Abstract
Background and objectives Adsorption of uremic solutes to activated carbon provides a potential means to limit dialysate volumes required for new dialysis systems. The ability of activated carbon to take up uremic solutes has, however, not been adequately assessed.
Design, setting, participants, & measurements Graded volumes of waste dialysate collected from clinical hemodialysis treatments were passed through activated carbon blocks. Metabolomic analysis assessed the adsorption by activated carbon of a wide range of uremic solutes. Additional experiments tested the ability of the activated carbon to increase the clearance of selected solutes at low dialysate flow rates.
Results Activated carbon initially adsorbed the majority, but not all, of 264 uremic solutes examined. Solute adsorption fell, however, as increasing volumes of dialysate were processed. Moreover, activated carbon added some uremic solutes to the dialysate, including methylguanidine. Activated carbon was particularly effective in adsorbing uremic solutes that bind to plasma proteins. In vitro dialysis experiments showed that introduction of activated carbon into the dialysate stream increased the clearance of the protein-bound solutes indoxyl sulfate and p-cresol sulfate by 77%±12% (mean±SD) and 73%±12%, respectively, at a dialysate flow rate of 200 ml/min, but had a much lesser effect on the clearance of the unbound solute phenylacetylglutamine.
Conclusions Activated carbon adsorbs many but not all uremic solutes. Introduction of activated carbon into the dialysate stream increased the clearance of those solutes that it does adsorb.
Introduction
There is increasing interest in the development of home and portable dialysis systems. Such systems generally use a limited amount of dialysate. Their requirement for dialysate can be reduced by using a sorbent to remove solutes from spent dialysate. The sorbent most commonly considered for this purpose has been activated carbon (1,2). Historically, activated carbon was used in a home dialysis system that recirculated a 6-L batch of dialysate (3). Currently, processing of spent dialysate by activated carbon is being considered to reduce the dialysate requirement for both hemodialysis and peritoneal dialysis. There has been, however, remarkably little study of the ability of activated carbon to remove different uremic solutes. Therefore, we used untargeted metabolomics to assess the ability of activated carbon to remove a wide range of uremic solutes from spent dialysate. We were particularly interested in the ability of activated carbon to adsorb uremic solutes that bind to plasma proteins. Additional studies, therefore, tested the ability of activated carbon to increase the clearances of such solutes with dialysate flows similar to those often used for home hemodialysis treatment.
Materials and Methods
The Uptake of Uremic Solutes by Activated Carbon Block
Spent dialysate was collected from patients maintained on hemodialysis at the Veterans Affairs Palo Alto Health Care System. Separate batches of spent dialysate were collected on nine occasions from two or three patients, as further detailed in Supplemental Table 1. Nine experiments were performed; in each experiment, spent dialysate from a different batch was passed through a carbon block containing approximately 200 g of activated carbon during four periods, as summarized in Table 1. The spent dialysate was sampled for chemical analysis at the beginning of each experiment and after passage through the block at the end of each period. Metabolomic analysis was performed by Metabolon, Inc., using a liquid chromatography/mass spectrometry platform to identify solutes and estimate their relative concentrations (4,5). Metabolites were identified by comparing masses, retention times, and fragmentation patterns with a chemical reference library including >4000 chemically confirmed metabolites (4⇓–6). The fractional removal of each solute was calculated as
where [Pre] and [Post] are the respective solute concentrations before and after passage through the block. Solute concentrations were estimated as proportional to metabolomic peak areas. Samples of spent dialysate diluted 1:5 and 1:20 were included in the analysis, and the lowest peak area identified among all of the samples for each solute was imputed when no peak was detected in an individual sample. The significance of solute removal was assessed by calculating false discovery rates using the Benjamini–Hochberg procedure from P values obtained using the paired t test to compare [Pre] and [Post] concentrations. A false discovery rate or “q value” of <0.05 was considered significant. Urea, indoxyl sulfate (IS), p-cresol sulfate (PCS), phenylacetyl glutamine (PAG), and methylguanidine (MG) concentrations were also measured using chemical standards, as previously described (7,8).
Passage of spent dialysate through activated carbon block
In Vitro Dialysis
In vitro dialysis experiments tested the ability of activated carbon block to increase solute clearances. Artificial plasma containing 4 g/dl albumin (12659-M; Sigma-Aldrich) was stirred in a 3.5-L reservoir and urea, PCS, IS, and PAG were added continuously to maintain concentrations of approximately 180, 2.4, 2.4, and 5 mg/dl, respectively. These PCS, IS, and PAG concentrations are similar to those we have observed in patients maintained on hemodialysis, whereas the urea concentration is higher (7). PCS and IS were chosen for study as the protein-bound uremic solutes that have been the most extensively investigated in patients on dialysis (9). PAG was chosen for comparison as an organic acid that, like PCS and IS, is cleared by secretion in the native kidney and accumulates in patients on dialysis but is not protein bound. Dialysis was performed using the circuit depicted in Figure 1, with a plasma flow of 240 ml/min and standard dialysate (Supplemental Table 2) running countercurrent to the plasma. Solute clearances were measured with and without the carbon block at the midpoint of the dialysate stream at dialysate flow rates of 200 and 600 ml/min in each of six experiments. The free fractions of PCS, IS, and PAG in the artificial plasma were calculated as the ratio of the concentrations measured in the plasma ultrafiltrate to the concentrations measured in the plasma (8). Solute clearances were calculated as
when the carbon block was not in the circuit, and
when the carbon block was in the circuit, where [P] is the solute concentration in the plasma and [DEND], [DMID], and [DCB] are, respectively, the solute concentrations in the dialysate at the end of the circuit, the midstream of the circuit, and after passage through the carbon block.
The in vitro dialysis circuit showing where the dialysate could be directed to pass through the carbon block or to bypass it. Artificial plasma was passed through two dialyzers in series, with dialysate flowing in the countercurrent direction. A valve indicated by the asterisk allowed the dialysate either to be passed through a carbon block between the two dialyzers or to flow directly from the first to the second dialyzer. [DEND], [DMID], and [DCB] indicate the points at which solute concentrations were measured in the dialysate. [DCB], solute concentration in the dialysate after passage through the carbon block; [DEND], solute concentration in the dialysate at the end of the circuit; [DMID], solute concentration in the dialysate at the midstream of the circuit.
The effect of the carbon block on solute clearances at each flow rate was analyzed using the paired t test. The effects of the two different flow rates on solute clearances with and without the carbon block were also analyzed using the paired t test. Using the Bonferroni correction, we considered P values <0.01 to indicate significance for each of the four comparisons we made. The effect of the carbon block on solute clearances was also mathematically modeled.
Experiments were done in accord with the Declaration of Helsinki; acquisition of waste dialysate did not require consent.
Results
The Uptake of Uremic Solutes by Carbon Block
Metabolomic analysis identified 458 solutes in the spent dialysate, as listed in Supplemental Table 3. Of these, 264 were classified as uremic on the basis of the finding of elevated plasma levels in a prior study using the same metabolomic platform (10). The plasma-free fraction for each of these uremic solutes was calculated as the average of metabolomic peak areas in the plasma ultrafiltrate relative to the plasma in 36 patients on maintenance hemodialysis included in that prior study. At the end of experimental period 2, spent dialysate was being pumped through the block at the rate of 300 ml/min and approximately 21 L had passed through. At this point, 216 of the 264 uremic solutes were taken up by the block, as depicted in Figure 2 and summarized in Supplemental Table 4. Passage through the block did not significantly affect concentrations of 45 other uremic solutes, whereas for three uremic solutes, the concentrations in dialysate exiting the carbon block were significantly higher than in the dialysate entering the block. The carbon block was notably effective in removing protein-bound solutes, as further depicted in Figure 2. The block took up a mean±SD of 93%±6% of 35 of 36 solutes with a plasma free fraction of <0.3 and had no significant effect on only one.
The extent to which uremic solutes were adsorbed from spent dialysate by carbon block. [Post]/[Pre] values are the ratios of solute concentrations after passage through the block ([Post]) to concentrations before passage through the block ([Pre]) at a flow rate of 300 ml/min and after passage of 21 L. The red triangles represent solutes for which the [Post]/[Pre] ratio was different from 1.0 with a q value of <0.05, whereas the filled blue circles represent solutes for which the q value for the difference in the [Post]/[Pre] ratio from 1.0 was >0.05. The left panel depicts data for all 264 uremic solutes and the right panel for 36 protein-bound uremic solutes with a free faction <0.30. Names of solutes and numeric values for their individual [Post]/[Pre] ratios are provided in Supplemental Table 3. carboxyethyl-GABA, carboxyethyl γ-aminobutyric acid.
The carbon block’s adsorptive capacity declined as increasing amounts of spent dialysate were processed. Among the 216 uremic solutes that exhibited significant uptake at the end of period 2, the block no longer had an effect on 62 and released 16 at the end of period 4, as shown in Supplemental Figure 1 and summarized in Supplemental Table 4. MG was the solute on which the effect of the block changed most after passage of an increased amount of dialysate. The MG concentration in dialysate exiting the block increased from an average of only 12%±14% of the concentration entering the block during period 2 to an average of more than three-fold greater than the concentration entering the block during period 4. Fifteen other uremic solutes were initially taken up but later released from the block, as listed in Table 2. Of note, the change in a solute’s adsorption after passage of an increased volume was widely variable and not readily predictable from its initial fractional adsorption, as shown in Supplemental Figure 1. The uptake of protein-bound solutes was better maintained, on average, but still often reduced. The block now accomplished significant uptake, averaging 78%±14% for 32 of the 36 solutes with a free fraction <0.3 and had no effect on four. Samples collected at the end of the first and third periods revealed that increasing the dialysate flow rate reduced the extent to which uremic solutes were taken up, as depicted in Supplemental Figures 2 and 3 and detailed in Supplemental Table 4.
Solutes released from activated carbon block after passage of 189 L of spent dialysate
A particularly notable finding of the metabolomic analysis was that the carbon block released some solutes into the spent dialysate. At the end of the fourth experimental period, when close to 190 L of spent dialysate had been processed, the carbon block released 20 solutes not classified as uremic and 18 uremic solutes, as summarized in Table 2. The majority of the solutes released into the dialysate after the processing of a large amount of spent dialysate had been taken up when a lesser amount of dialysate had been processed. There were, however, six solutes for which levels were significantly increased by passage through the block at both intervals, and several others for which levels were numerically higher after passage through the block at both intervals. The solute that appeared to be continually added to the dialysate in the greatest proportion was maleate. The carbon block also released several N-acetylated amino acids. The results obtained by metabolomic analysis for selected solutes were confirmed by assays using chemical standards, as shown in Supplemental Table 5. In particular, these measurements confirmed that MG was initially taken up and then released after a large quantity of spent dialysate had passed through the carbon block, as shown in Table 3.
The effect of activated carbon block on methylguanidine concentrations in spent dialysate
In Vitro Dialysis
The ability of activated carbon block to increase solute clearances during in vitro dialysis is summarized in Table 4. The introduction of the carbon block in the midpoint of the dialysate stream did not affect the clearance of urea but did increase the clearances of the organic anions PCS, IS, and PAG at a dialysate flow rate of 200 ml/min. The clearances for tightly bound solutes PCS and IS were increased by >70%, whereas the clearance of the largely unbound solute PAG was increased by only 30%. The introduction of the carbon block had a lesser effect at the higher dialysate flow rate of 600 ml/min. The clearances for tightly bound solutes PCS and IS were increased by only 35% and 30%, respectively, whereas the clearance of PAG was not increased.
Total solute clearances during in vitro dialysis
Accumulation of solutes in the dialysate accounted for the effects of the carbon block on solute clearances, as depicted in Figure 3. At a dialysate flow of 200 ml/min, the carbon block only slightly reduced the urea concentration in the dialysate and did not increase total urea removal. The block had a much larger effect on the protein-bound solutes PCS and IS. Without the block, the dialysate concentration approached the plasma-free concentration by the midpoint of the circuit so that little solute was removed in the second half of the circuit. In contrast, passage through the block reduced the concentration in the dialysate to zero. Additional solute was then removed as the dialysate concentration again approached the free concentration in the plasma at the end of the circuit. As was the case with PCS and IS, passage through the block reduced the concentration of PAG in the dialysate to zero. However, because a larger portion of the PAG in the plasma was removed without the carbon block, its removal could not be increased as much by regenerating the dialysate at the midpoint of the circuit.
The accumulation of solutes in the dialysate stream during in vitro dialysis with a dialysate flow of 200 ml/min. The lines depict the ratios ([D]/[Pf]) of solute concentrations in the dialysate to their free concentrations in the plasma at the dialysate inlet (0), midstream (0.5), and outlet (1.0) of the dialysate stream. The red lines depict [D]/[Pf] values with the carbon block in the circuit and the blue lines depict [D]/[Pf] values without the carbon block. Passage of the dialysate through the carbon block at the midpoint of the circuit (gray band) reduced the dialysate concentrations of PCS, IS, and PAG back to zero, while having little effect on the concentration of urea. The solute clearances are proportional to the sum of the successive increases in the solutes’ [D]/[Pf] ratios as dialysate flows through the two dialyzers. [D], solute concentrations in the dialysate; IS, indoxyl sulfate; PAG, phenylacetyl glutamine; PCS, p-cresol sulfate; [Pf] free solute concentrations in the plasma.
The carbon block was less effective at increasing solute removal at a dialysate flow of 600 ml/min, as depicted in Supplemental Figure 4. Introduction of the carbon block again reduced the dialysate concentrations of PCS, IS, and PAG effectively to zero. Solute removal was, however, greater without the carbon block at a dialysate flow of 600 ml/min, so that introduction of the block caused a proportionally lesser increase in the removal of PCS and IS and no increase in the removal of PAG. These effects were in close accord with the predictions of mathematic modeling, as described under Supplemental Figure 5.
Discussion
Calls to improve dialysis treatment have prompted renewed interest in the use of sorbents to remove uremic waste solutes. By reducing the amount of dialysate required, sorbents can make it easier to provide dialysis at home or on the road. Their use is essential in designs for wearable dialysis devices using fixed volumes of recirculating dialysate. The sorbent most often considered for these purposes is activated carbon, which is inexpensive and adsorbs a wide range of organic compounds (11). Activated carbon is incorporated in several published designs for new dialysis systems and in additional designs described in commercial presentations (12⇓⇓⇓–16).
The first aim of this study was to determine how well activated carbon adsorbs uremic solutes. Despite current interest in its use, this question has received remarkably little attention. The history of activated carbon’s use for home dialysis may have suggested that its ability to adsorb uremic solutes was well established. Activated carbon was originally used in the Redyx home hemodialysis system, which used a single 6-L batch of recirculating dialysate (2,3,17). The dialysate was regenerated by passage through a multielement cartridge in which granular activated carbon was said to remove solutes, including guanidines, indoles, organic acids, and phenols (18). We have, however, been unable to find documentation of the extent to which the Redyx cartridge removed these solutes. Patients maintained on the Redyx system were said to do well (3). It is not clear, however, if patients who were anuric were maintained on the system for long periods. Limited testing of the Redyx system may reflect its introduction at a time before medical devices were extensively regulated. The Redyx cartridge was redesigned for incorporation into the Allient portable dialysis system in the early 2000s, and the redesigned cartridge effectively adsorbed PCS in clinically relevant quantities (19,20). Commercial development of this system was, however, abandoned and its adsorbent capacity was not more widely tested.
Results of our metabolomic analysis suggest that further testing will be required if activated carbon is to be used again clinically. We found that activated carbon adsorbed many, but by no means all, of the organic uremic solutes identified using a large metabolomic platform. It was particularly effective in adsorbing uremic solutes that bind to plasma proteins. However, adsorption of these and other solutes declined irregularly as an increasing volume of dialysate passed through the carbon. A potentially greater problem revealed by our analysis was that activated carbon can release organic solutes into the dialysate. Reviews of sorbent dialysis have generally suggested that activated carbon is chemically inert and releases nothing. However, two prior studies demonstrated that activated carbon converts creatinine to MG, which was once considered an important uremic toxin (21⇓–23). We found significant release of a broader array of solutes after passage of spent dialysate over activated carbon in the form of a carbon block. The mechanism responsible for the release of these solutes is not clear. In most cases, they were initially adsorbed by the block and then released after passage of a larger volume of dialysate. This may reflect release of some compounds as other, more avidly bound compounds accumulate on the carbon. If so, use of a larger amount of carbon could limit the release of these solutes. However, the extent to which this is true remains to be determined. Activated carbon may also facilitate conversion of specific solutes to other solutes, as has been shown for the conversion of creatinine to MG (21,22). A few solutes were released from the carbon block throughout our experiments. We know little about the toxicity of these solutes and so cannot tell if their release would be a barrier to the clinical use of activated carbon.
The second aim of this study was to test whether activated carbon could increase the clearances of protein-bound uremic solutes in systems that use low dialysate flows. The desire to make hemodialysis available at home has motivated the development of several such systems (24⇓–26). All of these systems provide adequate treatment, as assessed by the clearance of urea. Dialysate flows that provide adequate urea clearances may, however, provide limited clearances of uremic solutes that bind to plasma proteins. Solute concentrations in the dialysate can rise only to the level of the free concentration in the plasma, so that the clearance for a bound solute is only a portion of the clearance for an unbound solute of the same size (27,28). The hemodialytic clearance of these solutes is thus highly dependent on the dialysate flow rate (27,29).
Our in vitro experiments showed that passing dialysate through a carbon block at the midpoint of a dialysis circuit greatly increased the clearance of protein-bound solutes at a dialysate flow of 200 ml/min. The carbon block increased the clearances of bound solutes without changing the clearance of urea. In effect, the carbon block partially regenerated the dialysate so that each dialyzer was supplied with fresh dialysate for the removal of bound solutes. The observed effects of regenerating dialysate with a carbon block closely matched those predicted by mathematic modeling. Activated carbon can adsorb unbound solutes and bound solutes. The increase in clearance achieved with a carbon block will, however, rise with the avidity of protein binding, as depicted in Supplemental Figure 5. For a tightly bound solute, dissociation of the solute from the binding proteins keeps the plasma-free concentration high as plasma flows through the dialysis circuit. Dialysate regeneration at the midpoint of the circuit will thus increase the solute’s clearance, as was observed for PCS and IS in our in vitro study. The plasma level of an unbound solute declines more as plasma flows, although the dialysate circuit and dialysate regeneration at the midpoint of the circuit can effect only a lesser increase in an unbound solute’s clearance, as we observed for PAG.
The design for partial regeneration of dialysate explored here bears comparison for other potential means to increase the clearances of protein-bound solutes. One means that has been explored in a clinical trial is to add a substance to the plasma stream that displaces bound solutes from albumin as the plasma passes through the dialyzer (30). It may also be possible to reduce solute binding to albumin by changing plasma pH or ionic strength (31,32). We have described adding powdered activated carbon to the dialysate, and others have explored adding liposomes (33,34). A potential advantage of partial regeneration of dialysate by passage through carbon block is that integration into existing dialysis circuits would be relatively simple. Carbon block technology is inexpensive and allows dense packing of very small activated carbon particles without the release of free particles. Protein-bound solute clearances could also be increased by adding activated carbon to the dialysate path within a single dialyzer (35). Demonstration that introducing a carbon block between standard dialyzers has a clinical benefit could further motivate development of such an advanced dialyzer.
Limitations of our work should be acknowledged. First, we do not know the relative toxicity of different uremic solutes and so do not know which solutes are most important to remove. Second, although the metabolomic platform we used identified hundreds of compounds, it is by no means complete. Finally, although spent dialysate was collected on nine different occasions, it was collected from a limited number of participants. The degree to which individual solutes are taken up by carbon block was, therefore, not precisely determined. Moreover, we may have missed uremic solutes that would have been detected if we had studied a wider range of participants with various diets, colon microbiomes, and other metabolic characteristics. Therefore, we undoubtedly failed to establish the effect of activated carbon on many uremic solutes.
In conclusion, dialysate processing with activated carbon provides a potential means to increase the clearance of uremic solutes in systems employing low dialysate flows. Bound solute clearances could be increased with existing systems by the introduction of an activated carbon block into the dialysate stream. Further testing will, however, be required to determine the amount of activated carbon needed to remove a broad range of uremic solutes through an entire treatment and to monitor release of solutes from activated carbon. Release of solutes from activated carbon could pose a particular barrier to the use of activated carbon in systems that continuously recirculate small volumes of dialysate.
Disclosures
T.W. Meyer reports receiving honoraria from Baxter; having consultancy agreements with Baxter and Daiichi Sankyo; serving on the editorial boards of JASN and Kidney International; receiving research funding from Outset Medical; being employed by the Veterans Administration; and applying for a patent to improve the dialytic removal of uremic solutes that bind to plasma proteins. N.S. Plummer reports being employed by Palo Alto Veterans Institute for Research and Veterans Affairs Medical Center Nephrology. T.L. Sirich reports having consultancy agreements with Baxter. All remaining authors have nothing to disclose.
Funding
S. Lee is supported by an American Society of Nephrology Ben J. Lipps Fellowship. Additional support for this work was provided by a Stanford University Translational and Clinical Award and by National Institute of Diabetes and Digestive and Kidney Diseases awards R01 DK101674 to T.W. Meyer and R01 DK118426 to T.L. Sirich. CB Tech, Inc. provided carbon blocks without cost but no additional funding.
Acknowledgments
The authors thank CB Tech, Inc. and its staff for provision of carbon blocks used in this study and for technical advice.
Author Contributions
I.J. Blanco was responsible for formal analysis; I.J. Blanco and T.L. Sirich provided supervision; S. Lee was responsible for visualization; S. Lee and T.W. Meyer conceptualized the study and wrote the original draft; S. Lee, T.W. Meyer, and N.S. Plummer were responsible for data curation; S. Lee, T.W. Meyer, and T.L. Sirich were responsible for funding acquisition; T.W. Meyer was responsible for investigation; T.L. Sirich reviewed and edited the manuscript and was responsible for validation; and all authors were responsible for methodology.
Data Sharing Statement
All data used in this study are available within this article.
Supplemental Material
This article contains the following supplemental material online at http://cjasn.asnjournals.org/lookup/suppl/doi:10.2215/CJN.01610222/-/DCSupplemental.
Supplemental Table 1. Sources of spent dialysate.
Supplemental Table 2. Composition of dialysate used for in vitro dialysis.
Supplemental Table 3. Solutes detected in spent dialysate by metabolomic analysis.
Supplemental Table 4. Removal of uremic solutes from spent dialysate by activated carbon.
Supplemental Table 5. Comparison of fractional removal values obtained by metabolomic analysis and by assays employing chemical standards.
Supplemental Figure 1. Removal of uremic solutes from spent dialysate by activated carbon after passage of different amounts of dialysate.
Supplemental Figure 2. Removal of uremic solutes from spent dialysate by activated carbon at different flow rates after passage of 18–21 liters of spent dialysate.
Supplemental Figure 3. Removal of uremic solutes from spent dialysate by activated carbon at different flow rates after passage of 186–189 liters of spent dialysate.
Supplemental Figure 4. Dialysate to plasma solute concentration ratios along the in vitro dialysis circuit.
Supplemental Figure 5. Mathematical modeling of the effect of introducing a carbon block at the midpoint of the dialysis circuit.
Footnotes
Published online ahead of print. Publication date available at www.cjasn.org.
See related editorial, “Novel Approaches for the Removal of Uremic Solutes,” on pages 1113–1115.
- Received February 7, 2022.
- Accepted May 30, 2022.
- Copyright © 2022 by the American Society of Nephrology
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