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Canrenone is a selective competitive inhibitor of the aldosterone receptor and a diuretic drug commonly used in the treatment of cirrhotic patients both with and without ascites.1 The aim of our observation was to determine if canrenone cross reacts with aldosterone in an immunoradiosorbent assay kit used for the hormonal scoring system according to the Child-Pugh classification for liver cirrhosis.2 Canrenone, for in vitro experiments, was a gift from GiEnne Pharma (Group Therabel) Diagnostics. Canrenone, potassium canrenoate, and spironolactone are often used with digoxin in clinical practice and can cause falsely low readings in common assays for digoxin (that is, AxSym MEIA-Abbott) due to negative cross reactivity, and falsely elevated serum digoxin concentrations with the fluorescence polarisation immunoassay for digoxin.
Human hepatic stellate cells, isolated from wedge sections of normal human liver unsuitable for transplantation, were separated, after digestion with collagenase/pronase, from other liver non-parenchymal cells by ultracentrifugation over gradients of stractan. Cells were cultured on plastic culture dishes in Iscove’s modified Dulbecco’s medium, supplemented as described elsewhere.3 Cells were plated (1×105 cells in well dishes) and incubated with increasing concentrations of canrenone or with no drug (SFIF or control conditions), after a 24 hour incubation period in serum free insulin free (SFIF) medium. After 24 hours, medium was removed and then lyophilised in a dry vacuum. Pellets were then resuspended in a total volume of 1 ml and aliquots of 200 μl were processed for aldosterone assay (Radim kit, Italy: KS17CT, RIA method). The results showed increasing linearity (0.007 (0.003); 0.018 (0.004); 0.027 (0.003) nmol/l (mean (SD)); n = 3). As values obtained in SFIF samples were below the lower limit of the assay (0.009 (0.001)), it is conceivable that these cells do not produce aldosterone under basal conditions.4
To further validate this observation, we then spiked sera with increasing concentrations of canrenone (10, 50, 100 μM) and, subsequently, aldosterone concentrations were determined. Sera were collected from patients with different degrees of liver disease (from acute hepatitis (n = 1), to non-alcoholic steatohepatitis (n = 1), or chronic active hepatitis with (n = 3) or without (n = 6) cirrhosis). Aldosterone concentrations were as follows: 0.32 (0.18), 0.63 (0.22), 0.85 (0.26), and 1.07 (0.35) nmol/l (n = 11). Comparing these concentrations with the increasing concentrations of canrenone spiked into sera, a statistically significance was found (from r = 0.874 to r = 0.988; p<0.001).
To date, only positive interferences leading to falsely high digoxin readings, including those due to spironolactone and canrenone, have been reported.5 Negative interference is much more dangerous. Toxic concentrations may remain undetected. Less severe negative interferences or interferences from clinically less significant cross reactants have been reported. Assay manufacturers should assess potential cross reactivity in the presence of the primary ligand. This difficulty may apply to immunoassays and cross reactants other than digoxin and canrenone. Pathologists and clinicians should be aware of negative interference so that intoxication due to drug dosing guided by monitoring of its concentration in serum can be avoided. For positive interference of a low molecular weight substance, such as in our observation, false positive test results for hormonal and electrolyte disturbances during liver cirrhosis can be avoided.
These in vitro results can be considered effective in measuring aldosterone concentrations in plasma under in vivo conditions using a more cautious approach, as cross reactivity could hardly affect biological determinations.
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