Chloride

Introduction

1. Chloride (Cl⁻) is the major extracellular anion in the body, essential for maintaining fluid balance, acid-base equilibrium, and electrical neutrality in cells.
2. The kidneys primarily regulate it.
3. Measuring chloride levels in serum, plasma, or urine helps diagnose and monitor electrolyte imbalances, kidney function, and acid-base disorders, such as metabolic acidosis or alkalosis.

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Principle

Chloride determination is based on the ability of chloride ions to form a complex or react with specific reagents:

1. Mercurimetric Titration (Schales and Schales Method): Chloride ions react with mercuric ions to form a soluble complex. A diphenylcarbazone indicator changes color when excess mercuric ions appear, marking the endpoint.
2. Colorimetric Method: Chloride reacts with a chromogenic reagent to form a colored complex, measured spectrophotometrically.
3. Ion-Selective Electrode (ISE) Method: Measures the activity of chloride ions directly using a specific electrode.

Methods

1. Mercurimetric Titration (Manual):
   • Based on the Schales and Schales method.

 

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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.

 

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Reagents

1. 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.
2. Diphenylcarbazone indicator: 100 mg/dl in 95% (v/v) ethanol. It is stable in an amber-colored bottle at 2 – 8aC
3. 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.

 

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Procedure

Prepare protein-free filtrate of the serum sample as follows:

In a centrifuge tube, pipette

1. 0 ml distilled water
2. 5 ml serum
3. 25 ml 2/3 N H,SO4
4. 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:

1. Pipette in a test tube 2.0 ml of protein-free filtrate.
2. Add one drop of the indicator. (0.05 ml)
3. Titrate against mercuric nitrate reagent. (Endpoint: colorless to violet-blue color).
4. Note the titration reading: X ml.
5. Dilute standard 1: l0 by using glass distilled water.
6. 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.
7. Note the titration reading: Y ml.

 

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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

 

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Clinical Significance

1. Increased Chloride (Hyperchloremia):
   • Dehydration
   • Metabolic acidosis (e.g., diarrhea, renal tubular acidosis)
   • Cushing’s syndrome
   • Excessive salt intake
2. Decreased Chloride (Hypochloremia):
   • Prolonged vomiting or nasogastric suction
   • Metabolic alkalosis
   • Addison’s disease
   • Congestive heart failure
   • Syndrome of inappropriate antidiuretic hormone secretion (SIADH)