published_or_final_version / Dentistry / Master / Master of Dental Surgery

Dietary fiber has been suggested as interfering with mineral utilization. The influence of broccoli florets and wastes, cauliflower heads and wastes, wheat bran and Metamucil on mineral uptake by the rat was determined using balance studies. All results were compared to control animals fed diets containing 10% hydrolyzed cellulose. Utilization of Ca, Mg, Cu, Zn and Fe were determined to be 62, 33, 23, 42, and 52% respectively for control animals. The uptake of Mg, Zn and Fe were significantly reduced to 12, 16 and 18% respectively from broccoli florets. Broccoli wastes decreased absorption of Zn to 14% and created a negative iron balance. Cauliflower heads improved Zn assimilation to 61%. Cauliflower wastes depressed Ca absorption to 26% and caused Mg balance to be negative. Wheat bran improved utilization of Ca and Zn to 88 and 54% respectively. Metamucil increased uptake of Ca to 88% and Mg to 53%. Copper balance was unaffected by any test diet. To better understand results of balance trials, the bioavailability of Fe from some sources was measured. The relative biological values (RBV) of endogenous Fe in broccoli, cauliflower, Brussel sprouts and wheat bran were determined to be 93, 88, 83, 98% respectively. These values were obtained using rats fed control diets containing 5% hydrolyzed cellulose and ferrous sulfate (RBV = 100). The efficiency - true bioavailability of utilizing dietary Fe is approximately one-half of the RBV. These values agree well with apparent uptake of iron by animals in the balance trials for all diets except broccoli. This exception may be due to differences in the amount and source of broccoli fed. High dietary levels of broccoli florets and wastes appear to interfere with utilization of Fe and Zn. Cauliflower wastes depress absorption of Ca and Mg. With the exceptions noted, dietary fiber from plant sources does not appear to decrease the utilization of minerals by the rat. The nature of mineral complexes in plants may be more important in defining the degree of its absorption. / Graduation date: 1980

Insulin, the principal secretory product of the /3-ce31s of the islets of Langerhans, occupies a central role in mammalian metabolism. The biosynthesis of this hormone involves two distinct proteolytic steps. Initially the N-terminal "leader" sequence is cleaved from preproinsulin (the primary translation product of the insulin gene) by a signal peptidase present in the membrane of the endoplasmic reticulum. Subsequently the product, proinsulin, is transported via the Golgi to nascent secretory granules, and is converted to insulin by the excision of the connectiong (C) peptide. This thesis describes a study of the enzymes involved in the conversion of proinsulin to insulin. Chapter 1 comprises a review of the literature relating to the biosynthesis of insulin in the pancreatic /?-cell, and of post-translational proteolysis in other secretory tissues. The tissue source for the majority of the experiments conducted in the present study was a transplantable rat isnulinoma. In chapter 2 the biosynthesis of insulin in this tissue is examined. It is demonstrated that the conversion of proinsulin to insulin follows a molecular pathway indistinguishable from that of pancreatic islet cells. This chapter also examines the subcellular location of acidic "carboxypeptidase B-like" enzymes in the tumour. Previously such an activity has been identified in insulinoma secretory granules. The present study identifies 2 activities capable of hy-drolysing a synthetic carboxypeptidase B substrate which are distinguished on the basis of subcellular location and inhibitor profiles. One activity is localized to lysosomes and the other identified as carboxypeptidase H (EC 3.4.17.10), and shown to be a component of the insulin secretory granule. Chapter 3 describes a procedure for the purification of the insulinoma caboxypeplidase H, and demonstrates that the purified enzyme is capable of converting the putative proinsulin conversion intermediates [seco 32/33]-proinsulin and [seco 65/66]-proinsulin to their respective desdibasic forms, and diarginyl insulin to insulin. In chapter 4 the subcellular and tissue distribution of immunoreactive carboxypeptidase H in the rat is described. Immunoreactive species were detected in the a and /?- cells of the islets of Langerhans, and in the pituitary. However immunoreactivity was not demonstrated in the adrenal medulla, although this tissue does contain enzymatic activity and was the tissue used for the purification of this enzyme by other workers. The possibility that multiple forms of the enzyme are present is discussed with regard to this and other data. Chapter 4 also describes the results of a study of the biosynthesis of carboxypeptidase H in insulinoma cells. It is shown that the enzyme is initially synthesized as a precursor of apparent molecular weight 56000 which is subsequently converted to the mature 54000 molecular weight form. It is proposed that the observed change in molecular weight results from post-translational modification of the enzyme's N-linked oligosaccharide chains. The results presented in chapter 5 demonstrate the presence of a novel Ca2+ -dependent acidic endopeptidase in insulin secretory granules which, in conjunction with carboxypeptidase H, is capable of converting proinsulin to insulin in vitro. The relationship of this activity to the enzymes previously implicated in proinsulin processing, and the compatibility of its molecular properties with the intragranular environment, are discussed. In chapter 6 procedures for the partial purification of the proinsulin processing endopeptidase are described. It is shown that there are actually two endopeptidases which each cleave proinsulin at one of the two processing sites. Preliminary characterization of these enzymes is presented, and the implications of these observations on granule biogenesis discussed. Chapter 7 comprises a general discussion of the work presented in this thesis. Attention is focussed on the contributions made by the present study to our understanding of secretory granule biogenesis, and on possible methods which might enable the successful purification of the proinsulin endopeptidases.

The suckling rat obtains about 70% of its energy from the catabolism of lipid, compared to only about 25% in the adult rat. In addition, lipids are in great demand for membrane synthesis in the rapidly growing tissues. Thus the suckling rat represents an important system in which to study lipid metabolism. In this dissertation research the following experiments were performed on rats during the suckling period: (1) Determination of the concentration and fatty acid composition of triacylglycerols, cholesteryl esters, phospholipids, and free fatty acids in dam's milk, and in suckling rat lymph, portal plasma, vena cava plasma and liver; (2) Determination of positional distribution of fatty acids among the three different positions in triacylglycerols of milk, lymph, plasma and liver; (3) Isolation of plasma and mesenteric lymph lipoproteins by density gradient centrifugation, and identification of the individual classes of lipoproteins by measurement of their density and by polyacrylamide gel electrophoresis; (4) Determination of the concentration of each lipoprotein by refractometry and determination of the concentration and fatty acid composition of different lipid components in each lipoprotein fraction; (5) Studies of physical properties of the lipoproteins using analytical ultracentrifugation; (6) Determination of apoprotein composition and concentration in each lipoprotein fraction. The results show that the suckling rat consumes a diet which is rich in medium chain fatty acids (35% of total milk fatty acids). Mesenteric lymph triacylglycerols carry a significant amount of these medium chain fatty acids, unlike the adult in which they would be transported as free fatty acids, unlike the adult in which they would be transported as free fatty acids via the portal vein. Medium chain fatty acids are enriched at the sn-3 position in all triacylglycerols. Lymph contains chylomicrons, very low density lipoproteins, and high density lipoproteins, but no low density lipoproteins. Plasma contains all classes of lipoproteins but at concentrations higher than found in the adult, especially low density lipoproteins which are present at levels six times that found in the adult. The plasma very low density lipoproteins have a lipid composition which resembles that of chylomicron remnants. Apoprotein, lipid composition, and electrophoresis patterns show that the plasma lipoproteins in the suckling rat are heterogeneous.

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