Acid and Alkaline Phosphatase activities in the small intestine of the Rat (Rattus norvegicus), Bat (Eidolon helvum) and Pangolin (Manis tricuspis)

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Gbenga A. Adefolaju M.Sc.
Anatomy Department
Faculty of Basic Medical Sciences
College of Health Sciences, University of Ilorin

Address:
Ilorin
Nigeria

Ademola E. Caxton-Martins Ph.D
Anatomy Department
Faculty of Basic Medical Sciences
College of Health Sciences, University of Ilorin

Address:
Ilorin
Nigeria

Bernard U. Enaibe M.Sc.
Anatomy Department
Faculty of Basic Medical Sciences
College of Health Sciences, University of Ilorin

Address:
Ilorin
Nigeria

Olukemi T. Alabi M.Sc.
Physiology Department
Faculty of Basic Medical Sciences
College of Health Sciences, University of Ilorin

Address:
Ilorin
Nigeria

Moyo S. Ajao
School of Anatomical Sciences
Faculty of Health Sciences
University of the Witwatersrand

Address:
Johannesburg
South Africa

Citation: G. A. Adefolaju, A. E. Caxton-Martins, B. U. Enaibe, O. T. Alabi & M. S. Ajao : Acid and Alkaline Phosphatase activities in the small intestine of the Rat (Rattus norvegicus), Bat (Eidolon helvum) and Pangolin (Manis tricuspis). The Internet Journal of Pharmacology. 2009 Volume 6 Number 2

Keywords: Rat | Bat | Pangolin | ALP | ACP | Small intestine

Abstract

Alkaline and Acid Phosphatase (ALP and ACP) activities were compared in the small intestine of the rat (Rattus norvegicus), bat (Eidolon helvum), and pangolin (Manis tricuspis) to observe possible modifications of their small intestine to different diets. Ten animals each of both sexes were used. Portions of their small intestine were homogenized and assayed spectrophotometrically for the activities of ALP, and ACP. ALP and ACP activities were significantly (at 95% confidence interval (p<0.05) and consistently higher in the bat than in the rat and pangolin. High ALP activities in the bat explain why chyle transit time in the bat gut is short because ALP facilitates transport across intestinal mucosa. A higher ACP activity in the bat is because they help to “mop up” the phosphate ions produced as a result of ATP hydrolysis. It can be concluded that the small intestine of the rat, bat and pangolin, utilizes the phosphatases to different degrees.

Introduction

General sections of the mammalian gut are usually adapted to suit the dietary requirements of the particular species. The mammalian gastrointestinal tract usually presents parts known to vary in enzyme systems due to their peculiar functions (Bannister et al, 1995, Sherwood, 2002).The bat is frugivorous, the pangolin is insectivorous while the rat is omnivorous. Fruits are a unique food source that combine a large proportion of well-protected seeds with a nutritious, easily digestible covering, (Levey and Duke,1992). Presumably, fruit-eating bats possess gut adaptations that allow them to efficiently process this mix of high and low digestibility component. Amongst mammals, bats are unique in their capacity for flapping flight. This mode of flight is extremely energy-expensive and it might be expected that strategies and adaptations might have evolved for meeting the high calorific demands. Some of these strategies and adaptations are known to occur in bats (Makanya et al, 1997).The intestine of bats display extremely short transit times and appears to be well adapted for accelerated digestive and absorptive activities. Although certain active transport systems may be lacking (Makanya et al, 1997), digestive enzyme distributions are extensive (Ogunbiyi and Okon, 1976).

Various other works have been undertaken in attempts to elucidate the form and functional characteristics of the GIT of the rat (Rattus norvegicus) and bat (Eidolon helvum), (Ogunbiyi and Okon,1976, Okon,1977; Stenling and Helander, 1981, Young et al, 2006). Ofusori and Caxton-Martins (2007) reported morphometric studies on the stomach of African pangolins (Manis tricuspis), however no work has been done comparing histological, histochemical and biochemical features of the small intestine of the rat (Rattus norvegicus), bat (Eidolon helvum), and Pangolin (Manis tricuspis).It is to be anticipated that the pangolin, which has no teeth unlike the rat, bat and some other mammals may possess certain morphological and functional features adapted by their alimentary tracts to make them suitable for these high chitinous diet. Phosphatases are present in a wide variety of animal tissues. They are responsible for the hydrolysis of organic phosphate esters. Alkaline phosphatase exhibits optimal activity at high pH values while Acid phosphatase exhibit optimal activity at low pH values (Bancroft, 1979). Alkaline phosphatases are activated by magnesium, manganese and cobalt ions, while their activity can be inhibited by cyanide and cysteine (Humason, 1979). In many cells, acid phosphatase appears to be associated almost exclusively with the lysosomes and considered to be a marker for them (Humason, 1979). Human prostatic acid phosphatase has been reported to directly stimulate collagen synthesis (Ishibe et al, 1991). This work was designed to comparatively evaluate the activities of Small intestinal Alkaline and Acid phosphatases in the rat, bat and pangolin.

Materials and methods

Experimental Animals

Ten (10) second generation bred Wistar rats (Rattus norvegicus) of both sexes were obtained from the Animal holdings of the department of Anatomy of the University of Ilorin. Ten (10) fruit bats (Eidolon helvum) were captured with the assistance of experts (who possess State permits) at the bats colony at Flower Gardens area of GRA (Government reserved area) of Ilorin Kwara State. Nigeria. They were kept in bird cages prior to sacrifice. Ten (10) Pangolins (Manis tricuspis) of both sexes were procured from Asejire area of Oyo State, Nigeria. They were brought to the Department of Anatomy, University of Ilorin Nigeria and kept overnight prior to sacrifice. All animals were handled in conformity with the rules and guidelines of the animal rights committee of the University of Ilorin. The study protocol was approved by the same committee. They were evaluated and judged presumably healthy, fit enough to use for the study. ACP and ALP kits were bought from Randox lab. Ltd. UK

Methods

Animals were transported live to research laboratories and after weighing, gastrointestinal tracts were obtained after cervical dislocation via a ventromedian incision in the abdominal wall. The oesophagus was severed cranial to the diaphragm, and the pelvic bones cut carefully to reveal the rectum. Gastrointestinal tracts were dissected free of mesenteries and immediately transferred to a 0.25M sucrose solution. The small intestine was separated into component parts and a 10% homogenate of the tissues in chilled sucrose solution was immediately prepared with Polter-Elvhjem homogenizer. The homogenate were centrifuged at 5000rmp for 10 minutes. The supernatants were immediately stored in the freezer (-20 ⁰ C) and assayed within 48hours. The activities of ACP and ALP were estimated by the methods of Richterich et al (1962) and Englehardt (1970) respectively.

Alkaline phosphatase activity was measured spectrophotometrically at 405nm. The reaction principle is

Sample absorbance was read against air.

Acid phosphatase activity was measured spectrophotometrically at 405nm. The reaction principle is

Sample absorbance was read against reagent blank.

Statistical analysis

Values were reported as mean ± S.E.M and data were analyzed using students t-test with the statistical software SPSS version13 at 95% confidence interval. A p<0.05 was considered statistically significant.

Results and Discussion

Alkaline phosphatase activity was found to be significantly higher (p<0.05) in the bat’s small intestine compared to the rat and the pangolin, but there was no significant difference in the ALP activity between the rat and the pangolin. Acid phosphatase activity was found to be significantly higher (p<0.05) in the bat’s small intestine compared to the rat and pangolin but the differences in ACP activity was not found to be significant in the rat and pangolin

Graph 1 showing the Activities of Alkaline Phosphatase (ALP) in the small intestine of rat, bat and pangolin(umol/min/mg)

Graph 2 showing the Activities of Acid Phosphatase (ACP) in the small intestine of rat, bat and pangolin(umol/min/mg)

Biochemical enzyme estimations reveal functional modifications of these mammals to diet. Alkaline Phosphatase (ALP) facilitates transport across intestinal mucosa. Intestinal biopsies from children classified as ‘failure to thrive’ revealed alkaline phosphatase activity patchily absent from the tips of the villi – a finding which was not so in control series. This was adduced to either defects in maturation of enterocytes or an accelerated degeneration rate. This was associated with some degree of malabsorption (Dawson, 1981). ALP also facilitates the breakdown of ATP to ADP and inorganic phosphate thereby making free energy available for metabolic processes (Murray et al., 2003). Its activity was found to be higher in bats than in rats and pangolin (p<0.05) which probably explains why transit time in the bat gut is short according to the work of Makanya et al, 1997. ALP activity is significantly (p<0.05) higher in the rat than pangolin which suggests long transit time in the pangolin gut. Acid Phosphatases (ACP) are lysosomal enzymes present within phagolysosomes of absorptive cells and in macrophages within the lamina propria (Dawson, 1981). ACP activity was found to be higher significantly (p<0.05) in bats than pangolin and rats. This is because ACP helps to “mop up” high energy bonds released in the bat gut.

The results of this study provides information on the biochemical peculiarities of the small intestine in the rat, bat and pangolin, revealing that the different mammals have evolved different mechanisms to cope with their different diets and suggesting that the rat and bat are more developed.

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