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72 Levels of calcium,sodium and potassium...

Received - 07.06.12Accepted - 17.07.13

Abstract

Influence of anticoagulants in thelevels of major electrolytes like calcium, sodiumand potassium in plasma was studied andcompared with their levels in serum. Forty twoblood samples were collected from sevenspecies namely human, elephant, cattle,sheep, goat, dog and rabbit. Heparin andPotassium Ethylene Diamine Tetra Acetate(EDTA) were used as anticoagulants. It wasfound that the levels of these minerals in theblood plasma got altered significantly by theaddition of heparin and EDTA. However, lessinterference was rendered by heparin thanEDTA, on comparison with the serum values.

Keywords:- EDTA, Heparin, Serumelectrolytes, Plasma electrolytes.

Anticoagulants are added in requiredquantity to inhibit the clotting of blood, therebyensuring that the concentration of substanceto be measured is changed as little as possiblebefore analysis (Guder, 2001). Heparin andEthylene Diamine Tetra Acetic acid (EDTA)exert their actions as anticoagulants byinhibiting thrombin in blood and chelating thecalcium ions. Even though serum fromcoagulated blood is the most preferredspecimen for clinical biochemistry, plasmaobtained with an appropriate anticoagulant canalso be served equally good. The mostpreferred anticoagulant meant for plasmaclinical biochemistry is heparin, while forhematological examination is either sodium orpotassium salt of EDTA. Since the harvest ofserum requires a minimum of 30 min wait forthe completion of coagulation before

LEVELS OF CALCIUM, SODIUM ANDPOTASSIUM IN PLASMA AS INFLUENCEDBY ANTICOAGULANTS

K.V. Pramina1, P. T. Mincy2, P. Anu Joseph3,V. Lisha4, K. A. Mercy5 and V. Ramnath 6*

Department of Veterinary Physiology,College of Veterinary and Animal Sciences,Mannuthy-680 651, Trichur, Kerala.

centrifugation, use of plasma is more specifiedin emergency situations. Furthermore, plasmayield from the given volume of whole blood isalways greater than the yield of serum (Youngand Bermes,1999).

The effects of various types ofanticoagulants on plasma biochemistry havebeen studied in man and various animals. Nocomprehensive information is available withrespect to levels of blood minerals like calcium,sodium and potassium in the serum andplasma collected using different anticoagulantsof farm animals. Thus, the study wasundertaken to compare the variations in theserum and plasma levels of calcium, sodiumand potassium in various farm animal speciesand human and to determine and recommendthe most suitable anticoagulant and its dosefor separation of plasma from blood.

Materials and Methods

About three ml of blood withoutanticoagulant was collected from clinicallyhealthy subjects belonging to each species,viz. human, elephant, cattle, sheep, goat, dogand rabbit and serum was separated as perstandard procedure. Anticoagulated bloodsamples (3ml) were collected simultaneouslyfrom each species with anticoagulant,dipotassium EDTA at concentrations of one,two, three and five mg per three ml blood aswell as with lithium heparin, 60 units per threeml blood. All samples were chilled immediatelyafter collection for 30 min, centrifuged at 1800g for 10 min. The levels of calcium in the serumand anticoagulant added plasma wereestimated spectrophotometrically using

1 to 4: Veterinary Graduates 5. Professor & Head, Dept.of Statistics;6. Associate professor and *corresponding author: 94446579057; drvrnath@gmail.com

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commercial kits following Arsenazo III method(Bauer, 1981). Sodium and potassium levelswere estimated spectrophotometrically usingcommercial kits by the modified methoddescribed by Maruna and Trinder (1958). Theresults were analysed statistically using one-way ANOVA method.

Results and Discussion

The serum and plasma levels ofcalcium, sodium and potassium of human andsix domesticated species of animals viz.elephant, cattle, sheep, dog, goat and rabbitwere estimated and compared with in group.It was found that the concentration of calciumin serum significantly (p<0.05) differed fromthat of plasma in all the species tested. Thelevel of calcium was found to be significantlymore in heparinised plasma compared toserum and other plasma samples (heparin andEDTA at varying concentrations). It was foundthat in cattle blood, addition of EDTA at therate of 1 mg/ 3 ml completely chelated calciumin the blood. However in other species, thisdose was not sufficient. Blood samples ofhuman and sheep showed zero concentrationof calcium when EDTA was added at the rateof 2mg/3ml. The blood from elephant andrabbit when collected with 5 mg EDTA/3ml,were left with trace amounts of free calcium in

the plasma (Table 1).

Addition of heparin as anticoagulantfor plasma separation resulted in significantly(p<0.05) high levels of sodium in the plasmasamples when compared to serum in allspecies. Addition of EDTA to blood resultedsignificantly (p<0.05) higher levels of sodiumin all the concentrations tested.. However, thevariation seen in sodium level was not muchrelevant with different EDTA concentrationsutilized (Table 2).

Heparinised plasma showed lesserdegree of variations in potassium levels whencompared to serum in all the species tested.However, significantly (p<0.05) higherincrease in plasma potassium level wasnoticed when EDTA at different concentrationswas used as anticoagulant (Table 3).

In the present study it was found thatthe levels of minerals like calcium, sodium andpotassium in the blood was alteredsignificantly by addition of naturalanticoagulant heparin and EDTA. Changes inelectrolyte contents induced by EDTA additionwere described earlier in man ,dog, cattle,horse and sheep (Morris et al., 2002; Mohri etal., 2007 a, b; Ceron et al., 2004). They havereported an increase in sodium level in

Table 1. Levels of calcium (mg/dl) in the serum and plasma.

Means with the same superscripts in a row did not differ significantly (P<0.05)

Table 2. Levels of sodium (mmol/l) in the serum and plasma.

Means with the same superscripts in a row did not differ significantly (P<0.05)

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74 Levels of calcium,sodium and potassium...

heparinised plasma, while a decrease inpotassium level as compared to the serumvalues. In the present study sodium as wellpotassium levels were on the higher side inthe plasma samples collected with EDTA.Serum and heparinised plasma yielded similarresults for sodium and potassium levels, whilethe calcium level in EDTA chelated bloodplasma was significantly low. The reason forincreased potassium level in heparinised andEDTA chelated blood plasma could beattributed to the activation of platelets.

EDTA acts as a chelating agentforming complexes with calcium which isessential for coagulation, is recommended forroutine haematology, since it provides a verygood staining quality of blood cells. It can beused as disodium, dipotassium or tripotassiumsalts. The latter two being preferred as theyare more soluble.

The use of serum or plasma in clinicalpathology remains debatable. Serum ispreferred by many laboratories for clinicalbiochemical tests, since it avoids the additionof anticoagulants which can interfere withsome analytical methods or change theconcentration of parameters being measured.Using EDTA as anticoagulant caused asignificant difference for concentrations ofurea, creatinine, total protein, calcium, andmagnesium and the activity of aspartateaminotransferase and alkaline phosphatase,in plasma comparing with serum (Mohri andRezapoor, 2008).

In the serum stored at lowtemperature, the chance of formation of fibrinstrands is lower when compared to plasmaand thus there is less risk of occlusion ofautomated analyzers. However the use ofplasma is preferred in some centres as it savestime because there is no necessity to wait until

coagulation is completed and also 15 to 20per cent more plasma can be obtained fromthe same volume of blood.

With the results of the study, it isrecommended that the relative amounts ofanticoagulants that can be used for separatingplasma using heparin is 20U/ml of blood forall species, while that of dipotassium EDTAfor cattle blood is 1mg/3ml, for sheep andhuman blood is 2mg/3ml, for goat and dogblood is 5mg/3ml and for rabbit and elephantblood is a minimum of 5mg/3ml or slightlyabove. Excess addition of EDTA and otheranticoagulants would result in changes in thelevels of other biochemical parameters.Results of the present study is also suggestiveof the less interference rendered by heparin inthe plasma electrolytes level.

References

Bauer, P. J.1981. Affinity and stoichiometry ofcalcium binding by Arsenazo III.Anal.Biochem. 110: 61

Ceron, J. J., Subiela, M. S., Hennemann, C.and Tecles, F. 2004. The effects ofdifferent anticoagulants on routinecanine plasma biochemistry. The Vet.J. 167: 294–301.

Guder, W. G. 2001. The quality of diagnosticsamples. Blood Gas News. 10: 18–24.

Maruna, R. F. L. and Trinder.P. 1958. Clin.Chem. Acta. 2: 581.

Mohri, M. and Rezapoor, H. 2009. Effects ofheparin, citrate and EDTA on plasmabiochemistry of sheep: Comparisonwith serum. Res.Vet. Sci.86: 111–114.

Mohri, M., Shakeri, H. and Zadeh, L. S. 2007.Effects of common anticoagulants(heparin,citrate and EDTA) on routine

Table 3. Levels of potassium (mmol/L) in the serum and plasma.

Means with the same superscripts in a row did not differ significantly (P<0.05)

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plasma biochemistry of cattle.Comp.Clin. Pathol. 16: 207-209

Mohri, M., Sharifi, K. and Eidi, S.2007.Hematology and serum biochemistryof Holstein dairy calves: Age relatedchanges and comparison with bloodcomposition in adults. Res.Vet. Sci.83:30–39.

Morris, D. J., Fernandez, J. M., Chapa, A. M.,Gentry, L. R., Thorn, K. E. and Weick,

T. M. 2002. Effects of sample handling, processing, storage, and hemolysis on measurements of key energymetabolites in ovine blood. SmallRuminant Res. 43: 157-166.

Young, D. S. and Bermes, E. W. 1999. Specimencollection and processing: sources ofbiological variation. Tietz Textbook ofClinical Chemistry. 3rd ed. W.B.Saunders, Philadelphia. pp. 42-72

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