SUMMARY AND EXPLANATION

Magnesium in the body is found primarily in bone with some soft tissue, and serum. Decreased levels have been observed in cases of diabetes, alcoholism. diuretics, hyperthyroidism, hypothyroidism, malabsorption, hyperalimenation, myocardial infarction, congestive heart failure and liver cirrhosis. Increased serum magnesium levels have been found in renal failure, diabetic acidosis, Addisons disease and vitamin D intoxication.

Serum magnesium measurement was first introduced in the 1920s using a precipitation procedure of Kramer and Tisdall (1), Briggs (2), and Denis (3). These were later followed by a variety of methods including complexometric EDTA titration procedure (4), fluorometric procedures involving chelates of magnesium (5,6) and dye absorption method based on the reaction of Titan Yellow with magnesium hydroxide to form a red-colored lake (7). Each of these procedures suffered from numerous technical difficulties which greatly affected the accuracy and precision of their results.

Atomic absorption remains the most accurate method for magnesium determinations. However, this method requires expensive instrumentation and uses large sample volumes which limit its usefulness for pediatric testing (8). More recently, colorimetric dye-complexing methods have been developed using such dyes as Calmagite, Eriochrome Black T. Magon and methylthymol blue (9).

The EAGLE MAGNESIUM PROCEDURE uses metallochromic dye Calmagite for a rapid, easy and accurate determination of magnesium in serum (10, 11). Some of the advantages of this method are: protein precipitation is unnecessary, interference from calcium or heavy metals is eliminated, and the method agrees very closely with values obtained by atomic absorption (11).

PRINCIPLE

Serum magnesium ions react with Calmagite in alkaline medium to produce a red complex which is measured spectrophotometrically at 530 nm. The intensity of the color produced is directly proportional to magnesium concentration. Calcium interference is eliminated by use of EGTA (12). Heavy metal interference is prevented by the presence of cyanide and a surfactant system is included to remove protein interference.

REAGENTS: FOR IN-VITRO DIAGNOSTIC USE

Reagent Set Cat. No. 3280 provides:

MAGNESIUM COLOR REAGENT - (Cat. No. 3281)

REACTIVE INGREDIENTS:

Calmagite 0.006% w/v; stabilizer and surfactant added.

PRECAUTION:

Causes irritation. Avoid contact with skin, eyes and clothing. In case of contact, wash with large amount of water. DO NOT PIPET BY MOUTH!

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date if sealed tightly and protected from contamination.

DETERIORATION:

Turbidity would indicate contamination and the reagent should not be uesd.

MAGNESIUM  BUFFER REAGENT - (Cat. No. 3282)

REACTIVE INGREDIENTS:

2-Ethylaminoethano, 6.0% w/v; EGTA 1018mM; Potassium cyanide 0.10% w/v.

PRECAUTION:

POISON/CAUSTIC AVOID ALL CONTACT! In case of contact, wash area with soap and large amount of water.

STORAGE AND STABILITY:

Store at 15-30ºC. Stable until expiration date if sealed tightly and protected from contamination.

DETERORIATION:

Reagent should be clear and colorless. Turbidity indicates deteroration and the reagent should not be used.

MAGNESIUM CALIBRATOR - ( Cat. No. 3283)

REACTIVE INGREDIENTS:

Equivalent to 2.0 mEq/L magnesium in an aqueous solution.

STORAGE AND STABILITY:

Store at 15-30ºC. Keep tightly sealed and protect from contamination.

DETERIORATION:

Failure to achieve assay values of freshly prepared control sera would indicate possible deterioration.

PREPARATION OF WORKING REAGENT:

Combine 10 volumes of Magnesium Color Reagent with one (1) volume of Magnesium Buffer Reagent in a disposable plastic container or an acid-washed glass container. Working reagent is stable for 48 hours at 15-25ºC and it is recommended that only enough reagent for one day of testing be prepared.

INSTRUMENTS

Use a spectrophotometer or colorimeter calibrated at 530 nm.

SPECIMEN COLLECTION

PRECAUTIONS:

1. Use fresh, unhemolyzed serum. Red cells contain twice the magnesium concentration as serum, therefore, a hemolyzed sample will falsely elevate results (13).

2. Grossly lipemic or icteric specimens should not be used.

3. Heparinized plasma may be used.

SAMPLE STORAGE:

Serum magnesium appears stable for 5 days at 2-8ºC.

ADDITIVES:

No special additives or preservatives are needed.

INTERFERING SUBSTANCES:

Hemolyzed, grossly icteric or lipemic specimens are unsuitable for this method. Young et al (14) have reviewed drug effects on serum magnesium levels.

PROCEDURE

MATERIALS PROVIDED:

MAGNESIUM COLOR REAGENT (Cat. No. 3281) and MAGNESIUM CALIBRATOR (Cat. No. 3283)

MATERIALS REQUIRED BUT NOT PROVIDED:

1. 0.01 mL micropipettor

2. 1.0 mL pipettor or dispenser

3. Timer, test tubes and rack.

REACTION CONDITIONS:

Wavelength 530 nm

Filter Selection 510-550 n

Reaction Type Endpoint

Reaction Time 10 minutes

Reaction Temperature Ambient

Sample Volume 0.01 mL

Reagent Volume 1.0 mEq/L

Low Normal 1.3 mEq/L

High Normal 2.5 mEq/L

Calibrator Value 2.0 mEq/L

AUTOMATED PROCEDURE:

Refer to specific instrument application for instructions.

MANUAL PROCEDURE:

PROCEDURAL NOTE: For instruments requiring a total volume of 1.0 mL, increase MAGNESIUM WORKING REAGENT to 2.0 mL and Sample Volume to 0.02 mL. Proceed as outlined.

1. Prepare a MAGNESIUM WORKING REAGENT as directed.

2. Dispense 1.0 mL of WORKING REAGENT into tubes labeled: Reagent Blank, Calibrator, Control, Sample 1, etc.

3. Place 0.01 mL Specimen into appropriately labeled tube and mix well. Use D-H20 as sample for Reagent Blank.

4. Incubate all tubes at Room Temperature for 10 minutes.

5. Zero instrument at 530 nm using Reagent Blank.

6. Read and record absorbencies for Calibrator, Control and Unknowns.

NOTE: For a direct read-out instrument, set read-out to concentration of Calibrator and read the Unknown concentrations directly.

STABILITY OF FINAL PRODUCT:

The test sample should be read within 15 minutes of color development.

CALIBRATION:

It is not necessary to perform a calibration curve with this procedure since the reaction is linear in the range o 0-4.0 mEq/L. However, a Calibrator and Reagent Blank must be determined with each set of Unknowns assayed. Use MAGNESIUM CALIBRATOR (Cat. No. 3283) which is provided with the reagent set or other suitable calibration material for this purpose. Specimens exceeding 4.0 mEq/L should be diluted with 0.9% sodium chloride solution and reassayed. Multiply the test result by the dilution factor to obtain final answer.

QUALITY CONTROL:

The reliability of test results should be monitored routinely using suitable quality control materials (normal and abnormal) analyzed in the same manner used for the Unknowns. EAGLE DIAGNOSTICS offers  CHEM-TROL NORMAL (Cat. No. 8100) and CHEM-TROL ELEVATED (Cat. No. 8200) for this purpose. Failure to achieve assayed values of freshly prepared control sera must be thoroughly investigated before patient results are reported.

CALCULATION OF RESULTS

The following equation is used to determine Unknown concentrations:

Unknown (mEq/L) =

Unk. Abs.

--------   X   Cal. Conc.  (mEq/L)

Cal.  Abs.

EXAMPLE:

A 2.0 mEq/L Calibrator had an Abs. = 0.120 while the Unknown Abs. = 0.140.The magnesium concentration of the Unknown is:

0.140

-----  X  2.0 mEq/L  = 2.3 mEq/L

0.120

NOTE:  To convert to mg/dL, multiply the mEq/L result by 1.21525.

LIMITATIONS

Hemolyzed, grossly icteric or lipemic specimens can not be assayed by this method.

EXPECTED RESULTS (15)

NEWBORNS 1.5 - 2.3 mEq/L

CHILDREN 1.4 - 1.9 mEq/L

ADULTS 1.3 - 2.5 mEq/L

This range is taken from literature reference. It is recommended that each laboratory establish its own range of expected values.

PERFORMANCE CHARACTERISTICS

LINEARITY:

This procedure is linear to 4.0 mEq/L.

PRECISION:

Normal and abnormal control sera were assayed 20 times each for within run precision and for 10 working days to establish run to run precision.

WITHIN RUN

MEAN / ST.DEV. / %CV

Normal 1.63 / 0.04 / 2.5

Abnormal 3.64 / 0.05 / 1.4

RUN TO RUN

Normal 1.60 / 0.04 / 2.5

Abnormal 3.67 / 0.05 / 1.4

SPECIFICITY:

A comparison of the MAGNESIUM PROCEDURE with another widely used commercial method showed a 99.8% correlation with samples in the normal and abnormal range.

SENSITIVITY:

This MAGNESIUM PROCEDURE has a sensitivity of 0.02 mEq/L per 0.001 absorbance unit.

REFERENCES

1. Kramer, B., Tisdall, F.F., J.Bio.Chem. 47:475 (1921).

2. Briggs, A.P., J. Biol. Chem. 52:349 (1922).

3. Denis, W., J. Biol. Chem 52:411 (1922).

4. Schwartzenbach, G., et al, Helvet Chim. Acta 29:811 (1946).

5. Schachter, D., J. Lab. and Clin. Med. 54:763 (1953).

6. Brien, M., Marshall, R.T., J. Lab and Clin. Med. 68:701 (1966).

7. Basinski, D.H., Standard Methods of Clinical Chemistry, 5, New York, Academic Press, p137 (1965).

8. Natelson, S., Techniques of Clinical Chemistry, 3rd. Ed., Springfield, IL, C.C., Thomas, p. 190 (1971).

9. Korbl, J., Pribl, R., Chem. Listy 51:1061 (1957) and Anal.Abstr. 5:10 (1958).

10. Lindstrom, F., Diel, H., Anal. chem. 32:11123 (1960).

11. Grindler, E.M., Heth, D.A., Clin. Chem. 17:662 (1971).

12. Klein, B.,Oklander, M. Clin. Chem 17:662 (1971).

13. Tietz, N. W., Fundamentals of Clinical Chemistry, Philadelphia, W.B. Saunders, p.918 (1976).

14. Young, D.S., et al, Clin. Chem. 21:1D (1975).

15. Tietz, N.W., Textbook of Clinical Chemistry, Philadelphia, W.B. Saunders, p.1328 (1994).