Application of atmospheric pressure chemical ionization liquid chromatography–mass spectrometry in identification of lymph triacylglycerols

Atmospheric pressure chemical ionization liquid chromatography–mass spectrometry was used in the identification of triacylglycerol molecular species in lymph samples from rats given either a structured lipid or safflower oil. The structured lipid was MLM-type (M, medium-chain fatty acid; L, long-chain fatty acid) and manufactured from caprylic acid (8:0) and the oil (safflower oil or high-oleic sunflower oil). The triacylglycerol composition of lymph varied significantly between structured triacylglycerols and safflower oil. Diacylglycerol fragment ions were found for all triacylglycerols and we could also observe the ammonium adduct molecular ion [M+NH₄]⁺ for all the triacylglycerols at the selected conditions. Protonated molecular ions were formed from triacylglycerols containing unsaturated fatty acids, and fatty acid fragment ions were also observed in the case of strong fragmentation. The lymph triacylglycerols were identified from their ammonium adduct molecular ions and diacylglycerol fragment ions. In addition to the intact MLM-type structured triacylglycerols, the MLL- and LLL-type triacylglycerols were also identified. The absorption pathway of MLM-type structured triacylglycerols is most likely the same as that of conventional long-chain triacylglycerols, i.e. they were hydrolyzed into 2-monoacylglycerol and medium-chain fatty acids, which were then used for resynthesis of triacylglycerols. The present study thereby also demonstrates the possibility to study the absorption pathway of triacylglycerol via identification of triacylglycerol species in biological samples.     

Plasma fatty acids and [13C]linoleic acid metabolism in preterm infants fed a formula with medium-chain triglycerides

Most preterm infant formulas contain medium-chain triacylglycerols (MCT), but the effects of MCT on polyunsaturated fatty acid status and metabolism are controversial. Thus, we studied the effects of MCT on linoleic acid metabolism using stable isotopes. Enterally fed preterm infants were randomized to receive for 7 days 40% of fat as MCT (n = 10) or a formula without MCT (n = 9). At study day 5, infants received orally 2 mg/kg body weight of (13)C-labeled linoleic acid. Fatty acids in plasma lipid classes and (13)C enrichment of phospholipid fatty acids were measured and tracer oxidation was monitored. Compared with the control group, the MCT group showed lower breath (13)CO(2) and higher plasma triacylglycerol contents of octanoic acid, of decanoic acid, and of total long-chain polyunsaturated fatty acids (57.1 +/- 4.4 micro mol/l vs. 37.9 +/- 4.8 micro mol/l, P < 0.01). Concentrations of several polyunsaturated fatty acids in plasma phospholipids and non esterified fatty acids were higher in the MCT group. (13)C concentrations in phospholipid n-6 fatty acids indicated no difference in the relative conversion of linoleic to arachidonic acid. We conclude that oral MCT effectively reduce polyunsaturated fatty acid and long chain polyunsaturated fatty acid oxidation in preterm infants without compromising endogenous n-6 long chain polyunsaturated fatty acid synthesis.     

Effect of randomly interesterified triacylglycerols containing medium- and long-chain fatty acids on energy expenditure and hepatic fatty acid metabolism in rats

In our previous studies, medium- and long-chain triacylglycerols (MLCT), randomly interesterified triacylglycerols containing medium-chain and long-chain fatty acids in the same glycerol molecule, significantly reduced body fat accumulation in humans and rats. To clarify mechanism(s) for this effect of MLCT, we measured energy expenditure and hepatic fatty acid metabolism in rats by comparison with long-chain triacylglycerols (LCT) or medium-chain triacylglycerols (MCT). MLCT, compared with LCT, showed significantly lower body fat accumulation, higher 24-h energy expenditure and acyl-CoA dehydrogenase activity measured using octanoyl-CoA as a substrate, and similar lipogenic activity. MCT, compared with LCT, showed significantly higher energy expenditure, but fat accumulation was comparable. Additionally, MCT exhibited significantly higher lipogenic activity than the other oils. These data suggest that enhancement of energy expenditure and medium-chain fatty acids (MCFA) oxidation without activating de novo lipogenesis are responsible at least for the lower body fat accumulation in rats fed MLCT. The activation of hepatic lipogenesis by excessive intake of MCFA might counteract their preventive effects on body fat accumulation.     

Intestinal absorption of specific structured triacylglycerols

To clarify the intestinal absorption pathway of medium-chain fatty acids from MLM-type structured triacylglycerols containing both medium- and long-chain fatty acids, we studied the lymphatic transport of 1,3-dioctanoyl-2-linoleoyl-sn-glycerol (8:0/18:2/8:0), 1,3-didecanoyl-2-linoleoyl-sn-glycerol (10:0/18:2/10:0), and 1,3-didodecanoyl-2-linoleoyl-sn-glycerol (12:0/18:2/12:0) in a rat model. Safflower oil was used in the absorption study in order to compare the absorption of medium-chain fatty acids and long-chain fatty acids. The triacylglycerol species of lymph lipids were separated on a reversed-phase high performance liquid chromatograph (RP-HPLC) and identified by atmospheric pressure chemical ionization mass spectrometry. The composition of triacylglycerols was quantified by RP-HPLC with evaporative light scattering detection. The intact MLM-type triacylglycerols were detected in the lymph lipids after administration of the specific structured triacylglycerols (STAG). The recoveries of 8:0/18:2/8:0, 10:0/18:2/10:0, and 12:0/18:2/12:0 were 0.6%, 12%, and 5%, respectively. Several new triacylglycerol species were detected in the lymph lipids, including MLL-, LLL-, and MMM-type triacylglycerols.From the present study we conclude that the medium-chain fatty acids from STAG, in addition to absorption into the portal blood as free fatty acids, are absorbed by the same pathway as the conventional long-chain triacylglycerols, that is, they are hydrolyzed into free fatty acids, absorbed and activated into CoA, and reacylated into triacylglycerols in the enterocyte. The hydrolysis of MLM-type STAG is predominantly partial hydrolysis, whereas part of the STAG can also be hydrolyzed to free glycerol and free fatty acids.     

Metabolic effects of intravenous LCT or MCT/LCT lipid emulsions in preterm infants

Most lipid emulsions for parenteral feeding of premature infants are based on long-chain triacylglycerols (LCTs), but inclusion of medium-chain triacylglycerols (MCTs) might provide a more readily oxidizable energy source. The influence of these emulsions on fatty acid composition and metabolism was studied in 12 premature neonates, who were randomly assigned to an LCT emulsion (control) or an emulsion with a mixture of MCT and LCT (1:1). On study day 7, all infants received [13C]linoleic (LA) and [13C]alpha-linolenic acid (ALA) tracers orally. Plasma phospholipid (PL) and triacylglycerol (TG) fatty acid composition and 13C enrichments of plasma PL fatty acids were determined on day 8. After 8 days of lipid infusion, plasma TGs in the MCT/LCT group had higher contents of C8:0 (0.50 +/- 0.60% vs. 0.10 +/- 0.12%; means +/- SD) and C10:0 (0.66 +/- 0.51% vs. 0.15 +/- 0.17%) than controls. LA content of plasma PLs was slightly lower in the MCT/LCT group (16.47 +/- 1.16% vs. 18.57 +/- 2.09%), whereas long-chain polyunsaturated derivatives (LC-PUFAs) of LA and ALA tended to be higher. The tracer distributions between precursors and products (LC-PUFAs) were not significantly different between groups. Both lipid emulsions achieve similar plasma essential fatty acid (EFA) contents and similar proportional conversion of EFAs to LC-PUFAs. The MCT/LCT emulsion seems to protect EFAs and LC-PUFAs from beta-oxidation.     

Effect of dietary medium chain triacylglycerols on plasma triacylglycerol levels in horses

The hypothesis tested was that the feeding of medium chain triacylglycerols (MCT) to horses would raise the level of plasma triacylglycerols by increasing the availability of glucose as lipogenic substrate, implying that the MCT effect would be greater with glucose in the diet instead of cellulose. A Latin square experiment was carried out with 4 horses and 4 dietary treatments. The experimental periods lasted 21 d. Blood samples were taken 16 h after feeding. The diets consisted of hay and experimental concentrates, differing in fat source (MCT or soybean oil) and carbohydrate source (corn starch plus glucose or cellulose). The dietary variables, MCT or soybean oil, provided on average 27% of total dietary net energy, while glucose plus cornstarch or cellulose provided 33%. The feeding of MCT versus soybean oil raised the level of plasma triacylglycerols significantly from 196.7 ± 30.2 to 427.3 ± 85.7 mmol/1 and that of VLDL cholesterol from 0.028 ± 0.01 to 0.069 ± 0.01 mmol/ml. As based on analysis of variance, for the four experimental diets there was no significant effect of carbohydrate source and no fat‐carbohydrate interaction. Thus, the hypothesis was rejected. When the diets contained soybean oil, cellulose versus starch plus glucose produced significantly greater increase plasma triacylglycerols. This carbohydrate effect was not seen when horses were fed the MCT diets. The experimental concentrates did not differently influence the concentrations of plasma glucose, total serum cholesterol, phospholipids, insulin, free fatty acids and the activity of post‐heparin lipoprotein lipase. We suggest that the MCT‐induced increase in plasma triacylglycerols is related to an increase in hepatic VLDL secretion, with the extra substrate for increased synthesis of triacylglycerols being the acetyl‐CoA derived from the hepatic oxidation of medium chain fatty acids.     

Use of structured triacylglycerols containing predominantly stearic and oleic acids to probe early events in metabolic processing of dietary fat.

Early events in the metabolic processing of dietary triacylglycerol may have an important impact on subsequent development of risk factors for coronary heart disease. We have used structured triacylglycerols containing predominantly stearic or oleic acids at the sn -2 position to probe aspects of the processing of dietary fatty acids presented to adipose tissue in chylomicron-triacylglycerol. Studies were conducted on 14 healthy women who were given meals containing 85 g carbohydrate and 60 g of either of the two structured triacylglycerols in random order. Systemic concentrations and arterio-venous differences across adipose tissue for plasma triacylglycerol and non-esterified fatty acids were measured, together with analysis of the fatty acid composition of the relevant fractions. The stereo-specific structure of the ingested triacylglycerol was largely preserved in chylomicron-triacylglycerol. Systemic concentrations of total and individual non-esterified fatty acids were not significantly different after ingestion of the two fats, nor were their rates of release across adipose tissue. The composition of non-esterified fatty acids released from adipose tissue changed after the meal to reflect more closely the composition of the triacylglycerol ingested, but again no significant differences were observed between the two test meals. There was no detectable release of monoacylglycerol from adipose tissue after either test meal.We conclude that the environment for lipoprotein lipase action in adipose tissue in vivo is likely to be highly organized, such that there is no release of monoacylglycerol, nor preferential uptake or release of fatty acids from chylomicron-triacylglycerol according to the nature or the position within triacylglycerol of the fatty acid.     

Production of structured lipids by lipase-catalysed interesterification in an ultrafiltration membrane reactor

The application of membranes to the enzymatic production of structured lipids has been investigated using an ultrafiltration membrane reactor for a reaction between medium chain triacylglycerols (MCT) and n-3 polyunsaturated fatty acids from fish oil. Lipozyme IM was used as the biocatalyst. The incorporation of polyunsaturated fatty acids into MCT was increased by about 15% over 80 h by simultaneous separation of the released medium chain fatty acids compared to control.     

Training affects muscle phospholipid fatty acid composition in humans.

Training improves insulin sensitivity, which in turn may affect performance by modulation of fuel availability. Insulin action, in turn, has been linked to specific patterns of muscle structural lipids in skeletal muscle. This study investigated whether regular exercise training exerts an effect on the muscle membrane phospholipid fatty acid composition in humans. Seven male subjects performed endurance training of the knee extensors of one leg for 4 wk. The other leg served as a control. Before, after 4 days, and after 4 wk, muscle biopsies were obtained from the vastus lateralis. After 4 wk, the phospholipid fatty acid contents of oleic acid 18:1(n-9) and docosahexaenoic acid 22:6(n-3) were significantly higher in the trained (10.9 +/- 0.5% and 3.2 +/- 0.4% of total fatty acids, respectively) than the untrained leg (8.8 +/- 0.5% and 2.6 +/- 0.4%, P < 0.05). The ratio between n-6 and n-3 fatty acids was significantly lower in the trained (11.1 +/- 0.9) than the untrained leg (13.1 +/- 1.2, P < 0.05). In contrast, training did not affect muscle triacylglycerol fatty acid composition. Citrate synthase activity was increased by 17% in the trained compared with the untrained leg (P < 0.05). In this model, diet plays a minimal role, as the influence of dietary intake is similar on both legs. Regular exercise training per se influences the phospholipid fatty acid composition of muscle membranes but has no effect on the composition of fatty acids stored in triacylglycerols within the muscle.     

Long- and medium-chain triacylglycerols in nutritional support of liver regeneration of partially hepatectomized rats

An appropriate choice for a suitable diet during liver regeneration still remains an enigma. To investigate the effect of isocaloric enteral feeding with medium-chain triacylglycerols (MCT) and long-chain triacylglycerols (LCT) supplement (MCT+LCT, 40%:60% w:w) (178 kJ/kg b.w./24 h), rat liver regeneration was studied 24 and 72 h after partial hepatectomy. The liver DNA synthesis 24 h after partial hepatectomy was significantly higher in the MCT+LCT-supplemented rats (30.2+/-8.2 x 10(3) dpm/mg liver DNA) compared to MCT-treated animals (18.1+/-5.7 x 10(3) dpm/mg liver DNA). Liver protein synthesis was non-significantly elevated both 24 and 72 h after surgery in MCT+LCT-supplemented rats (13.7+/-1.1 and 10.9+/-3.1 x 10(3) dpm/mg liver protein). Seventy-two hours after partial hepatectomy, the hepatocyte mitotic activity was significantly increased in MCT+LCT- supplemented group vs. LCT- or MCT-fed rats (3.3+/-0.7 vs. 1.9+/-0.7 or 1.0+/-0.6 mitoses per 1000 hepatocytes), thus exhibiting an increased proliferative potential. The results showed a qualitative difference according to the proportion of MCT to LCT in the enteral supplements. Overfeeding with MCT decreased body weight, increased liver weight by its fatty infiltration, increased rat mortality rate and reduced spontaneous caloric intake. We conclude that the balanced supplement of MCT+LCT (40%:60% w:w) preserves liver regeneration, whereas overfeeding with MCT seems to be deleterious.     

Fatty acid metabolism and ketone formation in the suckling rat

Rat milk triacylglycerols contain 35% medium-chain length fatty acids. About 70% of ingested medium-chain fatty acids are released from milk triacylglycerols in the stomach and small intestine and are absorbed directly into the portal venous system. Based on studies with the perfused suckling rat liver and in vivo studies with 2-tetradecylglycidic acid, an inhibitor of long-chain fatty acid oxidation, it is estimated that medium-chain fatty acids provide 75-80% of the substrate for ketogenesis. The preferential use of medium-chain fatty acids for ketogenesis spares long-chain fatty acids for complex lipid and membrane biosynthesis during this period of rapid growth. Although medium-chain fatty acids are the major substrate for ketogenesis, this pathway accounts for only 15% of the utilization of ingested medium-chain fatty acids, the rest presumably being oxidized directly in extrahepatic tissues.     

Preexercise medium-chain triglyceride ingestion does not alter muscle glycogen use during exercise.

This investigation determined whether ingestion of a tolerable amount of medium-chain triglycerides (MCT; approximately 25 g) reduces the rate of muscle glycogen use during high-intensity exercise. On two occasions, seven well-trained men cycled for 30 min at 84% maximal O(2) uptake. Exactly 1 h before exercise, they ingested either 1) carbohydrate (CHO; 0.72 g sucrose/kg) or 2) MCT+CHO [0.36 g tricaprin (C10:0)/kg plus 0.72 g sucrose/kg]. The change in glycogen concentration was measured in biopsies taken from the vastus lateralis before and after exercise. Additionally, glycogen oxidation was calculated as the difference between total carbohydrate oxidation and the rate of glucose disappearance from plasma (R(d) glucose), as measured by stable isotope dilution techniques. The change in muscle glycogen concentration was not different during MCT+CHO and CHO (42.0 +/- 4.6 vs. 38.8 +/- 4.0 micromol glucosyl units/g wet wt). Furthermore, calculated glycogen oxidation was also similar (331 +/- 18 vs. 329 +/- 15 micromol. kg(-1). min(-1)). The coingestion of MCT+CHO did increase (P < 0.05) R(d) glucose at rest compared with CHO (26.9 +/- 1.5 vs. 20.7 +/- 0. 7 micromol.kg(-1). min(-1)), yet during exercise R(d) glucose was not different during the two trials. Therefore, the addition of a small amount of MCT to a preexercise CHO meal did not reduce muscle glycogen oxidation during high-intensity exercise, but it did increase glucose uptake at rest.     

Effects of a single oral load of medium-chain triglyceride on serum lipid and insulin levels in man

Analysis of serum free fatty acids by gas-liquid chromatography showed high proportions (27-57%) of octanoic acid for up to 4 hr after the ingestion of a single oral load of medium-chain triglyceride (approximately 1 g/kg body weight) in four volunteers. The effects of a medium-chain triglyceride load on the concentrations of plasma free long-chain fatty acids, plasma glucose, serum insulin, and serum triglyceride were observed and compared with the effects of a glucose load. A rapid fall in the free long-chain fatty acids followed both loads but only a small rise in serum insulin was observed after medium-chain triglyceride. The fall in free long-chain fatty acids following ingestion of medium-chain triglyceride cannot therefore be caused mainly by the release of insulin and may be due to a direct action on adipose tissue. No medium-chain fatty acids were detected in the serum triglyceride after ingestion of medium-chain triglyceride, but there was a small but significant increase in the percentage of hexadecenoic acid in this fraction.     

Human absorption of fish oil fatty acids as triacylglycerols, free acids, or ethyl esters

The transient rise in plasma triacylglycerol fatty acids after single-dose ingestion of fish oil as triacylglycerols, free acids, or ethyl esters with linseed oil as an absorption standard was used to determine the relative absorption of fish oil fatty acids in eight men. As free acids, the fish oil fatty acids were well absorbed (greater than or equal to 95%). As triacylglycerols, eicosapentaenoic acid (1.00 g) and docosahexaenoic acid (0.67 g) were absorbed only 68% and 57% as well as the free acids. The ethyl esters were absorbed only 20% and 21% as well as the free acids. The incomplete absorption of eicosapentaenoic and docosahexaenoic acids from fish oil triacylglycerols correlates well with known in vitro pancreatic lipase activity.     

Oxaloacetate deficiency in MCT-induced ketogenesis.

This study was an attempt to discover whether a deficiency in hepatic oxaloacetate can explain the acceleration of ketogenesis observed after the ingestion of medium-chain triglycerides (MCT, constituent fatty acids from C8 to C12). The method of investigation used consisted in supplying oxaloacetate (by intraperitoneal injection of oxaloacetate, aspartate, or L-tryptophan) to rats that had ingested MCT. The indirectly given oxaloacetate caused a decrease in ketone body levels in the liver. The stimulation of ketogenesis induced by an exogenous supply of MCT is therefore at least partly due to a deficiency of oxaloacetate. The results show that this can be explained both by a leakage of this metabolite into the pathway of gluconeogenesis and by its reduction into malate. Since the acetyl-CoA derived from oxidized medium-chain fatty acids cannot enter into the Krebs cycle, it is diverted to the production of ketone bodies.     

The concentration of plasma triacylglycerols in horses fed diets containing either medium chain triacylglycerols or an isoenergetic amount of starch or cellulose

In a Latin square design, six horses were fed hay and concentrates with isoenergetic amounts of either starch, cellulose or medium chain triacylglycerols (MCT). The dietary variables provided on average 22% of total dietary net energy. Plasma triacylglycerols and other variables of lipid metabolism were determined. The experimental periods lasted 21 days. Blood samples were taken just before the morning meal and three and six hours later. The diet rich in MCT significantly raised the plasma level of triacylglycerols when compared to either the starch‐ or cellulose‐rich diet. The plasma concentrations of 3‐hydroxybutyrate, total cholesterol and phospholipids were significantly higher when the horses were fed the ration with MCT instead of either cellulose or starch. Postprandial.insulin concentrations were lowest for the MCT diet, and concentrations of free fatty acids were highest Lipoprotein lipase activity was not significantly different for the three diets. Our study does not support the idea that cellulose feeding generates sufficient acetic acid in the caecum and colon, so that it would enhance the provision of cytosolic acetyl‐CoA which in turn would stimulate hepatic fatty acid synthesis and then raise plasma triacylglycerols.     

Gastric lipolysis of milk lipids in suckling rats

Fatty acid composition of the major lipid classes in stomach contents of suckling rats at 1, 5, 10, 17 and 20 days of lactation was compared to that of milk lipids. In milk, 98% of fatty acids were in triacylglycerols at all lactation times. Medium-chain fatty acid concentrations increased from 8% in colostrum to 26% at day 5. Fatty acid composition of stomach acylglycerols at all lactation times was different from that of milk triacylglycerols, containing less medium-chain fatty acids, 8:0 and 10:0. This preferential hydrolysis was also shown by higher concentrations of medium-chain fatty acids in the free fatty acid fraction. The lipolysis of medium-chain fatty acids from triacylglycerols resulted in the appearance of di- and monoacylglycerols with 50-100% higher amounts of 14:0 and 16:0. The similar fatty acid composition of products suggests that considerable lipolysis occurred in stomachs of suckling rats even at 1 day of age. Although there was a 10-fold increase in milk consumption, the extent of lipolysis was similar throughout the suckling period because of a parallel rise in lingual lipase levels.     

Effects of long-term feeding of high-protein or high-fat diets on the response to exercise in the rat

The aim of this work was to find by which mechanisms an increased availability of plasma free fatty acids (FFA) reduced carbohydrate utilization during exercise. Rats were fed high-protein medium-chain triglycerides (MCT), high-protein long-chain triglycerides (LCT), carbohydrate (CHO) or high-protein low-fat (HP) diets for 5 weeks, and liver and muscle glycogen, gluconeogenesis and FFA oxidation were studied in rested and trained runner rats. In the rested state the hepatic glycogen store was decreased by fat and protein feeding, whereas soleus muscle glycogen concentration was only affected by high-protein diets. The percentage decrease in liver and muscle glycogen stores, after running, was similar in fat-fed, high-protein and CHO-fed rats. The fact that plasma glucose did not drastically change during exercise could be explained by a stimulation of hepatic gluconeogenesis: the activity of phosphoenolpyruvate carboxykinase (PEPCK) and liver phosphoenolpyruvate (PEP) concentration increased as well as cyclic adenosine monophosphate (AMPc) while liver fructose 2,6-bisphosphate decreased and plasma FFA rose. In contrast, the stimulation of gluconeogenesis in rested HP-, MCT- and LCT-fed rats appears to be independent of cyclic AMP.     

In vitro and in vivo synthesis of long-chain fatty acids from (1-14C) acetate in the renal papillae of rats.

1. The relationship between the rate of [1-14C] acetate incorporation into the fatty acids of renal papillary lipids and the acetate concentration in the medium has been measured. 2. [1-14C] acetate was incorporated mainly into fatty acids of phospholipids and triacylglycerols. Only a few per cent of the radioactivity was found in the free fatty acid fraction. 3. The major part of the [1-14C] acetate was found to be incorporated by a chain elongation of prevalent fatty acids. The major component of the poly-unsaturated fatty acids in triacylglycerols and the major product of fatty acid synthesis from [1-14C] acetate in vitro was demonstrated by mass spectrometry to be docosa-7,10,13,16-tetraenoic acid. 4. The radioactivity of docosa-7,10,13,16-tetraenoic acid accounted for 40% of total radioactivity in triacylglycerol fatty acids (lipid droplet fraction) and 20% of total radioactivity in membrane phospholipid fatty acids.