€53.4i553... 1. . .t. ztn, I: . I)1...t¢~..l$....; l . .x . . ,. .71: I; Ch. .53. i. :u 1 u {is}? I: )5 (h? 3.33.1.3: 3 HA .F... T x . .1. . .. ‘ r x .!.nu.z.u.l\.fi1.¢ .: u... .4... .3... :1... \. . .Iv I} u!- ..PIAT..I‘ 5...). .. 2:29.35... 3. 3.515.": :53 ‘ t ......:.l.>.r:.y. .. fax any... I- ‘ ._ ....,. , awmamimv THESIS Illl‘lllll llllulillllmlltl'lfilflllll ‘ LIBRARY 3 1293 02048 9070 Michigan State University This is to certify that the thesis entitled INHIBITORY EFFECTS OF TEA AND COFFEE ON 113 vitro AVAILABILITY OF ZINC AND IRON IN WHEAT PRODUCTS presented by Chia-Fen Chung has been accepted towards fulfillment of the requirements for Masters degree in Human Nutrition MW— Major professor Date June 27, 2000 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution PLACE IN RHURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 11/00 cJCIRC/DatoOuopss-p.“ INHIBITORY EFFECTS OF TEA AND COFFEE ON IN WYRO AVAILABILITY OF ZINC AND IRON IN WHEAT PRODUCTS By Chia-Fen Chung A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 2000 ABSTRACT INHIBITORY EFFECTS OF TEA AND COFFEE ON IN VITRO AVAILABILITY OF ZINC AND IRON IN WHEAT PRODUCTS By Chia-Fen Chung The effect of tea and coffee with or without milk on zinc and iron availability was determined using an in vitro method (Luten et al., 1996) which measures dialyzable zinc and iron following simulated gastric and intestinal digestion. Two extruded wheat products (white flour and whole wheat kernel) were used as the basis for the test meals composed of wheat product (5 g) with and without tea/coffee (40 g) or tea/coffee + milk (40 g tea/coffee, 5g milk). Both percent dialyzable zinc and iron were lower in the whole kernel wheat than in the white flour product. The addition of tea or coffee with or without milk to test meals composed of both extruded products resulted in decreased percent dialyzable zinc. The inhibitory effect of coffee on zinc dialyzability was greater than that of tea. A fiirther decrease in percent dialyzable zinc in the test meals was observed when milk was added to the tea or coffee. The addition of milk alone to white flour test meals resulted in a lower dialyzable zinc than tea with or without milk. An inhibitory effect of tea and coffee on percent dialyzable iron also was observed for the white flour but not the whole kernel wheat product. Addition of milk to both extruded products also resulted in a significant decrease in dialyzable iron. These data indicate that both zinc and iron bioavailability was significantly decreased by tea and coffee, and dialyzable zinc was lower when milk was add to the tea or coffee compared to tea or coffee alone. For my family ACKNOWLEDGMENTS I wish to give special thanks to my major advisor, Dr. Wanda L. Chenoweth, for her guidance, encouragement and support throughout my master’s studies. She is a very nice person and I appreciate all she did for me in the past couple of years. I also want to thank all the advice fiom the members of the guidance committee: Dr. Maurice R. Bennink and Dr. Jay Schroeder. I am deeply gratefiil to my best fiiend, Haiyan Zhang, for all the support and help through my studies. I also appreciate Shama Joseph for her technical advice and help,. and numerous staff and students in F SHN for their help and friendship. I would also like to thank to my friends, who always comfort me and cheer me up when I am low. Finally, I would like to express my deep appreciation to my family who always support me with their love. iv TABLE OF CONTENTS LIST OF TABLES .................................................................................. viii LIST OF FIGURES ................................................................................... ix INTRODUCTION ..................................................................................... 1 LITERATURE REVIEW ............................................................................. 3 Definition of bioavailability of trace elements ............................................... 3 Methods for determining bioavailability ..................................................... 3 Human studies ............................................................................. 3 Animal studies ............................................................................. S In vitro techniques ......................................................................... 7 The bioavailability of zinc ...................................................................... 8 Physiology and biochemistry .............................................................. 8 Factors that influence zinc bioavailability ............................................... 9 Protein ................................................................................ 10 Phytic acid ........................................................................... 11 Iron .................................................................................... 12 Calcium .............................................................................. 13 Other factors ......................................................................... l4 Phenolic compounds ........................................................................... 14 Chemistry of phenolic compounds ..................................................... l4 Tannins ..................................................................................... 18 Tea OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 20 Coffee ....................................................................................... 20 Phenolic compounds (tannins) as antinutrients ............................................. 21 Influence on the digestibility of macronutrients ...................................... 21 Influence of tannins on bioavailability of minerals .................................. 22 Justification ....................................................................................... 25 Hypotheses ...................................................................................... 26 MATERIALS AND METHODS ................................................................... 27 Research design ................................................................................ 27 Test meals ....................................................................................... 27 Chemicals ........................................................................................ 29 In vitro studies ................................................................................... 29 Analytical methods ............................................................................. 31 Methods of phenolic compounds analysis ............................................. 31 Vanillin assay ................................................................................ 31 Folin-Denis method31 Wet ashing and mineral analysis ........................................................ 32 Statistical Analysis ........................................................................ 32 RESULTS ............................................................................................. 34 Zinc, iron and total phenolic compounds analysis ......................................... 34 In vitro dialyzable zinc and iron .............................................................. 34 DISCUSSION ......................................................................................... 41 CONCLUSION ............................................................... . ....................... 49 FUTURE RESEARCH .............................................................................. 50 LIST OF REFERENCES ............................................................................. 52 vii LIST OF TABLES Table 1 Composition of test meals ............................................................. 2 Zn, Fe and tannic acid content of foods in the test meals ......................... 3a Percent dialyzable zinc and iron (meaniSEM) in test meals containing extruded wheat products with or without the addition of green tea or milk. . . . . . 3b Percent dialyzable zinc and iron (meaniSEM) in test meals containing extruded wheat products with or without the addition of coffee or milk... . . . . . . 4 Percent dialyzable zinc and iron (meaniSEM) in test meals containing extruded white flour with addition of milk ash or vitamin-free casein... . .. ... ......... viii Page ......... 28 ......... 35 .36 .38 .40 LIST OF FIGURES Figure Page 1 Structures ofcaffeic acid, quinic acid and chlorogenic acid... ......16 2 Structures of catechins, flavonols and basic flavonoid structure ......................... 17 3 Hydrolyzabletannins...........................................................................19 ix INTRODUCTION Deficiency of trace elements in humans may be a result of inadequate intake of minerals in the diet or impaired absorption in the presence of adequate dietary intakes. Factors that decrease or impair absorption include dietary constituents, such as phytate or fiber or other chemicals that may interact with the essential trace elements. Two beverages, tea and coffee, are widely consumed in the world. The most important and characteristic components of tea leaves and coffee beans are their phenolic compounds. Phenolic compounds are being studied to determine their antioxidant activities and potential role in prevention of chronic diseases such as cancer and cardiovascular disease. Flavonoids found in green tea have strong antioxidant activity, and there are recommendations to increase their daily intake. However, the condensed tannins, which are flavonoid condensation products, have been reported to be responsible for decreases in feed intake, grth rate, feed efficiency, net metabolizable energy and protein digestibility in experimental animals. Tannins form complexes with proteins, starch, and digestive enzymes to cause a reduction in the nutritional values of foods. Tannins are also believed to affect the utilization of vitamins and minerals. Numerous experiments in both humans and animals have shown that tea strongly inhibits iron absorption. Tannins are believed to form insoluble complexes with iron within the gastrointestinal tract and make the iron less available for absorption. Results of studies to determine the effect of tea on zinc bioavailability are conflicting. Reddy et al. (1990) found that in rats tea inhibited absorption of both non- heme iron and, to lesser extent, zinc. This effect was exacerbated by milk for iron but not zinc. In rats the ingestion of green tea (Zeyuan et al., 1998) has been shown to significantly decrease zinc absorption. Two other studies in rats showing an increase in zinc concentration in bone and in total blood with tea ingestion (Greger and Lyle, 1988; Hamdaoui et al., 1997) might suggest increased absorption of zinc. In humans, tea consumption was shown to have a small but not statistically significant adverse affect on zinc bioavailability in subjects consuming self-selected and laboratory-controlled diets (Ganji and Kies , 1994). Coffee is another beverage that is ofien consumed with meals and the inhibition of iron absorption has been shown when coffee was added to a test meal (Morck et al., 1983). Coffee and its major phenolic compounds, chlorogenic and caffeic acid, have been shown to significantly decrease zinc absorption in rats (Coudray et al., 1998). The purpose of this study was to determine the effect of phenolic-containing beverages, tea (tannins and catechins), and coffee (chlorogenic and caffeic acid), on the bioavailability of zinc in a control meal using an in vitro method. Because milk is frequently added to tea and coffee when it is consumed and because milk has been shown to influence mineral bioavailability, the effect of tea and coffee with and without added milk also was tested. LITERATURE REVIEW Definition of bioavailability of trace elements Bioavailability can be defined as the proportion of the total mineral in a food, meal or diet that is utilized for normal body fimctions (Fairweather—Tait, 1992). A variety of different dietary and physiological factors may affect bioavailability. The amount of a mineral that is available for absorption is dependent upon diet composition, gastro- intestinal secretions and luminal interactions (Mills, 1985). The factors that influence absorption include the amount of the minerals in the diet, the oxidation state of the mineral, the chemical form and the presence of interfering or enhancing factors (Tumlund, 1991). Methods for determining bioavailability The determination of excretion patterns of nutrients or metabolites in balance studies in humans or in animal models is the classical way to monitor nutrient interactions. Both fecal and urinary losses of minerals and their markers (either radioisotopes or stable isotopes) have been measured (Greger, 1988; Allen, 1982; Solomons, 1982). 1'1qu studies Although the best way to determine mineral or trace element bioavailability is believed to be in vivo studies in humans, such studies are time-consuming, expensive, often complex to perform and tend to give variable results. A number of investigators have tried to develop faster techniques. Several have fed “load dose" of zinc with test meals (Solomons, 1982; Meadows et al., 1983). The relative rise in serum zinc levels after a selected test substance, such as iron or tin, is added to a large dose of zinc has been used as an index of the effect of the test substance on zinc absorption. One problem with this technique is that a typical meal only contains 1-5 mg zinc; whereas the load doses of zinc (often 50 mg) are not physiological. Hence, absorptive mechanisms of zinc from these test meals and typical meals probably differ (Greger, 1989). More sophisticated techniques that utilize radioisotopes and stable isotopes also have been used. By using isotopic tracers as labels, it becomes possible to study the metabolic fate of minerals in individual foods or from meals that are intrinsically or extrinsically labeled. The first mineral isotopic tracers were radioisotopes. Radioactive iron was first used to study iron metabolism in humans in 1942 (Balfour et al.). Then a significant advance in the use of radioisotopes was made which was the use of extrinsic radiolabels as a valid technique for the measurement of food iron bioavailability (Cook et al., 1972). Radioisotopes were also used as tracers of other essential minerals, including copper, calcium, magnesium and zinc (Tumlund, 1989). A y—emitting zinc radioisotope (65 Zn) is commercially available. It has a half-life of 245 d, which gives it a low cost and facilitates its handling (Sandstrom B., 1997). The isotope can also be used to estimate absorption using the balance technique, is. by deducting fecal and urinary excretion from the administered dose over an appropriate time period. Measures of absorption or retention are ofien used to assess trace element bioavailability because of the lack of reliable and sensitive methods to determine the utilization of the element in the body. The drawback withthe technique is that the retention of the orally administered isotope can not be determined until the unabsorbed fraction has been excreted from the body. Measures of retention can only be equated with absorption when there is negligible excretion of absorbed isotope during the experimental period, which has been shown to be true for iron (King and Tumland, 1989). The advantage of radioisotopes is the easy detection and minimal sample preparation prior to analysis. However, the disadvantages of radioisotopes, i.e., hazards associated with exposure to ionizing radiation and rapid decay of some isotopes, have led to stable isotopes as a safe alternative to radioisotopes (Turnlund and Johnson, 1984), especially in studying certain groups, such as infants and pregnant women. Stable isotopes are naturally occurring nuclides of an element with the same atomic number but differing numbers of neutrons. They have similar chemical properties but differ in mass. Advantages of stable isotope studies are that multiple isotopes of the same mineral can be used simultaneously and multiple minerals can be studied simultaneously. Another major advantage is that they are safe and can be used to trace the metabolic fate of minerals with no exposure of the experimental subjects or researchers to radiation. The disadvantage of stable isotopes is that they usually cost more than radioisotopes and some isotopes are very expensive. Analysis of stable isotopes also is more difficult than that of radioisotopes. It takes longer, generally requires more extensive sample preparation and uses more expensive instrumentation than does analysis of radioisotopes. Mass spectrometry and neutron activation analysis are the two methods usually used for analysis of stable isotopes. Animal studies Knowledge of factors affecting zinc absorption is based mainly on animal studies because of the inherent difficulty in studying nutrient absorption in humans. In many studies, experimental animals are fed stock diets for long periods prior to the test periods. Experimental diets are usually formulated to provide at least a minimum supply of most nutrients and often contain excessive levels that are several times higher than the expected nutrient requirements. Such excessive dietary intakes of zinc are unlikely to occur in human. An increase in metallothionein synthesis in the mucosa] cell can be achieved by excess dietary zinc intake (Cousins, 1985), and as a consequence, the regulatory mechanisms for zinc absorption within the cell may differ from those operating during periods of normal zinc supply. A variety of experimental model systems have been developed to study the transfer of zinc across the intestinal mucosa (Sandstrom and Lennerdal, 1989). A system that is similar to the intact animals uses a segment of small intestine which is tied off or a loop of intestine which then is perfused with a solution of zinc. Measurements of changes in zinc concentration, transfer rates and kinetics of zinc transport can be determined. Another approach is to use everted gut sacs prepared by surgical removal of a segment of intestine, eversion of the segment, and placement in the test substance to allow perfusion into the sac. The problem with this method is the changes in the microenvironment such as the removal of blood vessels. A third system uses brush border membrane vesicles (BBMV) prepared by physically removing mucosa and selectively precipitating the BBMVs. The advantages of these approaches are that many conditions can be closely controlled and thus effects of pH, ionic strength, concentrations of ions, ligands, competitors and stimulatory factors can be investigated in detail. However, the methods have drawbacks such as absence of blood supply and intact innervation. Radiotracers can also be used in animals to study mechanisms involved in zinc absorption which would be difficult to do in humans. More pronounced deficiencies and excess of zinc as well as of other nutrients also can be studied, particularly during vulnerable periods such as pregnancy, lactation and infancy. In Vitro techniques As an alternative to human and animal in vivo studies, availability of minerals also has been estimated through simple, rapid in vitro methods (Miller et al.,l981; Hazel and Johnson, 1987; Hurrell et al., 1988; Wolters et al., 1993; Luten et al., 1996). The method of Miller et al. has been used as the basis for several in vitro methods for estimation of the bioavailability of iron and zinc. A good correlation has been shown between results using the in vitro dialyzable iron method of Miller et al. (1981) and in vivo studies of iron bioavailability in humans (Schricker et al., 1981; Hazel] and Johnson, 1987; Hurrell et al., 1988). Not all important physiological factors, however, can be simulated in the in vitro method, and the prediction of bioavailability is relative rather than absolute. The in vitro methods based on the method of Miller et al. (1981) involve a simulated gastrointestinal digestion with pepsin during the gastric stage followed by a digestion with pancreatin and bile salts in the intestinal stage. The proportion of the compounds diffusing across a semipermeable membrane during the intestinal stage is used as a prediction of the proportion of the element that is available for absorption. In vitro methods have been shown to be good predictors of many inhibitory/enhancing dietary factors, and have been used to examine the influence of various methods of food processing on mineral bioavailability from food. The bioavailability of zinc Certain groups of people appear to be at risk with regard to zinc nutrition. There are a number of reasons for their vulnerability, including inadequate intake or low bioavailability, malabsorption, elevated losses or elevated requirements to support growth, pregnancy and lactation (F airweather-Tait, 1988). Physiology and biochemistry of zinc Zinc is present in biological systems as the divalent cation complexed to organic ligands, rather than free in solution as metallic iron, and exists as loosely or firmly bound fractions. The organic ligands are mainly proteins, peptides, nucleic acids and amino acids (Vallee and Falchuk, 1993). The zinc content of foods varies widely. The major dietary sources of zinc are foods of animal origin such as meat, fish, shellfish, poultry, eggs, and dairy products. Processing may affect the amount of zinc in a food that is actually available for absorption. Heat treatment can cause food zinc to form complexes that are resistant to hydrolysis and therefore make zinc unavailable for absorption. The absorption of zinc occurs mainly in the duodenum and proximal small intestine and involves 1) influx of zinc into the enterocyte and through the basolateral membrane 2) transport into the portal circulation (Krebs, 2000). The first zinc transporter cloned, ZnT-l, fimctions mainly as a zinc exporter and may play a role in zinc homeostasis (McMahon and Cousins, 1998). Metallothionein, an intracellular metal binding protein, regulates zinc absorption. High dietary zinc concentration can stimulate gastrointestinal metallothionein synthesis. Animal and human studies indicate that uptake of zinc is enhanced with low dietary zinc intake or increased physiologic demands. However, the mechanism is unclear (King and Keen, 1994). Approximately 60-70% of plasma zinc is freely exchangeable and loosely bound to albumin and 30% is firmly bound to or-Z-macroglobulins. Two amino acids, histidine and cysteine, also loosely bind 2 to 8% of zinc to form a complex in the blood (Giroux, 1975) Zinc passing into the portal blood from the intestine is mainly transported to the liver where the mineral is initially concentrated. From the liver, zinc is distributed to other tissues including bone. The mechanism of zinc uptake by tissue is poorly understood. The zinc content of most soft tissues including muscles, brain, lung, and heart is relatively stable and does not respond to changes in dietary zinc intake (Cousins and Hempe, 1990; National Research Council, 1989). line plays a role in the fiJnction of a large number of metalloenzymes including carbonic anhydrase, carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, lactic dehydrogenase, and pyruvate carboxylase. It is also incorporated in a copper-containing enzyme, superoxide dismutase (Vallee and Falchuk, 1993). These enzymes participate in the synthesis and degradation of carbohydrates, lipids, protein and nucleic acids. Zinc also plays an essential role in the processes of gene expression. These biochemical functions of zinc give it a unique role for grth and development. Factors that influence zinc bioavailability Differences in bioavailability of zinc from various dietary sources may influence dietary adequacy. Zinc is less available for intestinal absorption from foods of vegetable origin than from foods of animal origin, which is believed to be largely due to the presence of phytate and dietary fiber frequently present in foods of vegetable origin (Knudsen et al., 1996). P_rqt_ei_t_t Higher bioavailability of zinc from meat is believed to be due to the presence of some types of enhancing factors in the meat (Perez-Llamas et al., 1996). In human adults, 80%-95% of dietary proteins are digested in the small intestine where the protein- bound zinc is probably rendered available for absorption (Sandstrom and Lonnerdal, 1989). Whether protein in general has a positive effect on the degree of zinc absorption is less clear. Greger and Snedeker (1980) observed significantly lower fecal zinc losses in adult males fed a high protein diet (150 g/d) than males consuming 50 g protein/d with similar zinc intakes. However, urinary zinc excretion was increased and the apparent retention of zinc was not statistically different. Different chemical properties of the proteins are a probable explanation for the less-efficient absorption of zinc from cow’s milk or soy-protein-based infant formulas compared with that from human milk (Sandstrom et al., 1983a, b). Differences in amino acid composition of proteins may be responsible for difference in absorption. Supplementing a low protein diet with sulphur- containing amino acids increased urinary zinc excretion in adult male subjects (Snedeker and Greger, 1981). When rats were fed a 45% lactalbumin diet or a 15% lactalbumin diet supplemented with histidine and cysteine, apparent absorption of zinc was higher than when a low-unsupplemented protein lactalbumin diet was fed (Snedeker and Greger, 1983). An improvement in zinc absorption was also observed when cysteine and methionine were added to a soy protein diet; absorption was similar to that from a lactalbumin-containing diet (Greger and Mulvaney, 1985). A much lower absorption of zinc from a soya-based infant formula compared with human milk or cow’s milk formula 10 has been observed in adults (Sandstrbm et al., 1983a), and growth studies of children recovering fi'om protein-energy malnutrition also suggest a poor availability of zinc from soya-based formulas (Golden and Golden, 1981). This low availability could be due to the phytic acid usually associated with such soya protein fractions but could also be attributed to properties of the soya protein itself. Phytic acid Chief among the inhibitors of zinc bioavailability is phytate (myo-inositol hexaphosphate) which occurs in the cereal grains and legumes (O’Dell and Savage, 1960). Phytate can be hydrolyzed prior to the intestinal site of zinc absorption. Hydrolysis may occur in the stomach either by phytase from plants, yeasts or bacteria. At low pH, phytate will be strongly negatively charged and phosphate groups on phytate have a strong potential to bind positively charged amino groups on proteins. These compounds could be formed only in the stomach. At neutral pH, the negatively charged phosphates and proteins form a complex with divalent metals (Wise, 1995). Numerous studies have demonstrated impairment of zinc absorption when phytate-rich diets have been given to animals (O’Dell and Savage, 1960; Davies and Olpin, 1979; Morris and Ellis, 1980; Forbes et al., 1984). Studies showing that phytate can act as a strong antagonist for zinc absorption also have performed in human using isotopes of zinc. Addition of phytate to a low-fiber diet (Turnlund et al., 1984), to white bread to the levels found in whole meal bread ( Navert et al., 1985) or to a cow’s-milk-based infant formula to the level found in a soya-bean formula (Lonnerdal et al., 1984) reduced the absorption of zinc significantly. Reduction of the phytate content of bran by yeast leavening in making bread (N avert et al., 1985) and fermentation (Gibson et al., 1998) significantly 11 improved zinc absorption. The molar ratio between phytate and zinc has been suggested as an index of zinc availability (Oberleas and Harland, 1981). It was proposed that absorption in humans is impaired significantly at molar ratios of phytate:zinc above approximately 10:1. A [phytate]x[Ca]/[Zn] molar ratio has been found in some studies to be a better predictor of zinc bioavailability than [phytate]/[Zn] molar ratio alone (Bindra et al., 1986; Davies et al., 1985). However, Wise (1995) indicated that although the [phytate]x[Ca]/[Zn] ratio has been used in animal experiments and has been applied in discussions about different human diets, there is no experimental or theoretical justification for its importance in humans. Even in animal studies, the relationship between the [phytate]x[Ca]/[Zn] ratio and zinc absorption has been found to differ. Zinc absorption may be more directly related to the other dietary components than to the [phytate]x[Ca]/[Zn] ratio. I_rog Several studies have described the effects of iron on zinc absorption. The negative effect of excess iron on zinc absorption reported in some of the studies raised concern about possible adverse effects of fortifying foods with iron as well as the effect of taking supplements on zinc absorption. In a study of iron and zinc interactions, Solomons and Jacob (1981) observed a reduced plasma zinc uptake after an oral dose of 25 mg zinc following administration of 25, 50, and 75 mg doses of elemental iron. No effect on zinc absorption was observed when heme iron was ingested in a 3:] iron to zinc ratio. Meadows et al. (1983) investigated the effect of daily oral supplements of 100 mg Fe, 50 mg Zn, and 350 ug folic acid for 14 din healthy nonpregnant adults and found a significant reduction in plasma zinc after the 14 d supplementation. The effect of iron on 12 zinc absorption has also been measured by radioisotopic labeling. Valberg et al. (1984) found that both inorganic iron and heme iron inhibited zinc absorption from 6 mg doses of 65211 as zinc chloride. In a recent study, the effect of iron fortification on the absorption of zinc was studied by using radioisotopic labeling of a single meal, followed by measurements of whole- body retention of “Zn (Davidsson et al., 1995). No significant differences in zinc absorption were observed in adult males consuming iron-fortified weaning cereal, bread rolls or infant formula when compared with those consuming the unfortified counterparts. No significant effect on zinc absorption had been observed previously in a metabolic balance study (Haschke et al., 1986) and a study using a dual-radioisotope method (5 5 Fe and ”Fe) ( Rossander-Hultén et al., 1991). Not all of the studies provide evidence for an interaction. Conflicting results in human studies have been reviewed by Solomons (1986). He concluded that the competitive inhibition of zinc uptake by excess iron in ratios of 2:1 or greater appears to have a measurable effect on human zinc nutriture when the total amount of ionic species (as the sum of zinc and iron species administered as a single oral dose in solution) is greater than 25 mg. Inorganic iron and zinc share some portion of a common absorption pathway based on the physiological competition of these chemically similar ions. geld—um The growing awareness of the need for high amounts of dietary calcium to prevent bone loss has resulted in a significant increase in the sale of calcium supplements and the introduction of many new calcium-fortified foods. High calcium intakes can reduce zinc absorption and exacerbate the signs of zinc deficiency in animals fed low-zinc diets l3 (Forbes, 1960). Animal studies have shown that high calcium levels can impair the intestinal absorption of zinc (Beth and Hoekstra, 1965). Metabolic balance studies in human subjects with calcium intakes of 200, 800 and 2000 mg/d did not show any effect of calcium level on zinc absorption or balance (Spencer et al., 1984). However a recent human study showed that zinc absorption was reduced by 50% when a calcium supplement (600 mg Ca) was given with the meal. This finding suggests that a high calcium diet can reduce net zinc absorption and balance and may increase the zinc requirement in adult humans (Wood and Zheng, 1997). Otherfifactors It has been suggested that other substances inhibit zinc absorption including fiber (Reinhold et al., 1976; Kelsay et al., 1979) and oxalic acid in spinach (Kelsay and Prather, 1981). Minerals with similar chemical properties often exhibit a biological competition. For example, a competitive interaction has been observed between zinc and other divalent cations such as copper (Sundaresan et al., 1996) and cadmium (Jaeger, 1990) Phenolic Compounds Chemistry of phenolic compounds Phenolic compounds are among of the most numerous and widely distributed substances in the plant kingdom. Natural phenolic compounds can range from simple molecules, such as phenolic acids, to highly polymerized compounds, such as tannins. They occur primarily in conjugated form, with one or more sugar residues linked to hydroxyl groups. The main classes of dietary phenolic compounds are phenolic acids, 14 flavonoids, and tannins (Bravo, 1998). Phenolic acids include hydroxybenzoic (e. g., gallic acid) and hydroxycinnamic acids (e.g., cafi‘eic acid). Caffeic and quinic acid combine to form chlorogenic acid (Figure 1). Although chlorogenic acid is widely distributed in fruits and vegetables, its relatively high concentration in coffee beans makes them the principal dietary source of chlorogenic acid. Hydroxycinnamic acid in cereal grains is located in the outer layers of the kernel (King and Young, 1999). The largest group of plant phenols, the flavonoids, are 2-phenyl benzopyran-based compounds (Figure 2) that are grouped into anthocyanins and anthoxanthins. Anthocyanins are molecules of red, blue and purple pigments. Anthoxanthins, which include flavonols, flavones, flavanols and isoflavones, are colorless or white to yellow compounds. F lavanols and flavonols, the main classes of phenolic compounds found in tea, constitute about 30% of the dry weight of the fresh leaf. Flavan-3-ols (the catechins) are the major flavonoids found in green tea and include: (-)-epicatechin (EC), (-)-epigallocatechin(EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3- gallate (EGCG) (Figure 2). Both green and black tea contain approximately 3% flavon-3- 015 such as quercetin, kaempferol, myrictin and their glycosides (Figure 2). During the manufacture of black tea, enzyme-catalyzed oxidation of the catechins leads to the formation of various catechin condensation products: theaflavins and thearubigins. Compositionally, the main difference between black tea and green tea is the relative levels of epicatechins and their oxidized condensation products (Dreosti, 1996; King and Young, 1999). 15 Caffeic acid + Quinic acid ———> Chlorogenic acid + H20 HO CH =CH-COOH H0 H0 H0 (le CH Caffeic acid HO CH C =0 l OH HO COOH HOOC OH HO Quinic acid Chlorogenic acid FIGURE 1 Structures of caffeic acid, quinic acid and chlorogenic acid (adapted from Salunkhe et al., 1990, p6). 16 Basic flavonoid structure Major tea flavan—3—ols (catechins) Major tea flavon-3-ols .H R1 OH OH o . o . R2 "'OR1 OGlycoslde OH OH 0 R1 R2 R1 R2 EC Epicatechin H H Kaempferol glycoside H H ECG Epicatechin gallate Gallate H Quercitin glycoside OH H EGC Epigallocatechin H OH Myn'citin glycoside OH OH EGCG Epigallocatechin gallate Gallare OH FIGURE 2 Structures of catechins, flavonols and basic flavonoid structure (adapted from Balentine et al., 1997, p695). l7 Mas Tannins are water-soluble polyphenols of intermediate to high molecular weight that contain sufficient phenolic hydroxyl groups to form insoluble complexes with carbohydrates and protein. Tannins are present in many plant foods. These include food grains such as sorghum, millets, barley, dry beans, faba beans, peas, carobs, pigeonpeas, winged beans, and other legumes. Fruits such as apples, bananas, black berries, cranberries, dates, grapes, peaches, pearsm persimmons, plums, raspberries, and strawberries contain an appreciable quantity of tannins. Tannins are also present in wines and tea (Chung et al., 1998). Tannins can be classified into two major groups: (1) hydrolyzable and (2) condensed tannins. HydrobIzable tannins contain a central core of glucose or other polyhydric alcohol which is esterified with gallic acid (gallotannins) or hexahydroxydiphenic acid (ellagitannins). These metabolites can oxidatively condense with other galloyl or hexahydroxydiphenic molecules and form high-molecular-weight polymers. These types of tannins are easily hydrolyzed by acid, alkali, or certain enzymes. The products of such hydrolysis are shown in Figure 3. The hexahydroxydiphenic acid of ellagitannins undergoes lactonization to produce ellagic acid (Figure 3). The best-known hydrolyzable tannin is tannic acid. Condensed tannins also are high-molecular-weight polymers and structurally more complex than hydrolyzable tannins. The most commonly described condensed tanins have molecular weights of approximately 5000 Da, although the structures of many of the compounds are yet to be determined. Condensed tannins do not break down readily under physiological conditions. Condensed tannins are widely present in fruits, vegetables, l8 Gallotannin + [120 ———+ Glucose + Gallic acid 0 :o‘OH OH HO OH OOC©OH HO 9 C00" 0 C @ @ Gallic acid HO OH HO OH Gallotannin Ellagitannins + H20 -—) Glucose + Gallic acid + Hexahydroxydiphenic acid Jr Lactonization OH Ellagic acid H0 HO OH H H on on H0 Ellagitannin o II -—o coon coon OH HO OH on no H H 0‘1} o Hexahydroxydiphenic acid Ellagic acid FIGURE 3 Hydrolyzable tannins (adapted from Chung et al., 1998, p423). l9 forage plants, cocoa, red wine and certain food grains, such as sorghum, finger millets and legumes (Chung et al, 1998; Bravo, 1998). Tea Tea is the fragrant brew prepared from the leaves of two varieties of the plant Camellia Sinesis:assamica and sinesis. The majority of tea beverage is prepared from two types of manufactured tea: black and green. Black tea, consumed typically in the United State, Europe, Africa and India, is made by crushing and drying from tea leaves to effect “fermentation” prior to final processing. During “fermentation”, some of the catechins combine to form complex theaflavins and other flavonoids, which provide distinctive flavor and color to black tea beverage. The fermentation process is initiated by the oxidation of catechins to reactive quinones, a process catalyzed by the enzyme polyphenol oxidase. While the gallocatechins, epigallocatechin, and epigallocatechin gallate are preferred, polyphenol oxidase can use any catechin as a substrate to form the complex polyphenolic constituents found in black and oolong teas. Oolong tea is partially fermented tea manufactured primarily in the People’s Republic of China and Taiwan. Green tea is prepared when the fresh leaves are processed rapidly to prevent fermentation. Consumption of green tea is highest in East Asian countries but is increasing worldwide (Wiseman et al., 1997; Balentine et al., 1997). Coffee The two important coffee species of commerce are Coflea arabica and Coflea canephora. The green coffee beans undergo the process of roasting, grinding, infusion with water and, in the case of the soluble or instant product, drying, followed eventually 20 by reconstitution to become the coffee beverage. Raw coffee beans contain about 7% dry weight chlorogenic acid and roasted coffee contain about 4-5% chlorogenic acid with an estimated 2% of other phenolic compounds (Sivetz and Desrosier, 1979). The roasting procedure decreases the concentration of chlorogenic acid depending on the roasting temperature. Phenolic compounds (tannins) as antinutrients Influence on the digestibility of macronutrients Tannins traditionally have been considered antinutrients because tannins form complexes with proteins, starch and digestive enzymes to cause a reduction in the nutritional values of foods. Highly polymerized tannins are the most effective precipitators of protein. Hagerman and Butler (1980) reported that under Optimal conditions sorghum tannin is capable of binding and precipitating at least 12 times its own molecular weight of proteins. High tannin sorghum grains contain more than enough tannins to bind to seed proteins and profoundly affect the availability of these proteins. Barley, rye, and common beans contains a low quantity of tannins and a high level of protein; therefore, the quality of these seed proteins is less affected by tannins (Price and Butler, 1980). Furthermore, tannins can bind endogenous proteins in the intestinal tract, such as digestive enzymes and inhibit them (Quesada et al., 1996; Longstaff and McNab, 1991; Carmona et al., 1991). This effect causes a reduction in the digestibility not only of protein (Jansman et al, 1994) but of other macronutrients, such as starch and lipid (Longstaff and McNab, 1991; Carmona et al., 1991). Tannins have been shown to inhibit virtually every digestive enzyme (Butler, 1989) and have also been reported to interfere 21 with the digestion and/or absorption of carbohydrates from sorghum (Blakeslee et al., 1979) and in vitro amylolysis of sorghum starch (Davis and Houseney, 1979). Influence of tannins on bioavailability of minerafi Tannins can form complexes with metal cations through their carboxylic and hydroxylic groups and thus interfere with the absorption of minerals in the intestine. The inhibition of iron absorption has been attributed to the galloyl and catechol groups of polyphenolic compounds (Brune et al., 1989). Catechins in green and herb teas (Brune et al., 1989; Gillooly et al., 1983; Reddy et al., 1991; South et al., 1997), phenolic acids in coffee (chlorogenic acid) (Brune et al., 1989), polymerized phenolic compounds in black tea and cocoa, (Hurrell et al., 1998), and wine polyphenols (Cook et al., 1995) have been shown to reduce iron bioavailability. Reduced copper absorption after consumption of tea also has been observed in human (Kies and Umoren, 1989). Kies and Umoren demonstrated that tannins in wheat bran and tea may contribute to decreased copper utilization, but this result conflicted with those in some rats studies. Vaquero et al. (1994) observed an increased absorption of 64Cu and an enhanced retention of copper in the liver of rats fed tea. Greger and Lyle (1988) showed in rats that ingestion of diets containing more than 1.17% black tea enhanced copper absorption leading to elevated liver copper levels. Ingestion of black tea by anemic rats elevated plasma ceruloplasmin activity and tended to elevate plasma copper compared to that of pair-fed control animals. The influence of tannins on zinc bioavailibility also has received attention. The effect of tea and catechin on tissue levels of zinc in rats was studied by Greger and Lyle (1988). A significant increase in the levels of zinc in tibias was shown. However, the 22 bone zinc levels were not correlated with apparent absorption of zinc. Rats fed ad Iibitum a tea decoction for 15 days showed a significant increase of 29.4% in total blood zinc (Hamdaoui et al., 1997). Tea significantly increased zinc, copper and magnesium concentration in the total blood but did not significantly raise the reserve of zinc or copper stored in the liver. In a 1991 abstract, Reddy et al. reported results of a study in which tea infusion mixed with water (4: 1) or milk (4:1) and a control solution of water and water and milk were administered by gavage to 16 day old rats. All solutions were labeled with ”FeCls and 65ZnSO4. Tea significantly decreased absorption of both iron and zinc. Addition of milk to tea had a further inhibitory effect on absorption of iron but not zinc. In a study in rats by Zeyuan et a1. (1998) addition of green tea to a control diet led to a decrease in apparent absorption of zinc whereas black tea enhanced absorption. In a human study using a dual-isotope technique, subjects were given an extrinsically labeled turkey test meal, and the results showed no significant effect on 65 Zn absorption from meals administered with tea (Flanagan et al., 1985). Ganji and Kies (1994) reported that tea consumption showed a small but statistically non-significant adverse effect on zinc utilization in humans consuming self-selected and laboratory-controlled diets. Decreased bioavailability of zinc in a mixed meal with addition of coffee was also observed in an in vitro study (Van Dyck et al., 1996). Ingestion of chlorogenic or caffeic acids was also reported to cause a decreased zinc absorption in rats (Coudray et al., 1998). Other factors may influence the bioavailability of minerals. The influence of adding milk to tea and coffee on minerals absorption has been investigated. Disler et al. (1975b) reported a similar reduction in the absorption of non-heme iron when tea with or without milk was added to the mixed meal in humans. Reddy et al. (1990) found that the 23 inhibitory effect of tea on iron absorption was exacerbated by milk. In contrast, Christian and Seshadri (1989a) found that milk added to a cereal meal completely counteracted the inhibitory effect of tea on in vitro iron availability. Coffee given with a meal containing milk was shown to inhibit the absorption of zinc in humans (Pécoud et al., 1975). 24 JUSTIFICATION Polyphenols, important components in tea and coffee, are believed to influence the bioavailability of trace mineral, especially iron (Disler et al., 1975a; Reddy and Cook, 1991; Hamdaoui et al., 1994, 1997; Zeyuan et al., 1998). An inhibitory effect of polyphenols in tea on iron absorption is believed to occur because of the formation of an unabsorbable “iron-tannin” complex in the gastro-intestinal lumen. Brune et al. (1989) concluded that the content of iron-binding galloyl groups might be a major determinant of the inhibitory effect of phenolic compounds on iron absorption. Although it is sometimes stated that tea has an inhibitory effect on other minerals such as zinc and copper, experimental data are limited and often conflicting. Studies in humans have shown no significant effect (Flanagan et al., 1985) or a small but not significant adverse effect on zinc bioavailability (Ganji and Kies, 1994). An inhibitory effect on zinc absorption in rats was reported for tea and the phenolic compounds, although in one study the effect was seen only for green tea and not black tea (Zeyuan et al., 1998). In contrast, results of other studies have shown increased bone and blood levels of zinc in rats fed diets containing tea, which might suggest enhanced absorption of zinc (Hamdaoui et al., 1997). Coffee is another beverage which is often consumed with meals. Its inhibitory effect on iron absorption has been observed in vitro (Van Dyck et al., 1996) and in human studies (Morck et al., 1983; Brune et al., 1989). However, the effect of coffee on zinc bioavailability has received little attention. Van Dyck et al. (1996) reported a negative effect on availability of zinc. Coudray et al. (1998) found zinc absorption was 25 significantly less in rats fed chlorogenic acid or caffeic acid, phenolic compounds found primarily in coffee, than in the control group. Numerous factors may contribute to the conflicting results including : the age of the rats (e. g., suckling or old rats); the tea or coffee concentration given to subjects or animals; the ways in which the tea is administered (e. g., self-selected versus test meals in humans or tea given as drinking fluid versus incorporation of tea leaves in the diet of animals); the measurement methods (e. g., isotope technique, metabolic balance measurement or the mineral content of organs). Polyphenols in tea and coffee, especially green tea, appear to have antioxidant and anticarcinogenic properties and consumption of green tea is being widely promoted. Similarly, green tea extracts are being added to other foods or used as supplements based on their potential health benefits. It is apparent, however, that additional research is need to clarify the potential effect of tea and other phenolic containing foods on bioavailability of trace minerals. HY POTHESES 1. Tea and coffee do not decrease the bioavailability of zinc in a food. 2. Addition of milk to tea and coffee does not modify their effect on the bioavailability of zinc in a food. 26 MATERIALS AND METHOD Research design The effects of tea and coffee on the in vitro bioavailability of zinc and iron in two wheat based products were determined. The effect of adding milk to tea also was determined. Test meals Extruded wheat products. Two extruded wheat products made from the same soft white wheat were used as the basis for the test meals. One product was made from white flour; the other was made from whole kernel wheat. Extrusion conditions were the same for both products (feed rate: 650 rpm; screw speed: 250 rpm; finished temperature, 160°C; moisture content: 28.3%). Products were ground to fine particles and dried in an oven over night before use. Preparation of tea decoction and coflee. The tea decoction and coffee were prepared fresh before each experiment. Tea was prepared by adding 240 g boiling double deionized water (DDW) to 2 g green tea leaves (Yamamotoyama Sen-Cha Green Tea, Yamamoto of Orient, Inc.) and the mixture then allowed to steep for 5 min. Coffee was prepared by adding 240 g boiling DDW to 4 g instant coffee (Folgers Coffee Crystals, F olger Coffee Company). Milk. Homogenized cow’s milk (2% fat) was purchased from a local grocery store. Water. Double deionized water was used throughout the experiment. Glassware. All glassware used in the experiments and analyses was acid-washed. Composition of test meals. Composition of individual test meals is shown in Table]. 27 .oohoo Eamon “535 w v 2 .255 “63623 03:3 wcfion w cvm wigs E 03:. 83 ootoo .EE n no.“ noon 8 326—? 93 823— aB 52w m N 3 $33 Buses—o 03:8 choa w ovm mafia 3 038 was 8...... m 2 -- m -- V.5): m 3 e. m -- 53+ caboose? -- an o... m -- 8:85? -- om .. m 1 833+ 32:5 359M 29.3 m 2 -- -- m 53+ m a. 2. -- m VE): 8:83? -- a 9. -- m 8:83? -- 8 -- -- m 333+ :5; 223 w w m m m 28:3 seam 223 so: 233 $2 38 30:8 5 a; 288a 83.5 as: use £on 38 no 5289950 fl HAM—4G. 28 The ratio of ingredients was equivalent to a 20 g portion of a dry cereal product consumed with 160 g of tea or coffee, with or without 20 g of milk. Chemicals Pepsin powder (from porcine stomach mucosa), pancreatin (from porcine pancreas), bile extract (porcine), HzOz. NaHCO3, vanillin (4-Hydroxy-3-methoxy-benzaldehyde), tungstic acid sodium salt dihydrate, phosphomolybdic acid and (+)catechin were obtained from Sigma Chemical Co ,St. Louis, MO. Trace metal grade HCl and HNOs were obtained from Fisher Scientific, Pittsburgh, PA. Absolute methanol and phosphoric acid were obtained from IT. Baker INC, Phillipsburg, NJ. Sodium carbonate anhydrous was obtained from Mallinckrodt Chemical Works. Pepsin suspension: 16 g pepsin powder suspended in 100 ml 0.1 M HCl. Pancreatin-bile extract mixture: 4 g pancreatin and 25 g bile extract dispersed in 0.1 M NaHC03 and brought to 1 L with 0.1 M NaHCOs. In vitro study A modification (Luten et al., 1996) of the in vitro method reported by Miller et al. ( 1981) was used. Dialysis tubing with molecular weight 6-8000 Da (Spectra/Pro 1) was used instead of 10,000 Da. Dialyzable mineral content of each test meal was determined eight times with each digestion performed in duplicate. Gastric stage. Ingredients for each meal were added to a 250 ml Erlenmeyer flask. The pH of the mixture was adjusted to 2.0 using 6 M HCl. After 15 min the pH was checked and readjusted to 2.0 if necessary. The freshly prepared pepsin solution (3 g) was 29 added and the flask content brought to total weight of 100 g with DDW. The flask was incubated at 37°C for 2 h in a shaking water bath. The water bath settings were 100-120 strokes/min and an arm movement of 2 cm. The gastric digests were stored in ice for 90 min during which time the titratable acidity was measured in an aliquot of the digest. T itratable acidity. The titratable acidity was determined on a homogeneous 20 g aliquot of the pepsin digest to which 5 g of the freshly prepared pancreatic-bile extract mixture was added. The pH was adjusted to 7.5 with 0.5 M NaOH. After an equilibrium period time of 30 min, the pH was checked and readjusted to pH 7.5 if necessary. The titratable acidity was defined as the number of equivalents of 0.5 M NaOH required to titrate the combined pepsin digest pancreatin-bile extract mixture to pH 7.5. Intestinal stage. Homogeneous gastric digest aliquots (20 g) were weighed and transferred to wide-necked 250 ml beakers, which were then placed in a water bath at 37°C for 5 min. A 25 cm segment of dialysis tubing sealed with a plastic clip at one end was filled with 25 g DDW and an amount of 0.5 N NaHC03 equivalent to the titratable acidity measured previously, sealed with a clip and then placed in the beakers containing the gastric digest. The beakers were sealed with parafilm and incubated for 30 min after which pancreatin-bile extract mixture (5 g) was added to each beaker. The incubation was continued for an additional 2 h. At the end of the incubation period, the dialysis tubes were removed, rinsed with DDW and the pH of the retentate measured. The content of each dialyzate was transferred into acid-washed containers, weighed and then analyzed for mineral content. 30 Analytical methods Methods of phenolic compounds analysis Vanillin assay The total phenolic compounds content of green tea and coffee was determined using the vanillin assay (Deshpande and Cheryan, 1985). One g of green tea leaves or instant coffee powder was extracted with 10 ml absolute methanol for 20 min with mechanical shaking. The sample was then centrifuged at 1500 x g for 10 min. One ml of the supernatant was added immediately to 5ml of the color reagent which was composed of a 1:1 mixture of 8% HCl/methanol and 1% vanillin/methanol. Another 1 ml of the supernatant was added to 5ml of 4% HCl/methanol for the blank. After 20 min, absorbance was measured at 500 nm using a spectr0photometer(UV-l601, UV-visible spectrophotometer, Shimadzu). A standard curve was prepared using catechin (0.05, 0.1, 0.2, 0.4, 0.5, 0.6, 0.8 mg catechin/1 ml methanol), and tannin concentrations were expressed as mg catechin equivalents on a dry weight basis. Folin-Denis method Total phenolic compounds content of the green tea decoction and dissolved coffee solutions were analyzed using the Folin—Denis colorimetric regent (AOAC, 1990). Folin-Denis reagent was prepared by adding 100 g Na2WO4o2Hz, 20 g phosphomolybdic, and 50 ml I-IsPOa to 750 ml HzO, refluxed 2 h, cooled and diluted to 1 L. A saturated Na2C03 solution was prepared by adding 35 g anhydrated NazCOs, to 100 ml I120. The solution was dissolved at 70-80°C and cooled overnight. 31 A standard curve was made by pipetting 0-10 ml aliquots standard tannic acid solution (20 mg tannic acid/l L HzO) into 100 ml volumetric flask containing 75 ml H20. Then 5 ml Folin-Denis reagent and 10 ml Na2C03 solution were added and diluted with I120 to 100 ml. The contents were mixed well and absorbance was determined at 760 nm after 30 minutes. One ml of sample solution was added to a 100 ml volumetric flask which contained 75 ml HzO, 5 ml Folin-Denis reagent and 10 ml Na2C03 solution. The contents were diluted to 100 ml, mixed well and absorbance was measured at 760 nm after 30 minutes. Wet ashing and mineral analysis Triplicate l g aliquots of the ground wheat products were weighed and placed in 25 ml Erlenmeyer flasks with 10 ml concentrated HNOs and 2 glass beads. The flasks were placed on a hot plate at low heat for 1 to 2 days until the samples were digested and dry. After the flasks were cool, 5 ml of 30% H202 was added; flasks then were placed on a hot plate at low heat until the samples were dry. If a white ash was not obtained, more H202 (5 to 10 ml) was added. When digestion was complete, samples were dissolved in 10 ml of 0.1 N HCl and zinc and iron content were analyzed using atomic absorption spectrophotometry (Perkin-Elmer 2380). National Institute of Standards & Technology (Gaithersburg, MD) standards (bovine liver and wheat flour) were used to confirm accuracy of analytical procedures. Statistical Analysis Data were evaluated by Student’s t test, and one-way analysis of variance followed 32 by Scheffe’s Test to test the significance of difference between all possible pairs of means (SAS version 8, 1999). If the variance was non-homogeneous, statistical analysis was performed using a non-parametric test, Kruskal-Wallis Test. Results are expressed as mean i SEM, and differences were considered to be significant when p<0.05. 33 RESULTS Zinc, iron and total phenolic compounds analysis The amount of zinc, iron and total phenolic compounds in foods used in the test meals are shown in Table 2. The zinc and iron content of 5 g white flour was 37.2 ug and 71.4 ug, respectively. Whole kernel wheat contained a much higher level of zinc (160.4 rig/5 g) and iron (213.9 rig/5 g). Total phenolic compounds were analyzed using Folin- Denis (AOAC, 1990) and vanillin (Deshpande and Cheryan, 1985) methods. Using the Folin-Denis assay, green tea decoction (2 g tea leaves in 240 ml DDW) contained 12.8 mg tannic acid equivalents per 40 g of tea decoction and coffee (4 g coffee powder in 240 ml DDW) contained 24.8 mg per 40 g of coffee solution. Using the vanillin assay, the total phenolic compounds were 189.6 mg catechin equivalents/g green tea leaves and 57.6 mg catechin equivalents/g coffee powder. The amount of zinc provided by other foods in the test meals was 5.8 ug, 3.6 ug and 22.5 ug fortea (40 g), coffee (40 g) and milk (5 g), respectively. The amount of iron provided by other foods in the test meals was 15.2 pg, 48 ug and 13.5 ug for tea (40 g), coffee (40 g) and milk (5 g), respectively. In vitro dialyzable zinc and iron Percent dialyzable zinc was lower in the whole kernel wheat than in the white flour product (39.3i1.3 vs. 14.5i0.6, Table 3a). This relationship was also observed in the test meals when tea or coffee, with or without milk was added. The addition of tea to the test meals composed of the extruded product made from white flour reduced dialyzable zinc 34 .AmEV amuse—give 5588.. 3 “08855 28 Swain—«bozo can 852339 Emma 5553 was: 3532: 203 3:38:56 ozone—E :38. e. 385353323 Box 35.8.. mm c8885 28 88— .U? 3% on 3% 5 282 ace 3:8 8 8&8 Mo .8363 05 3933 8 :23 $0255 «8:3 32:38 mew—5:8 £88 52 E AEm—mficwoev so: 98 0% 03333. Eoocom an HAM—4G. 38 Additional analyses were done adding the ash of milk or casein to white flour to determine the effect of these two factors present in milk on dialyzable zinc and iron. Dialyzable zinc was significant lower when milk ash (8.2i0.2%) and casein (16.9i2.3%) were added to the product compared to water control (3 9.3.421 .3%). However, dialyzable iron was significant higher with milk ash (10.2i1.7%) compared to the control (5.6i0.2%). When casein was added to the test meals, a higher but not statistically significant dialyzable iron was obtained (7.9i1.6%) compared to the control. 39 TABLE 4 Percent dialyzable zinc and iron (meaniSEM) in test meals containing extruded white flour with addition of milk ash or vitamin-free caseinl Test Meals Zn(%)2 Fe(%)3 Extruded White Flour +Water 393:1.3“ 5.61-0.23 +Milk Ash“ 8.2:t0.2b 10.2:1.7b +Casein5 16.9:23" 79:16“ lDialyzable mineral content of each meal was determined twice with each digestion performed in duplicate. 2 Within a column, values with unlike superscripts are significantly different (p<0.05), as determined by Scheffe’s Test after ANOVA. 3 Within a column, values with unlike superscripts are significantly different (p<0.05), on the basis of non-parametric Kruskal-Wallis Test. 4 Milk ash was obtained by wet- ashing 5 g of milk. 5 0.13 g casein was added. This amount was calculated on the basis of 2.6 g/dl cow’s milk (Fomon, 1993). 40 DISCUSSION Although tea and coffee generally are considered to have an inhibitory effect on iron absorption, results of studies to determine their effects on zinc have given conflicting results. Using an in vitro method which measures dialyzable zinc following simulated gastric and intestinal digestion, our results show that tea and coffee have an inhibitory effect on the availability of zinc in extruded products made from white flour and whole kernel wheat. Test meals used in the study were designed to resemble a simple meal composed of a typical size portion of cereal consumed with a cup of tea or coffee. Because milk is frequently added to these beverages, test meals containing tea and coffee with or without milk were compared. The whole kernel wheat product which contains phytate, a known inhibitor of zinc absorption (O'Dell and Savage, 1960; Davies and Olpin, 1979; Morris and Ellis, 1980; Forbes et al., 1984), was used to determine potential interactions between phytate and a possible inhibitory effect of tea or coffee. Foods that contain other known enhancers or inhibitors of zinc bioavailability such as vitamin C or meat protein, were not included in the test meals in order to avoid additional interactions that would made the evaluation of the effect of tea or coffee more complicated. In the extruded wheat products used in this experiment, percentage dialyzable zinc in white flour was 39.3 compared to 14.5 in whole kernel wheat. As expected dialyzable zinc was consistently found to be higher in all test meals containing white flour compared to those with whole kernel wheat. In our study, dialyzable zinc was reduced by 28% and 19% when tea was substituted for water in the test meals containing extruded white flour and whole kernel wheat, 41 respectively. These results are similar to some of those in human and rats studies. Ganji and Kies (1994) demonstrated that tea consumption had a small but not statistically significant adverse effect on zinc bioavailability in humans. Greger and Lyle (1988) showed that the zinc absorption was significantly reduced in rats given tea and catechin compared with the control. Reddy et al. (1990) found that infused tea significantly decreased “Zn absorption in rats. Another study similarly showed that green tea leaves and their water extracts significantly decreased the absorption of zinc in rats (Zeyuan et al., 1998). Dialyzable zinc was also lower than the control when coffee was added to the products. Dialyzable zinc was 55% and 90% lower when coffee was added to extruded white flour and whole kernel wheat, respectively. Coffee had a greater inhibitory effect on dialyzable zinc than tea when it was added to the test meals. Van Dyck et al. (1996) also observed a decrease in in vitro zinc bioavailability when coffee was added to wheat bran. In humans, when 50 mg zinc was given with coffee, serum zinc concentrations were much lower than when the same dose of zinc was given with water (Pécoud et al., 1975) Tea has been shown to have an inhibitory influence on iron bioavailability (Disler et al., 1975; Rossander et al., 1979; Hallberg and Rossander, 1982; Hamdaoui et al., 1994). Our results also showed a decrease in dialyzable iron when tea was added to white flour. However, there was no significant difference when tea was added to whole kernel wheat. There is no apparent explanation for this difference between white flour and whole kernel wheat, although all values for dialyzable iron in whole kernel wheat test meals were in the range where accurate detection was difficult. Reddy and Cook (1991) demonstrated 42 that single-strength (1.75 g tea/200 ml water) or triple-strength (5.25 g/200 ml water) tea fed with a test meal composed of mixed foods containing 4.1 mg of iron significantly reduced nonheme iron bioavailability in humans. Fairweather-Tait et al. (1991) reported that rats given tea or a low-iron diet containing tea leaves showed a greater degree of iron depletion. Hamdoui et al. (1997) demonstrated that tea decoction fed to rats significantly reduced total iron concentration in the blood by 31.8%. In an in vitro study, Christian and Seshadri (1989a) found a significant depressing effect on available iron by 35% when tea was added to a basal meal composed of wheat flour chapati. Brown et al. (1990) found that black tea and green tea reduced dialyzable iron in a cereal-milk meal by 43% and 63%, respectively. The results of the present study (63%) are similar to the observations of Brown et al. (1990). Results for the effect of coffee on dialyzable iron were similar to those observed for tea. A 63% decrease in dialyzable iron occurred when coffee was added to white flour, whereas coffee had no effect on dialyzable iron in the whole kernel wheat product. A similar negative influence of coffee on the availability of iron has been documented previously in in vivo and in vitro studies. Morck et al. (1983) used dual isotopes to evaluate the effect of coffee on nonheme iron absorption in iron replete human subjects and found that a cup of coffee (1 .5 g freeze-dried coffee/200 ml) reduced iron absorption from a hamburger meal by 39%. Similarly, Brune et al. (1989) reported an inhibitory effect (61%) of coffee in humans fed wheat rolls. Van Dyck et al. (1996) reported a significant decrease in iron availability (dialyzability) when coffee was added to a test meal composed of mixed foods. No significant difference was shown in dialyzable iron in test meals when tea or 43 coffee was added to whole kernel wheat compared to water control. Since percent dialyzable iron in whole kernel wheat is very low, it is difficult to demonstrate a change unless the factor has a pronounced effect, such as was observed in the case of milk. Tea and coffee contain structurally different phenolic compounds. Phenolic compounds have been considered to interfere with nonheme iron absorption by formation of a complex with iron in the gastro-intestinal lumen, making iron less available for absorption. Tannins extracted from tea leaves by hot water were shown to account for this inhibition (Disler et al., 1975b). Catechins, the monomers of tannins, are the major phenolic compounds in green tea leaves. Brune et al. (1989), however, reported no effect on iron absorption in humans when catechin was added to wheat rolls. These authors suggested a possible explanation is that the insolubility of the catechin molecule in water prevents formation of a complex between the catechol group of catechin and mineral ions in the gastro-intestinal lumen. The hydroxyls are the reactive part of the phenolic molecule. Molecules with aromatic rings bearing two hydroxyls (catechol group) or three hydroxyls (galloyl group) positioned at adjacent carbon atoms, have iron-binding properties. A relation between coffee inhibition on iron and its content of galloyl groups was also reported by Brune et al. (1989). They demonstrated that the inhibition can be explained by the content of galloyl groups in the coffee and the content of chlorogenic acid might be responsible for the rest of inhibitory effect. Chlorogenic acid has been reported previously to have an inhibitory action on intestinal iron absorption in rats (Brown et al., 1990). Chlorogenic acid is the major phenolic compound in coffee (Sivetz and Desrosier, 1979). The effect of catechin and chlorogenic acid on zinc absorption is still not clear. A study by Coudray et al. (1998) showed that the effect of catechin on zinc 44 absorption in rats was less than that of chlorogenic or caffeic acids. The results of the present study indicate that percent dialyzable zinc is decreased when milk is added to tea in the test meal. The effect of the addition of milk to tea or coffee on zinc and non-heme iron absorption has received minor consideration. Disler et al. (1975b) reported that tea with or without milk led to a similar reduction in the absorption of non-heme iron in humans. The ratio of tea to milk (200 ml/40 ml) was similar to the ratio used (40 ml/5 ml) in the current study. In contrast, Christian and Seshadri (1989a) found that 200 g milk added to 65 g cereal meal made from wheat flour chapati completely counteracted the inhibitory effect of tea on in vitro iron availability. In our study, no counteractive effect was shown, but the amount of milk in relation to the amount of cereal and tea was much lower. Layrisse et al. (1976) reported that the mean absorption ratios of iron from iron-fortified sugar given with coffee without or with milk was 0.30 and 0.15, respectively. In the present study addition of milk alone to white flour was shown to decrease dialyzable zinc. Pécoud et al. (1975) reported that zinc absorption following administration of a test dose of zinc was lower when dairy products (milk and cheese) were added to a control meal composed of brown or white bread. Although milk was shown to improve zinc absorption fiom a phytate-rich diet (Sandstrém et al., 1980), casein in milk has been reported to have a negative effect on zinc absorption. A study using the Caco-2 cell culture system showed that high levels of casein phosphopeptides, which form during digestion of casein, inhibited zinc binding and uptake (Hansen et al., 1996). The nature of zinc binding with casein phosphate may explain the lower bioavailability of zinc in bovine milk compared to human milk which has been shown in 45 many studies (Nelson et al., 1986; Nelson et al., 1987; Singh et al., 1989; Brushmiller et al,1989) Another possible explanation for the inhibitory effect of milk may be its content of calcium. In humans a high dietary calcium intake (1500 mg/day) was shown to reduce net zinc absorption and balance (Wood and Zheng, 1997). Increased milk consumption (468 mg of calcium) with test meals, however, was shown to have no effect on either zinc absorption or retention (Wood and Zheng, 1990). Spencer et al. (1984) and Dawson- Hughes et al. (1986) also found no effect on zinc absorption in human adults when they added large amounts of calcium to a meal. The mechanism by which high-calcium diets may influence net zinc absorption is poorly understood. A calcium-zinc antagonism in animals has been shown to occur only with high phytate in the diet (Oberleas et al., 1962; O’Dell and Savage, 1960). The formula of [Ca] x ([phytate]/[Zn]) ratio has been used as a predictor of zinc bioavailability. Davies et al. (1985) suggested that the [Ca] x ([phytate]/[Zn]) ratio would be a better index of available zinc than the ratio of phytate to zinc. The combination of phytate and calcium might explain why percent dialyzable zinc was negligible in the test meal containing whole kernel wheat and milk. In vitro studies have demonstrated that the addition of bovine milk to cereal meals enhanced the availability of iron. Christian and Seshardri (1989b) reported that the addition of 200 g of bovine milk to a cereal meal made from wheat flour chapati significantly increased the in vitro iron availability. Tumlund et al. (1990) reported that the amounts of soluble and ionizable iron in a cereal-based diet composed of mixed foods were increased two- and three- fold, respectively, when milk was added. However, in the 46 same study the authors found no inhibitory effect of milk on iron absorption in humans. They suggested that the absorption of iron from cereal-based diets is neither enhanced nor inhibited by the addition of milk. When 7.2 g of protein isolate from 200 g of bovine milk was added to a cereal meal made from wheat flour chapati, the available iron in the meal increased from 4.91% to 6.73% (Jonnalagadda and Seshadri, 1994). In the present study dialyzable iron was decreased when milk was added alone to white flour and whole kernel wheat. The addition of calcium in the form of milk or an inorganic salt to a meal has been shown to reduce percentage nonheme iron absorption in humans (Deehr et al., 1990). Hallberg et al. (1991) demonstrated that increasing the amount of calcium caused a dose- related reduction in nonheme iron absorption from a meal of wheat rolls prepared from unfortifred white wheat flour and containing 10 mg native calcium and 3.8 mg iron. Cook et al. (1991) found that when calcium salts commonly used as supplements were taken with a hamburger meal, they were all inhibitory at a dose of 600 mg. The mechanisms for this effect on iron absorption are poorly understood. Additional analyses were done to try to explain the effect of milk on dialyzable zinc and iron. The ash of milk (5 g) and vitamin-free casein (0.13 g) were added to the extruded white flour and digested with the same procedures. The amount of casein in 5 g milk was calculated on the basis of 2.6 g/dl cow’s milk (Fomon, 1993). Dialyzable zinc was decreased by 79.2 % and 57.1 % when milk ash and casein were added to the white flour, respectively. These results suggest that both the minerals and the protein in cow’s milk may have contributed to the reduction in dialyzable zinc. Milk ash tended to affect dialyzable zinc more than casein. In contrast to zinc, percent dialyzable iron was 47 increased 82 % when milk ash was added to the white flour. Based on previous studies showing a negative interaction between calcium and iron, this result was contrary to what we expected. In our study, dialyzable zinc was higher in the white flour test meals containing tea plus milk than containing milk alone. Although dialyzable zinc could not be measured accurately for the test meal containing whole kernel wheat with only added milk, results also were lower than those for the tea plus milk. A mechanism to explain these results is not clear. Phenolic compounds (condensed tannins) have a strong affinity for proteins, especially those with a high proline content such as caseins in milk, gelatin and salivary proline-rich proteins. The strong hydrophobic interactions and hydrogen bonding both determine protein-polyphenol complexation (Hollman et al., 1997). This reaction results in lower biological values and protein digestibilities. Binding of casein with phenolic compounds in tea might decrease formation of casein phosphate complexes with zinc thus reducing the inhibitory effect of milk on zinc dialyzability and explain why percent dialyzable zinc was higher when tea and milk were added to the white flour than milk alone. Future research is needed to test if such a mechanism may explain the results observed in this experiment. 48 CONCLUSION The addition of tea or coffee to test meals composed of extruded wheat products made from white flour and whole kernel wheat resulted in a decrease percent dialyzable zinc. An inhibitory effect of tea and coffee on percent dialyzable iron was also observed with the white flour but not the whole kernel wheat product. The inhibitory effect of coffee on dialyzable zinc was greater than the effect of tea. Because tea and coffee are rich sources of phenolic compounds or tannins which are known to bind iron and presumably other divalent cations, the inhibitory effect is likely due to their phenolic compounds. A further decrease in percent dialyzable zinc in the test meals was observed when milk was added to the tea or coffee. These results suggest an enhancement of the inhibitory effect of tea on zinc dialyzability by milk. However, addition of same amount of milk alone to the test meals resulted in a lower dialyzable zinc than tea or coffee with or without milk. Under these conditions, its appears that tea may have partially counteracted the inhibitory effect of milk. When the ash of milk or an amount of casein similar to that present in milk was added to the extruded white flour, the results showed a decrease in percent dialyzable zinc suggesting that both the minerals and protein in milk contributed to its inhibitory effect in zinc dialyzability. Addition of milk ash and casein, however, unexpectedly resulted in higher dialyzable iron. An explanation for this discrepancy between results with addition of milk compared to milk ash or protein is not readily apparent. 49 FUTURE RESEARCH Phenolic compounds in tea have been shown to inhibit the iron absorption in many studies. The in vitro method has been reported to have a good correlation with in vivo determinations of zinc and iron absorption. However, the prediction of absorption is only relative because it is not possible to simulate all the important physiological conditions in the stomach and the small intestine. The majority of previous research has been done in animals. Therefore, more research should be conducted in humans to confirm the effect of phenolic compounds on zinc absorption. Phenolic compounds are widely distributed in food plants such as legumes, fruits, wine and tea. When those phenolic-rich foods are consumed together with other foods, the bioavailability of zinc needs to be determined. Very little is known about the type and chemical characteristics of phenolic compounds which influence their ability to interfere with iron or zinc absorption. Although Brune et al. (1989) implied that tea inhibited iron absorption in proportion to their respective content of galloyl group, more research is needed to be done to establish the mechanism. Calcium and casein have been shown to inhibit zinc bioavailability. Although results of other investigators have shown no effect or a counteracting effect of milk, our results suggest that milk exacerbates the inhibitory effect of tea and coffee. 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