MSU RETURNING MATERIALS: P1ace in book drop to LJBRARJES remove this checkout from .—_-—. your record. FINES will » be charged if book is ‘- "web returned after the date stamped below. ‘=‘ :5 4 ;fP 2'773114 1:2,», 5 PANTOTHENIC ACID CONTENT OF VARIOUS RAT BLOOD COMPONENTS BY Cheryl Anne Bates 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 1988 ABSTRACT PANTOTHENIC ACID CONTENT OF VARIOUS RAT BLOOD COMPONENTS BY Cheryl A. Bates The blood profile and pantothenic acid (RA) content of red cells, white cells, platelets, and plasma in rats were studied in order to identify components which reflect PA intake sensitively and reliably. Fifty-four weanling Sprague- Dawley rats in three groups fed control diet, ad libitum, pantothenic acid-deficient diet, ad libitum, or pair-fed control diet were killed after 3, 6, or 9 wks of treatment. Blood was separated into its components by density gradient centrifugation. PA content of the cells was determined using radioimmunoassay. Rats fed a PA-deficient diet had significantly decreased total and free PA in whole blood and free PA in the plasma at 3, 6 and 9 wks (p<0.01). PA in isolated red cells, white cells and platelets did not change with treatment. Instead whole blood total and free PA did decrease with decreased intake of the vitamin, thereby establishing this parameter as a valid and reliable indicator of pantothenic acid intake. ACKNOWLEDGMENTS My deepest thanks are extended to Dr. Won Song. Her knowledge, encouragement and advice were key to the success of this thesis and to my overall graduate training. In addition, Dr. Song provided generous support in terms of her time, availability and desire for success in my career. In addition, I thank Dr. Chenoweth, Dr. Bennink, and Mrs. Thomas for their valuable input. Lastly, I would like to thank my husband, Richard for his continued love, encouragement and willingness to sacrifice to help me reach this goal. ii TABLE OF CONTENTS Introduction.......... .......... . .................. 1 Review of Literature..... ..... .. ........ .... ....... 5 History.........................................5 Pantothenic acid requirement in rats............6 Deficiency symptoms in animals..................7 Deficiency symptoms in humans...................9 Indicators of pantothenic acid status...........11 Coenzyme A metabolism...........................12 CoA synthesis......................... ....... 12 CoA degradation.................... ...... ....15 Hematopoiesis/Whole blood.......................15 Blood separation................................20 Erythrocytes....................................21 Leukocytes......................................25 Platelets.......................................29 P1asma..........................................30 Summary.........................................33 Materials and Methods..............................34 Experimental design.............................34 Blood separation............... ..... ............35 Pantothenic acid determination.......... ........ 37 Statistical analysis............................39 Results............................................u0 ‘Blood cell counts...............................HO Pantothenic acid content of cellular components of blood..................u4 Pantothenic acid distribution in blood.............. ........ . .............. 51 Discussion........................... .............. 53 Summary............... .................... ..... ...59 Conclusion.................. ....... .. ..... .........61 Recommendations....................................62 References.........................................63 Appendices.........................................7O Appendix A - Composition of USB biological pantothenic acid-deficient test diet for rats......................7O Appendix B - Food intake and weight gain of rats............................71 Appendix C - Individual raw data for blood cell counts, hematological indices and pantothenic acid content of blood components................72 iii Table Table Table Table Table Table Table Table LIST OF TABLES Reported human blood pantothenic acid (PA) content.................... ............ 18 Reported rat blood pantothenic acid (PA) content...........................19 Reported human red blood cell (RBC) pantothenic acid (PA) content ............... 2U Reported human plasma pantothenic acid (PA) content................................31 Reported rat plasma pantothenic acid (PA) content........................... ..... 32 Complete blood counts and hematological indices for Group I (Control), Group II (Deficient) and Group III (Pair-fed) rats...42 Free (FPA) and total (TPA) content of whole blood and blood components....... ..... ......45 Pantothenic acid content and distribution in blood cell components.......................52 iv Figure Figure Figure Figure Figure Figure Figure Figure LIST OF FIGURES Conversion of pantothenic acid to coenzyme A.................. ........... 1n Degradation of CoA to pantothenic acid......................................16 Weight gain of rats during the 9-week experimental period.... ............ U1 Total and free pantothenic acid content of whole blood....................46 Total and free pantothenic acid content of red blood cells (RBC). ......... “7 Total and free pantothenic acid content of white blood cells (WBC)........ “8 Total and free pantothenic acid content of p1atelets......................“9 Pantothenic acid content of plasma........ 50 INTRODUCTION Pantothenic acid is one of the B vitamins and, although an RDA level has not been set, it is well established that this vitamin is essential for humans and some animals for normal growth, reproduction, and physiological function (Robishaw and Neely, 1985). Currently, the Estimated Safe and Adequate Daily Dietary Intake of pantothenic acid is 4-7 mg/day for adults as established by the Food and Nutrition Board (1980). It is generally assumed that pantothenic acid intake is adequate because it is found in a wide variety of foods and no overt clinical deficiency symptoms have been reported in the free living population. However, recent studies show that sub-clinical deficiencies may exist (Chipponi et al., 1982). Physiological stress, such as pregnancy or alcoholism, may also lead to more rapid depletion of the vitamin (Cohenour and Galloway, 1972: Tao and Fox, 1976). Food sources include liver, eggs, beef, vegetables, and legumes. Pantothenic acid, along with ATP and cysteine, is needed to synthesize coenzyme A (CoA). CoA is necessary for the metabolism of carbohydrates, amino acids, and lipids through acetylation reactions. Most of the pantothenic acid in foodstuffs is present in the bound form as CoA. Since CoA cannot permeate cell membranes, it must be broken down to pantothenic acid or pantetheine to be absorbed. Phosphopantetheine is a component of acyl carrier protein 2 (ACP) which is essential for fatty acid biosynthesis (Robishaw and Neely, 1985). CoA and ACP together are necessary for over 100 metabolic pathways which occur in the body. Pantothenic acid deficiency has been induced in rats, but the clinical manifestations are rather non-specific, i.e. anorexia and lethargy (Nelson and Evans, 1946). Additional deficiency symptoms in rats and other animals include growth retardation, weight loss, a rough hair coat, gastrointestinal disorders, muscle weakness, and eventual death (Reibel et al., 1982; Sheppard and Johnson, 1957; Nelson, 1968). The methods currently employed to assess pantothenic acid nutriture include measurement of the vitamin in whole blood, plasma, and/or urine. These measurements, however, correlate poorly with dietary intake in well-nourished human subjects and thus are considered as insensitive and unreliable methods to assess pantothenic acid status or to detect sub-clinical deficiencies (Song et al, 1985; Kathman and Kies, 1984; Srinivasan et al, 1981). Furthermore, standards used for interpretation of the values are conflicting (Sauberlich et al., 1974; Baker and Frank, 1968). An insensitive test does not detect measurable parameters of) a deficiency until it has reached the most advanced stage in which overt clinical symptoms are apparent. An unreliable test does not accurately reflect pantothenic acid status in tissues. The sensitivity and reliability of the methods of assessment can be evaluated by feeding a pantothenic acid 3 deficient diet to rats and monitoring dependent biochemical indices. Once these methods are validated in the rat model, they can further be validated in humans. In the end, this information will be used to establish a nutrient requirement level and the pantothenic acid status of individuals can be monitored. The goal of this study was to examine current assessment methods and to further determine the PA content of blood components in order to determine if one component reflects PA intake more sensitively and reliably than others. The aim was to examine whether the concentration of free or bound forms of pantothenic acid in blood components reflect pantothenic acid intake. The specific objectives used to achieve this goal were to: 1. separate fresh rat blood quantitatively and qualitatively into its red blood cell, white blood cell, platelet, and plasma components on gradients of Percoll: 2. determine bound and free pantothenate in these blood components and whole blood by radioimmunoassay (RIA); 3. compare the bound and free pantothenic acid content in blood components of healthy control rats to those of rats on‘a pantothenic acid deficient diet: 4. determine which component of the blood will show the most sensitive change in bound and free forms of pantothenic acid when a diet deficient in pantothenic acid is fed; and 5. compare the amount of pantothenic acid measured in blood components to the amount of pantothenic acid measured in whole blood. It was hypothesized that pantothenic acid in red blood cells(RBC), white blood cells (WBC), platelets, and plasma would function independently and thus reflect pantothenic acid intake with varying sensitivities. Again, the pantothenic acid concentration in one particular component may vary with nutrient intake while overall whole blood pantothenic acid may remain unchanged. REVIEW OF LITERATURE HISTORY: Pantothenic acid was first reported by R. J. Williams and coworkers in 1933 (Novelli, 1953) as an acidic substance which served as a growth factor for yeast. Due to its wide distribution in the tissues of many species, the substance was named from the greek word "pantos" meaning everywhere. In 1937, Snell and coworkers isolated pantothenic acid from the lactic acid bacteria thereby providing a foundation for the use of lactic acid bacteria as assay organisms for the ' vitamin. In 1939, it was reported that the substance which had been called the chick anti-dermatitis factor and the liver filtrate factor in rats was indeed pantothenic acid (Novelli, 1953; Woolley et al., 1939). It was not until coenzyme A was discovered in 1945 that the biochemical function of this vitamin became known (Lipmann, 1945). Since its discovery, pantothenic acid has been measured in blood, plasma, and urine in an effort to link depressed levels to various disease states. In 1957, Slepyan and coworkers found low levels of bound pantothenic acid in the blood of persons with discoid and systemic lupus erythematosus and lymphoblastoma. Free pantothenic acid levels in the blood were not significantly different as compared to the healthy controls. Blood pantothenic acid was also low in patients with rheumatoid arthritis (Barton-Wright and Elliot, 1963), and continued to decrease with increased severity of symptoms. In patients with ulcerative colitis or granulomatous disease, blood and colonic mucosal pantothenic acid levels appear to be normal, however, coenzyme A activity was decreased (Ellestad-Sayed et al., 1976). Past studies indicated that during pregnancy, pantothenic acid levels in the blood decrease (Cohenour and Colloway, 1972; Song et al., 1985), and infants with low birth-weight or who are born prematurely also exhibit depressed levels (Baker et al., 1977). All of the studies mentioned above propose mechanisms for decreases in blood pantothenic acid or coenzyme A levels, which collectively indicates that the human body's requirement for this vitamin may increase during times of physiological stress and disease. PANTOTHENIC ACID REQUIREMENT IN RATS: In 1940, Klaus Unna conducted'a study in which he fed rats a diet deficient in pantothenic acid. Some rats received supplemental pantothenic acid in their diet while others received two compounds which together comprise the vitamin, alpha-hydroxy-beta-dimethyl-gamma-butyrolactone and beta-alanine. Unna found no improvements in deficiency symptoms when the two products were given singly, and that 80 ug per day of pantothenic acid in the diet appeared adequate to prevent external deficiency symptoms such as dermatitis 7 and porphyrin-caked whiskers. Early studies conducted by Unna and Richards (1942) were designed to determine the requirement level of pantothenic acid for the rat. Their study was based on the growth rate seen in rats fed various amounts of pantothenic acid in the diet. The requirement levels were set by observing the levels at which growth slowed (lower limit), and the level at which greater weight gain did not occur (upper limit). Using this research design, the authors succeeded in determining the level of pantothenic acid required for growth only. By measuring the ability of the animal to acetylate sulfonamides, the amount of pantothenic acid required for normal metabolic function could be determined in the absence of external signs of deficiency such as growth rate (Riggs and Halstead, 1948: Shils et al., 1949; and Barboriak et al., 1957). The use of growth rate as a determinant of requirement level may be appropriate in weanling (rapidly growing) rats, but in adult rats a more sensitive paramenter, such as ability to acetylate, should be used. As a result of these studies, the nutritional requirement for pantothenic acid has been set at 0.8-1.0 mg calcium pantothenate per 100 grams of diet. DEFICIENCY SYMPTOMS IN ANIMALS: Following its discovery, many studies were conducted to pinpoint symptoms associated with pantothenic acid deficiency in various species. In a number of studies, rats developed achromotrichia, scaly dermatitis, alopecia, and 8 porphyrin-caked whiskers (Henderson et al., 1942). Growth retardation (Moiseenok et al., 1987), adrenal necrosis, and congenital malformations in offspring were also shown with pantothenic acid deficiency in rats (Nelson et al., 1947). Mills and coworkers (1940) found that rats fed a purified diet deficient in pantothenic acid developed adrenal necrosis: however when a pantothenic acid supplement was given, the necrosis was completely prevented in 100% of the cases. Pantothenic acid has also been positively identified as a curative agent for nutritional achromotrichia in rats (Gyorgy and Poling, 1940). Chicks fed a pantothenic acid deficient diet develOped lesions on the corners of the mouth, swollen eyelids, hemorrhagic cracks on the feet, growth retardation, poor feathering and hatchability, paralysis, and fatty degeneration of the liver (Hegsted et al., 1940: Gries and Scott, 1972). Dogs developed fatty livers, mottled thymus, convulsions, nervous symptoms, and gastrointestinal tract disorders (Schaefer et al., 1942). When fed a pantothenic acid-deficient diet, pigs grew slowly, and developed a "goose-stepping" gate (Hughs, 1942). Female hogs developed fatty livers, enlarged adrenal glands, intramuscular hemorrhage, dilation of the heart, diminution of the ovaries, and improper development of the uterus (Ullrey et al., 1965). DEFICIENCY SYMPTOMS IN HUMANS: In humans, deficiency of pantothenic acid has not been observed except in cases of severe malnutrition where multiple vitamin deficiencies are present (Hodges et al., 1958). During World War II, prisoners in Japan and the Philippines developed "burning feet syndrome" believed to be a result of pantothenic acid deficiency (Glusman, 1947). Symptoms included abnormal skin sensations in the feet and lower legs. Bean and coworkers (1955) induced pantothenic acid deficiency in humans by using two vitamin antagonists: pantoyltaurine and omega-methyl pantothenic acid. It was found that pantoyltaurine was not able to induce deficiency ‘ over a twenty-month period, thus was not considered an adequate antagonist. After 25 days, subjects receiving omega-methyl pantothenic acid complained of numbness and tingling of the hands and feet, paresthesias, and upper respiratory infections. Hyperactive deep tendon reflexes, low blood pressure, increased insulin sensitivity, a high eosinophil count, and failure of ACTH to induce eosinopenia were also observed. A similar study conducted by Thornton and coworkers (1955) found a severe depression of gastric secretion; thus it was hypothesized that a lack of dietary pantothenic acid (or its bound form, CoA) may have a direct inhibitory effect on production of hydrochloric acid (HCl) from the parietal cells of the stomach. Other studies (Hodges et al., 1958; 1959) supported the work of Bean et al 10 (1955) with additional symptoms being observed: fatigue, headache, weakness, impaired motor coordination, muscle cramps, gastrointestinal distress, and tachycardia. It should be noted that human studies were conducted with the aid of metabolic antagonists on a minimal number of subjects and resulted in a large individual variation of symptoms. Vitamin deficiency, in general, occurs in three stages according to Chipponi and coworkers (1982). During the first stage of deficiency, blood and tissue levels are maintained by nutrient reserves in various tissue. During this stage it would be hard to detect a deficiency since tissue samples are not readily available for assessment. During the second stage of deficiency, the reserved stores become depleted, but overt clinical symptoms are not yet apparent. At this time it is possible to indirectly measure plasma, whole blood, urine, feces, and products of biochemical reactions to detect deficiency. During the third stage of vitamin deficiency, specific signs and symptoms can be seen in addition to biochemical changes. By detecting deficiency during the second stage, the expression of full-blown symptomatology can be avoided. Although pantothenic acid deficiency may be rare or non-existant in the general population, finding a sensitive indicator of pantothenic acid status may help in detecting sub-clinical deficiencies in some groups or individuals. Identifying the location of the greatest pantothenic acid concentration with the most sensitive change in whole 11 blood may aid in determining pantothenic acid status. Both bound and free forms of the vitamin can be found in whole blood. The cellular elements of the blood contain mostly the bound form (CoA), and the free form is more concentrated in the serum (Robishaw and Neely, 1985; Novelli, 1953). INDICATORS OF PANTOTHENIC ACID STATUS: Pantothenic acid nutritional status has been assessed by dietary intake, blood concentrations and urinary excretion of total pantothenic acid. Dietary intake can only be used as a good indicator of pantothenic acid status if a requirement for the vitamin is known, and if complete food composition data are available. The biochemical paramenters used to measure pantothenic acid status (i.e. blood and urinary pantothenate) are unreliable and insensitive. In addition, these parameters lack criteria for practical use and basic relevant information necessary to interpret the data. In a study conducted by Song et al (1985) of pregnant and lactating women, it was shown that the pantothenate levels in fasting whole blood, plasma, and 24-hour urine samples correlated poorly with the vitamin intake. A similar low correlation was shown between dietary intake and plasma in adolescents (Kathman and Kies, 1984) and between dietary intake and blood in the elderly (Srivivasan et al., 1981). The indices also showed large individual variation suggesting poor correlation coefficients and reliability. The parameters also have limited use because there is no 12 established normal or reference ranges. The concentrations of "normal" healthy humans reported in the literature range from 0.27 to 8.08 nmole/ml in whole blood (Pelczar and Porter, 1941: Cohenour and Calloway, 1972) and from 0.002 to 17.53 nmole/ml in serum (DeBari et al., 1984: Markkanen, 1973). Some of the variation was linked to methodology used in the preparation and analysis of samples. As a result, interpretation of data on adequacy of the pantothenate nutriture differs among researchers. A few studies reported that free pantothenate in blood or blood plasma pantothenate levels were decreased during deficiency (Kathman and Kies, 1984: Reibel et al., 1982). Yet others observed an elevated serum pantothenate level with a simultaneous reduction of urinary pantothenic acid during fasting (Reibel et al., 1981). The latter observation can be due to the redistribution process of tissue stores because CoA or other bound forms of pantothenate cannot cross cell membranes as such. Interpretation of these types of reported data is difficult due to the lack of basic understanding of pantothenate metabolism, tissue stores, distribution, total body pool and dose-response. COENZYME A METABOLISM: CoA Synthesis: A basic understanding of CoA metabolism will provide a basis for the analysis of blood cell concentration of 13 pantothenic acid. The synthesis of CoA from pantothenic acid, as proposed by Brown (Brown, 1959) and later confirmed by Abiko (1967), involves a series of reactions with five specific enzymes (Figure 1). The reaction involves the phosphorylation of pantothenic acid to 4’-phosphopantothenic acid by pantothenate kinase. ATP and Mg are required at this step. Next is the two-step conversion to 4'- phosphopantetheine. First 4’-pantothenic acid and cysteine are converted to 4'-phosphopantothenoyl-cysteine by formation of a peptide linkage. This is followed by decarboxylation of the cysteine to form 4'-phosphopantetheine, an ATP-requiring step. The enzymes in the pathways outlined above (pantothenate kinase, synthetase, and decarboxylase) are present exclusively in the cytosol of the cell. In the final two steps, the 4’-phosphopantetheine is adenylated to form dephospho-CoA, and this is then phosphorylated to form CoA. The 4'-phosphopantetheine may enter the mitochondria or remain in the cytosol for further conversion to CoA since the last two enzymes in the pathway (pyrophosphorylase and kinase) are present in both the mitochondria and the cytosol (Robishaw and Neely, 1985). CoA does not appear to penetrate the mitochondrial membrane once synthesized (Bremer et al., 1972; Haddock et al., 1970; Odell¥Wenger et al., 1978), and the 4'-phosphopantetheine and CoA produced appear to have a negative feedback effect on pantothenate kinase thereby regulating its synthesis. Skrede and Halvorsen (1979) reported that approximately 30% of the total activity of the 114 PANTOTHENIC ACID ‘1* Pantothenate kinase 4'-PHOSPHOPANTOTHENIC ACID Phosphopantothenoyl-cysteine synthetase 4’-PHOSPHOPANTOTHENOYLCYSTEINE PhosphOpantothenoyl-cysteine decarboxylase DEPHOSPHOPANTOTHEINE Pyrophosphorylase DEPHOSPHO-COA L Dephospho-CoA kinase COENZYME A Figure 1. Conversion of pantothenic acid to coenzyme A. *rate limiting step ' 15 last two enzymes is associated with the mitochondrial fraction. The unequal distribution of CoA between the cytosol and mitochondria may be related to the distribution of the enzymes required for metabolic activity. CoA Degradation: The pathway for coenzyme degradation is nearly the reverse of its synthetic pathway (Figure 2). The enzymes are mainly lysosomal, and are not specific for CoA but common to all nucleotide pyrophosphates. It is not fully understood how the lysosomal enzymes gain access to the CoA which is located mainly in the mitochondria. When exogenous CoA is administered parenterally, it is rapidly cleaved on the red blood cell or liver cell membrane. The pantotheine fragments can then enter the cell to be resynthesized to CoA, or undergo further degradation to form pantothenic acid depending on the CoA status of the host (Ono et al., 1974). HEMATOPOIESIS/WHOLE BLOOD: All blood cells, including erythrocytes (RBCs), leukocytes (WBCs), thrombocytes (platelets), and plasma cells originate from one multipotential stem cell found in the yolk sac during embryogenesis and in the bone marrow during adulthood. Differentiation of the stem cell to a specific blood cell type results from the hematopoietic inductive microenvironment. Further maturation of the cell occurs in 16 COENZYME A 1’ Phosphatase DEPHOSPHO-COA 1 Nucleotide pyrolphosphatase 4’-PHOSPHOPANTETHEINE Phosphatase PANTETHEINE Pantetheinase NV PANTOTHENIC ACID + CYSTEAMINE Figure 2. Degradation of CoA to pantothenic acid. 17 specified regions within the bone marrow under the influence of "poietins" such as erythropoietin, granulopoietin, and thrombopoietin. The rate of cell differentiation and maturation varies for the four types of blood cells, and varies according to the need of the host. Predictable changes in the blood cells occur during the course of maturation and can be useful in determining abnormalities in cell number, shape, and function. For example, as a red blood cell matures, the cell size decreases, the cytoplasm gradually changes from blue to red when stained, the density of the nuclear chromatin increases (pyknosis), and the nucleus decreases in size until it eventually disappears. Thus when many large polychromatic red cells are observed in a blood smear, one can speculate that immature cells are being released into the blood stream as a response to need (e.g. blood loss or destruction). Changes in the number of blood cells and the concentration of pantothenic acid within the blood during deficiency have been reported. By finding the amount of pantothenic acid present in the erythrocytes, leukocytes, platelets, and plasma individually, and the number of each of these cells, the concentration of the pantothenic acid present can be determined in separated blood. Whole blood pantothenic acid levels have been measured in a variety of species. Tables 1 and 2 list reported values for humans and rats, respectively. Table 1 Reported human blood pantothenic acid (PA) content. Subject Intake Blood PA Reference mg/d nmole/ml 17 adults N/A 0.27 Pelczar and Porter, 1941 11 adults N/A 0.88 Pearson, 1941 37 elderly 5.8 2.43 Srinivasan et al., 1981 47 females 4.8 2.44 Song et al., 1985 66 arthritic N/A 3.15 Barton-wright and Elliot, 1963 patients 20 adults N/A 2.10* Slepyan et al., 1957 0.53+ 11 lupus N/A 1.47* Slepyan et al., 1957 patients 1.11+ 5 teenagers 3.3 8.08* Cohenour and Calloway, 1972 0.27+ 32 females 4.14 1.57 Eissenstat et al., 1986 25 males 6.25 1.88 Eissenstat et al., 1986 50 females N/A 1.75 Baker et al., 1977 males: age Ishiguro et al, 1961 30-39 N/A 5.36 40-49 N/A 4.42 50-59 N/A 4.29 60-69 N/A 4.30 fenales:age Ishiguro et al., 1961 30-39 N/A 5.00 40-49 N/A 4.59 50-59 N/A 4.15 60-69 N/A 3.98 Blood PA values indicate mean total PA unless flagged otherwise N/A - information not available * free PA + bound PA 19 Table 2 Reported rat blood pantothenic acid (PA) contents. Subject Intake Blood PA Reference ug/d nmole/ml Hale-28d N/A 2.57 Hatano et al, 1967 Male-36d 390 4.84 Israel and Smith, 1986 Female-37d 390 2.46 ” " ' Hale-fasted 390 4.15 " " " Female-fasted 390 4.75 " " " Hale N/A 2.43 Ono et al, 1974 2.58 Blood PA values indicate mean total PA BLOOD SEPARATION: Density gradient centrifugation is an established method for the separation and purification of leukocytes (WBC), erythrocytes (RBC), platelets and plasma from whole blood. The density of gradient mediums used in the past have been far from the physiological norm in order to achieve optimal purity in the separation. Thus the degree of purity had to be compromised in order to maintain cellular integrity. Silica sols used in the past were evaluated by Pertoft and coworkers (1968, 1977) and Wolf (1975). They found that silica sols were unstable in salt solutions at physiological pH and that they were toxic to the cells. By adding dextran, polyethylene glycol (PEG) or polyvinylpyrrolidone (PVP), the stability of the sol could be increased and the toxic effects decreased. However, this required a large excess of free polymer to be present in the solution which increased the viscosity and the osmolality of the medium. Percoll (Pharmacia, Uppsala, Sweden) was developed to overcome the previously named problems. It consists of modified colloidal silica particles ranging in size from 15 to 30 nm in diameter. Each particle is coated with PVP, and the medium contains no free PVP as seen in other mediums. Percoll is non-toxic to cells, has a low viscosity, and has no adverse effects on assay procedures. The low viscosity of the medium allows for rapid banding of cells on the gradient and for thorough washing of the gradient from the cells upon separation. Percoll is also able to form a self- 21 generated density gradient by centrifugation for 10-30 minutes. Gradients ranging from 1.0-1.3 g/ml are possible. ERYTHROCYTES: Erythrocytes, or red blood cells (RBCs), originate in cords or islands between the vascular sinuses of the bone marrow. The cell matures close to the outside surface of the sinus so that rapid release is possible upon demand. Immature cells (normoblasts) form concentric circles around a macrophage from which they leach iron in a process termed rhopheocytosis. This central macrophage also functions to phagocytize the nucleus from mature cells and remove ’defective normoblasts prior to maturation. Iron received from the macrophage moves to the mitochondria of the normoblast to be incorporated into a porphyrin-ring via heme synthetase to form heme. When the heme is combined with globin, a protein synthesized on the ribosomes, hemoglobin is formed and serves as the oxygen carrier in the blood. Hemoglobin content of a mature RBC acts as one of the determinants of the number of mitotic divisions which the cell will undergo to reach maturity. Therefore, when hemoglobin content in the red cell is decreased due to inadequate synthesis of heme or globin, the immature cell will undergo more mitotic divisions in an attempt to maintian a normal hemoglobin concentration, i.e. the cell becomes microcytic. If faulty heme synthesis continues, the cell 22 will eventually be unable to maintain appropriate hemoglobin content and will become hypochromic also. Blunt and coworkers (1957) found evidence of microcytosis (low MCV and MCH) in pantothenic acid-deficient rats. In this study, Lister Institute black and white stock rats were given graded doses of pantothenic acid ranging from 0-100ug per day for 7- 11 weeks. In the group receiving no added pantothenic acid, the red cell count was higher, but the hemoglobin and hematocrit values remained constant for all groups. The MCHC decreased slightly in the deficient group, but the change was not significant. No differences were seen in any of the blood parameters in rats receiving 25ug pantothenic acid per day or 100ug per day, thus these authors concluded that 25ug pantothenic acid was adequate for normal hematopoiesis to occur. When considering the effects of pantothenic acid on the number, size, and function of the red blood cell, it is useful to examine the roles which the free vitamin and CoA play in cellular metabolism. During the course of maturation the erythrocyte loses its nucleus, mitochondria, and ribosomes, and thus its capacity to synthesize hemoglobin and undergo oxidative metabolism. Energy is obtained in the red cell by the conversion of glucose-6-phosphate (G-6-P) to glucose which enters glycolysis yielding 2 ATP molecules and one NADH. Glucose entering the pentose-phosphate shunt enhances the glycolytic pathway by returning 3- and 6-carbon sugars to it. The NADH produced is needed to reduce the 23 spontaneously generated oxidized form of hemoglobin, methemoglobin, to its functional reduced state. The pentose- phosphate shunt also produces reducing compounds such as NADPH and glutathione (GSH). The rate of glucose utilization is regulated by the concentration of hexokinase and G-6-P present (Rose and O’Connell, 1964). The TCA cycle appears to operate in the nucleated RBC (normoblast) as demonstrated by the presence of TCA cycle intermediates: however no TCA cycle intermediates were found in mature RBCs (Dajani and Orten, 1958). The intermediates, particularly succinate, formed by the nucleated RBC have been shown to be used in the formation of porphyrins via the succinate-glycine cycle (Shemin and Kumin, 1952; Shemin et al., 1955; and Dajani and Orten, 1958). The oxidation of a- ketoglutarate to a succinyl intermediate is CoA dependent. The succinyl-CoA then condenses with glycine to form each pyrole unit of the porphyrin in hemoglobin. Therefore, CoA must be present in the erythrocyte for the synthesis of hemoglobin to occur and for proper size and function of the cell. Once the red cell reaches maturity, hemoglobin synthesis no longer occurs and TCA intermediates are not needed. Coenzyme A and ACP are both required for fatty acid synthesis. Studies have also shown that fatty acid synthesis is possible in the young nucleated red cells and reticulocytes but not in the mature erythrocyte (Marks et al., 1960; Pittman and Martin, 1966). Weir and Martin (1973) 214 Table 3 Reported human red blood cell (RBC) pantothenic acid (PA) content. Subject Intake RBC PA Reference mg/d nmole/ml 29 colitis N/A 0.46* Ellestad-Sayed et al., 1976 patients 8.49+ 11 adults N/A 1.08! Pearson, 1941 32 females 4.14 1.38 Eissenstat et al, 1986 colitis N/A 0.27* Ellestad et al, 1967 patients 7.76+ RBC PA values indicate total PA unless flagged otherwise N/A - information not available * free PA I all cellular components of blood measured 25 showed that long-chain fatty acid synthesis from acetyl-CoA occured in reticulocytes in the presence of acetyl-CoA carboxylase. The enzyme acetyl-CoA carboxylase is needed to convert acetyl-CoA to malonyl-CoA, a key intermediate for long-chain fatty acid synthesis. The carboxylase enzyme is synthesized in the mitochondria and ribosomes of the cell, therefore the enzyme, the mitochondria, and the potential for fatty acid synthesis are lost as the red cell matures. Since the mature erythrocyte does not synthesize the long-chain fatty acids, the metabolic function of pantothenic acid in lipid synthesis in the mature RBC cannot be justified. Table 3 lists RBC pantothenic acid concentrations previously ' reported. LEUKOCYTES: Leukocytes, or white blood cells (WBCs), originate from the multipotential stem cell discussed previously. In general the white blood cells function to defend the body against disease. During the course of differentiation, the white cell mature into three types each serving a specific function: granulocytes (eosinopohils, basophils, and neutrophils), monocytes, and lymphocytes. The presence of pantothenic acid within the white cell is necessary for a number of normal metabolic processes to occur. Unlike the mature red cell, long-chain fatty acid synthesis does occur in the leukocyte (Marks et al, 1960). These free fatty acids can then be incorporated into 26 triglycerides or phospholipid esters (Rosenzweig and Ways, 1966). White cells have much higher metabolic acitivity as compared to the erythrocyte or the platelet, cell for cell (Barron and Harrop, 1929). This is most likely explained by the larger size of the cell and greater number and complexity of mitochondria in the white cell (Rosenzweig and Ways, 1966). Glycolytic activity occurs at a higher level in the polymorphonuclear leukocyte (PMN), a neutrophil, as compared to the lymphocyte,and is vital since the cell is often placed in conditions of very low oxygen tension (Barron and Harrop, 1929). In situations of ample oxygen supply, aerobic glycolysis is the preferred pathway for cellular metabolism, and normal leukocytes possess higher rates of respiration and glycolysis than leukemic cells (Beck and Valentine, 1953). In addition to active oxygen consumption, the PMN also shows indications of intact Krebs cycle activity (Martin et al., 1955). During pantothenic acid deficiency, specific changes in leukocyte number and function occur. Generally, the humoral antibody (HA) responsiveness diminishes, although there is no adverse impact on cell-mediated immunity. More specifically, impaired HA responses have been seen (Beisel, 1982; Axelrod, 1971). Similar responses are seen with vitamin B-6 deficiency: however different mechanisms have been proposed. Pyridoxine appears to be necessary for the production of "C1" units from serine, a requirement for the biosynthesis of nucleic acids, whereas pantothenic acid is involved in the 27 secretion of newly-synthesized proteins into the extracellular compartment (Axelrod, 1971). Leukopenia in pantothenic acid dieficiency, or an overall decrease in the number of white cells, has been reported by a number of authors (Dinning et al., 1954, 1955, 1962: Weir, 1953; Melampy et al., 1951). However, contradictions on the type of white cell responsible for the lowered count do exist. Dinning and coworkers (1954, 1962) reported that a decrease in leukocyte count was observed in rats with a pantothenic acid and methionine deficiency. This was specifically due to decreased lymphocyte production. It was also found that lymphocyte production could be brought back to normal by adding methionine, a sulfur-containing amino acid. Later, the decrease in lymphocytes was correlated with liver CoA levels by the same author (1955). This work suggests that pantothenic acid may require sulfur- containing amino acids for CoA production, and CoA may, in turn, play a significant role in lymphocyte production. Conflicting results were found by Weir (1953) who induced a pantothenic acid deficiency in mice. Granulocytosis without lymphopenia was seen in the mice. Melampy and coworkers (1951), also working with the mouse model, found leukopenia after 6 week of deficiency, followed by significant leukocytosis. Slight neutrophilia (granulocytosis) was seen throughout the study and insignificant lymphocytosis occurred after the 6 week mark. Melampy also noted a decrease in the amount of white pulp in the spleen, which consists mainly of 28 lymphoid tissue, accompanied by splenomegaly. Other researchers have found that a pantothenic acid deficient diet causes granulocytopenia (decreased granulocytes) often accompanied by anemia in rats (Daft et al., 1945; Ashburn et al., 1947). Bone marrow hypoplasia was also observed: however the red cell, which decreased slightly in number, were not affected as severely as the leukocytic line. Because of the relative increase in red cells compared to the marked decrease in granulocytes, the myeloid-erythroid ratio showed an extreme reversal, from a normal of 1.93 to 0.23. Upon treatment with folic acid, niacinamide, and pantothenic acid, a normal bone marrow hyperplastic response became evident. During this regenerative phase, proliferation and maturation of granulocytes took place. Under continued deficiency, the spleen once again showed a reduction in activity of lymphoid follicles. Differing responses due to a pantothenic acid-deficient diet are experienced with various species. Also, it is uncertain whether a true deficiency state was actually achieved in the mice and rats due to the intestinal microbial synthesis of the vitamin and failure to prevent them from coprophagy. Intestinal microbial synthesis of pantothenic acid has also been reported by Mameesh and coworkers (1959) in rats receiving antibiotics. Since pantothenic acid may play a significant role in lymphocyte formation, a decreased count may indicate poor pantothenic acid status. 29 PLATELETS: Originating from the multipotential stem cell, the platelet is a result of complete differentiation of the megakaryocyte in the bone marrow. Platelets function to form hemostatic plugs which restrict blood flow upon vessel damage, activate the coagulation system plasma proteins, and maintain the integrity of the vascular endothelium. Since there is a large number of platelets in the blood, most long- chain fatty acid synthesis probably occurs within platelets (Marks et al., 1960). These fatty acids can either be oxidized or incorporated into triglycerides and phospholipids. The platelet carries out active glycolysis and synthesis and utilization of large amounts of glycogen. The major energy source is glucose which is rapidly taken up from the plasma. The TCA cycle and the pentose-phosphate pathways are active in the platelet; however under normal conditions, glycolysis is more active than oxidation (Wintrobe et al., 1974). The platelet portion of the blood also appears to be affected both in number and function by the presence or absence of pantothenic acid. Pantothenic acid deficiency in rats has been reported to cause a decrease in the number of megakaryocytes resulting in a lowered production of circulating platelets. As a result, purpura on the abdominal skin and evidence of hemorrhage in the lymph nodes were observed upon autopsy (Ashburn et al., 1947). A recent study found that administration of pantetheine (300mg Pantetina, 30 four times per day) for hyperlipidemia in 20 humans resulted in a significant reduction in thromboxane A2 production and a decrease in platelet aggregation (Prisco et al., 1984). The authors proposed that pantetheine impaired thromboxane A2 synthesis by platelets by altering fatty acid metabolism and specifically the fatty acid composition of platelet phospholipids. This inturn, inhibited platelet aggregation. Quantitatively, Ashida and Abiko (1975) reported that pantetheine appeared to have a protective effect on rats with thrombocytopenia. Platelet count increased in these rats when pantetheine was administered prior to and during induction of the thrombocytopenia. Pantothenic acid caused a similar but less pronounced change in the rats. PLASMA: As mentioned previously, the plasma, or fluid portion of the blood, contains pantothenic acid almost exclusively in the free form. Ishiguro (1972) reported that 90% of pantothenic acid in the human blood cells is in the bound form and most pantothenic acid in the plasma is in the free form. This would suggest that the cell is capable of converting free forms to the bound forms within the cell for use in metabolic reactions or for reserve. Plasma pantothenic acid values in humans and rats are shown in Tables 4 and 5, respectively. 31 meme .cocuxxuax coma .Hu no Summon Head .comuoom mmma .Ho no mcom mama .Ho no Odom mums .Ha um omsamuoaumodam Home .cmq Home .cmu mama .mmom mama .mmom puma .caasuux meme .coanuax 4m amuou coo: macaqoca mosam> cm mamoan ma.d HB\Q~O§: mozmmmmflm (Q (Smtdm «\z <\z «\z m.v n.m (\2 mod ha o.m m.v v.m m.m O\OE Ni‘BZH madame mucoauon mamaaoac moans wasps monsaou moumlco: mauosou sensuoun macawuma manaaoo mm magnum oasaou m nuance odoaou m nuance mamaou o nuance mausou m nuance ma mucoOmoHooe Ha macabmam .ucoucoo A 4m flBmflHQ nn.o oo.N O~.o ov.~ oanouoouoocs cm.d mh.d vv.a Ha\oaoas O on omumnamonmaax O on c on <\z «\z ammo mx\ma coumauuosaz can: can: can: xaanoaaz oocououom 4m flfimfldm excusn uoofinsm .ucoucoo Acmv cave cacocuoucon camoam you couuoaom m wanna 33 SUMMARY: Current literature supports the fact that CoA is needed for many metabolic reactions to take place in each of the blood cell types described above. Generally, pantothenic acid can affect both the function and number of erythrocytes, leukocytes, and platelets. Upon accurate separation of blood cells, the distribution of pantothenic acid in whole blood can be determined via radioimmunoassay (RIA). From this, the specific effects of pantothenic acid status on blood component metabolism and function can be further studied. MATERIALS AND METHODS Experimental Design: Fifty-four Sprague-Dawley rats (Harlan-Davis, Indianapolis, IN) were individually housed in suspended mesh stainless steel cages to minimize coprophagy. The animal room was maintained at a controlled temperature (20-22°C) with a 12-hour light-dark cycle. Guidelines for the care and use of laboratory animals were followed as approved by MSU. After stabilization for 24 hours on water and stock diet, rats were divided randomly into three experimental study groups. Eighteen rats in the control group (group I) were fed ad libitum, the U.S. Biochemical Purified Pantothenic Acid-Deficient Test diet (Appendix A) with the addition of 12 mg D-calcium pantothenate per kg diet, ad libitum. The second group of 18 rats (group II) received, ad libitum, an identical diet without added pantothenic acid. The third group of 18 rats (group III) received the control diet but were pair-fed to the deficient group. Food intake of group I was determined once a week whereas those of groups II and III were determined each day to pair-feed. The weight of each rat was recorded weekly. At 3, 6 and 9 weeks, 5-6 rats from each group were anesthetized with ether, and 3 ml blood was collected via cardiac puncture into EDTA-treated evacuated blood collection tubes (Vacutainer - Becton/Dickinson). Prior to separation of the blood into various components, red cell, 311 35 white cell, and platelet counts were performed along with hemoglobin, hematocrit, MCV, MCH, and MCHC measurements using the ELT-8 electronic laser blood cell counter (Ortho Diagnostic Systems, Inc., Raritan, NJ). Blood Separation: Whole blood was separated by a modified density-gradient centrifugation method described by Jurd and Rickwood using Percoll (Pharmacia, Uppsala, Sweden). Percoll (polyvinylpyrrolidone-coated silica particles) was mixed with 1.5M NaCl (9:1 vol/vol) to adjust the osmolality to match physiological conditions. This mixture was identified as the stock solution (SIP). A self-generated gradient was formed by mixing 5.58 ml SIP with 2.42 ml 0.15M NaCl (69.75% SIP) in a 14 ml polycarbonate test tube (Nalgene Labware). Tubes were vortexed followed by centrifugation at 20,000xg and 20°C for 20 minutes. Two ml of EDTA-treated blood was loaded onto the gradient which was formed. Tubes were re-centrifuged at 1000xg and 4°C for 5 minutes to separate the plasma and platelets from the other blood components. This platelet- plasma layer was removed and placed in a small test tube for subsequent separation of platelets. The volume of plasma removed from the Percoll gradient was replaced with 40% SIP, the concentration of Percoll identical to the density of the plasma removed from the vial. The remaining fractions were centrifuged for 20 minutes at 20,000xg and 4°C. The WBC and RBC layers, distinctively separated, were transferred into 36 small test tubes for washing. Platelets were separated from the plasma by centrifugation for 5 minutes at 1000xg. The plasma was removed and frozen at -20°C. The platelet portion was washed three times with normal saline to remove plasma and any residual Percoll. Following the final wash procedure, all of the supernatant was carefully removed from the platelets leaving only the cell pellet. The cell pellet was resuspended in 200 ul normal saline and frozen at -20°C. The WBC samples were washed following the method for platelets described above. All supernatant was also removed from the RBC following the final wash cycle. The volume of RBC was measured, diluted 1:1 in saline and frozen. To determine if blood cell fractions were accurately separated, microscopic examination of each separated cell fraction was necessary. Microscope slides of cell suspensions were prepared and examined. The separated cell fractions were also quantified to determine percent recovery as compared to the whole blood cell counts. A hemocytometer was used to count each separated and washed cell fraction resuspended in 2 ml of normal saline, the original volume of blood from which the cells were taken. The recovery rates for red cells, white cells, and platelets were 100%, 35%, and 65%, respectivley. 37 Pantothenic acid determination: For pantothenic acid measurement, cellular samples, which had been separated, washed and quantified, were subjected to three quick freeze/thaw cycles to lyse the cells. Each of the cell fractions was divided into two aliquots, one aliquot for the determination of free pantothenic acid and the other for the determination of total pantothenic acid. To cleave the bound form of the vitamin for determination of total pantothenic acid, two enzymes were used as described by Song et a1 (1984). Ten units bovine intestinal alkaline phosphatase (Type VII-S, Sigma Chemical Co., St Louis, MO) and 20 units pantetheinase (compliments of Dr. Wittwer, University of Utah School of Medicine) were used. The enzymes, diluted in 100 uL of phosphate buffered saline (0.1 M PBS), were added to 100 uL whole blood, platelet, leukocyte, and 50 uL erythrocyte samples prepared as described above. One hundred uL of PBS was added to samples used for the determination of free pantothenic acid. Plasma samples were not subjected to the enzymatic treatment since only free pantothenic acid appears in this blood compartment. After addition of the enzymes, the samples were incubated at 37°C for 8 to 12 hours. Hydrolysis of bound pantothenic acid was terminated by the addition of saturated Ba(OH)2 and 10% ZnSO4 in equimolar concentrations. Samples were vortexed thoroughly followed by centrifugation at 4000xg for 10 minutes. The clear supernatant removed from the 38 remaining protein pellet was used for determination of pantothenic acid content by the radioimmunoassay method developed by Wyse et al (1979). Two preliminary studies were conducted to determine whether free pantothenic acid was transported into and out of the cell over time. The first study examined the degree of transport into the cell, and the second study examined the degree of transport out of the cell. In the first study study 3.4 nmole radioactive pantothenic acid was added to 4 fresh whole blood samples. Upon addition of the labelled pantothenic acid one sample was immediately divided into its plasma and cellular compartments by centrifugation. Results showed that the added pantothenic acid remained entirely in the plasma compartment and did not enter the cells immediately. The remaining three samples were divided into their plasma and cellular compartments 1 hour, 3 hours and 6 hours after the addition of the radioactive pantothenic acid. Results showed that after 1, 3 and 6 hours of incubation, 18%, 56%, and 64% of the added pantothenic acid had moved into the cellular compartment of the blood, respectively. Similarly, the second study suggested that pantothenic acid leaves the cell over time. In this study, rats were injected via tail vein with 13.7 nmole radioactive pantothenic acid. Twenty-four hours later blood was havrvested from the animals and the cellular portion separated by centrifugation. Normal saline was added to 4 samples to serve as a incubation medium. The first sample 39 was analyzed immediately to obtain a baseline level of pantothenic acid which had been incorporated into the cells in vivo. The remaining 3 samples were incubated for 1, 3 and 6 hours, respectively. Results of this study showed that 8%, 28% and 43% of the cellular pantothenic acid moved into the incubation medium at the three time points indicated, respectively. Because pantothenic acid appeared to move into and out of the cell over time, the separation process was completed as quickly as possible (maximum limit of 1 hour) after collection of blood to minimize inaccurate determination of pantothenic acid content of various rat blood components. Statistical Analysis: A one-tailed Dunnett’s t-test was used to detect significant differences in food intake and growth rate between groups. Results for group II (deficient group) were compared to those of group I (control group) and group III (pair-fed control group). The cell counts and pantothenic acid content of each cell type for each group were compared using a 2-way analysis of variance followed by a Dunnett's t- test to detect the effects of both treatment and time on measured parameters. RESULTS The amount of food consumed by group I and group II was significantly different (p<0.01) beginning at week 1 (77 vs. 699 diet/wk, respectively) (Appendix B). This difference was observed throughout the course of the study and by week 9, groups I and II were consuming 91 and 77g diet/wk, respectively. As shown in Figure 3, by week 2 significant differences were seen in the weight gains between all three groups (p<0.05). The group I rats exhibited the fastest weight gain and were the heaviest at the end of the study. Although group II and group III rats consumed the same amount of food, the group II rats weighed significantly less (p<0.05) at week 4 and remained smaller than group III throughout the course of the study. Blood cell counts: The RBC counts of rats fed the deficient diet were statistically higher than those of groups I and III (p<0.01 and p<0.05, respectively) at week 3. Although not statistically significant, the red cell count of group II remained higher than group I at 6 and 9 weeks (Table 6). The RBC counts increased in all groups over time. This observation was consistent with the trend normally seen in growing rats. Peripheral blood smears revealed a normal MO u: .1+. HHH asoec ago Amy H dsosu 0a ooemdsoo mm mo.ova mm cosmoaoca HH asono ca oo>eombo moocosouuao ocmoauacmam .>~o>fiuoodmon .mxoo: a one o .m as .mouusqmm. HHH ocm .ocoaofiuoa. HH masoec Low b one .m— .hp ocm .Hogucoo. H dsoso Lou m can o..>. u omen oaasmm .coHLod Hmbcosfigodxo xoozuo on» mcqsso mam; uo ncgobumd swam unwaoz .m unawam axon: aw Mw N. m. mu .v mu “w P Au — a q — q _ q q _ — Aum. Aqu nxur nxum“ Aummw .nxumw (9) :ufitan TABLE Couplete blood counts and henatoloqical indices for Group I (Control), Group II (Deficient) and Group 6 III (Pair-fed) rats. 112 Group 0 RBC KGB HC‘I' MCV HCK MCHC WBC PL? II III II III I II III 106/111. q/dL 1 4 5.710.6” 11.311.o 2313 4 7.310.: 13.311.2 3314 s 6.410.6’ 12111.2 3310 5 6.011.3 10.912.1 2619 4 7.511.8 12.012.6 3214 5 6.311.6 11.212.8 2418 3 7.811.2 13.512.4 3417 5 8.011.3 ll.411.8 2914 4 8.710.6 14.310.4' 3612 :1. pg q/dl 103/111. 103/111. “e99 . 7712 2010.1 2610.3 2.111.02 9221114’ 7111 1810.4 2610.2- 1.310.4 6941133 7311* 1910.? 2610.4 2.110.7 8441132 6 weeks 7311” 1310.611 2510.4” 2.411.1 3131115 6212 1610.7* 2610.1' 24106 6711178 7011" 1810.451! 2510.5" 3.010.7 9031366 9 weeks 7011:: 1710.5112410.6:'3.510.4" 135175 5411+ 1410.2 2610.7 o.910.7 4511123 671111 1610.711 2510.51" 1.210.3 669159 values represent lean 1 5.11. RBC. 1101!: red blood cells: KGB: beloqlobin: HC'I': belatocrit: MCV: lean corpuscular volule: lean corpuscqu beloqlobin: MCHC: lean corpuscular benoqlobin concentration: 1130: white blood cells: and PM: platelets * treatIent effect p<0.05 "treatlent effect p<0.01 4+ tile effect p<0.05 ++tine effect p<0.01 ”3 number of nucleated RBCs (1-2 cells/100 WBC), and few polychromatic cells in all three treatment groups. The hemoglobin and hematocrit values of all groups were similar throughout the study. At 3, 6 and 9 weeks, group II had a lower mean corpuscular volume (MCV), or RBC size, compared to groups I and III (p<0.01); and over time the differences became more pronounced. In addition, the MCV decreased significantly over time in all three groups which is known to occur normally in rats of this age. The weight of hemoglobin in the red cell, as measured by the mean corpuscular hemoglobin (MCH), was significantly (p<0.01) lower in group II compared to groups I and III at all three time points. As with the MCV, the MCH decreased rover time in all groups. The mean corpuscular hemoglobin concentration (MCHC), or the concentration of hemoglobin within the red cell, was higher in group II at 6 and 9 weeks (p<0.01) as compared to groups I and III. This elevated value was within the normal range for rat MCHC, and thus was not physiologically significant. No significant differences were seen in the WBC counts of the three groups until 9 weeks. At this time, group II had a decreased count compared to the group I (p<0.01). An increase in the white cell count of group I at 9 weeks exaggerated the relative leukopenia of group II. Differential counts of the white cells were inconclusive in UM describing the source of leukopenia. In general, all groups exhibited lower WBC counts than normal rat profiles (Sanderson and Philips, 1981). The platelet count of group II was lower compared to those of groups I and III at all three time points. This decrease was significant (p<0.01) at 3 weeks only. Pantothenic acid content of cellular components of blood: Pantothenic acid content of whole blood and the cellular components of blood are presented in Table 7. Significant decreases (p<0.01) in total pantothenic acid content of whole blood in group II were observed at 3, 6 and 9 weeks; and free pantothenic acid content (p<0.01) at 3 and 6 weeks (Figure 4). At 9 weeks, a similar trend was noted in the free whole blood pantothenic acid, however it was not statistically significant due to the large standard deviation. No statitstical differences were seen in the free or total pantothenic acid of group II compared to groups I and III (Figure 5). 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The plasma pantothenic acid of group II was markedly decreased at all time points compared to the other 2 groups. As a result, the percentage of total pantothenic acid in plasma was lower and the RBC pantothenic acid percentage was higher in group II than it was in group I compared to the other two groups. The percentage of total pantothenic acid within the white blood cells also was higher in the rats receiving a pantothenic acid deficient diet as compared to groups I and III. Although the increase in the white cells percentage was not as pronounced as was seen in the red blood cells, a rise in percentage was noted at all three time points. The percentage of total pantothenic acid in the blood as contributed by the platelets was very small and was slightly elevated within group II. The percentage of total pantothenic acid found in the various blood components was similar between the control and pair-fed groups, thereby ruling out decreased food intake as a confounding variable, and establishing the effect of decreased pantothenic acid intake in group II. 52 .m.m~. communa-oo so. awe: eon-ac .....o 0. .m.a.e.... us. no... m..=°o ..66 as. .a..... as. no... ...oo...o. as... .6....o..o as...» m.....~>~ muse o: . .... ...o .oo.o .sos.o «.6 ...o ..~. m... ... s... ...O ..o.° .ooo.o m... m..o .... s..° .. ...m o... .oo.¢ .ooo.o ... ~N.¢ ..N. ...o. . mgmm: m a... .... ..o.¢ ~°os.o 2... -.° 2... ~..o ... N... ...o .mo.o mooo.o .... ...o a... s..° .. m... e... mo¢.° .ooo.o ... m..o a... ~m.¢ . exam: e . . . .oooo.° . o..° . ...s ... ..~. e..s s.°.s .ooos.o ...~ ...° .... o..° .. .... a... mo... .ooo.o m... .~.° ..e. ...o . mgmma n . so... 6.0.. .....°.. . so... 4.5.. .....o. . so... a...1 ..\o.°.. . .oo.. 4.5.. ..\o.°.. ..m... m.a.o.... u...u so... a...= m..ao as... .6. as... .muemeoa-oo HHmo coo.. a. =o..=...am.e can Lamugoo v.6. o.=ma.o.=~m o oHneu DISCUSSION A significant decrease in food intake and weight gain of pantothenic acid-deficient rats after 1 and 2 weeks of feeding has been reported previously (Barboriak et al.,1957:Moiseenok et al., 1987: Blunt et al., 1957: Dajani and Orten, 1958). Barboriak et al (1957) found that weanling rats receiving a diet with no added pantothenic acid ceased to grow after 2-3 weeks of feeding and died after 10 weeks. Rats receiving a sub-optimal amount of the vitamin (2 mg pantothenic acid/kg diet) grew at a slower rate compared to rats receiving 100 mg pantothenic acid/ kg diet. As the animals reached 2 years of age, the growth rate decreased for rats receiving the higher supplementation, but by the end of the 2-year study, the less supplemented rats reached the same final weight as their more highly supplemented counterparts. Moiseenok et al (1987) also observed a 17% decrease in growth rate of pantothenic acid-deficient animals as compared to controls after 3 weeks of feeding, and a 25% decrease after 6 weeks of feeding. In the present study, 12 mg pantothenic acid/kg diet appeared adequate to promote normal growth and hematopoiesis in these rats. The higher RBC counts, low MCV and MCH, and normal hemoglobin and hematocrit values seen in pantothenic acid deficient rats in this study were also reported by Blunt and coworkers (1957). During the course of red cell synthesis, the number of mitoses which an immature erythrocyte 53 54 (normoblast) will undergo is determined by the amount of hemoglobin synthesized within the cell to that point. More mitoses will occur if the hemoglobin content in the developing normoblast is depressed, thereby maintaining a normal concentration of hemoglobin within the smaller daughter cells. Biochemically, pantothenic acid plays an important role in the synthesis of porphyrin for hemoglobin via the succinate-glycine cycle (Shemin and Kumin, 1952: Shemin et al., 1955). Therefore, CoA must be present within the developing RBC for the synthesis of hemoglobin and for proper size and function of the red cell. It appeared that the red cells in the pantothenic acid-deficient group underwent more mitotic divisions to maintain the proper MCHC resulting in a higher red cell count. In this study, the hemoglobin of pantothenic acid deficient rats was able to be maintained due to the increase in the number of red cells despite the low MCV. Bone marrow can respond to erythropoietin stimulation by increasing the rate of synthesis and release of immature or nucleated red blood cells under various conditions. This does not, however, seem to occur in the case of pantothenic acid deficiency since an increased number of nucleated red cells and polychromatic cells was not observed in peripheral blood smears. The plasma and the red cells both lost total pantothenic acid during deficiency as supported by data collected on the pantothenic acid content of blood components; however the greater loss from the plasma resulted in a relatively higher 55 percentage in the red cells. A recent study done in hemodialysis patients revealed an increase in red cell total pantothenic acid as compared to healthy control subjects (DeBari et al., 1984). The authors suggested decreased clearance of the vitamin, inhibition of catabolizing or converting pathways for the vitamin, or the presence of intracellular sequestering agents as possible causes of the observed increase in red cell pantothenic acid. In the present study, an absolute increase in RBC pantothenic acid was not seen, rather the percentage of pantothenic acid within the cellular vs. plasma compartment was noted suggesting that the RBCs may preferentially conserved the vitamin during a period of inadequate pantothenic acid intake. In addition, human blood may respond differently than rat blood when inadequate pantothenic acid intake occurs. It appeared that the pantothenic acid was better retained in the cellular portion of the blood, especially the red cell compartment as compared to the plasma. Because the plasma serves as a shuttle or transport medium by which pantothenic acid travels from storage to the sites of greatest need, plasma pantothenic acid could show flucuations independent of pantothenic acid intake, and thus effects its use as an indicator of pantothenic acid status. The leukopenia observed in the pantothenic acid-deficient group at 9 weeks was consistent with previously reported studies in both mice and rats after 7 weeks of feeding a 56 pantothenic acid deficient diet (Dinning et al., 1954, 1955; Weir, 1953: Melampy et al., 1951; Daft et al., 1945). In general, all groups in this study exhibited a marked decrease in the number of white cells compared to published normal values for rats of 11,300/ul blood at 6 weeks of life, and 7,900/ul blOod at 10 weeks (Sanderson and Philips, 1981). Preliminary studies suggested that margination of white cells to the blood vessel wall due to the use of ether just prior to collection of the blood sample can depress the white count as much as 25%. In the future, the effects of various anesthetics on white cell counts should be studied to minimize such an effect however, all animals were treated the same and thus the effect should be equally seen in all groups. Leukopenia, in general, may occur as a result cf acute viral infection, sepsis, leukemia, and bone marrow damage. A recent study indicated that gradual degeneration of the bone marrow occurs during pantothenic acid deficiency, and after 12 weeks of deficiency the marrow is nearly completetly destroyed (unpublished data, Song and coworkers, 1988). Because the stem cells in the marrow normally give rise to all blood cells, and because white cells have a short lifespan, the gradual decrease in white cells seen in this study might be anticipated. In conjunction with the decreased number of white cells, the amount of pantothenic acid within each white cell remained relatively constant between all groups. The white cells were able to maintain their pantothenic acid content 6V 3C TC 57 even during times of deficient intake. Because pantothenic acid is required for synthesis of long-chain fatty acids and TCA cycle activity in the white cell (Marks et al., 1960; Rosenzweig and Ways, 1966; Martin et al., 1955), it would appear that the metabolic requirement for pantothenic acid in the white cell encouraged the level to be maintained. The absolute number of white cells collected and analyzed for pantothenic acid content was relatively small and may have had an effect of the pantothenic acid values obtained. The number of platelets in the pantothenic acid- deficient group was less than that in the control and pair- fed groups at all three time points, and was significantly (p<0.05) less at 3 weeks of treatment. This observation also reported by Prisco et al (1984) might have been due to damage to the stem cells in the bone marrow. Platelet pantothenic~ acid content in group II remained similar to that in groups I and III. Although pantothenic acid plays a key role in the metabolic functions of the platelet (Prisco et al., 1984; Wintrobe et al., 1974: Ashburn et al., 1947), the small size of the cell accounts for the small proportion of the pantothenic acid in blood contributed by platelets. Again, the absolute number of platelets collected was relatively small and may have had an effect on the pantothenic acid values obtained. Overall, a decrease in whole blood total and free pantothenic acid was noted as was a decrease in plasma free pantothenic acid. Individual examination of red cells, white 58 cell, and platelets did not produce any differences between control, deficient, and pair-fed groups, thus did not reflect pantothenic acid intake. Therefore this study suggests that whole blood free and total pantothenic acid is a valid and reliable indicator of pantothenic acid intake. In this study, however the number of rats was relatively small thereby limiting the statistical power of the results and the interpretations drawn from them. SUMMARY Food intake and weight gain of rats fed a pantothenic acid-deficient diet, ad libitum were significantly decreased (p<0.01 and p<0.05, respectively) compared to rats fed a control diet, ad libitum or pair-fed. Deficient and pair-fed rats consumed the same amount of food, however the deficient rats were significantly smaller after 4 weeks of feeding. Rats fed the pantothenic acid-deficient diet had higher RBC counts (p<0.01 and p<0.05 compared to groups I and III, respectively). Group II also had a significantly lower (p<0.01) MCV and MCH at 3, 6, and 9 weeks as compared to groups I and III. After 9 weeks of feeding, the deficient rats had significantly decreased (p<0.01) WBC counts as compared to group I. The platelet count was decreased in group II at all time points, and was significantly lower than controls at 3 weeks (p<0.05). Total and free whole blood pantothenic acid was significantly lower (p<0.01) at 3, 6 and 9 weeks in the deficient group compared to controls. RBC, WBC and and platelet free and total pantothenic acid in the deficient rats did not differ significantly between groups or over time. Plasma pantothenic acid was significantly decreased in the rats fed a deficient diet (p<0.01) at 3, 6 and 9 weeks. When calculated on a per 1ml whole blood basis, the red cell pantothenic acid of the deficient rats did not vary from 59 60 control values until 9 weeks, while plasma pantothenic acid was greatly decreased. Additionally, WBC and platelet pantothenic acid did not vary in the deficient rats. As a result, the percentage of pantothenic acid within 1 ml whole blood was greater within the cellular components than the plasma compartment of the deficient rats. CONCLUS ION The results of this study suggest that inadequate pantothenic acid intake may impair hemoglobin synthesis as evidenced by microcytic red cells, however overall hemoglobin values were maintained. The pantothenic acid content of red cells, white cells, and platelets did not change during deficiency and thus did not reflect pantothenic acid intake. Instead whole blood total and free pantothenic acid did decrease with decrease intake of the vitamin, thereby establishing this parameter as a valid and reliable indicator of pantothenic acid intake. 61 RECOMMENDATIONS Future studies can be recommended in this area as follows: 1. In order to accurately measure the white cell counts, a variety of anesthetics should be sampled to minimize margination of white cells. 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APPENDICES 70 Appendix A Composition of USB biological pantothenic acid-deficient test diet for rats.# Ingredient Amount Vegetable oil 10* Vitamin-free casein 18 Sucrose 68 Salt mixture No. 2 USP 4 Salt mixutre No.2 USP + calcium biphosphate 13.58 calcium lactate-5 H20 32.70 ferric citrate-5 H20 2.97 magnesium sulfate 13.70 potassium phosphate 23.98 sodium biphosphate-ZHZO 8.72 sodium chloride 4.35 Vitamin mixture ++ alpha tocopherol (100Lu/g) 5.0 L-ascorbic acid 45.0 choline chloride 75.0 inositol 5.0 menadione 2.25 niacin 4.5 PABA 5.0 pyridoxine HCl 1.0 # contains 434 kcal ME/lOOg diet * expressed as percent of total diet + expressed as percent of salt mixture ++ expressed as g/100 lb diet 71 Appendix B Food intake of rats over course of treatment.* Group I n Group II n g diet g diet Week 1 77 1 6 17 69 t 6# 17 Week 2 99 1 11 17 87 1 16+ 17 Week 3 93 i 21 17 73 i 11# 17 Week 4 95 1 11 17 70 i 10! 17 Week 5 100 i 15’ 10 73 i 13# 12 Week 6 88 i 17 10 68 t 10# 12 Week 7 92 f 16 5 69 1 7+ 6 Week 8 92 i 13 5 53 i 14# 6 Week 9 90 t 6 5 76 1 9+ 6 Group I (Control), Group II (Deficient) * Group III (Pair-fed) consumed amount diet reported for Group II + p<0.05 compared to group I # p<0.01 Values represent 1 S.D. Weight gain of rats over course of treatment. Group I n Group II n Group III n Week 0 4513 17 46:4 17 45:4 17 Week 1 10016 17 90:7 17 89:6 17 Week 2 134113 17 106i9+ 17 120110 17 Week 3 167123 17 120112+ 17 141111 17 Week 4 189120 10 130111+i 12 157115 12 Week 5 215133 10 142:13+# 12 171115 12 Week 6 214135 10 151114+I 12 180118 12 Week 7 235150 5 162111+# 6 192123 6 Week 8 249153 5 164il3+# 6 191117 6 Week 9 5 6 6 259160 169111+# values represent mean i S.D. Group I (Control), Group II (Deficient), and Group III (Pair-fed) + p<0.05 compared to group I I p<0.05 compared to group III 204122 '72 Appendix C Individual raw data for blood cell counts, hematological indices and pantothenic acid content of blood components. ’ RAT 0 6.00 7.00 9.00 10.00 8? 1.00 1.00 1.00 1.00 UK 3.00 3.00 3.00 3.00 DIET PA+ PA+ PA+ PA+ cat 1100103/01 1.40 1.30 3.60 . 1.60 m 10bit“. 4.34 6.12 6.10 5.82 HEB 9101 9.80 12.00 11.80 11.80 HCT 2 25.00 25.00 32.00 28.00 HCV 1L 78.00 75.00 76.00 79.00 MCH pg 20.20 19.60 19.30 20.30 NCHC g/dl 25.90 26.00 25.3 25.30 711 103/01 34.00 806.00 1,038.00 1,000.00 PA CONTENT 0681-? 1.19 1.21 1.30 .81 . 3.91 3.79 3.09 1988 rerun 1.39 1.16 1.62 1.93 1988 rerun2 ' 1988 rerun3 HhBl-T 3.72 1.81 1.66 2.22 1.54 2.89 04 1988 rerun 2.32 2.01 2.39 3.0 1988 rerun2 1988 rerun3 RBC-F .79 1.45 1.67 1.56 1.85 1.86 1.55 2.48 1988 rerun 1.01 .76 .87 1.32 RBC-1 7.74 7.66 6.68 7.25 3.92 6.38 3.93 3.54 1988 rerun 1.16 1.25 .58 1.36 NBC-F 79.41 112.20 77.46 NBC-T 136.61 90.95 139.93 PIT-T .14 .03 .10 .11 FLT-T .16 .05 .14 .16 PIS-F . 1,512.20 1,766.21 1,365.90 1,806.14 1988 rerun 2,040.00 1,191.00 2,388.00 2,196.00 CBC PA CONTENT APPENDIX C (con't) RAT 8 8? UK DIET 1101: 1300 RBC 111’qu 1168 9/41 HCT 1 11111 1L H13" 09 MCHC g/dl 717 103/01 8681-6 1988 rerun 1988 rerun2 1988 rerun3 8681-7 1988 rerun 1988 rerun2 1988 rerun3 RBC-F 1988 rerun RBC-T 1988 rerun RBC-F 880-7 PlT-F PtT-T PlS‘f 1988 rerun '73 24. 00 2.00 3.00 PA- 1.20 7.92 14.00 35.00 70.00 17.70 25.40 722.00 .27 1.01 .54 1.10 .92 102‘ 1.23 1.41 1.52 5.86 4.98 1.10 100.30 182.30 .06 .15 25.00 2.00 3.00 PA- 1.00 8.00 14.60 37.00 71.00 18.30 25.80 556.00 4 0‘ .62 .41 .26 .96 .63 1.34 1.95 1.23 2.36 6.61 .71 109.65 106.59 .21 .21 445.55 510.00 26.00 2.00 3.00 PA- .40 7.14 12.30 27.00 69.00 17.90 25.80 632.00 '2 e U .78 579.88 27. 00 2. 00 3. oo 70- 1.80 6.30 11.80 31.00 72.00 18.70 26.00 866.00 .53 .79 .75 .70 1.78 1.46 1.68 1.01 3.95 4.34 .90 67.72 118.72 .04 .05 493.43 570.00 03 4“) PA CONTENT RAT 8 8? 8K DIET 1101: 105/01 RBC 10*/.11 888 9701 1107 1 11011 0. "CH 119 110.110 g/dl PLT 103/01 HhBl-F 1988 rerun 1988 rerun2 1988 rerun3 8881-7 1988 rerun 1988 rerun2 1988 rerun3 RBC-F 1988 rerun RBC-T 1988 rerun BBC-F 880-7 FLT-f PLT-T PLS-f 1988 rerun 70. APPENDIX C (con't) 41.00 3.00 3.00 PA+PF 2.00 6.44 12.40 74.00 19.30 25.90 888.00 :- :° 313 1.16 67.32 88.99 .09 .13 42.00 3.00 3.00 PA+PF 1.40 6.08 11.80 73.00 19.40 26.60 626.00 .85 1.30 148.89 134.42 .17 I12 43.00 3.00 3.00 PA+PF 2.20 5.64 10.60 73.00 18.80 25.60 896.00 2.52 2.42 1.08 1.92 4.64 2.01 2.01 3.27 2.95 1.31 2.31 .96 3.53 4.15 .91 48.68 75.80 .11 .11 44.00 3.00 3.00 PA+PF 3.00 7.36 13.80 33.00 72.00 18.80 25.90 834.00 poo-bo— 4.0040 bacon.) 4.71 5.10 1.08 55.76 60.69 .03 .11 45.00 3.00 3.00 PA+PF .80 6.2 11.80 33.00 73.00 18.80 25.70 976.00 5.74 2.81 as 1: $3 23 3: 23 3% EB 88 23 mMo-ONNO-‘gnt—p... . C. C 139.60 .10 .11 PA CONTENT '75 APPENDIX C (con't) RAT 0 OP 8N DIET use 103/01 RBC 10“/u1. 808 g/dL HCT z 000 11 NC" 09 8080 g/dl PLT 103/01 UNOI-F 1988 rerun 1988 rerun2 1988 rerun3 8681-1 1988 rerun 1988 rerun2 1988 rerun3 RBC-F 1988 rerun RBC-T 1988 rerun 8OC-F 88C-T FLT-F PlT-T PLS-T 1988 rerun 1.00 1.00 6.00 PA+ 3.80 7.62 13.40 35.00 72.00 17.60 24.40 778.00 7.58 4.07 2.77 31.14 48.45 .29 .23 1,375.22 1,740.00 2.0 1.00 PA+ .40 5.14 9.80 18.00 75.00 19.10 25.30 680.00 2.38 1.88 .93 .03 .39 973.56 1,800.00 25.00 756.00 EDI-J «DO-eh. GQN 7.52 2.22 63.75 48.73 .04 .11 3,170.72 4.00 1.00 6.00 PA+ 2.60 6.04 11.20 26.00 73.00 18.50 25.30 930.00 .18 1,315.37 1,560.00 1.57 2.08 2.00 2.08 1.94 .19 7.58 4.05 1.40 103.82 .04 .07 :1 1,522.85 2,340.00 PA CONTENT '76 APPENDIX C (con't) RAT 4 8? 8K DIET 1131: 103/10 1231: 10*/01 "88 g/dL 807 2 11011 11. "CH 09 NCNC g/dl 711 103111 8h81-F 1988 rerun 1988 rerun2 1988 rerun3 8681-7 1988 rerun 1988 rerun2 1988 rerun3 RBC-F 1988 rerun RBC-T 1988 rerun NBC-F 886-7 FLT-F PLT-T 813-6 1988 rerun 13.00 2.00 6.00 PA- 2.40 8.56 13.40 34.00 63.00 15.70 24.90 938.00 .17 1.17 .33 453.53 810.00 20. 00 2.00 6.00 714- 3.00 8.54 13.60 36.00 61.00 15.90 26.2 566.00 .26 .39 .40 .93 1.60 3.29 .36 1.80 4.43 1.25 30.09 47.77 .21 .07 473.48 480.00 22.00 2.00 6.00 PA- 1.80 8.20 13.00 30.00 60.00 15.90 26.60 576.00 C. 62262 473.58 50.00 23.00 2.00 6.00 PA- .60 4.74 8.20 26.00 65.00 17.30 26.80 606.00 I N — (2:- m I“) ‘18 . an “cl .- u ‘ p. D-. o—o - a I o o M O L" .06 I, I 6 355.11 480.00 PA CONTENT ‘77 APPENDIX C (con't) RAT 8 GP 8N DIET 11111 103/01 131 10*/01 868 g/dL 317 z ncv 11 11611 79 8080 g/dl PLT 103/01 NNBI-F 1988 rerun 1988 rerun2 1988 rerun3 8681-T 1988 rerun 1988 rerun2 1988 rerun3 880-6 1988 rerun 880-1 1988 rerun NBC-F 888-7 FLT-F PLT-T 715-1 1988 rerun 35.00 3.00 6.00 PA+PF .60 8.32 14.40 35.00 68.00 17.30 25.50 630.00 2.17 1.86 1.51 6.36 6.21 .94 294.10 305.15 . .04 .06 36.00 3.00 6.00 PA+PF 2.40 3.96 6.80 12.00 71.00 17.20 24.30 1,536.00 2.76 1.37 1.50 2.79 1.63 6.49 9.68 2.38 78.63 81.81 .01 .27 2,036.77 37.00 3.00 6.00 PA+PF .80 6.30 11.40 25.00 71.00 18.10 25.30 858.00 .84 1.33 1.48 2.56 1.30 3.24 5.11 3.36 .17 .35 1,671.31 1,740.00 39.00 3.00 6.00 PA+PF 2.80 6.96 2.20 28.00 70.00 17.50 25.20 812.00 1.55 1.22 55.13 .29 .26 1,473.64 1,770.00 40.00 3.00 6.00 PA+PF o—aa‘Q . C $3.00) r30 1 . 22.00 72.00 18.00 25.10 680.00 ’3 .21 .34 743.47 1,110.00 PA CONTENT '78 APPENDIX C (con't) RAT 8 14.00 6? 1.00 88 9.00 DIET PA+ 1131 103/01 .60 1131 10%1 6.74 808 9701 11.20 801 2 26.00 608 [L 70.00 606 pg 16.60 6080 g/dl 23.80 P11 103/111 793.00 8681-? 1.86 1.86 1988 rerun 1.00 1988 rerunZ 1.05 1988 rerun3 2.27 8681-1 2.47 3.21 1988 rerun 2.71 1988 rerun2 3.80 1988 rerun3 1.75 RBC-f 1.39 1.16 1988 rerun 1.16 880-1 1.63 1988 rerun .79 880-1 886-1 FLT-1 .07 PLT-T .2 815-? 1,175.76 2,037.56 1988 rerun 1,410.00 16.00 1.00 9.00 PA+ ‘5'9 “In 9.10 16.00 40.00 71.00 17.60 24.90 930.00 1.69 1.53 1.24 1.11 2.22 _w hJ—‘o 4500- G O h) 8‘9 F) ‘4) I I C N "‘ 8') O O Q o ‘- co 0 A w‘ 0:) h) '- co m 3,.) k0 PC! I .32 31.40 71.40 .08 .17 1,026.72 3,626.91 3,111.00 17.00 1.00 9.00 PA+ 3.80 7.70 1340 35.00 70.00 17.40 24.90 926.00 1.57 3.14 1.39 .90 2.00 1.88 3.42 4.56 .1 V be! 3.14 1.47 3.76 .72 1.61 5.23 1.30 30.06 C" -‘E Joe :6) .05 .14 873.50 2,619.00 C8C PA CONTENT RAT 8 GP NK DIET 1131 103 101 331 10*/01 888 g/dL 011 z 610 11 ”CH 09 6080 131 P11 1 luL 8681-6 1988 rerun 1988 reru112 1988 rerun3 8681-1 1988 rerun 1988 rerun2 1988 rerun3 RBC-F 1988 rerun RBC-1 1988 rerun 880-1 880-1 PLT-F PLT-T PIS-F 1988 rerun '79 29.00 2.00 9.00 PA- 1.20 9.32 13.40 32.00 53. 00 14.40 26.90 348.00 1.89 1.28 .71 .76 .82 1.08 1.00 1.68 1.21 1.53 1.85 .83 1.23 3.57 .76 81.17 110.50 .19 .54 358.80 437.57 480.00 30.00 2.00 9.00 PA- 1.80 8.38 12.00 29.00 56.00 14.30 25.50 638.00 .75 .59 .95 .77 .98 .93 1.18 .7 .90 2.09 1.66 1.71 2.66 1.45 52.70 106.25 .09 .13 301.30 313.88 1,020.00 APPENDIX C (con't) 31.00 2.00 9.00 PA- .40 8.76 12.40 32.00 53.00 14.2 26.70 398.00 1.12 .90 no.3. . . . C O 03".“ ")th)”fi-‘—. . O O O mnmwnaonromammm comm-amalgam“ .12 .19 408.94 317.87 360.00 32.00 2.00 9.00 PA- .40 6.00 8.30 12.00 54.00 14.70 27.30 258.00 .77 .75 .80 .96 .93 1.62 .94 1.99 1.21 1.63 E 3042 1.3 270.30 .27 .36 506.00 674.31 510.00 33.00 2.00 9.00 PA- .20 7.44 10.60 23.00 54.00 14.20 26.50 422.00 1.12 .73 CBC PA CONTENT RAT 6 6? 8K DIET 1181 103 luL 201 109/01 868 g/dL 807 I HCV 1L "CH 119 NCHC g/dl PU 10’luL HhBI-F 1988 rerun 1988 rerun2 1988 rerun3 8881-T 1988 rerun 1988 rerun2 1988 rerun3 RBC-F 1988 rerun RBC-T 1988 rerun HBC-F 880-1 FLT-F FLT-T PLS-F 1988 rerun 8() APPENDIX C (con't) 47.00 3.00 9.00 PA+PF 33.00 2.02 2.01 2.30 2.47 4.21 2.66 1.65 2.74 y.) (,0 o». u 0 Chubto tom-q 2,820.93 870.00 48. 00 3.00 9.00 PA+PF 1.40 9.46 14.60 38.00 64.00 15.40 24.20 680.00 .74 .97 .64 1.61 .28 13 I31 .16 .26 1.64 1.17 3.00 3.02 4.06 61.20 96.53 .06 .26 . O (.18 a: 1,220.94 1,230.00 NMB)NFJ(JF‘ .00... MMBJNUIM mAmwo-or.) . Q" '4 ‘ p- 0-0 y—s III. I o o o a o . Lu m N m 4. '3' ('3 a: ‘18 (0 J1. o r.) 65.98 80.64 .06 .15 2,2 2.48 2,160.00 50.00 3.00 9.00 PA+PF O 1.0 8.5“ 14.40 38.00 68.00 16.90 24.90 612.00 ha I I.“ 4; _ D -. N mac—om.“ ubwfi-‘NN-NJ r) B) v—o h) h) h.) o- o o o o o b a 2,134.65 1,140.00 51.00 3.00 9.00 PA+PF 1.00 8.66 14.60 35.00 67.00 16.90 25.30 638.00 .50 1,782.20 1,290.00 "71111111111111.1111111