Introduction
- Chloride (Cl⁻) is the major extracellular anion present in the body and is one of the most important electrolytes required for maintaining normal physiological functions.
- It plays a crucial role in maintaining osmotic pressure, fluid balance, acid–base equilibrium, and electrical neutrality of body fluids.
- Chloride is mainly present in extracellular fluid and is closely associated with sodium metabolism.
- The kidneys regulate chloride concentration primarily through filtration and reabsorption mechanisms.
- Small changes in chloride concentration may significantly affect body fluid composition and acid–base status.
Approximately 88–90% of chloride is present in extracellular fluid, while the remaining amount is found within cells and bones.
The chloride test is commonly performed in clinical laboratories to:
- Evaluate electrolyte balance
- Assess kidney function
- Monitor acid–base disorders
- Diagnose dehydration
- Investigate metabolic abnormalities
- Monitor critically ill patients
Principle
Determination of chloride concentration is based on the ability of chloride ions to react with specific reagents or electrodes.
Several methods are available:
1. Mercurimetric Titration Method (Schales and Schales Method)
This classical manual method is based on the reaction between chloride ions and mercuric ions.
Principle
Chloride ions react with mercuric nitrate to form soluble mercuric chloride:
2Cl⁻ + Hg(NO₃)₂ → HgCl₂ + 2NO₃⁻
Diphenylcarbazone acts as an indicator.
When all chloride ions are consumed, excess mercuric ions react with diphenylcarbazone producing:
- Violet-blue color endpoint
Endpoint
Colorless → Violet-blue
2. Colorimetric Method
In this method:
- Chloride reacts with chromogenic reagents
- A colored complex forms
- Intensity of color is measured spectrophotometrically
Principle
Color intensity is directly proportional to chloride concentration.
3. Ion Selective Electrode (ISE) Method
Modern automated analyzers commonly use ISE.
Principle
- Chloride ions interact with chloride-selective membrane electrodes
- Electrical potential generated is proportional to chloride concentration
Advantages
- Rapid
- Highly accurate
- Automated
- Requires small sample volume
Methods
- Mercurimetric Titration (Manual):
- Based on the Schales and Schales method.
Samples
- Sample Type:
- Serum, plasma (collected in heparinized tubes), or urine.
- Sample Volume: ~0.5–1 mL.
- Collection Notes: Avoid hemolysis, as intracellular chloride can interfere. Do not use EDTA or citrate as anticoagulants, as they bind chloride.
- Storage:
- Serum/plasma samples: Store at 2–8°C and analyze within 48 hours.
- Urine: Can be stored at 2–8°C for 24 hours.
Reagents
- Mercuric nitrate reagent: Dissolve 2.9-3.0 g of mercuric nitrate in about 800 ml of distilled water, add 20 ml of 2N nitric acid, and make up to one liter. It is stable at room temperature in an amber-colored bottle.
- Diphenylcarbazone indicator: 100 mg/dl in 95% (v/v) ethanol. It is stable in an amber-colored bottle at 2 – 8aC
- Chloride standard: t00 mEq/l: It is prepared by dissolving 5.85 g of analar grade sodium chloride in one liter of glass distilled water. It is stable at 2 – 8’C.
Additional reagents
4) 2/3N sulturic acid
5) 10 g/dl, sodium tungstate.
Procedure
Prepare protein-free filtrate of the serum sample as follows:
In a centrifuge tube, pipette
- 0 ml distilled water
- 5 ml serum
- 25 ml 2/3 N H,SO4
- 25 ml I0 g/dl sodium tungstate
Mix thoroughly and centrifuge at 3000 R.P.M. for l0 minutes. The next procedure is as follows:
- Pipette in a test tube 2.0 ml of protein-free filtrate.
- Add one drop of the indicator. (0.05 ml)
- Titrate against mercuric nitrate reagent. (Endpoint: colorless to violet-blue color).
- Note the titration reading: X ml.
- Dilute standard 1: l0 by using glass distilled water.
- Pipette 2.0 ml of diluted standard in a test tube and titrate it against mercuric nitrate reagent. (same as for the test) by using a diphenylcarbazone indicator.
- Note the titration reading: Y ml.
Calculation
For the Schales and Schales method:
Chloride (mEq/L) = X ml / Yml x 100
Normal Range
- Serum/Plasma Chloride:
- Adults: 98–107 mEq/L
- Children: 95–105 mEq/L
- Urine Chloride (24-hour):
- 110–250 mEq/day
Clinical Significance
- Increased Chloride (Hyperchloremia):
- Dehydration
- Metabolic acidosis (e.g., diarrhea, renal tubular acidosis)
- Cushing’s syndrome
- Excessive salt intake
- Decreased Chloride (Hypochloremia):
- Prolonged vomiting or nasogastric suction
- Metabolic alkalosis
- Addison’s disease
- Congestive heart failure
- Syndrome of inappropriate antidiuretic hormone secretion (SIADH)