Comparison of Magnesium  by EDTA and  EGTA as chelating agent

D Jethva*, P Shruthi, S Patel, N Karbhari, N Savaliya

International Journal of Clinical Biochemistry

Research - International Journal of Clinical Biochemistry ( 2022) Volume 7, Issue 3

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Comparison of Magnesium by EDTA and EGTA as chelating agent

D Jethva^*, P Shruthi, S Patel, N Karbhari and N Savaliya

Department of Biochemistry, University of South Gujarat, Gujarat, India

^*Corresponding Author:
D Jethva, Department of Biochemistry, University of South Gujarat, Gujarat, India, Email: jethvadhvani807@gmail.com

Received: 01-Dec-2022, Manuscript No. IJCB-22-81949; Editor assigned: 05-Dec-2022, Pre QC No. IJCB-22-81949 (PQ); Reviewed: 20-Dec-2022, QC No. IJCB-22-81949; Revised: 27-Dec-2022, Manuscript No. IJCB-22-81949 (R); Published: 03-Jan-0023, DOI: 10.15651/IJCB.23.7.028

Abstract

Magnesium estimation in serum is done by various methods like Arsenazo III, Ortho Vitros Microslide System, Atomaticabsortption, Calmagite, Xylidyl blue, Methylthymol blue. One of the methods is Xylidyl blue in which chelating agents EDTA or EGTA can be used. In this study, Comparison is done between EDTA and EGTA as chelating agent. EGTA is a chelating agent with a much higher affinity for Ca²⁺ at pH 11.00. EDTA is a chelating agent for di and trivalent metal ions. EDTA binds to metals via four carboxylate and two amine groups. Magnesium is abundant cation in the human body. Majority is present in bone and muscle, Extra Cellular Fluid (ECF). Magnesium is a responsible for energy metabolism and the synthesis of proteins and nucleic acids. Methods-Measurement of magnesium is done by Xylidyl blue method using EGTA and EDTA as Chelating agent.

Keywords

Magnesium, Xylidyl blue, Quality control, Metal ions, Calibration

Introduction

Magnesium

Random Patient’s left over 50-serum samples are selected from indoor as well as outdoor of New Civil Hospital, Government Medical College Surat. Magnesium ions react with xylidyl blue in an alkaline medium to form a water soluble purple-red chelate, the colour intensity of which is proportional to the concentration of magnesium in the sample. Calcium is excluded from the reaction by complexing with EGTA and EDTA. Results and Statistical Analysis-Linear regression analysis using spreadsheet functions shows slope of 0.96 and r² of 0.91. Slope of 0.96 indicate that Magnesium with EDTA, on an average gives about 4% higher result as compared to Magnesium with EGTA. Bland altman plot of magnesium (EGTA) vs. difference (EDTA-EGTA) shows positive bias in Magnesium (EDTA) in comparison with Magnesium (EGTA).Conclusion-Results in patient’s sample as well as in quality control sera are comparable with acceptable accuracy and precision. As adequate research study are not available for magnesium with EDTA method till date so further study is required for validation and verification of this method.

Magnesium is the fourth most abundant cation in the human body. Majority is present in bone and muscle, only approximately 1% being present in the Extra Cellular Fluid (ECF). Magnesium is a cofactor in more than 300 enzyme-catalyzed reactions. It is responsible for energy metabolism and the synthesis of proteins and nucleic acids. It controls various trans-membrane ion channels and membrane excitability. Magnesium is widely distributed in foodstuffs, green vegetables being a particular rich source because it is a component of chlorophyll. The recommended daily intake is 420 mg (18 mmol) in men and 320 mg (10 mmol) in women (Bazydlo, 2014). It acts as a competitive inhibitor to calcium, preventing calcium entry into the presynaptic nerve and thus inhibiting the release of neurotransmitters. Deficiency of magnesium can lead to numerous metabolic and nervous-system pathology.

Deficiency

Hypomagnesemia can cause hypokalemia (through adverse effects on Na+, K+ -ATPase) and hypocalcemia (para-thyroid hormone is dependent on magnesium), and these may be responsible for some of its clinical manifestations.

Hypermagnesemia

Increased serum magnesium levels are associated with dehydration, severe diabetic acidosis and Addison's disease. Symptomatic hyper magnesemia is caused by excessive intake, resulting from administration of antacids, enemas, and parenteral fluids containing magnesium.

Magnesium estimation in serum is done by Arsenazo III, Ortho Vitros Microslide System, Atomaticabsortption, Calmagite, Xylidyl blue, Methylthymol blue, chlorophosphonazo III,eNZYMATICmethods. One of the methods is xylidyl blue in which chelating agents is used. In this study, Comaprision is done between EDTA and EGTA as chelation agent.

EGTA-(Ethylene Glycol-bis (β-aminoethyl Ether)- N,N,N′,N′-Tetraacetic Acid)

EGTA is a chelating agent with a much higher affinity for Ca²⁺ at pH 11.00

EDTA-Ethylenediaminetetraacetic Acid

EDTA is a chelating agent for di and trivalent metal ions. EDTA binds to metals via four carboxylate and two amine groups. Ethylenediaminetetraac etic acid is a chelating agent of divalent cations such as Ca²⁺ and Mg²⁺ that is particularly useful for hematologic examinations including transfusion medicine applications, measurement of intracellular drugs such as cyclosporine or tacrolimus, HbA 1c analysis isolation of genomicDNA (Nguyen, 2020).

Methodology

This study is planned for patients of New Civil Hospital, Government Medical College Surat; Random Patient’s left over serum samples are selected from indoor as well as outdoor Opd.

Inclusion Criteria: All the age group patient’s left over serum sample are randomly selected from inpatients and outpatients department (Rifai, 2017).

Exclusion criteria: We have made criteria for rejection sample with following condition.

• Haemolysed sample

• Sample collected in other than plain vacuttee

• Serum less than 2 ml

• Lipamic sample

Sample Preparation

Sample were identified and sorted for study. Samples were given unique Lab ID number and registration was done in Laboratory Information System (LIS). All samples were centrifuged for 10 minutes at 3000 rotation per minute speed.

Calibration and Quality Control: Calibration of magnesium reagent with EDTA AND EGTA was done first. Calibration was done by Randox Quality Control (RQC). 1 Cup of DI water, 1 cup of half diluted normal level quality control, 1 cup of normal level quality control ( Lot No-1502UN), 1 cup of 1:1 mixure of Normal level and high level Quality control (100 ul Normal QC+100 ul high QC ),1 cup of ( Lot No-1195UE) high level quality control are used for calibration. Each level is run 5 times and average OD is used and factor is calculated. All samples are analyzed after calibration with EGTA AND EDTA in single batch of 50 samples.

Principle: Magnesium ions react with xylidyl blue in an alkaline medium to form a water soluble purple-red chelate, the colour intensity of which is proportional to the concentration of magnesium in the sample. Calcium is excluded from the reaction by complexing with EGTA and EDTA Reagent Content: Mgnesium reagent and contain Tris base, Na₂Co₃, Xylidyle blue, Triton X-100, magnesium reagent contain EDTA Chelating agent and contain EGTA Chelating agent. Reagent preparationabove mentioned reagents is in-house reagents. All Glassware which used are being washed by 10% HCL or 10% acetic acid. Purpose of washing is minimizing the contamination of reagents from various unmeasured ions present on surface of the glassware. Fixed amount of chemicals were added in particular sequence. pH of reagent is most important matter to be maintained. All above reagents are stable on board for long time. Around 10 ml of R1 is being kept in sterile plastic container and placed in ERBA XL-1000 reagent tray for use (Ryan, 1998).

Magnesium test parameter in XL-1000: Magnesium measured by 2 point method, with sample-reagent ratio is 1:100 and reading will be taken by 505 nm wavelength (Staufenbiel, 2022).

Results and Discussion

Results of Magnesium obtained by both the method were entered in a spreadsheet. Following is the scatter plot of magnesium (EDTA) vs. magnesium (EGTA).

Linear regression analysis using spreadsheet functions shows slope of 0.96 and R² of 0.91 Slope of 0.96 indicate that magnesium with EDTA, on an average gives about 4% higher result as compared to magnesium with EGTA. Bland altman plot of magnesium (EGTA) vs difference (EDTA-EGTA) is shown below. It also shows positive bias in magnesium (EDTA) in comparison with magnesium (EGTA).

Bland Altman plot of magnesium average of EDTA and EGTA vs. difference (EDTA-EGTA) is shown below. It also shows positive bias in magnesium (EDTA) in comparison with magnesium (EGTA).

Comparison of both Bland Altman Plot shows that, there is relatively constant positive bias in magnesium (EDTA) with respect to magnesium (EGTA) (Stocco, 2019).

Magnesium estimation by xylidyl blue is well known method. Magnesium can be quantified through either an increase in absorption of chelate at 505 nm or a decrease in absorption of xylidyl blue at 600 nrn. In the xylidyl blue method, calcium in serum or urine is generally chelated selectively by GEDTA or EGTA (included in the reagent mix) prior to analysis of magnesium in the sample Currently commercially available magnesium estimation kit by xylidyl blue method is contained EGTA as chelating agent. In this study, EDTA is used as chelating agent instead of EGTA and comparison is done between two methods. Results are comparable between two methods. Intra-run average CV% of two level (normal and abnormal range) quality control for EDTA is 6.39% and for EGTA is 8.71%. No adequate research data and study are available for magnesium estimation with EDTA. More detailed study with larger sample size is needed to do to find out linearity, interfering and influencing factors (Valldecabres, 2022). Prior to analyzing the presence of magnesium in a sample using the xylidyl blue method, calcium in serum or urine is typically selectively chelated by GEDTA or EGTA (included in the reagent mix). In the currently available magnesium estimation kit, the chelating agent EGTA is used in the xylidyl blue method (Figures 1-3).

Figure 1. The scatter plot of magnesium (EDTA) vs. magnesium (EGTA).

Figure 2. Bland altman plot of magnesium (EGTA) vs. difference (EDTA-EGTA).

Figure 3. Bland Altman plot of Magnesium average of EDTA and EGTA vs. difference (EDTA-EGTA).

Conclusion

Comparison of serum magnesium level in patients and two level quality control sera by EDTA and EGTA as chelating agent is studied. It has been found that results in patient’s sample as well as in quality control sera are comparable with acceptable accuracy and precision. As adequate research study are not available for magnesium with EDTA method till date so further study is required for validation and verification of this method.