Adjusted Plasma Chloride and Bicarbonate Concentrations: An Approach to Identifying Acid–Base Disorders

Abstract

Background

Chloride disorders (hypochloremia, hyperchloremia) and bicarbonate disorders (hypobicarbonatemia, hyperbicarbonatemia) are common in clinical medicine and indicate metabolic and/or respiratory acid–base disorders. The normally inverse relationship between chloride and bicarbonate concentrations in the blood is altered, however, by changes in fluid balance, i.e., water excess or deficit, with resulting changes in sodium and other electrolyte concentrations and by anion gap metabolic acidosis, which lowers bicarbonate concentrations but not the chloride concentrations.

Methods

We used formulas derived over a decade ago that utilize dry slide laboratory technology to adjust plasma chloride and bicarbonate concentrations for changes in water balance, as reflected in changes in plasma sodium concentrations and in the plasma anion gap. We then prospectively validated these formulas in 736 consecutive adults, 499 having abnormal basic metabolic panel results and 237 having normal panel results, using modern wet laboratory technology.

Results

Plasma chloride and bicarbonate concentrations were inversely correlated (2-tailed P-value <0.0001), but the correlations were only modest (Spearman r: −0.48 for the abnormal group and −0.41 for the normal group). After adjusting the plasma chloride and bicarbonate concentrations using the 2 prior formulas, the inverse correlations were very high, with Spearman r: −0.998 for the abnormal group and −0.999 for the normal group.

Conclusions

Adjusting plasma chloride and bicarbonate concentrations for any water imbalance and anion gap alterations leads to very high inverse correlations between these 2 anions, allowing accurate assessment of either subtle or overt acid–base disorders.

Impact Statement

Plasma chloride concentrations adjusted for the sodium concentration and plasma bicarbonate concentrations adjusted for the sodium concentration and the anion gap are inversely correlated, allowing more accurate classification of patients with metabolic and/or respiratory acid–base disorders than simple use of measured (unadjusted) chloride and bicarbonate (total CO₂) concentrations.

Chloride (Cl⁻)² and bicarbonate (HCO₃  ⁻) are the major anions in plasma and extracellular fluid (ECF). The sum of their concentrations is on average 8–9 mmol/L less than the concentration of the predominant ECF cation, sodium (Na⁺). The difference, [Na⁺ − (Cl⁻ + HCO₃  ⁻)], represents the anion gap (AG) (1).

The acid–base status of an individual influences Cl⁻ and HCO₃  ⁻ concentrations. High Cl⁻ concentrations combined with low HCO₃  ⁻ concentrations are seen in nongap metabolic acidosis and in chronic respiratory alkalosis. Decreased Cl⁻ concentrations combined with high HCO₃  ⁻ concentrations indicate either metabolic alkalosis and chronic respiratory acidosis. In 2006, we reported that there was a significant inverse correlation between serum Cl⁻ and HCO₃  ⁻ concentrations, but the correlation was only modest (r = −0.46) (2). However, after adjusting (a) the serum Cl⁻ and HCO₃  ⁻ concentrations for any free water excesses (or deficits) that were present and (b) the serum HCO₃  ⁻ concentration for any deviation in the AG from the average concentration, there was an excellent inverse correlation between Cl⁻ and HCO₃  ⁻ concentrations in 135 hospitalized patients (2). At that time, our laboratory was using dry slide technology, using a solid layer on which the reaction with serum occurred, which was then read by a spectrometer or potentiometer (Vitros 950, Ortho Clinical Diagnostics). This technology has subsequently been replaced in our laboratory by wet chemistry technology in which liquid reagents are mixed together in a cuvette and allowed to react with plasma, followed by spectrometer readings or potentiometric reactions at different time intervals (Abbott Architect C 16000). The purpose of the current prospective study in 736 consecutive inpatients and outpatients was to validate the prior Cl⁻ and HCO₃  ⁻ adjustment formulas using data obtained from wet chemistry technology. We tested the 2 formulas prospectively in samples from a larger number of individuals with abnormal basic metabolic profiles (BMPs) than was previously tested (499 vs 135) and, for the first time, in 237 individuals with normal BMPs.

METHODS

Population sample

Our 2017 cohort included 736 consecutive unique individuals who were above the age of 18 years and who had had a BMP ordered for any reason. In total, 237 of them had BMP results that were each within the laboratory's references intervals at that time (Table 1). These data served as the internal control group for the remaining 499 individuals who had ≥1 abnormal BMP results (Table 1).

Table 1.

Average (±SD) plasma analyte concentrations in 499 individuals with abnormal BMP results and in 237 controls with normal BMP results.

Plasma analyte	Laboratory reference interval	Abnormal BMP (n = 499)	Normal BMP (n = 237)	P-valuea
Na+	135–145 mmol/L	137 (±4)	139 (±2)	<0.0001
K+	3.5–5.0 mmol/L	4.1 (±0.7)	4.1 (±0.4)	Not significant
Cl−	98–110 mmol/L	105 (±5)	105 (±3)	Not significant
HCO3  −	22–34 mmol/L	23 (±4)	25 (±2)	<0.0001
Creatinine	0.55–1.11 g/dL (women); 0.72–1.25 mg/dL (men)	1.38 (±1.69)	0.81 (±0.16)	<0.0001
Urea nitrogen	10–20 mg/dL (women); 9–25 mg/dL (men)	23 (±20)	13 (±4)	<0.0001
Glucose	65–139 mg/dL	128 (±56)	100 (±15)	<0.0001

^a

2-tailed Mann–Whitney U-test, abnormal BMP group vs normal BMP group.

Laboratory measurements

BMPs were performed on the Abbott Architect C 16000 chemistry analyzer using plasma samples collected in BD vacutainer lithium heparin tubes. Samples were run on an automated robotic line that centrifuged the specimens, decapped the vacutainer tubes, and ran the samples immediately. Sodium, potassium, and chloride were measured by ion selective electrode (indirect method) and the total carbon dioxide (CO₂) was measured by the phenylpyruvate carboxylase method and was expressed as bicarbonate, all in millimoles per liter. Laboratory results were imported from the clinical chemistry laboratory into a spreadsheet for further computations (Microsoft Excel 2016, Microsoft Corporation). All patient identifiers were removed by the laboratory personnel before sending the spreadsheets to the investigators, allowing the study to be exempted from approval by the Institutional Review Board. For each of the analytes in the BMP, the manufacturer's suggested reference intervals were verified by performing analysis of at least 40 venous plasma samples of apparently healthy individuals, as described in the CLSI document EP28-A3C (3).

Statistical methods

The formula we used to adjust each individual's plasma Cl⁻ concentration in mmol/L for their state of hydration was (2):

$$\text{Adjusted}\hspace{0.17em}{\text{Cl}}^{-}=\text{Measured}\hspace{0.17em}{\text{Cl}}^{-}+(\text{Measured}\hspace{0.17em}{\text{Na}}^{+}-\text{Average}{\text{\hspace{0.17em}Na}}^{+}\hspace{0.17em}\text{in}\hspace{0.17em}\text{controls})\times (\text{Average}\hspace{0.17em}{\text{Cl}}^{-}/{\text{Na}}^{+}\hspace{0.17em}\text{in}\hspace{0.17em}\text{controls})\mathrm{.}$$

In our prior study (2), the average (±SD) Na⁺ in 135 controls was 140 (±2) mmol/L, whereas it was 139 (±2) mmol/L in the current group of 237 controls. The average Cl⁻/Na⁺ ratio in the controls was 0.76 (±0.02) in the prior study (2) and in the current control group. Thus, the above Cl⁻ formula was rewritten for the present study as:

$$\mathrm{Adjusted}{\text{Cl}}^{-}=\mathrm{Measured}{\text{Cl}}^{-}+(\mathrm{Measured}{\text{Na}}^{+}-139)\times \mathrm{0.76.}$$

The formula used to adjust each individual's plasma HCO₃  ⁻ concentration in mmol/L for their state of hydration and for any deviation (Δ) in the plasma AG was (2):

$$\mathrm{Adjusted}{\text{HCO}}_3^{-}=\mathrm{Measured}{\text{HCO}}_3^{-}+(\mathrm{Measured}{\text{Na}}^{+}-\mathrm{Average}{\text{Na}}^{+}\mathrm{in}\mathrm{control}\text{s})\times (\mathrm{Average}{\text{HCO}}_3^{-}/{\text{Na}}^{+}\mathrm{\hspace{0.17em}in}\mathrm{control}\text{s})+\Delta \text{AG}\mathrm{.}$$

In our prior study (2), the average HCO₃  ⁻/Na⁺ ratio in 135 controls was 0.19 (±0.01), whereas it was 0.18 (±0.01) in the current group of 237 controls. ΔAG was the individual's AG minus the average AG in the controls. In our prior study (2), the average AG in the controls was 8 (±2) mmol/L, whereas in the current study, it was 9 (±2) mmol/L. Thus, the HCO₃  ⁻ formula above was rewritten for the present study as:

$$\mathrm{Adjusted}{\text{HCO}}_3^{-}=\mathrm{Measured}{\text{HCO}}_3^{-}+(\mathrm{Measured}{\text{Na}}^{+}-139)\times 0.18+(\text{AG}-9)\mathrm{.}$$

When correlating unadjusted (or adjusted) plasma Cl⁻ with unadjusted (or adjusted) plasma HCO₃  ⁻ concentrations, we used the Spearman's rank-order correlation (4). Group differences in patients with normal BMPs vs those with abnormal BMPs were tested for significance using the Mann–Whitney U-test (5). Two-tailed P values <0.05 were considered significant.

RESULTS

Overall results

Table 1 compares the average (±SD) plasma concentrations of the 7 analytes comprising the BMP in the 499 individuals with abnormal BMPs to those of the 237 controls with normal BMPs. The average (±SD) plasma Cl⁻ concentration in the abnormal BMP group was 105 (5) mmol/L, identical to the average concentration of 105 (3) mmol/L in the normal BMP group (Table 1). Plasma Cl⁻ concentrations were 85–128 mmol/L in the abnormal BMP group (reference interval: 98–110 mmol/L). The mean plasma HCO₃  ⁻ concentration in the abnormal BMP group was 23 (4) mmol/L, substantially lower than the concentration of 25 (2) mmol/L in the controls (Table 1). Plasma bicarbonate concentrations were 6–40 mmol/L in the abnormal BMP group (reference interval: 22–34 mmol/L).

The incidence of specific plasma electrolyte abnormalities among the 499 individuals with abnormal BMPs is shown in Table 1 of the Data Supplement that accompanies the online version of this article at http://www.jalm.org/content/vol2/issue6. Of the 8 possible abnormalities, the most common was hypobicarbonatemia, which was present in 46.7% of individuals, and the least common was hyperbicarbonatemia, which was present in 0.2% of individuals. Using our laboratory's current upper limit of the reference interval of 29 mmol/L, the prevalence of hyperbicarbonatemia would have been 2%. Hypochloremia and hyperchloremia each occurred in approximately 10% of the individuals with abnormal BMPs.

Correlations between unadjusted plasma Cl⁻ and HCO₃  ⁻ concentrations

In the group with abnormal BMP results, there were moderate inverse correlations between plasma Cl⁻ and HCO₃  ⁻ concentrations (Spearman r: −0.48) (Fig. 1A and Table 2). Plasma Na⁺ in this group was also correlated with plasma Cl⁻ and, to a lesser degree, with plasma HCO₃  ⁻ (Table 2). There was a weak inverse correlation between plasma Na⁺ and plasma potassium (K⁺) concentrations.

Plot relating measured (unadjusted) plasma chloride (Cl⁻) to measured (unadjusted) plasma bicarbonate (HCO₃  ⁻) concentrations in 499 individuals with abnormal BMPs (A). Spearman correlation coefficient was −0.48 (2p <0.0001). Plot relating adjusted plasma Cl⁻ to adjusted plasma HCO₃  ⁻ concentrations in the same 499 individuals (B).

Fig. 1.

Spearman correlation coefficient was −0.998 (2p <0.0001). Trendlines are shown.

Table 2.

Spearman rank-order correlations between plasma electrolyte pairs in individuals with abnormal BMPs and in controls with normal BMPs.

	Electrolyte	K+	Cl−	HCO3  −
Abnormal BMP (n = 499)	Na+	−0.14	0.58a	0.11a
	K+	–	−0.05	0.02
	Cl−	–	–	−0.48a
Normal BMP (n = 237)	Na+	−0.08	0.47a	0.30a
	K+	–	0.08	0.02
	Cl−	–	–	−0.41a

^a

2-tailed P-value < 0.05.

In the control group with normal BMPs, there were again moderate inverse correlations between plasma Cl⁻ and HCO₃  ⁻ concentrations (Spearman r: −0.41) (Fig. 2A and Table 2). Plasma Na⁺ was also significantly correlated with Cl⁻ and, to a lesser extent, with HCO₃  ⁻. Plasma K⁺ did not correlate with any electrolyte (Table 2).

Plot relating measured (unadjusted) plasma chloride (Cl⁻) to measured (unadjusted) plasma bicarbonate (HCO₃  ⁻) concentrations in 237 individuals with normal BMPs (A). Spearman correlation coefficient was −0.41 (2p <0.0001). Plot relating adjusted plasma Cl⁻ to adjusted plasma HCO₃  ⁻ concentrations in the same 237 individuals (B).

Fig. 2.

Spearman correlation coefficient was −0.999 (2p <0.0001). Trendlines are shown.

Correlations between adjusted plasma Cl⁻ and HCO₃  ⁻ concentrations

After adjustment of plasma Cl⁻ and HCO₃  ⁻ concentrations in the group with abnormal BMPs, the inverse correlation between plasma Cl⁻ and HCO₃  ⁻ was very high, with correlation coefficients near unity (Spearman r: −0.998) (Fig. 1B). Adjusted plasma Cl⁻ concentrations in this group were 84–115 mmol/L. Adjusted plasma HCO₃  ⁻ concentrations were 15–46 mmol/L.

Fig. 1 in the online Data Supplement plots the relationship between differences in plasma Cl⁻ and HCO₃  ⁻ concentrations from the mean control concentrations in the 499 individuals with abnormal BMP results. The Δ adjusted plasma Cl⁻ concentration was −21 mmol/L to 10 mmol/L, whereas the Δ adjusted plasma HCO₃  ⁻ was −10 mmol/L to 21 mmol/L.

After adjustment of the 237 controls' plasma Cl⁻ and HCO₃  ⁻ values for the relatively small variations from average concentrations, albeit still within the laboratory's reference intervals, the inverse correlation between plasma Cl⁻ and HCO₃  ⁻ improved to near unity (Spearman r: −0.999) (Fig. 2B).

DISCUSSION

The tonicity (osmolality) of the plasma and ECF space is maintained within a relatively narrow range, primarily by hypothalamic neurons that regulate water intake (thirst) and the release of vasopressin (antidiuretic hormone) from the posterior pituitary gland. Thus, plasma Na⁺ concentrations are normally maintained near 140 mmol/L. Disorders of water balance leading to hyponatremia or hypernatremia are common in clinical practice; in this study, either hyponatremia or hypernatremia occurred in 112 of 736 consecutive plasma samples (15%), with the former >6-fold higher than the latter (see Table 1 in the online Data Supplement). Such changes in water balance dilute or concentrate not only plasma cations such as Na⁺ but also plasma anions such as Cl⁻ and HCO₃  ⁻. In a patient with relative or absolute overhydration with hyponatremia, the plasma concentrations of Cl⁻ and HCO₃  ⁻ should decrease in proportion to their baseline concentrations. Because the baseline concentration of Cl⁻ is >4-fold greater than the HCO₃  ⁻ concentration (approximately 105 vs approximately 25 mmol/L), the absolute decrease in the Cl⁻ concentration in mmol/L will exceed the absolute decrease in the HCO₃  ⁻ concentration. Such an unequal decrease in the Cl⁻ vs HCO₃  ⁻ concentrations will erode the close inverse relationship between these two anions.

To assist busy clinicians, laboratories facilitate rapid assessment of circulating electrolyte concentrations by highlighting values that are above or below the reference interval. Nevertheless, a Cl⁻ or HCO₃  ⁻ concentration that is highlighted as being low or high may not yield additional useful information above that provided by a low or high plasma Na⁺ concentration. Moreover, a value that is not highlighted because it is within the reference interval may provide valuable diagnostic information, as discussed below.

As an example, if the measured plasma Na⁺ concentration is low (e.g., 129 mmol/L), the plasma Cl⁻ should fall by (129–139) mmol/L × 0.76, or by 7–8 mmol/L, to approximately 97 mmol/L. Should the measured plasma Cl⁻ in the same individual be in the middle of the laboratory's reference interval (e.g., 105 mmol/L), the plasma Cl⁻ concentration would in fact be 8 mmol/L higher than expected after considering the individual's water balance. This moderate 8 mmol/L plasma chloride excess would indicate the presence of a hidden hyperchloremic state (e.g., a nongap metabolic acidosis, a chronic respiratory alkalosis, or both; Table 3). As a second example, if the measured plasma Na⁺ concentration is high (e.g., 154 mmol/L), the plasma Cl⁻ would be expected to increase by (154–139) mmol/L × 0.76, or by 12 mmol/L, to approximately 117 mmol/L. If this same individual had a measured plasma Cl⁻ of 104 mmol/L (midreference interval), this considerably large (13 mmol/L) chloride deficit would indicate the presence of a hypochloremic state (e.g., metabolic alkalosis, chronic respiratory acidosis, or both; Table 3).

Table 3.

Possible acid–base disorders associated with highly correlated abnormalities in adjusted plasma Cl⁻ and HCO₃  ⁻ concentrations.

Abnormality	Possible Disorder(s)a
↑ in adjusted Cl− with a near equal and opposite ↓ in adjusted HCO3  − (high Cl−/Na+ ratio)	Hyperchloremic (non-gap) metabolic acidosis
	or
	Chronic respiratory alkalosis
	or
	Both disorders
↓ in adjusted Cl− with a near equal and opposite ↑ in adjusted HCO3  − (low Cl−/Na+ ratio)	Hypochloremic metabolic alkalosis
	or
	Chronic respiratory acidosis
	or
	Both disorders

^a

To distinguish between possibilities listed, a careful history, physical examination and, if necessary, arterial blood gas assessment with pH and pCO₂ measurements are recommended; the degree of the deviation (in mmol/L) from control concentrations can be used to classify the disorder as mild, moderate, or severe (marked). With any of these disorders, an anion gap metabolic acidosis may also be present.

In individuals with nongap (hyperchloremic) metabolic acidosis, usually due to diarrhea or renal tubular acidosis, as hydrochloric acid is added to the ECF, the plasma HCO₃  ⁻ falls proportional to the acid generated:

$$\text{HCl}+{\text{HCO}}_3^{-}\to {\text{Cl}}^{-}+{\text{CO}}_2+{\text{H}}_2\text{O}\mathrm{.}$$

The increase in plasma Cl⁻ will equal the decrease in plasma HCO₃  ⁻, with no change in the sum of the Cl⁻ and HCO₃  ⁻ concentrations and no change in the plasma anion gap. In contrast, in patients with anion gap metabolic acidosis from an accumulation of either a non-HCl inorganic acid (e.g., phosphoric acid, sulfuric acid) or an organic acid (e.g., lactic acid, β-hydroxybutyric acid, acetoacetic acid), the anion A⁻ of the acid accumulates as the acid's proton is neutralized by HCO₃  ⁻:

$$\text{HA}+{\text{HCO}}_3^{-}\to {\text{A}}^{-}+{\text{CO}}_2+{\text{H}}_2\text{O}$$

There is a proportional increase in A⁻ and a decrease in HCO₃  ⁻, with no change in plasma Cl⁻. Thus, the inverse relationship between Cl⁻ and HCO₃  ⁻ is altered by the accumulation of A⁻, with an increase in the anion gap and an equivalent decrease in HCO₃  ⁻.

The two formulas developed in 2006 (2) for adjusting serum Cl⁻ and HCO₃  ⁻ concentrations to account for the not-infrequent changes in water balance and anion gap alterations seen in clinical medicine were found in 2017 to be robust when evaluated prospectively using plasma samples and wet chemistry technology. Essentially, in patients with abnormal plasma electrolyte concentrations, the adjusted plasma Cl⁻ and adjusted plasma HCO₃  ⁻ concentrations gave identical and highly inversely correlated information regarding acid–base balance. Although it may seem redundant to perform 2 separate calculations to adjust both the plasma Cl⁻ and HCO₃  ⁻ concentrations, we often use both calculations as an extra step of validation and to detect the rare measurement error should the |ΔCl⁻| and |ΔHCO₃  ⁻| not be identical or nearly identical. With practice, such calculations can be done quite quickly. If only 1 formula is desired, we prefer the adjusted Cl⁻ formula due to its simplicity.

As a preliminary step to determine if the Cl⁻ formula is required, we calculate the plasma Cl⁻/Na⁺ ratio, which in controls with normal BMPs averaged 0.76 (0.02) in both our prior study (2) and in our current study. Using the nonparametric method advocated by Horowitz (6), the reference interval (central 95%) for the Cl⁻/Na⁺ ratio in our 237 controls with normal BMPs was 0.73–0.79, which is identical to the reference interval from our prior study (2). Plasma Cl⁻/Na⁺ ratios above or below this reference interval indicate a hyperchloremic or hypochloremic state, respectively (see Table 3).

We offer the following approach. First, calculate the Cl⁻/Na⁺ ratio, which will rapidly identify a hyperchloremic or hypochloremic state and its degree. To quantify the degree of adjusted hyperchloremia or hypochloremia present in mmol/L, use the adjusted Cl⁻ formula presented here, although each laboratory may choose to determine and use its own control concentrations, if available. Finally, if desired, use the adjusted HCO₃  ⁻ formula to “double check” the calculation of the adjusted plasma Cl⁻. Clinical interpretations are summarized in Table 3.

Perhaps the most novel finding of this study was the significant relationship between plasma Cl⁻ and HCO₃  ⁻ concentrations in the 237 controls with normal BMP results. In these 237 individuals, all 4 plasma electrolyte concentrations, as well as plasma creatinine, urea nitrogen, and glucose concentrations, were within the laboratory's reference intervals. Nevertheless, there was a moderate inverse correlation between measured (unadjusted) plasma Cl⁻ and HCO₃  ⁻ concentrations that was numerically similar to the inverse Cl⁻–HCO₃  ⁻ correlation seen in individuals with abnormal BMP results in this study and our prior study (2) (Spearman r values between −0.4 and −0.5). When plasma Cl⁻ and HCO₃  ⁻ concentrations in the 237 controls with normal BMPs were “adjusted,” the inverse correlation between plasma Cl⁻ and HCO₃  ⁻ was very high. This observation implies that even subtle changes in water balance and in the AG weaken the close inverse relationship between plasma Cl⁻ and HCO₃  ⁻ concentrations. Because it uncommon to know the actual (premorbid) baseline plasma electrolyte concentrations in a patient presenting for medical care, it is worthwhile considering using the adjustment formulas presented here, even in patients presenting with plasma electrolytes concentrations in the lower or upper parts of the laboratory's reference intervals. Such an approach could then be acted on by the astute clinician in keeping with the clinical presentation.

2 Nonstandard abbreviations

 
• Cl⁻

  chloride ion

 
• HCO₃  ⁻

  bicarbonate ion

 
• ECF

  extracellular fluid

 
• K⁺

  potassium ion

 
• Na⁺

  sodium ion

 
• AG

  anion gap

 
• BMP

  basic metabolic panel

 
• SD

  standard deviation

 
• A⁻

  anion.

Author Contributions:  All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Authors' Disclosures or Potential Conflicts of Interest:  No authors declared any potential conflicts of interest.

This work was supported by the William O. Tschumy, Jr. M.D. Chair of Internal Medicine (MF).

This work is original and has not appeared in prior abstracts, presentations, reports or publications.

Acknowledgments

The authors thank Margaret Preston and Andrea Sablica-Phillips for their valuable assistance.