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Department of Dairy Science University of Wisconsin, Madison 53706
Corresponding author:
R. R. Grummer; e-mail:
rgrummer{at}wisc.edu
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ABSTRACT |
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Key Words: fatty acids • hepatic uptake
The ruminant liver extracts between 12 to 25% of plasma NEFA during each pass through the liver under in vivo conditions (Emery et al., 1992). Uptake of NEFA is the predominant route by which fatty acids are supplied to the liver (Emery et al., 1992). However, few studies have measured hepatic uptake of individual long-chain fatty acids in ruminant animals. Thompson and Darling (1975) and Thompson et al. (1975) studied hepatic net uptake of palmitic, stearic, and oleic acids in sheep. These studies showed no differences between palmitic and oleic acid net uptake by the liver. However, hepatic net uptake of stearic acid was lower than palmitic and oleic acids when expressed as a percentage of the concentration reaching the liver. Hepatic net uptake of other long-chain fatty acids is unknown in ruminants. Therefore, the objective of the current study was to compare hepatic net uptake of various nonesterified long-chain fatty acids in the perfused caprine liver.
Four crossbred male goats between the ages of 4 and 10 mo were used. The goats were anesthetized with 1.5 g of sodium thiopental (Abbott Laboratories, North Chicago, IL) and the caudate lobe of the liver (39 ± 7.9 g; mean ± SD) was removed. After removal of the caudate lobe, the goats were euthanized with 10 ml of Beuthanasia-D Special (Schering-Plough, Union, NJ). The isolated caudate lobe was immediately flushed with 120 ml of Krebs-Ringer buffer and transported to a warm room maintained at 37°C. A catheter was inserted into the caudate lobe and buffer was perfused at a rate of 1 ml/min x g wet tissue. Buffer was oxygenated by continuous flow through a Hospal dialysis membrane (Gambro Renal Products, Lakewood, CO). After 2 min, the Krebs-Ringer buffer was replaced with 250 ml of Krebs-Ringer buffer containing 0.3 mM each of palmitic, stearic, oleic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids bound to albumin in a 4:1 fatty acid:albumin ratio. We chose 0.3 mM of each fatty acid because many fatty acids approach or exceed that concentration in serum near parturition (Rukkwamsuk et al., 2000). Having all the fatty acids at 0.3 mM, as opposed to different physiological concentrations of each fatty acid, allows for direct comparisons of fatty acids without the confounding effects of concentration. A 10-ml sample of the media was taken before the start of the perfusion and at 10-min intervals for the next 60 min.
Lipid was extracted from 4 ml of perfusion media according to Dole (1956) following the addition of butylated hydroxytoluene (0.01%) to each tube. Extracts were resuspended in 50 µl of chloroform:methanol (2:1) and 25 µl were spotted onto Adsorbosil Plus 1 silica gel plates (Alltech Associates Inc., Deerfield, IL), which were developed with hexane:diethyl ether:glacial acetic acid (90:10:1). Plates were dried under nitrogen and spots corresponding to fatty acids were visualized with 5% Rhodamine G in ethanol. The silica gels were extracted twice with chloroform:methanol (2:1), and the lipids were dried under nitrogen. Methyl esters were formed according to Sukhija and Palmquist (1988) and were analyzed by gas chromatography. Margaric acid (C17:0) was used as an internal standard for all samples.
Raw data were recorded as the amount of individual fatty acids in the perfusate. To account for differences in perfusate loss due to sampling, the total amount of each fatty acid in the perfusate was multiplied by the amount of preperfusion perfusate (250 ml) divided by the amount of perfusate remaining at the time of the individual sample. Data were analyzed using the mixed procedure of SAS (SAS, 1999). Individual slopes for the amount of fatty acid in the perfusate (dependent variable) over time were generated for each goat using a model that included fatty acid and time within fatty acid. The original model tested for linear and quadratic effects; however, the quadratic term was removed from the model due to lack of significance (P > 0.25). The slopes representing changes in the amount of fatty acid in the perfusate over time for each goat were then compared using a model containing the fixed effects of fatty acid and the random effects of goat. Treatment differences were tested using the Tukey-Kramer adjustment. To generate least-squares means for the amount of fatty acid in the perfusate over time, a separate model containing fixed effects of fatty acid, time within fatty acid, and the random effect of goat was used. When analyzing the above data, we included fatty acid in the model even though each fatty acid was not applied to a separate lobe. Therefore, an assumption in the analysis is that the presence of an individual fatty acid does not influence the uptake of another fatty acid. The experiment was designed and analyzed in this way because using individual caudate lobes for each fatty acid would be inefficient. Additionally, a mixture of fatty acids in the perfusate is more representative of physiological conditions than if the perfusate only contained one fatty acid. Positive slopes for the amount of fatty acid in the perfusate over time indicate net release and negative slopes indicate net uptake of fatty acids by the liver. Data are presented as an amount of the time zero (preperfusion) sample.
Preperfusion fatty acid concentrations were 296, 292, 298, 281, 284, 288, and 295 µM for palmitic, stearic, oleic, linoleic, linolenic, eicosapentaenoic and docosahexaenoic acid, respectively. The slopes for relative amounts of individual fatty acids contained in the perfusate over time are shown in Table 1
and Figure 1. All fatty acids, with the exception of stearic and oleic acids, had significant (P < 0.05) negative slopes thus indicating net uptake of fatty acids. Stearic acid had a positive, nonsignificant slope (P > 0.15), which was significantly different (P < 0.05) from all other fatty acids tested, with the exception of oleic acid. Oleic acid had a negative slope (i.e., net uptake), but the slope only tended to be different from zero (P < 0.15). The slope of oleic acid was not different (P > 0.15) from that of palmitic, linoleic, linolenic, eicosapentaenoic, and docosahexaenoic acids.
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Transport through the cell membrane is also thought to be a rate-limiting process in fatty acid uptake (Hamilton, 1998). The rates of desorption of fatty acids from phospholipid bilayers decreased with increasing chain length and increased with addition of each double bond (Zhang et al., 1996). Thus, it could be speculated that a low rate of removal from the cell membrane into the cytosol may impede stearic acid uptake, whereas uptake of the shorter or more unsaturated chain fatty acids are less hindered. In summary, changes in the amounts of fatty acid in the perfusate over time were similar among most long-chain fatty acids tested with the exception of stearic acid. The reason for different behavior of stearic acid is unknown, but it may be related to the hydrophobic nature of the acid.
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FOOTNOTES |
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Received for publication June 28, 2002. Accepted for publication November 28, 2002.
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