Principles

Accurate electrolyte testing is essential for diagnosing and managing a wide range of medical conditions

Analytical Principles

General Considerations
- Direct vs. Indirect Methods: Direct ISEs measure activity in undiluted samples, reducing interferences from proteins and lipids. Indirect ISEs require sample dilution
- Interferences: Be aware of common interfering substances and take appropriate steps to minimize their impact
Instrumentation
- Automated Chemistry Analyzers: Most electrolyte measurements are performed on automated chemistry analyzers using ISEs or spectrophotometric methods
- Blood Gas Analyzers: Measure electrolytes in whole blood using ISEs

Sodium and Potassium

Principle: Ion-Selective Electrodes (ISEs) are the most common method for measuring sodium and potassium
- Direct ISEs: Measure the activity of the ion in undiluted samples
- Indirect ISEs: Measure the activity of the ion after dilution
Electrodes
- Sodium Electrode: Contains a glass membrane that is selectively permeable to sodium ions (\(Na^+\))
- Potassium Electrode: Contains a valinomycin membrane that is selectively permeable to potassium ions (\(K^+\))
Process
1. The sample (serum, plasma, or whole blood) is introduced into the measuring chamber, where it comes into contact with the ISE
2. The ions of interest (sodium or potassium) diffuse across the membrane, creating a potential difference
3. The potential difference is measured by a high-impedance voltmeter and is proportional to the ion concentration
Reactions
- The ion-selective membrane selectively binds to the ion of interest, creating a potential difference
Detection: The ion concentration is displayed on the instrument readout
Advantages: Rapid, accurate, and precise measurements
Disadvantages: Susceptible to interferences from protein contamination (in some methods) and improper calibration

Chloride

Principle: Ion-Selective Electrodes (ISEs) are commonly used
Electrode: A chloride electrode contains a membrane that is selectively permeable to chloride ions (\(Cl^-\))
Process
1. The sample is introduced into the measuring chamber, where it comes into contact with the ISE
2. Chloride ions diffuse across the membrane, creating a potential difference
3. The potential difference is measured by a high-impedance voltmeter and is proportional to the chloride concentration
Reactions * The ion-selective membrane selectively binds to chloride ions, creating a potential difference
Detection: The chloride concentration is displayed on the instrument readout
Advantages: Rapid, accurate, and precise measurements
Disadvantages: Susceptible to interferences from certain medications and improper calibration
Amperometric-Chloridometric Titration
- Silver (\(Ag\)) ions are liberated at a known rate
- The \(Cl\) ions react with the silver
  - \(Cl^- + Ag^+ → AgCl (Solid)\)
- Once all the \(Cl\) ions are consumed, the excess of Silver can then be detected by amperometry

Total Carbon Dioxide (\(CO_2\))

Principle: Enzymatic methods or indirect ISEs are used to measure total carbon dioxide. Total \(CO_2\) includes bicarbonate (\(HCO_3^-\)), carbonic acid (\(H_2CO_3\)), and dissolved carbon dioxide (\(CO_2\))
Enzymatic Method
- Acidification of the Sample: \(CO_2\) is converted to \(H_2CO_3\)
  - \(CO_2 + H_2O ↔ H_2CO_3\)
- Conversion to Bicarbonate: Then it is converted to bicarbonate
  - \(H_2CO_3 ↔ H^+ + HCO_3^-\)
- Reaction with PEP Carboxylase: Bicarbonate will react with phosphoenolpyruvate (\(PEP\)) to produce oxaloacetate, which then converts to malate with \(NADH\)
  - \(HCO_3^- + PEP → Oxaloacetate\)
  - \(Oxaloacetate + NADH → Malate + NAD^+\)
- As \(NADH\) becomes \(NAD^+\), the change is measured spectrophotometrically
Indirect ISE Method
1. \(CO_2\) diffuses across a membrane into a bicarbonate buffer solution
2. The change in pH is measured by a pH-sensitive electrode, which is proportional to the \(CO_2\) concentration
Detection: The change in absorbance (enzymatic method) or the change in pH (ISE method) is measured
Advantages: Rapid and automated
Disadvantages: Susceptible to interferences from volatile acids and bases, improper calibration

Bicarbonate (\(HCO_3^-\))

Principle: Bicarbonate is not directly measured but is typically calculated from pH and Pa\(CO_2\) using the Henderson-Hasselbalch equation
Calculation
- The Henderson-Hasselbalch equation relates pH to the concentrations of bicarbonate (\(HCO_3^-\)) and carbon dioxide (\(CO_2\)):
- \(pH = pKa + log \left( \frac{[HCO_3^-]}{[CO_2]} \right)\)
- Since [\(CO_2\)] is proportional to Pa\(CO_2\), the equation can be written as:
- \(pH = pKa + log \left( \frac{[HCO_3^-]}{0.03 \times PaCO_2} \right)\)
- \([HCO_3^-] = 10^{(pH - pKa)} \times 0.03 \times PaCO_2\)
  - pKa = 6.1 (dissociation constant of carbonic acid)
  - 1. 03 = Solubility coefficient of \(CO_2\) in plasma
Advantages: Provides a clinically useful estimate of bicarbonate concentration
Disadvantages: Is subject to errors in pH and P\(CO_2\) measurements
Direct Measurement Methods
- ISE: Measured by this method using a bicarbonate-selective membrane
  - Similar to chloride methods, but using a bicarbonate selective electrode

Calcium (\(Ca^{2+}\))

Principle: Several methods are available, including spectrophotometry and ion-selective electrodes (ISEs)
Spectrophotometric Methods
- Complexation with a Dye: Dyes such as Arsenazo III or o-cresolphthalein complexone (\(OCPC\)) bind to calcium ions, forming a colored complex that is measured spectrophotometrically
Ion-Selective Electrode (ISE) Methods
- A membrane selectively permeable to calcium ions produces a potential difference related to the calcium concentration
- Measures ionized calcium
Reactions
- Spectrophotometric Methods: \(Ca^{2+} + Dye → Dye-Ca^{2+}\) Complex (Colored)
Detection: The absorbance of the colored complex (spectrophotometric methods) or the potential difference (ISE methods) is measured
Advantages: Both methods are widely available and automated
Disadvantages: Spectrophotometric methods are susceptible to interferences from hemolysis and lipemia; ISE methods require careful calibration and maintenance

Magnesium (\(Mg^{2+}\))

Principle: Several methods are available, including spectrophotometry and ion-selective electrodes (ISEs)
Spectrophotometric Methods
- Dye-Binding Methods: Magnesium reacts with a dye such as calmagite or methylthymol blue to form a colored complex
Ion-Selective Electrode (ISE) Methods
- Used to measure ionized magnesium
Reactions
- Spectrophotometric Methods: \(Mg^{2+} + Dye → Dye-Mg^{2+}\) Complex (Colored)
Detection: The absorbance of the colored complex (spectrophotometric methods) or the potential difference (ISE methods) is measured
Advantages: Both methods are widely available and automated
Disadvantages: Spectrophotometric methods are susceptible to interferences from calcium and other ions; ISE methods require careful calibration and maintenance

Phosphorus (\(P\))

Principle: Phosphorus (as phosphate) reacts with ammonium molybdate to form a phosphomolybdate complex, which is then reduced to molybdenum blue
Reactions * \(P + Ammonium Molybdate → Phosphomolybdate Complex\) * \(Phosphomolybdate Complex + Reducing Agent → Molybdenum Blue\)
Detection: The intensity of the molybdenum blue color is measured spectrophotometrically
Advantages: Relatively simple and widely available
Disadvantages: Susceptible to interferences from hemolysis, lipemia, and bilirubin

Iron (\(Fe\))

Principle: Iron is released from transferrin by an acidic buffer and reduced to \(Fe^{2+}\). The \(Fe^{2+}\) reacts with a chromogen to form a colored complex, which is measured spectrophotometrically
Reactions
- \(Transferrin-Fe^{3+} + Acidic Buffer → Transferrin + Fe^{3+}\)
- \(Fe^{3+} + Reducing Agent → Fe^{2+}\)
- \(Fe^{2+} + Chromogen → Colored Complex\)
Detection: The intensity of the colored complex is directly proportional to the iron concentration in the sample
Advantages: Widely available and relatively inexpensive
Disadvantages: Affected by diurnal variation and recent iron intake

Total Iron-Binding Capacity (TIBC)

Principle: Excess iron is added to saturate all binding sites on transferrin. The unbound iron is removed, and the total iron is then measured using a similar method to serum iron
Reactions
- \(Transferrin + Excess Fe^{3+} → Transferrin-Fe^{3+}\) (Saturated)
- Removal of Unbound \(Fe^{3+}\)
- \(Transferrin-Fe^{3+} + Acidic Buffer → Transferrin + Fe^{3+}\)
- \(Fe^{3+} + Reducing Agent → Fe^{2+}\)
- \(Fe^{2+} + Chromogen → Colored Complex\)
Detection: The intensity of the colored complex is directly proportional to the TIBC
Advantages: Provides an estimate of transferrin concentration
Disadvantages: More complex than serum iron measurement

Key Terms

Ion-Selective Electrode (ISE): An electrochemical sensor that responds selectively to specific ions
Potentiometry: A method to measure the electrical potential difference between two electrodes
Amperometry: A method to measure the electrical current flowing through an electrochemical cell
Spectrophotometry: A method to measure the absorbance of light by a solution
Chromogen: A substance that produces a colored product
Enzymatic Method: A laboratory method that uses enzymes to catalyze a reaction
ISE (Ion-selective electrode): Measures the concentration of specific ions through a membrane
ISE direct: Uses an undiluted sample
ISE indirect: Requires sample dilution
Coefficient of Variation: Measure of precision

Procedures

Precautions