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method does not involve hydrolysis of cholesteryl esters, the results, shown in Table I, indicate a good agreement between the methods (correlation coefficient 0.99). As in other methods (6), it is likely that marked differences in cholesteryl ester composition in some occasional samples may influence the results. Duplicate analyses by the present method gave percent mean (range) variations of -+ 1.2 (0-6.9) for the first batch and +- 0.81 (0-6.7) for the second batch. Since this method can determine as little as 5 /~g of cholesterol, the feasibility of measuring cholesterol on capillary blood samples was studied, and the results are compared with the analyses on venous blood samples of the same individuals. Plasma cholesterol levels, shown in Table II, indicate very good agreement between the capillary and venous blood samples. There was no interference in the color development by either bilirubin at 1-10 mg/100 ml levels or by hemoglobin at concentrations of 0.1-0.4 g/100 ml. Uranyl acetate is known to reduce interference by noncholesterol chromogens and eliminate intereference by bilirubin (5). Thus, the cholesterol assay system described above compared favorably with the reference method and offers several advantages. Elimination of solvent extraction and saponification steps and use of capillary blood specimens s h o u l d m a k e this procedure suitable for screening infants and small children, either in doctors' offices or in well-baby clinics. Also, the use of capillary blood would facilitate

repetitive sampling for clinical and experimental studies and allow inexpensive population screening. RAO R. B H A N D A R U SATHANUR R. SRINIVASAN PADMAKAR S. P A R G A O N K A R GERALD S. BERENSON Department of Medicine Louisiana State University Medical Center New Orleans, LA ACKNOWLEDGMENTS This work was supported by funds from The National Heart, Lung, and Blood Ins t i t ut e of The United States Public Health Service, Specialized Center of Research-Arteriosclerosis (SCOR-A), H L 0 2 9 4 2 and HL15103.

REFERENCES 1. Abell, L.L., B.B. Levy, B.B. Brodie, and F.E. Kendall, J. Biol. Chem. 195:357 (1952). 2. Zlatkis, A., and B. Zak, Anal. Biochem. 29:143 (1969). 3. Rudel, L,L., and M.D. Morris, J. Lipid Res. 14:364 (1973). 4. Lozsa, A., Clin. Chim. Acta 53:43 (1974). 5. Jung, D.H., H.G. Biggs, and W.R. Moorehead, Clin. Chem. 21:1526 (1975). 6. Manual of Laboratory Operations, Lipid Research Clinics Program, Vol. 1, Lipid and Lipoprotein Analysis, National Heart and Lung Institute, National Institutes of Health, Bethesda, MD, DHEW Publication No. (NIH) 75-628 (1974). [Received July 18, 1977]

Fatty Acid and Sterol Synthesis by Rat Small Intestine in vitro ABSTRACT

Slices of rat jejunum were incubated with [2-14C] pyruvate, [ 1-14C] acetate, or [ 3 H ] H 2 0 to determine lipogenic activity. Under all conditions studied, pyruvate acted as a better precursor than acetate for fatty acid synthesis but not for the synthesis of sterol. Exogenous glucose significantly (P~0.05) increased the conversion of both pyruvate and acetate to fatty acids. By contrast, fasting resulted in a decrease (p~~ 0.05. d i f f e r e n c e s reflect in part d i f f e r e n c e s in experim e n t a l c o n d i t i o n s , and w i t h r e s p e c t t o cholesterol synthesis, t h e p o r t i o n o f t h e small intestine used (2,4,5). However, such discrepancies could also i n d i c a t e t h a t a c e t a t e may n o t be t h e best precursor with which to measure intestinal lipogenesis. In an a t t e m p t t o resolve t h e discrepancies, t h e r e f o r e , this s t u d y c o m p a r e d t h e conversion to lipid o f [1-14C] a c e t a t e t o t h a t o f [2 -14C] p y r u v a t e b y slices p r e p a r e d f r o m rat j e j u n u m . T o investigate t h e possible e f f e c t s o f dietary m a n i p u l a t i o n on i n t e s t i n a l lipogenesis, one group of animals was fasted for 24 hr. Finally, t o establish an e s t i m a t e of t h e a b s o l u t e rate o f f a t t y acid s y n t h e s i s in t h e tissue preparat i o n , [3H] H 2 0 was used. EXPERIMENTAL PROCEDURES

Male Long-Evans rats weighing 2 5 0 - 3 0 0 g were used. Animals were h o u s e d u n d e r s t a n d a r d c o n d i t i o n s o f t e m p e r a t u r e (24 C) and lighting ( 0 6 0 0 - 1 8 0 0 light; 1800-0600 dark) and were allowed w a t e r and s t o c k ratio (Wayne Lab Blox) ad lib• F a s t e d animals w e r e w i t h o u t f o o d for 24 h r prior t o sacrifice. Animals were sacrificed b y cervical dislocat i o n and allowed t o b l e e d freely f r o m c a r o t i d arteries. I m m e d i a t e l y t h e r e a f t e r , t h e entire small i n t e s t i n e f r o m p y l o r u s t o c e c u m was excised and flushed w i t h c o p i o u s ice-cold 0.9% NaC1. A 10-12 cm l e n g t h was cut from t h e i n t e s t i n e i m m e d i a t e l y distal t o t h e d u o d e n u m . The s e g m e n t so o b t a i n e d was placed o n a cold glass plate, o p e n e d longitudinally along t h e m e s e n t e r i c b o r d e r leaving the m u c o s a l side up, and again rinsed w i t h ice-cold 0.9% NaC1. T h e n , w i t h a razor blade, 3 m m t h i c k slices were p r e p a r e d f r e e h a n d and t r a n s f e r r e d to cold Krebs-Henseleit b i c a r b o n a t e b u f f e r w h i c h had b e e n previously equilibrated at p H 7.3. Pre-

TABLE II Effect of Glucose Concentration on Incorporation of 3H from [3H]H20 into Fatty Acids and Sterol a Glucose (mM) 0 10 20 50

(ng atoms3H incorporated/g tissue/hr) Fatty acids Sterol 378 1036 1287 • 95 (4) 1390

114 249 350 • 87 (4) 302

aTwo hundred milligrams of tissue slices were incubated with [3H}H2 O (0.5 mCi) and glucose at the indicated concentrations in 2.0 ml Krebs-Henseleit bicarbonate buffer, pH %3 at 37 C in an atmosphere of 95% 02/5% CO2 for 2 hr. Results are presented as means of closely agreeing duplicate determinations, or as means + SE with the number of animals given in parentheses~ cisely weighed p o r t i o n s o f intestinal slices w e r e i n c u b a t e d u n d e r t h e c o n d i t i o n s detailed in t h e legends for t h e tables. M e t h o d s for d e t e r m i n i n g the c o n v e r s i o n o f labeled s u b s t r a t e s to f a t t y acids, n o n s a p o n i f i a b l e lipid, and CO2 were analagous to t h o s e previously d e s c r i b e d in detail for liver slice e x p e r i m e n t s (6). T h r o u g h out this paper, t h e t e r m " s t e r o l " refers t o t h e n o n s a p o n i f i a b l e lipid fraction. RESULTS AND DISCUSSION

Preliminary i n t e s t i n a l slice e x p e r i m e n t s i n d i c a t e d t h a t t h e c o n v e r s i o n o f labeled substrate t o lipids or CO2 was a p p r o x i m a t e l y linear for at least 2 hr. All s u b s e q u e n t e x p e r i m e n t s involved a 2 h r i n c u b a t i o n period. Previous r e p o r t s o f rat intestinal slice experim e n t s have s h o w n t h a t labeled acetate either w i t h (1,5,7) or w i t h o u t (2-4) a d d e d glucose will s u p p o r t f a t t y acid (1,2,4,7) o r sterol (1-5,7) LIPIDS, VOL. 12, NO. 12

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synthesis. The present study d e m o n s t r a t e d that pyruvate a c t e d as a b e t t e r precursor than acetate for fatty acid synthesis but not for the synthesis of sterols (Table I). F u r t h e r m o r e , added glucose, although not required for lipogenesis, significantly (p~

Fatty acid and sterol synthesis by rat small intestine in vitro.

SHORT COMMUNICATIONS 1080 method does not involve hydrolysis of cholesteryl esters, the results, shown in Table I, indicate a good agreement between...
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