n-6 Fatty Acids

The rat requires fatty acids from the n-6 family as a component of membranes, for optimal membrane-bound enzyme function, and to serve as a precursor for prostaglandin formation (Mead, 1984; Dupont, 1990; Clandinin et al., 1991). Linoleic acid [18:2(n-6)] cannot be synthesized endogenously but can be elongated and desaturated to form arachidonic acid [20:4(n-6)]; thus, the requirement for n-6 fatty acids can be met through dietary linoleic acid. Early estimates of the requirement for n-6 fatty acids were made by using growth and skin condition as response criteria. The requirement for growing male rats was estimated to be 50 to 100 mg/day and that of growing female rats to be 10 to 20 mg/day (Greenberg et al., 1950). That the requirement differs between sexes is in agreement with early observations that male rats are more susceptible than female rats to the development of EFA deficiency signs (Loeb and Burr, 1947). A series of later experiments indicated that other lipid components of the diet [e.g., oleic acid (Lowry and Tinsley, 1966) and cholesterol (Holman and Peiffer, 1960)] increased the need for n-6 fatty acids. Also, Holman (1960) demonstrated that more linoleic acid is needed in high-lipid diets versus low-lipid diets in order to meet the EFA requirement of the rat. Therefore it has become common practice to express the requirement as a percent of dietary ME rather than as a percent of diet weight. The Δ⁶ desaturase enzyme involved in the synthesis of long-chain polyunsaturated fatty acids uses the fatty acid substrates in the following order: n-3 > n-6 > n-9 (Johnston, 1985).

Holman (1960) found that the ratio of 20:3(n-9) to 20:4(n-6) (also called the triene:tetraene ratio) was relatively constant in tissues until clinical signs of EFA deficiency began to develop. Because this biochemical measure was both objective and relatively stable (the precise ratio varied slightly among tissues), Holman (1960) initiated the use of the triene:tetraene ratio to indicate inadequate EFA status. Diets high in n-3 fatty acids also lower the triene concentration (Morhauer and Holman, 1963; Rahm and Holman, 1964) because of the competition for desaturation mentioned above. Therefore the triene:tetraene ratio is a valid indicator of EFA status only when n-6 fatty acids are the principal unsaturated dietary fatty acids. Pudelkewicz et al. (1986) used this technique (in conjunction with dermatitis and growth) to estimate the linoleic acid requirement of growing female and male rats to be 0.5 percent and 1.3 percent of dietary ME, respectively.

The requirement for pregnancy is met by diets adequate for growth, while that for lactation is somewhat higher. Deuel et al. (1954) conducted two experiments to estimate these requirements. In the first experiment, 0, 10, 40, 100, 200, 400, and 1,000 mg cottonseed oil (approximately 50 percent linoleic acid) were fed daily. The number of litters born and the average number of pups per litter increased and then plateaued at 10 and 100 mg dietary cottonseed oil (e.g., 5 and 50 mg linoleate), respectively. When pure linoleate was fed as the fat source in a second experiment in which rats were provided with 0, 2.5, 5.0, 10, 20, 40, and 80 mg linoleate daily, the maximum litters born and average number of pups per litter increased and then plateaued at 2.5 and 20 mg linoleate, respectively. This higher amount of linoleic acid intake will be achieved if the pregnant rat consumes 18 g/day and the diet contains 0.25 percent of dietary ME as linoleic acid.

The requirement for lactation also can be estimated from these experiments. Indicators of successful lactation (the maximum average weight per pup in a litter at 21 days and minimum percent mortality between 3 and 21 days) reached a plateau between 100 to 200 mg cottonseed oil (50 to 100 mg linoleate) in the first experiment. In the second experiment, these criteria for successful lactation reached a maximum at 80 mg of linoleate. Because greater amounts of linoleate were not tested, it is not possible to determine whether the optimal requirement for lactation is more than 80 mg/day. In addition, the authors did not indicate the range of experimental error; therefore, a precise requirement cannot be established. Assuming a requirement of 100 mg linoleate/day, a lactating rat consuming 35 g diet/day will consume sufficient linoleate for lactation (100 mg/day) if the diet contains 0.68 percent of dietary ME as linoleate (approximately 0.30 percent of the diet).

Bourre et al. (1990) proposed that the n-6 fatty acid requirement be established as the percent of dietary linoleate that results in a constant concentration of tissue arachidonic acid. They found that constant concentrations of arachidonic acid were achieved at 150 mg (nerve tissue), 300 mg (testicle), 800 mg (kidney), and 1,200 mg (liver, lung, heart) linoleate/100 g diet. The minimal requirement then was expressed as 1,200 mg linoleic acid/100 g diet (or approximately 2.5 percent of dietary ME) because some tissues require a higher concentration of arachidonic acid to reach a plateau. Because the authors did not relate the arachidonic acid concentration plateau to any functional phenomena, the validity of this method of estimating the n-6 fatty acid requirement remains to be established.

n-3 Fatty Acids

The essentiality of the n-3 fatty acids has been equivocal until recently. It was initially demonstrated that the n-3 fatty acid, α-linolenic acid [18:3(n-3)], could substitute, in part, for the requirement of n-6 fatty acids (Greenberg et al., 1950). Tinoco et al. (1971) failed to show any change in growth of rats raised for three generations on diets lacking in n-3 fatty acids in comparison to litter mates fed diets containing 1.25 percent linoleate and 0.25 percent linolenate. However, the sequestering of n-3 fatty acids in specific tissues (retina, cerebral cortex, testis, sperm; Tinoco, 1982) and the tenacity with which they retain these fatty acids, despite the variation in dietary concentration (Crawford et al., 1976), led many researchers to speculate that n-3 fatty acids were required for some function in the body.

Bernsohn and Spitz (1974) fed rats lipid-free diets for 4 months and measured slightly decreased amounts (38 percent of control values) of monoamine oxidase and 5'-mononucleotidase in cerebral cortex that responded to dietary α-linolenic but not to linoleic acid. Retinal function may be negatively impacted in offspring of rats fed a low linolenate oil for two to three generations (Okuyama et al., 1987). Lamptey and Walker (1976) found reduced exploratory behavior in second generation rats fed safflower oil. A large percent of safflower oil is linoleic acid, and a very small percent is linolenic acid. This was confirmed in studies by Enslen et al. (1991) who showed a reduction in exploratory behavior in 16- to 18-week-old rats from dams fed safflower oil 6 weeks prior to mating and throughout gestation and lactation. These researchers found that when rats were switched to α-linolenic acid at weaning, they did not recover exploratory behavior, further suggesting a specific requirement for n-3 fatty acids during development.

Yamamoto et al. (1987, 1988) found a reduction in brightness discrimination learning in offspring from rats fed safflower oil through two generations in comparison to rats similarly fed perulla oil, a rich source of α-linolenic acid.

Bourre et al. (1989) measured impairment of nerve terminal Na⁺, K⁺ -ATPase activity, brain 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase in offspring from rats fed 1.8 percent sunflower oil in comparison to those from dams fed 1.9 percent soybean oil through two generations. They estimated the requirement for n-3 fatty acids as the least amount of α-linolenic acid in the diet that resulted in a higher concentration of brain n-3 fatty acids. Using this methodology, they estimated a requirement of 2 g/kg food (or 0.4 percent of the total dietary ME). These experiments indicate that n-3 fatty acids are required; further study is needed to determine the amount below which functional impairment occurs.

Although a requirement has not been defined, it may be advisable to include a source of n-3 fatty acids when dietary oils such as sunflower or safflower are fed through two or more generations. Homeostatic mechanisms appear to sequester n-3 fatty acids to protect the rat from n-3 deficiency during short-term dietary deprivation.

Calcium and Phosphorus

The dietary requirements for calcium and phosphorus are closely linked and depend on the availability of each mineral from the dietary source. In the 1978 edition of Nutrient Requirements of Laboratory Animals , the recommendation for the minimal concentration of calcium and phosphorus to maximize bone calcification during growth was 5 and 4 g/kg, respectively. This gives a Ca:P molar ratio of 0.96. However, Bernhart et al. (1969) had shown previously that adequate mineralization could be accomplished with smaller amounts of dietary calcium and phosphorus. They held the dietary molar ratio of Ca:P to 0.91 and noted that Sprague-Dawley rats attained maximum weight gain when fed diets containing as little as 1.6 g Ca/kg, but the rats required at least 3.5 g Ca/kg to attain maximum body accumulation of calcium. In the same experiment, the amount of dietary phosphorus required to maximize body weight was ³1.5 g/kg, and to maximize body phosphorus concentration it was ³3.5 g/kg. Several groups of investigators have observed normal growth and tissue concentrations of calcium and phosphorus in bones and other tissues of rats (RIVm:TOX and Sprague-Dawley strains) fed from 2.85 to 3.2 g P/kg diet (Kaup et al., 1991b; Shah and Belonje, 1991). Ritskes-Hoitinga et al. (1993) fed rats 2.0 and 4.0 g P/kg diet with 5.2 g Ca/kg diet and 0.6 g Mg/kg diet for three successive generations. They found that 2.0 g P/kg diet sustained reproduction but delayed bone mineralization in offspring. Kaup et al. (1991b) noted that growth and bone calcium concentrations in male Sprague-Dawley rats fed 2.8 g P/kg diet increased as dietary calcium concentrations increased from 2.1 to 3.4 g Ca/kg diet but were similar for rats fed 3.6 and 4.4 Ca/kg diet.

These studies suggest that dietary concentrations of calcium and phosphorus at 3.5 and 3.0 g/kg, respectively, with a molar ratio 0.9, would be sufficient. However, other studies have shown that a larger Ca:P ratio is required to prevent specific abnormalities in Sprague-Dawley rats. Draper et al. (1972), for example, showed that a molar ratio of 1.5 was better than 0.8 for the prevention of osteoporosis.

Variations in calcium and phosphorus intake have been associated with soft tissue calcification, especially nephrocalcinosis, in rats. However, a variety of other dietary factors can influence the development of nephrocalcinosis.

The recommendations for calcium and phosphorus intakes reflect the somewhat conflicting needs to maximize growth and maximize bone calcium and phosphorus concentrations without inducing nephrocalcinosis. A Ca:P molar ratio of 1.3 is needed to prevent nephrocalcinosis in female rats. However, a dietary phosphorus concentration of more than 2.0 g/kg diet is needed to prevent inadequate bone mineralization in successive generations. Therefore, the recommended concentrations of dietary calcium and phosphorus under normal conditions for growth and maintenance of nonlactating rats are 5.0 and 3.0 g/kg, respectively.

Considering the demands placed on the dams during lactation, it might be prudent to increase the amount of dietary calcium and phosphorus during this period. It has been estimated that a lactating dam will produce 70 mL of milk per day (Brommage, 1989). This amounts to approximately 200 mg calcium and 140 mg phosphorus transferred to milk in a 24-hour period. Brommage (1989) showed that this demand for calcium and phosphorus was compensated for by increases in food intake and dramatic increases in intestinal absorption of these minerals. Ritskes-Hoitinga et al. (1993) showed that 2 g P/kg diet, compared to 4 g P/kg, sustained reproductive performance but delayed growth and bone mineralization in successive generations of female rats. To help relieve some of the stress of lactation, it is recommended that the dietary calcium and phosphorus be increased by 25 percent (6.3 g Ca, 3.7 g P/kg diet) during this period. This could be especially helpful for those females used in continuous-breeding programs. It should be mentioned, however, that when lactating and nonlactating rats were given a choice of diets containing various concentrations of calcium and phosphorus, the lactating rats chose a diet that contained a Ca:P molar ratio of 2, whereas the nonlactating rats chose a diet that contained a ratio of 1.5 (Brommage and DeLuca, 1984).

Factors Affecting Calcium and Phosphorus Requirements Certain dietary factors can affect the biological availability of calcium and phosphorus and thus affect requirements for them in the diet. When these factors are present in the diet, appropriate adjustments to the dietary concentrations of calcium or phosphorus should be made. Low concentrations of vitamin D in the diet will reduce the absorption of calcium. Kaup et al. (1990) showed reduced phosphorus absorption in rats fed 10 g Ca/kg diet when compared to those fed only 2.5 g/kg. High dietary phosphorus will in turn reduce the apparent absorption of calcium (Schoenmakers et al., 1989) but may depend on the concentration of dietary magnesium (Bunce et al., 1965). Increasing the amount of fat in the diet from 5 to 20 percent reduced phosphorus absorption in older rats but not young rats (Kaup et al., 1990). High-fat diets also have been shown to decrease the absorption of calcium in mature rats but not young rats (Kane et al., 1949; Kaup et al., 1990). Calcium absorption is decreased in rats fed diets containing sources of oxalate (Weaver et al., 1987; Peterson et al., 1992), and phosphorus availability is reduced in diets containing phytate (inositol hexaphosphate) (Taylor, 1980; Moore et al., 1984). Some protein sources, including soybean protein isolates and other plant products, contain phytate. Phosphorus availability from these products should be considered when they are used in an animal's diet.

Other factors enhance calcium or phosphorus absorption. Bergstra et al. (1993) showed that the absorption of phosphorus was stimulated in rats fed diets high in fructose. Dietary disaccharides such as lactose and sucrose stimulate calcium absorption in rat intestine (Armbrecht and Wasserman, 1976). However, dietary lactose was better than sucrose in improving bone growth and development in intact vitamin D-deficient rats (Miller et al., 1988). Buchowski and Miller (1991) showed that 20 percent lactose added to diets containing a variety of calcium sources such as calcium carbonate, milk, and cheese significantly increased the amount of calcium in the tibia compared to rats fed diets without lactose. The enhancement was evident in 21-day-old rats fed the diets for 8 days but not in older rats.

Protein sources contain varying amounts of phosphorus, and the bioavailability of this phosphorus may not be the same in all sources. It is advisable, therefore, to analyze the source before using it in the diet and to be aware of whether the phosphorus is biologically available. Although few data are available in rats (Moore et al., 1984), data obtained for common feedstuffs in other species may be useful (National Research Council, 1988, 1994).

Signs of Calcium and Phosphorus Deficiency Boelter and Greenberg (1941, 1943) fed 0.1 g Ca/kg diet to young rats for 8 weeks. The rats exhibited growth retardation, decreased food consumption, increased basal metabolic rate, reduced activity and sensitivity, osteoporosis, rear leg paralysis, and internal hemorrhage. Males failed to mate; females did not lactate properly. Day and McCollum (1939) fed 0.17 g P/kg diet to young rats. The animals survived up to 9 weeks and exhibited lethargy, pain, and cessation of bone growth with massive losses of calcium in urine.

When Sprague-Dawley rats were fed diets with moderate restrictions in calcium (2.1 to 3.4 g Ca/kg diet) for 28 days, the rats grew normally but had reduced bone weight and bone calcium concentrations (Kaup et al., 1991b). Improved calcium absorption and reduced urinary calcium losses allowed the rats to compensate for these marginally low calcium intakes, but these mechanisms would not be sufficient to prevent growth retardation if lesser amounts of calcium had been fed.

Chloride

Voris and Thacker (1942) obtained a reduction of 25 percent in growth in a 10-week, paired-feeding comparison of rats fed 0.2 g Cl/kg diet as compared to 2.9 g Cl/kg diet. Picciano (1970) found no increase in weight gain of young rats fed 2 g Cl/kg compared to rats fed 0.5 g Cl/kg. Miller (1926) reported that 5 mg/day (0.17 to 0.25 g Cl/kg diet) was acceptable for reproduction and lactation. On the basis of these limited studies, the estimated requirement is 0.5 g Cl/kg diet, but future work may indicate this can be reduced.

Signs of Chloride Deficiency The rat tenaciously conserves its supply of tissue chloride by reducing drastically the urinary excretion within hours of consuming a diet deficient in the element. As a result, the signs of deficiency develop slowly. Rats fed 0.12 g Cl/kg diet exhibited poor growth, reduced efficiency of feed utilization, reduced blood chloride, reduced urinary chloride excretion, and increased blood CO₂ content (Greenberg and Cuthbertson, 1942). Rats fed 0.5 g Cl/kg diet for more than 70 days had reduced growth, reduced tissue chloride concentrations, and extensive kidney damage; but some rats survived for more than a year (Cuthbertson and Greenberg, 1945).

Signs of Chloride Toxicity Rats are also relatively insensitive to excess dietary chloride as judged by growth and tissue composition. Dahl (salt sensitive) and Sprague-Dawley rats fed excess chloride (15.6 to 26.6 g Cl/kg diet) as sodium or potassium chloride grew normally with unchanged chloride concentrations in kidneys and muscle (Whitescarver et al., 1986; Kaup et al., 1991a,c). However, the Sprague-Dawley rats fed 15.6 g Cl/kg had elevated blood pressure and enlarged kidneys (Kaup et al., 1991a,c). Dahl (salt sensitive) rats fed 4.86 g Cl/kg as NaCl also had elevated blood pressures, while rats fed a basal diet or a diet supplemented with NaHCO₃ did not (Kotchen et al., 1983).

Magnesium

Magnesium is required for numerous physiological functions in the rat. The amount required in the diet for adequate nutrition of the rat depends on numerous factors that affect availability of magnesium—the most important being the amount of dietary calcium, phosphorus, and vitamin D present. McAleese and Forbes (1961) found that a diet containing 0.1 g Mg/kg supported normal growth in weanling Sprague-Dawley rats. However, a diet containing 0.35 to 0.425 g/kg was required to maintain normal plasma magnesium concentrations of approximately 20 mg/L. More recently, Brink et al. (1991) found no significant difference in plasma magnesium between rats fed 0.4 and 0.6 g/kg diet; however, bone magnesium concentration was slightly (5 percent) but significantly higher in rats fed 0.6 g Mg/kg diet. On the other hand, rats fed only 0.2 g Mg/kg diet had significantly lower plasma and bone concentrations of magnesium than those fed 0.6 g/kg; 20 and 10 percent differences, respectively. Previously, Martindale and Heaton (1964) reported that 0.4 g Mg/kg was not sufficient to maintain normal bone and serum magnesium in adult Sprague-Dawley rats.

Other studies have shown questionable extremes for magnesium requirement. For example, Smith and Field (1963) estimated that a diet containing 0.05 g Mg/kg would replace endogenous losses in hooded Lister rats, but Clark and Belanger (1967) observed that a diet containing 2.5 g Mg/kg was needed for normal bone histology in Holtzman rats. These extremes may be the result of dietary factors that affect magnesium bioavailability. Brink et al. (1991) showed that magnesium absorption in rats fed diets with soybean protein was significantly less than in rats fed similar diets but with casein. Apparently this effect was caused by the presence of phytate in the soybean protein because when phytate was added to a casein diet, there was a similar reduction in absorption of magnesium. On the other hand, when lactose was added to the casein diet, magnesium absorption was enhanced.

Dietary calcium and phosphorus also affect magnesium requirement. O'Dell et al. (1960) showed that high-phosphorus diets enhanced the signs of magnesium deficiency in rats. Bunce et al. (1965) found that magnesium absorption was reduced by high dietary phosphorus. High dietary calcium also will depress magnesium absorption (O'Dell et al., 1960; Hardwick et al., 1987). Brink et al. (1992) have shown, however, that the effects of calcium and phosphorus on magnesium absorption is probably caused by the two minerals complexing magnesium in the gut lumen and rendering it insoluble. They showed that calcium was ineffective when phosphorus was not present and vice versa. [For an excellent review of factors affecting magnesium absorption, see Hardwick et al. (1991).]

Based on the review of these studies, and keeping the dietary calcium concentration at 5 g/kg and phosphorus at 3 g/kg, the dietary requirement for magnesium for growing and mature, nonpregnant rats is set at 0.5 g/kg diet. However, if diets contain factors, such as phytate, that might reduce the absorption of magnesium, a slightly higher dietary concentration might be required. In addition, because of the demands of lactation, an increase in dietary magnesium during this period is recommended. Wang et al. (1971) found that Sprague-Dawley rats were able to sustain gestation and lactation whether fed 0.8 g Mg/kg or 1.9 g Mg/kg diet.

Signs of Magnesium Deficiency Signs of magnesium deficiency in growing Sprague-Dawley rats include vasodilation, hyperirritability, cardiac arrhythmias, spasticity, and fatal clonic convulsions. Vasodilation occurred after about 1 week and often disappeared and reappeared spontaneously. Convulsions occurred between 21 and 30 days (Kunkel and Pearson, 1948; Ko et al., 1962). In Sprague-Dawley rats renal calcification was common and was detected within 2 days after initiating a markedly deficient diet (Reeves and Forbes, 1972).

Tufts and Greenberg (1938) reported that lactating females fed a deficient diet bred successfully but did not suckle their young. Hurley et al. (1976a,b) reported that magnesium-deficient Sprague-Dawley dams resorbed their fetuses or bore malformed pups; they suggested that the malformations resulted from a concomitant zinc deficiency.

Potassium

In two studies, rats grew normally when fed 1.7 to 1.8 g K/kg diet (Kornberg and Endicott, 1946; Grunert et al., 1950). Two other studies indicated that rats may require 5 to 6 g K/kg diet during lactation (Heppel and Schmidt, 1949; Nelson and Evans, 1961); however, diets designed by the American Institute of Nutrition (1977) that contain 3.6 g K/kg support adequate growth and reproduction. Studies to determine potassium requirements for the rat are limited. Until more extensive research has been done, the minimal requirement is estimated to be 3.6 g K/kg diet. The dietary concentration of potassium may be increased to 5 g/kg during lactation, but such an increase does not seem necessary based on available data.

The potassium requirement of different strains of rats may vary. Sato et al. (1991) showed that increasing the dietary potassium to 42 g/kg diet protected spontaneously hypertensive rats against elevation of blood pressure induced by the ingestion of 8 percent sodium chloride. However, Sprague-Dawley rats developed elevated blood pressure when fed 15.5 g Cl/kg diet either as sodium chloride or potassium chloride (Kaup et al., 1991a,b).

Signs of Potassium Deficiency Insufficient potassium markedly reduces appetite and growth. Animals become lethargic and comatose and may die within 3 weeks. They have an untidy appearance, cyanosis, short fur-like hair, diarrhea, distended abdomens with ascites, and are frequently hydrothoracic. Rats fed diets containing 1 g K/kg diet had a symmetrical loss of hair along the back and a 50 percent reduction in hair per follicular group (Robbins et al., 1965). Pathological lesions are widespread with potassium depletion (Schrader et al., 1937; Kornberg and Endicott, 1946; Newberne, 1964). The initial noninflammatory degeneration of myocardial fibers is followed by necrosis and cellular infiltration. Renal lesions include cast formation in proximal convoluted tubules, sloughing of tubular epithelium in the medulla, and accumulation of hyalin droplets in the epithelium of the collecting tubules.

Signs of Potassium Toxicity Pearson (1948) fed weanling rats 5 percent potassium in the diet as potassium bicarbonate, and after 3 weeks observed reduced growth rate. More than 60 percent mortality was observed when dietary magnesium was low and only 17 percent when magnesium was adequate. Drescher et al. (1958) showed electrocardiograph changes when potassium intake exceeded 10 mg/kg body weight. Potassium toxicity can cause hypertrophy of the adrenal zona glomerulosa, sodium depletion, and increased density of mitochondrial cristae in the kidney tubules (Hartroft and Sowa, 1964; Sealey et al., 1970; Pfaller et al., 1974).

Sodium

Grunert et al. (1950) estimated the sodium requirement of the rat to be 0.50 g Na/kg and to be independent of potassium intake. Forbes (1966) found that 0.48 g Na/kg diet was inadequate for maximum weight gain of weanling Sprague-Dawley rats over a 28-day period but that 2.20 g Na/kg gave maximum gains. Intermediate concentrations were not tested.

Pregnant Sprague-Dawley females fed low-sodium diets (0.3 g Na/kg diet) ate less food and showed languor and debility, particularly during the last week of pregnancy; however, reproduction was not seriously impaired (Kirksey and Pike, 1962). Ganguli et al. (1969a,b) suggested that the sodium requirement for gestation and lactation was 0.50 g Na/kg diet. The estimated requirement for growth, maintenance, gestation, and lactation is 0.5 g Na/kg diet.

Signs of Sodium Deficiency The classic sodium deficiency syndrome was described by Orent-Keiles et al. (1937). Rats fed a diet that contained 20 mg Na/kg diet exhibited growth retardation, corneal lesions, and soft bones. Males became infertile after 2 to 3 months, and sexual maturity was delayed in females. Death ensued in 4 to 6 months. At a concentration of 70 mg Na/kg diet, Kahlenberg et al. (1937) noted reduced appetite, poor growth, increased heat production, and reduced stores of energy, fat, and protein.

Signs of Sodium Toxicity Rats are relatively insensitive to excess sodium as indicated by growth and tissue composition. Sprague-Dawley rats fed 10.1 to 11.2 g Na/kg diet as chloride, sulfate, carbonate, and bicarbonate salts grew normally and had concentrations of sodium in bones and kidneys similar to rats fed a basal diet but had higher concentrations of sodium in bone than rats fed the potassium forms of these salts (Kaup et al., 1991a). However, ingestion of excess sodium as NaCl is associated with elevated blood pressure in several different rat strains, including Sprague-Dawley, Dahl (salt sensitive), Dahl (salt resistant treated with deoxycorticosterone acetate), and spontaneously hypertensive rats (SHR) (Kaup et al., 1991a,c; Tobian, 1991).

Copper

There is a general consensus that weanling and adult rats housed individually in wire-bottom stainless steel cages have a dietary copper requirement on the order of 5 to 6 mg/kg diet, the value used in the AIN-76A and AIN-93 diets. Johnson et al. (1993) fed weanling male Sprague-Dawley rats casein-starch-based purified diets containing copper concentrations ranging from 0.2 to 5.4 mg/kg for 5 weeks. They found that numerous functional measures of copper status (including platelet cytochrome c oxidase activity, serum ceruloplasmin activity, plasma copper, copper, and zinc-superoxide dismutase activity) were depressed in rats fed diets with copper concentrations at or below 3 mg/kg.

Studies by Klevay and Saari (1993) showed similar results. When weanling male rats were fed dietary copper ranging from 0.2 to 5.2 mg/kg diet for 5 weeks, the concentrations of copper in liver and heart and serum ceruloplasmin activity were depressed in rats fed copper concentrations at less than 4 mg/kg diet. However, Failla et al. (1988) could find no significant differences in copper status of Lewis rats fed an egg white-starch-based purified diet containing 2.1 or 7.0 mg Cu/kg diet. When similar rats were fed diets with sucrose instead of starch, indicators of copper status were significantly reduced at dietary concentrations up to 2.9 mg Cu/kg when compared to diets containing 7.1 mg Cu/kg. These studies suggest that the minimal dietary requirement for copper, to maintain adequate copper status in young growing rats of different strains and different dietary conditions, is more than 4 mg/kg diet.

Spoerl and Kirchgessner (1975a,b) reported that increasing dietary copper from 5 to 8 mg/kg during pregnancy and lactation resulted in an improvement in serum copper, serum ceruloplasmin activity, and liver copper concentrations in dams and their offspring. Cerklewski (1979) reported higher copper concentrations in milk on day 14 postpartum and in pup liver on day 21 postpartum when dams were fed diets containing 9 versus 6 mg Cu/kg diet during pregnancy and lactation. The functional significance of the higher tissue copper concentrations was not investigated.

Based on these data, a dietary copper concentration of 5 mg/kg diet is recommended for growth and maintenance for a variety of rat strains and under different dietary conditions. A dietary concentration of 8 mg Cu/kg diet is recommended for pregnancy and lactation. It should be noted that the requirement for growth and maintenance has not changed from that reported in the 1978 edition of this volume. However, the requirement recommended for pregnancy and lactation has increased from 5 to 8 mg Cu/kg diet. High dietary concentrations of zinc, cadmium, and ascorbic acid may increase the dietary requirement for copper (Davis and Mertz, 1987).

Signs of Copper Deficiency Copper deficiency develops rapidly in young rats and slowly in adult rats fed diets containing less than 1 mg Cu/kg. The copper deficiency develops more rapidly when fructose or sucrose is fed than when starch is fed, and males show a greater sensitivity to copper deficiency than females with respect to both the severity of signs and the duration of time until the onset of signs of deficiency (Fields et al., 1986; Koh, 1990; C.G. Lewis et al., 1990). Copper deficiency during early development can result in significant abnormalities in the cardiovascular, nervous, skeletal, reproductive, immune, and hematopoietic systems (Keen et al., 1982; Davis and Mertz, 1987). Weanling and adult rats fed copper-deficient diets can develop alternations in platelet function, impairments in both the acquired and innate arms of the immune system, altered exocrine pancreatic morphology and function, anemia (if dietary iron is marginal), alterations in thromboxane and prostaglandin synthesis, and impaired cardiovascular function (Cohen et al., 1985; Koller et al., 1987; Kramer et al., 1988; Dubick et al., 1989; Johnson and Dufault, 1989; Babu and Failla, 1990a,b; Allen et al., 1991; Medeiros et al., 1992; Saari, 1992). Weanling rats fed diets containing less than 2.7 mg Cu/kg show impaired neutrophil function within 5 weeks (Babu and Failla, 1990a).

Signs of Copper Toxicity Rats are particularly tolerant of high concentrations of dietary copper. Boyden et al. (1938) observed no adverse effects after feeding rats diets containing 500 mg Cu/kg, rats fed diets containing 2,000 mg/kg showed marked weight loss, and diets in excess of 4,000 mg/kg resulted in severe anorexia and starvation. Evidence of liver and kidney pathology in rats fed diets containing more than 1,000 mg/kg has been reported (Haywood, 1979; Czarnecki et al., 1984; Haywood, 1985).

Iodine

Iodine is regarded as essential for the rat. Utilization of dietary iodine is very high, and absorption occurs all along the gastrointestinal tract. It is concentrated by many tissues (Gross, 1962) but functions primarily as an integral part of the thyroid hormones. The few studies done to determine the iodine requirement generally agree that it is between 100 and 200 µg/kg diet (Levine et al., 1933; Remington and Remington, 1938; Halverson et al., 1945; Parker et al., 1951). Parker et al. (1951) found that 100 to 230 µg I/kg diet was satisfactory for reproduction; natural-ingredient diets that contained 330 µg/kg supported reproduction (Kellerman, 1934). The 1978 edition of this report set the iodine requirement at 150 µg/kg diet. Many commercially available natural-ingredient diets contain this amount and support adequate growth and reproductive performance in rats. There have been no recent studies to suggest that this concentration should be changed.

Signs of Iodine Deficiency The most obvious sign of iodine deficiency in the rat is enlargement of the thyroid glands with the formation of trabecular-type nodules (Taylor and Poulson, 1956). Iodine-deficient rats also had more coarse and less dense hair than controls. Iodine deficiency results in impaired reproduction (Feldmann, 1960). One biochemical sign of iodine deficiency is a decrease in serum concentrations of the thyroid hormone thyroxine (T₄) (Abrams and Larsen, 1973); another is a dramatic increase in the concentrations of serum thyrotropin (Pazos-Moura et al., 1991). An increase in the activity of liver type I iodothyronine deiodinase also occurs in iodine-deficient rats. Arthur et al. (1991) showed that the activity of this enzyme in rats fed less than 100 µg I/kg diet was 60 percent higher than in controls fed 1,000 µg/kg.

Signs of Iodine Toxicity The rat has a relatively high tolerance for dietary iodine. Adult female rats fed 500 to 2,000 mg/kg diet during pregnancy had increased neonatal mortality (Ammerman et al., 1964). However, when fed concentrations approaching 500 mg/kg diet, female rats had decreased milk production. The fertility of male rats fed as much as 2,500 mg I/kg diet for 200 days from birth was not affected.

Iron

For weanling and adult rats the iron requirement for growth and maintenance of maximum hemoglobin concentration is on the order of 35 mg/kg (McCall et al., 1962a; Ahlström and Jantti, 1969). For reproduction McCall et al. (1962b) reported that a diet containing 240 mg Fe/kg was adequate for maximum weight gain and hemoglobin concentrations. Ahlström and Jantti (1969) reported that considerably less iron was needed for reproduction: 28 mg Fe/kg for maximum hemoglobin concentration and 58 mg/kg for maximum iron stores in the offspring. Lin and Kirksey (1976) reported that growing, pregnant rats required between 10 and 50 mg Fe/kg diet for maximum hemoglobin concentration and between 50 and 250 mg/kg for maximum fetal liver iron stores. Shepard et al. (1980) reported that diets containing less than 10 mg/kg resulted in loss of embryos and fetuses. Kochanowski and Sherman (1983) have argued that although 35 mg Fe/kg diet is adequate to maximize maternal weight gain and hemoglobin concentrations during pregnancy and lactation, iron concentrations between 75 and 250 mg/kg diet are needed for optimal iron status at the end of lactation and between 150 and 250 mg Fe/kg are needed to maximize iron stores in the pups. Given the above, the recommended dietary iron concentration during pregnancy and lactation is 75 mg/kg diet.

Signs of Iron Deficiency In addition to anemia, iron-deficient rats can be characterized by multiple abnormalities including hyperlipidemia and low tissue carnitine concentrations (Bartholmey and Sherman, 1986), growth failure, elevated resting metabolic rate (Tobin and Beard, 1990; Borel et al., 1991), reduced exercise capacity (Willis et al., 1990), low milk folate concentrations (O'Connor et al., 1990), a compromised immune system including impaired phagocytosis and natural killer cell activity, and reduced antibody production (Hallquist et al., 1992; Kochanowski and Sherman, 1984, 1985).

Signs of Iron Toxicity The general term used to describe iron toxicity in animals is "iron overload." Chronic consumption of large amounts of dietary iron results in an accumulation of large quantities of iron in various cells and tissues, especially the liver. Wu et al. (1990) fed young (4 month old) and old (20 month old) rats 25 g elemental Fe/kg diet as finely powdered carbonyl iron and found detrimental effects on growth and maintenance of body weight. Within 2 weeks of feeding the high-iron diet, young rats had stopped gaining weight and old rats had lost 12 percent of their body weight. Weight of the old rats continued to decrease for up to 10 weeks of feeding. Large increases in the concentrations of iron in liver and spleen were seen in both age groups. Reductions in concentrations of serum, liver, and heart copper were also observed in the iron-overloaded rats. Britton et al. (1991) observed similar results in rats fed 30 g Fe/kg diet. These large amounts of tissue iron result in lipid peroxidation and cellular damage (Houglum et al., 1990; Wu et al., 1990).

Manganese

There is a paucity of studies that address manganese requirements in rats. Holtkamp and Hill (1950) reported that the optimal manganese intake for growth is between 2 and 5 mg/kg diet; when dietary manganese concentration was increased to 40 mg/kg, the average weight gain was less than in the group fed 5 mg/kg diet. In contrast, Anderson and Parker (1955) reported a faster growth rate in weanling rats fed 50 mg Mn/kg compared to rats fed 5 mg/kg. The manganese requirement for reproduction has not been firmly established. Diets containing 1 mg Mn/kg are inadequate for normal reproduction; litters from dams fed this concentration of manganese are characterized by ataxia (due to inner ear defects), skeletal defects, and a high incidence of early postnatal death (Hurley and Keen, 1987). Litters from dams fed diets containing 3 mg Mn/kg have normal survival and growth rates; however, depending on the strain, they can still be characterized by a high incidence of ataxia (Baly et al., 1986; Hurley and Keen, 1987). An increased incidence of ataxia was not observed in litters from Sprague-Dawley dams fed diets containing 5 mg Mn/kg (C. L. Keen, University of California, Davis, personal communication, 1992). The above data suggest that a dietary concentration of 5 mg Mn/kg is probably adequate for normal growth and development. However, because there is a significant difference in how different strains respond to dietary manganese intake (Hurley and Bell, 1974; Kawano et al., 1987), the requirement is set at 10 mg/kg. It should be noted that this is lower than the recommendation of 50 mg/kg (National Research Council, 1978); however, given the lack of data supporting the need for such a high concentration of manganese in the diet, coupled with the possible negative effects of excess manganese on iron metabolism (Davis et al., 1990), reduction in the manganese requirement is warranted.

High concentrations of dietary iron, calcium, phosphorus, and copper have been reported to increase the requirement for dietary manganese (Hurley and Keen, 1987; Johnson and Korynta, 1992).

Signs of Manganese Deficiency Diets containing less than 1 mg Mn/kg can result in reduced food consumption, poor growth, bone abnormalities, and early mortality. Reproduction can be impaired and is characterized by testicular degeneration in the male and by a delay in the opening of the vaginal orifice and defective ovulation in the female. If reproduction occurs, litters are characterized by ataxia, skeletal defects, marked abnormalities in glucose and lipid metabolism, and a high frequency of early postnatal death (Hurley and Keen, 1987). Manganese deficiency in weanling and adult rats can result in significant alterations in pancreatic exocrine and endocrine functions (Baly et al., 1985; Chang et al., 1990), impaired glucose transport and metabolism in adipose cells (Baly et al., 1990), an increase in tissue lipid peroxidation (Zidenberg-Cherr et al., 1983), abnormal lipoprotein metabolism (Davis et al., 1990; Kawano et al., 1987), decreased hepatic arginase activity (Brock et al., 1994), and marked inhibitions of osteoblast and osteoclast activities resulting in severe bone disease (Strause et al., 1987).

Signs of Manganese Toxicity The postnatal growth of rats is unaffected by dietary manganese intakes as high as 1,000 to 2,000 mg/kg diet, provided dietary iron is adequate. If dietary iron is low (820 mg/kg), dietary manganese concentrations in excess of 1,000 mg/kg can result in reduced weight gain and iron deficiency (Rehnberg et al., 1982). Diets in excess of 3,500 mg Mn/kg can result in severe growth retardation and mortality. Reproductive dysfunction resulting from long-term intake of excess manganese (>1,050 mg/kg) has been reported for both males and females (Laskey et al., 1982). Although the concentrations of dietary manganese needed for overt toxicity are quite high, weanling rats given water containing 55 µg Mn/mL for 3 weeks were reported to have reduced rates of brain RNA and protein synthesis (Magour et al., 1983). The mechanisms underlying the cellular toxicity of manganese have not been clearly identified but may involve manganese-initiated oxidative damage, disturbances in carbohydrate metabolism, and altered intracellular iron metabolism (Keen and Hurley, 1989).

Molybdenum

Molybdenum metabolism was reviewed by Mills and Davis (1989). Similar criteria used to establish selenium as an essential nutrient also can be used to establish the essentiality of molybdenum. Molybdenum is a cofactor for three known enzymes in the rat—xanthine oxidase/dehydrogenase (XDH), aldehyde oxidase (AO), and sulfite oxidase (SOX) (Rajagopalan, 1988). These enzymes catalyze redox reactions. When rats are fed diets with very low concentrations of molybdenum, activities of these enzymes in various tissues are depressed. However, it has not been demonstrated that this is detrimental to the animal. On the other hand, if rats are fed tungsten, an antagonist to molybdenum, the activities of molybdenum-dependent enzymes are scarcely measurable and signs of deficiency then become apparent. Genetic deficiencies of sulfite oxidase in humans have been shown to result in numerous pathologies (Mudd et al., 1967; Johnson et al., 1980; Abumrad et al., 1981).

Early studies used the effect of low dietary molybdenum on liver and intestinal XDH activities to establish the requirement for molybdenum. Studies by Higgings et al. (1956) concluded that 20 µg Mo/kg diet was sufficient to maintain normal growth and reproduction. Xanthine oxidase activity was impaired, however. Titration experiments showed that about 100 µg Mo/kg diet was required to maximize intestinal XDH activity. More recent studies showed that not all criteria affected by dietary molybdenum are maximized by the same concentrations of molybdenum. Wang et al. (1992) showed that 25 µg/kg diet satisfied growth requirements in female rats and that 50 µg/kg diet was the minimal concentration required to maintain maximum liver XDH and SOX activity as well as spleen and kidney molybdenum concentrations. It took 100 µg Mo/kg diet to maximize XDH activity in the intestine and 200 µg Mo/kg diet to maximize liver and brain concentrations. However, it may not be valid to use tissue concentra tion of a trace element as a criterion to establish requirements. In many instances, the element accumulates in the tissue above a certain concentration but does not have a physiological function. Based on these studies, the requirement for molybdenum is estimated to be about 150 µg/kg diet. There is a strong interaction among molybdenum, copper, and sulfur; thus the dietary requirement of molybdenum might depend on the amount of copper and sulfur in the diet.

Signs of Molybdenum Deficiency Outward signs of deficiency are difficult to produce when rats are fed purified diets with only molybdenum absent. Even when an antagonist of molybdenum, tungsten, was fed at a ratio of 2,000:1 (tungsten:molybdenum) there were no detrimental effects on weight gain, but the animals had very low activity concentrations of molybdenum-dependent enzymes in liver.

Signs of Molybdenum Toxicity The occurrence of signs of molybdenum toxicity when rats and other species are fed high concentrations depends on the amount of copper and sulfate in the diet. Gray and Daniel (1964) showed that as little as 10 mg Mo/kg diet caused weight loss in copper-deprived rats. This condition could be ameliorated with the addition of 3 mg Cu/kg diet. Miller et al. (1956) found that the reduction in growth rate of rats fed 100 mg Mo/kg diet could be prevented by sulfate supplementation. Molybdenum toxicity also causes elevated liver copper, decreased serum ceruloplasmin, and increased tissue concentrations of molybdenum. Most of these signs can be reversed by supplementing the diet with copper and/or sulfate.

Selenium

Selenium is found in living organisms as an integral part of selenoproteins (Sunde, 1990) in the form of selenomethionine or selenocystine. There are also selenium binding proteins. A number of important selenoenzymes have been discovered in mammalian systems—glutathione peroxidase (GSH-Px; Rotruck et al., 1973), selenoprotein P (⁷⁵SeP; Burk and Gregory, 1982), phospholipid hydroperoxide GSH-Px (Ursini et al., 1985), and hepatic type I iodothyronine 5'-deiodinase (ITD-I; Berry et al., 1991). GSH-Px is found in most tissues and cells and catabolizes hydrogen peroxide and other free and membrane associated hydro- and phospholipid peroxides. ITD-I is found in liver, kidney, and thyroid and catalyzes the generation of 3,5,3'-triiodothyronine (T₃), the metabolically active thyroid hormone, from T₄, the main circulating thyroid hormone. Other functions for selenium are probable but have not been adequately defined (Beckett et al., 1989; Kim et al., 1991). Since the 1978 edition of Nutrient Requirements of Laboratory Animals (National Research Council, 1978), there have been many excellent reviews on selenium metabolism (Burk, 1983; National Research Council, 1983; Combs and Combs, 1984; Levander and Burk, 1990; Sunde, 1990).

Dietary sources of selenium can be of two forms—inorganic, represented by selenite or selenate, and organic, represented by selenomethionine or selenocystine. Because of the complex metabolic fate of these different forms, and because of the influence of other possible antioxidants in the diet, it is difficult to establish an exact dietary requirement for selenium. Selenium is more readily transported across the intestinal cells as selenate than as selenite. Selenium from selenomethionine is more readily transported than either selenate or selenite (Vendeland et al., 1992).

Various criteria have been used to assess the requirement for selenium. Schwarz and Foltz (1957) showed that 40 µg Se/kg diet was required to prevent nutritional liver necrosis in rats that were also deficient in vitamin E. Hafeman et al. (1974) found that 50 µg Se/kg diet permitted maximum growth in rats, but 100 µg/kg was required to maintain maximum tissue activity of GSH-Px. More recent studies (Arthur et al., 1990) showed that less than 5 µg Se/kg diet permitted growth not different from that found when 100 µg Se/kg diet was fed. However, liver and plasma GSH-Px activities decreased significantly after only 2 weeks on low-selenium diets. The vitamin E content of these diets, supplied as α-tocopheryl acetate, was 200 mg/kg.

Yang et al. (1989) used the concentration of selenoprotein P (SeP) (Yang et al., 1987) in plasma and GSH-Px activities in plasma and liver to assess selenium nutriture in rats. Weanling rats were fed diets supplemented with selenium as sodium selenate ranging from 10 to 2,000 µg/kg diet. The SeP concentrations in plasma reached a plateau between 100 and 500 µg Se/kg of diet but they continued to rise when 2,000 µg Se/kg was fed. GSH-Px activities in plasma and liver were not maximized until dietary selenium had reached 500 µg/kg diet.

Whanger and Butler (1988) fed rats selenium, from 20 to 4,000 µg/kg, as sodium selenite. GSH-Px activities for numerous tissues, except red blood cells, were maximized at 200 µg Se/kg diet. Pence (1991) fed rats diets with selenium concentrations of 20, 120, and 520 µg/kg; Pence found that selenium-dependent GSH-Px as well as total GSH-Px activities in liver and colon of rats fed 120 µg/kg were only about 50 percent of the activities in those fed 520 µg/kg. L'Abbé et al. (1991) found that liver GSH-Px activity was about 20 percent higher in rats fed 1,000 µg Se/kg for 25 weeks than in those fed 100 µg/kg, but the difference was not significant. This may suggest, however, that 100 µg/kg diet is close to the minimum needed for maximum activity of GSH-Px and that more than 100 µg/kg would be optimal. In this regard Eckhert et al. (1993) found evidence that the microvasculature of rats may have a unique requirement for selenium. They fed male rats diets high in sucrose to induce an elevation in blood triglycerides and cholesterol—a feeding regimen used by Lockwood and Eckhert (1992) to cause insult to the microvascular system. To this diet 100 or 200 µg Se/kg was added. The results showed that dietary selenium concentration had no effect on GSH-Px activity in the erythrocytes; however, there was a marked effect on the microvasculature of the retinae. In two different experiments there were twofold increases in the number of acellular segments and in the number of vessels over the optic nerve head in rats fed 100 µg Se/kg diet compared to those fed 200 µg Se/kg diet. In addition, the inner retinal pericyte:endothelial cell ratio of the vessels was increased in rats fed the higher concentration of selenium. These authors interpreted these results to suggest that the higher concentration of dietary selenium protected the retinal microvasculature, particularly the pericyte cells, from sucrose-induced metabolic insult. These data suggest that a minimal dietary requirement for selenium is more than 100 µg/kg.

It has been suggested that GSH-Px activity might be the best criterion for establishing a dietary requirement for selenium. However, recent investigations by Sunde et al. (1992) and Evenson et al. (1992) showed that the concentrations of GSH-Px-mRNA and ⁷⁵Se incorporation into selenoproteins may also be used. They fed selenium (as selenite) in concentrations ranging from 7 to 200 µg/kg diet in a titration scheme to determine the amount of dietary selenium required to maximize GSH-Px activity, GSH-Px mRNA concentration, and ⁷⁵Se incorporation into liver selenoproteins of growing rats. In all three cases, 100 µg/kg was the minimal amount required. Vadhanavikit and Ganther (1993) used liver and thyroidal 5'-deiodinase (type I) activities as well as GSH-Px activities to determine selenium requirements for the rat. They fed rats diets containing 10, 50, 100, and 500 µg Se/kg for 20 weeks. Liver GSH-Px activity was significantly decreased in rats fed 10, 50, and 100 µg Se/kg than in those fed 500 µg Se/kg; however, liver 5'-deiodinase activity was significantly decreased (90 percent) only in rats fed 10 µg Se/kg. GSH-Px activity in the thyroid was decreased in rats fed 10 µg Se/kg but not in those fed other concentrations. Thyroidal 5'-deiodinase activity was not significantly affected even at the lowest concentration of dietary selenium.

Therefore, considering all the criteria mentioned for the establishment of the selenium requirement, it is suggested that 150 µg Se/kg diet is the minimal requirement for the growing rat. This concentration may also be used for maintenance.

Other investigators have provided evidence which suggests that the minimal selenium requirement in the form of selenite for pregnant and lactating rats might be higher than 150 µg/kg diet (Smith and Picciano, 1986, 1987). In one experiment, Smith and Picciano (1986) raised dams through pregnancy and lactation on four concentrations of dietary selenium: 25, 50, 100, and 200 µg/kg. At 15 days of gestation, erythrocyte selenium concentrations and GSH-Px activities of the dams were not different among the three highest concentrations of dietary selenium. At day 18 of lactation, erythrocyte GSH-Px activity was significantly higher in dams fed 200 µg Se/kg compared to those fed 100 µg Se/kg diet. At this period, selenium concentration and GSH-Px activity in the liver of nonpregnant controls was not different among the three highest concentrations of selenium. In the lactating females, however, liver selenium concentration and GSH-Px activity were different among all groups. The highest selenium concentrations and GSH-Px activities were in those lactating rats fed 200 µg Se/kg; however, this value was not as high as that from nonpregnant controls fed the same amount. GSH-Px activity in the liver of 18-day-old pups was 1.7 times greater when dams were fed 200 µg Se/kg diet than when they were fed 100 µg/kg diet.

Smith and Picciano (1987) also found that the form of dietary selenium could influence selenium bioavailability and dietary requirement. A concentration of 250 µg Se/kg diet as selenomethionine resulted in maximum GSH-Px activity in the tissues of both dams and pups. However, when selenium was supplied as sodium selenite, 500 µg Se/kg was necessary. Studies by Lane et al. (1991) showed that the livers of 14-day-old pups fed 150 µg Se/kg diet as selenomethionine had twice as much GSH-Px activity as livers from similar rats fed selenite. GSH-Px activity in the livers of dams was not different between the two sources. Whanger and Butler (1988) and Vendeland et al. (1992) also showed that selenium from selenomethionine was more available to rats than that from sodium selenite. Based on these criteria, it appears that when selenium is supplied as selenite, the minimal requirement during pregnancy and lactation is at least 400 µg Se/kg diet. If the dietary source of selenium is selenate or selenomethionine, the requirement could be less.

Signs of Selenium Deficiency Outward signs of selenium deficiency are difficult to produce in rats fed diets adequate in vitamin E. However, one report (McCoy and Weswig, 1969) demonstrated selenium deficiency signs in rats fed Torula yeast diets with adequate vitamin E. These signs included poor growth, sparse-hair coats, cataracts, and reproductive failure when the diets were fed for two generations. Caution should be exercised when interpreting these results, however. Male rats fed the Torula yeast diet with added selenium only gained about two-thirds as much as rats fed a commercial natural-ingredient diet during the first generation and less than one-half as much during the second. This indicates that the diet may have been lacking in other essential nutrients. Biochemical signs of selenium deficiency include the reduction of selenium-dependent enzyme activities in various tissues and the reduction of T4 deiodination in liver.

Signs of Selenium Toxicity Studies by Harr et al. (1967) and Tinsley et al. (1967) showed that 4,000 to 16,000 µg Se/kg diet caused ascites, edema, and poor hair quality in rats fed purified diets for long periods. Many of the animals did not live beyond 100 days. Liver toxicity and hyperplastic hepatocytes were found in rats receiving selenium as selenite or selenate supplemented at 500 to 2,000 µg/kg diet for 30 months.

Zinc

Weanling and adult rats housed individually in wire-bottom stainless steel cages have a dietary zinc requirement on the order of 12 mg/kg when egg white or casein is used as the primary protein source (Williams and Mills, 1970; Pallauf and Kirchgessner, 1971; Wallwork et al., 1981). The requirement is higher (18 mg/kg) when soybean protein is used. The increased requirement for zinc with soybean protein-based diets is primarily attributed to the phytic acid content of these diets (Berger and Schneeman, 1988).

For optimal growth and survival of the neonate, the dietary requirement for zinc for the pregnant and lactating rat has been estimated to be on the order of 25 mg/kg, even when a high-quality protein such as egg white is used (Rogers et al., 1984). An increased requirement for dietary zinc during pregnancy is also supported by the observation of Fosmire et al. (1977) that, at term, fetal weight and fetal zinc concentrations were higher in litters from dams given water containing 25 mg Zn/L compared to dams given water containing 11 mg Zn/L. Given the above, the recommended concentration of dietary zinc for pregnant and lactating dams is 25 mg Zn/kg diet.

The dietary requirement for zinc can be significantly influenced by an animal's housing conditions. It should be noted that high concentrations of dietary cadmium, iron, phosphorus, and tin have been reported to increase the requirement for dietary zinc (Hambidge et al., 1986; Johnson and Greger, 1984; Sandstrom and Lönnerdal, 1989).

Signs of Zinc Deficiency The pathologic signs of zinc deficiency depend on the length and severity of the deficiency, the age and sex of the animal, and environmental surroundings. An inadequate intake of zinc can be reflected by marked reductions in plasma zinc concentrations within 24 hours and by mild-to-severe anorexia within 3 days (Hambidge et al., 1986). Prolonged consumption of a zinc-deficient diet can result in continued anorexia, growth retardation/failure, abnormalities in platelet aggregation and hemostasis, alopecia, thickening of the epidermis, increased rates of cell membrane lipid peroxidation, hyperirritability, significant impairment of multiple components of the immune system, and alterations in lipid, carbohydrate, and protein metabolism (Hambidge et al., 1986; Hammermueller et al., 1987; Emery et al., 1990; Keen and Gershwin, 1990; O'Dell and Emery, 1991; Avery and Bettger, 1992). Esophageal lesions can occur in weanling rats within 7 days of the introduction of a zinc-deficient diet (Diamond et al., 1971). For weanling males, the prolonged consumption of a diet containing less than 0.5 mg Zn/kg can result in arrested spermatogenesis, atrophy of the germinal epithelium, and impaired growth of accessory sex organs (Diamond et al., 1971; Hambidge et al., 1986). Zinc deficiency in females results in a disruption of the estrous cycle, a reduced frequency of mating, and a low implantation rate if mating occurs.

The consumption of a zinc-deficient diet after mating can result in severe embryonic and fetal pathologies including prenatal death; a high incidence of central nervous system, soft tissue, and skeletal system defects; and abnormal biochemical development of the lung and pancreas. Zinc-deficient dams are characterized by severe parturition difficulties and the offspring are characterized by lower-than-normal growth rates, a high incidence of early postnatal death, and behavioral abnormalities (Apgar, 1985; Bunce, 1989; Keen and Hurley, 1989).

Signs of Zinc Toxicity Zinc is often considered to be relatively nontoxic, however, dietary zinc concentrations in excess of 250 mg/kg can induce a copper deficiency if dietary copper is low or marginal (L'Abbé and Fischer, 1984; Keen et al., 1985); and zinc in excess of 5,000 mg/kg diet can result in reduced growth rates, anorexia, anemia, and death even when dietary copper is considered adequate (Hambidge et al., 1986). It should be noted that investigators often use control diets that contain 100 mg Zn/kg. Although this amount of zinc is not thought to represent a "toxic" risk to rats, it is high enough that it may prevent the detection of important nutrient and/or drug-zinc interactions. It may be more accurate to classify 100 mg Zn/kg diet as a zinc-supplemented diet.

Phosphorus and Calcium

Rats (Sprague-Dawley, Wistar, RIVm:TOX, and Zucker; both males and females) fed purified diets with more than 5.0 g P/kg diet have, in a number of studies, been found to have elevated concentrations of calcium in their kidneys (Hitchman et al., 1979; Schaafsma and Visser, 1980; Woodard and Jee, 1984; Greger et al., 1987a; Ritskes-Hoitinga et al., 1989; Van Camp et al., 1990). Kidney calcification has also been noted in female rats (RIVm:TOX, Sprague-Dawley, and Wistar) fed 4.0 g P/kg diet (Shah et al., 1980; Mars et al., 1988; Schoenmakers et al., 1989; Henskens et al., 1991). For example, Schoenmakers et al. (1989) observed that 5-week-old female RIVm:TOX rats fed diets containing 4.3 g P/kg with 4.8 g Ca/kg and 0.4 Mg/kg accumulated 25-fold more calcium in their kidneys than rats fed 1.1, 1.9, or 2.8 g P/kg diet. Ritskes-Hoitinga et al. (1993) did long-term studies with three successive generations of rats fed casein-based purified diets containing 5.2 g Ca/kg, 0.6 g Mg/kg, and either 2.0 or 4.0 g P/kg diet. After 4 weeks of age, from 50 to 100 percent of the females fed the diet containing 4 g P/kg developed nephrocalcinosis, while only 2 of 54 rats fed 2.0 g P/kg had measurable nephrocalcinosis. Male rats were not affected.

The amount of calcium consumed and the ratio of dietary calcium to phosphorus are also important. Woodard and Jee (1984) found that ingestion of additional calcium (5.5 versus 3.5 g Ca/kg diet) by Sprague-Dawley rats fed moderately high concentrations of phosphorus (>5.5 g P/kg diet) increased the deposition of calcium in the kidneys. In contrast, Schaafsma and Visser (1980) observed that Zucker rats fed diets with low concentrations of calcium (2.0 g Ca/kg diet) were more sensitive to phosphorus-induced nephrocalcinosis than rats fed 6.0 g Ca/kg diet. Moreover, Hoek et al. (1988) observed that kidney calcium accumulation in RIVm:TOX rats dropped dramatically when dietary calcium was increased to 7.5 g Ca/kg diet. This may reflect the ratio of dietary calcium to phosphorus.

Investigators have noted that a dietary calcium:phosphorus molar ratio below 1.3 is associated with nephrocalcinosis in RIVm:TOX and Wistar strains of rats (Hitchman et al., 1979; Hoek et al., 1988). Ritskes-Hoitinga et al. (1991) compared the responses of Wistar rats to 10 commercial diets and found that the dietary ratios of calcium to phosphorus were inversely correlated to the degree of nephrocalcinosis, as determined by histological score. Reeves et al. (1993a) fed rats purified diets similar to the AIN-93G diet (Reeves et al., 1993b) but with varying amounts of calcium and phosphorus: 3.3, 5.9, and 6.7 g Ca/kg diet and 2.0, 3.0, and 4.0 g P/kg diet. The Ca:P molar ratio in all diets was 1.3. They found that the concentrations of calcium and phosphorus in the tibia of both male and female rats were similar to those in rats fed a commercial natural-ingredient diet that contained higher concentrations of calcium and phosphorus. In addition, there were no indications of nephrocalcinosis in female rats after they consumed these diets for 16 weeks.

Magnesium

Nephrocalcinosis is a sign of magnesium deficiency in laboratory rats. Several investigators have found that the addition of supplemental magnesium (above required or recommended amounts) reduced the accumulation of calcium in kidneys of rats (Sprague-Dawley and Wistar strains) fed higher concentrations of calcium and/or phosphorus (Goulding and Malthus, 1969; Shah et al., 1980; Ericsson et al., 1986; Shah et al., 1986). Although ingestion of generous amounts of phosphorus and/or calcium have been found to depress magnesium absorption (Greger et al., 1987b; Hoek et al., 1988) and sometimes serum magnesium concentrations (Ericsson et al., 1986) of rats (RIVm:TOX and Sprague-Dawley strains), kidney magnesium concentrations were not reduced. This suggests that the rats were not magnesium-deficient per se.

Protein

The ingestion of additional (25 or 30 percent versus 15 percent) protein was found to prevent phosphorus-induced nephrocalcinosis in rats in several studies (Hitchman et al., 1979; Van Camp et al., 1990). Similarly, Shah et al. (1986) observed that Sprague-Dawley rats fed purified diets containing recommended concentrations of phosphorus had less calcium deposited in their kidneys when the protein content of the diets was increased from 10 to 15 percent casein.

The substitution of lactalbumin for casein in semipurified diets, even if dietary phosphorus amounts are similar, also is associated with less accumulation of calcium in kidneys of Sprague-Dawley rats (Greger et al., 1987a,b). Zhang and Beynen (1992) found that an increased intake of protein, provided in the diet by soybean isolate or casein, reduced the incidence of calcinosis in female rats; however, protein from fish meal did not. They concluded that the antinephrocalcinogenic effect of the soybean protein was related to lower urinary phosphorus, and the effect of casein was the result of lower urine pH and elevated urinary magnesium.

Other Dietary Factors

Bergstra et al. (1993) showed that dietary fructose, as opposed to glucose, stimulated nephrocalcinosis in female rats. This was related to fructose stimulating greater concentrations of urinary phosphorus and magnesium and lowering the pH.

Levine et al. (1974) also found that chloride depletion stimulated nephrocalcinosis in Charles River rats but only in the presence of increased dietary phosphate or sulfate. Kootstra et al. (1991) observed that supplementing purified diets that contained 6 mg P/kg with ammonium chloride, but not ammonium sulfate, reduced the accumulation of calcium in the kidneys of female Wistar rats. Supplementation of diets with fluoride has also been observed to decrease the accumulation of calcium in the kidneys of Sprague-Dawley rats in several studies (Shah et al., 1980; Ericsson et al., 1986; Shah et al., 1986; Cerklewski, 1987).

Vitamin A

Vitamin A is essential for many critical functions of the body such as vision, which requires 11-cis-retinaldehyde bound to the photoreceptor pigments. Many cellular differentiation processes are mediated by all-trans-retinoic acid and 9-cis-retinoic acid bound to their respective nuclear receptors, RAR and RXR (Zelent et al., 1989; Mangelsdorf et al., 1992). 14-Hydroxy-4,14-retro-retinol also has been shown to be involved in signal transduction in B lymphocytes (Buck et al., 1991).

Retinol, the retinyl esters, and β-carotene are the main dietary compounds present in diets with vitamin A activity. Several plant carotenoids are precursor forms of vitamin A, and β-carotene is the most active carotenoid. The concentration of dietary intake influences the biopotency of β-carotene (Brubacher and Weiser, 1985) as shown in Table 2-10. β-Carotene is transformed to retinol in the intestinal mucosa. The retinol, irrespective of its source, is esterified primarily with palmitate or stearate. The esters are transported to the parenchymal cells of the liver as components of the chylomicrons. The esters are either hydrolyzed and transported out of the liver to the target tissues in combination with a specific transport protein, retinol-binding protein, or they may be transferred to the stellate cells of the liver for storage. Vitamin A can be stored in the liver in large amounts.

TABLE 2-10

Equivalence of β-Carotene and Retinol at Different Concentrations.

Rats are born with very low liver stores of vitamin A. As a consequence the vitamin A requirement in weanling rats varies according to the criteria used, overt signs of deficiency (e.g., epithelial keratinization), hepatic storage, or retinol kinetics. Maximum blood concentrations (about 2 µmol/L) were reached when liver deposition was moderate (140 µmol/kg liver) in Holtzman rats (Muto et al., 1972). An elevation in the cerebrospinal fluid pressure occurred when the serum retinol concentration dropped below 0.35 µmol/L in Sprague-Dawley rats (Corey and Hayes, 1972). Some of the different criteria for vitamin A requirements for repletion of deficient animals are presented in Table 2-11.

TABLE 2-11

Vitamin A Repletion of Vitamin A-Deficient Rats.

Guilbert et al. (1940) demonstrated that the need for vitamin A was related to body weight rather than energy intake. This concept is consistent with the vitamin's activity in maintaining integrity of the epithelia, which quantitatively directly correlate with body mass (Mitchell, 1950).

Takahashi et al. (1975) found that 56 nmol retinyl acetate/kg BW/day was sufficient to support gestation in Holzman rats with the delivery of pups of normal weight and with normal brain, liver, and kidney size. However, the mean number of live neonates was reduced to 4.8 compared to 9.5 for the controls, which received 1,400 nmol/kg BW/day. Sixty-three percent of the dams fed the marginal concentration of vitamin A delivered as compared to 100 percent of the dams fed the very high concentration. These low concentrations that supported gestation would not support optimal lactation, however. Davila et al. (1985) found that nursing Sprague-Dawley dams fed 2.1 µmol retinyl acetate/kg diet were healthy and their pups grew as well as the pups of dams fed 52 µmol/kg diet but had lower liver retinyl ester stores.

Vitamin A requirements are sensitive to other nutritional influences. Consumption of protein-deficient diets decreases the serum concentrations of vitamin A and its transport protein, retinol-binding protein (Peterson et al., 1974). Rates of depletion of liver and kidney reserves of vitamin A were linearly related to growth rate, which changed as dietary casein concentration varied from 0 to 18 percent (Rechcigl et al., 1962). In zinc-deficient rats, mobilization of vitamin A from the liver declined (Smith et al., 1973), but this effect was not confirmed by Apgar (1977). Vitamin E deprivation resulted in depletion of liver stores of vitamin A (Moore, 1957).

Age does not appear to have any significant effect on vitamin A requirement other than that related to differences in body weight. Suckling, young adult, and aged rats absorb retinol with about the same efficiency (Hollander and Morgan, 1979; Said et al., 1988).

Green and co-workers (1987) developed a computer model to describe the kinetics of vitamin A metabolism in male Sprague-Dawley rats and have studied the effects of vitamin A intake on vitamin A excretion. With diets containing 2.1 µmol/kg diet or less, the disposal was equal to the vitamin A intake. However, when the diets contained 2.4 µmol/kg diet the disposal rate was essentially the same as observed in the rats receiving 2.1 µmol/kg diet (Green and Green, 1991). The rats had a modest (»3.5 nmol/day) accumulation of vitamin A in the liver when they were fed 2.4 µmol/kg diet.

The estimated requirement for vitamin A, based on the kinetic studies of Green et al. (1987) and Green and Green (1991) and on the lactation study of Davila et al. (1985) is 2.4 µmol/kg diet (equivalent to 2,300 IU/kg) if retinol or retinyl esters are used. This requirement may be met by retinol at 0.7 mg/kg diet, retinyl acetate at 0.8 mg/kg diet, or retinyl palmitate at 1.3 mg/kg diet. At this low concentration β-carotene is used relatively efficiently, so the requirement would also be met by β-carotene at 12.4 µmol/kg diet (1.3 mg/kg diet). Because compounds with vitamin A activity are relatively unstable, the use of retinyl acetate or retinyl palmitate in gelatin coated beadlets is strongly recommended.

Several studies have shown that animals exposed to stress respond better at higher dietary concentrations of vitamin A than is recommended above. Gerber and Erdman (1982) reported that the strength of the scar tissue after a surgical incision was about twice as great in animals receiving 21 µmol retinyl acetate/kg diet or 13.4 µmol β-carotene/kg diet as it was in animals receiving 4.2 µmol retinyl acetate/kg diet. Demetriou et al. (1984) reported that rats fed a commercial diet containing 15 µmol retinol or retinyl esters per kg of diet and 12 µmol β-carotene/kg diet had only a 20 percent survival rate 72 hours after intra-abdominal sepsis. In contrast, when the diet was supplemented with an additional 525 µmol retinyl palmitate/kg, survival rose to 70 percent.

Signs of Vitamin A Deficiency The various procedures used to produce vitamin A-deficient rats have been reviewed in detail (Smith, 1990). The signs of vitamin A deficiency can be divided into six categories.

1.

Defect in vision. Because 11-cis-retinaldehyde is a necessary part of the visual pigments, a deficiency of vitamin A leads to a loss of vision through the lack of functional visual pigments (Wald, 1968).

2.

Bone defects. Vitamin A deficiency leads to improper bone cell differentiation, which causes retardation and disorganization of bone growth and failure of bone resorption during remodeling. The reduced size of the openings in the bones can cause a secondary compression of nerves (Underwood, 1984).

3.

Increase in cerebral spinal fluid pressure. The arachnoid villi, which release the fluid, become clogged with fibroblasts (Corey and Hayes, 1972).

4.

Reproductive failure. Cessation of spermatogenesis occurs in the male. In the female, severe and lethal deficiency causes cornification of the reproductive tract, which results in loss of reproductive function. With a more moderate deficiency, females will become pregnant but severe lethal fetal malformations and resorption are the most common result of the pregnancy (Wilson et al., 1953).

5.

Epithelial metaplasia and keratinization. All epithelia are sensitive to vitamin A deficiency to varying degrees. In early vitamin A deficiency, goblet cells and mucus formation decline in the intestine; squamous metaplasia followed by keratinization takes place in the trachea. Keratinization of the urogenital tract and the corneal epithelium combined with xerophthalmia and porphyrin deposits around the eyelids, and ultimate dissolution of the corneal stroma, takes place in severe vitamin A deficiency (Underwood, 1984).

6.

Growth failure. After 5 to 6 weeks of vitamin A deficiency, the weight of a weanling rat plateaus for about a week and then drops rapidly until the animal dies. Under germ-free conditions, the rat can survive at the weight plateau stage for several months (Rogers et al., 1971).

Signs of Vitamin A Toxicity Acute retinol toxicity occurs with an intake of 180 µmol/kg BW/day, although the long-chain retinyl esters were not toxic at this concentration of intake (Leelaprute et al., 1973). All-trans-retinoic acid is much more toxic; signs were found at 47 µmol/kg BW/day (Kurtz et al., 1984). The signs typically associated with vitamin A toxicity are weight loss, fatty liver, hyperlipidemia, calcification of soft tissues, mobilization of bone calcium, bone fractures, increased urinary excretion of 3-methylhistidine, and hemorrhage. Vitamin A is also teratogenic, causing cleft palate in fetuses at a retinyl palmitate intake of 40 µmol/kg BW/day given on days 9 to 12 of gestation (Nanda et al., 1970). Retinoic acid was much more teratogenic when low-protein diets (2.5 to 10 percent) were fed compared to a 20 percent protein diet (Nolen, 1972). Hemorrhage was caused by an interference in vitamin K metabolism and could be corrected by increasing vitamin K intake (McCarthy et al., 1989). α-Tocopherol has been shown to greatly reduce the toxicity of vitamin A (Jenkins and Mitchell, 1975). In contrast to the retinoids, ciency, the weight of a weanling rat plateaus for about a week and then drops rapidly until the animal dies. Under germ-free conditions, the rat can survive at the weight plateau stage for several months (Rogers et al., 1971). β-carotene was not toxic at doses up to 1,800 µmol/kg BW/day (Heywood et al., 1985).

Vitamin D

Vitamin D is an important precursor of the hormone 1,25-dihydroxycholecalciferol. The hormonal action is mediated by the interaction with a specific nuclear receptor and the corresponding responsive elements in the promotor regions of the genes that code for several proteins, many of which are involved in calcium metabolism. Although 1,25-dihydroxycholecalciferol is best known for its role in the regulation of calcium and phosphorus homeostasis, it may have significant roles in many other processes, including the synthesis of red blood cells, the proliferation of B and T lymphocytes, and the secretion of insulin and prolactin (Reichel et al., 1989). Vitamin D is hydroxylated in two positions before it becomes active. First, cholecalciferol is converted to 25-hydroxycholecalciferol in the liver. In the kidney 25-hydroxycholecalciferol is hydroxylated to form the active 1,25-dihydroxycholecalciferol. The formation of 1,25-dihydroxycholecalciferol is a tightly regulated process. Serum calcium concentration regulates the activity of the kidney 1-α-hydroxylase enzyme via the parathyroid gland. Low concentrations of serum phosphate directly increase the synthesis of this kidney enzyme. High dietary concentrations of both cause a decrease in the formation of 1,25-dihydroxycholecalciferol.

Information about the vitamin D requirements of the rat is surprisingly limited. The actual requirements are difficult to determine because the rat can absorb sufficient calcium and phosphorus to prevent the overt signs of rickets if the dietary ratio of calcium and phosphorus is about equal and they are present in adequate amounts. Also, cholecalciferol can be synthesized in the skin of the rat if it is exposed to ultraviolet light of appropriate wavelength (280 to 320 nm).

Relatively small amounts of vitamin D are needed to produce most of the biological responses, and the amount needed is a function of calcium and phosphorus intakes. A single injection of 0.13 µmol of either ergocalciferol (vitamin D₂) or cholecalciferol (vitamin D₃) promoted maximum active calcium absorption within 48 hours in vitamin D-deficient adult male rats of the Sherman strain fed a diet containing 5 g Ca/kg and 5 g P/kg (Schachter et al., 1961). Vitamin D is clearly required for maximum growth even when calcium and phosphorus are at concentrations typically considered optimal (Coward et al., 1932). However, vitamin D-deficient rats had normal bone mineralization and grew at the same rate as rats supplemented with vitamin D when they were continuously infused with calcium and phosphorus (Underwood and DeLuca, 1984). Male Sprague-Dawley rats fed a vitamin D-deficient diet containing 4.7 g Ca/kg and 3 g P/kg had low reproductive rates, but weekly injections of 5.2 µmol cholecalciferol/rat was sufficient to return reproductive function of deficient rats to normal (Kwiecinski et al., 1989). However, when male rats were fed a vitamin D-deficient diet containing 12 g Ca/kg and 3 g P/kg the fertility rate greatly improved (Uhland et al., 1992). Vitamin D-deficient female rats became pregnant and produced pups; nonetheless, the number of dams giving live births was only one-half that of dams given an oral dose of 1.6 µmol vitamin D/day (Halloran and DeLuca, 1980). Vitamin D-deficient rats were less likely to become pregnant, had more spontaneous abortions, and had a greater risk of death during parturition. In general, suckling pups grow poorly when nursed by vitamin D-deficient dams; however, this was the result of reduced milk production. When the litter size was reduced to two pups, the growth rate was normal (Mathews et al., 1986). Increasing the dietary calcium (16 g/kg) and phosphorus (14 g/kg) concentrations increased milk production so that suckling and weaned pups of vitamin D-deficient rats had normal growth and bone mineral content (Clark et al., 1987).

The rate of active absorption of calcium in the duodenum in middle-aged (12 to 14 months) and elderly rats (18 to 24 months) fed optimal amounts of calcium and phosphorus for a long period was reduced to about 70 percent of the rate observed in young rats (2 to 3 months) (Armbrecht, 1990). In contrast, the older rats actually absorbed cholecalciferol more efficiently than young rats (Hollander and Tarnawski, 1984). The activity of the kidney 1-α-hydroxy lase enzyme, however, was reduced in the older rats (Ishida et al., 1987). The concentration of the enzyme can be increased by a 4-month exposure to a low-calcium and vitamin D-deficient diet (Armbrecht and Forte, 1985). In contrast, young respond rapidly to this stimulus. There is no evidence that increasing the intake of vitamin D will have a beneficial effect on the absorption of calcium or phosphorus in adult rats.

In the absence of additional data, the estimated requirement of 1,000 IU vitamin D/kg diet recommended in the 1978 edition of this volume (National Research Council, 1978) is retained. This is equivalent to 65 nmol cholecalciferol/kg diet (25 µg/kg). This concentration, although adequate, may not represent the biological minimum.

Signs of Vitamin D Deficiency Vitamin D deficiency induces rickets. This disease is classically brought about in rats by a diet lacking vitamin D, adequate in calcium, and low in phosphorus. However, a low-calcium diet deficient in vitamin D has a more severe effect on growth rate and results in irritability, tetany, and decreased bone calcification (Steenbock and Herting, 1955). Bones of rachitic rats show decreased or absent calcification with wide areas of uncalcified cartilage at the junction of diaphysis and epiphysis. Bone ash may be less than half normal. A full description of histological changes in bones of vitamin D-deficient rats is given by Jones (1971).

Signs of Vitamin D Toxicity The first sign of vitamin D toxicity is usually elevated serum calcium followed by calcification of the kidneys (Potvliege, 1962). Soon the arteries become calcified, and then the liver and heart become calcified. The animals usually die from heart failure secondary to the uremia that comes from kidney failure. The animals also have a decreased growth rate and show considerable resorption of bone. Massive arteriosclerotic lesions developed in 100 percent of male rats fed a diet containing 78,800 µmol ergocalciferol/kg, 39 mmol cholesterol/kg, and 12 mmol cholic acid/kg for 6 weeks. Rats receiving this diet but containing only 63,000 µmol ergocalciferol/kg did not develop lesions, and rats fed ergocalciferol without cholesterol did not develop lesions (Bajwa et al., 1971). Vitamin D is also teratogenic. Treatment of pregnant dams with 2,500 µmol ergocalciferol/day reduced the growth rate of the fetuses and retarded the ossification of the long bones (Ornoy et al., 1968). All the pups died shortly after birth. When the dose was lowered to 1,250 µmol ergocalciferol/day the placenta was much smaller but the pups were normal. Nonpregnant rats receiving this lower dose had very high serum calcium concentrations. At 250 µmol/day the nonpregnant animals appeared normal. Shelling and Asher (1932) demonstrated that the toxicity of vitamin D was related to the calcium and phosphorus content of the diet. Rats fed diets containing 4.4 g Ca/kg and 17.8 g P/kg had severe toxicity signs at relatively low vitamin D intakes. However, the animals were able to tolerate high concentrations of vitamin D when they were fed diets with an optimal ratio of calcium to phosphorus.

Vitamin E

"Vitamin E is nature's best fat-soluble antioxidant" (Scott, 1978). In fact the only clearly defined function of vitamin E is as an antioxidant. Most of the signs of vitamin E deficiency can be prevented by feeding the antioxidant N,N'-diphenyl-p-phenylene diamine (DPPD).

Several compounds have vitamin E activity. The most active naturally occurring compound is RRR-α-tocopherol (formerly called D-α-tocopherol). The synthetic all-rac-α-tocopherol is a mixture of eight stereoisomers, and the other seven are less active than RRR-α-tocopherol. One mole of RRR-α-tocopherol has a biopotency equivalent to 1.36 moles of all-rac-α-tocopherol (U.S. Pharmacopeia, 1985). Although it does not occur naturally, tocopheryl acetate is frequently used in animal diets. The ester is hydrolyzed in the intestine, and tocopherol is released for absorption. However, having the alcohol group linked to the acetate prevents the tocopherol from being destroyed in the diet before it is consumed by the animal. In this section the concentrations of all compounds having vitamin E activity are expressed as the equivalent concentration of RRR-α-tocopherol.

Polyunsaturated fatty acids are labile to autoxidation. Each fatty acid free-radical that is oxidized damages about three other polyunsaturated fatty acid molecules, thus producing a geometrically expanding chain reaction (Chow, 1979). Vitamin E can readily donate hydrogen atoms to the free-radicals to terminate the chain reaction. As a result the requirement for vitamin E is related to the dietary and tissue concentrations of the polyunsaturated fatty acids (Witting and Horwitt, 1964). Adequate dietary selenium will greatly reduce the requirement for vitamin E. The selenium-containing enzyme, glutathione peroxidase, will convert the peroxides that are intermediates in this breakdown process to stable alcohols, thus reducing the requirement for vitamin E (Hoekstra, 1975).

Several criteria have been used to evaluate vitamin E status of the rat including: survival, growth, prevention of nutritional muscular dystrophy, prevention of creatinuria, prevention of fetal resorption, prevention of testicular degeneration, reduction of pentane expiration, reduction of malondialdehyde production, and prevention of the spontaneous hemolysis of red blood cells after they are diluted with saline.

In selenium-deficient rats, Hakkarainen et al. (1986) found that about 5.2 mg α-tocopheryl acetate/kg diet (11 µmol/kg) was required for survival. Gabriel et al. (1980) used plasma pyruvate kinase and glutamic oxaloacetic transaminase as indicators of myopathy of skeletal muscle. By feeding rats the antioxidant ethoxyquin (which is not stored) instead of vitamin E, they were able to rapidly produce vitamin E deficiency in rats from 12 to 68 weeks old by removing ethoxyquin from the diet. In all age groups the minimum vitamin E requirement to prevent myopathy of muscle was approximately 0.75 mg α-tocopheryl acetate/kg BW/day (1.6 µmol/kg BW/day). Jager and Houstsmuller (1970) found that preventing the spontaneous hemolysis of red blood cells required 13.2 mg RRR-α-tocopherol/kg diet (28 µmol/kg diet) when the diet contained 3.6 percent linoleic acid, but increasing the linoleic acid content to 13 percent increased the requirement to 18 mg/kg diet (38 µmol/kg). In a test of repletion of vitamin E-deficient rats, Bieri (1972) showed that hemolysis was prevented after feeding 20 mg RRR-α-tocopheryl acetate/kg diet (42 µmol/kg). The dietary lipid was a mixture of stripped corn oil and lard that provided 5.2 percent linoleic acid. At this percentage, α-tocopherol in the tissues reached a stable concentration within 8 weeks following the beginning of repletion. Buckingham (1985) studied the requirements of rats fed purified diets containing 20 percent lipid with polyunsaturated to saturated (P:S) ratios of 0.38, 0.82, and 2.30. Pentane expiration in the breath and production of malondialdehyde were reduced to normal levels when rats were fed a diet with 27 mg α-tocopherol/kg diet (62 µmol/kg). However, 44 percent spontaneous hemolysis was observed when the diet contained a 2.30 P:S ratio even with an α-tocopherol concentration of 67 mg/kg diet (156 µmol/kg).

Evans and Emerson (1943) investigated the rats' requirement for vitamin E during reproduction. As female rats aged, the requirement to maintain pregnancy became higher. To maintain pregnancy and optimal health in the suckling young, 0.57 mg α-tocopheryl acetate/rat/day (1.2 µmol/rat/day) was required. However, after the third pregnancy the suckling pups suffered slight muscular impairment at this concentration of intake. In young male rats 0.18 mg/rat/day (0.39 µmol/rat/day) was adequate to maintain reproduction; but at 9 months and older, the rats required 0.57 mg/rat/day (1.2 µmol/rat/day) to maintain fertility. Ames (1974) determined the amount of vitamin E necessary to maintain pregnancy in female rats. The dose to maintain 50 percent fetal viability increased about sevenfold from the first pregnancy (12 weeks) to the fourth pregnancy (60 weeks). This increase is more than can be explained by an increase in body weight. Gabriel et al. (1980) suggested that the increased requirement may be caused by accumulated toxic products from long-term exposure to very low intakes of antioxidants.

Autofluorescent pigment (lipofuscin) accumulates in the tissues of vitamin E-deficient animals and in old animals. Nonetheless the tissue distribution of the lipofuscin is different in vitamin E-deficient rats than in aging rats (Katz et al., 1984), thus the accumulation in aging rats does not appear to be the result of inadequate vitamin E intake. Although older rats may require more dietary vitamin E to maintain α-tocopherol concentrations in the cerebellum and brain stem (Meydani et al., 1986), the concentration needed has not been established. Hollander and Dadufalza (1989) have demonstrated that older rats absorb α-tocopherol more efficiently than younger rats.

Bendich et al. (1986) examined the vitamin E requirement of spontaneously hypertensive rats, which are more sensitive to vitamin E-deficient diets than the parent Wistar strain. Maintenance of normal growth required 7.5 mg all-rac-α-tocopheryl acetate/kg diet (16 µmol/kg diet); 15 mg/kg diet was required to prevent myopathy of muscle; and 50 mg/kg diet was required to prevent the spontaneous hemolysis of red blood cells. Optimal immune responses appeared to require slightly more than 50 mg/kg diet. The immune system response was the most sensitive indicator of vitamin E status.

The vitamin E requirement for most of the frequently used strains of rats is 18 mg RRR-α-tocopherol/kg diet (42 µmol/kg) when lipids comprise less than 10 percent of the diet. This corresponds to 27 IU/kg diet. When all-rac-α-tocopheryl acetate is used as the dietary source, this would be equivalent to 27 mg/kg diet (57 µmol/kg).

High intakes of either retinyl palmitate (42 µmol/kg diet) or β-carotene (89 µmol/kg diet) depressed plasma and liver concentrations of α-tocopherol to about one-half the normal concentrations (Blakely et al., 1990). It seems probable that high concentrations of vitamin A in the diet interfere with the absorption of vitamin E.

Signs of Vitamin E Deficiency Red blood cells from vitamin E-deficient rats show an increased hemolysis when diluted with saline or when treated with oxidizing agents (dialuric acid). Other signs are hyaline degeneration of skeletal muscle fibers with infiltration by histocytes, interfibrillar fat cells, and an increase in interstitial cells (Jager, 1972); accumulation of yellow pigment in smooth muscles; in the male, irreversible degeneration of the seminiferous epithelium of the testis, which occurs by age 40 to 50 days; in the female, induction of fetal abnormalities or intrauterine death and resorption; kyphoscoliosis (humped-back) (Machlin et al., 1977); rough coat; skin ulcers; neural lesions; and impaired learning ability (Sarter and Van Der Linde, 1987).

Signs of Vitamin E Toxicity In general vitamin E is relatively nontoxic. However, 2,000 mg RRR-α-tocopheryl acetate/kg BW (4,230 µmol/kg BW) prolonged the process of prothrombin production, and hemorrhagic diathesis developed in rats fed the AIN-76 diet (low vitamin K) (Abdo et al., 1986). The main metabolic product of vitamin E, tocopheryl quinone, inhibits the normal metabolism of vitamin K. In addition, Martin and Hurley (1977) found eye abnormalities in pups from dams receiving 1,600 mg α-tocopheryl acetate/kg BW/day (3,500 µmol/kg BW/day). Yang and Desai (1977) found a decreased implantation index in inseminated females fed a diet containing 7,300 mg α-tocopheryl acetate/kg diet (15,500 µmol/kg).

Vitamin K

The only known function of vitamin K in mammalian systems is the posttranslational conversion of glutamic acid to γ-carboxyglutamic acid (Gla) (Stenflo, 1976). Gla is found only in a limited number of proteins including the blood clotting proteins, prothrombin and factors VII, IX, and X; the anticlotting proteins, protein C and protein S; blood protein Z; the bone proteins, osteocalcin and matrix Gla-containing protein (MGP); and a few other Gla-containing proteins in the kidney and intestine (Suttie, 1991).

Phylloquinone (vitamin K₁) is the metabolically preferred source of vitamin K (Will and Suttie, 1992). Phylloquinone is also actively absorbed in the proximal portion of the small intestine (Hollander, 1973). The menaquinones (vitamin K₂) are a family of compounds synthesized by bacteria. The bacteria in the large intestine produce substantial amounts of menaquinones. Because the rat is coprophagous, excrement provides a substantial source of vitamin K activity. Essentially no absorption of the larger menaquinones, such as menaquinone-9, occurs in the large intestine; but they are effectively absorbed if recycled to the small intestine by coprophagy (Ichihashi et al., 1992). The smaller menaquinone-4 is absorbed to a limited extent in the colon, but menaquinone-4 is a minor product of the intestinal bacteria. The synthetic derivative of vitamin K, menadione, lacks the isoprenoid side chain of the natural compounds with vitamin K activity, and it must be converted to menaquinone-4 in the liver to be functional (Dialameh et al., 1971). Menadione is about one-tenth as active as phylloquinone. Some of the water-soluble derivatives of menadione, such as the menadione sodium bisulfite complex, are much more readily absorbed and are about equally as active as phylloquinone (Griminger, 1966).

The most prominent function of vitamin K is its role in blood clotting. Failure of blood to coagulate occurs in some rats that have been fed a vitamin K-deficient diet for 2 to 3 weeks. However, in most rats overt clotting problems only develop after they have been fed antibiotics or when coprophagy is prevented.

Estimation of the vitamin K requirement of rats is complicated by several factors: (1) the contribution from coprophagy, (2) the difficulty in producing vitamin K-free diets, (3) the large differences in requirements between strains, and (4) the problem of selecting which criteria to use to determine normal status.

The typical contribution from coprophagy is probably on the order of 4 to 9 µmol/rat/day (Mameesh and Johnson, 1960; Wostmann et al., 1963). Rats fed diets with a high nutrient density and high digestibility are less coprophagous than rats fed low-digestibility diets (Giovannetti, 1982; Mathers et al., 1990). Housing rats in a very cold environment (Smith and Borchers, 1972) will almost eliminate coprophagy. The contribution from coprophagy is difficult to estimate and may necessitate the use of germ-free animals for experimental work.

The purified milk proteins, casein and lactalbumin, usually contain substantial vitamin K that is difficult to extract (Matschiner and Doisy, 1965). Therefore, soybean proteins are frequently used as the source of protein when vitamin K metabolism is studied. If the soybean proteins are extracted with ethanol, diets can be formulated containing as little as 9 µg phylloquinone/kg (0.02 µmol/kg) (Kindberg and Suttie, 1989).

Many different criteria have been used to evaluate vitamin K status. Wostmann et al. (1963) used survival of germ-free male Lobund rats as a criterion and estimated that the diet must contain 0.44 µmol phylloquinone/kg to maintain survival. At 0.33 µmol/kg diet most of the rats died from hemorrhages. However, the minimum amount necessary to keep rats from bleeding to death may not be the optimal vitamin K intake. Most frequently, some estimate of clotting time is used to predict the vitamin K requirement. Mameesh and Johnson (1960) reported that male Sprague-Dawley rats required phylloquinone at 0.25 µmol/kg diet to maintain normal prothrombin activity when coprophagy was prevented by tail cups. Matschiner and Doisy (1965) found that male rats of the St. Louis strain required 0.55 µmol/kg diet to maintain normal prothrombin activity when fed a 21 percent soybean protein diet that was not extracted and coprophagy was not prevented.

More sensitive criteria of vitamin K status were used by Kindberg and Suttie (1989) to determine the requirement of male Holtzman and Sprague-Dawley rats. The rats were fed an extracted soybean protein diet and coprophagy was not prevented. A diet containing 1.1 µmol phylloquinone/kg was not adequate to bring liver carboxylase enzyme activity or plasma prothrombin to the concentrations observed in rats fed a 3.3 µmol/kg diet. Based on extrapolations of this data the optimal concentration appears to be about 2.2 µmol/kg diet. Even at higher doses the rats maintained small liver stores of vitamin K that were essentially depleted within 5 days. Consequently, rats depend on a continuous dietary supply of vitamin K to maintain optimal status.

When fed diets with suboptimal vitamin K activity, female rats develop signs of vitamin K deficiency much more slowly than do males (Matschiner and Bell, 1973). The lower requirement seems to reflect differences in the effects of estrogen and testosterone on the production of prothrombin (Matschiner and Willingham, 1974). In pregnancy the concentrations of prothrombin rise as a result of the increased estrogen concentrations. The requirement of female rats appears to be about three-fourths the requirement of males.

Mellette and Leone (1960) reported that rats 15 to 27 weeks old were about twice as likely to have a hemorrhagic death as rats 3 to 5 weeks old when fed irradiated beef diets low in vitamin K.

Warfarin-resistant rats have much higher vitamin K requirements than normal rats because they do not recycle vitamin K epoxide as efficiently as normal rats. Greaves and Ayres (1973) reported that the Wistar strain required 0.13 µmol phylloquinone/kg BW to maintain normal prothrombin activity, the Tolworth HS (Scottish) warfarin-resistant strain required 0.44 µmol/kg BW, and the Tolworth HW (Welsh) warfarin-resistant strain required 1.77 µmol/kg BW. The vitamin K was given by daily subcutaneous injections.

Roebuck et al. (1979) reported that male Wistar/Lewis rats developed hemorrhaging but had normal prothrombin activity when fed a modified AIN-76 diet for 8 to 20 weeks. The bleeding could be corrected by adding phylloquinone, 1.1 µmol/kg, to the diet. The AIN-76 diet (American Institute of Nutrition, 1977) was formulated to contain 0.18 µmol menadione sodium bisulfite/kg plus an undefined amount of vitamin K activity in the dietary casein and corn oil. Bieri (1979) confirmed that the amount of vitamin K activity in the AIN-76 diet was less than optimal for Fischer rats when the casein was replaced with other proteins; the American Institute of Nutrition recommended that the added menadione sodium bisulfite be increased to 1.8 µmol/kg diet (American Institute of Nutrition, 1980) and that the diet be designated AIN-76A.

For the most commonly used strains of rats (Fischer, Sprague-Dawley, and Wistar) a diet containing 1.00 mg phylloquinone/kg diet (2.22 µmol/kg) should satisfy the most sensitive criterion for vitamin K adequacy. Because the rat actively absorbs phylloquinone and gives preference to phylloquinone metabolically, phylloquinone is the recommended form to be used in diets. Menadione and its derivatives are not recommended.

High concentrations of dietary vitamin A and vitamin E have been shown to accelerate the onset of deficiency signs in rats fed vitamin K-deficient diets. As little as 5.2 µmol retinyl acetate/kg diet decreased the prothrombin activity in rats fed a diet low in vitamin K (Doisy, 1961). Supplementation of a low vitamin K diet with 12 µmol α-tocopherol orally twice a week increased hemorrhagic deaths (Mellette and Leone, 1960); hemorrhaging, however, could be corrected by phylloquinone supplementation. Vitamin E metabolites appear to interfere with the normal metabolism of vitamin K. The vitamin E metabolites tocopheryl hydroquinone and tocopheryl quinone are the most probable causes (Rao and Mason, 1975). Butylated hydroxytoluene (BHT, 1.2 percent of diet) has also been shown to induce hemorrhagic death when included in casein-based diets that were not supplemented with vitamin K. The simultaneous administration of 0.68 µmol phylloquinone/kg BW/day prevented the hemorrhages and maintained normal prothrombin concentrations (Takahashi and Hiraga, 1979).

Signs of Vitamin K Deficiency In vitamin K deficiency thrombin activity is depressed and liver carboxylase activity is greatly increased (Kindberg and Suttie, 1989). The first overt sign of vitamin K deficiency is usually a continuous oozing of blood from a minor injury such as a separated toenail or a small pin-prick on the tail. These injuries would not normally bleed longer than 30 seconds, but the vitamin K-deficient animal may continue to lose blood until death occurs. In an examination of specific-pathogen-free rats not supplemented with vitamin K, hemorrhages were found in the urogenital tract, the central nervous system, the chest cavity, the abdominal cavity, and under the skin (Fritz et al., 1968).

Signs of Vitamin K Toxicity Phylloquinone is essentially nontoxic when given orally. Rats given daily doses of 4,400 µmol phylloquinone/kg BW for 30 days did not show signs of toxicity, whereas 2,000 µmol menadione/kg BW was lethal (Molitor and Robinson, 1940). At 260 µmol/kg diet, menadione depressed the activity of several heme-containing enzymes in vitamin E-deficient rats (Hauswirth and Nair, 1975). This concentration is present in a popular commercial vitamin mix. At higher concentrations both menadione and menadione sodium bisulfite produce liver toxicity.

Vitamin B6

The vitamin B₆ compounds (pyridoxine, pyridoxal, and pyridoxamine) function as coenzymes for amino acid decarboxylases, racemases, transaminases, and other enzymes in amino acid, glycogen, and fatty acid metabolism (Baker and Frank, 1968). The coenzymes are formed by phosphorylation of the aldehyde and amine; nearly 50 percent of pyridoxal phosphate in the body is stored as coenzyme for muscle glycogen phosphorylase (Anonymous, 1975; Chen and Marlatt, 1975). Pyridoxal phosphate is involved in releasing steroid-hormone-complexes tightly bound to receptors (Compton and Cidlowski, 1986; Bender et al., 1989).

Phosphatase-mediated hydrolysis is the first step in the intestinal absorption of pyridoxal-5'-phosphate. Gastric acid secretions are important for the hydrolysis as intestinal alkaline phosphatase is activated at low pH (3.4) (Middle ton 1986). Products of digestion—amino acids and oligopeptides—inhibit hydrolysis (Middleton, 1990). Cellulose, pectin, or lignin did not alter the in vitro jejunal absorption rates of pyridoxine, pyridoxal, or pyridoxamine (Nguyen et al., 1983). Weanling Sprague-Dawley rats fed diets containing 1.5 mg vitamin B₆/kg diet or less had lower vitamin B6 concentrations in the intestinal mucosa than rats fed 3 to 100 mg/kg diet (Roth-Maier et al., 1982). Vitamin B6 aldehydes can reductively bind to food proteins as epsilonpyridoxyllysine complexes during processing and storage and, in this form, possess only 50 percent of the vitamin B6 activity (Gregory, 1980a,b). No differences were observed in urinary pyridoxic acid between germ-free and conventional rats, indicating that coprophagy does not alter requirements (Coburn et al., 1989).

Studies of the dietary requirement have been based on vitamin B6-dependent enzyme activities, body weight gain, tissue stores of pyridoxal phosphate, or reproductive performance. When male weanling rats were fed 1, 2, 4, or 8 mg vitamin B₆/kg diet, growth was the same in all groups; but liver, serum, and red blood cell alanine-aminotransferase was maintained only at dietary concentrations of 4 mg/kg and above (Chen and Marlatt, 1975). Concentrations of 1.0 to 1.6 mg/kg diet were required to achieve maximum growth (Roth-Maier and Kirchgessner, 1981; Van den Berg et al., 1982; Mercer et al., 1984). Alterations in red blood cell transaminase activity indicated a vitamin B₆ requirement of 6 to 7 mg/kg diet (Beaton and Cheney, 1965). Diets containing 7.0 mg pyridoxine-HCl/kg restored full activity of erythrocyte alanine aminotransferase and aspartate aminotransferase when fed to female Long-Evans rats that had been previously depleted of vitamin B6 (Skala et al., 1989).

Protein quality had no significant effect on urinary 4-pyridoxic acid, plasma pyridoxal phosphate or total hepatic vitamin B₆ concentrations in rats fed 0.2 and 7.0 mg vitamin B₆/kg diet (Fisher et al., 1984). Erythrocyte alanine aminotransaminase activity decreased with increasing dietary protein concentrations but was not altered when the quality of protein was changed (Dirige and Beaton, 1969).

Vitamin B6 is required by pregnant rats for normal development of their offspring. Vitamin B6-deficient offspring had retarded renal differentiation, abnormalities of cerebral lipids, and increased tissue and urinary concentrations of cystathionine (Kurtz et al., 1972; DiPaolo et al., 1974; Pang and Kirksey, 1974). Maternal weight gain and body and brain weight of offspring were normal when the diet contained 3 mg pyridoxine/kg and slightly, but not significantly, lower at 2 mg/kg/ 1 mg/kg was clearly inadequate. There was no significant difference between offspring of dams fed 3 mg/kg diet and those fed 6 mg/kg (Driskell et al., 1973). When female rats were reared on diets containing 1.2, 2.4, 4.8, 9.6, or 19.2 mg vitamin B₆/kg diet, maternal and fetal weights were reduced in the group fed 1.2 mg/kg; erythrocyte alanine aminotransferase activity coefficients were maintained at 2.4 mg/kg and above; and tissue vitamin B6 saturation concentrations indicated that 9.6 mg/kg was required for growth and 4.8 mg/kg for maintenance. However, 19.2 mg/kg diet did not achieve saturation concentrations of maternal liver, fetal brain, or carcass during gestation (Kirksey et al., 1975). Rats fed 8 or 40 mg/kg during pregnancy had similar activities of hepatic aspartate aminotransferase, erythrocyte alanine aminotransferase, and gastrocnemius muscle glycogen phosphorylase (Shibuya et al., 1990).

Female rats fed diets that contained 1.2 to 19.6 mg vitamin B₆/kg diet from weaning through breeding, gestation, and lactation bore offspring of normal weight at 2.4 mg/kg and above. Concentrations of vitamin B₆, protein, and cerebrosides in brains were significantly decreased at 1.2 and 2.4 mg/kg; however, brain protein concentration continued to increase up to the highest dietary vitamin B₆ concentration. Milk pyridoxine and erythrocyte transaminase were decreased when maternal diet was less than 4.8 mg/kg (Moon and Kirksey, 1973; Pang and Kirksey, 1974).

These studies indicate that vitamin B₆ requirements are met by 4 to 7 mg/kg diet, although higher tissue concentrations of pyridoxal phosphate may be achieved by higher dietary concentrations. The estimated vitamin B₆ requirement for maintenance, growth, and reproduction is set at 6 mg pyridoxine-HCl/kg diet.

Signs of Vitamin B₆ Deficiency Rats fed diets deficient in vitamin B₆ develop symmetrical scaling dermatitis on the tail, paws, face, and ears; microcytic anemia; hyperexcitability; and convulsions (Sherman, 1954). The amount of 3-hydroxykynurenine was increased in neonatal brain at 14 and 18 days of age but not in adult brain (Guilarte and Wagner, 1987). Amplitude of response to both acoustic and tactile stimuli was depressed by vitamin B₆ depletion (Schaeffer, 1987) as well as differences in angle and width of the hind-leg gait (Schaeffer and Kretsch, 1987; Schaeffer et al., 1990). Depleted rats demonstrated a taste preference for NaCl and excreted reduced concentrations of sodium (Mei-Ying and Kare, 1979). They had deficits in active and passive avoidance learning (Stewart et al., 1975). The pyramidal cells of the cerebral cortex of rats fed deficient diets for 2 or 3 months showed partial to nearly complete dendritic loss and axonal swelling in the hippocampus (Root and Longenecker, 1983). Reduced brain concentrations of vitamin B₆, dopamine, homovanillic acid, D-2 dopamine receptors, brain glutamic acid decarboxylase, gamma aminobutyric acid (GABA), and γ-aminobutyric acid transaminase have been measured in vitamin B₆-deficient rats (Driksell and Chuang, 1974; Aycock and Kirksey, 1976; Rajeswari and Radha, 1984; Guilarte, 1989). Cerebroside and ganglioside content and fatty acids (18:2, 20:1, 20:4, 22:6, and 24:0) and n-6 fatty acids (18:2, 20:4, and 22:4) are decreased in brain as a result of vitamin B₆ deficiency (Thomas and Kirksey, 1976a,b).

In deficiency, reproductive performance of both females and males was decreased; deficient production of insulin may occur (Huber et al., 1964). An 8-week deficiency resulted in decreases in hepatic alanine and aspartate aminotransferases, and the ability to use alanine for gluconeogenesis was impaired (Angel, 1980). The turnover of cytosolic, but not mitochondrial aspartate aminotransferase, was altered in vitamin B₆ deficiency (Shibuya and Okada, 1986); and the total activities of both aspartate aminotransferase and alanine transferase were reduced (Ludwig and Kaplowitz, 1980). Pyridoxine deficiency reduced the intestinal uptake of glucose, glycine, alanine, and leucine as well as the activities of mucosal sucrase, lactase, alkaline phosphatase, leucine aminopeptidase, and membrane synthesis (Mahmood et al., 1985). In rats fed a high protein diet deficient in vitamin B₆, urinary excretion of urea, free ammonia, and free amino acids were altered (Okada and Suzuki, 1974). Female rats fed vitamin B₆-deficient diets excreted less taurine than pair-fed controls (Lewis et al., 1982; Lombardini, 1986). Vitamin B₆-deficient rats had lower plasma homocystine in association with decreased and sporadic food intake (Smolin and Benevenga, 1984). Creatine in skeletal muscle and liver was higher and creatinine excretion lower in deficient rats (Loo et al., 1986). Vitamin B₆ deficiency produced a decrease in glucose-6-phosphate dehydrogenase activity in the periosteum and in the developing callus (Dodds et al., 1986) and a decrease in collagen cross-link formation (Fujii et al., 1979). Liver parenchymal ultrastructural changes after 7 weeks on a vitamin B₆-deficient diet included hyperplasia in the nucleoli and smooth endoplasmic reticulum and hypoplasia in the Golgi, rough endoplasmic reticulum, and mitochondria with reduced numbers of orthoperoxisomes (Riede et al., 1980). Vitamin B₆-deficient male rats had larger and a greater number of urinary calcium oxalate stones than females or castrated males (Gershoff, 1970). Changes in fatty acid oxidation and incorporation of the fatty acids into triglycerides, phospholipids, and cholesterol fractions have been observed (Dussault and Lepage, 1979).

Signs of Vitamin B₆ Toxicity Excess vitamin B₆ has been shown to have detrimental effects. Twelve-week-old rats fed 6 weeks on diets containing 1,400 mg vitamin B₆/kg had elevated red blood cell alanine aminotransferase activity and pyridoxal-5'-phosphate concentrations, but muscle pyridoxal-5'-phosphate concentrations were lower. Tissue pyridoxal concentrations were higher and muscle glycogen phosphorylase A activity was increased by the consumption of excessive dietary vitamin B₆ (Schaeffer et al., 1989). Rats injected intraperitoneally with 200 mg/kg BW developed gait ataxia with impaired oxidative metabolism in peripheral nerve tissue and an axonal degeneration of the sensory system fibers (Windebank et al., 1985).

Vitamin B12

In mammals, vitamin B₁₂ is required as a coenzyme for the transmethylation of homocystine to methionine, in utilizing 5-methyl-tetrahydrofolic acid, and in the conversion of methylmalonyl-CoA to succinyl-CoA (Weissbach and Taylor, 1970). The concentration of vitamin B₁₂ needed in the diet of the rat may vary with dietary content of choline, methionine, and folic acid. A number of conditions have been reported that impair vitamin B₁₂ absorption. Rats fed a diet with raw kidney bean (Phaseolus vulgaris) as 4 percent of dietary protein or 0.5 percent phytohemagglutinin developed vitamin B₁₂ malabsorption after only 3 days, and the condition was not correctable by giving intrinsic factor (Banwell et al., 1980). Highly fermentable fibers such as pectin, guar gum, and xylan fed as 5 percent of the diet increased urinary methylmalonic acid and depressed the oxidation of propionate to CO₂ (Cullen and Oace, 1989a). The half-life of vitamin B₁₂ was 58 days for rats fed fiber-free diets and 38 days for rats fed a 5 percent pectin diet (Cullen and Oace, 1989b). Vitamin B₁₂ deficiency has been induced by diets containing unheated soybean flour, but amino acid deficiencies occur also and unheated soybean flour contains other toxins; therefore, it has been suggested that its use for the study of vitamin B₁₂ deficiency is not justified (Edelstein and Guggenheim, 1971; Williams and Spray, 1973). Giardiasis also has been reported to impair vitamin B₁₂ absorption (Deka et al., 1981). The oxidation of acetaldehyde, generated from the metabolism of ethanol, by xanthine oxidase inhibited the ability of vitamin B₁₂ to bind to intrinsic factor (Shaw et al., 1990). Decreased vitamin B₁₂ concentrations have been reported in rats fed liquid diets with ethanol but not natural-ingredient diets with ethanol (Frank and Baker, 1980). Hypothyroidism slowed the rate of depletion of hepatic vitamin B₁₂ (Stokstad and Nair, 1988). Vitamin B₁₂ deficiency developed rapidly in rats exposed to nitrous oxide (Horne and Briggs, 1980; Muir and Chanarin, 1984).

The minimum requirement for vitamin B₁₂ has not been established; however, a dietary concentration of 10 µg vitamin B₁₂/kg seems to be inadequate based on urinary excretion of methylmalonic acid (Thenen, 1989). A concentration of 50 µg vitamin B₁₂/kg diet supported normal growth and reproduction (Woodard and Newberne, 1966). In the absence of more complete information, the requirement is currently set at 50 µg vitamin B₁₂/kg diet.

Signs of Vitamin B₁₂ Deficiency Induction of isolated vitamin B₁₂ deficiency in the rat, as well as in other experimental animals, is achieved with difficulty and generally does not reproduce the signs of human vitamin B₁₂ deficiency— megaloblastic blood cells and neurological lesions. Deficiency can be induced in rats fed vegetable rather than animal protein (which contains vitamin B₁₂). Female rats fed a diet that contained soybean protein supplemented with methionine and choline, but not vitamin B₁₂, grew normally or at a slightly decreased rate and bred and littered normally. The average weights of their offspring were decreased, and 10 percent of the litters were hydrocephalic. Hepatic content of vitamin B₁₂ was markedly decreased in both mothers and offspring. Deletion of choline from the diet increased the incidence of congenital abnormalities in the neonates. Supplementation of the diet with 50 µg vitamin B₁₂/kg supported normal growth in the mothers and prevented development of hydrocephalic offspring (Woodard and Newberne, 1966). When litters born to vitamin B₁₂-deficient females were fed diets deficient in vitamin B₁₂, growth was retarded by day 30 and the animals remained small throughout the 150 day experiment. Methionine (0.5 percent DL) reduced the growth retardation (Doi et al., 1989). Germ-free females fed soybean protein aborted, bore short-lived pups, or cannibalized their pups. The germ-free condition apparently enhanced vitamin B ₁₂ deficiency (Valencia and Sacquet, 1968). Vitamin B₁₂-deficient rats accumulated more odd-chained fatty acids in phosphatidylcholine of cerebrum and liver, more 18:2 acids in liver phosphatidylethanolamine, and arachidonate [20:4(n-6)] and 22:5 in liver phosphatidylcholine, but smaller amounts of 20:4(n-6) and 22:6(n-6) in cerebral phosphatidylcholine (Peifer and Lewis, 1979).

Biotin

Biotin is required by four carboxylase enzymes in mammalian systems: acetyl CoA carboxylase (fatty acid synthesis), pyruvate carboxylase (Gluconeogenesis), 3-methylcrotonyl CoA carboxylase (leucine catabolism), and propionyl CoA carboxylase (methionine, threonine, and valine catabolism).

Under normal conditions rats do not require biotin in the diet. Adequate biotin is provided by the intestinal microorganisms through coprophagy. Biotin deficiency can be produced in rats fed a biotin-free diet by (1) preventing coprophagy (Barnes et al., 1959b); (2) using germ-free animals (Luckey et al., 1955); (3) feeding sulfa drugs (Daft et al., 1942); or feeding raw egg white, which contains the biotin-binding protein avidin (Nielsen and Elvehjem, 1941).

Klevay (1976) fed 20 percent raw egg white diets to rats and determined that 2 mg d-biotin/kg diet (8.2 µmol/kg) was required to obtain optimal growth for 60 days. Several other investigators have given various amounts of biotin to animals to cure or prevent signs of deficiency, but these experiments do not permit conclusions to be made about requirements because the amounts given were insufficient to correct fully the deficiency, the animals did not grow at a rate equivalent to controls, controls were not used, or the biotin administration was erratic.

The AIN-76 diet (American Institute of Nutrition, 1977) and AIN-93G and AIN-93M diets (Reeves et al., 1993b) were formulated to contain 0.2 mg d-biotin/kg (0.82 µmol/kg). This concentration appears to be adequate when casein is the dietary protein; however, if spray-dried raw egg white is used as the dietary protein, the concentration of biotin should be increased to 2.0 mg/kg diet.

Signs of Biotin Deficiency Biotin deficiency causes rats to develop a progressive exfoliative dermatitis, "spectacle eye," achromotrichia (in black rats), and general alopecia. With severe deficiency many animals develop a spastic gait or assume a "kangaroo-like" posture. Immune responses were also depressed in biotin-deficient rats (Rabin, 1983).

Signs of Biotin Toxicity Biotin is relatively nontoxic. Paul et al. (1973) reported that 50 mg biotin/kg BW (205 µmol/kg BW) divided between morning and evening subcutaneous injections produced irregularities in the estrous cycle and a massive infiltration of leukocytes in the vagina. Mittelholzer (1976) did not observe problems in the reproduction of females given this dose.

Choline

Choline is a component of lecithin, of sphingomyelin, and of the neurotransmitter acetylcholine. Choline also has an important role in one-carbon metabolism.

Diets are usually formulated with either choline chloride or choline bitartrate. Although all choline compounds tend to be hygroscopic, choline bitartrate is substantially less hygroscopic than choline chloride, and does not add more chloride, so use of choline bitartrate is preferable for most practical diets.

Choline is required by the rat, but the requirement is a function of the methionine and lipid content of the diet. Diets containing about 14 percent casein require more choline than diets containing either more or less casein (Aoyama et al., 1971). Diets that contain 0.8 percent methionine or more do not require choline (Griffith, 1941; Newberne et al., 1969; Aoyama et al., 1971). Rats fed diets containing suboptimal amounts of methionine have been reported to require 0.6 to 2.0 g choline chloride/kg diet (4 to 14 mmol/kg) (Griffith and Wade, 1939; Griffith, 1941; Engel, 1942; Mulford and Griffith, 1942; Hale and Schaefer, 1951).

Using a diet that contained 38 percent sucrose, 20 percent lipid, and 36 percent protein (0.34 percent methionine), Chahl and Kratzing (1966) found that male Wistar rats housed at 21° C required 500 mg choline (free base) per kg diet (4.8 mmol/kg) to prevent lipid accumulation in the liver. However, when the rats were housed at 33° C the requirement was increased to 1,000 mg choline (free base) per kg diet (9.6 mmol/kg). In contrast, housing the rats at 2° C reduced the requirement to 250 mg choline (free base) per kg diet (2.4 mmol/kg).

Based on the studies of Chahl and Kratzing (1966), Mulford and Griffith (1942), and Griffith and Wade (1939), the requirement for choline is set at 750 mg/kg diet (7.2 mmol/kg diet). This is equivalent to 1.8 g choline bitartrate/kg diet. However, if diets with 5 to 15 percent and 20 to 40 percent fat are to be used, if the diets are deficient in folate or vitamin B₁₂, or if the rats are to be housed in a high environmental temperature, then the amount of choline may need to be increased.

Signs of Choline Deficiency Choline deficiency appears much more rapidly in male than in female rats (Patek et al., 1969). Droplets of triglycerides and abnormalities of the intracellular membranes appear in the livers of weanling male rate within 24 hours after they have ingested a choline-deficient diet. Long-term deficiency leads first to fatty liver, in which triglycerides may compose as much as 50 percent of the total wet weight of the liver and the liver cells are markedly distended with fat vacuoles, and then to cirrhosis, in which there is proliferation of fibrovascular tissue, vascular shunting, and hepatic failure (Zaki et al., 1963; Rogers and MacDonald, 1965). Sucrose was found to cause twice as great an increase in liver triglycerides as dextrin in choline-deficient diets (Chalvardjian and Stephens, 1970).

Choline deficiency has marked effects on both the kidney and the cardiovascular system in young rats. If male rats are fed a choline-deficient diet at weaning, 50 to 90 percent die within 10 days to 2 weeks of hemorrhagic renal necrosis. They may develop myocardial necrosis and atheromatous changes in arteries (Salmon and Newberne, 1962; Monserrat et al., 1974). Supplementation with about one-half the requirement will protect against renal damage and prevent cirrhosis of the liver, but it will not prevent the infiltration of the liver with triglyceride.

Signs of Choline Toxicity Sahu et al. (1986) reported that 100 mg choline chloride/kg BW/day (700 µmol/kg BW/day) depressed the rate of growth and caused pathological changes in the lungs and lymph nodes. The safety margin between the daily requirement (16 to 42 mg/kg BW or 115 to 300 µmol/kg BW) and the toxic concentration is relatively narrow with choline, so caution should be used in increasing the concentrations. The LD₅₀ of choline chloride has been reported to range from 0.28 to 0.75 g/kg BW/day (2,000 to 5,400 µmol/kg BW/day) depending on the concentration of the injected solution (Hodge, 1944; Ho et al., 1979; Sahu et al., 1986). Bell and Slotkin (1985) reported that a diet containing 5.0 percent soybean lecithin caused alterations in sensorimotor development and brain cell maturation.

Folates

Folates are composed of pteridines linked to p-aminobenzoic acid conjugated to one or more glutamic acid residues (Blakley and Benkovic, 1984; Blakley and Whitehead, 1986). Folates in most tissues exist primarily in the pentaglutamyl form. The principal function of folates is to transfer one-carbon units such as in thymidylate synthesis, purine synthesis, serine synthesis from glycine, and methionine synthesis from homocystine (Shane and Stokstad, 1985; Appling, 1991). Measurement of red blood cell, serum, and tissue folate content of the various folate forms as well as measurement of the urinary histidine metabolite, formiminoglutamic acid, is used to assess folate nutritional status (Shane and Stokstad, 1985; Clifford et al., 1989; Ward and Nixon, 1990; Varela-Moreiras and Selhub, 1992). Furthermore, folate biosynthesis by intestinal bacteria may meet much of the folate requirement (Rong et al., 1991). Diets inadequate in choline, methionine, and cobalamin may induce deficiency through the various interactions of these compounds (Thenen and Stokstad, 1973). Dietary concentrations between 0.5 and 10 mg/kg diet (1.1 and 23 µmol/kg) have been used. Studies by Clifford et al. (1989) have demonstrated that 2 mg folic acid/kg diet (4.5 µmol/kg) was no more effective than 1 mg/kg (2.3 µmol/kg) in young Sprague-Dawley rats that had been depleted of folic acid for 52 days. Thus, 1 mg/kg diet (about 2.3 µmol/kg) is recommended.

Signs of Folate Deficiency Induction of deficiency requires prolonged periods of feeding, usually with an amino acid-based diet and/or with an antibiotic to inhibit intestinal bacteria (Clifford et al., 1989; Ward and Nixon, 1990). Decreased growth rate, leukopenia, anemia, and formiminoglutamate excretion are reported.

Niacin

The only known roles of niacin are as components of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which function as electron carriers dehydrogenases in intermediary metabolism.

Both NAD and NADP can be synthesized from tryptophan in the liver of the rat, so pure niacin deficiency does not occur. Harris and Kodicek (1950) found that 24 moles of tryptophan were equivalent to 1 mole of niacin. Hundley (1947) found that rats fed a diet containing 15 percent casein required niacin for optimal growth, but rats fed a 20 percent casein diet did not require additional niacin.

Several investigators have reported that niacin-free diets containing fructose and low concentrations of tryptophan resulted in niacin deficiency. The amount of niacin required to restore normal growth was 15 mg/kg diet (120 µmol/kg) (Krehl et al., 1946; Henderson et al., 1947; Hundley, 1949). The requirement for niacin is much lower with diets containing glucose or starch than with high-sucrose or -fructose diets (Hundley, 1949). A high-fat (40 percent peanut oil) diet was found to decrease the urinary excretion of niacin metabolites, apparently because of an inhibition in the conversion of tryptophan to niacin (Shastri et al., 1968). Fleming and Barrows (1982) found that aging did not influence the absorption of nicotinic acid.

The AIN-93G diet (Reeves et al., 1993b) was formulated to contain 30 mg nicotinic acid/kg (244 µmol/kg). This provides an adequate amount for any experimental condition.

Signs of Niacin Deficiency Niacin deficiency causes reduced growth rate, rough coat, alopecia, and tissue concentrations of NAD and NADP are lowered. The concentrations of myelin in brain tissue are reduced as a result of a reduced synthesis of cerebrosides (Nakashima and Suzue, 1982, 1984).

Signs of Niacin Toxicity As the main excretory metabolites of nicotinic acid and nicotinamide are methylated, administration of high concentrations of niacin depletes the methyl pools of the body. Handler and Dann (1942) found that 10 g nicotinic acid/kg (80,000 µmol/kg) in a 10 percent casein diet caused an increase in fatty acids in the liver similar to a choline deficiency but without weight loss. A concentration of 2.5 g nicotinamide/kg diet (20,000 µmol/kg) caused the fatty liver and 10 g/kg diet (80,000 µmol/kg) decreased the rate of weight gain. The fatty livers could be prevented by including 1.5 g choline chloride/kg diet (11 mmol/kg), but choline alone would not support growth. Adding 0.6 percent methionine to the diet would correct the fatty liver and support growth. Growth could also be supported by 1.5 g choline chloride/kg and 0.6 percent homocystine in the diet. Jaus et al. (1977) noted that the injection of 0.50 g nicotinamide/kg BW (4,000 µmol/kg BW) twice daily caused enlarged liver cells and increased glycogen deposits. The effect was more pronounced in female rats. The injection of 0.6 g nicotinamide/kg BW (4,900 µmol/kg BW) per day resulted in reduced weight gain, and as little as 0.05 g/kg BW/day (400 µmol/kg BW/day) induced fatty livers (Kang-Lee et al., 1983; Sun et al., 1986). The LD₅₀ for nicotinic acid is 4.3 to 4.9 g/kg BW (35,000 to 40,000 µmol/kg BW), but 0.85 g/kg BW/day (6,900 µmol/kg BW/day) was tolerated for 40 days with no abnormal histology. Nicotinamide is more toxic, and the LD₅₀ is 1.7 to 3.0 g/kg BW (14,000 to 25,000 µmol/kg BW) (Unna, 1939; Brazda and Coulson, 1946).

Pantothenic Acid

Pantothenic acid functions as a constituent of coenzyme A and as a component of the acyl carrier protein in fatty acid synthesis. Pure pantothenic acid is an unstable viscous oil. Because pantothenic acid is difficult to handle, calcium pantothenate is normally used in diets. Only the d-pantothenic acid isomer has biological activity. The l-isomer does not have pantothenic acid activity and is actually a pantothenic acid inhibitor when its concentration is 100-fold greater than the d-isomer (Kimura et al., 1980). Some commercial preparations are equal molar mixtures of the d- and l-isomers and are only one-half as active as d-pantothenate. In this document the concentrations are expressed as the moles of d-pantothenic acid. One mole of calcium pantothenate is equivalent to 2 moles of d-pantothenic acid, and sodium pantothenate or hemicalcium pantothenate is only equivalent to 1 mole.

Unna (1940) found that 80 µg d-pantothenate/rat/day (0.36 µmol/rat/day) was necessary to maintain optimal growth. Henderson et al. (1942) found that only 40 µg/day (0.17 µmol/day) was needed to prevent the graying of black or hooded rats, that 80 µg/rat/day (0.36 µmol/rat/day) was required for optimal growth, and that about 100 µg/rat/day (0.42 µmol/rat/day) was necessary before pantothenate was excreted in the urine. Barboriak et al. (1957a) found 4 mg calcium pantothenate/kg diet (17 µmol pantothenic acid/kg diet) was needed to obtain optimal growth confirming the earlier values. This concentration would maintain pregnancy, but would not support lactation (Barboriak et al., 1957b). Nelson and Evans (1961) obtained normal growth of the suckling pups from dams receiving 10 mg calcium d-pantothenate/kg diet (42 µmol pantothenic acid/kg).

The AIN-76 (American Institute of Nutrition, 1977) and AIN-93G (Reeves et al., 1993b) diets were formulated to contain 15 mg calcium d-pantothenate/kg diet (63 µmol pantothenic acid/kg). The diets appear to contain an adequate safety margin because no problems related to pantothenic acid have been reported.

The antibiotics aureomycin, streptomycin, penicillin (Lih and Baumann, 1951; Sauberlich, 1952), and hygromycin (Barboriak and Krehl, 1957) delayed the appearance of the signs of pantothenic acid deficiency. In the later stages of the deficiency the signs develop in spite of the antibiotics. The effect seems to be related to changes in the intestinal microflora as the antibiotics did not delay the onset of deficiency when they were injected.

Signs of Pantothenic Acid Deficiency Pantothenic acid deficiency induces achromotrichia, exfoliative dermatitis, oral hyperkeratosis, necrosis, and ulceration of the gastrointestinal tract. Focal or generalized hemorrhagic necrosis of the adrenals may occur, and death results after 4 to 6 weeks of deficiency (Ralli and Dumm, 1953). Deficient rats had impaired antibody synthesis, decreased serum globulins, and decreased antibody forming cells in response to antigen. Restoration of antibody synthesis was achieved by parenteral administration of calcium pantothenate beginning 9 days before antigen injection (Lederer et al., 1975).

Signs of Pantothenic Acid Toxicity Pantothenic acid is relatively nontoxic. When excess amounts are consumed most of the excess is excreted in the urine. Daily administration of 185 mg (840 µmol) of pantothenate for 6 months did not produce any pathological changes in the rats (Unna and Greslin, 1941). The LD₅₀ for rats was 3 g/kg BW/day (13,700 µmol/kg BW/day).

Riboflavin

Riboflavin is the precursor of the flavin coenzymes and is stored in the liver primarily as flavin adenine dinucleotide (FAD) (Rivlin, 1970). The coenzymes function with many oxidation-reduction enzymes, e.g., cytochrome c reductase, xanthine oxidase, and diaphorase. They are required, as is vitamin B₆, for the conversion of tryptophan to nicotinic acid. Riboflavin is required for normal metabolism of vitamin B₆ and folate coenzymes (Baker and Frank, 1968; Rivlin, 1970; Tamburro et al., 1971). The requirement is influenced by the dietary content of carbohydrate. Starch increases intestinal synthesis of the vitamin and decreases the required amount in the diet; sucrose does the reverse. Rats fed high-fiber diets had lower red blood cell riboflavin concentrations (Brady et al., 1983).

Adult rats fed diets providing 31 kcal ME/day (130 kJ ME/day) and 0 or 12 mg riboflavin/kg diet were compared to groups provided energy supplements containing sucrose:starch:corn oil (10:3:1 by weight). Glutathione reductase activity coefficients in liver, gastrocnemius, and soleus muscles, and erythrocytes were increased by riboflavin deficiency but were unaffected by the supplemental energy intake. The concentration of riboflavin in muscle decreased in the energy-restricted rats (Turkki et al., 1989). In another study, rats were fed 0.6 or 1.8 g casein/day and 30 mg riboflavin and then progressively restricted to 30 percent of energy intake 6.2 kcal ME/day (26 kJ ME/day); muscle riboflavin concentrations decreased during energy deprivation, did not return to normal with riboflavin supplementation at 100 mg/day, and were unaffected by the amount of protein intake (Turkki and Degruccio, 1983). The effect of protein on the riboflavin requirement was shown to be related to the rate of growth and not to the protein intake per se (Turkki and Holtzapple, 1982; Turkki et al., 1986).

Maximum hepatic storage of flavins was found in rats fed 40 mg/day (equivalent to 2.7 mg/kg diet) and the maximum weight gain at 30 mg/day (equivalent to 2 mg/kg diet) (Bessey et al., 1958). A dietary content of 0.9 or 1.2 mg/kg produced hepatic storage in rats equivalent to that produced by 15.6 or 23.0 mg/kg (Gaudin-Harding et al., 1971). A dietary concentration of 3 mg/kg saturated the pools of riboflavin, flavin mononucletotide (FMN), and FAD in the retina (Batey and Eckhert, 1992). The effect of exercise has been studied in relation to riboflavin status in the rat. It did not increase the requirement in growing rats but did increase the concentration of flavins in the gastrocnemius and soleus muscles (Hunter and Turkki, 1987). Studies to determine the minimal requirement for riboflavin suggested that it may be as low as 3.2 mg/rat/day, the equivalent of 0.2 mg/kg diet; but the dietary concentration recommended was 17 mg/rat/day or about 1.2 mg/kg diet (Anonymous, 1972). The dietary requirement based on growth response and hepatic stores is 2 to 3 mg riboflavin/kg diet. The requirement can be expressed on a caloric basis as 0.6 to 0.8 mg riboflavin/1,000 kcal ME.

Offspring of dams fed a diet containing 1 mg riboflavin/kg after weaning had decreased body weight, brain weight, and brain DNA content compared to offspring of rats fed a diet containing 8 mg/kg. Correction of these defects in the riboflavin-deficient young was achieved by feeding their dams 2.7 mg/kg diet during lactation but not by supplementation of the diet of the offspring after weaning (Fordyce and Driskell, 1975). In another study rats were fed diets containing 0.40, 0.52, 0.65, or 15.4 mg riboflavin/kg diet 4 weeks before mating, throughout pregnancy, and up to day 15 of lactation (Duerden and Bates, 1985). Fetal resorption occurred in rats receiving 0.4 mg/kg, but those receiving 0.52 mg/kg had successful litters. Dams and their offspring receiving 0.52 or 0.65 mg/kg had higher erythrocyte glutathione reductase activity coefficients and their liver riboflavin concentrations were lower than those fed 15 mg/kg. The concentration of milk riboflavin in the rats fed 0.52 or 0.65 mg/kg was about one-eighth that of rats fed 15 mg/kg diet. The offspring of lactating dams fed diets containing 2, 4, 6, 8, 12, or 16 mg riboflavin/kg had blood glutathione reductase activity coefficients of 1.45, 1.12, 1.12, and 1.12, respectively, at 17 days postpartum. At day 34 the activity coefficients for those fed 4 and 8 mg/kg diet were 1.09 and 1.10 (Leclerc and Miller, 1987). In pregnant rats 4 mg/kg diet produced growth and hepatic riboflavin stores equivalent to 100 mg/kg diet (Schumacher et al., 1965). The intestinal transport of riboflavin is decreased during the period between 14 days to 3 months old and remained constant up to 26 months (Said et al., 1985; Said and Hollander, 1985). The requirement for normal reproduction is 3 to 4 mg/kg diet.

Signs of Riboflavin Deficiency The classical signs of riboflavin deficiency are dermatitis, alopecia, weakness, and decreased growth. Corneal vascularization and ulceration, cataract formation, anemia, and myelin degeneration may occur (Horwitt, 1954). The activity coefficient of lenticular glutathione reductase was increased and gamma crystallin lowered by deficiency (Bhat, 1982, 1987). Riboflavin-deficient rats may have fatty liver, abnormal hepatocyte mitochondria, and metabolic abnormalities of hepatocytes. The complex metabolic effects of riboflavin deficiency have been reviewed and summarized as (1) a decrease in flavoproteins involved in cellular oxidations; (2) increased protein turnover and an increased pool of free amino acids, which result in increased amounts of enzymes associated with amino acid metabolism, particularly enzymes of gluconeogenesis; and (3) a large decrease in mitochondrial respiration and adenosine triphosphate (ATP) synthesis (Garthoff et al., 1973). Reproductive performance is decreased in both males and females; offspring of deficient females may have congenital anomalies. On day 18 of gestation the iron-mobilizing activity in placental mitochondria was reduced. Maternal iron stores were higher and fetal tissue iron was unaffected presumably because of reduced fetal mass that limited maternal iron depletion and maternofetal iron transfer (Powers, 1987).

Riboflavin deficiency leads to a reduction in the storage of liver and spleen iron, transferring saturation, hemoglobin concentrations, plasma iron, iron absorption, and liver ferritin-Fe (Adelekan and Thurnham, 1986a; Yu and Cho, 1989; Shanghai, 1991). When deficiencies of riboflavin and iron were induced, riboflavin had a sparing effect on iron status because of the reduction in growth rate induced by the vitamin deficiency (Adelekan and Thurnham, 1986b). In rats fed purified diets deficient in riboflavin, both red blood cell and hepatic glutathione reductase was significantly decreased (Bamji and Sharada, 1972). Red blood cells of deficient rats have decreased fluidity and increased membrane bound acetylcholinesterase, increased glutathione peroxidase activity, and higher concentrations of peroxidation products (Levin et al., 1990). Increases in the amount of lipid peroxides were observed in serum and liver after a 5-week deficiency (Taniguchi, 1980). Rats fed riboflavin-deficient diets had decreased activities of hepatic flavokinase and FAD synthetase but not FMN phosphatase and FAD pyrophosphatase (Lee and McCormick, 1983). Rats deficient in riboflavin had depressed hepatic folate stores despite adequate or even increased intake of folate (Tamburro et al., 1971). Riboflavin-deficient rats prevented from coprophagy had lower hepatic methylene-tetrahydrofolate reductase activity but not lower dihydrofolate reductase activity (Bates and Fuller, 1986). Deficient rats had lower activities of the mitochondrial FAD-dependent straight-chain acyl-CoA dehydrogenases and the branched-chain acyl-CoA dehydrogenases (Veitch et al., 1988), NADPH-cytochrome c reductase (Taniguchi, 1980; Wang et al., 1985), ethoxycoumarin-0-deethylase and aryl hydrocarbon hydroxylase (Hietanen et al., 1980), and aflatoxin B₁ activation and DNA adduct formation (Prabhu et al., 1989). Plasma and tissue carnitine concentrations were reduced by deficiency (Khan-Siddiqui and Bamji, 1987). Riboflavin-deficient rats failed to increase their food intake when fed energy-diluted diets even when given insulin. However, cold exposure stimulated their intake (Matsuo and Suzuoki, 1982).

The activity coefficient of glutathione reductase in red blood cells, liver, and skin correlate with chronic marginal riboflavin deficiency (0, 0.5, 1.0, and 1.5 mg/kg diet). Hepatic and renal FAD is conserved at the expense of riboflavin and FMN. ATP:riboflavin 5-phosphotransferase was decreased in proportion to the concentration of dietary riboflavin, but ATP:FMN adenylyltransferase (FAD pyrophosphorylase) was increased in severely deficient rats. A reduction in succinate:(acceptor) oxidoreductase (succinate dehydrogenase) and NADH:(acceptor) oxidoreductase (NADH dehydrogenase) was tissue dependent (Prentice and Bates, 1981).

Signs of Riboflavin Toxicity Excessive concentrations of dietary riboflavin have been shown to decrease survivability of newborn rat pups. Mortality during the first week of life was increased 19 percent in a strain of Long-Evans rats by increasing the concentration from 6 to 12 mg/kg (Eckhert, 1987), and the percent of newborn Sprague-Dawley rats surviving to weaning was reduced 7 percent by increasing riboflavin from 8 to 80 mg/kg (Shirley, 1982). Chronic intakes of 12 mg/kg have been shown to cause photoreceptor damage (Eckhert et al., 1989, 1991).

Thiamin

Thiamin is the precursor of thiamin pyrophosphate, which is the storage form and the coenzyme for oxidative decarboxylation and other oxidative reactions. The requirement for thiamin in the diet of the rat depends in part on the quantity and source of dietary energy and is increased by increasing carbohydrate. It has been reported to decrease when dietary fat was increased (Scott and Griffith, 1957), but not in all cases (Murdock et al., 1974). Xylitol had a sparing effect on thiamin resulting from an increase in intestinal facultative bacteria that have the ability to synthesize the vitamin (Rofe et al., 1982). Diabetic rats may have an increased requirement for the vitamin as a result of the alterations in glucose turnover (Berant et al., 1988).

Male weanling rats fed a diet containing either 1.25 or 12.5 mg thiamin/kg diet did not differ in their feed efficiency ratio, but rats fed the higher concentration of thiamin grew faster (Mackerer et al., 1973). There was no significant difference in growth of rats fed either 5 or 50 mg thiamin/kg diet (Itokawa and Fujiwara, 1973). Growth curves of young male rats fed diets containing 0, 0.3, 0.6, 1.5, 6.0, 30.0, and 100.0 mg/kg were used to calculate the amount of thiamin required for maximum growth (Mercer et al., 1986). The theoretical maximum growth rate was 7.0 g/day, and a concentration of 3.68 mg/kg was sufficient to achieve 99 percent (6.93 g/day) of this rate. A concentration of 3.3 mg/kg supported retention of carcass thiamin in nonpregnant and pregnant female rats fed an 18.2 percent protein diet and 3.75 kcal ME/kg diet (15.7 kJ ME/kg) (Roth-Maier et al., 1990). The requirement for growth is 4 mg thiamin-HCl/kg diet.

In pregnant rats the concentration of plasma thiamin remained constant throughout the first 18 days of gestation, while erythrocyte thiamin reached a maximum at day 11 and then declined (Chen et al., 1984). Urinary excretion of thiamin in pregnant rats fed diets containing 2, 4, 6, or 8 mg/kg were stable up to day 16 of gestation and then decreased until parturition (Leclerc, 1991). Pregnant rats fed diets containing 0, 0.8, 1.7, 3.3, 6.7, 13.3, 20, 26.7, 100, 1,000, and 10,000 mg/kg were evaluated for organ thiamin retention (Roth-Maier et al., 1990). The amount of thiamin retained by the liver, muscle, brain, and whole carcass increased with each increase in dietary thiamin. Pregnant rats were fed diets that contained either 4 or 100 mg thiamin/kg. The hepatic stores of thiamin were higher at weaning in the offspring of dams fed the higher concentration, but growth was not affected (Schumacher et al., 1965). The requirement for pregnancy and lactation is 4 mg thiamin-HCl/kg diet.

Signs of Thiamin Deficiency Thiamin deficiency can be induced readily and produces anorexia and weight loss with an increase in food spillage (Tagliaferro and Levitsky, 1982) and in coprophagy (Fajardo and Hornicke, 1989). Thiamin-deficient rats avoid eating sucrose (Yudkin, 1979) and are slower to respond to tasks where food pellets are used for reinforcement (Hashimoto, 1981). Evaluations of behavior demonstrated that rats maintained on thiamin-deficient diets showed muricide aggression (Onodera et al., 1981; Onodera, 1987). After 7 days of deficiency there is a decrease in white and red blood cells and a drop in hemoglobin. After 30 days this is reversed and reticulocytes and plasma erythropoietin are increased, but red blood cell 2,3-diphosphoglycerate, membrane cholesterol, and phospholipids decrease (Hobara and Yasuhara, 1981). After 4 weeks there is a decrease in liver thiamin and an elevation in plasma branched-chain amino acids, α-ketoacids, and α-hydroxyacids (Shigematsu et al., 1989). Deficiency during pregnancy resulted in intrauterine growth retardation (Roecklein et al., 1985) and decreased activity of the thiamin-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase (Fournier and Butterworth, 1990) and gangliosides (Vaswani, 1985) in newborn rat brains. Acetylcholine was reduced in weanling rats of thiamin-deficient mothers (Kulkarni and Gaitonde, 1983).

In the adult, deficiency results in abnormalities of the central and peripheral nervous systems and the heart. Chronic thiamin deprivation leads to selective neuropathological damage in the brain. Rats become ataxic and a decrease in pyruvate dehydrogenase occurs in the midbrain and lateral vestibular nucleus (Butterworth et al., 1985). Conduction velocity in peripheral nerves was reduced, but axonal transport was increased (McLane et al., 1987). Treatment with pyrithiamine, a thiamine phosphokinase inhibitor, in combination with deficiency, resulted in a decrease in brain amino acids and monoamines (Langlais et al., 1988) and in the activities of glutamic acid decarboxylase and GABA-transaminase (Thompson and McGeer, 1985). Thiamin deficiency produced an increase in cardiac weight, a decrease in cardiac and renal ATP and pyruvate carboxylase, but no cardiac ultrastructural abnormalities (Schenker et al., 1969; McCandless et al., 1970). Cytochrome P-450 concentration and drug metabolizing ability are increased (Wade et al., 1983; Yoo et al., 1990). Thiamin deficiency resulted in an increase in liver nicotinamide methyltransferase and in the excretion of N-1-methylnicotinamide (Shibata, 1986).

Thiamin status has been reported to be influenced by folate deficiency. Folate-deficient rats had decreased absorption of low doses of thiamin, but large doses were absorbed normally (Howard et al., 1974). In an earlier study (Thomson et al., 1972), folate-deficient rats fed 22 mg/kg diet had a significant depletion of thiamin in blood and liver but not in brain. In a more recent study (Walzem and Clifford, 1988b), however, no differences were observed in either thiamin absorption or excretion as a result of folate deficiency.

The enzymatic activity of transketolase in blood and tissues of thiamin-deficient animals correlates with thiamin status, and it may or may not be restored in vitro by the addition of thiamin pyrophosphate (TPP). This may depend on the duration of the deficiency and the resultant instability of the apoenzyme (Brin, 1966; Pearson, 1967; Warnock, 1970; Bamji and Sharada, 1972; Walzem and Clifford, 1988a).

Chromium

Because supplemental trivalent chromium has been reported to have an enhancing effect on insulin and glucose metabolism, it has been suggested that chromium is essential for the rat and that chromium's function is to aid in the utilization of glucose. The work of Schwarz and Mertz (1959), Schroeder et al. (1963), Schroeder (1966), Roginski and Mertz (1967), Mertz et al. (1965), Mertz and Roginski (1969), and Roginski and Mertz (1969), using highly restrictive environmental conditions, often are cited as evidence for the essentiality of chromium for the rat. Whether this constitutes a beneficial physiological function is uncertain. Specificity is questioned because other heavy metals may initiate similar effects (Fagin et al., 1987; Pederson et al., 1989).

Other studies have failed to show positive effects of chromium on glucose tolerance or glucose utilization by tissues of rats (Woolliscroft and Barbosa, 1977; Flatt et al., 1989; Holdsworth and Neville, 1990). Woolliscroft and Barbosa (1977) fed 6-week-old Sprague-Dawley rats 30 percent torula yeast diets containing low-chromium concentrations (30 to 100 µg/kg, estimated) or diets that contained 5,000 µg Cr/kg. After 6 weeks, there was no significant difference in intravenous glucose tolerance between the two groups. Flatt et al. (1989) found no significant difference in food intake, body weight gain, glycosylated hemoglobin, plasma glucose, plasma insulin, glucose tolerance, or insulin sensitivity between two groups of weanling Wistar rats fed either 30 or 1,000 µg Cr/kg diet for 32 days. Differences in chromium concentrations in tissues between the two groups was variable—from no change in skeletal muscle to a 44 percent reduction in the pancreas.

Holdsworth and Neville (1990) found no effect of dietary chromium supplementation on glucose metabolism in rats. They fed weanling Wistar rats Torula yeast diets similar to those designed by Schwarz (1951) but supplemented with L-cystine, L-methionine, and L-histidine. These supplemented diets supported more rapid growth than the original diet and contained 100 (low-chromium diet) or 1,000 µg Cr/kg (high-chromium diet). A control group was fed a commercial natural-ingredient diet. After 5 weeks, the rats fed the Torula yeast diets gained 30 percent less weight than did the control rats, regardless of whether chromium was present. Those fed chromium-supplemented yeast diets did not grow at a significantly higher rate than those without supplemental chromium. The incorporation of glucose carbon into liver glycogen in the rats fed the low-chromium diet was only one-fifth that of the control rats, but was not different from that of rats given the chromium-supplemented yeast diet. Yeast was grown in media with or without chromium. Extracts from this yeast enhanced glucose incorporation into glycogen of hepatocytes isolated from rats fed low- or high-chromium diets regardless of whether chromium was present in the extract.

Others have reported lower sperm counts in rats fed low-chromium diets (<100 µg/kg) for 8 months than in rats fed high-chromium diets (2,000 µg/kg) (Anderson and Polansky, 1981). Effects of chromium supplementation on weight gain in rats are achieved only with restrictive environmental conditions (Schroeder et al., 1963). Under similar conditions, supplementation of the diet with other heavy metals such as cadmium and lead also enhance initial weight gain, suggesting a nonspecific pharmacological response rather than a nutritional response (Schroeder et al., 1963).

Although the earlier studies seemed to indicate that dietary chromium supplements enhanced glucose metabolism, the more recent studies did not. It could be argued that the duration of the experiments and environmental conditions in the later studies were not sufficient to allow chromium stores to be depleted and deficiency signs to be expressed. Signs of chromium deprivation might have been more evident if longer feeding periods or multiple-generation studies had been used.

Trivalent chromium salts, chromic oxide, and metallic chromium have low orders of toxicity; however, because of their oxidizing properties, chromium trioxide, chromates, and bichromates are potent poisons. A detailed discussion of tolerance concentrations for chromium in animals can be found in Mineral Tolerance of Domestic Animals (National Research Council, 1980).

Lithium

Pickett and O'Dell (1992) fed rats a low-lithium diet (5 µg/kg) through five successive generations and found that the weaning weights of the offspring were significantly lower than weaning weights from dams fed diets with 500 µg Li/kg. Other experiments showed that litter size and birth weights were decreased by feeding rats low-lithium diets through three generations. They also showed an interaction between lithium and sodium in that low-lithium effects were exaggerated in rats fed high-sodium diets. Earlier studies by this laboratory (Patt et al., 1978; Pickett, 1983) showed that second- and third-generation females fed low-lithium diets were less fertile than controls. These studies suggest that diets containing less than 10 µg Li/kg fed to rats through multiple generations could impair reproductive performance.

Signs of Lithium Toxicity Lithium in high doses can be toxic to the kidney. The minimal toxic concentration of dietary lithium is unknown, but rats fed 280 mg Li/kg diet from 0 to 65 weeks of age developed renal failure (Nyengaard et al., 1994). Nephrotoxicity occurred in rats given 14 mg Li/kg BW/day subcutaneously for 8 days (Qureshi et al., 1992). Rat embryos grown in rat serum with a lithium concentration of 0.6 mmol/L showed signs of toxicity. Until more is learned about the minimal toxic concentration of dietary lithium, it is recommended that the dietary concentration not exceed 1 mg/kg diet (Hansen et al., 1990).

Nickel

Nickel biochemistry plays a prominent role in the metabolism of anaerobic bacteria, plants, and tunicates. Many plant species contain the enzyme urease, which is nickel dependent (Cammack, 1988). Although investigators have shown some cause to believe that nickel is essential for animals, the results among experiments are not consistent. Lederer and Lourau (1948) first suggested that nickel was involved in hematopoiesis because it activated an enzyme required for this process. This began a series of nutritional experiments designed to show a relationship between dietary nickel and iron metabolism. Initial studies showed that nickel-deficient rats showed deficiency signs that could be alleviated by ingesting an adequate dietary concentration of iron (Schneggg and Kirchgessner, 1975a,b, 1976a,b, 1978). Subsequent studies by other investigators (Nielsen et al., 1979; Nielsen, 1980a,b, 1984) demonstrated that the apparent nickel-iron interaction in rats depended on the form of iron fed; however, these investigators could not show a consistent effect of nickel deprivation. They suggested that previously observed effects of nickel on iron metabolism were pharmacological rather than physiological because the amount of nickel supplementation was so high.

Studies with nickel have been carried through multiple generations. Nielsen et al. (1975) fed rats low-nickel diets (2 to 15 µg Ni/kg diet) for three generations and reported that this had no effect on growth of the offspring, but thriftiness and coat condition were worse in the nickel-deprived rats than in similar rats fed diets containing added nickel (3,000 µg/kg). They also found lower hematocrits in the deprived rats than in controls. These results strongly suggest that nickel is essential for the well-being of the rat, but the experiments have not been repeated or confirmed in other laboratories. Other studies have shown that higher concentrations of dietary nickel (20 µg Ni/kg diet) increased weight gain in F₁ and F₂ generation neonatal rats (Nielsen et al., 1979). However, growth rate in rats from weanling to 10 weeks old was not affected by this amount of nickel in the diet (Nielsen et al., 1984).

Studies to determine the possible interaction between nickel and other nutrients have not established with certainty that nickel is essential for growth or any known biochemical process in animal tissues (Nielsen et al., 1989). Given the question about pharmacological versus physiological actions (Nielsen et al., 1984), the lack of marked pathological effects with low dietary intakes of nickel, and the lack of defined biochemical functions in animals, it is not certain that nickel is essential.

Signs of Nickel Toxicity The amount of dietary nickel required to cause a toxic response in rats is relatively high. Numerous studies have demonstrated that rats have no adverse effects when fed 100 to 1,000 mg Ni/kg diet (Phatak and Patwardhan, 1950; Ambrose et al., 1976) or only lose weight (at 1,000 mg/kg diet; Whanger, 1973). Schnegg and Kirchgessner (1976b) found that rats fed 1,000 mg Ni/kg diet developed many abnormal physiological responses such as increased hematocrit, hemoglobin, and serum protein.

Silicon

Earlier work in two separate laboratories suggested that silicon was an essential nutrient for animals. Carlisle (1972) and Schwarz and Milne (1972) described the effects of silicon supplementation on growth of rats and chicks. Schwarz and Milne observed that the addition of silicon to diets at 500 mg/kg led to increased growth rates in rats fed added silicon as opposed those fed diets not supplemented with silicon. These data may be somewhat misleading because the maximal growth rate of the controls was only 25 to 50 percent of the rate expected for the strain of rat used. This suggests that the diets were generally deficient in some other nutrient(s). Carlisle (1972) showed similar results in chicks fed 100 mg Si/kg diet, but the maximal weight gain of the control chicks was relatively small, only 25 percent of the normal rate for chicks of this age. As in the experiments with rats, the diets seemed to be generally inadequate to support rapid growth.

Although these studies have been cited as establishing silicon essentiality, they have not been verified by subsequent studies. Elliot and Edwards (1991) found that weight gains were depressed in chicks fed 250 mg Si/kg diet compared to those fed diets with no added silicon. No significant effects on growth or any other measures were found in the animals fed the higher silicon concentration compared to those not receiving silicon in their diet. In this experiment the chicks grew at a rate expected for the age and strain.

A number of experiments have shown effects of dietary silicon supplementation on various physiological measures in rats, but none has been able to confirm that the effects are nutritional (Carlisle, 1970; Emerick and Kayongo-Male, 1990a,b; Carlisle et al., 1991).

Signs of Silicon Toxicity Large doses of silicon in the form of tetraethylorthosilicate (2 percent: »2,600 mg Si/kg diet) cause urolithiasis in the rat. Death occurred in some rats as a result of urethral obstruction. The lesion was enhanced as the dietary calcium concentration was increased (Schreier and Emerick, 1986).

Sulfur

Sulfur is required as an integral part of sulfur-containing amino acids and vitamins. Michells and Smith (1965) showed that dietary sulfate was readily incorporated into cartilage of Wistar rats and spared methionine for other purposes. Bernhart and Tomarelli (1966) reported a positive effect on growth of Sprague-Dawley rats with added sulfate in the diet. When fed an 8.8 percent lactalbumin diet with a mineral mix that met the requirements of the rat (National Research Council, 1962), growth was improved by inclusion of 1,000 mg sulfate/kg diet. No increased growth response was observed when sulfate was included in diets with adequate protein.

Jacob and Forbes (1969) found that weanling Sprague-Dawley rats grew slightly better when fed 15 percent casein diets supplemented with methionine and 350 mg S/kg than with a similar diet supplemented with methionine and 40 mg S/kg as sulfate. Smith (1973) reported that 200 mg inorganic sulfate/kg diet was optimal for adult Long-Evans rats because this amount of inorganic sulfate reduced expiration of ¹⁴CO₂ from a test dose of 1-¹⁴C-methionine. On the basis of these limited data, 300 mg S/kg diet as inorganic sulfate may be beneficial.

Vanadium

In the early 1970s there were several reports from different laboratories that led to the conclusion that vanadium was an essential trace mineral for the rat (Schwarz and Milne, 1971; Strasia, 1971; Hopkins and Mohr, 1974); however, this conclusion has not been supported by all studies (Williams, 1973). Later studies suggest that the results of the previous work demonstrated pharmacological rather than nutritional actions of vanadium (Nielsen, 1984; Nechay et al., 1986; Nielsen and Uthus, 1990).

Reports of the pharmacologic effects of vanadium are numerous. One of the most studied effects is on insulin action. Vanadium is an insulinomimetic agent in vitro and may be in vivo as well. Given in the drinking water, vanadium was shown to lower blood glucose and reduce the activity of phosphotyrosyl-protein phosphatase in the liver of mice (Meyerovitch et al., 1991). The concentration of vanadium given was many times higher than that found in a normal rodent diet, however. Others have shown that pervanadate mimics insulin action by activating the insulin receptor kinase (Fantus et al., 1989). Seaborn et al. (1992) found serum glucose significantly lower in male guinea pigs fed 500 µg V/kg diet compared to those fed less than 10 µg/kg. However, plasma cortisol in the vanadium-fed guinea pigs was elevated more than 100 percent over that of guinea pigs not fed vanadium, suggesting that vanadium in the diet might have stressed the animals. Other measurements were enhanced by vanadium in the diet, but because there were no criteria for normalcy in these studies, the results suggest that vanadium may have caused a toxic reaction rather than demonstrating nutritive value.

Because vanadium is known to be required by some haloperoxidases in lower life forms (Yu and Whittaker, 1989), it has been suggested that peroxidases involved in iodine metabolism in animals may be vanadium-dependent. Some studies have attempted to show an interaction between iodine and vanadium, but the findings were inconclusive and more definitive experiments have not been forthcoming (Uthus and Nielsen, 1990). Because of the lack of definitive and repeatable experiments on the nutritive response to vanadium, its essentiality for the rat or other mammals is uncertain.

Signs of Vanadium Toxicity The toxicity of vanadium is probably manifested through its effect on tissue enzyme activity. Vanadium has been shown to inhibit numerous enzymes that hydrolyze phosphate esters, including ribonuclease (Lindquist et al., 1973), acid and alkaline phosphatases (Lopez et al., 1976), and phosphotyrosyl-protein phosphatase (Swarup et al., 1982). Sodium, K-ATPase also is inactivated by vanadium (Nieder et al., 1979). Vanadium activates other enzymes such as adenylate cyclase (Grupp et al., 1979) and enhances the phosphorylation of the tyrosyl moiety on proteins. The latter is apparently involved in the overstimulation of membrane receptors, as seen when vanadium stimulates insulin action (Fantus et al., 1989). Rau et al. (1987) found that vanadate stimulated NADH oxidation in microsomes with an increased production of hydrogen peroxide and possible superoxide. Earlier work showed that as little as 25 mg V/kg diet caused visible signs of toxicity such as reduced growth and food utilization in the rat (Franke and Moxon, 1937; Hansard, 1975).

Ascorbic Acid

Rats do not require a dietary source of ascorbic acid. Enzymatic synthesis of this vitamin can occur via glucuronolactone or gulonolactone in the liver. However, ascorbic acid is a potentially beneficial dietary constituent.

Rats fed ascorbic acid store the vitamin as ascorbic acid and ascorbic acid 2-sulfate (Pillai et al., 1990). Ascorbic acid sulfotransferase was increased, whereas ascorbic acid-2-sulfate sulfohydrolase was reduced in activity in ascorbic acid-supplemented rats. When ascorbic acid was withdrawn from the diet, tissue ascorbic acid, ascorbic acid 2-sulfate, and the activity of ascorbic acid sulfotransferase were reduced and ascorbic acid-2-sulfate sulfohydrolase was increased (Pillai et al., 1990).

Ascorbic acid may be potentially beneficial in thiamin and vitamin B₁₂ deficiencies. Five percent ascorbic acid in the diet supported normal weight gain in thiamin-deficient rats and increased the fecal content of thiamin (Scott and Griffith, 1957; Murdock et al., 1974). The inclusion of 100 mg ascorbic acid/kg in a vitamin B₁₂-deficient diet raised liver vitamin B₁₂ concentrations in rats (Thenen, 1989).

Several interactions of ascorbic acid with minerals have been identified in rats. Magnesium deficiency reduced ascorbic acid concentration in liver and kidney, as well as the enzymatic synthesis of the vitamin from glucuronolactone or gulonolactone in liver (Hsu et al., 1983). Supplementation of the diet with high iron (5 mg/rat/day) decreased tissue, blood, and urinary concentrations of ascorbic acid (Majumder et al., 1975). However, dietary ascorbic acid increased the absorption of nonheme iron in rats but to a lesser extent than in humans (Reddy and Cook, 1991). Ascorbic acid fed at 1 percent of the diet decreased tissue copper and, in the presence of high iron (191 mg/kg), caused severe anemia and reductions in ceruloplasmin in copper-deficient rats (Johnson and Murphy, 1988). A 1 percent ascorbic acid diet decreased the efficiency of intestinal copper absorption. When copper was given intraperitoneally, however, the rate of copper excretion was decreased (Van den Berg et al., 1989). Lead exposure reduced brain ascorbic acid concentrations (Seshadri et al., 1982). Ascorbic acid given orally was as effective on a molar basis as was parenterally administered EDTA in removing lead from the central nervous system (Goyer and Cherian, 1979). In rats fed lead (500 mg/kg diet) the addition of ascorbic acid as 1 percent of the diet and of 400 mg Fe/kg diet decreased the accumulation of lead in the tissues and prevented growth depression, anemia, and food intake decreases (Suzuki and Yoshida, 1979).

Ascorbic acid may help protect against peroxidation and spare vitamin E. It has been shown to decrease expired pentane, used as a marker for lipid peroxidation (Dillard et al., 1984). Ascorbic acid supplementation reduced the elevation of liver thiobarbituric acid values and reversed decreases in hepatic pyruvate kinase, aspartate aminotransferase, plasma creatine phosphokinase, and vitamin E in vitamin E-deficient rats (Chen and Thacker, 1987). However, high concentrations of ascorbic acid (1.5 g/kg diet) increased in vitro erythrocyte hemolysis and liver lipid peroxidation while lowering reduced glutathione in plasma and erythrocytes (Chen, 1981).

A rat mutant (ODS) unable to synthesize ascorbic acid because of a lack of L-gluconolactone oxidase has been identified (Mizushima et al., 1984). Poor growth, muscle and leg joint hemorrhage, decreased cytochrome P-450, elevated serum and adrenal corticosterone, and lowered urinary excretion of hydroxyproline were prevented in the ODS rat by feeding them 300 mg ascorbic acid/kg diet (Horio et al., 1985). Concentrations of 1,000 to 3,000 mg ascorbic acid/kg diet were required to achieve maximum activities of several microsomal drug-metabolizing enzymes in the liver of these rats when exposed to polychlorinated biphenyls (PCBs) (Horio et al., 1986). A dietary concentration of 250 mg/kg increased survival time to at least 36 weeks following exposure to N-butyl-N-(4-hydroxybutyl)-nitrosamine to induce bladder cancer, while unsupplemented rats died within 4 weeks (Mori et al., 1988). Unsupplemented ODS rats developed an increase in ovarian aromatase activity (Tsuji et al., 1989). LDL cholesterol was higher in unsupplemented ODS rats (Horio et al., 1991).

Myo-inositol

Myo-inositol is not required by rats in conventional laboratory conditions, but Burton and Wells (1976) reported a requirement in lactating rats fed antibacterial drugs. Lactating rats fed phthalylsulfathiazole, to decrease myo-inositol contribution from the microflora, developed fatty livers with increased concentrations of cholesterol esters and triglycerides, but plasma lipoprotein lipid concentrations were depressed. These alterations in the lactating dam were corrected by supplementing the diet with 0.5 percent myo-inositol (Wells and Burton, 1978). The free myo-inositol content of milk is 80 mg/100 g in rat's milk and 4 mg/100 g in cow's milk. Myo-inositol concentration in milk was influenced by dietary intake (Byun and Jenness, 1982).

Galactose, when fed in high concentrations, results in an accumulation of polyol products, galactitol and sorbitol, which alter osmoregulation and deplete myo-inositol. The accumulation of polyols and depletion of myo-inositol can be prevented by feeding myo-inositol or aldose reductase inhibitors (Bondy et al., 1990). In diabetic rats the accumulation of sorbitol and depletion of myo-inositol in peripheral nerves reduces axonal transport of choline acetyltransferase, choline-containing lipids, and motor nerve conduction velocity. Maintaining the concentration of myo-inositol in tissue, either through ingesting myo-inositol or by the inhibition of aldose reductase, can prevent these changes (Greene et al., 1982; Tomlinson et al., 1986). Na⁺-K⁺-ATPase in diabetic rats was increased in nerve but not kidney tissue by ingesting dietary myo-inositol (Finegold and Strychor, 1988). A phospholipid-derived protein kinase C agonist that is myo-inositol dependent may be involved (J. Kim et al., 1991).

A comparison of rats fed diets containing 0 or 5 g myo-inositol/kg diet demonstrated that after 3 to 4 days, supplementation decreased the activities of liver fatty acid synthetase and acetyl-CoA followed by a return to unsupplemented concentrations (Beach and Flick, 1982). Liver triglyceride accumulation in rats fed diets devoid of myo-inositol only occurred in young rats and decreased with age (Andersen and Holub, 1980). Rats fed a liquid formula diet with myo-inositol supplementation (114 or 250 mg/100 g) did not exhibit any differences in weight gain, liver fat, or myelination of the brain; but supplemented rats had higher tissue free and lipid-bound myo-inositol concentrations (Burton et al., 1976).