In vitro and in vivo effects of probucol on hydrolysis of asymmetric dimethyl L-arginine and vasospasm in primates.
OBJECTIVE
Increased cerebrospinal fluid (CSF) levels of asymmetric dimethyl L-arginine (ADMA), an endogenous inhibitor of endothelial nitric oxide synthase (eNOS), are associated with delayed vasospasm after subarachnoid hemorrhage (SAH); however, the source, cellular mechanisms, and pharmacological inhibition of ADMA production following SAH are unknown.


METHODS
In an in vitro experiment involving human umbilical vein endothelial cells (HUVECs), the authors examined mechanisms potentially responsible for increased ADMA levels during vasospasm and investigated whether this increase can be inhibited pharmacologically. In a second study, an in vivo experiment, the authors used probucol, which effectively inhibited ADMA increase in HUVEC cultures in vitro, in a randomized double-blind placebo-controlled experiment in a primate model of delayed cerebral vasospasm after SAH. Oxidized low-density lipids (OxLDLs; positive control; p < 0.02) and bilirubin oxidation products (BOXes; p < 0.01), but not oxyhemoglobin (p = 0.74), increased ADMA levels in HUVECs. Probucol inhibited changes in ADMA levels evoked by either OxLDLs (p < 0.001) or BOXes (p < 0.01). Comparable changes were observed in cell lysates. In vivo probucol (100 mg/kg by mouth daily) did not alter serum ADMA levels on Days 7, 14, and 21 after SAH compared with levels before SAH, and these levels were not different from those observed in the placebo group (p = 0.3). Despite achieving therapeutic levels in plasma and measurable levels in CSF, probucol neither prevented increased CSF ADMA levels nor the development of vasospasm after SAH. Increased CSF ADMA and decreased nitrite levels in both groups were strongly associated with the degree of delayed vasospasm after SAH (correlation coefficient [CC] 0.5, 95% confidence interval [CI] 0.19-0.72, p < 0.002 and CC -0.43, 95% CI -0.7 to -0.05, p < 0.03, respectively).


CONCLUSIONS
Bilirubin oxidation products, but not oxyhemoglobin, increased ADMA levels in the HUVEC. Despite its in vitro ability to lower ADMA levels, probucol failed to inhibit increased CSF ADMA and decreased nitrite levels, and it did not prevent delayed vasospasm in a primate SAH model.
Pluta
Ryszard M
RM
Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. rysiek@ninds.nih.gov
Jung
Carla S
CS
Harvey-White
Judith
J
Whitehead
Anne
A
Shilad
Sabrina
S
Espey
Michael G
MG
Oldfield
Edward H
EH
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001120:Arginine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D001663:Bilirubin / Q000378:metabolism*; D018929:Cell Culture Techniques; D004311:Double-Blind Method; D042783:Endothelial Cells; D004791:Enzyme Inhibitors / Q000032:analysis / Q000378:metabolism*; D006868:Hydrolysis; D015227:Lipid Peroxidation; D008252:Macaca fascicularis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010084:Oxidation-Reduction; D010919:Placebos; D011341:Probucol / Q000494:pharmacology*; D011897:Random Allocation; D013345:Subarachnoid Hemorrhage / Q000150:complications / Q000662:veterinary; D013997:Time Factors; D014471:Umbilical Veins; D020301:Vasospasm, Intracranial / Q000503:physiopathology* / Q000662:veterinary
D000975:Antioxidants; D004791:Enzyme Inhibitors; D010919:Placebos; C018524:N,N-dimethylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D011341:Probucol; D001663:Bilirubin