Center for Liver, Digestive, and Metabolic Diseases, Pediatric Gastroenterology, Department of Pediatrics, University Hospital, 9700 RB Groningen, The Netherlands
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ABSTRACT |
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Essential
fatty acid (EFA) deficiency induces fat malabsorption, but the
pathophysiological mechanism is unknown. Bile salts (BS) and EFA-rich
biliary phospholipids affect dietary fat solubilization and chylomicron
formation, respectively. We investigated whether altered biliary BS
and/or phospholipid secretion mediate EFA deficiency-induced fat
malabsorption in mice. Free virus breed (FVB) mice received EFA-containing (EFA+) or EFA-deficient (EFA)
chow for 8 wk. Subsequently, fat absorption, bile flow, and bile
composition were determined. Identical dietary experiments were
performed in multidrug resistance gene-2-deficient
[Mdr2(
/
)] mice, secreting
phospholipid-free bile. After 8 wk, EFA
-fed wild-type
[Mdr2(+/+)] and
Mdr2(
/
) mice were markedly EFA deficient
[plasma triene (20:3n-9)-to-tetraene (20:4n-6) ratio >0.2]. Fat
absorption decreased (70.1 ± 4.2 vs. 99.1 ± 0.3%,
P < 0.001), but bile flow and biliary BS secretion increased in EFA
mice compared with EFA+
controls (4.87 ± 0.36 vs. 2.87 ± 0.29 µl · min
1 · 100 g body
wt
1, P < 0.001, and 252 ± 30 vs.
145 ± 20 nmol · min
1 · 100 g body
wt
1, P < 0.001, respectively). BS
composition was similar in EFA+- and EFA
-fed
mice. Similar to EFA
Mdr2(+/+)
mice, EFA
Mdr2(
/
) mice
developed fat malabsorption associated with twofold increase in bile
flow and BS secretion. Fat malabsorption in EFA
mice is
not due to impaired biliary BS or phospholipid secretion. We
hypothesize that EFA deficiency affects intracellular processing of
dietary fat by enterocytes.
fat absorption; multidrug resistance gene-2; ATP-binding cassette; polyunsaturated fatty acids; phospholipid; bile salt
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INTRODUCTION |
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ESSENTIAL FATTY
ACID (EFA) deficiency (EFA) has been associated
with several pathological consequences in humans and experimental animals, including fat malabsorption (1, 2, 15, 21, 28).
The pathophysiological mechanisms underlying EFA
-induced
fat malabsorption have not been elucidated. To address this issue, the
consecutive intraluminal and intracellular steps in the absorption
process have been investigated in control and EFA
rats,
i.e., lipolysis of dietary triglycerides by lipases, solubilization of
lipolytic products by bile components, uptake by the enterocyte and
intracellular reesterification to triglycerides and, finally, chylomicron formation and secretion into lymph (2,
21). Available data from rat models of EFA deficiency
have indicated that neither fat digestion nor fatty acid uptake by the
enterocyte is responsible for the fat malabsorption encountered in EFA
deficiency (2, 21). In rats, EFA deficiency resulted in
significantly decreased bile flow and biliary secretion of bile salts,
phospholipids, and cholesterol (20, 21). Acyl chain
analysis of biliary phosphatidylcholine (PC) from EFA
rats revealed that biliary PC contents of linoleic acid (18:2n-6) and
arachidonic acid (20:4n-6) were decreased to 10 and 26% of control
values, respectively, and were replaced by oleic acid (18:1n-9)
(2). In addition to alterations in bile, intracellular events, such as fatty acid reesterification and chylomicron production, were severely impaired (2, 21). It thus seems that EFA
deficiency in rats affects bile formation and/or intracellular
processing of fat by the enterocyte, resulting in fat malabsorption. In
a previous study (32), we demonstrated the role of biliary
PC in intestinal chylomicron formation, using multidrug
resistance gene-2 Mdr2(
/
) mice [gene
symbol: ATP-binding cassette-B4 (ABC-B4)] that are unable to secrete phospholipids into bile (26). We
reasoned that this animal model could be important to further delineate the pathophysiological mechanism of EFA
-induced fat malabsorption.
If altered biliary phospholipid secretion were involved in
EFA-associated fat malabsorption, one would expect fat
absorption in Mdr2(
/
) mice to be relatively
unaffected by changes in the EFA status. If, however, the
pathophysiological mechanism would not involve alterations in biliary
phospholipid secretion, EFA deficiency would be expected to equally
affect the process in Mdr2(
/
) and in
wild-type [Mdr2(+/+)] mice. In the present
study, we investigated the role of bile formation in general and of
biliary phospholipid secretion in particular in the pathophysiology of
EFA
-associated fat malabsorption. We first developed and
characterized a murine model for EFA deficiency with respect to fat
absorption and bile formation. We then compared fat absorption and bile
formation in EFA
and EFA-sufficient (EFA+)
Mdr2(
/
) mice.
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MATERIALS AND METHODS |
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Animals
Mice homozygous for disruption of the Mdr2(Experimental Procedures
Fat absorption and bile secretion in
EFA mice.
FVB mice (n = 14) were fed standard low-fat chow (6 weight% fat, 14 energy% fat) for standardization for 1 mo
after which they were anesthetized with halothane and a baseline blood
sample was obtained by tail bleeding for determination of EFA status. Blood was collected in microhematocrit tubes containing heparin, and
plasma was separated with an Eppendorf centrifuge at 9,000 rpm for 10 min and stored at
20°C until analysis. Subsequently, mice were
randomly assigned to either an EFA+ or an EFA
diet. The EFA+ and EFA
diets were isocaloric
and contained 16 weight% fat. The EFA+ chow contained 20, 34, and 46 energy% from protein, fat, and carbohydrate, respectively,
and had the following fatty acid profile: 32 mol% palmitic acid
(16:0), 6 mol% stearic acid (18:0), 32 mol% oleic acid (18:1n-9), and
30 mol% linoleic acid (18:2n-6) (custom synthesis; Hope Farms). The
EFA
diet had identical energy percentages derived from
protein, fat, and carbohydrate, and had the following fatty acid
composition: 41 mol% 16:0, 48 mol% 18:0, 8 mol% 18:1n-9, and 3 mol%
18:2n-6 (custom synthesis; Hope Farms). At intervals of 2 wk, blood
samples were taken in the manner described above. After 4 and 8 wk of experimental diet, a 72-h fecal fat balance was performed involving quantitative feces collection and determination of chow intake. At 8 wk, mice were anesthetized by intraperitoneal injection of Hypnorm
(fentanyl/fluanisone) and diazepam, and gallbladders were cannulated
for collection of bile for 1 h as described previously (31). At the end of bile collection, a large blood sample
(0.6-1.0 ml) was obtained by heart puncture.
Hepatic expression of Cyp7A and Cyp27.
A separate group of male FVB mice was fed EFA+ or
EFA chow for 8 wk (n = 6 mice per dietary
group). After 8 wk, animals were anesthetized with halothane, and blood
was collected by cardiac puncture for lipid analysis. Livers were
removed, and liver samples were immediately frozen in liquid nitrogen
and stored at
80°C for determination of mRNA levels of two key
enzymes in bile salt biosynthesis, cholesterol 7
-hydroxylase (Cyp7A)
and sterol 27-hydroxylase (Cyp27).
Plasma accumulation of oral [3H]triolein and
[14C]oleic acid after Triton WR-1339 injection.
In a separate experiment, male FVB mice fed EFA+ or
EFA chow for 8 wk (n = 5 per group) were
intravenously injected with 12.5 mg Triton WR-1339 (12.5 mg/100 µl
PBS) to block lipolysis of circulating lipoproteins in the blood.
Subsequently, an intragastric fat bolus was administered containing 200 µl olive oil, in which 10 µCi glycerol
tri-[9,10(n)-3H]oleate ([3H]triolein)
(Amersham, Arlington Heights, IL) and 2 µCi [14C]oleic
acid (NEN Laboratories, Boston, MA) were dispersed. Before (t = 0) and at 1, 2, 3, and 4 h after
label administration, blood samples (75 µl) were taken by tail
bleeding as described above. The content of 3H
{[3H]} and 14C concentrations
{[14C]} in plasma (25 µl) was measured by
scintillation counting (Packard Instruments, Downers Grove, IL).
Plasma appearance of retinyl palmitate after retinol
administration.
In addition to the absorption of fatty acids during EFA deficiency, the
plasma appearance of the less polar lipid molecule retinol (vitamin A)
was investigated. Retinol (5,000 IU) in an olive oil bolus (100 µl
per mouse) was administered intragastrically to EFA mice
and control mice (n = 7 per group), and blood samples
were taken at 0, 2, and 4 h after bolus administration.
Fat absorption and bile secretion in
EFA Mdr2(
/
) mice.
Mdr2(
/
) mice (n = 14) were
fed low-fat chow (6 weight% fat; 14 energy% fat) for standardization
for 1 mo. Baseline blood samples were taken for determination of plasma
EFA status, after which mice were randomly assigned to either the
EFA+ or the EFA
diet. Diets were identical to
those described above. After 8 wk of feeding the respective diets,
blood samples were obtained by tail bleeding under halothane
anesthesia. As in the Mdr2(+/+) mice,
fat absorption was measured by means of a 72-h fecal fat balance.
Retinol in a bolus of olive oil was administered intragastrically to
EFA
Mdr2(
/
) mice
(n = 7) and their EFA+ controls
(n = 7). Blood samples were taken at 0, 2, and 4 h
after bolus administration. Gallbladders were cannulated, and bile was collected for 1 h as described above (31). At the end
of bile collection, mice were killed after obtaining a large blood
sample (0.6-1.0 ml) by heart puncture.
Analytical Techniques
Fatty acid status was analyzed by extracting, hydrolyzing, and methylating total plasma lipids, liver homogenates, and biliary lipids according to the method described by Lepage and Roy (19). To account for losses during lipid extraction, heptadecaenoic acid (C17:0) was added to all samples as an internal standard before extraction and methylation procedures, and butylated hydroxytoluene was added as an antioxidant. Fatty acid methyl esters were separated and quantified by gas liquid chromatography on a Hewlett Packard model 6890 gas chromatograph, equipped with a 50 m × 0.2 mm Ultra 1 capillary column (Hewlett Packard, Palo Alto, CA) and a flame ionization detector. The injector and detector were set at 260 and 250°C, respectively. The oven temperature was programmed from an initial temperature of 160°C to a final temperature of 290°C in three temperature steps (160°C, held 2 min; 160-240°C, ramp 2°C/min, held 1 min; 240-290°C, ramp 10°C/min, held 10 min). Helium was used as a carrier gas with a constant flow rate of 0.5 ml/min. Individual fatty acid methyl esters were quantified by relating the areas of their chromatogram peaks to that of the internal standard heptadecaenoic acid (C17:0).Plasma lipid levels (cholesterol, HDL-cholesterol, triglycerides, free fatty acids, and phospholipids) were measured using commercially available kits (Roche Diagnostics, Mannheim, WAKO Chemicals, Neuss, Germany) according to the instructions provided. Cholesterol, cholesterol ester, and triglycerides in liver tissue were determined after Bligh and Dyer (4) lipid extraction, and biliary bile salt composition was measured as described previously. Total protein concentrations of liver homogenates were determined according to the method described by Lowry et al. (23).
Total RNA was isolated from liver tissue using TRIzol reagent (GIBCO-BRL, Grand Island, NY) according to the manufacturer's instructions. Single-stranded cDNA was synthesized from 4.5-µg RNA and subsequently subjected to quantitative real-time detection RT-PCR (12, 14). The following primers and probes were used: for Cyp7A (GenBank accession no. L23754): 5'-CAG GGA GAT GCT CTG TGT TCA-3' (forward primer), 5'-AGG CAT ACA TCC CTT CCG TGA-3' (reverse primer), 5'-TGC AAA ACC TCC AAT CTG TCA TGA GAC CTC C-3' (probe).
For Cyp27 (M73231, M62401): 5'-GCC TTG CAC AAG GAA GTG
ACT-3' (forward primer), 5'-CGC AGG GTC TCC TTA ATC ACA-3' (reverse primer), 5'-CCC TTC GGG AAG GTG CCC CAG-3' (probe); and for
-actin (M12481): 5'-AGC CAT GTA CGT AGC CAT CCA-3'
(forward primer), 5'-TCT CCG GAG TCC ATC ACA ATG-3' (reverse primer),
5'-TGT CCC TGT ATG CCT CTG GTC GTA CCA C-3' (probe). For each real-time
PCR, 4-µl cDNA was used in a final volume of 20 µl, containing (in nmol/l) 500 of forward and reverse primers and 200 of probe, 250 MgCl2, 10 deoxyribonucleoside triphosphate mix, and 5 µl
real-time PCR buffer (10×), and 1.25 U Hot GoldStar (Eurogentec).
Real-time detection PCR was performed on the ABI PRISM 7700 (PE Applied Biosystems) initialized by 10 min at 95°C to denature the
complementary DNA followed by 40 PCR cycles each at 95°C for 15 s and 60°C for 1 min.
Feces and chow pellets were freeze-dried and then mechanically homogenized. From aliquots of each, lipids were extracted, hydrolyzed, and methylated (19). Resulting fatty acid methyl esters were analyzed by gas chromatography for their fatty acid content as described above. Fatty acids were quantified using heptadacaenoic acid (17:0) as the internal standard.
Retinyl palmitate concentrations in plasma were determined after two extractions with hexane (5). Retinyl acetate was added to plasma samples as an internal standard before lipid extraction. Samples were resuspended in ethanol and analyzed by reverse-phase HPLC using a 150 × 4.6 mm Symmetry RP18 column (Waters, Milford, MA) (36). The peak area of retinyl palmitate was normalized to that of retinyl acetate. At each time point, concentrations were expressed as micromoles of retinyl palmitate per liter of plasma.
Calculations
Fatty acid status in plasma and in bile. Relative concentrations (mol%) of plasma, liver, and biliary phospholipid fatty acids were calculated using the summed areas of major fatty acid peaks (palmitic, stearic, oleic, linoleic acids) and then expressing the area of each individual fatty acid as a percentage of this amount. EFA deficiency was determined by calculating the triene-to-tetraene ratio (20:3n-9/20:4n-6) in plasma of mice. A ratio of >0.2 was considered deficient (16).
Fatty acid absorption using 72-h balance techniques. Absorption of major dietary fatty acids (palmitic, stearic, oleic, linoleic acids) was determined by subtracting the amount of each individual fatty acid excreted in feces in 72 h, from the amount of this dietary fatty acid ingested in 72 h (net fat absorption). This quantity was subsequently expressed as a percentage of the amount of fatty acid ingested in 72 h [coefficient of fat absorption; 100-fold the ratio between (amount ingested and the amount recovered in feces) and the amount ingested]. Net amount of fat absorption was calculated by subtracting the fecal loss of the four major fatty acids from the amount ingested.
Statistics
All results are presented as means ± SD for the number of animals indicated. Data were statistically analyzed using Student's two-tailed t-test. The level of significance for all statistical analyses was set at P < 0.05. Analyses were performed using SPSS for Windows software (SPSS, Chicago, IL). ![]() |
RESULTS |
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Body Weight and Chow Ingestion in
EFA and
EFA+
Mdr2(+/+)
Mice.
Essential Fatty Acid Status
Triene-to-tetraene ratio in plasma and liver.
The classic biochemical parameter describing EFA status, the
triene-to-tetraene ratio (20:3n-9/20:4n-6), was measured in plasma at
baseline and every 2 wk for 8 wk and in liver after 8 wk of feeding
EFA chow. The baseline plasma triene-to-tetraene ratio
was 0.02 ± 0.00, which is well below the cutoff value for EFA
deficiency (0.20). Already after the 2 wk on EFA
diet,
plasma triene-to-tetraene ratios approached the cutoff value for EFA
deficiency, i.e., 0.19 ± 0.08 for EFA
-fed mice vs.
0.01 ± 0.00 for controls. After 8 wk on EFA
chow,
mice had a pronounced EFA deficiency with triene-to-tetraene ratios of
0.66 ± 0.05 for plasma and 0.56 ± 0.09 for liver, in contrast to control mice (0.01 ± 0.00 for plasma;
P < 0.001; and 0.02 ± 0.00 for liver;
P < 0.001) (data not shown).
Characterization of EFA Deficiency in Mice
Because EFA deficiency has not been characterized before in a murine model, plasma and liver lipids were measured. No differences were found in cholesterol, HDL-cholesterol, free fatty acid or phospholipid concentrations in plasma between EFA+- and EFA
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Fat Absorption
Fecal fatty acid balance.
The fecal fat balance revealed a decreased absorption of total dietary
fat in EFA mice compared with their EFA+-fed
controls (P < 0.01) (Fig.
1). The absorption coefficient for
EFA
mice was 70.1 ± 1.6% compared with absorption
coefficients above 95% for mice fed EFA+ chow. Individual
fatty acid balances for palmitic, stearic, oleic, and linoleic acids
were calculated (Fig. 1). In EFA
mice, absorption of
saturated fatty acids (palmitic and stearic) was more affected than
absorption of unsaturated fatty acids (oleic and linoleic).
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Bile Secretion and Composition
Table 3 shows that bile flow and biliary secretion of bile salt, cholesterol, and phospholipids during a 1-h period immediately after interruption of the enterohepatic circulation were higher in EFA
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Cyp7A and Cyp27 mRNA Levels
To determine whether the increased bile salt secretion in EFA
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Plasma Accumulation of [3H]Triolein and [14C]Oleic Acid After Triton WR-1339 Administration
Figure 3 shows the time course of plasma [3H] and [14C] radioactivity after intragastric administration of [3H]triolein and [14C]oleic acid. EFA
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Plasma Appearance Of Retinyl-Palmitate After Retinol Administration
The absorption of dietary oleic acid, a polar lipid (class III, soluble amphiphile) (30) was only mildly impaired in EFA
|
Body Weight and Chow Ingestion in
EFA and
EFA+
Mdr2(
/
) Mice
EFA Status
Triene-to-tetraene ratio in plasma and liver.
The baseline triene-to-tetraene ratios (20:3n-9/20:4n-6) in plasma and
liver were significantly higher in Mdr2(/
)
compared with Mdr2(+/+) mice (0.035 ± 0.003 vs. 0.018 ± 0.001; P < 0.001) but were
still well below the cutoff value for EFA deficiency (0.2).
Mdr2(
/
) mice fed EFA
chow for
8 wk developed EFA deficiency according to triene-to-tetraene ratios in
plasma (0.46 ± 0.03) and in liver (0.38 ± 0.04) in contrast to the EFA+-fed Mdr2(
/
) controls
(0.01 ± 0.01 for plasma, P < 0.001; and
0.02 ± 0.00 for liver, P < 0.01).
Characterization of EFA deficiency in Mdr2(/
) mice.
Plasma concentrations of cholesterol and phospholipid were increased,
whereas the plasma triglyceride level was decreased in
EFA
Mdr2(
/
) mice compared with
their EFA+-fed controls (P < 0.05) (Table
5). Similar to the situation in
Mdr2(+/+) mice, a pronounced hepatic fat
accumulation characterized by increased levels of triglyceride,
unesterified and esterified cholesterol, was observed in
EFA
Mdr2(
/
) mice (Table
6).
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Bile Secretion and Composition
As in EFA
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DISCUSSION |
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We investigated the mechanism of fat malabsorption in EFA
deficiency in mice with particular emphasis on the possible role of
altered bile formation as proposed by Levy et al. (20, 21) based on studies in rats. To specify the contribution of biliary phospholipid secretion, studies were performed in EFA and
EFA+ Mdr2(+/+) and
Mdr2(
/
) mice; the latter are unable to
secrete phospholipids into their bile. Yet, we first had to develop and
characterize a murine model for EFA deficiency. Our data indicate that
dietary fat absorption is reduced in EFA
mice compared
with EFA+ controls. The mechanism underlying this EFA
deficiency-associated fat malabsorption does not likely involve
alterations in bile formation (including bile flow, bile salt secretion
rate, bile salt composition, or phospholipid secretion rate) nor
changes in fat digestion (lipolysis). Rather, our data strongly
indicate that EFA deficiency in mice affects intracellular events of
fat absorption that occur in the enterocyte.
Biochemical EFA deficiency conventionally defined by a molar ratio of
eicosatrienoic acid (20:3n-9)-to-arachidonic acid (20:4n-6) above 0.20 in plasma (16) was already reached after only 2 wk of
EFA diet feeding to mice. This rapidity of onset makes
the mouse an attractive and versatile model for studying EFA deficiency.
Similar to EFA rats (3, 34),
EFA
mice experienced changes in plasma and liver fat
contents. Specifically, EFA
mice have decreased plasma
triglycerides and increased hepatic triglyceride and cholesterol
levels. In EFA
rats, Wanon et al. (33)
observed alterations in HDL composition, with defective translocation
of HDL cholesterol into bile and concomitantly increased hepatic very
low-density lipoprotein (VLDL)-cholesterol secretion. Lipoprotein
abnormalities were also found by Levy et al. (22) in
EFA
cystic fibrosis (CF) patients. Compared with their
EFA+ counterparts, EFA
CF patients had
increased plasma triglyceride levels (specifically in the VLDL, LDL,
HDL2, and HDL3 fractions) and decreased plasma HDL- and LDL-cholesterol. Also, lipoprotein size was altered in these
EFA
patients, with larger VLDL, LDL, and HDL2
particles and smaller HDL3 particles. It could be
speculated that EFAs, as constituents of triglycerides, phospholipids,
and cholesterol esters, may be essential for regulation of lipoprotein
metabolism. Although the effects of EFA deficiency are species
specific, it appears that an adequate EFA status is required for
efficient intestinal and hepatic processing of lipoproteins.
In addition to the changes in plasma and liver lipids, EFA deficiency
in mice was associated with fat malabsorption. The coefficients of fat
absorption in EFA mice (60-70%) were somewhat lower
than corresponding values in EFA
rats (80-90%)
(2, 15, 28). In all of these studies, EFA deficiency was
induced by feeding the animals high-fat EFA
diets almost
entirely composed of saturated fatty acids. Apart from species
specificity, the difference in coefficients of fat absorption could be
related to the amount and type of fat in the diet. In the present
study, mice were fed high-fat chow (16 weight%), whereas Hjelte et al.
(15) used chow diets containing only 7 weight% fat. Levy
et al. (21) reported that lipolytic activity in
EFA
rats was unchanged compared with control rats. Our
present results in EFA
mice are compatible with this
observation. The appearance of [3H]triolein in plasma
after its intragastric administration was similar in EFA
mice and control mice. If anything, the [3H] label was
recovered from plasma of EFA
-fed mice even at higher
concentrations compared with EFA+-fed controls. The
explanation of this phenomenon may involve the choice of the lipid
oleic acid. The absorption of dietary oleic acid was only mildly
impaired during EFA deficiency (Fig. 1). In addition, a tracer effect
could not be excluded. When we investigated the absorption of a less
polar lipid (retinol) after its intragastric administration, both
EFA
Mdr2(+/+) and
EFA
Mdr2(
/
) mice showed
decreased plasma concentrations compared with their EFA+
controls, which underlines the occurrence of lipid malabsorption during
EFA deficiency.
Rather than differences in fat digestion (lipolysis), it could have
been expected that alterations in bile formation contributed to fat
malabsorption in EFA mice, in analogy to the situation in
EFA
rats (20). The pathophysiology of EFA
deficiency-associated fat malabsorption in mice could be due to
decreased biliary bile salt secretion rates, analogous to previous data
in rats. However, bile flow and biliary bile salt and phospholipid
secretion rates were increased during EFA deficiency. Robins and Fasulo
(27) reported that EFA
hamsters have
increased hepatic bile flow and biliary bile salt and cholesterol
secretion compared with controls. It is not known, however, whether
EFA
hamsters have a decreased coefficient of fat
absorption. Our observation in EFA
mice, together with
the available data on EFA
rats and hamsters, indicate
that the effects of EFA deficiency on bile formation are species
specific. Present data exclude the fact that EFA
fat
malabsorption is due to decreased rates of biliary bile salt secretion
in mice, in contrast to the situation in rats (20, 21).
Theoretically, an increase in the contribution of hydrophilic bile
salts (to total bile salts) could contribute to impaired solubilization
of dietary fats. Yet, biliary bile salt composition was virtually
unchanged in EFA
mice compared with controls.
The increased biliary secretion rate of bile salts, immediately after interruption of the enterohepatic circulation, strongly suggests an expansion of the bile salt pool size in EFA deficiency.
Bile salts negatively affect their own biosynthesis by repressing the
expression of Cyp7A via the FXR-SHP1-LRH1-Cyp7A pathway with
Cyp7 encoding the enzyme cholesterol 7A-hydroxylase that catalyzes the first step of the neutral pathway in bile salt synthesis (6-8, 24, 25). In our experiments, however,
Cyp7A and Cyp27 mRNA levels were similar in
livers from EFA+- and EFA
-fed mice. We
speculate that EFA deficiency in mice impairs the capacity of bile
salts to exert negative feedback inhibition on their own hepatic
biosynthesis, but the mechanism hereof remains unclear.
Not only biliary bile salts but also biliary phospholipids play a role
in dietary fat absorption, e.g., in supplying surface components for
the assembly of chylomicron particles in enterocytes (17,
29). Under physiological conditions, overall fat absorption in
Mdr2(/
) mice is only slightly decreased
compared with control mice (95 vs. 98%), based on 72-h fecal fat
balance measurements. On the other hand, kinetics of chylomicron
formation are clearly delayed in Mdr2(
/
)
mice (32). Therefore, a quantitative alteration in biliary phospholipid secretion was not likely to contribute to EFA
deficiency-associated fat malabsorption. Yet, fat malabsorption during
EFA deficiency could still be due to qualitative changes in biliary
phospholipid composition. Replacement of polyunsaturated acyl chains
(linoleoyl, arachidonoyl) by saturated or monounsaturated species could
theoretically be responsible for impaired chylomicron assembly and
secretion. In accordance with findings by Bennett Clark et al.
(2) in EFA
rats, the acyl chain composition
of biliary PC in EFA
mice showed less essential (i.e.,
18:2n-6, 20:4n-6) and more non-EFAs (i.e., 18:1n-9, 18:1n-7, 16:1n-7).
If acyl chain composition of bile phospholipids were important for fat
malabsorption during EFA deficiency, one would expect that EFA
deficiency would not or to a much lesser extent affect fat absorption
in Mdr2(
/
) mice. Present data, however,
clearly indicate that fat absorption in EFA
Mdr2(
/
) mice was affected similarly as in
EFA
Mdr2(+/+) mice. In
EFA
Mdr2(
/
) mice, as in
EFA
Mdr2(+/+) controls, bile flow
and biliary bile salt secretion were increased. On the basis of the
similar fat absorption coefficients we found in EFA
mice
with and without biliary phospholipid secretion, we conclude that the
effect of biliary PC acyl chain composition is not of pathophysiological relevance for EFA deficiency-associated fat malabsorption.
Rather than due to alterations in bile secretion, fat malabsorption
during EFA deficiency may be caused by other steps involved in fat
absorption. Intestinal mucosal phospholipids normally contain large amounts of 18:2n-6 and 20:4n-6, and during EFA deficiency, the levels of these fatty acids are markedly decreased (9, 10,
35). The resultant structural changes in membranes, and the
increased cellular turnover rate in the intestinal mucosa reported in
EFA rats (28) could be responsible for
decreased dietary fat absorption. Based on our study and previous
studies (2, 21) in EFA
rats, the
intraluminal events involved in fat absorption (i.e., lipolysis of
dietary triglyceride by pancreatic lipase, solubilization of lipolytic
products, and uptake by the enterocyte) seem to be relatively
undisturbed in EFA deficiency. By inference, it is, therefore, more
likely that defects in one of the several intracellular events (i.e.,
reesterification, chylomicron assembly, and/or chylomicron secretion)
are involved in EFA deficiency-associated fat malabsorption.
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ACKNOWLEDGEMENTS |
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We thank Renze Boverhof and Christian Hulzebos for excellent technical assistance.
![]() |
FOOTNOTES |
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* A. Werner and D. M. Minich contributed equally to this work.
Address for reprint requests and other correspondence: H. J. Verkade, Pediatric Gastroenterology, Dept. Pediatrics, Research Laboratory CMC IV, Room Y2115, PO Box 30001, 9700 RB Groningen, The Netherlands.
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.
10.1152/ajpgi.00094.2002
Received 11 March 2002; accepted in final form 3 June 2002.
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