Human Small Intestine

Gut: first published as 10.1136/gut.21.8.689 on 1 August 1980. Downloaded from

Gut, 1980, 21, 689-694

Studies on long chain fatty acid:CoA ligase from human small intestine

H BIERBACH* From the II Department of Medicine, University of Mainz, Federal Republic of Germany

SUMMARY Long chain fatty acid:CoA ligase (EC 6.2.1.3.) was examined in human small intestinal mucosa using the hydroxamate-trapping method. With optimal assay requirements using palmitate as substrate a significant difference of specific activities could be detected in the total homogenate from duodenum, 40.9 +1 16 nmol/min per mg protein versus upper jejunum, 51.9 ±13 7 (p < 005). The enzyme activity of the microsomal fraction of upper jejunum was 101.8 +44 nmol/min per mg protein. ATP, CoA, and Mg2+ were essential constituents of the reaction. A broad pH-optimum was observed between 6.75 and 7.75 with a maximum at a pH of 7.25. Whereas palmitate in the presence of albumin revealed a wide range of optimal concentration in supporting maximal enzyme activity, oleate was found to strongly inhibit the reaction. Where substrate specificity with both the total homogenate and the microsomal fraction was concerned, maximal reaction rates were obtained with palmitate for the long chain saturated fatty acids C12:0' C14:0' C16:0' and C18:0' and with oleate for the long chain unsaturated fatty acids Cl8:, C8:2' and C8:3' respectively. The highest specific activity of the enzyme was localised in the microsomal fraction. The kinetic data and properties of the long chain fatty acid:CoA ligase from human intestine are discussed with respect to the intestinal enzyme from other species. http://gut.bmj.com/

Three pathways of triacylglycerol biosynthesis in First demonstrated in guinea-pig liver by Korn- intestinal mucosa have been well established during berg and Pricer,5 this enzyme was subsequently the last two decades: the monoglyceride-, the shown to occur in the small intestinal epithelium of several species-that is, rabbit,6 rat,7-'0 pig, glycerolphosphate- and the dihydroxyacetone phos- on September 24, 2021 by guest. Protected copyright. phate pathway.' The monoglyceride pathway is the guinea-pig and cat.' Although indirect evidence quantitatively predominant reaction sequence for exists for the activity of fatty acid: CoA ligase in intestinal triacylglycerol biosynthesis in the rat,2 the human intestine from studies of triacylglycerol hamster3 and man.4 Whereas these three pathways biosynthesis,12 13 414 this enzyme has not been differ with respect to the acyl-acceptor involving directly determined in the intestinal mucosa of man. 2-monoglyceride, sn-glycerol-3-phosphate, and di- Thus the purpose of this study was to describe hydroxyacetone phosphate respectively, activated optimal kinetic conditions, to demonstrate the long chain fatty acids consisting of their CoA- substrate specificity using long chain saturated and derivatives serve as a common substrate. The unsaturated fatty acids and to reveal the intracellular enzyme catalysing the activation of long chain distribution of the long chain fatty acid :CoA fatty acids to their CoA-ester with the formation ligase from human small intestine. of a thiolester bond is the long chain fatty acid: CoA ligase (EC 6.2.1.3), a key enzyme in fatty acid Methods metabolism. Equation 1 shows the reaction catalysed by this enzyme: Fatty acid +CoA+ATP=Fatty Multiple histologically normal biopsies were obtained acyl-CoA +AMP+PPit. from the pars descendens duodeni of patients who had an oesophagogastroduodenoscopy for different t Inorganic pyrophosphate. indications, after an overnight fast usually between *Address for correspondence: Dr H Bierbach, II Department of and 12.00 a.m. Informed consent was obtained Medicine, University of Mainz, Langenbeckstr. 1, D-6500 MAINZ 1, 9.00 Federal Republic of Germany. from all individuals. The biopsies were stored at Received for publication 11 March 1980 -20°C and analysed within 14 days. This material 689 Gut: first published as 10.1136/gut.21.8.689 on 1 August 1980. Downloaded from

690 Bierbach

served to obtain normal values for the fatty acid :CoA iron-hydroxamate complex of 1.0 x 1021 X mmol-1x ligase. For all other purposes mucosal material mm-', as calculated by Kornberg and Pricer," was was gained from resected parts of the duodenum used. Specific activity was expressed as nmol and the proximal jejunum of patients operated product formed /min per mg protein. for peptic ulcer disease, carcinoma of the stomach Subcellular fractionation of human intestinal or pancreas, and chronic pancreatitis. Immediately mucosa was performed according to the method after removal, the resected parts of intestine were of Hiubscher et al.'8 As marker enzymes of the transferred to ice-cold 0.9% NaCl. Subsequently mitochondrial and microsomal fractions the succinic the macroscopically normal mucosa was scraped dehydrogenase with cytochrome c as electron off with the aid of tweezers. Biopsies and mucosal acceptor'9 and the glucose-6-phosphatase,l9a re- scrapings were homogenised in ice-cold 0.9% NaCl spectively, were assayed. using a Potter S-homogeniser (Braun Melsungen Germany) at 600 rpm. Protein was determined by Results the method of Lowry et al.1' using bovine serum albumin as standard. The optimal assay conditions were determined in The fatty acid:CoA ligase assay was based on the the whole homogenate using palmitic acid as hydroxamate-trapping method of Kornberg and substrate. Maximal palmitate activation was found Pricer (1953) as slightly modified by Rodgers et al.'6 to occur with concentrations of ATP above 5.0 mM, and followed under optimally determined conditions. CoA above 1l0 mM, and MgC12 above 5-0 mM, The principle of the hydroxamate method is based and hence concentrations of 10.0 mM, 1.5 mM, on the formation of red complex salts when hydroxa- and 100 mM respectively, were chosen for subse- mates react with ferric chloride. In the fatty acid: quent experiments. Addition of ATP, CoA, and CoA ligase assay hydroxamic acid derives from MgC12 was essential for the formation of palmitoyl- the reaction of the thiolester acyl-CoA with hydroxy- CoA with the subsequent development of hydroxa- lamine. Unless otherwise stated, the standard assay mate. Maximum reaction rates were reached with system contained, in a final volume of 2.0 ml, the 01 M Tris and between 1.0 and 1.5 M hydroxy- following concentrations of substances in the order lamine. given: a NH2OH-Tris buffer of pH 7T25 to provide 1.0 M and 0 1 M 10 mM http://gut.bmj.com/ for NH2OH Tris; MgCl2, C 1.5 mM CoA, 3.0 mM dithiothreitol, 10 0 mM 2 50 ATP, 6-0 mM palmitic acid, and 20 mg/ml bovine 0. serum albumin. Palmitic acid was dissolved in a E 40- small volume of 96% ethanol (final volume in the

test 0.04 ml) and vigorously mixed with the albumin c: 30. solution. Oleic acid was treated similarly but used on September 24, 2021 by guest. Protected copyright. at a concentration of 2-0 mM. After preincubation ~20-a10/~~~~~~ E at 37°C for five minutes, the reaction was started a 0 with the addition of mucosal homogenate or -v 10. microsomes in appropriate amounts, and the contents were incubated at 37°C for 15 minutes. A E control lacking ATP was always included as a blank 55 60 65 70 75 80 85 for each sample. pH The reaction was terminated by the addition of Fig. 1 pH-optimum oflong chain fatty acid:CoA 2-0 ml 96% ethanol and 0.4 ml 6.0 M HCL. Then ligase using palmitate as substrate under standard assay 15.0 ml light petroleum (b.p. 40-600C) were added conditions with total homogenate as enzyme source. and the contents of each stoppered tube vigorously Hydroxylamine-Tris served as buffer. agitated for one minute. After separation of the two layers, 12 ml of the light petroleum phase The pH optimum in this reaction system was were removed, taken to dryness by a rotation spread between 6-75 and 7-75 with a maximum at evaporator, and the residue redissolved in 10 ml 7.25 (Fig. 1) which was used for additional studies. chloroform-ethanol (1:1, by vol.). The iron-hydroxa- Under these optimal assay conditions, using whole mate complex was developed by the addition of homogenate as the source of enzyme, the rate of 2-0 ml 4% Hill's solution A17 freshly diluted from activation was proportional to the period of in- the stock solution with chloroform-ethanol (1:1, by cubation and the amount of protein. As shown in vol.). After 20 minutes, the absorbance was measured Fig. 2, there was a linear relationship between at 520 nm. A molar extinction coefficient of the enzyme activity and the duration of incubation over Gut: first published as 10.1136/gut.21.8.689 on 1 August 1980. Downloaded from

Studies on long chain fatty acid 691

Minutes 10 20 30 ~0 .60- a 0 E 0 0 °206~ E Polmitate Time/ ca 40- 04. Protein E a, I02:20.4 Xa 20- p a Oleate E 0 02 04 06 E 2 4 6 8 10 12 mg Protein Fatty acid concentration (mM) Fig. 2 Linearity of long chain fatty acid:CoA ligase assay with protein and time. Assays with total Fig. 4 Dependence of the long chain fatty acid:CoA homogenate were performed as described in the ligase activity on the fatty acid concentration. Methods section as a function ofprotein or time with Standard assay conditions were used with the total 0.51 mg ofprotein. The amount ofprotein shown on homogenate as enzyme source. the abscissa is given as mg protein per assay. BSA-concentration of 20 mg/ml reaction mixture a period of 30 minutes and the amount of protein was chosen for further experiments. With the whole up to 0-6 mg per assay. Using microsomes as up homogenate as enzyme source, optimal palmitate the enzyme source the reaction rate was linear concentrations were noted above 4.0 mM (Fig. 4) to 0.2 mg of protein per assay (not shown). Whole with no change in the reaction rate occurring up to homogenate boiled for five minutes revealed no 12-0 mM. In contrast, a marked difference was ob- enzyme activity. KF did not activate long chain served when oleic acid was used as substrate. fatty acids in human mucosa at concentrations up As demonstrated in Fig. 4, enzyme activity was to 10.0 mM. This was in accord with the behaviour maximal only at an oleic acid concentration of of fatty acid:CoA ligase in rat intestine.16 Further- 2.0 mM. At higher levels oleate was markedly more, fluoride added to the incubation medium results of and

inhibitory. Analogous palmitate http://gut.bmj.com/ at a concentration of 500 mM lowered palmitate oleate activation were obtained when microsomes activation by 90%. were used as the enzyme source. Optimum amounts for the saturated fatty acids C12:0, C14:O, and c C18, were identical with those for palmitate and 0.60 E Palmitate 120.

.cE L040. */ on September 24, 2021 by guest. Protected copyright. Oleate 100 E / 5>/95tn °_ 80 2 20 Microsomes 60 I E 10 20 30 40 E 40- Bovine serum albumin (mg/ml) 220- ~~~. ~~Total Fig. 3 Dependence of the long chain fatty acid:CoA homnogenatk ligase activity on bovine serum albumin concentration. G 120 140 160180 181 18 2 183 Total homogenate was used as enzyme and standard Number of carbon atoms and assays were performed as described in the Methods double bonds in fatty acid section. Fig. 5 Substrate specificity of long chain fatty the of various concentrations of acid:CoA ligase. Assays were performed as described Next, influence in the Methods section using total homogenate and albumin on hydroxamate formation was investi- microsomes as enzyme source. Saturated long chain in 3 albumin essentially gated. As shown Fig. fatty acids had a final concentration of6-0 mM, increased the enzyme activity, reaching a plateau unsaturated long chain fatty acids a final concentration at 12 mg/ml when both palmitic and oleic acid were of2-0 mM. Mean values ± SEM were given for nine used as substrate. No inhibition was observed at experiments (total homogenate) andfor seven concentrations of BSA up to 40 mg/ml and thus a experiments (microsomes). Gut: first published as 10.1136/gut.21.8.689 on 1 August 1980. Downloaded from

692 6Bierbach the unsaturated fatty acids C18:2 and C18:3 with microsomes, as can be seen by the distribution of those found for oleate (not shown). For subsequent glucose-6-phosphatase, a microsomal marker enzyme. assays, concentrations of 6-0 mM and 2.0 mM The rate of fatty acid:CoA ligase activity with were chosen for saturated and unsaturated fatty palmitate as substrate was determined in the total acids, respectively. homogenate of biopsies from the pars descendens With optimal assay conditions available, the fatty duodeni of 27 humans and in the total homogenate acid specificity of the enzyme was determined with of the mucosa of nine resected human upper various long chain saturated and unsaturated fatty jejunal segments. The mean enzyme activity from acids. As seen in Fig. 5, the pattern of reaction upper jejunum was significantly higher than from rates was similar for both enzyme sources. Setting the duodenum, 51.9±13 7 (1 SD) nmol/min per the specific activity of laurate activation as 100, mg protein versus 40.9±11.6 (P<0-05). A mean±1 the relative rates of activation of myristate, pal- SD of 101.8±440nmol/min per mg protein was mitate, stearate, oleate, linoleate, and linolenate found for microsomal fractions derived from the were respectively, 203: 697: 459: 228: 195: 147 for upper jejunal mucosa of seven patients. the microsomal fraction and 210: 625: 412: 383: 253: 165 for the total homogenate. Thus, maximum Discussion reaction rates were observed with palmitate for the saturated and with oleate for the unsaturated The present investigation of long chain fatty acid: fatty acids. With respect to the enzyme source, CoA ligase clearly demonstrated this enzyme in the oleate activation was markedly less in the micro- mucosa of human small intestine. Specific activity somal fraction than in the total homogenate. in the total homogenate of duodenal mucosa, 409 nmol/min per mg protein, at 37°C using Table Subcellular distribution of long chain fatty palmitate as substrate found by us was consider- acid:CoA ligase in mucosa ofhuman small intestine ably higher than that reported by Senior and Isselbacher7 for rat mucosa (1-4 nmol/min per mg Subcellular Fatty acid: Succinic Glucose-6- Protein protein), by Pande and Mead20 in rat mucosa fraction CoA ligase dehydrogenase phosphatase (mg) (0.7 nmol/min per mg protein), and by Brindley nmol/min per mg protein and Hiibscher1 in cat mucosa (22 nmol/min per mg Total 50.0 16-15 52.2 225 protein). Comparable specific activities in the total http://gut.bmj.com/ homogenate homogenate of rat jejunal mucosa were found by Nuclear and brush border Rodgers et al. (1972) who measured an activity fraction 58 3 (24.6) 26.65 (23.3) 81-4 (22 7) 32 of about 37 nmol/min per mg protein, also using Mitochondria 89-3 (38 0) 78-05 (690) 402 (11-4) 32-4 Microsomes 142-4 (32.8) 3.05 (1-4) 176 0 (26.9) 17-5 the hydroxamate-trapping method. With micro- 140 000 g somes from the upper jejunum as enzyme source, supernatant 2.5 (4.6) 1-65 (6.3) 31-8 (39 0) 140-8 were detected % recovery similar specific activities by Rodgers from total and Bochenek9 in the rat (119 nmol/min per mg on September 24, 2021 by guest. Protected copyright. homogenate 67-6 101 97-6 99.0 protein) and by us in man (102 nmol/min per mg a Subcellular fractions of intestinal mucosa were prepared and enzymes protein). The data of the present report showed assayed as described in the Methods section. The percentage of the significantly higher activity of palmitate-activating total activity recovered is shown in parentheses. Specific activities are enzyme in the total homogenate from upper jejunum expressed as nmol product formed/min per mg protein. versus duodenum. In rats, a similar finding was reported by Rodgers and Bochenekg for the micro- The Table presents the data for a typical experi- somal fatty acid-CoA ligase, 119±26 nmol/min ment ofthe subcellular fractionation and distribution per mg protein from the upper jejunum versus of long chain fatty acid:CoA ligase from human 106 ±20 from the duodenum, though this difference small intestinal mucosa. From these data it is was not significant. The possible reason for this evident that the highest specific activity of this physiological difference in man may be due to the enzyme was found in the microsomal fraction, fact that the proximal small bowel is the maximal followed by the mitochondrial and the nuclear and absorptive site for products of triacylglycerol brush border fractions. With regard to the distri- hydrolysis-that is, 2-monoglycerides and long chain bution of total activity it can be seen that the fatty fatty acids. Thus the greater enzyme activity in the acid :CoA ligase was equally associated with the upper jejunum may be the consequence of enzyme mitochondrial, microsomal, and the nuclear and induction by the natural substrates long chain brush border fractions. This might be partially fatty acids,9 which might be present in higher con- due to the contamination of the mitochondrial centrations in the upper jejunum than in the pars and the nuclear and brush border fractions with descendens duodeni. Gut: first published as 10.1136/gut.21.8.689 on 1 August 1980. Downloaded from

Studies on long chain fatty acid 693 The kinetic data of the palmitate-activating specificity of long chain fatty acid :CoA ligase enzyme from the human small intestine showed from the small intestine varies significantly from one many similarities with those reported by Pande and species to another. Maximal reaction rates were Mead8 for the rat liver and by Rodgers et al.16 observed in pig intestinal mucosa,1" in guinea-pig for the rat small intestine. Optimal concentrations and cat intestinal microsomes' with laurate, myri- of substrates and cofactors were found for the most state, and palmitate, respectively. No data of relative part to be within the same range. Fluoride caused activities concerning the enzyme of rat small no stimulatory effect, as observed for the enzyme intestine were available, although high reaction from rat liver8 and, as an inhibition was achieved rates were demonstrated with linoleate, oleate, with a concentration greater than 10.0 mM, it was and palmitate in the order mentioned.16 Where omitted from the assay. Bovine serum albumin, the fatty acid specificity profile of the human which markedly stimulated palmitate and oleate intestinal enzyme is concerned, the greatest accord- activation, was shown to be effective over a broad ance exists with the intestinal preparations from range up to 40 mg/ml in the total homogenate of the cat. In both human and cat intestinal mucosa human mucosa as opposed to microsomes from rat the highest activities were observed with palmitate liver8 and to the 14-fold purified enzyme from rat for the saturated and oleate for the unsaturated liver (Bar-Tana et al., 1971). With the two latter fatty acids. From the results of this investigation enzyme preparations albumin was effective within a it cannot be decided whether there are two different narrow concentration range only and became pro- enzymes, as has been suggested for rat liver, cata- foundly inhibitory over this limit. Albumin at low lysing the activation of long chain saturated and concentrations stimulated enzyme activity, pre- unsaturated fatty acids, respectively.8 sumably because of lowering the free concentration As concluded by Brindley,22 the predominant of the fatty acids, whereas albumin at high concen- site of triacylglycerol biosynthesis in the intestine trations was inhibitory, presumably because of is the endoplasmic reticulum. This conclusion was competition with the enzyme for the fatty acid based on several studies which demonstrated substrate.2' However, the mechanism by which the presence of fatty acid :CoA ligase and the albumin stimulated the fatty acid-CoA ligase is enzymes of the monoglyceride- and glycerol- unknown. The addition of CoA, ATP and Mg2+ phosphate pathway mainly in the microsomal was essential for acyl-CoA formation, as no value fraction. In agreement with these findings was the http://gut.bmj.com/ above the blank was observed without the addition subcellular distribution of the human gut fatty of these agents. acid-CoA ligase, whose highest specific activity The optimal requirements of long chain saturated was recovered in the microsomal fraction. The and unsaturated fatty acids for the human intestinal detection of the enzyme in the mitochondrial and enzyme were comparable with the findings of the nuclear and brush border fractions could be Brindley and Hubscher' for the intestinal enzyme of mainly accounted for by contamination with the guinea-pig and of Rodgers et al.16 for that of microsomes. Recently, the palmitic acid:CoA ligase on September 24, 2021 by guest. Protected copyright. the rat. In the presence of bovine serum albumin of rat small intestine was shown to be far more long chain saturated fatty acids C120, C1:40, C16:0 active in villi than in crypts.23 This special local- and C18:0 showed a wide range of optimal con- isation in accordance with a similar distribution centrations in supporting maximal enzyme activity. of the monoacylglycerol acyltransferase and the In contrast, the unsaturated long chain fatty glycerolphosphate acyltransferase emphasises the acids Cl8il, C18:2 and C18:3 exhibited a marked importance of this enzyme in triacylglycerol bio- substrate inhibition with a narrow range of optimal synthesis in the small intestine. The detection of concentration at 2.0 mM. The reason for this long chain fatty acid :CoA ligase in the human profound inhibition above 2.0 mM is so far gut and the description of optimal kinetic require- unknown. ments could be used as an interesting tool to study The substrate specificity of the enzyme from the triacylglycerol synthesis in the human intestinal total homogenate and the microsomal fraction was mucosa under various pathological conditions. similar except for oleate. Using the total homo- genate as enzyme source, the relative activity was markedly higher than in the microsomal fraction, The author gratefully acknowledges the expert which might be due to the fact that the substrate technical assistance of Mrs Th Wieser and the inhibition by oleate-and to a lesser degree by helpful cooperation of the medical staff, especially linoleate and linolenate-was overcome by the Dr H Brunner, from the Surgical Department, higher protein content of the total homogenate University of Mainz, in supplying resected material than that of the microsomal fraction. The substrate of the small intestine. Gut: first published as 10.1136/gut.21.8.689 on 1 August 1980. Downloaded from

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References and fatty acid esterification by intestinal mucosa from patients with Whipple's disease and non-tropical sprue. 'Brindley DN, Hubscher G. The effect of chain length Gastroenterology 1965; 48: 584-92. on the activation and subsequent incorporation of '4Sueur R, Cerf M, Di Costanzo G, Debray C. Quantita- fatty acids into glycerides by the small intestinal tive studies in vitro on uptake and esterification of mucosa. Biochim Biophys Acta 1966; 125: 92-105. palmitate into human and rat jejunal mucosa. Digestion 2Mattson FH, Volpenhein RA. The digestion and 1977; 15: 34-42. absorption of triglycerides. J Biol Chem 1964; 239: 15Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 2772-7. Protein measurement with the Folin phenol reagent. 3Kern F, Jr, Borgstrom B. Quantitave study of the J Biol Chem 1951;193: 265-75. pathways of triglyceride synthesis by hamster intestinal 16Rodgers JB, Jr Tandon R, Fromm H. Acyl-CoA syn- mucosa. Biochim Biophys Acta 1965; 98: 520-31. thetase for long chain fatty acids in rat small bowel and 'Kayden HJ, Senior JR, Mattson FH. The mono- the influence of diets containing different compositions glyceride pathway of fat absorption in man. J Clin of fatty acids on intestinal lipid reesterifying enzyme Invest 1967; 46: 1695-1703. activities. Biochim Biophys Acta 1972; 270: 453-62. 'Kornberg A, Pricer WE Jr. Enzymatic synthesis of the 17Hill UT. Colorimetric determination of fatty acids coenzyme A derivatives of long chain fatty acids. and esters Anal Chem 1947; 19: 932-33. J Biol Chem 1953; 204: 329-43. 18Hiubscher G, West GR, Brindley DN. Studies on 6Clark B, Hiubscher G. Biosynthesis of glycerides in the the fractionation of mucosal homogenates from the mucosa of the small intestine. Nature 1960; 185: 35-7. small intestine Biochem J 1965; 97: 629-42. 7Senior JR, Isselbacher KJ. Activation of long chain 19Green DE, Mii S, Kohout PM. Studies on the terminal fatty acids by rat-gut mucosa. Biochim Biophys Acta electron transport system. I. Succinic dehydrogenase. 1960; 44: 399-400. J Biol Chem 1955; 217: 551-67. 8Pande SV, Mead JF. Long chain fatty acid activation l9aBaginski ES, Foa PP, Zak B. Glucose-6-phosphatase. in subcellular preparations from rat liver. J Biol Chem In: Bergmeyer HU, ed. Methoden der enzymatischen 1968; 243: 352-61. Verlag Chemie: 'Rodgers JB, Bochenek W. Localisation of lipid re- Analyse 2. Aufl., Band I, Weinheim esterifying enzymes of the rat small intestine. Effects of 1970; 830-43. jejunal removal on ileal enzyme activities. Biochim 20Pande SV, Mead JF. Distribution of long chain fatty Biophys Acta 1970; 202: 426-35. acid-activating enzymes in rat tissues. Biochim Biophys '0Dejong JW, Hiulsmann WC. A comparative study of Acta 1968; 152: 636-8.

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palmitate-1-C14 by homogenates of intestinal mucosa. distribution of enzymes involved in glycerolipid on September 24, 2021 by guest. Protected copyright. J Clin Invest 1960; 39: 150-60. synthesis in rat small intestinal epithelium. Biochim "Brice RS, Owen EE, Tyor MP. Amino acid uptake Biophys Acta 1976; 450: 288-300.