Background

The concept of electroneutrality states that the total concentration of cations in a solution must equal the total concentration of anions. In extracellular fluid, sodium is the predominant cation, whereas chloride, bicarbonate, and albumin are the main anions. These electrolytes are essential for calculating the anion gap, which aids in the evaluation of acid-base imbalances.

The anion gap represents the difference between the main measured cations (sodium [Na⁺] and potassium [K⁺]) and the main measured anions (chloride [Cl⁻] and bicarbonate [HCO₃⁻]) in the serum. It is determined by using the following formula:

Serum anion gap = (Na⁺ + K⁺) − (Cl⁻ + HCO₃⁻)

In practice, a simplified formula is often applied because sodium is the predominant cation, and potassium (K⁺) is typically omitted from the calculation since it makes only a minor contribution to the extracellular cation pool (see also the Anion Gap calculator).

Serum anion gap = Na⁺ − (Cl⁻ + HCO₃⁻)

Alternatively, the serum anion gap can be defined as the difference between unmeasured anions (UA) and unmeasured cations (UC), which are not included in standard anion gap calculations [4], as illustrated below:

According to the principle of electroneutrality: the total of all cations equals the total of all anions.

[(Na⁺ + K⁺) + UC] = [(Cl⁻+ HCO₃⁻) + UA]

By rearranging the equation, (Na⁺ + K⁺) − (Cl⁻+ HCO₃⁻) = UA − UC

Hence, the anion gap can be expressed as UA − UC, allowing it to serve as an indirect measure of unmeasured anions or cations in the blood.

In a similar way, the anion gap can be applied to distinguish between cations and anions in urine, as demonstrated by the following equation:

UAG = Urinary [(Na⁺ + K⁺) + UC] − Urinary [(Cl⁻+ HCO₃⁻) +UA]

By rearranging the equation, Urinary (UA − UC) = Urinary (Na⁺ + K⁺) − Urinary (Cl⁻+ HCO₃⁻)

Since urinary bicarbonate (HCO₃⁻) is minimal when urine pH is below 6.6, it can be omitted from the urinary anion gap (UAG) calculation. Consequently, the frequently used formula for UAG is:

UAG = Urinary (Na⁺ + K⁺) − Urinary (Cl⁻)

UAG = UA − UC

In this equation, UA represents urinary unmeasured anions like sulfate, phosphate and various organic anions, while UC denotes unmeasured cations including NH₄⁺, Ca²⁺ and Mg²⁺. This formulation illustrates how changes in the levels of unmeasured anions, cations or both can affect the urinary anion gap (UAG).

Although NH₄⁺ is the primary unmeasured cation in urine, recent evidence indicates that in healthy individuals with normal kidney function, urinary NH₄⁺ excretion is determined by the daily acid load and does not show a consistent theoretical or empirical relationship with the UAG.

Indications/Applications

Anion gap is performed in patients with an altered mentals status, acute renal failure, unknown exposure and acute diseases.

Consideration

Laboratory errors should always be considered first when the clinical presentation does not match the test results. If the anion gap appears inconsistent, the initial step should be to re-evaluate the electrolyte measurements. Errors during sample collection or handling can affect the concentrations of electrolytes used to calculate the anion gap. Factors such as timing of collection, sample dilution, underlying renal disease, and small sample volumes can all interfere. For instance, delays in processing a blood sample allow ongoing leukocyte metabolism, which can lead to artificially elevated bicarbonate levels.

Factors affecting the anion gap include:

• Hyperlipidemia
• Chloride measurement errors: Some chloride ion-selective electrodes may misread bromide, iodide or high salicylate levels as chloride resulting in overestimation.
• Sodium measurement errors: Conditions like dehydration, hyperproteinemia or hyperlipidemia can cause underestimation of true sodium levels.

When an elevated anion gap is observed, further investigations like urine ketones, serum ketones (beta-hydroxybutyrate), serum lactate, urine and serum drug screens, salicylate levels, and creatine kinase should be conducted to determine the underlying cause of the anion gap acidosis.

Reference Range

The normal range for anion gap in blood:

16 ± 4 mEq/L (if calculation uses potassium)

12 ± 4 mEq/L (if calculation does not use potassium)

There is no normal range of urine anion gap (UAG) because of wide range of UAG. Different studies have showed a significant elevation in normal range for UAG in healthy individuals from 41 mEq/day to above 70 mEq/day.

Interpretation

Serum Anion Gap: Ions which contribute to serum anion gap involve serum cations (Mg²⁺, Ca²⁺, K⁺ and Na⁺) and serum anions (organic acid, OA⁻, SO₄²⁻, HPO₄²⁻, HCO₃⁻, and proteins or PR⁻). In normal physiological diseases, the serum Na⁺ levels above combined levels of bicarbonate and chloride ions. The anion gap calculated gives a positive value.

Urinary Anion Gap: Ions which contribute to UAG involve cations (Mg²⁺, Ca²⁺, K⁺ and Na⁺) and anions (organic anions, sulfate, phosphate, bicarbonate and chloride). Urinary HCO₃⁻ is negligible at urine pH of below 6.6. It is excluded in UAG equation. The UAG equation limiting factor is valid if urine sodium level is below 20 mEq/L.

The UAG is negative or positive and used when causes of normal anion gap acidosis are not clear. It helps to assess reduced plasma HCO₃⁻ levels (hypobicarbonatemia) because of chronic respiratory alkalosis or metabolic acidosis.

A positive UAG is observed in diseases of type 2 and type 1 renal tubular acidosis versus every other factor of normal anion gap acidosis (diarrhea). A UAG of above 20 mEq/L is seen in metabolic acidosis when kidneys are not able to excrete NH₄⁺ (like in renal tubular acidosis). UAG is positive because of reduced renal excretion of NH₄⁺ in chronic respiratory alkalosis. Elevated urinary levels of ketoanions can elevate UAG and make it positive.

If UAG is negative or zero but serum AG is positive. Increased urinary lithium level can cause negative or low UAG.

A reduced anion gap (below 6 mEq/L) can suggest:

Hypoalbuminemia as albumin is negatively charged plasma protein, reduced serum albumin is compensated by an elevation in the chloride anions which cause a decreased anion gap.

Plasma cell dyscrasia like multiple myeloma may give positively charged immunoglobulins.

Monoclonal gammopathy as light or monoclonal immunoglobulins are positively charged proteins. Specific diseases with increased positively charged polyclonal immunoglobulin involve cirrhosis, chronic kidney disease and human immunodeficiency virus (HIV).

Lithium intoxication or overload as increase lithium levels can cause concomitant compensatory elevation in Cl⁻ without impacting Na⁺ levels. 

Bromide intoxication

Prolonged gastic suction, chronic vomiting, hyperaldosteronism, hypoproteinemia

A normal range of anion gap (6 to 12 mEq/L) can be because of following:

Normal variant

Ureteral diversion surgery

Lysine and arginine in parenteral nutrition

Renal cause: Acute kidney injury, renal tubular acidosis, mineralocorticoid deficiency

Carbonic anhydrase inhibitor (topiramate, dorzolamide, acetazolamide)

Recovery from the diabetic ketoacidosis

Gastrointestinal cause: Loss of the bicarbonate rich pancreatic, duodenal or biliary fluid because of vesiculoenteric fistula or pancreatoenteric fistula, Ileostomy fluid loss. Loss of bicarbonate because of diarrhea is because of diminished colonic absorption and elevated fecal excretion.

Iatrogenic normal anion gap in metabolic acidosis because of elevated infusion with normal saline (NaCl: 0.9%). It is seen in patients treated with the therapeutic plasma exchange by using replacement fluid composed of 4 % of human albumin with increased chloride. An increased anion gap (below 12 mEq/L) can be because of following:

Uremia

Methanol

Diabetic ketoacidosis

Glycols: ethylene glycol and propylene glycol

Isoniazid intoxication

Salicylates toxicity

Renal failure or rhabdomyolysis

Alcoholic ketoacidosis

Pyroglutamic acidosis or 5-oxoproline seen in the malnourished chronic paracetamol or acetaminophen and individuals with the sepsis

Metformin: elevated metformin in patients with significantly impaired kidney function may cause accumulation of lactic acid with inhibition of mitochondrial respiratory chain complex 1.

Collection And Panels

To assess serum anion gap, all components is drawn in basic metabolic panel which involve carbon dioxide, calcium, chloride, glucose, creatinine, sodium, potassium and urea nitrogen

Sample type: Serum

Sample collection container: Green, serum or plasma separator tube

Sample storage or transport temperature: In refrigerated condition

Sample volume: 2 mL

Sample minimum volume: 1 mL

Unacceptable samples: Samples which are collected in ethylenediaminetetraacetic acid, oxalate or citrate

References

https://www.ncbi.nlm.nih.gov/books/NBK539757/