Bufodienolides in pig plasma and urine

Abstract

Water and electrolyte balance is regulated in large part by Na/K ATPase. Inhibition of this enzyme leads to cellular depolarization, a rise in extracellular K and intracellular Na, cellular swelling, and has been associated with shock, hypertension, bipolar disorder, schizophrenia, depression and diabetes. Recently, marinobufogenin, a bufodienolide, has been identified in human and rat plasma and is a strong inhibitor of Na/K ATPase. To determine what bufodienolides are in pig, 1000ml of pooled pig plasma and 500ml of urine was extracted using 1× volume ethyl acetate/ 4× volume acetonitrile, dried, de‐conjugated with glucoronidase/sulfatase, and extracted by affinity chromatography (Digibind®, GlaxoSmithKline). Extract was run on Reverse Phase High Pressure Liquid Chromatography (Beckman Coulter). Chromatographic peaks were identified as marinobufogenin, bufalin, resibufogenin and cinobufagen by 1) co‐elution of standards, 2) UV spectrum and 3) mass spectroscopy of collected fractions. These data show that bufodienolides, potent inhibitors of Na/K ATPase, exist in plasma and urine of pig. Supported by the ARMY MRMC.