N-acetylcysteine: a novel approach to methaemoglobinaemia in normothermic liver machine perfusion – Scientific Reports

This is the first series reporting the protective impact of NAC on the accumulation of methaemoglobin in extended NMP of donor livers. Severe methaemoglobinaemia is a progressive complication of extended NMP. This has previously been reported in shorter clinical perfusions, most notably in suboptimal donors, and is thought to be a universal complication during NMP¹¹. We encountered this problem during our development of a 5-days NMP model, where initial perfusions demonstrated universal accumulation of methaemoglobin beyond 48-h perfusions, with subsequent sharp rise in levels.

MetHb build up in our series was also accompanied with a progressive increase in carboxyhaemoglobin leading to a gradual decline in the oxyhaemoglobin (O₂Hb) and the impairment of essential oxygen delivery. Certainly, if left untreated, this gradual decline in oxyhaemoglobin would eventually lead to tissue hypoxia, graft injury, and potential failure. Livers 1–4 were stopped due to features of progressive organ failure (severe lactic acidosis, increasing vascular resistance, and parenchymal changes), whereas Livers 5–9 were stopped following meeting our perfusion time targets (initially 120 h, extended to 144 h and 168 h). The reasons for liver failure during machine perfusion has yet been unexplored and multifactorial with methaemoglobinaemia and poor oxygen delivery playing a role.

Interestingly, NAC was a component of the perfusion protocol in Tingle et al.¹¹ report of severe methaemoglobinaemia, and did not prevent its development. Presumably, the authors used a single bolus of acetylcysteine into the perfusate reservoir when commencing the perfusion. NAC has three main mechanisms of action: as a free radical scavenger; a precursor for glutathione biosynthesis; and a reducer of disulfide bonds¹⁴. A combination of both NAC’s anti-oxidant properties and replenishment of glutathione is hypothesised to subdue the MetHb accumulation seen.

It has been hypothesised that NAC does not require conversion to glutathione to exert its effects on MetHb¹⁹. This is not supported entirely by in-vitro studies, with evidence both for and against its effectiveness^20,21. The results in our study are mixed, with our in-vitro experiment highlighting the need for viable hepatocytes to facilitate the anti-oxidant benefit of NAC. We observed that even in the presence of NAC there was production of MetHb in line with packed red cells without treatment. When replicating our in-vitro study with glutathione (3 mg in 30 ml of research grade O^– packed red cells), we did not see significant difference in MetHb accumulation between no-glutathione and glutathione groups (data not included). Although unclear and undefined at present, the mechanism behind NAC’s effectiveness in suppressing MetHb accumulation is not solely due to its conversion to glutathione and requires the presence of healthy hepatocytes. Methaemoglobinaemia does not appear to be a relevant problem in the initial 24 h of NMP and we hypothesise that NAC supports an intrinsic protective pathway present within healthy hepatocytes.

NAC undergoes extensive first pass metabolism in the liver and kidneys leading to low levels in the circulating plasma^22,23. This supports our hypothesis that a continuous infusion rather than one-off bolus may be the preferred way to supplement NAC during extended NMP, even in the absence of continuous veno-venous haemofiltration. It has a low volume and there should be no risk of excessive dilution of the circuit.

This study has some limitations to consider when interpreting the results. One shortcoming is the evolution of the perfusion protocol alongside our learning curve. Following the landmark report by the Zurich group we added continuous veno-venous haemofiltration and methylprednisolone to our protocol (Supporting Table 1). This appeared to have an important role in increasing the success of the extended NMPs and to some extent this overlaps with the addition of NAC to our perfusion protocol.

Given the multiple constraints, including the access to discarded donor livers, financial resources, time, and labour intensity, it is not feasible in the real-world to perform a study to demonstrate the benefits of each specific component of the perfusion fluid. Other potentially usable anti-oxidant additives might be vitamin C, vitamin K, lipoic acid, and glutathione, having the ability to neutralise oxygen free radicals, however NAC has the additional benefit of cysteine replenishment allowing the manufacture of glutathione²⁴.

Although outside the scope of this manuscript, the benefits of NAC are likely to exceed suppression of methaemoglobin accumulation alone. This safe, cheap, and efficient drug has previously been used in some fluids developed for cold organ preservation to reduce ischaemia reperfusion injury. A major component of this process is the production of reactive oxygen species that aggravates tissue damage²⁵. NAC is likely to attenuate the pro-oxidant environment and damage cascade initiated during liver reperfusion, which might be of further benefit whilst using the NMP in extended criteria donors.