J. Env. Bio-Sci., 2016: Vol.

30 (1):13-19
(13) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

COMPARISON OF ABSORPTION RATES OF WATER AND ELECTROLYTES FROM

STANDARD ORS (S-ORS), HYDROLYZED STARCH ORS (HS-ORS)
AND HYDROLYZED STARCH WITH LIPOSOMAL SALTS ORS (LIPO-ORS)
IN THE SMALL INTESTINE OF RAT
R. Faruqui-1-2 , H. Khanum-1* , P. K. Bardhan-2, C. Mitchell-3, and D. A. Sack-2
1-
Department of Zoology, University of Dhaka.
2-
International Centre for Diarrhoeal Disease Research, Bangladesh and 3-CRM Corporation, USA.
[Corresponding Author E-mail1*: hamida_khanum@yahoo.com]

Received: 21-12-2015 Accepted: 20-01-2016

In the present study rats were selected by using the three types of Oral Rehydration Solutions (ORSs); standard ORS (S-ORS),
hydrolyzed starch ORS (HS-ORS) and Liposomal ORS (Lipo-ORS) for in vivo perfusion technique in the small intestine. The study
describes the absorption of electrolytes (Na+, K+, Cl-) and water from these three types of ORSs and compared among these
substances from each other. By using an in vivo perfusion technique, liposome based ORS was associated with significantly
greater Na+, K+, Cl- and water absorption compared to hydrolyzed starch ORS or standard ORS. This study observed the different
absorption rates of water and electrolytes between standard ORS or hydrolyzed starch ORS and liposome based ORS which was
significant (P=0.02) or was not significant (P=0.12). It can be concluded that liposomes enhance net electrolytes absorption from
a carbohydrate electrolytes solution in rat small intestine. These findings underscore the further experiment, on rat model which
would be cholera toxin treated secretory intestinal or 5-Fluorourasil treated mucosal injured intestinal diarrhoea by perfusion
technique with the help of liposome based electrolyte solution for rehydration.

Diarrhoeal disorders constitute a leading cause of morbidity
and mortality globally and continue to be a major concern,
particularly for developing countries1. Estimates show that
there are over 1 billion episodes of diarrhoea and 1.3 million
deaths in children less than 5 years of age in the developing
world2. Based upon the pathogenic mechanisms, the diarrhoeal
organisms may be broadly divided into two groups, secretory
and invasive. Vibrio cholera is the prototype pathogen causing
secretory diarrhoea through liberation of an enterotoxin3.
Cholera, is endemic in some parts of Asia, Africa and
Hispanola4-5. It is characterized by acute onset of vomiting,
profuse watery diarrhoea and development of dehydration that
might lead to death if not treated quickly.
Sufficient loss of fluids and electrolytes in patients with watery
diarrhoea leads to dehydration and thus the mainstay of
treatment of such patients is fluid replacement. The
development of ORS in the treatment of dehydration due to
diarrhea is one of the most significant therapeutic advances in
the history of medicine6-8. It is estimated that ORS alone can
successfully rehydrate 90% of patients with dehydration from
acute diarrhoea who previously would have required intra-venous
(i.v.) therapy9. However, Oral Rehydration Therapy (ORT) with
the present ORS formulation has certain limitations-ORT does
not reduce the volume, frequency or the duration of diarrhoea10.
These limitations prompted the concept of developing an
improved ORS (initially named 'super ORS')11. Conceptually,
an improved ORS should be able to exert some beneficial
effects: (a) reduce stool volume (by stimulating re-absorption
of fluid secreted into the small intestine), (b) shorten the
duration of diarrhoea (by reducing ideal effluent flow and
stimulating colonic salvage), and (c) reduce failure rate of ORT
particularly in patients with high purging rate.
Recently recommended reduced osmolarity ORS is the result
of an intensive search for such an improved ORS. The ORS
solution is similar to the original ORS but has a lower
concentration of sodium (75 mmol rather than 90) and lower
concentration of of glucose (75 mmol rather than 111 mmoles/
liter) yielding a solution with a total osmolality of 245 rather
than 311 (WHO-ORS, 2002)12. Though the new solution was
associated with less vomiting, the duration of diarrhoea was
not shortened, and there are still ORT failures in which patients
initially rehydrated and place on ORT, become dehydrated
again and require additional IV fluids13. Thus there is scope for
further improvements of ORT. One ingenious way may be

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 COMPARISON OF ABSORPTION RATES OF WATER AND ELECTROLYTES
delivering ORS in Liposomes to stimulate faster and more
efficient absorption. The basic component of all clinically useful
liposomes is the phospholipid molecule. Phospholipids are
amphiphilic molecules composed of a polar hydrophilic head
group and two hydrophobic fatty acid chains attached to a
three-carbon glycerol backbone. When phospholipids are
mixed with water, they spontaneously rearrange into concentric
bilayer structures, termed liposomes or vesicles, separated
by aqueous compartments14.
Incorporating ORS components into liposomes, as opposed
to simply having salts and substrate in solution, has several
potential advantages: (a) it may add an additional mechanism
of absorption to that already provided with glucose mediated
transport, (b) it may become especially important in patients
who have severe purging or who have damaged intestinal
epithelium (severe malnutrition, persistent diarrhoea), (c) the
solutions will have lower osmolarity and (d) the solutions also
will taste less salty. Such an ORS solution will also retain the
potential advantage of all cereal based ORSs, i.e. slow release
of substrate avoiding osmotic drag or load15.
The aim of the present study is to determine the in vivo effect
of absorption of water and electrolytes from liposome based
glucose-containing solution over the whole length of rat small
intestine. The whole length of rat small bowel was chosen to
obtain results close to those in an intact animal and therefore
relevant to the design of an improved ORS formulation16. This
study will be applied in the experimental study like cholera
toxin treated secretory intestine or 5-Fluorourasil treated
mucosal injured intestine and this will be helpful for clinical
trial.
MATERIAL AND METHODS
Thirty adult male Long Evans rats were selected for the present
experiment. The rat model was well established to study small
intestinal absorption of water and electrolytes and had been
extensively used to study 'Oral Rehydration Solution' (ORS)17.
In this study ORS was used as perfusion solution, prepared
with analytical grade chemicals. The electrolyte concentrations
of the three solutions are shown in Table-2. Polyethylene glycol
(PEG), Mol Wt 4,000 2g/L was used as the unabsorbable
marker. Three types of ORS were used as perfusion solution.
These were Standard glucose based ORS (S-ORS),
Hydrolyzed starch ORS (HS-ORS) and Hydrolyzed starch with
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(14)
Liposome based ORS (Lipo-ORS) (Table-1).
Experimental Procedure: Rats were grouped into three
experimental groups:(i) S-ORS group and (ii) HS-ORS group
(iii) Lipo-ORS group. Those were treated with one of three
types of oral rehydration solution (Table-2). The abdomen was
opened after a midline-incision (3-4 cm in length). Thereafter,
two incisions were performed into the intestine. The small
intestine was then cannulated with two polyvinyl tubes (2mm
in diameter) which were called proximal and distal cannula.
The isolated and cannulated small intestine was gently rinsed
to clear residual contents with the perfusion solution by gravity
drainage. The perfusion was performed by attaching the
proximal cannula to a constant infusion pump using a
measuring burette as a reservoir. Each solution was infused
at a constant rate of 0.5 ml/min. The distal cannula was
extended to aid drainage of effluent by gravity. After 30 minutes
of equilibration to achieve a steady state, the effluent was
collected for 3 consecutive 15 minute-collections (total 45
minutes) in Falcon tubes kept on ice. After completing
perfusion, aliquots of infusion and perfusion solutions were
stored at -50ºC for up to 48 hours before analysis of net water
and electrolyte movement. The perfused small intestinal
segment was removed and stripped of excess mesentery. The
segment length was taken by manually with measuring tape
and wet weight was taken by Metler Toledo, College digital
measuring scale (USA). After desiccation in an oven at 100ºC
for 18 hours the dry weight of the perfused segment was also
obtained by Metler Toledo, College digital measuring scale
(USA).
Analytical methods: Sodium (Na+), potassium (K+), chloride
(Cl-) were measured by flame emission spectroscopy and PEG
was measured by spectro-photometry18-20
Calculations21:
Net transport of water and electrolytes were calculated from
the changes in the PEG concentration and the solute
concentration22-23. The calculation for the net transport of water
and ions was done as follows:
Net Transport={F×([S1]-[S2]) ([PEG1]/[PEG2])}/ L or W
Where:
F = flow rate
S1 = solute concentration in the perfusate
 (15) FARUQUI , KHANUM , BARDHAN, MITCHELL AND SACK

Table-1. Composition of solution used to perfuse the rat intestines.

Table-2. Three groups of rats were treated with only one of three oral rehydration solutions as
shown.

Table-3. Comparative absorption rates of water and electrolytes from three types of ORSs in perfusion
study on whole small intestine of rat.
N a m e o f th e g r o u p E x p e r im e n ta l g r o u p
-A E xp e r im e n ta l g r o u p- B
( u s in g s e g m e n t le n g th ) ( u sin g se g m e n t d r y w e ig h t)

N o o f r a ts 10 10 10 10 10 10
L e n g th ( cm ) / D r y W e ig h t o f 8 5 .8 cm 9 1 .7 c m 8 8 .7 cm 1 .3 4 g m 1 .4 4 g m 1 .2 7 g m
s e g m en t ( g m )
T yp e s o f O R S K- W K-W
S a m p le co n ce n tr a tio n Te st Te st
µ l.cm - 1 .m i n - 1 o r µ l.g m - 1 .m in - 1 S ig S ig
S- O R S H S- O R S L ip o- O R S le ve l S -O R S H S -O R S L ip o- O R S l e ve l
& µ m o l.cm -1 .m in -1 o r µ m o l.g m -
1 .m in - 1 (P ) (P )

H2O Mean 0 .0 0 0 .0 0 0 .0 1 0 .1 4 0 .2 3 0 .2 5 0 .3 2 0 .0 7
Na+ Mean - 0 .0 7 -0 .0 2 - 0 .0 4 0 .0 3 -3 .6 7 -1 .1 4 - 2 .1 3 0 .1 4
K+ Mean 0 .0 3 0 .0 4 0 .0 3 0 .5 5 1 .6 6 2 .1 6 1 .8 9 0 .2 6

C l- Mean - 0 .0 5 0 .0 4 0 .0 1 0 .0 1 -1 .2 2 1 .9 2 1 .0 7 0 .0 1

Table-4. Comparative analysis between the absorption rates of water and electrolytes from S-ORS
and Lipo-ORS in perfusion study on whole small intestine of rat.
E x p e r im e n t a l g r o u p - A E x p e r im e n t a l g r o u p - B
N a m e o f t h e g ro u p
N o o f ra t s 10 10 10 10
L e n g t h o f s e g m e n t (c m )/ 91.7 c m 88.7 cm 1.34 gm 1.27 gm
D r y W e ig h t o f s e g m e n t ( g m )
Ty p es of O R S
S a m p le c o n c e n t r a t io n
µ l. c m - 1 . m in - 1 o r µ l. g m - 1 . m in - 1 P P
S -O R S L ip o - O R S (M -W ) S-OR S L ip o - O R S (M -W )
& µ m o l. c m - 1 . m in - 1 o r µ m o l. g m -
1 . m in - 1
H 2O M ean 0.00 0 .01 0.08 0.23 0.32 0.02
N a+ M ean -0 . 0 7 -0 . 0 4 0.04 -3 . 6 7 -2 .1 3 0.28
K+ M ean 0.03 0 .03 0.85 1.66 1.89 0.22
C l- M ean -0 . 0 5 0 .01 0.02 -1 . 2 2 1.07 0.02

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 COMPARISON OF ABSORPTION RATES OF WATER AND ELECTROLYTES (16)

Table-5. Comparative analysis between the absorption rates of water and electrolytes from HS-ORS
and Lipo-ORS in perfusion study on whole small intestine of rat.

Legend: S-ORS= Standard Oral Rehydration Solution

HS-ORS= Hydrolyzed Starch Oral Rehydration Solution
Lipo-ORS= Liposomal Oral Rehydration Solution
K-W=Kruskal-Wallis test and M-W=Mann-Whitney test

Graph-1: Net absorption of water from ORSs. Graph-2: Net absorption of Sodium ion from ORSs.

Graph-3: Net absorption of Potassium ion from ORSs. Graph-4: Net absorption of Chloride ion from ORSs.
Figure-1 .

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 (17) FARUQUI , KHANUM , BARDHAN, MITCHELL AND SACK
S2 = solute concentration in the effluent
PEG1 = PEG concentration in the perfusion fluid
PEG2 = PEG concentration in the effluent
L = length of the small intestinal segment used for perfusion.
W = dry weight of the small intestinal segment used for
perfusion.
Statistical analysis: Results were provided by mean and
values were expressed as µl.cm -1.min-1 (using length of
segment) or µl.gm -1.min-1 (using weight of segment) for water
and µmol.cm -1.min-1 (using length of segment) or µmol.gm -
1
.min -1 (using weight of segment) for electrolytes while
calculation performed on the dry weight of perfused segment.
Kruskal-Wallis Test was performed to test the statistical
significance of the differences among three the groups. Mann-
witney Test was used to test the statistical significance of the
differences between two groups. Significant P value is  0.05.
RESULTS AND DISCUSSION
Net transport of water and electrolytes were derived from the
changes in the PEG concentration and the solute
concentration22-23. Net sodium, potassium, chloride and water
absorption were significantly higher from the Lipo-ORS
compared to the other two solutions such as S-ORS and HS-
ORS in this experimental study. In case of water absorption,
present experiment showed that, absorption rates from Lipo-
ORS were highest than other two ORSs, whereas, the
differences among three ORSs were not statistically significant
(Table-3). Through, the statistical analysis Mann-Witney test
difference between two ORSs, S-ORS and Lipo-ORS was not
significant in the group-A, on the other hand difference between
these two ORSs was significant (P=0.02) in the group-B (Table-
4).
In all the experimental groups, secretion of sodium ion from
three ORSs were observed. In the study group-A, the
differences among the secretion rates from three ORSs was
statistically significant, P=0.03; while in the study group-B,
the differences among the secretion rates from three ORSs
was not statistically significant, P=0.14; (Table-3). In the study
group-A, the secretion rate was lower from Lipo-ORS and the
difference between two ORSs, S-ORS and Lipo-ORS was
significant (P=0.04), on the other hand the secretion rate was
also lower from Lipo-ORS whereas difference between these
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two ORSs was not significant (P=0.02) in the study group-B
(Table-4). Moreover, in the case of potassium ion for both in
the group-A and group-B, absorption was occurred from three
ORS. In the group-A and group-B, absorption of K+ from Lipo-
ORS was almost same like S-ORS and HS-ORS and the
difference was not statistically significant (Table-3). In the group-
A, there was no difference between S-ORS and Lipo-ORS
while, in the group-B, some differences between S-ORS and
Lipo-ORS were observed, and no statistical significance
observed (Table-4). In both groups, few difference between HS-
ORS and Lipo-ORS were observed, whereas, no statistical
significance observed (Table-5). In case of Cl- absorption on
both groups, the difference among three ORSs was statistically
significant, P=0.01 (Table-3). The difference between S-ORS
and Lipo-ORS was significant (Table-4), whereas, difference
between HS-ORS and Lipo-ORS was not significant (Table-
5). For all groups there were no difference between HS-ORS
and Lipo-ORS and the difference between these two ORSs
were not significant (Table-5).
The use of oral rehydration solution (ORS) has revolutionized
the management of acute diarrhoea. The implementation of
World Health Organization (WHO) ORS has resulted in
decreased mortality associated with acute diarrhoeal illnesses
in children, although in general stool volume and diarrhoea
durations were not reduced12. Several strategies were used to
develop and test for improved ORS. Though the new hypo-
osmolar ORS has advantages over the previously used
standard ORS, the duration of diarrhoea was still not shortened,
and there were failures with ORT24. Thus there was scope for
further improvements of ORT.
Various modifications to the standard ORS have been derived.
These m odification have included hypo-osm olar or
hyperosmolar solutions, use of rice-based ORS, and the use
of amino acids, including glycine, alanine, and glutamine25.
Some of these variations have been successful, some have
not, and others are still under investigation. ORS has also
been used to decrease intravenous (IV) fluid requirements in
patients with short bowel syndrome (SBS) who require
parenteral nutrition26.
In recent years, there have been certain cereal based
rehydration compositions that have starch sources. These
cereal-based oral rehydration compositions may contain a
 COMPARISON OF ABSORPTION RATES OF WATER AND ELECTROLYTES (18)

minimal concentration of fat or phospholipid naturally present Clinical studies showed improved absorption with alternative
in the cereal grain ( less than 0.05% on a solid basis). However, types of ORS. In a non-randomized open trial, children receiving
oral rehydration compositions do not contain fat, oil, or an oral rehydration salt solution with a lower concentration of
phospholipids as an added ingredient15. In this study author glucose and was hyptonic had reduced frequency of diarrhoeal
used liposome as an added ingredient of oral rehydration stools and could be discharged sooner than other children
compositions. Liposomes are artificially prepared vesicles made who received the standard ORS which was isotonic30. In 1996,
of lipid bilayer. They are simple microscopic vesicles in which Faruque et al compared a hypo-osmolar ORS with sucrose
an aqueous volume is entirely enclosed by a membrane replacing glucose (Na+ 60, K+ 15, Cl- 60, citrate 5, sucrose 58
composed of lipid molecule27. mmoll-1, calculated osmolality 198 mOsm kg-1) with mildly
hyperosmolar glucose ORS (WHO) in 46 children aged 6-30
Animal models have been extensively used to develop
months with acute diarrhoea and dehydration. In the hypo-
modifications in the formulation of ORS. In 1989, Rolston et al
osmolar sucrose ORS group (n=18) faecal output was 30%
investigated the effect of bicarbonate, acetate and citrate on
less during the initial 24 and 48 h compared with controls31. In
water and sodium transport in normal and also in secreting
a randomized controlled clinical trial, Dutta found that children,
(Cholera toxin-treated) rat small intestine using a single-pass
aged 2-10 years with severe cholera who were treated with a
perfusion technique. The observation suggest that those anions
rice-based hypo-osmolar ORS had reduced (p<0.05) stool
to oral rehydration solutions may had no beneficial effect with
output, ORS consumption and diarrhea duration than patients
regard to the promotion of water and sodium absorption in the
who received either WHO-ORS or glucose-based hypo-osmolar
secreting intestine during acute diarrhoeal states and could
ORS32. In another randomized controlled trial, in 2008,
actually be deleterious28. In 1990, Patra et al. , studied that
Ram akrishna tested a hypotonic ORS in which the
the effect of citrate on sodium, potassium, chloride and water
carbohydrate was an amylase resistant starch in adults with
absorption in the presence of glucose from the whole rat small
acute dehydrating diarrhoea. Compared to hypo-osmolar (HO-
intestine by an in vivo marker perfusion technique. Significantly
ORS) ORS, amylase resistant starch -ORS reduced diarrhoea
more sodium and water absorption occurred from the citrate-
duration by 55% and significantly reduced fecal weight after
containing solution than from the one without citrate29.
the first 12 hours of ORS therapy in adults with cholera like
The present study demonstrated that a liposome-based-ORS diarrhoea33.
induced a significantly greater electrolytes (Na+, K+ and Cl-)
Various liposome-based medications are already used in
and water absorption compared to standard-ORS and
diverse clinical situations. Many pharmacological agents of
hydrolyzed starch-ORS solution. The three types of ORSs
varying solubility and size (anti-tumour and antimicrobial
were then evaluated to determ ine their absorptive
agents, enzymes, peptides, hormones, vaccines and genetic
characteristics. In the experimentally treated rat's whole
materials) have already been encapsulated in either the aqueous
intestine, the liposome based ORS solution induced a
or the lipid phase of the liposomes14. Proteins and other non-
significantly greater water and electrolyte absorption compared
lipid molecules can be incorporated into the lipid membranes.
to other two ORSs. The highest absorption of water, K+ and
In fact, liposomes can be designed to satisfy particular needs
Cl- as well as lowest secretion of Na+ from Lipo-ORS were
in a variety of applications ranging from biochemical and
observed in the experimental rats which perfused with Lipo-
immunological assay kits and diagnostic reagents to
ORS. One of the interesting findings of this study was that the
therapeutic preparations for enteral and parenteral uses as
mean values of net Na+ transport were negative in all groups,
well as vaccines34-36.
suggesting net intestinal secretion; however the Na+ secretion
was observed lowest among the Lipo-ORS group. In contrast, In the present investigation liposomes incorporated into ORS
the mean values of net K+ transport were positive in all groups, for intestinal perfusion to stimulate absorption of water and
suggestive net intestinal absorption; again, the highest K+ electrolytes resulted in significantly enhanced absorption of
absorption was noted in the Lipo-ORS group. In case of chloride water, K+, Cl- and reduced secretion of Na+ when compared
ion absorption the difference among three ORSs was significant.

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 (19) FARUQUI , KHANUM , BARDHAN, MITCHELL AND SACK

with other ORSs. The improved lipsome based ORS (Lipo- 13.
ORS) may have potentials in reducing stool volume, duration
of diarrhoea and failure rate of ORT in patients with high purging 14.
rate.A promising outcome of the study in favour of liposomal-
ORS will prompt towards further experimental study on different 15.
types of rat model which would be caring secretory or mucosal
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Farthing, M. J. G. (1987). Journal of pediatric Gastroenterology
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