The effect of some different blood anticoagulants on rabbits plasma biochemical parameters under different temperatures

Ehssan N. Jasim Al-Obaidy, Muna Muhammed Ismail and Measer Zaid Muhi. The Effect of Some Different Blood Anticoagulants on Rabbits Plasma Biochemical Parameters under Different Temperatures. Cardiometry; Issue No. 30; February 2024; p. 396-40; DOI: 10.18137/cardiometry.2024.30.3640; Available from: effect-some-different-blood-anticoagulants

The blood samples preservation are related to the important role of anticoagulants which anble the blood samples analysis within more than several mints and hours of collection which respectively revealed the healthy status of animals according to routinely hematology and biochemistry (BuffEnstein et al., 2001). Both in vivo and invitro the clot formation prevented by anticoagulant resulting blood plasma which represent about 5% of total body volume, it characterized by presence of fibrinogen opposite to serum, pale yellow may changes according to total bilirubin, fats, lipdimea, hemoglobin, bacterial contamination and drugs(( Uchiike and Hirakawa, 2002Banfi et al.,2007(Guignard et al.,2012 (Cedrone,et al.,2018)).) .according to the importance of plasma to estimate many bio chemical tests( e.g glucose, total protein, cholesterol and urea) to evaluate the functions of body organs; therefore The aims of this study was to evaluate the chelating Ca anticoagulants (EDTA, citrate and oxalate)and antithrombin (heparin)anticoagulant usefulness for routine plasma glucose, total protein, cholesterol and urea analysis under room and extreme temperatures (-18°C&50°C). Calcium chelating anticoagulants agents EDTA, citrate and oxalate they produce their effect through binding with Ca ion and prevent cascade of clot formation: EDTA is a polyproticacid combined with four carboxylic group and two amino group with pair electrons that chelat calcium (Banfi, et al.,2007)(Farias et al.,2016). Citrate: is a organic acid isolated firstly from lemon act by binding with metal ion to form calcium citrate complex molecule that making the ion inactive (Oliveira etal.,2015;Gros etal.,2023). Oxalate: include many salts sodium -oxalate, potassium -oxalate and others also for insoluble prothrombin that thrombin will not form (Guder,2002). Heparin: it is a chain of monosac-charid, it is need cofactor to produce its activity, serine of thrombin and arginine reactive site on antithrombin molecule that lead to confrontational and convert antithrombin from slow to a very rapid thrombin inhibitor , also heparin bind to thrombocyte to induce or inhibit it aggregation depend on experimental condition, other mechanism also increase permeability of vessel wall, decrease the proliferation of vascular smooth muscle cell and suppress the osteoblast formation (Hirsh, et al.;2001;Onishi etal.,2016;Mathew et al.;2022; Zang et al.,2022). Blood plasma is a fluid part of whole blood, obtained by using anticoagulants and process of certification ; blood plasma expressed as blood with anticoagulant and fibrinogen while blood serum expressed as blood without anticoagulant and fibrinogen (Mathew et al.;2022; Hatami and Saadat-mand,2022).

MATERIALS AND METHODS

Experimental animals Five male rabbits weighting between 800-1000gm, put in the animal house /Col-lege of Veterinary Medicine/University of Diyala, under 12:12 dark light cycle with ad libtum food and water in individual cages (60cm,40cm) during November and December 2022.

Blood collection Four ml of blood samples were collected by cardiac puncture (23G needle) into four tubes contain anticoagulants: (EDTA, citrate, oxalate and heparin)one ml of blood for each tube; the the blood samples were kept at :room temperature 25C˚, waterbath50 C˚ (MeMMert 854 Schwabach), and refrigerator -18 C˚(Arecilik Turkey), for 1hr; then were put in centrifuge at 3000rpm for 15min to get plasma.

-Preparation of anticoagulants:

1-Heparin LEO 1,000IU/ml made in Danemark.

2-ethylene diamine tetra aceticacid (EDTA) 10%: 10gm powder of EDTA (Hi Midia Laboratories

3-Sodium citrate 3.8%:

38gm powder of sodium citrate (BDH- AnalaR, Chemicalls Ltd Poole England) dissolved in 1000ml distilled water.

4.sodium oxalate3.0%:

30gm powder of sodium oxalate(BDH- AnalaR, Chemicalls Ltd Poole England) dissolved in 1000ml distilled water.

Glucose determination: Enzymatic calorimetric-Test

Principle:(glucose kit- Biocon, Germany)

Determination of total protein: Biuret calorimetric method. Using Spectrophotometer LKB-Biochrom, England.

Principle:Protein –kit (Biomerieux, France) is a reagent for determination of total protein, .

Enzymatic calorimetric method

Principle: ( cholesterol –kit Biomerieux, France):m.

Determination of urea in the blood: by using Urease/Salycilate Enzymatic colorimetric method ENDPOINT.

RESULTS

Plasma glucose: levels of chelating Ca anticoagulants (EDTA, citrate, oxalate) and antithrombin anticoagulant (heparin) are present in table (1). The results showed that the all types of anticoagulants revealed no significant differences (P > 0.05) between them after 1hr at 25° C ;on the other hand the table (1) expressed the significant increase p<0.05 in plasma glucose concentration after 1hr at -18^O C and 50^O C of Ca chelating anticoagulants as compare with antithrombin.

Plasma total protein: plasma total protein levels of Ca chelating anticoagulants (EDTA, citrate, oxalate) and antithrombin are explained in table (2). The results revealed no significant differences (P > 0.05) in plasma total protein of both groups of anticoagulants (Ca chelating and antithrommbin anticoagulants) after 1hr at 25^O C ;while there was significantly increased ( p<0.05) occur after 1hr at -18^O C and 50^O C of plasma total protein in Ca chelating anticoagulants group as compared with antithrombin anticoagulant.

Plasma cholesterol:

Plasma cholesterol values of Ca chelating and antithrombin anticoagulants after 1hr at 25^O C , -18° C and 50° C are illustrated in table (3).plasma cholesterol levels appeared with un significantly differences (p>0.05) between both groups of anticoagulants(Ca chelating agent and antithrombin) after 1hr at 25^O C where as significantly increased (p<0.05)in Ca chelating group after 1hr at -18^O C and 50° C as compared to antithrombin (heparin) anticoagulant.

Plasma urea:

The data pertaining to plasma urea concentration of Ca chelating and antithrombin anticoagulants are depicted in table (4). According to statistical analysis

• Table (1)

The effect of Ca chelating (EDTA, citrate ,oxalate) and antithrombin (heparin) anticoagulants on plasma glucose concentration under different temperatures for 1hr

plasma Glucose mg/dL Time Anticoagulants EDTA After 1hr at 25^ 103±1.003 Aa After 1hr at -18^ 108±1.020 Bb After 1hr 50^ 109±1.000 Bb citrate 104±1.087 Aa 107±1.009 Bb 108±1.007 Bb oxalate 103±1.560 Aa 107±1.088 Bb 108±1.059 Bb Heparin 104±0.999 Aa 105±0.966 Aa 105±1.000 Aa plasma samples=24/anticoagulants type and 8/temperature; data expressed as mean ±SE , p<0.05; LSD= 1.5; capital litters denote differences between groups; small litters denote differences within groups.

• Table (2)

The effect of Ca chelating (EDTA, citrate , oxalate) and antithrombin (heparin) anticoagulants on plasma total protein concentration under different temperatures for 1hr

plasma Total Protein G/dL Time Anticoagulants After 1hr at 25^ After 1hr at -18^ After 1hr 50^ EDTA 5.9±0.136 Aa 86±0.210 Bb 7.7±0.233Bb citrate 6.0±0.129 Aa 6.9±0.222 Bb 7.4±0.211 Bb oxalate 6.0±0.122 Aa 16.9±0.201Bb 17.0±0.300 Bb Heparin 5.6±0.111Aa 5.7±0.211 Aa 5.9±0.299 Aa plasma samples=24/anticoagulants type and 8/time and temperature; data expressed as mean±SE , p<0.05; LSD=0.6;capital litters denote differences between groups; small litters denote differences within groups.

• Table (3)

The effect of Ca chelating (EDTA, citrate , oxalate) and antithrombin (heparin) anticoagulants on plasma cholesterol concentration under different temperatures for 1hr

Plasma Cholesterol mg/dL Time Anticoagulants After 1hr at 25^ After 1hr at -18^ After 1hr 50^ EDTA 205±1.077 Aa 211±0.988 Bb 217±0.696Bb citrate 206±0.999 Aa 213±0.909 Bb 217±0.709 Bb oxalate 205±1.000 Aa 211±0.975Bb 215±0.698 Bb Heparin 204±1.111Aa 205±0.989 Aa 206±0.699 Aa plasma samples=24/anticoagulants type and 8/time and temperature; data expressed as mean±SE , p<0.05; LSD= 5.9;capital litters denote differences between groups; small litters denote differences within groups.

• Table (4)

The effect of Ca chelating (EDTA, citrate , oxalate) and antithrombin (heparin) anticoagulants on plasma urea concentration under different temperatures for 1hr

Plasma Urea mg/dL Time Anticoagulants After 1hr at 25^ After 1hr at -18^ After 1hr 50^ EDTA 21±1.022 Aa 19±1.1012 Bb 19±1.189Bb citrate 20±0.999 Aa 18±1.098 Bb 18±1.032 Bb oxalate 21±1.101 Aa 18±1.071Bb 18±1.200 Bb Heparin 20±1.098Aa 205±0.989 Aa 20.7±1.077 Aa plasma samples=24/anticoagulants type and 8/time and temperature; data expressed as mean±SE , p<0.05;LSD=1.5;capital litters denote differences between groups; small litters denote differences within groups there was no significant differences (p>0.05) between Ca chelating and antithrombin anticoagulants in plasma urea after 1hr at 25OC. Plasma urea concentration significantly decreased (p<0.05)in Ca chelating group as compared with antithrombin anticoagulant after 1hr at -18OC and 50oC.

DISCUSSION

Plasma Glucose: The results of present study showed elevation of plasma glucose concentration in Ca chelating anticoagulants group with no change in antithrombin group. The bodies get energy from food specially glucose which is a monosac-charid with a chemical structure C6H12 O6 either from hydrolyse of fractose and galactose (Gurung et al.; 2022).

The glycolytic enzyme are present in erythrocytes and white blood cell therefore the glucose concentration decrease with time due to contentious cell consumed at 5%/hr or 40%/3hr.(McMilin, 1990).our result agree with (Bogan,et al.;2020 and Furtado, et al.2022) who found that heparin stablized glucose concentration within normal limits for 10 hr in yellow -spotted amazon river turtle and indigo snake which naturally present in the body (Bogan,et al.;2020 and Furtado, et al.2022). We can also related our result may be due to chemical structure of heparin which composed from monosaccarid chains; never the less the elevation of plasma glucose concentration of Ca chelating anticoagulant may be due to the extreme cool and hot condition lead to exhaust and stop glycolysis by red and white blood cell.

Plasma Total protein:The present study demonstrated elevation of plasma total protein in Ca chelating group as compared with antithrombin group of anticoagulants under -18°C and 50°C . Protein forms the major portion of dissolved substances in plasma they form the basic structural components of the body they constitutes the enzymes present in body and also act as secondary source of energy, The other function include distribution of water, buffering, transport of various compound , defense and blood coagulation (Heestermans et al.;2022). Plasma total protein concentration were not influenced using different anticoagulant in our study and agree with (Furtado,2022) but disagree with (Oliveira,2015) who record the using of Ca chelating anticoagulant lead to decrease plasma total protein due to the clot formation that retains protein. The elevation of total protein in our study may be come from the extreme condition which resulted in distraction of cellular part of blood consequently lead to elevated plasma protein.

Plasma Cholesterol: This experiment resulted in increase of plasma cholesterol of Ca chelating anticoagulants group with constant level in antithrombin group.The fluidity of plasma cell membrane is getting from cholesterol which represent one of the importanrt constituentes of cell membrane structure, steroid hormones in the body and bile synthesis; cholesterol transported in the blood as lipoprotein besid the erythrocytes composed about 50% of total blood volume therefore they carry large quantity of free cholesterol and they produce fragment of it or during exocytosis processes (Rifal ,et al.; 2000; Ohkawa et al.;2020).

Plasma Urea: In the present investigation, the plasma urea concentration decreased in Ca chelating anticoagulants group as compared with antithrombin anticoagulant. Urea synthesis occur in the liver and it is excreted by kidney after transported by blood with some little excreted through skin by sweating, some factor affected its level like protein consumption and health status of liver and kidney(Allison and Ojule, 2020).

CONCLUSIONS

AND RECOMMENDATIONS

Conclusions

• 1.    plasma parameters which used in this study was approximately equal under room temperature 25C˚ in both types of anti-coagulants Ca chelating (EDTA, citrate and oxalate) and antithrombin (heparin).

• 2.    antithrombin (heparin) anticoagulant was more effective than Ca chelating anticoagulant( EDTA, citrate and oxalate) in preserve plasma level samples within normal level under extreme cool and hot conditions.

Recommendations

• 1.study the effect of these anticoagulants on liver enzymes ….etc.

• 2.study the effect of stress like sun light on blood samples contain these anticoagulants on plasma parameters.

• 3.study the effect of extreme temperatures on serum biochemical parameters.