Research Article Vol:2, Issue:1
SAJMED.2017; 2(1):56-62 PDF Download
Potatoes are both tropical and temperate food crop but highly grown in the temperate. In Nigeria it is mostly available in the northern parts of the country particularly in Jos Plateau confirming the cold climatic preference of this food item. However, there are at least two varieties of the potatoes species, the pinkish coated type and the light brown. The former, is sweet whereas the light brown coated has no sweetness and such biochemical characteristics lies the health relevance of potatoes, (Graca, 2000). Perhaps this is why the high rate of consumption of the popular light brown coated potatoes which is also small in size called, the Irish potatoes. Of course not only consumed by the Irish people but by the whole world. It is best taken when fried and serve with tomatoes sauce and chicken and very easily handled as “take away” snacks. It is rich in carbohydrate. With varied glucose levels, (Ekpo, 2016), (Soh, 1999) (Englyst 1992) (Englyst (1987). Apart from the nutritional values of Irish potatoes. It is also a very good source of biochemical substance, a protein (Enzyme) called apyrase (Molnar 1961) used for platelet storage and as washing medium for physiologic functioning of this blood cell invitro. Platelet is an important blood cell to study due mainly to its small size, complicated features but very responsive in its functional state to bleeding situation though, the mechanism of platelet activation during injury is not fully understood, but its functions are fully understood in adhesion, and aggregation potentated by ADP, CAMP, calciums ions and thromboxane but regulated by its receptors, (Williams, 2010) for final arrest of bleeding (Donna 2001) (Thompson et al 1983). However, platelet alone cannot arrest bleeding during tissue damage without interplay of the coagulation system. But even with this system platelet is very paramount in ensuring perfect haemostasis and in clinical management of bleeding (Shan, 2016). This is measured by the bleeding time and correlation with platelet count. Bleeding will be less with adequate platelet count and increase in low platelet count with structural defects, (Hoffbrand 1991). Thus, platelet is a very relevant cell in understanding haemostatic mechanics. Such relevance led to the search for a non defective physiologic medium for the invitro study of this blood cell also in a physiologic state. Platelet has short half life outside the body and without adequate medium to keep it in a normal state platelet studies invitro would be difficult. This necessitated sourcing for locally available raw material for the preservation of platelet in apyrase enzyme from potatoes. The study was also for the preservation of an important metabolic enzyme, Glucose 6-phosphate dehydrogenase (G6PD). G6PD Glucose-6 phosphate dehydrogenase is an enzyme that is present in virtually all cells. It catalyses the first and rate limiting steps of the hexose monophosphate pathway. The pathway generates nicotinamide adenine dinucleotide phosphate (NADPH) due to the presence of G6PD enzyme. Lipid production in platelets depends on NADPH though the platelet has the capacity for denovo synthesis of lipids as a result of their content of acetyl COA, (Jack et al 1974). For example an important lipid with coagulant activity called platelet factor three ((PF3a) depends on NADPH for its production showing the relevance of G6PD enzyme in the biochemistry activities of platelet (Hoffmans, 1981). In platelet G6PD deficiency where NADPH generation is impaired, haemostatic disorder(s) from impaired PF3a many result, (Duangdao, 2012). The invitro study of platelet biochemical activities with apyrase from locally available potatoes actually enhances the examination of this important blood cell outside the body. But importantly, the study demonstrates the research potentials in sourcing for materials in our environment such which compete qualitatively with the imported apyrase.
Materials And Methods
Extraction and Purification of Apyrase:
The method of Molnar (1961) was used. But briefly 5kg of Irish potatoes were homogenized in a blender after peeling the coats and washed for 10 mins with 1/10 volume of distilled H2O (500ml). It was stirred for 20mins and pressed through cheese cloth. The homogenate was then centrifuged at 450g at room temperature for 10mins in order to remove the sedimenting starch. All consecutive steps were carried out at 0-100c. Five hundred and twenty millitre (520ml) of the extract was brought to 0.024 McalCl2 by addition of appropriate volume of IMcaCl2 solution. The mixture was stirred for 10mins and any precipitate of calcium phosphate was allowed to settle for 1 hour. A dark colour formed was decanted and the sediment centrifuged for 20mins at 1000g. It was suspended in IMcalCl2 (1/10 volume) of the original extracting volume (520ml) and was stirred for 1 hour. The procedure resulted in the elution of the apyrase enzyme from the calcium phosphate precipitate and was centrifuged at 1000g for 20mins. The supernatant (40ml) was dialysed against 0.1MkCl for 20 hours, an aspect of purification. A small amount of precipitate formed after dialysis was removed by further centrifugation. Thirty millilitre (30ml) of the enzyme was added to tyrode solution and used for platelet washing and suspension.
5 – 10ml of venous blood was collected from human subject’s age range 15-40yrs.
Extraction of Platelets from Human Whole Blood: Whole blood of 10ml was centrifuged at 120g for 10mins, to obtain platelet-rich plasma (PRP). The PRP was further centrifuged at 6000g for 30mins to obtain pure platelets.
G6pd Activity in Platelet Stored In Apyrase and Those without Apyrase:
Both platelets stored in apyrase and those without the enzyme were lysed using triton x – 100. The lysing resulted in the elution of G6PD enzyme examined in the study. Two tests were carried out to confirm the presence of G6PD enzyme (a) fluorescence spot test (b) electrophoresis.
Preparation for Electrophoresis Tank /Fluorescence Spot Test and Sample Application:
250ml of Tris buffer pH 8.6 was measured into each compartment of the tank. The shoulder piece was adjusted to obtain a gap of 5cm. Filter paper pads were used as wicks. They were impregnated with the buffer solution and placed over the Perspex-shoulder pieces with one edge immersed in the catholic and anodic compartments of the buffer solution. The titan III paper was soaked for 10mins in the Tris buffer and set firmly in place using tension rod for maximum contact. It was set at a voltage of 260V and ImA current per centimeter of the strip. The lysates of platelets and red blood cells as control were applied on the cellulose acetate strip using multiple applicators that deliver appropriately 2ul aliquot of each sample and electrophoresis done for 20 mins.
Fluorescence Spot Test: 10ml of blood was added to 100ml of G6PD screening mixture in a test tube. A drop of this mixture was made on a filter paper using Pasteur pipette and observed under UV light for fluorescence. This served as control for platelet G6PD which same preparation as that of red blood cells was done.
Preparation of G6PD screening mixture for G6PD Spot Test:
Materials: D.Glucose-6 Phosphate, NADP, saponin, Tris Hcl, buffer, oxidized glutathione (GSSG), uv.light, blood, whatman No.1 paper.
(a) 10mmol/L of D. Glucose-6 phosphate (b) 305mg of dissodium salt or potassium salt was dissolved in 100ml of water.
(c) 60mg of NADP and 60mg of dissodium salt was dissolved in 10ml of water.
(d) 750mmol/L of saponin was prepared
(e) Tris Hcl buffer (200mmol/L
(f) 24.23 of Tris hydroxymethyl amino methane was added to 250 of mater, add 1mol/L of Hcl and make up to 1 litre with water for the pH OF 7.8.
(g) 49mg of GSSG was dissolved in 10ml of water
(h) The prepared reagents were mixed as follows.
(1) 2ml of G6P(Glucose 6-phosphate)
(2) 1ml of NADP
(3) 2ml of saponin
(4) 30ml of buffer
(5) 1ml of GSSG( Reduced glutathione)
(6) 2ml of water. This is called screening or reagent mixture
(7) 10ml of whole blood was added to 100ml of reagent mixture.
The lysed samples were applied on titan paper iii for electrophoresis and migration of the platelet G6PD was observed with red cell G6PD as control indicating the presence of the enzyme in platelets.
Platelets stored in apyrase for up to 48hrs extracted from Irish potatoes showed G6PD activity under spot test and electrophoresis, whereas the one stored without apyrase enzyme for such a time did not show G6PD activity. Also there was no difference in activity with the commercially available apyrase and the locally extracted one as tested by the activity of the G6PD enzyme in the platelets.
In the study, the extracted apyrase from potatoes proved its potency by maintaining the physiologic activity of the platelet invitro as demonstrated by the active presence of the G6PD enzyme in a functional state. However, platelet without apyrase had no G6PD activity. The report is in keeping with platelet G6PD kinetics in apyrase (Kosow 1974). The difference with this report is the quantitative and qualitative methods (spot test) and electrophoresis, applied in platelet G6PD studies and comparative potency of apyrase enzyme. Platelet storage in long duration for transfusion especially in bleeding disorders are with complications without adequate medium particularly with the short half life of this blood cell. In the study, platelets were stored for 48 hours with intact physiologic activity in apyrase as compared with imported apyrase. Imported apyrase is expensive and takes a long time to arrive when ordered.Besides, import duties are high and thus a source of capital flight with effects on the economy. Irish potatoes are highly available and in large quantities in our environment and will form a good source of foreign exchange earning if exploited at the manufacturing level. But very importantly will be the removal of out of stock syndrome in our health institutions particularly the tertiary ones when this chemical is produced locally. Such availability will enhance health care delivery and promote cellular researches in bleeding diathesis.