No differences in pH, pCO2, and HCO3− values were observed between extracorporeal blood obtained at baseline and that obtained during equilibration phases. Nor were such differences observed in the ultrafiltrate. This served as proof of reversal of the effects of acids infusion during the equilibration phases (see Table 1, Supplemental Digital Content, http://links.lww.com/ASAIO/A65).
During citric acid infusion, a significant reduction of ionized calcium concentration (iCa++) was observed in the extracorporeal blood (Figure 5). The iCa++ was significantly reduced (p < 0.001) in post-Acid, post-ML, and outlet samples as well as in ultrafiltrate samples compared with the inlet blood (1.071 ± 0.144 to 0.549 ± 0.139, 0.496 ± 0.095, and 0.511 ± 0.098 mMol/L, respectively). Moreover, iCa++ was lower in ultrafiltrate than in post-acid samples (p < 0.05). In contrast, iCa++ concentration of samples collected from the extracorporeal circuit did not vary after infusion of acetic or lactic acid (p = 0.40 and p = 0.09, respectively; see Table 2, Supplemental Digital Content, http://links.lww.com/ASAIO/A65).
Arterial blood gas analyses and physiologic variables are presented in Table 2. Physiologic variables (to include temperature, HR, mean arterial pressure, and mean pulmonary artery pressure) were not altered significantly during any of the acid infusions. No statistically significant difference was observed between systemic arterial pH at baseline and during any of the acid infusions. Arterial pCO2 during citric acid infusion was lower than at baseline (p < 0.05). There was no statistically significant difference between arterial HCO3− at baseline and during acid infusion.
No difference was observed between arterial pH, pCO2, or HCO3− obtained at baseline and during equilibration phases (see Table S3, Supplemental Digital Content, http://links.lww.com/ASAIO/A65). Moreover, there was no statistically significant difference in RR and VCO2NL during acid infusion. Changes in MV were not statistically significant after post-hoc analysis.
No difference in ACT between experimental phases was detected; ACT was 224 ± 12, 218 ± 36, and 219 ± 17 during acetic, citric, and lactic acids infusions, respectively.
We compared the effects of regional extracorporeal acidification with three different metabolizable acids during ECCO2R. Equivalent infusions of acetic, citric, and lactic acids upstream from the ML of an ECCO2R device resulted in similar levels of enhancement of VCO2ML and ML CO2 removal efficiency of approximately 35%. However, we did not observe changes in MV or in systemic arterial blood gas values in these spontaneously breathing sheep.
In previous studies,6,7,11 regional acidification led to a significant increase in CO2 removal with an extracorporeal BF as low as 250 ml/min. Likewise, in the current study, BF of 250 ml/min was used. This should be compared with other studies, not employing acidification, in which BF of 470 ml/min was used to produce a VCO2ML of 70 ml/min.3 With regional acidification as proposed here, ECCO2R may be achieved with smaller sized extracorporeal cannulas (e.g., 13–14 Fr, percutaneously introduced, double-lumen catheters). Consequently, ECCO2R may develop into a procedure with the same footprint and invasiveness of continuous renal replacement therapy (CRRT) making it widely available to larger cohorts of patients. One of the questions that remains to be answered is the choice of optimal acid for regional acidification. In previously published studies,6,7 lactic acid was used. In this study, we evaluated other metabolizable acids as well.
In this study, we demonstrated that equivalent infusions of acetic, citric, and lactic acids are comparably effective in raising VCO2ML. This was subsequent to their similar effects on extracorporeal blood. Indeed, the different acids caused equivalent degrees of acidification; for all the acids, the drop of post-acid extracorporeal blood pH was consistently around 0.25 points. Likewise, reductions in BE and increments in pCO2 (i.e., 15–20 mm Hg) did not differ between the three treatments. Passage of blood throughout the ML affected gas analyses similarly during the different experimental phases. Interestingly, we observed the absolute pH values of all the extracorporeal blood samples to be slightly higher during acetic acid infusion, when compared with citric and lactic acids infusion. Arguably, because sheep metabolize acetate more efficiently than citrate or lactate,12 the pH of venous blood (i.e., inlet blood) may be less impacted during acetic acid infusion than during citric or lactic acid infusions.
We have shown that the enhancement in VCO2ML is dependent on blood acidification, regardless of the compound used, and therefore, identification of the best compound to be utilized requires consideration of their potential risks and benefits (see Table 3).
Lactic acid is an indispensable substrate for the intermediary metabolism of mammalian cells.13 It is nontoxic,14 and its concentration is readily measurable by point-of-care testing devices. Lactic acid metabolism is rapid and well studied in humans.15 These reasons make lactic acid particularly suited for extracorporeal blood acidification in humans. When infused, lactic acid is readily cleared from the bloodstream, undergoing gluconeogenesis or direct oxidation to ATP and CO2.16 In a healthy swine model, lactic acid caused a slight increase in total CO2 production compared with an equicaloric glucose infusion.9 If completely oxidized, compared with acetic and citric acids, lactic acid has a more favorable caloric profile. Indeed, dosages of lactic acid similar to the one used in this experiment (1.5 mEq/min) would provide a significant fraction of a patient’s caloric requirement (up to 700 kcal/day), limiting the necessary caloric input and CO2 production originating from other sources. Consequently, if using lactic acid for regional acidification, a thorough evaluation of energy balance is necessary.
Despite being widely used as a constituent of dialysis replacement fluids and balanced solutions, little is known about metabolism of acetate in humans. Acetic acid does not constitute a common caloric input in humans.17 When infused in high doses (up to 2.5 mmol/min), it is readily oxidized by the liver18 as well as by muscle tissues to CO2 and water,19 supplying as much as 65% of the necessary caloric requirements of an adult.20 Part of the infused acetate is not oxidized and is used for lipid synthesis, leading to thermogenesis.21 This thermal effect is associated with augmented CO2 production and has been demonstrated to be greater than the thermal effect of lactate infusion.22 Efficacy of regional acidification with acetic acid may be influenced by this thermal effect. The possible toxicity associated with long-term acetate infusion23 warrants more in-depth studies.
Citric acid has various interesting characteristics. While acetic and lactic acids are monocarboxylic acids, citric acid is a tricarboxylic one. Therefore, a completely dissociated mole of citric acid provides three equivalents of H+. The finding that pH reduction during infusion of 0.5 mmol/min (i.e., 1.5 mEq/min) of citric acid was similar to that during infusion of 1.5 mmol/min of acetic and lactic acids is suggestive of the complete dissociation of all three citric acid carboxylic moieties. Consequently, the same level of acidification obtained with 1 mole of acetic or lactic acid is attainable with one-third mole of citric acid. Metabolism of citric acid leads to less CO2 production and provides a lower caloric input than the other two acids. In humans, the clearance of citric acid from bloodstream is slow24 and strongly dependent on hepatic function.25 Its use in patients with altered liver function could be prohibitive because of toxicity.26
At the same time, citric acid is a calcium chelator and, as such, has anticoagulant properties. Indeed, citric acid salts (e.g., sodium citrate) are commonly used during CRRT as a locoregional anticoagulant. We infused citric acid at a rate of 0.5 mEq/min, which is well within the range used for locoregional anticoagulation.27 Previously, citrate infusion was tested during ECCO2R. Unfortunately, measurements of iCa++ concentration in the extracorporeal circuitry were not reported.28 In this study, we documented significant iCa++ chelation in blood samples after citric acid infusion. Indeed, iCa++ blood levels were reduced to approximately 0.5 mmol/L, which is at the upper limit for optimal anticoagulation (i.e., 0.25–0.5 mmol/L).29 In light of this consistent reduction in extracorporeal blood levels of iCa++, we speculate that citric acid could be used to achieve regional acidification and anticoagulation. This may enable low-flow ECCO2R without systemic anticoagulation. Techniques such as thromboelastography would be necessary to confirm the effectiveness of citric acid in anticoagulating blood during ECCO2R.30 This goes beyond the scope of this study and further experiments are necessary to confirm this hypothesis.
Infusion of acids may have deleterious effects on blood components. In our work, an innovative technique was utilized to provide regional acidification. A hemofilter was positioned after the ML, and ultrafiltrate was generated with a peristaltic pump. This ultrafiltrate was acidified and then recirculated before the ML. The novel circuit configuration used in this study allowed highly concentrated hyperosmolar acids to be injected into the recirculating ultrafiltrate. This ensured safe extracorporeal blood acidification by avoiding damage of blood components and unnecessary free water infusion. Direct injection of concentrated acids (4.4 M = 8,800 mOsm/L, pH 1) into the blood would have caused hemolysis.31 On the other hand, a high volume of free water (i.e., 600 ml/hour) would have been necessary to infuse isotonic acids (i.e., 300 mOsm/L), causing severe electrolyte derangements. By contrast, our innovative setup allowed for safe infusion of concentrated acids without excessive free water infusion. Such circuit setup was previously tested by us and proved not to alter CO2 removal capabilities of the ML.10 With that experiment, we excluded the possibility of bicarbonate recycling by means of ultrafiltrate recirculation. Notably, introduction of a hemofilter into an extracorporeal circuit may increase the hemostatic and inflammatory responses because of the blood contact with artificial surfaces. Nevertheless, in this circuit setup, hemodilution of the blood subsequent to ultrafiltrate recirculation may mitigate these effects.32 The hemofilter enables renal replacement therapy along with lung support. With this extracorporeal circuit setup, we envision the advent of modular multiorgan support technology (MOST).10
During acid infusion, no major systemic complications were documented. No hemodynamic alterations were observed. No residual effect of the acids on arterial blood gas analyses was observed after the equilibration period, suggesting complete washout of the acids.
This is the first documented experience of the application of regional acidification in spontaneously breathing animals. Despite providing significant enhancements of VCO2ML, none of the acids resulted in a statistically significant reduction in MV, RR, or VCO2NL. Similarly, no reduction in systemic arterial pCO2 was observed. Previously, animals of another species (swine) were paralyzed and sedated while arterial pCO2 was kept constant throughout the experimental phases by changing ventilation settings.6,7
The current study was neither powered nor designed to detect effect of acids infusion on ventilation; however, to explain these findings, several possibilities exist. First, in this experimental setting, the caloric input subsequent to the infusion of the acids may have increased metabolic expenditure and CO2 production when compared with the baseline phase when animals had no caloric input and were fasting for 12 hours. Therefore, the enhanced VCO2ML and expected benefits on ventilation may have been masked by augmented metabolic rate and CO2 production. Use of special techniques (e.g., isotopic carbon-labeled glucose or lactate and direct calorimetry) that go beyond the objectives of this study would be necessary to investigate the metabolic fate of infused acids. Second, infused acids, although metabolized, may cause systemic metabolic acidosis, which may trigger a compensatory respiratory alkalosis, as shown by a reduction in pCO2 during citric acid infusion. Third, ventilation of spontaneously breathing animals may have been influenced by factors other than CO2 clearance (e.g., physical activity). Finally, the absolute augmentation in VCO2ML (+15 ml/min) may have been too small, given their VCO2tot was about 165 ml/min. The absolute VCO2ML during both the baseline and the acidification phases was limited by the low venous content of CO2 of the healthy sheep, rather than by poor device efficiency. Arguably, in a clinical setting, VCO2ML would be higher, and the effects on ventilation would be more pronounced. For instance, hypercapnic patients suffering from chronic obstructive pulmonary disease (COPD) usually have venous HCO3− and pCO2 much higher than sheep (i.e., 35 mEq/L and 60 mm Hg, respectively).33,34 In this scenario, standard low-flow ECCO2R devices are capable of VCO2ML as high as 80 ml/min with a BF of 350 ml/min.35 Accordingly, we speculate that the rise in efficiency of the ML (+35%) obtained by extracorporeal blood acidification may lead to a VCO2ML of 110 ml/min.
Further limitations of our study are subsequent to the low number of animals used, which restricts the strength of the results, especially with regards to evaluation of respiratory effects of acid infusion. Extrapolation of these results to humans should be done with caution, given that the systemic effect of acid infusion is influenced by the differences in these acids metabolism between ruminants (i.e., sheep) and non-ruminants (e.g., humans).12,17,36–38 Healthy animals were utilized for this experiment. The impact of extracorporeal blood acidification on injured subjects with respiratory or metabolic acidosis must be evaluated as well. The feasibility of extracorporeal acidification at higher blood flow ranges and the effects of recirculation of ultrafiltrate on coagulation need to be further studied. Finally, additional in-depth studies are warranted to assess the effectiveness of citric acid anticoagulation during ECCO2R support.
In conclusion, using extracorporeal infusion of acetic, citric, and lactic acids, we demonstrated comparable significant enhancement of carbon dioxide removal by a ML of 35%. Citric acid also provided consistent calcium chelation, suggesting the possibility of achieving regional anticoagulation as well as acidification. Future studies will be necessary to further evaluate the effects of regional acidification on ventilatory status in spontaneously breathing subjects.
1. Terragni PP, Del Sorbo L, Mascia L, et al. Tidal volume lower than 6 ml/kg enhances lung protection: role of extracorporeal carbon dioxide removal. Anesthesiology. 2009;111:826–835
2. Pesenti A, Pelizzola A, Mascheroni D, et al. Low frequency positive pressure ventilation with extracorporeal CO2 removal (LEPPV-ECCO2R) in acute respiratory failure (ARF): technique. Trans Am Soc Artif Intern Organs. 1981;27:263–266
3. Batchinsky AI, Jordan BS, Regn D, et al. Respiratory dialysis: reduction in dependence on mechanical ventilation by venovenous extracorporeal CO2 removal. Crit Care Med. 2011;39:1382–1387
4. Karagiannidis C, Kampe KA, Sipmann FS, et al. Veno-venous extracorporeal CO2 removal for the treatment of severe respiratory acidosis: pathophysiological and technical considerations. Crit Care. 2014;18:R124
5. Kolobow T, Gattinoni L, Tomlinson T, White D, Pierce J, Iapichino G. The carbon dioxide membrane lung (CDML): a new concept. Trans Am Soc Artif Intern Organs. 1977;23:17–21
6. Zanella A, Patroniti N, Isgrò S, et al. Blood acidification enhances carbon dioxide removal of membrane lung: an experimental study. Intensive Care Med. 2009;35:1484–1487
7. Zanella A, Mangili P, Redaelli S, et al. Regional blood acidification enhances extracorporeal carbon dioxide removal: a 48-hour animal study. Anesthesiology. 2014;120:416–424
8. Zanella A, Mangili P, Giani M, et al. Extracorporeal carbon dioxide removal through ventilation of acidified dialysate: an experimental study. J Heart Lung Transplant. 2014;33:536–541
9. Zanella A, Giani M, Redaelli S, et al. Infusion of 2.5 mEq/min of lactic acid minimally increases CO2 production compared to an isocaloric glucose infusion in healthy anesthetized, mechanically ventilated pigs. Crit Care. 2013;11:6
10. Scaravilli V, Kreyer S, Linden K, et al. Modular extracorporeal life support: effects of ultrafiltrate recirculation on the performance of an extracorporeal carbon dioxide removal device. ASAIO J. 2014;60:335–341
11. Cressoni M, Zanella A, Epp M, et al. Decreasing pulmonary ventilation through bicarbonate ultrafiltration: an experimental study. Crit Care Med. 2009;37:2612–2618
12. Sabine JR, Johnson BC. Acetate metabolism in the ruminant. J Biol Chem. 1964;239:89–93
13. Gladden LB. A lactatic perspective on metabolism. Med Sci Sports Exerc. 2008;40:477–485
14. Leverve XM. Lactate in the intensive care unit: pyromaniac, sentinel or fireman? Crit Care. 2005;9:622–623
15. Gladden LB. Lactate metabolism: a new paradigm for the third millennium. J Physiol. 2004;558(Pt 1):5–30
16. Jenssen T, Nurjhan N, Consoli A, Gerich JE. Failure of substrate-induced gluconeogenesis to increase overall glucose appearance in normal humans. Demonstration of hepatic autoregulation without a change in plasma glucose concentration. J Clin Invest. 1990;86:489–497
17. Ballard FJ. Supply and utilization of acetate in mammals. Am J Clin Nutr. 1972;25:773–779
18. Pouteau E, Piloquet H, Maugeais P, et al. Kinetic aspects of acetate metabolism in healthy humans using [1-13C] acetate. Am J Physiol. 1996;271(1 Pt 1):E58–E64
19. Mittendorfer B, Sidossis LS, Walser E, Chinkes DL, Wolfe RR. Regional acetate kinetics and oxidation in human volunteers. Am J Physiol. 1998;274(6 Pt 1):E978–E983
20. Skutches CL, Sigler MH, Teehan BP, Cooper JH, Reichard GA. Contribution of dialysate acetate to energy metabolism: metabolic implications. Kidney Int. 1983;23:57–63
21. Burnier P, Tappy L, Jéquier E, Schneeberger D, Chioléro R. Metabolic and respiratory effects of infused sodium acetate in healthy human subjects. Am J Physiol. 1992;263(6 Pt 2):R1271–R1276
22. Chioléro R, Mavrocordatos P, Burnier P, et al. Effects of infused sodium acetate, sodium lactate, and sodium beta-hydroxybutyrate on energy expenditure and substrate oxidation rates in lean humans. Am J Clin Nutr. 1993;58:608–613
23. Veech RL. The toxic impact of parenteral solutions on the metabolism of cells: a hypothesis for physiological parenteral therapy. Am J Clin Nutr. 1986;44:519–551
24. Bauer E, Derfler K, Joukhadar C, Druml W. Citrate kinetics in patients receiving long-term hemodialysis therapy. Am J Kidney Dis. 2005;46:903–907
25. Kramer L, Bauer E, Joukhadar C, et al. Citrate pharmacokinetics and metabolism in cirrhotic and noncirrhotic critically ill patients. Crit Care Med. 2003;31:2450–2455
26. Apsner R, Schwarzenhofer M, Derfler K, Zauner C, Ratheiser K, Kranz A. Impairment of citrate metabolism in acute hepatic failure. Wien Klin Wochenschr. 1997;109:123–127
27. Lanckohr C, Hahnenkamp K, Boschin M. Continuous renal replacement therapy with regional citrate anticoagulation: do we really know the details? Curr Opin Anaesthesiol. 2013;26:428–437
28. Cardenas VJ Jr, Miller L, Lynch JE, Anderson MJ, Zwischenberger JB. Percutaneous venovenous CO2 removal with regional anticoagulation in an ovine model. ASAIO J. 2006;52:467–470
29. Oudemans-van Straaten HM, Ostermann M. Bench-to-bedside review: citrate for continuous renal replacement therapy, from science to practice. Crit Care. 2012;16:249
30. Alexander DC, Butt WW, Best JD, Donath SM, Monagle PT, Shekerdemian LS. Correlation of thromboelastography with standard tests of anticoagulation in paediatric patients receiving extracorporeal life support. Thromb Res. 2010;125:387–392
31. Snider MT, Chaudhari SN, Richard RB, Whitcomb DR, Russell GB. Augmentation of CO2 transfer in membrane lungs by the infusion of a metabolizable organic acid. ASAIO Trans. 1987;33:345–351
32. Ogawa S, Ohnishi T, Hosokawa K, Szlam F, Chen EP, Tanaka K a. Haemodilution-induced changes in coagulation and effects of haemostatic components under flow conditions. Br J Anaesth. 2013;111:1013–1023
33. Kelly AM, Kerr D, Middleton P. Validation of venous pCO2 to screen for arterial hypercarbia in patients with chronic obstructive airways disease. J Emerg Med. 2005;28:377–379
34. Razi E, Moosavi GA. Comparison of arterial and venous blood gases analysis in patients with exacerbation of chronic obstructive pulmonary disease. Saudi Med J. 2007;28:862–865
35. Burki NK, Mani RK, Herth FJ, et al. A novel extracorporeal CO(2) removal system: results of a pilot study of hypercapnic respiratory failure in patients with COPD. Chest. 2013;143:678–686
36. Hanson RW, Ballard FJ. The relative significance of acetate and glucose as precursors for lipid synthesis in liver and adipose tissue from ruminants. Biochem J. 1967;105:529–536
37. Muramatsu M, Ambo K, Tsuda T. ATP citrate lyase activity in the liver of newborn lambs. J Biochem. 1970;67:727–729
38. Langer T, Ferrari M, Zazzeron L, Gattinoni L, Caironi P:. Effects of intravenous solutions on acid-base equilibrium: from crystalloids to colloids and blood components. Anaesthesiol Intensive Ter. 2014;46(5):350–360
extracorporeal circulation; carbon dioxide; lactic acid; citric acid; acetic acid
Supplemental Digital Content
Copyright © 2015 by the American Society for Artificial Internal Organs