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Essential fatty acid deficiency and the small intestineLukovac, Sabina

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CHAPTER 6

GELUCIRE®44/14 IMPROVES FAT ABSORPTION IN RATS WITH

IMPAIRED LIPOLYSIS

S. Lukovac1, K.E.G. Gooijert

1, P.C. Gregory

2, G. Shlieout

2, F. Stellaard

1, E.H.H.M.

Rings1, H.J. Verkade

1

(1) Pediatric Gastroenterology, Department of Pediatrics, Beatrix Children’s Hospital,

Groningen University Institute for Drug Exploration (GUIDE), Center for Liver, Digestive

and Metabolic Diseases, University of Groningen, University Medical Center Groningen,

Groningen, The Netherlands.

(2) Solvay Pharmaceuticals GmbH, Hannover, Germany.

Manuscript conditionally accepted for publication in Biochimica et Biophysica

Acta (BBA) Molecular and Cell biology of lipids

CHAPTER 6

112

ABSTRACT

Clinically relevant fat malabsorption is usually due to impaired intestinal fat digestion

(lipolysis) and/or to impaired solubilization of the lipolytic metabolites. We hypothesized

that Gelucire®44/14 –a semi-solid self-micro-emulsifying excipient– could increase fat

absorption. In relevant rat models for impaired lipolysis or for impaired solubilization we

tested whether administration of Gelucire®44/14

enhanced fat absorption. Rats with

impaired lipolysis (lipase inhibitor Orlistat-diet) and rats with reduced solubilization

(permanent bile diversion) underwent a 72h fat balance test to assess fat absorption.

The absorption kinetics of a stable isotope-labeled fatty acid was assessed in rats with

reduced solubilization, in the presence or absence of Gelucire®44/14. Gelucire

®44/14

improved fat absorption in rats with impaired lipolysis (from 70% to 82%, p<0.001). In

rats with reduced solubilization, Gelucire®44/14 did not increase fat absorption nor did it

reconstitute the absorption kinetics of 13

C-labeled palmitate, compared with control rats

administered buffer without Gelucire®44/14.

The present data show that Gelucire®44/14 might enhance fat absorption under

conditions of impaired lipolysis, but not during impaired solubilization. We speculate that,

due to its self-micro-emulsification properties, Gelucire®44/14 stabilizes and improves

residual lipolytic enzyme activity in vivo, which could be of therapeutic value in clinical

conditions of fat malabsorption due to impaired lipolysis.

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INTRODUCTION

Dietary fat is mainly composed of triglycerides, which undergo several intraluminal

processes before their absorption in the form of fatty acids and monoacylglycerides by

the small intestinal enterocytes.1 The first step of fat absorption involves emulsification

and hydrolysis by gastric lipase, which results in partial hydrolysis of triglycerides into

free fatty acids and diglycerides. The remaining partially digested and undigested,

triglycerides are digested by pancreatic lipases in the small intestine, which leads to

lipolysis into free fatty acids and monoglycerides. Subsequent steps involve micellar

solubilization with bile salts, phospholipids and cholesterol, transport to the enterocytes,

and translocation of the fatty acids and monoacylglycerides across the apical brush

border of the enterocytes. Under physiological conditions the pancreas produces

sufficient amounts of pancreatic lipases. However, under conditions of severe pancreatic

insufficiency, lipolysis may be incomplete and fat malabsorption occurs.2

Cystic fibrosis (CF), a common autosomal recessive disorder, is a condition in which

pancreatic secretory function is frequently affected.3 On top of impaired lipolysis, CF

patients often have solubilization defects, which may be due to changes in bile

production and composition, impaired pancreatic and intestinal bicarbonate secretion,

and/or changes in the intestinal microclimate.4,5,6

The combination of impaired lipolysis

and solubilization in CF patients can lead to severely reduced absorption of dietary fats

and to essential fatty acid deficiency.7,8

Several attempts to correct for low fat absorption,

for example with pancreatic enzyme replacement therapies and linoleic acid

supplementations, have shown variable effects in CF patients.9,10,11,12

Gelucire®44/14 is a semi-solid, self-emulsifying excipient frequently used in the

pharmaceutical industry as an enhancer of absorption of poorly soluble and poorly

bioavailable drugs.13,14

Gelucire®44/14 is composed of surfactants (mono- and diesters

of polyethylene glycol), co-surfactants (monoglycerides), and an oily phase (di- and

triglycerides). In vitro, Gelucire®44/14 has been shown to maintain the activity of

pancreatic enzymes under unfavorable conditions at low pH (Patent WO 2005/092370).

Moreover, Fernandez et al. have demonstrated that Gelucire®44/14 is a good substrate

for digestive enzymes.15

However, it remains unclear whether the efficacy of

Gelucire®44/14 is exclusively related to increasing the acid stability of pancreatic lipase,

and thereby to increasing lipolysis of dietary lipids. Alternatively, the mechanism of fat

malabsorption in CF is not exclusively related to impaired lipolysis. Thus it seems

feasible that Gelucire®44/14 might enhance net fat absorption by increasing the

solubilization of the lipolytic metabolites (free fatty acids and monoglycerides) and

thereby improve present CF therapy. Therefore, studies have been performed to test the

effects of Gelucire®44/14 on fat absorption in animals with induced fat malabsorption. In

order to address the potential, specific roles of Gelucire®44/14 in lipolysis and

solubilization of fat in vivo, we used validated rat models for either impaired lipolysis or

for severely reduced solubilization. Rats fed the lipase-inhibitor Orlistat (Xenical®) have a

selectively inhibited hydrolysis of dietary triglycerides, but unaffected

solubilization.16,17,18,19

On the other hand, rats with permanent bile diversion (BDD rats)

are a well characterized model to assess fatty acid uptake under condition of

(exclusively) reduced solubilization.18,20

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114

MATERIAL AND METHODS

Compounds

Taurocholate, phosphatidylcholine and cholesterol were purchased by Sigma Chemical

(St. Louis, USA). 13

C-labeled palmitic acid (C16:0) was purchased from Isotec Inc.

(Matheson, USA). Gelucire®44/14 was a generous gift from Solvay Pharmaceuticals

GmbH (Hannover, Germany). Orlistat (tetrahydrolipstatin, Xenical®) was obtained as

capsules containing 120 mg active compound from Roche Nederland B.V. (Mijndrecht,

The Netherlands).

Animals and diets

Male Wistar rats (Harlan, Zeist, The Netherlands), weighing 300-350 g, were housed in a

light-controlled (lights on 7 AM - 7 PM) and temperature-controlled facility with free

access to food and tap water, and, in the case of bile-diverted rats, saline (0.9% NaCl

w/v). The experimental protocol was approved by the Ethics Committee for Animal

Experiments, Faculty of Medical Sciences, University of Groningen, The Netherlands.

A semi-synthetic high-fat diet containing 16 weight% fat (4141.07) and the same diet

containing Orlistat (4141.07 + 200 mg/kg Orlistat) were produced by Arie Blok BV

(Woerden, The Netherlands). The diet contained 35 energy% fat and 16.2 wt% long-

chain fatty acids (fatty acid composition (in mol%): palmitic acid (C16:0), 39.0%; stearic

acid (C18:0), 4.0%; oleic acid (C18:1n-9), 31.7%; linoleic acid (C18:2n-6), 22.9%).

Gelucire®44/14 (1 wt% or 2 wt%) was mixed into the semi-synthetic high-fat diet or into

the semi-synthetic high-fat diet containing Orlistat.

Infusates and intraduodenal infusions

Infusates and bolus were prepared as described previously.20

Buffer contained 10 mM

HEPES and 135 mM NaCl (negative control). Model bile contained 60 mM taurocholate,

8 mM phosphatidyl choline and 1 mM cholesterol (positive control). Gelucire®44/14-

infusates contained buffer with 0.1% or 0.5% Gelucire®44/14.

Bolus (500 μl) was administered intraduodenally and composed of olive oil (25%),

medium chain triglyceride oil (75%; (composed of extracted coconut oil and synthetic

triglycerides; fatty acid composition: 6:0, 2%; 8:0, 50-65% max.;10:0, 30-45%; 12:0, 3%

max.) and 10 mg of 13

C-labeled palmitic acid (> 99% enriched) per 300 g body.20,18

Fat balance study in rats with impaired lipolysis

After a run-in period of two weeks on the semi-synthetic high-fat diet (4141.07) the fat

absorption was assessed during a 72 hours period in individually housed rats.

Subsequently, the rats fed the Orlistat containing diet for two weeks. At the end of the

two weeks, fat absorption test was performed again. Consistent with previous studies

with Orlistat feeding in rats, two weeks of Orlistat-diet (200 mg/kg) was sufficient to

decrease the net fat absorption.17

Next, one group of rats received the Orlistat-diet with

additional 1 wt% Gelucire®44/14 and another group of rats received the Orlistat-diet with

2 wt% Gelucire®44/14 for one additional week. At the end of the experimental week food

intake was determined and feces were collected for the assessment of the fat absorption

upon Gelucire®44/14 feeding. Net fat absorption was determined by measuring the fatty

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acid intake and excretion by fatty acid methylation as described in section “Analytical

techniques”. The experimental set up is shown in detail in Figure 1a.

Fat balance study in rats with reduced solubilization

Rats were individually housed and were fed a semi-synthetic high-fat diet (4141.07) for a

run-in period of 1 week. At the end of the first week, a 72h fat balance was performed

(intact EHC; control situation). During this 72h period, feces were collected and chow

intake was measured. Subsequently, rats received a permanent bile duct catheter, by

means of a surgical procedure as previously described by Kuipers et al.21

After the

recovery period of three days, the rats were fed the same diet as before the surgery and

after one week of diet 72h fat balance was performed (BDD; interrupted EHC).

Afterwards, rats with BDD were separated into 2 different groups receiving the same diet

as before the surgery supplemented with either 1 wt% or 2 wt% Gelucire®44/14, and

after two weeks of diet fat balance was determined (BDD 1% Gelucire®44/14 and BDD

2% Gelucire®44/14). Subsequently, the rats who first received 1% concentration were

fed 2% concentrations of Gelucire®44/14, and vice versa, for additional two weeks. A

72h fat balance was repeated at the end of these two last weeks of diet. The described

72h fat balances were performed in each rat individually. The experimental set up is

indicated in detail in Figure 1b.

Figure 1 Experimental set up of the experiments performed to determine the effects of Gelucire

®44/14 on fat absorption in vivo. (a) Experimental scheme of the fat balance study in rats

with impaired lipolysis. (b) Experimental scheme of the fat balance study in rats with reduced solubilization. (c) Experimental scheme of the kinetics experiment of fat absorption in rats with reduced solubilization.

CHAPTER 6

116

Kinetics of fat absorption in rats with reduced solubilization

Rats were fed standard chow and received permanent catheters in bile duct and

duodenum as previously described by Kuipers et al.21

After the surgery, bile duct and

duodenum catheters were connected with each other at the skull of the rat for at least

three days to restore the enterohepatic circulation, in order to allow the rats to recover

from surgery. Subsequently, catheters of bile duct and duodenum were chronically

interrupted, resulting in permanent intestinal bile-deficiency. On the day of the

experiment, rats were infused intraduodenally for 7 hours (flow rate 1.5 ml/h) with buffer

(negative control), model bile (positive control), and with Gelucire®44/14-buffer

containing 0.1% or 0.5% Gelucire®44/14. The infusion rate and concentrations of bile

components were selected to reflect the physiological rates of bile flow and of the

intestinal delivery of specific bile components in adult Wistar rats. After starting the

intraduodenal infusion, 500 μl of fat per 300 gram body weight was administered slowly

as a bolus (olive oil, medium chain triglyceride oil and 13

C-labeled palmitic acid) via the

intraduodenal catheter. Medium chain triglyceride oil was included in the bolus in order to

obtain a reliable, reproducible vehicle for the quantitative administration of the labelled

compound, without introducing a profound increase in the intake of long-chain fatty

acids.22

The fat bolus represented approximately 15% of the daily fat intake of the semi-

synthetic high-fat diet. Blood samples (approximately 200 μl) were taken from the tail

vein at base line and every hour for 6 hours after administration of the fat bolus. The

baseline sample was taken prior to the administration of the fat bolus. Plasma and

erythrocytes were separated by centrifugation (2000 rpm, 10 min at 4˚C) and afterwards

stored at -20˚C until further analysis. Rats were used as their own controls during the

experiment, which was performed four times during the two weeks subsequent to the

chronic interruption of the bile duct (with different infusates); the above described

intraduodenal infusion conditions were performed in each rat individually. The

experimental set up is shown in detail in Figure 1c.

Analytical methods

Fatty acid analysis in chow and feces

Feces and chow were freeze-dried and homogenized mechanically. From aliquots of

feces and chow, lipids were extracted, hydrolyzed and methylated according to Muskiet

et al.23

Resulting fatty acid methyl esters were analyzed by gas chromatography to

calculate ingestion and fecal excretion of major fatty acids. Fatty acids were quantified

using heptadecanoic acid (C17:0) as internal standard. Total fecal fat excretion was

calculated from the daily fat intake and the daily fecal fat excretion and expressed as a

percentage of the daily fat intake as indicated in the following formula:

Fat intake (g day-1

) – Fecal fat output (g day-1

)

Percentage of total fat absorption = x100%

Fat intake (g day-1

)

Plasma lipids

Total lipids of plasma samples were extracted, hydrolyzed and methylated for gas-

chromatographic analysis of fatty acid profile as described by Muskiet et al.23

13

C

enrichment of fatty acid methyl esters was determined on a gas chromatography

GELUCIRE®44/14 AND FAT ABSORPTION

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combustion isotope ratio mass spectrometer (GC-C-IRMS). The concentration of 13

C

fatty acid in plasma at each time point was calculated from the fatty acid concentration

and 13

C enrichment and expressed as the percentage of the dose administered per ml

plasma (%dose/ml).20

Statistical analysis

Statistical analyses were performed using analysis of variance (One-Way ANOVA)

followed by post-hoc analysis (Bonferroni) using the SPSS version 12.0.2 software

(Chicago, IL, USA). For all experiments, p-values below 0.05 were considered

statistically significant.

RESULTS

After bile diversion, rats transiently lost up to 10% of their body weight. However, body

weights returned to normal within days (data not shown). There was no significant

difference in body weight between rats fed Orlistat and rats fed control high fat diet, or

between rats fed Orlistat and rats fed Orlistat with additional Gelucire®44/14 (data not

shown).

Dietary Gelucire®44/14 increases food intake in rats with reduced lipolysis and

solubilization, but increases fecal fat excretion exclusively in rats with reduced

solubilization

Food ingestion and feces production slightly increased in rats fed Orlistat, which is

consistent with previous findings in Gunn rats fed Orlistat.17

Feces production was

approximately 14% lower in rats with impaired lipolysis fed 2% Gelucire®44/14 diet

compared to rats fed Orlistat diet alone, but the difference did not reach statistical

significance (Figure 2b, NS). Gelucire®44/14 (1% and 2%) significantly increased food

ingestion in rats with impaired lipolysis compared to rats fed Orlistat alone (+23 and

+19%, respectively, each p<0.001, Figure 2a).

As expected, bile diversion significantly enhanced the amount of feces produced per day

by 76% compared to the condition in the same rats before bile diversion (p<0.001,

Figure 2d). Comparable to Orlistat-treated rats, Gelucire®44/14 increased food intake in

bile diverted rats (by 35% and 41%, in 1% and 2% Gelucire®44/14 diet fed rats,

respectively; each p<0.001, Figure 2c). Simultaneously, Gelucire®44/14 (both doses)

increased feces production by ~30%, compared to bile diverted rats fed the control diet

(p< 0.05, Figure 2d).

Dietary Gelucire®44/14 enhances absolute absorption of fat in rats with impaired

lipolysis, and to a lesser extent in rats with permanent bile diversion

The ingestion of fatty acids in absolute terms (mmol/day) was unchanged upon bile

diversion, but excretion was significantly increased, resulting in a lower absolute amount

of fat absorbed (Table 1). Inclusion of 1% or 2% Gelucire®44/14 in the diet increased the

absolute amount of fatty acids excreted in these rats (Table 1). Therefore, the absolute

amount of fat absorbed per day in rats with bile diversion fed Gelucire®44/14 was slightly

increased, but still significantly lower than in rats with intact enterohepatic circulation.

CHAPTER 6

118

Orlistat feeding increased absolute amounts of fatty acids ingested in two weeks (Table

2). Gelucire®44/14 decreased the amounts of fatty acids excreted in the feces of rats

with impaired lipolysis (Table 2). Accordingly, Gelucire®44/14 (both doses) increased the

absolute amounts of fatty acids absorbed daily in rats with impaired lipolysis, even to a

higher level than observed in rats with normal lipolysis (Table 2).

Figure 2 Data are represented as means ± SD of (a, b) 6-18 rats per group for impaired lipolysis study and (c, d) 5-9 rats per group in impaired solubilization study. (a, b) Significant difference in (a) ingestion and (b) excretion between control rats on high-fat diet () versus rats fed Orlistat ( for two weeks) or Orlistat with Gelucire

®44/14 ( 1% and 2%) is indicated as * p<0.05. Significant

difference between rats fed Orlistat diet with additional Gelucire®44/14 ( 1% and 2%) versus

rats fed only Orlistat ( for two weeks) is indicated as #p<0.05. (c, d) Significant difference in ingestion (c) and excretion (d) between control rats with intact EHC () versus bile diverted rats on control diet (), bile diverted rats on 1% Gelucire

®44/14 diet () or 2% Gelucire

®44/14 diet () is

indicated as *p<0.05. Significant difference between bile diverted rats on control diet () versus bile diverted rats on Gelucire

®44/14 diets ( 1% or 2%) is indicated as #p<0.05.

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Gelucire®44/14 (2%) increases net fat absorption in rats with impaired lipolysis,

but has no effect on net fat absorption in rats with permanent bile diversion

We analyzed the relative fat absorption from the fat balance. Orlistat administration significantly decreased net fat absorption in rats already after one week from 88% in control rats to approximately 70%, attributed to reduced lipolytic activity of lipases (data not shown). An additional week of Orlistat feeding did not further reduce net fat absorption (Figure 3a), neither did an additional 2 weeks (data not shown). This was in agreement with previous findings in Gunn rats.

17

Table 1 Absolute dietary fat ingestion, excretion and absorption in rats with intact EHC (control), bile diverted rats on control diet (BDD) and bile diverted rats on diet supplemented with 1% or 2% Gelucire

®44/14 (BDD 1% Gelucire

®44/14 and BDD 2% Gelucire

®44/14, respectively). Data are

means ± SD of 5-9 rats per group. Mean values represent the average of 72h per rat. *p<0.05 versus control rats with intact EHC. #p<0.05 versus BDD rats on control diet.

The 1% dose of Gelucire®44/14 did not significantly affect net fat absorption in rats fed

Orlistat (72%, NS), but 2% Gelucire®44/14 significantly increased the net fat absorption

reaching close to physiological values (82%, p<0.001, Figure 3a). In accordance with

previous observations, bile diversion lowered net fat absorption to 45% (Figure 3b).22

However, in these rats net fat absorption was not significantly altered by either dosage of

Gelucire®44/14 compared to fat absorption in the same rats on control diet without

Gelucire®44/14 (both 52%, NS, Figure 3b).

Table 2 Absolute dietary fat ingestion, excretion and absorption in control rats, rats fed control diet with Orlistat for one week or two weeks, and rats fed control diet with Orlistat for three weeks with additional Gelucire

®44/14 during the last week of treatment. Data are means ± SD of 6-18 rats per

group. Mean values represent the average of 72h per rat. *p<0.05 versus rats on control diet. #p<0.05 versus rats on Orlistat diet for two weeks.

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Gelucire®44/14 increases absorption of saturated and unsaturated fatty acids in

rats with reduced lipolysis, but does not affect absorption of fatty acids in rats

with impaired solubilization

In order to determine whether the positive effects of Gelucire®44/14 on fat absorption

were selective for specific fatty acid species, we assessed the net fat absorption of the

four major dietary fatty acids (linoleic, oleic, stearic and palmitic acid). Both impaired

lipolysis (Orlistat feeding, Figure 4a, 4b) and impaired solubilization (bile diversion,

Figure 4c, 4d) reduced the net absorption of all of the major fatty acids.

Figure 3 The effect of Gelucire

®44/14 on total net fat absorption in rats with (a) impaired lipolysis

and (b) impaired solubilization. Data are represented as means ± SD of (a) 6-18 rats per group for impaired lipolysis study and (b) 5-9 rats per group in impaired solubilization study. (a) Significant difference between control rats on high-fat diet () versus rats fed Orlistat ( for two weeks), rats fed Orlistat diet with additional Orlistat with 1% Gelucire

®44/14 () or 2% Gelucire

®44/14 () is

indicated as *p<0.05. Significant difference between rats fed Orlistat diet with additional 1% Gelucire

®44/14 () or 2% Gelucire

®44/14 () versus rats fed only Orlistat ( for two weeks) is

indicated as #p<0.05. (b) Significant difference between control rats with intact EHC () versus bile diverted rats on control diet (), bile diverted rats on 1% Gelucire

®44/14 diet () or 2%

Gelucire®44/14 diet () is indicated as *p<0.05. No significant difference was found between bile

diverted rats fed Orlistat diet with additional Gelucire®44/14 and bile diverted rats fed Orlistat diet

without Gelucire®44/14.

In rats with impaired lipolysis, Gelucire

®44/14 dose-dependently improved the uptake of

saturated fatty acids (Figure 4b). The absorption of unsaturated fatty acids was also

increased, but only in rats fed 2% Gelucire®44/14 diet (Figure 4a). In rats with permanent

bile diversion neither of the two Gelucire®44/14 dosages significantly affected the

absorption of unsaturated fatty acids (Figure 4c, 4d).

Intraduodenal administered Gelucire®44/14 does not reconstitute plasma

appearance of 13

C-labeled palmitic acid in rats with permanent bile diversion

Gelucire®44/14 caused a weak but significant increase in absorption of saturated fatty

acids in rats with permanent bile diversion. We therefore determined whether

Gelucire®44/14 affected the kinetics of fat absorption during impaired solubilization. We

assessed the absorption of 13

C-labeled palmitic acid for six hours after its intraduodenal

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administration in rats with permanent bile diversion. Figure 4a shows the time course of

plasma enrichment of 13

C-labeled palmitic acid after intraduodenal administration of a

bolus with buffer (negative control), model bile (positive control) or buffer supplemented

with 0.1 or 0.5% Gelucire®44/14. Bile diverted rats infused with model bile had a

significantly increased plasma concentration of 13

C-labeled palmitic acid at all time points

after the administration (Figure 5a). The plasma appearance of 13

C-labeled palmitic acid

Figure 4 The effects of Gelucire

®44/14 on net fat absorption of the major dietary (a, c) unsaturated

linoleic and oleic fatty acids and (b, d) saturated stearic and palmitic fatty acids in (a, b) rats with impaired lipolysis and (c, d) rats with impaired solubilization. Data are represented as means ± SD of (a, b) 6-18 rats per group for impaired lipolysis study and (c, d) 5-9 rats per group in impaired solubilization study. (a, b) Significant difference between control rats on high-fat diet () versus rats fed Orlistat ( for two weeks), rats fed Orlistat diet with 1% Gelucire

®44/14 () or 2%

Gelucire®44/14 () is indicated as *p<0.05. Significant difference between rats fed Orlistat diet with

additional 1% () or 2% Gelucire®44/14 () versus rats fed only Orlistat ( for two weeks) is

indicated as #p<0.05. (c, d) Significant difference between control rats with intact EHC () versus bile diverted rats on control diet (), bile diverted rats on 1% Gelucire

®44/14 diet () or 2%

Gelucire®44/14 diet () is indicated as *p<0.05. (c) No significant difference was found between bile

diverted rats on control diet versus bile diverted rats fed with additional 1% or 2% Gelucire®44/14.

(d) Significant difference between bile diverted rats on 2% Gelucire®44/14 () versus bile diverted

rats on control diet () is indicated as #p<0.05.

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122

did not differ between bile diverted rats infused with buffer only or with buffer

supplemented with 0.1 or 0.5% Gelucire®44/14 (Figure 5a). Figure 5b shows area under

the curves of Figure 5a which reflect the amounts of 13

C-labeled palmitic acid absorbed

during the six hours after administration of the bolus. In accordance with the

observations in Figure 5a, bile diverted rats receiving model bile infusion had a higher

area under the curve than bile diverted rats administered buffer alone (Figure 5b).

Intraduodenal administration of buffer supplemented with either 0.1% or 0.5%

Gelucire®44/14 did not increase the amount of the label absorbed (Figure 5b).

Figure 5 The effect of Gelucire

®44/14 on kinetics of fat absorption in rats with impaired

solubilization. (a) Curves of plasma concentrations of 13

C-palmitic acid during 6 hours after administration and (b) cumulative areas under the curves in rats with permanent bile diversion administered an intraduodenal fat bolus containing

13C-palmitic acid in combination with buffer ()

as negative control, 0.1% Gelucire®44/14 in buffer (), 0.5% Gelucire

®44/14 in buffer () or model

bile as positive control (). Data are represented as means ± SEM of 3-6 rats per group. Significant difference between bile diverted rats administered model bile and bile diverted rats administered buffer, 0.1% Gelucire

®44/14 in buffer or 0.5% Gelucire

®44/14 in buffer is indicated as *p<0.05. No

significant difference was found between BDD rats administered buffer with Gelucire®44/14 and

buffer alone.

DISCUSSION

Novel role for Gelucire®44/14 in fat absorption

Previous studies revealed a role for Gelucire®44/14 as an efficient emulsifier for

improvement of dissolution and absorption of poorly water soluble drugs.15,24,25

Hamid et

al. showed by means of an in vitro diffusion chamber and an in situ closed-loop

technique using rat intestinal tissue, that the use of Gelucire®44/14 can be considered

safe and that it does not lead to any intestinal membrane damage.26

Recently,

Fernandez et al. described in vitro effects of different lipases on Gelucire®44/14 and

showed that in particular gastric lipase was able to lipolyse Gelucire®44/14.

13 However, it

was not clear whether Gelucire®44/14 in turn appears to exert effects on lipases, namely

to enhance the lipolytic activity of lipases. We now show that Gelucire®44/14 might

improve the absorption of fatty acids in vivo under conditions of impaired lipolysis. These

findings could be of value for improving the nutritional status of patients with reduced

activity of pancreatic lipase, e.g. due to cystic fibrosis or chronic pancreatitis. Our data

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show that Gelucire®44/14 might act as an enhancer of lipolysis, rather than a solubilizer

under conditions of impaired fat malabsorption.

Dietary Gelucire®44/14 as enhancer of impaired lipolysis

In accordance with previous studies, our rat models of impaired lipolysis and impaired

solubilization show signs of fat malabsorption.17,18,22

Rats fed Orlistat show more

preserved fat absorption (71% after one week and 70% after two weeks of Orlistat

feeding) compared with rats with reduced solubilization (45% two weeks after the

operation). These percentages of net absorption coefficients are in agreement with

previously published data in similar rat models.17,18,27

However, rats fed Orlistat show

lower levels of inhibition of fat absorption (20% reduction) compared with human subject

who received lower amount of Orlistat (120 mg per meal; 30-40% reduction in fat

absorption).28,29,30

It appears that human subject received 0.59% of Orlistat per grams of

fat ingested, while the rats in our study received 0.89% of Orlistat per gram of fat

ingested. Present and other studies performed in rats fed Orlistat show that rats seem to

compensate the fecal fat loss by increasing their food intake during Orlistat feeding.17,19

To our knowledge human subjects fed Orlistat do not compensate for fecal fat loss by

increased food intake and, therefore, these observations might explain the discrepancy

between the effects of Orlistat fat absorption in rats and humans.

In vitro studies of Subramanian and Wasan suggested that Gelucire®44/14 might inhibit,

rather than improve, lipolytic activity.31

However, these data are not conclusive since no

significant difference was found in lipolytic activity between untreated lipases and lipases

treated with increasing concentrations of Gelucire®44/14.

31

One could have anticipated that Gelucire®44/14 would have had the largest effect on fat

absorption in bile diverted rats in which the fat malabsorption was most severely

affected. However, this would only be the case if the effects of Gelucire®44/14 could

restore (to some extent) the solubilization. Our data show that Gelucire®44/14 improves

total net fat absorption to a much larger extent in rats with impaired lipolysis; indeed, the

coefficient of fat absorption returned to almost normal values after one week of 2%

Gelucire®44/14 diet in Orlistat fed rats. In contrast, there was no effect of Gelucire

®44/14

on the solubilization of unsaturated fatty acids and only a minimal effect on the

solubilization of saturated fatty acids in rats with bile diversion. This suggests that

Gelucire®44/14 has only a slight effect on fat absorption under conditions of fat

malabsorption exclusively due to impaired solubilization.

Both rat models showed increased food ingestion of Gelucire®44/14 diets, while rats with

bile diversion also showed increased feces production upon Gelucire®44/14 feeding. The

underlying mechanism of the increased food intake by Gelucire®44/14 in these ad libitum

fed rats remains to be elucidated. We cannot exclude the possibility that difference in the

texture of the granules (which were manually made for Gelucire®44/14 diets and custom

made for the other diets) contribute to increased food ingestion in rats fed

Gelucire®44/14 diets. It has been described previously by Sako et al. that texture of food

plays an important role in food selection behavior in rats.32

Another possibility would be

that pre-digested lipolysis products (monoglycerides) directly derived from the ingested

Gelucire®44/14 might have a positive effect on food intake in these animals. The

elucidation is relevant for several reasons, including an explanation for the observed

CHAPTER 6

124

increase in net absorption of fatty acids by Gelucire®44/14. Gelucire

®44/14 may directly

enhance the activity of pancreatic lipases on fat digestion in the small intestine or retard

their degradation, for example by pancreatic proteases. However, it is unlikely that an

increase in food ingestion due to softer food pellets is the main cause of increased fat

absorption. Food intake was similarly increased during impaired lipolysis and reduced

solubilization, while the net absorption of fat was only enhanced under conditions of

impaired lipolysis and moreover was not improved in rats fed Orlistat with 1%

Gelucire®44/14. Therefore other factors must be responsible for enhanced absorption of

fat in rats with impaired lipolysis. It is possible that due to the emulsification properties of

Gelucire®44/14 an increase in the specific surface area of the fat could enable the lipase

to be more effective in vivo.

To expand our insights in the (possible) effects of Gelucire®44/14 on the kinetics of fat

absorption, we additionally measured the absorption kinetics of the saturated fatty acid

palmitate. Direct duodenal infusion of Gelucire®44/14 did not result in increased plasma

appearance of 13

C-labelled palmitate, indicating that there is no significant effect of the

compound on the kinetics of palmitate absorption. The ratio of Gelucire®44/14 to the total

amount of fat administered was equivalent to the ratio of Gelucire®44/14 to the total

amount of fat ingested in diet so that it is unlikely that the concentration of

Gelucire®44/14 infused was too low to exert an effect. The possibility remains however,

that by intraduodenal administration of the bolus, preduodenal lipolysis of Gelucire®44/14

is bypassed and that this limited its biological activity.

The exact reason for the higher specificity of Gelucire®44/14 on saturated fat compared

to unsaturated fatty acid absorption remains to be elucidated, but it seems that there

could be an effect on both solubilization and lipolysis with saturated fats but only an

effect on lipolysis for unsaturated fats. We cannot exclude, however, that some of the

absorbed palmitate and stearate in rats fed Orlistat with additional Gelucire®44/14 are

derived directly from Gelucire®44/14 itself. Fatty acids within Gelucire

®44/14 are mainly

incorporated within monoglycerides and are absorbed independently by the lipase

activity. Since absolute absorption rates of palmitic and stearic fatty acids in rats fed

Orlistat and Gelucire®44/14 exceed the amount of fatty acids that is present within

Gelucire®44/14 (data not shown), we expect that the contribution of increased fatty acid

absorption directly by fatty acids derived from Gelucire®44/14 is very low.

Future studies on the effects of Gelucire®44/14 on fat absorption

The main focus of the present study was to determine whether dietary supplementation

of Gelucire®44/14 enhances the absorption of fatty acids in vivo in relevant rat models for

different types of fat malabsorption. The underlying mechanisms might include direct

effect on lipolysis leading to increased fat absorption or indirect improvement of fat

absorption by means of enhanced emulsification properties of fat absorption. Our

findings support the concept that Gelucire®44/14 indeed enhances fat absorption in rats

in vivo. Concerning the improved absorption of saturated palmitic (C16:0) and stearic

(C18:0) acids, it would be interesting to measure the solubility of saturated and

unsaturated fatty acids in Gelucire®44/14. It is possible that saturated fatty acid have a

better solubility in a hydrogenated vegetable oil, which is the excipient in Gelucire®44/14.

Furthermore, it would be interesting to perform similar studies as presented here in a

GELUCIRE®44/14 AND FAT ABSORPTION

125

6

CH

AP

TE

R

mouse model for cystic fibrosis, where both lipolysis and solubilization are impaired and

lead to fat malabsorption. We have shown that there seems to be a dose dependent

effect of Gelucire®44/14 on fat absorption in rats with impaired lipolysis. It would be

interesting to further characterize this dose dependency using different diets and animal

models. Moreover, since unsaturated fatty acids may undergo microbial hydrogenation in

the large intestine,33

future studies would also measure absorption of fatty acids at the

level of the terminal ileum to clarify the differential effects of Gelucire®44/14 on

absorption of saturated and unsaturated fatty acids.

Overall conclusion

Dietary supplementation of Gelucire®44/14 to rats with impaired lipolytic activity corrects

the net total fat absorption. If Gelucire®44/14 would similarly improve fat absorption in

patients with impaired lipolysis, such as in CF patients or patients with chronic

pancreatitis, it could constitute a major improvement in the current therapy. However,

essential fatty acid deficiency during CF may remain prominent, even under dietary

Gelucire®44/14 supplementation, since this compound mainly improves the absorption of

saturated fatty acids. Studies in e.g. a mouse model of CF should reveal if

Gelucire®44/14 can normalize fat absorption and can be used in combination with other

compounds to improve the absorption of saturated, but also of essential fatty acids in

clinical conditions of pancreas insufficiency.

ACKNOWLEDGEMENTS

The authors would like to thank Rick Havinga for his excellent technical assistance

during the studies in bile diverted rats. Furthermore, we would like to thank Theo Boer

for his technical assistance during GC-C-IRMS measurements.

GRANTS

Part of this study was supported by an unrestricted grant of Solvay Pharmaceuticals

GmbH (Hannover, Germany) and by the Dutch Digestive Foundation.

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