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Electrolyte measurement - myths and misunderstandings- Part II

Shailesh Bihari, Steven Galluccio, Shivesh Prakash

PII: S0883-9441(20)30587-6
DOI: https://doi.org/10.1016/j.jcrc.2020.06.004
Reference: YJCRC 53553

To appear in: Journal of Critical Care

Please cite this article as: S. Bihari, S. Galluccio and S. Prakash, Electrolyte measurement
- myths and misunderstandings- Part II, Journal of Critical Care (2019), https://doi.org/
10.1016/j.jcrc.2020.06.004

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Electrolyte measurement - myths and misunderstandings- Part II

Shailesh Bihari1,2 , Steven Galluccio1 and Shivesh Prakash1,2

1. Department of ICCU, Flinders Medical Centre, Bedford Park South Australia 5042

2. College of Medicine and Public health, Flinders University Bedford Park South Australia
5042

Address for correspondence

Shailesh Bihari
Department of ICCU, Flinders Medical Centre, Bedford Park South Australia 5042

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Phone number: +61 8 82044247

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Fax number: +61 8 82045751
Email: Shailesh.Bihari@sa.gov.au ; biharishailesh@gmail.com
Conflict of interest: None
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All authors contributed equally to the manuscript
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Abbreviations
AG: anion gap
BGAs: blood-gas analysers
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ECF: extracellular fluid

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ISEs: ion-selective electrodes

LAAs: laboratory auto-analysers
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LOX: lactate oxidase

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LDH: lactate dehydrogenase

NADH: nicotinamide adenine dinucleotide hydride
POC: point of care
SID: strong-ion difference
TCO2: total carbon dioxide content
WB: whole blood
[Cl-]: Chloride
[HCO3-]: bicarbonate concentration
[lactate]: lactate concentration

Electrolyte measurement: myths and misunderstandings- Part II

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Bicarbonate

Significance

Perturbed acid-base homeostasis is a fundamental consideration in critical illness. It is

beyond the scope of this review to affirm or refute the importance of bicarbonate (HCO 3 -) as
independent or dependent determinant of acid-base status (1). Nonetheless, the measurement
of bicarbonate concentration ([HCO3 -]) remains very useful for the bedside clinician in
describing acid-base status and managing any disorder thereof (2,3).

Measurement

[HCO3-] can be directly measured, typically by Laboratory Auto-Analyzers (LAA) using

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enzymatic methods (4). More precisely, auto-analysers do not “measure” [HCO3-], but total
carbon dioxide content (TCO 2 ). TCO 2 is comprised of approximately 95% HCO 3 -, but also
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includes dissolved CO 2 , carbonate ions and carbamino compounds. In adults, the normal
range can be instrument dependent, but is usually cited as 22 to 32 mmol/L. The most
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commonly employed measurement method involves alkalinising the specimen to convert all
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CO 2 and carbonic acid into HCO3- (4). Through a series of enzymatic reactions,
nicotinamide adenine dinucleotide hydride (NADH) is consumed, which in turn, acts as a
spectrophotometric indicator of TCO 2 . Reduced NADH leads to decreased absorbance,
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monitored in the ultraviolet range of 320 nm to 400 nm, with the rate of change in absorbance
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being directly proportional to the TCO 2 . Less commonly, a strong acid is added to the initial
sample, which liberates CO 2 , which is then measured using volumetric, manometric or ion-
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specific electrode techniques (5).

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In contrast, BGAs derive [HCO 3 -] by firstly measuring pCO 2 and pH using potentiometric
methods. CO 2 and pH electrodes measure the potential difference due to the ion concentration
gradient across a membrane (made of silicone/Teflon and pH sensitive glass, respectively) of
ISEs. The change in potential is a measure of ion activity, and is converted to concentration
using a modified Nerst equation. Finally, the Henderson-Hasselblach equation is applied to
calculate [HCO 3 -]. Under most clinical scenarios, the two methodologies show acceptable
levels of agreement. However, under certain conditions, both the dissociation constant for
carbonic acid (pK1’) and solubility coefficient for CO 2 (α) cannot be assumed to be constant,
and hence the [HCO 3 -] calculated by BGA will be affected. Such situations can include
extremes of temperature, as well as altered water, salt and protein content of the sample (6,7).
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Clinical relevance

[HCO 3 -] measurement using LAAs appears to be relatively insensitive to common pre-

analytical errors (8). This can include delays from sample collection to analysis (serum clot-
contact time) of up to 6 hours (9) , notwithstanding that such delays would be undesirable in
relation to critically-unwell patients. For either technique, exposure to air should be avoided,
which can result in reduced sample pCO 2 concentration, and therefore, spuriously increased
[HCO 3 -]. BGA samples should employ solid-state (lyophilised) heparin, in order to avoid
aberrant results due to dilution (10). Haemolysis is not thought to effect [HCO 3 ] measurement
in a clinically meaningful way, as can occur in relation to other commonly-measured
parameters such as potassium (11).

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In most clinical situations, despite the methodological differences, LAA and BGA
[HCO 3 -] have acceptable interchangeability. However, [HCO 3 -] can be spuriously low when
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measured by LAA using enzymatic methodologies. This phenonenom has been reported in
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association with hyperlipidaemia (12) and paraproteinaemia (13). Conversely, elevated serum
lactate dehydrogenase concentration can result in spuriously elevated [HCO 3 -] (14). These
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limitations do not apply to BGAs using ISEs. In such occasional clinical situations where
there is a discrepancy in [HCO 3 -], the result by enzymatic/photometric methodology (LAA)
should be ignored, and the result by electrode-based methodology (BGA) trusted.
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Chloride
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Significance
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Chloride is the most abundant anion in the ECF. Like other ions, the cited normal range can
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vary between laboratories, but is usually quoted in the vicinity of 98 to 110  mmol/L (5).
Outside the context of acid-base analysis, [Cl-] will usually concur with [Na+] and move in
parallel fashion to changes in [Na+]. Historically, [Cl-] has not been regarded as clinically
important per se, but renewed interest is changing this sentiment (15). In any case, when
characterizing and managing disorders of acid-base homeostasis, consideration of [Cl-] is
critically important.

Measurement

Both LAAs and BGAs measure [Cl-] by ISE methodologies. However, akin to [Na+]
measurement above, the difference of sample dilution becomes important when the solid
phase of plasma is increased. With hyperlipidaemia or hyperproteinaemia, indirect ISE will
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underestimate the true plasma water [Cl-], whereas direct ISE will remain accurate. It is the
electrolyte concentration of the plasma water, rather than total plasma volume, which is
thought to be the physiologically relevant entity (16,17). The same relationship is not as well
described when the solid-phase of plasma is decreased. Hypoalbuminaemia, as commonly
seen during critical illness, is not correlated with differences between [Cl-] measurement by
direct and indirect ISE methodologies, unlike the case with [Na+] (16). Haemolysis does not
appear to effect measured [Cl-] (5). Indirect ISE modules have been associated with
significant inaccuracies in the presence of interfering anions. Most LAAs utilize quaternary
ammonium salt anion exchangers in constructing the Cl- ISE. These ISE are not entirely
“specific”; any ion with an equivalent or higher hydration energy than Cl- can potentially

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cause interference. Such anions include the other halides iodide and bromide (18,19).
Sulphates, thiocyanate, salicylates and heparin are also known to cause interference with [Cl-]
measurement (19-21).

Clinical relevance
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Acid-base disturbances can be conceptualized in different ways. Bedside clinicians
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incorporate different models and rules of thumb when interpreting such disorders, including
the anion-gap (AG) or strong-ion difference (SID) using the Stewart physiochemical
formulation. In all cases, [Cl-] is a parameter of paramount importance.
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Ironically, [Cl-] measurement may be particularly affected when [HCO 3 -] is either

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abnormally high or low, since [HCO 3 -] itself can act as an interfering anion. Described almost
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30 years ago, several LAA ISEs for [Cl-] are particularly susceptible to [HCO 3 -] interference
(22,23). That is, when [HCO 3 -] is low, measured [Cl-] will be spuriously low, and spuriously
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high when [HCO 3 -] is high. Modern LAAs seem to be afflicted by the same flaw (24,25). At
extremes of [HCO 3 -], as typically seen in critically ill patients, the differences between
measured [Cl-] by LAA and BGA, and hence anion-gap (or SID), can be significantly large to
impact clinical management (26-29). The irony applies in the sense that the AG is usually of
most interest to the clinician in the setting of abnormal [HCO 3 -]. Yet, interference effects
involving quaternary-ammonium based ISEs due to low [HCO 3 -] can result in an AG (or SID)
exaggerated by > 10 mmol/L. The clinician could then either misdiagnose the nature of the
acid-base disturbance, or at least, underappreciate any “non-anion-gap” component. This
well-known laboratory fact is perhaps not so well-appreciated by clinicians. A clinical
example of this phenomenon was recently observed by the authors in a patient with severe
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anorexia nervosa. She had presented to hospital with altered mental status. Serum electrolytes
(by LAA) revealed [Na+] 116 mmol/L, [HCO 3 -] 6 mmol/L and [Cl-] 83 mmol/L.
Simultaneous venous BGA revealed pH 6.86, identical [Na +] at 116 mmol/L, similar [HCO 3 -]
of 5 mmol/L but discrepant [Cl-] at 102 mmol/L. Hence, the AG was calculated at 27 with
LAA versus 9 by BGA. The BGA correctly signaled the presence of a significant “non-anion
gap” component to the severe metabolic acidosis, presumably related to large gastrointestinal
losses of bicarbonate due to her eating disorder and associated laxative misuse. Judicious
sodium bicarbonate infusion was commenced with improvement in her condition over several
days. Relying on the LAA result could have resulted in unnecessary investigation of the
spuriously raised anion-gap or delays to definitive treatment. When the presence of

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interfering anions is suspected, including either very high or very low [HCO 3 -], BGAs or

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LAAs using alternative electrodes, such as those based on silver/silver chloride, should be
used to measure [Cl-] and calculate the AG to guide subsequent management (30).

Lactate
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Significance
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Lactate concentration ([lactate]) is well validated for prognostication and monitoring in

critically ill patients (31). In addition, it has diagnostic value in various syndromes
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encountered in critical care practice. Lactate exists in in two stereoisoforms: L-lactate and D-
lactate. The widely available measurement techniques utilize enzymes which are specific for
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L-lactate, the predominant form in humans. Extremely small amount of D-lactate found in
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humans, may result from exogenous source such as bacterial fermentation in the gut or diet
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(yoghurts, soured cream etc) or from methylgloxal pathway (a minor off-shoot pathway of
glycolysis).

Measurement

[Lactate] is measured using biosensors which involve enzymatic degradation of lactate. These
are either lactate oxidase (LOX) based (POC BGA machine) or lactate dehydrogenase based
(central laboratories) (32). The LOX enzyme method involves production of peroxide which
gets reduced at the platinum electrode to produce a current which is proportional to the
sample [lactate].

The LDH method mostly relies on the spectrophotometric measurement to assess the amount
of NADH formed, as lactate is metabolized. This is used to derive [lactate].
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Clinical relevance

The clinical relevance of the measurement technique originates from interferences with the
enzymatic measurement process that leads to factitious reading or sample collection or
processing factors that lead to elevated ‘specimen’ [lactate]. Both of these are detailed in the
Table. An important clue towards factitious reading is significantly elevated [lactate] (>5
mmol/L) without co-existing raised AG or acidosis.

An interference of clinical significance is that induced by the glycolic acid metabolites

(glyoxalate and glycolate) in ethylene glycol poisoning. Patients with Ethylene glycol
poisoning have spuriously elevated L-lactate concentration as measured by most point of care

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analysers using LOX enzyme methods (33). Analyzers using LDH are unaffected, and even
some using lactate oxidase are also free of interference (34). The glycolic acid metabolites

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(glyoxalate and glycolate) of ethylene glycol, serve as a substrate for LOX enzyme giving
rise to falsely elevated readings. This problem can be overcome by measuring [lactate] using
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the LDH enzyme-method of LAAs. In suspected cases of poisoning with elevated [lactate] by
POC testing, concurrent measurement using the LDH technique by LAA is recommended.
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Any such difference in [lactate] or “lactate gap” is particularly useful in late presentations of
ethylene glycol poisoning; not only are these patients potentially sicker, but may present a
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diagnostic challenge as metabolism of ethylene glycol will normalize both the plasma
concentration and osmolar gap. Moreover, most laboratories can measure ethylene glycol but
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not its metabolites, so the lactate gap can serve as a useful surrogate.
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D-lactate is not measured by the routinely available assays. Thankfully, the only pathology
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where D-lactate levels could rise enough to cause acidosis is short-bowel syndrome (35).
Mechanism involves large carbohydrate load delivered to colon, where bacterial fermentation
results in D-Lactate production. Even then, at least hundred-fold increase in D lactate
concentration (> 3mmol/L) is required before it can cause clinical concerns. A clinical
vignette would be a patient with unexplained high anion gap acidosis and altered conscious
state in the setting of previous bowel resection. Mechanism of neurological disturbances in
these patients is unclear and appear unrelated to D-Lactate.

Table: Measurement errors and pitfalls of the common electrolytes – bicarbonate,

chloride, and lactate.
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Analyte Common measurement pitfalls
Bicarbonate

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ISE, ion-selective electrode; BGA, blood-gas analyser; LAA, laboratory auto-analysers; LDH, lactate
dehydrogenase.
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Errors specific to LAAs

 Increased serum LDH – spuriously increases [HCO 3 -] (14)
 Hyperlipidemia – spuriously decreases [HCO3 -] (12)
Errors common to LAAs and BGAs
 Exposure to air – spuriously increases [HCO 3 -] (5)
 Exposure to heparin (non solid-state) – spuriously decreases
[HCO3 -] (5)

Chloride Measurement artefacts generally confined to indirect ISE

methodologies.
Plasma solid artefacts:
 Increased plasma solids associated with decreased [Cl-] (16,
17)
 Decreased plasma solids not as well associated with changes
in [Cl-] (16)
Interfering anions artefacts (quaternary-ammonium based ISEs)

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 Low [HCO3 -] – associated with decreased [Cl-] (22-25)

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 High [HCO3 -] – associated with increased [Cl-] (22-25)
 Halides (iodide, bromide) (18, 19)
 Others: sulphates, salicylates, thiocyanate, heparin (19-21)

Lactate Sampling:
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 Venous lactate concentrations are higher (0.18 to 1.06
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mmol/L) than arterial blood (36)
 Fist clenching with tight tourniquet
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 Delayed transport to lab raises [lactate] by about

0.7mmol/L/hr (37)
Interference
Molecules that interfere with amperometric biosensors for lactate
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(38) (More commonly reported with LOX enzyme method versus

LDH method):
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 Glycolic acid metabolites (glyoxalate and glycolate) in

ethylene glycol poisoning
 Hydroxyurea (if > 0.92mmol/L)
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 Mannitol
Bilirubin (if > 274 μmol/L)
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 Ascorbate (if > 568 μmol/L)

 Bromide (not at therapeutic levels)
 Thiocyanate
Citrate
 Oxalate
 Triglycerides (>11.3 mmol/L).
Non-specific interference with spectrophotometric biosensors
Lipemic or icteric sample

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