Assay of malondialdehyde and other alkanals in biological fluids by gas chromatography-mass spectrometry.

The method described in this chapter allows the accurate measurement of MDA in diverse biological samples and can be extended to measurements of other alkanals. The use of GC/MS-NCI ensures specificity and sensitivity, and the ability to prepare samples without heating limits oxidation artifact. Although the widely used TBA assay for MDA does not require sophisticated equipment, its results may be of limited value as the assay is hindered by the possibility of cross reactivity and by heat-induced oxidation artifact. This GC-MS technique offers the additional advantages of efficient processing of large numbers of samples and the elimination of recovery errors by inclusion of an internal standard.

DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine.

Oxidative DNA damage is important in aging and the degenerative diseases of aging such as cancer. Estimates commonly rely on measurements of 8-oxo-2'-deoxyguanosine (oxo8dG), an adduct that occurs in DNA and is also excreted in urine after DNA repair. Here we examine difficulties inherent in the analysis of oxo8dG, identify sources of artifacts, and provide solutions to some of the common methodological problems. A frequent criticism has been that phenol in DNA extraction solutions artificially increases the measured level of oxo8dG. We found that phenol extraction of DNA contributes a real but minor increase in the level of oxo8dG when compared, under equivalent conditions, with a successful nonphenol method. A more significant reduction in the baseline level was achieved with a modification of the recently introduced chaotropic NaI method, reducing our estimate of the level of steady-state oxidative adducts by an order of magnitude to 24,000 adducts per cell in young rats and 66,000 adducts per cell in old rats. Of several alternative methods tested, the use of this chaotropic technique of DNA isolation by using NaI produced the lowest and least variable oxo8dG values. In further studies we show that human urinary 8-oxo-guanine (oxo8Gua) excretion is not affected by the administration of allopurinol, suggesting that, unlike some methylated adducts, oxo8Gua is not derived enzymatically from xanthine oxidase. Lastly, we discuss remaining uncertainties inherent both in steady-state oxo8dG measurements and in estimates of endogenous oxidation ("hit rates") based on urinary excretion of oxo8dG and oxo8Gua.

Adrenocortical function in the very low birth weight infant: improved testing sensitivity and association with neonatal outcome.

OBJECTIVE: To evaluate adrenocortical function in ill preterm infants and investigate potential relationships between plasma cortisol concentrations and major neonatal outcomes. STUDY DESIGN: Randomized trial of adrenocorticotropic hormone (1-24ACTH) stimulation testing, followed by a chart review. SETTING: Two level III neonatal intensive care units, Sacramento, Calif. PARTICIPANTS: Sixty-seven very low birth weight infants, born at 32 weeks of gestation or earlier weighing 1500 gm or less, who had endotracheal intubation and indwelling arterial access. RESULTS: Most infants (76%) had baseline cortisol concentrations < 414 nmol/L (15.0 micrograms/dl), and of those, only 36% responded to stimulation with 1-24ACTH, 0.1 microgram/kg. Raising the 1-24ACTH dose to 0.2 microgram/kg resulted in a response rate of 67% (p = 0.09) but decreased the sensitivity of the test. An elevated mean 11-deoxycortisol/cortisol ratio indicated that decreased 11 beta-hydroxylase activity may limit cortisol production in some infants. Infants with baseline cortisol concentrations less than 414 nmol/L (15.0 micrograms/dl) were more likely to have chronic lung disease (p < 0.002) and less likely to have severe intraventricular hemorrhage (p < 0.02). Response to 1-24ACTH was not associated with a detectable difference in outcome. CONCLUSION: Many very low birth weight infants have low cortisol and ACTH concentrations and are unable to mount a cortisol response to physiologic doses (0.1 microgram/kg) of 1-24ACTH. These findings suggest that delayed maturation of adrenal response may result in physiologically inadequate cortisol concentrations in stressed very low birth weight infants. This delayed maturation may contribute to the development of chronic lung disease.

N2-methyl-8-oxoguanine: a tRNA urinary metabolite--role of xanthine oxidase.

DNA damage produces a series of oxidation products including 8-oxo-2'-deoxyguanosine and 8-oxoguanine, whose urinary excretion have been used to estimate in vivo oxidative injury. A monoclonal antibody to 8-oxoguanine was used to measure these adducts in urine taken from ill infants. In the process of this investigation we observed a large chromatographic peak that did not correspond to any of the known 8-oxoguanine adducts. A combination of liquid chromatography with electrochemical detection and gas chromatography/mass spectrometry allowed isolation and identification of the previously undescribed oxidation product, N2-methyl-8-oxoguanine. The excretion of this compound is increased in ill and growing infants but is also found in the urine of adult rats and humans. Experiments with allopurinol, a xanthine oxidase inhibitor, show that in humans, N2-methyl-8-oxoguanine is formed from the tRNA modified base N2-methylguanine. This is in contrast to the nonmethylated bases, which are converted to 8-oxo derivatives as a result of oxidative damage to nucleic acids and do not appear to be substrates for xanthine oxidase.

Assay of malondialdehyde in biological fluids by gas chromatography-mass spectrometry.

Malondialdehyde (MDA) is assayed in femtomole quantities in biological samples by gas chromatography-mass spectrometry (GC-MS). The MDA trapped in protein as a Schiff base is released by H2SO4, the protein precipitated using Na2WO4, and the MDA derivatized with pentafluorophenylhydrazine to form the stable adduct, N-pentafluorophenylpyrazole. Negative chemical ionization (NCI) capability allows the sensitive detection of this MDA adduct in biological samples at a level of 5 nM on-column. A stable-isotope-labeled MDA, [2H2]MDA, was used as an internal standard for quantitation. MDA recovery from plasma was 76%. This assay provides two forms of confirmation of the analyte, retention time and mass ion, thus minimizing error due to interfering compounds. The commonly used thiobarbituric acid assay for MDA overestimates the MDA levels by over 10-fold, possibly resulting from cross-reactivity with other aldehydes and artifactual oxidation due to 100 degrees C temperature conditions. In our assay, all steps were performed at room temperature thereby suppressing artifactual oxidation of the sample. We have successfully applied this assay to biological samples including plasma, tissue homogenates, and sperm.

Toxic hydroperoxides in intravenous lipid emulsions used in preterm infants.

The unsaturated fatty acids that make up a large component of the lipid emulsion Intralipid are highly susceptible to peroxidation, and the products of this reaction could explain the toxicity that has been associated with the administration of some emulsions. Lipid peroxidation produces hydroperoxides, which can alter arachidonic acid metabolism or react to form organic free radicals, which then stimulate a cascade of damage to endogenous lipids. The lipid hydroperoxides and their breakdown products are also mutagens and carcinogens. To determine the degree of lipid peroxidation in Intralipid, we measured the lipid hydroperoxide content of three lots of 20% Intralipid using high-performance liquid chromatography with chemiluminescence detection. The average concentration was 290 +/- 29 mumol/L (SEM) lipid hydroperoxides (n = 15), a large portion of which was made up of trilinoleate derivatives. Measurements made on Intralipid samples collected from the end of the intravenous tubing after a 20-hour infusion cycle were not significantly different from measurements made on newly opened bottles. The lipid hydroperoxide content of some lipid emulsions may represent a clinically significant risk to premature infants, particularly those with preexisting lung disease.

Ascorbic acid protects against endogenous oxidative DNA damage in human sperm.

Damage to the DNA of germ cells can lead to mutation, which may result in birth defects, genetic diseases, and cancer. The very high endogenous rate of oxidative DNA damage and the importance of dietary ascorbic acid (AA) in preventing this damage has prompted an examination of these factors in human sperm DNA. The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine (8-oxo-7,8-dihydro-2'-deoxyguanosine; oxo8dG), 1 of approximately 20 major products of oxidative damage to DNA, was measured in DNA isolated from human sperm provided by healthy subjects and compared to the seminal fluid AA levels. This relationship was studied in two groups. In a group of 24 free-living individuals 20-50 years old high levels of oxo8dG were correlated with low seminal plasma AA. The endogenous level of oxo8dG in this group was 13 fmol per microgram of DNA or approximately 25,000 adducts per sperm cell. The second group of individuals was maintained on a controlled diet that varied only in AA content. When dietary AA was decreased from 250 to 5 mg/day, the seminal fluid AA decreased by half and the level of oxo8dG in sperm DNA increased 91%. Repletion of dietary AA for 28 days (from 5 mg/day to 250 or 60 mg/day) caused a doubling in seminal fluid AA and reduced oxo8dG by 36%. These results indicate that dietary AA protects human sperm from endogenous oxidative DNA damage that could affect sperm quality and increase risk of genetic defects, particularly in populations with low AA such as smokers.