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Differential diagnosis of maldigestion and malabsorption of fat. II. Comparison of 131I-triolein with 14C-triolein in normal, pancreatic juice-deficient, short bowel and bile-deficient dogs.

To determine whether or not purified 131I-triolein produces the same behavior as 14C-triolein in the intraluminal phase, and to assess whether or not 0.10 +/- 0.02 of the Digestion-Absorption Index (DAI) obtained from clinical cases is an adequate value for differentiating maldigestion from malabsorption of fat, dogs, pancreatic juice-deficient, short bowel and bile-deficient were prepared. Both the pancreatic juice-deficient and bile-deficient groups indicated a high fecal excretion of 14C- and 131I-radioactivity, and the short bowel group indicated a moderate fecal excretion of fat. Daily fecal fat levels in each group were in parallel to the 3-day fecal excretion of radioactivity. However, the Digestive-Adsorption Index of the pancreatic juice-deficient group of 0.602 in 14C and 0.620 in 131I, and indicated that a considerable portion of the ingested triolein was excreted into the feces without hydrolysis. DAI in the short bowel group were 0.020 in 14C and 0.022 in 131I, and in the bile-deficient group, the indices were 0.031 in 14C and 0.021 in 131I. Both latter groups showed a malabsorptive defect. Additionally, the Digestion-Absorption Index of the pancreatic juice-deficient group was higher than 0.10 +/- 0.02 of the clinical borderline mentioned in the foregoing paper, and that of the other two groups was lower. Therefore, this borderline may represent adequate values for the differentiation of fat malassimilation. On the other hand, it was confirmed that 14C-triolein and 131I-triolein showed similar behavior in the intraluminal phase, and that the 131I-triolein test was adequate for clinical testing for fat digestion and absorption.

Animals↗

Mechanism of action of lipoprotein lipase on triolein particles: effect of apolipoprotein C-II.

Triolein particles stabilized by a phosphatidylcholine monolayer were used to study the lipoprotein lipase (LpL) reaction. They were prepared in two different sizes and with triolein and phosphatidylcholine in the molar ratios of 0.9-1.2 : 1 (small particles) and 8-17 : 1 (large particles). The rate of hydrolysis by LpL of phosphatidylcholine on the surface of both lipid particles was only 1/20 as much as that of triolein, even if it was activated to the maximum by apolipoprotein C-II (apoC-II). Thus, the phospholipase activity of LpL was low enough to measure the initial rate of hydrolysis of triolein without causing a gross change of the surface of the lipid particle. When the hydrolysis of triolein by LpL was monitored, fatty acid was released at a constant rate until all of the triolein molecules were hydrolyzed. The enzyme required 220 +/- 17 and 66 +/- 9 nM apoC-II for its half-maximal activity (Km (apoC-II] with small and large particles as a substrate (1.15 mM triolein for small and 2.13 mM triolein for large particles), respectively, using various concentrations of LpL. The Km(apoC-II) values for these two substrates became similar when LpL activity was analyzed with respect to the density of apoC-II on the phosphatidylcholine monolayer at the surface of the particles (bound apoC-II/phosphatidylcholine). The concentration of substrate particles did not affect the Km(apoC-II) values. The presence of an adequate amount of apoC-II increased the maximal activity of LpL (Vmax(triolein)) from 0.48 +/- 0.21 to 6.81 +/- 0.45 and from 0.32 +/- 0.04 to 7.13 +/- 0.64 mmol/h/mg with a slight decrease in the apparent Michaelis constant (Km(triolein)) for small (from 90 to 54 microM triolein) and large (from 1.00 to 0.65 mM triolein) particles, respectively. Although the apparent Km for triolein in large particles was about ten times greater than that in small particles, the values became similar when they were corrected for the concentration of phosphatidylcholine (50-100 microM phosphatidylcholine), which corresponded to the surface area of the substrate particles. It was suggested that bound apoC-II molecules were transferred relatively slowly to other lipid particles while LpL molecules moved rapidly among the lipid particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Triolein-induced pulmonary embolization and increased microvascular permeability in isolated perfused rat lungs.

BACKGROUND: The pathophysiologic mechanism of the fat embolism syndrome is poorly understood. This study was designed to determine the effects of fat emboli on pulmonary vasculature. METHOD: Triolein was infused into isolated rat lungs perfused with Krebs-Henseleit buffer. Pulmonary arterial pressure and microvascular permeability (Kf) were measured at baseline and 20 minutes after the triolein infusion. RESULT: The 99% triolein produced dose-dependent increases in both pulmonary arterial pressure and Kf. The 65% triolein, containing free fatty acid, resulted in a greater increase in Kf. Pretreatment with indomethacin attenuated the increase in Kf after 65% triolein but not after 99% triolein. CONCLUSION: Pure triolein induced mainly embolization in the pulmonary vasculature, and 65% triolein caused embolization and subsequently increased vascular permeability, which are, at least in part, mediated by the action of cyclooxygenase products. Free fatty acids might induce permeability edema by means of a cyclooxygenase-dependent mechanism. We conclude that triolein-induced increases in pulmonary arterial pressure and Kf in isolated rat lungs provides a useful model of acute lung injury by fat embolism.

1-Methyl-3-isobutylxanthine↗

Solubilization and localization of triolein in phosphatidylcholine bilayers: a 13C NMR study.

Cosonicated mixtures of egg phosphatidylcholine and small amounts (less than 5% wt/wt) of triolein have been studied by 13C NMR spectroscopy. The 50.3-MHz 13C NMR spectrum of vesicles preparations containing 90% isotopically substituted [1-13C]triolein showed two carbonyl resonances at chemical shift values that indicate hydrogen bonding of H2O molecules with the carbonyl groups. The extent of hydration, estimated from the chemical shift values that indicate hydrogen bonding of H2O molecules with the chemical shift values (173.07 ppm and 172.39 is approximately 50%. The data suggest that the triolein is located in the bilayer with the three carbonyl groups at the aqueous interface. The acyl chains are extended toward the bilayer interior, with a conformation of the glyceryl region such that the primary (alpha) carbonyls are closer to the aqueous medium than is the secondary (beta) carbonyl. Thus, triolein is present in the bilayer in an orientation appropriate for enzymatic hydrolysis, with the second substrate (H2O) in close proximity to the hydrolytic site, and with a conformation that could explain, in part, enzymatic specificity for hydrolysis at the alpha position. Spectra of vesicles containing greater than or equal to 3% triolein showed two additional carbonyl peaks characteristic of pure (neat) triolein. This allowed a determination of the maximum solubility (approximately 2.8%) of surface-oriented triolein in the bilayer phase. Beyond this limit all excess triolein partitions into a separate oil phase.

Hydrogen Bonding↗

Solubilization of triolein and cholesteryl oleate in egg phosphatidylcholine vesicles.

The incorporation of cholesteryl oleate and triolein into phospholipid vesicles was studied in cosonicated mixtures of 94 weight % egg phosphatidylcholine and 6 weight % neutral lipid (0-6% triolein and 6-0% cholesteryl oleate). 13C NMR spectroscopy was used to quantitate both neutral lipids in vesicles containing 90% isotopically substituted [carbonyl-13C]cholesteryl oleate and [carbonyl-13C]triolein. Vesicles were also prepared with radiolabeled cholesteryl oleate and triolein and the composition of ultracentrifugal subfractions determined by chemical and radioisotopic methods. For a given starting composition, the incorporation of neutral lipids into vesicles was similar for vesicles prepared and analyzed by the two methodologies. The maximum solubility in vesicles prepared at 55 degrees C with a single neutral lipid was 3.1 weight % triolein (2.8 mol %) and 2.3 weight % cholesteryl oleate (2.8 mol %). In sonication mixtures with both triolein and cholesteryl oleate, the incorporation of each lipid into vesicles was proportional to the starting concentration; the total incorporation of neutral lipid was less than or equal to 4.0% (weight or mole per cent). The solubility limits were intermediate between the theoretical cases of complete additivity and complete competition. The [13C]carbonyl chemical shifts showed that the carbonyl groups of the vesicle-solubilized neutral lipids were close to the vesicle surface and that excess triolein and cholesteryl oleate partitioned into an oil phase containing both triolein and cholesteryl oleate.

Cholesterol Esters↗

Uptake, incorporation and metabolism of (3H)triolein in the isolated perfused rabbit heart.

The purpose of these experiments was to study the uptake and metabolism of exogenous triglyceride in the isolated perfused rabbit heart. When infused into the rabbit heart, [9,10-3H(N)]triolein was retained and incorporated into a lipid fraction that had the chromatographic mobility of authentic triolein. Incorporation of labeled triolein was not likely to be the result of a lipoprotein lipase-mediated lipolysis/resynthesis cycle, since: (i) The distribution of radioactivity following administration of [3H]oleic acid was markedly different from the distribution of radioactivity following the administration of [3H]triolein; (ii) heparin was administered to the rabbits at the time of sacrifice; and (iii) the hearts were perfused with a protein-free buffer for 20 min prior to the labelling period. When isoproterenol was administered to hearts labelled with [3H]triolein, there was an increased output of total radioactivity, composed of labelled free fatty acids, diacylglycerol and monoacylglycerol. In these same hearts, there was an increased output of glycerol in response to isoproterenol. However, following the administration of bradykinin or angiotensin II, neither the radioactivity nor the glycerol content of the perfusate was changed. These data suggest that [3H]triolein is selectively incorporated into the triglyceride pool of the isolated perfused rabbit heart. Furthermore, this [3H]triolein is available to hormonally-activated lipolytic enzymes.

Angiotensin II↗

Estimation of 14C-triolein assimilation from postprandial serum radioactivity of 14C.

In an attempt to establish a test of lipid assimilation, based on the measurement of the postprandial serum radioactivity of 14C from ingested 14C-triolein, the activity of 14C was measured in serum samples, drawn 1,2,3,4,6 and 9 h after ingestion in 48 consecutive patients suspected of malassimilation. Simultaneously, faecal excretion of 14C was measured to estimate 14C-triolein assimilation (F-14C-Ass). F-14C-Ass served as reference of 14C-triolein assimilation. The sum of the 2- and 4-h serum concentration of 14C (S-(2 + 4)14C) was found to be most useful as an estimate of 14C-triolein assimilation, with regard to both the diagnostic value and applicability. At a level of 1.0% of dose/l serum, S-(2 + 4)14C correctly discriminated between normal and reduced 14C-triolein assimilation in 83% of the patients (95% confidence limits 70-93%). A significant correlation between 14C-triolein assimilation and S-(2 + 4)14C was found (r = 0.91, P less than 0.001). Since 14C-triolein assimilation correlates closely with the assimilation of dietary lipids, S-(2 + 4)14C seems to provide in a simple way sufficient information about lipid assimilation to be useful as a clinical test.

Carbon Radioisotopes↗

The [14C]-triolein breath test is not valid as a test of fat absorption.

The [14C]-triolein breath test is used as a test of fat absorption. However, its validity has not been established. The aim of this study was to investigate, whether the absorption of [14C]-triolein could be estimated from the breath test, and whether the breath test could be useful as a clinical test. The [14C]-triolein absorption was estimated from faecal measurements, using 51CrCl3 as non-absorbable marker. The breath test was done according to the standard technique with hourly estimations of the 14CO2 expiration. Fifty-one patients participated. A nearly perpendicular, curvilinear relation between the 6-h cumulative 14CO2 expiration and the [14C]-triolein absorption was found, and no obvious cut-off level for normal 14CO2 expiration could be identified. Accordingly, the diagnostic sensitivity of the breath test was 80% at the expense of a specificity of 45%. In 19 patients duplicate measurements were done. A high intra- and inter-individual variation in the fraction of absorbed [14C]-triolein, expired within 6 h, was found. It is concluded that expiration of 14CO2 is influenced by factors other than the absorption of [14C]-triolein, and that the [14C]-triolein breath test is not useful as test of fat absorption.

Breath Tests↗

Non-volatile products of triolein produced at frying temperatures characterized using liquid chromatography with online mass spectrometric detection.

Oxidation products from triolein under model heated frying conditions have been analyzed using liquid chromatography with an evaporative light scattering detector and atmospheric pressure chemical ionization (APCI) mass spectrometric detection. Triolein was heated at 190 degrees C with 2% water added each hour, to simulate the moisture of a frozen product, until polar components reached approximately 30%. The samples were separated using reversed-phase high-performance liquid chromatography with APCI-MS detection. Triolein oxidation products included hydroperoxides, epoxides and a ketone. Other products were formed by shortening of an acyl chain on the intact triolein. Normal and oxygen-containing products formed by the dimerization of triolein were also observed. Other products included chain addition products formed by addition of acyl chain subunits to intact triolein to form higher molecular weight products.

Chromatography, High Pressure Liquid↗

Rapid inhibition by intragastric triolein of the re-activation of glucose utilization and lipogenesis in the mammary gland during the starved-refed transition in lactating rats. Evidence for a direct effect of oral lipid on mammary tissue.

1. Oral administration of triacylglycerol (triolein) to starved/chow-refed lactating rats suppressed the lipogenic switch-on in the mammary gland in vivo. 2. A time-course study revealed that triolein, administered at 30 min after the onset of refeeding, had no influence on lipogenic rate in the mammary gland between 30 and 60 min, but markedly decreased it between 60 and 90 min. Glucose uptake by the mammary gland (arteriovenous difference) increased by 30 min of refeeding, as did lactate production. Between 30 and 90 min glucose uptake remained high in the control animals, but glucose uptake and net C3-unit uptake were decreased in the triolein-loaded animals by 90 min. 3. Triolein increased [glucose 6-phosphate] in the gland and simultaneously decreased [fructose 1,6-bisphosphate], indicative of a decrease in phosphofructokinase activity. This cross-over occurred at 60 min, i.e. immediately before the inhibition of lipogenesis, and by 90 min had reached 'starved' values. 4. Triolein had no effect on plasma [insulin] nor on whole-blood [glucose], [lactate] or [3-hydroxybutyrate]; a small increase in [acetoacetate] was observed. 5. Infusion of the lipoprotein lipase inhibitor, Triton WR1339, abolished the suppression of mammary-gland lipogenesis by triolein and the increase in the [glucose 6-phosphate]/[fructose 1,6-bisphosphate] ratio, suggesting a direct influence of dietary lipid on mammary-gland glucose utilization and phosphofructokinase activity.

Animals↗

Regulation of cholesteryl oleate and triolein miscibility in monolayers and bilayers.

The miscibility of triolein and cholesteryl oleate with 1-palmitoyl-2-oleoyl phosphatidylcholine was studied at the argon-buffer interface. The surface phase behavior of the system was analogous to that for cholesteryl ester-phospholipid mixtures in that both monolayer and double layer surface phases were formed. By considering the bulk properties of cholesteryl oleatetriolein mixtures and the two-dimensional phase rule, the entire system could be described. Double layer properties suggest that it consists of mostly triolein and phospholipid in the layer adjacent to the aqueous phase. The monolayer phase shows the formation of complexes between the neutral lipids and the phospholipid with stoichiometries nearly identical with those reported for bilayers (Hamilton, J. A., Miller, K. W., and Small, D. M. (1983) J. Biol. Chem. 258, 12821-12826). A second complex with a 3:1 stoichiometry is formed between triolein and cholesteryl oleate independently of interactions with phospholipid. Upon interaction with phospholipid, the triolein-cholesteryl oleate complex loses proportionately more area than either lipid alone. Because the area of complexes with phospholipid is constant, overall neutral lipid miscibility in such complexes is enhanced by the cholesteryl oleate-triolein interaction. Thus, our data explain the apparently nonideal mixing of cholesteryl oleate, triolein, and phospholipid in monolayers and in bilayers.

Cholesterol Esters↗

Human plasma carboxyl esterase-catalyzed triolein hydrolysis. Existence of promoting factor in serum.

The possibility that some factor in serum changes the substrate specificity of purified human plasma carboxyl esterase, which hydrolyzes the short chain fatty acid ester, tributyrin, was investigated. The purified carboxyl esterase from human plasma hydrolyzed 48 mmol of tributyrin/mg of protein/h, monoolein at 1560 mumol of released fatty acids/mg of protein/h, diolein at 133 mumol of released fatty acids/mg of protein/h, and triolein at less than 10 mumol of released fatty acids/mg of protein/h. When human serum was applied to phenyl-Sepharose, a triolein hydrolysis-promoting factor (THPF) for purified carboxyl esterase was bound to the gel and was eluted with water. This partially purified human serum THPF enhanced carboxyl esterase-catalyzed triolein hydrolysis about 30-fold, diolein hydrolysis 2-fold, and monoolein hydrolysis 1.5-fold. Hydrolysis of triolein in very low density lipoproteins (d less than 1.006) and intermediate lipoproteins (1.006 less than d less than 1.019) by carboxyl esterase was also enhanced by addition of THPF. THPF activity was reduced by treatment of delipidation, but resistant to trypsin treatment or heating at 50 degrees C. These results indicated that serum carboxyl esterase can hydrolyze the long chain fatty acid ester, triolein, in the presence of triolein hydrolysis-promoting factor in serum.

Carboxylic Ester Hydrolases↗

Hydrolysis of triolein in phospholipid vesicles and microemulsions by a purified rat liver acid lipase.

An acid lipase was purified from rat liver lysosomes. Lipase purification involved affinity chromatography, gel filtration, and stabilization of the purified preparation using ethylene glycol and Triton X-100. A molecular weight of 67,000-69,000 was determined independently using density gradient centrifugation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gel filtration. To study enzyme action, model substrates were prepared by incorporating radiolabeled triolein into either unilamellar vesicles or microemulsions. Substrates were prepared by cosonicating aqueous dispersions of lecithin and triolein. Formation of vesicles or emulsions depended on the relative amount of each lipid and on sonication conditions. Vesicles were prepared at molar ratios between 70:1 and 26:1 (lecithin:triolein) and the microemulsion preparation at a molar ratio of 1:1. The substrate particles were of similar size (220-250 A) as determined by Bio-Gel A-15m chromatography. Hydrolysis of triolein contained in vesicles or emulsions was similar with respect to pH, temperature, and reaction products. Kinetic studies on vesicles with increasing triolein content showed progressively greater Vmax values (0-0.6 mumol/min/mg), and Vmax for the emulsion was 3.1 mumol/min/mg. Addition of human very low or low density lipoprotein produced a dose-dependent inhibition with both substrates. The results show that synthetically prepared microemulsions are stable and effective substrates for the acid lipase and indicate that surface-oriented triolein is hydrolyzed in both preparations.

Animals↗

Estimation of assimilation of simultaneously ingested 14C-triolein and 3H-oleic acid as a test of pancreatic digestive function.

The ratio between assimilation of simultaneously ingested 14C-triolein and 3H-oleic acid was investigated as a test of pancreatic digestive function in 48 consecutive patients suspected of malassimilation. Faecal excretion of 14C-triolein and 3H-oleic acid was measured by means of a combustion/liquid scintillation counting technique. Compared with the patients in whom the clinical investigation showed normal lipid assimilation (no. = 31) the assimilation of both 14C-triolein and 3H-oleic acid was significantly reduced in the patients with malabsorption (no. = 6) and maldigestion (no. = 11). However, in all except one patient with maldigestion the assimilation of 14C-triolein was more reduced than that of 3H-oleic acid, whereas there was no difference in 14C-triolein and 3H-oleic acid assimilation in the patients with malabsorption and normal lipid assimilation. Measurement of the postprandial serum radioactivities of 14C and 3H, like the faecal measurements, indicated more severely reduced assimilation of 14C-triolein than of 3H-oleic acid in the patients with maldigestion than in the other patient groups. In fact, the 2-h postprandial 3H/14C ratio at a level of 1.3 correctly classified digestive function in 47 of the 48 patients.

Digestion↗

Enrichment of selected serum fatty acids after a small oral dosage of (1-13C)- and (8-13C)triolein in human volunteers analysed by gas chromatography/combustion isotope ratio mass spectrometry.

In this study the fate of 100 mg orally administered (1-13C)- and (8-13C)triolein was traced in the serum lipids of four healthy human subjects. After an overnight fast the subjects consumed hourly meals of a liquid formula diet over 12 h. Ninety minutes after the first meal in the first study (1-13C)triolein was given and in the repeat study the same subject received (8-13C)triolein. Triacylglycerol (TG), phospholipid (PL) and cholesterol ester (CE) were isolated from serum sampled prior to and in intervals after isotope administration. Fatty acid composition of serum lipids was measured using gas chromatography/mass spectrometry. 13C enrichment in palmitic, stearic, oleic and linoleic acids of these fractions was determined by gas chromatography/isotope ratio mass spectrometry. With (8-13C)triolein a significantly higher enrichment (peak +17.1 +/- 14.3/1000 delta 13C) was found in the oleic acid of TG fraction than with (1-13C)triolein (peak -7.1 +/- 4.2/1000), which may be due to a faster elimination of (1-13C)oleic acid from serum TG. 13C enrichments in the other fatty acids of the TG fraction as well as of PL and CE fractions were in the range of natural 13C abundance (-25 to -32/1000).

Adult↗

Effects of triolein or oleic acid on lymphatic recovery of docosahexaenoic acid given as ethyl ester and their intramolecular distribution in lymph triglyceride of rats.

Effects of oleic acid or triolein on lymphatic recovery of docosahexaenoic acid (DHA) given as an ethyl ester were examined in rats with cannulated thoracic ducts. Lymphatic recovery of ethyl DHA given with oleic acid or triolein was significantly higher than in rats given ethyl DHA alone. DHA distributed almost exclusively at the 1- and 3-position of triglyceride in lymph collected at 0-3 h after the administration, when it was given with oleic acid or triolein. A small part of DHA distributed at the 2-position when ethyl DHA was the sole fatty acid given. Oleic acid given as free acid or triolein with ethyl DHA was a major fatty acid at the 2-position. Intramolecular distribution of DHA and oleic acid in lymph triglyceride was similar when ethyl DHA was given with oleic acid or triolein.

Absorption↗

Effect of free cholesterol on incorporation of triolein in phospholipid bilayers.

Triacylglycerols are the major substrates for lipolytic enzymes that act at the surface of emulsion-like particles such as triglyceride-rich lipoproteins, chylomicrons, and intracellular lipid droplets. This study examines the effect of cholesterol on the solubility of a triacylglycerol, triolein, in phospholipid surfaces. Solubilities of [carbonyl-13C]triolein in phospholipid bilayer vesicles containing between 0 and 50 mol % free cholesterol, prepared by cosonication, were measured by 13C NMR. The carbonyl resonances from bilayer-incorporated triglyceride were shifted downfield in the 13C NMR spectra from those corresponding to excess, nonincorporated material. This enabled solubilities to be determined directly from carbonyl peak intensities at most cholesterol concentrations. The bilayer solubility of triolein was inversely proportional to the cholesterol/phospholipid mole ratio. In pure phospholipid vesicles the triolein solubility was 2.2 mol %. The triglyceride incorporation decreased to 1.1 mol % at a cholesterol/phospholipid mole ratio of 0.5, and at a mole ratio of 1.0 for the bilayer lipids, the triolein solubility was reduced to just 0.15 mol %. The effects of free cholesterol were more pronounced and progressive than observed previously on the bilayer solubility of cholesteryl oleate (Spooner, P. J. R., Hamilton, J. A., Gantz, D. L., & Small, D. M. (1986) Biochim. Biophys. Acta 860, 345-353]. As with cholesteryl oleate, we suggest that cholesterol also displaces solubilized triglyceride to deeper regions of the bilayer.

Carbon Isotopes↗