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True absorption of selenium in dairy cows : stable isotope tracer methodology and effect of dietary copper Koenig, Karen Marie 1988

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TRUE ABSORPTION OF SELENIUM IN DAIRY COWS: STABLE ISOTOPE TRACER METHODOLOGY AND EFFECT OF DIETARY COPPER. By KAREN MARIE KOENIG B.Sc, The University of B r i t i s h Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Animal Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1988 w Karen Marie Koenig, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives, it is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Animal Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 31, 1988 ABSTRACT Gas chromatography mass spectrometry (GCMS) and inductively coupled plasma mass spectrometry (ICPMS) were evaluated for the measurement of selenium (Se) and Se stable isotope r a t i o s . GCMS and ICPMS were found to be accurate for quantitative Se analysis i n b i o l o g i c a l matrices by isotope d i l u t i o n using Se-78 and Se-76 as i n t e r n a l standards, respectively. A higher p r e c i s i o n was obtained for ICPMS than GCMS enabling a smaller quantity of the tracer to be administered to subjects i n l a b e l l i n g experiments. The isotopes of choice for metabolic tracers were Se-76 when sample analysis was by GCMS and Se-77 and Se-82 when analysis was by ICPMS. The influence of copper (Cu) on endogenous f e c a l Se excretion and true absorption of Se i n nonlactating Holstein cows was examined by the use of Se stable isotopes as tracers. The method involved the application of conventional balance techniques i n conjunction with i s o t o p i c enrichment of the body Se pools. Selenium i n several tissues following o r a l and intravenous routes of isotope administration were evaluated as the precursors of endogenous f e c a l Se. Two cows fed a Se d e f i c i e n t d i e t (0.035 mg kg - 1 ) were administered 4 mg Se-76 o r a l l y , d a i l y , for 5 d. After a 10-d e q u i l i b r a t i o n period t o t a l c o l l e c t i o n of feces was made d a i l y for two 5-d periods. The animals were then s a c r i f i c e d and samples obtained from a l l major tissues and f l u i d s . Se-7 6 enrichment - i i i -(tracer/tracee mass percent, TTMP) i n tissues was variable (< 0.56 - 13.4). However, enrichment was s i m i l a r (9.8 - 12.9) i n the tissues considered as p o t e n t i a l contributors to endogenous f e c a l Se (serum, epithelium of the stomach, l i v e r , b i l e , pancreas, small i n t e s t i n e and colon). Enrichment i n serum and l i v e r was used to calculate endogenous f e c a l Se. Apparent absorption of Se i n the two cows was negative (-37 and -147 \q d - 1 ) . Correction of apparent absorption for the f e c a l Se of endogenous o r i g i n gave a true Se absorption (% of intake) of 10 and 16%. The percentage of t o t a l f e c a l Se of endogenous o r i g i n was 23 and 36%. In two t r i a l s , 5 or 6 cows were assigned to one of two Cu-supplemented treatment d i e t s : 0 mg k g - 1 or 17 mg k g - 1 . The basal d i e t contained 0.19 mg Se k g - 1 and 13 mg Cu k g - 1 . To each cow ~4.6 mg Se-77 and ~1.3 mg Se-82 were administered by o r a l and intravenous routes, respectively. After a 14-d e q u i l i b r a t i o n period, t o t a l c o l l e c t i o n of feces and urine were made d a i l y for two 5-d periods. Serum was co l l e c t e d on the f i r s t , t h i r d and f i f t h days of each period. Liver biopsies were taken 2 d following the completion of the balance periods. The estimates of endogenous f e c a l Se ( d - 1 ) from enrichment i n the serum (256) and l i v e r (235) following o r a l administration of the tracer and from enrichment i n serum (241) following intravenous administration were not s i g n i f i c a n t l y d i f f e r e n t (P>0.05) but were higher than the estimate from the enrichment i n l i v e r (197) (P<0.05). No s i g n i f i c a n t differences (P>0.05) were present when - i v -true absorption ( /g d~ ) was determined from enrichment i n serum (290) or l i v e r (268) following o r a l administration or from enrichment i n serum (274) or l i v e r (230) following intravenous administration. I t was concluded the analysis of serum or l i v e r with o r a l administration or the analysis of serum with intravenous administration of the tracer would provide r e l i a b l e methods for estimation of endogenous f e c a l Se and true absorption. There was no e f f e c t of Cu on endogenous f e c a l Se excretion or true absorption of Se. Apparent and true absorption were 3.2 and 11%, respectively. Approximately 90% of the t o t a l Se excreted was i n the feces, of which, 9.7% was of endogenous o r i g i n . The use of Se stable isotopes as metabolic tracers i n dairy c a t t l e provided a safe a l t e r n a t i v e to the use of radioactive tracers and enabled experiments requiring m u l t i - i s o t o p i c enrichment to be performed. -V-TABLE OF CONTENTS Page Abstract i i Table of Contents v L i s t of Tables v i i i L i s t of Figures x L i s t of Appendix Tables x i Acknowledgements x i i 1. INTRODUCTION 1 2. LITERATURE REVIEW 7 2.1. Selenium metabolism i n ruminants 7 2.2. Stable isotopes as tracers for the study of mineral metabolism 45 3. MATERIALS AND METHODS 52 3.1. A n a l y t i c a l techniques 52 3.1.1. Gas chromatography mass spectrometry 5 3 3.1.2. Inductively coupled plasma mass spectrometry 56 3.2. Calculation of Se stable isotope enrichment . . 59 3.3. T r i a l I 63 3.3.1. Animals 6 3 3.3.2. Diet 64 3.3.3. Single isotope enrichment of the whole body Se pool 64 3.3.4. Balance periods 65 3.3.5. Tissue c o l l e c t i o n 67 - v i -Page 3.4. T r i a l II and III 68 3.4.1. Animals 68 3.4.2. Experimental diets 68 3.4.3. Double isotope enrichment of the whole body Se pool 70 3.4.4. Balance periods 7 3 3.4.5. Liver biopsy 73 3.5. Calculations for apparent absorption, balance, endogenous f e c a l Se and true absorption . . . . 74 3.6. Experimental design and s t a t i s t i c a l analysis. . 75 4. RESULTS 7 8 4.1. GCMS a n a l y t i c a l technique 7 8 4.2. ICPMS a n a l y t i c a l technique 8 3 4.3. T r i a l I 90 4.3.1. Animals and die t 90 4.3.2. Selenium content of tissues and f l u i d s 90 4.3.3. D i s t r i b u t i o n of Se-76 i n tissues and f l u i d s 9 3 4.3.4. Absorption and endogenous f e c a l excretion of selenium 100 4.4. T r i a l II and I I I . 104 4.4.1. Experimental diets 104 4.4.2. Feed and water intake and urine and feces excretion 104 4.4.3. Selenium concentration i n serum and l i v e r 108 4.4.4. Isotope enrichment i n tissues and excrement 108 - v i i -Page 4.4.5. Apparent absorption and balance of selenium I l l 4.4.6. Endogenous f e c a l excretion and true absorption of selenium 113 DISCUSSION 119 5.1. Selenium stable isotopes as tracers 119 5.2. Selenium content of the experimental d i e t s . . . 124 5.3. Tissue d i s t r i b u t i o n of selenium 125 5.4. Method for measurement of true absorption . . . 128 5.5. Apparent and true absorption of selenium. . . .136 5.6. Selenium excretion 139 5.7. E f f e c t of copper on endogenous f e c a l excretion and true absorption of selenium 140 6. CONCLUSIONS 145 7. BIBLIOGRAPHY 148 8. APPENDIX 162 - v i i i -LIST OF TABLES Table Page 1 Selenium concentration of tissues and f l u i d s i n c a t t l e of adequate selenium status 23 2 Selenium concentration i n tissues i n d i c a t i n g marginal and d e f i c i e n t selenium status i n c a t t l e 24 3 Stable isotope composition of chemical elements of concern to mineral n u t r i t i o n 47 4 Description of tissue and f l u i d samples co l l e c t e d ( T r i a l I) 69 5 Mass of Se-77 and Se-82 administered to each cow i n T r i a l II and III 72 6 Selenium determination of standard reference materials by GCMS ( & g" 1) 81 7 Precision of selenium stable isotope enrichment determined by GCMS 82 8 Analysis of standard reference materials for selenium by ICPMS 88 9 Precision of selenium stable isotope enrichment determined by ICPMS 89 10 Composition of orchardgrass hay (dry matter basis) ( T r i a l I) 91 11 Selenium concentration i n tissues and f l u i d s of cow 8219 and cow 7939 95 12 Estimated selenium content i n tissues, f l u i d s and the whole body of non-lactating dairy cows 96 13 Selenium content i n the placenta and tissues of the 5-month old fetus from cow 8219 97 14 Feed intake and feces and urine excretion i n Period 1 and 2 ( T r i a l I) 101 15 Intake and feces excretion of selenium ( \q d - 1 ) ( T r i a l I) 102 16 Apparent and true absorption of selenium ( d _ 1 ) ( T r i a l I) 103 - i x -Table Page 17 Composition of the basal d i e t used i n T r i a l II and III (dry matter basis) 105 18 Copper concentration of the experimental diets (dry matter basis) ( T r i a l II and III) 106 19 Body weight, feed and water intake and feces and urine excretion for cows on Treatment 1 and 2 ( T r i a l II and III) 107 20 Selenium concentration ( yg mL - 1) i n serum 1 day p r i o r to the t r i a l and during Period 1 and 2 ( T r i a l II and III) 109 21 Se-77 and Se-82 enrichment (TTMP) i n serum, l i v e r , feces and urine ( T r i a l II and III) 110 22 Apparent absorption and balance of selenium ( /jg d - 1 ) ( T r i a l II and III) 112 23 Endogenous f e c a l selenium ( /jg d _ 1 ) ( T r i a l II and I I I ) . 115 24 True absorption of selenium ( /jg d _ 1 ) ( T r i a l II and III) 116 25 E f f e c t of route of isotope administration and tiss u e analyzed on the c a l c u l a t i o n of endogenous f e c a l selenium (jg d - 1 ) ( T r i a l II and III) 117 26 E f f e c t of route of isotope administration and tiss u e analyzed on the c a l c u l a t i o n of true absorption of selenium ( /jg d - 1 ) ( T r i a l II and III) 118 -x-LIST OF FIGURES Figure Page 75 1 Excretion of Se during a 12-day period following o r a l and intravenous administration of the isotope by lambs with varying selenium intakes 19 2 Selenide metabolism 3 3 3 Possible mechanism of glutathione peroxidase 40 4. C a l i b r a t i o n curve for selenium standards enriched with Se-76 plus 5 l e v e l s of Se-78 ranging from TTMP 7 8 0 to 8 3.9 7 9 5 C a l i b r a t i o n curve for selenium standards enriched with Se-78 plus 5 l e v e l s of Se-76 ranging from TTMP„ 0 to 11.8 80 / b 6 E f f e c t of HCl and Cu on the in t e n s i t y of the Se sig n a l 84 7 Se-76 enrichment (TTMP) i n tissues and f l u i d s of cow 8219 and cow 7939 98 8 Se-7 6 enrichment (TTMP) i n the placenta and tissues of the 5-month old fetus from cow 8219 99 - x i -LIST OF APPENDIX TABLES Table Page 1 Isotopic composition of natural abundance selenium and of the enriched selenium stable isotope preparations (atom percentage) 162 - x i i -ACKNOWLEDGEMENTS I would es p e c i a l l y l i k e to thank to Dr. W. T. Buckley, Research Station, Agassiz, for his invaluable advice and guidance provided throughout the conduct of t h i s research. Many thanks to Dr. J . A. Shelford, Department of Animal Science, U.B.C., for his input and support. I also wish to express my gratitude to D.V. Godfrey and E.G. Wilson, Research Station, Agassiz for t h e i r assistance with the chemical analysis of samples and to S. Hainstock, Research Station, Agassiz, for her assistance with the animal experiments. The research was supported i n part by a grant from Agriculture Canada and from a G.R.E.A.T. award #69(GC-5) from the Science Council of B r i t i s h Columbia. -1-1. INTRODUCTION The most c r i t i c a l time for meeting the selenium (Se) requirements of the dairy cow extends from l a t e gestation through early postpartum. I t i s during t h i s period when conditions due to Se deficiency can develop i n both the dam and o f f s p r i n g (Jenkins et a l . 1974; Harrison et a l . 1984). In the mature dairy cow retained placenta, the f a i l u r e of the f e t a l placenta to separate from the maternal placenta, i s an expression of Se deficiency (Trinder et a l . 1969; J u l i e n et a l . 1976a, b). Prepartum Se supplementation by i n j e c t i o n (Trinder et a l . 1969; J u l i e n et a l . 1976b) or i n the d i e t ( J u l i e n et a l . 1976a) has been reported to reduce the incidence of placental retention. The e f f i c a c y of the Se treatment appears to be dependent on the Vitamin E (Vit E) status of the animal. A "sparing e f f e c t " or s y n e r g i s t i c r e l a t i o n s h i p between Se and V i t E acts i n the prevention of many of the various Se responsive disorders (Jenkins and Hidiroglou 1972). Selenium deficiency during the prepartum period has been reported to predispose the dairy cow to increased r i s k of m e t r i t i s and c y s t i c ovaries (Harrison et a l . 1984). There i s some i n d i c a t i o n that d e f i c i e n c i e s of V i t E and possibly Se elevate the incidence of mastitis from enviromental pathogens (Smith et a l . 1984). A dietary deficiency of Se was found to increase the duration of the c l i n i c a l symptoms of mastitis (Smith et a l . 1984). -2-In dairy c a t t l e of a l l ages a Se responsive condition known as u n t h r i f t i n e s s or i l l t h r i f t may develop. At the s u b c l i n i c a l l e v e l there i s a f a i l u r e to achieve optimal growth rate and production. Progression of the condition can lead to c l i n i c a l symptoms such as a rapid loss of weight and mortality (Underwood 1981) . An inadequate Se intake by the dam increases the s u s c e p t i b i l i t y of the developing c a l f to n u t r i t i o n a l muscular dystrophy (NMD) (Jenkins et a l . 1974). NMD, also known as white muscle disease, i s a cardiac and s k e l e t a l myopathy which usually af f e c t s the young bovine i n the f i r s t 1 to 3 months of l i f e . A congenital form may also be found i n animals at b i r t h and i n fetuses that have been aborted (Jenkins and Hidiroglou 1972). There i s increasing evidence of the importance of the involvement of Se i n the immune system. Boyne and Arthur (1979) have shown that polymorphonuclear neutrophils of Se d e f i c i e n t c a t t l e were unable to k i l l ingested c e l l s of the microorganism Candidia albicans. C i r c u l a t i n g neutrophils play an important role i n the defense of animals against microbial i n f e c t i o n s . The role of Se i n t h i s function may underlie the mechanism whereby Se adequate dairy cows better r e s i s t the onset of c l i n i c a l uterine infe c t i o n s and mastitis (Harrison et a l . 1984; Smith et a l . 1984). In addition to impaired neutrophil function and resistance to microbial and v i r a l i n f e c t i o n s , Se deficiency has also been shown to i n h i b i t antibody production, p r o l i f e r a t i o n of T and B lymphocytes i n response to mitogens, and cytodestruction -3-of T lymphocytes and natural k i l l e r c e l l s (Kiremidjian-Schumacher and Stotzky 1987). The current recommended dietary intake of Se by dairy c a t t l e i s 0.1 mg kg" 1 feed dry matter (DM) (NAS-NRC 1978). I f feed containing < 0.1 mg Se k g - 1 i s fed to dairy c a t t l e , Se/Vit E responsive disorders may develop i n varying degrees with a greater incidence occurring when the Se concentration drops below 0.05 mg k g - 1 (NAS-NRC 1983). In a survey of feedstuffs grown i n B r i t i s h Columbia, Miltimore et a l . (1975) reported that the percentage of samples with a Se concentration below 0.1 mg k g - 1 were: wheat, 12%; barley and oats, 32%; legumes, 22%; grasses, 21%; and corn s i l a g e , 76%. Selenium analysis of dairy c a t t l e feed i n the Upper Fraser Valley revealed lower Se values for a l l classes of feed than those found by Miltimore et a l . (1975) (Cathcart et a l . 1980). Eighty percent of the commercially prepared concentrates and only 2% of a l l other feedstuffs including pasture, grass s i l a g e , orchardgrass hay and corn s i l a g e were above 0.1 mg Se k g - 1 (Cathcart et a l . 1980). The B r i t i s h Columbia Ministry of Agriculture and Fisheries reported 14% of dry roughages and silages grown i n the Fraser Valley contain > 0.1 mg Se k g - 1 (Soder 1984). Interaction of Se with other dietary nutrients can also p r e c i p i t a t e Se deficiency i n animals. Copper (Cu), s i l v e r , tellurium, zinc, arsenic, cadmium, lead, mercury and under some circumstances s u l f u r can induce t y p i c a l lesions of Se/Vit E deficiency i n animals fed diets containing amounts of Se -4-o r d i n a r i l y considered adequate (Van Vleet 1980; Puis 1981). Many of the dairy c a t t l e feedstuffs produced i n B r i t i s h Columbia contain l e v e l s of Cu below the recommended dietary l e v e l (10 mg k g - 1 , NAS-NRC 1978) thereby leaving c a t t l e susceptible to Cu deficiency. In addition to low l e v e l s of Cu i n the d i e t , higher than normal contents of s u l f u r , molybdenum, cadmium, iron and zinc can induce Cu deficiency (Puis 1981). In an e f f o r t to meet requirements and avoid problems with i n t e r a c t i n g nutrients, Cu supplementation may reach quantities many times greater than the recommended dietary l e v e l . High dietary Cu l e v e l s have been shown to be e f f e c t i v e i n overcoming the e f f e c t s of Se t o x i c i t y ( H i l l 1974; Jensen 1975a). The existence of an i n t e r a c t i o n between Cu and Se at high Cu and adequate Se intakes however, i s not as c l e a r . High l e v e l s of dietary Cu (800 - 1600 mg k g - 1 ) when added to diets adequate i n Se produce Se deficiency lesions i n chicks (Jensen 1975b) and ducklings (Van Vleet and Boon 1980). The Cu was thought to reduce the a v a i l a b i l i t y of Se i n tissues for synthesis of the selenoenzyme glutathione peroxidase by i n t e r f e r i n g with Se absorption and/or by formation of insoluble i n t r a c e l l u l a r Se compounds. In contrast to these studies, White et a l . (1981) found no e f f e c t of lower l e v e l s of Cu (10 mg k g - 1 ) on the metabolism of radiolabelled selenomethionine i n sheep. There was however, a tendency for Cu to decrease the t o t a l retention of radioactive Se. In view of the importance of Se to the health and well-being -5-of the dairy cow, an understanding of Se n u t r i t i o n and metabolism and the possible i n t e r a c t i n g e f f e c t s of other dietary nutrients are paramount for ensuring Se requirements are met. Experimental investigations of Se n u t r i t i o n and metabolism i n ruminants have been car r i e d out by measuring t o t a l elemental Se and/or using 7 5 7 5 radioactive Se. Se has been used successfully as a metabolic tracer i n ruminant animals (Handreck and Godwin 1970; Kincaid et a l . 1977; Symonds et a l . 1981a, b) but i t does present l i m i t a t i o n s for use i n metabolism studies with mature dairy c a t t l e . The isotope has a r e l a t i v e l y long h a l f - l i f e and the associated radiation hazards can make handling and disposing of excrement, f l u i d s and carcasses a problem. The use of stable isotopes as tracers overcomes these disadvantages associated with using radioisotopes i n large animals. In addition there are six stable isotopes of Se thus enabling m u l t i - i s o t o p i c studies to be performed. To aid i n the understanding of Se metabolism and n u t r i t i o n through the application of non-invasive, non-hazardous tracer methodology, t h i s research was conducted to evaluate the use of Se stable isotopes as tracers i n dairy cows. The measurement of Se and Se stable isotope ra t i o s by gas chromatography mass spectrometry (GCMS) and inductively coupled plasma mass spectrometry (ICPMS) with sample introduction by hydride generation were evaluated. The e f f e c t of Cu on true absorption of Se i n dairy cows was determined by u t i l i z i n g a method combining Se f e c a l balance with measurements of endogenous f e c a l -6-excretion i n cows with an i s o t o p i c a l l y enriched whole body pool of Se. Oral and intravenous routes of isotope administration and the analysis of several tissues were evaluated for the estimation of endogenous f e c a l excretion and true absorption of Se. The tissues analyzed were selected based on the tissu e d i s t r i b u t i o n of an enriched Se stable isotope. -7-2. LITERATURE REVIEW 2.1. Selenium metabolism i n ruminants Interest i n the b i o l o g i c a l s i g n i f i c a n c e of Se began with the recognition of i t s toxic and possible carcinogenic properties. I t was not u n t i l 1957 that work by two independent groups demonstrated the e s s e n t i a l physiological role of Se. Schwarz and F o l t z (1957) f i r s t demonstrated supplementation of Se to rats on c e r t a i n diets would prevent the development of l i v e r necrosis. In the same year Patterson et a l . (1957) showed Se also prevented exudative diathesis i n chicks. This sparked research and i n v e s t i g a t i o n into the n u t r i t i o n and metabolism of Se i n a number of animal species i n many areas of the world. I t led to the recognition of a number of Se/Vit E responsive diseases i n a l l classes of l i v e s t o c k and poultry. Disorders associated with Se and/or V i t E deficiency include: exudative diathesis, encephalomalacia, myopathy, and decreased egg productivity and h a t c h a b i l i t y i n poultry; breast, heart, i n t e s t i n a l and gizzard (white gizzard disease) muscle dystrophy and poor reproductive performance i n turkeys; myocardial degeneration and necrosis (mulberry heart disease), hepatic necrosis (hepatosis d i e t e t i c a ) , n u t r i t i o n a l muscular dystrophy, c i r c u l a t o r y f a i l u r e , g a s t r i c u l c e r a t i o n , m a s t i t i s - m e t r i t i s - a g a l a c t i a and reproductive problems i n swine (Jenkins and Hidiroglou 1972; Underwood 1977). C l i n i c a l symptoms -8-of Se deficiency i n sheep include embryonic death, peridontal disease and scouring. In c a t t l e Se/Vit E deficiency conditions a r i s e as s k e l e t a l myopathy (p a r a l y t i c anemia), placental retention, m e t r i t i s , c y s t i c ovaries and m a s t i t i s . Another Se responsive condition appearing i n sheep, beef and dairy c a t t l e i s general u n t h r i f t i n e s s , a condition of slow growth. The most widely recognized Se/Vit E responsive disorder i n calves and lambs i s NMD, also refered to as white muscle disease and s t i f f lamb disease. NMD i s a degenerating disease of the s k e l e t a l and cardiac muscle most commonly a f f e c t i n g lambs and beef calves 1 to 3 months of age. A congenital form may also be found i n animals at b i r t h and i n fetuses that have been aborted (Jenkins and Hidiroglou 1972). In 1974-75 Keshan disease was i d e n t i f i e d as the f i r s t human disease associated with Se deficiency (Yang 1985). Keshan disease i s a cardiomyopathy a f f e c t i n g primarily young peasant women and children l i v i n g i n mountainous and r u r a l areas of a region extending from northeast to southwest China (Levander 1987). Selenium provided o r a l l y as sodium selenate i s now used on a large scale i n China as a preventative measure against the disease. Not a l l features of the disease are explained s o l e l y on the basis of Se deficiency and i t appears other factors are involved (Yang 1985). Another disease possibly associated with Se deficiency i s Kashin-Beck disease. This i s a d i s a b l i n g p o l y a r t i c u l a r degenerative j o i n t disease that occurs i n northern China, North Korea and eastern Siber i a (Levander 1987). Most -9-nutrient solutions administered parenterally are very low i n Se (Levander 1987). A Se responsive condition i n a patient receiving t o t a l parenteral n u t r i t i o n has been described by Van Rij et a l . (1979). The patient developed muscular discomfort i n the quadricep and hamstring muscles which was a l l e v i a t e d by Se supplementation. In animals and man exh i b i t i n g Se responsive conditions subnormal t i s s u e and f l u i d Se l e v e l s and glutathione peroxidase (GSHPx) (glutathione: hydrogen-peroxide oxidoreductase, EC 1.11.1.9) a c t i v i t i e s are present along with a number of other biochemical changes. It i s important to recognize and d i s t i n g u i s h between the various chemical forms of Se for the discussion of the n u t r i t i o n a l and metabolic aspects of t h i s element. The chemical forms of i n t e r e s t include: elemental Se, inorganic Se such as 2- 2-s e l e n i t e (Se0 3 ) and selenate (Se0 4 ), selenoamino acids such as selenomethionine (Se-Met) and selenocysteine (Se-Cys) and other organic selenocompounds. Metabolic s i m i l a r i t i e s do ex i s t but there are also some important differences between these chemical forms. Rumen microorganisms Se content of rumen microorganisms The Se concentration and i n v i t r o Se metabolism of rumen microorganisms are influenced by the previous d i e t of the host animal. Whanger et a l . (1978) reported a variable Se -10-concentration ranging from 0.040 - 1.90 mg kg i n rumen microorganisms iso l a t e d from sheep on various dietary regimes. 75 In radioisotopic studies the incorporation of Se into bacteria i n v i t r o was inversely proportional to the previous dietary intake of Se by the host animal (Hidiroglou et a l . 1968). Whanger et a l . (1978) reported a s u l f u r deficiency i n a p u r i f i e d d i e t fed to sheep decreased the Se and nitrogen concentration of rumen microbes. Changes i n microbial populations have also been observed following changes i n dietary Se supplementation of a p u r i f i e d d i e t low i n Se and V i t E (Hidiroglou et a l . 1968). Metabolism of Se by rumen microorganisms i n v i t r o L i t t l e i s known of the products formed following b a c t e r i a l incorporation of selenocompounds. Paulson et a l . (1968) reported 7 5 that 68.0% of the r a d i o a c t i v i t y added as [ Se]Se-Met was found i n the TCA-insoluble f r a c t i o n . Radioactivity i n the TCA-insoluble f r a c t i o n was assumed to indicate radioactive Se associated with the b a c t e r i a l protein f r a c t i o n . Following a 3-h 75 incubation period of rumen microorganisms with [ Se]Se-Met, approximately 60% of the r a d i o a c t i v i t y incorported into the TCA-insoluble f r a c t i o n was i d e n t i f i e d by paper chromatography and 75 ion-exchange chromatography as [ Se]Se-Met (Paulson et a l . 1968). Hidiroglou et a l . (1974) reported that rumen bacteria 75 75 metabolized [ Se]Se-Met to [ Se]Se-cystine and incorporated both selenoamino acids into b a c t e r i a l protein. Paper chromatography of hydrolyzed rumen b a c t e r i a l protein 75 2 — following a 24-h incubation of rumen microbes with [ Se]SeO, -11-revealed r a d i o a c t i v i t y co-chromatographing with Se-Met (Hidiroglou et a l . 1968). In addition, smaller amount were found associated with taurine, homocystine and 75 selenocystine. Identical f r a c t i o n a t i o n patterns of Se 75 2-following incubation of rumen microbes with [ Se]Se0 3 and 7 5 2 — [ Se]Se0 4 indicated that selenate was reduced to s e l e n i t e by rumen microorganisms (Paulson et a l . 1968). Thus, rumen 75 75 2-microorganisms incubated i n v i t r o with [ Se]Se-Met, [ Se]Se0 3 75 2 — and [ Se]Se0 4 metabolize these chemical forms of Se and incorporate them into microbial protein (Hidiroglou et a l . 1968, 1974) . Metabolism of Se i n the rumen Se administered to the rumen becomes quickly associated with the b a c t e r i a l f r a c t i o n . In sheep dosed intraruminally with 75 [ Se]Se-Met, 50% of the l a b e l i n the rumen l i q u o r was i n the b a c t e r i a l f r a c t i o n 6 h after dosing (Hidiroglou et a l . 1974). Approximately 66% of the l a b e l i n the b a c t e r i a l f r a c t i o n was protein bound. Radiolabelled selenocompounds i d e n t i f i e d i n the b a c t e r i a l protein f r a c t i o n 2 h following intraruminal 75 administration of [ Se]Se-Met included Se-Met, selenocystine and elemental Se. Unidentifed compounds constituted 40 - 50% of the r a d i o a c t i v i t y (Hidiroglou et a l . 1974) Inorganic Se i s also rapidly metabolized by rumen bacteria. One hour following the intraruminal administration of 75 2-[ Se]Se0 3 , 30% of the rumen l i q u o r a c t i v i t y was bound to b a c t e r i a l protein (Hidiroglou et a l . 1968). The highest l e v e l of -12-r a d i o a c t i v l t y associated with the protein f r a c t i o n was 71% at 4 h. Chromatographic separation of hydrolyzed b a c t e r i a l protein revealed patterns s i m i l a r to that found af t e r i n v i t r o studies, with most of the r a d i o a c t i v i t y i d e n t i f i e d as Se-Met and smaller amounts of the r a d i o a c t i v i t y associated with selenocystine, homocystine and taurine (Hidiroglou et a l . 1968). The findings obtained both i n v i t r o and i n vivo indicate rumen bacteria are capable of metabolizing inorganic Se to organic Se compounds. Metabolism of dietary Se by rumen microorganisms w i l l thus influence the chemical form of Se available to the host animal. Absorption S i t e of Se transport across the g a s t r o i n t e s t i n a l t r a c t The everted-gut sac technique has been used i n i n v i t r o investigations to compare the rate of absorption of various selenocompounds and to elucidate the mechanisms of transport across the i n t e s t i n a l c e l l . McConnell and Cho (1965) using everted i n t e s t i n a l sacs of the hamster demonstrated the rate of transport of Se-Met was highest i n the d i s t a l jejunum, intermediate i n the terminal ileum and lowest i n the proximal jejunum and proximal ileum. In sheep, Hidiroglou and Jenkins (1973b) reported Se-Met was absorbed primarily from the mid jejunum. Radioisotopic studies showed only small amounts of 75 [ Se]Se-Met were transported across the rumen wall into the blood (Hidiroglou and Jenkins 1973a). A d i f f e r i n g pattern of results were observed i n chicks. Humaloja and Mykkanen (1986) -13-used an i n vivo l i g a t e d i n t e s t i n a l loop procedure to study the absorption of l a b e l l e d compounds from the d i f f e r e n t g a s t r o i n t e s t i n a l segments of the chick and found a more e f f i c i e n t 75 transfer of [ Se]Se-Met from the duodenal segments than from the more d i s t a l segments. The v a r i a t i o n i n results with regards to the s i t e of transport of Se-Met i n the various sections of the small i n t e s t i n e may r e f l e c t differences i n animal species or may have arisen from the experimental procedures. 2 -Absorption of Se0 3 from the small i n t e s t i n e occurs primarily i n the duodenum with s l i g h t l y smaller amounts absorbed from the jejunum and ileum (Whanger et a l . 197 6; Humaloja and Mykkanen 1986) Wright and B e l l (1966) studied the net absorption of Se from the i n t a c t g a s t r o i n t e s t i n a l t r a c t of ruminant and 75 2-monogastric animals using [ Se]Se0 3 and a non absorbable 75 marker, chromium oxide. Net absorption of Se occurred from the d i s t a l f o u r - f i f t h s of the small i n t e s t i n e i n both species. I t was not absorbed from the rumen or abomasum of sheep nor was i t absorbed from the stomach of swine. There was also no absorption occurring from the cecum or colon. Paulson et a l . (1966) reported the primary s i t e for Se0 4 absorption i n the ewe was the small i n t e s t i n e . Wolffram et a l . (1985) using an i n vivo perfusion technique i n rats found Se0 4 was absorbed at decreasing rates from the ileum, proximal jejunum, cecum and colon. Mechanisms of Se transport across the g a s t r o i n t e s t i n a l t r a c t There i s l i t t l e information regarding the mechanisms of Se -14-absorption from the g a s t r o i n t e s t i n a l t r a c t , but there appear to be d i f f e r e n t pathways involved for the d i f f e r e n t molecular forms. In experiments using the everted i n t e s t i n a l t r a c t of the hamster, 75 McConnell and Cho (1965) demonstrated [ Se]Se-Met was ac t i v e l y transported across the small i n t e s t i n e . The i n h i b i t i o n by Met of the transport of Se-Met and vice-versa suggested that the transport system for Se-Met was the same as that for the su l f u r analogue, Met (McConnell and Cho 19 65). I t has been suggested that high protein d i e t s , e s p e c i a l l y diets with high Met contents, may o f f e r a protective e f f e c t against Se t o x i c i t y through i n h i b i t i o n of the i n t e s t i n a l absorption of Se-Met by Met (McConnell and Cho 1967). McConnell and Cho (1965) reported Se0 3 was absorbed across the small i n t e s t i n e by simple d i f f u s i o n . The transport of sel e n i t e and selenocystine were not inh i b i t e d by s u l f i t e and cystine, respectively, i n d i c a t i n g there i s no common transport mechanism shared by these selenocompounds and the corresponding s u l f u r analogues. Wolffram et a l . (1985) found Se0 4 absorption to be concentration dependent, and concluded that Se0 4 was absorbed by a saturable carrier-mediated transport mechanism i n the i l e a l 2-mucosa. The absorption of Se0 4 from the ileum was not affected 2_ by a 100-fold higher concentration of S0 4 . On the other hand, Cardin and Mason (1975, c i t e d by Wolffram et a l . 1985) found 2- 2- 2-Se0 4 and Mo04 i n h i b i t e d S0 4 transport by the everted sacs of rat ileum, suggesting a common mechanism for absorption of -15-these and other anions. This mechanism however, i s probably not V 2-that important i n ruminants as most of the SeO. i s l i k e l y reduced i n the rumen. Absorption of Se i n laboratory animals and man In monogastric species Se i s well absorbed from the d i e t . Experiments conducted with laboratory animals have established inorganic Se s a l t s (selenite and selenate) are almost as well absorbed from the int e s t i n e as selenoamino acids. In the rat 2-i n t e s t i n a l absorption of Se0 3 was only s l i g h t l y less than for Se-Met (Thomson and Stewart 1973). The i n t e s t i n a l absorption following o r a l administration was estimated to be 91-9 3% for 2-Se0 3 and 95-97% for Se-Met. In rats fed torula yeast diets containing 0, 0.5 or 4 mg Se k g - 1 (as sodium s e l e n i t e ) , 95 to 100% of the Se was absorbed. Under most experimental conditions Se i s r e l a t i v e l y well absorbed from the g a s t r o i n t e s t i n a l t r a c t of man. In general 2 — Se-Met i s more completely absorbed than Se0 3 , with Se contained i n food intermediate between the two (Barbezat et a l . 1984). In young women Se-Met was found to be more completely absorbed (95.5 - 97.3% of administered dose) ( G r i f f i t h s et a l . 1976) than S e 0 3 2 _ (70, 64 and 44% of the administered dose) (Thomson and Stewart 2_ 1974). Similar results were obtained for Se0 3 absorption i n young adult males with 68 and 76% of the dose absorbed (Janghorbani et a l . 1982a). Estimates of true i n t e s t i n a l absorption of food Se by New Zealand women was 7 6 to 8 3% of intake with a mean of 79%. True i n t e s t i n a l absorption of food Se -16-was higher than apparent absorption which was 49 to 60% (mean 55%) (Stewart et a l . 1978). In pregnant and non-pregnant women consuming a semi-synthetic di e t with egg albumin contributing the majority of Se, apparent absorption of Se was approximately 80% (Swanson et a l . 1983). Absorption of Se i n ruminants There e x i s t r e l a t i v e differences between the e f f i c i e n c y of Se absorption by ruminants and monogastrics. Wright and B e l l (1966) reported the t o t a l net absorption of Se i n sheep represented approximately 35% of the Se ingested with 85% absorbed by swine when rations containing 0.35 and 0.50 mg Se k g - 1 , respectively, were consumed. The lower e f f i c i e n c y of Se absorption by ruminant animals i s attributed to the action of the rumen microoganisms and the conditions within the rumen which a l t e r the chemical form of Se ingested and thereby influence the chemical form of Se absorbed. Inorganic s a l t s of Se (selenite and selenate) are l i k e l y reduced to insoluble forms such as elemental Se or insoluble metal selenides (Butler and Peterson 1961; Cousins and Carney 1961; Peterson and Spedding 1963) and thereby may be made less available for absorption than organic forms of Se which occur natu r a l l y i n feeds. Peter et a l . (1982, c i t e d by Peter et a l . 75 1985) reported the apparent absorption of Se was 12% to 15% higher for [ Se]Se-Met than [ Se]Se0 3 i n sheep of a low Se status receiving low Se d i e t s . Conrad (1985) reported the apparent absorption of Se i n -17-non-lactating dairy c a t t l e was 41% of the dietary Se intake. When Se was provided to dairy c a t t l e from natural feedstuffs i t s apparent absorption was greatest at dietary calcium l e v e l s of 0.8% of dry matter intake. Amounts of dietary calcium less or greater than 0.8% resulted i n a reduction of the apparent Se absorption (Harrison and Conrad 1984b). Quantitative information on the true absorption of Se i n ruminants i s s t i l l lacking. Excretion Selenium i s excreted i n feces, urine and expired a i r . The major route of excretion i s a function of the animal species, the Se status of the animal, the mode of Se administration and the nature of the d i e t . In non-ruminant animals most dietary Se i s excreted i n urine. In ruminant animals the primary route of excretion i s v i a the feces when dietary l e v e l s of Se are low to adequate. As dietary Se l e v e l s increase the urinary route of excretion may equal or exceed f e c a l Se excretion (Butler and Peterson 1961; Cousins and Cairney 1961; Lopez et a l . 1969). In non-lactating dairy c a t t l e consuming 400 - 3100 /g Se d _ 1 , 50 -83% of the d a i l y selenium intake was excreted i n the feces and 7 - 14% i n the urine (Harrison and Conrad 1984a). Lopez et a l . (1969) examined the excretory patterns of 7 5 S e after o r a l and intravenous dosing i n lambs with varying l e v e l s of 7 5 Se intake. The major factor a f f e c t i n g the f e c a l loss of Se was the route of isotope administration, with much higher quantities of Se excreted i n feces following o r a l admistration (Fig. l ) . -18-Roughly equal quantities of the radioisotope were excreted i n the feces i n a l l groups dosed intravenously, i n d i c a t i n g a small but constant endogenous secretion independent of dietary Se intake. The dietary Se le v e l s were ref l e c t e d i n urinary and v o l a t i l e Se 75 excretion. Urinary and respiratory excretion of Se increased with increasing dietary Se l e v e l , i n p a r t i c u l a r when the isotope was intravenously administered (Fig. 1). 7 5 The pattern of Se excretion i s also influenced by the chemical form of the Se administered depending on the route of isotope administration. Hidiroglou and Jenkins (1972) report no s i g n i f i c a n t differences i n t o t a l r a d i o a c t i v i t y i n urine or feces when radioselenium was administered o r a l l y as organic (Se-Met and 2- 2-Se-Cys) or inorganic ( S e 0 3 a n <3 S e 0 4 ) forms to sheep fed a low Se d i e t (0.02 mg k g - 1 ) . There was however, greater r a d i o a c t i v i t y excreted i n the urine following intraabomasal administration of radioselenate than following intraabomasal administration of the other chemical forms. Symonds et a l . 75 (1981a) also found diffences i n excretory patterns of Se following intravenous administration of radi o l a b e l l e d Se0 4 and 2- 75 2-Se0 3 . More of the Se injected as Se0 4 was excreted i n feces and urine during the f i r s t 24 h afte r dosing than of the 75 2 — 7 5 Se injected as Se0 3 . The cumulative excretion of Se from these two chemical forms i n urine 14 d after intravenous dosing was equal to 10% of the i n i t i a l dose. The cumulative excretion of 7 5 S e from [ 7 5 S e ] S e 0 4 2 ~ and [ 7 5 S e ] S e 0 3 2 ~ i n feces was 17% and 9.5% of the dose respectively. -19-o o o X Hi o o Q T3 O o *•> (0 c E < O a. 70 60 50 40 30 20 10 0 Intravenous 7 5 S e Administration J 50r 40 30 20 10 0 Oral 7 5 S e Administration 0.014 0.264 0.514 5.014 Dietary Selenium Levels (mg kg"1) I I Feces ^ U r i n e ••Volati le Se F i g . 1. Excretion of 7 5 S e during and intravenous administration of varying selenium intakes (Source, a 12-day period following oral the isotope by lambs with Lopez et a l . 1969) -20-Selenium i n the feces of ruminants includes l a r g e l y Se which i s unabsorbed from the die t and which has probably undergone reduction i n the rumen to unavailable forms such as elemental Se and metal selenides. Selenium i s secreted i n b i l e , pancreatic and other g a s t r o i n t e s t i n a l secretions a l l of which may not be completely reabsorbed and thus also contribute to f e c a l Se (Dejneka et a l . 1979; Symonds et a l . 1981b; Langlands et a l . 1986). Trimethyselenonium ion (TMSe) [(CH 3) 3Se +] i s the most well-characterized metabolite excreted i n the urine (Palmer et a l . 1970). In rats fed or injected with r e l a t i v e l y high l e v e l s 2-of Se0 4 , Se-Met, Se-cystine, Se-methylselenocysteine and seleniferous wheat, 20 - 50% of the urinary Se was i d e n t i f i e d as TMSe (Palmer et a l . 1970). A second major urinary metabolite referred to as U-2 accounted for 11 - 28% of the t o t a l urinary Se. There i s some controversy as to the importance of TMSe as a urinary metabolite at lower l e v e l s of Se intake. Palmer et a l . (1969) found TMSe to be a major metabolite accounting for 40% of 2-urinary Se from Se0 3 at low phys i o l o g i c a l doses. In contrast, Nahapetian et a l . (1983) reported TMSe was a major urinary 2_ metabolite for near toxic doses of Se0 3 and selenoamino acids but was only a minor metabolite at low doses. The major v o l a t i l e form of Se i n respiratory gases i s dimethyl selenide (DMSe) [(CH 3) 2Se)]. Expiration of Se by animals with physiological intakes of Se does not reach s i g n i f i c a n t quantities (Ganther et a l . 1966). Handreck and -21-Godwin (1970) reported only 1% of the Se excreted i n ewes from 7 5 Se l a b e l l e d rumen p e l l e t s (elemental Se) appeared i n expired a i r . At toxic dietary Se intakes the exhalation of Se becomes an important route of excretion. In rats up to 6 2% of a large dose of Se was expired (McConnell and Roth 1966). The expiration of Se may also be influenced by other factors. In the rat high dietary Met and protein l e v e l s increased the expiration of Se (Ganther et a l . 1966). 7 5 Lopez et a l . (1969) found the formation of Se l a b e l l e d v o l a t i l e compounds by sheep on a low Se d i e t was greater following o r a l administration of the dose than following intravenous administration, and suggested there was formation of v o l a t i l e or gaseous Se products within the rumen. D i s t r i b u t i o n i n tissues and f l u i d s The predominant chemical form of Se i n animal tissues i s Se-Cys. In the rat 80 to 85% of the t o t a l body Se was i n protein i n the form of Se-Cys (Hawkes et a l . 1985; Tappel 1987). Of the t o t a l body Se i n the rat, one t h i r d was present as the selenoenzyme GSHPx (Tappel 1987). Selenium i s present throughout the animal body at varying concentrations depending on the c e l l , t i s s u e and f l u i d (Underwood 1977). Listed i n Table 1 are t y p i c a l t i s s u e Se concentrations for c a t t l e of adequate Se status. The highest concentration of Se i s found i n the kidney with high l e v e l s also found i n the l i v e r and glandular tissues, e s p e c i a l l y i n the pancreas and -22-adrenal glands. Intermediate l e v e l s of Se are found i n the i n t e s t i n e , lung and cardiac muscle and low l e v e l s i n the s k e l e t a l muscle, bones and blood. Even lower Se concentrations are found i n the adipose t i s s u e . In the whole blood of dairy c a t t l e the c e l l u l a r component contains approximately 7 3% of the t o t a l Se with the remainder i n plasma (Scholz and Hutchinson 1979). Grace and Watkinson (1985) estimated the t o t a l body Se i n a 50-kg sheep with 2.9 kg fleece when maintained on a low Se d i e t (0.042 mg k g - 1 ) was 1.45 mg. The muscle, digestive t r a c t , bone, kidney and l i v e r contained respectively 40%, 12%, 10%, 7%, and 6% of the t o t a l body Se. Tissue Se l e v e l s r e f l e c t the dietary intake of Se over a wide range. Thompson et a l . (19 81) monitored the response of several body components to changes i n dietary Se concentration i n calves transferred between low and high Se pastures. Liver and plasma Se concentrations responded most rapidly. I t was concluded l i v e r and plasma Se provided the best indicator of the current Se status of c a t t l e . Table 2 l i s t s t i s s u e Se l e v e l s i n l i v e r , serum, kidney and muscle which may serve as indicators for the diagnosis of Se deficiency. 75 Retention of Se by tissues and f l u i d s 7 5 The retention of Se by the tissues i s influenced by the Se l e v e l s of the d i e t . In almost a l l tissues, percentage retention 75 of Se decreased with increasing dietary Se l e v e l (Lopez et a l . 1969; Kincaid et a l . 1977). In calves fed a p r a c t i c a l d i e t containing 0.3 mg k g - 1 of natural Se with 0.0 (control), 0.1, and -23-Table 1. Selenium concentration of tissues and f l u i d s i n c a t t l e of adequate selenium status Tissue Selenium concentration ( IQ g" 1 DM) l i v e r 0.800 - 1.750 kidney 1.370 - 2.700 lung 0 . 815 heart 0.733 - 0.770 s k e l e t a l muscle 0.208 - 0.583 pancreas 1. 300 spleen 0 . 940 adrenal glands 1.405 - 1.576 thymus 0.613 brain 0.594 uterus 0.149 f ovary 0.230 f testes 1.585 adipose tissue 0.031 - 0.043 bone 0.100 hair 0.400 - 1.340 whole blood 0.110 - 0.194 % plasma 0.034 - 0.112 % f g wet weight X A§ mL - 1 (Compiled from Perry et a l . 1976; Kincaid et a l . 1977; Ullr e y et a l . 1977; Doyle 1979; Puis 1981; and Scholz et a l . 1981a) -24-Table 2. Selenium concentration i n tissues i n d i c a t i n g marginal and d e f i c i e n t selenium status i n c a t t l e Liver Kidney(cortex) Muscle Serum IB g"11 IB g - 11 m g - 11 m Marginal 0.420-0.875 1.40-3.50 0.175-0.245 0.020-0.040 Defici e n t 0.07-0.595 0.630-1.40 0.035-0.175 0.002-0.008 | Dry matter basis. (Source, Puis 1981). -25-1.0 mg kg supplemental Se (as selenite) the s p e c i f i c a c t i v i t y i n the kidney decreased 29% and 69% respectively i n the Se 75 supplemented groups 48 h a f t e r o r a l dosing of Se. Selenium s p e c i f i c a c t i v i t i e s were also reduced by supplemental Se i n the heart, l i v e r , and blood but not s i g n i f i c a n t l y i n the muscle or pancreas (Kincaid et a l . 1977). 7 5 There i s a wide range of Se retention i n tissues and f l u i d s following the administration of the radioisotope (Lopez et a l . 1969; Handreck and Godwin 1970; Dejneka et a l . 1979; Scholz 7 5 et a l . 1981a). Based on Se concentration the highest retention i s found i n the kidney cortex followed by i n descending order: kidney medulla, testes, l i v e r , spleen, lung and heart. High l e v e l s are also retained i n the glandular t i s s u e i n p a r t i c u l a r by the pancreas and the p i t u i t a r y , pineal, adrenal and s a l i v a r y 75 glands. Lower l e v e l s of Se are retained i n the smooth muscle of the g a s t r o i n t e s t i n a l t r a c t , with concentrations i n the small i n t e s t i n e higher than i n the four stomach parts, large i n t e s t i n e and cecum. The lowest l e v e l of r a d i o a c t i v i t y i s measured i n s k e l e t a l muscles, hide, bone, adipose tiss u e and parts of the eye. 7 5 The t i s s u e retention of Se, i n addition to being influenced by the Se l e v e l i n the d i e t , i s also influenced by the chemical form of the element and i t s route of administration. In 7 5 sheep there i s a greater retention of Se by the tissues following intravenous (Lopez et a l . 1969) or abomasal (Hidiroglou and Jenkins 1972) administration than following o r a l -26-administration of the radioisotope. 75 Hidiroglou and Jenkins (1972) administered [ Se]Se-Met and 7 5 [ Se]Se-cystine to the abomasum of sheep and found higher lev e l s of r a d i o a c t i v i t y i n the kidney, l i v e r , and heart i n those animals 7 5 administered the former [ Se]selenoamino acid. These differences were not found following the administration of the selenoamino acids to the rumen. Differences i n the main s i t e of r a d i o a c t i v i t y incorporation also ex i s t between these two selenoamino acids when administered to the abomasum. In animals 75 receiving [ Se]Se-Met the highest l e v e l of r a d i o a c t i v i t y was i n 75 the pancreas whereas i n animals receiving [ Se]Se-cystine the highest l e v e l was i n the kidney. 7 R o 7 R o When [ Se]Se0 3 and [ Se]Se0 4 were administered to the abomasum of sheep, the l a t t e r resulted i n higher r a d i o a c t i v i t y i n tis s u e of the rumen, omasum, abomasum, duodenum, cecum, l i v e r , and pancreas (Hidiroglou and Jenkins 1972). The tissue retention 75 of Se from these two inorganic Se sources were not d i f f e r e n t when they were administered to the rumen. 75 A comparison of the tissue retention of Se from organic and inorganic sources y i e l d s d i f f e r i n g results depending on the tissue examined. Peter et a l . (1985) reported the s p e c i f i c 75 a c t i v i t y and retention of Se i n the heart, lung, spleen, kidneys, whole blood and plasma to be si m i l a r i n sheep 75 75 2-administered [ Se]Se-Met and [ Se]Se0 3 by intravenous or abomasal routes. In contrast, there was a s i g n i f i c a n t l y higher s p e c i f i c a c t i v i t y and retention i n muscle tissu e of animals -27-7 5 receiving [ Se]Se-Met by either route of administration. Studies i n rats have also demonstrated a greater deposition of 7 5 Se i n the muscle tissue following o r a l (Cary et a l . 1973) and int r a p e r i t o n e a l ( B e i l s t e i n and Whanger 1986) administration of 75 75 2-[ Se]Se-Met compared to [ Se]Se0 3 . No differences were found 75 for the deposition of Se i n the l i v e r , testes, erythrocytes, hair and skin. 7 5 Despite s i m i l a r i t i e s reported for c e r t a i n t i s s u e Se a c t i v i t y l e v e l s following [ Se]Se-Met and [ Se]Se0 3 administration, a closer look at the predominant chemical form of 75 Se indicates some differences i n t h e i r intermediary metabolism. B e i l s t e i n and Whanger (1986) reported the predominant form of 75 Se i n erythrocyte protein lysate was Se-Cys i n rats injected 7 5 2 — 7 R with [ Se]Se0 3 . In the rats injected with [ Se]Se-Met two u n i d e n t i f i a b l e compounds were recovered. In hemoglobin 75 75 [ Se]Se-Met was i d e n t i f i e d from rats injected with [ Se]Se-Met 75 2-but not from rats injected with [ Se]Se0 3 . In acid 75 hydrolysates of l i v e r , Se was recovered primarily as 75 7 5 2 — [ Se]Se-Cys from rats injected with [ Se]Se0 3 . In animals 75 75 injected with [ Se]Se-Met, Se i n the l i v e r was present 75 75 i n i t i a l l y as [ Se]Se-Met but aft e r 5 d the majority of Se was 75 as [ Se]Se-Cys ( B e i l s t e i n and Whanger 1986). Whole body turnover of Se The whole body turnover of Se i n lambs was described by two f i r s t order processes with d i f f e r i n g rate constants (Lopez et a l . 1969). The i n i t i a l slope covering a 48-hr period was greater -28-than the f i n a l slope. As the dietary Se intake of the animals increased so did the f i n a l slope and thus the e f f e c t i v e h a l f - l i f e 7 5 of Se decreased. This described the pattern following o r a l and 7 5 intravenous Se administration. The i n i t i a l slope describing 75 the f i r s t component of Se clearance was considered to represent 75 excretion of "unequilibrated" Se i n animals given Se o r a l l y . 75 I t appeared to consist of unabsorbed Se as well as absorbed 75 Se excreted i n the urine, expired a i r or v i a the g a s t r o i n t e s t i n a l t r a c t with l i t t l e or no e q u i l i b r a t i o n with the main Se pools of the body. The second component appeared to represent r e l a t i v e l y slow turnover of Se involved i n e s s e n t i a l metabolic functions or storage. In dairy cows administered [ Se]Se0 3 or [ Se]Se0 4 7 5 intravenously or Se-labelled barley o r a l l y , the whole body turnover was also described by two exponential components (Symonds et a l . 1981a). In animals where the f i r s t component of clearance was measurable, i t was calculated to equal 1 - 1 . 9 days. The second component of clearance was 60.7 ±3.9 days for a l l animals. S i m i l a r i e s i n the response of the second component suggested that intravenously administered inorganic Se and o r a l l y administered organic Se were incorporated into the same metabolically active pool (Symonds et a l . 1981a). Plasma Se turnover The decay of r a d i o a c t i v i t y i n plasma aft e r intravenous 75 2-i n j e c t i o n of [ Se]Se0 3 occurs i n four phases (McMurray and 7 5 Davidson 1985). Immediately following i n j e c t i o n Se i s rapidly -29-taken up by the erythrocytes. The Se i s modified by the erythrocyte and excreted as possibly hydrogen selenide (H 2Se) or another reduction product of selenodiglutathione (GS-Se-SG) (Sandholm 1973; Gasiewicz and Smith 1978) and i s transferred to 7 5 other ti s s u e s . The f i r s t phase represents the decay of Se bound to plasma proteins. Symonds et a l . (1981a) demonstrated 75 the uptake of Se by the l i v e r of the cow was concomitant with the f i r s t phase of plasma radioactive decay. During the second 75 phase there was an increase i n the Se a c t i v i t y i n the plasma 7 5 due to the release of protein bound Se from the l i v e r (Symonds et a l . 1981a). There was a s h i f t of the protein bound 7 5 S e i n plasma from albumin to the alpha and gamma globulins which was also attributed to the action of the l i v e r (Symonds et a l . 1981a). The l a s t two phases represent the slow biphasic disappearance of various selenoproteins from the plasma (McMurray and Davidson 1985). Up to twenty d i f f e r e n t selenoproteins i n plasma have been separated (Davidson and McMurray 1987). The pattern of clearance and reappearance of r a d i o a c t i v i t y 75 of Se i n plasma i s dependent on the form i n which Se i s administered. Radioactivity from the i n j e c t i o n of [ 7 5 S e ] S e 0 4 2 ~ was cleared from the systemic c i r c u l a t i o n at a lower rate and was not released as rapidly back into the c i r c u l a t i o n as when 7 5 S e 2— 2 — was injected i n the form of Se0 3 , suggesting Se0 3 i s more 2 — readi l y metabolized than SeO. (Symonds et a l . 1981a). -30-Intermediary metabolism of Se The chemistry of Se resembles s u l f u r (S) and the metabolism of Se and S i n animals i s si m i l a r under some circumstances, however, there are important biochemical differences between them. In both ruminants and non-ruminants inorganic forms of Se (selenate and selenite) undergo reduction, i n contrast to the inorganic forms of S (sulphide and sulphite) which undergo oxidation (NAS-NRC 1983). 2-The proposed mechanism of Se0 4 reduction involves i t s a c t i v a t i o n by ATP and ATP sulfurylase to form adenosine-5'-selenophosphate which subsequently undergoes non-enzymatic cleavage catalyzed by glutathione (GSH) to form - 2-th i o s e l e n i c acid (GSSe03 ) and then Se0 3 (Ganther 1984). Selenite i s reduced to H 2Se by the glutathione reductase pathway i n the cytosol of the c e l l (Combs and Combs 1984): 4GSH GSSG+3H-0 2NADPH 2NADP++GSH 2NADPH 2NADP++GSH H„SeO ? >GSSeSG ^ GSSeH ^,Se GSH GSH reductase reductase Selenite f i r s t reacts non-enzymatically with glutathione followed by c a t a l y t i c reduction by glutathione reductase (EC 1.6.4.2) with NADPH to y i e l d H 2Se (Ganther 1984). This i s analogous to the reduction of su l f a t e and s u l f i t e to hydrogen s u l f i d e i n plant and microbial systems (Wilson and Bandurski 1958; Dilworth and 2-Bandurski 1977). The Se0 3 taken up by erythrocytes i s probably reduced by t h i s mechanism and released from the c e l l as H 0Se. -31-At p h y s i o l o g i c a l pH hydrogen selenide exists as HSe and plays an important central role i n Se metabolism (Fig. 2). It may be oxidized to elemental Se, incorporated into or intera c t with plasma and c e l l u l a r proteins or become methylated. Hydrogen selenide i s methylated i n the c y t o s o l i c and microsomal fractions with methyl groups provided by S-adenosylmethionine. Selenium i s excreted as methylated compounds, the best known being dimethyl selenide and the trimethylselenonium ion. DMSe i s an intermediate i n the pathway for the production of TMSe. The methylation of DMSe to TMSe i s a rate l i m i t i n g step, thus when a large dose of Se i s presented to the excretory pathway DMSe accumulates and i s exhaled (Palmer et a l . 1969). Ganther (1987) has proposed the t h i r d methyl group of TMSe might be derived from a carbon chain rather than through the methylation of DMSe. This offered an explanation as to why the administration of arsenite to animals had no e f f e c t on TMSe formation but yet in h i b i t e d the synthesis of DMSe (Ganther 1987). Selenomethionine i s believed to follow the metabolic pathways of methionine. Selenide may be released from Se-Met by tra n s u l f u r a t i o n producing Se-Cys or by transamination forming methylselenol. Selenocysteine i s produced by reduction of selenocystine i n a manner analagous to the reduction of cystine. Esaki et a l . (1982) have i d e n t i f i e d an enzyme, selenocysteine- /Myase which s p e c i f i c a l l y catalyzes the decomposition of Se-Cys producing alanine and H 2Se. The mechanism of Se incorporation into the Se-Cys of GSHPx -32-or other selenoproteins has not yet been determined. There are two proposed mechanisms, t r a n s l a t i o n a l and po s t - t r a n s l a t i o n a l , i n both of which H 2Se i s an intermediate. Biosynthesis of Se-Cys may ar i s e by transfer of H 2Se to an acceptor such as o-acetyl serine. Incorporation of Se-Cys into selenoproteins could then occur t r a n s l a t i o n a l l y through a tRNA s p e c i f i c for Se-Cys (Hawkes et a l . 1982). On the other hand, Sunde and Hoekstra (1980) believe Se-Cys i s formed p o s t - t r a n s l a t i o n a l l y by i n s e r t i o n of Se into an amino acid residue, possibly serine and/or cysteine present i n the appropriate p o s i t i o n of the peptide backbone. Hydrogen selenide may also form complexes with e l e c t r o p h i l i c metabolites (X +, i . e . metals) which may then undergo methylation thereby reducing the t o x i c i t y and/or b i o l o g i c a l a v a i l a b i l i t y of the components involved (Ganther 1984). Glutathione Peroxidase Glutathione peroxidase was shown by Rotruck et a l . (1973) to be a Se-containing enzyme and i s the only well-characterized selenoenzyme i n higher animals at t h i s time. I t i s a homologous tetramer of molecular weight 75,000 to 98,000, depending on tis s u e and animal species (Combs and Combs 1984). Each subunit of GSHPx contains one Se atom (Epp et a l . 19 83) i n the form of Se-Cys (Forstrom et a l . 1978). The number of active s i t e s per tetramer has not been determined. In contrast to other peroxidases, GSHPx contains no heme or f l a v i n moeity. The d i s t r i b u t i o n and a c t i v i t y of GSHPx varies considerably among -33-GSHPx (or other selenoproteins) elemental Se pre-GSHPx X-Se-CH 3« o-acetyl serine^, Se-Cys-/5-lyase t h i o l - S -transferase Se-Cys CH3SeH methylselenol (CH 3) 2Se dimethyl selenide \ trans-s u l f u r a t i o n \ Se-Met trans-amination > exhaled + (CH 3) 3Se trimethylselenonium urine F i g . 2. Selenide metabolism -34-tissues and animal species. In tissues of the c a l f , the highest l e v e l of GSHPx a c t i v i t y was i n the erythrocytes followed i n descending order by the testes, kidneys, adrenal glands, heart, lung and l i v e r . The lowest l e v e l of a c t i v i t y was i n brain, s k e l e t a l muscle, adipose tissue and blood plasma (Scholz et a l . 1981a). In contrast, the highest GSHPx a c t i v i t y i n the rat was in the hepatic tiss u e followed by the erythrocytes. Lower leve l s were found i n kidney, adrenal glands, heart, lung and testes (Lawrence et a l . 1974). High l e v e l s of GSHPx a c t i v i t y were also found i n the c e l l s with phagocytic a c t i v i t y (Scholz et a l . 1981a). The majority of GSHPx a c t i v i t y i n whole blood i s associated with the c e l l u l a r f r a c t i o n with only about 1% associated with the blood plasma (Scholz and Hutchinson 1979). GSHPx accounts for approximately 75% of the t o t a l Se i n erythrocytes of sheep (Oh et a l . 1974), 100% i n rats and 10% i n humans ( B e i l s t e i n and Whanger 1987). In the erythrocyte of humans the majority of Se i s associated with hemoglobin ( B e i l s t e i n and Whanger 1987). Tissue a c t i v i t i e s of GSHPx vary d i r e c t l y with the l e v e l of dietary Se when supplemented at low l e v e l s to low Se d i e t s . The a c t i v i t i e s i n most tissues were reported by Oh et a l . (1976a,b) to plateau at dietary concentrations of about 0.1 mg Se k g - 1 , while tiss u e Se concentrations continued to r i s e above that dietary Se l e v e l . Moksnes and Norkeim (1983) reported the a c t i v i t y of GSHPx i n tissues of lambs continued to increase u n t i l dietary Se l e v e l s reached 0.23 mg k g - 1 . Above t h i s l e v e l , GSHPx -35-a c t i v i t y i n the tissues approached a plateau with the exception of blood GSHPx. The d i s p a r i t y between the a c t i v i t y of GSHPx and the concentration of Se i n tissues i n animals consuming Se adequate diets i s the re s u l t of non-specific incorporation of selenium into tissue proteins which becomes more quantit a t i v e l y s i g n i f i c a n t with increasing Se intake (Combs and Combs 1984). A n u t r i t i o n a l deficiency of Se results i n a decrease i n tissue GSHPx a c t i v i t y . Plasma GSHPx a c t i v i t y responds rapidly to changes i n dietary Se concentration and has been suggested as an indicator for assessment of Se status (Thompson et a l . 1981). The value of plasma GSHPx a c t i v i t y for assessing Se status, i s however, questionable due to i t s unknown o r i g i n and functional metabolic s i g n i f i c a n c e . In addition, bovine plasma GSHPx does not correlate well with plasma Se concentration (Scholz and Hutchinson 1979). Hepatic GSHPx has also been suggested as an indicator of short term Se status i n some animal species (Scholz and Hutchinson 1979). The a c t i v i t y of GSHPx i n eythrocytes i s highly correlated with whole blood Se over a wide range of blood Se values i n sheep and c a t t l e (Wilson and Judson 19 76; Scholz and Hutchinson 1979; Thompson et a l . 1981). Se i s incorporated into erythrocyte GSHPx during erythropoeisis and remains there throughout the l i f e of the c e l l . The l i f e s p a n of the bovine erythrocyte i n c i r c u l a t i o n i s 135 to 162 days (Kaneko et a l . 1971). C e l l s are continually being produced and removed from c i r c u l a t i o n and as a re s u l t an increase i n the dietary l e v e l of Se i s not immediately reflected -36-by an increase i n erythrocyte GSHPx a c t i v i t y (Thompson et a l . 1980, 1981). Likewise a decrease i n Se intake i s not immediately followed by a decrease i n erythrocyte GSHPx a c t i v i t y . For t h i s reason measurement of erythrocyte GSHPx i s considered useful for assessment of an animal's long term Se status (Thompson et a l . 1981). Glutathione-S-transferases catalyze the conjugation of a large number of xenobiotics and endogenous toxins. One or more of these enzymes also demonstrate glutathione peroxidase a c t i v i t y (Prohaska and Ganther 1977) and thus are sometimes referred to as Se-independent GSHPx. They catalyze the reduction of organic hydroperoxides but not hydrogen peroxide. I t i s therefore important to use hydrogen .peroxide as the peroxide substrate when using GSHPx assays to estimate Se status from tissues having the Se-containing GSHPx and the glutathione transferases with GSHPx a c t i v i t y . Tissues of the c a l f having both enzymes include l i v e r , lungs, adrenal glands, testes, kidney medulla and kidney cortex. The hepatic tis s u e contains the highest percentage of Se-independent GSHPx a c t i v i t y . Tissues with only the Se-containing GSHPx include spleen, cardiac muscle, erythrocytes, brain, thymus, adipose tissue and s t r i a t e d muscle (Scholz et a l . 1981b). Role of glutathione peroxidase i n the oxidant defence system  of the c e l l In b i o l o g i c a l systems oxygen radi c a l s are produced as a normal process. Examples of such reactions occur i n the terminal -37-oxidases of the mitochondrial electron-transport system, the microsomal cytochrome P-450 containing and cytochrome b._-containing electron-transfer systems, and i n the adrenal b mitochondrial system, responsible for steroid hydroxylation that also contains cytochrome P-450 species (Diplock 1987). Should production of the oxygen metabolites go uncontrolled damage to important b i o l o g i c a l macromolecules including unsaturated phospholipids of membranes, DNA and protein may r e s u l t . The reduction of molecular dioxygen to water i n b i o l o g i c a l systems occurs by a process involving the sequential addition of 4 electrons (Diplock 1985) and i s summarized below: e~ 0 2 1 0 2 * superoxide anion e ~ 2-0 2 • 1 0 2 peroxy anion 2- H + - H + 0 2 ^ H 02 e H2°2 hydrogen peroxide e~ H 20 2 y OH + OH • hydroxyl r a d i c a l e~ OH • ^ OH The superoxide anion r a d i c a l (0 2 ~)and the hydrogen peroxide (H 20 2) are both capable of inducing peroxidation of polyunsaturated phospholipids i n b i o l o g i c a l membranes. The product of greater concern i s the more damaging species, the hydroxyl r a d i c a l (OH *). Under circumstances leading to the increased concentration of the 0 9 ~ and H„0 9 these species can -38-react to produce s i g n i f i c a n t amounts of OH • by the reaction: °2 ~ • • + H2°2 * °2 + 0 H ' + 0 H ~ The production of the OH • i s catalyzed by the presence of iron (II) by the Fenton reaction: H 20 2 + F e 2 + ^ OH * + OH" + Fe 3 + 2 + The Fe i s regenerated by the reaction: - 3+ 2+ 0 2 • + Fe vO + Fe and: H 20 2 + Fe 3 + >02 ~ + 2H + + F e 2 + The Fenton reaction may also be catalyzed by other 2+ redox-active divalent cations such as Cu . A large portion of the Cu within b i o l o g i c a l systems however i s t i g h t l y bound to protein and whether c a t a l y t i c quantities of free Cu i n the c e l l e xists i s questionable (Diplock 1987). H a l l i w e l l and Gutteridge (1984, c i t e d by Diplock 1987) report a f e r r y l r a d i c a l (Fe0 2 +) i s a more l i k e l y species than the hydroxyl r a d i c a l for i n i t i a t i n g peroxidation of phospholipids i n b i o l o g i c a l membranes. The f e r r y l r a d i c a l i s produced through the -39-2 + int e r a c t i o n of Fe and H 20 2 by the reaction: F e 2 + + H 20 2 ^ e 0 2 + + OH" Iron can also catalyze l i p i d peroxidation as iron complexes (Diplock 1987) : 2+ - 3 + Lipid-OOH + (Fe complex) Hjipid-0 * + OH + (Fe complex) Lipid-OOH + ( F e 3 + complex) Hjipid-00 • + H + + ( F e 2 + complex). Selenium, manganese, copper, zinc and Vitamin E function i n a multicomponent oxidant defense system to prevent the formation of hydroxyl r a d i c a l s and f e r r y l r a d i c a l s by maintaining low le v e l s of the reactive species (Diplock 1987). The 0 2 *~ i s reduced to H 20 2 i n the mitochondria by a manganese containing superoxide dismutase (EC 1.15.1.1) and i n the c y t o s o l i c compartment by a superoxide dismutase requiring copper and zinc for c a t a l y t i c a c t i v i t y . The r e s u l t i n g H 20 2 i s i n turn reduced to water i n the cytosol and mitochondrial matrix catalyzed by the Se-containing GSHPx. In peroxisomes H 20 2 i s reduced to water and oxygen by the enzyme catalase (EC 1.11.1.6). Vitamin E i s located l a r g e l y within i n t r a c e l l u l a r membranes. I t functions to l i m i t the p r o l i f e r a t i o n of free r a d i c a l damage by scavenging l i p i d peroxy r a d i c a l s . I t thereby terminates the propagation of membrane l i p i d peroxidation chain reactions l i m i t i n g the area of -40-damage within the membrane (Diplock 1984). Mechanism of glutathione peroxidase action Glutathione peroxidase catalyzes the reduction of H 20 2 using reducing equivalents derived from glutathione by the the reaction (Underwood 1977) : GSHPx 2GSH + H 20 2 • GSSG + 2H20 GSH reductase GSSG + 2NADPH h 2GSH + 2NADP Selenium i s required for the a c t i v i t y of GSHPx but the mechanism of i t s action remains unresolved. Under physiological conditions the enzyme i s la r g e l y i n the reduced state. The enzyme undergoes c y c l i c oxidation and reduction possibly v i a selenol (Enz-SeH) and selenenic acid (Enz-SeOH) by the proposed scheme (Fig. 3): selenol Enz-SeH selenenic acid s e l e n i n i c acid +ROOH +ROOH -+ Enz-SeOH +GSH -GSSG -ROH Enz-SeSG +GSH +GSH -H20 -GSSH -ROH Enz-SeOSG -v Enz-SeOOH +GSH -H20 Fi g . 3. Possible mechanism of glutathione peroxidase (Source, Ganther 1975) The enzyme i s oxidized by the peroxide substrate (ROOH) followed by the release of the corresponding alcohol (ROH). A selenosulfide intermediate species [Enz-SeSG] i s formed during -41-the reduction of the enzyme by glutathione. The enzyme i s further reduced by glutathione followed by the release of oxidized glutathione (GSSG). A cycle involving Enz-SeOH and s e l e n i n i c acid (Enz-SeOOH) with a s e l e n i n y l - s u l f i d e intermediate [Enz-SeOSG] has also been proposed. Iodoacetate and other a l k y l a t i n g agents inactivate reduced GSHPx suggesting the presence of a selenol i n the reduced enzyme (Ganther 1987). I t i s possible however, that both cycles could operate depending on the r e l a t i v e concentrations of the o x i d i z i n g and reducing substrates (Ganther 1975). The role of GSHPx i n the metabolism of organic hydroperoxides i n the c e l l i s not well defined. Fatty acid hydroperoxides, the major organic hydroperoxides formed i n the c e l l s , are reduced by GSHPx when present i n an u n e s t e r i f i e d form. In the c e l l however, f a t t y acid hydroperoxides e x i s t l a r g e l y i n e s t e r i f i e d phospholipids and do not seem to be available to the enzyme. Other selenoproteins Several non-GSHPx selenoproteins i s o l a t e d from various tissues indicate there may be other important roles of Se i n animals. Pedersen et a l . (1972) i d e n t i f i e d a 10,000 dalton Se-containing protein i n tissues of Se supplemented lambs which was absent i n the heart and muscle cytosol from lambs with white muscle disease. This protein i s now being referred to as G-protein. -42-Selenoprotein P was isola t e d from the plasma of the rat. It consists of two subunits: one with a molecular weight of 53,000 containing approximately 5 atoms of Se and one with a molecular weight of 35,000 containing no Se (Motsenbocker, c i t e d by Tappel 1987). I t i s believed to be synthesized i n the l i v e r and may function to transport Se from the l i v e r to extrahepatic tissues (Motsenbocker and Tappel 1982b). B e i l s t e i n et a l . (1984) reported 85% of plasma Se i s associated with selenoprotein P i n monkey plasma. Selenocysteine i s the chemical form of Se i n selenoprotein P (Motsenbocker and Tappel 1982a,b) and G-protein ( B e i l s t e i n et a l . 1981). Se i n the testes i s required for the formation of spermatozoa (Brown and Burk 197 3; Wu et a l . 197 3) and may also function i n the Leydig c e l l s (Behne et a l . 1987). Se i s concentrated i n the mid-piece of the sperm i n a cy s t e i n e - r i c h structure of the outer membrane of the mitochondria. I t i s present as a s p e c i f i c selenoprotein with a molecular weight of 15,000 to 20,000 and i s believed to have a s t r u c t u r a l role (Calvin et a l . 1981). Interactions of Se with Cu H i l l (1974) investigated the e f f e c t of cupric sulphate added to the die t to test i t s effectiveness i n overcoming Se t o x i c i t y i n chicks. The toxic e f f e c t of selenium dioxide (Se0 2) when included at 40 mg k g - 1 of the d i e t was p a r t i a l l y a l l e v i a t e d and mortality decreased by the presence of 32 and 500 mg Cu k g - 1 . -43-Reaction between Cu and Se within the i n t e s t i n a l t r a c t leading to the production of insoluble cupric selenides was believed responsible ( H i l l 1975). Jensen (1975a) studied the effects of high l e v e l s of copper sulphate on the response of chicks to toxic l e v e l s of dietary Se. The addition of very high l e v e l s of Cu (1000 mg k g - 1 ) improved the growth rate and decreased mortality of chicks receiving 40 and 80 mg Se k g - 1 as sodium s e l e n i t e . Analysis of the l i v e r showed s i g n i f i c a n t l y higher l e v e l s of Se accumulation upon the addition of Cu to the d i e t . The high l e v e l s of Cu reduced the a v a i l a b i l i t y of Se by the formation of insoluble i n t r a c e l l u l a r Se compounds and to a lesser degree by interference with Se absorption. To determine i f Se deficiency could be induced i n chicks by high l e v e l s of Cu i n the d i e t , 800 or 1'600 mg Cu k g - 1 was added to a basal d i e t containing 0.2 mg Se k g - 1 (Jensen 1975b). The r e s u l t was high mortality and a high incidence of both exudative diathesis and muscular dystrophy. When the basal d i e t was supplemented with an additional 0.5 mg Se k g - 1 , no signs of Se deficiency were observed i n the chicks regardless of the l e v e l of added Cu. Van Vleet and Boon (1980) also found high l e v e l s of Cu (1500 mg k g - 1 ) added to a d i e t produced a high incidence of Se/Vit E deficiency i n ducklings. The lesions of Se/Vit E deficiency were characterized by white areas of necrosis with or without c a l c i f i c a t i o n i n the gizzard, i n t e s t i n e , s k e l e t a l muscle and heart. In contrast to the results of Jensen (1975b) and Van -44-Vleet and Boon (1980) which indicated that Cu induced Se deficiency, Whanger and Weswig (1978) found l i v e r necrosis was not promoted i n rats receiving V i t E and Se d e f i c i e n t diets when the d i e t contained subtoxic l e v e l s of Cu. Gooneratne and Howell (1981) found sheep s u f f e r i n g from chronic Cu t o x i c i t y to have a s i g n i f i c a n t increase i n Se concentration and GSHPx a c t i v i t y i n the l i v e r . White et a l . (1979, c i t e d by Gooneratne and Howell 1981) reported a s i m i l a r e f f e c t i n sheep receiving increasing dietary Cu concentrations. I t was suggested the increased Se retention i n the sheep was a response to ti s s u e damage caused by Cu accumulation. White et a l . (1981) investigated the e f f e c t of Cu (10 mg k g - 1 ) , Mo (10 mg kg - 1) and Cu + Mo (10 mg Cu k g - 1 + 10 mg Mo — 1 75 kg ) on the metabolism of [ Se]Se-Met administered 7 5 intraruminally. There was no s i g n i f i c a n t e f f e c t on [ Se]Se-Met excretion or retention but there was a tendency for Cu, Mo, and Cu + Mo to decrease the t o t a l retention of Se. In view of these studies there appears to be an i n t e r a c t i o n within the tissues between Se and Cu. The e f f e c t of Cu on Se absorption however i s not as clear. Studies suggesting a Cu-Se in t e r a c t i o n i n the g a s t r o i n t e s t i n a l t r a c t of simple stomached animals were investigating the e f f e c t of Cu on inorganic Se. The study investigating the e f f e c t of Cu on Se absorption i n ruminants reported no antagonistic e f f e c t of Cu when Se was provided as an organic complex. These c o n f l i c t i n g r e s u l t s may be due to species differences and/or the form of Se provided i n the (organic verses inorganic). Stable isotopes as tracers for the study of mineral  metabolism The application of radioisotopes as tracers has made a major contribution to the understanding of mineral n u t r i t i o n i n man and animals. However, inherent with the use of radioisotopes i s the serious issue of radiation exposure, p a r t i c u l a r l y with the radiotracers with long b i o l o g i c a l h a l f - l i v e s . In metabolic studies with large animals, handling and disposing of radioactive excrement, f l u i d s , and carcasses can present problems. A non-invasive, safe a l t e r n a t i v e to the use of radioisotopes i s the use of stable isotopes. Stable isotopes of an element contain the same number of protons but d i f f e r e n t numbers of neutrons i n t h e i r n u c l e i . Naturally occuring chemical elements are present as either mono-isotopes (single isotopes) or multi-isotopes (a mixture of several isotopes). Table 3 l i s t s the number of stable isotopes for chemical elements of in t e r e s t i n n u t r i t i o n . In p r i n c i p l e the stable isotope method i s applicable to elements with two or more isotopes. The natural pattern of stable isotope occurance or "natural abundance" i s expressed as atomic percentage. Stable isotope natural abundances are c h a r a c t e r i s t i c of the element. Selenium consists of s ix stable isotopes a l l of which are commercially available as highly enriched preparations. In contrast, there i s -46-only one radioactive isotope of Se ( Se) commonly available for l a b e l l i n g experiments thus l i m i t i n g the scope of studies that may require simultaneous multiple l a b e l l i n g techniques. The fundamental application of stable isotope tracers for metabolic studies requires an understanding of the potentials, l i m i t a t i o n s and measurement methodology. In p r i n c i p l e , the application of stable isotopes as non-radioactive tracers i s analogous to the application of radiotracers. In practice however, there are important differences between the two techniques. The a v a i l a b i l i t y of radioisotopes of high s p e c i f i c a c t i v i t y and the absence of a background l e v e l for most radiotracers of mineral elements i n b i o l o g i c a l matrices, enables extremely small amounts of radiotracers to be administered experimentally. In contrast, stable isotopes are n a t u r a l l y present and t h e i r use requires t h e i r presence i n excess of t h e i r natural i s o t o p i c abundance. The a b i l i t y of the instrumentation to measure small amounts of excess enrichment on top of the natural abundance is o t o p i c background w i l l determine the degree of i s o t o p i c enrichment necessary. The quantity of the isotope administered experimentally i s , therefore, dependent on i t s natural abundance, the p r e c i s i o n of the i s o t o p i c analysis, and the degree of enrichment i n the commercial preparation. I t may be many times greater than the amount required for a s i m i l a r study using radiotracers. The quantity of the enriched stable isotope can become a l i m i t a t i o n i n c e r t a i n studies where the required amount adds s i g n i f i c a n t l y to the p h y s i o l o g i c a l l e v e l of -47-Table 3. Stable isotope composition of chemical elements of concern to mineral n u t r i t i o n Elements Number of stable isotopes Be, F, Na, A l , P, 1 Mn, Co, As, I, CI, K, V, Cu, Br, 2 Rb, Ag Mg, S i , S, Ca, Cr, 3 or Fe, Ni, Zn, Se, Sr, Mo, Cd, Sn, Ba, W, Hg, Pb (Source, Janghorbani 1984) -48-the element. Such a s i t u a t i o n may be overcome by administering the required dose over several days or, i f possible, by increasing the precision of the isotope measurement. The stable isotope technique has been used extensively for the in v e s t i g a t i o n of amino acid, carbohydrate and l i p i d 13 15 2 metabolism using C, N, and H l a b e l l i n g and to a limi t e d extent for mineral metabolism. Measurement of stable isotopes Methodology should be capable of absolute and r e l a t i v e i s o t o p i c abundance measurements i n the matrices of in t e r e s t with the required degree of accuracy and prec i s i o n and at the lev e l s r e s u l t i n g from physiological l e v e l s of intake of isotope. There are two general methods available for stable isotope measurement, neutron a c t i v a t i o n analysis (NAA) and mass spectrometry (MS). NAA i s based on the interactions of thermal neutrons with the n u c l e i of stable isotopes. The nuc l e i capture neutrons to y i e l d radiotracer n u c l e i with various h a l f - l i v e s . The decay of these radioisotopes results i n the emission of c h a r a c t e r i s t i c gamma radiatio n which i s measured with a high-resolution gamma spectrometry system. To date NAA has been more widely applied to investigations of mineral n u t r i t i o n and metabolism than has MS. NAA has been successfully applied for measurement of three of the 74 76 80 stable isotopes of selenium, Se, Se, and Se i n such matrices as feces, plasma, red blood c e l l s and urine of humans (Janghorbani et a l . 1981). An a n a l y t i c a l p r e c i s i o n and accuracy -49-of 5-10% was reported for routine measurement of these isotopes which was considered s a t i s f a c t o r y for experiments concerned with g a s t r o i n t e s t i n a l absorption i n human subjects. MS i s well established i n geochemistry and related applications but i t s application for mineral n u t r i t i o n has been l i m i t e d . A lack of routine procedures and methods for trace element i s o t o p i c measurements of b i o l o g i c a l matrices has been a factor i n i t s l i m i t e d use. Thermal i o n i z a t i o n mass spectrometers provide high precision measurements of isotopic r a t i o s for the majority of mineral elements. Its application to stable i s o t o p i c tracer studies i s limit e d however, due i n part to the high instrument cost, the degree of technical s k i l l required and the slow sample throughput. Another instrument with an extremely high measurement pre c i s i o n i s the isotope r a t i o mass spectrometer. This i s used for gas analysis of i s o t o p i c a l l y enriched H, C, 0, and N. A method for stable isotope tracer studies involving complex matrices i s GCMS. The sample components of in t e r e s t must be converted to thermally stable v o l a t i l e complexes. The ions measured may be produced from the entir e derivatized molecule or from suitable fragment ions. Despite some successes, the application of GCMS suffers from problems related to the preparation of suitable chelates, l i m i t a t i o n s due to overlapping of minor isotopes of C, H, 0 i n the chelate, and lower precision. Methodology for the application of GCMS for measurement of double -50-iso t o p i c enrichment of Se i n b i o l o g i c a l samples has been reported by Reamer and V e i l l o n (1983). Two newer approaches being explored for the measurement of stable isotopes are fast atom bombardment mass spectrometry (FABMS) and ICPMS. Possibly the greatest p o t e n t i a l for precise measurement of stable isotopes over a wide range of chemical elements l i e s with the development of ICPMS. The technique offers rapid analysis and pot e n t i a l s i m p l i c i t y of chemical manipulations. I t provides advantages over GCMS applied to metal chelates, by the avoidance of overlap of minor isotopes of the organic component. In the t r a d i t i o n a l application of mass spectrometry, the method measures only isotope r a t i o s . Absolute quantities of elements are determined by application of p r i n c i p l e s of isotope d i l u t i o n with mass spectrometric measurement or by an independent elemental analysis technique. Isotope d i l u t i o n eliminates the need for additional sample preparation usually required for elemental analysis and permits an accurate determination of the trace element content of a p a r t i c u l a r sample as well as allowing stable isotopes to be used as tracers, isotope d i l u t i o n i s based on the addition to the sample of an exact known quantity of an enriched stable isotope of the element to be analyzed (referred to as an i n t e r n a l standard or spike isotope). Chemical processing of the sample must then render the i n t e r n a l standard and endogenous element i n the same chemical form. Given t h i s , the enriched i n t e r n a l standard isotope serves as an " i d e a l " -51-st and a rd with incomplete recoveries a f f e c t i n g the analyte and the in t e r n a l standard i n the same manner and offers the advantage that quantitative recovery i s not required. The addition of the in t e r n a l standard to the sample a l t e r s the isotope abundances of the element i n the sample. From the altered isotope r a t i o , mass of the sample, mass of the added i n t e r n a l standard and additional data, the concentration of the element o r i g i n a l l y present i n the sample can be calculated. The application of t h i s technique for quantitation of an element i n an i s o t o p i c a l l y enriched sample requires the element to consist of at l e a s t three isotopes, because of the need for two isotopes of unaltered natural abundance i n enriched samples. This l i m i t s i t s application for studies involving elements with only two isotopes such as copper and for some studies involving multiple i n vivo l a b e l l i n g . I t i s possible to analyze samples both before and after spiking i n these cases, but t h i s requires twice the number of analyses. -52-3. MATERIALS AND METHODS The research for t h i s project was conducted using the laboratory f a c i l i t i e s and dairy herd at the Agriculture Canada Research Station i n Agassiz, B. C. Cows of the Research Station's dairy herd normally receive i n j e c t i o n s of a commercial preparation containing Se and V i t E at the time of drying o f f . The animals used for the purpose of t h i s research did not receive the i n j e c t i o n s . I t was also ensured the animals had never received enriched Se stable isotopes at any time p r i o r to the t r i a l s . 3.1. A n a l y t i c a l techniques The notation used when r e f e r r i n g to the enriched isotope preparations i s , e.g. "Se-76". The enriched stable isotopes, Se-76, Se-77, Se-78 and Se-82 were purchased from Oak Ridge National Laboratory (ORNL), Oak Ridge, TN. The i s o t o p i c composition of the preparations ( i d e n t i f i e d by ORNL sample no.) were supplied by ORNL (Appendix Table 1). Elemental Se (Se p e l l e t s , 99.9999%) was purchased from A l d r i c h Chemical Co., Inc., Milwaukee, WI. Instra-analyzed hydrochloric acid (HCl) and n i t r i c acid (HN03) and Ultrex grade HN03 were purchased from J. T. Baker Chemical Co., P h i l l i p s b u r g , NJ. Magnesium n i t r a t e (Mg(N0 3) 2 -6H20), AnalaR grade was from BDH Chemicals, Vancouver, B.C.; and magnesium (Mg) metal from either BDH Chemicals (as ribbon) or Morton Thiokol Inc., A l f a Products, Danvers, MA (as -53-grignard turnings, 99.99%). Toluene and chloroform from Caledon Laboratories, Georgetown, Ont. ( d i s t i l l e d i n glass) was r e d i s t i l l e d before use. A l l p l a s t i c and glassware was soaked i n 10% HN03 for a minimum of l hour and rinsed several times with deionized water (deionized to a r e s i s t i v i t y of 18 megaohms cm - 1). A l l water used was deionized. Analysis for Se and Se stable isotope ra t i o s for samples of T r i a l I was by GCMS and for samples of T r i a l II and III by ICPMS. 3.1.1. Gas chromatography mass spectrometry Instrumentation: The instrument was a Hewlett Packard Model 5985 quadrupole GCMS with select ion monitoring (SIM) (Department of C i v i l Engineering, UBC, Vancouver, B.C.). The following GCMS parameters were used: column - DB-5, 0.32 mm ID x 25 m s p l i t l e s s i n j e c t i o n time - 0.5 min temperature program - 50 - 270 C (1 min hold, 10 C m i n - 1 ramp) i n j e c t i o n port temperature - 280 C interface temperature - 280 C ion source temperature - 200 C SIM dwell time/mass - 20 ^ e c . Der i v a t i z i n q reagent: The d e r i v a t i z i n g reagent was 4-nitro-l,2-phenylenediamine (4NPD) (Aldrich Chemical Co., Inc., Milwaukee, WI) and was prepared as described by Reamer and -54-V e i l l o n (1981). N i t r i c acid-magnesium n i t r a t e ashing aid: The ashing aid was prepared by di s s o l v i n g 40 g Mg(N0 3) 2 "6H20 per 100 mL HN03 by s l i g h t heating. Sample preparation: F i f t e e n mL of ashing aid was added to samples of siz e 1 - 1.5 g or 5 - 10 mL i n 200 mL Berzelius beakers. Quantification of Se was by the app l i c a t i o n of stable isotope d i l u t i o n using Se-78 as the i n t e r n a l standard. To each sample 260.3 ng Se-78 (ORNL sample 199901) i n 1% HN03 was added. Non-enriched samples of the same or s i m i l a r b i o l o g i c a l matrix and reagent blanks were included with each batch of samples. The beakers were heated on a hot plate for 60 min at 105 C followed by 30 - 90 min ( u n t i l samples were digested) at 115 C. Samples were taken to dryness on the hotplate then ashed for 4 h at 500 C (Isotemp Programmable Ashing Furnace Model 497, Fisher S c i e n t i f i c , Vancouver, B.C.). The ash was wetted with 5 mL of water, and dissolved i n 15 mL HCl. Samples were boiled gently for ~ l min to reduce a l l Se(Vl) to Se(IV). I t was important for a l l Se to be converted to Se(IV), as the d e r i v a t i z i n g reagent reacts only with Se i n that valence state. After cooling the contents of each beaker were quantit a t i v e l y transferred to separatory funnels with the aid of 20 mL water. The hydrochloric s a l t of 4NPD was prepared as described by Reamer and V e i l l o n (1981). One mL of 1% 4NPD (wt/vol) was added and samples l e t stand for 2 h at room temperature under subdued l i g h t . The reaction between 4NPD and Se(IV) produced the -55-thermally stable compound, 5-nitropiazselenol (NPS). The NPS formed was extracted into 5 mL toluene by shaking for 8 min on a mechanical shaker. The toluene extracts were separated from the aqueous phase, transferred to 16 x 150 mL te s t tubes and evaporated to dryness by an evapomix (Model 3-2100, Buchler Instruments, Fort Lee, NJ). The residues were dissolved i n 2 x 100 fL chloroform, transferred to r e a c t i - v i a l s (0.3 mL capacity, Pierce Chemical Co., Rockford, IL) and dried under a stream of N 2. Samples were sealed under N 2 and refrigerated u n t i l analyses. Analysis: Samples were taken up i n toluene with gentle heating to ensure complete d i s s o l u t i o n of the complex and injected into the GCMS. With selected ion monitoring (SIM) the MS was tuned for 225, 227 and 229 mass to charge r a t i o (m/z). Accuracy and precision: The accuracy of the measurement of Se stable isotopes was determined by the analyses of two sets of double isotope c a l i b r a t i o n standards. Standards were prepared by the addition of f i v e l e v e l s of Se-76 and Se-78 over ranges of 0.0 to 1.229 /g and 0.0 to 8.676 /jg respectively, i n a l l possible combinations to 10.055 /£f of natural abundance Se ( n a S e ) . Blank standards were also included. The accuracy of the method for quantitative Se analysis was evaluated by the analyses of standard reference materials of bovine l i v e r (1577) and orchard leaves (1571) (U.S. National Bureau of Standards (NBS), Washington, D.C.). The NBS standards were prepared as described for the samples. Natural abundance Se standards containing ~5 /g Se were analyzed i n t r i p l i c a t e on each day of sample analysis and the r e s u l t s used for c a l c u l a t i o n of the p r e c i s i o n . The detection l i m i t was defined as the l e v e l of enrichment above which the p r o b a b i l i t y of obtaining a measure of natural abundance Se was < 0.05 for a single analysis of a sample. The dynamic range was defined as the d i l u t i o n factor that a tracer i n a t i s s u e may undergo and s t i l l remain detectable based on an i n i t i a l tracer enrichment equal to 10% of the natural Se. 3.1.2. Inductively coupled plasma mass spectrometry Instrumentation: The s e n s i t i v i t y of the analysis for Se i s enhanced many orders of magnitude when sample introduction i s by hydride generation i n place of nebulization. A continuous flow hydride generator described by Buckley et a l . (1987) was coupled to a Perkin-Elmer model ELAN 250 ICPMS equipped with mass flow c o n t r o l l e r s on i n j e c t o r and a u x i l i a r y gas flows. The instrument parameters were: argon flow rates - plasma 2 L m i n - 1 i n j e c t o r 1.4 - 1.6 L m i n - 1 a u x i l i a r y 1.5 L m i n - 1 power - incident power 1.0 kWatt ref l e c t e d power 0 Watt ion optics - B lens 40 P lens 10 E l lens 85 -57-S2 lens 31 detector - channel electron m u l t i p l i e r 3600 V deflector - +4500 V machine configuration - sample delay 90 s wash delay 60 s Hydrochloric acid concentration: The in t e r a c t i o n between HCl concentration, Cu concentration and the i n t e n s i t y of the Se signal was investigated by the analyses of a series of samples containing 20 ng n a S e mL - 1 i n 4 - 10 M HCl with Cu additions ranging from 0 to 400 /g mL - 1. N i t r i c acid-magnesium ashing aid: Three g Mg was dissolved per 100 mL of HN03. Sample preparation: Samples of siz e (max 1.5 g dry matter or 10 mL volume) containing 8 - 600 ng Se were weighed or dispensed into 200 mL Berzelius beakers. Forty ng Se-76 (ORNL sample 194802) i n 1% HN03 and 15 mL of n i t r i c acid-magnesium ashing aid was added to each sample. Non-enriched samples of the same b i o l o g i c a l matrix and reagent blanks were included with samples. Samples were placed on a preheated aluminum block for 30 min at a sample temperature of 75 C followed by 2 h at a sample temperature of 115 C. Samples were taken to dryness then ashed for 4 h at 500 C. After cooling the ash was wetted with 5 mL water and dissolved i n 15 mL HCl. The contents of each beaker were transferred to 20 mL s c i n t i l l a t i o n v i a l s for analysis by ICPMS. -58-Analysis: A 1% (wt/vol) sodium borohydrate solution i n 0.1 M sodium hydroxide was prepared d a i l y for generation of v o l a t i l e Se hydrides. The ICPMS was tuned to measure ion i n t e n s i t y at m/z 76, 77, 78 and 82. Reagent blanks were pooled and analyzed after every 3 samples to correct for instrument d r i f t . The natural abundance samples or standards were analyzed i n t r i p l i c a t e during sample analysis, each day. Accuracy and precision: The accuracy and p r e c i s i o n were evaluated as described for GCMS with any changes noted below. Single isotope c a l i b r a t i o n standards were prepared for each of Se-76 (ORNL sample 194802), Se-77 (ORNL sample 194901) and Se-82 (ORNL sample 195201) alone, over a range of enrichment l e v e l s expected i n samples. Another set of isotope c a l i b r a t i o n standards were also prepared for each isotope over the same range of enrichment to which was added the highest l e v e l of the other two Se isotopes (refered to as secondary Se isotopes). Standards were prepared by adding the appropriate quantity of Se-76 (0 -325.1 ng), Se-77 (0 - 19.3 ng) and Se-82 (0.0 - 19.8 ng) to ~200 ng Se. Blanks were included with the standards. NBS standard reference materials of bovine l i v e r (1577), bovine serum (8419), freeze dried urine (2670) and r i c e f l o u r (1568) were prepared as described for samples. The p r e c i s i o n for the measurements of Se-76, Se-77 and Se-82 were determined from the analyses of natural abundance serum, feces and urine. -59-3.2. Calculation of Se stable isotope enrichment Stable isotope enrichment was expressed as TTMP equal to 100 x the mass of the tracer divided by the mass of the tracee; i . e . tracer/tracee mass percentage. The tracer was defined as the enriched stable isotope of the element of i n t e r e s t : Se-76, Se-77 and Se-82. The tracee i s the naturally occuring element, Se. The equations for TTMP for solution of up to three simultaneously enriched isotopes of Se were derived by Buckley (1987). The sets of simultaneous equations for two and three enriched isotopes are given below. Reference isotope the isotope ion chosen as the denominator for ion in t e n s i t y r a t i o s (GCMS, 80; ICPMS, 78). v, w, x and y subscripts r e f e r r i n g to stable isotopes of an element and designating mass/charge (m/z) rati o s of the isotope ion or the nominal mass of the isotope. When v i s i n parentheses i t refers to the i n t e r n a l standard enriched i n the indicated stable isotope. When x and y are i n parentheses they refer to the tracer enriched i n the indicated stable isotope. Isotope w i s always the reference isotope. n, m subscripts designating tracee (natural Se) or a mixture of tracer and tracee respectively. M exact atomic mass for substance indicated i n parentheses (e.g. M ^ 7 6 j = exact atomic mass of Se-76. H ion abundance of the designated isotope for the substance indicated i n parentheses (e.g. H 7 6 ( n ) = ion in t e n s i t y at m/z for isotope 76 i n natural Se divided by the t o t a l ion in t e n s i t y for natural Se). -60-ion i n t e n s i t y r a t i o of the designated isotope over isotope w for the substance indicated i n parentheses (e.g. 6_,,_. = c 3 76(m) ion i n t e n s i t y at m/z for isotope 76 divided by inte n s i t y at m/z for isotope w for a mixture of natural Se and t r a c e r ) . B mass of tracee (ng) . D mass of tracer (ng) . Two enriched isotopes (v and x) M ( v ) H w ( n ) ^ P + u r> TTMPV = 100 [ ' ( ' M ( n ) H w ( v ) < u s " hP> M ( x ) H w ( n ) ( r h + TTMP = 100 K ' K ' X M ( n ) H w ( x ) ( u s " hP> Three enriched isotopes (v, x and y): M / v \ H w m ^ e i P + SdP + cjq + equ + gru - c i r ) TTMP. = ioo ( v) w< n> M ( n ) H w ( v ) ( g s u " a q u " c i s " g h p ~ c h q " a l P ) M / x i H w f n i ( e h ^ + g h r + a i r + eis + gjs - ajq) TTMPx = 100 W ( n ^ X M ( n ) H w ( x ) ( g S U " a q u " C i s " g h p " C h q " a i P ) M. .H . .(ajp + aru + chr + cjs + esu - ehp) TTMPy = 100 m ( > M ( n ) H w ( y ) ( g s u " a q u " C i s " g h p " c h q " a i P ) -61-Where: a = ey(v) • 6y(m) c = ey(x) • 9y(m) e = ey(n) " 9y(m) g = 9y(m) ' ey(y) h = 9x(v) - 6x(m) i = 9x(y) • 9x(m) ex(n) • 6x(m) P = 9v(x) • " 6v(m) q = ev(y) " 6v(m) r = 9v(n) • 9v(m) s = 9v(m) ' 9v(v) u = 9x(m) " 9x(x) 9 , , , 9 , ^ , 9 . . , 9 , . , 9 . . and 9 were obtained v(m)' v(n)' x(m)' x(n)' y(m) y(n) from mass spectrometric analysis a f t e r subtraction of reagent blank ion i n t e n s i t i e s . The remaining factors i n the equations were calculated from s p e c i f i c a t i o n s supplied with the purchased isotopes (Appendix Table 1) and from the Handbook of Chemistry and Physics (1970). The organic component of the NPS ion contains 6 carbon atoms (C), 3 nitrogen atoms (N), 2 oxygen atoms (0) and 3 hydrogen 13 atoms (H). Of these, 1.11% of the C atoms are C, 0.36% of the 15 18 17 N atoms are N, 0.2% and 0.04% of the 0 atoms are 0 and 0, 2 and 0.015% of the H atoms are H. The occurrence of these " s a t e l l i t e " isotopes i n the organic component of the ion a l t e r s the 7 6Se-NPS/ 8 0Se-NPS and 7 8Se-NPS/ 8 0Se-NPS ra t i o s from 7 6 S e / 8 0 S e 7 8 80 and Se/ Se. Based on the p r o b a b i l i t i e s of occurrence of each of the s a t e l l i t e isotopes, the proportion of molecules (NPS) with each possible molecular mass was calculated using the elementary laws of p r o b a b i l i t y (Pickup and McPherson 1976). The proportions -62-of the arrangements contributing to the same molecular mass were summed enabling the p r o b a b i l i t y of occurrence, P 1, P 2 and P 3 i n the mass spectrum for a given molecular mass with m/z, m/z+1 and m/z+2, respectively, to be determined. The p r o b a b i l i t i e s of occurrence were used to adjust the ion abundances of the enriched isotope preparations and of natural Se by the equations: H225 = P1 A76 + P3 A74 227 = P1 A78 + P2 A77 229 = P1 A80 + P3 A78 where A i s the atomic abundance of the isotope indicated by the subscript. The adjusted ion abundances were used to calculate the factors i n the TTMP equations which were not obtained from GCMS analysis. The quantity of tracee (B) i n a sample i s calculated from the r e l a t i o n s h i p : D TTMP = x 100 B thus B = x 100 TTMP„ -63-Knowledge of B and TTMPx Q r enables the quantity (ng) of tracer (D ) i n the sample to be determined from the x o ir y rela t i o n s h i p : T T M P x or y = ° J L f L J L X 1 0 0 thus T T M P x or y x B  x o r y 100 Total selenium (ng) equals the sum of D x a n ( j and B, 3.3. T r i a l I 3.3.1. Animals Two non-lactating, non-pregnant Holsteins (8219 and 7939), cul l e d from the herd for f a i l u r e to conceive (believed to be unrelated to Se nu t r i t i o n ) were used. The cows had been dry for about 3 months p r i o r to the t r i a l . The cows were weighed on two consecutive days at the beginning and end of the t r i a l period to obtain an estimate of t h e i r true weight. The test cows were housed i n box s t a l l s for the f i r s t 13 days. On the 14th day the cows were moved into stanchion s t a l l s where they remained for 13 days. -64-3.3.2. Diet P r i o r to the t r i a l the test cows were maintained on orchardgrass pasture and received no supplemental Se. The experimental d i e t was low-Se orchardgrass hay. I t was chopped to f a c i l i t a t e the weighing of feed and weighback necessary during the balance t r i a l s . The die t was offered free choice with a minimum weighback of 2 kg each day. Water was provided free choice to each animal from i n d i v i d u a l water bowls with flow meters. Cobalt-iodized s a l t was provided ad libitum. The test cows were adapted to the experimental d i e t over a 15-d period (Days 1-15). After d i e t adaptation, two 5-d balance periods were run consecutively, during which the the tes t cows were i n d i v i d u a l l y fed the experimental d i e t . The weight of the experimental d i e t fed and weighback, and the water intakes were recorded d a i l y at 10:00 am. 3.3.3. Single isotope enrichment of the whole body Se pool  Preparation of Se-76 tracer dose: In a 100 mL volumetric flask 44.47 mg of the Se-76 isotope (ORNL sample 194802) was dissolved i n 5 mL of HN03 (Ultrex) overnight. I t was dil u t e d to volume with water to y i e l d a solution of 444.7 Se-76 mL - 1. Dissolution i n HN03 oxidized the Se to selenious acid (H 2Se0 3), changing the valence state from 0 (elemental Se) to IV (s e l e n i t e ) . The d a i l y tracer dose of 4.00 mg Se-76 was prepared by d i l u t i n g 9 mL of the Se-76 solution to 250 mL with water. -65-Administration of Se-7 6 tracer: The Se-76 tracer dose was administered to the rumen by stomach tube. One dose (4.00 mg Se-76) was administered to each cow each day for a t o t a l of 5 days (Days 1-5). The bottle containing the dose was thoroughly rinsed with approximately 1 L of water and the washings also administered to the rumen. Administration of the tracer took place at 9:30 am. Based on an estimated whole body Se pool equal to 70 mg and retention of 30% of the dose, the administration of 20.00 mg Se-76 was predicted to r e s u l t i n a whole body enrichment of Se-76 equivalent to 10% of the whole body pool s i z e . 3.3.4. Balance periods Two days p r i o r to the balance periods the test cows were prepared for urine and f e c a l c o l l e c t i o n s . A thick layer of a rubber based glue was applied surrounding the anal and vulval area of the cows. Once the glue had p a r t i a l l y set (~1 h) a p l a s t i c mesh and another layer of glue were applied. The glue was allowed to dry overnight. The end of a rubber hose (~2.5 m long) was stretched over a wire shaped to e n c i r c l e the vulva, and stitched to the p l a s t i c mesh. Urine was channelled down the hose into a covered s t a i n l e s s s t e e l container. C o l l e c t i o n boxes were positioned i n the gutter behind the cows for f e c a l c o l l e c t i o n . Feces were transferred several times a day from the c o l l e c t i o n box into covered p l a s t i c buckets. Two 5-d t o t a l c o l l e c t i o n balance periods were run -66-consecutively following the dietary adaptation (Period 1, days 15-20; Period 2, days 20-25). Complete c o l l e c t i o n s of feces and urine were made every 24 h and the d a i l y outputs of each were recorded. The feces was mechanically mixed for ten minutes. A "500 g sample was transferred to an aluminum tray, weighed and frozen. A subsample of ~450 mL of urine was transferred to a p l a s t i c b o t t l e , a c i d i f i e d with HN03 (Ultrex) and frozen. Three feed and water samples were coll e c t e d over both periods. Water samples were a c i d i f i e d with HN03 (Ultrex). Dry matter percentages of feed and feces samples were determined by l y o p h i l i z a t i o n . Feed and feces samples were ground to pass through a 1 mm sta i n l e s s s t e e l screen. The three feed and water samples were each composited. Pooled samples of feces and urine were prepared for each of the 5-d balance periods. Blood samples were obtained by jugular vein puncture at the beginning of each balance period and at the end of the second balance period. F i f t y mL of blood was col l e c t e d i n 2 X 25 mL tubes without anticoagulant (Sarstedt Canada, Inc., Que.). The blood samples were allowed to c l o t and centrifuged at 4000 x g for 10 min to separate the serum. Serum was decanted o f f and frozen. One 24-h t o t a l c o l l e c t i o n of feces and urine was made from a dry cow which had never before received enriched Se stable isotopes. This was begun at the same time as the s t a r t of the f i r s t balance t r i a l with the test cows. These samples were to determine isotope ra t i o s of natural abundance Se i n these sample -67-matrices. The feed was analyzed for protein by a modification of the Kjeldahl procedure with colorimetric determination (AOAC 1980); acid detergent f i b e r (Van Soest 1963); calcium, magnesium, potassium, i r o n , manganese, and zinc by atomic absorption spectrometry ( B r i t i s h Columbia Ministry of Agriculture and Fis h e r i e s (BCMAF), S o i l , Feed and Tissue Testing Laboratory, Kelowna, B.C.); phosphorus by colorimetry (BCMAF); and s u l f u r , molybdenum, and copper by ICPMS following sample decomposition with n i t r i c and p e r c h l o r i c acids. Feed, water, feces, urine and serum were analyzed for t o t a l Se and Se stable isotope ra t i o s by mass spectrometry. 3.3.5. Tissue c o l l e c t i o n The t e s t cows were s a c r i f i c e d following completion of the second balance period. Test cow 8219 was slaughtered at Scott's Meats (Agassiz, B.C.) and cow 7939 at the Veterinary Pathology Laboratory (Abbotsford, B.C.). Samples of a l l major tissues and f l u i d s of the cows were obtained (Table 4). Large samples and i n some cases whole organs were co l l e c t e d , placed i n i n d i v i d u a l p l a s t i c bags on ice and transported immediately back to the lab to be subsampled. Stainless s t e e l instruments used for subsampling the tissues were soaked i n saturated ethylenediaminetetraacetic acid (EDTA) and rinsed with water. Where possible samples were obtained from the i n t e r i o r of tissues to avoid contact with benches, other body -68-f l u i d s etc. Further notes on sampling i n d i v i d u a l tissues are given i n Table 4. The tissues were rinsed with water and drained on ashless f i l t e r paper. Samples of 200 - 300 g fresh weight were transfered to tared specimen cups, weighed and dry matter percentage determined by l y o p h i l i z a t i o n . Tissue samples were analyzed for t o t a l Se and Se stable isotope r a t i o s by mass spectrometry. 3.4. T r i a l II and III As only small numbers of animals were manageable by one person i t was necessary to conduct the second phase of the experiment as two t r i a l s ( T r i a l II and III) of six animals each. 3.4.1. Animals Two groups of six non-lactating, pregnant Holstein cows i n t h e i r second, t h i r d or fourth dry period were used. The cows were weighed on two consecutive days at the beginning and end of the t r i a l . They were maintained within stanchion s t a l l s for the duration of the experiment (27 days). 3.4.2. Experimental diets The animals i n each t r i a l were randomly assigned to one of two dietary Cu treatments: Treatment l - control, no supplemental Cu Treatment 2 - 20 mg k g - 1 DM of supplemental Cu. -69-Table 4. Description of tissu e and f l u i d samples collected Tissue Description l i v e r Individual samples were taken from l e f t and right dorsal, l e f t and ri g h t ventral and caudal lobes. kidney Cortex and medulla were separated. spleen pancreas lung heart s k e l e t a l muscle Samples were taken from the semimembranosus semitendinosus, biceps femoris, longissimus d o r s i , s u p e r f i c i a l and deep pectoral muscles. rumen Epithelium and smooth muscle were separated. reticulum See rumen. omasum See rumen. abomasum See rumen. small i n t e s t i n e Lumen was rinsed repeatedly with water. colon See small i n t e s t i n e . udder bone Samples were taken of femur, humerus, and r i b . hide A l l hair was removed with a s c a l p e l . uterus Epithelium and smooth muscle were separated. p e r i r e n a l f a t thoracic f a t b i l e Collected i n tared specimen cup d i r e c t l y from the g a l l bladder. -70-The basal d i e t was cubed orchardgrass hay f o r t i f i e d with Se and Zn (Treatment 1 and 2) and Cu (Treatment 2). The Se (sodium s e l e n i t e ) , Zn (zinc sulphate) and Cu (copper sulphate) were dissolved i n water and mixed with molasses. The mineral-molasses mixture was sprayed on ~200 kg batches of chopped orchardgrass hay (~4% molasses wt/wt) and the hay mixed. The hay was then cubed to f a c i l i t a t e handling. The dietary adaptation period was 14 days. 3.4.3. Double isotope enrichment of the whole body Se pool The administration of Se-77 (intrarumen) and Se-82 (intravenous) was predicted to achieve a t o t a l whole body enrichment of 3 - 4%. Dosing took place on the same day as the commencement of the dietary adaptation period. Preparation and administration of Se-77: An exact mass of Se-77 (ORNL sample 194901) ( T r i a l I I , 29.881 mg; T r i a l I I I , 28.186 mg) was dissolved i n 10 mL HN03 (Ultrex) and dil u t e d to volume with water i n a 100 mL volumetric f l a s k . The tracer dose was prepared by d i l u t i n g 16 mL of the Se-77 solution to 250 mL. One dose was administered intraruminally to each animal (See t r i a l I, 3.3.3.). The exact mass of the isotope administered to each animal i s l i s t e d i n Table 5. Preparation of Se-82 infusate: An exact mass of Se-82 (ORNL sample 195201) ( T r i a l I I , 8.718 mg; T r i a l I I I , 7.963 mg) was dissolved i n 3.5 mL HNO~ (Ultrex) and 3.5 mL water. The -71-s o l u t i o n was q u a n t i t a t i v e l y transferred with 4 x 5 mL water washings to a tared 50 mL autoclavable b o t t l e , d i l u t e d to ~30 mL water and the exact weight recorded. Capped with a t e f l o n faced septum and aluminum seal, the solution was s t e r i l i z e d by autoclaving at 15 psig steam pressure for 20 min. The bottle was weighed before and after autoclaving to check for loss of s o l u t i o n . Using s t e r i l e technique and 5 cc disposable syringes, "5 mL of the Se-82 solution was injected into 1-L bags of s t e r i l e s a l i n e (0.9% NaCl) l a b e l l e d for each cow. The exact mass of the solution transferred to each bag was determined by weighing the syringe before and after i n j e c t i o n . The weight of each bag of s a l i n e was recorded following the addition of Se-82 and following intravenous administration. Intravenous infusion of Se-82: A 14 gauge needle was inserted into the jugular vein and approximately 2 feet of a t e f l o n catheter was fed down the vein toward the heart. Using a 5 cc disposable syringe, a few mL of s t e r i l e heparinized saline was injected i n the catheter then drawn back u n t i l blood appeared i n the tube to test the catheter implant and remove c l o t s . The administration of saline was begun and once a l l animals were connected to s a l i n e , the saline bag was switched to the Se-82 containing infusate and the infusion started. Infusions took place over a 3 - 4 h period. When approximately 50 mL of the experimental infusate remained, the bag was switched back to s a l i n e solution for 20 - 30 min to rinse the catheter tube. The mass of Se-82 infused into each animal i s l i s t e d i n Table 5. -72-Table 5. Mass of Se-77 and Se-82 administered to each cow i n T r i a l II and III Cow No. T r i a l No. Se-77 (194901) mg Se-82 (195201) mg 8222 8228 8328 8022 8238 8337 8334 8227 8329 8240 8307 8331 II II II II II II III III III III III III 4.7810 4.7810 4.7810 4.7810 4.7810 4.7810 4.5098 4.5098 4.5098 4.5098 4.5098 4.5098 1.2380 1.4035 1.3749 1.3793 1.3483 1.4065 1.2336 1.2174 1.1902 1.2341 1.1889 1.2411 -73-3.4.4. Balance periods The t o t a l output of urine was co l l e c t e d using indwelling catheters (size 24 FR balloon catheter, balloon s i z e 75 mL, Rusch, Scarborough, Ont.). Catheters were inserted by a veterinarian according to the procedure of C r u t c h f i e l d (1968), two days p r i o r to the commencement of the balance periods. During these two days the cows were observed for any sign of discomfort and hematuria. The catheters remained i n place for the duration of the balance periods. Twice d a i l y , the vul v a l area was washed with warm external d i s i n f e c t a n t and an a n t i b i o t i c ointment applied. Two 5-d t o t a l c o l l e c t i o n periods were run following the dietary adaptation period. The record of-output for feces and urine, record of intake for feed and water and sample c o l l e c t i o n and analysis were as described for T r i a l I (Refer to 3.3.4.). Blood samples were collected one day p r i o r to the t r i a l and on the f i r s t , t h i r d and f i f t h day of each balance period. Blood was col l e c t e d and analyzed as described for T r i a l I. 3.4.5. Liver biopsy Liver biopsies were taken by a veterinarian two days after the completion of the balance periods. The biopsy instrument used was as described by Buckley et a l . (1986). The preparation of the i n s e r t i o n s i t e and the biopsy procedure were according to the method of Pearson and Craig (1980). To obtain approximately 0.5 g of l i v e r tissue (wet weight) two or three -74-penetrations of the l i v e r with withdrawl of sample were necessary. The l i v e r core samples were transferred from the cannulae into 20 mL s c i n t i l l a t i o n v i a l s containing heparinized 0.9% NaCl. Within the v i a l s the l i v e r samples were washed several times with heparinized 0.9% NaCl u n t i l the wash solution was c l e a r . The samples were dried by patting with f i l t e r paper. The sample was transferred to clean 20 mL v i a l s and dry matter determined by l y o p h i l i z a t i o n . 3.5. Calculations for apparent absorption, balance, endogenous  f e c a l Se and true absorption Apparent absorption (AA, /g d - 1 ) and balance (BA, /g d - 1 ) of Se were computated by equations (1) and (2), respectively. Equations (3) and (4) define endogenous f e c a l Se (EF, /g d _ 1 ) and true absorption (TA, /g d - 1 ) . AA = C - F (1) BA = C - F - U (2) TTMP feces EF F X (3) TTMP serum or l i v e r TA C F + EF (4) where: C = d a i l y dietary Se consumption ( /.g day ) -75-F = d a i l y f e c a l Se excretion ( /g day - 1) U = d a i l y urinary Se excretion ( /g d a y - 1 ) . TTMP^ , TTMP and TTMP.. = tracer feces serum l i v e r enrichment i n feces, serum and l i v e r , respectively 3.6. Experimental design and s t a t i s t i c a l analysis The data for endogenous f e c a l Se and true absorption were subject to ANOVA by computer using the VHM version of SAS 5.0 3 by the general l i n e a r models procedures. A l l observations were interpreted by the appropriate F tes t on the basis of an error p r o b a b i l i t y of P < 0.05. The mathematical model employed was: Y. ijklmn - " + T R i + C j + T R x C i j + A k ( i j ) + P l + TRXP^ + CxP^ + TRxCxP..k + A x P k l ( i j ) + I m + T + IxT. + TRxI. + Cxi. TRxCxI . . + n mn im jm ijm Pxl, + TRxPxI.. + CxPxI.. + TRxCxPxI. . -. + lm llm jlm 13lm TRxT. + CxT. + TRxCxT.. + PxT. + i n jn. xjn In TRxPxT ., + CxPxT . -i + TRxCxPxT... + j In j In i ] l n TRxIxT. + CxIxT. + TRxCxIxT. . + PxIxT-. imn jmn ijmn lmn + TRxPxIxT.-, + CxPxIxT.-, + TRxCxPxIxT. limn jlmn IJlmn + W ( ( i j ) l ) -76-Where: Y i j klmn represents the measured dependent variable the o v e r a l l mean the t r i a l e f f e c t where i = 1, 2 C_. = the dietary copper treatment e f f e c t where j = l , 2 = the animal e f f e c t where k = 1...11 P-^  = the period e f f e c t where 1 = 1,2 I = the route of isotope administration e f f e c t where m = 77, 82 T = the tissue e f f e c t where n = l i v e r , serum The other terms represent interactions between the main factors TR, C, A, P, I and T. The same model excluding the main ef f e c t s of isotope and tissue and the corresponding interactions was used for analysis of the data for apparent absorption and balance. 1cmn( ( i j )1) = residual error The analysis of variance was as follows: Source of v a r i a t i o n degrees of freedom Total 87 T r i a l 1 Copper 1 T r i a l x copper 1 A n i m a l ( t r i a l x copper) 7 Period 1 Period x t r i a l 1 period x copper 1 Period x t r i a l x copper 1 Animal x period ( t r i a l x copper) 7 Isotope 1 Tissue 1 Isotope x tiss u e 1 Isotope x t r i a l 1 Isotope x copper 1 Isotope x t r i a l x copper 1 Isotope x period 1 Isotope x period x t r i a l 1 Isotope x period x copper 1 Isotope x period x t r i a l x copper 1 Tissue x t r i a l 1 Tissue x copper 1 Tissue x t r i a l x copper 1 Tissue x period 1 Tissue x period x t r i a l 1 Tissue x period x copper 1 Tissue x period x t r i a l x copper 1 Isotope x tiss u e x t r i a l 1 Isotope x t i s s u e x copper 1 Isotope x t i s s u e x t r i a l x copper 1 Isotope x tiss u e x period 1 Isotope x tiss u e x period x t r i a l 1 Isotope x tiss u e x period x copper 1 Isotope x t i s s u e x period x t r i a l x copper 1 Residual error 42 -78-4. RESULTS 4.1. GCMS a n a l y t i c a l technique The parent ion cl u s t e r of the mass spectrum of NPS contained a series of ions corresponding to the six stable isotopes of Se plus the stable isotopes of C, N and 0. Ion i n t e n s i t i e s at m/z 225, 227 and 229 were determined employing the selected ion monitoring feature. The m/z values represented the most intense 7 6 78 80 ions for Se, Se and Se, respectively. Data from the analyses of the stable isotope c a l i b r a t i o n sets are plotted as predicted (X, independent variable) verses observed TTMP (Y, dependent variable) i n F i g . 4 and F i g . 5 for Se-76 and Se-78, respectively. For each c a l i b r a t i o n set there i s a strong l i n e a r r e l a t i o n s h i p and the slope and intercept do not d i f f e r from unity and zero (P > 0.05). This indicates the presence of a secondary Se isotope up to an enrichment l e v e l of TTMP = 83.9 does not i n t e r f e r e with the analysis of a sample and that there i s a minimum of bias i n the analysis. The accuracy of the method for quantitative Se determination was evaluated by the analyses of two NBS standard reference materials: bovine l i v e r (1577) and orchard leaves (1571). Good agreement between the c e r t i f i e d and observed values was found (Table 6). The p r e c i s i o n of Se stable isotope enrichment was better for the measurement of Se-76 than Se-78 (Table 7). The detection l i m i t for the measurement of isotope enrichment i n a sample for -79-12 £ 1 0 Q. I-h-Q LU > DC UJ (/) CO o 8 6 4 2 Oh MEAN ±SD(n=10) A ,i Y = 0.9657X - 0.1837 r2=0.9716 J L 8 10 12 PREDICTED T T M P 7 6 l i 9 ; f i 4 ; i 1 1o a i 1 ? r a t i l 0 n S U r v e f o r s e l e n ^ m standards enriched with Se-76 plus 5 l e v e l s of Se-78 ranging from TTMP 7 8 o to 83.9. The broken l i n e indicates slope = l.o and intercept =0.0. - a u -80 MEAN ± SD (n = 10) I 60! Q LU > DC LU </) QQ o 40 201 * Y = 1.0045 X + 0.1927 r2= 0.9976 20 40 60 80 PREDICTED TTMP 7 Q ? p 9 - ; f t 5 ; i 1 1 S a i i ? r a t i i 0 n S U r v e f o r s e l e n i u m standards enriched with Se-78 plus 5 l e v e l s of Se-76 ranging from TTMP 0 to 11.8. The broken l i n e indicates slope = 1.0 and intercept = 0 . 0 . -81-Table 6. Selenium determination of standard reference materials by GCMS ( /g g _ 1 ) . C e r t i f i e d Se concentration Observed Se Material f J- estimate of concentration uncertainty ± SD (n = 3) Bovine l i v e r 1.1 =0.1 1.19 ±0.053 (NBS 1577) Orchard leaves 0.08 0 .01 0.089 J-o.004 (NBS 1571) | U.S. National Bureau of Standards (NBS), Washington DC. -82-Table 7. Precision of selenium stable isotope enrichment determined by GCMS. f TTMP 7 6 TTMP Selenite (sample si z e ~5 /£f) Detection l i m i t £ 0.555 0.892 Dynamic Range § 18 11 "One sample was analyzed i n t r i p l i c a t e on each day for 15 days. "Detection l i m i t = t , n o c w „ -, . x (within day variance ^ (0.95)(n-1) v 1 0 5 component + between day variance component) ' . § The d i l u t i o n factor that a tracer may undergo i n a tissue or f l u i d and remain detectable [Dynamic range = (TTMP=10/detection l i m i t ) ] . -83-Se-76 and Se-78 was TTMP = 0.56 and TTMP = 0.89, respectively. Following an i n i t i a l tracer enrichment of TTMP = 10, Se-76 could undergo an 18-fold d i l u t i o n and remain detectable i n a sample. 4.2. ICPMS a n a l y t i c a l technique A considerable amount of time was spent on method development for the determination of Se and multiple stable isotope enrichment of Se i n b i o l o g i c a l materials by ICPMS. The resu l t s reported by Buckley et a l . (1987) pertinent to decisions with regards to tracer enrichment i n animals and sample preparation are included here. The i n t e r a c t i o n between HCl concentration, Cu concentration and the in t e n s i t y of the Se sign a l were investigated i n samples containing 20 ng Se mL - 1 of 4, 5, 6, 7, 8, 9, and 10 M HCl concentrations with Cu additions of 0, 25, 50, 100, 200 and 400 mL - 1. The l e v e l s of Cu were selected based on what might be expected i n b i o l o g i c a l samples. The Se sign a l remained v i r t u a l l y unaffected by Cu i n preparations of 9 M and 10 M HCl (Fig. 6). It was concluded for optimum s e n s i t i v i t y and freedom from interference by Cu, the optimum concentration of HCl for sample preparation was 9 M HCl. Preparation of samples i n 9 M HCl also afforded complete reduction of Se(VI) to Se(IV). The generation of Se hydrides requires Se to be i n the tetravalent state. Usually the most abundant isotope of an element i s selected to serve as the reference isotope for isotope r a t i o determination. However, interference from an A r _ + ion at m/z 80, -84-Cu in sample (jug) F i g . 6. E f f e c t of HCl and Cu on the i n t e n s i t y of the Se s i g n a l -85-the most abundant isotope of Se, prevented i t s determination. Se-78 was therefore selected as the reference isotope. 7 6 7 8 82 Interference i n the measurement of Se, Se and Se by other ions was removed by blank subtraction. A r e l a t i v e l y high background at m/z 76 suggested Se-76 would be unsuitable as a tracer. The amount of an in t e r n a l standard added to a sample for quantitative determination i s several times greater than the expected l e v e l of tracer enrichment. Therefore, Se-76 was used as the i n t e r n a l standard. The accuracy of selenium stable isotope enrichment was analyzed s t a t i s t i c a l l y by l i n e a r regression analysis of predicted verses observed isotope enrichment for Se-76, Se-77 and Se-82 The predicted TTMP (Pred TTMP) was the independent variable (X) and was fixed at graded l e v e l s of enrichment. The highest l e v e l of enrichment represented what might be expected i n tracer experiments (TTMP = 10) or i n samples to which an i n t e r n a l standard i s added for quantitative analysis (TTMP = 160). The observed TTMP (Obsv TTMP) was the independent variable (Y). There was no interference of secondary Se isotope enrichment on the observed TTMP for Se-76, Se-77 and Se-82 over the ranges studied. The intercepts and slopes were pooled from the single isotope and t r i p l e isotope c a l i b r a t i o n sets for each isotope and yielded the following l i n e a r regression equations: -86-Se-76, Obsv TTMP„, / b r 2 = 0.9998 (0.9536 x Pred TTMP_C) - 0.5592 Se-77, Obsv TTMP ? 7 r 2 = 0.9998 (0.9491 x Pred TTMP ? y) 0.0327 Se-82, Obsv TTMPg2 r 2 = 0.9996 (1.0721 x Pred TTMPQ_) + 0.0508 Student's t - t e s t s were employed to determine i f the regression c o e f f i c i e n t s (slopes) and y-intercepts d i f f e r e d from unity and zero respectively. A deviation of the regression c o e f f i c i e n t from unity was present for a l l isotopes. The intercept was not s t a t i s t i c a l l y d i f f e r e n t from zero for Se-77 and Se-82, but did deviate from zero for Se-76. To improve the accuracy for the determination of isotope enrichment and t o t a l Se, the regression equations were rearranged to solve for Pred TTMPs and used to adjust a l l TTMP data: Se-76, Pred TTMP 76 Obsv TTMP_C + 0.5592 ib 0.9536 Se-77, Pred TTMP 77 Obsv TTMP 77 0.9491 Se-82, Pred TTMP 82 Obsv TTMP 1.0720 82 -87-The accuracy of the method for quantitative Se analysis by the a p p l i c a t i o n of stable isotope d i l u t i o n using Se-76 as the in t e r n a l standard was determined by analysis of NBS standard reference materials (Table 8). There was good agreement between the observed Se concentration and the c e r t i f i e d value for a l l sample materials investigated. In sample matrices of serum, feces and urine the best p r e c i s i o n was obtained for the measurement of TTMP ? 7 followed i n decending order by TTMPft9 and TTMP , (Table 9). -88-Table 8. Analysis of standard reference materials for selenium by ICPMS C e r t i f i e d Se concentration Observed Se No. of ±estimate of concentration Material f samples uncertainty ± SD Units Bovine l i v e r (NBS 1577) 1.1 ±0.1 1.1 ± 0.0004 /g g -1 Bovine serum (NBS 8419) 16 ± 2 15 ±0.3 ng mL -1 Freeze dried 3 urine (NBS 2670) 30 J- 8 30 J- 2.2 ng mL -1 Rice f l o u r 6 0.4 * 0.1 0.32 ± 0.007 /g g (NBS 1568) "|"U.S. National Bureau of Standards (NBS), Washington DC. -89-Table 9. Precision of selenium stable isotope enrichment determined by ICPMS t TTMP 7 6 TTMP y 7 T T M P 8 2 Serum (sample size = 430 ng) Detection l i m i t + Dynamic range § 0.145 69 0 .050 200 0 .078 128 Feces (sample si z e = 244 ng) Detection l i m i t J 0.355 0.039 Dynamic range § 28 256 0 .070 142 Urine (sample s i z e = 438 ng) Detection l i m i t J Dynamic range § 0 . 699 14 0.017 588 0.069 145 f Three samples were prepared and analyzed each day for 4 (urine) or 7 days (serum and feces). Se concentrations i n serum, feces and urine were 86 ng mL - 1, 244 ng g - 1 and 73 ng mL - 1, respectively. t Detection l i m i t = t.„ n v x (within day variance (0.95)(n-1) x J 0 5 component + between day variance component) ' . § The d i l u t i o n factor that a tracer may undergo i n a tiss u e or f l u i d and remain detectable [Dynamic range = (TTMP=10/detection l i m i t ) ] . -90-4.3. T r i a l I 4.3.1. Animals and d i e t Cow 8219 and 7939 weighed 642 and 711 kg respectively. Cow 7939 remained standing during much of the f i r s t balance period. During the second period the cow appeared weak and developed a s l i g h t elevation of temperature. The cow was moved into a box s t a l l where i t continued to receive the experimental d i e t . I t recovered over the next two days, and so remained on t r i a l for the c o l l e c t i o n of tissues and f l u i d s . Balance data for t h i s animal was obtained for period 1 only. The amounts of most components i n the orchardgrass hay met the recommended l e v e l s for dry pregnant cows (NAS-NRC 1978) (Table 10). The exceptions were Se, Cu, and Zn whose dietary concentrations f e l l below the current recommended dietary l e v e l s of 0.1 mg k g - 1 , 10 mg k g - 1 and 40 mg k g - 1 , respectively. The Se concentration of the orchardgrass hay was 0.035 ±0.002 mg k g - 1 . The concentration of Se i n the water was 1.0 3 /£j L - 1 . 4.3.2. Selenium content of tissues and f l u i d s At the time of slaughter, cow 8219 was found to be carrying a fetus of ~5 months of age. The whole fetus and several tissues were c o l l e c t e d , subsampled and analyzed as described for tissues of the mature animals. Despite the d i f f e r e n t physiological states of the two non-lactating cows (pregnant and non-pregnant) the Se -91-Table 10. Composition of orchardgrass hay (dry matter basis) ( T r i a l 1) Component Dry matter (%) f 89.3 ±0.7 Crude protein (%)"(• 12.3 ±0.96 Acid detergent f i b e r (%) f 34.8 ±0.33 Calcium (%) 0.36 Phosphorus (%) 0.24 Magnesium (%) 0.16 Sulfur (%) -Iron (mg k g - 1 ) 116 Copper (mg k g - 1 ) f 6.2 ±0.5 Manganese (mg k g - 1 ) 90 Zinc (mg k g - 1 ) 16 Molybdenum (mg k g - 1 ) j" 0.63 * 0.007 Selenium (mg k g - 1 ) | 0.035 ± 0.002 t Mean ± SD, n=3. -92-concentrations of the tissues and f l u i d s were s i m i l a r (Table 11). Based on the mean for the two animals (Table 12), the highest concentration of Se was found i n the kidney cortex (8.07 (jg g - 1 ) . Intermediate l e v e l s (0.87 - 1.82 /g g _ 1 ) were found i n the kidney medulla, spleen, pancreas, small i n t e s t i n e , heart, lung and l i v e r . The Se concentration of the cardiac muscle (1.0 ig g - 1 ) was greater than that of the smooth muscle of the g a s t r o i n t e s t i n a l t r a c t and uterus (0.40-0.65 /g g - 1 ) and the s k e l e t a l muscle (0.44 /g g _ 1)« The lowest Se concentrations were found i n the b i l e (0.06 \q g 1 ) , bone and adipose t i s s u e (0.02 /g g - 1 ) . The serum contained 0.048 /g mL - 1. Of the f e t a l tissues analyzed (Table 13), the highest Se concentration was found i n the l i v e r (1.7 /g g - 1 ) . The Se concentration i n the f e t a l l i v e r and hide was greater than twice that of the corresponding tissues i n the dam (8219). In contrast, values for the f e t a l kidney and muscle were lower than the corresponding tissues i n dam. The quantities of Se associated with the various tissues (Table 12) were estimated from the mean tissue Se concentrations and from data of anatomical structures for Holstein cows (Matthews et a l . 1975) and estimates of carcass composition (R. J. Forrest, personal communication: subdivision of t o t a l carcass weight into : muscle, 55%; adipose tissue, 25%; and bone, 20%) The weight of the uterus was estimated from the weight of the fetus (5.1 kg) and placenta (1.6 kg) and the r e l a t i v e weights of the conceptus and associated tissues i n sheep (Langlands et a l . -93-1982; Table 2, 5th stage). The estimate of t o t a l Se for hair and hide was calculated from the Se concentration of the hide (0.22 /g g - 1 ) found i n the present study. Perry et a l . (1977) reported Hereford steers fed a di e t containing 0.08 mg Se k g - 1 had hair Se concentrations of 0.30 /g g - 1 . Puis (1981) reported the Se concentration of hair ranged from 0.06 to 0.2 3 /g g - 1 for c a t t l e on diets containing <0.10 mg Se kg"*1. The animals i n the present study were consuming a di e t containing 0.0 35 mg Se k g - 1 , therefore, 0.22 /g Se g - 1 was assumed to provide a representative estimate of the Se concentration i n the hai r . The Se concentration i n blood was estimated from the Se concentration i n serum and from Scholz and Hutchinson (1979) whom reported 72.8% of whole blood Se i n dairy cows was i n the c e l l u l a r component. Summation of the estimated quantities of Se associated with the various tissues and f l u i d s , yielded a t o t a l body Se content of 44.4 mg for the non-lactating dairy cows consuming a low Se di e t (0.035 ±0.002 mg k g - 1 ) . Of the t o t a l body Se, about 47%, 17%, 6%, 5% and 3% was associated with s k e l e t a l muscle, hide and ha i r , digestive t r a c t , l i v e r , and kidney, respectively. The blood accounted for 6% of the t o t a l body Se. The Se content of the 5-month old fetus was 0.3 mg and made up 0.6% of the body Se of the mature cow (Table 13). The f e t a l l i v e r and kidney contained 11% and 3%, respectively, of the f e t a l body Se. 4.3.3. D i s t r i b u t i o n of Se-76 i n tissues and f l u i d s Se-76 enrichment i n tissues and f l u i d s was variable as -94-indicated i n F i g . 7. The enrichment of Se-76 i n the adipose tissu e and bone was below the detection l i m i t for GCMS analysis (detection l i m i t , TTMP = 0.56). The tissues and f l u i d s considered i n f l u e n t i a l and/or contributors to endogenous f e c a l Se are: serum, the epithelium of the stomach (including the rumen, reticulum, omasum and abomasum), l i v e r , b i l e , pancreas, small i n t e s t i n e and colon. There existed l i t t l e v a r i a b i l i t y of tracer enrichment amongst these tissues and f l u i d s with TTMPs of 9.9 -12.4 and 8.7 - 13.3 for cow 8219 and 7939, respectively. The uterus (Fig. 7), placenta and tissues of the fetus (Fig. 8) from cow 8219 show a high l e v e l of tracer enrichment with TTMPs ranging from 11.2 to 13.4. The tracer enrichment i n the s k e l e t a l muscle of the fetus (TTMP = 12.2) was > 6 times the l e v e l present i n the dam (TTMP = 1.8). The hide of the fetus (TTMP = 11.9) was also enriched with Se-76 to a greater degree than the hide of the dam (TTMP = 8.6). -95-Table 11. Selenium concentration i n tissues and f l u i d s of cow 8219 and cow 7 9 39 (dry matter basis) No. Of Tissue s i t e s Cow 8219 Cow 7939 Se ( & g" 1 ) SD f Se ( m g - 1 ) SD f kidney cortex 1 8.949 7.191 kidney medulla 1 1.868 1 . 775 spleen 1 1.491 1.219 pancreas 1 0 . 890 1. 369 lung 1 0.952 0.952 l i v e r 5 0.773 0 .026 0.966 0 .042 udder 1 0.705 0.625 uterus epithelium 1 0.713 uterus smooth muscle 1 0 .401 stomach epithelium 4 0.606 0 .022 0 .716 0.078 stomach smooth muscle 4 0 .586 0 .182 0 . 709 0.071 small i n t e s t i n e 1 1.155 1.090 colon 1 0 .512 0.948 s k e l e t a l muscle 6 0.443 0 .025 0.433 0.033 adipose 2 0.014 0.004 0 .022 0 .007 bone 3 0 .021 0 .002 0 .025 0 .001 b i l e 1 0 .045 0 .077 serum 1 1 0 .041 0 .054 fSD for sample s i t e s . X & mL_1 -96-Table 12. Estimated selenium content i n tissu e s , f l u i d s and the whole body of non-lactating dairy cows Mean Se Estimated Percent of concentration t o t a l Se t o t a l body Tissue ( $ g" 1 DM) (mg) Se Kidney 4.95 1.5 3.3 cortex 8.07 medulla 1. 82 Spleen 1.36 0.3 0.7 Pancreas 1.13 0.2 0.4 Heart 1.0 0.6 1. 3 Lung 0 .95 0.9 2.1 Liver 0.87 2.1 4.8 Udder 0.67 1.5 3.4 Uterus 0 .56 0 . 3 0.7 Digestive t r a c t 0.75 2.8 6.2 Skeletal muscle 0 .44 20.8 46.8 Adipose 0.02 1 . 2 2.8 Bone 0.02 1.8 4.1 Hide and hair 0.22 f 7.4 16.7 Serum 0 .048 0 . 7 1. 7 Blood 2.75 2.7 6.2 | Mean Se concentration for hair. -97-Table 13. Selenium content i n the placenta and tissues of the 5-month old fetus from cow 8219 Tissue Se ( fjg g" 1 DM) Estimated t o t a l Se (mg) Percent of dam's t o t a l body Se Placenta 0 .52 0.08 0.2 Kidney (whole) 1.47 Liver 1.77 Skeletal muscle j" 0.28 * 0.037 Hide 0 .45 Fetus (whole) 0.30 X 0.3 0.6 f Mean* SD of longissimus d o r s i , biceps femoris and s u p e r f i c i a l pectoral. X Se concentration of whole fetus less kidneys, l i v e r , and sections of muscle and hide. -98-F i g * 7 ^ , S e - 7 6 enrichment (TTMP) i n tissues and f l u i d s of cow 8219 (HI) and cow 7939 . The SD are for the sample s i t e s . (E = epithelium; M = muscle; K = kidney; DL = detection l i m i t TTMP =0.56) ' -99-4* F i g . 8. Se-76 enrichment (TTMP) i n the placenta and tissues of the 5-month old fetus from cow 8219. -100-4.3.4. Absorption and endogenous f e c a l excretion of selenium Incomplete c o l l e c t i o n of urine resulted i n urine contamination of the 24-h f e c a l c o l l e c t i o n s for cow 8219 on day 1 of Period 1 and day 4 of Period 2, and for cow 7939 on day 4 of Period 1. Therefore, the number of samples composited for feces and urine for each balance period was reduced from f i v e to four. The average d a i l y feed intake, and f e c a l and urine excretion for each period are given i n Table 14. The average dry matter percent of the feces was 17.3%. Mechanical f a i l u r e of the regulator valves of the water bowls prevented the measurement of water intakes. The average d a i l y Se intake and f e c a l Se excretion for the two cows are given i n Table 15. Selenium was unable to be determined i n the urine by GCMS analysis. The endogenous f e c a l excretion of Se by cow 8219 was 109 - 111 /g d - 1 and contributed 22 - 23% of the t o t a l Se excreted i n the feces (Table 15). For the non-pregnant cow (79 39) with a lower Se intake, the d a i l y endogenous f e c a l excretion of Se was 183 /g d - 1 , amounting to 36% of the t o t a l f e c a l Se (Table 15). Apparent absorption of Se for the two cows was negative (Table 16). True absorption expressed as a percentage of the Se intake by cow 8219 was 15 - 16% (Table 16). There was very good agreement between the measurements taken i n each period for t h i s animal. The true absorption of Se by cow 7939 was lower and equal to 10% of the t o t a l Se intake (Table 16). -101-Table 14. Feed intake and feces and urine excretion i n Period 1 and 2 f ( T r i a l I) Feed intake § Feces output § Urine output Cow No. PeriodJ (kg d - 1 ) (kg d - 1 ) (L d _ 1 ) 8219 1 12.49 J- 1.43 4.58 4-0.07 15.14 J-0.83 8219 2 13.13 J-1.36 4.68 J-0.50 12.27 ±1.92 7939 1 10.04 J-2.07 3.80 ±0.17 17.45 J-1.84 Mean J- SD, n=4. Period 1 = days 15-20 and Period 2 = days 20-25. Dry matter basis. -102-Table 15. Intake and feces excretion of selenium ( /g d ) ( T r i a l I) Se Feces Endogenous Endogenous Intake f Se f e c a l Se f e c a l Se Gow No. Period X (C) (F) (EF) (% of intake) 8219 1 448 ± 51 484 109 23% 8219 2 471 ± 49 509 111 22% 7939 1 360 ± 74 507 183 36% "" Mean * SD, n=4. J Period 1 = days 15-20 and Period 2 = days 20-25. -103-Table 16. Apparent and true absorption of selenium ( jg d 1) ( T r i a l I) Se Apparent Se True Se True Se Intake f Absorption Absorption Absorption Cow No. PeriodJ (C) (AA) (TA) (% of intake) 8219 1 448 ± 51 -36 73 16% 8219 2 471 ± 49 -38 73 15% 7939 1 360 ± 74 -147 36 10% Mean * SD, n=4. Period 1 = days 15-20 and Period 2 = days 20-25 -104-4.4. T r i a l II and III In T r i a l II one cow (8337) was diagnosed by a veterinarian with pneumonia. This animal was removed and the t r i a l continued with 5 animals. 4.4.1. Experimental diets The basal d i e t provided the recommended dietary allowance for a l l nutrients analyzed (Table 17), with the exception of zinc (37 mg k g - 1 ) which was below the recommended concentration of 40 mg k g - 1 (NAS-NRC 1978). The Se concentration of the d i e t was 0.188 ±0.006 mg k g - 1 . The l e v e l of Cu supplementation achieved (Table 18) was 15 - 17 mg k g - 1 . The t o t a l Cu concentration of Treatment 2 was 29 - 30 mg k g - 1 . The Cu content of Treatment 1 (control) was expected to be below 10 mg kg 1 , but contained 13 -14 mg Cu k g - 1 . The concentration of Se i n the water was 1.24 ± 0.21 /g l " 1 . 4.4.2. Feed and water intake and urine and feces excretion The mean body weight of the cows (Table 19) of Treatment 2 (681 ±31) was greater than that of Treatment 1 (651 ± 3 2 ) . The larger body weight of the cows on Treatment 2 i s re f l e c t e d i n greater d a i l y feed and water intakes and f e c a l and urine outputs (Table 19). -105-Table 17. Compositon of basal d i e t used i n T r i a l II and III (dry matter basis) | Component Dry matter (%) 91.1 0.4 Crude protein (%) 16.8 0.1 Acid detergent f i b e r (%) 33 . 3 0.5 Calcium (%) 0.48 0.02 Phosphorus (%) 0.31 0.01 Magnesium (%) 0.26 0.01 Potassium (%) 1.66 0.9 Sulfur (%) 0.36 0 .005 Iron (mg k g - 1 ) 586 70 Manganese (mg k g - 1 ) 109 ± 2 Zinc (mg k g - 1 ) 37 X 1 . 3 Molybdenum (mg k g - 1 ) 0.42 ± 0 .001 Selenium (mg k g - 1 ) 0.188 0 .006 | Mean * Pooled SD for Treatment l and 2 of T r i a l II and I I I , n=4. -106-Table 18. Copper concentration of the experimental diets (dry matter basis) f ( T r i a l II and III) Copper (mg kg 1) T r i a l II Treatment 1 (control) 14 ±1.8 Treatment 2 29 ±1.9 T r i a l III Treatment l (control) 13 ±0.4 Treatment 2 30 J- 2 .0 •f Mean J- SD, n=3. Table 19. Body weight, feed and water intake, and feces and urine excretion for cows on Treatment 1 and 2 j" ( T r a i l II and III) Body Feed Water Feces Urine Weight Intake £ Intake Output ^ Output Treatment n (kg) (kg d _ 1 ) (L d - 1 ) (kg d _ 1 ) (L d" 1) 1 5 651 i-32 12.54 40.88 62.19 4-3.94 5.13 40.41 12.70 40.83 2 6 681 4.31 14.94 40.42 72.19 42.98 6.01 40.20 14.32 40.40 t Mean 4. SEM. | Dry matter basis. -108-4.4.3. Selenium concentration i n serum and l i v e r The concentration of Se ( /g mL - 1) i n the serum of the animals of Treatment l (0.051 ±0.002) was higher than that of animals of Treatment 2 (0.046 ±0.001) one day p r i o r to the t r i a l (P < 0.05). The Se concentration during Period 1 and 2 were also higher for those animals on Treatment 1 (Table 20). No difference (P > 0.05) was found between periods for Treatment 1 or Treatment 2. The mean serum Se concentration for the periods for both Treatment 1 (0.059 ±0.001) and Treatment 2 (0.054 ± 0.001) were higher than the corresponding concentrations p r i o r to the t r i a l (P < 0.05) . The Se concentration i n the l i v e r for animals on Treatment 1 and Treatment 2 were 1.0 + 0.05 and 0.97+0.03 /g g - 1 , respectively, and were not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05). 4.4.4. Isotope enrichment i n tissues and excrement There was greater enrichment of Se-82 i n a l l the materials (serum, l i v e r , feces and urine) i n accordance with dosage and route of administration (Table 21). The values for the l i v e r are for the enrichment of Se-77 and Se-82 occurring 2 days following the completion of Period 2. Cu did not influence the l e v e l of enrichment of either isotope (P > 0.05). There was an e f f e c t of period (P < 0.05) re s u l t i n g i n lower isotope enrichment i n serum, feces and urine i n Period 2. -109-Table 20. Selenium concentration ( /g mL ) 1 day p r i o r to the t r i a l and during Period 1 and 2 f ( T r i a l II and I I I ) . 1 day Mean of pr i o r to t r i a l Period 1J Period 2 J Period 1+2 Treatment 1 0.051 ±0.002a 0 .058 =0 .002c 0.059 =0 . 002c 0.059 ±3.001 Treatment 2 0.046 ±0.001b 0 .054 ±0 . OOladO . 054 ±0 . 002ad 0.054 ±0.001 " Mean J-SEM, n=5 for Treatment 1 and n=6 for Treatment 2. X Period 1 = days 15-20 and Period 2 = days 20-25. a,b,c,d Means with the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). -110-Table 21. Se-77 and Se-82 enrichment (TTMP) i n serum, l i v e r feces and urine j* ( T r i a l II and III) Se-77 Se-82 Tissue Treatment Period 1£ Period 2 J Period 1 | Period 2 £ Serum 1 1.56=0.12 1.27=0.10 2.34=10.09 1.99=0.08 2 1.66=10.07 1.33 =10.05 2.38 =10.05 1.96 =0.05 Liver § 1 1.38 =0.15 2.36 =0.13 2 1.47 =0.06 2.45 =0.05 Feces 1 0.17 =0.013 0.15 =0.019 0.23 =0.02 0.20 =0.03 2 0.17=0.007 0.12 =0.008 0.23 =0.02 0.18=0.004 Urine 1 1.08=0.12 0.88 =0.11 1.59 =0.10 1.37 =0.13 2 1.03J0.04 0.85 =0.03 1.53 =0.03 1.27 =0.02 Mean * SEM, n=5 for Treatment 1 and n=6 for Treatment 2. T Period 1 = days 15-20 and Period 2 = days 20-25. f Isotope enrichment 2 days following the completion of Period 2. Means for Se-77 and Se-82 enrichment i n serum, feces and urine for periods are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). -111-4.4.5. Apparent absorption and balance of selenium The Se intake from the water amounted to ~3.2% of the o v e r a l l mean Se intake from the feed and was not included i n absorption or balance c a l c u l a t i o n s . The measurements of apparent absorption were variable as indicated i n Table 22. The mean apparent absorption (% of intake) for the animals receiving the supplemental Cu (5.1 ± 1.5%) was greater than for those receiving the control d i e t (0.9 ±11.5%) but was not s i g n i f i c a n t l y d i f f e r e n t (P > 0.05). There was no e f f e c t of period (P > 0.05) although the mean for Period 1 (5.4 ±1.8%) was greater than for Period 2 (1.1 ±1.2%) The o v e r a l l mean for apparent absorption of Se (% of intake) was 3.2 ± 1.2%. There was no e f f e c t of Cu or period on Se balance (P > 0.05) (Table 22). The cows on t h i s experiment were i n a negative balance with an o v e r a l l mean loss of 175 ±31 /g Se d _ 1 from the body Se stores. There was l i t t l e v a r i a t i o n between treatments or periods for the d a i l y urinary Se excretion (Table 22) which accounted for an o v e r a l l mean loss of 267 ±10 /g d _ 1 . Of the t o t a l Se excreted, 10.6 % was excreted i n the urine. -112-Table 22. Apparent absorption and balance of selenium ( fg d" 1) | ( T r i a l II and III) Treatment 1 Treatment 2 Overall Period 1X Period 2 X Period 1X Period 2 X Mean Se Intake (C) 2398 ±239 2328 4211 Fecal Se (F) 2350 =225 2317 =191 Apparent Se Absorption (AA) 48 ±72 Apparent Se Absorption § 11 ±35 1.8 ±2.8 0.05 =1.6 Urinary Se (U) 269 =31 258 =34 Se Balance(BA) -221 =58 -247 =43 2859 ±107 2736 ±104 2600 ±91 2624 ±129 2683 ±122 2508 =34 234 ±50 53 ±49 8 . 3 ±1. 7 2 . 0 ±1. 8 269 ±11 268 ±3 -35 =50 -215 =53 92 ±31 3 . 2 ±1. 2 267 ±10 -175 ±31 Mean ± SEM, n=5 for Treatment l and n=6 for Treatment 2. T Period 1 = days 15-20 and Period 2 = days 20-25. g % of intake. Means for treatments and for periods were not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). -113-4.4.6. Endogenous f e c a l excretion and true absorption of Se The endogenous f e c a l Se was determined from the analysis of serum and l i v e r enriched with Se-77 (oral route of administration) and Se-82 (intravenous route of administration) y i e l d i n g 4 estimates. These estimates of endogenous f e c a l Se were then used to calculate 4 estimates of true Se absorption (TA = C - F + EF). The period e f f e c t was not s i g n i f i c a n t (P > 0.05) for either route of administration when endogenous excretion (Table 23) and true absorption (Table 24) were estimated from enrichment of the tracers i n serum. I t was s i g n i f i c a n t when estimated from the enrichment of the tracers i n the l i v e r . The estimate for T T M P l i v e r for Period 1 was higher than for Period 2 for both routes of administration (P < 0.05). The l i v e r sample was taken 2 days following the completion of the second period and therefore the tracer enrichment did not accurately r e f l e c t that which would have been present i n Period 1. As natural Se continues to enter the system the quantity of the tracer i n tissues becomes d i l u t e d . Thus, the tracer enrichment i n the l i v e r at the end of the two periods would be lower than that which would have been present during the f i r s t period. The re s u l t i s a larger r a t i o of T T M P f e c e s / T T M P i i v e r a n ^ a n overestimation of the endogenous f e c a l Se excreted i n Period 1. For t h i s reason, the e f f e c t of route of isotope administration and the tissu e index on the estimate of endogenous f e c a l Se (Table 25) and true absorption (Table 26) were determined from -114-the observations of Period 2. The ANOVA showed no e f f e c t of Cu on the endogenous f e c a l Se excreted and on true absorption. There was no difference between the estimates of endogenous fe c a l Se ( d _ 1 ) when determined from tracer enrichment i n the serum (256 ±13) or l i v e r (235 ±14) following o r a l administration of the tracer (Se-77) and when determined from enrichment i n serum (241 ±15) following intravenous administration of the tracer (Se-82). The mean of these three measures for endogenous f e c a l Se excretion was 244 ±6 /g d _ 1 . This represented 9.7% of the t o t a l d a i l y f e c a l Se excreted. The estimate of endogenous f e c a l Se determined from the enrichment i n the l i v e r (197 ±11) following intravenous administration of the tracer was s i g n i f i c a n t l y less (P < 0.05) than the estimates obtained from the other three methods of measurement. There were no s i g n i f i c a n t differences (P > 0.05) among the four estimates of true Se absorption for each treatment (Table 26). True absorption determined from the enrichment i n l i v e r following intravenous administration, did however, tend to be lower than the means determined from enrichment i n serum or l i v e r following o r a l administration and from enrichment i n serum following intravenous administration of the tracer. The o v e r a l l mean for true absorption (% of intake) of Se i n the dairy c a t t l e of T r i a l II and III was 11 ±1.5%. -115-Table 23. Endogenous f e c a l selenium ( /jg d 1) j-( T r i a l II and III) Treatment 1 Treatment 2 Route of Administration Tissue Period 1J Period 2 £ Period 1J Period 2 £ Oral (Se-77) Serum 256 ±32a 258 ±13a 267 ±23a 254 ±23a Liver 294 J41a 242 ±20b 300 ±26a 229 JElb Intravenous Serum 239 ±34a 231 ±29a 255 ±29a 248 ±17a (Se-82) Liver 238 434a 194-Gib 248 J27a 199 J-14b "Mean ± SEM, n=5 for Treatment 1 and n=6 for Treatment 2. f Period 1 = days 15-20 and Period 2 = days 20-25. a,b Means followed by the same l e t t e r within a row are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). -116-Table 24. True absorption of selenium ( /g d ) (Values i n parentheses are means for true absorption of selenium expressed as % of selenium intake) | ( T r i a l II and III) Treatment 1 Treatment 2 Route of Administration Tissue Period 1J Period 2 J Period l J Period 2 J Oral (Se-77) Serum 304 ±73a 269 ±41a 501 J61a 307 ±54a (12.4*2.2) (11.4 ±1.1) (17.6 ±1.8) (11.2 ±2.0) Liver 342 ±78a 253 ±42b 534 ±51a 281 ±50b (13.9=2.1) (10.6=1.2) (18.8 =1.8) (10.3=1.8) Intravenous Serum 287 J67a 242 J56a 490 ±31a 251 J43a (Se-82) (11.7 ±2.1) (10.2 ±1.9) (17.2 ±1.2) ( 9 . 2 ±1. 6 ) Liver 286 ±70a 205 ±49b 482 ±30a 301 ±42b (11.6 J2.1) (8.6-1.7) (17.0 ±1.2) (11.0 ±1.6) "Means ± SEM, n=5 for Treatment 1 and n=6 for Treatment 2. J Period 1 = days 15-20 and Period 2 = days 20-25. a,b Means followed by the same l e t t e r within a row are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). -117-Table 25. E f f e c t of route of isotope administration and tissue analyzed on the c a l c u l a t i o n of endogenous f e c a l selenium ( /jg d - 1 ; (Values i n parentheses are means for endogenous f e c a l selenium expressed as % of f e c a l selenium) f ( T r i a l II and III) Route of Administration Tissue Treatment l Treatment 2 Overall Mean Oral (Se-77) Serum 258 J- 13a 254 J- 23a 256 J- 13 (11.4 ±0.8) (9.5 ±0.8) (10.3 ±0.6) Liver 242 ± 20a 229 ± 21a 235 ± 14 (10.6 ±0.8) (8.5 ±0.6) (9.4 ±0.6) Intravenous Serum 231 •*• 29a 248 J- 17a 241 x 15 (Se-82) (10.2 ±1.2) (9.2 ±0.4) (9.7 ±0.6) Liver 194 ± 21b 199 ± 14b 197 ± 11 (8.5 ±0.9) (7.4 ±0.2) (7.9 ±0.4) f Mean J- SEM for Period 2 (days 20-25) , n=5 for Treatment 1 and n=6 for Treatment 2. a,b Means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). -118-Table 26. E f f e c t of route of isotope administration and tissue analyzed on the c a l c u l a t i o n of true absorption of selenium ( /g d - 1 ) (Values i n parentheses are means for true absorption of selenium expressed as % of selenium intake) f ( T r i a l II and III) Route of Administration Tissue Treatment 1 Treatment 2 Overall Mean Oral (Se-77) Serum 269 ± 41 307 ± 54 290 33 (11.4 - 1.1) (11.2 ±2.0) (11.3 1.2) Liver 253 J- 42 281 J- 50 268 X 32 (10.6 ±1.2) (10.3 ±1.8) (10 .4 ± 1.1) Intravenous Serum 242 ± 56 301 ± 42 274 34 (Se-82) (10.2 ±1.9) (11.0 ±1.6) (10.7 1.2) Liver 205 4. 49 252 J- 43 230 j. 31 (8.6 ±1.7) (9.2 ±1.6) (8.9 ± 1.1) | M e a n ± SEM for Period 2 (days 20-25), n=5 for Treatment 1 and n=6 for Treatment 2. Means for true absorption of selenium are not s i g n i f i c a n t l y d i f f e r e n t (P>0.05). -119-5. DISCUSSION 5.1. Selenium stable isotopes as tracers A minor systematic bias was present i n the analysis of Se stable isotopes by ICPMS. This was refl e c t e d by the deviation of the regression c o e f f i c i e n t from unity for a l l isotopes, and the deviation of the intercept from zero for Se-76. The systematic bias observed i n the c a l i b r a t i o n l i n e s was not due to cross-contamination or memory eff e c t s from previously run samples (Buckley et a l . 1987). To improve the accuracy of Se stable isotope measurements by ICPMS the bias was removed i n the analysis of b i o l o g i c a l samples and NBS reference materials by applying the appropriate correction factors based on the regression equations: Se-76, Pred TTMP 76 Obsv TTMP_, + 0.5592 / b 0 . 9536 Se-77, Pred TTMP 77 Obsv TTMP 77 0.9431 Se-82, Pred TTMP 82 Obsv TTMP 1.0720 82 The technique of hydride generation i s subject to interferences between the hydride forming elements themselves and from other matrix elements. The production of gaseous hydrides of Se are chemically i n h i b i t e d by the presence of commonly -120-occurring metals such as Cu. Vijan and Leung (1980) reported that i n samples of high HCl concentration, chlorocomplexes were formed with the metals (Cu, Ni and heavy metals) r e s u l t i n g i n the v i r t u a l elimination of interference. They reported an acid concentration of 7.5 M HCl was necessary to achieve optimum s e n s i t i v i t y and freedom from interference by heavy metals. In the present study 9 M HCl was necessary to eliminate the interference by Cu on the Se signal i n t e n s i t y (Fig. 6). The need for a higher acid concentration for the elimination of interference found i n the present study may be due to the higher concentration of Cu i n the samples. The samples contained up to 8 mg Cu mL - 1 whereas the samples of Vijan and Leung (1980) contained up to 30 mg Cu mL - 1. The preparation of samples with 9 M HCl also offered the advantage of complete reduction of Se(Vl) to Se(IV), the chemical form of Se required for the production of Se hydrides. This eliminates the need for further sample processing, such as heating, for the conversion of Se to the appropriate valency state. The elemental compositions of the sample matrices of i n t e r e s t d i f f e r e d s u f f i c i e n t l y such that interference problems associated with each matrix were unique. The interference resulted i n minor deviations of the Se i s o t o p i c r a t i o s from the published values for natural i s o t o p i c r a t i o s . I t i s therefore advisable to measure the i s o t o p i c ratios of natural Se i n a p a r t i c u l a r matrix and use the measured values, rather than the published values, to calculate tracer enrichment i n unknown -121-samples of the same matrix. A n a l y t i c a l chemistry of Se i n matrices of i n t e r e s t i s r e l a t i v e l y complex because of a number of issues which include: the multivalent state of Se and the r e l a t i v e ease of conversion among oxidation states; the l i k e l y existence of multiple forms of Se and t h e i r r e l a t i v e l i a b i l i t y and v o l a t i l i t y ; and the need for conversion of a l l Se moieties into a s i m i l a r chemical species (Se(IV) i n t h i s case) (Janghorbani et a l . 1985). The methods described herein for the quantitative analysis of Se were found to be accurate as indicated by good agreement between the c e r t i f i e d values of NBS reference materials and the observed values (Table 6 and Table 8). Janghorbani et a l . (1982b) reported some chemical forms of Se (e.g. trimethylselenonium) i n urine were re s i s t a n t to digestion and conversion to Se(IV). The method for the preparation and analysis of samples by ICPMS proved capable of overcoming any such resistance as demonstrated by the results of the analyses of NBS freeze dried urine (Table 8). In contrast, Se was not detected i n urine samples analyzed by GCMS. The digestion aid used i n the preparation of the samples for GCMS analysis was prepared with hydrated magnesium n i t r a t e . The presence of water i n the digestion aid may have decreased the strength of the aid for oxidation of Se i n the urine sample. I t could have also been possible that reactions between Se and chemical interferents i n the matrix of the urine prevented the d e r i v a t i z a t i o n of Se. The 4NPD d e r i v a t i z i n g reagent i s s p e c i f i c towards i t s reaction with Se (Reamer and -122-V e i l l o n 1983), thus i t i s u n l i k e l y 4NPD was combining with other elements i n the matrix. The measurement of Se i n urine was not necessary for quantitation of the endogenous f e c a l excretion of Se or true absorption and therefore no further attempt was made to measure Se i n urine by GCMS. GCMS analysis should be capable of measuring a l l six stable isotopes of Se. In the present study the measurement by GCMS of only three of the six stable isotopes of Se was investigated. Using the most abundant isotope of Se as the reference isotope 80 ( Se), Se-76 and Se-78 served s a t i s f a c t o r i l y as the metabolic tracer and the i n t e r n a l standard respectively. Reamer and V e i l l o n (1983) have also reported the successful application of a 8 0 method of GCMS analysis based on Se as the reference isotope, Se-76 as the metabolic tracer and Se-82 as the i n t e r n a l standard. They reported p r e c i s i o n of the measurement, expressed as a r e l a t i v e standard deviation, was 1.4% for 20 determinations of samples of about 40 ng Se mL, and was independent of the sample type (Reamer and V e i l l o n 1983). In t h i s study ICPMS proved capable of measuring four of the six stable isotopes of Se ( 7 6Se, 7 7 S e , 7 8 S e and 8 2 S e ) . 8 0 S e could not be measured by ICPMS because of interference by A r 2 + 74 ion. Se was not investigated as the Se-74 isotope preparation costs $100-200 US/mg. The cost of t h i s isotope preparation makes i t an u n l i k e l y choice as a tracer for experiments with dairy c a t t l e . Successful application of the stable isotope approach for -123-the study of Se metabolism depends on the degree of achieved isotope enrichment i n r e l a t i o n to the a n a l y t i c a l p r e c i s i o n with which i s o t o p i c measurements can be made. The pr e c i s i o n of Se stable isotope enrichment measured by GCMS was better for Se-76 than for Se-78 (Table 7). Thus for experiments involving a single tracer, Se-7 6 would be the isotope chosen for enrichment and Se-78 for quantitative measurement. The prec i s i o n obtained for measurement of Se stable isotopes was greater for sample analysis by ICPMS than by GCMS. The best pr e c i s i o n obtained from ICPMS analysis (Table 9) was for the measurement of Se-77 and therefore, i t would be the isotope of f i r s t choice for enrichment purposes. For double isotopic enrichment, Se-77 and Se-82 are the isotopes of choice with Se-76 as the in t e r n a l standard. Tracer l e v e l s of stable isotopes are t y p i c a l l y only a few percent above natural i s o t o p i c l e v e l s , thus, i f stable isotopes are to be used as tracers, t h e i r concentration must be determined with high p r e c i s i o n . Under the experimental protocol of T r i a l I the l e v e l of prec i s i o n obtained for GCMS analysis was s u f f i c i e n t to measure Se-76 enrichment i n the samples of in t e r e s t . However a reduction i n the quantity of the isotope administered (20 mg) may be necessary under some circumstances. For k i n e t i c analysis i t i s important to produce a minimum perturbance to the ki n e t i c s of a tracer pool and i t i s advisable to add no more than about 10% of the pool si z e as tracer (Buckley et a l . 1982). I t i s also desirable to work with smaller quantities of tracer, such as those administered to the animals i n T r i a l II and III -124-(Se-77, ~4.6 mg; Se-82, ~1. 3 mg), for economic reasons. I t may be possible to administer smaller quantities of tracers as the p r e c i s i o n of the isotope r a t i o determination i s expected to improve with continued method development of the ICPMS technique. The p r e c i s i o n obtained with GCMS analysis however, would not permit the tracer enrichment i n the feces to be measured following administration of s i m i l a r quantities of isotope as i n T r i a l II and I I I . 5.2. Selenium content of the experimental diets The dietary concentration of Se has been shown to influence 7 5 the concentration of Se retained by the tissues following both o r a l and intravenous routes of isotope administration (Lopez et a l . 1969). As dietary Se intake increased the s p e c i f i c a c t i v i t y 75 of Se i n tissues decreased i n d i c a t i n g reduced Se retention or increased Se turnover (Lopez et a l . 1969; Kincaid et a l . 1977). T r i a l I was a f i r s t attempt at measuring the endogenous f e c a l excretion and true absorptin of Se and therefore only approximations could be made with regard to the quantity of tracer required to obtain measurable l e v e l s of tracer enrichment i n the b i o l o g i c a l samples of i n t e r e s t . Therefore i n T r i a l I i t was desirable to provide a low-Se d i e t to the two cows i n order to maximize the uptake and enrichment of the whole body Se pool with the Se-76 tracer. For T r i a l II and III the target Se concentration for the experimental diets was 0.2 mg kg 1 . The dietary Se concentration -125-was chosen i n a n t i c i p a t i o n of a proposed r e v i s i o n to the Nutrient Requirements of Dairy Cattle (NAS-NRC 1988) that would increase the recommended Se intake to 0.2 mg kg 1 . Selenite was the chemical form of the Se added to the experimental diets as t h i s i s the form used by feed companies i n B r i t i s h Columbia. The close association between Se and V i t E i n preventing deficiency conditions has been recognized for many animal species (Jenkins and Hidiroglou 1972). The quantity of V i t E considered adequate to meet the dairy cow's requirements l i e s between 10 and 15 mg k g - 1 dietary DM (ARC 1980). The V i t E content i n orchard grass hay i s ~190 mg k g - 1 (Ensminger and Olentine 1978). Therefore the V i t E consumed by the cows from the d i e t i n t h i s study was considered adequate. 5.3. Tissue distrubution of selenium In ruminants of an adequate Se status the highest concentration of Se i s i n the kidney, with the cortex containing a higher concentration than the medulla (Scholz et a l . 1981a). High concentrations are also reported i n the small i n t e s t i n e , lung, l i v e r , pancreas, adrenal and p i t u i t a r y glands, and spleen (Kincaid et a l . 1977; Scholz et a l . 1981a). Handreck and Godwin (1970) report that higher l e v e l s of Se are found i n the cardiac muscle than i n the s k e l e t a l muscle. The lowest l e v e l s of Se are reported found i n the bone, adipose t i s s u e and plasma. The d i s t r i b u t i o n of Se i n the tissues and f l u i d s of the two cows i n t h i s study were consistent with the results above. -126-The concentration of Se i n the l i v e r and serum have been suggested as useful indicators for the assessment of the Se status of c a t t l e (Thompson et a l . 1980, 1981). Based on data from studies involving c a t t l e of adequate Se status (Table 1) the Se concentration of the l i v e r and plasma ranged from 0.80-1.75 /g g - 1 and 0.034-0.112 /g mL - 1. The mean concentration of Se i n the l i v e r was 0.87 and 0.9 9 /g g - 1 and i n serum was 0.048 and 0.049 /g mL - 1 for the cows of T r i a l I and T r i a l II and I I I , respectively. These values f a l l within the ranges reported above and indicate the cows at the time of tissue sampling were of an adequate Se status. Had the animals of T r i a l I continued to consume the low Se d i e t (0.035 mg kg - 1) i t i s l i k e l y t h e i r tissue Se l e v e l s would have declined to below the normal ranges for Se concentration. There was a large range of Se-76 enrichment i n the tissues and f l u i d s (Fig. 7) as has been reported by other authors for 75 Se concentrations (Lopez et a l . 1969; Kincaid et a l . 1977; Dejneka et a l . 1979). Lopez et a l . (1969) reported the 7 5 S e 7 5 concentration i n tissues of lambs 12-15 days a f t e r Se administration i n descending order were as follows: kidney, l i v e r , spleen, lung, b i l e , brain, and heart. In the present study the mean Se-76 enrichment was s i m i l a r i n kidney and l i v e r . The differences i n the patterns of tracer d i s t r i b u t i o n i n the kidney and l i v e r may be due to to an e f f e c t of dietary Se l e v e l . In another experiment, Lopez et a l . (1969) found lambs fed a -1 75 rat i o n containing 0.014 mg Se kg retained a higher Se -127-concentration i n the kidney than i n the l i v e r . When lambs received additional Se i n the ration (0.264 - 5.014 mg Se kg - 1) the retention by these tissues was reversed. Lopez et a l . (1969) reported i n lambs receiving additional Se, more than 99% of the 75 Se a c t i v i t y i n the kidney was present i n the cortex with a neglegible amount present i n the medulla. In the present study Se-76 enrichment i n the kidney cortex and medulla were s i m i l a r . The p o s i t i o n of the spleen i n the pattern of descending order of tracer enrichment also d i f f e r e d from that reported by Lopez et a l . (1969). Se-76 enrichment i n descending order was lung, b i l e , heart and spleen. Lopez et a l . (1969) reported accumulations of 75 Se i n the p e l t , gastrocnemius muscle and femur bone were about the same and were higher than i n adipose ti s s u e . The enrichement of Se-76 was higher i n the hide than i n s k e l e t a l muscle. Se-76 enrichment was not measurable i n bone or adipose tiss u e as i t was below the detection l i m i t for GCMS analysis. The detection l i m i t and therefore the Se-76 enrichment i n bone, was less than one-third the l e v e l of enrichment i n the s k e l e t a l muscle. Some 7 5 of the differences found between the patterns of Se and Se-76 d i s t r i b u t i o n may be explained by the age and stage of development of the experimental animals (growing lambs verses mature dairy cows). This was evident from the comparison of Se-76 enrichment of the f e t a l tissues (Fig. 8) with that of the dam (Fig. 7). The f e t a l l i v e r and kidney were enriched to l e v e l s comparable to those of the corresponding tissues i n the dam, however, the enrichment i n the f e t a l muscle was ~6 times the l e v e l i n the -128-muscle of the dam. 5.4. Method for measurement of true absorption The evaluation of the b i o a v a i l a b l i t y of a nutrient includes an assessment of i t s a v a i l a b i l i t y for i n t e s t i n a l absorption and i t s subsequent u t i l i z a t i o n . In monogastric species Se i s regarded as well absorbed and the a v a i l a b i l i t y for i n t e s t i n a l absorption as e s s e n t i a l l y complete. These studies have lead to the b e l i e f that there i s l i t t l e or no homeostatic regulation of Se at the g a s t r o i n t e s t i n a l l e v e l . More recently, studies with experimental animals have focused on monitoring only changes i n the a v a i l a b i l i t y of Se for incorporation into the biochemically active form of GSHPx i n plasma, erythrocytes and occasionally l i v e r and other tissues (Barbezat et a l . 1984). In ruminant animals however, the a v a i l a b i l i t y of Se for i n t e s t i n a l absorption plays a s i g n i f i c a n t role i n the o v e r a l l a v a i l a b i l i t y of t h i s element to the animal. The conditions e x i s t i n g within the rumen tend to reduce the Se to less available chemical forms which pass from the digestive t r a c t v i a the feces. The nature of t h i s action and the p o t e n t i a l for multiple dietary interactions remain l a r g e l y unknown. In addition there i s a lack of quantitative experimental data on the various aspects of g a s t r o i n t e s t i n a l absorption of Se, including the contribution from endogenous components to f e c a l Se. Influencing factors on the g a s t r o i n t e s t i n a l absorption and endogenous secretion of Se are of fundamental importance for a more complete -129-understanding of g a s t r o i n t e s t i n a l function and i n r e l a t i o n to dietary management. Apparent or net absorption determined by conventional balance techniques provides only a rough estimate of absorption. This measure does not make any reference to the o r i g i n of the nutrient i n the feces and accordingly does not correct for the endogenous f e c a l l o s s . Endogenous excretion of many elements makes a considerable contribution to the f e c a l output and for such elements balance studies underestimate the true extent of absorption. True absorption measures the proportion of a nutrient i n food which moves from the i n t e s t i n a l lumen through the mucosal c e l l and into the body. To measure true absorption the f e c a l excretion of the nutrient i s corrected for the endogenous f e c a l l o s s . Endogenous losses of elements v i a the feces may originate from hepatobiliary transport, secretion by the pancreas and glands of the alimentary t r a c t , sloughing of i n t e s t i n a l e p i t h e l i a l c e l l s and d i r e c t secretion from blood into the g a s t r o i n t e s t i n a l t r a c t (Gregus and Klaassen 1986). In t h i s study quantitation of the endogenous f e c a l excretion of Se and true absorption were determined following the administration of Se stable isotopes i n conjunction with conventional metabolic balance techniques. This procedure has been applied successfully to measure true absorption of Zn with radioisotopes i n rats (Evans et a l . 1979) and stable isotopes i n humans (Jackson et a l . 1984). The endogenous f e c a l Se was estimated i n d i r e c t l y based on -130-the tracer enrichment i n a reference or index tissue which was assumed to r e f l e c t the enrichment of endogenous f e c a l Se during the balance period. Prospective indices were evaluated by examination of the d i s t r i b u t i o n of the Se-76 i n tissues and f l u i d s following a period to allow e q u i l i b r a t i o n of the tracer with natural Se i n the body. The length of time required for tracer e q u i l i b r a t i o n w i l l depend upon the element, the route of the isotope administration and the subject species. In rats 65 dosed intramuscularly with radioactive Zn, balance periods were begun 9 days following isotope administration (Evans et a l . 1979). In the experiment by Jackson et a l . (1984) the balance study commenced 2 days subsequent to the intravenous administration of the enriched stable isotope Zn-67 i n humans. The bioeffectiveness and metabolism of trace elements may be influenced by several dietary and host factors. One key factor a f f e c t i n g the a v a i l a b i l i t y of Se i s the chemical form. In many of the experimental investigations involving the use of tracers to study trace element metabolism the route of isotope administration i s v i a i n j e c t i o n . A major underlying assumption i s that the administration route does not a f f e c t the r e s u l t s . Trace elements, however, enter the animal body from the digestive t r a c t and may undergo transformations during t h i s process. To ensure data obtained from experimental investigations with intravenous administration of the tracer r e f l e c t s the metabolism of the element under p r a c t i c a l s i t u a t i o n s , the tracer should be administered i n the same chemical form as the absorbed element. -131-In ruminants the chemical form of Se ingested and hence the chemical form of Se absorbed i s influenced by the rumen environment. I t i s not known what the chemical form(s) are of the Se that i s absorbed. Thus to ensure the Se-76 tracer would be under s i m i l a r influences as dietary Se, the tiss u e d i s t r i b u t i o n of the tracer was determined following o r a l administration. 7 5 Symonds et a l . (1981a) reported that the clearance of Se from the whole body of dairy cows was described by two exponential components. The f i r s t component of clearance 75 75 consisted of unabsorbed Se as well as absorbed Se excreted i n urine and the g a s t r o i n t e s t i n a l t r a c t with l i t t l e or no e q u i l i b r a t i o n with the main Se pools of the body. The rentry of 7 5 the absorbed Se into the g a s t r o i n t e s t i n a l t r a c t during t h i s time would not be quant i t a t i v e l y the same as the endogenous secretions as t h i s equivalency does not take place u n t i l isotopic e q u i l i b r a t i o n occurs within those body pools responsible for endogenous secretions. At 8 days afte r o r a l dosing with 7 5 7 5 Se-labelled barley the contribution of Se from the f i r s t component of clearance was not detectable and the subsequent clearance from the body was considered to be by endogenous loss (Symonds et a l . 1981a). Based on t h i s information and the need for a s u i t a b l e length of time for dietary adaptation, the e q u i l i b r a t i o n period i n the present study was 10 ( T r a i l I) and 14 days ( T r a i l II and I I I ) . I f e q u i l i b r a t i o n of Se-76 i n the tissues was not complete -132-p r i o r to obtaining balance measurements, Se-76 from the f i r s t component of clearance would be expected to contribute to and elevate the t o t a l Se-76 enrichment i n the feces. The e f f e c t of the elevated Se-7 6 enrichment i n feces on the estimation of the endogenous f e c a l Se would depend also, on the behavior of Se-76 75 i n the index t i s s u e . Langlands et a l . (1986) measured Se (counts min 1 mass Se - 1) i n feces and plasma c o l l e c t e d 1 to 45 75 2-days afte r intravenous administration of [ Se]Se0 3 . Using 75 75 t h i s data, the calculated r a t i o of Se i n the feces to Se i n plasma, tended to be higher during days 1 to 10. I t i s suspected there would be a tendancy to overestimate the contribution of the endogenous Se to the t o t a l f e c a l Se and hence underestimate the true absorption of Se i f determinations were made from data c o l l e c t e d p r i o r to e q u i l i b r a t i o n of the tracer with the body Se. Twenty-two days following the administration of Se-76 (10 days e q u i l i b r a t i o n , 10 days balance), the tissue and f l u i d s considered as possible contributors to the endogenous f e c a l Se were found to be enriched to a si m i l a r degree. These tissues included the serum, epithelium of the stomach (including the rumen, reticulum, omasum and abomasum), l i v e r , b i l e , pancreas, small i n t e s t i n e and colon (Fig. 7). The range of enrichment i n these tissues (mean of two cows) was TTMP 9.8 to 12.9. In ruminant animals s a l i v a may also make a considerable contribution to the endogenous secretion of an element. In the present study the enrichment of Se-76 i n the s a l i v a r y glands was 75 not measured. But Dejneka et a l . (197 9) measured Se -133-concentration i n tissues and f l u i d s of sheep and reported a high l e v e l of enrichment i n the parotid s a l i v a r y gland. The l i v e r has been i d e n t i f i e d as playing a key role i n Se metabolim (Symonds et a l . 1981a; McMurray and Davidson 1985). Se i s believed to be assimilated by the l i v e r where a number of selenocompounds and s p e c i f i c selenoproteins are synthesized and released (McMurray and Davidson 1985). The l i v e r also makes a d i r e c t contribution to the f e c a l Se v i a b i l a r y Se excretion. It has been hypothesized that one of several of the selenoproteins released by the l i v e r into the plasma act as transport proteins to d i s t r i b u t e selenocompounds to other tissues (Motsenbocker and Tappel 1982a; McMurray and Davidson 1985). Thus the serum because of i t s possible transport function provides a medium for exchange of Se between the tissues including the g a s t r o i n t e s t i n a l t r a c t , l i v e r , pancreas and s a l i v a . The l i v e r and serum play a ce n t r a l and i n t e r r e l a t e d role i n Se metabolism, d i s t r i b u t i o n and excretion and therefore these tissues were believed to best represent the enrichment of the Se tracer of endogenous f e c a l o r i g i n . The use of l i v e r or serum as tissu e indices for the c a l c u l a t i o n of endogenous f e c a l Se also o f f e r an advantage from a p r a c t i c a l standpoint. With the development of routine procedures for obtaining l i v e r biopsy samples and the a c c e s s i b i l i t y of blood, these tissues can be e a s i l y sampled. The e f f e c t of the choice of the tissue sampled ( l i v e r or serum) on the estimation of endogenous f e c a l Se excretion and true absorption were evaluated i n T r i a l II and I I I . There was -134-close agreement between the l e v e l of enrichment measured i n serum and l i v e r (Table 21) following o r a l administration of Se-77 and hence the estimation of endogenous f e c a l Se from these two indices were equivalent (Table 25). In addition to the evaluation of the choice of tissu e sampled on the estimation of endogenous f e c a l Se and true absorption, the e f f e c t of o r a l and intravenous routes of tracer administration were also evaluated. The advantage to using the intravenous route of tracer administration i s that l e s s isotope i s required for tissue enrichment reducing the cost of the experiment. When Se-82 was administered intravenously a difference was apparent i n the degree of enrichment i n serum and l i v e r (Table 19). Enrichment i n the l i v e r was higher than that measured i n the serum. As a re s u l t the estimation of endogenous f e c a l Se following intravenous administration with analysis of the l i v e r was lower than that determined from the analysis of serum. A comparison of the estimates of endogenous f e c a l Se from o r a l and intravenous routes of administration with serum and l i v e r analysis revealed a l l estimates were equivalent except for the estimate determined from enrichment of l i v e r following intravenous administration of the isotope. Major issues related to d i f f e r e n t i a l metabolism of Se administered v i a o r a l and intravenous routes are s t i l l unresolved. However the s i m i l a r i t y between the estimates for endogenous f e c a l Se from the o r a l route of isotope administration with the analysis of serum or l i v e r and -135-the intravenous route of isotope administration with the analysis of serum indicate that i f d i f f e r e n t i a l metabolism e x i s t s , i t does not a f f e c t the determination of endogenous f e c a l Se by these methods. There does however, appear to be differences i n the metabolism of Se by the l i v e r depending on the route of administration. Therefore, for estimation of endogenous f e c a l Se, the o r a l route of tracer administration with the analysis of serum or l i v e r and the intravenous route of tracer administration with the analysis of serum were found suitable. The absolute values and the associated v a r i a t i o n for the d a i l y Se intake and the t o t a l f e c a l Se were large r e l a t i v e to that for the estimation of endogenous f e c a l Se. As a r e s u l t apparent absorption of Se varied widely among animals and when corrected for the contribution of endogenous Se i n the feces to ca l c u l a t e the true absorption, differences among the 4 estimates of true absorption were not s t a t i s t i c a l l y s i g n i f i c a n t (Table 26). Thus i t would appear true absorption of Se could be determined from the o r a l or intravenous route of isotope administration with analysis of serum or l i v e r . The values for the 4 estimates of true absorption however, do show a trend s i m i l a r to the values for the estimate of endogenous f e c a l Se. That i s , the estimates of true absorption determined from the o r a l route of tracer administration with serum and l i v e r analysis and from the intravenous route of administration with serum analysis were more cl o s e l y comparable and the estimate from the intravenous route of administration with l i v e r analysis was lower. I f circumstances -136-prevailed where the endogenous f e c a l Se loss contributed a greater portion to the o v e r a l l f e c a l Se excretion (such as i n T r i a l I (Table 15) where the animals were consuming a low Se d i e t ) , i t i s expected s i g n i f i c a n t differences between the 4 estimates for true absorption would p a r a l l e l that for the 4 estimates of endogenous f e c a l Se. Therefore i t i s recommended that true absorption of Se be determined from one of the approaches suggested for determination of endogenous f e c a l Se l o s s : i . e . the o r a l route of tracer administration with the analysis of serum or l i v e r or the intravenous route of tracer administration with the analysis of serum. 5.5. Apparent and true absorption of selenium Harrison and Conrad (1984a) measured apparent absorption of Se i n non-lactating dairy cows with dietary Se intakes ranging from 437 to 3136 /g d - 1 . The quantity of Se absorbed ( /g d - 1 ) increased with increasing Se intake and was described by the 2 l i n e a r r e l a t i o n s h i p : Y = 0.51X - 132, r =0.98. Extrapolation of the l i n e a r regression l i n e to zero absorption indicated cows consuming below 259 /g d - 1 would be i n a negative Se absorptive state. In T r i a l I the two cows were found to be i n a negative Se absorptive state when consuming 360 - 460 /g Se d _ 1 . The p r a c t i c a l s i g n i f i c a n c e of the data from these two animals and that from Harrison and Conrad (1984a) suggested that non-lactating cows consuming a d i e t with a Se concentration <_ 0.0 35 mg k g - 1 DM would be i n a negative Se balance. Therefore, -137-i t i s important for non-lactating pregnant cows consuming low Se diets to receive Se supplementation at a time when adequate provision of Se i s necessary for the reproductive health of the cow (Harrison and Conrad 1984a). Apparent absorption and true absorption of Se by non-lactating cows consuming a d i e t with a Se concentration of 0.19 mg k g - 1 was 3% (Table 22) and 11% (Table 26) of the Se intake (2600 \q d - 1 ) , respectively. Harrison and Conrad (1984a) have reported much higher values of 46% and 50% for apparent absorption by cows consuming 2701 and 3136 /g Se d - 1 . Several factors may be responsible for the d i s p a r i t y between the results reported here and those of Harrison and Conrad (1984a). These include the source of Se and possible interactions a r i s i n g between Se and other dietary components. Work by Peter et a l . (1982, c i t e d by Peter et a l . 1985) indicated organic Se was absorbed to a greater extent than inorganic Se. These authors reported sheep of a low Se status fed a low Se d i e t and infused 2-intraruminally with Se0 3 or Se-Met, absorbed 12% to 13% more Se 2-from Se-Met than Se0 3 . The major Se compounds i n seeds or forages consumed by livestock are organic and include Se-Met, Se-Cys, selenocystine and Se-methylselenomethionine (NAS-NRC 1983). The Se i n the rations of the study by Harrison and Conrad (1984a) were provided a l l or i n a large part by natural sources and therefore, the Se was primarily i n an organic form. In t r i a l II and III the Se was provided primarily as s e l e n i t e , an inorganic form. However, a comparison of the results for true -138-absorption of Se from the cows of T r i a l II and III (11%) which consumed primarily se l e n i t e , with the results from the two cows of T r i a l I (10% and 16%) which received Se from a natural source, suggested the absorption of Se from feedstuffs and s e l e n i t e were s i m i l a r . Differences i n the composition of the diets i n the study of Harrison and Conrad (1984a) and the present study, r e s u l t i n g i n differences i n possible interactions among Se and other dietary factors may also be involved. Harrison and Conrad (1984b) reported a maximum Se absorption i n cows of 40% to 50% when the dietary Ca was 0.8% of dry matter intake. At dietary Ca intakes of 0.4% to 0.5% of dry matter intake, Se absorption decreased by ~20 %. The concentration of Ca i n t h i s study was 0.48%, and therefore, might have contributed to the r e l a t i v e l y low rates of absorption. Selenite has been reported to be less stable than the other common form of inorganic Se, selenate (Ammerman and M i l l e r 1974). The reduction of s e l e n i t e to elemental Se forms may be observed upon exposure to organic matter. This can cause p r a c t i c a l problems when se l e n i t e i s incorporated into diets as a premix with reducing sugars (Ganther 1984). Molasses contains >_4 6% t o t a l sugars (Church 1984), however, these sugars e x i s t primarily as sucrose which i s a non-reducing sugar. Thus, reduction of s e l e n i t e to the less available elemental form upon i t s addition to molasses was not believed to be a contributing factor towards the low values for Se absorption found i n t h i s study. -139-5.6. Selenium excretion Butler and Peterson (1961) reported that the main pathway of Se excretion i n ruminants i s v i a the feces when dietary Se lev e l s are below 0.01 mg k g - 1 . When Se supply i s adequate Se i s excreted v i a the urine and feces i n about equal proportions (Lopez et a l . 1969). As the Se supply i s increased the urinary excretion of Se also increases and may even exceed f e c a l Se excretion at high l e v e l s of administration. In the present study a greater proportion of the dietary Se intake was excreted i n the feces. The excretion of Se i n the feces constituted ~90% of the t o t a l d a i l y Se excreted, with the remainder of Se excreted i n the urine ( T r i a l II and I I I , Table 22). The endogenous f e c a l Se was 9.7% of the t o t a l f e c a l Se ( T r i a l II and I I I , Table 23). In cows consuming a low Se d i e t (0.035 mg k g - 1 ) , the contribution of endogenous Se to the t o t a l f e c a l Se was increased to 23% and 36% (Table 15). The excretion of Se i n expired a i r was considered to be i n s i g n i f i c a n t and was not measured i n t h i s experiment. Handreck and Godwin (1970) reported sheep receiving 0.5 to 1.3 mg Se d _ 1 excreted only about 1% i n expired a i r . There i s very l i t t l e information regarding the quantity of Se contributed by the various sources to the endogenous f e c a l Se 75 pool. In sheep dosed intravenously with [ Se]Se-Met, the r a d i o a c t i v i t y 28 d post i n j e c t i o n i n the anterior section of the small i n t e s t i n e was ~100 times that i n the feces suggesting a large flow of endogenous Se into the small i n t e s t i n e (Langlands et a l . 1986). In another experiment, Langlands et a l . (1986) -140-reported approximately 300 to 400 \q Se entered the anterior portion of the small i n t e s t i n e of sheep each day; equal to two to three times the quantity of Se ingested, most of which was subsequently reabsorbed. 75 Gregus and Klaassen (1986) administered Se intravenously to rats and by comparison of the b i l a r y and f e c a l Se excretions were able to predict whether the Se was excreted or reabsorbed by the i n t e s t i n e . The 2-h b i l a r y excretion was higher than the 4-d f e c a l excretion suggesting Se underwent i n t e s t i n a l reabsorption. Langlands et a l . (1986) reported the Se concentration i n b i l e of sheep averaged 0.0086 /g Se mL - 1, which represented a d a i l y excretion of 13.8 /g or 28% of the Se consumed. In t h i s study the excretion of Se from b i l e into the g a s t r o i n t e s t i n a l t r a c t based on an estimated b i l a r y secretion of 525 mL h - 1 (Symonds et a l . 1981b) and a b i l a r y Se concentration of 0.0061 fg mL - 1 ( T r i a l I) was 769 fjg d - 1 . This i s 1.5 times the the d a i l y f e c a l Se excretion and also suggests reabsorption i s taking place within the i n t e s t i n e . Recycling of Se would not have affected the estimation of endogenous f e c a l Se i n the present study as the method described herein measured the net amount of unabsorbed Se of endogenous o r i g i n i n the f e c a l pool. 5.7. E f f e c t of copper on the endogenous f e c a l excretion and true  absorption of selenium Major economic losses i n animal production may ari s e from d e f i c i e n c i e s or imbalances i n trace element intake. This becomes -141-p a r t i c u l a r l y important with regard to Se where problems exi s t i n the diagnosis of s u b c l i n i c a l deficiency. Further problems are evident i n defining the animals dietary requirements, as situations under p r a c t i c a l conditions have developed where animals respond to Se supplementation despite receiving what was considered adequate. The involvement of many factors i n the Se/Vit E responsive conditions seem apparent from the wide v a r i a t i o n i n what i s reported to be selenium deficiency i n c a t t l e (Fenimore et a l . 1983). Fenimore et a l . (1983) reported beef c a t t l e i n southeastern B r i t i s h Columbia fed moderate to low Se l e v e l s i n hay responded to Se supplementation with the disappearance of a variety of c l i n i c a l observations including weak calves, neonatal diarrhea, pneumonia, poor growth, placental retention and poor reproductive performance. Fundamental quantitative data are necessary for a comprehensive understanding of Se metabolism and the complex series of host and dietary interactions which are l i k e l y to be involved i n influencing the Se economy. Selenium and s u l f u r show si m i l a r chemical c h a r a c t e r i s t i c s and Se shares with S an a f f i n i t y for heavy metals such as cadmium, s i l v e r , mercury and copper. An increase i n the intake of S, either as sul f a t e or an organic form can reduce l i v e r and blood Cu concentrations i n sheep. I t i s believed that Cu absorption i s decreased through an i n t e r a c t i o n i n the alimentary 2-t r a c t , possibly involving the formation of S i n the rumen and the formation of insoluble CuS, a r e l a t i v e l y non-available form -142-of Cu (Bremner and Davies 19 80). The chemical environment i n the 2-rumen would probably lead to the formation of Se and the formation of metal selenides (Peterson and Spedding 1963). A p a r a l l e l between the formation of CuS and the formation of insoluble CuSe l i k e l y exists within the rumen. Therefore i t was hypothesized that an i n t e r a c t i o n between Cu and Se i n the rumen might r e s u l t i n the formation of CuSe and thereby reduce the a v a i l a b i l i t y of Se for absorption. Interaction between Cu and inorganic Se have been demonstrated i n chicks ( H i l l 1974; Jensen 1975a) where high dietary Cu (500 - 1000 mg k g - 1 ) was found to counteract Se t o x i c i t y . High dietary Cu concentrations (800 - 1600 mg kg - 1) have also been reported to induce Se deficiency i n chicks (Jensen 1975a) and ducklings (Van Vleet and Boon 1980). I t was suggested that Cu formed insoluble complexes with Se within the g a s t r o i n t e s t i n a l t r a c t and tissues thereby reducing the a v a i l a b i l i t y of Se. In ruminants, high l e v e l s of Cu supplementation were found to increase the apparent Se retention (White et a l . 1979; Gooneratne et a l . 1981). Gooneratne et a l . (1981) reported the accumulation of Cu i n the l i v e r of copper loaded sheep was associated with an increase i n l i v e r Se concentration and GSHPx a c t i v i t y . I t was suggested that increased Se retention i n sheep was a response to tiss u e damage caused by Cu accumulation (White et a l . 1979). Thus there appears to be a metabolic i n t e r a c t i o n between Se and Cu when excessively high concentrations of Cu are used. I t i s however, -143-less clear whether the interactions between Cu and Se have s i g n i f i c a n t metabolic or pathological consequences when Cu concentrations are more t y p i c a l of those incurred i n p r a c t i c a l s ituations a r i s i n g from variations of supplemental Cu added to the d i e t . Abdel Rahim et a l . (1986) investigated the e f f e c t of lower concentrations of dietary Cu (1.3 - 200 mg k g - 1 ) on Se u t i l i z a t i o n by the rat. Cu supplementation did not a f f e c t absorption and total-body retention of Se from [ Se]Se0 3 . Only when the dietary Cu concentration reached 200 mg k g - 1 was 7 5 there an influence on the organ d i s t r i b u t i o n of Se i n tissues. 75 This occurred when Se was administered o r a l l y and i n t r a p e r i t o n e a l l y . There was a reduction i n the Se concentration i n l i v e r , kidneys and whole blood and reduced GSHPx a c t i v i t i e s i n l i v e r , t e s t i s , kidney and whole blood. Increasing the Cu concentration of the diets did not r e s u l t i n s i g n i f i c a n t l y higher Cu concentrations i n the tissues and i t was suggested that the 200 mg Cu k g - 1 d i e t may have resulted i n the appearance of a novel Se-reactive form of Cu or a higher rate of turnover of an e x i s t i n g reactive pool of Cu (Abdel Rahim et a l . 1986). Fehrs et a l . (1981) studied the e f f e c t s of supplementing diets with 0 and 100 mg Cu k g - 1 on the Se metabolism i n calves. Calves were fed diets containing 0 or 1.0 mg Se k g - 1 . 75 75 Forty-eight hours following an o r a l dose of Se, Se i n blood, 75 kidney and s p i n a l cord was lower, and more Se was excreted i n the urine and feces of calves receiving the high Cu and Se d i e t s , -144-i n d i c a t i n g a lower Se retention. In contrast supplementation of the low Se d i e t with 100 mg Cu k g - 1 did not a l t e r the 75 75 excretion of Se i n feces and urine or the d i s t r i b u t i o n of Se. White et a l . (1981) also reported no s i g n i f i c a n t e f f e c t of supplemental Cu (10 mg kg - 1) on the absorption or the excretion 75 and t i s s u e retention of Se 7 days following the o r a l 75 administration of [ Se]Se-Met. In the present study with dairy c a t t l e offered a p r a c t i c a l d i e t , there was no s i g n i f i c a n t e f f e c t of 17 mg k g - 1 of supplemental Cu on the excretion and true absorption of Se. In addition Cu supplementation did not influence the d i s t r i b u t i o n of Se-77 and Se-82 i n serum or l i v e r . These results are i n agreement with Fehrs et a l . (1981) and White et a l . (1981) and suggest there i s no s i g n i f i c a n t Cu-Se in t e r a c t i o n or metabolic consequence a r i s i n g when the dietary concentrations of Se and Cu are t y p i c a l of those found under p r a c t i c a l conditions. -145-6. CONCLUSIONS 1. ICPMS and GCMS both proved to be accurate for quantitative analysis of Se by isotope d i l u t i o n as indicated by good agreement between measured and c e r t i f i e d Se values i n standard reference materials (Table 6 and 8). 2. GCMS was found to be suitable for the measurement.of Se-76 and Se-78 enrichment and ICPMS for the measurement of Se-76, Se-77 and Se-82 enrichment. The isotopes of choice for metabolic tracers were Se-76 when samples were analyzed by GCMS and Se-77 and Se-82 when analyzed by ICPMS. 3. Greater p r e c i s i o n for measurement of Se stable isotope enrichment i n b i o l o g i c a l materials was obtained for ICPMS than for GCMS (Table 7 and 9). The advantage to using the most precise method of analysis i s that a smaller quantity of the tracer may be administered to the dairy cow thereby minimizing the p o s s i b i l i t y of a l t e r i n g Se metabolism as well as reducing the cost of the experiment. 4. The combination of conventional metabolic balance techniques with i s o t o p i c enrichment of the body Se pools was considered to y i e l d r e l i a b l e estimates of the endogenous f e c a l excretion of Se and true absorption of Se i n dairy cows. The r e l i a b i l i t y of the method was evaluated by investigation of the enrichment of Se -146-pools which were poten t i a l contributors to the endogenous f e c a l Se and by studying the e f f e c t of route of administration of the tracer. The application of t h i s technique using Se stable isotopes as tracers w i l l enable further experimental investigations to increase the understanding of ways i n which the e f f i c i e n c y of Se absorption may vary and thereby further improve the a b i l i t y to estimate the dietary requirements for Se i n dairy c a t t l e . 5. Tracer enrichment was s i m i l a r (TTMP 9.8 to 12.9) i n tissues considered to be p o t e n t i a l contributors to the endogenous f e c a l Se (serum, epithelium of the stomach, l i v e r , b i l e , pancreas, small i n t e s t i n e and colon). 6. Serum and l i v e r were selected as t i s s u e indices for the c a l c u l a t i o n of endogenous f e c a l Se and true Se absorption. The s e l e c t i o n of the index tissue was dependent on the route of tracer administration. To ensure an accurate measurement of endogenous f e c a l Se and true absorption of Se under a variety of conditions i t i s recommended these estimates be made from the analysis of serum or l i v e r with an o r a l route of tracer administration or from analysis of serum with an intravenous route of administration. 7. In dairy c a t t l e consuming a Se-supplemented d i e t containing 0.19 mg Se k g - 1 (primarily as selenite) apparent absorption and -147-true absorption was 3.2 and 11% of Se intake, respectively. In two cows fed a Se d e f i c i e n t d i e t (0.035 mg k g - 1 ) apparent absorption of Se was negative (-8 and -41%). 8. In dairy cows consuming the Se-supplemented d i e t the percentage of the f e c a l Se of endogenous o r i g i n was 9.7%. In the cows fed the Se-deficient d i e t the f e c a l Se of endogenous o r i g i n was higher and equal to 2 3 and 36% of the t o t a l f e c a l Se. 9. There was no e f f e c t of supplemental dietary Cu (17 mg kg - 1) on the endogenous f e c a l Se excretion or the true absorption of Se i n dairy cows. Thus there appears to be no Cu-Se in t e r a c t i o n occurring i n the g a s t r o i n t e s t i n a l t r a c t which would cause a reduction of the absorption of Se supplemented as sodium s e l e n i t e . -148-7. BIBLIOGRAPHY Abdel Rahim, A. G., Arthur, J. R. and M i l l s , C. F. 1986. Effects of dietary copper, cadmium, iron, molybdenum and manganese on selenium u t i l i z a t i o n by the rat. J. Nutr. 116: 403-411. Agriculture Research Council. 1980. Pages 249-250 i n The nutrient requirements of li v e s t o c k . ARC, Commonwealth A g r i c u l t u r a l Bureaux, Slough, United Kingdom. Ammerman, C. B. and M i l l e r , S. M. 1974. Selenium i n ruminant n u t r i t i o n : a review. J . Dairy S c i . 58: 1561-1577. Association of O f f i c i a l A n a l y t i c a l Chemists. 1980. O f f i c i a l methods of analysis. 13th ed. AOAC, Washington, D.C. Barbezat, G. O., Casey, C. E., Reasbeck, P. G., Robinson, M. F. and Thomson, C. D. 1984. Selenium. Pages 231-258 i n N. W. Solomons and I. H. Rosenberg, eds. Absorption and malabsorption of mineral nutrients. Alan R. L i s s , Inc., New York. Behne, D., Hofer-Bosse, T. and Elger, W. 1987. Selenium and hormones i n the male reproductive system. Pages 733-739 i n G. F. Combs, J r . , 0. A. Levander, J. E. Spallholz and J. E. O l d f i e l d , eds. Selenium i n biology and medicine. Part B. Van Nostrand Reinhold Co., New York. B e i l s t e i n , M. A., Tripp, M. J. and Whanger, P. D. 1981. Evidence for selenocysteine i n ovine tissue organelles. J . inorg. Biochem. 15: 339-347. B e i l s t e i n , M. A., Butler, J. A. and Whanger, P. D. 1984. 75 Metabolism of Se-selenite by rhesus monkeys. J . Nutr. 114: 1501-1509. B e i l s t e i n , M. A. and Whanger, P. D. 19 86. Chemical forms of selenium i n rat tissues after administration of s e l e n i t e or selenomethionine. J . Nutr. 116: 1711-1719. B e i l s t e i n , M. A. and Whanger, P. D. 1987. D i s t r i b u t i o n of selenium i n erythrocyte fractions of humans and animals. Pages 265-277 i n G. F. Combs, J r . , 0. A. Levander, J . E. Spallholz and J . E. O l d f i e l d , eds. Selenium i n biology and medicine. Part A. Van Nostrand Reinhold Co., New York. Boyne, R. and Arthur, J. R. 1979. Alterations of neutrophil function i n selenium d e f i c i e n t c a t t l e . J. Comp. Pathol. 89: 151-158. -149-Bremner, I. and Davies, N. T. 1980. Dietary composition and absorption of trace elements by ruminants. Pages 409-427 i n Y. Ruckebusch and P. Thivend, eds. Digestive physiology and metabolism i n ruminants. MTP Press Ltd., Lancaster, England. Brown, D. G. and Burk, R. F. 1973. Selenium retention i n tissues and sperm of rats fed a torula yeast d i e t . J . Nutr. 10 3: 102-108. Buckley, W. T., Huckin, S. N. and Budac, J. J . 1982. Mass spectrometric determination of a stable tracer for copper i n b i o l o g i c a l materials. Anal. Chem. 54: 504-510. Buckley, W. T., Eigendorf, G. K. and Dorward, W. J. 1986. A l i v e r biopsy instrument for large animals. Can. J . Anim. S c i . 66: 1137-1140 Buckley, W. T., Godfrey, D. V., Koenig, K. M. and Shelford, J. A. 1987. Determination of selenium and multiple stable isotope enrichment of selenium i n b i o l o g i c a l materials by inductively coupled plasma mass spectrometry. In L. S. Hurley, C. L. Keen, B. Lonnerdal and R. B. Rucker, eds. Trace elements i n man and animals - TEMA 6. Plenum Publishing Co., New York. In press. Buckley, W. T. 1987. The use of stable isotopes i n studies of mineral metabolism. Proc. Nutr. Soc. In press. Butler, G. W. and Peterson, P. J. 1961. Aspects of f e c a l excretion of selenium by sheep. N. Z. J. Agric. Res. 4: 484-491. Calvin, H. I., Cooper, G. W. and Wallace, E. 1981. Evidence that selenium i n rat sperm i s associated with a cy s t e i n e - r i c h s t r u c t u r a l protein of the mitochondrial capsule. Gamete Res. 4: 139-149. Cardin, C. J . and Mason, J. 1975. Sulphate transport by rat ileum. E f f e c t of molybdate and other anions. Biochim. Biophys. Acta. 394: 46-54. Cary, E. E., Allaway, W. H. and M i l l e r , M. 1973. U t i l i z a t i o n of d i f f e r e n t forms of dietary selenium. J . Anim. S c i . 36: 285-292. Cathcart, E. B., Shelford, J. A. and Peterson, R. G. 1980. Mineral analyses of dairy c a t t l e feed i n the Upper Fraser Valley of B r i t i s h Columbia. Can. J. Anim. S c i . 60: 177-183. Church, D. C. 1984. Pages 123-126 i n Livestock feeds and feeding. 2nd ed. 0 and B Books, Inc., C o r v a l l i s , Oregon. -150-Combs, G. F., J r . and Combs, S. B. 1984. The n u t r i t i o n a l biochemistry of selenium. Ann. Rev. Nutr. 4: 257-280. Conrad, H. R. 1985. The role of selenium and vitamin E i n bovine reproduction. Pages 26-30 i n Selenium responsive diseases i n food animals. Veterinary Learning Systems Co., Inc., United States. Cousins, F. B. and Cairney, I. M. 1961. Some aspects of selenium metabolism i n sheep. Aust. J . Agric. Res. 12: 927-943. C r u t c h f i e l d , W. 0. 1968. A technique for placement of an indwelling catheter i n the cow. Vet. Med./Sm. Anim. C l i n . Dec. pp. 1141-1144. Davidson, W. B. and McMurray, C. H. 1987. D e f i n i t i o n of plasma selenium proteins using two-dimensional electrophoresis. Pages 278-282 i n G. F. Combs, J r . , 0. A. Levander, J. E. Spallholz and J. E. O l d f i e l d , eds. Selenium i n biology and medicine. Part A. Van Nostrand Reinhold Co., New York. Dejneka, J . , Nowosad, R. and Simoni, J. 1979. Badania nad 75 rozmieszczeniem radioselenu Se w tkankach i t r e s c i zwacza oraz jego wydalaniem z z o l c i a , moczem i kalem u owiec. Polskie Archiwum Weterynaryjne 21: 237-248. Dilworth, G. L. and Bandurski, R. S. 1977. A c t i v a t i o n of selenate by adenosine-5'-triphosphate sulfurylase from Saccharomyces  cere v i s i a e . Biochem. J. 163: 521-529. Diplock, A. T. 1984. Vitamin E, selenium, and free r a d i c a l s . Med. B i o l . 62: 78-80. Diplock, A. T. 1985. The role of selenium i n the control of oxygen-induced c e l l u l a r damage. Nutr. Res. (Suppl. I ) : 175-179. Diplock, A. T. 1987. Metabolic defences against oxygen and xenobiotic t o x i c i t y : biochemical i n t e r r e l a t i o n s h i p s . Pages 90-103 i n G. F. Combs, J r . , 0. A. Levander, J. E. Spallholz and J. E. O l d f i e l d , eds. Selenium i n biology and medicine. Part A. Van Nostrand Reinhold Co., New York. Doyle, J . 1979. Toxic and e s s e n t i a l elements i n bone - a review. J . Anim. S c i . 49: 482-497. Ensminger, M. E. and Olentine, C. G., J r . 1978. Pages 1256-1257 i n Feeds and n u t r i t i o n - complete. Ensminger Publishing Co., C l o v i s , C a l i f o r n i a . -151-Epp, 0., Ladenstein, R., and Wendel, A. 1983. The refined structure of the selenoenzyme glutathione peroxidase at 0.2-nm resolution. Eur. J. Biochem. 133: 51-69. Esaki, N., Nakamura, T., Tanaka, H. and Soda, K. 1982. Selenocysteine lyase, a novel enzyme that s p e c i f i c a l l y acts on selenocysteine. J. B i o l . Chem. 257: 4386-4391. Evans, G. W., Johnson, E. C. and Johnson, P. E. 197 9. Zinc absorption i n the rat determined by radioisotope d i l u t i o n . J . Nutr. 109: 1258-1264. Fehrs, M. S., M i l l e r , W. J . , Gentry, R. P., Neathery, M. W., Blackmon, D. M. and Heinmiller, S. R. 1981. E f f e c t of high but non toxic dietary intake of copper and selenium on metabolism i n calves. J . Dairy S c i . 64: 1700-1706. Fenimore, R. L., Adams, D. S. and Puis, R. 19 83. Selenium l e v e l s of beef c a t t l e i n southeastern B r i t i s h Columbia r e l a t i v e to supplementation and type of pasture. Can. Vet. J. 24: 41-45. Forstrom, J . W., Zakowski, J . J. and Tappel, A. L. 1978. I d e n t i f i c a t i o n of the c a t a l y t i c s i t e of rat l i v e r glutathione peroxidase as selenocysteine. Biochem. 17: 2639-2644. Ganther, H. E., Levander, 0. A. and Baumann, C. A. 1966. Dietary control of selenium v o l a t i l i z a t i o n i n the rat. J . Nutr. 88: 55-60. Ganther, H. E. 1975. Selenoproteins. Chemica Scripta 8A: 79-84. Ganther, H. E. 1984. Selenium metabolism and function i n man and animals. Pages 3-24 i n Trace element a n a l y t i c a l chemistry i n medicine and biology. Vol. 3. Walter de Gruyter and Co., B e r l i n . Ganther, H. E. 1987. Chemistry and metabolism of selenium. Pages 53-65 i n G. F. Combs, J r . , 0. A. Levander, J . E. Spallholz and J . E. O l d f i e l d , eds. Selenium i n biology and medicine. Part A. Van Nostrand Reinhold Co., New York. Gasiewicz, T. A. and Smith, J. C. 1978. The metabolism of s e l e n i t e by i n t a c t rat erythrocytes i n v i t r o . Chem. B i o l . Interact. 21: 299-313. Gooneratne, S. R. and Howell, J. McC. 1981. Selenium i n copper t o x i c i t y i n sheep. Pages 468-470 i n J. M. Gawthorne, J. McC. Howell and C. L. White, eds. Trace elements i n man and animals - TEMA 4. Springer-Verlage, New York. -152-Grace, N. D. and Watkinson, J. H. 1985. The d i s t r i b u t i o n and amounts of Se associated with various tissues and liveweight gains of grazing sheep. Pages 490-493 i n C. F. M i l l s , I. Bremner and J . K. Chesters, eds. Trace elements i n man and animals - TEMA 5. Commonwealth A g r i c u l t u r a l Bureaux, Slough, United Kingdom. Gregus, Z. and Klaassen, C. D. 1986. Disposition of metals i n rats: a comparative study of f e c a l , urinary and b i l a r y excretion and tissu e d i s t r i b u t i o n of eighteen metals. Tox. Appl. Pharmacol. 85: 24-38. G r i f f i t h s , N. M., Stewart, R. D. H. and Robinson, M. F. 1976. The 7 5 metabolism of [ Se]selenomethionine i n four women. Br. J. Nutr. 35: 373-382. H a l l i w e l l , B. and Gutteridge, J. M. C. 1984. Oxygen t o x i c i t y , oxygen r a d i c a l s , t r a n s i t i o n metals and disease. Biochem. J. 219: 1-14. Handbook of chemistry and physics, 5 l s t ed. 1970. R. C. Weast, ed. Chemical Rubber Co., Ohio. Handreck, K. A. and Godwin, K. 0. 1970. D i s t r i b u t i o n i n the sheep 75 of selenium derived from Se-labelled ruminal p e l l e t s . Aust. J . Agric. Res. 21: 71-84. Harrison, J . H., Hancock, D. D. and Conrad, H. R. 1984. Vitamin E and selenium for reproduction of the dairy cow. J. Dairy S c i . 67: 123-132. Harrison, J. H. and Conrad, H. R. 1984a. E f f e c t of selenium intake on selenium u t i l i z a t i o n by the nonlactating dairy cow. J . Dairy S c i . 67: 219-223. Harrison, J . H. and Conrad, H. R. 1984b. E f f e c t of dietary calcium on selenium absorption by the nonlactating dairy cow. J. Dairy S c i . 67: 1860-1864. Hawkes, W. C , Lyons, D. E. and Tappel, A. L. 19 82. I d e n t i f i c a t i o n of a selenocysteine-specific aminoacyl transfer RNA from rat l i v e r . Biochim. Biophys. Acta. 699: 183-191. Hawkes, W. C , Wilhelmsen, E. C. and Tappel, A. L. 1985. Abundance and tissue d i s t r i b u t i o n of selenocysteine-containing proteins i n the rat. J. Inorg. Biochem. 23: 77-92. -153-Hidiroglou, M., Heaney, D. P. and Jenkins, K. J. 1968. Metabolism of inorganic selenium i n rumen bacteria. Can. J . Physiol. Pharmacol. 46: 229-232. Hidiroglou, M. and Jenkins, K. J . 1972. Le sort du radioselenium administre dans l e rumen ou dans l a c a i l l e t t e du mouton. Ann. B i o l . Anim. Bioch. Biophys. 12: 599-616. Hidiroglou, M. and Jenkins, K. J. 1973a. Absorption of 7 5 Se-selenomethionine from the rumen of sheep. Can. J . Anim. S c i . 53: 345-347. Hidiroglou, M. and Jenkins, K. J. 1973b. Fate of 75 Se-selenomethionine i n the g a s t r o i n t e s t i n a l t r a c t of sheep. Can. J. Anim. S c i . 53: 527-536. Hidiroglou, M., Jenkins, K. J. and Knipf e l , J. E. 1974. Metabolism of selenomethionine i n the rumen. Can. J. Anim. S c i . 54: 325-330. H i l l , C. H. 1974. Reversal of selenium t o x i c i t y i n chicks by mercury, copper and cadmium. J . Nutr. 104: 593-598. H i l l , C. H. 1975. Interrelationships of selenium with other trace elements. Fed. Proc. 34: 2096-2100. Humaloja, T. and Mykkanen, H. M. 1986. I n t e s t i n a l absorption of 75 Se-labeled sodium selenite and selenomethionine i n chicks: e f f e c t of time, segment, selenium concentration and method of measurement. J. Nutr. 116: 142-148. Jackson, M. J . , Jones, D. A., Edwards, H. T., Coleman, M. L. and Swainbank, I. G. 19 84. A new stable isotope technique for the measurement of zinc absorption and g a s t r o i n t e s t i n a l excretion i n man. Nutr. Res. (Suppl. I ) : 88-91. Janghorbani, M., Ting, B. T. G. and Young, V. R. 1981. Use of stable isotopes of selenium i n human metabolic studies: development of a n a l y t i c a l methodology. Am. J. C l i n . Nutr. 34: 2816-2830. Janghorbani, M., Christensen, M. J . , Nahapetian, A. and Young, V. R. 1982a. Selenium metabolism i n healthy adults: 7 4 2 — quantitative aspects using stable isotope S e 0 3 • J« C l i n . Nutr. 35: 647-654. Janghorbani, M., Ting, B. T. G., Nahapetian, A. and Young, V. R. 1982b. Conversion of urinary selenium to selenium(IV) by wet oxidation. Anal. Chem. 54: 1188-1190. Janghorbani, M. 1984. Stable isotopes i n n u t r i t i o n and food science. Progress Food Nutr. S c i . 8: 303-332. Janghorbani, M., Young, V. R. and Ehrenkranz, R. A. 1985. Isotopic methods i n the study of mineral metabolism of infants with sp e c i a l reference to stable isotopes. Pages 6 3-85 i n R. K. Chandra, ed. Trace elements i n n u t r i t i o n of children. Nestle N u t r i t i o n , Vevey/Raven Press, New York. Jenkins, K. J . and Hidiroglou, M. 1972. A review of selenium/vitamin E responsive problems i n l i v e s t o c k : a case for selenium as a feed additive i n Canada. Can. J . Anim. S c i . 52: 591-620. Jenkins, K. J . , Hidiroglou, M., Wauthy, M. and Proulx, J. E. 1974. Prevention of n u t r i t i o n a l muscular dystrophy i n calves and lambs by selenium and vitamin E additions to the maternal mineral supplement. Can. J. Anim. S c i . 54: 49-60. Jensen, L. S. 1975a. Modification of a selenium t o x i c i t y i n chicks by dietary s i l v e r and copper. J. Nutr. 105: 769-755. Jensen, L. S. 1975b. P r e c i p i t a t i o n of a selenium deficiency by high dietary l e v e l s of copper and zinc (38754). Proc. Soc. Exp. B i o l . Med. 149: 113-116 J u l i e n , W. E., Conrad, H. R., Jones, J. E. and Moxon, A. L. 1976a. Selenium and vitamin E and incidence of retained placenta i n parturient dairy cows. J . Dairy S c i . 59: 1954-1959. J u l i e n , W. E., Conrad, H. R. and Moxon, A. L. 1976b. Selenium and vitamin E and incidence of retained placenta i n parturient dairy cows. I I . Prevention i n commercial herds with prepartum treatment. J. Dairy S c i . 59: 1960-1962. Kaneko, J . J . , Zinkle, J. G. and Keeton, K. S. 1971. Erythrocyte porphyrin and erythrocyte s u r v i v a l i n bovine erythropoietic porphyria. Am. J. Vet. Res. 32: 1981-1985. Kincaid, R. L., M i l l e r , W. J . , Neathery, M. W., Gentry, R. P. and Hampton, D. L. 1977. E f f e c t of added dietary selenium on metabolism and tissue d i s t r i b u t i o n of radioactive and stable selenium i n calves. J. Anim. S c i . 44: 147-151. Kiremidjian-Schumacher, L. and Stotzky, G. 1987. Reveiw. Selenium and immune response. Environ. Res. 42: 277-30 3. Langlands, J . P., Bowles, J. E., Donald, G. E., Smith, A. J . , P a u l l , D. R. and Davies, H. I. 1982. Deposition of copper, manganese, selenium and zinc i n the ovine foetus and associated t i s s u e s . Aust. J . Agric. Res. 33: 591-605. -155-Langlands, J. P., Bowles, J. E., Donald, G. E. and Smith, A. J. 1986. Selenium excretion i n sheep. Aust. J. Agric. Res. 37: 201-209. Lawrence, R. A., Sunde, R. A., Schwartz, G. L. and Hoekstra, W. G. 1974. Glutathione peroxidase a c t i v i t y i n rat lens and other tissues i n r e l a t i o n to dietary selenium intake. Exp. Eye Res. 18: 563-569. Levander, 0. A. 1987. A global view of human selenium n u t r i t i o n . Ann. Rev. Nutr. 7: 227-250. Lopez, P. L., Preston, R. L. and Pfander, W. H. 1969. Whole-body retention, tissue d i s t r i b u t i o n and excretion of selenium-75 a f t e r o r a l and intravenous administration i n lambs fed varying selenium intakes. J . Nutr. 97: 123-132. Matthews, C. A., Swett, W. W. and McDowell, R. E. 1975. External form and i n t e r n a l anatomy of holsteins and jerseys. J. Dairy S c i . 58: 1453-1475. McConnell, K. P. and Cho, G. J . 1965. Transmucosal movement of selenium. Am. J. Physiol. 208: 1191-1195. McConnell, K. P. and Roth, D. M. 1966. Respiratory excretion of selenium. Proc. Soc. Exp. B i o l . Med. 123: 919-921. McConnell, K. P. and Cho, G. J . 1967. Active transport of selenium i n the everted i n t e s t i n e of the hamster. Pages 329-343 i n 0. H. Muth, J . E. O l d f i e l d and P. H. Weswig, eds. Selenium i n biomedicine. AVI Publishing Co., Inc., Westport, Connecticut. McMurray, C. H. and Davidson, W. B. 1985. Towards a selenium model - time course of selenium binding to plasma and red c e l l s i n sheep blood aft e r intravenous administration of 75 Se s e l e n i t e . Pages 474-480 i n C. F. M i l l s , I. Bremner and J . K. Chesters, eds. Trace elements i n man and animals -TEMA 5. Commonwealth A g r i c u l t u r a l Bureaux, Slough, United Kingdom. Miltimore, J . E., Ryswyk, A. L., Pringle, W. L., Chapman, F. M. and Kalnin, C. M. 1975. Selenium concentrations i n B r i t i s h Columbia forages, grains and processed feeds. Can. J. Anim. S c i . 55: 101-111. Moksnes, K. and Norheim, G. 1983. Selenium and glutathione peroxidase l e v e l s i n lambs receiving feed supplemented with sodium se l e n i t e or selenomethionine. Acta. Vet. Scand. 24: 45-58. -156-Motsenbocker, M. A. and Tappel, A. L. 1982a. Selenocysteine-containing proteins from rat and monkey plasma. Biochim. Biophys. Acta. 704: 253-260. Motsenbocker, M. A. and Tappel, A. L. 1982b. A selenocysteine-containing selenium-transport protein i n rat plasma. Biochim. Biophys. Acta. 719: 147-153. Nahapetian, A. T., Janghorbani, M. and Young, V. R. 198 3. Urinary trimethylselenonium excretion by the rat. J . Nutr. 113: 401-411. National Academy of Sciences - National Research Council. 1978. Nutrient requirements of dairy c a t t l e . 5th ed. NAS-NRC, Washington, D.C. National Academy of Sciences - National Research Council. 1983. Selenium i n n u t r i t i o n . NAS-NRC, Washington, D.C. National Academy of Sciences - National Research Council. 1988. Nutrient requirements of dairy c a t t l e . 6th ed. NAS-NRC, Washington, D.C. In press. Oh, S. H., Ganther, H. E. and Hoekstra, W. G. 1974. Selenium as a component of glutathione peroxidase i s o l a t e d from ovine erythrocytes. Biochem. 13: 1825-1829. Oh, S. H., Pope, A. L. and Hoekstra, W. G. 1976a. Glutathione peroxidase response to selenium intake i n lambs fed a torula yeast-based a r t i f i c i a l milk. J . Anim. S c i . 42: 977-983. Oh, S. H., Pope, A. L. and Hoekstra, W. G. 1976b. Dietary selenium requirement of sheep fed a practic a l - t y p e d i e t as assessed by tissue glutathione peroxidase and other c r i t e r i a . J . Anim. S c i . 42: 984-992. Palmer, I. S., Fischer, D. D., Halverson, A. W. and Olson, 0. E. 1969. I d e n t i f i c a t i o n of a major selenium excretory product i n rat urine. Biochem. Biophys. Acta. 177: 336-332. Palmer, I. S., Gunsalus, R. P., Halverson, A. W. and Olson, O. E. 1970. Trimethylselenonium ion as a general excretory product from selenium metabolism i n the rat. Biochim. Biophys. Acta. 208: 260-266. Patterson, E. L., Milstrey, R. and Stokstad, E. L. R. 1957. E f f e c t of selenium i n preventing exudative diathesis i n chicks. Proc. Soc. Exp. B i o l . Med. 95: 617-620. Paulson, G. D., Baumann, C. A. and Pope, A. L. 1966. Fate of a phys i o l o g i c a l dose of selenate i n the l a c t a t i n g ewe: ef f e c t of s u l f a t e . J . Anim. S c i . 25: 1054-1058. -157-Paulson, G. D., Baumann, C. A. and Pope, A. L. 1968. Metabolism 75 75 75 of Se-selenite, Se-selenate, Se-selenomethionine 35 and S-sulfate by rumen microorganisms i n v i t r o . J. Anim. S c i . 27: 497-504. Pearson, E. G. and Craig, A. M. 1980. The diagnosis of l i v e r disease i n equine and food animals. Mod. Vet. Pract. March pp. 233-237 and A p r i l pp. 315-320. Pedersen, N. D., Whanger, P. D., Weswig, P. H. and Muth, 0. H. 1972. Selenium binding proteins i n tissues of normal and selenium-responsive myophathic lambs. Bioinorg. Chem. 2: 33-45. Perry, T. W. , Caldwell, D. M. and Peterson, R. C. 1976. Selenium content of feeds and e f f e c t of dietary selenium on hair and blood serum. J. Dairy S c i . 59: 760-763. Peter, D. W., Whanger, P. D., Lindsay, J. P. and Busc a l l , D. J. 1982. Excretion of selenium, zinc and copper by sheep receiving continuous intraruminal infusions of se l e n i t e or selenomethionine. Proc. Nutr. Soc. Aust. 7: 178-181. Peter, D. W., Young, P., Buscall, D. J . and Whanger, P. D. 1985. Selenium retention and concentrations i n sheep given s e l e n i t e or selenomethionine - anomalies, an apparent explanation and implications. Pages 484-487 i n C. F. M i l l s , I. Bremner and J. K. Chesters, eds. Trace elements i n man and animals - TEMA 5. Commonwealth A g r i c u l t u r a l Bureaux, Slough, United Kingdom Peterson, P. J . and Spedding, D. J . 196 3. The excretion by sheep 75 of Se incorporated into red clover (Trifolium pratense L.): the chemical nature of excreted selenium and i t s uptake by three plant species. N. Z. J. Agric. Res. 6: 13-23. Pickup, J . F. and McPherson, K. 1976. Theoretical considerations i n stable isotope d i l u t i o n mass spectrometry for organic analysis. Anal. Chem. 48: 1885-1890. Prohaska, J . R. and Ganther, H. E. 1977. Glutathione peroxidase a c t i v i t y of glutathione transferases p u r i f i e d from rat l i v e r . Biochem. Biophys. Res. Commun. 76: 437-445. Puis, R. 1981. Veterinary trace mineral deficiency and t o x i c i t y information. Publ. 5139. Agriculture Canada, Ottawa, Ontario. Reamer, D. C. and V e i l l o n , C. 1981. Determination of selenium i n b i o l o g i c a l materials by stable isotope d i l u t i o n gas chromatography-mass spectrometry. Anal. Chem. 53: 2166-2169. -158-Reamer, D. C. and V e i l l o n , C. 1983. A double isotope d i l u t i o n method for using stable selenium isotopes i n metabolic tracer studies: analysis by gas chromatography/mass spectrometry (GC/MS). J . Nutr. 113: 786-792. Rotruck, J . T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G. and Hoekstra, W. G. 197 3. Selenium: biochemical role as a component of glutathione peroxidase. Science 179: 588-590. Sandholm, M. 1973. The i n i t i a l fate of a trace amount of intravenously administered s e l e n i t e . Acta. Pharmacol, et. Toxicol. 33: 1-5. Scholz, R. W. and Hutchinson, L. J. 1979. D i s t r i b u t i o n of glutathione peroxidase a c t i v i t y and selenium i n the blood of dairy cows. Am. J. Vet. Res. 40: 245-249. Scholz, R. W., Todhunter, D. A. and Cook, L. S. 1981a. Selenium content and glutathione peroxidase a c t i v i t y i n tissues of young c a t t l e fed supplemented whole milk d i e t s . Am. J . Vet. Res. 42: 1718-1723. Scholz, R. W., Cook, L. S. and Todhunter, D. A. 1981b. D i s t r i b u t i o n of selenium-dependent and nonselenium-dependent glutathione peroxidase a c t i v i t y i n tissues of young c a t t l e . Am. J . Vet. Res. 42: 1724-1728. Schwarz, K. and F o l t z , C. M. 1957. Selenium as an i n t e g r a l part of factor 3 against dietary necrotic l i v e r degeneration. J. Am. Chem. Soc. 79: 3292-3293. Smith, K. L., Harrison, J. H., Hancock, D. D., Todhunter, D. A. and Conrad, H. R. 1984. E f f e c t of vitamin E and selenium supplementation on incidence of c l i n i c a l mastitis and duration of c l i n i c a l symptoms. J. Dairy S c i . 67: 1293-1300. Soder, M. 1984. Average analyses of B r i t i s h Columbia Feeds 1969-1984. D i s t r i c t : Fraser Valley. B r i t i s h Columbia Ministry of Agriculture and Food, V i c t o r i a , B r i t i s h Columbia. Stewart, R. D. H., G r i f f i t h s , N. M., Thomson, C. D. and Robinson, M. F. 1978. Quantitative selenium metabolism i n normal New Zealand women. Br. J. Nutr. 40: 45-54. Sunde, R. A. and Hoekstra, W. G. 1980. Incorporation of selenium from s e l e n i t e and selenocysteine into glutathione peroxidase i n the isola t e d perfused rat l i v e r . Biochem. Biophys. Res. Commun. 93: 1181-1188. -159-Swanson, C. A., Reamer, D. C., V e i l l o n , C., King, J . C. and Levander, 0. A. 1983. Quantitative and q u a l i t a t i v e aspects of selenium u t i l i z a t i o n i n pregnant and nonpregnant women: an ap p l i c a t i o n of stable isotope methodology. Am. J. C l i n . Nutr. 38: 169-180. Symonds, H. W., Sansom, B. F., Mather, D. L. and Vagg, M. J. 1981a. Selenium metabolism i n the dairy cow: the influence of the l i v e r and the e f f e c t of the form of Se s a l t . Br. J. Nutr. 45: 117-125. Symonds, H. W., Mather, D. L. and Vagg, M. J. 1981b. The excretion of selenium i n b i l e and urine of steers: the influence of form and amount of Se s a l t . Br. J. Nutr. 46: 487-493. Tappel, A. L. 1987. Glutathione peroxidase and other selenoproteins. Pages 122-132 i n G. F. Combs, J r . , 0. A. Levander, J . E. Spallholz and J . E. O l d f i e l d , eds. Selenium i n biology and medicine. Part A. Van Nostrand Reinhold Co., New York. Thomson, C. D. and Stewart, R. D. H. 1973. Metabolic studies of 7 5 7 5 [ Se]selenomethionine and [ Se]selenite i n the rat. Br. J. Nutr. 30: 139-147. Thomson, C. D. and Stewart, R. D. H. 1974. The metabolism of 75 [ Se]selenite i n young women. Br. J. Nutr. 32: 47-57. Thompson, K. G., Fraser, A. J . , Harrop, B. M. and Kirk, J. A. 1980. Glutathione peroxidase a c t i v i t y i n bovine serum and erythrocytes i n r e l a t i o n to selenium concentrations of blood, serum and l i v e r . Res. Vet. S c i . 28: 321-324. Thompson, K. G., Fraser, A. J . , Harrop, B. M., Kirk, J . A., Bul l i a n s , J . and Cordes, D. 0. 1981. Glutathione peroxidase a c t i v i t y and selenium concentration i n bovine blood and l i v e r as indicators of dietary selenium intake. N. Z. Vet. J. 29: 3-6. Trinder, N., Woodhouse, C. D. and Renton, C. P. 1969. The ef f e c t of vitamin E and selenium on the incidence of retained placentae i n dairy cows. Vet. Rec. 85: 550-553. Ul l r e y , D. E., Brady, P. S., Whetter, P. A., Ku, P. K. and Magee, W. T. 1977. Selenium supplementation of diets for sheep and beef c a t t l e . J . Anim. S c i . 46: 559-565. Underwood, E. J. 1977. Pages 302-346 i n Trace elements i n human and animal n u t r i t i o n . 4th ed. Academic Press, New York. -160-Underwood, E. J. 1981. Selenium. Pages 149-166 i n Mineral n u t r i t i o n of liv e s t o c k . 2nd ed. Commonwealth A g r i c u l t u r a l Bureaux, Age Bros. (Norwich) Ltd., London. Van R i j , A. M., Thomson, C. D., McKenzie, J. M. and Robinson, M. F. 197 9. Selenium deficiency i n t o t a l parenteral n u t r i t i o n . Am. J. C l i n . Nutr. 32: 2076-2085. Van Soest, J . P. 1963. A rapid method for determination of f i b e r and l i g n i n . J . Assn. O f f i c a l . Anal. Chem. 46: 829. Van Vleet, J . F. 1980. Current knowledge of selenium-vitamin E deficiency i n domestic animals. J. Am. Vet. Med. Assn. 176: 321-325. Van Vleet, J . F. and Boon, G. D. 1980. Evaluation of the a b i l i t y of dietary supplements of s i l v e r , copper, cobalt, tellurium, cadmium, zinc, and vanadium to induce lesions of selenium-vitamin E deficiency i n ducklings and swine. Pages 366-372 i n J. E. Spallholz, J . L. Martin and H. E. Ganther, eds. Selenium i n biology and medicine. AVI Publishing Co., Inc., Westport, Connecticut. Vijan, P. N. and Leung, D. 1980. Reduction of chemical interference and speciation studies i n the hydride generation-atomic absorption method for selenium. Analytica Chimica Acta. 120: 141-146. Whanger, P. D., Pedersen, N. D., H a t f i e l d , J. and Weswig, P. H. 1976. Absorption of selenite and selenomethionine from l i g a t e d digestive t r a c t segments i n rats. Proc. Soc. Exp. B i o l . Med. 153: 295-297. Whanger, P. D. and Weswig, P. H. 1978. Influence of 19 elements on development of l i v e r necrosis i n selenium and vitamin E d e f i c i e n t rats. Nutr. Rep. Inter. 18: 421-428. Whanger, P. D., Weswig, P. H. and O l d f i e l d , J . E. 1978. Selenium, s u l f u r and nitrogen l e v e l s i n ovine rumen microoganisms. J. Anim. S c i . 46: 515-519. White, C. L., Cloninger, R. W., Hansen, J. A., Hoekstra, W. G. and Pope, A. L. 1979. Species variations i n copper-selenium in t e r a c t i o n s . Proc. Nutr. Soc. Aust. 4: 148. White, C. L., Hoekstra, W. G. and Pope, A. L. 1981. The e f f e c t of 75 copper and molybdenum on Se-selenomethionine metabolism i n sheep. Pages 561-563 i n J. M. Gawthorne, J . McC. Howell and C. L. White, eds. Trace elements i n man and animals -TEMA 4. Springer-Verlage, New York. -161-Wilson, L. G. and Bandurski, R S. 1958. Enzymatic reactions involving s u l f a t e , s u l f i t e , selenate and molybdate. J. B i o l . Chem. 223: 975-981. Wilson, P. S. and Judson, G. J. 1976. Glutathione peroxidase a c t i v i t y i n bovine and ovine erythrocytes i n r e l a t i o n to blood selenium concentration. Br. Vet. J. 132: 428-434. Wolffram, S., Arduser, P. and Scharrer, E. 1985. In vivo i n t e s t i n a l absorption of selenate and s e l e n i t e by rats. J. Nutr. 115: 454-459. Wright, P. L. and B e l l , M. C. 1966. Comparative metabolism of selenium and tellurium i n sheep and swine. Am. J . Physiol. 211: 6-10. Wu, S. H., O l d f i e l d , J . E., whanger, P. D. and Weswig, P. H. 1973. E f f e c t of selenium, vitamin E, and antioxidants on t e s t i c u l a r function i n rats. B i o l . Reprod. 8: 625-629. Yang, G. Q. 1985. Keshan disease: an endemic selenium-related deficiency disease. Pages 273-290 i n R. K. Chandra, ed. Trace elements i n n u t r i t i o n of children. Nestle N u t r i t i o n , Vevey/Raven Press, New York. -162-8. APPENDIX Table 1. Isotope composition of natural abundance Se | and enriched isotope preparations % (atomic percentage) Se-76 Se-77 Se-78 Se-82 Isotope n a S e (194802) (194901) (199901) (195201) 7 4 S e 0.87 0.08 0.03 0.06 0.12 7 6 S e 9.02 96.48 1.20 0.77 0.24 7 7 S e 7.58 0.83 94.75 0.37 0.53 7 8 S e 23.52 1.06 2.37 97.27 0.74 8 0 S e 49.82 1.38 1.49 1.42 1.70 8 2 S e 9.19 0.17 0.16 0.11 96.66 From the Handbook of Chemistry and Physics (1970). Spe c i f i c a t i o n s for enriched stable isotopes supplied by Oak Ridge National Laboratory, Oak Ridge, TN. 

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