Relationship Between Serum Total Carbon Dioxide Concentration and Bicarbonate Concentration in Patients Undergoing Peritoneal Dialysis

Background Few studies have assessed the relationship between serum total carbon dioxide (CO2) and bicarbonate ion (HCO3−) concentration in patients undergoing peritoneal dialysis. We determined the agreement between serum total CO2 and HCO3− concentration and the diagnostic accuracy of serum total CO2 for the prediction of low (HCO3− <24 mEq/L) and high (HCO3− ≥24 mEq/L) bicarbonate concentrations in patients on peritoneal dialysis. Methods We collected 245 samples of venous blood from 51 patients on peritoneal dialysis. Independent factors that correlated with the HCO3− concentration were analyzed using multiple linear regression analysis. The diagnostic accuracy of serum total CO2 was evaluated by receiver operating characteristic (ROC) curve analysis and a 2×2 table. Agreement between serum total CO2 and HCO3− concentration was assessed by Bland-Altman analysis. Results Serum total CO2 was independently correlated with HCO3− concentration (β = 0.354, p < 0.001). The area under the curve of serum total CO2 for the identification of low and high bicarbonate concentrations was 0.909. The diagnostic accuracy of serum total CO2 for the prediction of low and high bicarbonate concentrations was: sensitivity, 91.5%; specificity, 74.7%; positive predictive value, 53.5%; negative predictive value, 96.5%; and accuracy, 78.8%. Bland-Altman analysis showed a moderate agreement between serum total CO2 and HCO3− concentration. Conclusion Serum total CO2 correlated closely with the HCO3− concentration in patients undergoing peritoneal dialysis. Serum total CO2 might be useful for predicting low and high bicarbonate in peritoneal dialysis patients.


Introduction
Metabolic acidosis is a commonly observed complication in patients with chronic kidney disease (CKD), including those undergoing peritoneal dialysis, and is associated with bone mineral loss, protein energywasting, insulin resistance, and higher mortality risk [1][2][3][4]. It also contributes to a rapid decline in residual renal function [5]. Therefore, early detection and accurate diagnosis of metabolic acidosis is important to preserve residual renal function and improve prognosis in patients undergoing peritoneal dialysis.
In Japan, blood-gas analyzers are available in most hospitals. Therefore, bicarbonate ion (HCO 3 − ) measured using arterial/venous blood gas samples has been widely used for the assessment of metabolic acidosis in peritoneal dialysis patients [6]. A lower HCO 3 − concentration has been reported to be associated with increased mortality in patients undergoing peritoneal dialysis [7]. Because the HCO 3 − concentration is an important predictor of mortality, a specific device measurement and syringe are necessary, in addition to the blood samples used for blood-gas analyses [8].
The serum total carbon dioxide (CO 2 ) concentration represents the total amount of carbon dioxide in the serum. It can be readily measured, along with creatinine, urea, and electrolytes, using a biochemical analyzer in clinical settings [9]. Furthermore, serum total CO 2 has been shown to be correlated strongly with the HCO 3 − concentration in both patients with CKD not undergoing renal replacement therapy [10] and patients undergoing hemodialysis [11]. However, few studies have investigated the relationship between serum total CO 2 and the HCO 3 − concentration in patients undergoing peritoneal dialysis. We analyzed the relationship between serum total CO 2 and the HCO 3 − concentration in peritoneal dialysis patients.

Ethics approval
The study was approved by the ethics committee of Saitama Medical Center, Jichi Medical University (S17-052), and was conducted according to the principles contained within the Declaration of Helsinki. The requirement of informed consent was waived and an opt-out method was used because of the retrospective design of the study.

Study design
This was a single-center, retrospective, cross-sectional study. We analyzed the patient data obtained from medical records from the Division of Nephrology, Saitama Medical Center, between April 2017 and March 2019. The laboratory data of blood tests and venous blood-gas tests obtained simultaneously were used for analyses. The relationship between serum total CO 2 and the HCO 3 − concentration was analyzed using Pearson's correlation coefficient. Independent factors correlated with the HCO 3 − concentration were analyzed using multiple linear regression analysis. The diagnostic accuracy of serum total CO 2 for low and high bicarbonate was analyzed using receiver operating characteristic (ROC) curve analysis and a 2×2 table.
The correlation between serum total CO 2 and HCO 3 − concentration was analyzed using Bland-Altman analysis.

Laboratory methods
Blood and urinary parameters were determined by the Department of Clinical Laboratory, Saitama Medical Center. Samples of venous blood were collected in EDTA-containing tubes from the antecubital vein in all patients and centrifuged within 15 minutes to obtain serum. Serum total CO 2 was measured within 15 minutes after centrifugation using an automated biochemical analyzer (JCA-BM6070; JEOL, Tokyo, Japan), as were biochemical parameters (hemoglobin, total protein, serum albumin, blood urea nitrogen, serum creatinine, sodium, potassium, chloride, calcium, phosphate, magnesium, and glucose). Serum total CO 2 was determined by an enzymatic method using a commercial kit (Toyobo, Osaka, Japan) in an automated biochemical analyzer. Total weekly urea clearance (Kt/V) was measured by calculating the sum of the residual renal and peritoneal clearances of urea and converting this to a weekly value [12]. Residual renal urea clearance was determined using 24-hour urine urea divided by plasma urea concentration. Total body water volume was estimated from height, weight, age, and gender using Watson's formula [13].
Samples of venous blood for gas analyses were collected in a heparinized blood-gas syringe from the brachial vein simultaneously with samples for other blood tests and analyzed within 10 minutes to obtain values for pH and the partial pressure of carbon dioxide (pCO 2 ). The pH and pCO 2 of blood were measured using a blood-gas analyzer (Rapidlab-1265; Siemens Healthcare Diagnostics, Tarrytown, New York). The HCO 3 − concentration was calculated from measured pH and pCO 2 using the Henderson-Hasselbalch equation [14]:

Statistics
Statistical analyses were performed using JMP v11 (SAS Institute, Cary, North Carolina). Continuous variables were expressed as mean ± standard deviation when they were normally distributed and as median and interquartile range when non-normally distributed. Categorical variables were expressed as numbers and percentages. The peritoneal dialysis duration was not normally distributed; therefore, this variable was transformed using a natural logarithm. The relationships between two variables were evaluated using Pearson's correlation coefficient. Linear regression analysis was used to identify parameters that independently correlated with HCO3− concentration. The parameters that significantly correlated with HCO 3 − concentration in simple linear regression analyses were included in subsequent multiple linear regression analysis. The diagnostic accuracy of serum total CO 2 was determined using ROC curve analysis and a 2×2 table. The area under the curve (AUC), sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were calculated for the identification of low (HCO 3 − <24 mEq/L) and high (HCO 3 − ≥24 mEq/L) bicarbonate concentrations. The cut-off value for HCO 3 − was set at 24 mEq/L based on a previous study [15]. Agreement between serum total CO 2 and HCO 3 − concentration was assessed using the Bland-Altman method. P < 0.05 was considered to represent statistical significance.

Patient characteristics
Patients' characteristics and medications are shown in Table 1  Relationship between serum total CO 2 and HCO 3 − concentration Figure 1 shows the correlation between serum total CO 2 and HCO 3 − concentration. Serum total CO 2 was correlated with HCO 3 − concentration significantly and closely (r = 0.80; p < 0.001).

Correlation between serum total CO 2 and HCO 3 − concentration
Bland-Altman analysis showed moderate agreement between serum total CO 2 and HCO 3 − concentration.
The mean difference was -1.64 ± 3.66, and 95.1% of the points were included within the limits of agreement (the mean difference between the two methods ± 2 standard deviation [95% confidence interval]) ( Figure 3).

Discussion
In the present study, we investigated the relationship between serum total CO 2 and HCO 3 − concentration in peritoneal dialysis patients and found that serum total CO 2 closely correlated with HCO 3 ˗ concentration. We also found that serum total CO 2 has high diagnostic accuracy for predicting low bicarbonate and high bicarbonate in peritoneal dialysis patients.
Serum total CO 2 is a total concentration of all forms of CO 2 in a serum sample, including HCO 3 − , carbonate, and dissolved CO 2 . Serum total CO 2 value is known to be approximately equivalent to HCO 3 − concentration because most of CO 2 exists as HCO 3 − in blood [9]. In fact, serum total CO 2 has been reported to have a close correlation with HCO 3 − concentration in both pre-dialysis CKD patients [10] and hemodialysis patients [11].
However, a discrepancy between serum total CO 2 and HCO 3 − concentration is sometimes observed, and temperature and acidity [16] are considered one of the causes of discrepancy in patients without renal impairment [17]. In the present study, serum albumin, calcium, chloride, sodium, phosphate, blood urea nitrogen, and total weekly Kt/V in addition to serum CO 2 were independently correlated with HCO 3 − concentration in serum.
Serum albumin represents the nutritional status of patients and is reportedly associated with dietary protein intake in peritoneal dialysis patients [18]. Protein intake is associated with metabolic acidosis because amino acids into which dietary proteins are broken down release hydrogen ions [19]. Increased serum albumin was reported to be associated with metabolic acidosis in pre-dialysis CKD patients [20]. The weak acidity of albumin has also been considered as the cause of this phenomenon [21]. These findings are consistent with our result, showing a negative correlation between serum albumin and HCO 3 − concentration.
It has been reported that HCO 3 − was positively correlated with calcium concentration in hemodialysis patients [22]. In the present study, HCO 3 − concentration was positively correlated with calcium concentration in peritoneal dialysis patients. These findings suggest that serum HCO 3 − concentration might be positively correlated with calcium concentration in patients with end-stage renal disease. There were differences in calcium concentrations among peritoneal dialysis solutions used in the present study. The possibility remains that these differences might affect the results of our study.
HCO 3 − concentration has been shown to decrease along with an increase in chloride concentration through following equilibrium with HCl and NaHCO 3 : H + + Cl -+ Na + + HCO 3 − = Na + + Cl -+ H 2 CO 3 [23]. In the present study, chloride concentration was negatively correlated with HCO 3 − concentration, which is consistent with the findings of previous reports [10][11].
A cross-sectional study of peritoneal dialysis patients reported that sodium concentration was lower in patients with HCO 3 − < 22 mEq/L than in patients with 22 ≤ HCO 3 − < 28 mEq/L [24]. In the present study, sodium concentration was positively correlated with HCO 3 − concentration. These results suggest that sodium concentration is positively associated with HCO 3 − concentration in peritoneal dialysis patients.
Phosphate and blood urea nitrogen were shown to be associated with daily protein intake in patients with end-stage renal disease [25]. Protein intake is negatively associated with bicarbonate, as the amino acids into which dietary proteins are broken down release hydrogen ions [19]. Phosphate and blood urea nitrogen were reported to be negatively correlated with bicarbonate in peritoneal dialysis patients [24], which is consistent with the findings of our study.
Currently available peritoneal dialysis fluids contain alkaline anions of 35-40 mmol/L as lactate or/and bicarbonate [6]. The influx of alkaline anions from the peritoneal dialysis fluid into the blood occurs during peritoneal dialysis because the alkaline anion concentration in serum is usually lower than that in peritoneal dialysis fluid [24]. A previous study reported that dialysis adequacy assessed by daily Kt/V was positively correlated with serum bicarbonate level [26]. In the present study, the total weekly Kt/V was positively correlated with HCO 3 − concentration. These results suggest that peritoneal dialysis dose-dependently increases serum HCO3− concentration caused by the influx of bicarbonate from the peritoneal dialysis fluid into the blood.
In the present study, serum total CO 2 was closely correlated with HCO 3 − concentration and showed high accuracy for the differentiation of low or high bicarbonate concentrations. Therefore, serum CO 2 may be a good predictor of bicarbonate concentration and useful to predict whether this is low or high. However, the correlation between serum total CO 2 and HCO 3 − concentration in the present study (β = 0.323) was weaker as compared with that of hemodialysis patients (β = 0.858) [11]. The number of clinical parameters correlated with HCO 3 − concentration was greater in the present study than in the previous one [11] (eight vs three), which might explain the lower correlation between serum total CO 2 and HCO 3 − in this study. The correlation between serum total CO 2 and HCO 3 − concentration might be attenuated in peritoneal dialysis patients. Further studies are necessary to confirm the close correlation between serum total CO 2 and HCO 3 − concentration and the usefulness of serum total CO 2 for the diagnosis of low or high bicarbonate concentrations in peritoneal dialysis patients.
The measurement of serum total CO 2 has two advantages as compared with blood-gas analyses. First, the cost of a blood gas-syringe can be saved and the amount of blood required will be reduced using serum total CO 2 instead of a blood-gas test. Second, serum total CO 2 can be used to predict low bicarbonate and high bicarbonate without the use of a blood-gas analyzer. Therefore, the measurement of serum total CO 2 could reduce some of the burden on peritoneal dialysis patients and laboratory staff.
Our study had four limitations. First, it was a retrospective, observational study; therefore, selection bias could not be completely eliminated. Second, the study was performed at a single center, which limits the external validity of the results. Third, the study cohort was small, which restricts the generalizability of our findings. Fourth, we used venous blood samples for the analyses. The results might have been different if arterial blood samples had been used. Therefore, further prospective, large-scale, multicenter studies are necessary to confirm our findings.

Conclusions
Serum total CO 2 correlated closely with HCO 3 − concentration in peritoneal dialysis patients. Serum total CO 2 might be useful for predicting low and high bicarbonate in peritoneal dialysis patients.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Ethics committee of Saitama Medical Center, Jichi Medical University issued approval S17-052. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.