Original Contribution KETOSIS (ACETOACETATE) CAN GENERATE OXYGEN RADICALS AND CAUSE INCREASED LIPID PEROXIDATION AND GROWTH INHIBITION IN HUMAN ENDOTHELIAL CELLS SUSHIL K. JAIN,KRISHNASWAMY KANNAN, and GIDEON LIM Departments of Pediatrics and Medicine, Louisiana State University Medical Center, Shreveport, LA, USA (Received 23 April 1998; Revised 27 May 1998; Accepted 1 June 1998) Abstract—Elevated level of cellular lipid peroxidation can increase the incidence of vascular disease. The mechanism by which ketosis causes accelerated cellular damage and vascular disease in diabetes is not known. This study was undertaken to test the hypothesis that elevated levels of ketone bodies increase lipid peroxidation in endothelial cells. Human umbilical venous endothelial cells (HUVEC) were cultured for 24 h at 37 o C with ketone bodies (acetoacetate, -hydroxybutyrate). Acetoacetate, but not -hydroxybutyrate, caused an increase in lipid peroxidation and growth inhibition in cultured HUVEC. To determine whether ketone bodies generate oxygen radicals, studies using cell-free buffered solution were performed. They showed a significant superoxide dismutase (SOD) inhibitable reduction of cytochrome C by acetoacetate, but not by -hydroxybutyrate, suggesting the generation of superoxide anion radicals by acetoacetate. Additional studies show that Fe 2+ potentiates oxygen radical generation by acetoacetate. Thus, elevated levels of ketone body acetoacetate can generate oxygen radicals and cause lipid peroxidation in endothelial cells, providing a possible mechanism for the increased incidence of vascular disease in diabetes. © 1998 Elsevier Science Inc. Keywords—Lipid peroxidation, Endothelial cells, Ketosis, Diabetes, Free radical INTRODUCTION Endothelial cells play a major role in preserving the architecture and function of blood vessels [1–3]. Differ- ent studies have shown that lipid peroxidation in endo- thelial cells causes increased cell permeability, reduced cell viability, foam cell formation, and adhesion to monocytes [4 –11]. These endothelial cell dysfunctions and chemical modifications of plasma lipoproteins are risk factors in the development of vascular disease [1,11– 15]. It is known that diabetics with frequent episodes of ketosis have an increased incidence of vascular disease, morbidity, and mortality [16]. In severe diabetes, the level of ketone bodies begins to rise in the blood (ke- tonemia) because body fuel is mainly derived from fat [17]. The blood concentration of ketone bodies may reach 10 mM in diabetic patients with severe ketosis compared with concentrations of less than 0.5 mM in normal volunteers [18,19]. The biochemical mechanisms by which ketone bodies cause accelerated cellular dam- age are not known. For example, it is not known whether ketone bodies cause lipid peroxidation damage, which may in turn promotes the vascular disease of diabetes. This study was undertaken to test the hypothesis that ketone bodies can cause lipid peroxidation in human endothelial cells. METHODS Human umbilical venous endothelial cells (HUVEC) were used to study the effects of acetoacetate and -hy- droxybutyrate on endothelial cells [3]. HUVEC and the media were obtained from Clonetics Inc. (San Diego, CA, USA). Cells were at passage 1 o or 2 o and cultured as per the protocol recommended by the supplier. These cells were certified to be free from mycoplasma contam- ination, and tested negative for HIV, HBV, and HCV virus infections or contamination by PCR results. All the experiments reported in this paper were carried out on cells between passages 3 and 7. Cells were maintained in Address correspondence to: Dr. Sushil K. Jain, Department of Pe- diatrics, LSU Medical Center, 1501 Kings Highway, Shreveport, LA 71103, USA; Tel: (318) 675-6086; Fax: (318) 675-6059. Free Radical Biology & Medicine, Vol. 25, No. 9, pp. 1083–1088, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/98/$–see front matter PII S0891-5849(98)00140-3 1083