Institute for Liver, Digestive, and Metabolic Diseases, Department of Pediatrics, University Hospital Groningen, 9700 RB Groningen, The Netherlands
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ABSTRACT |
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We investigated in
bile duct-ligated (BDL) and sham-operated control rats whether the
frequent presence of essential fatty acid deficiency in cholestatic
liver disease could be related to linoleic acid malabsorption, altered
linoleic acid metabolism, or both. In plasma of BDL rats, the
triene-to-tetraene ratio, a biochemical marker for essential fatty acid
deficiency, was increased compared with controls (0.024 ± 0.004 vs. 0.013 ± 0.001; P < 0.05). Net and percentage
of dietary linoleic acid absorbed were decreased in BDL rats compared
with control rats (1.50 ± 0.16 mmol/day and 81.3 ± 3.3%
vs. 2.08 ± 0.07 mmol/day and 99.2 ± 0.1%, respectively;
each P < 0.001). At 24 h after
[13C]linoleic acid administration, BDL rats had a similar
ratio of plasma [13C]arachidonic acid to plasma
[13C]linoleic acid concentration compared with control
rats. 6-Desaturase activity was not significantly
different in hepatic microsomes from control or BDL rats. At 3 h
after [13C]linoleic acid administration, plasma
appearance of [13C]linoleic acid and cumulative
expiration of 13CO2 were decreased in BDL rats,
compared with controls (by 54% and 80%, respectively). The present
data indicate that the impaired linoleic acid status in cholestatic
liver disease is mainly due to decreased net absorption and not to
quantitative alterations in postabsorptive metabolism.
essential fatty acid; enterohepatic circulation; cholestasis; stable isotope
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INTRODUCTION |
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ESSENTIAL FATTY ACIDS (EFAs) and their polyunsaturated fatty acid (PUFA) metabolites are major components of structural lipids in all tissues and modulate cell membrane fluidity and function. The availability of long-chain PUFA (LCPUFA; >18 carbon atoms), such as arachidonic acid [20:4(n-6)], is important for early human growth and for the production of eicosanoids, which mediate immune and vascular functions (6, 23). A suboptimal status of EFAs and LCPUFAs is frequently noted in patients with impaired bile formation, such as cholestatic liver disease (1, 9, 10, 24). Low plasma concentrations of these fatty acids have been speculated to contribute to the morbidity and mortality of these patients.
Theoretically, impaired status of EFAs and LCPUFAs in plasma may
be related to malabsorption, increased metabolism (i.e., desaturation
and elongation, -oxidation), and/or tissue redistribution of these
fatty acids. Because patients with cholestatic liver disease have
impaired bile secretion into the intestinal lumen, it would seem
reasonable that they malabsorb a substantial amount of their dietary
lipids. Kobayashi et al. (14) have reported that dietary
lipid absorption was reduced to 30% in patients with biliary atresia.
Recently, we (19) found that rats with permanent biliary
drainage had decreased percentages of linoleic acid absorption; however, by ingesting more diet (~ 40%), they compensated for fecal
losses of linoleic acid. As a result, their net uptake of dietary
linoleic acid was not different from control rats (19). In
addition to impaired bile formation, the pathophysiology of cholestasis
is characterized by retention of bile-destined compounds such as bile
salts in the body (11). It may be that the retention of
bile compounds contributes to impaired status of EFAs and LCPUFAs in
cholestasis, for example, by enhancing EFA metabolism (preferential oxidation, increased elongation/desaturation). Indirect indications that the efficacy of hepatic elongation/desaturation is altered during
cholestatic liver disease in infants were recently reported by Socha et
al. (24, 25). It could be conceived that linoleic acid is
metabolically more readily utilized for energy supply, i.e.,
-oxidized, under conditions of impaired fat absorption.
In the present study, we aimed to simulate the clinical condition of cholestasis by using an experimental animal model, the bile duct-ligated (BDL) rat, which is characterized both by intestinal bile deficiency and by accumulation of biliary compounds in the body. The absorption and metabolism of linoleic acid was investigated in short-term (1 wk) BDL rats.
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MATERIALS AND METHODS |
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Animals. Male Wistar rats (Central Animal Laboratory, Groningen, The Netherlands), weighing 250-350 g (mean ± SD: 277 ± 37 g), were kept in an environmentally controlled facility with diurnal light cycling and free access to diet (standard rodent chow, Hope Farms, Woerden, The Netherlands) and tap water. Experimental protocols were approved by the Ethical Committee for Animal Experiments, Faculty of Medical Sciences, University of Groningen.
Labeled substrates. [1-13C]palmitic acid and [U-13C]linoleic acid were purchased from Isotec. Both stable isotopes were >99% enriched.
Experimental procedures. Rats were individually housed in metabolic cages and fed a high-fat diet [35 en% lipid, 4,283 kcal/kg diet; major long-chain fatty acid composition as measured by gas chromatography: 16:0, 31.5%; 18:0, 7.3%; 18:1(n-9), 31.2%; and 18:2(n-6), 30.0%] (Hope Farms). After 1 wk of feeding, rats were equipped with permanent catheters in jugular vein and duodenum, as described by Kuipers et al. (17). Bile duct ligation was performed on one group of rats (n = 6), and the other group was sham operated (n = 6). The experimental model allows for physiological studies in unanesthetized rats with bile duct ligation without the interference of stress or restraint. Animals were allowed to recover from surgery for 7 days.
On day 7, 1.67 ml lipid/kg body wt was slowly administered as a bolus via the duodenal catheter to one-half of the rats in each group. Medium-chain triglyceride oil was included in the bolus to have a sufficient amount to allow a reliable, reproducible delivery. Specifically, the lipid bolus was composed of olive oil [25% vol/vol; fatty acid composition: 16:0, 14%; 18:1(n-9), 79%; and 18:2(n-6), 8%] and medium-chain triglyceride oil (75% vol/vol; composed of extracted coconut oil and synthetic triglycerides; fatty acid composition: 6:0, 2%; 8:0, 50-65% maximum; 10:0, 30-45%; 12:0, 3% maximum) and contained 6.7 mg [U-13C]linoleic acid and 6.7 mg [1-13C]palmitic acid/kg body wt. The lipid bolus represented <10% of the daily lipid intake for control and BDL rats. Blood samples (0.2 ml) were taken from the jugular cannula at baseline and hourly for 6 h after administration of the label and were collected into tubes containing heparin. A blood sample for quantification of [13C]linoleic acid and [13C]arachidonic acid was taken at 24 h after label administration. Plasma was separated by centrifugation (10 min, 2,000 rpm, 4°C) and stored atAnalytical techniques. Plasma ALT, AST, and bilirubin were determined by routine clinical procedures. Plasma bile salts were measured enzymatically (17). Lipids from plasma (n = 6/group) were extracted and methylated according to Lepage et al. (18). After freeze-drying and mechanical homogenization, aliquots of diet and feces were subjected to the same procedure (18). Duplicate aliquots of liver homogenate (n = 3/group) were extracted (2) and methylated (18) as described previously. Resulting fatty acid methyl esters from all biological samples were analyzed by gas chromatography to measure total and individual amounts of major fatty acids and, for plasma, liver and feces, by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) to measure the 13C enrichment of palmitic and linoleic acids and linoleic acid metabolites.
Gas liquid chromatography. Fatty acid methyl esters in plasma were separated and quantified by gas liquid chromatography as detailed by Kalivianakis et al. (13) using heptadecanoic acid (17:0) as internal standard.
GC-C-IRMS. 13C enrichment of palmitic, linoleic, and arachidonic methyl esters was determined by using a Finnigan MAT Delta S IRMS interfaced to a Varian 3400 gas chromatograph via a capillary oxidation furnace (Finnigan MAT, Bremen, Germany), according to the method described by Minich et al. (20).
IRMS.
13C enrichment in aliquots of breath samples was determined
by means of continuous flow isotope ratio mass spectrometry (Finnigan Breath MAT). The 13C abundance of breath CO2
was expressed as the difference from the reference standard Pee Dee
Belemnite limestone (13CPDB,
). The
proportion of 13C label excreted in breath CO2
was expressed as the cumulative percentage of administered
13C label recovered per indicated time point.
6-Desaturase assay.
The metabolism of [13C]linoleic acid to
[13C]arachidonic acid was measured in vivo using the
ratio between [13C]arachidonic acid and
[13C]linoleic acid in plasma at 24 h after
[13C]linoleic acid bolus administration.
6-Desaturase activity was measured in hepatic microsomes
from BDL and control rats in vitro. Hepatic microsomes were isolated in conformance with the procedures described by Smit et al.
(22). The conditions for the (unlabeled) desaturation
assay were described by Su and Brenna (26), using
incubation media made according to de Antueno et al. (5).
After isolation of hepatic microsomes, the activity of
6-desaturase was determined by measuring the conversion
of linoleic acid [18:2(n-6)] to gamma-linolenic acid [18:3(n-6)],
during 1 h in a shaking water bath at 37°C. The reaction was
terminated by addition of 10% (wt/vol) potassium hydroxide in ethanol,
after which lipids were extracted, methylated, and analyzed by gas
chromatography, as described above.
6-Desaturase
activity was expressed as the change in mass of 18:3(n-6) calculated
directly from the quantitative gas chromatography results. Results
obtained from a blank, non-incubated sample were subtracted from those
from the incubated sample.
Calculations. The triene-to-tetraene ratio was calculated for plasma and liver by dividing the concentration of 20:3(n-9) by that of 20:4(n-6). In plasma and liver, n-6 fatty acid status was calculated using the sum of the area of major fatty acids (>90%) [16:0, 16:1(n-7), 18:0, 18:1(n-9), 18:1(n-7), 18:2(n-6), 18:3(n-6), 20:3(n-6), 20:3(n-9), and 20:4(n-6)] and then expressing the area of each individual n-6 fatty acid as a percentage of the total amount. Absorption of major dietary fatty acids (palmitic, stearic, oleic, linoleic acids) and of [13C]palmitic and linoleic acids was measured using balance techniques as described by Minich et al. (19).
Statistics. Values represent means ± SE for the indicated number of animals per group. Using SPSS version 6.0 statistical software (Chicago, IL), we calculated significance of differences with the two-tailed Student's t-test for normally distributed, unpaired data or a Mann-Whitney U-test for data that were not normally distributed. Variance among data was determined using Levene's test for equality of variances. P < 0.05 was considered significant.
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RESULTS |
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Body weight and food ingestion. Between BDL and control rats, there were no significant differences in body wt (277.5 ± 14.9 vs. 290.3 ± 12.2 g, respectively) or in amount of food ingested (recorded values for 3 days: 14.2 ± 1.0 vs. 16.3 ± 0.6 g, respectively).
Cholestatic markers in plasma. Compared with control rats, BDL rats had increased activities in plasma of the liver enzymes ALT (34.0 ± 3.0 vs. 74.5 ± 10.3 U/l, P < 0.01) and AST (98.0 ± 7.0 vs. 273.7 ± 59.0 U/l, P < 0.05), and higher concentrations of total bilirubin (4.2 ± 0.2 vs. 172.3 ± 10.7 µmol/l, P < 0.01) and bile salts (9.4 ± 2.0 vs. 229.5 ± 20.8 µmol/l, P < 0.01), respectively, in accordance with the presence of cholestasis. Liver weights were similar between BDL and control rats (13.9 ± 1.0 vs. 11.3 ± 0.4 g; not significant).
Absolute and relative fatty acid concentrations in plasma and in
liver.
Total plasma lipid analysis revealed a similar cumulative concentration
of major fatty acids between BDL and control rats (7.11 ± 0.18 vs. 6.90 ± 0.33 mM; not significant). Plasma concentrations of
the individual fatty acids, palmitic, linoleic, and arachidonic acid,
were similar between both groups (data not shown). However, stearic
acid plasma concentration decreased and oleic acid plasma concentration
increased in BDL vs. control rats (stearic acid, 0.79 ± 0.05 vs.
1.04 ± 0.04 mM; P < 0.01; oleic acid, 1.39 ± 0.06 vs. 0.98 ± 0.07 mM, respectively; P < 0.01). Accordingly, the molar percentages (Fig.
1) for stearic acid and oleic acid were decreased and increased, respectively, in plasma of BDL rats compared with that of control rats (11.08 ± 0.24% vs. 15.13 ± 0.40%, P < 0.001; 19.63 ± 0.71% vs. 14.22 ± 0.71%, P < 0.001). The triene-to-tetraene ratio
[20:3(n-9)/20:4(n-6)], a biochemical indicator of EFA deficiency, was
increased in plasma of BDL rats compared with control rats (0.024 ± 0.004 vs. 0.013 ± 0.001; P < 0.05).Yet, both
values were still considerably below 0.2, the generally accepted
threshold value for EFA deficiency in humans (12).
Relative fatty acid concentrations in liver were similar between
control and BDL rats for all major fatty acids (data not shown).
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Dietary fatty acid balance profile.
In Table 1, dietary fatty acid balance
data (ingestion, fecal excretion, and net absorption) for control and
BDL rats are shown. BDL rats excreted significantly more fatty acids
into feces compared with control rats (P < 0.001). The
overall lipid absorption percentage was significantly decreased in BDL
rats compared with control rats (53.7 ± 5.0% vs. 94.2 ± 0.6%; P < 0.001). On comparison of the percent uptake
of individual fatty acids from the diet (Fig.
2), the absorption of saturated fatty
acids (palmitic acid, stearic acid) appeared considerably more affected
by bile duct ligation than that of unsaturated species (oleic acid,
linoleic acid). Given the similar amounts of diets ingested by the two groups of rats, BDL rats had significantly decreased net fatty acid
absorption (Table 1, P < 0.001).
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Plasma 13C-fatty acid concentrations.
Absorption kinetics of saturated and unsaturated fatty acids were
studied by determining the appearance of 13C-fatty acids in
plasma after their duodenal administration to control and BDL rats
(Fig. 3). In control rats, plasma
[13C]linoleic acid and [13C]palmitic acid
concentrations increased within 1 h, reaching apparent maximum
values of 0.082 ± 0.014 and 0.068 ± 0.009% dose/ml plasma
at 6 and at 4 h after bolus administration, respectively. On bile
duct ligation, plasma 13C-fatty acid concentrations for
either [13C]palmitic acid or [13C]linoleic
acid were significantly lower than in controls (P < 0.001). Area under the curve (AUC) for the plasma appearance of [13C]linoleic acid was significantly lower in BDL rats
than in controls (after 3 h: 0.031 ± 0.019 vs. 0.157 ± 0.080% administered
dose · h1 · ml
1,
respectively; P < 0.05). A similar, equally
significant difference between BDL and control rats was observed for
the AUC for the plasma appearance of [13C]palmitic acid
(Fig. 3B).
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Metabolism of [13C]linoleic acid.
Cholestasis by bile duct ligation may not only affect linoleic acid
status via its intestinal absorption of EFA, it could also induce
alterations in EFA metabolism. The metabolism of linoleic acid can be
largely discriminated into its elongation and desaturation to LCPUFA
and its -oxidation. An in vivo estimation of elongation and
desaturation activity during bile duct ligation was obtained by
measuring plasma concentrations of [13C]arachidonic acid
and [13C]linoleic acid at 24 h after intraduodenal
administration of [13C]linoleic acid. At 24 h after
[13C]linoleic acid administration, BDL rats had a similar
ratio of plasma [13C]arachidonic acid to plasma
[13C]linoleic acid concentration compared with control
rats (0.53 ± 0.19 vs. 0.60 ± 0.13; not significant). In
accordance with these in vivo data,
6-desaturase
activity in vitro appeared not significantly different in hepatic
microsomes from control or BDL rats (32.9 ± 5.7 vs. 32.9 ± 9.2 pmol · min
1 · mg
protein
1, n = 5 and 4/group, respectively).
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DISCUSSION |
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The association between cholestatic liver disease and impaired status in plasma of EFA and LCPUFA is well known. However, the pathophysiological relationship between the two has remained unclear. From the aspect of cholestatic liver disease, it is not clear whether its effects on EFA and LCPUFA status are due to the decreased availability of intestinal bile, the (toxic) accumulation of compounds normally secreted into bile (e.g., bile salts) in the body, or both. Thus it has remained unresolved whether the mechanism(s) behind impaired EFA status involves decreased intestinal absorption of EFA, increased metabolism, or both. In the present study, we aimed to determine the influence of cholestatic liver disease on the intestinal absorption and metabolism of the major EFA, linoleic acid, in an animal model. One-week-old BDL rats had a significantly decreased percent absorption of dietary fat (54%). BDL rats did not ingest more food to compensate for their increased fecal lipid excretion, and the net uptake of dietary linoleic acid was decreased by ~28% compared with control rats. Our present data also indicate that postabsorptive metabolism of linoleic acid (elongation/desaturation, oxidation) is not profoundly affected in short-term (1 wk) BDL rats, compared with control rats.
The cholestatic condition is characterized both by decreased availability of intestinal bile and by (toxic) accumulation in the body of compounds that are normally secreted into bile, such as bile salts. The present data, in combination with our previous study (19), allow us to qualify the contributions of each of these two pathophysiological processes to EFA absorption during cholestasis. We (19) recently determined linoleic acid absorption in a rat model of chronic bile diversion. Bile diversion results in intestinal bile deficiency but, in contrast to bile duct ligation or cholestasis, it is not associated with the toxic accumulation of bile compounds in the body. In bile-diverted rats, the percentage of dietary linoleic acid absorption from the diet was 78% (19), a value very similar to the percent absorption presently found in BDL rats (82%). From these quantitative data, it can be inferred that the intestinal bile deficiency per se, either with (bile duct ligation) or without (bile diversion) toxic accumulation of bile components in the body, is responsible for the impaired efficiency of linoleic acid absorption.
The percent linoleic acid absorption from the diet differed for the various fatty acid species. Absorption percentage and net uptake decreased in BDL rats in the following order: linoleic acid > oleic acid > palmitic acid > stearic acid. These results are in accordance with those of Demarne et al. (8), who reported a decrease in the percentage of dietary palmitic acid and stearic acid absorption relative to that of oleic acid and linoleic acid in 10-day-old BDL rats fed diets containing 10% peanut oil. The selectivity of fatty acid absorption in bile duct ligation was confirmed by the decreased fecal excretion and, accordingly, increased net absorption of intraduodenally administered [13C]linoleic acid compared with [13C]palmitic acid in BDL rats. Despite the relative preservation of linoleic acid absorption, however, its quantitative uptake (in mmol/day) was reduced in BDL rats compared with controls. Whereas bile-deficient rats in our previous study (19) managed to maintain a net similar quantitative uptake of linoleic acid, at least in part by the ingestion of increased amounts of food, BDL rats appear not to compensate for the decreased efficiency of fat absorption. It was demonstrated previously (7) that appetite and chemosensory function are altered in human liver disease, although the underlying mechanisms have not been elucidated. It is tempting to speculate that the present observations in rats refer to an analogous phenomenon.
In addition to fecal balance data, the appearance of
13C-fatty acids in plasma was measured after their
intraduodenal administration. The plasma concentrations of either
[13C]linoleic acid or [13C]palmitic acid
were significantly decreased in BDL rats compared with controls. Given
the relative preservation of linoleic acid absorption measured by
balance techniques (Table 2), it can be speculated not only that the absorption is not limited to the proximal
segments of the intestine of BDL rats but also that more distal
segments are involved in its absorption, giving rise to altered plasma
kinetics (3, 4). To exclude the (theoretical) possibility
that absorbed linoleic acid would be distributed differently over the
various lipid classes in plasma (for example, triacylglycerols, phospholipids, free fatty acids), an analytical method was applied in
which all fatty acids present in plasma, either esterified or
unesterified, were determined simultaneously in a pooled fashion.
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As stated above, a quantitatively altered metabolism (i.e.,
desaturation/elongation, -oxidation) could contribute to the frequently observed impaired EFA and LCPUFA status in cholestatic patients. Socha et al. (24) suggested that the hepatic
microsomal desaturase/elongase activity needed for LCPUFA synthesis is
altered in patients with cholestatic liver disease. In the presently
used rat model of bile duct ligation, the conversion of
[13C]linoleic acid to [13C]arachidonic acid
appeared to be not different quantitatively compared with control rats.
At 24 h after its duodenal administration, the ratio between
[13C]arachidonic acid and [13C]linoleic
acid concentrations in plasma was similar in BDL and control rats. In
agreement with these in vivo data,
6-desaturase activity
was not significantly different as determined in hepatic microsomes
isolated from BDL and control rats. It is possible that the cholestatic
(BDL) condition affects the EFA status by quantitatively altering the
utilization of linoleic acid for energy purposes, i.e.,
-oxidation.
After administration of [13C]linoleic acid to BDL rats,
however, the plasma appearance and cumulative expiration of
13CO2 were affected to a similar extent (
80%
and
54%, respectively), indicating that
-oxidation of absorbed
linoleic acid was not greatly altered. In long-term BDL rats, a
decreased capacity in hepatic
-oxidation has been reported
(15, 16).
In summary, we conclude that a cholestatic condition in rats (short-term bile duct ligation) is associated with decreased net uptake of linoleic acid but with quantitatively rather unaffected metabolism of absorbed linoleic acid. Although differences between our experimental model and humans with cholestatic liver disease may apply, the present observations are in agreement with a decreased net uptake of linoleic acid in patients with chronic cholestatic disease, not as much due to a greatly decreased efficacy of its absorption per se, but to the associated decrease in food and linoleic acid ingestion (7, 21). A protracted decrease in the net uptake of dietary linoleic acid can be expected to eventually result in decreased linoleic acid levels. An implication of the present findings may be that the occurrence of EFA deficiency in patients with cholestatic liver disease can be counteracted by nutritional means, given the relatively preserved absorption efficiency of EFA. It remains to be demonstrated in patient studies, however, whether these implications can indeed be extrapolated from our present results in the experimental animal model used.
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ACKNOWLEDGEMENTS |
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H. J. Verkade is a Fellow of the Royal Netherlands Institute for Arts and Sciences.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. J. Verkade, Dept. of Pediatrics, Laboratory Center CMC IV, Rm. Y2115, Univ. Hospital Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands (E-mail: h.j.verkade{at}med.rug.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 January 2000; accepted in final form 16 June 2000.
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