Chloride

Physiology

Chloride is the major extracellular anion, found together with sodium. Chloride is important for osmolality and acid-base balance, because there are chloride-containing acids in the body, including gastric hydrochloric acid (HCl) and renal ammonium chloride (NH₄Cl = NH₃ + HCl) that can be lost or gained. Because chloride is found in a 1:1 ratio with sodium (although is slightly lower than sodium in plasma), changes in chloride should always be interpreted with sodium so that you can recognize acid-base disturbances by looking at these two strong ions. Changes in water with or without electrolytes and gain or loss of chloride-containing acids are the major mechanisms for a change in chloride concentration.

• A loss or gain of electrolyte-poor or -rich fluid is expected to change sodium and chloride concentrations proportionally. 
• A selective loss or gain of a chloride-containing acid (or loss of bicarbonate, which is equivalent to gain of a chloride-containing acid) will change chloride concentrations without altering sodium, and result in an acid-base disturbance. By Stewart’s acid-base principles, chloride is a strong anion and an independent variable that influences acid-base status. So if a chloride-containing acid is gained, e.g. distal renal tubular acidosis, chloride will increase without a concomitant increase in sodium, with these electrolyte changes indicating a metabolic acidosis. Conversely, if a chloride-containing acid is lost, e.g. vomiting of gastric contents, then chloride will decrease without a similar decrease in sodium concentration, which helps us recognize the presence of a metabolic alkalosis.

To determine if a change in chloride is due to water or an acid-base disturbance, we can do several things.

1. Calculate a strong ion difference: We can calculate the strong ion difference, following Stewart’s principles, using this formula: Strong ion difference = (Na+K)-Cl. A low strong ion difference indicates an acidosis (hyperchloremic acidosis due to retention of a chloride-containing acid or a dilutional acidosis from low sodium) whereas an increased strong ion difference indicates an alkalosis, usually due to loss of a chloride-containing acid, although there could be a contraction alkalosis. This calculation is not part of routine chemistry panels and reference intervals are not provided or are not routinely available (see the strong ion approach for reference intervals from our laboratory). Thus, other techniques can be used, i.e. evaluating whether the changes in chloride are due to changes in fluid balance (or sodium) or not (which we term, “disproportionate” changes in chloride with respect to sodium). Since we have reference intervals for sodium and chloride for most domestic species, the latter approach can be easier, versus relying on incomplete published literature for strong ion difference reference intervals.
2. Taking into account the sodium concentration: This can be done in 2 ways.
   1. Eyeballing results: This approach can be used when the sodium and chloride are tracking in the same direction (even if the sodium concentration is normal). Assess the degree of change in sodium using the upper and lower limits of the reference interval for both, i.e. degree of change = measured sodium minus appropriate reference limit (if decreased, use the lower limit of the interval; if increased, use the upper limit of the interval). Then assess the degree of change in chloride with respect to the reference interval limits for chloride (measured chloride minus appropriate reference limit) and determine if it is roughly similar to the change in chloride (remember, that chloride is normally found in slightly lower concentrations than sodium in plasma, so the degree of change does not have to be exactly equal; it is quite rare to have such clear-cut changes with biological data). This is the preferred method of assessment (or calculation of the strong ion difference can be done), because correcting the chloride for changes in sodium can be misleading, even when both analytes are tracking in the same direction. See the example below for eyeballing the changers.
      1. A chemistry panel reveals a sodium of 139 mEq/L (reference interval, 142-150 mEq/L). The chloride is 80 mEq/L (reference interval, 105-118 mEq/L). Eyeballing the results, the degree of change (or decrease) in sodium from the lower limit of the reference interval is -3 mEq/L (139-142 mEq/L). The degree of decrease in chloride is -25 mEq/L. The change in chloride is far greater than the change in sodium (-25 versus -3 mEq/L) indicating a disproportionate decrease in chloride with respect to sodium. Since a decreased chloride indicates loss of an acid, this result indicates a metabolic alkalosis (alkalosis = loss of acid and/or gain of base). Thus, this change should be accompanied by evidence of a metabolic alkalosis (high bicarbonate) as long as a mixed acid base disturbance is not concurrently present (which would affect bicarbonate concentrations). The low sodium in this example also indicates a concurrent loss of electrolyte-rich fluid but also gain of free water (and a dilutional acidosis by Stewart’s principles). In this case, the dog was vomiting resulting in loss of HCl in gastric secretions with concurrent loss of sodium (but far less than chloride) and ADH-mediated retention of water and drinking resulting in dilution of sodium concentrations.
   2. Correcting the chloride: Instead of eyeballing results, the chloride concentration can be “corrected” for changes in free water (or the sodium concentration). This should only be done if the sodium and chloride are tracking in the same direction (i.e. both above or below the reference interval, but common sense should still be applied to look at the degree of changes in both analytes.
      The chloride can be corrected using the following formula:

Corrected Cl^– = (normal Na⁺/measured Na⁺) x measured Cl^–
where, normal Na⁺ is the midpoint of the sodium reference interval

The corrected chloride is then interpreted based on the provided chloride reference interval.
Using the same example above: The normal sodium is 146 mEq/L (142 + 150/2). The corrected chloride is 84 mEq/L (146/139 x 80 mEq/L, which is still far below the reference interval (105-118 mEq/L), indicating a corrected hypochloridemia or a loss of chloride in excess of sodium with the same interpretation as above.

Note, that small disproportional changes (decreases or increases) in chloride with respect to sodium should not be over-interpreted as indicating an acid-base disturbance. All laboratory data should always be interpreted with respect to the patient and if there is no evidence of an acid-base disturbance or no disease process that could result in an acid-base abnormality, then you should not give the animal one based on laboratory data alone.

Methodology

Serum or plasma concentrations of these major electrolytes can be measured by ion-specific electrodes or flame photometry. Measurement of electrolytes by ion-specific electrodes is called potentiometry. There are two types of potentiometry: direct and indirect. Direct potentiometry is utilized by blood gas machines and does not involve sample dilution. Indirect potentiometry is utilized by automated chemistry analyzers, such as the ones used at Cornell University, and involves sample dilution before analysis. This distinction is important because endogenous interferents such as lipemia may falsely decrease electrolyte concentrations with indirect, but not direct, potentiometry.

Technique used at Cornell

Direct (blood gas machine) or indirect (chemistry analyzer) potentiometry.

Procedure

With this technique, an electrode containing an internal electrolyte solution is immersed in the patient sample, which is separated from the internal solution by a membrane that can detect the electromotive force (EMF) generated by the ions in both solutions. This EMF is determined by the difference in concentration of the test ion in the test solution and internal filling solution (test ion at fixed concentration). The EMF is predicted by the Nernst equation (see Techniques for more details on the method). For testing purposes with the chemistry analyzer, the sample is diluted 1:32 before analysis (indirect potentiometry).

Units of measurement

Chloride concentration is measured in mEq/L (conventional units), mg/dL (conventional units), or mmol/L (SI units). The unit conversion formulas are shown below:

mEq/L x 1 = mmol/L
mg/dL ÷ 3.55 = mmol/L

Sample considerations

Sample type

Serum, plasma, or urine

Anticoagulant

Heparin or EDTA

Stability

The Cl^– concentration in human plasma or serum is stable for several days when separated from red blood cells and stored at 2 – 8°C. Samples of urine should be stored at 4°C.

Interferences

• Lipemia: Moderate to severe lipemia due to chylomicrons will falsely decrease chloride concentrations measured with indirect potentiometry (chemistry analyzer) but not direct (blood gas) potentiometry. Since indirect potentiometry is the main method used to determine electrolyte concentrations on chemistry panels, lipemia may affect our chloride results to a similar extent as sodium.
• Hemolysis and Icterus: No effect.

Test interpretation

A normal chloride concentration with an abnormally high or low sodium concentration may still reflect an acid-base disturbance, depending on the degree and direction of change in both analytes.

High chloride (hyperchloridemia)

In general, a high chloride concentration that cannot be explained by changes in sodium is associated with a strong ion metabolic acidosis, which could be primary or compensatory for a primary respiratory alkalosis. Thus, bicarbonate concentrations should track low (unless there is a concurrent primary metabolic alkalosis). Exceptions do apply (e.g. anticonvulsant-associated error in chloride measurement).

• Artifact
  • Anticonvulsant medication: The ion-selective electrode used to measure chloride is, unfortunately, not specific for chloride. It also detects other anions, such as bromide, and measures them as “chloride”. Thus, chloride concentrations measured by potentiometry (direct or indirect) will be falsely increased in animals on bromide medication for seizures. In these settings, we measure chloride using flame photometry, which provides more accurate chloride measurements. We have noticed a similar artifact in dogs on zonisamide therapy for seizures. The sodium concentration is unaffected and the anion gap in such cases is usually low, which is a clue as to the presence of an error in measurement of the analytes that are used to calculate the anion gap (sodium, potassium, bicarbonate, and chloride).
• Iatrogenic
  • Drug administration: Administration of Cl-containing fluids (hypertonic saline, ammonium chloride; both acidifying solutions). Note that 0.9% NaCl is also considered an acidifying solution, because the sodium and chloride are in a 1:1 ratio, whereas chloride is normally lower than sodium.
  • Diet: Providing dairy cattle with negative dietary cation-anion diets or supplementing their diet calcium chloride leads to acidification of the plasma, with an appropriate renal corrective response (aciduria) (Tucker et al 1988, Sampson et al 2009, Melendez et al 2021)
• Pathophysiologic causes
  • Gain of electrolyte-rich fluids or decreased body water: These conditions, e.g. salt intoxication, diabetes insipidus with lack of drinking, primary adipsia, will increase sodium and chloride to similar extents (refer to the sodium page for more causes).
  • Acid-base abnormalities: Here, the chloride will not be increased to the same degree or even in the same direction as sodium (i.e. strong ion difference will be low).
    • Primary normal anion gap (hyperchloremic) metabolic acidosis: This acid-base disturbance is due to
      • Loss of bicarbonate with retention of a chloride-containing acid, e.g proximal renal tubular acidosis, secretory diarrhea.
      • Retention of a chloride-containing acid, i.e. NH₄Cl by the kidney,  or a defect in the H⁺ antiporter in the distal tubules and collecting duct resulting in a distal renal tubular acidosis. 
    • Compensatory metabolic acidosis to a primary respiratory alkalosis (hypocapnea or hyperventilation): This compensatory response is associated with loss of bicarbonate (filtered via the glomerulus and actively excreted by type B intercalated cells in the distal tubules) with corresponding retention of a chloride-containing acid by the kidney.

Low chloride (hypochloremia)

A low chloride concentration that cannot be attributed to changes in sodium or fluid shifts/losses is associated with a chloride-responsive (or chloride-depleted) metabolic alkalosis (chloride behaves as an acid), whether a primary acid-base disturbance or a compensatory response to a primary respiratory acidosis. Bicarbonate concentrations are expected to be increased with a normal anion gap. However, the disease state that results in a primary metabolic alkalosis (e.g. vomiting, abomasal outflow obstruction) frequently also produces a lactic acidosis (due to hypovolemia), hence bicarbonate concentrations may not be increased as expected and the anion gap may be high (reflecting accumulation of L-lactic acid, an unmeasured anion). The bicarbonate concentration and pH will be affected in opposite directions by a primary metabolic alkalosis and primary metabolic acidosis; the dominating disturbance will determine the final result. For example, if acidosis is dominating, the pH may be acidic or trending towards acidemia and bicarbonate concentration will be decreased (below the reference interval) or trending low. The reverse would be the case if the primary metabolic alkalosis is dominating. 

• Iatrogenic
  • Drug administration: Administration of sodium-rich fluids and diuretics. 
    • Loop diuretics – these inhibit the Na-K-2Cl carrier in the loop of Henle, hence two chloride ions are lost per one sodium, which can result in a primary metabolic alkalosis.
    • Thiazide diuretics inhibit the NaCl cotransporter in the early distal tubule; although these diuretics are expected to result in proportional losses of sodium and chloride, the increased sodium delivered to the collecting tubule may be absorbed by principal cells with concurrent stimulation of acid excretion and chloride being lost with the acid, resulting in primary metabolic alkalosis.
• Pathophysiologic causes:
  • Loss of chloride in excess of sodium: This is associated with development of metabolic alkalosis.
    • Gastrointestinal:
      • Loss of chloride-rich fluid: Vomiting of gastric contents in small animals, gastric reflux in horses, large colon or cecal diarrhea in horses (chloride is absorbed in the ileum and colon of horses, e.g. Potomac horse fever), gastrointestinal ulcers (horses). In humans, a deficiency or abnormality in the ileal transporter for chloride results in severe chloride loss and hypokalemia (Gennari et al 2011).
      • Sequestration of chloride-rich fluid: Displaced abomasum, abomasal atony (a common finding in sick cattle), gastric rupture, gastric dilation-volvulus (dogs), proximal intestinal ileus (horses).
    • Renal: Loop diuretics (see above), stimulation of the H⁺ATPase pump in the distal tubules (lose HCl) by aldosterone in primary hyperaldosteronism could result in a primary metabolic alkalosis. In a salt-losing nephropathy in cattle, sodium and chloride losses are expected to be proportional.
      • Compensatory metabolic alkalosis to a primary respiratory acidosis: Enhanced renal excretion of NH₄Cl (from increased ammoniagenesis) resulting in a compensatory hypochloremic metabolic alkalosis is an appropriate compensatory response to a primary respiratory acidosis. It is also a corrective response to a primary metabolic acidosis, as long as renal function is normal.
    • Cutaneous: Excess sweating in horses (loss of KCl).