full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1338 -1345 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
1341
directly collected from the mucosal
compartment.
Biochemical analysis
Estimation of protein: To 0.1 ml of homogenized
jejunal sac, 0.9 ml of water and 4.5 ml of alkaline
copper reagent were added and kept at room
temperature for 10 minutes, then 0.5 ml of Folin’s
reagent was added and the color developed was
read after 20 minutes at 640 nm (14). The level
of protein was expressed as mg/5 cm of jejunal
segment.
Estimation of glucose: Glucose was estimated
in the serosal fluid of the everted intestinal sac
by the glucose oxidase/peroxidase (GOD/POD)
method (15) by the AGAPPE reagent kit, India.
Glucose is oxidized by glucose oxidase into
gluconic acid and hydrogen peroxide. Hydrogen
peroxide in presence of peroxidase (POD)
oxidizes the chromogen 4-aminoantipyrine
compound to a red colored compound. The
intensity of the red colored compound is
proportional to the glucose concentration and is
measured at 505 nm. The final color is stable for
2 hours.
Estimation of Pyruvate: Pyurvate levels was
estimated in the serosal fluid of the everted
intestinal sac by DNPH method (16) based on
reaction of pyruvate with DNPH which produces
a color and the color was read at 520 nm.
Estimation of lactate dehydrogenase: Lactate
dehydrogenase was estimated in the serosal fluid
of the everted intestinal sac by using UV kinetic
method by commercially available AUTOPAK
kit, India. Lactate dehydrogenase catalyzes the
conversion of pyruvate to lactate and NADH to
NAD. Lactate dehydrogenase activity in sample
is directly proportional to the rate of decrease in
the absorbance of NADH at 340 nm.
Statistical Analysis: Data was analyzed by one
way analysis of variance (ANOVA) followed by
Tukey’s multiple comparison. The significant
level was fixed at p