IHIWMHW 1W THlILiIHI K" 3:523“:th This is to certify that the thesis entitled Effects of Diet and_Coprophagy on the Body Pool Size and Balance of Pantothenic Acid in Rats presented by Mary Lyn Greeley has been accepted towards fulfillment of the requirements for _Ma_S_t_ELS_degree in Human Nutrition t/MWW/ profér Date—JJLlLJ._1.9.9_Q_ 0—7639 MS U is an Affirmative Action/Equal Opportunity Institution ~._k, ._ +7‘. 4 , 7, A“. __ #. PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. A DATE DUE DATE DUE DATE DUE H m 05 us: I \., 'i____ fif MSU I: An Affirmative Action/Equal Opportunity Institution cMcWWS-DJ EFFECTS OF DIET AND COPROPHAGY ON THE BODY POOL SIZE AND BALANCE OF PANTOTHENIC ACID IN RATS BY Mary Lyn Greeley A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1990 颢~ 073/93 ABSTRACT EFFECTS OF DIET AND COPROPHAGY ON THE BODY POOL SIZE AND BALANCE OF PANTOTHENIC ACID IN RATS BY Mary Lyn Greeley Pantothenic acid (PA) balance and body pool were examined in rats to understand why PA deficiency is rare and develops slowly. In Study 1, 33 rats were fitted with tail cups and fed a PA deficient diet, ad lib, or a PA control diet, pairfed. In Study 2, 29 rats were fed a PA deficient diet ad lib, fitted with tail cups, or pairfed, without tail cups. In both studies urine and feces were collected daily and rats were killed at the beginning and end of the study for determination of whole body PA pool. The whole body PA pool was significantly decreased in the deficient diet, tail cup group; maintained in the deficient diet, pairfed, no tail cup group; and significantly increased in the control diet, tail cup group. Rats fed the deficient diet excreted significantly less urinary PA than control diet rats. Fecal PA loss was significantly less than urinary PA loss in all groups and did not differ between groups in each study. The pantothenic acid pool was thus affected by both diet and coprophagy. ACKNOWLEDGEMENTS I would like to express my appreciation to Dr. Won Song. Her constant support and encouragement have allowed me to succeed in my graduate program. The members of my committee have also given valuable input: Drs. Bergen, Chenoweth and Romsos. I have also greatly appreciated the friendships of Cheryl, Dana, Debbie, Leslie, Mary, Ruven and Sherri. Finally I would like to thank my husband Will for his love and unending faith in my abilities. TABLE OF CONTENTS List of Tables .............................. ........ v List of Figures ..................................... vi IntrOduction O.......OOOOOOOO......O...’ ..... O ...... O 1 Review of Literature Discovery of Pantothenic Acid .................... Pantothenic Acid Function ........................ Deficiency Signs and Symptoms .................... Metabolic Effects of Deficiency .................. Requirement for Pantothenic Acid ................. 1O Pantothenic Acid Intake .......................... 12 Pantothenic Acid Absorption ...................... 14 Uptake and Synthesis of CoA ...................... 15 Pantothenic Acid Excretion ....................... 18 Tissue Content and Body Pool Size of Pantothenic Acid .............................. 23 flat-hub Pantothenic Acid Balance ...... ......... . ....... .. 25 Materials and Methods Preliminary Experiments .................. ..... ... 31 Research Design .................................. 35 Sample Collection ................................ 38 Sample Preparation ............................... 39 Pantothenic Acid Determination ................... 41 Statistical Analysis ............................. 43 Results Food Intake and Weight Gain ...................... 44 Pantothenate Intake .............................. 46 Tissue Pantothenate Concentration and Whole Body Pool .............................. 48 Pantothenate Excretion: Urinary ................. 51 Pantothenate Excretion: Fecal ................... 53 Pantothenate Balance: Intake - Excretion ........ 57 Pantothenate Balance versus Measured Change in the Whole Body Pool .................... 59 Pantothenate in Circulation: Whole Blood ........ 62 Discussion .......................................... 68 Conclusions ......................................... 75 Recommendations ..................................... 76 Appendices A. Sample Collection Protocols .................. 77 B. Individual Raw Data for Rats ................. 85 List of References ..................................108 iv Table Table Table Table Table Table Table Table Table Table Table LIST OF TABLES Reported Urinary Excretion of Pantothenic Acid in Humans ................ Reported Urinary Pantothenic Acid with Varying Intakes in Humans ............ Reported Human Balance Studies ............ AIN-76 Semipurified Pantothenate Deficient Diet Composition ................ Tissue Pantothenate Concentration and Whole Body Pool ....................... Pantothenate Excretion: Urinary and Fecal ................................. Feces Collected ........................... Fecal Pantothenate Concentration .......... Pantothenate Balance ...................... Balance versus Measured Pool Change .................................... Pantothenate Content in Blood 19 21 26 36 49 52 55 56 58 61 63 LIST OF FIGURES Figure 1: Dietary Intake of Rats ........ ...... ..... 45 Figure 2: Growth Pattern of Rats ................... 47 Figure 3: Correlation of Whole Blood and Tissue Pantothenic Acid Content .......... 65 Figure 4: Correlation of Whole Blood and Whole Body Pantothenic Acid .............. 66 Figure 5: Correlation of Whole Body and Tissue Pantothenic Acid Content .......... 67 vi INTRODUCTION Pantothenic acid (pantothenate), a B vitamin, is an essential nutrient for humans and many animals for growth, reproduction and normal physiological function (Robishaw and Neeley, 1985). At the cellular level the vitamin is a precursor for two compounds, Coenzyme A (CoA) and the phosphopantetheine in acyl carrier protein. CoA and acyl carrier protein are essential for more than 100 metabolic pathways in the body (Robishaw and Neeley, 1985). Although pantothenate is necessary for metabolic processes, a deficiency of the vitamin is rarely reported. While some clinical symptoms of deficiency have been reported after long term dietary depletion of the vitamin, no overt deficiency symptoms have been reported in free living populations (Hodges et al., 1958). Signs of pantothenic acid deficiency observed in rats include growth retardation, a rough hair coat, muscle weakness and eventual death (Reibel et al., 1982). These signs are rather general in nature and take a long time to develop, thus there is the possibility of a large body pool size and a slow loss of the vitamin. Also to be considered is the possibility of other sources of pantothenic acid available to the rat maintained on a 1 2 deficient diet. Sources of the vitamin may include any trace amount of pantothenic acid in the deficient diet, pantothenic acid obtained through coprophagia or microbial synthesis of pantothenic acid in the gut. A number of studies have evaluated the distribution of pantothenic acid in rat body organs (Hatano, 1962; Srinivasan and Belavady, 1976). When coprophagia was not controlled, rats fed a pantothenic acid deficient diet had a maintained or decreased organ pantothenate concentration but an apparent maintainance or increase in pantothenate as estimated from total organ pantothenic acid content (Hatano, 1962). We questioned whether this increase in organ pantothenate content was a reflection of actual changes in the pantothenate body pool or if it resulted from a shift of pantothenate from organs not analyzed. We also questioned how much of the organ pantothenate pool changes could be explained by the balance of the vitamin. The goal of this study was to address why pantothenic acid deficiency is rare and develops slowly by examining the pantothenate balance and body pool size in rats, controlling for diet and coprophagia. We hypothesized three relationships: 1) the body pool size of pantothenate in rats is altered in deficient rats by changing the pantothenate intake from the diet; 2) the changes in the pantothenate body pool size are explained by the pantothenate balance: 3) coprophagia is a significant source of pantothenate in deficient rats. 3 Specific objectives of the study are as follows: 1) 2) 3) 4) 5) To determine the changes in the body pool size of pantothenate over time in rats fed a pantothenate deficient or a sufficient diet To estimate the pantothenate balance by quantitating pantothenate intake and excretion in urine and feces To compare the changes in the pantothenate body pool size with the pantothenate balance over time To evaluate the significance of c0prophagia as a pantothenate source To determine the correlations between pantothenate blood concentration with the pantothenate whole body pool, and pantothenate blood concentration with pantothenate tissue concentration REVIEW OF LITERATURE DISCOVERY OF PANTOTHENIC ACID Pantothenic acid, a B vitamin, was first discovered in 1933 by Williams et al. as a growth factor for yeast. In 1939 Jukes recognized its importance as a chick antidermatitis factor and Wooley et a1. (1939) as a liver filtrate factor of rats. The importance of pantothenic acid in humans was unquestionably realized in 1947 when its primary biochemically active form was found to be CoA (Lipman, et al., 1947), an element necessary for carbohydrate, fat, and protein metabolism. PANTOTHENIC ACID FUNCTION Pantothenic acid is comprised of beta-alanine and a dihydroxy acid (pantoic acid). Biochemically pantothenic acid functions as part of CoA and phosphopantetheine of acyl carrier protein. CoA and acyl carrier protein are key cofacors in the body, functioning in over one hundred reactions (Robishaw and Neely, 1985). Pantothenic acid, in the form of CoA, is important 4 5 in carbohydrate metabolism. Prior to the citric acid cycle, CoA acts as an acyl acceptor for pyruvate in its conversion to acetyl CoA. Within the citric acid cycle it plays a similar role in the conversion of alpha- ketoglutarate to succinyl CoA. In the process of gluconeogenesis, CoA is necessary for the activity of pyruvate carboxylase, an enzyme needed for the conversion of pyruvate into glucose. In protein metabolism branched chain amino acids require CoA for their breakdown to acetyl, propionyl, or succinyl CoA which can be used for energy. Acetyl CoA and succinyl CoA enter into the citric acid cycle directly while propionyl CoA must be metabolized to methylmalonyl CoA and then succinyl CoA before its entry. In the oxidation of fatty acids in the mitochondria pantothenate plays a role as a part of CoA. Prior to entry into the mitochondria fatty acids are activated in a reaction involving CoA. Via a special transport mechanism involving carnitine, the activated fatty acids are transported into the mitochondria. The fatty acyl CoAs are degraded in a continuous B-oxidation process involving CoA, where one shortened acyl CoA, and one acetyl CoA are produced. When fatty acids are used as a major energy source, ketone bodies are formed, via activation with CoA. Acetyl CoA is converted to acetoacetate and then to hydroxybutyrate and acetone. 6 Pantothenic acid is also involved in the synthesis of many compounds. As a structural component of acyl carrier protein, the pantothenic acid is required in the elongation of the fatty acid chain by the sequential addition of two carbon units. The acetyl CoA pool is used in the production of prostaglandins, cholesterol and steroid hormones. DEFICIENCY SIGNS AND SYMPTOMS Pantothenic acid deficiency signs have been defined in many experimental animals, including rats and chickens. Symptoms observed in rats were: a failure to grow (Morris and Lippincott, 1941), scaley dermatitis (Gyorgy, et al., 1939), inhibition of the immune system (Alexrod, et al., 1947) and adrenal insufficiency (Cowgill, et al., 1952: Eissenstein, 1957). Chicks fed a pantothenic acid deficient diet have shown symptoms of retarded growth, skin lesions and nervous derangements (Gries and Scott, 1972). In man, symptoms of spontaneous pantothenic acid deficiency have not been observed. Deficiency signs like gastrointestinal discomfort and personality changes did begin however after six weeks on a synthetic diet devoid of pantothenic acid (Hodges, et al., 1958). A study by Fry et al. (1976), with ten males consuming a diet completely completely deficient in pantothenic acid for 7 ten weeks produced symtoms of fatigue and listlessness in the men, but no further specific clinical symptoms of pantothenic acid deficiency. When a synthetic diet devoid of pantothenic acid was consumed along with the pantothenic acid antagonist, omega-methylpantothenic acid, deficiency symptoms were produced after two weeks (Bean and Hodges, 1954). Four healthy men consuming the antagonist complained of fatigue, muscular weakness, vomiting, depression, and insomnia. Thus through long term vitamin depletion or use of an antagonist, various nonspecific symtoms of pantothenic acid deficiency have been observed, but no specific, clinical signs reported. METABOLIC EFFECTS OF DEFICIENCY Pantothenic acid plays a key role in many biochemical reactions in the body, thereby altering several metabolic pathways when deficient. Pathways affected include: CoA levels, pyruvate utilization, gluconeogenesis and lipid metabolism. A number of authors have found decreased CoA levels in tissues under the condition of dietary pantothenic acid deficiency (Olson and Kaplan, 1948: Srinivasan and Belavady, 1976). Olson and Kaplan (1948) reported that thirty day old rats maintained on a pantothenic acid-free 8 diet for up to nine weeks had a normal CoA content in heart, liver, kidney and adrenal tissues after three weeks but showed a gradual depletion to a level 35-40% of normal by nine weeks. Srinivasan and Belavady (1976) found that weanling rats fed a diet without pantothenate for a period of six weeks had less than 50% of the pantothenate and CoA in their livers, compared to control rats. Reibel et al. (1982) fed rats a pantothenic acid deficient diet (less than 1 mg pantothenic acid per kilogram diet) for four and eight weeks. The levels of pantothenic acid in the heart, kidney, gastrocnemius muscle, liver, and testes were lowered by at least seventy percent, while CoA levels were maintained in each of the organs. The apparent contradiction between the study by Reibel and the earlier studies may be due in part to three factors. First, Reibel's rats were provided with a small amount of pantothenic acid in their diet while the earlier studies specified pantothenate free diets. Second, Olson and Kaplan (1948) and Reibel et al. (1982) did not indicate the type of housing rats were caged in, to attempt to control for coprophagy. Srinivasan and Belavady (1976) however reported that their rats were housed in individual, raised, wire bottom cages. Finally, Olson and Kaplan (1948) and Srinivasan and Belavady (1976) used younger animals than did Reibel et al. (1982) and the pantothenic acid requirement has been found to decrease with age. Although Reibel et a1. (1982) found that the deficient rats were able to maintain CoA levels, they did not grow at normal rates. Thus pantothenic acid may affect metabolism, specfically growth, in some fashion other than through the function of CoA, such as acyl carrier protein. Mice maintained on a pantothenate deficient diet for 9 to 15 weeks had significantly (p<.05) lower CoA levels than controls in liver, kidney, spleen, heart and leg muscle (Smith et al, 1987). The effect of pantothenic acid deficiency upon pyruvate utilization was studied in rat and duck tissues (Olson and Kaplan, 1948). After nine weeks on a diet devoid of pantothenic acid, liver slices from deficient rats metabolized pyruvate more poorly (no significance indicated) than control rat’s tissues. Methodology included incubating liver slices from sacrificed rats with or without sodium pyruvate for two hours and then determining oxygen consumption and total pyruvate utilization. The impairment of gluconeogenesis in pantothenic acid deficiency (Srinivasan and Belavady, 1976) has also been reported. In this case the activity of hexosediphosphatase (fructose 1,6-diphosphatase), a key enzyme in the gluconeogenic pathway, in rat liver tissue showed a significant (p<.01) reduction in rats fed no 10 pantothenate for six weeks. Hexosediphosphatase activity was measured in a 105,000 x g liver supernatant fraction. Also liver glycogen contents and blood glucose levels were lower in deficient animals. Lipid metabolism is also reported to be affected by a deficiency in pantothenic acid. After six weeks of deprivation of the vitamin, Klein and Lipmann (1953) noted a decrease in the rate of synthesis of fatty acids and cholesterol in rat liver slices incubated with carboxyl- labelled acetate. Another study found that in the presence of octanoate, liver slices from 8 rats maintained on a synthetic deficient diet for an average of eighty days had significantly (p<.01) lower oxygen uptake and a lower ketone-body accumulation than 13 rats fed a supplement of pantothenic acid (Carter and Hockaday, 1962). This impairment in the capacity of the liver to oxidize octanoate would seem to indicate an inefficiency in catabolism. REQUIREMENT FOR PANTOTHENIC ACID Weanling rats consuming 5.3 g food daily required 80 to 100 ug of pantothenate for optimal growth. In contrast, a 10-week old rat with a food consumption of 15.5 gm had a daily requirement of approximately 25 ug pantothenate for optimal growth and prevention of characteristic deficiency lesions (Unna and Richards). 11 Thus the pantothenic acid requirement of the rat appeared to decrease with age. In a study by Barboriak et al. (1956) rats with initial weights of 50 g were fed 0, .2, .4, .8, 1 or 10 mg of calcium pantothenate per 100 g of diet. The rats on the deficient diet ceased to grow after 14 to 20 days and all rats in this group were dead by the 75th day of the study. The animals receiving .2 mg of calcium pantothenate per 100 grams of diet showed significantly lower weight gains than the rats receiving more pantothenate during the first one half year of the experiment. An acetaylation study was done after one year of the experiment (Barboriak et al., 1956). Each rat was injected intraperitoneally with 3 mg of sulfanilamide. For the following 24 hours urine was collected and analyzed for total and free sulfanilamide. The animals receiving the lowest vitamin level showed the lowest acetylation value, 33.6%. Those receiving 8 mg pantothenate per kg of diet had the highest acetylation percentage, 76.1%. The 1 and 10 mg pantothenate per kg diet groups acetylated 71 and 67%, respectively. These data would seem to indicate that adult rats required a minimum of 8 mg pantothenate per kg of diet for optimal metabolic function. The lower pantothenate requirement of adult rats, indicated by Unna and Richards (1942) was related only to growth. Thus the period in which rats may 12 be most sensitive to decreased pantothenic acid intake is during the period of rapid growth but for optimum metabolic function a minimum of 8 mg of calcium pantothenate per kg of diet is required throughout the life of the rat. PANTOTHENIC ACID INTAKE No reports of spontaneous pantothenic acid deficiency have been reported in the free living human population, partly due to the good sources of pantothenic acid available. Sources include: eggs, liver, milk, peanuts, bran and baker's yeast (Orr, 1969). The National Research Council’s current Estimated Safe and Adequate Daily Dietary Intake (ESADDI) for pantothenic acid is 4-7 mg (RDA, 1989). Currently there is not enough quantitative evidence to establish an official Recommended Dietary Allowance for pantothenic acid. The daily intake of sixty-three adolescents, as determined by a four-day self reported dietary record, ranged from 1.7 to 12.7 mg pantothenate/day. The average intake of pantothenate was 4.14 mg/day for females and 6.25 mg/day for males (Eissenstat, et al., 1986). No significant difference was found however in the nutrient densities between sexes, which were 2.17 mg pantothenate/1000 kcal for females and 2.34 mg pantothenate/1000 kcal for males. Kathman and Kies (1984) 13 reported similar results with pantothenic acid intakes of adolescents averaging 5.55 mg/day. Pantothenic acid intake of institutionalized and noninstitutionalized elderly persons was recorded as 5.8 and 5.9 mg/day respectively (Srinivasan and Belavady, 1981). In contrast with these groups of elderly and adolescents, whose intakes reached at least the minimal ESADDI of 4 mg, at least three studies have identified populations whose pantothenate intake did not meet the ESADDI. First, a study of pregnant and lactating women indicated that members of this group had intakes less than the ESADDI when they did not take pantothenic acid supplements (Song, et al., 1985). The second study, by Cohenour and Galloway (1972) similarly reported that pantothenic acid status was unsatisfactory in both pregnant and nonpregnant female teenagers, as assessed by dietary intake. In the third study, by Emmons (1986), four weekly 24-hour recalls were taken from 76 white and black low-income families. With the midpoints of the suggested ESADDI ranges used as a standard, pantothenate intake was found to be 75% of the ESADDI. Thus while overt pantothenic acid deficiency symptoms are rarely seen there are populations who's intake would be considered less than optimal when compared to the ESADDI for pantothenic acid. 14 PANTOTHENIC ACID ABSORPTION Pantothenic acid is mostly present in the bound (CoA) form in foods. The bound form cannot be directly absorbed through the mucosal cells and thus must be hydrolyzed to free pantothenic acid (Kaplan and Lipmann, 1948) before absorption can occur. This hydrolysis takes place in the intestinal lumen as follows: CoA ; dephosphoCoA )phosphopantetheine pantetheine > pantothenate Intestinal tissue absorbs pantetheine and pantothenate but transports only pantothenate. Pantetheinase, the enzyme which converts pantetheine to pantothenate is found in the intestinal lumen and tissue (Ono, et al., 1974). One model for pantothenic acid absorption is that of simple diffusion (Shibata, et al., 1983; Turner and Hughes, 1962). In in vivo experiments Shibata et al. perfused sodium [1-14C] pantothenic acid into the isolated upper 25-35 cm of the rat small intestine. Labelled pantothenic acid concentrations from 7.5 uM to 90 mM were injected into the lumen. After 20, 40 and 60 minutes the intestine was removed and the contents rinsed with water. The amount of 1-14C pantothenic acid in the intestinal contents and the intestinal tissue was determined. The 15 results indicated that the absorption process is not saturable and therefore absorption occurs by simple diffusion. Using a substrate concentration of 100 uM Turner and Hughes (1962) likewise did not detect transport of pantothenic acid against a concentration gradient in everted sacs of rat intestine. They too concluded that absorption occurs by simple diffusion. In contrast to the simple diffusion theory, Fenstermacher and Rose (1986) indicated there is a saturable, energy-dependent absorption mechanism, specific for pantothenate. At a low concentration, .9 uM, [3H]pantothenate was absorbed by a specific transport mechanism which can be described as a Na-dependent, secondary active transport process. The movement of pantothenate was seen in only one direction across the membrane of the jejunum. Since Fenstermacher and Rose used pantothenic acid concentrations lower than those in Shibata et al. (1985) and Turner and Hughes (1962) experiments there appears to be two separate absorption mechanisms, depending on the pantothenate concentration in the lumen. UPTAKE AND SYNTHESIS OF CoA Once pantothenic acid is absorbed and reaches the bloodstream, it travels in that form until it reaches the 16 tissues where cellular uptake is apparently affected by metabolic and hormonal factors (Reibel et al., 1981; 1982). Radiolabelled pantothenic acid injected into the rat was found in highest concentration in the kidneys, liver and heart. This was true both one hour and one week after a single dosage of radiolabelled sodium pantothenate was administered (Pietrzik and Horning, 1980). At the tissues, CoA can again be synthesized from pantothenate, L-cysteine and ATP according to the following scheme (Smith and Savage, 1980): pantothenate ——) 4'phosphopantothenate -—¢>4'phosphopanto- thenylcysteine -—-94’phosphopantethine )dephospho-CoA CoA Glucagon, dibutyryl cyclic AMP and dexamethasone have been reported to increase the rate of production of coenzyme A in adult rat liver parenchymal cells (Smith and Savage, 1980). This demonstrates a mechanism whereby the biosynthesis of CoA in the liver could be regulated through hormones. The primary site of control in the production of CoA is the first step, a panothenate kinase-catalyzed reaction (Robishaw, et al., 1982). In isolated, perfused rat heart the rate of the pantothenate to 4'phosphopantothenate reaction is inhibited by glucose, pyruvate, fatty acids, 17 and beta-hydroxybutyrate. Insulin also was a strong inhibitor in the presence of glucose. This again illustrates how the biosynthesis of CoA is controlled by many factors. Fasting and diabetes also affect pantothenic acid and CoA metabolism. In rat liver, fasting and diabetes resulted in accelerated rates of (14C) pantothenic acid uptake, higher tissue concentrations of pantothenic acid, increased incorporation of 14C pantothenic acid into CoA and an elevated tissue level of CoA (Reibel, et al., 1981). In the heart of diabetic rats, Lopaschuk (1988) found that insulin, in the presence of glucose, significantly (p<.05) increased pantothenate uptake. Beinlich et al. (1989) studied CoA synthesis in hearts from diabetic and control rats in vitro. When a perfusate of pantothenate, cysteine and dithiothreitol was available, CoA synthesis was stimulated in control hearts. In diabetic hearts the rate was reduced due to a decreased rate of pantothenate phosphorylation and decreased pantothenate transport. Reibel et al. (1981) went a step further, noting that in cardiac muscle placed under fasting and diabetic conditions, increased incorporation of (14C) pantothenic acid into CoA and increased CoA levels were associated with a decrease in both pantothenic acid uptake and tissue pantothenic acid. Both liver and heart issues indicate the hormonal 18 control of CoA synthesis, but in the heart higher CoA levels and accelerated pantothenate incorporation into CoA were seen when tissue concentration of pantothenate was lower (as compared to control tissues). This observation indicates that in the heart synthesis of CoA or the level of CoA are controlled by some mechanism other than the increased availability of pantothenate. Thus, while the importance of pantothenic acid for CoA structure is clear, its rate of incorporation into CoA is independent of pantothenate concentration. PANTOTHENIC ACID EXCRETION Urinary excretion of pantothenic acid generally parallels intake in normal healthy populations who consume self-selected American diets. In a study addressing pantothenic acid excretion on three different levels of daily pantothenate intake, 2.8, 7.8, and 12.8 mg, pantothenic acid excretion responded to changes in intake (Fox and Linkswiler, 1961). Subjects consumed each of the three pantothenate level diets for two five day periods and consumed a self-chosen diet (6.7 mg pantothenic acid/day), for two five day periods, for a total of forty days (Table 1). 19 Table 1. Reported Urinary Excretion of Pantothenic Acid in Humans Intake (mg/day) Urine (mg/day) 6.7 i 2.1* 3.9 t 1.5 2.8 i 0 3.2 i 0.8 7.8 i 0 4.5 i 1.0 12.8 :i: 0 5.6 i- 0.6 Reported by Fox and Linkswiler, 1961 * Values represent mean 1 standard deviation An experiment concerning the pantothenic acid status of adolescents showed urinary excretion of pantothenic acid to be highly correlated (r = 0.60) with a dietary intake range of 1.7 to 12.7 mg/day (Eissenstat, et al., 1986). In contrast, the Kathman and Kies study (1984), with an intake range of 4.03 to 7.93 mg/day for adolescents did not find a correlation between intake and excretion. Both studies utilized four-day food records and 24-hour urine collections. The differences between the two studies can be explained by several factors. Methodological differences include the method used for calculation of dietary intake. Kathman used the Orr (1969) handbook values while Eissenstat used the NUTREDFO computer data base, developed 20 at Utah State University, for which the original data sources were not indicated. Also, Kathman determined pantothenic acid content of the urine microbiologically, while Eissenstat's analysis was done through radioimmunoassay. The subjects in the two studies differed, Kathman studied 11 subjects while Eissenstat had 63. The mean ages of the subjects also differed. Eissenstat's study also included greater variation in pantothenic acid intake (1.7 - 12.7 mg/day versus 4.0 - 7.9 mg/day in Kathman and Kies study), which could be a better model for prediction of the relationship between the biochemical variables and dietary intake. The amount of counseling provided to the clients concerning the dietary records and the completeness of the 24-hour urine collections could also have contributed to the different results in the two studies. Tarr et al. (1981) reported that healthy males who had consumed low amounts of pantothenic acid, 3.1-3.5 mg/day, for 35 days, showed little or no increase in urinary pantothenic acid when provided with a high pantothenic acid diet, 8.2 mg/day, for 35 days (Table 2). Thus it could be speculated that 3.1 to 3.5 mg/day was below the requirement and led to a depletion of tissue storage which, upon repletion, was restored so an increased pantothenate excretion in urine was not seen. In contrast, subjects initially on the high pantothenic acid diet (6.6 to 8.2 mg/day), when switched to the low 21 intake diet (4.6 mg/day), decreased their pantothenic acid excretion by greater than 50%. In this case it appears that in order to maintain tissue stores excretion decreased (the drop in intake prompted a drop in excretion). Table 2. Reported Urinary Pantothenic Acid with Varying Intakes in Humans Number of Pantothenate Pantothenate Subjects Intake Excretion ( rug/day) (mg/day ) 4 1st phase 3.1-3.5 1.3-1.6 2nd phase 8.2 1.2-2.2 4 lst phase 6.6-8.2 3.2-4.4 2nd phase 4.6 1.5-1.6 Reported by Tarr et al. (1981) Fry, et al. (1976) sudied urinary pantothenate excretion in two groups of adult male subjects for 63 days. One group was on a diet void of pantothenic aicd, the other on the same diet supplemented with 10 mg pantothenic acid/day. While pantothenic acid excretion dropped from 3.05 to .79 mg in subjects on the pantothenic 22 acid deficient diet over a period of nine weeks, the four subjects who consumed the supplements diet excreted 3.95 mg pantothenate at first but 5.84 mg pantothenic acid at the ninth week of the study. Both groups were then administered 100 mg pantothenic acid/day for one week. After the first day of the the 100 mg supplementation, previously unsupplemented subjects retained 63% of the test dose, while the supplemented group retained 48%. By the end of the seven days of supplementation both groups retained approximately 40% of the pantothenic acid (40 mg/day), suggesting that the body is capable of storing large amounts of the vitamin, regardless of previous status. Thus urinary excretion responds to both intake and body stores. The transport and metabolism mechanisms of urinary pantothenic acid excretion have been studied in the rat kidney (Karnitz, et al., 1984). At physiological pantothenate plasma concentrations (2 uM), filtered pantothenic acid was largely reabsorbed. In contrast, at higher concentrations of 5.0, 10.0, 20.0, and 30.0 uM pantothenic acid, pantothenic acid underwent tubular secretion to a greater extent. Uptake of pantothenic acid across the brush-border membrane of the proximal tubule was found to be a Na+-dependent, active transport process when the final concentration of pantothenic acid in the incubation tube was 2 uM. Along with the urinary and fecal routes of 23 pantothenic acid excretion, other avenues of pantothenate excretion may exist, such as respiratory pantothenate excretion, as suggested by Pietrzik and Horning, 1980. 1-14C pantothenate (specific activity 34.45 mCi/mmol) was dissolved in water to a final concentration of 100 uCi/ml and each rat was given 200 ul of this solution by stomach tube. Two hundred-twenty gram rats were fed a commercial diet and placed in metabolic chambers. By suction with an electric pump air passed through the metabolic chamber and the respiratory carbon dioxide was absorbed by ethanol amine. To determine the radioactivity a 500 ul aliquot from the absorption flasks was added to a mixture of ethanol and scintillator solution and counted. The respiratory route was found to contain 13-15% of the administered dose of 1-14C pantothenate. TISSUE CONTENT AND BODY POOL SIZE OF PANTOTHENIC ACID A number of studies have addressed to concentration of pantothenic acid in organs when varying amounts of the vitamin are supplied in the diet. A study specifically designed to estimate the entire body pool size of pantothenic acid has not preiously been reported however. The body pool size is the total amount of pantothenic acid within the body. Currently only rough estimates of the body pool size (and how that varies with the pantothenic acid content of the diet) are possible through the 24 summation of tissue pantothenate contents. Hatano (1962) determined the total amount of pantothenate in nine organs (liver, kidney, lung, spleen, brain, heart, testis, adrenal, and thymus) of weanling rats consuming diets deficient (less than .08 mg of pantothenate per kg of diet) or sufficient (40 mg of calcium pantothenate added per kg of the deficient diet) in pantothenic acid. After 32 days the pantothenate concentration (bound and free) in all organs decreased an average of 46% in the deficient rats, as compared to the control rats. By summing the total amount of pantothenic acid in nine organs a rough estimate of the organ pool size was made. After two and a half weeks on the deficient diet the nine organ pool size in the three rats average 321 i 3 ug pantothenate (mean i standard deviation), after four and a half weeks the pool size had reached 337 i.12 ug pantothenate. In comparison, the three rats consuming the sufficient diet had an organ pool size of 797 ug after four and a half weeks. One large tissue, muscle, which may be a considerable pantothenate reserve simply due to its size was not included in the study however. 25 PANTOTHENIC ACID BALANCE The body pool size of vitamins can be indirectly estimated over time from their balance if the initial body pool is known. By measuring the dietary intake and output of a nutrient (through the urine and feces), provided the vitamin is not synthesized or degraded within tissues of the body the body pool can be determined. If synthesis or degradation could occur controls must be made to account for the possible appearance or disappearance of the vitamin through these routes. The balance could be complicated by an extra source of the vitamin, such as would be available through intestinal microbial synthesis of the vitamin (Henderson et al., 1942). Three human balance studies have reported the intake and output of pantothenic acid (Table 3). The subjects of Oldham et al. (1946) and Denko et al. (1946) consumed typical American diets while the subjecs of Gardner et al. (1943) consumed a quart of milk at each meal and caramel candy ad libitum to supplement caloric intake. With intake levels of 3.3 and 6.9 mg pantothenate/day the intake approximated output in urine and feces (Oldham et al., 1946 and Gardner et al., 1943, respectively). When intake was very low, a negative balance (pantothenate intake - output) of pantothenic acid resulted (Denko et al., 1946). 26 Table 3: Reported Human Balance Studies Author Sample Average Intake Average Output Balance Size (mg PA/day) (mg PA/day) Urine Fecal Oldham,1946 12 3.3 i 1.1 2.3:.8 1.1:.3 -0.1 Gardner,1943 3 6.9* 6.01.7 .4i.3 0.6 Denko,1946 5 1.1* 1.0* 2.8* -2.7 Values represent mean 1 SD * No SD indicated When two of the seven subjects in Denko et al.’s study (1946) consumed supplements of 7.2 mg of pantothenic acid per day with their meals, fecal pantothenic acid excretion did not change, while urinary excretion returned to excretion levels found on the control diet (4.7 mg pantothenic acid/day). Thus while a significant amount of pantothenic acid was excreted in the feces, the excretion was independent of pantothenic acid levels in the diet. The availability of pantothenic acid synthesized by intestinal microflora may be questioned as the urinary 27 output of Denko et al.'s subjecs (1946) did not exceed the pantothenate intake. If the vitamin synthesized by microflora were a significant source of pantothenate (and was available to the host), a urinary output higher than intake may be expected. In rat balance studies, with daily intakes of 20 - 150 ug of pantothenic acid per day (80 to 100 ug of pantothenic acid are required per day for maximum growth), rat urinary pantothenic acid excretion paralleled intake. Urinary excretion of pantothenate was at no time greater than the amount consumed in the diet (Henderson et al., 1942). The percentage of dietary pantothenic acid excreted in the urine varied between 7 and 36%, with the most deficient rats excreting the least. Although a rat consuming deficient amounts of pantothenic acid retains a greater percentage of the dietary pantothenic acid, ultimately pantothenic acid deficiency signs will develop as the quantity of pantothenic acid retained is not adequate to maintain body processes or stores. In contrast to urinary pantothenic acid, the amount of pantothenic acid excreted in the feces of rats was almost independent of the diet: rats suffering from severe pantothenic acid deficiency excreted as much pantothenate as those receiving adequate pantothenate (Henderson et al., 1942). A balance study in rats is complicated by coprophagy. Rats housed in cages with wire screen floors consume 35 - 28 50% of excreted feces (Barnes et al., 1957). Daft et al. (1963) studied the effect of corprophagy on pantothenate excretion by feeding pantothenic acid-free diets for 10 to 44 days to rats maintained with or without tail cups. The rats without tail cups excreted an average of 5.6 ug (2.7 - 8.5 ug) pantothenic acid per day in the feces while those with tail cups voided 3.9 ug (3.3 - 4.5 ug) per day. Urinary pantothenate excretion was slightly higher in the rats without tail cups, averaging 1.3 (1.1 - 1.6) ug/day versus 0.9 (0.7 - 1.1) ug/day in the rats with tail cups. Dietary intake of the rats and fecal excretion were not reported. Deficient rats with tail cups failed to gain weight as rapidly as those without tail cups. After five weeks on a panothenic acid deficient diet rats with tail cups averaged a weight gain of 50 g while the rats without tail cups averaged a weight gain of 66 g (Mameesh et al., 1959). The apparent extra source of pantothenic acid available via coprophagy was not sufficient however to prevent pantothenic acid deficiency in the rats with access to their feces. Rats maintained without tail cups, fed a diet containing no pantothenic acid for four to six weeks, developed pantothenic acid deficiency signs including adrenal necrosis, a condition which was prevented by as little as 5 ug of pantothenate per day (Mills et al., 1940). An analysis of pantothenate in segments of the 29 intestinal tract of severely pantothenate deficient rats revealed over ten times as much fecal pantothenate in the in the cecum and colon as in the area from the stomach to the cecum (Henderson et al., 1942). Thus the synthesis of pantothenate by intestinal microflora is clearly supported but the availability of the pantothenate to the host may be questioned. Mameesh et al. (1959) studied the possibility and the availability of pantothenate produced endogenously. Weanling male Sprague-Dawley rats were housed in wire- bottom cages and fed pantothenate-limiting (1.0 mg pantothenate/kg diet) and pantothenate-adequate (20 mg pantothenate/kg diet) diets. The rats were further divided into groups such that half of the rats received 100 mg of oxytetracycline per kg of diet, an antibiotic shown to stimulate the growth of rats fed diets limiting in pantothenic acid. Although the exact mechanism of this growth stimulation is not known, it is generally agreed that the antibiotic increases the amount of limiting vitamin available to the rat. The rats were further divided, with one half of the rats fitted with tail cups to prevent coprophagy. Two experiments were carried out for a period of five weeks each, with six rats per group in Experiment 1 and five rats per group in Experiment 2. The results of the two experiments were in subsantial agreement. Oxytetracycline stimulated the growth of the rats fed 30 the pantothenate limiting diet, regardless of coprophagy, but did not affect the growth of the rats fed adequate pantothenate. Thus oxytetracycline had a sparing effect on the growth of pantothenate deficient rats with or without the practice of coprophagy. This indicated that microbially synthesized pantothenate was absorbed directly from the intestinal tract during its first passage through. Whether oxytetracycline acted by increasing the synthesis of pantothenate, increasing the absorption of pantothenate or by some other mechanism, could not be determined. In contrast, Daft et al., 1963, did not find that the antibiotic penicillin stimulated growth of rats fed a pantothenic acid deficient diet. Tail cups and penicillin were used in conventional rats fed diets very low in pantothenic acid. The three cupped animals experienced growth failure typically seen in rats fed a pantothenate deficient diet and died after 80 days on the deficient diet. With the studies by Mameesh et al., 1959 and Daft et al., 1963, reaching different conclusions regarding coprophagy, the significance of coprophagy as a pantothenic acid source is not known at this time. MATERIALS AND METHODS PRELIMINARY EXPERIMENTS A number of preliminary experiments were carried out to validate several methodological steps necessary for completion of this study. The first experiment was to determine the extent of destruction of pantothenic acid in tissues and organs by autoclaving. The second series of experiments were to determine the optimal activities of enzymes necessary to maximally release the bound forms of pantothenic acid from the feces and whole body carcasses. The third experiment was carried out to determine if daily feces samples, randomly divided into two equal aliquots by weight, contain equivalent amounts of pantothenate. The effect of fasting upon the blood pantothenate concentration was studied in the fourth experiment. Finally, the pantothenic acid body pool size of rats was studied in control and deficient rats. The effect of autoclaving on pantothenic acid was studied by the addition of known quantities of pantothenic acid to blood samples. Half of the samples were autoclaved for 60 minutes at 16.5 pounds of pressure and half of the samples were not autoclaved. The pantothenic 31 32 acid content of the blood was determined by radioimmunoassay (RIA) (Wyse et al., 1978). Recovery of pantothenic acid in the autoclaved blood was greater than 90%. Pantetheinase (provided by Dr. Carl Wittwer, University of Utah) and alkaline phosphatase (type VII-S, P-5521, Sigma Chemical Co.) were added in varying quantities to fecal and whole body carcass slurries. Graded amounts of alkaline phosphatase, 0, 10, 20, 30, 60, 60 and 120 units, with 0, 20, 10, 60, 30, 60, and 120 units of pantetheinase, respectively, were added to duplicate samples of 200 and 400 ul of whole body slurries. Whole body slurries were made by autoclaving the rat carcass after sacrifice, adding distilled water and homogenizing the carcass to a homogenous state. Fecal slurries were similarly autoclaved and homogenized. Graded amounts of alkaline phosphatase, 0, 30, 60, 120, 240 and 360 units with 0, 30, 60, 120, 240, 360 units of pantethenaise, respectively, were combined with duplicate samples of 200 ul of fecal slurries. The whole body and fecal samples, with the added enzymes, were incubated (37°C) for seven hours in a shaker water bath, deproteinized with equimolar concentrations of saturated Ba(OH)2 and 10% ZnSO4, centrifuged at 4,000 x g for 10 minutes, and the supernatants saved for pantothenic acid analysis. Samples were analyzed by radioimmunoassay in 33 duplicate. A combination of 20 units pantetheinase and 10 units alkaline phosphatase with 200 or 400 ul of whole body slurry, and 30 units of pantetheinase and 30 units alkaline phosphatase with 200 ul of fecal slurry maximally released pantothenate. Additional enzyme did not result in the release of significantly more pantothenate. In the blood at the various concentrations, the following amounts of pantothenic acid were released, respectively: .028, .095, .094, .054, .053, .047, .035 nmol pantothenic acid/50 ul of blood supernatant. In the feces at the various concentrations, the following amounts of pantothenic acid were released, respectively: .998, 1.75, 1.71, 1.59, 1.93, 1.84 nmol pantothenic acid/50 ul fecal supernatant. In the next preliminary experiment daily fecal excretions from rats were randomly divided into two equal aliquots by weight. Each aliquot was then separately processed in duplicate and the fecal PA content determined by the procedure described above. No differences in the total pantothenate amount were noted between the two aliquots (42.5 i 26.7 and 43.4 i 21.8 nmol pantothenic acid/g feces). Seven adult, male Sprague-Dawley rats fed a chow diet were used to determine the effect of fasting upon blood pantothenate concentration. Blood was removed from the tail veins of the rats on four different days. The first day nonfasted blood was collected, five days later 34 after a 12 hour fasting period blood was collected. After another two days nonfasted blood was collected and finally blood was again collected after a 12 hour fasting period five days later. The blood was hemolyzed, enzyme hydrolyzed, and deproteinized by a method previously established by Wyse et al., 1978. Analysis of the pantothenic acid content by radioimmunoassay revealed that a 12 hour period of fasting did not affect blood pantothenate concentration (fasted: .40 i .15 and nonfasted: .47 i .19). Finally, a study was done to investigate if there was a difference in the pantothenic acid body pool size of rats fitted with tail cups, fed a semipurified diet deficient or sufficient in pantothenate for thirty days. The study allowed for practice in fitting the tail cups, a technique to be used in studies to follow and established that diet did affect the body pool size of pantothenic acid. 35 RESEARCH DESIGN STUDY 1 Study 1 was done to establish the effect of diet on the body pool size of pantothenic acid in rats. Thirty- three weanling, male Sprague-Dawley rats (Supplier: Harlan, Indianapolis, Indiana) were housed in stainless steel metabolism cages in a room with a 12 hour light, 12 hour dark schedule. The temperature was maintained at 20- 22'C. The animals were fed, ad libitum, for two days the AIM-76 control semipurifed diet (Table 4), with an adequate amount of pantothenate (12 mg/kilogram of diet). Water was available to the rats at all times. After two days (Day 0 of the study) ten rats were sacrificed for determination of the baseline whole body pantothenate pool. The remaining twenty-three rats were divided into two groups: (1) Deficient group: 8 rats, fed ad libitum an AIM-76 purified diet deficient in pantothenate (+0 mg/kg diet) and (2) Control group: 15 rats, pairfed the AIM-76 purified diet sufficient in pantothenate (+12mg/kg diet) with the deficient ad libitum group. Rats in both groups were fitted with tail cups for the prevention of coprophagia. Tail cups were constructed of 20 ml polypropylene scintillation cocktail bottles with the bottoms cut off. The tail of the rat was put through 36 Table 4. AIM-76 semipurified pantothenate deficient diet composition Ingredient % by weight Sucrose 50.0 Casein (vitamin free) 20.0 Corn Starch 15.0 Fiber - Celufil 5.0 Corn Oil 5.0 AIN Mineral Mix 3.5 AIN Vitamin Mix (without pantothenate) 1.0 DL-Methionine .3 Choline Bitartrate .2 Control diet supplemented with 12 mg D-Calcium Pantothenate/ kilogram diet 37 the tail cup so the bottom of bottle was against the rat's body and the neck of the bottle could be taped to the rat's tail. The deficient diet, ad libitum, group with tail cups will hereafter be referred to as the DEF, TC group and the control diet, pairfed, group with tail cups will be called the CON, PF, TC group. STUDY 2 Study 2 was done to establish the effect of coprophagy on the body pool size of rats fed a diet deficient in pantothenic acid. Twenty-nine weanling, male Sprague Dawley rats were housed and fed as outlined in Study 1. After two days (Day 0 of the study) nine rats were sacrificed for determination of the baseline whole body pantothenate pool. The remaining twenty rats were divided equally into two groups, paired individually by weight: (1) Tail cup group: 10 rats fed ad libitum the AIM-76 purified diet deficient in pantothenate (+0 mg/kg diet) and (2) No tail cup group: 10 rats pairfed to the tail cup group and fed the AIN-76 purified diet deficient in pantothenate (+0 mg/kg diet). The tail cup group was fitted with tail cups for the prevention of coprophagia and will hereafter be referred to as the DEF, TC group. The no tail cup group will be called the DEF, PF, NO TC group. 38 SAMPLE COLLECTION All detailed sample collection protocols are included in Appendix A. Food intake was measured daily, accounting for food spilt in the metabolism cages. Rat weights were recorded every five days. Daily urine excretion was collected into urine collection bottles containing .5 ml of an antibacterial agent (1% Sodium Azide). The daily samples were diluted to 50 ml with distilled water and frozen at -20°C until analysis. In Study 1 urine samples from each rat were pooled into one pooled sample every five days for determination of urinary PA. In Study 2 all thirty days were pooled, for an estimate of urinary PA excretion throughout the study. Urinary pantothenate excretion of the rats was determined in duplicate samples via radioimmunoassay. Feces were collected from the tail cups or the grates of the metabolism cages, dependent on the treatment group of the rats, daily and the tail cups replaced or retaped as needed. In Study 1 determination of fecal pantothenate content was carried out in feces which were combines into five day pools after homogenization. In Study 2 daily feces were divided into two equal aliquots by weight, with one half from each rat kept as daily excretion and the other half from each rat pooled for determination of fecal pantothenate excretion throughout the study. All samples were kept frozen at -70°C. 39 Sacrifice of ten rats in Study 1 and nine rats in Study 2 for data on the baseline body pool size occurred on Day 0 of each study. In Study 1 five CON, PF, TC group rats were sacrificed on Days 20, 25 and 30. All DEF, TC group rats in Study 1 were sacrificed on Day 30. In Study 2 rats in both the DEF, TC and DEF, PF, NO TC groups were sacrificed on Day 30. All rats were fasted 12 hours prior to their killing. Rats were anesthetized with methoxyflurane, weighed, and killed by bleeding via cardiac puncture. The blood was collected into EDTA vacutainers, the volume measured, and then was frozen at - 70°C until analysis. Contents of the stomach, small intestine, cecum and large intestine were removed. Carcasses were placed in preweighed canning jars, weighed, and autoclaved for 60 minutes at 16.5 pounds of pressure. SAMPLE PREPARATION Whole body carcasses were homogenized with a volume of distilled water equal to the weight of the rat (after the gastrointestinal contents were removed). Three fourths of the distilled water was added to the canning jar containing the rat carcass. Homogenization was done with a Polytron (Brinkmann Instruments Co., type PT 1020 3500), set at speed 7.5 for three minutes. The resultant uniform slurry was transferred to a graduated cylinder. The remaining one fourth of the water was used to rinse 40 off the Polytron probe and jar and was quantitatively transferred to the graduated cylinder for determination of the final volume. The entire volume of slurry was strained through a large strainer to remove hair and was frozen (-70.C) until enzyme hydrolysis, followed by pantothenate analysis. Whole body samples were defrosted and mixed well. Duplicate 200 ul whole body samples were added to 10 units alkaline phosphatase and 20 units pantetheinase. The samples were incubated at 37°C for 7 hours in a shaker water bath. Hydrolysis was terminated by the addition of equimolar concentrations of saturated Ba(OH)2 and 10% ZnSO4 (approximately 200 ul total volume). The ratio of Ba(OH)2 and 10% ZnSO4 was determined by titration using phenolphthalein as an indicator. The samples were then centrifuged (Sorvall Superspeed RC2-B Automatic Refrigerated Centrifuge) at 4,000 x g for 10 minutes. The supernatants were separated and frozen at -70’C until analysis via radioimmunoassay. Whole blood samples were processed in duplicate according to procedures previously determined (Wyse et al., 1978). Blood was hemolyzed by three quick freeze/thaw cycles and 100 ul samples were incubated for seven hours in a 37°C shaker water bath with 10 units alkaline phosphatase and 20 units pantetheinase. Deproteinization and storage of samples were carried out as outlined for the whole body samples. 41 The feces were autoclaved for 60 minutes at 16.5 pounds of pressure. For Study 1 homogenization of the feces was accomplished with the Polytron set at speed 3, with the addition of 3 ml of distilled water to each daily fecal sample. All daily samples were made up to constant homogeneous volumes of 10 ml. Two ml were taken from each daily homogenate to form 5-day fecal pools for each rat. In Study 2, for each rat, one pooled fecal sample was homogenized for the entire 30-day study. The pool from each rat was made up to a constant volume of 300 ml. Duplicate 200 ul samples of the pooled fecal homogenates were incubated for seven hours in a 37°C shaker water bath with 30 units of alkaline phosphatase and 30 units of pantetheinase. Hydrolysis of the samples was terminated by the addition of 300 ul total volume of equimolar concentration of saturated Ba(OH)2 and 10% ZnSO4. The samples were centrifuged at 4,000 x g for 10 minutes. The supernatants were separated and frozen at - 70°C until analysis for pantothenate content. PANTOTHENIC ACID DETERMINATION All analyses of pantothenic acid content of the various samples were carried out via radioimmunoassay (RIA) (Wyse et al., 1979). 50 ul supernatants of whole body, blood, and feces were used for the RIA while 100 ul 42 of the pooled diluted urine samples were used. Samples were combined in a 5 ml polypropylene vial with a mixture of 1.5% rabbit serum albumin-phosphate buffered saline (PBS), antibody and 14-C pantothenic acid. The rabbit antisera was diluted to obtain a binding of 20 to 30 percent in the blank. The radiolabelled pantothenic acid purchased from New England Nuclear (Boston, Massachusetts) was diluted to deliver approximately 5000 dpm for each sample. The mixture of sample, rabbit serum albumin in PBS, antisera and radiolabelled pantothenate was vortexed and agitated at room temperature for fifteen minutes. 325 ul of saturated ammonium sulfate (a volume equivalent to the sum of the sample, rabbit serum albumin in PBS, antisera and radiolabelled pantothenate) was added to each vial, the samples were vortexed and centrifuged at 11,750 x g for 15 minutes. The supernatant was suctioned off and 500 ul of 50% saturated ammonium sulfate added to the pellet. The pellet was resuspended and centrifuged at 11,750 x g for 15 minutes. The supernatant was removed by suction and 30 ul of tissue solubilizer (Soluene 350, Packard Co, Inc.) was added to the pellet. After vortexing, the samples were agitated in a 60°C water bath for 30 minutes to disolve the pellet. 3 ml of Picoflour-40 (Packard Canberra Co, Inc.) were added to each vial to aid in counting in the liquid scintillation counter. Radioactivity of the samples was measured by the 43 Packard 4000 series liquid scintillation counter, five minutes for each vial. Pantothenic acid content of the samples was determined through the use of a standard curve ranging from 0 to 10 nmol of pantothenic acid. STATISTICAL ANALYSIS Statistical analysis was performed in both studies using the analysis of variance (ANOVA) to test the effects of diet, time and diet and time interactions. Differences between treatments were further tested by the Bonferroni t-test, a conservative measure of significance which is appropriate for research exploratory in nature. Standard errors of the means were calculated for each repeated measure, to determine the variance over time. Correlations between the blood and tissue pantothenate concentration, and between the blood and pantothenate pool were calculated using Pearson’s product-moment correlations. RESULTS FOOD INTAKE AND WEIGHT GAIN A preliminary study established that diet did affect the body pool size of rats. After thirty days rats fed a control diet ad lib, had three times the body pool size of the rats fed the deficient diet, ad lib. The raw data from the preliminary study are included in Appendix B. In Study 1 and Study 2 two groups of rats in each study were pairfed. In Study 1 the CON, PF, TC rats were pairfed to the level of food intake of the DEF, TC rats. The average food intake over the thirty day period was 9 grams per day (Figure 1). In Study 2 the DEF, PF, NO TC rats were pairfed to the intake level of the DEF, TC rats. The average daily food intake in Study 2 was 8.5 grams. In both studies the food intake of all rats thus represents the intake of the DEF, TC rats. The total food intake (mean 1 SEM) did not differ between the Study 1 and 2 (270 i 15 and 255 i 15 grams), respectively. All individual rat data on food intake, weight gain and other variables are reported in Appendix B. In Study 1 there was a significant (p<.05) diet effect upon weight gain. At Day 30 the rats in the DEF, 44 45 om-mw «>85 E 26:5 I mN-PN .EMm ooHoom A name ucomoumou mosam> mumm mo oxmucH mumuofla “H wusmfim m> om mm - p — - mumm mo suoupmm nusono _>g§n 0N mw or m p p — . — p _ p "m musmflm Am sesame us Am sesame 09 oz .mm AH sesame 69 AH sesame 09 .mm 1} .mmn .mmo IIInTIII. .amo .Iliorlll .ZOU IIILUIIII 0N 0v om om 00F GNP (o) iHoiaM 4:8 pantothenate from the diet. The control diet utilized in Study 1 had 12 mg calcium pantothenate/kg diet. The American Institute of Nutrition recommendation of 12 mg calcium pantothenate per kg diet is slightly above the recommendation of 10 mg pantothenate/kg diet by the National Research Council. Thus over the thirty day study the CON, PF, TC rats in Study 1 were expected to consume an average of 11,255 nmol pantothenate. TISSUE PANTOTHENATE CONCENTRATION AND WHOLE BODY POOL Prior to this study the effects of diet and coprophagy on the entire tissue pantothenic acid concentration and whole body pool of pantothenic acid were not known. In this study tissue pantothenate concentration and whole body pantothenate pool were found to decrease over time in the rats in the DEF, TC group. Rats in the DEF, PF, NO TC group similarly had a decrease in tissue pantothenate concentration over time but were able to maintain the whole body pantothenate pool. Rats in the CON, TC group maintained their pantothenate tissue concentration and had an increase in whole body pool. In Study 1 the tissue pantothenate concentration (nmol pantothenate/g tissue) at Day 0 was 125 i 4 nmol (Table 5). By Day 30, the tissue pantothenate concentration of the DEF, TC group had decreased 49 .Hoo.v6**«.nso=um some genes; Anv anotm ocwpmmmn one sow; cowstmasoo as use Aav mazotm ommtwmg on» coozomn mocmowmwcmwm Fmowpmwumpm .Aazotm Lou mums Co Lmnszcucv sumacmms pcmmmtamt mmst> azo Fem» oemnmmeo nAoHucv mace 0: .umtttma .pmte pcmwottmo anooenoomm ...nxohucv muse ago Peso .smse seesawtwo ... ommummsm ... Amuse mmomfi was_mmmm N suzpm noowuoeofifi ammucv mmoofl on sea *4. ooofims_o_ ... Amuse mfiomfi mm sea Neeflmmmo_ Amuse mmoefi om sec ago Fem» .ometwma .umwo Potpcoo amhmmmmwm A. Amuse mumm s36 some .3m_o 3=m_ottmo ...... 2:83 ...... 575 ES 2:83 H seapm omg\mpm:m;poucma Poe: mammwp m\mumcmzuoocma Foe: pcosummth cowpmtpcmocou Foo; Seem upon: mpmcmgoopcma 63mm?» pcosotzmmmz Fooa Soon mpogz ocm cowumtucmocoo mumcoguoucma msmmws .m m_nme 50 significantly (p<.001) to 37 i.3 nmol, while the CON, PF, TC group did not change from Day 0. Comparing the two groups at Day 30, the tissue pantothenate concentration of the CON, PF, TC group was significantly (p<0.001) higher than that of the DEF, TC group. In Study 2 the Day 0 tissue pantothenate concentration was 150 :35. The DEF, TC and the DEF, PF, NO TC groups had a significantly decreased (p<.001) concentration at Day 30 (40 i 5 and 60 i 5 nmol, respectively), as compared to that at Day 0. The two groups were not significantly different from each other however. Thus coprophagy did not affect the tissue concentration of pantothenate when a pantothenic acid deficient diet was fed. The whole body pool of pantothenate at Day 0 in Study 1 was 4420 i 175 nmol. The DEF, TC rats had a significantly lower body pool at Day 30 than at Day 0 (65% of Day 0, 2895 i 275 nmol). The CON, PF, TC rats had a significant increase in their whole body pool (p<.001), to 11640 i 800 nmol (265% of Day 0). The diet effect was evident in the whole body pool as the CON, PF, TC rats had a significantly larger pool than the DEF, TC at Day 30. In Study 2, the pantothenate pool at Day 0, 5755 i 280 nmol was comparable to that of Study 1. The DEF, TC pool decreased significantly (p<.001) by Day 30 to 55% of the Day 0 body pool (3200 i 400 nmol). The DEF, PF, NO TC rats meanwhile did not show any change in their pools from Day 0 to Day 30 (6425 i 240 nmol). The DEF, PF, NO TC 51 rats had a significantly larger body pool size than the DEF, TC rats (p<.001), demonstrating the effect of the tail cup use on pantothenic acid body pool size. The two deficient groups in Study 1 and 2 were not significantly different in the changes in their body pool sizes from Day 0 to Day 30. The changes in their tissue concentrations from Day 0 to Day 30 were also similar. In Study 1 the rats in the DEF, TC group decreased to 29% of Day 0 by Day 30, while the rats in the DEF, TC group in Study 2 decreased to 27% of Day 0 tissue concentration by Day 30. PANTOTHENATE EXCRETION: URINARY The amount of pantothenate lost in the urine of the rats fed the deficient diet with or without tail cups was small, while the rats fed the control diet excreted in their urine the equivalent of 35% of their net pantothenate intake. In Study 1 the DEF, TC group urinary excretion varied little over time, from 25 to 65 nmol pantothenate per five day pool (Table 6). Over the entire study the amount of pantothenate excreted via urine by the DEF, TC rats was equivalent to 6% of their pantothenate stores at Day 0. The urinary excretion of the CON, PF, TC group did change over time however, increasing from 55 nmol per five day pool at the beginning of the study to 2610 nmol 52 Hoo.vaes4 ”soaom comm senor; maaotm on» cmmzpmn mocmowcscmwm Pmowum_umpm 2mmwcmos pcmmotaot mmzsm> azo Pwmu ofinomfi oc .umetwma .pmwn pcmsosemo mflnomfi qzo Prep .pmwo pcmwo_wwo N sugpm APOECV smoom gnu Fees mfiwomm o: .oomtwma .pmwu “cowosemo mHHmoH ago —wmp .pmwu “cowowcmo ... N sespm A—oscv Stuart: ommmkm mwmm whom mumm mums mnmm ofinmm ago _smo .cmcewma .omwu _01ocoo mm+m_m mumm mnmm memm meme whom oflnom ago some .umse pcmwuwemo H sospm Aposcv Pmoom clonmomo oosuofiem mkfluoems omsnoemfl msnmwm mammmh mumm Q36 _1mp .emtt_mq .pmtu _013cou o_ummm mnom mnmm whee snow «Hem ohms ago P_mp .pmwu pcmwowemo sf... «:3. «in... «:3. .1... H Aozum “coaumote Aposcv Success omrom mNuHm omuofi mHuHH oflro mnfi Pesos mxmo Paco» use Saucer: ”corpoaoxm momcmguoucma .m oFDQH 53 pantothenate per five day pool at the end of the study. At no point was the urinary pantothenate output of the CON, PF, TC rats significantly greater than the pantothenate intake in the diet however. The effect of diet on urinary excretion is evident beginning at the Days 16—20 pool, when there was significantly (p<0.01) more pantothenate in the urine of the CON, PF, TC rats than the DEF, TC. The urinary pantothenate excretion also showed diet and time interaction effects (p<.001). That is, the rats fed the control diet showed increasing pantothenate excretion over time while the deficient rats pantothenate excretion was consistently low. In Study 2 the DEF, TC rats excreted 105 i 15 nmol pantothenate in their urine over the thirty day study, a quantity equivalent to 2% of their Day 0 pantothenate pool. In contrast, the DEF, PF, NO TC group excreted 230 i 15 nmol pantothenate during the study. Thus over the course of the study the amount of pantothenate that the DEF, PF, NO TC group lost in their urine was equivalent to 4% of their Day 0 pantothenate pool. PANTOTHENATE EXCRETION: FECAL Over the entire study period, rats in the CON, PF, TC group excreted a significantly smaller (p<.001) amount of pantothenate (375 i 30 nmol) in feces (Table 6) as compared to that in urine (6,565 i 610 nmol). Deficient 54 rats, in contrast, excreted small quantities of pantothenate in their urine as well as in their feces (285 .i 10 nmol and 315 i 25 nmol, respectively). In Study 1 the DEF, TC rats fecal pantothenate excretion was consistent over time, with 35.: 5 to 75 i 15 nmol pantothenate excreted per five day pool. The total fecal excretion of the DEF, TC rats, 315 i 25 nmol, was equivalent to 7% of their Day 0 pantothenate pool. The fecal pantothenate excretion of the CON, PF, TC group was likewise consistent over time, with a range of 35 i 5 to 85 i 5 nmol pantothenate excreted. The total loss of 375 i 30 nmol pantothenate in the feces by the CON, PF, TC group was equivalent to 3% of their dietary intake of pantothenate. The total fecal PA loss of the DEF, TC and the CON, PF, TC rats were not different. While diet, time and diet and time interactions (p<.05) were noted in the fecal pantothenate excretion, no consistent trends were noted over time. The two groups in Study 1 consumed the same amount of food, but the quantity of feces excreted (Table 7) was significantly higher (p<.001) in the CON, PF, TC group beginning at Days 16-20. This difference could be due to the increased size of the contol rats, as compared to the rats fed the deficient diet. While the quantity of feces collected was higher in the CON, PF, TC group, the fecal pantothenate concentration (nmol pantothenate/g feces) (Table 8) was not significantly different between the two groups at any 55 go.oa.. ”sogom somo cwgooz mggOLm ecu cmmzooo mocoowewcmwm Fmowomwooom 2mmucome pcmmotgot mmgro> ago Pen» 0: .ooCLVmg m.eo.mfi N.onm.m N.oam.m N.ono.m H.onm.N H.0NN.N N.oum.H .omwo “cowowmmo go Fwop N.HHN.NH e.onm.N m.oem.N m.ouo.N N.onm.N N.onm.H m.ono.N .omwo pcmwowwoo N sogom ago Frau .omcewog m.FHm.em m.onfi.m m.onm.e m.onm.e N.OHo.e N.onm.m m.ono.m .omwm Forecou go Pogo H.HHH.ON m.oum.m e.onm.m H.0nm.N N.oee.m e.ofim.m m.ons.m .oowo “cowowemo .3. .3. .3. >. 3 H o om macaw ocosoootk Pope» om-mN mmufim omuofi mSoo mH-HH ofiuo m-H omoomppoo mmomm .m o_nop 56 sumacaos ocmmmtame mmgpa> ago Pwoo o: HHHH .oootwaa .oowo ocowowmmo NHmH ago Foo» .oowo oco_os+oo N soaom ago Fray mama mama «HRH mama enoa mama cams .omCLVNa .pmwo _otocoo ago Pwoo HHmH Nam mHeH enafi enafi mama mHeH .omso ocmwowemo H sogom m\#osc ocmsoaot» oaaeo>< omumm mN-HN om-oH oxen mH-HH oH-o m-H cowomtocoocoo moacmcooocoa Fooma .m mpnas 57 time. Thus the groups did not differ in quantity of pantothenate excreted in the feces over the course of the study. In Study 2 the total amount of pantothenate excreted in the feces did not differ between the DEF, TC and the DEF, PF, NO TC groups. The DEF, TC and the DEF, PF, NO TC groups excreted 180 i 15 and 180 i 10 nmol pantothenate, respectively. For both groups, this fecal excretion was equivalent to 3% of their Day 0 pantothenate pool. There were no significant differences noted between the groups in Study 2 in quantity of feces collected or concentration of fecal pantothenic acid. PANTOTHENATE BALANCE: INTAKE - EXCRETION The pantothenate balance (Table 9) was calculated as the difference between pantothenate intake from food and excretion in urine and feces. The net pantothenic acid balance throughout the entire study was hypothesized to be comparable to the change in the pantothenate body pool size from Day 0 to Day 30. In Study 1 the pantothenate balance over time in the DEF, TC rats was consistently negative, ranging from -135 i 5 to -75 1.5 nmol per five day pool. Over the thirty day study period, the DEF, TC rats lost 600 t 35 nmol of pantothenate which would be equivalent to 14% of their pantothenate pool at Day 0. In contrast, the CON, PF, TC Hoo. ya xxx .fio.oa *4 .mo..va¥ “Sogum comm crcowz magOLm on» cooZpon mocoowwwcmwm —aowom_ooom Amhmm 58 ago Pomp o: .ooetwaa omnoHe- .pmwo “cowowmmo ago Poop mmnomN- .oowo “cowowcmo N sogom ago Peep .omwtwaa oemnooam mmflaomfln oofinmee omfinomm oanommH menooom oenmme .pomo _otpcou go Pwmo mmnooo- ofiaom- mama- oHHom- mammfi- muoofi- mHmHH- .oowo “cowowmmo ¥¥ k. ¥¥ ¥¥ {.k. ¥¥ H XUDHW Poe: “cospomtp .1 om-oN mN-HN 0N-oH mo-oo ag-o m-o _oooo mesa mocopon opmcmguoucoa .m mFQMP 59 rats had a positive balance of 6,760 i 240 nmol over the entire study period (equivalent to 60% of the pantothenate in their diet). The positive balance, however, decreased over time, from a positive 1,985 i 35 nmol per five day pool to a negative balance of 180 i 155 nmol per five day pool. This trend was directly inverse to the urinary pantothenic acid excretion which started at 55,: 5 nmol per five day pool and ended at 2,610 i 160 nmol per five day pool. The overall balance was significantly greater in the CON, PF, TC group, as compared to the DEF, TC group (p<.05). In Study 2 the total pantothenate balance was negative in both the DEF, TC group (-290 i 25 nmol) and the DEF, PF, NO TC group (-410 i 20 nmol). These negative balances are equivalent to 5 and 7% of the Day 0 pantothenate pools of the DEF, TC and DEF, PF, NO TC group, respectively. The two deficient groups in Study 2 were not different in their overall balances however. PANTOTHENATE BALANCE VERSUS MEASURED CHANGES IN WHOLE BODY POOL There were losses in the pantothenate body pool in the DEF, TC group in both studies that can no be totally accounted for by the balance data. In contrast, the gain in the measured whole body pantothenate pool by the CON, PF, TC rats in Study 1 was approximately the sum of 60 pantothenate gain expected from the balance data. The DEF, PF, NO TC rats in Study 2 had a measured gain of pantothenate which was greater than expected from the balance calculation. This gain may be partially explained by the pantothenic acid available from the feces through the practice of coprophagy and bacterial synthesis of the vitamin. In Study 1 the measured change in the pantothenic acid body pool size from Day 0 to Day 30 in the DEF, TC rats was -1,525 i 270 nmol. A loss of 600 i 35 nmol would have been expected from the pantothenate balance (Table 10). Thus there was an unexpected loss of 925 i 295 nmol, equivalent to 20% of the Day 0 pool, over the course of the entire study. In Study 2 the measured change in the body pool size from Day 0 to Day 30 in the DEF, TC rats was -2,560 i 400 when a loss of 290 i 25 was expected from the balance. Thus the rats had an unexpected loss of - 2,270 i 410 nmol over the course of the study (equivalent to 40% of the Day 0 pool). In the CON, PF TC rats in Study 1 the change in the measured body pool size from Day 0 to Day 30 was 7,220 i 1,010 nmol, whereas a retention of 6,760 i 420 nmol was expected from the balance calculation. Contrary to the deficient groups, the control group rats amassed pantothenate in the body pool to the extent expected from the balance data. The measured body pool size of DEF, PF, NO TC rats in 61 Table10. Balance versus measured pool change 2 Measured Unexplained Balance Pool3 gain/loss Change Treatment nmol pantothenate Study 1 Deficient diet, tail cup -600:35 -1525i245 *** -925i295 Control diet, pairfed, 6760:240 7220:1010 D tail cup Study 2 *** Deficient diet, tail cup -290i25 -2560:4OD -2270:410 Deficient diet, pairfed, -410:20 670:235 1080:240 no tail cup Values represent mean:SEM 2BALANCE = DIETARY PANTOTHENATE - (URINARY PANTOTHENATE + FECAL PANTOTHENATE) 3MEASURED POOL CHANGE = BODY POOL AT DAY 30 - BODY POOL AT DAY 0 Statistical significance in body pool between day 0 and day 30: *** p<.001 62 Study 2 increased 670 i 235 nmol from Day 0 to 30, which was a statistically insignificant change from baseline. From the balance calculation, however, a loss of 410 i 20 nmol of pantothenate was expected. Thus the rats allowed coprophagy had an unexplained gain of 1,080 i 240 nmol pantothenate over the course of the study (equivalent to 20% of baseline stores). While the DEF, TC rats had an unexplained loss of pantothenate, the DEF, PF, NO TC rats had an unexplained gain of pantothenate, possibly attributable to the practice of coprophagy, enteromicroflora synthesis of the vitamin, or a combination of factors. PANTOTHENATE IN CIRCULATION: WHOLE BLOOD In Study 1 the blood pantothenic acid concentration at Day 0 was 1.30 :_.10 nmol/ml blood (Table 11). The DEF, TC group rats decreased significantly (p<.001) to .30 ‘i .03 nmol pantothenate/ml blood at Day 30. The blood concentration of the CON, PF, TC group rats did not change from Day 0 and was significantly greater (p<.001) than the DEF, TC group at Day 30. In Study 2 the Day 0 blood pantothenic acid concentration was .85 1 .13 nmol/ml blood. Both the DEF group rats in Study 2 had significantly less blood pantothenate at Day 30 than at Day 0 (p<.001). The two DEF group rats were not significantly different from each 63 Table 11. Pantothenate content in blood Blood pantothenate Treatment nmol/ml blood Study 1 Baseline 1.30:.10b *** Deficient diet, tail cup .30:.03 9 Control diet, pairfed, tail cup 1.10:.05 Study 2 Baseline .85i.13 *** Deficient diet, tail cup .28:.01b*** Deficient diet, pairfed, no tail cup .26:.01 Values represent meaniSEM Statistical significant between the pairfed groups (p) and in comparison with the baseline group (b) within each study: ***p<.001 64 other however. Figures 3, 4 and 5 show the relationships between blood, tissue concentration and whole body pool. With all rats included (Baseline, Control and Deficient groups) the relationships are linear. The correlations between blood and tissue concentration and blood and whole body pool were positive, with the higher correlation seen between whole blood and tissue concentration, 0.75. The correlation between blood and whole body pantothenic acid was 0.60. The relationship between tissue concentration and whole body pool was also positive, 0.50. 65 co_poepcoocoo mgmmah mgmto> ooosm ”m megawa so. 3 BEE cozmoocoocoo ozmmt. cow co F o 3-685.... + N833 . u N (Iw / IowU) pooua 66 cu Hooa zoom mgmcw> ooofim ”e wcgm_a so. \ .223 .02. Sam or o e 0‘ '0 fie 3-823 + 53.3 a a (1w / IowU) poms 67 cowoococmocoo ogmm_H mgmco> Looa soom ”m ocgmwa so. 3 .225 223.3538 2.3:. ggN _ gov . g xommmm . maven . a 0N (191 / mum) lood Apog DISCUSSION The results of a preliminary study showing that diet did affect the body pool size of rats prompted the investigations described here. The balance and body pool size differences were studied in rats fed a pantothenic acid deficient or control diet, pairfed, and in rats fed a deficient diet with or without control of coprophagy. Rats fed a deficient diet weighed significantly less (p<.05) than rats pairfed the control diet for thirty days. This growth promoting effect of pantothenate has been reported by many other investigators. Srinivasan and Belavady (1976), reported that the weights of rats fed diets deficient and sufficient (pairfed) in pantothenate were significatnly different (p<.001) after eight weeks. While in this study the deficient group rats were able to grow over the thirty day study (although not optimally) the concentration of pantothenic acid in tissue decreased. Prior to this study the changes in the tissue concentration in the whole body had not been addressed. The reduction in pantothenic acid concentration of specific organs has been reported by a number of authors however. Reibel et al. (1982) reported that in adult rats fed a pantothenic acid deficient diet for 4 - 8 weeks the levels of free pantothenate in several tissues decreased at least seventy percent as compared to rats fed a regular 68 69 diet. Srinivasan and Belavady (1976) found that rats fed panothenic acid deficient diets for six weeks had significantly (p<.01) lower levels of both free pantothenic acid and CoA in their livers when compared to rats fed pantothenic acid sufficient diets. Neither of these studies, however, addressed how the organ pantothenate concentration was related to the whole body pantothenic acid pool. In the rats fed the deficient diet, the pantothenate body pool was affected by diet but did not decrease to the extent that the pantothenate tissue concentration did. The body pool sizes of the rats on the deficient diets with tail cups decreased over time while the rats fed a pairfed control diet, with tail cups, increased. Previous studies have not been done on the whole body pantothenate pool, but similar trends were discovered in the data of Hatano (1962) when the organ pantothenate pool was summed in the nine organs he studied. Hatano measured the organ pantothenate concentration and organ weight of rats fed diets deficient and sufficient in pantothenate. The rats fed a deficient diet had organ pools of 1,540 i 30 nmol after 32 days while rats fed diets sufficient in pantothenate had 3,640 i 180 nmol pantothenate in their organ pool. No estimates were given of the body pool of the rats at Day 0. In Study 1 the pattern of urinary pantothenate excretion by the control group rats suggests that younger 70 rats may need more pantothenate than older rats. The control rats retained in their bodies an equivalent of 98, 94, 81, 35, 17 and 0% of their dietary intake at Days 1-5, 6-10, 11-15, 16-20, 21-25, and 26-30, respectively. The pantothenate urinary output did not begin to parallel the pantothenate intake until Days 16-20, thus the recommendation of 12 mg pantothenate per kg diet may not be sufficient for the young rat. These findings are consistent with Unna and Richards (1942) who previously noted that the pantothenic acid requirement, as detemined by growth and prevention of deficiency lesions, decreases with age in growing rats. While the urinary excretion increased over time in the control rats it never significantly exceeded the amount of pantothenic acid consumed from the diet, as also reported by Henderson et al. (1942). The pantothenate urinary excretion was also never absolutely zero, suggesting a minimal obligatory loss of the vitamin. Karnitz et a1. (1984) concluded that the only conservation mechanism for pantothenate was tubular reabsorption in the kidney. Thus a small amount of pantothenate was not . reabsorbed, as also observed by Fox and Linkswiler (1961) in human studies. The total pantothenate lost in the urine and feces of the deficient animals over the thirty day study represented 5 to 14% of their Day 0 pantothenate pools. Thus if the rate of pantothenate loss did not change over 71 time pantothenate would be lost from the body relatively slowly. The balance calculation in this study was limited to pantothenate intake, as calculated from the dietary intake, and pantothenate output, as calculated from fecal and urinary pantothenate. The rats fed a deficient diet were consistently in small negative pantothenic acid balance while the rats fed the control diet had an overall large positive balance. Changes in the pantothenic acid body pool could not be totally explained by the pantothenic acid balance over time. The primary weakness of using a balance study to estimate changes in the body pool of a vitamin over time are technical in nature. First, it is difficult to ensure the complete estimates of all avenues in and out of the animal. All vitamin sources into the animal must be quantitated and complete sample collections must be made of all avenues of excretion. Secondly, any metabolism or synthesis of the vitamin within the animal must be detected. In this study several assumptions were made in the calculation of the pantothenate balance. First it was assumed that when tail cups were used to prevent coprophagy the only source of the vitamin was the pantothenate in the diet. Secondly, it was assumed that urine and feces were the only routes of pantothenate excretion. Pietrzik (1980) reported that 13-15% of a 1- 72 14C pantothenate dose injected via a stomach tube was excreted via the respiratory route in the form of carbon dioxide. The exhaled label may have been the result of pantothenate metabolized by the microflora in the large intestine. Also assumed in the balance calculation was the complete collection of all urinary and fecal samples. Every attempt was made to insure complete collection of all samples however the loss of very small amounts of pantothenate may have been possible through incomplete sample collections. In calculating the pantothenate balance the assumption was made that pantothenic acid was not metabolized within the rat body. A small portion of pantothenate may have been diverted to acyl carrier protein. Pietrzik (1980) indicated that the vitamin was excreted in urine in the free form but also as 4- phosphopantothenate. While 4-phosphopantothenate is not assayed by our methods the portion of pantothenate thought to be in that form in the body is usually considered to be insignificant. Finally, the assumption was made in the balance calculation that pantothenate was not synthesized by the enteromicroflora. In the eighteen deficient rats with tail cups the average loss of pantothenate, as determined by the measured pantothenate body pool, was significantly (p<.01) larger than that estimated by the balance data. 73 Explanations for the pool loss exceeding the balance loss include first, the incomplete collection of all urine and feces, causing an underestimated balance loss. Second, the unexpected loss of pantothenate could result from another route of pantothenate excretion such as the respiratory pathway, as speculated by Pietrzik and Horning, 1980. Finally, a diversion of pantothenate to other forms, not measured by our assay, such as acyl carrier protein, could result in a loss of the vitamin. The ten deficient rats allowed coprophagy gained more pantothenate, as determined by the measured pantothenate body pool, than expected from the balance data. A possible extra source of pantothenate to the rat includes pantothenate consumed through coprophagy, although it has been shown in this study that the pantothenate content of the feces is small in comparison to the body pool of the vitamin. Another source of pantothenate is enteromicroflora synthesis of the vitamin. Mameesh et al. (1959) proposed that microbially synthesized pantothenate was absorbed directly from the intestinal tract. Because the body pool of the rats fed a deficient diet, allowed to practice coprophagy, increased from Day 0 to Day 30 while the pantothenate pool of the rats prevented from ceprophagy significantly decreased, coprophagy significantly affects the pantothenate body pool in rats. The body pool size difference between the two groups was approximately 3,000 nmol pantothenic acid. 74 From this study the precise mechanism of how coprophagy affects the body pool was not determined. The mechanism may include both the extra source of pantothenate available in the feces and/or the promotion of synthesis of pantothenate by enteromicroflora in a form available to the rat. An assessment of how well blood predicts the tissue concentration and whole body pool of pantothenate revealed that whole blood most closely predicts tissue concentration but it was also a good indicator of the whole body pool. CONCLUSIONS Based on the results of this study the following conclusions can be made: 1) 2) 3) 4) 5) The body pool size of pantothenate in rats is altered by the pantothenate intake from the diet. The rats fed the deficient diet were consistently in negative pantothenic acid balance throughout the study while the rats fed the control diet were in positive balance for the first 25 days of the study. The changes in the pantothenic acid body pool cannot be totally explained by the balance data. Coprophagy is a significant source of pantothenate in rats, as determined by the body pool size of the vitamin. The correlations between blood pantothenate and pantothenate tissue concentration and between blood pantothenate and the whole body pantothenate pool are both positive, with the stronger relationship being between blood and tissue concetration. 75 RECOMMENDATIONS 1) 2) 3) The effect of coprophagy on the body pool size of deficient rats needs further study. While this initial study determined coprophagy to be a significant source of pantothenic acid, additional experiments, with larger sample sizes, would solidify the importance of coprophagy as a pantothenic acid source . The diversion of pantothenic acid to forms other than CoA within the rat body, under conditions of deficiency, also needs to be identified using tools such as High Performance Liquid Chromatography. Finally a more comprehensive study of the loss of the vitamin from the body, specifically the respiratory loss of pantothenic acid, needs to be completed. Such an assessment would help explain the difference between the balance and body pool size of pantothenic acid noted in this study. 76 APPENDICES 77 APPENDIX A SAMPLE COLLECTION PROTOCOLS 1) 1. 2. 3. 4. 5. FOOD INTAKE Remove food jars from cages of all rats Weigh food jars of rats that are fed ad libitum, record Shake all food jars to insure rat can get to the food Subtract today' 5 weight of jar + food from yesterday’ 5 to measure food consumed Feed pairfed rats the amount of food the pair in the ad libitum group ate (put food jar on scale without lid or follower, tare, add correct amount of food) Every 5 days, after weighing food of ad libitum rats, dump out food and refill jar with new food Reweigh jar + food and record If level of food in jar géts less than 2/3 full before 5 days are up add new food on top of old and record new weight Weigh and record food spilt, subtract that amount from daily intake 2) 1) 2) 78 URINE SAMPLE COLLECTION AND PREPARATION FOR RIA Sample Collection 1. 2. 3. 4. 5. 11. 12. Add .5 ml 1% Na Azide to each urine collection bottle Move rack of rats into room with sink, take urine bottle and funnel from bottom of each cage down Remove feces from grate, put into feces test tube Spilt food should be dumped on to a clean sheet of paper, labelled, and saved to be weighed Put funnel on top of bottle and rinse with distilled water to insure all urine gets into the bottle Clean funnel by rinsing with water, remove any solid residue from funnel with brush Pour urine from bottle into 50 m1 volumetric flask, rinse collection bottle with distilled water, pour urine into flask Dilute urine to 50 ml with distilled water Mix by inverting at least 4 times, save one vial full of diluted urine, discard rest of urine Label vial: m4 1: .1... Rinse volumetric flask with distilled water before beginning next rat 0 Store diluted urine samples in -20 C freezer Sample Preparation After defrosting and mixing by inversion, put 2 m1 of each sample of diluted urine into a pooled sample for each rat Save the remaining daily sample in a plastic scintillation vial Store all samples in -20°C freezer until pantothenate analysis by RIA 3) 1) 2) 79 FECES SAMPLE COLLECTION AND PREPARATION FOR RIA Sample Collection 1. 2. CDQO‘ LII-#1...) 10. 11. 12. Remove rat from cage Place rat in large bowl, tip tail cup to remove feces, use spoon to remove any feces caught in tail cup Retape or replace tail cup as necessary Return rat to cage Pour feces from bowl into appropriate feces test tube Re eat above procedure with remaining rats Weigh rats every 5 days without tail cups, record Tare weight of measuring boat on scale, pour feces from test tube into bowl, record weight Divide feces weight in half - record. By weight put one half of feces into large baggy for pooled feces and the other half into small baggy for daily pool Repeat above procedure for all rats Store feces in -20°C freezer Sample Preparation 1. 2. 3. 4. 5. 6. At the end of the study autoclave the pooled collections from all rats for 60 minutes at 250°F and 16.5 lb. pressure Add 90 ml of distilled water to each pooled sample Homogenize with the Polytron, beginning at speed 3 for three minutes Transfer each pooled sample to a graduated cylinder, rinse the Polytron with additional d stilled water, add that sediment to the graduated cylinder Make up the samples to a constant volume of 300 ml Add 30 units of alkaline phosphatase, 30 units of pantetheinase and PBS to a total volume of 200 ul to 200 ul of fecal slurry for enzyme hydrolysis Vortex and put in shaker bath at 37 C for seven hours Deproteinize by adding 200 ul total volume of Ba(OH)2 and ZnSO4 (equimolar concentrations) to each tube Vortex and centrifuge at 4,000 rpm for 10 minutes Remove and store supernatants in the freezer for RIA 4) 1. 2. 10. 11. 12. 13. 14. 15. 16. 8O Sacrifice of Rats for Whole Body Analysis Fast rats overnight prior to sacrifice Supplies needed:jar for whole rat and a nose cone (for anesthesia) metofane gauze gloves 21 g needles (green) EDTA (purple) blood collection tube (vacutainer) scissors (large and small) test tube rack en ars for carcases ce Put a rat in a large jar with metofane soaked gauze until rat is unconscious Weigh rat Place rat on it's back, tape four paws down to surgery plate Put head of rat into nose cone (with gauze soaked in metofane) Prepare needle and collection tube Make incision in lower abdomen with large scissors and then a cut is made all the way up to the diaphragm, then cut right a small distance Use small scissors to make sure liver is down and cut through diaphragm until see the heart Insert the needle into the heart and push container down to get vacuum, if more than one tube of blood is needed, leave needle in place and insert another tube into needle holder, remove needle slowly giaggre quantity of blood removed and label tubes of 0 Keep the test tubes containing blood on ice Remove contents of stomach, small intestine cecum, and large intestine, rinse with deo, until "clean" Place carcasses in weighed jars with head and tail down Weigh rat + jar (subtract jar weight to get weight of rat) Autoclave immediately, following Autoclave outline 3. 4. 8. 9. 10. 81 AUTOCLAVING OF CARCASSES FOR WHOLE BODY ANALYSIS Place rats in canning jars of appropriate size depending on size of rat with head and tail down Place jars in a rack and place in the autoclave, lids should be slightly loosened, with seal upside down Tighten lower handle on the door of the autoclave Set time for 60 minutes, temperature will reach 250°F and 16 1/2 lb pressure Set on liquid setting, this cycle will take approximately 45 minutes to cool As dial moves to sterilize, close door tightly by tightening upper handle on the door of the autoclave Sign record sheet, buzzer will go off when pressure is down and items can be removed Remove jars from autoclave with gloves Weigh jars + rats Cool and store carcasses in -20°C freezer until ready to homogenize 6) 2. 3. 4. 5. 82 HOMOGENIZATION OF CARCASSES FOR WHOLE BODY ANALYSIS Measure out a volume of dHZO, equal to weight of rat (e.g. rat weighs 400 g, use 400 ml dH20) Add approximately 3/4 of water while homogenizing Homogenize at speed 7.5 for three minutes Once slurry is uniform, transfer to a graduated cylinder Clean off Pol tron with remaining 1/4 water, add to graduated cylinder, measure volume, record value Strain entire volume of slurry through large strainer to remove hair, strain into a large beaker Take samples for enzyme hydrolysis immediately after straining or rehomogenize to insure a uniform sample Freeze slurries in -20°C freezer 7) 2. 3. 4. 83 ENZYME HYDROLYSIS AND DEPROTEINIZATION OF CARCASSES FOR WHOLE BODY ANALYSIS Pipet 200 ul of whole body slurry into test tubes Add 10 u of alkaline phosphatase, 20 u of pantetheinase and enough PBS to make 200 ul of enzyme solution to each test tube Vortex Cover each tube with parafilm twice and place in shaker bath at 37°C for approximately 8 hours Deproteinize by adding a total volume of 200 ul of Ba(OH)2 and ZnSO4 (equimolar concentrations) to each tube. Vortex Centrifuge at 4,000 rpm for 10 minutes Remove and store supernatant in -70°C freezer for RIA. 8) 3. 4. 5. 6. 8. 9. 10. 84 PREPARATION OF WHOLE BLOOD FOR ANALYSIS Collect fasted blood samples into EDTA (purple topped) vacutainer. Hemolyze frozen whole blood by three quick freeze/thaw cycles. Pipet .1 m1 of blood into sample tubes. Add 10 units of alkaline phosphatase, 20 units of pantetheinase + enough PBS to make up 200 ul of solution to each tube. Vortex Cover each tube with parafilm twice and place in shaker bath at 37°C for 7 to 8 hours. Terminate hydrolysis by adding equimolar concentrations of sat'd Ba(OH)2 and 10% ZnSO4 (total volume of 200 ul) to each tube. Vortex Centrifuge at 4,000 x g for 10 minutes Separate clear supernatant for RIA, freeze at -70°C until analysis. APPENDIX B INDIVIDUAL RAW DATA FOR RATS STUDY 1 BLOOD, RAT! DGQO‘thUNP ..o 0 AVERAGE: SD: SE: ' BASELINE GROUP TISSUE CONTENT AND WHOLE BODY PANTOTHENATE NHOL PA/ NMOL PA/NMOL PA/ HI. BLOOD G RAT 1.50 1.00 1.80 1.20 1.20 1.20 1.40 1.00 1.29 0.27 0.10 113.91 105.89 128.22 145.39 143.10 116.77 115.62 128.22 136.23 117.91 125.13 13.22 4.18 85 war (G) 46.14 36.99 34.46 28.91 25.99 43.83 32.39 33.08 34.45 41.63 35.79 6.41 2.03 RAT 5255.81 3916.87 4418.46 4203.22 3719.17 5118.03 3744.93 4241.52 4693.12 .4908.59 4421.97 553.42 175.13 86 STUDY 1 DEF, TC GROUP RAT WEIGHT (G) RAT 4 ARRIVAL DAY 5 DAY 10 DAY 15 DAY 20 DAY 25 DAY 30 16 46.10 62.30 74.90 77.10 82.60 92.20 95.00 18 38.00 51.40 66.00 69.00 81.90 87.00 90.00 19 37.70 55.30 61.70 61.00 73.90 79.50 88.40 20 47.80 65.30 73.40 73.60 83.20 86.20 93.60 21 31.40 43.30 49.30 49.50 49.10 53.30 58.00 22 35.60 46.60 56.60 54.70 66.10 73.30 75.50 23 51.80 74.00 86.10 93.20 108.80 116.50 121.40 24 29.00 39.00 47.30 48.10 53.70 56.00 58.60 MEAN: 39.68 54.65 64.41 65.78 74.91 80.50 85.06 SD: 7.66 11.27 12.64 14.61 17.93 19.23 19.73 SE: 2.71 3.98 4.47 5.16 6.33 6.80 6.97 STUDY 1 DEF, TC GROUP BLOOD, TISSUE CONCENTRATION AND WHOLE BODY PANTOTHENIC ACID NMOL RA/ ML BLOOD RAT 4 16 18 19 20 21 22 23 24 AVERAGE: SD: SE: 16 18 19 20 21 22 23 24 AVERAGE: SD: SE: 16 18 19 20 21 22 23 24 AVERAGE: SD: SE: 0.25 0.31 0.23 0.32 0.24 0.33 0.43 0.30 0.07 0.03 1-5 41.99 39.19 42.43 46.27 37.30‘ 38.50 50.84 35.95 41.56 4.99 1.76 1-5 65.12 83.61 71.43 40.90 59.00 89.00 42.50 53.50 63.13 17.71 6.26 87 33.54 37.55 30.68 49.91 45.45 35.95 30.57 30.11 36.72 7.37 2.60 FOOD INTAKE DAYS 6-10 11-15 44.45 41.08 43.45 42.82 40.49 32.74 45.94 38.01 41.55 40.25 39.68 41.46 52.90 49.87 35.17 31.93 42.95 39.77 5.21 5.74 1.84 2.03 NMOL PA/ CARCASS NMOL PA/ GRAM RAT war (G) RAT 89.82 3012.56 85.99 3228.92 80.95 2483.55 85.10 4247.34 54.34 2469.75 71.68 2576.90 112.98 3453.80 56.15 1690.68 79.63 2895.44 19.07 772.34 6.74 272.91 (G/S DAYS) 16-20 21-25 46.84 52.67 50.30 51.12 50.04 51.22 51.91 49.24 44.54 37.60 51.69 53.13 65.01 72.80 46.70 43.18 50.88 51.37 6.29 10.19 2.22 3.60 URINARY PANTOTHENATE (NHOL/S DAYS) DAYS 6-10 57.50 40.00 42.50 50.00 35.00 52.50 57.50 65.00 50.00 10.19 3.60 11-15 70.00 58.75 37.50 95.00 58.75 30.00 60.00 60.00 58.75 19.74 6.97 16-20 38.43 52.50 30.00 24.50 18.25 12.25 82.50 49.00 38.43 22.73 8.03 ' 21.25 32.25 29.50 23.00 18.00 12.75 11.75 32.50 25.00 23.09 8.28 2.92 FINAL- INITIAL BODY POOL -1409.41 -1193.05 -1938.42 -174.63 -1952.22 -1845.07 -968.17 -2731.29 -1526.53 772.34 244.41 26-30 TOTAL 40.02 267.05 44.89 271.77 37.04 253.96 49.34 280.71 33.53 234.77 41.71 266.17 53.39 344.81 34.63 227.56 41.82 268.35 7.04 35.92 2.49 12.69 26-30 TOTAL 45.00 308.30 52.50 316.86 45.00 249.43 60.00 288.40 30.00 213.75 47.50 243.00 82.50 357.50 42.50 295.00 50.63 284.03 15.47 46.37 5.47 16.38 STUDY 1 DEF, TC GROUP RAT 4 1“5 16 44.88 18 51.67 19 24.66 20 67.80 21 18.90 22 49.15 23 108.60 24 47.70 AVERAGE: 51.67 SD: 27.73 SE: 9.80 6-10 30.60 78.00 37.80 42.60 41.40 30.00 85.20 36.90 47.81 21.43 7.57 FECAL PANTOTHENATE (NHOL/S DAYS) 21-25 - 65.92 62.95 60.30 52.99 44.40 40.95 '62.37 43.28 54.14 10.08 3.56 26-30 TOTAL 41.70 44.07 35.64 38.16 37.80 29.91 43.89 23.40 36.82 7.16 2.53 311.59 376.85 249.00 324.25 283.20 283.01 443.73 265.28 317.11 64.78 22.89 PANTOTHENATE BALANCE: INTAKE - (URINARY + FECAL EXCRETION) (NMOL) RAT # 16 18 19 20 21 22 23 24 AVERAGE: SD: SE: RAT 16 18 19 20 21 22 23 24 AVERAGE : SD: SE: 1-5 “110.00 “135.28 “96.09 “108.70 “77.90 “138.15 “151.10 “101.20 “114.80 24.63 8.70 1“5 3.16 3.44 4.28 5.12 2.17 3.11 4.70 3.48 3.68 0.96 0.34 6.10 “88.10 “118.00 “80.30 “76.40 “82.50 “142.70 “101.90 “97.81 22.57 7.98 6-10 2.24 3.05 1.96 2.34 2.08 2.01 4.82 3.58 2.76 1.01 0.36 DAYS 11-15 16-20 99.99 28.50 76.80 63.36 60.00 30.60 69.00 53.70 72.60 68.10 73.90 59.10 78.00 65.67 60.90 53.10 73.90 52.77 12.51 15.30 4.42 5.41 DAYS 11“15 16“20 “169.99 “66.93 “135.55 “115.86 “97.50 “60.60 “164.00 “78.20 “131.35 “86.35 “103.90 “71.35 “138.00 “148.17 “132.65 “91.20 25.70 29.51 9.08 10.43 11-15 3.97 3.43 2.78 3.56 2.38 6.53 4.39 3.08 3.77 1.29 0.46 16-20 2.72 3.26 2.13 2.79 2.46 2.57 3.07 3.36 2.80 0.42 0.15 '21-25 “98.17 “92.45 “83.30 “70.99 “57.15 “52.70 “94.87 “68.28 “77.24 17.51 6.19 FECES COLLECTED (G/5 DAYS) DAYS 21-25 3.37 4.52 4.99 4.72 1.72 2.31 4.00 2.58 3.53 1.22 0.43 26-30 TOTAL “86.70 “96.57 “80.64 “67.80 “77.41 “126.39 “65.90 “87.45 19.70 6.96 26-30 3.28 5.01 5.01 5.04 3.75' 3.06 3.51 2.69 3.92 0.96 0.34 ' “619.89 “693.71 “498.43 “612.65 “496.95 “526.01 “801.23 “560.28 “601.14 105.52 37.29 TOTAL 18.74 22.71 21.15 23.57 14.56 19.59 24.49 18.77 20.45 3.23 1.14 89 STUDY 1 CON, pr, TC GROUP RAT WEIGHT (0) RAT 4 ARRIVAL DAY 5 DAY 10 DAY 15 DAY 20 DAY 25 DAY 30 1 ' 46.20 63.30 83.50 86.70 96.00 2 38.00 44.10 45.50 61.50 76.00 3 37.40 54.70 73.50 86.10 93.50 4 34.60 49.00 67.30 60.60 72.00 83.60 5 48.60 64.70 85.30 88.30 79.10 76.40 6 30.00 43.00 61.10 73.30 76.10 80.90 7 32.50 44.30 62.00 80.70 89.70 107.80 124.00 8 49.70 71.50 101.00 113.50 121.80 142.50 161.10 9 27.80 40.60 54.40 57.50 70.00 81.70 88.40 10 27.00 42.50 61.00 69.20 77.50 11 23.50 35.80 54.50 64.60 75.50 12 49.40 61.80 79.30 87.20 93.30 105.70 13 46.40 60.50 76.10 78.10 85.10 99.40 14 44.40 54.80 73.60 77.40 83.90 98.50 110.80 15 45.50 61.30 79.30 83.80 89.00 99.90 104.00 MEAN: 38.73 52.79 70.49 77.90 85.23 97.64 117.66 SD: 8.83 10.51 14.10 14.06 12.75 18.55 24.81 SE: 2.28 2.72 3.64 3.63 3.29 5.87 11.08 90 STUDY 1 CON, RAT # NNOI. PA/ NNOL PA/ NNOI. PA/ NNOL PA/ NMOL PA/ NNOL PA/ WGT (G) NL BLOOD NL BLOOD NL BLOOD G RAT G RAT G RAT . DAY 20 DAY 25 DAY 30 DAY 20 DAY 25 DAY 30 DAY 20, 1 1.63 127.75 90.81 2 1.14 138.01 71.79 3 1.45 140.29 88.28 4 1.20 136.30 5 1.30 120.33 6 1.50 110.41 7 1.11 108.21 8 0.97 95.70 9 1.14 106.99 10 1.11 143.15 73.67 11 1.64 151.13 69.60 12 2.00 115.66 13 2.10 115.66 14 1.14 107.44 15 1.28 93.76 AVERAGE: 1.39 1.62 1.13 140.07 119.67 102.42 78.83 SD: 0.26 0.41 0.11 8.50 9.95 7.08 9.94 SE: 0.11 0.18 0.05 3.79 4.44 3.16 4.44 FOOD INTAKE (G/s DAYS) DAYS RAT : 1-5 6-10 11-15 16-20 21-25 26-30 TOTAL 1 41.99 44.45 41.08 46.84 174.36 2 41.99 44.45 41.08 46.84 174.36 3 39.19 43.45 42.82 50.30 175.76 4 42.43 40.49 32.74 50.04 51.22 216.92 5 46.27 45.94 38.01 51.91 49.24 231.37 6 37.30 41.55 40.25 44.54 37.60 201.24 7 38.50 39.68 41.46 51.69 53.13 41.71 266.17 8 50.84 52.90 49.87 65.01 72.80 53.39 344.81 9 35.95 35.17 31.93 46.70 43.18 34.63 227.56 10 41.56 42.95 39.77 50.88 175.16 11 41.56 42.95 39.77 50.88 175.16 12 41.56 42.95 39.77 50.88 51.37 226.53 13 41.56 42.95 39.77 50.88 51.37 226.53 14 41.56 42.95 39.77 50.88 51.37 41.82 268.35 15 41.56 42.95 39.77 50.88 51.37 41.82 268.35 AVERAGE: 41.59 43.05 39.86 50.61 51.27 35.56 223.51 SD: 3.51 3.68 4.05 4.54 8.93 18.85 48.29 SE: 0.91 0.95 1.05 1.17 2.83 8.41 12.48 PF, TC GROUP BLOOD, TISSUE CONTENT AND WHOLE BODY PANTOTHENATE 91 STUDY 1 CON, PF, TC GROUP BLOOD, TISSUE CONTENT AND WHOLE BODY P001. 01" PANTOTHENATE NNOL PA/ FINAL-4 442.12 WGT (G) WGT (G) NMOL PA/ NHOL PA/ FINAL- FINAL- RAT RAT RAT INITIAL INITIAL INITIAL DAY 25 DAY 30 DAY 20 DAY 25 DAY 30 BODY POOLBODY POOLBODY POOL DAY 30 DAY 25 DAY 20 11600.98 7178.98 9907.74 5435.74 12384.80 7952.30 78.65 10720.00 6298.00 73.34 8825.00 4403.00 76.17 8409.93 3987.93 121.41 13137.78 8715.78 152.90 14632.53 10210.53 84.44 9034.24 4612.24 10545.86 6123.86 10518.65 6096.65 101.27 11712.89 7290.89 97.06 11225.96 6803.96 104.94 11274.75 6852.75 108.01 10127.02 5705.02 85.30 114.34 10991.61 10178.75 11641.26 7219.26 5756.75 6569.61 12.91 25.34 990.36 1477.77 2262.90 2262.90 1477.77 990.36 5.76 11.31 659.72 1010.22 1010.22 659.72 442.12 STUDY 1 CON, 2 UOQOUMFUNH UOQGUI-hUND-l PF, TC GROUP 1-5 .2114.62 2114.62 1973.61 2136.77 2330.16 1878.43 1938.86 2560.30 1810.44 2092.96 2092.96 2092.96 2092.96 2092.96 2092.96 2094.37 176.67 45.65 25.90 48.41 34.66 47.25 117.75 49.00 31.00 .86.75 27.00 39.30 22.45 105.75 58.25 52.25 60.00 53.71 28.65 7.40 PANTOTHENATE DAYS 6-10 11-15 2238.50 2068.79 2238.50 2068.79 2188.14 2156.42 2039.08 1648.79 2313.54 1914.18 2092.46 2026.99 1998.28 2087.93 2664.04 2511.45 1771.16 1607.99 2162.96 2002.82 2162.96 2002.82 2162.96 2002.82 2162.96 2002.82 2162.96 2002.82 2162.96 2002.82 2168.10 2007.22 185.47 203.86 47.92 52.68 92 INTAKE (NNOL/s DAYS) 16-20 2358.86 2358.86 2533.11 2520.01 2614.19 2243.03 2603.11 3273.90 2351.81 2562.32 2562.32 2562.32 2562.32 2562.32 2562.32 2548.72 228.72 59.10 21-25 2579.44 2479.73 1893.54 2675.63 3666.21 2174.54 2586.99 2586.99 2586.99 2586.99 .2581.71 449.77 142.33 26-30 2100.52 2688.72 1743.97 2106.06 2106.06 2149.06 340.23 151.89 URINARY PANTOTHENATE (NMOL/ 5 DAYS) 6-10 197.50 52.50 97.50 150.00 130.00 155.00 85.00 192.50 115.00 165.00 70.00 187.50 80.00 100.00 240.00 134.50 54.59 14.10 DAYS 11-15 317.50 502.50 195.00 352.50 447.50 132.50 120.00 1057.50 142.50 87.50 50.00 750.00 667.50 352.50 580.00 383.67 286.10 73.93 16-20 2132.50 945.00 2307.50 1940.00 1900.00 957.50 1110.00 1222.50 900.00 760.00 337.50 2725.00 1577.50 1927.50 2356.50 1539.93 698.34 180.45 21-25 1160.00 1392.50 2032.50 1417.50 3000.00 1557.50 2000.00 1450.00 2015.00 2360.00 1838.50 553.85 175.27 26-30 2675.00 3100.00. 2650.00 2527.50 2107.50 2612.00 356.02 158.94 TOTAL 8780.77 8780.77 8851.27 10924.09 11651.79 10134.45 13404.32 17364.63 11459.92 8821.06 8821.06 11408.05 11408.05 13514.11 13514.11 11255.90 2432.13 628.46 TOTAL 2673.40 1548.41 2634.66 3649.75 3987.75 3326.50 5438.50 8659.25 5392.00 1051.80 479.95 5768.25 3833.25 6974.75 ‘7704.00 4208.15 2409.44 622.59 STUDY 1 CON, IuT# UGQGU‘DUNH raw vac HHHH mauu AVERAGE: SD: sm PANTOTHENATE BALANCE: NW1! UOQGUIbUMt-l PF, TC GROUP 54.18 22.20 74.31 107.97 116.64 10.20 42.54 86.94 20.04 14.94 35.52 43.29 71.01 53.10 69.69 54.84 32.63 8.43 1-5 2034.54 2044.01 1864.64 1981.55 2095.77 1819.23 1865.32 2386.61 1763.40 2038.72 2034.99 1943.92 1963.70 1987.61 1963.27 1985.82 143.93 37.19 6-10 44.70 45.00 35.70 44.10 33.90 25.50 44.10 29.70 28.80 21.60 48.60 23.10 14.10 33.10 25.80 33.19 10.30 2.66 6-10 1996.30 2141.00 2054.94 1844.98 2149.64 1911.96 1869.18 2441.84 1627.36 1976.36 2044.36 1952.36 2068.86 2029.86 1897.16 2000.41 178.26 46.06 % DAYS 11-15 16-20 41.70 44.40 45.90 37.80 54.45 34.92 104.40 47.70 100.50 60.60 53.10 51.00 80.40 43.89 102.90 48.96 39.90 29.10 100.80 97.80 52.50 71.40 53.40 80.64 25.50 52.80 44.70 47.40 60.00 102.30 64.01 56.71 26.37 21.86 6.81 5.65 DAYS 11“15 16-20 1709.59 181.96 1520.39 1376.06 1906.97 190.69 1191.89 532.31 1366.18 653.59 1841.39 1234.53 1887.53 1449.22 1351.05 2002.44 1425.59 1422.71 1814.52 1704.52 1900.32 2153.42 1199.42 “243.32 1309.82 932.02 1605.62 587.42 1362.82 103.52 1559.54 952.07 263.24 731.29 68.02 188.96 21-25 110.31 61.80 56.70 94.50 92.09 86.10 66.24 86.46 65.52 73.32 79.30 17.15 5.43 21-25 0.00 0.00 0.00 1309.13 1025.43 “195.66 1163.63 574.12 530.94 0.00 0.00 520.75 1050.53 506.47 153.67 442.60 506.16 160.18 FECAL PANTOTHENATE (NNOL/S DAYS) 26-30 86.58 99.00 71.94 97.44 62.01 83.39 16.15 7.21 26-30 0.00 0.00 0.00 0.00 0.00 0.00 “661.06 “510.28 “977.97 0.00 0.00 0.00 0.00 “518.88 “63.45 “182.11 345.77 154.36 TOTAL 184.98 150.90 199.38 414.48 373.44 196.50 392.01 459.59 275.88 235.14 208.02 266.67 249.87 341.26 393.12 289.42 97.46 25.18 INTAKE “ (URINARY + FECAL EXCRETION) (NMOL) TOTAL 5922.39 7081.46 6017.23 6859.86 7290.60 6611.45 7573.81 8245.79 5792.04 7534.12 8133.09 5373.13 7324.93 6198.10 5416.99 6758.33 933.60 241.24 94 STUDY 2 BASELINE GROUP BLOOD, TISSUE CONTENT AND WHOLE BODY PANTOTHENATE RAT # NMOL PA/ NMOL PA/ WGT (G) NMOL PA/ ML BLOOD G RAT RAT 1 115.10 40.21 4628.17 2 0.91 165.00 41.65 6872.25 3 1.71 174.60 40.73 7111.46 4 1.03 158.90 40.26 6397.31 5 0.58 118.80 41.46 4925.45 6 0.79 154.00 35.22 5423.88 7 0.49 160.90 33.97 5465.77 8 0.82 161.40 33.83 5460.16 9 0.43 150.00 36.85 5527.50 MEAN: 0.85 150.97 38.24 5756.88 SD: 0.40 20.47 3.26 850.25 SE: 0.13 6.82 1.09 283.42 DAY 5 95 RAT WEIGHT (G) DAY 10 DAY 15 DAY 20 DAY 25 DAY 30 STUDY 2 DEF, TC GROUP RAT 4 DAY 0 12 45.80 13 51.80 14 43.00 15 40.50 16 41.80 17 49.20 19 38.90 20 43.00 21 38.70 22 44.30 MEAN 43.70 SD 4.25 SE 1.34 42.20 71.30 57.40 40.00 51.70 51.70 37.80 52.80 35.50 45.50 48.59 10.72 3.39 41.80 80.20 81.20 40.40 52.10 49.60 38.00 71.70 36.10 57.90 54.90 17.19 5.44 49.10 96.50 92.20 42.80 49.70 49.10 39.70 81.50 37.00 73.00 61.06 22.45 7.10 55.90 105.60 99.40 48.90 48.50 48.00 56.50 90.60 41.30 86.50 68.12 24.38 7.71 66.20 117.80 100.50 53.00 63.50 46.50 72.00 97.70 53.70 104.40 77.53 25.21 7.98 77.50 119.40 106.50 55.70 75.30 46.40 77.00 106.10 62.50 100.80 82.72 24.30 7.69 STUDY 2 DEF, TC GROUP BLOOD, RAT # 12 13 14 15 16 17 19 20 21 22 MEAN: SD: SE: RAT # 12 13 14 15 16 17 19 2O 21 22 MEAN: SD: SE: 12 13 14 15 17 19 20 21 22 MEAN: SD: SE: 0.26 0.27 0.27 0.30 0.33 0.30 0.30 0.24 0.27 0.23 0.28 0.03 0.01 NMOL PA/ URINE 96.00 141.00 179.00 53.00 66.00 48.00 78.00 105.00 132.00 175.00 107.30 47.69 . 15.09 1-5 28.01 44.77 39.98 32.15 41.18 39.72 27.30 40.97 30.85 32.87 35.78 6.20 1.96 G RAT 58.78 54.67 31.96 37.04 36.92 54.66 32.04 39.58 35.79 29.85 41.13 10.69 3.38 3.20 4.70 5.97 1.77 2.20 1.60 2.60 3.50 4.40 5.83 3.58 1.59 0.50 6.10 31.24 51.65 50.54 34.56 41.30 40.04 29.97 44.87 27.89 43.41 39.55 8.37 2.65 96 NNOL PA/ NNOL PA/ DAY IN U . FECES NMOL PA/ NMOL PA/ CARCASS NMOL PA/ ML BLOOD TISSUE CONCENTRATION AND WHOLE BODY PANTOTHENATE FINAL“ INITIAL WGT (G) RAT BODY POOL 74.52 4380.00 “1376.88 110.97 6067.00 310.12 101.50 3244.00 “2512.88 52.21 1934.00 “3822.88 69.96 2583.00 “3173.88 44.33 2423.00 “3333.88 72.82 2333.00 “3423.88 100.43 3975.00 “1781.88 60.66 2171.00 “3585.88 96.82 2890.00 “2866.88 78.42 3200.00 “2556.88 22.76 1273.42 1273.42 7.20 402.98 402.98 NNOL PA/ APPARENT G FECES BALANCE 110.80 11.08 “206.80 151.20 8.73 “292.20 205.80 12.35 “384.80 153.80 10.28 “206.80 181.00 23.91 “229.00 130.00 13.10 “208.00 254.40 14.79 “359.40 135.00 14.24 “267.00 214.64 13.77 “389.64 181.36 ' 15.40 “288.66 55.18 7.03 75.24 17.46 2.23 23.81 FOOD INTAKE (G/5 DAYS) DAYS 11“15 16“20 21'25 26“30 42.22 46.22 47.96 45.19 59.30 53.59 57.53 49.17 57.19 60.70 55.08 53.86 35.09 36.59 35.75 41.71 38.88 33.95 37.32 43.44 41.57 39.33 36.91 33.65 39.42 40.86 40.35 35.93 54.23 54.53 55.72 50.21 30.08 35.31 46.66 40.53 50.13 48.06 52.51 45.01 44.81 44.91 46.58 43.87 9.81 9.14 8.45 6.25 3.10 2.89 2.68 1.98 TOTAL 240.84 316.01 317.35 215.85 236.07 231.22 213.83 300.53 211.32 271.99 255.50 42.30 13.39 STUDY 2 TC GROUP DEF, RAT # 1-5 1.00 3.55 2.32 1.91 1.90 1.40 1.54 2.98 1.18 1.86 1.96 0.80 0.25 6-10 0.86 2.14 1.77 1.05 0.66 1.24 0.56 1.53 0.94 2.47 1.32 0.64 0.20 97 DAYS 11-15 16-20 2.38 2.10 3.44 2.54 2.02 1.15 2.11 3.11 1.59 2.94 2.34 0.70 0.22 1.91 1.93 3.30 2.87 0.26 1.89 1.07 2.83 1.32 2.49 1.99 0.92 0.29 FECES COLLECTED (G/5 DAYS) 21-25 2.09 3.09 1.91 3.36 0.89 1.46 1.94 2.98 2.01 2.63 2.24 0.77 0.24 26-30 1.76 4.50 3.93 3.23 2.99 0.43 2.70 3.77 2.44 3.20 2.90 1.16 0.37 TOTAL 10.00 17.31 16.67 14.96 8.72 7.57 9.92 17.20 9.48 15.59 12.74 3.90 1.24 STUDY 2 DEF, PF, RAT!’ HHOOOU‘AUNH PO 2 SD SE NO TC GROUP DAY 0 46.10 54.20 44.80 40.80 43.20 47.50 40.50 44.50 35.00 46.90 44.35 5.07 1.60 DAY 5 56.60 75.30 63.20 53.40 59.30 65.60 51.90 64.10 47.90 62.00 59.93 7.89 2.50 98 67.70 93.20 85.80 70.00 76.10 81.30 64.90 79.40 56.80 79.80 75.50 10.71 3.39 RAT WEIGHT (G) DAY 10 DAY 15 DAY 20 DAY 25 DAY 30 91.00 115.00 100.00 80.00 87.00 92.00 83.00 95.00 68.50 97.00 90.85 12.50 3.96 101.50 128.20 107.40 84.50 85.20 95.10 90.50 109.40 72.20 105.30 97.93 15.83 5.01 128.10 143.60 120.00 100.00 101.70 110.00 99.00 118.70 91.00 123.00 113.51 16.02 5.07 131.60 152.50 126.60 109.20 105.80 101.50 98.10 125.90 102.60 133.50 118.73 17.81 5.64 STUDY 2 DEF, BLOOD, RAT # HOWQGUIJDUNH s ..g 5'35?" P“ HO UQGMbUNH HH PF, NO TC GROUP TISSUE CONTENT AND WHOLE BODY PANTOTHENATE 99 FINAL“ NHOL PA/ NMOL PA/ CARCASS NMOL PA/ INITIAL NL BLOOD G RAT WGT (G) RAT BODY POOL 0.17 58.90 122.00 7185.80 1428.92 0.27 39.40 145.21 5721.27 “35.61 0.28 64.42 120.27 7747.79 1990.91 0.27 56.35 102.64 5783.76 26.88 0.26 67.16 99.72 6697.20 940.32 0.23 61.46 96.77 5947.48 190.60 0.30 74.57 92.79 6919.35 1162.47 0.30 55.76 120.78 6734.69 977.81 0.30 54.96 96.74 5316.83 “440.05 0.20 49.39 125.48 6197.46 440.58 0.26 58.24 112.24 6425.16 668.28 0.04 9.67 16.94 754.05 754.05 0.01 3.06 5.36 238.62 238.62 NHOL PA/ NHOL PA/ NMOL PA/ NMOL PA/ BALANCE URINE DAY IN U. FECES G FECES 267.00 8.90 174.24 12.80 “441.24 272.00 9.07 232.60 11.61 “504.60 267.00 8.90 191.60 10.48 “458.60 317.00 10.57 163.80 11.59 “480.80 139.00 4.63 185.00 12.94 “324.00 153.00 5.10 163.00 10.13 “316.00 193.00 6.43 200.80 11.92 “393.80 230.00 7.67 174.00 11.75 “404.00 230.00 7.67 154.40 9.50 “384.40 -231.10 7.70 179.50 11.27 “410.60 55.37 1.85 24.01 1.18 61.77 17.52 0.58 7.60 0.37 19.55 FOOD INTAKE (G/S DAYS) DAYS 1“5 6“10 11“15 16“20 21“25 26“30 28.01 31.24 42.22 46.22 47.96 45.19 44.77 51.65 59.30 53.59 57.53 49.17 39.98 50.54 57.19 60.70 55.08 53.86 32.15 34.56 35.09 36.59 35.75 41.71 41.18 41.30 38.88 33.95 37.32 43.44 39.72 40.04 41.57 39.33 36.91 33.65 27.30 29.97 39.42 40.86 40.35 35.93 40.97 44.87 54.23 54.53 55.72 50.21 30.85 27.89 30.08 35.31 46.66 40.53 32.87 43.41 50.13 48.06 52.51 45.01 35.78 39.55 44.81 44.91 46.58 43.87 6.20 8.37 9.81 9.14 8.45 6.25 1.96 2.65 3.10 2.89 2.68 1.98 TOTAL 240.84 316.01 317.35 215.85 236.07 231.22 213.83 300.53 211.32 271.99 255.50 42.30 13.39 STUDY 2 PF, NO TC GROUP DEF, RAT 4 12 13 14 15 16 17 19 20 21 22- MEAN: SD: SE: 1-5 1.24 2.61 2.80 1.29 1.98 2.40 1.73 1.36 1.53 1.17 1.81 0.60 0.19 6-10 1.78 2.81 2.71 2.04 1.64 2.42 1.76 2.48 2.03 2.14 2.18 0.41 0.13 100 2.07 3.43 2.87 2.82 2.39 2.62 2.50 3.05 2.32 2.27 2.63 0.41 0.13 16-20 2.90 3.87 3.19 2.35 2.80 2.87 3.29 2.69 2.39 3.32 2.97 0.46 0.15 FECES COLLECTED (G/5 DAYS) DAYS 11-15 21-25 2.50 3.70 3.04 2.43 2.79 2.33 3.10 3.38 2.24 3.55 2.91 0.53 0.17 26-30 3.12 3.61 3.67 3.20 2.70 3.45 3.21 3.88 4.30 3.81 3.50 0.46 0.14 TOTAL 13.61 20.03 18.28 14.13 14.30 16.09 15.59 16.84 14.81 16.26 15.99 1.99 0.63 101 PRELIMINARY STUDY BASELINE GROUP BLOOD, TISSUE CONTENT AND WHOLE BODY PANTOTHENATE RAT # NMOL PA/NMOL PA/ WGT NMOL PA/ ML BLOOD G RAT (G) RAT 2 0.79 100.93 52.02 5250.38 3 0.57 110.69 50.53 5593.17 4 1.11 103.90 47.03 4886.42 5 0.89 108.27 44.67 4836.42 6 0.95 113.30 47.50 5381.75 7 1.11 117.69 51.43 6052.80 8 0.76 122.28 54.09 6614.13 9 1.08 122.67 46.35 5685.75 10 0.81 106.48 41.40 4408.27 AVERAGE: 0.90 111.80 48.34 5412.12 SD: 0.18 7.80 4.01 670.92 SE: 0.06 2.60 1.34 223.64 PRELIMINARY STUDY DEFICIENT GROUP RAT # Arrival 40.80 40.50 36.25 38.05 42.10 48.20 35.50 37.10 37.75 40.70 39.70 3.70 1.17 102 RAT WEIGHT Day 5 Day 10 51.04 62.19 49.19 65.49 56.74 63.69 71.49 72.09 63.34 69.69 65.84 80.79 58.49 74.59 60.84 70.89 66.29 78.89 63.19 72.59 60.64 71.09 6.94 6.12 2.19 1.94 (G) Day 15 77.10 73.80 75.40 78.80 76.30 92.80 83.00 77.60 90.20 89.40 81.44 6.93 2.19 Day 20 93.60 75.50 80.50 91.60 85.50 109.50 91.80 88.70 101.00 101.90 91.96 10.24 3.24 Day 25 101.50 83.20 93.90 114.80 96.00 126.00 108.80 102.50 119.00 116.90 106.26 13.20 4.18 Day 30 97.69 84.69 92.19 125.49 97.69 129.89 109.19 108.49 116.49 115.69 107.75 14.61 4.62 PRELIMINARY STUDY DEFICIENT GROUP 103 BLOOD, TISSUE CONTENT AND WHOLE BODY PANTOTHENATE RAT 4 13 14 15 17 18 19 20 21 22 23 SD: SE: 13 14 15 17 18 19 20 21 22 23 AVERAGE: SD: SE: RAT 8 13 14 15 17 18 19 20 21 22 23 0.35 0.19 0.23 0.34 0.13 0.17 0.34 0.10 0.11 0.19 0.22 0.09 0.03 39.00 37.80 40.50 45.80 44.00 44.50 43.00 43.00 49.50 44.70 43.18 3.44 1.09 29.40 31.80 16.60 26.00 45.00 39.50 26.80 23.60 45.30 10.10 29.41 11.57 3.66 NMOL PA/ NMOL PA/ ML BLOOD GRAM RAT 64.00 68.00 62.00 76.00 70.00 70.00 71.00 63.00 72.00 67.00 68.30 4.51 1.43 6-10 43.00 50.20 41.90 42.00 44.70 51.90 50.70 48.60 50.50 55.90 47.94 4.80 1.52 6-10 36.20 14.80 24.50 22.40 24.40 15.50 24.70 18.20 19.90 18.80 21.94 6.22 1.97 WGT (G) 95.30 79.20 89.50 119.10 94.10 122.30 100.30 100.80 107.40 110.30 101.83 15.17 4.80 11-15 50.90 51.90 44.10 47.80 44.00 48.35 47.40 55.10 53.90 52.50 49.60 3.90 1.23 DAYS 11-15 13.30 3.20 6.60 3.00 4.40 6.60 10.00 4.60 5.30 5.60 6.26 3.20 1.01 RAT 6135.00 5351.00 5508.00 9104.00 6585.00 8498.00 7075.00 6307.00 7783.00 7396.00 6974.20 1401.05 443.37 FOOD INTAKE (G/5 DAYS) DAYS 16-20 56.80 47.60 42.80 47.20 43.50 56.80 54.30 48.70 63.70 53.00 51.44 6.67 2.11 16-20 15.00 8.90 19.80 10.70 15.80 12.20 11.90 17.60 16.30 17.90 14.61 3.55 1.12 NMOL PA/ FINAL- INITIAL BODY POOL 722.00 “61.00 96.00 3692.00 1173.00 3085.00 1663.00 895.00 2371.00 1984.00 1562.00 1400.95 443.34 21-25 61.20 51.00 51.10 55.10 47.90 63.20 58.50 53.30 7 63.80 56.00 56.11 5.49 1.74 URINARY PANTOTHENATE (NNOL/s DAYS) 21-25 9.80 2.20 4.20 5.00 6.00 15.40 9.70 5.50 4.00 8.80 7.06 3.91 1.24 26-30 53.30 44.70 48.10 57.40 49.80 60.50 54.20 53.10 58.70 54.40 53.42 4.89 1.55 26-30 16.80 10.30. 14.30 19.00 12.80 13.00 13.00 13.40 11.00 15.00 13.86 2.60 0.82 TOTAL 304.20 283.20 268.50 295.30 273.90 325.25 308.10 301.80 340.10 316.50 301.69 22.62 7.16 TOTAL 120.50 71.20 86.00 86.10 108.40 102.20 96.10 82.90 101.80 76.20 93.14 15.44 4.89 PRELIMINARY STUDY DEFICIENT GROUP SD: SE: PANTOTHENATE BALANCE: RAT! 13 14 15 17 18 19 20 21 22 23 MEAN: SD: SE: 1“5 114.00 75.00 69.00 63.00 42.00 117.00 69.00 81.00 88.00 162.00 88.00 34.63 10.96 1“5 “143.00 “107.00 “86.00 “89.00 “87.00 “156.00 “105.00 “133.00 “172.00 31.46 9.95 6“10 37.80 45.30 41.60 41.30 32.70 42.60 37.30 34.50 32.70 45.60 39.14 4.88 1.54 6-10 “60.00 “66.00 “64.00 “57.00 “58.00 “62.00 “53.00 “53.00 “61.10 6.42 2.03 104 DAYS 11“15 87.30 51.90 58.20 42.00 59.10 66.70 51.00 96.90 85.20 68.70 66.48 18.97 6.00 DAYS 11-15 16-20 46.20 43.50 55.90 82.80 44.90 77.10 50.00 52.20 69.30 88.80 61.07 17.04 5.39 16-20 “61.00 “93.00 “61.00 “89.00 “62.00 “70.00 “86.00 “107.00 -75.79 17.66 5.59 21-25 38.40 22.60 39.00 78.00 60.60 105.00 41.60 64.00 59.30 72.20 58.07 23.99 7.59 21“25 “48.00 “25.00 “43.00 “83.00 “67.00 “120.00 “51.00 “70.00 “63.00 “81.00 “65.10 26.41 8.36 FECAL PANTOTHENATE (NNOL/s DAYS) 26-30 69.80 58.90 83.70 86.00 104.00 122.00 87.50 82.80 99.50 121.50 91.57 20.57 6.51 “87.00 “69.00 “98.00 “105.00 “117.00 “135.00 “100.00 “96.00 “136.00 “105.30 20.67 6.54 TOTAL 393.50 297.20 347.40 393.10 343.30 530.40 336.40 411.40 434.00 558.80 404.55 84.79 26.83 INTAKE “ (URINARY + FECAL EXCRETION) (NMOL) 26-30 TOTAL “514.00 “368.00 “434.00 “479.00 “453.00 “631.00 “495.00 “634.00 “497.50 85.82 PRELIMINARY STUDY CONTROL GROUP 10 CQGAUNH‘ HF! rec AVERAGE SD SE Arrival 37.05 42.50 40.05 38.70 40.30 38.80 37.25 40.06 39.50 35.70 38.99 1.85 0.59 105 Day 5 55.89 65.24 66.04 68.14 67.39 65.49 53.19 71.29 70.19 49.59 63.25 7.16 2.27 RAT WEIGHT (G) Day 10 69.69 88.14 81.94 92.59 92.79 91.39 75.29 99.69 98.49 68.99 85.90 10.74 3.40 Day 15 70.80 113.21 110.20 120.55 121.55 122.80 94.80 128.30 114.10 91.60 108.79 16.92 5.35 Day 20 71.70 138.40 137.40 145.50 147.40 151.50 123.60 151.80 114.00 115.70 129.70 23.46 7.43 Day 25 75.00 170.90 164.80 171.90 180.70 185.90 145.50 183.90 153.10 142.10 157.38 31.13 9.85 Day 30 73.50 186.80 176.70 191.50 192.50 205.20 158.40 201.50 179.40 149.00 171.45 36.72 11.62 PRELIMINARY STUDY CONTROL GROUP BLOOD, RAT fl QNObHN 10 AVERAGE: SD: SE: RAT # ONOAMN 10 AVERAGE: SD: SE: RAT 0 OVOMAUN 10 AVERAGE: SD: SE: 0.80 1.10 1.10 0.60 1.30 0.80 1.30 1.00 0.27 0.10 1-5 2713.03 2323.24 2586.66 2205.85 2266.79 2347.95 2481.91 2417.92 179.77 69.14 1'5 53.87 46.13 51.36 43.80 45.01 46.62 49.28 48.01 3.57 1.37 NHOL PA/ NMOL PA/ ML BLOOD GRAN RAT 106 NHOL PA/ RAT 21554.40 24663.53 25761.50 26445.60 24931.80 20725.50 20455.70 23505.43 2467.96 949.21 16-20 3752.02 3485.14 3865.39 3769.62 3945.89 3527.92 2953.80 3614.25 329.53 126.74 16-20 74.50 69.20 76.76 74.85 78.35 70.05 58.65 71.77 6.54 2.52 HGT (G) 124.24 173.50 152.81 161.40 145.25 177.40 145.17 182.20 127.32 195.80 111.29 186.20 120.25 170.10 132.33 178.09 15.15 11.05 5.83 4.25 DAYS 6-10 11-15 3349.12 3888.04 2759.84 3286.21 2996.58 3966.11 2775.00 3392.28 2470.30 3391.90 2754.88 3240.81 3059.58 3205.62 2880.76 3481.57 276.09 307.29 106.19 118.19 FOOD INTAKE (8/5 DAYS) DAYS 6-10 11-15 66.50 77.20 54.80 65.25 59.50 78.76 55.10 67.36 49.05 67.35 54.70 64.35 60.75 63.65 57.20 69.13 5.48 6.10 2.11 2.35 TISSUE CONTENT AND WHOLE BODY PANTOTHENATE FINAL- INITIAL BODY POOL 16142.28 19251.41 20349.38 21033.48 19519.68 15313.38 15043.58 18093.31 2467.96 949.21 PANTOTHENATE INTAKE (NMOL/5 DAYS) 21-25 4094.45 3447.32 4016.47 3837.68 4089.49 3885.51 3991.29 3908.89 220.93 84.97 21-25 81.30 68.45 79.76 76.20 81.21 77.15 79.26 77.62 4.39 1.69 26-30 4177.58 3792.28 4079.38 3752.02 4336.25 3956.00 4026.49 4017.14 202.76 77.98 26-30 82.95 75.30 81.00 74.50 86.11 78.55 79.95 79.77 4.03 1.55 RAT fl OVOOUN 10 AVERAGE: SD: SE: RAT 4 QVObUN 10 AVERAGE: SD: SE: PANTOTHENATE BALANCE: RAT I 2 3 4 6 7 0 10 AVERAGE: SD: SE: 1-5 247.20 188.40 188.88 181.20 183.60 188.88 144.00 188.88 29.72 11.43 1-5 49.97 157.03 82.70 46.10 57.90 90.00 90.20 81.99 37.28 14.34 1-5 2415.86 1977.81 2315.08 1978.55 2025.29 2069.07 2247.71 2147.05 173.98 66.91 DAYS 6-10 11-15 16-20 21-25 26-30 63.00 60.70 138.00 3649.20 5024.80 117.20 118.20 187.20 2893.20 4948.80 173.20 81.50 200.40 3974.40 5749.20 46.00 237.20 1429.80 5412.00 2400.00 40.00 168.00 588.00 4237.20 3549.60 47.30 106.40 427.07 5373.60 3174.00 108.70 67.40 187.87 4256.60 4141.07 85.06 119.91 451.19 4256.60 4141.07 48.79 62.06 452.74 885.99 1156.73 18.76 23.87 174.13 340.77 444.90 FECAL PANTOTHENATE (NMOL/S DAYS) DAYS 6-10 11-15 16-20 21-25 26-30 36.00 39.50 55.80 66.00 104.40 58.20 43.70 60.00 45.00 41.20 29.70 73.50 72.00 99.00 81.50 58.80 69.80 25.80 51.00 80.02 43.20 40.20 50.70 162.00 111.60 44.70 60.80 19.20 108.00 74.30 38.40 37.20 33.00 36.00 67.10 44.14 52.10 45.21 81.00 80.02 10.76 15.20 19.13 44.00 23.02 4.14 5.85 7.36 ,16.92 8.85 INTAKE - (URINARY + FECAL EXCRETION) (NMO DAYS 6-10 11-15 16-20 21-25 26-30 3250.12 3787.84 3558.22 379.25 -951.62 2584.44 3124.31 3237.94 509.12 “1197.72 2793.68 3811.11 3592.99 -56.93 -1751.32 2670.20 3085.28 2314.02 -1625.32 1272.00 2387.10 3183.70 3307.19 ~309.71 675.05 2662.88 3073.61 3081.65 -1596.09 707.70 2912.48 3101.02 2732.93 -301.31 -181.68 2751.56 3309.55 3117.85 -428.71 '203.94 269.20 330.36 450.71 849.47 1113.44 103.54 127.06 173.35 326.72 428.25 107 URINARY PANTOTHENATE (NHOL/S DAYS) LIST OF REFERENCES 108 LIST OF REFERENCES Adams, J. 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