Using a precise technique for measuring authentic plasma lipid hydroperoxides (ROOHs), we show that individuals with non-insulin-dependent diabetes mellitus (NIDDM) have higher levels of ROOH than do control subjects. ROOHs were measured by the ferrous oxidation with xylenol orange assay coupled with the selective ROOH reductant triphenylphosphine. Formation of the ferric xylenol orange complex was determined at 560 nm and calibrated against H2O2. For 22 individuals with NIDDM, a concentration of 9.04 +/- 4.3 mumol/l (mean +/- SD) ROOH was recorded. This concentration was higher (P < 0.0005 by separate-variance t test) than that of plasma ROOHs from control subjects (3.76 +/- 2.48 mumol/l). There was no difference between concentrations of plasma malondialdehyde measured as thiobarbituric acid-reactive material (TBARM) in NIDDM or control subjects (1.00 +/- 0.70 vs. 1.21 +/- 0.62 mumol/l, respectively; P > 0.1). A trend to lower vitamin E levels in the NIDDM group (9.03 +/- 3.31 vs. 10.31 +/- 5.02 micrograms/ml in control subjects) failed to achieve significance at the 95% confidence level. Plasma ROOHs in the diabetic group did not correlate with total plasma cholesterol, triglyceride, fasting glucose, HbA1, vitamin E, or TBARM levels. These data indicate that measurement of authentic ROOHs shows NIDDM to be associated with oxidative stress, which may be unrelated to abnormalities in lipid metabolism and glycemic control.
High-density lipoprotein (HDL) has been proposed as the principal carrier of hydroperoxides in plasma, based upon data gathered with an HPLC-chemiluminescence technique. To test this hypothesis we have measured total lipid hydroperoxides in native plasma using the ferrous oxidation in Xylenol Orange (FOX) assay and then fractionated plasma into very-low-density lipoprotein (LDL) and HDL fractions. Hydroperoxides were found to accumulate principally (more than 65%) in LDL, as judged by hydroperoxide content per amount of protein or cholesterol, or expressed as a proportion of total hydroperoxide in plasma. Plasma was also incubated at 37 degrees C in the presence and absence of 2,2'-azo-bis-(2-amidinopropane) hydrochloride (AAPH), an azo-initiator of lipid peroxidation. The majority of hydroperoxides generated in plasma were recovered in the LDL fraction. Furthermore, when isolated lipoproteins were subject to oxidation initiated by AAPH, very-low-density lipoprotein and LDL showed the greatest propensity for hydroperoxide accumulation, whereas HDL seemed relatively resistant. Estimates for plasma and LDL peroxidation based upon techniques which measure total lipid hydroperoxides suggest that levels of hydroperoxides in plasma and LDL are far higher than that those estimates generated by ostensibly more selective techniques. Higher levels of hydroperoxides in LDL than those reported by HPLC-chemiluminescence also seem in greater accordance with other available data concerning LDL oxidation.
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