well nourished adults showed a marked impairment in both intensity and duration of activity. About 40% of the reduction was attributable to a decrease in actual costs of performing tasks, whereas 60% of the reduction was due to a decrease in tasks undertaken. In previously well-nourished semistarved adults, behavioral reduction in voluntary activity seems to be quantitatively more important. Analysis of the pattern of an individual’s physical activity during a voluntary reduction in food intake shows that the behavioral responses were associated with a distinct change in activity pattern. Physiological changes in the physical work capacity of undernourished young men are also difficult to demonstrate, and the overwhelming evidence seems to support the view that differences, if any, are largely due to changes in body composition and not to adaptive differences in cell function. Spurr summarized the results of several of his studies in Colombia, which demonstrated that maximal oxygen uptake (Vo2 max) was lower in malnourished young adults; the degree of reduction being related to the progressive severity of undernutrition. He was also able to demonstrate that 80% of the reduction in Vo2 max in moderate and severe categories of undernutrition was accounted for by differences in muscle cell mass. Assessment of endurance at 70–80% of the Vo2 max in the undernourished also failed to demonstrate any differences in the maximum endurance time. However, assessment of productivity in agricultural environments shows that work productivity is affected indirectly by nutritional status, through its influence on stature, body weight, body composition, and Vo2 max. Chronically undernourished adults are likely to demonstrate increased ergonomic or ‘real life’ efficiency. By this is meant a reduction in the effort needed to do any piece of physical work. It is reasonable to suppose that tradition and experience have enabled people living on marginal intakes and hence likely to be chronically undernourished to find the most economical methods of doing the tasks they have to do. This manifestation of increased efficiency might be regarded as a training effect, quite distinct from the behavioral adaptation that accompanies undernutrition, which is mainly related to how individuals allocate time and energy to different productive and leisure activities, with inevitable biological and economic consequences. In undernutrition, more time is given to work activities, while leisure and home production activities are reduced; this is an important form of behavioral adaptation. Marginally undernourished individuals tend to become more sedentary at the expense of decreased social interactions and discretional noneconomic activities. Latham showed that when energy-deficient individuals are forced over a period of time to limit their activities, they forego activities to conserve energy, some of which they do consciously and wilfully, some they do unconsciously. Thus, restricting physical activity or performing it more efficiently is an important coping strategy for undernourished individuals and may form part of the behavioral adaptive response to a lowered intake of food energy. Adaptation to Low Protein Intakes Most of our knowledge on this subject has been derived from experimental studies on man. Adaptation to low protein intakes has two proximate functions: to secure nitrogen balance and to maintain lean body mass (LBM). As regarding balance, there is an obligatory loss of nitrogen from the body which has been estimated in male Caucasian adults to amount to about 55–65 mg of nitrogen per kilogram per day and which has to be balanced by the intake. There is little evidence that this loss is lower in people long accustomed to low protein intakes, or to an intake mainly from vegetable sources, so there does not seem to be much opportunity for adaptation at this point. There is, however, evidence, that on lower protein intakes or in children recovering from malnutrition, the efficiency of utilization of food protein may be increased above the usual level of about 70%. This effect may be regarded as a response to depletion, i.e., loss of body nitrogen, but is none the less an adaptive response aimed at conserving body nitrogen. When a person moves from a normal intake, providing say 1.5 g of protein (250 mg of nitrogen) per kilogram per day to an intake close to the obligatory loss, the nitrogen output falls to a new low level in 7– 10 days in the human adult, 1–2 days in the infant and about 30 h in the rat. This is the first stage of adaptation. During this stage, there is a small loss, amounting to 1–2% of body N, which probably has no physiological significance. The main variable in this adaptation is the urinary excretion of urea. Urea production, which is a measure of amino acid oxidation, is related to nitrogen intake, although at the present time, there is some controversy about the strength of the relationship. Only part of the urea produced is excreted in the urine; the remainder passes into the colon, where it is hydrolyzed by gut bacteria to ammonia. A relatively small part of this ammonia is recycled to urea. The rest of it enters the amino acid pool, and there is increasing evidence that microbes in the gut are 0010 0011 0012 ADAPTATION – NUTRITIONAL ASPECTS 0013 0014 0015 capable of using it to synthesize indispensable as well as dispensable amino acids. In the normal individual, on an adequate nitrogen intake and in a steady state, these reactions are essentially exchanges, and there is no net gain of nitrogen. However, with a deficient intake or an increased demand for growth, amino acids derived from the colonic hydrolysis of urea can make a significant contribution to the body’s nitrogen economy. Hence, the term ‘urea salvage,’ introduced by Jackson is appropriate, salvage representing an important component of adaptation. Since the proportion of urea hydrolyzed to that excreted increases on a low protein intake, it follows that the maintenance of nitrogen balance involves control of the rate of hydrolysis. It is thought that this control may be exerted by a urea transporter, which is sensitive to the protein level of the diet. A second phase of adaptation comes into play if the protein intake is inadequate to cover the obligatory losses, so that there is a prolonged negative nitrogen balance. This inevitably leads to a loss of body protein. Since the magnitude of the obligatory loss is determined by the body protein mass, as this mass decreases, the loss will decrease until eventually the nitrogen balance is restored. This would represent an adaptation at the expense of a certain loss of lean body mass. Whether that loss is important will be discussed below. An example of such an adaptation is provided by the poor Indian laborers, studied by Shetty’s group in Bangalore, whose lean body mass was substantially less (13%) than that of taller controls with the same body mass index (BMI). An important finding was that in these men, the main deficit was of muscle rather than of visceral mass. Presumably, this adaptation has its cost in terms of reduced muscular capacity, but it seems justifiable to regard it as a successful adaptation, since these men could live reasonable lives. The metabolism of plasma albumin provides an interesting example of adaptation to low protein intake. In children with protein-energy malnutrition, one of the most constant findings is a reduction in plasma albumin concentration. This is accompanied by a fall in the rate of albumin catabolism, as if in an effort to maintain the concentration in plasma. The same effect has been shown in adults on experimental low protein intakes; the relative change in the rate of albumin breakdown was much greater than the change in albumin concentration. Thus, the breakdown rate would provide a much more sensitive measure of the state of protein nutrition than the albumin concentration; unfortunately, it is not a measurement that is practical on a large scale. In real life, it is in famines, refugee camps, or concentration camps that we are faced with the 21 question: what are the limits of adaptation to a food supply that is inadequate in both energy and protein – in other words, to semistarvation? Nowadays, the response is generally measured by the level of the body mass index (BMI ¼ weight (kg)/height2 (m)). Factors that affect the response of the BMI are the degree of deficiency, its duration, and the relative deficiencies of energy and protein. In total starvation, of which, as already mentioned, there have been a number of experimental studies, no steady state can be achieved, and no adaptation is possible. In the famous Minnesota semistarvation experiment, subjects were fed half their normal intakes of energy and protein; after 24 weeks, their BMI had fallen to about 16 from an initial level of about 22, and they showed severe functional and psychological impairment. This was in marked contrast to the Indian laborers referred to above who had a similarly low BMI. It seems that by life-long exposure to presumably inadequate food intakes they had adapted to a steady state of what would be currently described as ‘chronic energy deficiency,’ yet, their vital functions of energy and protein turnover were well maintained. Some cases of semistarvation present with edema, which is quite commonly seen in famines and in refugee camps. Although the cause of the edema is controversial, it is a reasonable hypothesis that it results from a particular deficiency of protein in relation to energy, although there may be other deficiencies as well. In one study in a refugee camp, subjects with edema had a higher BMI, as might be expected from the accumulation of fluid, than those without edema, but they also had a substantially higher mortality rate. Women adapted better than men; this is apparent in several accounts. It appears, therefore, that when protein is particularly deficient, the capacity for adaptation is reduced. From a physiological point of view, if the requirement for successful adaptation is the maintenance of LBM within ‘normal’ limits, it becomes crucial to define those limits. There are many difficulties. The BMI is a crude estimate of LBM, since it does not separate fat from lean tissue. However, the fat content of the body has a bearing on the capacity for adaptation, since it has been shown, not surprisingly, that in starvation, the loss of LBM is inversely related to the size of the initial fat stores. A low BMI with loss of muscle mass would explain the association mentioned above with decreased maximal oxygen consumption and reduced work capacity. However, it does not explain other associations that have been found, such as reduced resistance to infections and low birth weight of infants. Interestingly, there is no effect on breast-milk output, suggesting that this function, basic for the survival of the race, is well 0016 0017 22 ADAPTATION – NUTRITIONAL ASPECTS protected. What, then, are the normal limits? Is there a threshold or cutoff point of LBM, as assessed by BMI, above which function is normal and below which it falls off? Some evidence from epidemiological studies suggest that there is no threshold, but a steady fall-off with falling BMI. However, because BMI is influenced by many factors beyond physiological homeostasis, it is difficult to establish with certainty the limits within which adaptation may be regarded as successful. Adaptation to Variations in Micronutrient (Mineral and Vitamin) Intakes 0018 0019 One of the major processes by which adaptation to changes in nutrient intakes occurs, particularly that of micronutrients, is by changes in gastrointestinal function. The gastrointestinal tract has extensive potential for adaptation. For instance, following intestinal resection, the residual intestine is capable of a considerable increase in size and absorptive capacity. This is achieved by dilatation and an increase in rugosity and by hypertrophy of the villi and microvilli. This increases the available surface area of contact with the nutrients and thus increases the absorptive capacity. The enzyme activities and the turnover of cells are also increased. The ileal part of the intestines adapts better than the jejunum. Changes in the function of the intestines, such as slowing down the transit, also helps the process of adaptation by increasing absorptive capacity. These adaptive changes are maximized by the mucosal exposure to nutrients and by the role played by several key hormones. Intestinal adaptation is, however, limited by inadequate blood supply or poor nutritional status. Calcium represents the best example of a micronutrient whose absorption by the gastrointestinal tract is modulated to demonstrate adaptation. The physiological need for calcium changes throughout the lifecycle, i.e., growth, puberty, pregnancy, lactation, and menopause. Calcium intakes are also highly variable world-wide, with a more than fourfold difference between the lowest intake and the highest. Hence, the absorption of calcium from the diet must be adaptable and responsive to both dietary and physiological circumstances. This process of adaptation and physiological plasticity is largely orchestrated by vitamin D, which stimulates intestinal calcium absorption by both genomic and nongenomic mechanisms. The renal output of dihydroxy vitamin D3, which is regulated, reflects the perceived needs of the organism for calcium, which in turn influences the tightly regulated process of intestinal calcium absorption. The latter regulation occurs both by genomic receptor mediated action (i.e., through calbindin) and by nongenomic mechanisms (through transcaltachia). There are other social and behavioral adaptations, too, which influence the individuals’ choice of diet and determine what is available for intestinal absorption. It is hence believed that vitamin D-mediated calcium absorption by the intestines satisfies the requirement for it to be considered as an adaptive function. One would expect that the requirements of most micronutrients are amenable to adaptation when intakes are lowered, although the evidence for such changes is not readily available. See also: Calcium: Properties and Determination; Physiology; Energy: Intake and Energy Requirements; Energy Expenditure and Energy Balance; Famine, Starvation, and Fasting; Protein: Digestion and Absorption of Protein and Nitrogen Balance Further Reading Benedict FG, Miles WR, Roth P and Smith HM (1919) Human Vitality and Efficiency Under Prolonged Restricted Diet. Publication No. 280. Washington, DC: Carnegie Institute of Washington. Blaxter KL and Waterlow JC (eds) (1985) Nutritional Adaptation in Man. London: John Libbey. Ferro-luzzi A (1985) Range of variation in energy expenditure and scope of regulation: In: Proceedings of XIIIth International Congress of Nutrition, pp. 393–399. London: Libbey. Jackson AA (1968) Salvage of urea nitrogen in the large bowel: functional significance in metabolic control and adaptation. Biochemical Society Transactions 26: 231–236. James WPT and Ralph A (eds) (1994) Functional significance of low body mass index. European Journal of Clinical Nutrition 48 (supplement 3). James WPT and Shetty PS (1982) Metabolic adaptation and energy requirements in developing countries. Human Nutrition: Clinical Nutrition 36: 331–336. Keys A, Brozeck J, Henschel A, Mickelson O and Taylor HL (1950) In: The Biology of Human Starvation. Minneapolis, MN: University of Minneapolis Press. Latham MC (1989) Nutrition and work performance, energy intakes and human wellbeing in Africa. In: Proceedings of XIVth International Congress of Nutrition. London: Libbey. Norman AW (1990) Intestinal calcium absorption: a vitamin D-hormone-mediated adaptive response. American Journal of Clinical Nutrition 51: 290–200. Shetty PS (1990) Physiological mechanisms in the adaptive response of metabolic rates to energy restriction. Nutrition Research Reviews 3: 49–74. Shetty PS (1993) Chronic undernutrition and metabolic adaptation. Proceedings of the Nutrition Society 52: 267–284. 0020 ADIPOSE TISSUE/Structure and Function of White Adipose Tissue Spurr GB (1993) Nutritional status and physical activity work capacity. Yearbook of Physical Anthropology 26: 1–35. Spurr GB (1987) The effects of chronic energy deficiency on stature, work capacity and productivity. In: Schurch B and Scrimshaw NS (eds) Chronic Energy Deficiency: 23 Causes and Consequences, pp. 95–134. Lausanne, Switzerlnd: IDECG. Waterlow JC (1990) Nutritional adaptation in man: General information and concepts. American Journal of Clinical Nutrition 51: 259–263. ADIPOSE TISSUE Contents Structure and Function of White Adipose Tissue Structure and Function of Brown Adipose Tissue Structure and Function of White Adipose Tissue R G Vernon and D J Flint, Hannah Research Institute, Ayr, UK Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Distribution and Structure of Adipose Tissue 0001 0002 White adipose tissue is quantitatively the most variable component of the body, ranging from a few percent of body weight to over 50% in obese animals and people. In mammals, adipose tissue is found within the abdominal cavity, under the skin, within the musculature where it is found between muscles (intermuscular) and within muscles (intramuscular) (e.g., marbling of meat) and in a few highly specialized locations such as the eye socket. Within these locations, the tissue occurs in discrete depots (e.g., perirenal, epididymal, omental, popliteal); there are about 16 in most species. Comparative studies have revealed that the distribution of adipose tissue depots evolved early in mammalian evolution and has been retained in most species. In some species (e.g., pigs, whales) subcutaneous depots have become enlarged and have fused to form a continuous layer; this also occurs in obese individuals. Adipose tissue depots are also found in birds, reptiles, and amphibians. White adipose tissue is a soft tissue, devoid of rigidity, and is well supplied with capillaries and nerve endings from the sympathetic nervous system. In mature animals, adipocytes (fat cells) comprise about 90% of the mass of the tissue but only 25% or less of the total cell population. The 75% or so nonadipocytes are often termed the stromal–vascular fraction and comprise mainly endothelial cells of blood vessels and adipocyte precursor cells. Adipocytes vary enormously in size from several picolitres to about 3 nl in volume, depending on the amount of lipid present. The mature fat cell is essentially a lipid droplet surrounded by a film of cytoplasm (containing mitochondria, endoplasmic reticulum, etc.) and bounded by a plasma membrane; the nucleus is pushed to the periphery and appears as a blip on the surface of the cell. Within a depot, there will be fat cells of various sizes so that it is usual to refer to the ‘mean fat cell volume’ of a tissue; this varies amongst adipose tissue depots in an individual. The adipocyte mean cell volume also varies with size of the animal, larger animals having larger fat cells; this occurs both within and between species. Functions of Adipose Tissue The major function of white adipose tissue is the storage of energy as triacylglycerol (fat, lipid). Fat is a highly efficient form of energy storage, not only because of its high energy content per unit weight, but also because it is hydrophobic. Hence, 1 g of adipose tissue may contain about 800 mg of triacylglycerol and about 100 mg of water. In contrast, glycogen not only has a lower energy content per unit weight than fat, but also is much more hydrated. The development of copious stores of fat was probably very important for the evolution of homeothermy in mammals and birds. Homeotherms have a much higher basal metabolic rate and so need a more substantial energy reserve than poikilotherms (reptiles, fish, and amphibians). The ability to accrue copious amounts of adipose tissue was also essential for exploitation of habitats where food supply is 0003 24 ADIPOSE TISSUE/Structure and Function of White Adipose Tissue 0004 scarce (e.g., deserts) or seasonal (e.g., arctic). Northern species such as polar bears and reindeer build up substantial depots of fat during the summer to provide reserves of nutrients during the winter. Such species thus have substantial seasonal fluctuations in the amount of adipose tissue in their bodies. Additional reserves of adipose tissue are also accumulated during pregnancy in most species to help support the development of the fetus during the later stages of pregnancy and to facilitate milk production. The use of adipose tissue lipid is very important during early lactation in dairy cows, for example, in which appetite increases more slowly than milk production at the beginning of lactation. It is also important for milk production in some species of bears and seals that fast during lactation. It is now apparent that adipose tissues are not solely a store of fat. Subcutaneous adipose tissue will act as insulation; adipose depots in the eye socket may have a protective function. More importantly perhaps, adipose tissue produces a number of biologically active substances, e.g., prostaglandins, insulin-like growth factor 1 and binding proteins, adipsin, cytokines (e.g., tumor necrosis factor a), estrogens (primarily estrone), and leptin. Some of these substances are probably important for adipose tissue function and development, but some have other roles. Adipose tissue is the major source of estrogens in postmenopausal women. The mammary gland grows in a bed of adipose tissue and is thought to require factors secreted by adipose tissue for its development. Lymph nodes are located in adipose tissue depots and in some species (e.g., guinea-pigs), at least, there is an interaction between adipocytes and lymphoid cells. Adipose tissue may have another role in defense systems of the body as it secretes adipsin and several other proteins involved in an alternative pathway of complement production. Another important protein produced by adipocytes is the cytokine tumor necrosis factor-a; production of this factor is normally low, but it is markedly increased during obesity, when it appears to play a major role in the development of insulin resistance in the tissue, and hence noninsulindependent diabetes. Perhaps the most important and interesting protein secreted by adipocytes is leptin, which has a key role in appetite control and energy balance (Figure 1). Leptin was discovered only recently through studies on the basis of a genetically obese strain of mice (ob/ ob mice); these mice produce a nonfunctional form of leptin. Leptin is released into the blood and travels to the brain, where there are leptin receptors in discrete areas involved in appetite control. Low levels of leptin in the blood increase appetite, whereas administration of high doses inhibit appetite. Leptin not only modulates appetite, but also increases energy expenditure, stimulating thermogenesis in brown adipose tissue, suggesting a key role in the control of energy balance in the body. Leptin synthesis is regulated by insulin, glucocorticoids, and catecholamines, but most interestingly, the concentration of leptin in the blood in the fed state is proportional to the amount of fat in the body; this led to the idea that leptin acts as a ‘lipostat,’ matching appetite to adiposity. However, the leptin concentration in the blood is decreased by fasting, and leptin is involved in the changes in secretion of several pituitary hormones during fasting. Thus, it has been suggested that the major Hypothalamus LEPTIN RECEPTORS Adipocyte LEPTIN (−ve) (+ve) Catecholamines CNS, pituitary gland (+ve) Glucocorticoids Insulin Immune system Food intake Thermogenesis Energy balance fig0001 Figure 1 Leptin production and function. CNS, central nervous system. Reproductive system 0005 ADIPOSE TISSUE/Structure and Function of White Adipose Tissue 0006 0007 role of leptin may be in adaptation to fasting and acting as a signal of too little rather than too much adipose tissue. Leptin appears to be required for normal functioning of the immune system and also for reproductive function. Indeed, a lack of leptin may well be the main reason for the failure of the menstrual cycle in anorexics and very lean athletes. This makes good physiological sense as it insures that females do not become pregnant, unless they have adequate reserves of adipose tissue lipid. Adipose tissue thus has a variety of functions, in addition to being an energy store. While the accumulation of adipose tissue lipid reserves provides a buffer against starvation, and some degree of adiposity is important for the various other functions of the adipose tissue described above, there is a cost in that additional body mass decreases speed and agility and so increases the chance of succumbing to predation. Thus, in most wild animals for which food is generally plentiful, there are usually only small amounts of adipose tissue (predation rather than starvation being the greatest threat to mortality). In such species, it seems likely that the leptin system, and probably other systems, will be acutely tuned to maintain the minimal amounts of adipose tissue needed. In general, it is only species living in environments where the availability of food is erratic or seasonal that accumulate large amounts of adipose tissue since, for these species, starvation is a greater threat than predation. In such species, the leptin system must be modulated to allow the accumulation of adipose tissue lipid. It would also appear that the leptin system can be readily subverted in humans and also domestic pets for excess adiposity is becoming a major problem. In addition to white adipose tissue, there is also another form, brown adipose tissue, which differs morphologically and biochemically, and has an important role in thermogenesis. Development of Adipose Tissue 0008 Adipose tissue develops both by accretion of lipid in adipocytes and by increases in the number of adipocytes. Mature adipocytes are thought to be unable to divide; rather, they are produced from a pool of precursor cells within the tissue. The sequence of events in the formation of mature adipocytes (Figure 2) is still partly speculative, and much has been gleaned from studies of certain cell lines (e.g., ob17 and 3T3 L1 cells), which will differentiate and develop into adipocytes in cell culture. Current thinking envisages a pluripotent stem cell that can give rise to muscle and bone cells as well as adipocytes. Once committed to adipocyte formation, this cell is termed an adipoblast. 25 Stem cell Commitment Muscle-cell precursors Adipocyte precursors (adipoblasts) Bone-cell precursors Proliferation Differentiation Lipid accumulation Mature, fat-filled adipocytes Figure 2 Adipocyte development. This is envisaged (it has not been isolated) as an undifferentiated cell, devoid of lipid droplets but able to proliferate. At some point, these cells begin to differentiate, acquiring, in stages, the enzymes and other proteins characteristic of adipocytes. Once differentiated, these cells can begin to accumulate lipid, which appears at first as a series of small droplets within the cell. As these become larger, they fuse to form the single lipid droplet characteristic of mature adipocytes. Both differentiating cells and cells with several small lipid droplets (multilocular phase) are often referred to as preadipocytes, the term adipocyte usually being used to describe cells with a single lipid droplet. Multilocular adipocytes are very similar in appearance to mature brown adipocytes, and it was once thought that the brown adipocyte was a stage in the development of the white adipocytes. It is now recognized that this view is incorrect, except possibly for a few special cases (e.g., the perirenal adipose tissue depot of newborn lambs). Adipocytes begin to appear in the fetus about half way through gestation, developing in small clumps around blood vessels. Within a depot, both the number and size of adipocytes increase in phases (Figure 3). In addition, it is now clear that development is not synchronized in all depots; abdominal depots in general develop earlier than those fig0002 0009 26 ADIPOSE TISSUE/Structure and Function of White Adipose Tissue Number of subcutaneous adipocytes / sheep ( 10−9) Deposition and Mobilization of Fat The synthesis of triacylglycerol (esterification) requires a supply of fatty acids and glycerol 3-phosphate (Figure 4). The latter is mostly synthesized from glucose. Fatty acids, however, may be synthesized de novo within the cell or obtained from blood triacylglycerols. Fatty acids can be synthesized in adipocytes from a variety of precursors, including glucose, acetate, lactate, and some amino acids. Glucose is quantitatively the most important in man and some laboratory species (e.g., rats, mice), whereas acetate is most important in ruminants. Liver is also an important site of fatty acid synthesis in many mammals and is the major site of fatty acid synthesis in birds (avian adipocytes have essentially no capacity for fatty acid synthesis) and also in humans on a typical Western diet. Some of the fatty acids synthesized in the liver are incorporated into very-low-density lipoprotein 0011 ADIPOCYTE 6 1000 3 500 0 0 B 200 400 Triacylglycerol Glycerol 3phosphate Fatty acids Fatty acids Glucose Glycerol Acetate 600 Days fig0003 in site-specific development of adipose tissue. Derivatives of arachidonic acid (an essential fatty acid) such as 15-deoxy-D12,14-prostaglandin J2 are also thought to have a major role in adipogenesis, acting via the recently discovered (and inappropriately named!) peroxisome proliferator-activated receptor-g. Growth hormone has a complex role, stimulating insulin-like growth factor 1 production in adipose tissue and hence proliferation of preadipocytes and in addition may be required for the cells to become ‘committed’ to differentiation. In addition to positive effectors, tumor necrosis factor a and transforming growth factor b can inhibit differentiation. In contrast to hyperplasia, much more is known about the control of hypertrophy, for this is dependent on the metabolic rates of the pathways of lipid synthesis and degradation. 1500 9 Mean cell volume (pl) 0010 associated with the musculature. In most species, the fetal stage is a period of active proliferation but little hypertrophy, so that cells are small at birth (about 10 pl in volume). The suckling period usually results in rapid hypertrophy and hyperplasia; this is followed by a more quiescent period when muscle growth predominates. When the rate of muscle growth begins to slacken, nutrients are diverted into adipose tissue, and the fattening phase begins. This phase is associated with marked hypertrophy, due to lipid deposition, in most depots and further hyperplasia, especially in the carcass depots. During the fattening phase, depot-specific differences in adipocyte size appear. Adipocytes do not increase in size indefinitely; once a maximum is reached (about 1–3 nl, depending on species), this seems to trigger the formation of new adipocytes from the precursor pool. The view prevalent in the 1970s that all hyperplasia occurred in young animals, including humans, is now thought to be invalid. A great deal of research has gone into identifying the hormones and other factors that promote the proliferation and differentiation of adipocyte precursor cells. At present, the picture is far from clear, in part because of probable species differences and also because much of the work has involved the use of cell lines that do not all appear to have identical hormonal requirements for development. A variety of peptide growth factors (e.g., insulin-like growth factor 1, fibroblast growth factor, platelet-derived growth factor, epidermal growth factor) can stimulate preadipocyte proliferation, whereas insulin, thyroid hormones, and glucocorticoids appear to be important for differentiation of preadipocytes into adipocytes in a variety of species. Glucocorticoid hormones and also testosterone are thought to have important roles Figure 3 Developmental changes in adipocyte number (broken line) and mean cell volume (solid line) of sheep subcutaneous adipose tissue from 25 days before birth (B) until 600 days after birth. Fatty acids VLDL, Chylomicron BLOOD Figure 4 Pathways for synthesis and hydrolysis of triacylglycerol in adipocytes. VLDL, very-low-density lipoprotein. fig0004 ADIPOSE TISSUE/Structure and Function of White Adipose Tissue 0012 (VLDL) triaclyglycerols for transport to adipocytes and other tissues. Dietary fatty acids are also incorporated into triacylglycerols in the intestinal cells and secreted as another form of lipoprotein, called chylomicrons. Triacylglycerols are essentially insoluble in water and so cannot be taken up directly by adipocytes from blood lipoproteins; thus, the fatty acids are released by the action of the enzyme lipoprotein lipase. This enzyme is synthesized in adipocytes and then secreted, after which it migrates to the inner surface of the cells lining the blood capillaries. Whereas most of the fatty acids released by the action of lipoprotein lipase are taken up by the adipocytes, some are released into the blood and used by other tissues. The relative importance of de novo synthesis and lipoprotein lipase activity as a source of fatty acids for fat synthesis depends on the diet and the species. When animals are fed high-fat diets, chylomicron lipids are the major source. When animals are fed diets rich in carbohydrates, the major source becomes VLDL lipids or de novo fatty acid synthesis in adipocytes, depending on whether adipocytes or the liver are the major site of fatty acid synthesis in the species. Once synthesized within the adipocyte, triacylglycerols are stored in the lipid droplet. Fatty acids are released from them when required by the action of the enzyme hormone-sensitive lipase (distinct from lipoprotein lipase). This enzyme cleaves two molecules of fatty acids to yield a monoacylglycerol that is then hydrolyzed to glycerol and fatty acid by a separate enzyme. Essentially all the glycerol is released from the cell as it cannot be metabolised by adipocytes. Some fatty acids, however, are usually reesterified, and so the ratio of fatty acid to glycerol leaving the cell is normally less than the theoretical 3:1. Released fatty acid is bound to albumin in the blood and transported to the liver and other tissues. Fatty acid esterification and triacylglycerol hydrolysis (lipolysis) occur continuously, i.e., there is a continual turnover of adipocyte triacylglycerol. Net accretion or loss of lipid thus depends on the relative rates of these two processes. Regulation of Adipose Tissue Metabolism 0013 Both lipid synthesis and hydrolysis are under complex hormonal control. Hormones regulate the amounts of key enzymes and other proteins involved, as well as their activities. In addition, the ‘signal transduction’ systems (a series of reactions transmitting hormoneinduced signals to targets in the cell), through which hormones achieve their effects, are also subject to endocrine control themselves, and changes in the ability of adipocytes to transmit such signals are an 27 important part of the adaptations to some physiological states (e.g., lactation). Regulation of fatty acid synthesis depends on the precursor. For glucose, control begins at the point of entry into the cell where its transport is dependent on a specific carrier protein (transporter); the major glucose transporter of adipocytes is called ‘glut 4.’ Insulin stimulates glucose transport both by promoting recruitment of glut 4 into the plasma membrane and by increasing its activity. Within the cell, glucose is initially phosphorylated and then metabolized by a long series of reactions, some in the cytosol, some in the mitochondria, to produce acetyl coenzyme A (CoA) in the cytosol. Several enzymes, in particular phosphofructokinase and pyruvate dehydrogenase, have key roles in controling this flux. Insulin, for example, activates pyruvate dehydrogenase. For acetate, the control is much simpler as its initial reaction results in the production of acetyl CoA. The conversion of acetyl CoA to fatty acid is catalyzed by two enzymes, acetyl CoA carboxylase and fatty acid synthetase. The former is thought to be the most important enzyme controling flux. Both the amount of acetyl CoA carboxylase and its activation status (it is an enzyme that exists in active and inactive forms in the cell) change markedly with physiological, nutritional, and pathological condition. The amount and activity, for example, are decreased by fasting, highfat diets, diabetes, and lactation. Insulin increases both the amount and activity of the enzyme. These effects of insulin are antagonized by growth hormone. Catecholamines and glucagon also cause inactivation of the enzyme and hence a fall in the rate of fatty acid synthesis. Insulin increases the synthesis and secretion of lipoprotein lipase; this effect is accentuated by glucocorticoids. Gastric inhibitory polypeptide also increases lipoprotein lipase activity; this effect is likely to be important for promoting fat deposition in animals eating high-fat diets as such diets stimulate secretion of this hormone. Thus, insulin and certain gut hormones increase fat synthesis by increasing the supply of fatty acids for esterification. Insulin also promotes glycerol 3-phosphate formation, in part at least, by increasing glucose uptake by adipocytes. The rate of fatty acid esterification itself may not be stimulated directly by hormones but varies directly with fatty acid availability. Curiously, adipocytes secrete adipsin and two related proteins, which interact in the presence of chylomicrons, to produce acylationstimulating protein, which then acts on adipocytes to stimulate esterification and glucose uptake. The enzyme controling lipolysis, hormone-sensitive lipase, exists in active and inactive states in the fat cell. Glucagon and adrenaline (epinephrine), and also 0014 0015 0016 28 ADIPOSE TISSUE/Structure and Function of White Adipose Tissue Adrenaline Noradrenaline β-Adrenergic receptor α2-Adrenergic receptor Prostaglandin E Insulin Prostaglandin E receptor Insulin receptor Adenylate cyclase Cyclic AMP phosphodiesterase Cyclic AMP Hormone-sensitive lipase Triacylglycerol fig0005 3 fatty acids + glycerol Figure 5 Control of triacylglycerol hydrolysis (lipolysis) by the catecholamines (adrenaline and noradrenaline) and insulin. AMP, adenosine monophosphate; ", #, activity/concentration increased or decreased by stimulus, respectively. noradrenaline (norepinephrine) (which is released from nerve endings of the sympathetic nervous system within the tissue itself), interact with specific receptor proteins in the plasma membrane (Figure 5). This causes activation of a key enzyme, adenylate cyclase, which synthesizes cyclic adenosine monophosphate (cAMP). Increased concentrations of cAMP both activate hormone-sensitive lipase and promote its movement from the cytosol to the surface of the lipid droplet, resulting in increased lipolysis. This stimulatory mechanism is attenuated by several inhibitory systems. Adenosine and prostaglandin E2, which are both produced within adipose tissue, interact with their own receptors, leading to inhibition of adenylate cyclase. Curiously, adrenaline and noradrenaline can both activate and inhibit adenylate cyclase. They activate adenylate cyclase by interacting with b-adrenergic receptors and inhibit by interacting with a2-adrenergic receptors. The effect of adrenaline and noradrenaline on lipolysis will thus depend in part on the relative number of b- and a2adrenergic receptors in the adipocytes. There is considerable site- and gender-specific variation in the ratio of a2- to b-adrenergic receptor number of adipocytes in some species. For example, in women, intraabdominal adipocytes have a ratio of about 1:1, whereas subcutaneous femoral and gluteal adipocytes have a ratio of about 10:1 a2-:b-adrenergic receptors. This ratio is thought to be responsible for the very poor lipolytic response to catecholamines of these subcutaneous adipocytes in women and hence the relatively large size of these cells compared with adipocytes elsewhere in the body. In addition to the above, insulin activates the enzyme, cAMP-phosphodiesterase, which catalyzes the degradation of cAMP and so reduces its concentration. The rate of lipolysis then will depend on the concentration of a whole range of hormones, locally produced factors, and neurohumoral transmitters (substances, such as noradrenaline, which are released by nerve endings in tissues). In addition, the ability of the ‘signal transduction’ system to transmit signals varies with age and with physiological state. For example, during lactation, when fat is often mobilized to support milk production, the system can become more responsive to agents that promote lipolysis. Thyroid hormones, glucocorticoids, sex steroids, and growth hormone all act on one or more components of the signal transduction system, altering its ability to respond to stimulatory and/or inhibitory agents. Adipose tissue metabolism is thus under complex control. In general, insulin promotes fat synthesis and inhibits lipolysis, whereas catecholamines and glucagon inhibit synthesis and promote lipolysis. In addition, steroid hormones, thyroid hormones, and growth hormone act to modulate the effects of insulin and catecholamines, in part at least, by modifying the ability of the signal transduction systems to transmit signals. 0017 Composition of Stored Fat Triacylglycerols comprise about 95% of adipose tissue lipid; the remainder includes diacylglycerols, phospholipids, unesterified fatty acids, and cholesterol. The fatty acid composition of the triacylglycerols shows species variation (Table 1), but oleic and 0018 ADIPOSE TISSUE/Structure and Function of Brown Adipose Tissue tbl0001 Table 1 Fatty acid composition triacylglycerols (representative values) of adipose tissue Fatty acids (g per 100 g of total fatty acids) 0019 Fatty acid Humans Pig Sheep Chicken Myristic Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Other 4 23 5 6 49 9 1 3 1 26 3 13 42 13 2 3 22 4 20 39 3 2 7 1 26 6 7 40 19 1 palmitic acids are major components in all species. The proportions of polyunsaturated fatty acids (linoleic and linolenic) are usually low in adipose tissue from ruminant animals and higher in chicken and pig adipose tissue. This reflects the dietary supply; as described above, fatty acids are derived both from dietary lipid (via chylomicrons) and from de novo synthesis (which produces palmitic acid). There is some capacity for chain elongation of palmitic acid to produce stearic acid, and for desaturation, which converts palmitic to palmitoleic and stearic to oleic acids, but the tissue cannot synthesize linoleic or linolenic acids. In simple-stomached species, such as humans and pigs, varying the fatty acid composition of the diet will alter the fatty acid composition of adipose tissue lipids. For ruminant animals, however, dietary polyunsaturated fatty acids are mostly hydrogenated in the rumen to produce oleic and stearic acids. The small amount of linoleic and linolenic acids escaping this fate is conserved for essential functions (membrane synthesis, prostaglandin production), so that adipose tissue lipids (and milk fat) normally contain little linoleic or linolenic acids. This is ironic, for linolenic acid is the major fatty acid of the ruminant diet. If hydrogenation in the rumen is avoided (e.g., by coating dietary lipid with formaldehyde-treated casein), large quantities of these polyunsaturated fatty acids are absorbed, producing adipose tissue rich in linoleic and linolenic acids. Minor changes in the fatty acid composition occur during development, and there are minor differences between adipose tissue depots, but these are small compared with the changes that can be elicited by dietary manipulation. See also: Fats: Production of Animal Fats; Fatty Acids: Properties; Hormones: Adrenal Hormones; Pituitary Hormones; Obesity: Etiology and Diagnosis; Fat Distribution 29 Further Reading Bjorntorp P (1991) Adipose tissue distribution and function. International Journal of Obesity 15: 67–81. Flier JS (1995) The adipocyte: storage depot or node on the information superhighway? Cell 80: 15–18. Flint DJ and Vernon RG (1993) Hormones and adipose tissue growth. In: Pang PKT, Scanes CG and Schreibman MP (eds) Vertebrate Endocrinology: Fundamentals and Biomedical Implications, pp. 469–494. Orlando, FL: Academic Press. Friedman JM and Halaas JL (1998) Leptin and the regulation of body weight in mammals. Nature 395: 763–770. Gregoire FM, Smas CM and Sul HS (1998) Understanding adipocyte differentiation. Physiological Reviews 78: 783–809. Mohammed-Ali V, Pinkey JH and Coppack SW (1998) Adipose tissue as an endocrine and paracrine organ. International Journal of Obesity 22: 1145–1158. Pond CM (1992) An evolutionary and functional view of mammalian adipose tissue. Proceedings of the Nutrition Society 51: 367–377. Spiegelman BM and Flier JS (1996) Adipogenesis and obesity – rounding out the big picture. Cell 87: 377–389. Vernon RG (1992) Control of lipogenesis and lipolysis. In: Buttery PJ, Boorman KN and Lindsay DB (eds) The Control of Fat and Lean Deposition, pp. 59–80. Oxford: Butterworth-Heinemann. Vernon RG, Barber MC and Travers MT (1999) Present and future studies on lipogenesis in animals and human subjects. Proceedings of the Nutrition Society 58: 541–549. Structure and Function of Brown Adipose Tissue M J Stock*, St George’s Hospital Medical School, Tooting, London, UK S Cinti, Universita degli Studi di Ancona, Ancona, Italy Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Brown Adipose Tissue Brown adipose tissue (BAT), or brown fat, is a small but highly specialized tissue, the main function of which is to produce heat (thermogenesis). This function requires a good blood supply and a dense population of mitochondria – two features that account for its reddish brown color and distinguish it from white adipose tissue (WAT) (see Figure 1). It is found in most mammals, particularly in the neonate, and plays an important role in the control of body temperature during exposure to the cold. There is *Author deceased. 0001 30 ADIPOSE TISSUE/Structure and Function of Brown Adipose Tissue N P CAP L L m m CAP m L P P fig0001 Figure 1 Electron micrograph of brown adipose tissue showing the typical features of a highly thermogenic tissue, i.e., a dense population of well-developed mitochondria, lipid droplets, rich nerve (sympathetic) and blood (capillaries) supply. m, mitochondria; L, lipid droplets; CAP, capillary; N, nerve fiber; P, precursor. evidence indicating that it is also involved in the regulation of energy balance. The tissue was first described some 300 years ago, but its thermogenic function was not recognized until the early 1960s, and only during the 1980s did its capacity for thermogenesis and its unique metabolism come to be fully appreciated. (See Thermogenesis.) BAT has been identified histologically in human adults up to the age of 80 years or more, and biochemical tests suggest that it might retain its thermogenic activity. BAT depots often contain white adipocytes, and some WAT depots may contain brown adipocytes, but these can be difficult to see. Histology and Development Location 0002 BAT is most obvious in small mammals, hibernators, and neonates, and is usually found around the kidneys, heart and aorta, along the intercostal muscles and sternum, in the axilla, in the subcutaneous inter- and subscapular regions, and deep within the neck, around the main arteries and veins. This distribution suggests that the tissues act as a jacket to heat the major organs and warm the blood passing from the periphery into the trunk. The distribution varies considerably between species, and some (e.g., dog, human) have little or no interscapular BAT, whereas in others (e.g., rodents), the interscapular depot may account for 20–30% of the total. BAT rarely exceeds 2–3% of body mass, and is present in such small quantities in large adult mammals that it is often impossible to detect visually. In spite of this, Brown adipocytes appear polygonal under the microscope, with a diameter of 10–25 mm, compared with 20–150 mm for white adipocytes. The adipocytes are organized in discrete lobules, surrounded by connective tissue, extensive blood vessels and numerous sympathetic nerves terminating on the adipocytes and blood vessels. Unlike white adipocytes, the nuclei are spherical and located centrally, and the lipid is stored in small, multilocular droplets. Between the droplets and packing the cytoplasm are numerous, well-developed mitochondria that possess distinctive and regular cristae, often traversing the width of the mitochondrion. The endoplasmic reticulum (particularly the rough reticulum) and Golgi apparatus are relatively small, and lysosomes, peroxisomes, and clusters of glycogen granules are often present; adjacent cells are usually connected by gap junctions. 0003 ADIPOSE TISSUE/Structure and Function of Brown Adipose Tissue 0004 Cytogenic studies indicate that brown adipocytes are derived from stem cells closely associated with vascular structures, and it is now generally agreed that these are distinct from stem cells that give rise to white adipocytes. Mature brown adipocytes cannot undergo mitosis, and the recruitment (hyperplasia) seen during cold adaptation occurs by cytogenesis and mitosis of newly differentiated brown adipocytes. The first appearance of differentiated BAT cells varies between species, and in some neonates (e.g., guineapig, rabbit, puppy, lamb), the tissue is well developed and functional at birth. In other species (e.g., rats, mice), the tissue is not fully functional at birth, but becomes thermogenically active within a few days. By contrast, the Syrian hamster is born without BAT, and it takes about 2 weeks for the tissue to develop, during which time, the animal is essentially poikilothermic. Morphology is highly dependent on age, strain, environment, and various physiological and pathological conditions. Brown adipocytes will transform gradually into what look like white adipocytes during prolonged inactivity. Innervation 0005 The innervation of BAT is another feature that distinguishes it from WAT, since the metabolic activity of the tissue is almost entirely determined by the release of noradrenaline at sympathetic nerve terminals on the brown adipocytes. In some depots (e.g., rodent interscapular BAT), the sympathetic nerves enter as obvious bundles. This makes experimental techniques such as surgical sympathectomy and nerve stimulation and recordings relatively easy to undertake, although there can be problems in distinguishing between effects on adipocytes and those on the vascular supply. The parenchymal sympathetic fibers innervating adipocytes and arterioles release mainly noradrenaline, and this explains why the tissue content and turnover of noradrenaline are high; noradrenaline turnover is a good index of sympathetic activation in response to various environmental and dietary stimuli. Apart from noradrenaline, histamine, adenosine, and various peptides may modulate the sympathetic activation of BAT. Neuropeptide-Y (NPY) is found colocalized with noradrenaline in perivascular sympathetic nerve endings, and the depletion of sensory peptides – CGRP (calcitonin gene-related peptide) and Substance P – by capsaicin suggests that the tissue contains afferent fibers. Blood Supply 0006 The high oxygen supply required to support thermogenesis is provided by an extensive network of vessels, 31 estimated to be four to six times denser than that in white adipose tissue. The vascular supply can support a blood flow in excess of 20 ml per gram of tissue per minute; during maximal stimulation in cold-adapted rodents, this relatively small mass of tissue can receive over 30% of cardiac output. Blood flow increases result partly from the vasomotor activity of the sympathetic nerves, but also from autoregulatory increases caused by sympathetic activation of metabolism and the release of metabolites. Aerobic heat production can be so intense that the oxygen supplied in arterial blood is almost completely extracted, and the venous blood appears desaturated. The small amounts of oxygen remaining probably represent blood that bypassed the capillary network via arteriovenous anastomoses (i.e., vascular shunts). These vascular shunts, of which there are many, probably act to convect the heat generated away from the tissue, thereby avoiding thermal damage (BAT temperatures can rise to over 44
C). The thermogenic capacity of BAT can be determined from measurements of blood flow and oxygen extraction, and estimates of up to 500 W kg1 can be compared with values of only 60 W kg1 for the maximal aerobic power of skeletal muscle. (See Exercise: Muscle.) Metabolism The exceptional heat-producing capacity of BAT is due to its mitochondria, which possess a 32-kDa polypeptide called uncoupling protein (UCP). This is now known as UCP1, since two other, similar mitochondrial proteins (UCP2 and UCP3) have been discovered, but UCP1 is unique to BAT mitochondria and is responsible for the only significant, physiological example of uncoupled oxidative phosphorylation in mammalian metabolism. UCP forms a proton conductance channel in the mitochondrial inner membrane, and dissipates the proton electrochemical gradient generated by oxidation of substrates via the electron transport system. This has the effect of uncoupling oxidation from the phosphorylation of ADP (adenosine diphosphate) to ATP (adenosine triphosphate), thereby dissipating the energy released as heat, as well as increasing the rate of oxidation due to the loss of respiratory control. The proton conductance pathway is under inhibitory control by purine nucleotides (e.g., ADP, ATP, GDP), which bind to UCP, and is activated following sympathetic activation of the adipocyte b-adrenergic receptors, which also stimulate lipolysis and the release of free fatty acids from the triglyceride droplets. These fatty acids provide the principal fuel for thermogenesis. The rapid activation of the proton conductance pathway following sympathetic 0007 0008 32 ADIPOSE TISSUE/Structure and Function of Brown Adipose Tissue 0009 0010 stimulation can be detected by measuring the mitochondrial binding of purine nucleotides – usually GDP (guanosine diphosphate) – in vitro, whereas chronic, adaptive changes in thermogenic capacity depend on immunoassay of mitochondrial UCP concentrations. High rates of oxidation in any tissue require adequate levels of all the enzyme systems of intermediary metabolism, and BAT is particularly well endowed with those required for glycolysis, the tricarboxylic acid cycle, and the mitochondrial electron conductance chain. Since fatty acids are the main fuel for thermogenesis, adenyl cyclase activity and the subsequent cascade that leads to the intracellular release of fatty acids from stored triglyceride are prominent features of BAT metabolism. However, the lipid stored in the multilocular droplets is not sufficient to sustain thermogenesis for long periods, and brown adipocytes then rely on their remarkable capacity for lipogenesis. In cold-adapted rats and mice, the lipogenic capacity of BAT is high enough to account for a major fraction of the amount of dietary carbohydrate that the animal converts to lipid. As well as the fatty acids supplied de novo by lipogenesis, the high level of lipoprotein lipase allows BAT to take up fatty acids released by the hydrolysis of circulating triglycerides. In addition to the normal complement of respiratory enzyme systems, brown fat cells also contain peroxisomes, and these proliferate during chronic stimulation of the tissue. Peroxisomal oxidation of substrates is not linked to phosphorylation, and could therefore make a contribution to cellular thermogenesis. However, the contribution is probably very small, and their function may be more to do with controling levels of free radicals as well as the cytosolic metabolism of fatty acids that are not preferentially metabolized by mitochondria. Another interesting feature of BAT metabolism is the presence of an enzyme, 50 -deiodinase, that converts thyroxine (T4) to the physiologically active hormone, triiodothyronine (T3). The enzyme is under sympathetic control, and its activity can increase several hundred-fold in cold-adapted animals. The T3 produced is more than sufficient to saturate the nuclear receptors, and it is possible that much of the T3 is exported and exerts effects on other tissues. (See Hormones: Thyroid Hormones.) Functions of BAT Thermoregulation 0011 Shivering is an acute response to cold exposure and not a particularly effective mechanism for protecting the body against hypothermia. As a consequence, many animals resort to a form of heat production called nonshivering thermogenesis (NST), which, unlike shivering, can be sustained without fatigue and disruption of locomotor activity or sleeping behavior. NST appears as an adaptive response to chronic cold exposure in many mammals, but particularly in small animals where heat losses are greater due to the large surface area relative to body mass. The high degree of surface heat loss and immature neuromuscular development also explain why the neonates of most mammalian species (including humans) depend on NST to maintain body temperature until shivering, locomotor activity and other behavioral thermoregulatory responses develop. A third group is the hibernators, who rely on NST for the rapid rewarming that occurs during arousal. Depending upon the species, NST can raise heat production by 100–300% above that in a warm, thermoneutral environment, and is associated with large increases in the activity of the sympathetic nervous system. Pharmacological blockade (particularly with b-adrenergic antagonists) can inhibit completely the cold-induced rise in heat production, and demonstrates the dominant role of the sympathetic nervous system in mediating NST. The effector tissue is BAT, and a considerable body of evidence now exists to link BAT function to NST. For example, the capacity for NST is inversely proportional to age, bodyweight, and acclimation temperature, and this coincides with histological, physiological, and biochemical indices of BAT activity. Conversely, deacclimation and decreased NST is associated with a parallel decline in BAT activity. Perhaps the most convincing evidence comes from in vivo measurements of BAT oxygen consumption, which, in spite of enormous technical difficulties, have shown that the tissue can account for well over 60% of NST. Even this may be an underestimate, since it is not possible to measure the contribution of all the numerous, small and diffuse BAT depots. 0012 Energy-balance Regulation Evidence linking BAT to energy-balance regulation comes mainly from studies on laboratory rodents that represent examples of two extremes of metabolic efficiency. At one extreme, there are normal, young rats and mice that fail to become obese in spite of an excessive energy intake, and at the other extreme, there are examples of obesity developing in rats and mice (e.g., genetic and hypothalamic obesities), even when energy intake is normal. The explanation for these differences appears to depend on an adaptive form of heat production called diet-induced thermogenesis (DIT), which is absent or defective in obese 0013 ADIPOSE TISSUE/Structure and Function of Brown Adipose Tissue 0014 animals, but provides a mechanism whereby normal animals can adjust energy expenditure to compensate for energy consumed in excess of requirements. DIT can produce increases in total heat production of 60–70%, and account for up to 90% of the excess energy consumed by hyperphagic rats. In rats feeding normally, the level of DIT is low, but sufficient to control energy balance by compensating for errors in the control of energy intake. The control and metabolic origins of DIT are identical in almost every respect to NST, although cold is a more potent stimulus and produces more dramatic changes than dietary stimuli. As a consequence, the changes in sympathetic activity, BAT hypertrophy and hyperplasia, mitochondrial proliferation, guanosine diphosphate binding and UCP concentration in rats exhibiting DIT are smaller than those seen in cold-adapted rats. However, these changes in BAT function are sufficient to account for up to 80% of the diet-induced changes in thermogenic capacity seen in hyperphagic rats. By contrast, BAT is usually atrophied and relatively inactive in obese rodents, although it will respond to exogenous noradrenaline, and the animals retain the capacity to adapt to the cold and exhibit NST. This suggests that the defective DIT in these obese rodents is due to a failure of the sympathetic activation of BAT, rather than a defect in BAT itself. This contrasts with what is seen in a transgenic mouse bearing a ‘toxigene’ that causes a genetic ablation of BAT. These mice fail to exhibit NST and DIT, and become obese – sometimes without eating any more than normal. (See Obesity: Etiology and Diagnosis.) Other Functions 0015 0016 In addition to cold- and diet-induced thermogenesis, there are several pathological conditions in which BAT has been implicated as a source of increased heat production. Fever, sepsis, and cancer cachexia are three examples where increased sympathetic activation of BAT is thought to be at least partly responsible for the hypermetabolic response seen in animal models of these conditions, and often involve cytokines such as the interleukins. Patients with pheochromocytoma (adrenomedullary tumor) have very high circulating levels of adrenaline and noradrenaline, and it is thought that the elevated heat production in this condition is due to the stimulatory effect of these catecholamines on BAT; the best examples of active BAT in human adults have been seen in patients with pheochromocytoma. In spite of increased energy intakes, pregnant rats and mice show little or no change in BAT activity, but during lactation, the tissue atrophies, and its sympathetic activation and thermogenic capacity decline to 33 levels seen after sympathectomy or fasting. Similar reductions can be seen in warm-adapted nonlactating animals, which suggests that BAT thermogenesis declines to compensate for the elevated heat production associated with milk synthesis in the lactating mammary glands. Increased heat production during exercise could also account for the lower BAT activity seen in exercise-trained animals. This is particularly noticeable in cold environments, where exercise can prevent many of the changes in BAT function associated with NST. Control of BAT Neural The control over the sympathetic supply to the various BAT depots originates from the hypothalamus, which receives afferent information on thermal and nutrient status from the periphery, as well as having its own receptor mechanisms and pathways. One of the main thermosensitive and thermoregulatory areas is the preoptic/anterior hypothalamus (POAH), but this is thought to modulate BAT thermogenesis via inhibitory pathways that descend to the lower brainstem. The area that appears to exert a major influence over BAT is one that has been classically associated with the control of energy intake – the ventromedial hypothalamus (VMH), often loosely referred to as the ‘satiety center’. Electrical stimulation of the VMH increases BAT thermogenesis, whereas lesions cause the tissue to atrophy, and the latter observation helps explain why VMH-lesioned animals can become obese without overeating. There are connections between the VMH and other hypothalamic areas concerned with feeding behavior (e.g., lateral hypothalamus, paraventricular nucleus), and with the POAH, which provide a neural basis for integrating information on energy intake and body temperature, and modulate the level of NST and DIT accordingly. 0017 Hormonal Adrenaline stimulates BAT thermogenesis, but it is not as potent as noradrenaline, and in most physiological situations, the circulating levels of adrenaline are probably not sufficient to activate the tissue’s b-adrenoceptors. However, views may change on this in the light of recent, more sensitive measurements that show that circulating levels of adrenaline may have been previously underestimated. Although thyroid hormones (T3 and T4) are necessary to maintain BAT function, and T3 is itself produced by the tissue, hyperthyroidism suppresses BAT activity. This is probably due to reduced sympathetic activation 0018 34 ADOLESCENTS 0019 0020 compensating for high levels of heat production in other, thyroid-sensitive tissues. Glucocorticoids exert little or no direct effects on BAT, even though the tissue has glucocorticoid receptors. However, these adrenocorticoids have central inhibitory actions on the sympathetic outflow to BAT, which are particularly noticeable in genetically obese rodents. Adrenalectomy in these rodents completely restores sympathetic activity, BAT thermogenesis, and DIT to levels seen in lean animals. Only very low replacement doses of glucocorticoids are required to reverse the effects of adrenalectomy, suggesting that these obese animals are hypersensitive to the inhibitory actions of glucocorticoids. The glucocorticoids are thought to inhibit thermogenesis via feedback inhibition of hypothalamic corticotrophinreleasing factor (CRF). In addition to its effects on the pituitary, CRF is a potent stimulus for the sympathetic activation of BAT. Insulin exerts direct, metabolic effects on BAT that are essentially similar to those that it exerts on white adipose tissue, but it also influences the tissue indirectly via its actions on the glucoreceptors of the VMH. A recent and exciting discovery has been the identification of the lipostatic hormone, leptin. In addition to influencing energy balance (i.e., fat stores) via the hypothalamic control of food intake, leptin also causes sympathetic activation of BAT thermogenesis. This is likely to prove a complex relationship because increased sympathetic activity decreases the expression and release of leptin from both WAT and BAT. Pharmacological 0021 For many years, the b-adrenergic receptor subtype responsible for activation of BAT lipolysis and thermogenesis was thought to be the b1-adrenoceptor. However, there is much recent evidence to suggest that the receptor is atypical (neither b1 nor b2), and it is now classified as the b3-adrenoceptor. Much of this evidence came from experiments using novel thermogenic drugs developed for the treatment of obesity, and which were designed to be highly selective agonists of BAT thermogenesis, with little or no effect on b1-or b2-mediated functions. Subsequent to the pharmacological identification, the gene sequence for the b3-adrenoceptor was identified in the human genome. Treatment with novel b3-adrenoceptor agonists results in significant weight loss (mainly as fat) without affecting food intake in both genetic and dietary obese rodents. However, because the rodent b-adrenoceptor is different, these agonists are not effective in humans, and trials with human b3adrenoceptor agonists have yet to be undertaken. BAT also contains a-adrenergic receptors, which are known to be important for activating the conversion of T4 to T3 by the 50 -deiodinase, and may also play a minor, facilitatory role in thermogenesis. (See Obesity: Treatment.) See also: Exercise: Muscle; Hormones: Thyroid Hormones; Obesity: Etiology and Diagnosis; Treatment; Thermogenesis Further Reading Cinti S (1999) The Adipose Organ. Milan: Editrice Kurtis. Himms-Hagen J (1990) Brown adipose tissue thermogenesis. In: Schonbaum E and Lomax P (eds) Thermoregulation and Biochemistry, pp. 327–414. New York: Pergamon Press. Rothwell NJ and Stock MJ (1997) Classics in obesity: A role for brown adipose tissue in diet-induced thermogenesis. Obesity Research 5: 650–656. Trayhurn P and Nicholls DG (eds) (1986) Brown Adipose Tissue. London: Edward Arnold. ADOLESCENTS J J B Anderson, University of North Carolina, Chapel Hill, NC, USA Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Introduction 0001 Adequate amounts of energy from carbohydrates, fats, and high-quality protein are considered the critical nutrients for the hormone-driven processes of growth and development of the skeleton and the other organ systems of the body. Developmental sequences vary by gender; Tanner staging is frequently used to assess skeletal development. The needs for nutrients other than those that provide energy and essential amino acids (and N) continue to undergo evaluation as new data are published, and several micronutrients have new recommendations (see below). It has become clear in recent years that adolescents in the USA are not consuming the recommended numbers of servings of foods each day from the basic food groups, which suggests that a very ADOLESCENTS 0002 0003 high percentage are not meeting daily nutrient requirements. Because of the wide availability of healthful foods but limited physical activities, overweight and obesity have reached epidemic proportions in the US and many technologically advanced nations. The energy imbalance in children as well as in adolescents results from changing societies that value time, speed, and technological advances in their daily lives over physical activities and sports, e.g., physical work, walking, climbing stairs, and other physically demanding chores. It has been estimated in the 1990s that between 30 and 50% of North American adults are overweight (body mass index or BMI > 25). Lower percentages of adolescents are overweight, but they represent a much greater prevalence than is desirable because of the tracking of body weight into adulthood and the potential risks for diabetes mellitus type 2, hypertension, and cardiovascular diseases. Obesity in children favors the development of elevated blood pressure and total cholesterol. This trend toward more overweight and obesity in adolescents is facing many nations, especially those in Asia, which have plentiful food available for their youth and declining levels of physical activity. This review covers the nutritional needs for adolescents, and it also highlights the problem nutrients that are either too little consumed or ingested in excess. Other issues of adolescent nutrition are also addressed. Nutrient Requirements 0004 Nutrient requirements are divided into macronutrients and micronutrients. Phytomolecules, which are technically not classified as nutrients and which are consumed only in plant foods, may have significant, but under-appreciated, health benefits. Energy-providing Nutrients 0005 0006 Fats and carbohydrates, when metabolized, generate approximately 85% of the energy used by cells; the other 15% is obtained from the metabolic degradation of the organic backbone of amino acids. Fats also provide the essential fatty acids that exist in plant foods and in fish, but only very minimally in other animal products. The current adolescent Recommended Dietary Allowances (RDAs) for energy, as kilocalories or kilojoules, are listed in Table 1. As these RDAs are currently under review, it is likely that they will be altered, especially because of the problem of overconsumption of food energy relative to daily energyexpending activities (see above). 35 Proteins Proteins supply the essential amino acids, as well as nonessential amino acids; all the amino acids also generate N that is used in the cellular synthesis of many organic molecules, such as nucleic acids. Table 1 lists the recommended intakes of protein, assuming high-quality protein food sources (protein digestibility and absorption of approximately 75%). 0007 0008 Micronutrients More than 20 essential micronutrients have been established for humankind, and RDAs exist for most of them. A few micronutrients have become classified as having less scientific basis for establishing RDAs; these micronutrients have therefore been given Adequate Intakes (AIs), even though they may previously have had RDAs. Calcium is a good example. In the 10th edition of the RDAs, calcium RDAs for adolescent girls and boys were 1200 mg per day from 13 to 19 years of age. In the 1997 edition, the calcium AIs have been increased to 1300 mg per day for the same age range. Other micronutrients with AIs include vitamin D, fluoride, and choline. Table 2 lists the recommended intakes of the micronutrients. 0009 0010 0011 Water Often a forgotten nutrient, water carries no specific recommendation. Aside from thirst, a general guide to water consumption is the drinking of 1–1.5 l of water and/or juices, but not counting caffeinated or alcoholic beverages because they have mild diuretic effects. Athletes and those with physically demanding work will need more than the amounts given. The goal is to maintain water balance and, therefore, to avoid dehydration. 0012 Dietary Fiber No specific recommendation has been made for dietary fiber, but food-labeling guidelines in the US suggest that 25 g of fiber (total of both soluble and insoluble fibers) per day should be consumed with a 2000-kcal diet and 30 g of fiber with a 2500-kcal diet. It is generally assumed that the vast majority of adolescents in the USA consume only 10–15 g of fiber per day. 0013 Phytochemicals The broad group of phytochemicals, which are not nutrients per se, have become increasingly recognized as being important components of our diets because of their numerous roles, such as antioxidants, in 0014 36 ADOLESCENTS tbl0001 Table 1 Food and Nutrition Board, National Academy of Sciences – National Research Council Recommended Dietary Allowancesa, revised 1989 (abridged), designed for the maintenance of good nutrition of practically all healthy people in the USA Category Age (year) or condition 0.0–0.5 0.5–1.0 Children 1–3 4–6 7–10 Males 11–14 15–18 19–24 25–50 51þ Females 11–14 15–18 19–24 25–50 51þ Pregnant Lactating 1st 6 months 2nd 6 months Infants Weight b Height b 6 9 13 20 28 45 66 72 79 77 46 55 58 63 65 60 71 90 112 132 157 176 177 176 173 157 163 164 163 160 Protein VitaminA Vitamin E Vitamin K Vitamin C Iron (mg) Zinc Iodine Selenium (mg RE)c (mg a-TE)d (mg) (mg) (mg) (mg) (mg) (kg) (lb) (cm) (in) (g) 13 20 29 44 62 99 145 160 174 170 101 120 128 138 143 24 28 35 44 52 62 69 70 70 68 62 64 65 64 63 13 14 16 24 28 45 59 58 63 63 46 44 46 50 50 60 65 62 375 375 400 500 700 1000 1000 1000 1000 1000 800 800 800 800 800 800 1300 1200 3 4 6 7 7 10 10 10 10 10 8 8 8 8 8 10 12 11 5 10 15 20 30 45 65 70 80 80 45 55 60 65 65 65 65 65 30 35 40 45 45 50 60 60 60 60 50 60 60 60 60 70 95 90 6 10 10 10 10 12 12 10 10 10 15 15 15 15 10 30 15 15 5 5 10 10 10 15 15 15 15 15 12 12 12 12 12 15 19 16 40 50 70 90 120 150 150 150 150 150 150 150 150 150 150 175 200 200 10 15 20 20 30 40 50 70 70 70 45 50 55 55 55 65 75 75 a The allowances, expressed as average daily intakes over time, are intended to provide for individual variations among most normal persons as they live in the USA under usual environmental stresses. Diets should be based on a variety of common foods in order to provide other nutrients for which human requirements have been less well defined. See text for a detailed discussion of allowances and of nutrients not tabulated. b Weights and heights of Reference Adults are actual medians for the USA population of the designated age, as reported by NHANES II. The median weights and heights of those under 19 years of age were taken from Hamill et al. (1979) (see pp. 16–17). The use of these figures does not imply that the height-to-weight ratios are ideal. c Retinol equivalents. 1 retinol equivalent ¼ 1 mg of retinol or 6 mg of b-carotene. See text for calculation of vitamin A activity of diets as retinol equivalents. d a-Tocopherol equivalents. 1 mg of d-a tocopherol ¼ 1 a-TE. See text for variation in allowances and calculation of vitamin E activity of the diet as a-tocopherol equivalents. ß Copyright 1998 by the National Academy of Sciences. All rights reserved. This table does not include nutrients for which Dietary Reference Intakes have recently been established [see Dietary Reference Intakes for Calcium, Phosphorus, Magnesium,Vitamin D, and Fluoride, 1997 and Dietary Reference Intakes for Thiamin, Riboflavin, Niacin,Vitamin B6, Folate,Vitamin B12, Pantothenic Acid, Biotin, and Choline, 1998]. promoting health. Because these nonnutrient molecules come exclusively from plant foods, adolescents who avoid fruits and vegetables will consume little of these molecules and not receive their beneficial effects. Genistein, a soy-derived isoflavone, has been shown to have several possible benefits on health. Nutrition and Skeletal Growth 0015 Growth of the skeleton (height) is typically completed during adolescence by both girls and boys, and bone mass is practically maximized at this same time. For a large majority of girls in the USA, the bone mass achieved during this adolescent period is considered to be less than optimal because of limited selections of nutrient-dense foods and preferences for low nutrient-dense foods. Figure 1 illustrates the declining consumption of milk and the increasing intake of soft-drink beverages over the last two decades in the USA. Concern has arisen that girls, and possibly boys, are not getting sufficient calcium and other essential nutrients from milk and related dairy products, because so many of these nutrients are essential for the growth of skeletal tissue, and also that adolescent females may be consuming excessive amounts of phosphorus, which alters the calcium:phosphorus ratio in a deleterious way (see Calcium and Phosphorus sections). Although not the only nutrient required for the extracellular mineral phase of bone, calcium may be the limiting nutrient for mineralization. This divalent cation is provided in the diet by only a few foods that are commonly used and available in most countries. In the USA, dairy foods (exclusive of butter) and calcium-fortified foods provide the bulk of the cation, i.e., approximately 60–70%, in the diets of adolescents. Dark green leafy vegetables, such as broccoli, provide modest, but important, amounts of calcium. The problem of a shortfall in calcium in the presence of adequate, or possibly, excessive phosphorus consumption is that the low calcium:phosphorus ratio potentially produces a persistent elevation in parathyroid hormone (PTH) that in turn prevents adequate mineralization of the skeleton during the 0016 0017 tbl0002 Table 2 Food and Nutrition Board, Institute of Medicine – National Academy of Sciences Dietary Reference Intakes: recommended levels for individual intake Life-stage group Infants 0–5 months 6–11 months Children 1–3 years 4–8 years Males 9–13 years 14–18 years 19–30 years 31–50 years 51–70 years >70 years Females 9–13 years 14–18 years 19–30 years 31–50 years 51–70 years >70 years Pregnancy 18 years 19–30 years 31–50 years Lactation 18 years 19–30 years 31–50 years a Calcium (mg per day) Phosphorus (mg per day) Magnesium (mg per day) 30* 75* D (mg per day)a,b Niacin (mg per day)c B12 (mg per day) Pantothenic acid (mg per day) 65* 80* 0.4* 0.5* 1.7* 1.8* 0.5 0.6 150 200 0.9 1.2 Fluoride (mg per day) Thiamin (mg per day) Riboflavin (mg per day) B6 (mg per day) 5* 5* 0.01* 0.5* 0.2* 0.3* 0.3* 0.4* 2* 3* 0.1* 0.3* 0.7* 1* 0.5 0.6 0.5 0.6 6 8 Folate (m g per day)d 210* 270* 110* 275* 500* 800* 460 500 80 130 5* 5* 1300* 1300* 1000* 1000* 1200* 1200* 1250 1250 700 700 700 700 240 410 400 420 420 420 5* 5* 5* 5* 10* 15* 2* 3* 4* 4* 4* 4* 0.9 1.2 1.2 1.2 1.2 1.2 0.9 1.3 1.3 1.3 1.3 1.3 12 16 16 16 16 16 1.0 1.3 1.3 1.3 1.7 1.7 300 400 400 400 400 400 1300* 1300* 1000* 1000* 1200* 1200* 1250 1250 700 700 700 700 240 360 310 320 320 320 5* 5* 5* 5* 10* 15* 2* 3* 3* 3* 3* 3* 0.9 1.0 1.1 1.1 1.1 1.1 0.9 1.0 1.1 1.1 1.1 1.1 12 14 14 14 14 14 1.0 1.2 1.3 1.3 1.5 1.5 1300* 1000* 1000* 1250 700 700 400 350 360 5* 5* 5* 3* 3* 3* 1.4 1.4 1.4 1.4 1.4 1.4 18 18 18 1300* 1000* 1000* 1250 700 700 360 310 320 5* 5* 5* 3* 3* 3* 1.5 1.5 1.5 1.6 1.6 1.6 17 17 17 Biotin (mg per day) Cholinee (mg per day) 5* 6* 125* 150* 2* 3* 8* 12* 200* 250* 1.8 2.4 2.4 2.4 2.4f 2.4f 4* 5* 5* 5* 5* 5* 20* 25* 30* 30* 30* 30* 375* 550* 550* 550* 550* 550* 300 400g 400g 400g 400g 400g 1.8 2.4 2.4 2.4 2.4f 2.4f 4* 5* 5* 5* 5* 5* 20* 25* 30* 30* 30* 30* 375* 400* 425* 425* 425* 425* 1.9 1.9 1.9 600h 600h 600h 2.6 2.6 2.6 6* 6* 6* 30* 30* 30* 450* 450* 450* 2.0 2.0 2.0 500 500 500 2.8 2.8 2.8 7* 7* 7* 35* 35* 35* 550* 550* 550* As cholecalciferol. 1 mg of cholecalciferol ¼ 40 IU vitamin D. In the absence of adequate exposure to sunlight. c As niacin equivalents. 1 mg of niacin ¼ 60 mg of tryptophan. d As dietary folate equivalents (DFE). 1 DFE ¼ 1 mg of food folate ¼ 0.6 mg of folic acid (from fortified food or supplement) consumed with food ¼ 0.5 mg of synthetic (supplemental) folic acid taken on an empty stomach. e Although AIs have been set for choline, there are few data to assess whether a dietary supply of choline is needed at all stages of the life cycle, and it may be that the choline requirement can be met by endogenous synthesis at some of these stages. f Since 10–30% of older people may malabsorb food-bound B12, it is advisable for those older than 50 years to meet their RDA mainly by consuming foods fortified with B12 or a B12-containing supplement. g In view of evidence linking folate intake with neural tube defects in the fetus, it is recommended that all women capable of becoming pregnant consume 400 mg of synthetic folic acid from fortified foods and/or supplements in addition to intake of food folate from a varied diet. h It is assumed that women will continue consuming 400 mg of folic acid until their pregnancy is confirmed and they enter prenatal care, which ordinarily occurs after the end of the periconceptional period – the critical time for formation of the neural tube. ß Copyright 1998 by the National Academy of Sciences. All rights reserved. This table presents Recommended Dietary Allowances (RDAs) in bold type and Adequate Intakes (AIs) in ordinary type followed by an asterisk (*). RDAs and AIs may both be used as goals for individual intake. RDAs are set to meet the needs of almost all (97–98%) individuals in a group. For healthy breastfed infants, the AI is the mean intake. The AI for other life stage groups is believed to cover their needs, but lack of data or uncertainty in the data prevent clear specification of this coverage. b 38 ADOLESCENTS adolescent growth phase, and also earlier during prepuberty. The net result is suboptimal skeletal development and peak bone mass accrual by the end of adolescence that is less than maximal. So, low dietary calcium intakes, especially in relation to elevated phosphorus intakes, may be disadvantageous for young girls and also for young boys. The problem may be more widespread than previously thought because of the possibility that the fracture rate in adolescent girls may be underestimated. 70 Percentage 60 50 40 30 20 10 fig0001 1977−1978 NFCS 1989−1991 CSF II Most of the mineral micronutrients reviewed below exist in the body in solutions as cations (positively charged) or anions (negatively charged). The vitamins, however, exist as organic molecules that may carry a slight electrical charge that permits their binding, usually for only brief times, to other molecules. 1994−1996 CFS II Figure 1 Percentages of US individuals in USDA National Food and Consumption Surveys (NFCS and CSF) consuming milk soft drinks between 1977–1978 and 1994–1996. Note the decline in milk consumption while soft-drink consumption increased, according to percentage of consumers. Low iron consumption by females is one of the most common nutrient problems throughout the world. Normal Bone mineral density (g cm−2) fig0002 Low PBM Menopause Fracture risk range 30 40 0019 Iron Peak bone mineral density 0 0018 Calcium Inadequate consumption of calcium in the diets of adolescents, especially females, in the USA has gained them the label, ‘the lost generation.’ Calcium intakes have reached an all-time low, and it has been predicted that the present generation of girls, and perhaps boys, are placing themselves at increased risk for skeletal fractures late in life, as longevity is increasing in the USA and most other nations. The concern about increased age-adjusted rates of osteoporosis assumes that tracking, or following the same relative path as age increases, of bone mass and density occur, similar to the tracking of high blood pressure with increasing age. Therefore, an individual who has less bone at peak bone development is, during aging, assumed to follow a path, i.e., track, at the relatively lower level of bone mass to reach critically low bone values sooner, e.g., after the menopause, than an individual who develops a greater peak bone at the end of the skeletal growth phase (Figure 2). 80 0 Specific Problem Micronutrients: Deficits and Excesses 50 Age (years) 60 70 Figure 2 Tracking of bone mass of females from early adulthood to postmenopausal life. Adapted from Anderson JJB (2000) Nutrition in osteoporosis, in Mahan K and Escott-Stump S (eds) Krause’s Food, Nutrition, and Diet Therapy, 10th edn. Philadelphia, PA: Saunders with permission. PBM ¼ peak bone mass. 0020 ADOLESCENTS Adolescent females are particularly likely to develop iron deficiency, and even the more severe irondeficiency anemia, if they have little red meat in their diets. In the USA, iron-fortified foods, especially ready-to-eat breakfast cereals, have reduced the severity of the two forms of iron deficits. Zinc 0021 Too little zinc consumption by adolescents results from poor intakes of plant foods. Red meats have good amounts of zinc, as well as iron, but these would not be consumed by many adolescents who avoid red meats. Magnesium 0022 Insufficient magnesium typically signifies poor selection of plant foods, especially dark green leafy vegetables. Although magnesium intakes by adolescents are lower than recommended, it has been difficult to pinpoint real physiologic problems, except possibly that optimal heart function may be compromised. Potassium 0023 Too little potassium consumption reflects a limited selection of fruits and vegetables, the highest potassium-containing sources of this monovalent cation. Both potassium and magnesium have intracellular roles, and in part, adequate intakes of potassium are needed to counter the high intakes of sodium. Therefore, a dietary balance between potassium and sodium is needed to maintain efficient cellular functions that require potassium ions. Renal mechanisms tend to conserve sodium at the expense of potassium and magnesium, which are excreted in increased amounts when sodium intakes are higher than recommended. Sodium 0024 Excessive amounts of sodium in the diet may contribute to higher blood pressure levels, but still within the normal range, even in adolescents. Sodium consumption by adolescents is a concern, because the taste for this mineral, or perhaps sodium sensitivity, becomes established early in life and continues into adolescence. Higher blood pressure is typically associated with excessive intakes of sodium throughout adolescence tracks with age. Phosphorus 0025 Excessive consumption of phosphorus from foods and soft drinks that contain phosphoric acid reduces the calcium:phosphorus ratio in the total diet and, thereby, may increase the circulating level of PTH, though the elevated PTH concentration usually remains within the upper range of normality. The 39 net effect is increased calcium losses, i.e., a negative calcium balance. In the USA, food-labeling regulations do not require that information on the phosphorus content of foods be included. This omission means that consumers cannot estimate their daily phosphorus consumption or calculate a calcium: phosphorus ratio of a food or their entire daily intake. In addition, a food may include hidden amounts of phosphorus because of the wide use of phosphate additives in food processing. The magnitude of this potential problem of excessive phosphorus has not been investigated by governmental agencies. Folic Acid Recent advances in understanding this nutrient have focused on obtaining sufficient intakes of folic acid to prevent neural tube defects in offspring, but other important roles of this B vitamin exist in cell divisions, e.g., in red blood cell formation, and gut epithelial turnover of cells. In addition, adequate intakes of folate also maintain lower circulating levels of homocysteine, which is now considered a risk factor for coronary heart disease. 0026 Vitamin D The hormonal form of vitamin enhances intestinal calcium absorption, when calcium intakes are low, and also it increases osteoblastic bone-forming activities. This hormonal metabolite of vitamin D also stimulates osteoblast cells in bone to make new organic matrix (osteoid) and therefore new bone tissue. This metabolite, along with PTH, also contributes to calcium homeostasis, i.e., regulation of blood calcium concentration. Too little intake of vitamin D from the few foods that contain it, and too little sun exposure for skin biosynthesis of this vitamin, puts individuals at risk for reduced functions in the gut and bone tissues. Adolescent intakes of vitamin D are low in the USA because the consumption of milk that is fortified with this vitamin has declined (see above). 0027 Vitamin K An established requirement for vitamin K exists for both blood clotting and bone health. The daily amount needed for the production of mature osteocalcin may be higher than that needed for the bloodclotting factors, but insufficient information has been generated to be certain. Vitamin K intakes may be insufficient in adolescents because of limited consumption of dark green leafy vegetables and legumes. 0028 Problem Areas A few problem areas involving meeting nutrient requirements by adolescents need special attention 0029 40 ADOLESCENTS because of their potentially adverse effects on nutritional status. Adolescent Athletes 0030 0031 The female athlete triad of disordered eating, amenorrhea or oligomenorrhea, and osteopenia or low bone mass, but generally not osteoporosis, has a marked adverse effect on skeletal mass that has been shown to increase the risk of stress fractures of athletes, especially those participating in cross-country running and other endurance events involving running or repetitive jumping as in volleyball. The risk of fracture, however, is reduced if estrogen is replaced in these athletes. However, Dutch investigators have suggested that physical activity during adolescence may be even more important for bone development than calcium intake, so long as dietary calcium is in the adequate range, i.e., approximately 1000 mg per day. This conclusion has also been reached for female gymnasts, even amenorrheic gymnasts, because of the positive effects of weight-loading on the skeleton. Consumption of Alcoholic Beverages 0032 Over-consumption of alcoholic beverages by adolescents in the USA has become a big problem. The adverse effects of excessive alcohol consumption on the nutritional status of adolescents may result from insufficient food consumption, too many empty calories from low-nutrient dense alcoholic beverages, and unwise food choices. Strict Vegetarians 0033 Vegans or strict vegetarians place themselves at risk for deficits of several micronutrients if supplements are not taken. Fortunately, most vegans know about the pitfalls of this practice of eating, and they do not develop serious deficiencies. However, a few problems, such as iron, zinc, and cobalamin deficiencies, may arise because of the avoidance of all animal foods. Vegans may also not be able to consume sufficient amounts of calcium or of omega-3 polyunsaturated fatty acids. One of the potential disadvantages of a vegetarian diet is a low estrogen status across the life cycle in females. The lower estrogen status may have an adverse effect on the skeleton, although it may be protective against estrogen-dependent cancers and other chronic diseases. Nutrition Education 0034 Despite the fairly high level of knowledge about nutrition by youth in the US, health practices, including food selection, are too often detrimental to the support of continued skeletal growth. Part of the reason for insufficient calcium consumption by adolescent girls is their limited knowledge about daily calcium allowances and the amounts of calcium in foods. Adolescents, especially females, clearly do not eat the way they should and even, perhaps, know how to eat. These unhealthy eating practices result primarily from bad attitudes toward specific foods, negative reactions to parental influences, and peer pressure. The serious problem in skeletal development in the more technologically advanced nations relates to the unwise food and drink choices being made by our children, especially after about 10 years of age. In the USA, adolescent females are not only not getting sufficient amounts of calcium, but consuming too little of the nutrient-dense foods in general. The practices of meal-skipping, grazing, snack/convenience food selection, and soft-drink consumption insure suboptimal intakes of several micronutrients in addition to calcium. Surveys of food-consumption patterns of girls demonstrate that inadequate mean consumption exists for the following micronutrients: calcium, magnesium, iron, zinc, and both fat-soluble and water-soluble vitamins, especially E, folate, cobalamin, and other B vitamins. Vitamins D and K typically have not been assessed in these surveys. Mean sodium and phosphorus intakes were higher than recommended. Avoidance of dairy products by young girls has become widespread, at least in the USA, for fear of weight gain, despite the fact that low-fat milks and no-fat yogurts are widely available. Thus, dairy foods can be consumed with little or no fat. Education of the appropriate fats and amounts of daily fat intake for these females is either lacking or not appropriately highlighted. Some fat is clearly needed for skeletal growth, so that extremely low intakes may be deleterious. Of course, some mono- and polyunsaturated fats found in salad dressings and cooking oils are now being recommended for overall balance within the types of fatty acids and also for optimal health outcomes, but young females are barely aware of these important needs. Finally, nutrition education messages about the consumption of soft drinks should target both parents and children to limit the number of servings because they displace nutritious beverages, such as milk and fruit juices (see Figure 1), especially when two or more 400-ml (12-ounce) soft drinks are consumed in a day (more than 22% of one adolescent population in the USA). Since many of the soft drinks contain phosphoric acid as the acidulant, the calcium:phosphorus ratio becomes even further skewed away from a recommended ratio of 0.70–0.75. The widespread use of soft drinks, in place of milk, has contributed to 0035 0036 0037 ADOLESCENTS the increase in phosphorus consumption and the low calcium:phosphorus dietary ratio of USA adolescents. How serious this trend of increased phosphorus consumption is cannot be resolved without a better understanding of the overall phosphorus intake from processed foods, lowly processed foods, and soft drinks that contain phosphoric acid. See also: Alcohol: Alcohol Consumption; Calcium: Properties and Determination; Dietary Fiber: Properties and Sources; Exercise: Metabolic Requirements; Folic Acid: Properties and Determination; Iron: Physiology; Functional Foods; Magnesium; Phosphorus: Properties and Determination; Obesity: Epidemiology; Plant Antinutritional Factors: Characteristics; Protein: Requirements; Sodium: Properties and Determination; Vegetarian Diets; Vitamin K: Properties and Determination Further Reading Alaimo K, McDowell MA, Briefel R et al. (1994) Dietary Intakes of Vitamins, Minerals, and Fiber of Persons Ages 2 Months and Over in the United States: Third National Health and Examination Survey, Phase 1, 1988–91. Advance Data No. 258 (November 3, 1994). Washington, DC: National Center for Health Statistics, CDC, PHS, USHHS. Anderson JJB (1999) Plant-based diets and bone health: nutritional implications. American Journal of Clinical Nutrition 70 (supplement): 539S–542S. Anderson JJB (2000) Nutrition in osteoporosis. In: Mahan K and Escott-Stump S (eds) Krause’s Food, Nutrition, and Diet Therapy, 10th edn. Philadelphia, PA: Saunders. Anderson JJB, Anthony M, Messina M and Garner SC (1999) Effects of phytooestrogens on tissues. Nutrition Research Reviews 12: 75–116. Anderson JJB and Garner SC (eds) (1995) Calcium and Phosphorus in Health and Disease. Boca Raton, FL: CRC Press. Anderson JJB and Miller CP (1998) Lower lifetime estrogen exposure among vegetarians as a possible risk factor for osteoporosis: a hypothesis. Vegetarian Nutrition 2: 4–12. Calvo M and Park YK (1995) Changing phosphorus content of the US diet: Potential for adverse effects on bone. Journal of Nutrition 126: 1168S–1180S (1996). Goulding A, Cannan R, Williams SM et al. (1998) Bone mineral density in girls with forearm fractures. Journal of Bone and Mineral Research 13: 143–148. 41 Harel Z, Riggs S, Vaz R, White L and Menzies G (1998) Adolescents and calcium: what they do and do not know and how much they consume. Journal of Adolescent Health 22: 225–228. Harnack L, Stang J and Story M (1999) Soft drink consumption among US children and adolescents: nutritional consequences. Journal of the American Dietetic Association 99: 436–441. Hetzel BS and Berenson GS (1987) Cardiovascular Risk Factors in Childhood: Epidemiology and Prevention. Amsterdam: Elsevier Science. Institute of Medicine, Food and Nutrition Board (1997) Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: National Academy Press. Institute of Medicine, Food and Nutrition Board (1998) Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: National Academy Press. McMurray RG, Harrell JS, Levine AA and Gansky SA (1995) Childhood obesity elevates blood pressure and total cholesterol independent of physical activity. International Journal of Obesity 19: 881–886. Munoz KA, Krebs-Smith SM, Bvallards-Barbash R and Cleveland LE (1998) Food intakes of US children and adolescents compared with recommendations. Pediatrics 100 (3 Pt 1): 323–329. National Research Council, Food and Nutrition Board(1989) Recommended Dietary Allowances, 10th edn. Washington, DC: National Academy Press. Simopoulos A (1999) Essential fatty acids in health and disease. American Journal of Clinical Nutrition 70 (supplement): 560S–569S. Tanner JM (1962) Growth in Adolescence, 2nd edn. Oxford: Blackwell Scientific. Thrash L and Anderson JJB (2000) The female athlete triad. Nutrition Today 35: 168–174. Weaver CM, Peacock M and Johnston CC Jr. (1999) Adolescent nutrition in the prevention of postmenopausal osteoporosis. Journal of Clinical Endocrinology and Metabolism 84: 1939–1843. Weaver CM, Proulx WR and Heaney R (1999) Choices for achieving adequate dietary calcium with a vegetarian diet. American Journal of Clinical Nutrition 70 (supplement): 543S–548S. Welten DC, Kemper HCG, Post GB et al. (1994) Weightbearing activity during youth is a more important factor for peak bone mass than calcium intake. Journal of Bone and Mineral Research 9: 1089–1095. Adrenal Hormones See Hormones: Adrenal Hormones; Thyroid Hormones; Gut Hormones; Pancreatic Hormones; Pituitary Hormones; Steroid Hormones 42 ADULTERATION OF FOODS/History and Occurrence ADULTERATION OF FOODS Contents History and Occurrence Detection History and Occurrence M Tsimidou and D Boskou, Aristotle University of Thessaloniki, Thessaloniki, Greece Copyright 2003, Elsevier Science Ltd. All Rights Reserved. Introduction 0001 0002 Food is a basic prerequisite for human survival and also for social and economic welfare and progress. Problems related to food have varied from one period of history to another, from continent to continent, and from country to country. The problem of food adulteration has been a major one and the protection of the consumer has occupied the attention of civil authorities from ancient times. Food is considered adulterated if it contains poisons or other substances which may render it injurious to the health of the consumer, or if it contains filth or it is decomposed; if it contains a coloring agent or other food additive, that is not approved or contains materials that disguise inferior quality; if any important constituent has been wholly or in part abstracted or any specified ingredient has been substituted by a nonspecified ingredient; if it contains any substance that increases its weight and bulk or changes its strength to improve appearance. A food is misbranded if it is illicitly labeled or it is a food for which standards of identity have been written and it fails to comply with these standards. History 0003 Food has been liable to adulteration to a greater or lesser extent since very early times. Mosaic and Egyptian laws made provision for preventing contamination of meat, while several centuries before the time of Christ India had regulations prohibiting the adulteration of grain and edible fats. Athens had its public inspector of wines. Adulteration was also common during the Roman period. Evidence for this is given in Apicius’ famous cookbook (De Re Coquinaria). During the Middle Ages in England, pepper and other costly spices imported from the east were adulterated by mixing with ground nutshells, local seeds, and olive pits. Between the 13th and 16th centuries, bread, wine, beer, spices, and valuable natural coloring materials were often adulterated. In England in 1319, a meatmarket overseer succeeded in putting a butcher in the pillory for selling unsound beef. Wines were ‘sophisticated’ (adulterated) with burnt sugar, juices, starch and gums, and other substances. Such practices may have reduced the quality of the wine but they were not injurious to health. After the 18th century, however, food adulteration became dangerous. Vinegar was often adulterated with sulfuric acid, wine with preservatives containing lead salts, green vegetables in vinegar with copper (to improve color), essential oils with oil of turpentine, confectionery products with colorings containing lead and arsenic, chocolate with Venetian red (ferric oxide), and red pepper with vermilion (mercury sulfide). ‘Black extract’, obtained by boiling poisonous berries of Cocculus indicus in water and concentrating the fluid, was used in beer. This extract imparted flavor, but also narcotic properties and intoxicating qualities to the beverage. Bread was not only the basic item of diet for many centuries but also the one most subjected to adulteration. The incorporation of sieved boiled potatoes, chalk, or bone ashes in the flour was a common fraudulent practice, but the most serious example was the addition of alum, which whitened flour of inferior quality. Recipe books of the 18th century contain instructions, which today would cause alarm. The recipe for preserving green color in pickles is characteristic: ‘To render pickles green, boil them with a halfpenny or allow them to stand for 24 hours in copper brass pans.’ It is not surprising that records of that period list a number of deaths from copper poisoning. Frederic Accum, writing from London in 1820, gives a vivid description of the adulteration of bread, alcoholic drinks, tea, coffee, and many other foods. Accum claimed that: ‘Indeed, it would be difficult to mention a single article of food which is not to be met 0004 0005 0006 0007 ADULTERATION OF FOODS/History and Occurrence 0008 with in an adulterated state; and there are some substances which are scarcely ever to be genuine.’ Even if these accusations are somewhat overstated, the seriousness of intentional adulteration of food, which prevailed between 1800 and the early 1900s, is undisputed. The second decade of the 19th century marks the beginning of the second period in the history of food adulteration. In the first period (ancient times to about 1820) food was procured from small enterprises and individuals who were, to a certain extent, responsible for their own transactions. In the second period (early 1800s to 1900s), the methods of food production changed significantly. Large-scale food production became necessary because of the industrial revolution, which led to a move of population from country to city. This created conditions that were conducive to large-scale adulteration of food. In the second half of the 19th century the first publicly supported analytical laboratories for food inspection were established in Germany and the USA. Intentional adulteration of food remained a serious problem until the beginning of the 20th century. Regulatory pressures and the effectiveness of analytical methods reduced the frequency and magnitude of food adulteration. Further improvements have been achieved up to the present time and, owing to strict legal standards and also to the growth of an increasingly critical public, deliberate adulteration in industrialized countries has become less serious. Of course, fraudulent practices still continue in most countries, especially those which lack adequate means to insure that laws and regulations are enforced. Food Additives 0009 The excessive use of additives and the use of nonpermitted ones present a serious threat to food safety. It could be argued that a