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Department of Animal Science, North Carolina State University, Raleigh, NC 27695
ABSTRACT
INTRODUCTION
ESTABLISHED DOGMA OF MCT DIGESTION, ABSORPTION AND METABOLISM
NEW INSIGHTS INTO MCT UTILIZATION FROM A PIGLET MODEL
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Because of their unique digestive and metabolic properties, medium-chain triglycerides (MCT) are used in a variety of nutritional settings, including use as a readily digestible energy source for the neonate. This review examines recent findings from our laboratory related to MCT digestion and metabolism that are drawn from a neonatal piglet model, but which may be clinically relevant to human infants. We have shown that MCT utilization improves rapidly with postnatal age (within 24 h), which is likely due to the ontogeny of pancreatic lipase. Additional data delineate the dramatic effects of emulsification and fatty acid chain length (within the medium-chain family) on utilization, with the suggestion that triacylhexanoate is utilized at the highest rate. Again, these effects are likely mediated via an increase in the kinetics of digestion rather than metabolism. Indeed, using both in vitro and in vivo radiotracer techniques, we were unable to detect metabolic differences among even-chain fatty acid homologues. However, studies with isolated hepatocytes have shown greater oxidation rates of odd-chain fatty acids compared with even-chain homologues, in part as a reult of the anaplerotic potential of propionyl-CoA arising from odd-carbon fatty acid oxidation. In vivo radiotracer studies also showed an improvement in octanoate oxidation to CO2, with a concomitant reduction in urinary dicarboxylic acid excretion when colostrum-deprived piglets were supplemented with L-carnitine. Further metabolic research led to the novel finding that piglets have a very limited hepatic capacity to synthesize ketone bodies, and that acetate may be a relatively important product of hepatic fatty acid oxidation in this species.
KEY WORDS: medium-chain triglyceride · fatty acid metabolism · carnitine · swineINTRODUCTION
Research examining the nutritional and metabolic properties of medium-chain triglycerides (MCT)3 spans a period of 40 y. Following early studies of the 1950s (Bloom et al. 1951
), which described effects of fatty acid chain length on the portal vs. lymphatic routes of absorption, research focused on delineating the unique features of MCT digestion and metabolism in contrast to the established dogma for long-chain triacylglycerols (LCT). Since then, because of their rapid rate and extent of digestion, accelerated rate of portal absorption and obligatory oxidation (detailed below), MCT have been examined as a specialized energy source within a wide variety of clinical nutrition settings. These include treatment of pancreatic and biliary insufficiency (e.g., cystic fibrosis), gastroenteritis, chylothorax, obesity and diabetes. They also have been employed in total parenteral nutrition and preterm infant formulas. Recent development of reduced-calorie, structured lipids containing medium-chain fatty acids also represents an exciting new application (Finley et al. 1994, Webb et al. 1993).
Given this immense and heterogeneous literature base, it is well beyond the scope of this review to provide a comprehensive survey of the MCT literature. Rather, the intent is to provide a brief overview of the unique attributes of MCT utilization to enhance the reader's appreciation for their application in neonatal nutrition and then to summarize the new findings from our laboratory, which have employed the increasingly popular neonatal piglet model (Reeds and Odle 1996).
ESTABLISHED DOGMA OF MCT DIGESTION, ABSORPTION AND METABOLISM
, Bach and Babayan 1982, Borum 1992, Greenberger & Skillman 1969, Hamosh et al. 1991, Jensen & Jensen 1992, Kaunitz and Johnson 1968, Lima 1989, Roy et al. 1981) that review in detail the experiments supporting the basic tenets of MCT utilization.
NEW INSIGHTS INTO MCT UTILIZATION FROM A PIGLET MODEL ). Preduodenal lipases (Hamosh 1990) also hydrolyze MCT preferentially, and the resulting MCFA can be absorbed in part through the stomach mucosa.
Fig. 1.
Metabolism of medium- and long-chain triglycerides and fatty acids by intestine, liver and adipose tissue. Abbreviations are defined within the figure. In contrast to LCT, MCT are more rapidly digested, and the resulting MCFA are more rapidly absorbed via portal circulation than the corresponding LCFA which are re-esterified and packaged into chylomicrons for lymphatic absorption. Furthermore, MCFA are predominantly oxidized, whereas LCFA may be directly deposited into adipose triglyceride stores. (Adapted from Greenberger and Skillman 1969.)
[View Larger Version of this Image (33K GIF file)]
).
) that MCFA may be elongated to LCFA by enzymes housed in the mitochondria, the major metabolic fate of MCFA is oxidation. Very little MCFA are re-esterified. Long-chain fatty acids are activated to their CoA-thioesters by enzymes located in the endoplasmic reticulum and in the outer mitochondrial membrane (Groot et al. 1976). These enzymes have maximal activity toward LCFA (i.e., peak activity toward C12:0 and C16:0 fatty acids), but can activate fatty acids of medium-chain length. In contrast, medium-chain acyl-CoA synthetase is located within the mitochondrial matrix, suggesting that a portion of MCFA entry into the mitochondria may be independent of the carnitine acyltransferase system. This has at least three important ramifications for the neonate. First, MCFA oxidation may be less carnitine dependent. If so, this would facilitate their use by newborns that may have low carnitine levels (Baltzell et al. 1987). Second, it represents a bypass of carnitine palmitoyltransferase I, which represents an important control point in LCFA oxidation (McGarry and Foster 1980). Malonyl-CoA, an intermediate in the lipogenic pathway, allosterically inhibits carnitine palmitoyltransferase I and thereby decreases transport into the mitochondria, limiting oxidation. This control serves to limit energy loss from wasteful recycling of fatty acid carbon. Because MCFA can by-pass this control, they are extensively oxidized regardless of the physiological state of the animal. Third, the rapid and "uncontrolled" entry into the liver mitochondria results in more pronounced ketone body production from MCFA than from LCFA. Although mild hyperketonemia would not pose a problem to the neonate (Williamson 1982), higher levels could result in serious metabolic acidosis. A quantitatively less important oxidation pathway (not shown) involves oxidation at the 
-carbon, which results in the production of dicarboxylic acids (Gregersen et al. 1983).
, an increasing number of laboratories have used the perinatal piglet as a pediatric model for studies of nutrition, metabolism and growth biology (Reeds and Odle 1996). In many respects, the piglet is an excellent model for pediatric research. Piglets exhibit accelerated postnatal growth and development (compared with human infants) and therefore are very sensitive to environmental variables (i.e., nutritional inputs and pharmaceutical drug testing). From a digestive physiology standpoint, they have many similarities to humans (see Moughan et al. 1992), and with birth weights ranging from 0.7 to 3.0 kg, they are of a size that permits both reduction science methodologies (e.g., muscle, liver and intestinal-tissue studies) and also whole-animal, preclinical investigations that involve such techniques as catheterization and multiple blood sampling. Furthermore, because they are a litter-bearing species, with natural variation in birth weight, they provide a good model for the study of intrauterine growth retardation wherein low-birth-weight piglets can be compared with their larger littermates.
). Furthermore, this dose was marginally capable of sparing muscle glycogen (Benevenga et al. 1989), reducing nitrogen excretion and maintaining blood glucose in fasted piglets (Odle et al. 1989). In addition to sparing carbohydrate oxidation, fatty acid oxidation may directly support gluconeogenesis in the piglet (Lepine et al. 1989 and 1993).
Factors affecting MCT digestion and absorption
Effects of fatty acid chain length. As discussed previously, a plethora of studies have identified the stark differences in digestion and absorption of medium- and long-chain triacylglycerols (e.g., Chiang et al. 1990). In contrast, few studies have examined differences within the medium-chain family. We therefore established a simple in vivo screening protocol (Odle et al. 1991a) to examine the relative rates of utilization of MCT of varying fatty acid chain length. The protocol involved gastric intubation of piglets with various nonemulsified MCT oils, followed by serial sampling of peripheral blood for analysis of MCFA by HPLC. Figure 2A shows the profound effect of fatty acid chain length within the medium-chain family. The peak systemic fatty acid concentration at 1 h after feeding was 50-fold higher in piglets fed tri-C7:0 than in those fed tri-C10:0, with C8:0 and C9:0 concentrations being intermediate. These data suggested that although the route of absorption may be similar for all MCFA, the rate of digestion and absorption might differ considerably. Using a similar protocol, several follow-up studies (Fig. 2B) consistently indicated that tri-C6:0 yielded the highest blood fatty acid profile (Wieland et al. 1993a and 1993b). To extend these collective observations, radiolabeled triglycerides were synthesized from 14C-hexanoate and 14C-octanoate and fed to piglets housed in respiration chambers capable of trapping expired 14CO2 (Odle et al. 1994). Total collections of CO2 at 20-min intervals allowed for detailed kinetic analysis of relative utilization rates (Fig. 3A). The peak rate of 14CO2 expiration occurred 3.5 h after feeding, and the corresponding maximal utilization rate of tri-C6:0 exceeded tri-C8:0 by 37%. However, total utilization over the 24-h collection was not significantly affected by chain length (P < 0.05). In complementary studies (Odle et al. 1992) involving intravenous infusion of radiolabeled fatty acids, no differences in whole-animal oxidation of fatty acids ranging in chain length from C7:0 to C10:0 were detected, thus suggesting that differences in the kinetics of digestion and absorption were likely responsible for the previously observed effects of chain length. Indeed, when we subsequently examined chain-length specificity of pancreatic lipase in developing piglets (unpublished observations), we observed greater rates of hydrolysis of tri-C6:0 when presented as an equimolar composite mixture with other MCT (containing C4:0, C6:0, C8:0 and C10:0 fatty acids). Collectively, based on these data, we contend that tri-C6:0 may offer additional clinical utility beyond that afforded by MCT composed of tri-C8:0/C10:0 under cases of severe pancreatic/biliary insufficiency and steatorrhea.
, respectively.)
and Wieland et al. 1993a, respectively.)
Effects of emulsification. Given the putative constraint on digestion and absorption inferred from our previous studies, we expected that emulsification would enhance utilization of the MCT oils. Thus, we prepared 30% oil-in-water emulsions using various emulsifying agents (Wieland et al. 1993a
). When piglets were gavaged with a 30% (v/v) Tween-80 emulsion, peak circulating MCFA were elevated as much as 14-fold (Fig. 3B) over concentrations observed in controls fed equimolar, nonemulsified MCT. Similarly, when piglets in respiration chambers were fed emulsified 14C-MCT (Odle et al. 1994), both the maximal rate and extent of utilization were enhanced by 20% (Fig. 3A). Additional experiments indicated that utilization of MCT of all chain lengths was enhanced to a similar extent by emulsification (Wieland et al. 1993b). Notably, toxicity was observed when too much readily digestible emulsified MCT was administered. Circulating MCFA concentrations in excess of 15 mmol/L were observed in some piglets. Even when concentrations exceeded 3 mmol/L, piglets lapsed into a transient narcoleptic state, but would recover as the absorbed MCFA were metabolized. This prompted further dose-response studies to establish safe intake levels (i.e., < 6.5 mmol MCT/kg0.75 per dose) for piglets (Wieland et al. 1993a). The narcoleptic properties of MCFA have long been recognized (Samson et al. 1956) and therefore require caution when MCT are used in various clinical applications. Collectively, these studies support the use of emulsified MCT to maximize the rate and extent of utilization, with the provision that appropriate doses be given to avoid toxicity.
Effects of piglet age.
When considering the practical application of these research findings in production agriculture, it was important to examine the ontogenic aspects of MCT utilization. Because the majority of postnatal piglet death losses occur during the first 3 d following parturition (USDA 1991), our attention has necessarily focused on utilization within the first 48 h of life. In an initial experiment, piglets were gavaged with MCT at 6, 18 or 48 h of age, after which the circulating MCFA concentrations were measured. Peak concentrations were up to fivefold higher in piglets gavaged at 18 h compared with those gavaged at 6 h, but no further increase occurred between 18 and 48 h. Subsequent research (Dicklin et al. 1995) also showed that emulsification enhanced circulating MCFA concentrations in both 5- and 29-h-old piglets. Examination of lingual, gastric and pancreatic lipase activities revealed that the last mentioned has primary importance in the piglet. Interestingly, lipase activity per gram wet weight of pancreas decreased steadily from birth to 18 h of age, likely because the rate of enzymic discharge by the pancreas exceeded the rate of synthesis. Activity in 48-h-old piglets rebounded, and levels exceeded those at birth when expressed on a whole-organ basis.
, Lepine et al. 1989) using nonemulsified C8/C10 MCT given at multiple and higher doses have not demonstrated improved piglet survival.
Factors affecting metabolism of medium-chain fatty acids
Effects of fatty acid chain length. Given the dramatic effect of chain length on the rate of digestion/absorption, one might expect some effect on the rate of metabolism as well. To examine this, day-old piglets were surgically fitted with indwelling catheters (via umbilical arteries) and were infused systemically with increasing amounts of 14C-medium-chain fatty acids ranging in chain length from C7:0 to C10:0 (Odle et al. 1992), thereby eliminating effects mediated via differences in rate of digestion/absorption. Rates of oxidation to 14CO2 were proportional to infusion rates, but were unaffected by fatty acid chain length (Fig. 4A). Interestingly, only 60% of infused radiolabel could be recovered in expired CO2. Although the metabolic fate of the remaining 40% is unknown, Carnielli et al. (1994) have documented significant incorporation of octanoate-carbon into long-chain fatty acids in preterm infants.
and Odle et al. 1991a, respectively.)
-oxidation of odd-carbon fatty acids (Guisard et al. 1973). Theoretically, this effect might be greatest in hepatic tissue because the gluconeogenic potential of propionyl-CoA could increase carbohydrate flux, and its anaplerotic potential (i.e., ability to increase tricarboxylic-acid-cycle carbon) could diminish ketogenic flux. In support of this, Linseisen and Wolfram (1993a, 1993b and 1993 c) reported elevated plasma glucose, lactate, pyruvate and hepatic glycogen, but reduced ketone bodies in rabbits maintained on total parenteral nutrition containing tri-C9:0. Using the piglet model, we did not observe the anticipated improvement in blood glucose concentrations in piglets given odd- vs. even-chain MCT orally (Odle et al. 1989). Circulating ketone body concentrations were surprisingly low in all piglets, but were higher in piglets given MCT containing a mixture (3:1, mol/mol) of C8:0 and C10:0 than in piglets fed MCT containing C9:0 (Odle et al. 1989). To examine effects on hepatic metabolism directly, hepatocytes were isolated from newborn piglets and incubated with 14C-fatty acids ranging from C7:0 to C10:0 (Odle et al. 1991a). Accumulation of fatty acid carbon in both CO2 (Fig. 4B) and acid-soluble products showed a "zig-zag" pattern, which was higher for odd-carbon than for even-carbon fatty acids. Rates of oxygen consumption followed a similar pattern. Further examination of the acid-soluble products by radio-HPLC (Lin et al. 1996) showed much greater accumulation of 14C in various tricarboxylic-acid-cycle intermediates when hepatocytes were incubated with radiolabeled C7:0 compared with C8:0. Collectively, these data indicate that odd-chain MCT may be a promising alternative fuel for use in enteral and parenteral nutrition.
). These studies showed minor effects of birth weight, but major effects of postnatal age (6 vs. 48 h of age) on fatty acid oxidation. The effects were congruent with developmental increases in metabolic rate. Further study of low-birth-weight piglets (Odle et al. 1995) provided evidence consistent with normal regulation of fatty acid oxidation via carnitine palmitoyltransferase I, despite low rates of ketogenesis. The attenuated ketogenic capacity of neonatal piglets appears to stem from limited expression of mitochondrial HMG-CoA synthase (Duée et al. 1994). Although capacity appears to develop somewhat with age (Adams and Odle 1993), it remains far below other species such as rats and rabbits. We have estimated that at maximum concentrations observed physiologically, -hydroxybutyrate contributes no more that 3-4% of the piglets' basal energy needs (Tetrick et al. 1995). Most recent radio-HPLC evidence (Fig. 5) suggests that acetate, rather than ketone bodies, may represent a major product of fatty acid oxidation in piglet hepatocytes (Lin et al. 1996, Odle et al. 1995). Of the fatty acid carboxyl-carbon accumulating in acid-soluble products, 40-70% was found in acetate. The degree to which acetogenesis replaces ketogenesis and the subcellular origin (mitochondrial vs. peroxisomal) of acetogenesis in the porcine species remain to be elucidated.
and Lin et al. 1996, respectively.)
Effects of supplemental L-carnitine. When viewed from the prescribed dogma (discussed previously), MCFA oxidation should be in part independent of carnitine, because of the ability of MCFA to freely diffuse across the mitochondrial membranes (thus bypassing carnitine palmitoyltransferase I) and to be activated to CoA esters via medium-chain CoA synthetases located within the mitochondrial matrix. This is likely true for hepatic tissue, because when isolated piglet hepatocytes were incubated with supplemental L-carnitine, octanoate oxidation was unaffected (Odle et al. 1991 and 1995), whereas oleate oxidation was increased and esterification was reduced (Odle et al. 1995
). However, other in vitro investigations (Groot and Hulsmannn 1973) have reported carnitine stimulation of MCFA oxidation by muscle tissue. More recently, in vivo studies (Kempen and Odle 1992, Rebouche et al. 1990, Rössle et al. 1990) have documented alterations in plasma and urinary concentrations of carnitine esters following ingestion of MCT. These studies have suggested that carnitine status might influence MCFA metabolism (and visa versa), especially in the colostrum-deprived neonate whose carnitine status is marginal (Baltzell et al. 1987). To test this hypothesis, we parenterally infused varying levels of 1-14C-octanoate into colostrum-deprived piglets (Kempen and Odle 1993 and 1995). After a steady-state oxidation rate was attained (at ~l6 h), piglets received a primed continuous co-infusion of L-carnitine, and the effect on octanoate -oxidation to CO2 and -oxidation to dicarboxylic acids was monitored (Fig. 6A, B). The rate of octanoate oxidation to CO2 was increased by 5-20% when carnitine was supplemented, and the stimulation was proportional to the octanoate infusion rates beyond 50% of the piglets' metabolic rate. A concurrent 8-48% reduction in suberate (i.e., 8-carbon dicarboxylic acid) excretion in the urine also was observed. The mechanism(s) by which carnitine affects MCFA oxidation remains unclear, but it may be related to buffering (lowering) of the acyl-CoA to free CoA ratio, which could be particularly important in a species limited in ketogenic capacity.
, Reeds and Odle 1996), the extrapolation of findings across species with known idiosyncrasies (such as the deficit of ketogenesis in piglets) should be made with caution. As illustrated in our studies, the piglet model can provide invasive, in vitro data of interest, but is also amiable to whole-animal studies which can closely simulate the clinical environment of the preterm infant. Clearly, no single animal model is perfect, and thus care must be used when relating findings from these models to human clinical situations. Breadth in our understanding of several animals models coupled with clinical observations on infants will afford the best balance toward improving clinical treatments.
ACKNOWLEDGMENTS
FOOTNOTES
Manuscript received 26 December 1996. Initial reviews completed 23 January 1997. Revision accepted 17 February 1997.
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