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Carnitine and carnitine acetyltransferase; their role in development Seccombe, David William 1981

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CARNITINE AND CARNITINE ACETYLTRANSFERASE: THEIR ROLE IN DEVELOPMENT by DAVID WILLIAM SECCOMBE, B . A . , M.Sc. Department of Physiology, Un ivers i ty of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in Physiology We accept th is thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA FEBRUARY , 1 9 8 1 Copyright: David Seccombe In present ing t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I fur ther agree that permission for extensive copying of t h i s t h e s i s for s c h o l a r l y purposes may be granted by the head of my department or by h i s or her representa t ives . It i s understood that copying or p u b l i c a t i o n of t h i s thes is for f i n a n c i a l gain s h a l l not be allowed without^my wr i t ten permiss ion. Department of /Wys/0/.. <D G y The Un ivers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date /?)/?-/ 3 / / f / /7Q\ ABSTRACT Carni t ine ( ;ytrimethylamino-3-hydroxybutyrate) , a betaine der ivat ive which is widely d is t r ibuted in mammalian t i ssues , serves to f a c i l i t a t e the passage of act ivated fat ty acids into mitochondria. The carn i t ine acyl transferases (ace ty l - , oc tanoy l - , pa lmi ty l - and possibly others) es te r i fy t issue acyl-CoA to carn i t ine producing acy lcarn i t ine which read i ly passes into mitochondria. Once i n s i d e , the fat ty acids undergo j3 -oxidat ion. The purpose of this study was threefo ld: to examine changes that occur in blood levels of f ree , a c y l - and to ta l carn i t ine with starvat ion and diets varying in l i p i d content; to compare and contrast changes in blood levels of carn i t ine during the per inata l period in sheep, ra t , rabb i t , guinea pig and man; and to measure the a c t i v i t y of carn i t ine acetyl t ransferase in mitochondria and microsomes isolated from l i v e r and brown adipose t issue taken during the per inata l period from ra t , rabbit and guinea p i g . Methods for determining serum levels of f ree , a c y l - and to ta l carn i t ine were developed and used. Short-term starvat ion, in man and ra t , resul ted in a s i g n i f i c a n t decrease in serum levels of free carn i t ine and a s ign i f i can t increase in acy lca rn i t ine . Free carn i t ine levels correlated negatively with serum concentrations of D-3-hydroxybutyrate and free fat ty ac ids . In man, the renal clearance of free carn i t ine decreased while the clearance of acy lcarn i t ine increased. In the ra t , to ta l serum carn i t ine decreased s i g n i f i c a n t l y w i t h i n 24 hours of s t a r v a t i o n . By 48 hours, i t had increased s i g n i f i c a n t l y . A s i m i l a r decrease i n t o t a l serum c a r n i t i n e occurred with a long-chain t r i g l y c e r i d e d i e t . Although t o t a l c a r n i t i n e remained unchanged with a medium-chain t r i g l y c e r i d e d i e t , a c y l c a r n i t i n e l e v e l s were twice, and 3~D~hydroxybutyrate l e v e l s s i x times, higher than with the long-chain t r i g l y c e r i d e d i e t . In l a t e g e s t a t i o n , t o t a l serum c a r n i t i n e l e v e l s i n the maternal sheep and r a t were s i g n i f i c a n t l y higher than i n the f e t u s . There was no d i f f e r e n c e between maternal and f e t a l values i n the guinea p i g and r a b b i t . At day one, t o t a l c a r n i t i n e had increased s i g n i f i c a n t l y i n a l l species except the r a b b i t . L i v e r and brown adipose t i s s u e m i t o c h o n d r i a l a c t i v i t y of c a r n i t i n e a c e t y l t r a n s f e r a s e was very high i n the f e t a l r a b b i t and guinea p i g and very low i n the r a t . A c t i v i t y i n the r a t increased n e o n a t a l l y reaching a peak at day one i n the case of l i v e r and at day 10 i n the case of brown f a t . F e t a l r a b b i t and guinea p i g l e v e l s of a c t i v i t y remained unchanged at day one. In r a t , l i v e r microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y remained at low f e t a l values u n t i l weaning at which time the a c t i v i t y began to increase reaching adult l e v e l s by day 30. The data obtained i n t h i s study i n d i c a t e that the o x i d a t i o n of f a t t y acids i s r e f l e c t e d i n serum l e v e l s of f r e e and a c y l c a r n i t i n e and i n l i v e r m i t o c h o n d r i a l c a r n i t i n e a c e t y l t r a n s f e r a s e _ a c t i v i t y . ACKNOWLEDGEMENTS During the course of these studies I have had the opportunity to work with many excel lent invest igators and c lose f r i e n d s . Without the i r encouragement, guidance and patience th is work could not have been achieved. I wish to thank the Department of Physiology for the i r co-operat ion and support of th is interdepartmental study. I am indebted to Dr. Lioy and a l l the members of my advisory committee for the i r time and construct ive c r i t i c i s m s . I want to acknowledge the fol lowing ind iv idua ls for the i r • p a r t i c i p a t i o n in co l l abora t i ve s tudies: Dr. J . F roh l i ch Dr. M. Novak Dr. D. Sch i f f Dr. M. Towell A spec ia l thanks to Nada Hahn and Freda-Anne Smale for the i r excel lent t echn ica l ass is tance . To Dr. Peter Hahn, my advisor , mentor and f r iend I w i l l always be indebted. His enthusiasm, encouragement and guidance w i l remain with me forever . Without my w i f e ' s continuing love and encouragement I wou never have been able to complete th is work. • TABLE OF CONTENTS 4 ABSTRACT 1 ACKNOWLEDGEMENTS 3 LIST OF TABLES . . 7 LIST OF FIGURES 8 LIST OF ABBREVIATIONS 11 INTRODUCTION 14 REVIEW OF PERTINENT LITERATURE 25 Carnit ine 26 The synthesis of carn i t ine 26 The metabolism of carn i t ine 28 Tissue leve ls of carn i t ine 30 Carnit ine uptake by t issues 33 Blood leve ls of carn i t ine 34 Acy.lcarnit ines 36 Urinary carn i t ine 40 Carnit ine palmity l t ransferase . 42 Carnit ine palmity l t ransferase and development 44 Factors regulat ing carn i t ine palmityl t ransferase a c t i v i t y 44 Carnit ine acetyl t ransferase . . 45 Mitochondrial carn i t ine acetyl t ransferase 46 Properties of carn i t ine acetyl t ransferase 47 Extra-mitochondrial carn i t ine acetyl t ransferase 48 Carnit ine acety l t ransferase: Its development and regulat ion 49 5 METHODS 51 An automated method for the determination of L -carn i t ine in serum 52 The determination of carn i t ine using [1-1^C] acetyl-CoA 64 Determination of free carn i t ine according to the method of McGarry and Foster (189) 66 The ef fect of buffer and protein on the determination of carn i t ine in serum 67 Deproteinizat ion and hydrolysis of acy lcarn i t ine esters in serum 73 Determination of D-8-hydroxybutyrate 77 Determination of free fa t ty acids 78 Tissue preparation 79 Succinate dehydrogenase determination 82 Carni t ine acetyl t ransferase determination 86 Protein determination . 94 Iso la t ion of peroxisomes 99 RESULTS AND DISCUSSION 107 PART I Ef fect of fast ing on free carn i t ine levels in human serum and ur ine: cor re la t ion with serum leve ls of free fat ty acids and D-B-hydroxybutyrate 109 Effect of fast ing on serum levels and renal clearance of acy lcarn i t ine in man 117 The ef fect of prolonged starvat ion on serum levels of f ree , e s t e r i f i e d and to ta l carn i t ine in the rat 123 The ef fects of diet on D-/3-hydroxybutyrate and carn i t ine . serum levels in the rat 126 6 PART II Feta l and neonatal serum levels of carn i t ine and D-S-hydroxybutyrate in ra t , sheep, guinea pig and rabbit 134 Placental transfer of carn i t ine in the sheep and the guinea pig 140 Serum leve ls of free and e s t e r i f i e d carn i t ine during development in the rat . 160 Plasma carni t ine levels during intravenous feeding of the neonate 163 PART III Carnit ine acetyl t ransferase a c t i v i t y in l i v e r and brown adipose t issue of developing animals 171 CONCLUDING REMARKS 187 REFERENCES 196 7 LIST OF TABLES Table I . The Composition o f Mammalian M i l k s 22 Table I I . L - C a r n i t i n e C o n c e n t r a t i o n s i n Serum o f Normal A d u l t s 61 Ta b l e I I I . W i t h i n - r u n P r e c i s i o n Study: R e s u l t of 20 Assays of Three Aqueous L - C a r n i t i n e Standards on the Same Day 63 Table IV. The E f f e c t o f B u f f e r and Serum P r o t e i n on the L e v e l o f Background A c t i v i t y i n the D e t e r m i n a t i o n o f C a r n i t i n e A c c o r d i n g to the Method o f McGarry and F o s t e r (189) 74 Table V. The E f f e c t o f D i f f e r e n t Methods o f M i t o c h o n d r i a l S e p a r a t i o n by D i f f e r e n t i a l C e n t r i f u g a t i o n 81 Table V I . Serum and U r i n a r y C a r n i t i n e and Serum D-3-Hydroxybutyrate and Free F a t t y A c i d L e v e l s i n a Group o f V o l u n t e e r s I l l Table V I I . Changes i n the C r e a t i n i n e and Free C a r n i t i n e C l e a r a n c e s and i n the T u b u l a r R e a b s o r p t i o n o f Free L - C a r n i t i n e w i t h F a s t i n g 118 Ta b l e V I I I . E f f e c t of F a s t i n g on Serum L e v e l s and Renal H a n d l i n g o f A c y l c a r n i t i n e 120 Ta b l e IX. The E f f e c t o f S t a r v a t i o n on Serum C a r n i t i n e L e v e l s i n the Rat 125 Ta b l e X. The E f f e c t s o f D i e t on D - 3-Hydroxybutyrate and C a r n i t i n e L e v e l s i n the Rat 129 Table X I . Serum C a r n i t i n e and D-3-Hydroxybutyrate L e v e l s i n 4 Species 136 Ta b l e X I I . T r a n s f e r of L - C a r n i t i n e from Mother to Fetuses i n the Guinea P i g 159 Ta b l e X I I I . The C o n c e n t r a t i o n of Serum C a r n i t i n e i n the Rat a t D i f f e r e n t Stages o f Development 162 Table XIV. Plasma L e v e l s of C a r n i t i n e i n Newborn I n f a n t s During Intravenous and M i l k Feeds 167 Table XV. C a r n i t i n e A c e t y l t r a n s f e r a s e A c t i v i t y i n L i v e r and Brown Fat M i t o c h o n d r i a and Microsomes of R a b b i t s and Guinea P i g s 184 8 LIST OF FIGURES F i g . 1. S p e c t r a l Scan of DTNB w i t h M e r c a p t o e t h a n o l and A f t e r L - C a r n i t i n e D e t e r m i n a t i o n 59 F i g . 2. Standard Curve f o r the D e t e r m i n a t i o n of Free L - C a r n i t i n e A c c o r d i n g to the Method o f McGarry and F o s t e r (189) 68 F i g . 3. D e t e r m i n a t i o n of Free L - C a r n i t i n e i n the Presence o f 100 uM A c e t y l - d l - C a r n i t i n e A c c o r d i n g to the Method of McGarry and F o s t e r (189) 70 F i g . 4. The E f f e c t s o f P r o t e i n on the Non-Enzymatic Re d u c t i o n o f INT by PMS 84 F i g . 5. S u c c i n a t e Dehydrogenase: A c t i v i t y as a F u n c t i o n o f the Amount of M i t o c h o n d r i a l P r o t e i n P r e s e n t i n the Assay 87 F i g . 6. The E f f e c t o f EDTA, pH, DTNB, L - C a r n i t i n e and A c e t y l - C o A on the C a r n i t i n e A c e t y l t r a n s f e r a s e R e a c t i o n 90 t F i g . 7 The E f f e c t of M i t o c h o n d r i a l P r o t e i n C o n c e n t r a t i o n on C a r n i t i n e A c e t y l t r a n s f e r a s e A c t i v i t y 92 F i g . 8. L i n e a r i t y o f the C a r n i t i n e A c e t y l t r a n s f e r a s e Assay w i t h Time 95 F i g . 9. L i n e a r i t y o f the C a r n i t i n e A c e t y l t r a n s f e r a s e Assay w i t h M i t o c h o n d r i a l P r o t e i n 97 F i g . 10. F r a c t i o n a t i o n o f Rat L i v e r by I s o p y c n i c C e n t r i f u g a t i o n 100 F i g . 11. F r a c t i o n a t i o n o f Rat Brown Adipose T i s s u e by I s o p y c n i c C e n t r i f u g a t i o n 104 F i g . 12. R e l a t i o n s h i p Between Serum Free F a t t y A c i d s and Free C a r n i t i n e L e v e l s D uring S t a r v a t i o n i n Man 113 F i g . 13. R e l a t i o n s h i p Between Serum D-3-Hydroxybutyrate and Free C a r n i t i n e L e v e l s During S t a r v a t i o n i n Man 115 F i g . 14. R a t i o o f A c y l c a r n i t i n e to Free C a r n i t i n e C o n c e n t r a t i o n i n Sera of S i x F a s t i n g Humans 121 9 F i g . 15. Blood L e v e l s of F r e e , A c y l - and T o t a l C a r n i t i n e i n a Pregnant Ewe I n j e c t e d w i t h 500 mg of L - C a r n i t i n e 143 F i g . 16. Content of R a d i o a c t i v i t y i n M a t e r n a l and F e t a l Blood of the Sheep F o l l o w i n g M a t e r n a l I n j e c t i o n of (methyl-^^C) C a r n i t i n e 1 4 5 F i g . 17. R a d i o a c t i v i t y Recovered i n F e t a l and M a t e r n a l T i s s u e s of the Sheep F o l l o w i n g M a t e r n a l I n j e c t i o n of (methyl-^^C) C a r n i t i n e 147 F i g . 18. M a t e r n a l and F e t a l Serum L e v e l s of T o t a l and Free C a r n i t i n e i n the Sheep During a 59 Minute I n f u s i o n of L - C a r n i t i n e i n t o the Ewe 150 F i g . 19. F e t a l and M a t e r n a l Serum L e v e l s of Free, A c y l - and T o t a l C a r n i t i n e A f t e r I n t r a v e n o u s I n j e c t i o n o f 200 mg of D L - A c e t y l c a r n i t i n e i n t o the M a t e r n a l J u g u l a r V e i n of the Ewe 153 F i g . 20. M a t e r n a l Serum L e v e l s of Free, A c y l - and T o t a l C a r n i t i n e A f t e r the I n j e c t i o n o f 600 mg o f D L - A c e t y l c a r n i t i n e i n t o the M a t e r n a l J u g u l a r V e i n of the Ewe 155 F i g . 21. F e t a l Serum L e v e l s of Free, A c y l - and T o t a l C a r n i t i n e A f t e r the I n j e c t i o n o f 600 mg o f D L - A c e t y l c a r n i t i n e i n t o the J u g u l a r V e i n o f a Mother Sheep 157 F i g . 22. The E f f e c t o f D i e t a r y Supply o f C a r n i t i n e on the Serum L e v e l s of A c y l - and T o t a l C a r n i t i n e i n a 990 g Newborn I n f a n t 168 F i g . 23. C a r n i t i n e A c e t y l t r a n s f e r a s e A c t i v i t y i n L i v e r M i t o c h o n d r i a and Microsomes at D i f f e r e n t Stages of Development of the Rat I 7 4 F i g . 24. C a r n i t i n e A c e t y l t r a n s f e r a s e A c t i v i t y of Brown Adipose T i s s u e M i t o c h o n d r i a and Microsomes at D i f f e r e n t Stages of Development o f the Rat 17 6 10 F i g . 25. The R a t i o o f C a r n i t i n e A c e t y l t r a n s f e r a s e and S u c c i n i c Dehydrogenase A c t i v i t i e s i n M i t o c h o n d r i a to those i n Microsomes of L i v e r and Brown Adipose T i s s u e D u r i n g Development of the Rat 178 F i g . 26. C a t a l a s e A c t i v i t y i n Rat L i v e r and Brown Adipose T i s s u e D u r i n g Development 181 LIST OF ABBREVIATIONS A absorbance ACTH a d r e n o c o r t i c o t r o p h i c hormone ATP adenosine triphosphate Ba(0H)2 barium hydroxide BAT brown adipose t i s s u e BOH D-B-hyroxybutyrate C clearance Ca calcium CaCl2 calcium c h l o r i d e Car c a r n i t i n e CAT c a r n i t i n e a c e t y l t r a n s f e r a s e CoA coenzyme A CPT c a r n i t i n e p a l m i t y l t r a n s f e r a s e Cre c r e a t i n i n e CV c o e f f i c i e n t of v a r i a t i o n d o l 0 d e n s i t y at 0°C - 10°C DPM d i s i n t e g r a t i o n s per minute DTNB 5 , 5 ' - d i t h i o b i s - ( 2 - n i t r o b e n z o i c acid) EBM expressed breast milk EDTA ethylenediaminetetraacetate FFA f r e e f a t t y acids g grams HCL h y d r o c h l o r i c a c i d HEPES N-2-hydroxyethypiperazine-N 1-2-ethanesulphonic a c i d HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A Hr hours INT 2 - ( p - i o d o p h e n y l ) - 3 - ( p - n i t r o p h e n y l ) - 5 - p h e n y l t e t r a z o l i u m c h l o r i d e I.V. intravenous K potassium KCN potassium cyanide KOH potassium hydroxide MCT medium chain t r i g l y c e r i d e Mg magnesium^ MIC microsomes Min minutes MIT mitochondria Na sodium NaCl sodium c h l o r i d e NADPH dihydronicotinamide adenine d i n u c l e o t i d e phosphate NaOH sodium hydroxide NEM N-ethylmaleimide (NH4)2SC>4 ammonium sulphate PMS phenazine methosulphate SD standard d e v i a t i o n SDH s u c c i n a t e dehydrogenase SEM standard e r r o r of the mean SH s u l p h y d r y l T-1/2 turnover time TCA t r i c h l o r a c e t i c a c i d TRC tubular r e a b s o r p t i o n of c a r n i t i n e TRIS tris(hydroxymethyl)aminomethane TX100 t r i t o n X 100 U u r i n e U.B.C. U n i v e r s i t y of B r i t i s h Columbia V volume w/v weight to volume INTRODUCTION 15 There has been an increasing awareness that the type of d iet to which a newborn animal i s exposed can have long-range ef fects on i t s subsequent heal th . It has been suggested that d i e t s , other than breast milk, somehow a l te r the metabolic bias of the animal so that, when i t i s stressed by the same diet in la ter l i f e , an inappropriate metabolic response may occur. In time, th is response may resul t in pathology. It is evident that the short and long-term ef fects of d i f ferent d iets on the animal can only be determined af ter f i r s t establ ish ing the normal dietary patterns of the developing animal and the biochemical responses that accompany these dietary patterns. This study focuses on these biochemical responses in rat and, when poss ib le , in man. Emphasis is given to the per inata l period because at this time a major change in dietary pattern is f i r s t encountered by both of these species. The pattern sh i f t involves a switch from a high fe ta l carbohydrate diet to a high l i p i d diet (breast milk) neonatal ly . The e f f i c i e n t u t i l i z a t i o n of th is neonatal l i p i d d iet requires a funct ional l i p i d transport system which ensures i n t r a c e l l u l a r de l ivery of the dietary l i p i d to s i tes of biosynthesis and energy production. In this regard, the c a r n i t i n e - c a r n i t i n e acyl transferase system plays an important ro le and, therefore, has been selected for study in the present work. For purposes of organizat ion, the thesis has been divided into three par ts . Part I i s concerned with changes in serum of f ree , a c y l - and to ta l carn i t ine that occur, in rat and man, in response to dietary l i p i d . Part II considers s imi lar serum parameters during the per inata l period in ra t , rabb i t , guinea p i g , sheep and, when 16 p o s s i b l e , man. Part I I I deals with the l e v e l s of a c t i v i t y of c a r n i t i n e a c e t y l t r a n s f e r a s e i n mitochondria and microsomes i s o l a t e d from l i v e r and brown f a t . These studies were c a r r i e d out i n r a t , r a b b i t and guinea p i g , during the p e r i n a t a l p e r i o d . PART I C a r n i t i n e ( -trimethylamino-3-hydroxybutyrate) i s a substance which plays a major r o l e i n the o x i d a t i o n of l i p i d by f a c i l i t a t i n g the entry of a c t i v a t e d f a t t y acids i n t o mitochondria where they undergo 3 - o x i d a t i o n (109,188). By forming an est e r with c a r n i t i n e , the f a t t y acids are able to pass through the inner membrane of the mitochondrion and can then be f u r t h e r processed f o r the production of energy. During s t a r v a t i o n , when the r a t e of o x i d a t i o n of f a t t y acids' i s high, there i s a l a r g e increase i n the r a t e of generation of acetyl-CoA w i t h i n the l i v e r . In a d d i t i o n , t i s s u e l e v e l s of both long-chain and s h o r t - c h a i n a c y l c a r n i t i n e i n c r e a s e * . Under t h i s c o n d i t i o n , the content of c a r n i t i n e i n the l i v e r i ncreases i n some species (188,250) which suggests that there i s an increased demand f o r c a r n i t i n e by t h i s organ when the r a t e of l i p i d o x i d a t i o n i s high. The r a t e of turnover of c a r n i t i n e i n l i v e r i s high (50,63) and i t i s conceivable that l i v e r c a r n i t i n e may exchange *For the purpose of t h i s t h e s i s s h o r t - c h a i n a c y l c a r n i t i n e i s any ester of c a r n i t i n e i n which the a c y l - moiety contains up to, but not i n c l u d i n g , s i x t e e n carbon atoms. Esters i n which the a c y l - group contains s i x t e e n or more carbon atoms i s r e f e r r e d to as long-chain a c y l c a r n i t i n e . When no d i s t i n c t i o n i s made the term a c y l c a r n i t i n e i s used. 17 with the c a r n i t i n e that i s found i n blood. Yet, no s t u d i e s have been p u b l i s h e d as to whether or not t h i s i s t r u e . Few s t u d i e s are a v a i l a b l e on the e f f e c t of s t a r v a t i o n on serum l e v e l s of f r e e , a c y l -and t o t a l c a r n i t i n e . Part I examines changes in blood l e v e l s of f r e e , a c y l - and t o t a l c a r n i t i n e under c o n d i t i o n s of i n c r e a s e d f a t u t i l i z a t i o n by the body. Two such c o n d i t i o n s are s t a r v a t i o n and d i e t . I n i t i a l l y , the e f f e c t of a short-term f a s t i n man on serum l e v e l s of f r e e c a r n i t i n e , f r e e f a t t y a c i d s , and D-3-hydroxybutyrate was s t u d i e d . In p a r t i c u l a r , the f o l l o w i n g questions were posed. Is there any r e l a t i o n s h i p between these three blood parameters during s t a r v a t i o n ? What i s the e f f e c t of a short-term f a s t on the r e n a l c l e arance of f r e e c a r n i t i n e ? Are these parameters a f f e c t e d during the f a s t by an o r a l dose of c a r n i t i n e ? Next, the e f f e c t of a short-term f a s t , i n man, on the l e v e l s of a c y l c a r n i t i n e i n serum was examined. In a d d i t i o n , the r e n a l clearance of a c y l c a r n i t i n e was compared to that of f r e e c a r n i t i n e . It was f e l t that these data might help to i n d i c a t e whether or not serum c a r n i t i n e exchanges with t i s s u e c a r n i t i n e during short-term s t a r v a t i o n i n man. The e f f e c t of long-term s t a r v a t i o n was then s t u d i e d i n the r a t . The f o l l o w i n g questions were i n v e s t i g a t e d . Do the changes i n serum f r e e , a c y l - and t o t a l c a r n i t i n e that occur i n the r a t with prolonged s t a r v a t i o n m i r r o r the serum changes that occur during a short-term f a s t i n man? Do the serum l e v e l s of t o t a l c a r n i t i n e 18 change w i t h p r o l o n g e d s t a r v a t i o n and, i f so, what i s the time c o u r s e of such a change? F i n a l l y , the e f f e c t on serum c a r n i t i n e l e v e l s o f d i e t s v a r y i n g i n l i p i d c o n t e n t and c o m p o s i t i o n was s t u d i e d . One was r i c h i n c a r b o h y d r a t e , a n o t h e r r i c h i n medium-chain f a t t y a c i d t r i g l y c e r i d e s , and the l a s t was r i c h i n l o n g - c h a i n f a t t y a c i d t r i g l y c e r i d e s . The u t i l i z a t i o n o f the c a r b o h y d r a t e - r i c h d i e t by the a n i m a l would be e x p e c ted to be v i r t u a l l y c a r n i t i n e independent. The d i e t t h a t i s r i c h i n medium-chain t r i g l y c e r i d e s i s r a p i d l y absorbed from the gut d i r e c t l y i n t o the b l o o d stream. The s h o r t - c h a i n l e n g t h f a t t y a c i d s i n t h i s type of t r i g l y c e r i d e are o x i d i z e d i n the l i v e r by a c a r n i t i n e independent pathway (230). T h e i r u t i l i z a t i o n r e s u l t s i n v e r y h i g h l e v e l s o f a c e t y l - C o A i n the l i v e r and ketone b o d i e s ( a c e t o a c e t a t e and D - 3 - h y d r o x y b u t y r a t e ) i n serum (287). In c o n t r a s t to s t a r v a t i o n , the k e t o s i s g e n e r a t e d w i t h t h i s medium-chain t r i g l y c e r i d e d i e t tends to be h i g h e r . By examining the e f f e c t o f t h i s d i e t on serum a c y l c a r n i t i n e l e v e l s , some i n s i g h t may be g a i n e d as to whether or hot the serum c a r n i t i n e l e v e l i s a l t e r e d by the o x i d a t i o n o f t h i s s u b s t r a t e . Such a change might i n d i c a t e t h a t t h e r e i s an exchange between t i s s u e and serum c a r n i t i n e , an exchange w h i c h r e s u l t s i n the i n c r e a s e of serum a c y l c a r n i t i n e c o n t e n t . I f t h e r e i s such an exchange, i t may be f u n c t i o n i n g to r e p l e n i s h the s u p p l y of reduced i n t r a c e l l u l a r coenzyme A. In c o n t r a s t to the medium-chain t r i g l y c e r i d e d i e t , the d i e t t h a t i s r i c h i n l o n g - c h a i n t r i g l y c e r i d e r e q u i r e s i n t r a c e l l u l a r c a r n i t i n e f o r i t s o x i d a t i o n . A t tempts were made to answer a number of q u e s t i o n s about the e f f e c t s o f t h i s type o f d i e t on serum c a r n i t i n e l e v e l s . When an animal i s on such a d i e t , i s there an increased requirement f o r c a r n i t i n e ? I f so, how i s t h i s requirement met? What i s the impact of such a d i e t on serum l e v e l s of c a r n i t i n e ? How do the changes i n serum c a r n i t i n e patterns that occur on the t r i g l y c e r i d e - r i c h d i e t compare with those of the medium-chain t r i g l y c e r i d e d i e t ? PART II L i p i d , because of i t s high c a l o r i c value could represent an i d e a l s ubstrate f o r the r a p i d l y growing mammal both i n utero and ne o n a t a l l y . The u t i l i z a t i o n of l i p i d presupposes that i t i s r e a d i l y a v a i l a b l e and that the growing animal has the c a p a c i t y to use i t . The a v a i l a b i l i t y of maternally derived l i p i d to the fetus i s q u i t e v a r i a b l e from species to specie's. Whereas p l a c e n t a l t r a n s f e r of l i p i d i s high i n the guinea p i g (186,137,30) and r a b b i t (93,275), i t i s low i n the r a t (158,143) and sheep (274,95). Although the s i t u a t i o n i n the primate i s not as c l e a r , some supply of maternal l i p i d to the fetus does occur (223,235,94). On the b a s i s of these data, one could speculate that the metabolic c a p a c i t y of the fetus to u t i l i z e l i p i d would be grea t e r i n the guinea p i g , r a b b i t and primate than i n the r a t and sheep. Since c a r n i t i n e and the c a r n i t i n e t r a n s f e r a s e enzymes a s s o c i a t e d with c a r n i t i n e ( c a r n i t i n e a c e t y l t r a n s f e r a s e , o c t a n o y l t r a n s f e r a s e , p a l m i t o y l t r a n s f e r a s e and p o s s i b l y others) p l a y a p i v o t a l r o l e i n f a c i l i t a t i n g the entry of a c t i v a t e d f a t t y a cids i n t o mitochondria, i t seems reasonable to conclude that t h e i r a v a i l a b i l i t y i s e s s e n t i a l f o r the optimum u t i l i z a t i o n of l i p i d by the f e t a l guinea p i g , r a b b i t and primate. The need f o r the c a r n i t i n e system i n the f e t a l r a t and sheep, 20 however, seems q u e s t i o n a b l e i n view of the s m a l l amounts o f l i p i d t h a t are a b l e to pass through t h e i r p l a c e n t a s . Few s t u d i e s have examined the f e t a l a v a i l a b i l i t y o f c a r n i t i n e i n a l l of the above s p e c i e s . The f o l l o w i n g q u e s t i o n s have not been addressed: How do f e t a l l e v e l s o f c a r n i t i n e compare w i t h m a t e r n a l l e v e l s ? I s t h e r e any r e l a t i o n s h i p between serum l e v e l s o f c a r n i t i n e i n the f e t u s and the r e l a t i v e p e r m e a b i l i t y of i t s p l a c e n t a to l i p i d ? Are t h e r e d i f f e r e n c e s i n the p e r m e a b i l i t y o f the p l a c e n t a s o f these s p e c i e s t o c a r n i t i n e ? I s t h e r e any d i f f e r e n c e i n the p l a c e n t a ' s p e r m e a b i l i t y to c a r n i t i n e ( X - t r i m e t h y l a m i n o - 3 - h y d r o x y b u t y r a t e ) and D-y3-hydroxybutyrate, a m o l e c u l e which i s q u i t e s i m i l a r to c a r n i t i n e i n i t s s t r u c t u r e ? I n the immediate n e o n a t a l p e r i o d , , sheep, g u i n e a p i g , r a b b i t , r a t and man r e c e i v e m a t e r n a l b r e a s t m i l k . Once a g a i n , d i f f e r e n c e s are seen i n the c o n t r i b u t i o n t h a t l i p i d makes t o the d e v e l o p i n g a n imal (see Table I ) . I n c o n t r a s t to the f e t a l p e r i o d , the r a t and sheep now r e c e i v e a d i e t t h a t i s r i c h i n l i p i d and they t h e r e f o r e have a g r e a t e r requirement f o r c a r n i t i n e than they d i d i n u t e r o . I s t h i s i n c r e a s e d requirement r e f l e c t e d i n an i n c r e a s e i n serum l e v e l s o f c a r n i t i n e i n these two s p e c i e s ? I f so, what i s the source of t h i s c a r n i t i n e ? The d i e t t h a t i s s u p p l i e d to the g u i n e a p i g and r a b b i t c o n t i n u e s to be r i c h i n l i p i d i n the n e o n a t a l p e r i o d . Does exposure to t h i s new d i e t a l t e r the serum l e v e l s of c a r n i t i n e i n these two s p e c i e s ? I n man, over 507o o f the c a l o r i c content o f h i s n e o n a t a l d i e t i s d e r i v e d from l i p i d (see T able I ) . I s t h e r e a requirement f o r c a r n i t i n e above t h a t found i n the f e t a l p e r i o d ? I f so, what i s the source of t h i s c a r n i t i n e ? I s t h e r e any r e l a t i o n s h i p between serum c a r n i t i n e l e v e l s and growth? 21 Based on dietary l i p i d supply, the rat would be expected to have a low carn i t ine requirement in utero, a high requirement as a neonate and a low requirement in adulthood. Serum leve ls of f ree , a c y l - and to ta l carn i t ine were followed throughout development to determine whether or not they re f lected these projected requirements and to determine the age at which they reach adult l e v e l s . Very few studies even mention f e t a l serum carn i t ine and none have measured f e t a l serum acy lcarn i t ine l e v e l s . Only one study has looked at the permeabil ity of the placenta to carn i t ine (128). The rat was the species that was studied and the resul ts indicated that there is only a l imited transfer of carn i t ine from the mother to the fetus in late gestat ion. No data are ava i lab le on the other species mentioned e a r l i e r . In view of the species var ia t ion in maternal - feta l l i p i d supply, a la te gestat ion comparison of f e t a l and maternal serum levels of f ree , a c y l - , to ta l carn i t ine and D-jB-hydroxybutyrate seemed warranted. The ra t , sheep, guinea pig and rabbit were studied. In a f i n a l study, plasma levels of carn i t ine were determined during intravenous feeding of the human neonate. No data are avai lable on the n u t r i t i o n a l requirements for carn i t ine at d i f fe rent stages of development in man. It appears p a r t i c u l a r l y important to estab l ish i f there is a carn i t ine requirement, since l i p i d is a major source of energy in the newborn per iod. The need may be even greater in the very small infant who has to re ly on intravenous alimentation for s u r v i v a l . To this end, a study was undertaken to measure serum levels of f ree, a c y l - and to ta l carn i t ine in neonates receiv ing intravenous alimentations which varied in carn i t ine content. TABLE I The Composition of Mammalian M i l k s The data were adapted from: Metabolism. A B i o l o g i c a l Handbook of the F e d e r a t i o n of American S o c i e t i e s f o r E x p e r i m e n t a l B i o l o g y , compiled and e d i t e d by: P.L. Altman and D.S. D i t t m e r , 1968. p. 1-3. For any g i v e n s p e c i e s the mean of the r e p o r t e d v a l u e s was used. I n c a l c u l a t i n g the c a l o r i c c o n t e n t s o f the m i l k a c a l o r i c d e n s i t y o f 9 c a l . was used f o r f a t , 4 c a l . f o r car b o h y d r a t e and p r o t e i n . S pecies Fat P r o t e i n T o t a l C a l . Per 100 g Whole M i l k P e r c e n t C a l . Due to Fat Fat C a l . as a P e r c e n t of Fat P l u s Carbohydrate (g) (g) (%) C a l . (7c) Man Rat R a b b i t Guinea P i g Sheep 3.84 1 2.58 13.10 7.2 5.48 6.84 3.32 1 .93 2.68 4.68 1 .43 9.17 12.27 7.39 5.48 67.64 1 63.12 174.7 105.1 89.9 51 .1 69.4 67.5 61 .7 54.8 55.8 89.5 93.9 85.8 72.5 PART I I I 23 As suggested i n the p r e c e d i n g d i s c u s s i o n , t h e r e i s , i n a l l l i k e l i h o o d , c o n s i d e r a b l e v a r i a t i o n from s p e c i e s to s p e c i e s i n the requirement f o r c a r n i t i n e i n both the f e t a l and n e o n t a l p e r i o d s o f development. S t u d i e s have h e l p e d t o c l a r i f y the b i o c h e m i c a l r o l e p l a y e d by a l l o f the c a r n i t i n e a c y l t r a n s f e r a s e s , except c a r n i t i n e a c e t y l t r a n s f e r a s e . I t s ex a c t r o l e and f u n c t i o n are unknown i n s p i t e o f the many s p e c u l a t i o n s t h a t have been made (219,69,26,219,22,42, 169). A renewed i n t e r e s t i n the f u n c t i o n o f c a r n i t i n e a c e t y l t r a n s f e r a s e has r e s u l t e d from the r e c e n t f i n d i n g s t h a t t h i s enzyme i s not o n l y p r e s e n t i n m i t o c h o n d r i a , but a l s o i n microsomes and peroxisomes o f l i v e r (177,179). I t s presence i n per o x i s o m e s , a c a t a l a s e - r i c h o r g a n e l l e w h i c h i s c a p a b l e o f o x i d i z i n g l i p i d (162,163), i s o f p a r t i c u l a r i n t e r e s t . The p e r o x i s o m a l o x i d a t i o n sytem, u n l i k e the system t h a t i s found i n m i t o c h o n d r i a , can gen e r a t e hydrogen p e r o x i d e which may f u r t h e r be degraded to water by the a c t i o n o f c a t a l a s e (162). T h i s l a t t e r r e a c t i o n i s e x o t h e r m i c . I t i s worthy o f n o t e , t h a t some drugs, used c l i n i c a l l y f o r l o w e r i n g b l o o d l e v e l s o f l i p i d , have been shown t o cause marked i n c r e a s e s , not o n l y i n the c o n t e n t o f l i v e r p e r o xisomes, but a l s o i n the a c t i v i t y o f the c a r n i t i n e a c e t y l t r a n s f e r a s e enzyme whi c h i s a s s o c i a t e d w i t h t h i s o r g a n e l l e (181,256,254). Whether o r not the p e r o x i s o m a l o x i d a t i o n system i s p r e s e n t i n o t h e r t i s s u e s i s unknown. But, i f i t were p r e s e n t i n brown a d i p o s e t i s s u e , a t h e r m o g e n i c a l l y a c t i v e organ, the exo t h e r m i c d e g r a d a t i o n o f hydrogen p e r o x i d e might c o n t r i b u t e t o the heat p r o d u c t i o n i n t h i s t i s s u e . 24 heat production in this t i ssue . The present study was designed to measure carn i t ine acetyl t ransferase a c t i v i t y levels in l i v e r and brown adipose t issue during development. The separation of microsomes, mitochondria, and peroxisomes was attempted in these two t issues and the a c t i v i t y of carn i t ine acetyl t ransferase was determined. Three species were fol lowed, the' rabb i t , guinea pig and ra t . The former two species were selected because of the i r re l iance on maternally derived l i p i d as a substrate for development in la te gestat ion. The rat does not re ly on maternally derived l i p i d during this period but begins to u t i l i z e large amounts of l i p i d neonatally and therefore, at th is time, i t would probably have an increased requirement for both carn i t ine and the carn i t ine acyl t ransferase enzymes. Therefore i t i s a good animal model for studying the developmental induction of the c a r n i t i n e / c a r n i t i n e acyl t ransferase system. Few developmental studies have been published on carn i t ine acety l t ransferase. No data are ava i lab le on the a c t i v i t y of th is enzyme in microsomes and peroxisomes isolated from f e t a l and neonatal l i v e r and brown fa t . The developmental patterns of carn i t ine acety l t ransferase, in r a t , rabbit and guinea p ig , were compared in an attempt to shed some l i g h t on the function of this enzyme, not only in the developing, but also in the adult animal. REVIEW OF PERTINENT LITERATURE 26 CARNITINE C a r n i t i n e ( y - t r i m e t h y l a m i n o - 8 - h y d r o x y b u t y r a t e ) was d i s c o v e r e d i n d e p e n d e n t l y by two d i f f e r e n t groups i n 1905 (161). One o f the groups (119) i s o l a t e d i t from mammalian muscle and a s s i g n e d i t the e m p i r i c a l f o r m u l a C 7 H 1 5 N O 3 which was shown to be c o r r e c t i n 1926 (269). I n 1948, F r a e n k e l et a l . (100) d i s c o v e r e d a growth f a c t o r r e q u i r e d by the l a r v a of the b e e t l e T e n e b r i o M o l i t o r and named t h i s f a c t o r v i t a m i n B T (T f o r T e n e b r i o ) . V i t a m i n B T was i d e n t i f i e d f o u r y e a r s l a t e r as c a r n i t i n e ( 56). A crude b i o l o g i c a l assay was developed (101), which u t i l i z e d the growth requirement of the Tenebrio M o l i t o r l a r v a f o r c a r n i t i n e as an i n d i c a t i o n o f c a r n i t i n e c o n c e n t r a t i o n i n the assayed m a t e r i a l . F r a e n k e l (102,103) used t h i s assay to measure the c o n c e n t r a t i o n o f c a r n i t i n e i n a wide v a r i e t y of b i o l o g i c a l m a t e r i a l s taken from many d i f f e r e n t organisms. The b i o l o g i c a l f u n c t i o n o f c a r n i t i n e was s t i l l unknown u n t i l F r i t z (107) demonstrated t h a t muscle e x t r a c t s s t i m u l a t e d the o x i d a t i o n of p a l m i t i c a c i d i n l i v e r s l i c e s and s u b s e q u e n t l y showed t h a t c a r n i t i n e had a s i m i l a r e f f e c t on the o x i d a t i o n of l o n g - c h a i n f a t t y a c i d s i n the l i v e r (108). I t soon became apparent t h a t c a r n i t i n e was n e c e s s a r y f o r the optimum o x i d a t i o n of l o n g - c h a i n f a t t y a c i d s and/or t h e i r acyl-CoA e s t e r s by m i t o c h o n d r i a (109). E a r l i e r s t u d i e s i n d i c a t e d t h a t the h y d r o x y l group of the c a r n i t i n e m o l e c u l e was, i n a l l l i k e l i h o o d , n e c e s s a r y f o r t h i s f u n c t i o n (19). THE SYNTHESIS OF CARNITINE E a r l y s t u d i e s i n the r a t (166,167) demonstrated t h a t - b u t y r o b e t a i n e ( 4 - t r i m e t h y l a m i n o b u t y r a t e ) c o u l d be h y d r o x y l a t e d to form 1 c a r n i t i n e by an enzyme t h a t was p r e s e n t i n the s o l u b l e f r a c t i o n o f r a t l i v e r . The r e a c t i o n had a requirement f o r m o l e c u l a r oxygen and f e r r o u s i r o n and was s t i m u l a t e d by a s c o r b a t e and NADPH. I t i s i n t e r e s t i n g t h a t these e a r l y workers demonstrated t h a t h i g h l e v e l s o f c a t a l a s e , when added to the assay system, markedly s t i m u l a t e d t h i s h y d r o x y l a t i o n r e a c t i o n (167). The r e c e n t f i n d i n g t h a t c a t a l a s e p o s i t i v e p a r t i c l e s i n the l i v e r (peroxisomes) c o n t a i n c a r n i t i n e a c e t y l t r a n s f e r a s e (178) r a i s e s the q u e s t i o n as to a p o s s i b l e r o l e f o r peroxisomes i n the s y n t h e s i s o f c a r n i t i n e by the l i v e r . The l i v e r , and t o a l e s s e r e x t e n t the t e s t e s , are the o n l y organs s t u d i e d so f a r i n the r a t which are capable of t h i s h y d r o x y l a t i o n of Y - b u t y r o b e t a i n e + (130,27) to c a r n i t i n e ( (CH3)3N-CH2-CH-CH2-COO" ). OH Most o f the s t u d i e s which o u t l i n e the b i o s y n t h e t i c pathways i n v o l v e d i n the s y n t h e s i s of c a r n i t i n e have been c a r r r i e d out i n the r a t . I t has been shown t h a t m e t h i o n i n e i s a source o f the 4-N-methyl groups of c a r n i t i n e (284,40). The b u t y r a t e carbon c h a i n of c a r n i t i n e i s d e r i v e d from l y s i n e (75). P r o t e i n - b o u n d l y s i n e i s m e t h y l a t e d . S-adenosylmethionine i s the source o f the methyl groups f o r t h i s r e a c t i o n (75,211). The p r o d u c t , t r i m e t h y l l y s i n e , i s f u r t h e r m e t a b o l i z e d to both c a r n i t i n e and y - b u t y r o b e t a i n e (76,261). L y s i n e methylase i s p r e s e n t i n many t i s s u e s o f the r a t (212) and i s ca p a b l e of m e t h y l a t i n g o n l y those l y s i n e m olecules which are p r o t e i n - b o u n d (211). R e c e n t l y , i t has been r e p o r t e d (141) t h a t t r i m e t h y l l y s i n e undergoes a , 3 - h y d r o x y l a t i o n r e a c t i o n forming 3-hydroxy-6-N-t r i m e t h y 1 l y s i n e b e f o r e i t i s co n v e r t e d to / - b u t y r o b e t a i n e . The m i t o c h o n d r i a l enzyme c a t a l y z i n g t h i s r e a c t i o n i s p r e s e n t i n both l i v e r and k i d n e y (141,236). S e v e r a l t i s s u e s have the c a p a c i t y to con v e r t t r i m e t h y l l y s i n e to K - b u t y r o b e t a i n e (55,262). Recent evidence 28 indicates that the kidneys are the major source of Y -butyrobetaine and that the l i v e r i s the p r i n c i p a l s i t e for the hydroxylat ion of this substance to carn i t ine ( 5 5 ) . Many of the f indings outl ined above are i n d i r e c t l y demonstrated by dietary s tudies . Concentrations of carn i t ine in t issues were decreased in rats fed a choline def ic ient diet ( 2 5 9 ) . They were normal when such diets were supplemented with methionine. S ign i f icant reduct ions, in heart and ske le ta l muscle carn i t ine concentrat ions, occurred when rats were fed a diet which was def ic ient in lys ine ( 2 6 0 ) . The l i v e r s from lys ine def ic ient animals accumulated l i p i d ( 2 6 3 ) which, in part , was at tr ibuted to the reduction in l i v e r carn i t ine l e v e l s . THE METABOLISM OF CARNITINE Most of the studies dealing with the metabolism of carn i t ine have been carr ied out in the ra t . Reported values of the carn i t ine body pool content in normal animals vary widely. One group has reported values ranging from 138 to 159 umol. per 100 g body weight ( 2 6 6 , 1 9 2 ) which tends to support the 224 umol. per 100 g body weight value quoted by Wolf and Berger ( 2 8 4 ) . These f igures are probably high since the color imetr ic bromophenol method ( 1 9 0 ) was used for the determination of carni t ine and a one compartment model was used to estimate the body pool . More recent data ( 5 0 , 6 3 , 2 7 2 ) indicate that a two compartment metabolic model is more appropriate for interpret ing carn i t ine pool turnover data. When the l a t te r approach and the more precise enzymatic method for carn i t ine determination are u t i l i z e d , the to ta l body content of carn i t ine in the r a t i s 35-40 umol. per 100 g body weight (50,63). F o l l o w i n g the i n j e c t i o n o f D L - ( m e t h y l - ^ C ) c a r n i t i n e i n t o r a t s , a l l t i s s u e s come i n t o e q u i l i b r i u m , a t v a r y i n g r a t e s , w i t h b l o o d plasma c a r n i t i n e ( 5 0 ) . There i s a r e l a t i v e l y r a p i d exchange between the plasma and s p l e e n , and between the k i d n e y and l i v e r , whereas the exchange i s much slower between h e a r t and s k e l e t a l muscle (50) . Cederblad and L i n d s t e d t (63) u t i l i z e d a two compartment model to i n t e r p r e t t h e i r d a ta and suggested t h a t the c a r n i t i n e c o ntent of the s m a l l e r compartment r e p r e s e n t s e x t r a c e l l u l a r water space p l u s those t i s s u e s h a v i n g a h i g h t u r n o v e r r a t e o f c a r n i t i n e such as the l i v e r . The l a r g e r c a r n i t i n e compartment i n c l u d e s those t i s s u e s which have a slower r a t e o f t u r n o v e r l i k e s k e l e t a l muscle. I n 24 h o u r s , a p p r o x i m a t e l y 7% o f the t o t a l body p o o l o f c a r n i t i n e i s e x c r e t e d (50,63). U t i l i z i n g these f i g u r e s , the d a i l y s y n t h e t i c r a t e f o r c a r n i t i n e was c a l c u l a t e d to be 1.8 umol. per 100 g body weight ( 6 3 ) . T h i s v a l u e i s supported by the f i n d i n g s of T s a i et a l . (271). The q u e s t i o n as to whether or not c a r n i t i n e i s m e t a b o l i z e d to o t h e r compounds i n the r a t i s not c o m p l e t e l y r e s o l v e d as y e t , but the evidence does tend to fa v o u r t h a t i t i s n o t . I n 1961 i t was r e p o r t e d t h a t t r i m e t h y l a m i n e o x i d e was the o n l y m e t a b o l i t e o f c a r n i t i n e found i n the u r i n e o f r a t s (166). These workers suggested t h a t t h i s substance was most l i k e l y a d e g r a d a t i o n product of c a r n i t i n e which was generated i n the i n t e s t i n e by b a c t e r i a l a c t i o n . K h a i r a l l a h and Wolf (151) found t h a t , i n r a t , 19% o f an i n j e c t e d dose o f r a d i o l a b e l l e d c a r n i t i n e c o u l d be r e c o v e r e d , 48 hours l a t e r , i n the u r i n e as 8 - m e t h y l c h o l i n e . These same workers r e p o r t e d (152) t h a t an enzyme was p r e s e n t , i n r a t l i v e r , which d e c a r b o x y l a t e d c a r n i t i n e to 3 - m e t h y l c h o l i n e . A c c o r d i n g l y , i t was suggested t h a t under some 30 p h y s i o l o g i c a l c o n d i t i o n s (e.g. pregnancy, p r o t e i n r i c h d i e t s , s t r e s s ) c a r n i t i n e undergoes d e c a r b o x y l a t i o n t o 8 - m e t h y l c h o l i n e which i s e x c r e t e d i n the u r i n e (266,192,152,193). These f i n d i n g s have not been s u b s t a n t i a t e d by o t h e r s (288, 165,284,166,104,50,63). TISSUE LEVELS OF CARNITINE I n r e v i e w i n g the p u b l i s h e d v a l u e s f o r t i s s u e c a r n i t i n e l e v e l s , the wide d i s t r i b u t i o n and v a r i a b i l i t y i n t i s s u e c o n c e n t r a t i o n s soon becomes e v i d e n t . The e a r l i e s t d a t a were p u b l i s h e d by F r a e n k e l i n 1953 and 1954 (see 104 f o r a comprehensive l i s t i n g ) . The a n a l y s i s o f t i s s u e c a r n i t i n e content should i n c l u d e f r e e c a r n i t i n e as w e l l as a c y l c a r n i t i n e l e v e l s (185). On the b a s i s o f t h i s e a r l y work, s e v e r a l t i s s u e s c o u l d be c o n s i d e r e d to have v e r y h i g h contents o f c a r n i t i n e , namely h e a r t , brown adipose t i s s u e , s k e l e t a l muscle, t e s t i s , and a d r e n a l glands (49,185). T h i s l i s t seems to be v a l i d today (50,196,124) except i n one n o t a b l e case. E a r l i e r workers had r e p o r t e d t h a t the a d r e n a l glands had v e r y h i g h l e v e l s of c a r n i t i n e (49,2) but t h i s has not been conf i r m e d by more r e c e n t work (50). There i s a tendency f o r 1 - c a r n i t i n e to i n c r e a s e i n l i p i d - a r t e r i o s c l e r o t i c a o r t i c t i s s u e (155) o f man. A s i m i l a r i n c r e a s e i n r a b b i t a o r t a has been r e p o r t e d when animals are fed a s t o c k d i e t t h a t i s supplemented w i t h 570 l a r d and 1% c h o l e s t e r o l (117). I t has been decrease and those o f r e p o r t e d t h a t serum l e v e l s of f r e e c a r n i t i n e a c y l c a r n i t i n e i n c r e a s e w i t h i n 10 minutes of the 31 i n i t i a t i o n of m y o c a r d i a l i s c h e m i a . Upwards to 50% o f the t o t a l c a r n i t i n e o f the h e a r t can be l o s t i n t o the bloodstream ( p r o b a b l y as a c e t y l c a r n i t i n e ) i f i s c h e m i a c o n t i n u e s f o r 60 minutes or more (245). These o b s e r v a t i o n s emphasize the importance o f c a r n i t i n e i n the m e t a b o l i c responses of the c a r d i o v a s c u l a r system to p h y s i o l o g i c a l s t r e s s e s . R e c e n t l y , M i t c h e l l (196) has summarized the p u b l i s h e d v a l u e s f o r c a r n i t i n e c o n c e n t r a t i o n i n normal human s k e l e t a l muscle. In c h i l d r e n t h e r e i s no c o r r e l a t i o n between age or sex and muscle c a r n i t i n e c o n c e n t r a t i o n (145). A s i m i l a r f i n d i n g was r e p o r t e d f o r a d u l t samples (60) and i t was concluded t h a t muscle c a r n i t i n e l e v e l s remain f a i r l y c o n s t a n t w i t h i n any g i v e n i n d i v i d u a l . S i n c e the c a p a c i t y of s k e l e t a l muscle f o r l o n g - c h a i n f a t t y a c i d o x i d a t i o n i s r e l a t e d to muscle c a r n i t i n e c o n c e n t r a t i o n ( 6 2 ) , i t i s not s u r p r i s i n g t h a t l i p i d d r o p l e t s accumulate i n muscle f i b e r s which have reduced l e v e l s o f c a r n i t i n e (276). S e v e r a l c a r n i t i n e r e l a t e d myopathies have been r e p o r t e d i n the l i t e r a t u r e (see 195). I n g e n e r a l , these myopathies are c h a r a c t e r i z e d by l i p i d i n f i l t r a t i o n o f s k e l e t a l muscle f i b e r s ( p a r t i c u l a r l y type I ) , muscle cramps, weakness, and a reduced c a p a c i t y to o x i d i z e l o n g - c h a i n f a t t y a c i d s . I t i s e v i d e n t t h a t the maintenance of normal l e v e l s o f c a r n i t i n e i n both red and w h i t e s k e l e t a l muscle i n the r a t r e q u i r e s an i n t a c t i n n e r v a t i o n (149). C a r n i t i n e added to the d i e t does not i n c r e a s e f a t t y a c i d t u r n o v e r i n e x e r c i s e d r a t s (9) nor does e x e r c i s e i n c r e a s e the c o n c e n t r a t i o n o f c a r n i t i n e i n muscle (115). One of the t i s s u e s h a v i n g a h i g h c a r n i t i n e content i s brown adipose t i s s u e (185,124). T h i s t i s s u e p l a y s an important r o l e i n the 32 thermogenic response of the newborn mammal (7 9 ) . The n o r e p i n e p h r i n e r e l e a s e d i n t h i s t i s s u e , i n response to the abrupt change i n temperature e x p e r i e n c e d w i t h b i r t h , a c t i v a t e s adenyl c y c l a s e (248) which r e s u l t s i n i n c r e a s e d l e v e l s o f c y c l i c AMP (adenosine 3 ' : 5 ' - c y c l i c monophosphate) and a c t i v a t i o n of hormone s e n s i t i v e l i p a s e . The r e s u l t i n g h y d r o l y s i s o f s t o r e d t r i g l y c e r i d e s r e l e a s e s f a t t y a c i d s which are o x i d i z e d i n the m i t o c h o n d r i a (87) to generate heat. O p t i m a l o x i d a t i o n o f these f a t t y a c i d s depends on an adequate su p p l y o f c a r n i t i n e and ATP (86,122). Cold exposure f o r 20 days, of 18 day o l d r a t s , r e s u l t s i n an i n c r e a s e i n the c a r n i t i n e content o f brown f a t (124). Cold a d a p t a t i o n i n the r a t s i g n i f i c a n t l y i n c r e a s e s the c a r n i t i n e c o n t e n t o f l i v e r (82) and muscle (265) and reduces the h a l f - l i f e and t u r n o v e r time of c a r n i t i n e to o n e - h a l f o f t h a t found f o r c o n t r o l r a t s housed at 25°C (265). U n f o r t u n a t e l y , these s t u d i e s have expressed c a r n i t i n e c o n t e n t on the b a s i s o f t i s s u e wet w e i g h t . I t i s w e l l known t h a t under c e r t a i n p h y s i o l o g i c a l c o n d i t i o n s (e.g. s t a r v a t i o n ) (136) organ weights can change d r a m a t i c a l l y . I t t h e r e f o r e remains to be shown whether or not c o l d a d a p t a t i o n i n c r e a s e s t o t a l body c a r n i t i n e c o n c e n t r a t i o n . I t has been shown t h a t l i v e r c a r n i t i n e content i n c r e a s e s i n the r a t w i t h s t a r v a t i o n (188). I n t h i s same s p e c i e s t o t a l body c a r n i t i n e c o n t e n t i n c r e a s e s d u r i n g pregnancy (150). I t has not y e t been determined i f the i n c r e a s e , i n the c a r n i t i n e content of the l i v e r , found w i t h s t a r v a t i o n i s due to an i n c r e a s e i n s y n t h e s i s or t o an i n c r e a s e i n m o b i l i z a t i o n from o t h e r t i s s u e s (54,36). Diabetes m e l l i t u s i n the sheep markedly enhances the r a t e o f c a r n i t i n e s y n t h e s i s i n the l i v e r (250). I n man, i t has been shown t h a t c h r o n i c a l l y i l l p a t i e n t s and c i r r h o t i c s who have been i n a p r o l o n g e d 33 catabol ic state have decreased levels of carn i t ine in thei r ske le ta l muscle ( 3 2 , 2 3 4 ) . Other studies (64) have establ ished that carn i t ine content in the heart is depleted in d iph ther i t i c myocardi t is . CARNITINE UPTAKE BY TISSUES Carni t ine transport and the mechanisms regulat ing t issue uptake of carn i t ine have been studied by several workers. Marquis and F r i t z (184) were the f i r s t to show that androgens were required for the maintenance of normal levels of carn i t ine and carn i t ine acetyl t ransferase in the rat epididymis and t e s t i s . Later ( 2 9 ) , i t was shown that the uptake of radioact ive carn i t ine by the rat epididymis was induced by testosterone and suppressed by estrogens. C a s i l l a s (57) demonstrated that the uptake of carn i t ine by bovine caput spermatozoa was l inear with time, carn i t ine concentrat ion, and number of spermatozoa. He found no evidence for the hydroxylation of ^-butyrobetaine to carn i t ine in the tes t i s nor was he able to detect carni t ine in spermatozoa. He detected acety lcarn i t ine in caudal sperm and observed that the levels of this der ivat ive of carn i t ine increased markedly when the sperm were incubated with fructose ( 5 7 ) . More recently ( 1 5 6 ) , i t has been reported that seminal plasma carn i t ine levels are tenfold greater than those found in blood and that 507o of this carn i t ine is in the form of ace ty lca rn i t ine . In i n f e r t i l e human semen to ta l carn i t ine levels are normal whereas the leve ls of acety lcarn i t ine are s i g n i f i c a n t l y depressed ( 1 5 6 ) . Studies dealing with the uptake of carn i t ine by cultured human heart c e l l s (31) demonstrated that, although the c e l l s release carn i t ine , the rate of release is exceeded by the rate of uptake. In these c e l l s t r a n s p o r t was shown to be an a c t i v e process which i s temperature dependent and i s not l i n k e d to amino a c i d and g l u c o s e uptake, or to Na+/K+ ATPase a c t i v i t y . T r a n s p o r t f a l l s o f f at a pH above 8.0 and i s i n h i b i t e d by a c y l c a r n i t i n e s (198). S i m i l a r d a t a were found f o r c a r n i t i n e uptake by r a t k i d n e y s l i c e s . Uptake was l i n e a r w i t h time, energy dependent, and the p r o c e s s c o u l d be s a t u r a t e d (144). P r i o r i n c u b a t i o n of the s l i c e s w i t h cAMP s t i m u l a t e d the uptake o f c a r n i t i n e (144). I n s p i t e o f the evidence which suggests t h a t a t i s s u e c a r n i t i n e b i n d i n g p r o t e i n may b e " i n v o l v e d and c o u l d t h e r e f o r e account f o r the wide v a r i a t i o n s i n t i s s u e c o n c e n t r a t i o n s of c a r n i t i n e , no such p r o t e i n has been found (290). Pande (213) has suggested t h a t h e a r t m i t o c h o n d r i a possess a t r a n s l o c a s e system which a l l o w s the t r a n s p o r t o f c a r n i t i n e and a c y l c a r n i t i n e s a c r o s s the i n n e r m i t o c h o n d r i a l membrance by exchange d i f f u s i o n . Whether or not t h i s system i s i n v o l v e d i n d e t e r m i n i n g t i s s u e l e v e l s o f c a r n i t i n e i s u n c e r t a i n . I n 1966 (47) B r e s s l e r and W i t t e l s r e p o r t e d t h a t , i n the guinea p i g , d a i l y i n j e c t i o n s o f t h y r o x i n e f o r a p e r i o d o f 10 days r e s u l t e d i n a s i g n i f i c a n t e l e v a t i o n of c a r d i a c c a r n i t i n e as w e l l as a marked h y p e r t r o p h y o f the h e a r t . Other workers (188) have demonstrated t h a t l i v e r c a r n i t i n e c o n c e n t r a t i o n i s i n c r e a s e d by e l e v a t i o n o f plasma glucagon l e v e l s . BLOOD LEVELS OF CARNITINE As p o i n t e d out i n a r e c e n t r e v i e w on c a r n i t i n e metabolism i n man (196), i t i s d i f f i c u l t to e v a l u a t e p u b l i s h e d data on b l o o d 35 l e v e l s of c a r n i t i n e . T h i s i s m a i n l y due to the l a c k o f i n f o r m a t i o n on sex, d i e t a r y s t a t u s , and age o f the s u b j e c t s s t u d i e d . I n a d d i t i o n , the methodologies used to measure c a r n i t i n e v a r i e d . The s i t u a t i o n has now been c o m p l i c a t e d f u r t h e r by our p u b l i s h e d r e s u l t s (243,116) and those of o t h e r s (37) which have demonstrated t h a t c a r n i t i n e can and does e x i s t i n b l o o d not o n l y i n a f r e e , but a l s o , i n an a c y l form. D i f f e r e n t methodologies are r e q u i r e d to determine these two forms. I t has been r e p o r t e d (28) t h a t the l e v e l of f r e e c a r n i t i n e i n the serum decreases w i t h Crohn's d i s e a s e , a n o r e x i a n e r v o s a , and h e m o d i a l y s i s , and t h a t i t i n c r e a s e s w i t h h e a r t f a i l u r e and k i d n e y d i s e a s e . L e v e l s o f f r e e c a r n i t i n e have been r e p o r t e d to be lower than normal i n plasma from p a t i e n t s w i t h K w a s h i o r k o r , marasmus (153) and s c h i s t o s o m i a s i s (194). L e v e l s of c a r n i t i n e r e t u r n to normal v a l u e s w i t h t r e a t m e n t . I n the r a b b i t , plasma f r e e c a r n i t i n e decreases w i t h f a s t i n g and w i t h a h i g h f a t d i e t (117). The l e v e l s o f r e e c a r n i t i n e i n c r e a s e d when 1 % c h o l e s t e r o l was added to the h i g h f a t d i e t (117). I n the r a t ( 3 4 ) , no d i f f e r e n c e i n plasma l e v e l s o f c a r n i t i n e are seen between male and female up to the age o f weaning but plasma l e v e l s i n a d u l t males are h i g h e r than i n females. I n man females tend to have s l i g h t l y lower t o t a l c a r n i t i n e l e v e l s than male (6 1 ) . Y e t , t h e r e i s no c o r r e l a t i o n between plasma c a r n i t i n e l e v e l s and body c e l l mass. S i m i l a r l y , no c o r r e l a t i o n can be demonstrated between plasma c a r n i t i n e l e v e l s and muscle c a r n i t i n e c o n c e n t r a t i o n s i n r a t s (63). D e v e l o p m e n t a l l y , i t has been shown t h a t the p r i m a r y source o f n e o n a t a l c a r n i t i n e f o r the r a t , over the f i r s t 2-3 days postpartum, i s m a t e r n a l m i l k (229). I t i s not know at what age the r a t develops the c a p a c i t y to s y n t h e s i z e c a r n i t i n e but i t appears t h a t a d i e t a r y source i s r e q u i r e d d u r i n g the n e o n a t a l p e r i o d . I t i s a l s o not known whether or not t h i s i s the case i n man, however, human b r e a s t m i l k i s v e r y r i c h i n c a r n i t i n e (209) and i n f a n t s on a soy-bean based m i l k d i e t , which c o n t a i n s a l l the n e c e s s a r y p r e c u r s o r s f o r c a r n i t i n e s y n t h e s i s but no c a r n i t i n e per se, have s i g n i f i c a n t l y lower c a r n i t i n e b l o o d l e v e l s (208) than do s u c k l e d i n f a n t s . A r e c e n t r e p o r t on plasma c a r n i t i n e l e v e l s i n the r a t (35) i n d i c a t e s t h a t these l e v e l s are r e g u l a t e d by a product from the p i t u i t a r y as w e l l as one from the o v a r i e s and t e s t e s . ACYLCARNITINES Subsequent to the p u b l i s h e d f i n d i n g s t h a t l i v e r l o n g - c h a i n acyl-coenzyme A l e v e l s are e l e v a t e d by s t a r v a t i o n and d i a b e t e s m e l l i t u s (33,273), Bohmer et a l . (24) determined the r e l a t i v e amounts of f r e e c a r n i t i n e , a c e t y l c a r n i t i n e and l o n g - c h a i n a c y l c a r n i t i n e s i n the h e a r t , l i v e r and k i d n e y s o f male r a t s under s i m i l a r c o n d i t i o n s . The r e l a t i v e p r o p o r t i o n o f l o n g - c h a i n a c y l c a r n i t i n e s to f r e e c a r n i t i n e i n c r e a s e d d u r i n g s t a r v a t i o n , f a t f e e d i n g , and d i a b e t e s and decreased when g l u c o s e was g i v e n . Bohmer et a l . noted t h a t these changes p a r a l l e l e d those seen f o r the l o n g - c h a i n acylcoenzyme A l e v e l s . I n the k i d n e y the r e l a t i v e p r o p o r t i o n of a c e t y l c a r n i t i n e to f r e e c a r n i t i n e i n c r e a s e d w i t h f a s t i n g , d i a b e t e s , and f a t - f e e d i n g , whereas, i n the h e a r t and l i v e r , a c e t y l c a r n i t i n e l e v e l s d i d not 37 change s i g n i f i c a n t l y . These same au t h o r s (25) and o t h e r s (219) subs e q u e n t l y r e p o r t e d t h a t the r a t i o o f a c e t y l c a r n i t i n e to f r e e c a r n i t i n e i n the l i v e r , i n c r e a s e d w i t h s t a r v a t i o n . T h i s r a t i o has a l s o been found to i n c r e a s e i n the l i v e r o f s t a r v e d guinea p i g s (97) and sheep (249). I t has been noted t h a t the c o n c e n t r a t i o n o f a c e t y l c a r n i t i n e , i n the k e t o t i c guinea p i g l i v e r ( 9 7 ) , d i d not change, i n a b s o l u t e terms, w i t h s t a r v a t i o n but t h a t i t s T-1/2 decreased as d i d the l e v e l o f f r e e c a r n i t i n e . When the r a t h e a r t (219) i s p e r f u s e d w i t h f a t t y a c i d s o f an even number o f carbons, p y r u v a t e o r f>-hydroxybutyrate, c a r d i a c l e v e l s o f a c e t y l c a r n i t i n e i n c r e a s e ; y e t , the a c e t y l c a r n i t i n e to f r e e c a r n i t i n e r a t i o i n t h i s same organ i s u n a f f e c t e d by s t a r v a t i o n (219,24). I t has been demonstrated t h a t , w i t h i n 2 hours o f f e e d i n g a cor n o i l d i e t t o r a t s , the r a t i o o f l o n g - c h a i n a c y l c a r n i t i n e s to f r e e c a r n i t i n e i n c r e a s e s i n the h e a r t ( 2 5 ) . More r e c e n t l y (168), i t has been shown t h a t c a r n i t i n e e s t e r s , i n v o l v i n g the C6 -C14 ^ - o x i d a t i o n i n t e r m e d i a t e s o f p a l m i t a t e , accumulate i n i s o l a t e d l i v e r m i t o c h o n d r i a d u r i n g the o x i d a t i o n o f t h i s f a t t y a c i d . Brass and Hoppel (38 ) , u n l i k e o t h e r s (187,188), concluded t h a t the f o r m a t i o n o f a c y l c a r n i t i n e s does not p l a y an impo r t a n t ( r e g u l a t o r y ) r o l e i n k e t o g e n e s i s . . T h e i r c o n c l u s i o n was based upon the o b s e r v a t i o n t h a t , i n the fed r a t , e l e v a t i o n o f l i v e r l o n g - c h a i n a c y l c a r n i t i n e s , to l e v e l s seen w i t h f a s t i n g , does not r e s u l t i n an e l e v a t i o n o f plasma P-hydroxybutyrate as i t does i n the f a s t i n g a n i m a l . A f t e r i t had been e s t a b l i s h e d t h a t c a r n i t i n e had a g e n e r a l a c y l - c a r r y i n g f u n c t i o n (41,114), attempts were made to determine the r o l e t h a t a c y l c a r n i t i n e s p l a y e d i n i n t e r m e d i a r y metabolism. I t was 38 shown t h a t a c y l c a r n i t i n e s are o x i d i z e d by i s o l a t e d m i t o c h o n d r i a i n p r e f e r e n c e to p y r u v a t e (43) s u g g e s t i n g t h a t the a v a i l a b i l i t y of m i t o c h o n d r i a l coenzyme A may, i n p a r t , r e g u l a t e the d e c a r b o x y l a t i o n o f p y r u v a t e . The f o r m a t i o n of a c y l c a r n i t i n e s c o u l d i n f l u e n c e t h i s a v a i l a b i l i t y by r e p l e n i s h i n g the s u pply of coenzyme A and thus a c t i n g as an acetyl-coenzyme A b u f f e r i n g system (219,97,249,69). Other workers showed (202) t h a t a c e t y l c a r n i t i n e and p r o p i o n y l c a r n i t i n e were formed i n m i t o c h o n d r i a from o i-ketoacids which l e d to the s u g g e s t i o n (26) t h a t p r o p i o n y l c a r n i t i n e may a l s o be the end-product of odd-carbon f a t t y a c i d metabolism. Choi et a l . (70) have i s o l a t e d and c h a r a c t e r i z e d w a t e r - s o l u b l e a c y l c a r n i t i n e s from r a t h e a r t , l i v e r , s k e l e t a l muscle, and t e s t e s . Of the a c y l c a r n i t i n e s i s o l a t e d , a a c e t y l c a r n i t i n e was the most p r e v a l e n t . These workers and o t h e r s (219) suggest t h a t a c e t y l c a r n i t i n e may serve as a r e a d i l y a v a i l a b l e energy source d u r i n g p e r i o d s o f i n c r e a s e d energy demand. The appearance of c o n s i d e r a b l e q u a n t i t i e s of b r a n c h e d - c h a i n a c y l c a r n i t i n e s , i n t i s s u e s from r a t s f a s t e d f o r 6-8 days, has i n d i c a t e d a r o l e f o r c a r n i t i n e i n the metabolism of v a l i n e , l e u c i n e , and i s o l e u c i n e (22). A d d i t i o n o f c a r n i t i n e to homogenates of l i v e r and s k e l e t a l muscle s t i m u l a t e d the o x i d a t i o n of b r a n c hed-chain amino a c i d s (217). T h i s f u r t h e r suggests t h a t c a r n i t i n e may p l a y a r o l e i n the o x i d a t i o n o f amino a c i d s . A l t h o u g h i s was r e p o r t e d (191) t h a t embryonic t i s s u e s of c h i c k s and r a t s c o n t a i n e d h i g h c o n c e n t r a t i o n s of p h o s p h a t i d y l c a r n i t i n e , o t h e r s t u d i e s concluded t h a t e i t h e r i t d i d not e x i s t (113,4) or t h a t , i f i t d i d e x i s t i n embryonic t i s s u e s , i t d i d so i n v e r y s m a l l amounts (252) . I t has been suggested t h a t a c y l c a r n i t i n e s p l a y a r o l e i n the s y n t h e s i s of complex l i p i d s i n 39 microsomes (42) and are a source o f a c e t y l groups f o r f a t t y a c i d s y n t h e s i s (169) i n r a t l i v e r and mammary g l a n d . The importance o f a c e t y l c a r n i t i n e , i n the s y n t h e s i s o f f a t t y a c i d s i n a d u l t r a t l i v e r , i s q u e s t i o n a b l e s i n c e c i t r a t e and a c e t a t e have been found to i n c o r p o r a t e i n t o f a t t y a c i d s to a much g r e a t e r e x t e n t than the a c e t y l group from a c e t y l c a r n i t i n e (169). In the r a t , the " c i t r a t e - m a l a t e s h u t t l e " i s , i n a l l l i k e l i h o o d , the major r o u t e whereby a c e t y l - C o A , g enerated i n the m i t o c h o n d r i a , i s c a r r i e d t o the c y t o s o l (131) where f a t t y a c i d s y n t h e s i s o c c u r s . The adequate s u p p l y o f a c e t y l - C o A from c i t r a t e depends, i n p a r t , on the presence o f A T P - c i t r a t e l y a s e w h i c h i s v e r y a c t i v e i n a d u l t r a t l i v e r (215). C o n c e i v a b l y , the r o l e of a c y l c a r n i t i n e s , i n f a t t y a c i d s y n t h e s i s , may be r e l a t e d not o n l y t o s p e c i e s , organ, and stage of development, but a l s o i n v e r s e l y r e l a t e d to the a c t i v i t y o f the " c i t r a t e - m a l a t e s h u t t l e " found i n a g i v e n s p e c i e s and t i s s u e a t a p a r t i c u l a r stage of development. Thus, i t has been shown t h a t c a r n i t i n e s t i m u l a t e s the i n c o r p o r a t i o n o f p y r u v a t e , g l u c o s e , and a c e t a t e i n t o f a t t y a c i d s i n the a d u l t g u i n e a p i g l i v e r ( 4 6 ) , an organ i n which, A T P - c i t r a t e l y a s e i s v i r t u a l l y absent (215). I n ' a d i p o s e t i s s u e of the a d u l t g u i n e a p i g , the a c t i v i t y o f A T P - c i t r a t e l y a s e i s v e r y h i g h (215), and one c o u l d s p e c u l a t e t h a t the c o n t r i b u t i o n made by a c e t y l c a r n i t i n e to f a t t y a c i d s y n t h e s i s would be v e r y much l e s s than t h a t i n the l i v e r . The r e v e r s e s i t u a t i o n may be t r u e i n mice and man, s p e c i e s i n which i t has'been shown t h a t , a l t h o u g h c i t r a t e i s an e f f e c t i v e e x t r a m i t o c h o n d r i a l c a r r i e r of a c e t y l - C o A i n the l i v e r , i t i s a much l e s s e f f e c t i v e c a r r i e r i n adipose t i s s u e (231,232,99,246). I t has been suggested t h a t a c e t y l c a r n i t i n e may ser v e as a source o f a c e t y l groups f o r the s y n t h e s i s o f a c e t y l c h o l i n e (267). 40 Other workers have suggested t h a t p a l m i t y l c a r n i t i n e may p l a y a r e g u l a t o r y r o l e i n d e t e r m i n i n g Na+/K+ ATPase and Mg2+, Ca2+-ATPase a c t i v i t y i n s a r c o p l a s m i c r e t i c u l u m of c a r d i a c and s k e l e t a l muscle (286,73). However, the exact r o l e o f these e s t e r s remains to be determined. I if 1 964, Hulsmann et a l . (142) r e p o r t e d t h a t c a r n i t i n e s t i m u l a t e d the o x i d a t i o n o f a c e t o a c e t a t e i n i s o l a t e d m i t o c h o n d r i a l p r e p a r a t i o n s from r a t h e a r t and k i d n e y . Hahn and S k a l a (124) suggested t h a t c a r n i t i n e may have a s i m i l a r r o l e i n f a c i l i t a t i n g ketone body u t i l i z a t i o n by brown adipose t i s s u e . Other s t u d i e s (257), which used e v i s c e r a t e d and nephrectomized r a t s concluded t h a t c a r n i t i n e a d m i n i s t r a t i o n does not i n c r e a s e p e r i p h e r a l t i s s u e uptake of ketone b o d i e s ; y e t , i t does reduce f a s t i n g k e t o s i s i n r a b b i t s (228), dogs ( 4 8 ) , and man (118). I t i s not known how c a r n i t i n e does t h i s . I n s i g h t i n t o the problem i s f u r t h e r c o m p l i c a t e d by the f a c t t h a t c a r n i t i n e s t i m u l a t e s the m o b i l i z a t i o n o f l i p i d (3,207). I t has been suggested t h a t acetoacety1-CoA may e x i t from h e p a t i c m i t o c h o n d r i a d u r i n g s t a r v a t i o n (114,45,233) i n the form of a c e t o a c e t y l c a r n i t i n e . T h i s compound has been t e n t a t i v e l y i d e n t i f i e d i n l i v e r under c o n d i t i o n s of s t a r v a t i o n (45,97). URINARY CARNITINE I n p u b l i s h e d s t u d i e s , the problems t h a t were encountered w i t h the i n t e r p r e t a t i o n of c a r n i t i n e l e v e l s i n blo o d were a l s o encountered w i t h the i n t e r p r e t a t i o n o f c a r n i t i n e l e v e l s i n u r i n e (196). The g r e a t e s t problem i n v o l v e s the techniq u e s used. The presence o f a c y l c a r n i t i n e s i n the u r i n e has been v i r t u a l l y i g n o r e d i n these s t u d i e s (58,176,175). One study r e p o r t e d t h a t males e x c r e t e d a l a r g e r amount o f f r e e c a r n i t i n e per day than women (58). The a u t h o r s noted the tremendous day to day v a r i a t i o n i n u r i n a r y c o n c e n t r a t i o n s o f c a r n i t i n e w i t h i n a g i v e n s u b j e c t . They a t t r i b u t e d these f l u c t u a t i o n s to d i f f e r e n c e s i n d i e t and amount of muscular a c t i v i t y (58). The l a r g e s t number of s t u d i e s on u r i n a r y c a r n i t i n e s has been done by a Japanese l a b o r a t o r y (176,175) u s i n g a c o l o r i m e t r i c method f o r the d e t e r m i n a t i o n of c a r n i t i n e (106). Because of d i f f e r e n c e s between the Japanese d i e t and the North American d i e t , i t i s d i f f i c u l t to draw c o n c l u s i o n s . These workers r e p o r t e d (175,176) t h a t u r i n a r y c a r n i t i n e e x c r e t i o n i n c r e a s e s when t h e r e i s an i n c r e a s e i n the r a t e o f l i p o l y s i s (eg. f a s t i n g , a f t e r ACTH i n j e c t i o n s ) . I n c o n t r a s t to Cederblad and L i n d s t e d t (58 ) , these workers r e p o r t e d t h a t c a r n i t i n e e x c r e t i o n , o f a f e d s u b j e c t , d i d not v a r y from day to day nor v a r y s i g n i f i c a n t l y between day and n i g h t (75). The content o f c a r n i t i n e , i n the d i e t o f these s u b j e c t s , was not r e p o r t e d but i t i s known (58) t h a t o r a l c a r n i t i n e can have a marked e f f e c t on the c o n c e n t r a t i o n of t h i s substance i n the u r i n e . I n the r a t k i d n e y , t h e r e i s a r e n a l t r a n s p o r t maximum f o r c a r n i t i n e (289). When the L and DL isomers o f c a r n i t i n e are i n j e c t e d i n t o r a t s , more DL - c a r n i t i n e i s r e c o v e r e d i n the u r i n e than L - c a r n i t i n e (165). I t i s e v i d e n t t h a t t h e r e i s a need f o r f u r t h e r r e s e a r c h on the impact of d i e t and p h y s i o l o g i c a l s t a t e on u r i n a r y c a r n i t i n e l e v e l s (both f r e e c a r n i t i n e and a c y l c a r n i t i n e s ) b e f o r e any d e f i n i t i v e statements can be made. From the p r e c e d i n g , i t i s apparent t h a t c a r n i t i n e i s 42 i n v o l v e d i n the s h u t t l i n g of a c y l r e s i d u e s a c r o s s the i n n e r m i t o c h o n d r i a l membrane; however, i t may a l s o have o t h e r f u n c t i o n s . I t has been found t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e and o c t a n o y l t r a n s f e r a s e are l o c a t e d not o n l y i n the m i t o c h o n d r i a o f l i v e r but a l s o i n peroxisomes and microsomes (177,179,180); t h a t h i g h l e v e l s o f o c t a n o y l t r a n s f e r a s e a c t i v i t y are p r e s e n t i n many t i s s u e s o t h e r than l i v e r (181,98,255); t h a t a c e t y l c a r n i t i n e and o t h e r s h o r t - c h a i n a c y l c a r n i t i n e s are found i n muscle (219,22); and t h a t the a c t i v i t i e s of h e p a t i c c a r n i t i n e a c e t y l t r a n s f e r a s e and o c t a n o y l t r a n s f e r a s e i n c r e a s e i n response to c l o f i b r a t e a d m i n i s t r a t i o n (a plasma l i p i d l o w e r i n g agent) (1 47,1 99,254). The f o l l o w i n g s e c t i o n r eviews the l i t e r a t u r e on c a r n i t i n e a c y l t r a n s f e r a s e s w i t h s p e c i a l emphasis on c a r n i t i n e a c e t y l t r a n s f e r a s e . Friedman and F r a e n k e l (105) d e s c r i b e d the presence of an enzyme, i n crude pigeon l i v e r e x t r a c t s , which t r a n s f e r r e d the a c e t y l group from acetylcoenzyme A to c a r n i t i n e . T h i s was f o l l o w e d by r e p o r t s t h a t two o t h e r such enzymes, c a r n i t i n e p a l m i t y l t r a n s f e r a s e (111) and o c t a n o y l t r a n s f e r a s e (253), c a t a l y z e the t r a n s f e r of l o n g - c h a i n and medium-chain a c y l groups to c a r n i t i n e r e s p e c t i v e l y . Most s t u d i e s , to da t e , have been concerned w i t h the c a r n i t i n e a c e t y l t r a n s f e r a s e and p a l m i t y l t r a n s f e r a s e enzymes. CARNITINE PALMITYLTRANSFERASE C a r n i t i n e p a l m i t y l t r a n s f e r a s e i s l o c a t e d e x c l u s i v e l y i n m i t o c h o n d r i a (178,177) and c a t a l y z e s the f o l l o w i n g r e a c t i o n : l o n g - c h a i n acyl-coenzyme A + c a r n i t i n e <--> l o n g - c h a i n a c y l c a r n i t i n e + CoA. The r e a c t i o n occurs on the o u t e r s u r f a c e of the i n n e r m i t o c h o n d r i a l membrane and the r e v e r s e r e a c t i o n occurs on the i n n e r s i d e o f the i n n e r m i t o c h o n d r i a l membrance. There appear to be two d i s t i n c t y e t i n t e r c o n v e r t i b l e forms of the same enzyme (280,51,157, 139). The o u t e r form which i s r e a d i l y s o l u b i l i z e d i s d e s i g n a t e d as CPT-1 and i s i r r e v e r s i b l y i n h i b i t e d by 2-bromopalmitoyl-CoA (280) whereas the i n n e r form, CPT-II, which i s bound t i g h t l y to the i n n e r membrane, i s n o t . The i n n e r form r e q u i r e s p r e - i n c u b a t i o n w i t h coenzyme A b e f o r e i t m a n i f e s t s a c t i v i t y (157). The o u t e r form of the enzyme i s r e a d i l y s e p a r a t e d from the i n n e r membrane by treatment w i t h d e t e r g e n t s . I t has been suggested t h a t , i n v i t r o , CPT-I can be c o n v e r t e d to CPT-II (157). West et a l . (280) have shown t h a t the Km f o r acyl-CoA decreases w i t h i n c r e a s i n g c h a i n l e n g t h o f the a c y l moiety. T h i s suggests t h a t the g r e a t e r the c h a i n - l e n g t h o f the acyl-CoA s u b s t r a t e the g r e a t e r the a f f i n i t y f o r the enzyme. The presence of CPT I and CPT I I , on d i f f e r e n t s i d e s of the i n n e r m i t o c h o n d r i a l membrane, gave r i s e to the v e c t o r i a l - t r a n s f e r a s e h y p o t h e s i s of f a t t y a c y l t r a n s f e r a c r o s s the i n n e r m i t o c h o n d r i a l membrane. I t was h y p o t h e s i z e d t h a t the " o u t e r " CPT I enzyme c a t a l y z e d the f o r m a t i o n o f a c y l c a r n i t i n e from c y t o s o l i c c a r n i t i n e and acyl-CoA, and the " i n n e r " CPT I I enzyme c a t a l y z e d the t r a n s f e r of the a c y l group from the o u t e r formed a c y l c a r n i t i n e to m a t r i x CoA (139). T h i s h y p o t h e s i s suggested t h a t , not o n l y was the i n n e r enzyme not a c c e s s i b l e t o c y t o s o l i c acyl-CoA but t h a t c y t o s o l i c and m i t o c h o n d r i a l c a r n i t i n e p o o l s were s e p a r a t e . However, i t has been shown t h a t t h e r e i s a r a p i d exchange between e x t e r n a l and m a t r i x c a r n i t i n e i n a 1:1 r a t i o and t h a t the exchange i s s p e c i f i c , s a t u r a b l e , and v e r y temperature dependent (226,225). A p p a r e n t l y , CPT I and CPT I I do not p l a y an enzymic r o l e i n the exchange (226). S i m i l a r f i n d i n g s have been r e p o r t e d by workers i n v e s t i g a t i n g b l o w f l y f l i g h t muscle (78) and 44 spermatozoa from b u l l , boar, ram and r a t . Thus, i t appears t h a t f a t t y a c y l t r a n s p o r t proceeds by a proc e s s o f f a c i l i t a t e d exchange d i f f u s i o n i n which t h e r e i s an a c y l c a r n i t i n e - c a r n i t i n e exchange s t e p . CARNITINE PALMITYLTRANSFERASE AND DEVELOPMENT The a c t i v i t y o f c a r n i t i n e p a l m i t y l t r a n s f e r a s e i n c r e a s e s , d u r i n g the n e o n a t a l p e r i o d , i n r a t (127) and p i g (21) l i v e r and r a t h e a r t (278,285). I n p i g s the i n c r e a s e occurs f a s t e r i n a fed than a f a s t e d a n i m a l . I t has been r e p o r t e d t h a t t h i s enzyme i s pr e s e n t i n the t i s s u e s o f the monkey f e t u s (129), human f e t u s (125), and human p l a c e n t a (204,148). I n human p l a c e n t a , the a c t i v i t y has been found to be comparable to t h a t i n r a t l i v e r m i t o c h o n d r i a . FACTORS REGULATING CARNITINE PALMITYLTRANSFERASE ACTIVITY I t has been r e p o r t e d t h a t c a r n i t i n e p a l m i t y l t r a n s f e r a s e a c t i v i t y i n the l i v e r o f r a t s i n c r e a s e s w i t h f a s t i n g , d i a b e t e s , and f a t f e e d i n g (203,1) and t h a t the i n c r e a s e i n a c t i v i t y i s not i n h i b i t e d by commonly used i n h i b i t o r s o f p r o t e i n s y n t h e s i s (203). On the o t h e r hand, the a c t i v i t y of the l i v e r enzyme does not i n c r e a s e when s u c k l i n g r a t s are p r e m a t u r e l y weaned to a h i g h f a t d i e t or g i v e n a h i g h c a r b o h y d r a t e d i e t (127). Mole et a l . (197) have r e p o r t e d t h a t c a r n i t i n e p a l m i t y l t r a n s f e r a s e i n c r e a s e s i n muscle w i t h e x e r c i s e . The c o n v e r s i o n of p a l m i t y l - C o A i n t o p a l m i t y l c a r n i t i n e i s r a t e - l i m i t i n g f o r both p a l m i t a t e and p a l m i t y l - C o A o x i d a t i o n , i n r a t l i v e r m i t o c h o n d r i a (244), but o n l y when the l e v e l of p a l m i t y l c a r n i t i n e formed i s low ( 4 4 ) . At h i g h e r p a l m i t y l c a r n i t i n e c o n c e n t r a t i o n s the a c e t o a c e t a t e forming system seems to be r a t e l i m i t i n g ( 4 4 ) . Normann et a l . (201) r e c e n t l y demonstrated t h a t , i n m i t o c h o n d r i a i s o l a t e d from guinea p i g brown adipose t i s s u e , the " o u t e r " (CT-I) a c y l t r a n s f e r a s e r e a c t i o n i s r a t e l i m i t i n g i n the t r a n s f e r o f l o n g - c h a i n a c y l groups a c r o s s the i n n e r m i t o c h o n d r i a l membrane. Wood and Claycomb (285) r e p o r t e d t h a t , i n the h e a r t and l i v e r o f the n e o n a t a l r a t , m i t o c h o n d r i a l a c t i v i t y o f c a r n i t i n e p a l m i t y l t r a n s f e r a s e can be i n c r e a s e d by dexamethasone and t h a t t h i s i n c r e a s e can be prevented by p r i o r i n j e c t i o n o f a c t i n o m y c i n D. I n d i r e c t evidence suggests t h a t glucagon may i n f l u e n c e l i v e r c a r n i t i n e p a l m i t y l t r a n s f e r a s e a c t i v i t y . I t has been shown (71) t h a t , i n l i v e r c e l l s i s o l a t e d from c a r b o h y d r a t e - r e f e d r a t s , glucagon s t i m u l a t e s the o x i d a t i o n o f p a l m i t a t e and markedly i n c r e a s e s the s y n t h e s i s of l o n g - c h a i n a c y l c a r n i t i n e s . CARNITINE ACETYLTRANSFERASE C a r n i t i n e a c e t y l t r a n s f e r a s e has been found i n sheep and pigeon l i v e r e x t r a c t s (105) . The enzyme has been p a r t i a l l y p u r i f i e d (110) and i t s p r o p e r t i e s s t u d i e d (110,66). C a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y has been r e p o r t e d to occur i n m i t o c h o n d r i a and/or homogenates o f r a t l i v e r , k i d n e y , s k e l e t a l muscle, h e a r t ( 1 6 ) , brown adipose t i s s u e , t e s t i s , b r a i n (183), and i n the mammary gland and l i v e r o f goat and guinea p i g (11). Only one study r e p o r t e d p l a c e n t a l a c t i v i t y and t h i s o c c u r r e d i n the mouse (279). Others have r e p o r t e d t h a t the enzyme i s p r e s e n t i n pigeon b r e a s t muscle and h e a r t ( 1 6 ) , locust f l i g h t muscle ( 1 7 ) , spermatozoa ( 1 8 3 ) , frog ske le ta l muscle ( 6 ) , in the l i v e r of ox and sheep ( 2 3 7 ) and in f e t a l human and monkey t issues ( 1 2 5 , 1 2 9 ) . Most of these e a r l i e r studies found the a c t i v i t y of carn i t ine acetyl t ransferase to be associated with mitochondria ( 1 6 , 1 8 3 , 1 3 3 , 1 7 , 2 0 5 , 1 1 , 2 7 7 ) ; however, recent ly i t was reported that , in rat l i v e r , approximately 5 2 7 o of the t i s s u e ' s carn i t ine acetyl t ransferase a c t i v i t y was mitochondrial , 1 4 % peroxisomal and 3 4 7 > microsomal ( 1 7 7 , 1 7 9 ) . Extra-mitochondrial a c t i v i t y has also been reported to occur in pig l i v e r ( 1 7 7 ) and brown adipose t issue ( 2 1 8 ) . MITOCHONDRIAL CARNITINE ACETYLTRANSFERASE It has been postulated that mitochondrial carn i t ine acetyl t ransferase a c t i v i t y consists of two pools on either side of the inner mitochondrial membrane ( 1 7 , 1 1 2 ) in a fashion analogous to carn i t ine palmi ty l t ransferase. This dual l o c a l i z a t i o n of carn i t ine acetyl t ransferase would f a c i l i t a t e the transport of a c e t y l - and short -chain acyl groups between the cytoplasm and mitochondrial matrix ( 1 7 , 3 9 ) . It has been concluded from cytochemical evidence ( 1 3 8 ) that carn i t ine acetyl transferase is si tuated on the inner surface of the outer mitochondrial membrane and/or on the outer surface of the inner membrane. Using a d ig i ton in method for removal of the outer mitochondrial membrane iso la ted from rat l i v e r , Brdiczka et a l . ( 3 9 ) concluded that at least 2 5 7 o of the to ta l mitochondrial a c t i v i t y of th is enzyme i s loca l i zed in the outer mitochondrial compartment. Other studies in which mitochondria were iso la ted from the l i v e r and mammary gland of goat, guinea pig and rat and from f e t a l bovine heart ( 2 7 7 , 1 1 ) , concluded that very l i t t l e , i f any, c a r n i t i n e a c e t y l t r a n s f e r a s e i s a v a i l a b l e to a c e t y l - C o A o u t s i d e i n t a c t m i t o c h o n d r i a . Edwards et a l . (91) used m i t o c h o n d r i a i s o l a t e d from v a r i o u s t i s s u e s and s p e c i e s and concluded t h a t t h e r e i s a s i n g l e c a r n i t i n e a c e t y l t r a n s f e r a s e which i s bound to the i n n e r m i t o c h o n d r i a l membrane which c a t a l y z e s o n l y the t r a n s f e r o f s h o r t - c h a i n a c y l groups between m i t o c h o n d r i a l m a t r i x coenzyme A and e x t e r n a l c a r n i t i n e . T h i s c o n c l u s i o n was supported by the work of S o l b e r g (256) who found t h a t t h e r e was no s h o r t - c h a i n t r a n s f e r a s e a c t i v i t y i n the " o u t e r " t r a n s f e r a s e p o o l i n l i v e r m i t o c h o n d r i a i s o l a t e d from r a t and mouse l i v e r . I n c o n t r a s t to these s p e c i e s , c a l f l i v e r m i t o c h o n d r i a had s i g n i f i c a n t " o u t e r " p o o l t r a n s f e r a s e a c t i v i t y f o r s h o r t - c h a i n s u b s t r a t e s . The presence o f an " o u t e r " s h o r t - c h a i n t r a n s f e r a s e seems to be q u i t e s p e c i e s dependent and the exact f u n c t i o n . o f t h i s t r a n s f e r a s e remains to be determined. PROPERTIES OF CARNITINE ACETYLTRANSFERASE C a r n i t i n e a c e t y l t r a n s f erase ( a c e t y l - C o A - c a m i t i n e a c e t y l t r a n s f e r a s e , EC 2.3.1.7) c a t a l y z e s the r e v e r s i b l e r e a c t i o n : a c e t y l - L - c a r n i t i n e + CoAsIl <--> L - c a r n i t i n e + a c e t y l - C o A . T h i s r e a c t i o n has an e q u i l i b r i u m c o n s t a n t of 0.6 (110) at pH 7.0 and 35°. F r i t z et a l . (110), u s i n g a p a r t i a l l y p u r i f i e d enzyme i s o l a t e d from p i g h e a r t , demonstrated t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e t r a n s f e r s the a c y l groups of a c e t y l - C o A , p r o p i o n l y - C o A and b u t y r y l - C o A to c a r n i t i n e a t a p p r o x i m a t e l y the same r a t e . The a c y l group of p a l m i t y l - C o A was not t r a n s f e r r e d and o n l y a slow r a t e o f t r a n s f e r was found w i t h octanoyl-CoA and decanoyl-CoA (110). The enzyme has a broad pH range (6 7 ) . Removal of one of the m ethyl groups from c a r n i t i n e does not a b o l i s h the a c t i v i t y (110). 48 The f i n d i n g s of Chase and Tubbs (66) , who examined pigeon b r e a s t muscle enzyme, suggest t h a t the enzyme has two independent b i n d i n g s i t e s f o r L - c a r n i t i n e and CoASH. I t has been p o s t u l a t e d t h a t a t h i r d s i t e ( 68), which i s hydrophobic i n n a t u r e , a l s o e x i s t s on the enzyme. The i n t e r a c t i o n of an acyl-CoA hydrocarbon s i d e c h a i n , which c o n t a i n s e i g h t o r more carbon atoms, w i t h t h i s s i t e , reduces the b i n d i n g o f c a r n i t i n e s thereby e f f e c t i v e l y i n h i b i t i n g the a c t i v i t y of the enzyme. This i n h i b i t i o n i s c o m p e t i t i v e and r e v e r s i b l e . D - c a r n i t i n e and a c e t y l - D - c a r n i t i n e are known to be c o m p e t i t i v e i n h i b i t o r s o f the enzyme (114,66). I t has a l s o been demonstrated t h a t v a r i o u s s u l f h y d r y l reagents (eg. DTNB) i n h i b i t the enzyme (114) and p r i o r treatment w i t h a c e t y l - C o A p r e v e n t s t h i s i n h i b i t i o n . EXTRA-MITOCHONDRIAL CARNITINE ACETYLTRANSFERASE A renewed i n t e r e s t i n c a r n i t i n e a c e t y l t r a n s f e r a s e has been generated by the f i n d i n g t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i s present" i n l i v e r peroxisomes and microsomes (1 7 7 , 1 7 9 ) . C l o f i b r a t e [ 2 - ( p - c h l o r o p h e n o x y ) - 2 - m e t h y l p r o p i o n i c a c i d ] , which i s used c l i n i c a l l y t o lower b l o o d l e v e l s o f t r i g l y c e r i d e s and c h o l e s t e r o l ( 2 1 0,135), has been shown to cause marked i n c r e a s e s i n c a r n i t i n e a c e t y l t r a n s f e r a s e i n both peroxisomes and m i t o c h o n d r i a (181,256,254). Peroxisomes, c h a r a c t e r i z e d by t h e i r c o n tent of H2O2 p r o d u c i n g o x i d a s e s and c a t a l a s e ( 8 1 ) , have r e c e n t l y been shown to c o n t a i n the enzymes of / 3 - o x i d a t i o n (1 62 , 1 63) (acyl-CoA dehydrogenases, enoyl-CoA h y d r a t a s e , 8-hydroxyacyl-CoA dehydrogenase and t h i o l a s e ) . These enzymes c a t a l y z e d the / 3 - o x i d a t i o n o f l o n g - c h a i n f a t t y a c i d s . The a d d i t i o n o f p a l m i t o y l - C o A to p u r i f i e d r a t l i v e r peroxisomes r e s u l t s i n 0 2 consumption, H202 p r o d u c t i o n and 0 2-dependent NAD r e d u c t i o n (162). C l o f i b r a t e i n c r e a s e d the a c t i v i t y of t h i s system by a p p r o x i m a t e l y one or d e r o f magnitude. T h i s i n c r e a s e i n a c t i v i t y was shown to be cyanid e i n s e n s i t i v e . I n d i r e c t evidence (162) suggests t h a t the ^ 8 - o x i d a t i o n system o f peroxisomes i s s i m i l a r to t h a t found i n glyoxysomes (74) i n t h a t the f i r s t dehydrogenase t r a n s f e r s i t s e l e c t r o n s to 0 2 p r o d u c i n g H 2 O 2 . The hydrogen p e r o x i d e t h a t i s generated i s c o n v e r t e d to water and oxygen by c a t a l a s e . A c e t y l - C o A accumulates as the end-product of ^ - o x i d a t i o n i n peroxisomes. The f a t e o f t h i s a c e t y l - C o A i s not known (163). The mechanism, whereby c l o f i b r a t e lowers serum l i p i d s and causes p e r o x i s o m a l p r o l i f e r a t i o n i n the l i v e r , i s unknown. The drug has no e f f e c t on l i v e r HMG-CoA r e d u c t a s e a c t i v i t y i n young swine (154). C l o f i b r a t e s upresses a r g i n i n e induced i n s u l i n r e l e a s e , i n the r a t , and p o t e n t i a t e s the r e l e a s e of glucagon ( 8 9 ) . I t a l s o has an a n t i - l i p o l y t i c e f f e c t on adipose t i s s u e (14) as measured by f r e e f a t t y a c i d r e l e a s e i n v i t r o . The r o l e t h a t c a r n i t i n e and c a r n i t i n e a c e t y l t r a n s f e r a s e p l a y i n the serum l i p i d l o w e r i n g e f f e c t s o f t h i s drug i s not known. I t i s i n t e r e s t i n g to note however, t h a t c l o f i b r a t e causes the a c t i v i t y o f the " i n n e r p o o l " o f m i t o c h o n d r i a l c a r n i t i n e a c e t y l t r a n s f e r a s e to i n c r e a s e 13 f o l d (256). The impact t h a t i t has on the " o u t e r p o o l " o f c a r n i t i n e a c e t y l t r a n s f e r a s e i n ot h e r s p e c i e s , l i k e the c a l f , which have s i g n i f i c a n t amounts o f "o u t e r p o o l " a c t i v i t y (256), i s not known. CARNITINE ACETYLTRANSFERASE: ITS DEVELOPMENT AND REGULATION Few s t u d i e s have been c a r r i e d out on the development and r e g u l a t i o n of c a r n i t i n e a c e t y l t r a n s f e r a s e . Most of these s t u d i e s 50 were concerned w i t h a c t i v i t y i n the m i t o c h o n d r i a . Lee and F r i t z (164) found t h a t , on a wet weight b a s i s , c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y , i n r a t l i v e r , was h i g h e r i n the neonate than i n the f e t u s . The a c t i v i t y peaked a t 4 days o f age and remained e l e v a t e d up t o 10 days of age. I t i s not known what happens from day 10 to weaning; however, Lee and F r i t z found t h a t a d u l t l e v e l s o f a c t i v i t y were s i g n i f i c a n t l y lower than n e o n a t a l v a l u e s . M i t o c h o n d r i a l and microsomal a c t i v i t i e s were not determined. Hahn and S k a l a (124) measured c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n m i t o c h o n d r i a i s o l a t e d from l i v e r , b r a i n , h e a r t , and brown adipose t i s s u e i n r a t s a t d i f f e r e n t stages o f development. I n each case, the a c t i v i t y was g r e a t e r i n the e a r l y n e o n a t a l p e r i o d than i n the f e t a l p e r i o d . The n e o n a t a l l e v e l s were s i m i l a r to a d u l t l e v e l s i n each t i s s u e except brown f a t i n which the a c t i v i t y d e c l i n e d at the time o f weaning. I n both the h e a r t and l i v e r of the monkey, a c e t y l t r a n s f e r a s e a c t i v i t y was found t o i n c r e a s e w i t h age (129) whereas, i n brown adipose t i s s u e and muscle, the a c t i v i t y remained unchanged. When r a t s are pre m a t u r e l y weaned, a t 18 days o f age, to a h i g h c a r b o h y d r a t e d i e t , c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n the l i v e r decreases ( 2 6 ) . I n m i t o c h o n d r i a o f brown f a t , i t has been found t h a t c o l d a d a p t a t i o n r e s u l t s i n a s i g n i f i c a n t i n c r e a s e o f c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y (124) and p e r o x i s o m a l content (5) but does not a f f e c t p a l m i t y l t r a n s f e r a s e a c t i v i t y ( 1 2 4 ) . I t has been suggested t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e i s p r e s e n t i n peroxisomes o f brown f a t and t h a t i t s a c t i v i t y i n c r e a s e s w i t h c o l d a d a p t a t i o n (218). The exact r o l e o f c a r n i t i n e a c e t y l t r a n s f e r a s e i n the mammal s t i l l remains to be determined. 51 METHODS AN AUTOMATED METHOD FOR THE DETERMINATION OF L-CARNITINE IN SERUM In the i n i t i a l s t a g e s o f t h i s p r o j e c t , we were i n t e r e s t e d i n d e v i s i n g a method f o r L - c a r n i t i n e d e t e r m i n a t i o n t h a t would be s u i t a b l e f o r m a s s - s c r e e n i n g p r o j e c t s . The p r e v i o u s l y d e s c r i b e d e nzymatic method o f Marquis and F r i t z (182) was not s u i t a b l e f o r t h i s purpose because o f a t e d i o u s d e p r o t e i n i z a t i o n s t e p and because o f the i n s t r u m e n t a t i o n i n v o l v e d . An automated method was developed w h i c h u t i l i z e d the c a r n i t i n e a c e t y l t r a n s f e r a s e (EC 2.3.1.7) r e a c t i o n as d e s c r i b e d by M a r q u i s and F r i t z (182) to measure the c o n c e n t r a t i o n o f L - c a r n i t i n e i n d e p r o t e i n i z e d serum. The b a s i s o f the r e a c t i o n i s as f o l l o w s : L - c a r n i t i n e + a c e t y l CoA <--> a c e t y l c a r n i t i n e + CoA. The f r e e coenzyme A, g e n e rated i n the r e a c t i o n , combines w i t h 5 , 5 1 - d i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB) and forms t h i o p h e n o l a t e i o n (92) w hich has a maximum absorbance a t 412nm. The r e a c t i o n i s a l l o w e d to go to c o m p l e t i o n and the i n c r e a s e i n absorbance i s r e c o r d e d . A s t a n d a r d curve i s d e v e l o p e d , from known s e t s o f s t a n d a r d s , and t h i s curve i s used to c a l c u l a t e the c o n c e n t r a t i o n o f l - c a r n i t i n e i n the unknown samples. The o r i g i n a l method of M a r q u i s and F r i t z was a l t e r e d by use o f membrane cone f i l t r a t i o n f o r d e p r o t e i n i z a t i o n o f samples and an automated b i c h r o m a t i c a n a l y z e r t o determine change i n absorbance. T h i s method was found to be s u i t a b l e f o r high-volume q u a n t i t a t i v e a n a l y s i s o f serum and u r i n e . E s s e n t i a l l y , t h i s procedure was found to save time and r e a g e n t s w i t h o u t s a c r i f i c i n g the s p e c i f i c i t y and a c c u r a c y o f the o r i g i n a l method. In a d d i t i o n , automated p i p e t t i n g and m i x i n g h e l p e d to improve the p r e c i s i o n o f the method. 53 M a t e r i a l s A l l c h e m i c a l s were o b t a i n e d from Sigma Chemical Co., S t . L o u i s , Mo. 63178, except where no t e d . A l l r e a g e n t s were p r e p a r e d f r e s h l y f o r each assay and k e p t on i c e d u r i n g the assay. T h i s ensured the s t a b i l i t y o f the rea g e n t mix over the assay p e r i o d . DTNB (16.0 mg) and 480.0 mg o f t r i s ( h y d r o x y m e t h y l ) methylamine ("Trizma") were d i s s o l v e d i n 10.0 ml o f d i s t i l l e d w a t e r . The pH was a d j u s t e d to 7.5 (at 24°C) w i t h c o n c e n t r a t e d HCl and d i l u t e d t o 20.0 ml w i t h d i s t i l l e d w a t e r . The f i n a l c o n c e n t r a t i o n was 0.2 mmol o f DTNB and 0.2 mol o f Tr i z m a per l i t e r . The sodium s a l t o f S - A c e t y l coenzyme A (76.8 mg, 95% pure) was d i s s o l v e d i n 30.0 ml o f d i s t i l l e d w a t e r . The f i n a l c o n c e n t r a t i o n was 2.0 m m o l / l i t e r . C a r n i t i n e a c e t y l t r a n s f e r a s e from pigeon b r e a s t muscle, c r y s t a l l i n e s u s p e n s i o n i n 3.2 m o l / l i t e r (NH4)2SC>4 s o l u t i o n (5 mg o f p r o t e i n per m i l l i l i t e r ; 105 U/mg o f p r o t e i n ) was used. S e q u e n t i a l d i l u t i o n s were p r e p a r e d from a 1mM s t o c k s o l u t i o n o f L - c a r n i t i n e t o g i v e s t a n d a r d s o l u t i o n s o f 100, 50, 25, 12.5, 6.25, and 3.125 u m o l / l i t e r . To 15.0 ml o f the DTNB/Trizma s o l u t i o n , 3.0 ml o f the S - a c e t y l coenzyme A s o l u t i o n were added and the volume brought up to 30.0 ml w i t h d i s t i l l e d , w ater. F i n a l l y , 30 u l o f the enzyme s u s p e n s i o n (about 105 U/ml) was added t o 14 ml o f the m i x t u r e . T h i s amount o f m i x t u r e was s u f f i c e n t to measure t e s t , r e a g e n t , and wat e r b l a n k s on 30 samples (see Procedure f o r d e t a i l s ) . 54 I f two or t h r e e assays (60 o r 90 specimens) were to be c a r r i e d o u t , 40 and 60 ml o f the m i x t u r e were used and an a p p r o p r i a t e amount o f enzyme s u s p e n s i o n was added. The f i n a l c o n c e n t r a t i o n s o f the i n d i v i d u a l components of the m i x t u r e i n the c u v e t t e ( w i t h s a m p l e - t o - m i x t u r e r a t i o o f 1/10, e.g. 25 u l / 2 5 0 u l ) , were: CoA, 0.2 mM; DTNB, 0.1 mM; and enzyme (when added), about 0.3 U. Membrane f i l t e r cones (CF50A), c e n t r i f u g e tubes and c o n i c a l s u p p o r t s (CST1) were from Amicon Corp., L e x i n g t o n , Mass. 02173. The Spectrophotometer (SOP-800A) was from Unicam In s t r u m e n t s L t d . , Cambridge, England and the B i c h r o m a t i c a n a l y z e r (ABA-100) from A b b o t t L a b o r a t o r i e s , N. C hicago, 111. 60064. S u b j e c t s and Sample P r e p a r a t i o n F o r t y - s e v e n a p p a r e n t l y h e a l t h y l a b o r a t o r y employees [27 women and 20 men, average age 30 t 7.5 (SD) y e a r s ] v o l u n t e e r e d to g i v e b l o o d a f t e r a minimum o f 12 hr o v e r n i g h t f a s t . Blood was c o l l e c t e d i n t o evacuated t u b e s , e i t h e r p l a i n or w i t h h e p a r i n or o x a l a t e f l u o r i d e p r e s e n t as a n t i c o a g u l a n t s . Serum ( a f t e r c l o t t i n g ) o r plasma was s e p a r a t e d and e i t h e r a n a l y z e d i m m e d i a t e l y or s t o r e d f r o z e n at -40°C f o r one to 30 weeks. No s i g n i f i c a n t d i f f e r e n c e s were r e c o r d e d i n v a l u e s o b t a i n e d from serum and plasma. S t o r a g e a t _40°C f o r 3 0 weeks d i d not a l t e r c a r n i t i n e l e v e l s . P rocedure D e p r o t e i n i z a t i o n : About 0.5 t o 1 ml o f serum were c e n t r i f u g e d i n a membrane f i l t e r cone at. 2000 rpm f o r 20 min at 10°C. Only new cones were used. A l i q u o t s (100 p l ) of the u l t r a f i l t r a t e were used i n the assay. Unknowns, aqueous c a r n i t i n e s tandards and a l i q u o t s of pooled serum ( q u a l i t y c o n t r o l ) were t r e a t e d i d e n t i c a l l y . Automated assay: The Abbott ABA-100 was f i t t e d w i t h a 415/550 mm f i l t e r , the i n c u b a t o r s e t to 37°C, a n a l y s i s time to 10 min, r e a c t i o n d i r e c t i o n to "Down", and k i n e t i c s to " E n d p o i n t " . The machine was s w i t c h e d to " T e s t " mode and c a l i b r a t e d to f o u r d e c i m a l p l a c e s (.000). A 1:11 s y r i n g e p l a t e was a t t a c h e d and f l u s h e d manually s e v e r a l times w i t h the reagent m i x t u r e w i t h o u t enzyme. The cups w i t h 100 u l o f specimen (or standards and q u a l i t y c o n t r o l ) were p l a c e d i n t o the c a r o u s e l . Each specimen cup was f o l l o w e d by a cup c o n t a i n i n g 100 u l o f d i s t i l l e d water (reagent b l a n k s ) . Specimens (and water b l a n k s ) were disp e n s e d i n the f i r s t c y c l e and the reagent m i x t u r e w i t h o u t the enzyme i n the second c y c l e . None of the m i x t u r e was added i n t o c u v e t t e no. 1. The change i n absorbance, A 4 1 5 n m , was read a f t e r 10 min (reagent b l a n k s ) . Next, the reagent m i x t u r e was r e p l a c e d by the m i x t u r e c o n t a i n i n g the enzyme. The t u b i n g was f l u s h e d and the whole assay r e p e a t e d . C a l c u l a t i o n 1 • M l 5 nm °f t n e specimen i n the run w i t h the enzyme -A41 5 n r n o f the b l a n k i n the run w i t h the enzyme = X. 2 . X - ( A 4 1 5 n m o f the specimen i n the run w i t h o u t the enzyme - A 4 1 5 n m o f the b l a n k i n the run w i t h o u t the enzyme) = f i n a l A 4 1 5 n m o f the specimen. 56 R e s u l t s I t has been r e p o r t e d (83) t h a t i n c o n s i s t e n t r e s u l t s were o b t a i n e d f o r c a r n i t i n e d e t e r m i n a t i o n s i n whole serum, and d e p r o t e i n i z a t i o n o f the samples has been recommended. With t h e d e p r o t e i n i z a t i o n procedure d e s c r i b e d above, the o r i g i n a l p r o t e i n c o n t e n t (75 t 10 g / l i t e r ) o f serum was decreased to 0.70 t 0.10 g / l i t e r - more than 98% o f the p r o t e i n was removed. With t h i s d e p r o t e i n i z a t i o n p rocedure, 45-50% o f the o r i g i n a l volume was l o s t . The above v a l u e s were o b t a i n e d w i t h new cones. When cones were washed and re-used as o u t l i n e d by the manufacturer, the e f f i c i e n c y o f p r o t e i n removal was not a l t e r e d but the volume r e c o v e r e d decreased t o 20-25% o f the o r i g i n a l . T h e r e f o r e , new cones were used f o r the d e p r o t e i n i z a t i o n o f s m a l l samples. The e f f i c i e n c y o f the above d e p r o t e i n i z a t i o n procedure was compared to a stan d a r d barium h y d r o x i d e and z i n c s u l f a t e method. The c o n d i t i o n s f o r d e p r o t e i n i z a t i o n by the l a t t e r method were t e s t e d , and the f o l l o w i n g procedure proved to be o p t i m a l : An 80 m m o l / l i t e r s o l u t i o n o f z i n c s u l f a t e , 0.25 ml, was added t o 0.5 ml o f serum and mixed t h o r o u g h l y . Subsequently, 0.25 ml of barium h y d r o x i d e (80 m m o l / l i t e r s o l u t i o n ) was added and the r e s u l t i n g p r e c i p i t a t e removed by c e n t r i f u g a t i o n (4000 rpm, 15 min). A 0.5 ml a l i q u o t o f the supernate was taken and the a d d i t i o n s were both r e p e a t e d . A l l o f the above was done at.0-4°C. The average serum p r o t e i n c o ntent decreased to about 27-33 g / l i t e r , a f t e r the f i r s t p r e c i p i t a t i o n s t e p , and to about 1.5-2.5 g / l i t e r a f t e r the second. Only the second supernate was s a t i s f a c t o r y f o r the s p e c t r o p h o t o m e t r i c assay of c a r n i t i n e . The t h r e e f o l d d i l u t i o n i n c u r r e d by c h e m i c a l 57 deproteinizat ion severely l imited the s e n s i t i v i t y of the assay procedure at lower concentrat ions. Another important considerat ion was the di f ference in ana ly t i ca l recovery of carn i t ine between the two deprote in izat ion procedures. In the case of the zinc sulfate/barium hydroxide p r e c i p i t a t i o n , a n a l y t i c a l recovery was v i r t u a l l y 1 0 0 7 o . In contrast , recovery of P^C] carn i t ine from sera by the cone procedure was 8 4 . 2 + 1 . 9 7 o . In s imi lar experiments in which water standards were used, recovery of [^C] carn i t ine was 8 7 . 9 t 1 . 2 7 , . Thus binding of carn i t ine to the membrane cone was not re lated to the presence of prote in . To compensate for carn i t ine loss on the membrane, we "deproteinized", in exactly the same manner, a l l standards, qua l i ty cont ro ls , and serum samples before they were' assayed. Assay Conditions The optimal concentrations of reagents were determined by assaying with various concentrations of DTNB ( 0 . 0 2 - 4 mmol / l i te r ) , acety l CoA ( 0 . 0 4 - 0 . 2 mmol / l i te r ) , and enzyme ( 1 0 - 2 4 0 U/ml) present. The optimal concentrations were found to be 0 . 2 and 2 . 0 mmol/ l i ter for DTNB and acetyl -CoA, respect ive ly . The presence of increasing amounts of enzyme always resulted in an increase of nonspeci f ic absorbance and necessitated the introduct ion of an "enzyme blank" (see Procedure). The apparent K m of the enzyme for acetyl CoA i s 4 x 1 0 ~ 5 mmol. Figure 1 shows the absorption spectrum for DTNB, with 57 d e p r o t e i n i z a t i o n s e v e r e l y l i m i t e d the s e n s i t i v i t y o f the assay procedure a t lower c o n c e n t r a t i o n s . Another i m p o r t a n t c o n s i d e r a t i o n was the d i f f e r e n c e i n a n a l y t i c a l r e c o v e r y o f c a r n i t i n e between the two d e p r o t e i n i z a t i o n p r o c e d u r e s . In the case o f the z i n c s u l f a t e / b a r i u m h y d r o x i d e p r e c i p i t a t i o n , a n a l y t i c a l r e c o v e r y was v i r t u a l l y 100%. In c o n t r a s t , r e c o v e r y o f [^C] c a r n i t i n e from s e r a by the cone procedure was 84.2 t 1.9%. In s i m i l a r e x p e r i m e n t s i n which water s t a n d a r d s were used, r e c o v e r y o f [ ] c a r n i t i n e was 87.9 j? 1.2%. Thus b i n d i n g o f c a r n i t i n e t o the membrane cone was not r e l a t e d to the presence o f p r o t e i n . To compensate f o r c a r n i t i n e l o s s on the membrane, we " d e p r o t e i n i z e d " , i n e x a c t l y the same manner, a l l s t a n d a r d s , q u a l i t y c o n t r o l s , and serum samples b e f o r e they were assayed. Assay C o n d i t i o n s The o p t i m a l c o n c e n t r a t i o n s o f r e a g e n t s were determined by a s s a y i n g w i t h v a r i o u s c o n c e n t r a t i o n s o f DTNB (0.02-4 m m o l / l i t e r ) , a c e t y l CoA (0.04-0.2 m m o l / l i t e r ) , and enzyme (10-240 U/ml) p r e s e n t . The o p t i m a l c o n c e n t r a t i o n s were found to be 0.2 and 2.0 m m o l / l i t e r f o r DTNB and a c e t y l - C o A , r e s p e c t i v e l y . The presence o f i n c r e a s i n g amounts o f enzyme always r e s u l t e d i n an i n c r e a s e o f n o n s p e c i f i c absorbance and n e c e s s i t a t e d the i n t r o d u c t i o n o f an "enzyme b l a n k " (see P r o c e d u r e ) . The apparent K m o f the enzyme f o r a c e t y l CoA i s 4 x I O " 5 mM. F i g u r e 1 shows the a b s o r p t i o n spectrum f o r DTNB, w i t h 58 mercaptoethanol p r e s e n t (A) and a f t e r L - c a r n i t i n e d e t e r m i n a t i o n ( B ) . D e t e r i o r a t i o n o f the "reagent m i x t u r e " l e a d s to a change i n A415/550 nm* I n both curves (A and B ) , the d i f f e r e n c e between absorbance a t 412 n m (optimum absorbance) and 4-15 n m i s n e g l i g i b l e ; hence, use o f the c o m m e r c i a l l y a v a i l a b l e 415/550 n m f i l t e r does not decrease the s e n s i t i v i t y of the method. At the temperature used (37°C), the r e a c t i o n i s completed i n 10 min. Comparison w i t h the Manual Method (182) P a r a l l e l assays were r u n on s e r a , w i t h v a r i o u s c a r n i t i n e c o n c e n t r a t i o n s , by both the automated and the manual methods. The r e s u l t s gave a c o r r e l a t i o n c o e f f i c i e n t o f 0.989 f o r 11 p a i r s . Normal Values Values f o r c a r n i t i n e , i n the serum and plasma of h e a l t h y a d u l t v o l u n t e e r s as assayed by the automated method, are summarized i n Table I I . The mean and the range agree w e l l w i t h d a t a from o t h e r l a b o r a t o r i e s , shown i n Table I I . The q u a l i t y c o n t r o l serum used i n our assay was prepared by p o o l i n g s e r a from about 200 i n - p a t i e n t s . I t s average c a r n i t i n e c o n c e n t r a t i o n (51.1 p m o l / l i t e r ) was v e r y c l o s e to the mean f o r normal s u b j e c t s . A n a l y t i c a l V a r i a b l e s A c c u r a c y : Based on the degree of enzyme s p e c i f i c i t y , the use of aqueous L - c a r n i t i n e s t a n d a r d s , and the a n a l y t i c a l r e c o v e r y 59 FIGURE 1 S p e c t r a l scan of 5 , 5 ' - d i t h i o b i s - ( 2 - n i t r o b e n z o i c acid) with mercaptoethanol (A) and a f t e r L - c a r n i t i n e determination (B). 60 TABLE I I L - C a r n i t i n e C o n c e n t r a t i o n s i n Serum o f Normal A d u l t s L - c a r n i t i n e Q/.M) x ± SD Range Ref. No, 49.4 + 12.2a 25.0-73.8 46.2 + 14.2b 17.8-74.6 ( 58) 43.1 + 9/Oc 25.3-60.9 ( 60) 51.6 d 29.9-66.4 (109) 46.0 + 13.0e 20.0-72.0 ( 96) a Samples from 47 a p p a r e n t l y normal a d u l t s , c o l l e c t e d a f t e r o v e r n i g h t f a s t b, c, d, e Val u e s from o t h e r l a b o r a t o r i e s 62 from serum-based standards, t h i s m o d i f i c a t i o n i s at l e a s t as accurate as that of the o r i g i n a l method. S e n s i t i v i t y : The lower l i m i t of s e n s i t i v i t y f o r t h i s method i s 1 0 . 0 u m o l / l i t e r . Intra-assay v a r i a t i o n : Within-run studies showed a CV of 3.87o which was based on 20 measurements of low, normal, and high concentrations o f aqueous c a r n i t i n e standards (Table I I I ) . Inter-assay v a r i a t i o n s : Forty-nine day-to-day assays o f the above-mentioned pooled serum gave a CV of 8.97>, f o r serum with an average value of 5 1 . 1 u m o l / l i t e r , and a CV of 207, f o r the same c o n t r o l d i l u t e d t h r e e f o l d with p h y s i o l o g i c a l - s a l i n e (average value, 1 6 . 8 u m o l / l i t e r ) . S a l i c y l a t e , o x a l a t e , and heparin i n t e r f e r e n c e : L - c a r n i t i n e , at three d i f f e r e n t concentrations ( 2 0 , 6 0 , 80 u m o l / l i t e r ) , was added to sera c o n t a i n i n g 80 and 700 mg of s a l i c y l a t e per l i t e r (from p a t i e n t s ' specimens). The a n a l y t i c a l recovery o f c a r n i t i n e was determined to be 9 9 , 9 9 , and 1 007., r e s p e c t i v e l y . I t was concluded that s a l i c y l a t e or i t s metabolites do not i n t e r f e r e i n a s i g n i f i c a n t way with the determination of L - c a r n i t i n e . Oxalate and heparin d i d not i n t e r f e r e with the determination of c a r n i t i n e . In some instances during the e a r l y stages of t h i s study, L - c a r n i t i n e was determined using the manual spectrophometric method of Marquis and F r i t z ( 1 8 2 ) . The method used was i d e n t i c a l to the o r i g i n a l method, except that the co n c e n t r a t i o n of acetylcoenzyme A 63 TABLE I I I Within-Run P r e c i s i o n Study: Result of 20 Assays of Three Aqueous L - C a r n i t i n e Standards on the Same Day L - c a r n i t i n e (jiH) Absorbance (x t SD) CV,% 25 0.0537 + .0029 5.4 50 0.0642 + .0021 3.3 100 0.0873 + .0024 2.7 64 was doubled. The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of 0.8 u n i t s of c a r n i t i n e a c e t y l t r a n s f e r a s e (Sigma Chem. Co.) and allowed to run to completion at 25°C. A l l samples were r o u t i n e l y d e p r o t e i n i z e d before they were assayed. THE DETERMINATION OF CARNITINE USING (1-1 ^ C) acetyl-CoA Cederblad and L i n d s t e d t were the f i r s t to p u b l i s h a method f o r the determination of L - c a r n i t i n e using (1-1^C) acetyl-CoA (5 9). Although the assay had the c a p a c i t y to measure values i n the picomole range, i t was not without problems. The p r i n c i p l e of t h e i r assay was s i m i l a r to that of the spectrophotometric methods except that i t u t i l i z e d (1-1 ^ C)acetyl-CoA and generated (1-1 ^C) a c e t y l c a r n i t i n e . The r a d i o a c t i v e acetyl-coenzyme A, which was not incorporated, was separated from the product of the r e a c t i o n by passing the r e a c t i o n mixture through an anion exchange r e s i n column (Dowex 2-X8, i n the c h l o r i d e form). This column e f f e c t i v e l y r e t a i n e d the (1-1^C) acetyl-CoA and allowed the r a d i o l a b e l l e d a c e t y l c a r n i t i n e to pass through. This was l a t e r q u a n t i f i e d by l i q u i d s c i n t i l l a t i o n counting. The r e a c t i o n may be summarized as f o l l o w s : l - c a r n i t i n e + (1-1^C)acetyl-CoA <--> ( 1 - 1 4 C ) a c e t y l c a r n i t i n e + CoA This r e a c t i o n i s c a t a l y z e d by c a r n i t i n e a c e t y l t r a n s f e r a s e which has an apparent e q u i l i b r i u m constant of 0.6 at pH 7.0 and 35°C (111). Cederblad and L i n d s t e d t reported that standard curves were n o n - l i n e a r unless there was a l a r g e excess of acetyl-CoA over c a r n i t i n e . They chose to accept n o n - l i n e a r standard curves and adjusted the c o n c e n t r a t i o n o f a c e t y l - C o A a c c o r d i n g to the c o n c e n t r a t i o n range o f c a r n i t i n e b e i n g measured. A second problem encountered w i t h the low e q u i l i b r i u m c o n s t a n t o f the r e a c t i o n , was the d i l u t i o n o f the r a d i o - l a b e l l e d a c e t y l - C o A p o o l w i t h c o l d a c e t y l - C o A when the sample b e i n g measured c o n t a i n e d a c e t y l c a r n i t i n e . The r e a c t i o n i s an e q u i l i b r i u m r e a c t i o n so t h a t the a c e t y l c a r n i t i n e p r e s e n t c o u l d be h y d r o l y z e d t o c a r n i t i n e and ac e t y l - C o A . The a c e t y l - C o A so produced would change the s p e c i f i c a c t i v i t y of the r e a c t i o n mix, t h e r e b y c a u s i n g a lower n e t DPM per u n i t o f c a r n i t i n e c o n c e n t r a t i o n than would be seen i n a sample which d i d not c o n t a i n a c e t y l c a r n i t i n e . A l t h o u g h the assay was c o n s i d e r a b l y more s e n s i t i v e than the s p e c t r o p h o t o m e t r y a s s a y s , the s e r i o u s l i m i t a t i o n s o u t l i n e d made i t s use somewhat q u e s t i o n a b l e . I n 1974 these l i m i t a t i o n s were p a r t i a l l y overcome (28) by the a d d i t i o n , to the assay m i x t u r e , o f DTNB which f u n c t i o n e d as a t r a p f o r the coenzyme A r e l e a s e d d u r i n g the r e a c t i o n . Even though t h i s h e l p e d to reduce the r e v e r s i b i l i t y of the assay, a r a t i o o f ac e t y l - C o A to c a r n i t i n e i n excess o f 5:1 was r e q u i r e d f o r l i n e a r i t y . DTNB tended t o i n h i b i t the enzyme c a r n i t i n e a c e t y l t r a n s f e r a s e ; however the i n h i b i t i o n c o u l d be overcome by p r i o r i n c u b a t i o n w i t h a c e t y l - C o A (114). I n 1976, McGarry and F o s t e r (189) p u b l i s h e d an improved and s i m p l i f i e d r a d i o i s o t o p i c assay, f o r the d e t e r m i n a t i o n o f f r e e and e s t e r i f i e d c a r n i t i n e , based on the same p r i n c i p l e as t h a t p u b l i s h e d by Cederblad and L i n d s t e d t (59). Four major m o d i f i c a t i o n s were r e p o r t e d . These authors used sodium t e t r a t h i o n a t e , i n t h e i r assay, to o x i d i z e the r e l e a s e d coenzyme A which helped to p u l l the r e a c t i o n to c o m p l e t i o n . In c o n t r a s t to the f i n d i n g s o f o t h e r s ( 2 1 4 ) , they found t h a t t h i s s ubstance d i d not i n h i b i t the c a r n i t i n e a c e t y l t r a n s f e r a s e enzyme and was a much more e f f e c t i v e agent than DTNB f o r the removal o f coenzyme A. In a d d i t i o n , these a u t h o r s r e p o r t e d t h a t serum samples d i d not have to be d e p r o t e i n i z e d b e f o r e they were measured. They used a TRIS-HCL b u f f e r both i n the a s s a y and, a t a h i g h e r c o n c e n t r a t i o n , i n t h e i r h y d r o l y s i s p r o c e d u r e . The f i n a l m o d i f i c a t i o n t h a t McGarry and F o s t e r made was the a d d i t i o n o f Dowex ( f o r the removal of n o n - i n c o r p o r a t e d [ 1 - 1 ^ C ] a c e t y l - C o A ) , as a s l u r r y , d i r e c t l y to the assay i n s t e a d o f the passage o f the r e a c t i o n m i x t u r e through a column. Because o f the s m a l l sample s i z e t h a t our s t u d i e s i n v o l v e d and the markedly i n c r e a s e d s e n s i t i v i t y o f the r a d i o i s o t o p i c a s s a y , the e a r l i e r s p e c t r o p h o t o m e t r i c methods were abandoned i n f a v o u r o f the r a d i o a c t i v e a s s a y . The f o l l o w i n g r e l a t e s to s e v e r a l s t u d i e s c a r r i e d out w i t h the McGarry and F o s t e r method f o r c a r n i t i n e d e t e r m i n a t i o n . C o r r e c t i o n s f o r quench were made u t i l i z i n g an e x t e r n a l standard- and c h a n nels r a t i o method. DETERMINATION OF FREE CARNITINE ACCORDING TO THE METHOD OF McGARRY AND FOSTER C a r n i t i n e s t a n d a r d s were prepa r e d u s i n g l - c a r n i t i n e HCl which was n e u t r a l i z e d w i t h KOH d u r i n g the p r e p a r a t i o n o f the s t a n d a r d . A l i q u o t s o f the s t a n d a r d s were f r o z e n and s t o r e d a t -40°C u n t i l used. The assay f o r f r e e c a r n i t i n e was conducted, as o u t l i n e d by McGarry and F o s t e r (189), w i t h one e x c e p t i o n . At the t e r m i n a t i o n o f the r e a c t i o n , the c o n t i n u o u s l y s t i r r e d s l u r r y o f Dowex 1-X10 a n i o n exchange r e s i n (30%, w/v) was added i n two a l i q u o t s o f 0.5 ml each, r a t h e r than i n t h r e e a l i q u o t s o f 0.3 ml each. The r e a c t i o n m i x t u r e c o n t a i n e d , i n a volume o f 1.05 ml, the f o l l o w i n g : 120 umoles of T r i s - H C l b u f f e r , pH 7.3; 2 umoles o f sodium t e t r a t h i o n a t e ; 25 nmoles (0.025 u C i ) o f ( 1 - 1 4 C ) a c e t y l - C o A and l - c a r n i t i n e s t a n d a r d . The r e a c t i o n was s t a r t e d w i t h the a d d i t i o n o f 1 U o f c a r n i t i n e a c e t y l t r a n s f e r a s e (0.050 m l ) . The r e a c t i o n was a l l o w e d to proceed a t room temperature f o r 30 m i n u t e s , at which time 0.5 ml o f the Dowex 1-X10 s l u r r y was added. The c o n t e n t s o f the assay tube were mixed t h o r o u g h l y and l e f t on i c e f o r 10 minutes b e f o r e the second 0.5 ml a l i q u o t o f the Dowex s l u r r y was added. The tubes were l e f t f o r a f u r t h e r 10 minutes and then c e n t r i f u g e d f o r 10 min at 2000 rpm. S u b s e q u e n t l y , an a l i q u o t (0.8 ml) o f the r e s u l t i n g s u p e r n a t a n t was t r a n s f e r r e d t o 10 ml o f ACS s c i n t i l l a n t and the r a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n c o u n t i n g . A t y p i c a l s t a n d a r d c u r v e i s p r e s e n t e d i n F i g u r e 2. A second s t a n d a r d c u r v e was run i n the presence o f 100 uM a c e t y l - d l - c a r n i t i n e . (see F i g u r e 3 ) . There i s v i r t u a l l y no d i f f e r e n c e i n the s l o p e s o f t h e s e two curves which s u g g e s t s t h a t t h e r e has been l i t t l e to no c o n v e r s i o n o f the a c e t y l - d l - c a r n i t i n e p r e s e n t to c a r n i t i n e and a c e t y l - C o A . T h i s suggests t h a t the r e v e r s a l problem encountered i n e a r l i e r methods c o u l d l a r g e l y be overcome w i t h the a d d i t i o n o f sodium t e t r a t h i o n a t e to the assay. THE EFFECT OF BUFFER AND PROTEIN ON THE DETERMINATION OF CARNITINE IN SERUM As mentioned e a r l i e r , a McGarry and F o s t e r method f o r the been r e p o r t e d t h a t t h i s b u f f e r i s T r i s - H C l b u f f e r was used i n d e t e r m i n a t i o n o f c a r n i t i n e , a c e t y l a t e d by c a r n i t i n e the I t has 68 FIGURE 2 Standard curve f o r the d e t e r m i n a t i o n o f f r e e l - c a r n i t i n e a c c o r d i n g to the method o f McGarry and F o s t e r (189). Each tube c o n t a i n e d 0.05 ml a l i q u o t o f the a p p r o p r i a t e l - c a r n i t i n e s t a n d a r d . C a r n i t i n e was determined as o u t l i n e d i n the methods s e c t i o n . A water blank was i n c l u d e d and the net r a d i o a c t i v i t y , generated i n a 0.8 ml a l i q u o t o f the r e s u l t i n g s u p e r n a t a n t s , was.determined. L-Carnitine (juM) 70 FIGURE 3 D e t e r m i n a t i o n o f f r e e l - c a r n i t i n e i n the presence of 100 uM a c e t y l - d l - c a r n i t i n e a c c o r d i n g to the method o f McGarry and F o s t e r (189). Each assay tube c o n t a i n e d a 0.05 ml a l i q u o t of the a p p r o p r i a t e l - c a r n i t i n e s t a n d a r d and 100 uM a c e t y l - d l - c a r n i t i n e . The r e a c t i o n was c a r r i e d out as o u t l i n e d i n the methods s e c t i o n . A water b l a n k was i n c l u d e d and the net r a d i o a c t i v i t y , generated i n a 0.8 ml a l i q u o t o f the r e s u l t i n g s u p e r n a t a n t s , was determined. L-Carnitine ( J U M ) a c e t y l t r a n s f e r a s e i n the presence of a c e t y l - C o A ( 7 2 ) . A s e r i e s o f experiments were conducted i n which the a c e t y l a t i o n o f T r i s - H C l b u f f e r by the McGarry and F o s t e r assay m i x t u r e was s t u d i e d . S i n c e these authors r e p o r t e d t h a t i t was not ne c e s s a r y to d e p r o t e i n i z e samples p r i o r to b e i n g assayed, the n o n - s p e c i f i c b i n d i n g of ( 1 - ^ C ) a c e t y l - C o A by serum p r o t e i n s was a l s o examined. The assay m i x t u r e was prepared as o u t l i n e d i n the o r i g i n a l procedure w i t h the e x c e p t i o n t h a t , the amount o f r a d i o a c t i v i t y per assay tube was doubled. A f t e r the Dowex had been added as a su s p e n s i o n , the tubes were c e n t r i f u g e d , f o r 10 min a t 2000 rpm, and the r a d i o a c t i v i t y , i n an 0.20 ml a l i q u o t o f the s u p e r n a t a n t , was determined by l i q u i d s c i n t i l l a t i o n c o u n t i n g . The a c e t y l a t i o n o f T r i s - H C l b u f f e r was compared to t h a t f o r HEPES ( N - 2 - h y d r o x y e t h y p i p e r a z i n e - N 1 - 2 - e t h a n e s u l p h o n i c a c i d ) b u f f e r . A l l tubes had the same volume and the co n t e n t s were v a r i e d as o u t l i n e d i n Table IV. For the h y d r o l y s i s p r o c e d u r e , 0.05 ml o f water was added to 0.1 ml of 1 M T r i s base and 0.05 ml of 0.4N KOH ( r e s u l t a n t pH a p p r o x i m a t e l y 13). The co n t e n t s were l e f t f o r 1 h r at 37°C a f t e r which 0.2 ml o f 0.575 N HCl was added to r e t u r n the pH t o 7.3. The re m a i n i n g components were added, as o u t l i n e d i n Ta b l e IV, b r i n g i n g the f i n a l volume to 1.05 ml. From T a b l e IV, i t can be seen t h a t the background count i n a 0.20 ml a l i q u o t o f the s u p e r n a t a n t , o b t a i n e d a f t e r the a d d i t i o n of Dowex, i s 387 co u n t s . When the enzyme c a r n i t i n e a c e t y l t r a n s f e r a s e i s p r e s e n t , t h i s v a l u e i n c r e a s e s s l i g h t l y to 497 co u n t s . When the T r i s - H C l b u f f e r i s used i n s t e a d of HEPES b u f f e r , the count i n c r e a s e s t o 1601 when c a r n i t i n e a c e t y l t r a n s f e r a s e i s p r e s e n t . From t h i s and from the r e s u l t s of o t h e r s ( 7 2 ) , i t appears e v i d e n t t h a t T r i s - H C l b u f f e r i s a c e t y l a t e d i n the assay mix. When r a t serum i s p r e s e n t , the background count i n c r e a s e s from 387 t o 1007 which suggests t h a t the p r o t e i n i n the serum b i n d s some of the r a d i o a c t i v i t y . When the r e p o r t e d h y d r o l y s i s procedure i s f o l l o w e d , the background count i n c r e a s e s f u r t h e r from t h a t o b t a i n e d w i t h T r i s - H C l b u f f e r and enzyme (1601) to 5792 DPM. T h i s , must be due, i n p a r t , to the i n c r e a s e d c o n c e n t r a t i o n o f T r i s - H C l b u f f e r t h a t i s p r e s e n t i n samples which have been h y d r o l y z e d . I n o r d e r to reduce any volume f l u c t u a t i o n s encountered when Dowex i s added as a s u s p e n s i o n , i t was decided to remove non-i n c o r p o r a t e d (1 -14-C)acetyl-CoA by the a d d i t i o n o f 200 mg of Dowex i n an a c t i v a t e d and dry form r a t h e r than i n the form o f a s u s p e n s i o n . The r e s u l t s o f t h i s study i n d i c a t e d t h a t T r i s - H C l b u f f e r s h o u l d not be used i n the assay, t h a t a l l serum samples should be d e p r o t e i n i z e d b e f o r e b e i n g assayed, and t h a t an a l t e r n a t i v e method f o r h y d r o l y z i n g the c a r n i t i n e e s t e r s should be found. DEPROTEINIZATION AND HYDROLYSIS OF ACYLCARNITINE ESTERS IN SERUM A l l samples (s t a n d a r d s and s e r a ) were r o u t i n e l y d e p r o t e i n i z e d w i t h e q u a l t i t r a t e d volumes of 0.087M ZnSo4 and 0.083M Ba(0H)2 ( r e s u l t i n g pH 7.3) b e f o r e they were assayed f o r f r e e c a r n i t i n e . I n o r d e r to h y d r o l y z e the s h o r t - c h a i n a c y l c a r n i t i n e e s t e r s , the sequence of a d d i t i o n o f the two substances was r e v e r s e d . F o l l o w i n g the a d d i t i o n of 0.4 ml Ba(0H)2 to 0.1 ml serum, the tube was stoppered and heated at 37°C f o r 1 h r ( r e s u l t a n t pH g r e a t e r than 12.0). Subsequently, the tube was c o o l e d on i c e and ZnS04 was 74 TABLE IV The E f f e c t of Bu f f e r and Serum P r o t e i n on the Level of Background A c t i v i t y i n the Determination of C a r n i t i n e According to the Method of McGarry and Foster (189) Data are the mean of two determinations. *The h y d r o l y s i s procedure of the o r i g i n a l method was followed i n o b t a i n i n g t h i s data. The b a s i c r e a c t i o n mixture contained i n a f i n a l volume of 1.05 ml, 120 umoles of T r i s - H C l or HEPES b u f f e r , pH 7.3; 2 A m o l e s sodium t e t r a t h i o n a t e ; 25 nmoles (0.050 uCi) of (1- 1^C)acetyl-CoA; 1U of c a r n i t i n e a c e t y l t r a n s f e r a s e and sample. When a substance was added or subtracted the appropr i a t e volume adjustment was made with water i n the assay tube. A c t i v a t e d Dowex 1-X10 anion exchange r e s i n (30%, w/v, 0.50 ml) was added from a continuously s t i r r e d s l u r r y . The contents o f the tube were mixed and l e f t on i c e f o r 10 min. This step was then repeated, the contents" l e f t f o r a f u r t h e r 10 min i n t e r v a l and subsequently, the tubes c e n t r i f u g e d . A 0.20 ml a l i q u o t of the r e s u l t i n g supernatant was placed i n t o ACS (10 ml) and r a d i o a c t i v i t y determined by l i q u i d s c i n t i l l a t i o n counting. ADDITIONS MIX HEPES TRIS WATER TO VOLUME ENZYME RAT SERUM X DPM (0.05 ml) (0.20 ml) + + - + + + - + + - + + + + - + + - +* + 387 + - 497 + - 1601 + 1007 + - 5792 75 added. The p r e c i p i t a t e formed was removed by c e n t r i f u g a t i o n and an a l i q u o t of the supernatant was used f o r the determination of c a r n i t i n e . The value f o r s h o r t - c h a i n a c y l c a r n i t i n e e s t e r s was obtained by s u b t r a c t i n g the value obtained f o r f r e e c a r n i t i n e from that obtained f o l l o w i n g h y d r o l y s i s of the e s t e r s . Using t h i s h y d r o l y s i s procedure f o r the determination of f r e e c a r n i t i n e and i t s s h o r t - c h a i n a c y l c a r n i t i n e esters i n 100 uM standards of DL-acetyl, DL-butyryl, DL-octanoyl, DL-decanoyl, D L - l a u r o y l , DL-myristoyl, and DL-palmityl c a r n i t i n e c h l o r i d e s , the c a l c u l a t e d a c y l c a r n i t i n e recovery was, i n a l l cases, g r e a t e r than 957. of t h e o r e t i c a l . The assay f o r the determination of c a r n i t i n e was improved f u r t h e r by i n c o r p o r a t i n g the recommendations of P a r v i n and Pande ( 2 1 4 ) . These authors found thart NEM (N-ethylmaleimide) was s u p e r i o r to sodium t e t r a t h i o n a t e i n b i n d i n g CoA and preventing a r e v e r s a l of the c a r n i t i n e a c e t y l t r a n s f e r a s e r e a c t i o n . In a d d i t i o n , they demonstrated that NEM d i d not i n h i b i t c a r n i t i n e a c e t y l t r a n s f e r a s e . A second improvement in v o l v e d the use of a c h a r c o a l s e p a r a t i o n method f o r removal of the non-incorporated ( 1 - 1^C)acetyl-CoA from the ( 1 - 1 ^ C ) a c e t y l c a r n i t i n e , generated i n the r e a c t i o n . At the termination of the r e a c t i o n , 0 .6 ml of the f o l l o w i n g a c t i v a t e d c h a r c o a l s l u r r y (which i s s t i r r e d continuously on a magnetic s t i r r e r ) was added: a c t i v a t e d c h a r c o a l ( n e u t r a l i z e d ) 4 . 0 g absolute ethanol 54 .5 ml 857o phosphoric a c i d 1.25 ml water 4 .25 ml 76 The f i n a l procedure u t i l i z e d f o r the determination of f r e e c a r n i t i n e and s h o r t - c h a i n a c y l c a r n i t i n e s was as f o l l o w s : a l l d e p r o t e i n i z a t i o n and assay procedures were c a r r i e d out i n disposable p o l y s t y r e n e c o n i c a l tubes (12 x 75 mm). Each sample of serum and standard (0.1 ml) was d e p r o t e i n i z e d as o u t l i n e d e a r l i e r . In a c l e a n tube, a 0.1 ml a l i q u o t of the d e p r o t e i n i z e d supernatant was placed with 0.1 ml of an assay mixture which contained 20 umoles HEPES b u f f e r , pH 7.6, 0.8 umoles EDTA, 2.0 umoles NEM and 2.5 nmol ( 1 - 1 4 C ) a c e t y l - C o A ( s p e c i f i c a c t i v i t y 0.02 u C i per nmol). The r e a c t i o n was i n i t i a t e d by the a d d i t i o n of enzyme (0.05 ml c o n t a i n i n g 1 u n i t of c a r n i t i n e a c e t y l t r a n s f e r a s e ) and then mixed g e n t l y , capped, and l e f t at room temperature f o r 60 minutes. The r e a c t i o n was terminated by the a d d i t i o n of 0.6 ml of the a c t i v a t e d c h a r c o a l s l u r r y to the assay tube. The contents were mixed w e l l and then c e n t r i f u g e d at 3000 rpm f o r 15 minutes i n a c l i n i c a l c e n t r i f u g e . The r e s u l t i n g supernatant was poured o f f i n t o a clean tube and c e n t r i f u g e d a second time. An a l i q u o t (0.5 ml) of the supernatant from t h i s c e n t r i f u g a t i o n was placed i n t o a s c i n t i l l a t i o n v i a l with 0.4 ml of water and 5 ml ACS s c i n t i l l a n t . A d i s p o s a b l e mini-counting v i a l system was used to reduce c o s t s . For each assay, a p p r o p r i a t e standards of known concentrations of c a r n i t i n e were included with a q u a l i t y c o n t r o l serum sample and a water blank at the beginning and at the end of the assay. Each standard was d e p r o t e i n i z e d and hydrolyzed i n a manner i d e n t i c a l to that used f o r the unknowns. The assay was l i n e a r up to 150 JUM concentrations of c a r n i t i n e i n the o r i g i n a l sample. More concentrated samples could be measured f o l l o w i n g a p p r o p r i a t e 77 d i l u t i o n . The i n t e r - a s s a y CV f o r t h i s a s s a y , based on the v a l u e s o b t a i n e d f o r the q u a l i t y c o n t r o l , was 4.3% (n = 2 5 ) . DETERMINATION OF D-3-HYDROXYBUTYRATE D-3-Hydroxybutyrate was determined a c c o r d i n g to P e r s s o n (221). T h i s enzymatic f l u o r o m e t r i c micromethod r e q u i r e s t h a t a l l serum samples be d e p r o t e i n i z e d p r i o r to a s s a y . The ZnS04 and Ba(0H)2 d e p r o t e i n i z a t i o n p r o c e d u r e , used f o r the d e t e r m i n a t i o n o f f r e e c a r n i t i n e , was a l s o s u i t a b l e f o r the d e t e r m i n a t i o n o f D-3-h y d r o x y b u t y r a t e . F o l l o w i n g the d e t e r m i n a t i o n o f f r e e c a r n i t i n e , an a l i q u o t o f the d e p r o t e i n i z e d sample's s u p e r n a t a n t was decanted and s t o r e d at -40°C f o r f u t u r e d e t e r m i n a t i o n o f D - 3 - h y d r o x y b u t y r a t e . A l l assays were c a r r i e d out i n d i s p o s a b l e c o n i c a l p o l y s t y r e n e t u b e s . The d e p r o t e i n i z e d s u p e r n a t a n t (0.2 ml) was p l a c e d i n t o the a s s a y tube w i t h water (0.1 ml) and 0.150 ml o f an assay mix which c o n t a i n e d 75 umoles sodium b i c a r b o n a t e b u f f e r , pH 9.4, 3.0 umoles o f NAD and 0.1 u n i t s o f D-3-hydroxybutyrate dehydrogenase. The r e a c t i o n proceeded a t room temperature (22°C) f o r 90 minutes and was then t e r m i n a t e d by the a d d i t i o n o f 1.55 ml o f 0.01 N NaOH c o n t a i n i n g 1 mM EDTA. The c o n t e n t s o f the tubes were mixed and the NADH p r e s e n t was d e t e r m i n e d i n a F.K. Turner and A s s o c i a t e s f l u o r o m e t e r . The p r i m a r y f i l t e r was 360 nm and the secondary f i l t e r 460 nm. Standards o f known c o n c e n t r a t i o n o f D - 3 - h y d r o x y b u t y r a t e , a q u a l i t y c o n t r o l serum, and a water b l a n k were r o u t i n e l y i n c l u d e d i n each a s s a y and t r e a t e d i n a manner which was s i m i l a r to t h a t used f o r the unknowns. 78 U t i l i z i n g the above assay c o n d i t i o n s , the r e a c t i o n was found to go to completion i n 6 0 minutes at room temperature. No d i f f e r e n c e was found to occur when a TRIS-hydrazine hydrate b u f f e r ( 2 8 2 ) was s u b s t i t u t e d f o r the sodium bicarbonate b u f f e r . The assay was found to be l i n e a r at values i n excess of 1 . 0 umole per ml ( o r i g i n a l s o l u t i o n ) but, because of s c a l e l i m i t a t i o n s on the fluorometer, samples having a c o n c e n t r a t i o n of D - ^3-hydroxybutyrate i n excess of 1 . 5 mM had to be d i l u t e d p r i o r to being assayed. DETERMINATION OF FREE FATTY ACIDS Free f a t t y a c i d s were determined according to the method of Novak ( 2 0 6 ) . The f r e e f a t t y a c i d s were ex t r a c t e d from 0 . 0 5 ml of serum according to Dole ( 8 4 ) , and complexed with c o b a l t n i t r a t e to form c o b a l t soaps. The c o b a l t soaps were extracted i n t o an upper phase from the c o b a l t n i t r a t e aqueous phase and an a l i q u o t used f o r the determination of f r e e f a t t y a c i d s . This l a t t e r e x t r a c t i o n step overcomes many of the t r o u b l e s encountered i n some of the e a r l i e r p ublished methods f o r the determination of f r e e f a t t y a cids ( 1 3 , 8 8 ) . This method i s f r e e from i n t e r f e r e n c e by l a c t a t e and b i l i r u b i n and was found to be l i n e a r when the assay tube contained 0 . 1 uEq of f a t t y a c i d . The method followed was e x a c t l y as p u b l i s h e d by Novak ( 2 0 6 ) w i t h the exception that the i n d i c a t o r s o l u t i o n was changed to 0 . 4 7 o 2 - n i t r o s o - 1 - n a p h t h o l i n 9 6 7 o ethanol. This s o l u t i o n was d i l u t e d by a f a c t o r of 1 2 . 5 before use. This change i n the i n d i c a t o r s o l u t i o n increases the s e n s i t i v i t y of the assay (Novak, personal communciation). 79 TISSUE PREPARATION I s o l a t e d t i s s u e s were homogenized i n 10 volumes o f a b u f f e r e d s u c r o s e s o l u t i o n (0.25 M s u c r o s e , 1 mM EDTA, 3.4 mM HEPES, pH 7.4). The HEPES b u f f e r was s e l e c t e d because i t s pKa (7.55) i s c l o s e to the d e s i r e d w o r k i n g pH and i t i s not a c e t y l a t e d by c a r n i t i n e a c e t y l t r a n s f e r a s e . I t s major d i s a d v a n t a g e i s t h a t i t i n t e r f e r e s w i t h s t a n d a r d methods used i n the d e t e r m i n a t i o n o f p r o t e i n ( 2 2 2 ) . Two s t r o k e s o f a l o o s e - f i t t i n g T e f l o n p e s t l e ( c l e a r a n c e - 0.041 cm) were used at a moderate h o m o g e n i z a t i o n speed to prepare the 10% (weight to volume) t i s s u e homogenates. S u b s e q u e n t l y , the homogenates were c e n t r i f u g e d , a t 600 g f o r 10 min, and the r e s u l t i n g s u p e r n a t a n t was decanted. At t h i s s t a g e , the s u p e r n a t a n t was passed through a s i n g l e l a y e r o f M i r a c l o t h i f i t c o n t a i n e d a l a r g e amount o f l i p i d . The f i l t e r e d s u p e r n a t a n t was then c e n t r i f u g e d a t 3,500 g f o r 10 min. The m i t o c h o n d r i a l - r i c h p e l l e t was kept f o r f u r t h e r p r o c e s s i n g and the s u p e r n a t a n t used f o r the p r e p a r a t i o n o f a m i c r o s o m a l - r i c h p e l l e t . The m i t o c h o n d r i a l p e l l e t was washed by r e - s u s p e n d i n g the m i t o c h o n d r i a l - r i c h p e l l e t i n 4.0 ml o f the homogenizing b u f f e r and c e n t r i f u g i n g a t 3,500 g f o r 10 min. The r e s u l t i n g s u p e r n a t a n t was c a r e f u l l y decanted so as not to d i s t u r b the p e l l e t and the washing procedure was r e p e a t e d a second t i m e . A f t e r washing, the m i t o c h o n d r i a l - r i c h p e l l e t was re-suspended i n a f i n a l b u f f e r s o l u t i o n (0.25 M s u c r o s e , 1 mM EDTA, 0.4 M KC1, 3.4 mM HEPES, pH.7.4) and s t o r e d a t 4°C f o r l a t e r e n z y m a t i c a n a l y s i s . A c e n t r i f u g a l f o r c e o f 3,500 x g was s e l e c t e d f o r g e n e r a t i n g the m i t o c h o n d r i a l p e l l e t , i n o r d e r to m i n i m i z e 80 c o n t a m i n a t i o n o f the m i t o c h o n d r i a w i t h peroxisomes. A study was undertaken, u s i n g r a t l i v e r and brown adipose t i s s u e , to study d i f f e r e n t methods of m i t o c h o n d r i a l s e p a r a t i o n by d i f f e r e n t i a l c e n t r i f u g a t i o n . An assessment o f these d i f f e r e n t methods was made by examining the f o l l o w i n g parameters i n each f r a c t i o n : p r o t e i n c o n t e n t , a c t i v i t y l e v e l s o f s u c c i n a t e dehydrogenase and c a r n i t i n e a c e t y l t r a n s f e r a s e . The d e t a i l s and r e s u l t s o f the study a re pr e s e n t e d i n Table V. The m i t o c h o n d r i a l p e l l e t s were ge n e r a t e d , a t 3,500 x g and 10,000 x g, from both l i v e r and brown adipose t i s s u e o f 20 day o l d r a t s . The o n l y s i g n i f i c a n t d i f f e r e n c e i n the t h r e e parameters under study o c c u r r e d w i t h the l i v e r m i t o c h o n d r i a l f r a c t i o n t h a t was prepared a t 10,0.00 x g. The p r o t e i n c o n t e n t o f t h i s f r a c t i o n was s i g n i f i c a n t l y h i g h e r than t h a t o b t a i n e d a t 3,500 x g, and the s p e c i f i c a c t i v i t y o f s u c c i n a t e dehydrogenase (a m i t o c h o n d r i a l marker enzyme) was s i g n i f i c a n t l y lower. No s i g n i f i c a n t d i f f e r e n c e s were found i n the t h r e e parameters, i n brown adipose t i s s u e , r e g a r d l e s s of the method o f m i t o c h o n d r i a l p r e p a r a t i o n . Because the da t a i n d i c a t e d t h a t the l i v e r and brown f a t m i t o c h o n d r i a l p e l l e t s , o b t a i n e d at 3,500 x g, were as good a s , i f not b e t t e r ( i n the case o f l i v e r ) than, those o b t a i n e d a t 10,000 x g and, because the h i g h e r f o r c e (10,000 x g) would cause g r e a t e r c o n t a m i n a t i o n o f the p e l l e t w i t h peroxisomes, the lower f o r c e (3,500 x g) was s e l e c t e d . T i s s u e microsomes were prepared i n the f o l l o w i n g manner. The supernatant t h a t was generated w i t h the f i r s t c e n t r i f u g a t i o n , a t 3,500 g f o r 10 min, was t r a n s f e r r e d to a c l e a n s e t o f tubes and c e n t r i f u g e d at 25,000 g f o r 10 min. The upper t w o - t h i r d s of the 81 TABLE V The E f f e c t o f D i f f e r e n t Methods o f M i t o c h o n d r i a l S e p a r a t i o n by D i f f e r e n t i a l C e n t r i f u g a t i o n Means + S.E.M. The number o f f r a c t i o n s s t u d i e d i s g i v e n i n p a r e n t h e s e s . Brown a d i p o s e t i s s u e (BAT) and l i v e r were removed from 20 day o l d r a t s , homogenized i n 10 volumes o f su c r o s e b u f f e r , and c e n t r i f u g e d a t 600 x g f o r 10 min (see Methods s e c t i o n ) . The m i t o c h o n d r i a l p e l l e t s were g e n e r a t e d u s i n g the f o r c e s i n d i c a t e d i n the T a b l e . Each f r a c t i o n was washed t w i c e and then s u b s e q u e n t l y re-suspended i n a known volume o f b u f f e r . P r o t e i n s were de t e r m i n e d f o l l o w i n g p r e c i p i t a t i o n w i t h t r i c h l o r a c e t i c a c i d . S u c c i n a t e dehydrogenase and c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t i e s were determined as o u t l i n e d i n the Methods s e c t i o n . F r a c t i o n P r o t e i n S u c c i n a t e Dehydro-(mg/ml) genase (umoles/mg p r o t e i n per min) C a r n i t i n e A c e t y l t r a n f e r a s e (nmoles/mg p r o t e i n per min)  L i v e r (3) 3,500 g x 10' L i v e r (3) 10,000 g x 10' BAT (3) 3,500 g x 10' BAT (3) 10,000 g x 10' 1.72 t 0.06 1.97+ 0.03* 0.56 t 0.02 0.63 + 0.03 0.09 + 0.002 0.075 t 0.003* 0.27 + 0.002 0.27 + 0.013 25.9 t 2.1 26.3 t 0.6 258.0 + 10.5 278.1 + 6.5 *p<.01 f o r l i v e r 10,000 g x 10' vs 3,500 g x 10' 82 r e s u l t i n g s u p e r n a t a n t were removed and a 2.0 ml a l i q u o t was p l a c e d i n t o a c e l l u l o s e n i t r a t e tube (7.9 x 49 mm). The m i c r o s o m a l - r i c h f r a c t i o n was o b t a i n e d by c e n t r i f u g i n g t h i s a l i q u o t a t 105,000 g f o r 45 min. The s u p e r n a t a n t was c a r e f u l l y decanted and d i s c a r d e d and the tube c o n t e n t s were l i g h t l y r i n s e d t w i c e w i t h homogenizing b u f f e r . D u ring t h i s r i n s i n g p r o c e d u r e , c a r e was taken not to d i s t u r b the microso m a l p e l l e t i n the tube. The p e l l e t was then re-suspended i n the same f i n a l s a l t - c o n t a i n i n g b u f f e r s o l u t i o n t h a t was used f o r r e s u s p e n s i o n o f the m i t o c h o n d r i a . S u c c i n a t e dehydrogenase a c t i v i t y was determined on f r e s h l y p r e p a r e d samples and c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y d e t e r m i n e d on samples t h a t had been l e f t a t 4°C f o r 24 h r . SUCCINATE DEHYDROGENASE DETERMINATION S u c c i n a t e dehydrogenase was determined a c c o r d i n g to the method o f Nachlas et a l . (200) w i t h the f o l l o w i n g m o d i f i c a t i o n s . In the o r i g i n a l method, the a u t h o r s used PMS as an e l e c t r o n c a r r i e r between s u c c i n a t e dehydrogenase and the t e t r a z o l i u m s a l t , INT. On r e d u c t i o n , INT forms a c o l o u r e d formazan product which has a molar e x t i n c t i o n c o e f f i c i e n t a t 490 nm o f 20.1 x 10^ (220) when e x t r a c t e d i n e t h y l a c e t a t e . S i n g e r and L u s t y (247) have suggested the use o f low c o n c e n t r a t i o n s o f CaCl2 t o i n c r e a s e the p e r m e a b i l i t y o f the m i t o c h o n d r i a l membranes to the dyes used i n the as s a y . These same a u t h o r s suggested t h a t any i n h i b i t i o n o f s u c c i n a t e dehydrogenase by o x a l o a c e t a t e can be removed by the i n c l u s i o n o f c y s t e i n e s u l f i n i c a c i d i n the assay . C y s t e i n e s u l f i n i c a c i d c o n v e r t s o x a l o a c e t a t e to 3 - s u l f i n y l - p y r u v a t e w h i c h s p o n t a n e o u s l y breaks down to p y r u v a t e and s u l f i t e . Both CaCl2 and c y s t e i n e s u l f i n i c a c i d were i n c l u d e d i n the 83 p r e s e n t assay. E a r l y a t t e m p t s a t e s t a b l i s h i n g o p t i m a l c o n d i t i o n s f o r the assay demonstrated t h a t INT i s n o n - e n z y m a t i c a l l y reduced by PMS and t h a t t h i s r e d u c t i o n i s , i n p a r t , i n f l u e n c e d by the presence o f p r o t e i n (see F i g . 4 ) . In a d d i t i o n , INT i s reduced by p r o t e i n a l o n e . T h i s r e d u c t i o n i s s t a b l e and d i r e c t l y r e l a t e d to the c o n c e n t r a t i o n o f p r o t e i n p r e s e n t ( F i g . 4 ) . By e l i m i n a t i n g the use o f PMS, the a c t i v i t y o f the enzyme i s reduced by a p p r o x i m a t e l y 2/3 (200) but the i n t e r f e r e n c e s e n c o u n t e r e d are l a r g e l y removed (220). The i n t e r a c t i o n o f INT w i t h p r o t e i n can be c o r r e c t e d f o r by the i n c l u s i o n o f a p p r o p r i a t e b l a n k s i n the a s s a y . In the p r e s e n t s t u d y , s i n c e s u c c i n a t e dehydrogenase was b e i n g used as a marker enzyme i t was f e l t t h a t the c o n d i t i o n s o f the a s s a y s h o u l d be e s t a b l i s h e d so as to e l i m i n a t e i n t e r f e r i n g r e a c t i o n s eventhough t h i s might r e s u l t i n some r e d u c t i o n i n obse r v e d a c t i v i t y . The b u f f e r f o r the as s a y c o n s i s t e d o f 100 mM po t a s s i u m phosphate, 2 mM KCN, 100 mM s u c r o s e , 2 mM c y s t e i n e s u l f i n i c a c i d , 1.5 mM CaCl2 w i t h the pH a d j u s t e d to 7.5. A 0.10 ml a l i q u o t o f a 0.5 M s o l u t i o n o f s u c c i n a t e (pH 7.5) and 0.50 ml o f the assay b u f f e r was added to each a s s a y tube. An a p p r o p r i a t e volume o f the enzyme s o l u t i o n was added, and the f i n a l volume brought up to 0.90 ml w i t h w ater. The tubes were mixed w e l l and l e f t to i n c u b a t e a t 37°C f o r 10 min. S u b s e q u e n t l y , the as s a y was s t a r t e d by the a d d i t i o n o f 0.1 ml o f a 0.1% (w/v) f r e s h l y made s o l u t i o n o f INT. The r e a c t i o n was stopped, 10 min l a t e r , w i t h the a d d i t i o n o f 1.0 ml o f a 10% TCA s o l u t i o n . The formazan p r o d u c t was e x t r a c t e d i n t o 4.0 ml o f e t h y l a c e t a t e and the A 4 9 0 n m o f the e x t r a c t r e a d . 84 FIGURE 4 The e f f e c t s o f p r o t e i n on the non-enzymatic r e d u c t i o n o f INT by PMS. The assay c o n d i t i o n s f o r s u c c i n a t e dehydrogenase were those as o u t l i n e d i n the t e x t w i t h the e x c e p t i o n t h a t the s u b s t r a t e , s u c c i n i c a c i d , was o m i t t e d . M i t o c h o n d r i a were f r e s h l y i s o l a t e d from female r a t l i v e r f o l l o w i n g the i s o l a t i o n methods as o u t l i n e d i n the t e x t . Data are the mean o f two d e t e r m i n a t i o n s . V - INT o n l y p r e s e n t a - INT and PMS p r e s e n t m - INT and PMS p l u s i n c r e a s i n g amounts o f albumin (0.25, 0.5, 1.0 and 2.5 mg r e s p e c t i v e l y ) .05 0.1 0.15 2.0 Mitochondrial Protein (mg) The s p e c i f i c a c t i v i t y o f s u c c i n a t e dehydrogenase can be c a l c u l a t e d by the i n c l u s i o n o f a p p r o p r i a t e b l a n k s i n the a s s a y and by d e t e r m i n i n g the p r o t e i n c o n t e n t i n the o r i g i n a l sample assayed. For r e p r o d u c i b l e r e s u l t s , i t i s i m p o r t a n t t h a t the assay tubes not be exposed to d i r e c t s u n l i g h t . The r e a c t i o n was found to be l i n e a r w i t h m i t o c h o n d r i a l p r o t e i n (see F i g . 5 ) . CARNITINE ACETYLTRANSFERASE DETERMINATION The method o f M a r k w e l l et a l . (177) was used f o r the s p e c t r o p h o t o m e t i c d e t e r m i n a t i o n o f c a r n i t i n e a c e t y l t r a n s f e r a s e . T h i s method, which i s based on a e a r l i e r r e p o r t by F r i t z and S c h u l t z (114), can be summarized as f o l l o w s : Acetyl-coenzyme A + L - c a r n i t i n e + DTNB A l \,y c a r n i t i n e a c e t y l t r a n s f e r a s e A c e t y l c a r n i t i n e + DTNB-coenzyme A ( t h i o p h e n o l a t e i o n - absorbs m a x i m a l l y a t 412 nm) In t h i s a s s a y , each sample i s measured t w i c e , once i n the presence o f l - c a r n i t i n e and once i n i t s absence (volume adjustments b e i n g made w i t h w a t e r ) . The l - c a r n i t i n e independent r a t e f u n c t i o n s as a c o n t r o l f o r any a c e t y l - C o A h y d r o l a s e a c t i v i t y (20) t h a t may be p r e s e n t i n the sample and f o r any p r o t e i n s u l p h y d r y l - DTNB i n t e r a c t i o n s t h a t may o c c u r d u r i n g the r e a c t i o n . At the t e r m i n a t i o n o f the assay, the change per u n i t time i n absorbance generated i n the f i r s t two minutes o f the a s s a y , i s used to c a l c u l a t e the c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y . The e x t i n c t i o n c o e f f i c i e n t f o r the t h i o p h e n o l a t e i o n i s taken as 13.68 mM (9 2 ) . F I G U R E 5 S u c c i n a t e d e h y d r o g e n a s e : A c t i v i t y as a f u n c t i o n o f t h e amount o f m i t o c h o n d r i a l p r o t e i n p r e s e n t i n t h e a s s a y The a s s a y c o n d i t i o n s w e r e a s o u t l i n e d i n t h e t e x t . A f r e s h l y p r e p a r e d s u s p e n s i o n o f r a t l i v e r m i t o c h o n d r i a was u s e d and a p p r o p r i a t e b l a n k s w e r e i n c l u d e d i n t h e a s s a y t o a c c o u n t f o r n o n - e n z y m a t i c r e d u c t i o n o f INT b y p r o t e i n . No PMS was p r e s e n t . 88 .05 .10 .15 .20 .25 .30 Mitochondrial Protein (mg) 89 The conditions for the present assay were establ ished u t i l i z i n g mitochondria prepared from female rat l i v e r fol lowing the method outl ined ea r l i e r in this text . Typica l resul ts from one ser ies of experiments are presented in F i g . 6. The o r i g i n a l assay condit ions (177) were held constant while one var iable was a l te red . Once establ ished, th is parameter was held constant while a second parameter was var ied . In the f i n a l assay, each tube contained 116 mM TRIS buf fer , pH 7.8 containing 0.2% TX-100, 0.5 mM EDTA (pH adjusted to 7 . 8 ) , 2.5 mM l - c a r n i t i n e (when i t was present) , 0.25 mM DTNB ( f reshly prepared), and 0.2 mM acetyl-CoA in a f i n a l volume of 0.4 ml ( including sample). The resu l t ing assay was l inear up to 0.2 mg of mitochondrial prote in (see F i g . 7 ) . The in teract ion of DTNB with protein sulphydryl groups as wel l as i t s reported inh ib i to ry e f fect on carn i t ine acety l t ransferase (114) , imposed serious l imi ta t ions on the spectrophotometric method. In addi t ion , the assay was l imi ted in i t s s e n s i t i v i t y . These problems were overcome by the development of a radioact ive method for the determination of carn i t ine acetyl t ransferase a c t i v i t y based on the method of Parvin and Pande (214) . These authors reported that NEM prevents the reversa l of the carn i t ine acety l t ransferase react ion by binding coenzyme A and does not i n h i b i t the enzyme. As was the case with the spectrophotometric method, each sample has to be assayed twice. The react ion system, in a f i n a l volume of 0.1 ml, contained 116 mM HEPES buffer (pH 7 . 8 ) , 2.5 mM l - c a r n i t i n e (when i t was present) , 0.5 mM EDTA (pH adjusted to 7 . 8 ) , 3.0 mM NEM, 0.2% TX-100, 0.4 mM (14c) acetyl-CoA (spec i f i c a c t i v i t y of 1.35 nCi per 90 FIGURE 6 The e f f e c t o f EDTA, pH, DTNB, L - c a r n i t i n e and a c e t y l - C o A on the c a r n i t i n e a c e t y l t r a n s f e r a s e r e a c t i o n The i n c u b a t i o n s and assay c o n d i t i o n s were those d e s c r i b e d i n the Methods w i t h the e x c e p t i o n t h a t the c o n c e n t r a t i o n o f the component under i n v e s t i g a t i o n was v a r i e d as i n d i c a t e d i n the F i g u r e . M i t o c h o n d r i a were prepared from r a t l i v e r as d e s c r i b e d i n the Methods. The data are the mean o f two d e t e r m i n a t i o n s . nmol Coenzyme A/min o — p r -o bi o o In o > 1 1 i r FIGURE 7 Effect of mitochondrial protein concentration on carn i t ine acetyl t ransferase a c t i v i t y Assays and incubations were performed using the assay conditions as described in the Methods sect ion with the exception that the concentration of mitochondrial protein was varied as indicated in the Figure. 93 94 nanoraole) and sample. The assay was c a r r i e d out at 30°C f o r 10 min and then t e r m i n a t e d by adding 1.0 ml o f a c o n s t a n t l y s t i r r e d c h a r c o a l s u s p e n s i o n (8g a c t i v a t e d c h a r c o a l , 109 ml a b s o l u t e e t h a n o l , 2.5 ml of 14 M o r t h o p h o s p h o r i c a c i d and 4.25 ml w a t e r ) . The tubes ( c o n i c a l p o l y s t y r e n e , 12 x 75 mm) were mixed immediately and l e f t on i c e . They were then c e n t r i f u g e d f o r 10 min a t 800 g, the sup e r n a t a n t was poured o f f i n t o a c l e a n s e t o f tubes and the c e n t r i f u g a t i o n was re p e a t e d . F o l l o w i n g the second c e n t r i f u g a t i o n , a 0.5 ml a l i q u o t o f the supernatant was p l a c e d i n t o a c o u n t i n g v i a l w i t h 0.4 ml of water and 5.0 ml o f ACS s c i n t i l l a n t . An a l i q u o t o f the r e a c t i o n m i x t u r e ( l e s s sample) and a 0.5 ml a l i q u o t o f the p o s t - c h a r c o a l s u p e r n a t a n t of an assay water b l a n k were counted. The net DPM, generated by a g i v e n sample i n 10 min, was used t o c a l c u l a t e the a c t i v i t y o f t h e c a r n i t i n e a c e t y l t r a n s f e r a s e enzyme p r e s e n t . A d u l t female r a t l i v e r m i t o c h o n d r i a were prepa r e d as o u t l i n e d e a r l i e r and used to assess l i n e a r i t y o f the c a r n i t i n e a c e t y l t r a n s f e r a s e assay w i t h time and m i t o c h o n d r i a l p r o t e i n (see F i g . 8 and 9 ) . The assay showed good l i n e a r i t y w i t h r e s p e c t to these two parameters. Values o b t a i n e d w i t h t h i s method d i d not d i f f e r s i g n i f i c a n t l y from those o b t a i n e d w i t h the s p e c t r o p h o t o m e t r i c method. PROTEIN DETERMINATION Compounds t h a t can i n t e r f e r e w i t h the d e t e r m i n a t i o n o f p r o t e i n (170) i n c l u d e HEPES b u f f e r (222), EDTA and TX-100 (1 4 6 ) , suc r o s e and potassi u m i o n s (see 18). I n o r d e r to e l i m i n a t e these i n t e r f e r e n c e s , p r o t e i n s were p r e c i p i t a t e d a c c o r d i n g to the method of FIGURE 8 L i n e a r i t y of the c a r n i t i n e a c e t y l t r a n s f e r a s e assay with time Mitochondria were i s o l a t e d , from female r a t l i v e r , as o u t l i n e d i n the Methods s e c t i o n . The assay tubes, which contained the same amount of m i t o c h o n d r i a l p r o t e i n , were incubated f o r d i f f e r e n t time periods and, a f t e r the r e a c t i o n was terminated, the number of nanomoles of (^C) a c e t y l - l - c a r n i t i n e , formed w i t h i n a given time pe r i o d , were determined. Time (min) 97 FIGURE 9 L i n e a r i t y of the c a r n i t i n e a c e t y l t r a n s f e r a s e assay with m i t o c h o n d r i a l p r o t e i n Mitochondria were i s o l a t e d from r a t l i v e r as o u t l i n e d i n the Methods s e c t i o n . The assay tubes, which contained v a r y i n g amounts of m i t o c h o n d r i a l p r o t e i n , were a l l incubated f o r 10 min and, a f t e r the r e a c t i o n was terminated, the number of nanomoles of (14rj) a c e t y l - l - c a r n i t i n e was determined. 98 0.233 r 2 4 6 8 10 12 14 16 18 20 22 24 26 /ng Mitochondrial Protein 99 Bensadoun and W e i n s t e i n ( 1 8 ) . F o l l o w i n g the p r e c i p i t a t i o n of both s t a n d a r d s and unknowns, p r o t e i n was determined a c c o r d i n g to Lowry (170). A s t a n d a r d c u r v e was p l o t t e d a c c o r d i n g to S t a u f f e r (258) and the amount o f p r o t e i n c a l c u l a t e d . ISOLATION OF PEROXISOMES D u r i n g the c o u r s e o f t h i s p r o j e c t , s t u d i e s appeared i n the l i t e r a t u r e w hich i n d i c a t e d t h a t peroxisomes c o n t a i n e d c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y (177,179). The a c t i v i t y i n these o r g a n e l l e s was found to i n c r e a s e d r a m a t i c a l l y w i t h serum l i p i d l o w e r i n g agents such as C l o f i b r a t e (181,254,256). Other s t u d i e s suggested t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e was p r e s e n t i n s i m i l a r c a t a l a s e p o s i t i v e m i c r o b o d i e s o f brown a d i p o s e t i s s u e and t h a t the a c t i v i t y o f t h i s enzyme, i n these p a r t i c l e s , i n c r e a s e d w i t h c o l d a d a p t a t i o n (218). In l i g h t o f these r e p o r t s , attempts were made to se p a r a t e peroxisomes and m i t o c h o n d r i a by i s o p y k n i c c e n t r i f u g a t i o n . The d e n s i t i e s o f these two o r g a n e l l e s , i n a d u l t l i v e r , a r e s i m i l a r ( m i t o c h o n d r i a l d^Q = 1.16 - 1.18 g/ml and peroxisomes d^Q = 1.24 - 1.26 g/ml). By u s i n g a l a r g e volume g r a d i e n t (50 ml) and an adequate s u p p l y o f t i s s u e (1 g or more), a good s e p a r a t i o n o f these o r g a n e l l e s can be a c h i e v e d (177). Because o f the l i m i t e d t i s s u e a v a i l a b i l i t y i n most d e v e l o p m e n t a l s t u d i e s , an attempt was made to s c a l e down the above s e p a r a t i o n . t e c h n i q u e . T i s s u e s were homogenized i n 10 volumes o f the f o l l o w i n g b u f f e r : 0.05 M TRIS, pH 7.6, 1 mM EDTA, 0.1% e t h a n o l , and 0.4M 100 FIGURE 10 • F r a c t i o n a t i o n o f r a t l i v e r from 14 d a y - o l d r a t s by i s o p y k n i c c e n t r i f u g a t i o n i n a l i n e a r s u c r o s e d e n s i t y g r a d i e n t For d e t a i l s see Methods s e c t i o n . • — • Protein (mg/ml) o b o b o o o o o • • i 1 1 1 ' L 1 o Catalase (mmoles/mg protein/min) to H ^ . . SDH (nmoles/mg q protein/min) 1 — ' — I — I — l ^ 1 J, 1 1 1 i 1 I ^ k3 IO K> iO JO U 8 8 8 £ 8 § 8 Density (g/ml) 5°C 102 s u c r o s e . The e t h a n o l was i n c l u d e d to s t a b i l i z e c a t a l a s e , the p r i n c i p a l marker enzyme o f peroxisomes ( 6 5 ) . The sucrose c o n c e n t r a t i o n was s e l e c t e d to maximize the y i e l d o f peroxisomes because, a t t h i s c o n c e n t r a t i o n , s u c r o s e s t a b i l i z e s these o r g a n e l l e s (268). L i n e a r s u c r o s e g r a d i e n t s (12 ml) were prepared which had a c u s h i o n o f s u c r o s e a t the bottom (0.8 ml o f a s u c r o s e s o l u t i o n , d e n s i t y 1.30 g/ml a t 5°C). The range of l i n e a r i t y f o r the g r a d i e n t was 1.14 to 1.30 g/ml a t 5 ° C T i s s u e s were homogenized, by a s i n g l e s t r o k e , u s i n g a l o o s e - f i t t i n g ( c l e a r a n c e 0.041 cm) t e f l o n p e s t l e and s l e e v e . The homogenate was then c e n t r i f u g e d a t 600 g f o r 10 m i n u t e s . The s u p e r n a t a n t was g e n t l y poured o f f and c e n t r i f u g e d a t 25,000 g f o r 15 m i n u t e s . The p e l l e t was resuspended i n b u f f e r (1.5 ml) and c a r e f u l l y l a y e r e d onto the g r a d i e n t s . The g r a d i e n t s were developed, at 100,000 g ( f o r c e g e n e r a t e d a t the m i d - p o i n t o f the g r a d i e n t ) f o r 2.5 h r , i n a SB-283 Swi n g i n g Bucket r o t o r (Beckman Instruments I n c . , Palo A l t o , C a l i f o r n i a ) . The bottom of the g r a d i e n t tube was p e r f o r a t e d and the g r a d i e n t c o l l e c t e d i n t o 20 e q u a l f r a c t i o n s . A f t e r c o l l e c t i o n , each f r a c t i o n was assayed f o r p r o t e i n , s u c c i n a t e dehydrogenase a c t i v i t y (see Methods s e c t i o n ) , and c a t a l a s e a c t i v i t y (171). The d e n s i t y o f each f r a c t i o n was determined by r e f r a c t o m e t r y . A r e p r e s e n t a t i v e g r a d i e n t t h a t was o b t a i n e d u s i n g r a t l i v e r (14 days o l d ) i s p r e s e n t e d i n F i g . 10. The m i t o c h o n d r i a l peak o c c u r r e d a t a d e n s i t y o f a p p r o x i m a t e l y 1.21 g/ml ( f r a c t i o n 12) whereas the p e r o x i s o m a l peak (as i n d i c a t e d by c a t a l a s e a c t i v i t y ) o c c u r r e d a t an approximate d e n s i t y o f 1.25 g/ml ( f r a c t i o n 7 ) . I t s h o u l d be n o t e d t h a t t h e r e i s c o n s i d e r a b l e o v e r l a p , i n the a c t i v i t i e s 103 of c a t a l a s e and s u c c i n a t e dehydrogenase, between f r a c t i o n s 7 and 12. On r e p e a t e d g r a d i e n t s , the v a r i a b i l i t y i n t h i s degree o f o v e r l a p was found to be l a r g e . T h i s n e c e s s i t a t e d a complete c h a r a c t e r i z a t i o n o f the enzyme p r o f i l e i n every g r a d i e n t b e f o r e i t c o u l d be used. Because o f the low p r o t e i n c o ntent i n the p e r o x i s o m a l f r a c t i o n , a r a d i o a c t i v e method f o r the d e t e r m i n a t i o n o f c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y had to be developed. I n a d d i t i o n , t h i s low p r o t e i n c o n t e n t s e v e r e l y l i m i t e d the number of assays t h a t c o u l d be c a r r i e d out on t h i s f r a c t i o n . The g r a d i e n t s e p a r a t i o n o f brown adipose t i s s u e i s p r e s e n t e d i n F i g . 11. The m i t o c h o n d r i a from t h i s t i s s u e are spread over a g r e a t e r d e n s i t y range than those from l i v e r . T h i s f i n d i n g , t o g e t h e r w i t h the lower d e n s i t y o f brown ad i p o s e t i s s u e c a t a l a s e p o s i t i v e m i c r o b o d i e s (218), suggests t h a t the p r e s e n t procedure w i l l not a d e q u a t e l y s e p a r a t e m i t o c h o n d r i a and peroxisomes i n brown f a t . Because of the h i g h s p e c i f i c a c t i v i t y of c a r n i t i n e a c e t y l t r a n s f e r a s e i n brown ad i p o s e t i s s u e m i t o c h o n d r i a , the s l i g h t e s t c o n t a m i n a t i o n o f a c a t a l a s e r i c h f r a c t i o n w i t h m i t o c h o n d r i a w i l l have d r a m a t i c e f f e c t s on the measured a c t i v i t y l e v e l s i n t h i s f r a c t i o n . From the r e s u l t s p r e s e n t e d i n F i g . 11, the h i g h e s t s p e c i f i c a c t i v i t y o f c a t a l a s e was found a t the top o f the g r a d i e n t , a t a d e n s i t y o f a p p r o x i m a t e l y 1.18 g/ml. T h i s f r a c t i o n c o n t a i n s enzymes t h a t have been s o l u b i l i z e d d u r i n g the i s o l a t i o n p r o c e d u r e . I t i s apparent, from the p r e s e n t d a t a , t h a t c o n s i d e r a b l e amounts o f c a t a l a s e were r e l e a s e d from brown ad i p o s e t i s s u e o r g a n e l l e s d u r i n g the i s o l a t i o n procedure s u g g e s t i n g t h a t these o r g a n e l l e s are not as s t a b l e as those found i n l i v e r . FIGURE 11 F r a c t i o n a t i o n o f brown adipose t i s s u e from 14 d a y - o l d r a t s by i s o p y c n i c c e n t r i f u g a t i o n i n a l i n e a r s ucrose d e n s i t y g r a d i e n t For d e t a i l s see Methods s e c t i o n . • Protein (mg/ml) o C a t a l a s e ( m m o l e s / m g protein/min) S D H (nmoles/mg protein/min) I—1 o 106 These r e s u l t s i n d i c a t e d t h a t b e f o r e d e v e l o p m e n t a l s t u d i e s o f c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n peroxisomes from brown adipose t i s s u e and l i v e r can be u n d e r t a k e n , a p p r o p r i a t e s e p a r a t i o n t e c h n i q u e s i n v o l v i n g more t i s s u e and l a r g e r volume g r a d i e n t s would have to be u t i l i z e d . R E S U L T S A N D D I S C U S S I O N 108 PART I 109 EFFECT OF FASTING ON FREE CARNITINE LEVELS IN HUMAN SERUM AND URINE: CORRELATION WITH SERUM LEVELS OF FREE FATTY ACIDS AND D-fi-HYDROXYBUTYRATE. I n t r o d u c t i o n An experiment was designed to study the e f f e c t o f f a s t i n g , i n man, on f r e e c a r n i t i n e l e v e l s i n serum and u r i n e . I n a d d i t i o n , serum l e v e l s o f FFA and D-B-hydroxybutyrate were measured to determine whether or not they would c o r r e l a t e w i t h changes i n serum f r e e c a r n i t i n e l e v e l s . The e f f e c t o f o r a l a d m i n i s t r a t i o n o f l - c a r n i t i n e , on these parameters and on i t s r e n a l h a n d l i n g , was a l s o s t u d i e d . E x p e r i m e n t a l Design Two experiments, i n v o l v i n g a p p a r e n t l y h e a l t h y v o l u n t e e r s , were conducted. A l l the s u b j e c t s were l a b o r a t o r y workers who were f u l l y informed about the study. Experiment 1 i n v o l v e d 6 i n d i v i d u a l s (2 f e m a l e s , 4 males ages 21-54 y r . ) and experiment 2 i n v o l v e d i n d i v i d u a l s (ages 18-54 y r s ; 4 females and 7 m a l e s ) . Blood was c o l l e c t e d a t 0900 h r s on day 1, 1 h r a f t e r b r e a k f a s t . Another b l o o d specimen was o b t a i n e d a f t e r 12 and 24 h r s o f f a s t i n g . A f t e r 24 h r s , n e u t r a l i z e d L - c a r n i t i n e was a d m i n i s t e r e d o r a l l y t o 7 i n d i v i d u a l s and a p l a c e b o (an e q u a l volume o f a 0.97, NaCl s o l u t i o n ) t o 4 i n d i v i d u a l s . Serum and u r i n e were c o l l e c t e d 1, 4 and 5 1/2 h r s l a t e r . The u s u a l amount o f a c t i v i t y was a l l o w e d d u r i n g the experim e n t s . Blood was c o l l e c t e d i n t o v a c u t a i n e r tubes and a l l o w e d t o c l o t a t room temperature b e f o r e a n a l y s i s or s t o r a g e . Serum and u r i n e were e i t h e r a n a l y z e d immediately or s t o r e d f o r a maximum of 2 110 wks a t -40°C. No changes i n the parameters under s t u d y were observed to o c c u r i n specimens s t o r e d under these c o n d i t i o n s . Free L - c a r n i t i n e was d e t e r m i n e d by the automated method and D-3-hydroxybutyrate and f r e e f a t t y a c i d s as d e s c r i b e d i n the methods s e c t i o n . C r e a t i n i n e was determined a c c o r d i n g to s t a n d a r d p r ocedures (264). C r e a t i n i n e ( e r e ) and c a r n i t i n e ( c a r ) c l e a r a n c e s (C) as w e l l as the p e r c e n t a g e t u b u l a r r e a b s o r p t i o n o f c a r n i t i n e (% TRC) were c a l c u l a t e d from the serum (S) and u r i n a r y (U) l e v e l s (V, u r i n a r y volume) a c c o r d i n g to the f o l l o w i n g f o r m u l a e : C U V ere = ere x S ere C U V c a r = c a r x S c a r %TRC = 1 - C c a r x 100 C ere S t a t i s t i c a l a n a l y s i s ( S t u d e n t ' s t t e s t and c a l c u l a t i o n o f c o r r e l a t i o n c o e f f i c i e n t r) was performed u s i n g a Gemsac computer programmed a c c o r d i n g to B a r n e t t ( 1 2 ) . R e s u l t s and D i s c u s s i o n There was no s i g n i f i c a n t d i f f e r e n c e i n the data o b t a i n e d i n experiments 1 and 2, the f i n d i n g s w i l l be d i s c u s s e d t o g e t h e r . The r e s u l t s o f experiment 2 are summarized i n Table V I . Both the serum and u r i n a r y l e v e l o f f r e e l - c a r n i t i n e decreased s i g n i f i c a n t l y w i t h f a s t i n g . F o l l o w i n g the i n g e s t i o n o f 1 g o f f r e e l - c a r n i t i n e , the u r i n a r y c o n c e n t r a t i o n o f t h i s substance i n c r e a s e d markedly as measured 1 and 4 hr p o s t - i n g e s t i o n . Serum l e v e l s o f f r e e f a t t y a c i d s I l l TABLE VI Serum and U r i n a r y C a r n i t i n e and Serum BOH and FAA L e v e l s i n a Group o f V o l u n t e e r s  C a r n i t i n e Serum U r i n e Serum FFA Serum BOH ( p M / l i t e r ) (pM/hr) ( m e q / l i t e r ) ( m M / l i t e r ) Experiment 2 1 h r a f t e r b r e a k f a s t 57. 9 + 8. 3 8.9 + 4.5 0 .43 + 0.15 0 .115 + 0.1 30 1 2 h r f a s t 46. 1 + 10. 6 3.2 + 3.3 0 .80 + 0.24 0 .520 + 0.365 24 h r f a s t 40. 7 + 13. 8* 0.4 + 0.4* 1 .22 + 0.49* 1 .060 + 0.845* 1 h r a f t e r c a r n i t i n e 47. 3 + 13. 8 3.3 + 2.3 1 .59 + 0.47 0 .960 + 0.235 1 h r a f t e r p l a c e b o 42. 6 + 12. 7 3.5 + 3.7 1 .73 + 0.39 1 .100 + 0.140 4 h r a f t e r c a r n i t i n e 54. 0 + 17. 8 16.6 + 12.0 1 .54 + 0.54 1 .540 + 0.340 4 hr a f t e r p l a c e b o 35. 4 + 15. 0 1.8 + 2.6 1 .67 + 0.50 1 .705 + 0.260 5 1/2 h r a f t e r c a r n i t i n e 40. 4 14. 8 4.9 + 5.9 1 .81 + 0.51 1 .750 + 0.375 5 1/2 h r a f t e r p l a c e b o 23. 9 + 16. 5 0.2 + 0.1 1 .80 + 0.408 2 .060 + 0.450 Means + SD are shown. Free f a t t y a c i d s (FFA). D-p-hyroxybutyrate (BOH). * S i g n i f i c a n t l y d i f f e r e n t (p<0.01) from the base v a l u e a f t e r b r e a k f a s t . and D -3-hydroxybutyrate were not a f f e c t e d by the i n g e s t i o n o f l - c a r n i t i n e . Of p a r t i c u l a r note was the f i n d i n g t h a t serum f r e e c a r n i t i n e l e v e l s showed a s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n w i t h serum f r e e f a t t y a c i d s and D-3-hydroxybutyrate l e v e l s (see F i g . 12 and F i g . 13) . Combined d a t a on c l e a r a n c e s and the t u b u l a r r e a b s o r p t i o n o f f r e e l - c a r n i t i n e are g i v e n i n Table V I I . A l t h o u g h the c l e a r a n c e o f c r e a t i n i n e and f r e e c a r n i t i n e tended to decrease over the 24 and 36 hr o f f a s t i n g , o n l y the decrease i n l - c a r n i t i n e c l e a r a n c e was s t a t i s t i c a l l y s i g n i f i c a n t . From the d a t a , i t i s e v i d e n t t h a t serum f r e e l - c a r n i t i n e l e v e l s i n man d e c r e a s e w i t h f a s t i n g . There are a number o f p o s s i b l e e x p l a n a t i o n s f o r t h i s d e c r e a s e : i n t e r r u p t i o n o f the exogenous s u p p l y ; an i n c r e a s e d uptake o f c a r n i t i n e by muscle and o t h e r t i s s u e s ; i n c r e a s e d g a s t r o i n t e s t i n a l l o s s e s ; or d e c r e a s e d s y n t h e s i s from l y s i n e (260). At l e a s t a p o r t i o n o f the d e c r e a s e i n l - c a r n i t i n e c o u l d be accounted f o r by i t s use i n the f o r m a t i o n o f s h o r t - c h a i n a c y l c a r n i t i n e s such as a c e t y l - , a c e t o a c e t y 1 - , 3 - h y d r o x y b u t y r y l -and/or o t h e r a c y l c a r n i t i n e s . U r i n a r y f r e e c a r n i t i n e e x c r e t i o n and i t s r a t e o f c l e a r a n c e a l s o d e c l i n e d w i t h f a s t i n g (see T a b l e s VI and V I I ) . In two v o l u n t e e r s , who f a s t e d f o r 5 days, f r e e c a r n i t i n e e x c r e t i o n markedly decreased by 36 h r o f f a s t i n g and remained a t v i r t u a l l y u n d e t e c t a b l e l e v e l s f o r the remainder of the f a s t ( d a t a not shown). These r e s u l t s are i n c o n t r a s t to the f i n d i n g s o f Maebashi et a l . (175), who d e s c r i b e d an i n c r e a s e i n u r i n a r y c a r n i t i n e e x c r e t i o n i n f a s t i n g 1 1 2 FIGURE 12 R e l a t i o n s h i p between serum FFA and f r e e L - c a r n i t i n e . Serum v a l u e s f o r these c o n s t i t u e n t s a t 12, 24 and 36 hr o f f a s t i n g are shown: e, experiment 1; o, experiment 2. The r e g r e s s i o n e q u a t i o n (shown by the s t r a i g h t l i n e ) i s B = -24.85.A + 2038, r = -0.607, p<0.001. 114 Serum Free L-Carnitine (uM/l) 115 FIGURE 13 R e l a t i o n s h i p between serum B - h y d r o x y b u t y r a t e and f r e e L - c a r n i t i n e . The same specimens as those shown i n F i g . 12 were a n a l y z e d . The r e g r e s s i o n e q u a t i o n i s B = -0.021 A = 1.495, r = -0.567, p<0.001. 116 117 s u b j e c t s . However, i t i s not c l e a r whether t h e i r c h e m i c a l method f o r the d e t e r m i n a t i o n o f c a r n i t i n e measured f r e e o r t o t a l c a r n i t i n e . These data i n d i c a t e t h a t the r e n a l h a n d l i n g of l - c a r n i t i n e i s c h a r a c t e r i s t i c o f a t h r e s h o l d substance. The d e c r e a s i n g serum l - c a r n i t i n e l e v e l c o r r e l a t e d w e l l w i t h the decreased urinary-e x c r e t i o n . A p p r o x i m a t e l y 10% o f the l - c a r n i t i n e i n g e s t e d a t the end o f the f a s t i n g p e r i o d s , was e x c r e t e d i n the u r i n e w i t h i n the f i r s t 3-6 h r . T h i s f i n d i n g i s s i m i l a r t o the f i n d i n g s of o t h e r s (58,175). F o l l o w i n g c a r n i t i n e a d m i n i s t r a t i o n , f r e e l - c a r n i t i n e e x c r e t i o n r o s e f a r out o f p r o p o r t i o n to the r i s e i n serum l e v e l s , which suggests t h a t t h e r e i s an i n c r e a s e d c l e a r a n c e and a t u b u l a r maximum f o r r e a b s o r p t i o n . EFFECT OF FASTING ON SERUM LEVELS AND RENAL CLEARANCE OF ACYLCARNITINE IN MAN I n t r o d u c t i o n The p r e v i o u s data i n d i c a t e t h a t , w i t h s t a r v a t i o n i n man, serum l e v e l s of f r e e c a r n i t i n e decrease and l e v e l s of f r e e f a t t y a c i d s and D-8-hydroxybutyrate i n c r e a s e . The decrease i n f r e e c a r n i t i n e l e v e l s d u r i n g s t a r v a t i o n may r e f l e c t an i n c r e a s e d uptake of c a r n i t i n e by t i s s u e s . An a l t e r n a t i v e e x p l a n a t i o n i s t h a t serum f r e e c a r n i t i n e l e v e l s decrease because serum c a r n i t i n e i s b e i n g used f o r the p r o d u c t i o n o f a c y l c a r n i t i n e . I f t h i s i s the case and i f t o t a l serum c a r n i t i n e l e v e l s do not change d u r i n g s t a r v a t i o n , then a decrease i n f r e e c a r n i t i n e may merely r e f l e c t a s h i f t i n the c i r c u l a t i n g forms o f c a r n i t i n e i n f a v o u r of a c y l c a r n i t i n e . 118 TABLE V I I Changes i n the C r e a t i n i n e and Free C a r n i t i n e C l e a r a n c e s and i n the T u b u l a r R e a b s o r p t i o n o f Free L - C a r n i t i n e W i t h F a s t i n g  T u b u l a r R e a b s o r p t i o n Hours o f F a s t i n g C r e a t i n i n e F r e e C a r n i t i n e o f Free C a r n i t i n e ( % ) 1 (11) 93.1 + 13.2 1.21 + 0.34 98.7 + 1 .4 12(17) 94.3 + 14.3 1 .22 + 0.39 98.7 + 1 .5 24(17) 89.1 + 17.3 .89 + 0.34* 99.0 + 1.4 36(6) 82.2 + 19.1 .66 + 0.21 99.2 + 0.8 Va l u e s a r e means + SD. The d a t a from e x p e r i m e n t s 1 and 2 have been combined, 12 and 3T4 h r o f f a s t i n g . Number o f s u b j e c t s i s g i v e n i n p a r e n t h e s e s . * S i g n i f i c a n t (p<0.01) vs 1 h r . 119 The assay t e c h n i q u e , used i n the p r e v i o u s s t u d y , i s capable o f d e t e c t i n g o n l y f r e e c a r n i t i n e . I n c o r p o r a t i n g a p p r o p r i a t e changes i n methodology, so t h a t a c y l - and t o t a l c a r n i t i n e c o u l d be d e t e r m i n e d , an experiment was d e s i g n e d to answer the f o l l o w i n g q u e s t i o n s . Can the d e c r e a s e i n f r e e c a r n i t i n e be accounted f o r by a c o n c o m i t a n t i n c r e a s e i n serum a c y l c a r n i t i n e ? I f serum a c y l c a r n i t i n e l e v e l s do i n c r e a s e , does the r e n a l c l e a r a n c e o f a c y l c a r n i t i n e a l s o i n c r e a s e ? E x p e r i m e n t a l Design S i x v o l u n t e e r s (1 female and 5 males; ages 22-55 y r ) f a s t e d from 2100 h r o f day 1 t o 1200 h r on day 3, a t o t a l o f 39 h r . Blood specimens were o b t a i n e d a f t e r 12, 36, and 39 hr o f f a s t i n g . U r i n e was c o l l e c t e d every 12 h r and over the l a s t t h r e e hr of the e x p e r i m e n t . C a r n i t i n e e s t e r s were q u a n t i f i e d by s u b s t r a c t i n g f r e e c a r n i t i n e from t o t a l c a r n i t i n e v a l u e s . The l a t t e r were o b t a i n e d f o l l o w i n g m i l d a l k a l i n e h y d r o l y s i s (see Methods s e c t i o n ) . R e s u l t s and D i s c u s s i o n The r e s u l t s are p r e s e n t e d i n Table V I I I and F i g . 14. D u r i n g the f a s t the c o n c e n t r a t i o n o f c a r n i t i n e e s t e r s ( T a b l e V I I I ) and the r a t i o o f serum c a r n i t i n e e s t e r s to f r e e c a r n i t i n e i n c r e a s e d (see F i g . 14). The r e n a l c l e a r a n c e o f c a r n i t i n e e s t e r s and o f " t o t a l c a r n i t i n e " (the sum o f f r e e c a r n i t i n e p l u s i t s e s t e r s ) a l s o i n c r e a s e d over the 39 h r f a s t (see Table V I I I ) w h i l e the r e n a l c l e a r a n c e o f f r e e c a r n i t i n e d e c r e a s e d . The d a t a i n d i c a t e t h a t , w i t h 120 TABLE V I I I E f f e c t o f F a s t i n g on Serum L e v e l s and Renal H a n d l i n g o f A c y l c a r n i t i n e Serum C o n c e n t r a t i o n C l e a r a n c e T u b u l a r Hours o f F a s t i n g ( u M / l i t e r ) (ml/min) R e a b s o r p t i o n (%) 12(6) 13.5 +11.1 1.1 + 0.6 98.8 + 8.5 36(6) 28.9 + 10.8 2.9 + 2.0 96.5 + 2.7 39(6) 26.6 + 8.2 * 2.9 + 1.1** 96.4 + 2.7 Values a re means + SD Number o f s u b j e c t s i s g i v e n i n par e n t h e s e s *p<0.001, v e r s u s 1.2 hr o f f a s t ( p a i r e d t t e s t ) **p<0.05, v e r s u s 12 h r o f f a s t FIGURE 14 R a t i o o f a c y l c a r n i t i n e to f r e e l - c a r n i t i n e c o n c e n t r a t i o n s e r a o f s i x f a s t i n g s u b j e c t s . 1 2 2 123 s t a r v a t i o n i n man, the r e l a t i v e d i s t r i b u t i o n between f r e e L - c a r n i t i n e and a c y l c a r n i t i n e i n the serum c a r n i t i n e p o o l undergoes marked changes. The r a t i o o f a c y l c a r n i t i n e to f r e e c a r n i t i n e , which may be used as a i n d i c a t o r o f the e x t e n t o f t h i s s h i f t , i n c r e a s e s . THE EFFECT OF PROLONGED STARVATION ON SERUM LEVELS OF FREE, ESTERIFIED AND TOTAL CARNITINE IN THE RAT I n t r o d u c t i o n The p r e v i o u s d a t a i n man showed t h a t , w i t h s h o r t - t e r m s t a r v a t i o n , l e v e l s o f serum f r e e c a r n i t i n e d e c r e a s e , those, o f a c y l c a r n i t i n e i n c r e a s e and t o t a l c a r n i t i n e l e v e l s tend to remain unchanged. The e f f e c t s o f s h o r t - t e r m s t a r v a t i o n may be q u i t e d i f f e r e n t from those seen w i t h l o n g - t e r m s t a r v a t i o n . In a d d i t i o n , the changes i n serum c a r n i t i n e l e v e l s d u r i n g s t a r v a t i o n may be q u i t e v a r i a b l e from s p e c i e s to s p e c i e s . For those reasons a d u l t r a t s were f a s t e d f o r 96 h r . Answers to the f o l l o w i n g q u e s t i o n s were sought. Are the changes i n serum f r e e , a c y l - and t o t a l c a r n i t i n e , d u r i n g p r o l o n g e d s t a r v a t i o n i n the r a t , s i m i l a r to those seen i n man d u r i n g a s h o r t - t e r m f a s t ? Do the serum l e v e l s o f t o t a l earn i t i n e . change w i t h p r o l o n g e d s t a r v a t i o n ? I f they do change, when does t h i s change occ u r ? E x p e r i m e n t a l D e sign A d u l t male W i s t a r r a t s ( w e i g h i n g 300 to 350 g) were used th r o u g h o u t . Animals had f r e e access to water d u r i n g the s t a r v a t i o n p e r i o d . Free c a r n i t i n e , a c y l c a r n i t i n e and t o t a l c a r n i t i n e were 124 during the s t a r v a t i o n p e r i o d . Free c a r n i t i n e , a c y l c a r n i t i n e and t o t a l c a r n i t i n e were determined as o u t l i n e d i n the Methods s e c t i o n . Results and D i s c u s s i o n The r e s u l t s obtained during a 96 hr s t a r v a t i o n p e r i o d are presented i n Table IX. Following 24 hr of s t a r v a t i o n i n the r a t , serum f r e e c a r n i t i n e l e v e l s decreased, a c y l c a r n i t i n e l e v e l s increased and t o t a l serum c a r n i t i n e l e v e l s decreased s i g n i f i c a n t l y over values obtained at time 0. This i n i t i a l decrease i n t o t a l serum c a r n i t i n e was followed by a s i g n i f i c a n t i n c r e a s e ( a f t e r 48 hr of s t a r v a t i o n ) which was maintained throughout the remainder of the s t a r v a t i o n p e r i o d . The r a t i o of a c y l c a r n i t i n e to f r e e c a r n i t i n e increased s i g n i f i c a n t l y during the f i r s t 24 hr of s t a r v a t i o n , remained ele v a t e d at t h i s l e v e l up to 48 hr, and t h e r e a f t e r tended to d e c l i n e . The decrease i n t o t a l c a r n i t i n e l e v e l s , a f t e r 24 hr of s t a r v a t i o n , may r e f l e c t e x c r e t o r y losses or increased t i s s u e uptake of c a r n i t i n e . I t i s not c l e a r whether the increase, a f t e r 48 hr of s t a r v a t i o n , r e f l e c t s increased m o b i l i z a t i o n of c a r n i t i n e from t i s s u e s or an increased s y n t h e t i c c a p a c i t y by the l i v e r , these data i n d i c a t e t h a t , with short-term s t a r v a t i o n , the p a t t e r n of change i n serum f r e e and a c y l c a r n i t i n e l e v e l s i s s i m i l a r i n both r a t and man; however, a 48 hr pe r i o d of s t a r v a t i o n i n the r a t i s equivalent to a much longer p e r i o d i n man. For t h i s reason, i t i s d i f f i c u l t to know whether prolonged s t a r v a t i o n i n man would a l s o r a i s e t o t a l c a r n i t i n e l e v e l s . In c o n t r a s t to Maebashi et a l . (175) who claimed that no a c y l c a r n i t i n e i s present i n serum, the present data i n d i c a t e that not 1 2 5 TABLE IX The E f f e c t o f S t a r v a t i o n on Serum C a r n i t i n e L e v e l s i n the Rat R a t i o , S t a r v a t i o n Free T o t a l A c y l c a r n i t i n e P e r i o d C a r n i t i n e A c y l c a r n i t i n e C a r n i t i n e Free (h) (uM) (uM) (uM) C a r n i t i n e 0 ( 5 ) 65.8 + 3.2 17.6 + 3.2 83.4 + 3.7 0.274 + 0.053 24 ( 5 ) 33.4 + 2.9* 28.8 + 3 . 5 * * 62.2 + 2.8* 0.905 + 0.140' 48 ( 5 ) 60.4 + 9.9 46.3 + 9.2 106.7 + 5 # 2 * * * 0.922 ~-Z 0.259 72 ( 5 ) 69.8 + 7.9 3 2 . 5 + 9.3 102.4 + 6.8 0.525 + 0.1 71 96 ( 5 ) 75.8 + 2.7 40.8 + 7.8 116.6 + 6.7 0.547 + 0.114 Mean + S.E. The number o f a n i m a l s s t u d i e d i s g i v e n i n p a r e n t h e s i s . A d u l t male r a t s were used. * p<0.01 v s . time 0 ** p<0.05 v s . time 0 *** p<0.01 v s . time 24 h r 126 o n l y i s a c y l c a r n i t i n e p r e s e n t i n serum but a l s o t h a t i t s l e v e l s change markedly d u r i n g s t a r v a t i o n . The d i f f e r e n c e s between the p r e s e n t f i n d i n g s and those of Maebashi et a l . (175) are p r o b a b l y due to the i n h e r e n t d i f f e r e n c e s between our enzymatic method and the c h e m i c a l method used by these authors f o r the d e t e r m i n a t i o n o f c a r n i t i n e . THE EFFECTS OF DIET ON D-6-HYDROXYBUTYRATE AND CARNITINE SERUM LEVELS IN THE RAT I n t r o d u c t i o n The p r e v i o u s data i n d i c a t e t h a t , w i t h s t a r v a t i o n i n both r a t and man, serum l e v e l s o f f r e e , a c y l - and t o t a l c a r n i t i n e change q u i t e d r a m a t i c a l l y . Fat i s u t i l i z e d as an energy source both d u r i n g s t a r v a t i o n and when r a t s are f e d a h i g h f a t d i e t . The d i f f e r e n c e between these two s t a t e s i s i n the s u p p l y o f c a l o r i e s . During s t a r v a t i o n l i p i d s are m a i n l y used to produce energy w h i l e d u r i n g f e e d i n g o f a h i g h f a t d i e t they are not o n l y o x i d i z e d but a l s o used f o r s y n t h e t i c purposes ( l a y i n g down f a t e t c . ) . An experiment was designed to examine the f o l l o w i n g q u e s t i o n s : Are the changes seen i n serum c a r n i t i n e l e v e l s unique to s t a r v a t i o n o r can a d i e t t h a t i s r i c h i n l i p i d b r i n g about the same changes? Do these changes occur when s h o r t - c h a i n f a t t y a c i d s are the p r i n c i p a l energy source f o r the animal? Can the i n c r e a s e d p r o d u c t i o n o f ketone b o d i e s by a c a r n i t i n e independent pathway, a f f e c t the serum l e v e l s of c a r n i t i n e ? Three d i e t s , which d i f f e r e d i n l i p i d c ontent and c o m p o s i t i o n , were f o r m u l a t e d . One d i e t was r i c h i n ca r b o h y d r a t e and i t s u t i l i z a t i o n by the animal would t h e r e f o r e be v i r t u a l l y independent o f i n t r a c e l l u l a r c a r n i t i n e . The second d i e t was r i c h i n medium-chain l e n g t h f a t t y a c i d t r i g l y c e r i d e s . These t r i g l y c e r i d e s which are ' r a p i d l y m e t a b o l i z e d by the animal would r e s u l t i n marked i n c r e a s e s i n serum l e v e l s o f ketone b o d i e s . The u t i l i z a t i o n o f the s e f a t t y a c i d t r i g l y c e r i d e s i s r e p o r t e d to be independent o f i n t r a c e l l u l a r c a r n i t i n e (230). The t h i r d d i e t was r i c h i n l o n g - c h a i n f a t t y a c i d t r i g l y c e r i d e s . The u t i l i z a t i o n of t h i s d i e t would be expected to r e q u i r e i n t r a c e l l u l a r c a r n i t i n e . EXPERIMENTAL DESIGN White W i s t a r r a t s were mated and pregnancies dated by v a g i n a l smears. L i t t e r s were reduced to e i g h t animals each and r a i s e d w i t h the mother a t 23°C w i t h a 12 h r l i g h t and 12 h r dark c y c l e . Weaned animals had f r e e access to s t a n d a r d r a t chow ( P u r i n a Chow, S t . L o u i s , Mo., f a t content 4%) and water. Animals aged 20 to 22 days were p l a c e d on t h e i r r e s p e c t i v e d i e t s f o r 48 h r , p r i o r to b e i n g s a c r i f i c e d . The samples were s t o r e d at -40^C u n t i l a n a l y z e d . D-^3-hydroxybutyrate and c a r n i t i n e were determined as o u t l i n e d i n the Methods s e c t i o n . The h i g h f a t d i e t was f o r m u l a t e d i n the f o l l o w i n g manner: p r o t e i n , 120 g ( c a s e i n h y d r o l y s a t e - e n z y m a t i c , U n i t e d S t a t e s B i o c h e m i c a l Corp., C l e v e l a n d , Ohio, USBC); c o t t o n - s e e d o i l , 176 g; t o t a l v i t a m i n supplement t r i t u r a t e d i n d e x t r o s e , 1.11 g (USBC); s a l t mix (Rogers-Harper, USBC), 5.55 g and water 556 g. G e l a t i n (7g/500 ml d i e t ) was added to pre v e n t s e t t l i n g o f the d i e t . The medium-chain 128 t r i g l y c e r i d e d i e t was f o r m u l a t e d i n the same manner as the h i g h f a t d i e t except the o i l component was r e p l a c e d w i t h a l i p i d f r a c t i o n o f coconut o i l c o n s i s t i n g p r i m a r i l y of the t r i g l y c e r i d e s o f Cg and C-JQ s a t u r a t e d f a t t y a c i d s ( o b t a i n e d from Drew Chemical Co., as MCT o i l ) . The h i g h c a r b o h y d r a t e d i e t was s i m i l a r l y f o r m u l a t e d except the o i l component was r e p l a c e d w i t h 360 g d e x t r o s e . The weight g a i n s on these d i e t s were w i t h i n the normal range except i n the case o f the medium c h a i n t r i g l y c e r i d e d i e t . Animals on t h i s d i e t d i d not g a i n weight a t the same r a t e as d i d animals on the o t h e r two d i e t s . T h i s o c c u r r e d i n s p i t e of an adequate food i n t a k e . A l l t h r e e d i e t s had the same c a r n i t i n e c o n c e n t r a t i o n (9.4pM). R e s u l t s and D i s c u s s i o n The r e s u l t s a re p r e s e n t e d i n Table X. Both of the h i g h f a t d i e t s produced a s i g n i f i c a n t i n c r e a s e i n the l e v e l of serum D-B-hydroxybutyrate. As expected (287), the k e t o s i s was v e r y much more pronounced w i t h the medium-chain t r i g l y c e r i d e d i e t . Free c a r n i t i n e l e v e l s were lower i n r a t s f e d the f a t d i e t s than i n those f e d the c a r b o h y d r a t e d i e t . Only the d i e t t h a t was r i c h i n medium-chain t r i g l y c e r i d e s r e s u l t e d i n s i g n i f i c a n t l y e l e v a t e d serum a c y l c a r n i t i n e l e v e l s . No change i n t o t a l serum c a r n i t i n e c o n tent was found w i t h the h i g h c a r b o h y d r a t e and medium-chain t r i g l y c e r i d e d i e t s ; however, w i t h the l o n g - c h a i n t r i g l y c e r i d e d i e t , t o t a l serum c a r n i t i n e decreased s i g n i f i c a n t l y . R a t s , f e d the h i g h f a t d i e t s , had a c y l - to f r e e c a r n i t i n e r a t i o s which were s i g n i f i c a n t l y g r e a t e r than those fed the c a r b o h y d r a t e d i e t . The medium-chain t r i g l y c e r i d e d i e t r e s u l t e d i n an a c y l - to f r e e c a r n i t i n e r a t i o which was s i g n i f i c a n t l y h i g h e r than t h a t produced w i t h e i t h e r o f the o t h e r two d i e t s . A p o s i t i v e TABLE X The E f f e c t s o f D i e t on ^B-Hydroxybutyrate and C a r n i t i n e Serum L e v e l s i n the Rat  Free T o t a l A c y l c a r n i t i n e 8-Hydroxybutyrate C a r n i t i n e A c y l c a r n i t i n e C a r n i t i n e Free D i e t _ (mM) (uM) (uM) (uM) C a r n i t i n e Carbohydrate (8) 0.043 + 0.007 39.2 + 2.2 12.3 + 1.0 51.6 +2.9 0.316 + 0.021 Long-chain t r i g l y c e r i d e (8) 0.838 + 0.073* 20.0 + 1.9* 14.3 + 1.3 34.4 + 1.9* 0.784 + 0.110* Medium-chain t r i g l y c e r i d e (10) 5.543 + 1.13*,** 19.4 + 1.1* 28.1 + 1.6*,** 47.5 + 2.5** 1.464 + 0.076*, Mean + S.E. The number o f ani m a l s s t u d i e d i s g i v e n i n parentheses. A l l animals were 20-22 days o l d and were p l a c e d on t h e i r r e s p e c t i v e d i e t s f o r 48 h b e f o r e s a c r i f i c e . C o r r e l a t i o n c o - e f f i c e n t between f r e e c a r n i t i n e and 8 - h y d r o x y b u t y r a t e : r = -0.410, P<0.02, n = 26. A c y l c a r n i t i n e / f r e e c a r n i t i n e r a t i o and ^3-hydroxybutyrate: r= 0.583, P<0.01, n = 26. *P<0.001 v s . ca r b o h y d r a t e d i e t **P<0.001 v s . l o n g - c h a i n t r i g l y c e r i d e d i e t 130 correlation was found to exist between the acyl- to free carnitine r a t i o and serum D-^-hydroxybutyrate content. A negative correlation existed between free carnitine levels and D-B-hydroxybutyrate. It should be noted that a similar correlation was found with starvation in man. It i s evident that dietary l i p i d can alter the di s t r i b u t i o n of free, acyl- and t o t a l carnitine in serum. With a strongly ketogenic diet (MCT), serum acylcarnitine levels increase in a manner similar to that seen with short-term (24 to 48 hr) starvation. "~ However, t o t a l serum carnitine levels do not increase after 48 hr on the MCT diet whereas they do increase s i g n i f i c a n t l y after 48 hr of starvation. The increase in t o t a l carnitine after 48 hr of . starvation may r e f l e c t an increased requirement for carnitine, by the l i v e r and other tissues, for the oxidation of l i p i d during starvation. This i s not the case when the animal i s fed the MCT diet. The findings of Rossi and Gibson (230) support this explanation. They demonstrated that short-chain (Cg to C-JQ) fatty acids are d i r e c t l y activated to fatty acyl-CoA inside l i v e r mitochondria by a GTP s p e c i f i c thiokinase and, therefore, do not require carnitine and carnitine acyltransferase for their oxidation. Others (283) have supported these findings by demonstrating that long-chain fatty acids are oxidized in the l i v e r by a carnitine dependent pathway whereas octanoate and other short-chain fatty acids are not. In the present study, t o t a l carnitine in the serum decreased s i g n i f i c a n t l y below levels found with the high carbohydrate diet when long-chain fatty acids were the major energy substrate. A 131 s i m i l a r decrease i n t o t a l serum c a r n i t i n e l e v e l was seen a f t e r 24 h r of s t a r v a t i o n i n the r a t . I n both o f these c a s e s , i t seems l i k e l y t h a t t h i s decrease i s due to an i n c r e a s e i n the uptake of c a r n i t i n e by the t i s s u e s i n o r d e r to maximize the o x i d a t i o n o f l i p i d . I f t h i s i s the case, then i t i s l i k e l y t h a t t h i s serum c a r n i t i n e i s b e i n g t r a p p e d , w i t h i n the t i s s u e s , i n the form o f l o n g - c h a i n a c y l c a r n i t i n e s . I t i s i n t e r e s t i n g to note t h a t , a f t e r 48 h r o f "A s t a r v a t i o n , t o t a l serum c a r n i t i n e l e v e l s i n the r a t i n c r e a s e d s i g n i f i c a n t l y above those v a l u e s found i n the fed r a t (see p r e v i o u s s e c t i o n ) . A p o r t i o n o f t h i s i n c r e a s e may r e p r e s e n t c a r n i t i n e which has been f r e e d by an i n c r e a s e d r a t e o f u t i l i z a t i o n o f l o n g - c h a i n a c y l e s t e r s o f c a r n i t i n e . The magnitude o f the i n c r e a s e suggests t h a t t h e r e has a l s o been an i n c r e a s e i n the r a t e o f s y n t h e s i s of c a r n i t i n e or an i n c r e a s e i n the r a t e o f m o b i l i z a t i o n o f c a r n i t i n e from t i s s u e s r i c h i n t h i s substance. I t i s i n t e r e s t i n g to s p e c u l a t e t h a t t h i s i n c r e a s e i n t o t a l c a r n i t i n e i s not o n l y r e q u i r e d f o r the p r o d u c t i o n o f a c y l c a r n i t i n e but a l s o needed to compensate f o r r e n a l l o s s e s o f c a r n i t i n e d u r i n g s t a r v a t i o n . A l t e r n a t i v e l y , the decrease i n t o t a l serum c a r n i t i n e , when the animals a re on a d i e t r i c h i n l o n g - c h a i n f a t t y a c i d t r i g l y c e r i d e s , might be e x p l a i n e d by an i n c r e a s e i n r e n a l e x c r e t i o n o f c a r n i t i n e . I t i s noteworthy t h a t , i n man d u r i n g s t a r v a t i o n , as serum a c y l c a r n i t i n e l e v e l s i n c r e a s e d the r e n a l c l e a r a n c e o f these e s t e r s a l s o i n c r e a s e d whereas the c l e a r a n c e o f c a r n i t i n e d e c r e a s e d . One might, t h e r e f o r e , expect an i n c r e a s e i n the e x c r e t i o n o f c a r n i t i n e ( i n the form o f a c y l c a r n i t i n e ) when a c y l c a r n i t i n e l e v e l s are e l e v a t e d . When the animals were on a d i e t , r i c h i n l o n g - c h a i n f a t t y a c i d s , b l o o d a c y l c a r n i t i n e l e v e l s d i d not i n c r e a s e to a 1 3 2 s i g n i f i c a n t e x t e n t . F o l l o w i n g the l i n e o f r e a s o n i n g j u s t g i v e n , i t seems u n l i k e l y t h a t the e x c r e t i o n o f c a r n i t i n e i n the form of a c y l c a r n i t i n e had i n c r e a s e d when the animals were on the l o n g - c h a i n t r i g l y c e r i d e d i e t . I f t h i s i s the case, a c c o u n t i n g f o r the decrease on the b a s i s o f an i n c r e a s e d r e n a l e x c r e t i o n seems unwarranted. Whether or not the o x i d a t i o n o f s h o r t - c h a i n f a t t y a c i d s i s c o m p l e t e l y c a r n i t i n e independent (230,283) cannot be determined by the p r e s e n t study. C e r t a i n l y , when s h o r t - c h a i n f a t t y a c i d s are the p r i m a r y energy s o u r c e , serum l e v e l s o f a c l y c a r n i t i n e i n c r e a s e markedly. I t i s p o s s i b l e t h a t c a r n i t i n e p l a y s a secondary r o l e i n the o x i d a t i o n o f s h o r t - c h a i n f a t t y a c i d s , by s e r v i n g as a b u f f e r through which excess a c e t y l - C o A , generated w i t h i n the l i v e r , can be removed. T h i s removal, which would be i n the form of a c e t y l c a r n i t i n e , would s e r v e to r e g e n e r a t e coenzyme A and thus f a c i l i t a t e the f u r t h e r o x i d a t i o n o f the a v a i l a b l e l i p i d . The e l e v a t e d a c y l c a r n i t i n e l e v e l s seen w i t h s t a r v a t i o n may be a r e f l e c t i o n o f the same phenomenon. I t i s apparent t h a t l i p i d can have marked e f f e c t s on serum c a r n i t i n e l e v e l s . I t causes a decrease i n t o t a l c a r n i t i n e as w e l l as an i n c r e a s e i n a c y l c a r n i t i n e l e v e l s . The exact n a t u r e of serum a c y l c a r n i t i n e , i t s s o u r c e , and i t s f u n c t i o n a l s i g n i f i c a n c e , remain t o be determined. 133 PART II 134 FETAL AND NEONATAL SERUM LEVELS OF CARNITINE AND D-B-HYDROXYBUTYRATE IN RAT, SHEEP, GUINEA PIG AND RABBIT I n t r o d u c t i o n F e t a l demands f o r c a r n i t i n e may be q u i t e v a r i a b l e from one s p e c i e s to another, depending upon the e x t e n t to which the d e v e l o p i n g animal r e l i e s upon m a t e r n a l l y d e r i v e d l i p i d as an energy s o u r c e . I n s p e c i e s such as the r a b b i t (93,275) and guinea p i g (30,137,186), where p l a c e n t a l t r a n s f e r o f m a t e r n a l l i p i d i s much g r e a t e r than i n the r a t (143,158) and sheep (15,95,274), a h i g h e r f e t a l requirement f o r c a r n i t i n e may be presupposed (assuming t h a t the t r a n s f e r r e d l i p i d i s o x i d i z e d by the f e t u s ) . Few s t u d i e s have examined the f e t a l a v a i l a b i l i t y o f c a r n i t i n e i n these s p e c i e s . An attempt was made to g a i n some i n s i g h t i n t o the r e l a t i v e p e r m e a b i l i t y o f the p l a c e n t a s i n these s p e c i e s to c a r n i t i n e and D-8-hydroxybutyrate by s t u d y i n g the changes i n these serum parameters t h a t occur i n l a t e g e s t a t i o n and i n the e a r l y n e o n a t a l p e r i o d and by comparing them to m a t e r n a l v a l u e s . An a d d i t i o n a l purpose was to seek evidence i n support o f the s u p p o s i t i o n t h a t the f e t u s e s o f those s p e c i e s which r e c e i v e l a r g e amounts of t r a n s p l a c e n t a l l i p i d would a l s o have h i g h e r t o t a l c a r n i t i n e l e v e l s than the f e t u s e s o f those s p e c i e s i n which l i t t l e l i p i d c r o s s e s the p l a c e n t a . E x p e r i m e n t a l Design P r e g n a n c i e s were timed i n r a t , sheep, g u i n e a p i g and r a b b i t . F e t a l and m a t e r n a l samples were c o l l e c t e d i n the l a s t 25% o f g e s t a t i o n . F o l l o w i n g c o l l e c t i o n , the samples were a l l o w e d to c l o t at room temperature, and serum was then s e p a r a t e d by, c e n t r i f u g a t i o n . 135 Samples were s t o r e d a t -40°C u n t i l they were assayed f o r c a r n i t i n e and D-^3-hydroxybutyrate (see Methods s e c t i o n ) . Blood was c o l l e c t e d a f t e r d e c a p i t a t i o n , from 1 day o l d r a t s , r a b b i t s and guinea p i g s and, by venous p u n c t u r e , from 1 day o l d sheep. The b l o o d from a l l these s p e c i e s was processed i n a s i m i l a r manner. R e s u l t s and D i s c u s s i o n The r e s u l t s are p r e s e n t e d i n Table X I . As mentioned e a r l i e r , the r a t and sheep r e c e i v e v e r y l i t t l e m a t e r n a l l i p i d i n l a t e g e s t a t i o n i n c o n t r a s t to the guinea p i g -and r a b b i t . These s p e c i e s have been grouped a c c o r d i n g l y f o r e a s i e r comparison. I t i s w e l l known t h a t , i n the r a t (240), a m a t e r n a l h y p e r l i p e m i a develops i n l a t e g e s t a t i o n and t h a t t h i s m e t a b o l i c s t a t e i s accompanied by an i n c r e a s e d tendency to develop k e t o s i s (241). F e t a l u t i l i z a t i o n of t h i s l i p i d i s l i m i t e d by the r e l a t i v e i m p e r m e a b i l i t y o f the r a t p l a c e n t a to l i p i d (143,158). I t should be noted t h a t the r a t ' s p l a c e n t a i s f r e e l y permeable to ketone b o d i e s (239,242) which can s e r v e as a s u b s t r a t e f o r l i p o g e n e s i s i n the f e t u s (242). From the data i n T a b l e X I , i t i s c l e a r t h a t , i n the r a t , t h e r e was no s i g n i f i c a n t d i f f e r e n c e , i n l a t e g e s t a t i o n , between f e t a l and m a t e r n a l l e v e l s o f D-8-hydroxybutyrate. T h i s supports e a r l i e r s t u d i e s (239,242) which found t h a t the r a t p l a c e n t a i s permeable to ketone b o d i e s . M a t e r n a l a c y l c a r n i t i n e l e v e l s were s i g n i f i c a n t l y h i g h e r (P<.005) than f e t a l l e v e l s . The a c y l - to f r e e c a r n i t i n e r a t i o i s h i g h e r i n the mother than i n the f e t u s e s . • On the b a s i s o f t h i s , TABLE XI Serum C a r n i t i n e and D-3-Hydroxybutyrate L e v e l s i n 4 Species S p e c i e s F r e e C a r n i t i n e (pM) A c y l - T o t a l Mean R a t i o A c y l / F r e e C a r n i t i n e D-B-Hydroxybutyrate ' (mM)  Rat Fetus (20) 1 day (8) Mother (8) Sheep Fetus (4) 1 day (8) Mother (4) Guinea P i g Fetus (7) 1 day (6) Mother (4) R a b b i t Fetus (12) 1 day (6) Mother (3) 15.0 + 0 .8 5.4 + 0.8 20.3 + 1.3 0.360 0.192 + 0. 05 27.3 + 0 .8 16.4 + 1 .1 43.6 + 1 .4 0.601 1 .300 + 0. 10 24.2 + 1 .3 13.3 + 2.0 37.5 + 2.3 0.550 0.168 + 0. 10 9.2 + 0. 5 4.3 + 0.2 13.5 + 0.6 0.467 17.9 + 0. 7 8.3 + 1 .1 26.2 + 1 .1 0.464 20.3 + 1. 0 28.0 + 0.1 48.4 + 2.0 1 .379 34.3 + 1 . 9 18 .0 + 3.6 53.0 + 5.1 0 .525 28.4 + 4. 9 45 .9 + 3.2 74.3 + 3.2 1 .616 21 .1 + 6. 6 39 .0 + 9.6 58.7 + 14.7 1 .848 19.6 + 1.3 31 .3 + 1.6 50.8 + 5.1 1 .597 13.5 + 0.7 17.6 + 1 .2 32.2 + 2.1 1 .304 20.3 + 3.0 28.1 + 4.0 48.4 + 6.0 1 .384 0.259 + 0. 03 1 . 080 + 0. 20 0. 820 + 0. 03 0. 256 + 0. 04 1 . 236 + 0. 22 0. 748 + 0. 19 0. 256 + 0. 02 0. 454 + 0. 17 1 . 767 + 0. 03 — Mean - S.E. u n l e s s s t a t e d o t h e r w i s e , i n p a r e n t h e s e s . The number of animals s t u d i e d i s g i v e n 137 one would expect m a t e r n a l l e v e l s o f D-8-hydroxybutyrate to be s i g n i f i c a n t l y h i g h e r than f e t a l l e v e l s . Y e t , t h i s i s not the c a s e . The h i g h e r a c y l - t o f r e e c a r n i t i n e r a t i o i n the m a t e r n a l serum i s p r o b a b l y i n d i c a t i v e o f an i n c r e a s e d u t i l i z a t i o n o f l i p i d as an energy so u r c e by m a t e r n a l t i s s u e s i n l a t e g e s t a t i o n . I t seems l i k e l y t h a t , i n s p i t e o f the e l e v a t i o n i n m a t e r n a l a c y l c a r n i t i n e l e v e l s and the p r o b a b l e i n c r e a s e i n the p r o d u c t i o n of D-8-hydroxybutyrate, the expected m a t e r n a l e l e v a t i o n i n serum l e v e l s o f t h i s ketone does not o c cur because of the r a p i d f e t a l u t i l i z a t i o n o f t h i s m a t e r n a l l y d e r i v e d s u b s t r a t e (242). M a t e r n a l l e v e l s o f t o t a l c a r n i t i n e were s i g n i f i c a n t l y h i g h e r than f e t a l l e v e l s (P<.005) which suggests t h a t the r a t p l a c e n t a i s not f r e e l y permeable to c a r n i t i n e . T e l e o l o g i c a l l y , the need f o r c a r n i t i n e i n the f e t a l r a t s h ould be low, s i n c e the r a t p l a c e n t a i s not v e r y permeable to l i p i d (143,158). T h i s s i t u a t i o n changes d r a m a t i c a l l y a t day 1, f o r the animal i s then exposed to a d i e t t h a t i s r i c h i n f a t . P a r t of the a d a p t a t i o n to t h i s d i e t i n c l u d e s an i n c r e a s e i n the a v a i l a b i l i t y o f c a r n i t i n e . T o t a l c a r n i t i n e l e v e l s i n c r e a s e s i g n i f i c a n t l y from f e t a l l e v e l s by day 1 (P<.005) a l o n g w i t h the a c y l - to f r e e c a r n i t i n e r a t i o . I n a d d i t i o n , serum l e v e l s o f D-^3-hydroxybutyrate i n c r e a s e s i g n i f i c a n t l y (P<.005). The sheep p l a c e n t a , l i k e t h a t of the r a t , i s r e l a t i v e l y impermeable to m a t e r n a l l y d e r i v e d l i p i d (15,95,274) which suggests t h a t the c o n t r i b u t i o n t h i s s u b s t r a t e makes to the d e v e l o p i n g f e t u s i s p r o b a b l y s m a l l . I t i s apparent (Table XI) t h a t , i n l a t e g e s t a t i o n , l e v e l s o f a c y l c a r n i t i n e (P<.005) and D-yS-hydroxybutyrate (P<.005) ar e s i g n i f i c a n t l y h i g h e r i n the ewe than i n the f e t u s . The a c y l - to f r e e 13 8 c a r n i t i n e r a t i o i s a l s o h i g h e r i n the mother. T h i s suggests t h a t the sheep p l a c e n t a i s not p a r t i c u l a r l y permeable to e i t h e r D-3-hydroxybutyrate or c a r n i t i n e . In the case o f D-3-hydroxybutyrate, t h i s f i n d i n g s u p p o r t s e a r l i e r r e p o r t e d o b s e r v a t i o n s ( 1 5 ) . On the f i r s t p o s t n a t a l day i n the sheep, t h e r e i s a s i g n i f i c a n t i n c r e a s e i n serum l e v e l s o f f r e e (P<.005), a c y l - (P<.005) and t o t a l c a r n i t i n e (P<.005) over l e v e l s r e c o r d e d f o r the f e t u s . The a c y l - to f r e e c a r n i t i n e r a t i o remained unchanged i n s p i t e o f the s i g n i f i c a n t i n c r e a s e i n serum D - 3 - h y d r o x y b u t y r a t e (P<.005) t h a t o c c u r r e d . T h i s r e p r e s e n t s another i n s t a n c e when changes i n the a c y l -to f r e e c a r n i t i n e r a t i o do not p a r a l l e l those o f serum D-3-hydroxybutyrate. T h i s i s p r o b a b l y due to a u t i l i z a t i o n o f serum a c y l c a r n i t i n e by the neonate (see sheep i n f u s i o n experiments i n the next s e c t i o n ) . The g u i n e a p i g p l a c e n t a , u n l i k e t h a t o f the r a t and sheep, i s permeable to f a t t y a c i d (30,137,186). But evi d e n c e i n d i c a t e s t h a t much o f t h i s t r a n s f e r r e d f a t t y a c i d i s d i r e c t l y i n c o r p o r a t e d i n t o f e t a l l i p i d s r a t h e r than b e i n g o x i d i z e d ( 3 0 ) . I t i s noteworthy, t h a t ( i n c o n t r a s t to the r a t ) m a t e r n a l l e v e l s o f D-3-hydroxybutyrate are h i g h e r (P<.05) than f e t a l l e v e l s , w hich s u g g e s t s t h a t the g u i n e a p i g p l a c e n t a i s r e l a t i v e l y impermeable to ketone b o d i e s . A l s o , m a t e r n a l l e v e l s o f t o t a l , a c y l - and f r e e c a r n i t i n e are not s i g n i f i c a n t l y d i f f e r e n t from f e t a l l e v e l s ( i n c o n t r a s t to the r a t and sheep) wh i c h suggests t h a t the p l a c e n t a i n the g u i n e a p i g i s f r e e l y permeable to c a r n i t i n e . I t i s p o s s i b l e t h a t the f e t a l g u i n e a p i g can u t i l i z e c a r n i t i n e i n some c a p a c i t y , o t h e r than f o r the. o x i d a t i o n o f l i p i d , 139 and hence has a requirement f o r i t i n l a t e g e s t a t i o n . The r i s e i n the a c y l - to f r e e c a r n i t i n e r a t i o i n d i c a t e s t h a t the o x i d a t i o n o f l i p i d i n the g u i n e a p i g i n c r e a s e s at day 1 . There was a l s o a s i g n i f i c a n t i n c r e a s e i n a c y l c a r n i t i n e (P<.005), t o t a l c a r n i t i n e (P<.005) and D-S-hydroxybutyrate (P<.005) l e v e l s over those l e v e l s r e c o r d e d i n the f e t a l p e r i o d . The data f o r the r a b b i t are s i m i l a r , i n many r e s p e c t s , to those seen i n the guinea p i g . There was no s i g n i f i c a n t d i f f e r e n c e between m a t e r n a l and f e t a l l e v e l s o f f r e e , a c y l - and t o t a l c a r n i t i n e which suggests t h a t the r a b b i t p l a c e n t a i s f r e e l y permeable to c a r n i t i n e . L i k e w i s e , m a t e r n a l l e v e l s of D-3-hydroxybutyrate (P<.005) were s i g n i f i c a n t l y h i g h e r than f e t a l l e v e l s which i n d i c a t e s t h a t the r a b b i t p l a c e n t a i s not f r e e l y permeable to ketone b o d i e s . I t i s noteworthy t h a t the a c y l - to f r e e c a r n i t i n e r a t i o i n the f e t a l r a b b i t was h i g h , and y e t l e v e l s of D-3-hydroxybutyrate were low. This f i n d i n g may i n d i c a t e t h a t the f e t a l r a b b i t u t i l i z e s m a t e r n a l l y d e r i v e d a c y l c a r n i t i n e as a b i o s y n t h e t i c s u b s t r a t e i n l a t e g e s t a t i o n . As a r e s u l t , f e t a l a c y l c a r n i t i n e l e v e l s may r e f l e c t a b i o s y n t h e t i c , r a t h e r than an o x i d a t i v e , event. An a l t e r n a t i v e e x p l a n a t i o n would be t h a t the i n c r e a s e d a c y l - to f r e e c a r n i t i n e r a t i o does indeed r e p r e s e n t i n c r e a s e d o x i d a t i o n of l i p i d by the f e t u s w i t h a concomitant i n c r e a s e i n the p r o d u c t i o n o f ketone b o d i e s by the f e t a l l i v e r ; b u t , because the r a t e o f u t i l i z a t i o n of the produced ketones matches the r a t e o f p r o d u c t i o n , f e t a l serum l e v e l s o f D-y3-hydroxybutyrate do not i n c r e a s e . R e g a r d l e s s of the e x p l a n a t i o n , t hese d a t a r a i s e the p o s s i b i l i t y t h a t serum l e v e l s o f a c y l c a r n i t i n e may r e f l e c t b i o c h e m i c a l events o t h e r than the o x i d a t i o n o f l i p i d . I n the one day o l d r a b b i t , a s i m i l a r p a t t e r n was seen w i t h the e x c e p t i o n 140 t h a t f r e e , a c y l - and t o t a l c a r n i t i n e l e v e l s were s i g n i f i c a n t l y lower than f e t a l l e v e l s (P<.005). Once a g a i n , the a c y l - to f r e e c a r n i t i n e r a t i o was i n c r e a s e d ; y e t , l e v e l s o f D-yS-hydroxybutyrate d i d not i n c r e a s e s i g n i f i c a n t l y from the low l e v e l s seen i n the f e t u s . I t appears from these data t h a t , i n c o n t r a s t to the r a t , sheep and guinea p i g , the o x i d a t i o n o f l i p i d does not i n c r e a s e a p p r e c i a b l y i n the n e o n a t a l r a b b i t over l e v e l s seen i n the f e t u s . T h i s c o n c l u s i o n i s s u p p o r t e d , i n d i r e c t l y , by the s i g n i f i c a n t decrease seen i n a c y l c a r n i t i n e and t o t a l c a r n i t i n e l e v e l s i n the n e o n a t a l p e r i o d . These d a t a a l s o i n d i c a t e t h a t the d i e t o f t h e . n e o n a t a l r a b b i t , u n l i k e t h a t o f the r a t and man, may be low i n c a r n i t i n e . However, i t i s n e c e s s a r y to remember t h a t newborn r a b b i t s are f e d o n l y once a day i n c o n t r a s t to the o t h e r s p e c i e s and t h e r e f o r e c o n s i d e r a b l e d i u r n a l v a r i a t i o n s may r e s u l t . PLACENTAL TRANSFER OF CARNITINE IN THE SHEEP AND THE GUINEA PIG I n t r o d u c t i o n The data from the p r e v i o u s experiment i n d i c a t e d t h a t the sheep p l a c e n t a was r e l a t i v e l y impermeable to c a r n i t i n e i n c o n t r a s t to t h a t o f the guinea p i g . Y e t , some serum c a r n i t i n e was p r e s e n t i n the f e t a l sheep i n l a t e g e s t a t i o n . The q u e s t i o n a r i s e s as to the source of t h i s c a r n i t i n e . Could t h e r e i n f a c t be a t r a n s f e r o f c a r n i t i n e from the ewe to the f e t u s ? I s t h i s t r a n s f e r i n the form of a c y l c a r n i t i n e r a t h e r than f r e e c a r n i t i n e ? An experiment was designed to determine whether or not f r e e c a r n i t i n e can be t r a n s f e r r e d through the sheep p l a c e n t a . I n a d d i t i o n , the r a t e o f t r a n s f e r f o r f r e e c a r n i t i n e was compared to t h a t f o r a c y l c a r n i t i n e . Another study 1 4 1 examined the t r a n s f e r of m a t e r n a l l y i n j e c t e d f r e e c a r n i t i n e i n the guinea p i g . E x p e r i m e n t a l Design Three pregnant ewes ( S u f f o l k ) were o b t a i n e d from the Department o f Animal S c i e n c e a t U.B.C. T h e i r body weights ranged from 65-80 kg. They were used between the 120th and 130th day o f g e s t a t i o n . They had been prepared s u r g i c a l l y f o r c h r o n i c e x p e r i m e n t a t i o n a t l e a s t one week p r i o r to t h e i r use i n t h i s s t u d y . The s u r g i c a l t e c h n i q u e has been d e s c r i b e d p r e v i o u s l y (270). Blood (0.5 ml f o r each sample) was c o l l e c t e d from c h r o n i c c a t h e t e r s i n the f e t a l and m a t e r n a l f e m o r a l a r t e r i e s and c a r n i t i n e was i n j e c t e d i n t o the j u g u l a r v e i n o f the ewe. A t o t a l o f 4 experiments were performed. I n two, D L - a c e t y l c a r n i t i n e was i n j e c t e d i n t o the j u g u l a r v e i n over a p e r i o d o f two minutes. I n one experiment, L - c a r n i t i n e was i n j e c t e d t o g e t h e r w i t h D L - ( m e t h y l - ^ C ) c a r n i t i n e , and i n the l a s t experiment L - c a r n i t i n e was i n f u s e d v i a a j u g u l a r c a t h e t e r over a p e r i o d of 59 minutes. D e t a i l s o f these experiments are g i v e n i n the a p p r o p r i a t e f i g u r e s . I n another experiment, pregnant guinea p i g s i n the l a s t q u a r t e r o f g e s t a t i o n were i n j e c t e d , w i t h L - c a r n i t i n e (100 mg per 100 g body w e i g h t , s u b c u t a n e o u s l y ) and the animals s a c r i f i c e d 1 h r l a t e r . Blood was r a p i d l y c o l l e c t e d from the mother and f e t u s e s f o l l o w i n g d e c a p i t a t i o n . Serum l e v e l s o f c a r n i t i n e were determined, on a l l sheep and guinea p i g samples, by the r a d i o a c t i v e method f o r c a r n i t i n e d e t e r m i n a t i o n (see Methods s e c t i o n ) . 142 R e s u l t s and D i s c u s s i o n A ewe was i n j e c t e d , on the 134th day o f g e s t a t i o n , w i t h a bo l u s o f 500 mg L - c a r n i t i n e and 50 u C i o f D L - ( m e t h y l - ^ ^ C ) c a r n i t i n e (45 mCi/mmol) i n 10 ml of s a l i n e over a p e r i o d o f 2.0 minutes. The b l o o d l e v e l o f t o t a l c a r n i t i n e r o s e t o 400 pM. There was a l s o a v e r y r a p i d r i s e i n the a c y l c a r n i t i n e c o n t e n t (see F i g . 15). However, the v e r y low b l o o d l e v e l s o f the f e t u s were not a f f e c t e d and remained unchanged up to 400 minutes a f t e r the i n j e c t i o n ( d a t a not shown). The r a d i o a c t i v e c o n t e n t o f the ma t e r n a l b l o o d f o l l o w e d a s i m i l a r p a t t e r n (see F i g . 16). V i r t u a l l y no r a d i o a c t i v i t y c o u l d be d e t e c t e d i n f e t a l b l o o d ( a c t i v i t y was never more than 8 DPM/0.1 ml of b l o o d ) . N e v e r t h e l e s s , on s a c r i f i c e (24 h r a f t e r the i n j e c t i o n ) the l a b e l was found i n f e t a l muscle, l i v e r and the p l a c e n t a (see F i g . 17). The a c t i v i t y i n mat e r n a l muscle and l i v e r was c o n s i d e r a b l y h i g h e r than i n the c o r r e s p o n d i n g f e t a l t i s s u e s . These data i n d i c a t e t h a t t h e r e may be a slow r a t e of p l a c e n t a l t r a n s f e r o f c a r n i t i n e from the m a t e r n a l sheep to the f e t u s . T h i s judgement i s made s o l e l y on the b a s i s o f the appearance of r a d i o a c t i v i t y , i n f e t a l t i s s u e s , 24 hr f o l l o w i n g i n j e c t i o n o f t h e r a d i o - l a b e l l e d c a r n i t i n e i n t o the ewe. I t must be s t r e s s e d t h a t the exact n a t u r e o f t h i s f e t a l r a d i o a c t i v i t y was not determined and i t i s p o s s i b l e t h a t i t may r e p r e s e n t a d e c a r b o x y l a t e d form of c a r n i t i n e ( ^ - methyl-choline) (151). Before a d e f i n i t i v e c o n c l u s i o n can be drawn, i t i s e s s e n t i a l t h a t the n a t u r e o f t h i s f e t a l r a d i o a c t i v i t y be determined. 143 FIGURE 15 Blood l e v e l s o f f r e e , t o t a l and a c y l c a r n i t i n e i n a pregnant ewe i n j e c t e d w i t h 500 mg L - c a r n i t i n e HC1 i n 10 ml s a l i n e , t o g e t h e r w i t h 50 u C i o f DL ( m e t h y l - 1 ^ C ) c a r n i t i n e ( s p e c i f i c a c t i v i t y 45 mCi/mmole). The s o l u t i o n was adjus'ted to pH 7 w i t h s o l i d sodium b i c a r b o n a t e and was i n j e c t e d over a p e r i o d o f 2 min. Blood was c o l l e c t e d b e f o r e , and 2, 5, 10 e t c . minutes ( a b s c i s s a ) a f t e r the i . v . i n j e c t i o n . 145 FIGURE 16 Content o f r a d i o a c t i v i t y i n m a t e r n a l and f e t a l b l o o d . For d e t a i l s , see F i g . 15. Heavy b l a c k c u r v e : DPM/0.1ml m a t e r n a l b l o o d . Weak b l a c k s t r a i g h t l i n e : DPM/0.1ml f e t a l b l o o d . A c t i v i t y was never more than 8 DPM and the l i n e i s drawn to i n d i c a t e t h i s v e r y low a c t i v i t y . Dotted l i n e : DPM/u mole c a r n i t i n e . 147 FIGURE 17 R a d i o a c t i v i t y r e t a i n e d i n muscle and l i v e r o f f e t u s and mother (see F i g . 1 5 ) and i n f e t a l h e a r t and p l a c e n t a 24 hours a f t e r i n j e c t i o n o f the l a b e l . F e t a l v a l u e s are the mean o f 2 d e t e r m i n a t i o n s , one on each t w i n . They d i d not d i f f e r by more than 1 0 7 o . M = mother; F = f e t u s ; H = h e a r t ; P.L. = p l a c e n t a . 1 4 9 The b o l u s i n j e c t i o n of L - c a r n i t i n e d i d not r e s u l t i n any-s i g n i f i c a n t i n c r e a s e i n serum c a r n i t i n e l e v e l s i n the f e t u s . T h i s may have been due to the s h o r t p e r i o d of time d u r i n g which h i g h m a t e r n a l c a r n i t i n e b l o o d l e v e l s had been m a i n t a i n e d . Thus, a f u r t h e r ewe was i n f u s e d w i t h a s a l i n e s o l u t i o n of L - c a r n i t i n e (50 mg/ml) at a r a t e o f 0.96 ml/min f o r 58 minutes. The d ata p r e s e n t e d i n F i g . 18 demonstrate t h a t a s u s t a i n e d e l e v a t i o n of L - c a r n i t i n e i n m a t e r n a l b l o o d d i d not l e a d to a s i g n i f i c a n t change i n f e t a l b l o o d l e v e l s o f f r e e or a c y l - c a r n i t i n e . I t i s a l s o worthy of note t h a t m a t e r n a l l e v e l s o f L - c a r n i t i n e remained v e r y h i g h f o r a t l e a s t 2 hours a f t e r the end o f the i n f u s i o n . I t was thought p o s s i b l e t h a t a c y l c a r n i t i n e might be t r a n s p o r t e d more e a s i l y a c r o s s the p l a c e n t a than c a r n i t i n e i t s e l f . Hence, two ewes were i n j e c t e d w i t h a c e t y l c a r n i t i n e . One r e c e i v e d 200 mg, the o t h e r 600 mg i n t r a v e n o u s l y w i t h i n two minutes. F i g . 19 and 20 show t h a t , a f t e r the i n i t i a l r i s e i n t o t a l and a c y l c a r n i t i n e c o n t e n t s , the l e v e l s o f b o th substances f a l l r a p i d l y a f t e r the end o f the i n j e c t i o n . However, i n b oth ewes, the f r e e c a r n i t i n e c o ntent c o n t i n u e d to i n c r e a s e f o r at l e a s t 60 minutes and o n l y s t a r t e d to d e c l i n e between the 1 s t and 2nd hours a f t e r the end o f the i n j e c t i o n . A g a i n , no t r a n s f e r o f f r e e or e s t e r i f i e d c a r n i t i n e to the f e t u s c o u l d be demonstrated (see F i g . 21). I t i s noteworthy t h a t , a f t e r the i n j e c t i o n s o f a c e t y l c a r n i t i n e to the mothers, the d isappearance of a c y l c a r n i t i n e from the m a t e r n a l serum i s , at f i r s t , accompanied by a r i s e i n f r e e c a r n i t i n e (see F i g . 19 and 2 0 ) . T h i s suggests t h a t the a c y l - moiety o f the serum a c y l c a r n i t i n e can be u t i l i z e d as a s u b s t r a t e by m a t e r n a l t i s s u e s . Depending upon the r a t e of t u r n o v e r of the serum a c y l c a r n i t i n e p o o l , i t i s p o s s i b l e t h a t serum 150 FIGURE 18 f" M a t e r n a l and f e t a l sheep serum l e v e l s o f t o t a l and f r e e c a r n i t i n e a f t e r the i n f u s i o n i n t o the mother o f L - c a r n i t i n e HCl (pH 7.0) i n s a l i n e a t a r a t e o f 50 mg/ml/min. i . v . f o r 59 m i n u t e s . L e f t o r d i n a t e and upper two c u r v e s : R i g h t o r d i n a t e and lower two c u r v e s : mother f e t u s 151 160 140 120 § 100 I : ° o* Total o» o* 80 60 a | 40 a 20 OL L 30 60 90 F r > o Total Free 20 _ ~o 16 | 12 8 4 a 120 _L 150 180 J J o Minutes 152 s h o r t - c h a i n a c y l c a r n i t i n e c o u l d make a s i g n i f i c a n t m e t a b o l i c c o n t r i b u t i o n to the sheep. I t remains to be determined whether o r not t h i s i s so i n the o t h e r s p e c i e s s t u d i e d . I t sh o u l d be mentioned t h a t , i n the p r e s e n t s t u d y , f e t a l and m a t e r n a l l e v e l s o f D-3-hydroxybutyrate were u n a f f e c t e d i n a l l experiments. / o f c a r n i t i n e i n the gui n e a p i g . The r e s u l t s a r e p r e s e n t e d i n Ta b l e X I I . I n c o n t r a s t t o f i n d i n g s i n the sheep, the t r a n s f e r o f c a r n i t i n e was found to be v e r y r a p i d from mother to f e t u s . W i t h i n one hour of ma t e r n a l i n j e c t i o n o f L - c a r n i t i n e (100 mg/100 g body w e i g h t ) , the serum l e v e l s o f c a r n i t i n e i n the f e t u s e s had r i s e n to l e v e l s t h a t were not s i g n i f i c a n t l y d i f f e r e n t from the v e r y h i g h m a t e r n a l l e v e l s t h a t had been a c h i e v e d . These data support the d a t a , p r e s e n t e d e a r l i e r i n t h i s s t u d y , which i n d i c a t e d t h a t the guinea p i g p l a c e n t a i s r e l a t i v e l y permeable to L - c a r n i t i n e . One experiment was c a r r i e d out to assess p l a c e n t a l t r a n s f e r 153 FIGURE 19 F e t a l and m a t e r n a l sheep serum l e v e l s o f t o t a l , f r e e and a c y l c a r n i t i n e a f t e r i . v . i n j e c t i o n i n t o the m a t e r n a l j u g u l a r v e i n , o f 200 mg D L - a c e t y l c a r n i t i n e (pH 7.0). The a c e t y l c a r n i t i n e was c o n t a i n e d i n 10 ml s a l i n e and i n j e c t e d over a p e r i o d o f 2 m i n u t e s . The upper t h r e e c u r v e s r e p r e s e n t m a t e r n a l v a l u e s and the lower t h r e e curves f e t a l v a l u e s . 154 120 r 100 80 60 40 20 0 ' 0 ' M . . . , . „ ( '"•••o.„ qOO-I '•o . ^ L F e t o i 0 20 40 60 120 180 Minutes •o Total Carnitine •• Free Carnitine •A Acylcarnitine 360 155 FIGURE 20 M a t e r n a l sheep serum l e v e l s o f t o t a l f r e e and a c y l c a r n i t i n e a f t e r i n j e c t i o n o f 600 mg o f D L - a c e t y l c a r n i t i n e i n 10 ml s a l i n e over a p e r i o d o f 2 m i n u t e s . 156 Minutes FIGURE 21 Serum l e v e l s o f c a r n i t i n e i n the f e t u s of the mother ewe own i n F i g . 20. 158 20 r 16 12 8 4 0 cP^ ^ ,l0......... ° To ta' * * ^sL"'o» '^2'^! " ° ' " i Free K ******* * * ' * A c e t y l r i —i 1 — i 1 — ! 0 60 120 180 240 300 400 Minutes 159 TABLE X I I T r a n s f e r o f L - C a r n i t i n e from Mother to Fetuses i n the Guinea P i g Mean + S.E. The number o f animals s t u d i e d i s g i v e n i n p a r e n t h e s e s . Pregnant guinea p i g s ( f e t a l w e i g h t s r a n g i n g from 123 t o 128 g) were i n j e c t e d , s u b c u t a n e o u s l y , w i t h L - c a r n i t i n e (100 mg/100 g body w e i g h t ) . The animals were s a c r i f i c e d 1 hr l a t e r . Blood was c o l l e c t e d and serum l e v e l s of L - c a r n i t i n e were determined as o u t l i n e d i n the Methods s e c t i o n . Free C a r n i t i n e A c y l c a r n i t i n e T o t a l C a r n i t i n e Sample £uM) <^ uM) (uM) B e f o r e I n j e c t i o n F e t a l (4) 30.5 + 1.8 13.5 + 2.7 45.8 + 4.4 M a t e r n a l (3) 30.5 + 2.1 47.0 + 10.0 76.0 + 15.0 A f t e r I n j e c t i o n F e t a l (11) 275.0 +8.0 - 276.0 + 10.0 M a t e r n a l (3) 284.0 + 3.0 - 285.0 +11.0 160 SERUM LEVELS OF FREE AND ESTERIFIED CARNITINE DURING DEVELOPMENT IN THE RAT I n t r o d u c t i o n I t has been shown i n the r a t t h a t , p e r i n a t a l l y , t h e r e i s a r i s e i n the c o n t e n t o f c a r n i t i n e i n some t i s s u e s ( 1 24), and i n the a c t i v i t y o f c a r n i t i n e a c y l t r a n s f e r a s e i n l i v e r ( 1 0 ) , h e a r t and brown adipose t i s s u e (124). However, l i t t l e to no data are a v a i l a b l e on the c o n t e n t o f c a r n i t i n e and i t s e s t e r s i n serum o f f e t a l , newborn and i n f a n t mammals. The p r e s e n t study was undertaken to determine the changes t h a t occur i n serum c a r n i t i n e l e v e l s d u r i n g development o f the r a t . E x p e r i m e n t a l Design White W i s t a r r a t s (Woodlyn L a b o r a t o r i e s , Guelph, O n t a r i o ) were mated and pr e g n a n c i e s were dated by v a g i n a l smear. F e t a l animals were d e l i v e r e d by s e c t i o n on day 21 o f g e s t a t i o n ( f u l l term 22 d a y s ) . L i t t e r s were reduced to e i g h t animals and r a i s e d w i t h the mother a t 23°C w i t h a 12 h r l i g h t and 12 h r dark c y c l e . Weaned animals had f r e e access to s t a n d a r d r a t chow ( P u r i n a Chow, S t . L o u i s , Mo., f a t c o n t e n t 4 7 o ) and water. Animals were s a c r i f i c e d and c a r n i t i n e was measured as o u t l i n e d i n the Methods s e c t i o n . R e s u l t s and D i s c u s s i o n The r e s u l t s are p r e s e n t e d i n Table X I I I . Between the 21st day o f g e s t a t i o n and the 1 s t p o s t n a t a l day, a h i g h l y s i g n i f i c a n t i n c r e a s e i n the serum l e v e l s o f f r e e , a c y l - and t o t a l c a r n i t i n e was 161 o b s e r v e d . T h i s i s the t i m e , d u r i n g development, when the r a t ' s h i g h c a r b o h y d r a t e d i e t rn u t e r o i s changed to a l i p i d - r i c h d i e t (mother's m i l k ) a f t e r b i r t h . Once the a n i m a l s b e g i n to s u c k l e , t h e r e i s a d r a m a t i c i n c r e a s e i n the b l o o d l e v e l s o f f r e e f a t t y a c i d s and ketone bo d i e s ( 1 2 1 ) . The c a r n i t i n e l e v e l s i n the p r e s e n t study remained e l e v a t e d u n t i l weaning commenced ( r a t s b e g i n n i b b l i n g the P u r i n a Chow d i e t when aged 15-18 days and are c o m p l e t e l y weaned when aged 30 d a y s ) . T o t a l c a r n i t i n e c o n t e n t reached a peak on day 18. F o l l o w i n g day 18, serum t o t a l and a c y l c a r n i t i n e c o n t e n t d e c r e a s e d . The r a t i o o f a c y l c a r n i t i n e to f r e e c a r n i t i n e r o s e s i g n i f i c a n t l y a f t e r b i r t h and then decreased as the ani m a l s s t a r t e d to consume h i g h c a r b o h y d r a t e s o l i d food ( P u r i n a r a t chow) i n a d d i t i o n to m i l k . I t i s w e l l e s t a b l i s h e d t h a t m i l k l i p i d i s the p r i m a r y energy source o f the s u c k l i n g r a t . I t i s d u r i n g the s u c k l i n g p e r i o d t h a t ketone b o d i e s have been shown to be a major energy and l i p o g e n i c s o u r c e f o r the d e v e l o p i n g b r a i n ( 9 0 ) . I t i s apparent from the d a t a t h a t , as the a n i m a l ' s d i e t changed from the c a r b o h y d r a t e - r i c h d i e t i n  u t e r o to the l i p i d - r i c h d i e t n e o n a t a l l y , the l e v e l o f serum c a r n i t i n e i n c r e a s e d d r a m a t i c a l l y . Under these c o n d i t i o n s o f i n c r e a s e d f r e e f a t t y a c i d t u r n o v e r and k e t o g e n e s i s , both serum a c y l c a r n i t i n e l e v e l s and the r a t i o o f a c y l c a r n i t i n e to f r e e c a r n i t i n e i n c r e a s e d . The e l e v a t e d serum a c y l c a r n i t i n e l e v e l s may r e f l e c t r i s i n g a c y l c a r n i t i n e l e v e l s which a r e found i n the l i v e r when a h i g h f a t d i e t i s f e d ( 7 2 ) . The i n c r e a s e i n t o t a l c a r n i t i n e l e v e l s , seen on day 1, over f e t a l l e v e l s s u p port the c o n c l u s i o n o f R o b l e s - V a l d e s et a l (229) t h a t r a t m i l k i s the major source o f c a r n i t i n e i n the newborn a n i m a l . I t TABLE X I I I The C o n c e n t r a t i o n o f Serum C a r n i t i n e i n the Rat at D i f f e r e n t Stages of Development Mean + S.E. The number o f anim a l s s t u d i e d i s g i v e n i n p a r e n t h e s e s . F e t a l s a m p l i n g s r e p r e s e n t p o o l e d samples taken from two or more f e t u s e s . T o t a l R a t i o , Age Free C a r n i t i n e A c y l c a r n i t i n e C a r n i t i n e A c y l c a r n i t i n e : (Days) ( u M> (uM) (uM) Free C a r n i t i n e F e t a l (5) 13.7 + 1.8 4.7 + 1.2 18.3 + 1.8 0.346 + 0.10 1 (8) 27.3 + 0.80*,*** 16.4 + 43.6 + 1 .4* 0.603 + 0.04**,*** 7 (8) 38.3 + 2.3 14.3 + 1 .*3 52.6 + 1 #g*** 0.390 + 0.05 12 (8) 34.5 + 1 .1 12.9 + 1 .2 47.4 + 113 0.378 + 0.04 18 (6) 49.9 + 2.6 21 .3 + 2.1 71 .3 + 3.5 0.446 + 0.07 22 (5) 52.1 + 2.9 9.8 + 1 .0 61.9 + 2.8 0.191 + 0.03 27 (8) 38.2 + 2.3 5.0 + 1.5 42.9 + 1.7 0.148 + 0.05 36 (9) 36.1 + 2.1 5.2 + 1.5 40.8 + 1.6 0.163 + 0.06 *p<0.01 v s . f e t a l *p<0.05 v s . f e t a l *p<0.01 v s . 36 days 163 i s i n t e r e s t i n g to note t h a t on day 18, when the animals began to n i b b l e on r a t chow and to s u c k l e l e s s , serum a c y l c a r n i t i n e l e v e l s decreased. T h i s p r o b a b l y r e f l e c t s the lower content of l i p i d i n t h e i r d i e t . The i n c r e a s e i n t o t a l c a r n i t i n e , seen as weaning i s due to the combined c o n t r i b u t i o n of c a r n i t i n e from both d i e t s . I n a d d i t i o n , an i n c r e a s e i n the c a r n i t i n e s y n t h e t i c c a p a c i t y of t h e l i v e r may o ccur at t h i s age. I t i s e v i d e n t , however, t h a t , as the l i p i d c o n t r i b u t i o n to the d i e t decreases w i t h weaning, a c y l c a r n i t i n e l e v e l s decrease as w e l l , so t h a t by day 27 a c y l c a r n i t i n e l e v e l s have reached a d u l t v a l u e s . I n t h i s s tudy, no attempt was made to examine s e x u a l d i f f e r e n c e s i n serum c a r n i t i n e l e v e l s . R e c e n t l y , o t h e r s have r e p o r t e d (72) t h a t , i n the r a t , t h e r e are no s e x u a l d i f f e r e n c e s i n serum l e v e l s o f f r e e c a r n i t i n e b e f o r e weaning. PLASMA CARNITINE LEVELS DURING INTRAVENOUS FEEDING OF THE NEONATE I n t r o d u c t i o n I n man (209) and the r a t (see p r e v i o u s d a t a ) , the l e v e l s o f t o t a l and a c y l c a r n i t i n e i n c r e a s e s i g n i f i c a n t l y a f t e r the f i r s t day of l i f e . I n b o th s p e c i e s , the i n c r e a s e i n b l o o d l e v e l s o f t o t a l c a r n i t i n e i s l a r g e l y due to an adequate d i e t a r y source of c a r n i t i n e (209,229). I n b oth premature and f u l l - t e r m i n f a n t s plasma l e v e l s o f c a r n i t i n e and a c y l c a r n i t i n e are s i g n i f i c a n t l y lower w i t h a d i e t of soybean based f o r m u l a c o n t a i n i n g no c a r n i t i n e than w i t h a b r e a s t m i l k d i e t . 164 At the time t h a t t h i s study was undertaken w i t h Dr. S c h i f f i n Edmonton, no r e p o r t e d d a t a were a v a i l a b l e on the metabolism o f c a r n i t i n e i n newborn i n f a n t s and the e f f e c t o f d i e t a r y c a r n i t i n e on the b l o o d l e v e l s o f t h i s substance i n neonates. S i n c e many i n f a n t s a r e r e c e i v i n g i n t r a v e n o u s a l i m e n t a t i o n which may or may not have c a r n i t i n e i n them, i t seems p e r t i n e n t to ask whether or not c a r n i t i n e s h o u l d be p r o v i d e d i n the d i e t o f the i n f a n t s . i C a r n i t i n e i s p r e s e n t i n human b r e a s t m i l k (39-63 uM) and i n S i m i l a c (60-90 uM) (209). The advent o f i n t r a v e n o u s a l i m e n t a t i o n , and the use o f I n t r a l i p i d as a f a t source has r a i s e d s e v e r a l q u e s t i o n s . I s f a t t y a c i d o x i d a t i o n compromised i n i n f a n t s r e c e i v i n g t h i s t reatment (7)? Do these d i e t s c o n t a i n adequate amounts o f c a r n i t i n e ? I f n o t , c o u l d the l i m i t e d c a p a c i t y of these i n f a n t s to o x i d i z e l i p i d be due, i n p a r t , t o a r e d u c t i o n i n the a v a i l a b i l i t y o f d i e t a r y c a r n i t i n e ? E x p e r i m e n t a l Design A t o t a l o f 33 i n f a n t s a d mitted to the N e o n a t a l I n t e n s i v e Care U n i t o f the U n i v e r s i t y o f A l b e r t a H o s p i t a l , who r e q u i r e d p e r i p h e r a l i n t r a v e n o u s a l i m e n t a t i o n f o r v a r i o u s r e a s o n s , were s t u d i e d . The i n f a n t s ranged i n g e s t a t i o n a l age from 26 to 40 weeks w i t h a mean g e s t a t i o n a l age o f 31.3 weeks. The mean weight was 1525.0 g w i t h a range o f 800 t o 4300 g. Samples of blo o d were o b t a i n e d from the i n f a n t s when they were m a i n t a i n e d s o l e l y on i n t r a v e n o u s f e e d i n g and when they were f e d o r a l l y . The i n t r a v e n o u s f e e d i n g s i n c l u d e d T r a v a s o l which i s a m i x t u r e of c r y s t a l l i n e amino a c i d s . The amount o f the amino a c i d s o l u t i o n r e c e i v e d was i n c r e a s e d g r a d u a l l y from 0.5 g per Kg per day to a t o t a l of 2.5 g per Kg per day. The f a t source was I n t r a l i p i d . The i n i t i a l dose o f I n t r a l i p i d was 0.5 g per Kg per day and was g r a d u a l l y i n c r e a s e d to a maximum o f 4 g per Kg per day depending on the plasma t r i g l y c e r i d e l e v e l s which were m a i n t a i n e d below 100 mg/dl. The i n t r a v e n o u s s o l u t i o n s of T r a v a s o l and I n t r a l i p i d c o n t a i n e d l e s s than 1.0 uM c a r n i t i n e . I n f a n t s t h a t were f e d o r a l l y r e c e i v e d e i t h e r expressed b r e a s t m i l k •p (EBM) or S i m i l a c . Venous b l o o d samples were c o l l e c t e d i n t o tubes c o n t a i n i n g EDTA and s u b s e q u e n t l y , plasma was se p a r a t e d by c e n t r i f u g a t i o n and s t o r e d a t -40°C u n t i l assayed f o r c a r n i t i n e . R e s u l t s and D i s c u s s i o n The r e s u l t s a re pr e s e n t e d i n Ta b l e XIV. When the i n f a n t s r e c e i v e d a d i e t t h a t was r i c h i n c a r n i t i n e , t h e r e was a s i g n i f i c a n t i n c r e a s e i n plasma l e v e l s o f f r e e , a c y l - and t o t a l c a r n i t i n e over those l e v e l s seen w i t h a d i e t t h a t was c a r n i t i n e d e f i c i e n t . These d a t a document t h a t b l o o d c a r n i t i n e l e v e l s i n i n f a n t s a r e , to a l a r g e e x t e n t , dependent on an e x t e r n a l source of c a r n i t i n e . However, even when e s s e n t i a l l y no c a r n i t i n e was g i v e n ( i n t r a v e n o u s f e e d ) , the b l o o d l e v e l s of c a r n i t i n e remained r e l a t i v e l y c o n s t a n t , a l t h o u g h v e r y low. Thi s suggests t h a t c a r n i t i n e may a l s o be s u p p l i e d from endogenous s o u r c e s . The s y n t h e s i s of c a r n i t i n e r e q u i r e s l y s i n e , m e t h i o n i n e and/or c h o l i n e (75) a l l o f which are p r e s e n t i n T r a v a s o l R i n s u f f i c i e n t amounts to ensure b i o s y n t h e s i s . Whether or not the source of t h i s c a r n i t i n e i s due to s y n t h e s i s by the l i v e r or to r e l e a s e from t i s s u e s t o r e s , remains to be determined. But i t has been e s t a b l i s h e d t h a t , even i n the absence o f a d i e t a r y source, c a r n i t i n e i s s t i l l p r e s e n t i n b l o o d , a l b e i t a t low l e v e l s . F u r t h e r support f o r the 166 impact t h a t d i e t a r y c a r n i t i n e can have, on b l o o d l e v e l s o f t h i s substance i n the newborn, came from the study o f an i n f a n t who had two bouts of n e c r o t i z i n g e n t e r o c o l i t i s (see F i g . 22). Treatment r e q u i r e d two courses o f i n t r a v e n o u s a l i m e n t a t i o n w i t h EBM f o l l o w e d by P two attempts a t o r a l a l i m e n t a t i o n , w i t h S i m i l a c . During both p e r i o d s o f i n t r a v e n o u s a l i m e n t a t i o n , the c a r n i t i n e l e v e l s f e l l t o extremely low levels' 4 and d u r i n g b oth p e r i o d s of o r a l a l i m e n t a t i o n the l e v e l s r o s e s t r i k i n g l y . I t i s i n t e r e s t i n g to note t h a t , when the i n f a n t s r e c e i v e d a d i e t which was r i c h i n c a r n i t i n e , not o n l y d i d b l o o d l e v e l s o f t o t a l c a r n i t i n e i n c r e a s e , but a l s o those of a c y l c a r n i t i n e . E a r l i e r s t u d i e s have i n d i c a t e d a p o s i t i v e c o r r e l a t i o n between l e v e l s o f a c y l c a r n i t i n e and D-3-hydroxybutyrate which suggests t h a t serum l e v e l s of a c y l c a r n i t i n e may be r e l a t e d t o the r a t e o f o x i d a t i o n o f f r e e f a t t y a c i d s . Whether or not the i n c r e a s e i n plasma l e v e l s o f a c y l c a r n i t i n e , i n the p r e s e n t study, r e p r e s e n t s an i n c r e a s e d c a p a c i t y o f the i n f a n t t o o x i d i z e l i p i d remains to be e s t a b l i s h e d . Reports have appeared on the t o x i c e f f e c t s o f I n t r a l i p i d i n v e r y s m a l l i n f a n t s (224). I t i s p o s s i b l e t h a t t h i s t o x i c i t y may be due to a d i e t a r y d e f i c i e n c y i n c a r n i t i n e . A l t h o u g h i t has not been e s t a b l i s h e d t h a t any c l i n i c a l problems occur when newborn i n f a n t s are f e d a d i e t which i s d e f i c i e n t i n c a r n i t i n e , the p r e s e n t study r a i s e s the q u e s t i o n o f the p o s s i b l e t h e r a p e u t i c b e n e f i t s o f supplementing c a r n i t i n e t o the i n t r a v e n o u s l y a l i m e n t e d c h i l d . TABLE XIV Plasma L e v e l s o f C a r n i t i n e i n Newborn I n f a n t s d u r i n g I n t r a v e n o u s and M i l k Feeds Mean + S.D. A t o t a l o f 33 newborn i n f a n t s were s t u d i e d . I n t r a v e n o u s f e e d c o n s i s t e d o f T r a v a s o l and I n t r a l i p i d w i t h a c a r n i t i n e content o f l e s s than 1 /iM. M i l k feed c o n s i s t e d o f e i t h e r expressed b r e a s t m i l k o r S i m i l a c ^ w i t h a c a r n i t i n e c o ntent o f a p p r o x i m a t e l y 50 uM. C a r n i t i n e was determined by the r a d i o a c t i v e metnod as o u t l i n e d i n the Methods s e c t i o n . Feed Free C a r n i t i n e OuM) A c y l c a r n i t i n e (uM) T o t a l C a r n i t i n e (jiM) I n t r a v e n o u s 10.5 + 3.6 4.9 + 2.0 15.9 + 5.0 M i l k 19.4 + 6.9* 7.5 + 3.1** 29.1 + 8.7* —-*p<0.001 vs I n t r a v e n o u s Feed **p<0.005 vs I n t r a v e n o u s Feed 168 FIGURE 22 I n f a n t g i r l F was born at 30 weeks g e s t a t i o n w e i g h i n g 990 grams. Her major c l i n i c a l problem was apnea. She developed n e c r o t i z i n g e n t e r o c o l i t i s on two s e p a r a t e o c c a s i o n s seven days a p a r t . D u r i n g each of these p e r i o d s , she r e c e i v e d T r a v a s o l and I n t r a l i p i d f o r a t o t a l o f 43 days. O r a l a l i m e n t a t i o n s were i n - t h e form of expressed b r e a s t m i l k (EBM) or S i m i l a c . C a r n i t i n e was determined as o u t l i n e d i n the Methods s e c t i o n . 170 PART I I I CARNITINE ACETYLTRANSFERASE ACTIVITY IN LIVER AND BROWN ADIPOSE TISSUE OF DEVELOPING ANIMALS I n t r o d u c t i o n R e c e n t l y , c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y has been found not o n l y i n m i t o c h o n d r i a , but a l s o i n microsomes and peroxisomes o f l i v e r (177,179). Others have r e p o r t e d t h a t the enzyme i s p r e s e n t i n c a t a l a s e p o s i t i v e m i c r o b o d i e s i n brown adipose t i s s u e (5) and t h a t i t s a c t i v i t y i n c r e a s e s w i t h c o l d a d a p t a t i o n (124). I t has been suggested t h a t the f u n c t i o n o f c a r n i t i n e a c e t y l t r a n s f e r a s e , i n t h e s e 3 c e l l p a r t i c l e s , may not be the same (181). I n o r d e r to g a i n some i n s i g h t i n t o the r o l e o f c a r n i t i n e a c e t y l t r a n s f e r a s e , the p e r i n a t a l development o f the a c t i v i t y o f t h i s enzyme i n m i t o c h o n d r i a , microsomes and peroxisomes was examined i n the l i v e r and brown adipose t i s s u e o f the r a t , guinea p i g , and r a b b i t . The l i v e r and brown adipose t i s s u e o f the r a t were s e l e c t e d because they u t i l i z e f a t a v i d l y i n the p e r i n a t a l p e r i o d . The guinea p i g and r a b b i t were s e l e c t e d because t h e i r p l a c e n t a s , u n l i k e t h a t i n the r a t , are r e l a t i v e l y permeable t o l i p i d i n l a t e g e s t a t i o n (30,93,137,186,275) and because c a r n i t i n e passes more r e a d i l y through t h e i r p l a c e n t a s than i n the r a t (see e a r l i e r d a t a ) . These two s p e c i e s appear to u t i l i z e m a t e r n a l l y d e r i v e d l i p i d as a s u b s t r a t e i n l a t e g e s t a t i o n and, hence, would be expected to have a requirement f o r c a r n i t i n e and c a r n i t i n e a c e t y l t r a n s f e r a s e . E x p e r i m e n t a l Design White W i s t a r r a t s were bred and t h e i r p r e g n a n c i e s dated by v a g i n a l smear. Pregnant guinea p i g s and r a b b i t s were o b t a i n e d from 172 the U n i v e r s i t y a n i m a l farm. Rats and g u i n e a p i g s were s a c r i f i c e d by d e c a p i t a t i o n and r a b b i t s by the r a p i d i n t r a v e n o u s i n j e c t i o n o f a i r (approx. 20.0 m l ) . F e t u s e s were r a p i d l y removed from the u t e r u s , d e c a p i t a t e d , and brown ad i p o s e t i s s u e and l i v e r r a p i d l y e x c i s e d . T i s s u e s were i m m e d i a t e l y p l a c e d on i c e , weighed and s u b s e q u e n t l y homogenized i n 10 volumes o f i c e - c o l d s u c r o s e b u f f e r . The t i s s u e s were p r o c e s s e d and c e l l f r a c t i o n s s e p a r a t e d as o u t l i n e d i n the Methods s e c t i o n . The m i t o c h o n d r i a l c o n t e n t o f the d i f f e r e n t f r a c t i o n s was t e s t e d by d e t e r m i n i n g s u c c i n a t e dehydrogenase (E.C.1.3.99.1) a c t i v i t y i n f r e s h l y p r e p a r e d samples. This enzyme was used as a marker f o r m i t o c h o n d r i a . C a t a l a s e (E.C.1.11.1.6) was determined a c c o r d i n g to (216) and c a r n i t i n e a c e t y l t r a n s f e r a s e was determined f o l l o w i n g the r a d i o a c t i v e p r o c e d u r e i n the Methods s e c t i o n . R e s u l t s and D i s c u s s i o n As mentioned e a r l i e r (see Methods s e c t i o n ) , attempts at i s o l a t i n g peroxisomes from the l i v e r s and brown ad i p o s e t i s s u e of n e o n a t a l r a t s were u n s u c c e s s f u l . Others (159), w o r k i n g w i t h n e o n a t a l r a t l i v e r , have r e c e n t l y r e p o r t e d s i m i l a r d i f f i c u l t i e s . I t i s a p p a r e n t t h a t m i t o c h o n d r i a l c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y , i n both l i v e r and brown adipose t i s s u e , r o s e r a p i d l y a f t e r b i r t h (see F i g s . 23 and 2 4 ) . T h i s s u p p o r t s the f i n d i n g s o f o t h e r s (124,164). In a d d i t i o n to m i t o c h o n d r i a l a c t i v i t y , m icrosomal c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y was found i n both t i s s u e s . In the l i v e r , m icrosomal a c t i v i t y i n c r e a s e d at the time of weaning whereas i n brown adipose t i s s u e i t i n c r e a s e d soon a f t e r b i r t h . I t s h o u l d be noted t h a t the a c t i v i t y o f c a r n i t i n e a c e t y l t r a n s f e r a s e showed a s i m i l a r developmental p a t t e r n i n both the m i t o c h o n d r i a and microsomes o f brown adipose t i s s u e . T h i s may i n d i c a t e t h a t the microsomal a c t i v i t y i n t h i s t i s s u e i s a r e f l e c t i o n o f m i t o c h o n d r i a l c o n t a m i n a t i o n o f t h i s f r a c t i o n r a t h e r than microsomal a c t i v i t y per se. I n o r d e r to a s c e r t a i n whether c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y , i n the microsomal f r a c t i o n o f brown adipose t i s s u e and l i v e r , was not due to m i t o c h o n d r i a l c o n t a m i n a t i o n , s u c c i n a t e dehydrogenase a c t i v i t y was determined i n the m i t o c h o n d r i a l and microsomal f r a c t i o n s prepared from each t i s s u e . I t was d i s c o v e r e d t h a t m i t o c h o n d r i a l c o n t a m i n a t i o n undoubtedly o c c u r r e d ; y e t , i t i s apparent from F i g . 25 t h a t , a t a l l ages, c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n microsomes cannot be a s c r i b e d to m i t o c h o n d r i a l c o n t a m i n a t i o n o n l y . I n f e t a l l i v e r , where the m i t o c h o n d r i a l to microsomal r a t i o o f s u c c i n a t e dehydrogenase a c t i v i t y i s lowest ( f i v e ) , the r a t i o f o r c a r n i t i n e a c e t y l t r a n s f e r a s e was o n l y 2. At a l l o t h e r ages, t h e r e was 30 t o 60 times as much s u c c i n a t e dehydrogenase a c t i v i t y i n m i t o c h o n d r i a as i n microsomes. The c o r r e s p o n d i n g f i g u r e s f o r c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y reached a peak o f 6.5, s i x days a f t e r b i r t h . C o n d i t i o n s are l e s s obvious i n brown adipose t i s s u e . The d i s t r i b u t i o n o f the s u c c i n a t e dehydrogenase marker i n t h i s t i s s u e i n d i c a t e s t h a t the microsomal f r a c t i o n was h e a v i l y contaminated w i t h m i t o c h o n d r i a . Y e t , a t a l l ages the r a t i o was a g a i n h i g h e r f o r s u c c i n a t e dehydrogenase than f o r c a r n i t i n e a c e t y l t r a n s f e r a s e . T h i s 174 FIGURE 23 C a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y o f h e p a t i c m i t o c h o n d r i a (MIT) and microsomes (MIC) d u r i n g development of the r a t . F = f e t u s Means + S.E.M. (6 to 12 animals f o r each p o i n t ) 175 LIVER Age (days) FIGURE 24 C a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y o f brown a d i p o s e t i s s u e (BAT) m i t o c h o n d r i a (MIT) and microsomes (MIC) d u r i n g development o f the r a t . F = f e t u s Means + S.E.M. (each p o i n t r e p r e s e n t s the mean of 6 to 12 r a t s ) BAT Age (days) 178 FIGURE 25 The r a t i o o f c a r n i t i n e a c e t y l t r a n s f e r a s e (CAT) and s u c c i n i c dehydrogenase (SDH) i n m i t o c h o n d r i a to t h a t i n microsomes o f l i v e r ( t o p ) and brown a d i p o s e t i s s u e (bottom) d u r i n g development i n r a t s . Brown a d i p o s e t i s s u e (BAT) F= f e t u s A b s c i s s a : age i n days Mean (6 t o 12 r a t s f o r each p o i n t ) 179 -1 Days 180 suggests t h a t the microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n brown f a t can not t o t a l l y be a t t r i b u t e d to c o n t a m i n a t i o n o f the f r a c t i o n by m i t o c h o n d r i a . C a t a l a s e a c t i v i t y was determined on a per g wet weight b a s i s i n l i v e r and brown ad i p o s e t i s s u e of the r a t . The d a t a are p r e s e n t e d i n F i g . 26. I n the l i v e r , t h e r e was a steep r i s e i n c a t a l a s e a c t i v i t y a t the time o f weaning. T h i s was a l s o found by o t h e r s (216). On the o t h e r hand, i n brown adipose t i s s u e , where c a t a l a s e p o s i t i v e m i c r o b o d i e s have been found (218), a c t i v i t y i n c r e a s e d to a peak on day 6 and then decreased a g a i n . I t should be noted t h a t the l i v e r c o n t a i n s 1.5 t o 20 times as much c a t a l a s e as brown f a t . These d a t a suggest, but do not prove, t h a t peroxisomes may be more numerous i n l i v e r than i n brown adipose t i s s u e . Based on the low l e v e l s o f c a t a l a s e t h a t are p r e s e n t n e o n a t a l l y , i t seems u n l i k e l y t h a t peroxisomes are i n v o l v e d i n the thermogenic response o f brown f a t . Y e t , the developmental p r o f i l e o f c a t a l a s e a c t i v i t y peaks at day 6, when the thermogenic response of t h i s t i s s u e would be expected to be a t a maximum, and d e c l i n e s t h e r e a f t e r . Indeed, r e l a t i v e to the a c t i v i t y i n l i v e r , the a c t i v i t y o f c a t a l a s e i n brown f a t i s v e r y low; y e t , a t i t s peak the a c t i v i t y does r e a c h i n excess o f 7 mmoles of hydrogen p e r o x i d e c o n v e r t e d per g wet weight per minute. Table XV shows c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n brown adipose t i s s u e and l i v e r o f f e t a l , newborn and m a t e r n a l r a b b i t s and guinea p i g s . I n c o n t r a s t to t h a t i n the r a t , m i t o c h o n d r i a l a c t i v i t y of. c a r n i t i n e a c e t y l t r a n s f e r a s e i n the l i v e r o f these two s p e c i e s was v e r y h i g h and d i d not appear to i n c r e a s e d u r i n g the 181 FIGURE 26 C a t a l a s e a c t i v i t y i n r a t l i v e r and brown f a t d u r i n g development. F = f e t u s Note the d i f f e r e n t o r d i n a t e s f o r l i v e r and BAT. Mean + S.E.M. (6 t o 12 r a t s f o r each p o i n t ) Brown Adipose Tissue Catalase • -(nmoles/min/g weight) 183 p o s t - n a t a l p e r i o d . T h i s l a c k of a p o s t - n a t a l i n c r e a s e p r o b a b l y r e f l e c t s the f a c t t h a t the r a b b i t and guinea p i g are a l r e a d y u t i l i z i n g l i p i d i n the f e t a l p e r i o d and, t h e r e f o r e , r e q u i r e h i g h e r f e t a l l e v e l s o f c a r n i t i n e and c a r n i t i n e a c e t y l t r a n s f e r a s e than the f e t a l r a t . I n f e t a l r a b b i t and guinea p i g , the a c t i v i t y o f c a r n i t i n e a c e t y l t r a n s f e r a s e i n brown f a t m i t o c h o n d r i a i s v e r y h i g h and does not change s i g n i f i c a n t l y a t 1 day of p o s t n a t a l age, whereas i n r a t i t i s low a t b i r t h and i n c r e a s e s n e o n a t a l l y r e a c h i n g a peak by day 8. Assuming t h a t t h i s enzyme p l a y s some r o l e i n thermogenesis of brown f a t , i t i s p u z z l i n g why i t s l e v e l i s so h i g h i n the f e t a l r a b b i t and guinea p i g and y e t so low i n the r a t f e t u s . The h i g h l e v e l s o f c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n brown adipose t i s s u e o f f e t a l r a b b i t and guinea p i g may r e f l e c t the advanced m a t u r i t y o f those two s p e c i e s i n comparison to the r a t . I f one assumes t h a t t h i s enzyme i s i n v o l v e d i n the thermogenic f u n c t i o n o f brown adipose t i s s u e , then i t i s d i f f i c u l t , from a t e l e o l o g i c a l p o i n t of view, to e x p l a i n why the a c t i v i t y i s so h i g h i n r a b b i t and guinea p i g brown f a t i n l a t e g e s t a t i o n . I t i s p o s s i b l e t h a t , i n these two s p e c i e s , i t has a d i f f e r e n t f u n c t i o n i n l a t e g e s t a t i o n . I n agreement w i t h the f i n d i n g s f o r the r a t , the r a t i o of microsomal to m i t o c h o n d r i a l c a r n i t i n e a c e t y l t r a n s f e r a s e i s much g r e a t e r i n brown f a t than i n l i v e r i n both the guinea p i g and the r a b b i t . As was the case i n the r a t , brown adipose t i s s u e microsomal a c t i v i t y o f c a r n i t i n e a c e t y l t r a n s f e r a s e was v e r y h i g h i n comparison to the l i v e r . There i s l i t t l e doubt t h a t t h i s f r a c t i o n i n brown adipose t i s s u e was h e a v i l y contaminated by m i t o c h o n d r i a . Whether or TABLE XV C a r n i t i n e A c e t y l t r a n s f e r a s e A c t i v i t y i n L i v e r and Brown Fat M i t o c h o n d r i a and Microsomes o f R a b b i t s and Guinea P i g s Means + S.E.M. The number o f animals i s g i v e n i n p a r e n t h e s e s . Fetuses were used two days b e f o r e term. Mother animals were those of the f e t u s e s . Brown Adipose T i s s u e (BAT). C a r n i t i n e A c e t y l t r a n s f e r a s e A c t i v i t y (nmoles/min per mg p r o t e i n ) Fetus 1 Day Mother R a b b i t L i v e r M i t o c h o n d r i a 179.0 + 3.4 (6) 150.0 + 6.0 (4) 100.0 + 14.0 (4) L i v e r Microsomes 2 1 . 4 + 1 . 0 8 . 7 + 0 . 9 * 1 1 . 0 + 5 . 0 BAT M i t o c h o n d r i a 1 7 8 . 0 + 8 . 0 1 8 3 . 0 + 5 . 0 BAT Microsomes 91.4+ 9.0 68.0 + 3.6 Guinea P i g L i v e r M i t o c h o n d r i a 117.0 + 3.7 (8) 124.0 + 12.0 (4) 123.0 + 4.2 (4) L i v e r Microsomes 9 . 0 + 0 . 3 1 2 . 0 + 0 . 8 1 4 . 6 + 1 . 4 BAT M i t o c h o n d r i a 1 9 8 . 0 + 1 7 . 0 2 0 0 . 0 + 1 2 . 0 BAT Microsomes 1 0 8 . 0 + 8 . 0 9 0 . 0 + 1 0 . 0 *p<.01 vs f e t u s 185 not c a r n i t i n e a c e t y l t r a n s f e r a s e i s p r e s e n t i n the microsomes o f brown adipose t i s s u e i s o l a t e d from the r a b b i t and guinea p i g i s u n c e r t a i n . L i v e r microsomal a c t i v i t y o f c a r n i t i n e a c e t y l t r a n s f e r a s e i n the f e t a l and n e o n a t a l r a b b i t and guinea p i g i s h i g h e r than i n s i m i l a r l y aged r a t s . I t i s o n l y at the time of weaning i n the r a t t h a t l i v e r microsomal a c t i v i t y l e v e l s r e a c h those v a l u e s seen p e r i n a t a l l y i n r a b b i t and g u i n e a p i g . I n the r a t , the i n c r e a s e i n l i v e r microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y t h a t o c c u r r e d at weaning was accompanied by a c o i n c i d e n t i n c r e a s e i n the a c t i v i t y o f c a t a l a s e . The s i g n i f i c a n t decrease (p<.01), seen i n l i v e r microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n the r a b b i t at day 1, i s d i f f i c u l t to e x p l a i n (see T a b l e XV). I t may i n d i c a t e t h a t c a r n i t i n e a c e t y l t r a n s f e r a s e i s b e i n g s t o r e d i n the lumen of the microsomes i n the f e t u s and then i n c o r p o r a t e d i n t o newly formed peroxisomes and m i t o c h o n d r i a a t b i r t h . A l t e r n a t i v e l y , i t may i n d i c a t e a change i n the b i o s y n t h e t i c events i n r a b b i t microsomes at b i r t h . The e xact r o l e o f l i v e r microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e i s unknown. C o n c e i v a b l y , i t may o n l y be f u n c t i o n i n g as a s t o r e f o r the enzyme so t h a t i t w i l l be a v a i l a b l e f o r i n c o r p o r a t i o n i n t o newly formed peroxisomes and m i t o c h o n d r i a . I n t h i s r e g a r d , i t i s i n t e r e s t i n g to note t h a t at the time of weaning i n the r a t t h e r e i s not o n l y a marked i n c r e a s e i n h e p a t i c microsomal l e v e l s o f c a r n i t i n e a c e t y l t r a n s f e r a s e , but a l s o i n l e v e l s o f h e p a t i c c a t a l a s e . A l t e r n a t i v e l y , l i v e r microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e may p l a y a r o l e i n b i o s y n t h e t i c events t h a t take 186 p l a c e i n the microsomes, by f a c i l i t a t i n g the a v a i l a b i l i t y and i n c o r p o r a t i o n o f s h o r t - c h a i n a c y l m o i e t i e s i n t o s y n t h e t i z e d p r o d u c t s such as s t e r o i d s and f a t t y a c i d s . CONCLUDING REMARKS 188 From the d ata o f the p r e s e n t study, i t i s e v i d e n t t h a t serum l e v e l s o f f r e e , a c y l - and t o t a l c a r n i t i n e change when the r a t e o f l i p i d u t i l i z a t i o n by the a n i mal changes. T h i s need not be the o n l y f a c t o r c a p a b l e o f b r i n g i n g about such changes i n serum c a r n i t i n e l e v e l s and undoubtedly o t h e r f a c t o r s w i l l be found w i t h time. The p o s i t i v e c o r r e l a t i o n between serum a c y l c a r n i t i n e l e v e l s and D-3-hydroxybutyrate, seen d u r i n g s t a r v a t i o n , suggests t h a t serum a c y l c a r n i t i n e l e v e l s are r e l a t e d i n some way to the o x i d a t i o n o f l i p i d . T h i s i n c r e a s e i n serum a c y l c a r n i t i n e may be the r e s u l t of an exchange pr o c e s s between serum f r e e c a r n i t i n e and i n t r a c e l l u l a r a c y l c a r n i t i n e . Depending on the mechanism of the exchange, such a p r o c e s s c o u l d , under c o n d i t i o n s o f i n c r e a s e d 3 - o x i d a t i o n , f u n c t i o n to r e l i e v e " a c e t y l - C o A p r e s s u r e " and r e g e n e r a t e coenzyme A. During the course o f prolonged s t a r v a t i o n i n the r a t , s i g n i f i c a n t changes i n t o t a l serum c a r n i t i n e were r e c o r d e d . I n i t i a l l y , t o t a l serum c a r n i t i n e d ecreased, r e a c h i n g a n a d i r a t 24 h r . Subsequently, l e v e l s i n c r e a s e d r e a c h i n g a maximum at 48 h r . The i n i t i a l decrease i n t o t a l c a r n i t i n e may have been caused by an i n c r e a s e d t i s s u e uptake of serum c a r n i t i n e r e q u i r e d to meet s h o r t - t e r m demands u n t i l the a v a i l a b i l i t y o f c a r n i t i n e i n c r e a s e d a t 48 h r . A l t h o u g h the source of t h i s i n c r e a s e d s u p p l y o f c a r n i t i n e i s not known, the 48 h r . l a g p e r i o d suggests t h a t i t may be due to an i n c r e a s e i n the r a t e o f c a r n i t i n e s y n t h e s i s by the l i v e r . I n man, the r e n a l c l e a r a n c e of a c y l c a r n i t i n e i n c r e a s e d w i t h s t a r v a t i o n . I f a s i m i l a r s i t u a t i o n o c c u r s i n the r a t then, undoubtedly, p a r t of the i n c r e a s e i n t o t a l c a r n i t i n e , t h a t o ccurs a f t e r 48 h r . o f s t a r v a t i o n , c o u l d be d i r e c t e d toward compensating f o r t h i s l o s s . T h i s c o n c l u s i o n i s based upon the assumption t h a t c o n t i n u e d r e n a l l o s s e s would 189 e v e n t u a l l y d e p l e t e the a n i mal of i t s c a r n i t i n e s t o r e s and hence s e v e r e l y reduce i t s c a p a c i t y to o x i d i z e l i p i d w i t h p r o l o n g e d s t a r v a t i o n . By i n c r e a s i n g the r a t e of s y n t h e s i s of c a r n i t i n e the a n i m al c o u l d ensure i t s e l f o f an adequate c a r n i t i n e s u p p l y i n s p i t e o f c o n t i n u e d r e n a l l o s s e s . I t i s c o n c e i v a b l e t h a t the r e n a l c l e a r a n c e o f a c y l c a r n i t i n e may i n c r e a s e i n e a r l y s t a r v a t i o n and then, s u b s e q u e n t l y decrease as the p e r i o d o f s t a r v a t i o n i s p r o l o n g e d . I f t h i s i s the case i n the r a t , then t o t a l serum c a r n i t i n e l e v e l s would be expected to decrease i n i t i a l l y and then s u b s e q u e n t l y i n c r e a s e as the p e r i o d o f s t a r v a t i o n l e n g t h e n s . S i n c e these changes i n t o t a l c a r n i t i n e would be due to changes i n the r e n a l c l e a r a n c e s of a c y l c a r n i t i n e , then one would expect the serum l e v e l s o f a c y l c a r n i t i n e to i n i t i a l l y d ecrease and then s u b s e q u e n t l y i n c r e a s e . I n the p r e s e n t study, serum a c y l c a r n i t i n e l e v e l s d i d not show any f l u c t u a t i o n , beyond the i n c r e a s e r e c o r d e d at 24 h r . The m a j o r i t y of the i n c r e a s e i n t o t a l c a r n i t i n e t h a t was r e c o r d e d a t 48 h r . was not i n the a c y l c a r n i t i n e f r a c t i o n , but r a t h e r i n the f r e e c a r n i t i n e f r a c t i o n . Assuming t h a t the r e n a l c l e a r a n c e of a c y l c a r n i t i n e does decrease w i t h p r o l o n g e d s t a r v a t i o n , one would have expected the serum l e v e l s o f a c y l c a r n i t i n e to i n c r e a s e a t 48 h r . The i n c r e a s e i n f r e e c a r n i t i n e t h a t o c c u r r e d a t 48 h r . may r e f l e c t a change i n the c a p a c i t y o f the animal to u t i l i z e a c y l c a r n i t i n e as an energy s u b s t r a t e . Under these c o n d i t i o n s , the t u r n o v e r r a t e o f a c y l c a r n i t i n e would i n c r e a s e . I f the r a t e o f u t i l i z a t i o n matched the r a t e o f p r o d u c t i o n , serum l e v e l s o f a c y l c a r n i t i n e would remain r e l a t i v e l y unchanged. Assuming t h a t t h e r e i s an i n c r e a s e d r a t e o f a c y l c a r n i t i n e t u r n o v e r , then serum l e v e l s o f f r e e c a r n i t i n e would l i k e l y i n c r e a s e , p a r t i c u l a r l y i f 190 c a r n i t i n e were r e l e a s e d back i n t o the bl o o d stream subsequent to the removal o f the a c y l m oiety. When animals were p l a c e d on a d i e t t h a t was r i c h i n medium-chain f a t t y a c i d t r i g l y c e r i d e s , serum l e v e l s o f 3-h y d r o x y b u t y r a t e and a c y l c a r n i t i n e i n c r e a s e d w h i l e t o t a l c a r n i t i n e l e v e l s remained unchanged. A s i m i l a r p r o f i l e was seen w i t h 48 h r . o f s t a r v a t i o n i n the r a t , w i t h the e x c e p t i o n t h a t t o t a l serum c a r n i t i n e d i d i n c r e a s e . T h i s suggests t h a t the serum l e v e l s o f c a r n i t i n e are adequate f o r meeting the demands o f the animal on an MCT d i e t but not when the animal i s s t a r v e d . T h i s d i f f e r e n c e may be due to an i n c r e a s e d demand f o r i n t r a c e l l u l a r c a r n i t i n e i n the s t a r v e d a n i m a l . I n a d d i t i o n , the data may r e f l e c t d i f f e r e n c e s i n the r a t e of u t i l i z a t i o n and r e n a l c l e a r a n c e o f a c y l c a r n i t i n e i n these two m e t a b o l i c s t a t e s . Both s t a r v a t i o n and the MCT d i e t r e s u l t e d i n marked i n c r e a s e s i n serum l e v e l s o f a c y l c a r n i t i n e . The r e l a t i v e i n c r e a s e was g r e a t e r w i t h the MCT d i e t . I t i s r e a s o n a b l e to suggest t h a t , i n t h i s case, c a r n i t i n e i s b e i n g used to b u f f e r the i n c r e a s e d " a c e t y l - C o A p r e s s u r e " which i s generated d u r i n g the metabolism of t h i s d i e t . The f o r m a t i o n o f a c y l c a r n i t i n e would s e r v e to r e g e n e r a t e the s u pply o f a v a i l a b l e coenzyme A. T h i s b u f f e r i n g c a p a c i t y would be l o s t once the a v a i l a b l e s u p p l y o f c a r n i t i n e was d e p l e t e d . I n o r d e r to p r e v e n t t h i s from happening, a c o n s t a n t s u p p l y o f c a r n i t i n e would be n e c e s s a r y . T h i s c o u l d be a c h i e v e d by i n c r e a s i n g the r a t e o f s y n t h e s i s o f c a r n i t i n e , by i n c r e a s i n g the r a t e of r e l e a s e o f c a r n i t i n e from c a r n i t i n e - r i c h t i s s u e s , or by r e g e n e r a t i n g c a r n i t i n e through the u t i l i z a t i o n o f a c y l c a r n i t i n e as an energy and/or / 191 b i o s y n t h e t i c s u b s t r a t e . T o t a l serum c a r n i t i n e l e v e l s d i d not i n c r e a s e when animals were f e d the MCT d i e t . T h i s suggests t h a t the s y n t h e s i s and r a t e o f r e l e a s e o f t i s s u e c a r n i t i n e p r o b a b l y do not i n c r e a s e on t h i s d i e t . I f t h i s i s the case, then the s u p p l y o f c a r n i t i n e i s l i k e l y r e p l e n i s h e d through the u t i l i z a t i o n of a c y l c a r n i t i n e as an energy and/or b i o s y n t h e t i c s u b s t r a t e . No i n f o r m a t i o n i s a v a i l a b l e on the r e n a l c l e a r a n c e o f a c y l c a r n i t i n e when animals a re on the MCT d i e t . T h i s i n f o r m a t i o n w i l l be e s s e n t i a l t o the u n d e r s t a n d i n g o f the changes t h a t are seen i n serum c a r n i t i n e l e v e l s when animals are fed t h i s d i e t . The source and f a t e of the a c y l c a r n i t i n e , generated d u r i n g s t a r v a t i o n , may be q u i t e d i f f e r e n t from t h a t seen w i t h the MCT d i e t . I t i s c o n c e i v a b l e t h a t a p o r t i o n o f the serum a c y l c a r n i t i n e i n s t a r v a t i o n may be f u n c t i o n i n g to t r a n s p o r t p e r i p h e r a l l y m o b i l i z e d l i p i d to o t h e r t i s s u e s f o r u t i l i z a t i o n . Another p o r t i o n may be gener a t e d , through the b u f f e r i n g o f " a c e t y l - C o A p r e s s u r e " , i n a manner s i m i l a r to t h a t seen w i t h the MCT d i e t . As mentioned e a r l i e r , t h e r e appears to be an i n c r e a s e d demand f o r c a r n i t i n e i n the s t a r v e d a n i m a l , as r e f l e c t e d by a s i g n i f i c a n t i n c r e a s e i n t o t a l c a r n i t i n e l e v e l s a f t e r 48 h r . o f s t a r v a t i o n . T h i s i n c r e a s e i n demand may r e s u l t from l o s s e s i n c u r r e d through e x c r e t i o n by the k i d n e y s as w e l l as from an i n c r e a s e d t r a p p i n g o f c a r n i t i n e w i t h i n t i s s u e s i n the form o f l o n g - c h a i n a c y l c a r n i t i n e s . The q u e s t i o n a r i s e s as to whether or not the s t a r v e d animal can u t i l i z e serum a c y l c a r n i t i n e as an energy s u b s t r a t e . I t i s 192 u n l i k e l y t h a t a c y l c a r n i t i n e i s used b i o s y n t h e t i c a l l y d u r i n g s t a r v a t i o n . I t i s tempting to s p e c u l a t e t h a t serum a c y l c a r n i t i n e r e p r e s e n t s an a d d i t i o n a l energy s u b s t r a t e f o r the s t a r v e d a n i m a l . -I t s importance may be p a r t i c u l a r l y s i g n i f i c a n t to those t i s s u e s , which are o b l i g a t e g l u c o s e u t i l i z e r s . I n the b r a i n , p r o l o n g e d s t a r v a t i o n r e s u l t s i n an i n c r e a s e d c a p a c i t y to u t i l i z e ketone b o d i e s as an energy s u b s t r a t e . T h i s a d a p t a t i o n i s g r a d u a l and o c c u r s o n l y w i t h severe s t a r v a t i o n . A c y l c a r n i t i n e may r e p r e s e n t an a d d i t i o n a l s u b s t r a t e t h a t can be u t i l i z e d by the b r a i n u n t i l the a d a p t a t i o n to ketone body u t i l i z a t i o n has o c c u r r e d . F u r t h e r d a t a r e g a r d i n g the exact n a t u r e of these a c y l c a r n i t i n e s as w e l l as t h e i r r a t e of t u r n o v e r would h e l p to i n d i c a t e whether or not serum a c y l c a r n i t i n e i s an i m p o r t a n t energy s u b s t r a t e f o r the s t a r v e d a n i m a l . P e r f u s i o n data taken from the pregnant ewe i n d i c a t e t h a t , i n t h i s s p e c i e s , serum a c e t y l c a r n i t i n e may be u t i l i z e d as a s u b s t r a t e . When the i n f u s i o n o f a c e t y l - d l - c a r n i t i n e was t e r m i n a t e d , the serum l e v e l s o f a c y l c a r n i t i n e began to decrease, a t the same time, f r e e c a r n i t i n e l e v e l s i n c r e a s e d and t o t a l serum c a r n i t i n e l e v e l s remained unchanged. These d a t a suggest t h a t , once the a c y l moiety o f a c y l c a r n i t i n e was u t i l i z e d by the ewe, the f r e e c a r n i t i n e t h a t remained was r e l e a s e d back i n t o the serum. How i m p o r t a n t i s a c y l c a r n i t i n e as an energy source f o r the animal? With the l i m i t e d d a t a a v a i l a b l e from the p r e s e n t s t u d y , one would have to conclude t h a t i n a l l l i k e l i h o o d i t i s not a v e r y i m p o r t a n t energy source. T h i s c o n c l u s i o n i s based on the f a c t t h a t serum l e v e l s of a c y l c a r n i t i n e are a p p r o x i m a t e l y 1000 f o l d lower than l e v e l s o f ketone b o d i e s and f r e e f a t t y a c i d s . T h i s b e i n g the case, the r a t e o f t u r n o v e r of a c y l c a r n i t i n e would have to be v e r y h i g h 193 indeed, f o r i t to make a s i g n i f i c a n t c o n t r i b u t i o n to the energy needs of the a n i m a l . Perhaps, t h i s i s the case but a d e f i n i t i v e answer must await f u r t h e r s t u d i e s . Could serum a c y l c a r n i t i n e be s e r v i n g as a means by which l i v e r c a r n i t i n e i s t r a n s p o r t e d t o p e r i p h e r a l t i s s u e s ? Under such a scheme, as the r a t e o f f a t t y a c i d o x i d a t i o n i n c r e a s e d i n the l i v e r so would the f l u x o f a c y l c a r n i t i n e out o f the l i v e r . T h i s f l u x would not o n l y decrease the content o f c a r n i t i n e i n the l i v e r but a l s o c o u l d cause, by a n e g a t i v e feedback mechanism, an i n c r e a s e i n the r a t e of s y n t h e s i s of c a r n i t i n e w i t h i n the l i v e r . The a c y l c a r n i t i n e r e l e a s e d i n t o the serum would be removed by p e r i p h e r a l t i s s u e s , the a c y l moiety u t i l i z e d as an energy s o u r c e , and the f r e e c a r n i t i n e , t h e r e b y r e l e a s e d , used by the t i s s u e f o r the g e n e r a t i o n of a c y l c a r n i t i n e l o c a l l y . I n a l l s p e c i e s s t u d i e d , except the r a b b i t , t o t a l serum c a r n i t i n e l e v e l s i n the s u c k l i n g neonate were h i g h e r than i n the f e t u s . The serum c a r n i t i n e l e v e l s , seen w i t h s u c k l i n g , soy bean and I.V. f e e d i n g suggest t h a t a d i e t a r y source o f c a r n i t i n e i s r e q u i r e d by the n e o n a t a l r a t , sheep and man. I n man, when neonates are f e d a c a m i t i n e - f r e e soy bean d i e t , serum c a r n i t i n e l e v e l s remain low but do not decrease to z e r o . T h i s s u g g e s t s , t h a t at l e a s t i n man, the neonate has some c a p a c i t y f o r endogenous s y n t h e s i s o f c a r n i t i n e . Whether or not t h i s i s so f o r the o t h e r s p e c i e s s t u d i e d i s not known. There was no s i g n i f i c a n t d i f f e r e n c e between m a t e r n a l and f e t a l t o t a l c a r n i t i n e l e v e l s i n those s p e c i e s i n which m a t e r n a l l i p i d 194 i s r e a d i l y t r a n s f e r r e d to the f e t u s ( r a b b i t and guinea p i g ) • T h i s suggests t h a t , i n these s p e c i e s , c a r n i t i n e may pass through the p l a c e n t a s w i t h r e l a t i v e ease. I n f e t a l r a t and sheep, two s p e c i e s i n which m a t e r n a l l y d e r i v e d l i p i d i s not f r e e l y a c c e s s i b l e to the f e t u s , c a r n i t i n e was p r e s e n t i n the serum; however, l e v e l s were v e r y much lower than m a t e r n a l l e v e l s . T h i s suggests t h a t the p l a c e n t a s i n these two s p e c i e s may be r e l a t i v e l y impermeable to c a r n i t i n e . The marked i n c r e a s e i n the a c t i v i t y o f c a r n i t i n e a c e t y l t r a n s f e r a s e , i n l i v e r m i t o c h o n d r i a i s o l a t e d from the r a t a t day one, r e f l e c t s the changing d i e t o f the r a t a t t h i s time. P r e l i m i n a r y d a t a ( d a t a not shown) i n d i c a t e t h a t d i e t a r y l i p i d i s r e q u i r e d f o r t h i s n e o n a t a l i n c r e a s e i n the a c t i v i t y o f c a r n i t i n e a c e t y l t r a n s f e r a s e . I t i s not known what the f a c t o r s r e g u l a t i n g t h i s i n d u c t i o n a r e , or whether the a c t i v i t y can be induced p r e m a t u r e l y i n the f e t u s d u r i n g l a t e g e s t a t i o n . I t i s noteworthy t h a t microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n c r e a s e d at the same time t h a t t i s s u e c a t a l a s e a c t i v i t y i n c r e a s e d . Workers have suggested t h a t peroxisomes ar e d e r i v e d from "outbuddings" o f microsomes. The c o n j u n c t i o n o f these two events makes p l a u s i b l e the s u g g e s t i o n t h a t microsomal c a r n i t i n e a c e t y l t r a n s f e r a s e r e p r e s e n t s a s t o r e o f the enzyme p r i o r t o i t s i n c o r p o r a t i o n i n t o peroxisomes. I t seems incongruous t h a t the r a t which has h i g h l e v e l s of a c y l c a r n i t i n e n e o n a t a l l y has ten times l e s s c a r n i t i n e a c e t y l t r a n s f e r a s e a c t i v i t y i n l i v e r m i t o c h o n d r i a than r a b b i t or guinea p i g . T h i s suggests t h a t the enzyme may be p e r f o r m i n g 195 d i f f e r e n t f u n c t i o n s i n these s p e c i e s . C e r t a i n l y , i n the case of the g u i n e a - p i g , i t i s c o n c e i v a b l e t h a t the l i v e r c a r n i t i n e a c e t y l t r a n s f e r a s e may be p r o v i d i n g " a c e t y l " groups f o r e x t r a - m i t o c h o n d r i a l s y n t h e s i s o f l i p i d (46,215) whereas i n the r a t t h i s f u n c t i o n i s b e i n g subserved by the " c i t r a t e - m a l a t e " s h u t t l e (131,215). Whether or not t h i s accounts f o r the l a r g e d i f f e r e n c e i s not known, but c e r t a i n l y i t seems l i k e l y t h a t i t i s o n l y p a r t of the t o t a l e x p l a n a t i o n . Brown f a t a c t i v i t y of c a r n i t i n e a c e t y l t r a n s f e r a s e , i n m i t o c h o n d r i a i s o l a t e d from f e t a l r a b b i t s and guinea p i g s , was v e r y much h i g h e r than the a c t i v i t y l e v e l s seen i n the f e t a l r a t . Assuming t h a t t h i s enzyme i s r e q u i r e d f o r the thermogenic response of t h i s t i s s u e , i t seems odd t h a t the a c t i v i t y s h ould be so h i g h w h i l e the animals were s t i l l i n u t e r o . Y e t , when one c o n s i d e r s the m a t u r i t y o f the r a b b i t and guinea p i g at b i r t h ( r e l a t i v e to the r a t ) , i t seems r e a s o n a b l e t h a t these two s p e c i e s , u n l i k e the r a t , would r e q u i r e a more f u n c t i o n a l l y a c t i v e t i s s u e at b i r t h . T h i s study has p r o v i d e d new i n s i g h t s i n t o the changing p a t t e r n s of serum c a r n i t i n e seen w i t h d i f f e r e n t d i e t s and at d i f f e r e n t stages o f development. I t has demonstrated t h a t a c y l c a r n i t i n e i s p r e s e n t i n serum and t h a t d i e t a r y l i p i d can p l a y an i m p o r t a n t r o l e i n r e g u l a t i n g l e v e l s o f t h i s substance. 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