1 Department of Nephrology and Dialysis, A. Manzoni Hospital, Lecco and 2 Provincial Hospital, Camposampiero, Italy
Correspondence and offprint requests to: Vincenzo La Milia, MD, Department of Nephrology and Dialysis, A. Manzoni Hospital, Via DellEremo 9/11, 23900 Lecco, Italy. Email: v.lamilia{at}ospedale.lecco.it
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Abstract |
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Methods. We performed peritoneal equilibration tests (PETs) in 44 peritoneal dialysis patients and calculated the NaR. We also calculated D/PNa during the test; plasma and dialysate sodium concentrations were measured by F, D-ISE and I-ISE.
Results. NaR was lower (P<0.001) with D-ISE (69±29 mmol) than with F (81±29 mmol) or I-ISE (79±28 mmol). D/PNa was also lower at baseline (0.92±0.02 vs 0.95±0.02 and 0.95±0.02; P<0.001), after 60 min (0.87±0.03 vs 0.90±0.03 and 0.90±0.03; P<0.001) and at the end of PET (0.88±0.04 vs 0.92±0.04 and 0.92±0.04; P<0.001) when measured by D-ISE in comparison with F and I-ISE, respectively.
Conclusions. NaR and D/PNa were lower when measured by the D-ISE method compared with the F and I-ISE methods. NaR and D/PNa were similar when measured by F or I-ISE. I-ISE can be used reliably in the evaluation of NaR and D/PNa in everyday clinical practice of peritoneal dialysis.
Keywords: flame photometry; ion-selective electrode; peritoneal dialysis; sodium concentration; sodium removal; sodium gradient
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Introduction |
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The dialysate/plasma sodium concentration ratio (D/PNa) is a useful means of studying the hydraulic permeability of the peritoneal membrane, and an indirect index of transcellular water transport by aquaporin channels [57]. As a result of free water transport into the peritoneal cavity, D/PNa decreases markedly during hypertonic dwell (sodium sieving) [57], and nearly 4050% of ultrafiltration during hypertonic dwell occurs through the aquaporin channels [8]. Dialysate sodium concentration at the end of the peritoneal equilibration test (PET) is also of importance because it can be used to assess transport characteristics across the peritoneal membrane [9].
The sodium concentration of biological fluids is typically measured using flame photometry (F), or ion-selective electrode (ISE) methods. The ISE methods include both direct (D-ISE) and indirect (I-ISE) methodologies [1013]. In normal plasma from healthy subjects, the sodium concentrations are the same on average when measured by F, D-ISE or I-ISE. However, in the plasma of non-healthy subjects, such as uraemic patients, the results obtained by F, D-ISE and I-ISE may diverge [13]. In PD, sodium concentration has been assessed using all three methods [6,7,1416] but, unlike in the setting of haemodialysis [17,18], the method-related differences in measurement have not yet been completely clarified. Furthermore, the peritoneal dialysate has properties distinct from thos of the plasma, and sodium concentrations measured with the three methods can be different in this fluid.
In plasma, F measures total sodium concentration (i.e. the ionic concentration plus the amount complexed with anions) after the aspiration of a known plasma volume. However, nearly 6% of plasma consists of colloidal proteins and lipids, whose physical structure occupies space that is not penetrated by water, and so the sodium concentration measured by F is in 0.94 l of plasma water. For example, a plasma sodium concentration of 140 mmol/l measured by F (NaFp) corresponds to a total sodium concentration in plasma water of 149 mmol/l (NaFpw), a value that is obtained by correcting the observed concentration for the volume of plasma water (i.e. 0.94). Indeed, any time we add protein or lipid to a saline solution, the sodium concentration measured by F decreases in proportion to the amount of protein or lipid added [12]. A certain amount of NaFpw (nearly 7 mmol/l) is complexed with anions (in particular, proteins and bicarbonate) [17]. The ionic sodium concentration in plasma water (Na+pw) can be directly measured by D-ISE, which detects the chemical activity of sodium ions in a solution, but not the volume in which they are dissolved. The sodium activity is then converted into sodium concentration using the activity coefficient of the solution [18,19]. It follows that an Na+pw of 142 mmol/l detected by D-ISE corresponds to an NaFp of 140 mmol/l and an NaFpw of 149 mmol/l.
Given that peritoneal dialysate is almost free of proteins and lipids, it can be assumed that F measures its total sodium concentration (NaFD), whereas D-ISE only measures its sodium activity, i.e. its ionized sodium concentration (Na+D) [16,17]. However, the pH and the concentration of other ions (carbonate, bicarbonate, etc.) of the peritoneal dialysate can change the activity coefficient of the solution [18,19].
I-ISE, a method of sodium measurement that is used increasingly in clinical laboratories, is an auto-analyser that aspirates a known plasma volume, dilutes it with a buffered solution (to bring the activity coefficient near to 1.0) and presents the diluted sample to a sodium electrode for activity measurement. These indirect readings are calibrated against reference values determined by F [18].
The aim of this study was to evaluate the differences in the three methods of measurement on NaR and D/PNa in PD patients.
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Subjects and methods |
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We used a modified PET, which differed from the classical method [20] insofar as the PD solution contained a 3.86% instead of 2.27% concentration of anhydrous glucose. Lactate (35 mEq/l) was used as the buffer, with a nominal pH of 5.06.5 and a nominal sodium concentration of 132134 mmol/l.
The overnight dwell before PET was performed using a PD solution containing a glucose concentration of 1.36%, with lactate as the buffer; the overnight dialysate was instilled in the evening before the test (11.00 p.m.) and drained at 7 a.m.
Blood samples were taken at the start of the test (P0), after 60 min (P60) and at the end of the test (P240). The overnight dialysate (DNight) samples were taken from the bag; the fresh PD fluid (D0) samples were taken from the bag at the end of the infusion. After the complete infusion of the PD solution, and after having flushed back 30 ml of dialysate, 20 ml dialysate samples were taken after 1, 60, 120, 180 and 240 min (D1, D60, D120, D180 and D240).
The patients were instructed to sit up or move about in bed before the collection of each dialysate sample; otherwise, they remained recumbent throughout the 4-h investigation.
After 240 min, the dialysate was gravity collected for at least 20 min. The volumes of the infused fresh peritoneal dialysis solution and the drained dialysate were measured by weighing the bag and then subtracting the weight of the empty bag; no corrections were made for the differences in the specific weight of the solutions.
Sodium measurements
Plasma and dialysate sodium concentrations were analysed using an IL 943 flame photometer (Instrumentation Laboratory, Milan, Italy), a Stat Profile M direct ion-selective electrode (Nova Biomedical Corp., Waltham, MA) and a Hitachi 717 indirect ion-selective electrode (Hitachi Ltd, Tokyo, Japan). Each method was calibrated and performed according to the manufacturer's specifications via internal (ISO 9001) and external (Regional Interlaboratory) quality controls. The coefficient of variation was 1% for IL 943, 0.5% for Stat Profile M and 1% for Hitachi 717. The accuracy, i.e. the difference of agreement with the target values of an internal quality control, was 0.15% for IL 943, 0.06% for Stat Profile M and 0.48% for Hitachi 717.
The pH of the peritoneal dialysis fluid and dialysate was measured by means of the Stat Profile M.
NaR was calculated as follows:
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where V is the volume of the peritoneal dialysis solution expressed in litres, and DNa the dialysate sodium concentration expressed in mmol/l.
D/PNa was calculated at the start (D1/PNa0), after 60 min (D60/PNa60) and at the end of the test (D240/PNa240).
Statistical analysis
The data were expressed as mean values±1 SD, together with their 95% confidence intervals (95% CIs). Analysis of variance (ANOVA) was used to compare the three approaches with the estimation of NaR according to the method of sodium measurement, with the NaR calculated according to the F method being considered the reference. If the total F-test was statistically significant, two specific contrasts were tested: NaR measured by F vs NaR measured by D-ISE, and NaR measured by F vs NaR measured by I-ISE. Repeated measures ANOVA was used to evaluate the differences between the three measurements of sodium in the plasma and dialysate samples, their ratio, the effect of the time course of PET on the same variables, and the effect of their interactions. The F method of sodium measurement was considered the reference for both the plasma and the dialysate samples. Bonferroni's adjustment was used for multiple comparisons at the different times of PET, which were expressed as mean differences and 95% CIs. Correlations between the different methods of sodium measurement were estimated using Pearson correlation coefficients. BlandAltman plots were used to assess agreement between the different methods of sodium measurement visually. This statistical method compares two measurement techniques by plotting the absolute differences against the averages of the two techniques.
A P-value of 0.05 was considered significant. All of the statistical analyses were made using SPSS for Windows statistical software (release 11.0).
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Results |
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Figure 1 shows the mean plasma sodium concentrations measured by F, D-ISE and I-ISE. The mean plasma sodium concentrations measured by D-ISE were significantly higher than those measured by F and I-ISE (P<0.001), which were similar.
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The correlation coefficient between D1/PNa0 measured by F and D-ISE was 0.767 and the mean difference was 0.023±0.015 (95% CI 0.0180.028). The correlation coefficient between D1/PNa0 measured by F and I-ISE was 0.743 and the mean difference was 0.002±0.015 (95% CI 0.007 to 0.003). The correlation coefficient between D60/PNa60 measured by F and D-ISE was 0.905 and the mean difference was 0.033±0.013 (95% CI 0.0290.037). The correlation coefficient between D60/PNa60 measured by F and I-ISE was 0.861 and the mean difference was 0.001±0.016 (95% CI 0.004 to 0.006). The correlation coefficient between D240/PNa240 measured by F and D-ISE was 0.954 and the mean difference was 0.045±0.011 (95% CI 0.0410.048). The correlation coefficient between D240/PNa240 measured by F and I-ISE was 0.883 and the mean difference was 0.003±0.019 (95% CI 0.009 to 0.002). Figure 4 shows the BlandAltman plots for D/PNa measured by F, D-ISE and I-ISE. Similarly to Figure 3, the visual inspection of the plots underlines the bias when the D/PNa was measured by D-ISE: D/PNa is clearly underestimated when measured with D-ISE (on the y-axis; the solid line representing the absolute difference between F and D-ISE is far from zero).
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Discussion |
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Given the importance of the degree of sodium removal on patient outcome, and the relevance of the sodium gradient across the peritoneal membrane, sodium concentrations must be assessed rigorously. In contrast to normal healthy subjects, the plasma of patients with end-stage renal disease who receive PD contains a number of abnormal substances (known and unknown uraemic toxins). As already demonstrated in the setting of haemodialysis [1719], this can give different values of sodium concentrations compared with normal healthy subjects when different methods of sodium measurement are used. Besides, the dialysate displays properties different from those of the plasma (lower pH, higher glucose concentration, very low protein concentration) and these properties change throughout the dwell. These characteristics of the dialysate are to be kept in mind when evaluating the data of PET according to the method of sodium measurement used.
In PD, the sodium concentrations have been measured traditionally using F, D-ISE and I-ISE; the differences in the values obtained have not yet been clearly defined [3,4,6,7,1416]. We previously demonstrated that NaR and D/PNa were different when measured by F and an old D-ISE method [21], but most laboratories now use I-ISE to analyse sodium concentrations because it is easy and simple to use. We therefore compared I-ISE with F and D-ISE, in order to verify the impact of the differences in sodium concentration measurement on NaR and D/PNa.
The results of this study confirm that, in PD patients, NaR and D/PNa vary significantly depending on the method used to measure sodium concentrations. The plasma sodium concentrations measured by D-ISE were higher than those measured by F or I-ISE, which is in accord with the results of previous studies [17,18]. As D-ISE directly measures sodium activity (i.e. the amount of ionized sodium), the obtained concentration is the amount of diffusible sodium. F measures the total amount of sodium (i.e. ionized sodium plus the sodium complexed with anions), but does not take into account the fact that nearly 6% of plasma is occupied by proteins. I-ISE measures sodium activity but, because it aspirates a known plasma volume and dilutes it with a buffered solution (to bring the activity coefficient near to 1.0), the sodium concentration values obtained were similar to those obtained by means of F (we found similar plasma sodium concentrations as measured by F and I-ISE at all of the time points).
Unlike plasma, dialysate is almost free of proteins, and so F measures the total sodium concentration, whereas D-ISE only measures its sodium activity, i.e. its ionized sodium concentration. This explains why D-ISE showed lower dialysate sodium concentrations than F at all of the PET time points, whereas those measured by F and I-ISE were similar. I-ISE only measures ionized sodium but, because it dilutes the aspirate with a buffered solution, to bring the activity coefficient near to 1.0, the sodium concentrations in the peritoneal dialysate were similar to those obtained by means of F.
The difference in the sodium concentrations in peritoneal dialysate measured by F and D-ISE is minimal in a fresh dialysis solution that contains lactate as a buffer and has an acidic pH, but progressively increases during the dwell in the peritoneal cavity and is maximal at the end of the night dwell, which lasts nearly 8 h. At the end of this dwell, the difference is similar to that observed in the dialysate used for haemodialysis [17,18] (Figure 2). This is explained by the fact that the pH of the dialysate progressively increases during the dwell (Figure 5), thus favouring the binding of sodium with other anions (mainly bicarbonate). The variation of the pH of peritoneal dialysate during the PET did not influence the measurement of the sodium concentration by means of I-ISE.
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We found that the D/PNa measured by F and I-ISE was not statistically different, whereas that measured by D-ISE was lower. This can be explained by the differences in the sodium concentration measurements provided by the three methods. The sodium concentrations measured by D-ISE are higher in plasma and lower in dialysate (after a few minutes of PET) than those measured by F and I-ISE, and the difference in dialysate sodium concentrations progressively increases with longer times of dialysate permanence in the peritoneal cavity.
In comparison with F, I-ISE is being used increasingly in clinical laboratories because it allows the immediate measurement of various electrolytes (sodium, potassium, calcium, etc.), does not require a gas supply and because of its ease of handling and simple daily maintenance. Most of the studies of PD have assessed NaR and D/PNa using I-ISE [7,14,15], but no previous study compared the results either with I-ISE or with F. We did not find any differences in NaR and D/PNa when sodium concentrations were measured by F or I-ISE. This finding has important practical implications: for clinical purposes, I-ISE can replace F (which is unwieldy and available in only a few laboratories) in evaluating sodium concentrations, D/PNa and NaR.
In conclusion, the method of sodium measurement significantly influences the obtained values of NaR and D/PNa, with D-ISE differing significantly from F and I-ISE. A clear understanding of the differences in sodium concentrations in uraemic plasma of PD patients and in the dialysate according to the method of measurement is very important when comparing results of different studies. Furthermore, these differences are not only to be kept in mind when building complex mathematical models of the hydraulic permeability of the peritoneal membrane in the experimental setting, but they should also be considered in everyday clinical practice, in order to assess and understand NaR and D/PNa correctly. These two indexes give us important information on transport characteristics through the peritoneal membrane. This has become of particular interest since it has been supposed that a high transport status allows less removal of sodium and water and this can be an independent risk factor for mortality, and causes decreased technique survival [22].
Conflict of interest statement. None declared.
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References |
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