Measurement of endogenous Na+,K+‐ATPase inhibitors in human plasma and urine using high‐performance liquid chromatography

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Abstract
This study was undertaken to assess endogenous Na+,K+‐ATPase inhibitors in both plasma and urine in the same subjects. Samples were chromatographed on reverse‐phase HPLC using an acetonitrile gradient and the eluent screened using Na+,K+‐ATPase inhibition and cross‐reaction with anti‐digoxin antibodies. The donors were divided into inhibiting and non‐inhibiting subjects using a previously described method, plasma action on ouabain binding and on Na+,K+‐ATPase activity. Three Na+,K+‐ATPase inhibitors (IP, 2P and 3P) were detectable in plasma; the antibodies cross‐reaction of the peaks 2P and 3P were larger than that of peak IP. The peaks 2P and 3P were significantly higher in inhibiting subjects as compared to non‐inhibiting subjects. The 24‐h urine is resolved into two peaks inhibiting Na+,K+‐ATPase activity (1U and 2U). Peak 2U cross‐reacted with anti‐digoxin antibodies to a greater extent than peak 1U and is significantly larger in inhibiting subjects in terms of Na+,K+‐ATPase inhibition. These data support the heterogeneity of human Na+,K+‐ATPase inhibitor in both plasma and urine.