Evaluation of a urinary multi-parameter biomarker set for oxidative stress in children, adolescents and young adults

The involvement of reactive oxygen species (ROS) and oxidative stress in pediatric diseases is an important concern, but oxidative stress status in healthy young subjects and appropriate methods for its measurement remain unclear. This study evaluated a comprehensive set of urinary biomarkers for oxidative stress in healthy children, adolescents and young adults. Results show that urinary excretion of acrolein-lysine, 8-hydroxy-2'-deoxyguanosine (8-OHdG), nitrite/nitrate and pentosidine were highest in the youngest subjects and decreased to constant levels by early adolescence. Urinary acrolein-lysine, 8-OHdG, nitrite/nitrate and pentosidine showed significant inverse correlations with age, but pyrraline did not change significantly with age. No significant differences in biomarkers were apparent between males and females. Younger subjects grow rapidly and sustain immune activation, and are probably exposed to high concentrations of ROS and nitric oxide. Consequently, they are more vulnerable to oxidation of lipids, proteins, DNA and carbohydrates. Normal reported values in this study are a basis for future studies of disease mechanisms involving oxidative stress and for future trials using antioxidant therapies for oxidative stress-related diseases in the pediatric field.     

Oxidative stress and altered antioxidant defenses in children with acute exacerbation of atopic dermatitis

The underlying mechanisms of skin inflammation in atopic dermatitis (AD) are not completely understood. The purpose of the present study was to examine the involvement of oxidative stress and antioxidant defenses in children with acute exacerbation of AD. We studied 13 children who were hospitalized for acute exacerbation of AD with purulent skin infection by Staphylococcal aureus (age, 1.5 to 10.0 years), and 28 age-matched healthy subjects (controls). Urine samples obtained from the patients on admission, on 2nd and 7th-9th hospital days, as well as from the controls were analyzed for 8-hydroxy-2'-deoxyguanosine (8-OHdG) (a marker of oxidative DNA damage), acrolein-lysine adducts (a marker of lipid peroxidation), bilirubin oxidative metabolites (BOM) (a marker of antioxidant activity of bilirubin under oxidative stress) and nitrite/nitrate (NO(x)(-)) (a marker of endogenous nitric oxide production). Of these, urinary concentrations of 8-OHdG, acrolein-lysine adducts and BOM, but not NO(x)(-), were significantly higher in AD children on admission than those in control subjects. Response to treatment was associated with significant falls in the concentrations of 8-OHdG and acrolein-lysine adducts. Urinary concentrations of acrolein-lysine adducts, but not 8-OHdG, were still significantly higher in AD patients on the 7th-9th hospital day relative to the control. Urinary BOM remained almost constant and significantly high in AD children during hospitalization. Our findings indicate that oxidative stress and altered antioxidant defenses are involved in the pathophysiology of acute exacerbation of AD, and that suppression of oxidative stress might be a potentially useful strategy for the treatment of AD.     

Increased oxidative stress in childhood atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease of unknown etiology. To examine the involvement of impaired homeostasis of oxygen/nitrogen radicals in childhood AD, we compared the levels of urinary 8-hydroxy-2'-deoxyguanosine (marker of oxidative stress), nitrite/nitrate (marker of nitric oxide synthesis) and selenium (marker of selenium store) in 27 children with AD to those of 25 healthy control children. Urinary 8-hydroxy-2'-deoxyguanosine was significantly higher and nitrite/nitrate levels were significantly lower in patients with AD than in the control. Urinary selenium levels were similar in both groups. Our findings suggest that impaired homeostasis of oxygen/nitrogen radicals and increased oxidative stress are involved in the pathophysiology of childhood AD, and indicate that suppression of oxidative stress might be a potentially useful strategy for the treatment of AD.     

Evaluation of a urinary multi-parameter biomarker set for oxidative stress in children,adolescents and young adults

The involvement of reactive oxygen species (ROS) and oxidative stress in pediatric diseases is an important concern, but oxidative stress status in healthy young subjects and appropriate methods for its measurement remain unclear. This study evaluated a comprehensive set of urinary biomarkers for oxidative stress in healthy children, adolescents and young adults. Results show that urinary excretion of acrolein–lysine, 8-hydroxy-2′-deoxyguanosine (8-OHdG), nitrite/nitrate and pentosidine were highest in the youngest subjects and decreased to constant levels by early adolescence. Urinary acrolein–lysine, 8-OHdG, nitrite/nitrate and pentosidine showed significant inverse correlations with age, but pyrraline did not change significantly with age. No significant differences in biomarkers were apparent between males and females. Younger subjects grow rapidly and sustain immune activation, and are probably exposed to high concentrations of ROS and nitric oxide. Consequently, they are more vulnerable to oxidation of lipids, proteins, DNA and carbohydrates. Normal reported values in this study are a basis for future studies of disease mechanisms involving oxidative stress and for future trials using antioxidant therapies for oxidative stress-related diseases in the pediatric field.     

Pyridoxal-aminoguanidine adduct is more effective than aminoguanidine in preventing neuropathy and cataract in diabetic rats.

We examined the ability of a pyridoxal-aminoguanidine adduct with both antiglycation and antioxidant activities in vitro to protect against neuropathy and cataract in streptozotocin-diabetic rats and compared the result with that of aminoguanidine. In vivo antiglycation and antioxidant activities were also compared between the adduct and aminoguanidine. Diabetic rats were given either of the compounds in their drinking water (9 mM) for 7 weeks. Neither compound affected body weight, blood glucose level or urine volume. The adduct, but not aminoguanidine, significantly improved motor nerve conduction velocity. The time to develop cataract was longer in adduct-treated rats than in untreated and aminoguanidine-treated rats. The increase in opacification of lenses in culture medium containing high glucose levels (55.5 mM) was more efficiently attenuated by the adduct than by aminoguanidine. Adduct and aminoguanidine similarly lowered glycated hemoglobin levels. The level of urinary 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, and the level of liver malondialdehyde plus 4-hydroxy-2-alkenals, a marker of tissue lipid peroxidation, both of which were elevated by diabetes, were significantly reduced by the adduct but not by aminoguanidine. These findings indicate that the pyridoxal-aminoguanidine adduct is superior to aminoguanidine in preventing diabetic neuropathy and cataracts, and we suggest that this may be at least partly due to the higher antioxidant activity of the former.     

Biomarkers of oxidative damage in cigarette smokers: Which biomarkers might reflect acute versus chronic oxidative stress?

Cigarette smoking predisposes to the development of multiple diseases involving oxidative damage. We measured a range of oxidative damage biomarkers to understand which differ between smokers and nonsmokers and if the levels of these biomarkers change further during the act of smoking itself. Despite overnight abstinence from smoking, smokers had higher levels of plasma total and esterified F(2)-isoprostanes, hydroxyeicosatetraenoic acid products (HETEs), F(4)-neuroprostanes, 7-ketocholesterol, and 24- and 27-hydroxycholesterol. Levels of urinary F(2)-isoprostanes, HETEs, and 8-hydroxy-2'-deoxyguanosine were also increased compared with age-matched nonsmokers. Several biomarkers (plasma free F(2)-isoprostanes, allantoin, and 7β-hydroxycholesterol and urinary F(2)-isoprostane metabolites) were not elevated. The smokers were then asked to smoke a cigarette; this acute smoking elevated plasma and urinary F(2)-isoprostanes, plasma allantoin, and certain cholesterol oxidation products compared to presmoking levels, but not plasma HETEs or urinary 8-hydroxy-2'-deoxyguanosine. Smokers showed differences in plasma fatty acid composition. Our findings confirm that certain oxidative damage biomarkers are elevated in smokers even after a period of abstinence from smoking, whereas these plus some others are elevated after acute smoking. Thus, different biomarkers do not measure identical aspects of oxidative stress.     

Oxidant and antioxidant activities in childhood meningitis

Animal studies have provided substantial evidence for a key role of reactive oxygen species, nitric oxide and its related compounds in the complex pathophysiology of bacterial meningitis. However, there is little information on changes in the redox status in human meningitis. In the present study, we evaluated the redox status and oxidative stress in the central nervous system of children with meningitis. Oxidant and antioxidant activities were assessed from cerebrospinal fluid levels of acrolein-lysine adducts (a marker of lipid peroxidation), nitrite (a marker of nitric oxide production) and bilirubin derivatives (a marker of antioxidant activity of bilirubin). All these markers were several times higher in children during the early phase of bacterial meningitis compared with those of children without meningitis and patients with aseptic meningitis. In the bacterial meningitis group, the levels of bilirubin derivatives correlated significantly with those of acrolein-lysine adducts and nitrite. Acrolein-lysine adducts and nitrite decreased significantly as the patients started to respond to treatment but bilirubin derivatives remained elevated. In conclusion, our data indicate the enhancement of both oxidant and antioxidant activities in the central nervous system of children with early bacterial meningitis, but not in those with aseptic meningitis. Clinical and laboratory improvement may be associated with a decrease in oxidant activities in the central nervous system.     

Assays for 8-hydroxy-2'-deoxyguanosine: a biomarker of in vivo oxidative DNA damage.

HPLC with electrochemical detection (HPLC-EC) is a highly sensitive and a selective method for detecting 8-hydroxy-2'-deoxyguanosine (oh8dG), a biomarker of oxidative DNA damage that is formed from hydroxyl radical attack of guanine residues in DNA. We propose that the noninvasive measurement of oh8dG in urine can be used to estimate in vivo oxidative damage. Application of this assay to urine samples obtained from rats of different ages and various species provide examples of the utility of this assay. The measurement of steady-state levels of oh8dG in DNA combined with the urinary excretion rates of oh8dG and oh8Gua, offer a powerful approach for estimating oxidative DNA damage and its repair. This method will be useful for studies designed to investigate the relationship of oxidative stress in DNA damage and the role of this damage in aging and cancer.     

Patients with dystrophinopathy show evidence of increased oxidative stress

Duchenne muscular dystrophy (DMD) is associated with an increase in oxidative stress. We measured 24 h 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion in 24 patients with MD (DMD + Becker's MD), 23 with myotonic dystrophy, and 34 healthy controls. The 8-OHdG/creatinine ratio was higher in patients with dystrophinopathy ( upward arrow 48%, p <.01) but not myotonic dystrophy, as compared to healthy controls. These results indicate that 8-OHdG excretion can be used as a marker of oxidative stress in clinical trials with dystrophinopathy.     

DNA repair: insights from urinary lesion analysis

Due to various confounding factors, namely dietary contribution and cell death, measurement of urinary 8-oxo-2'-deoxyguanosine (8-oxodG) has long been considered to be no more than a marker of generalised oxidative stress. Indeed, the action of no single enzyme has been reported to excise 8-oxodG from DNA. However, analysis of recent research has suggested that these confounders may be circumvented, which, combined from work from the authors' laboratory, indicates that urinary 8-oxodG has the potential to become a most important marker of oxidative damage to, and repair of, DNA.     

Effect of docosahexaenoic acid and eicosapentaenoic acid supplementation on oxidative stress levels during pregnancy

Docosahexaenoic acid (DHA) is an indispensable component of cell membranes with high requirements during pregnancy. DHA supplementation is thought to enhance oxidative stress because of increased likelihood of lipid peroxidation. We estimated the oxidative stress levels in two groups of pregnant women who received daily supply of required vitamins with (n = 23) or without (n = 23) 500 mg of DHA and 150 mg of eicosapentaenoic acid (EPA) from 20 weeks of gestation to the time of delivery. Urinary excretions of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage and of malondialdehyde (MDA), a marker of lipid peroxidation, were measured at 20, 30 weeks and at the time of delivery. Urinary MDA excretion remained unchanged throughout the study period in both groups. Urinary 8-OHdG excretion at delivery was significantly higher than at 20 and 30 weeks (p < 0.05), but there were no group differences at the three time points. There were no differences between the two groups in plasma a-tocopherol levels. We conclude that under the conditions studied, a daily supplementation of 500 mg DHA and 150 mg EPA with vitamins to pregnant women did not enhance lipid peroxidation or oxidative DNA damage.     

Quantitative determination of urinary 8-hydroxy-2'-deoxyguanosine level in healthy Japanese volunteers

8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.     

PARP inhibition alleviates diabetes-induced systemic oxidative stress and neural tissue 4-hydroxynonenal adduct accumulation: correlation with peripheral nerve function

This study evaluated the role of poly(ADP-ribose) polymerase (PARP) in systemic oxidative stress and 4-hydoxynonenal adduct accumulation in diabetic peripheral neuropathy. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor, 1,5-isoquinolinediol, 3 mg kg(-1) day(-1), for 10 weeks after an initial 2 weeks. Treatment efficacy was evaluated by poly(ADP-ribosyl)ated protein content in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons and nonneuronal cells (fluorescence immunohistochemistry), as well as by indices of peripheral nerve function. Diabetic rats displayed increased urinary isoprostane and 8-hydroxy-2'-deoxyguanosine excretion (ELISA) and 4-hydroxynonenal adduct accumulation in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord and neurons and glial cells of the dorsal root ganglia (double-label fluorescence immunohistochemistry), as well as motor and sensory nerve conduction velocity deficits, thermal hypoalgesia, and tactile allodynia. PARP inhibition counteracted diabetes-induced systemic oxidative stress and 4-hydroxynonenal adduct accumulation in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons (perikarya, fluorescence immunohistochemistry), which correlated with improvement of large and small nerve fiber function. The findings reveal the important role of PARP activation in systemic oxidative stress and 4-hydroxynonenal adduct accumulation in diabetic peripheral neuropathy.     

Gender differences in steady-state levels of oxidative damage to DNA in healthy individuals

Oxidative damage to DNA has often been used as a biomarker for oxidative stress and more specifically for cancer risk. Indeed, the measurement of oxidative damage to DNA, particularly of 8-hydroxyguanine (8OHG) and 8-hydroxy-2'-deoxyguanosine (8OHdG), has been adopted as a method for establishing the effects of antioxidant supplementation towards protection from certain cancers, cardiovascular and neuro-degenerative diseases, both in patients and healthy individuals. However, reported levels of 8OHdG or 8OHG vary considerably, possibly due to the different methodologies used, and only few data are available for the non-smoking and the female population. In this paper, steady-state levels of oxidative damage to DNA measured in a group of 20 males and 19 females are reported. Significant gender differences in levels of modified DNA bases such as 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine), 8-hydroxyadenine (8OHA) and 5-hydroxycytosine (5OHC), measured by gas chromatography-mass spectrometry (GC/MS), were observed. The results are discussed in relation to the Vitamin C and iron status of the subjects and to the existing, yet limited, literature data. The role of gender in predisposition to oxidative damage to DNA needs to be addressed in future studies.     

Evidence for attenuated cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine removal in cancer patients

Measurement of the products of oxidatively damaged DNA in urine is a frequently used means by which oxidative stress may be assessed non-invasively. We believe that urinary DNA lesions, in addition to being biomarkers of oxidative stress, can potentially provide more specific information, for example, a reflection of repair activity. We used high-performance liquid chromatography prepurification, with gas chromatography-mass spectrometry (LC-GC-MS) and ELISA to the analysis of a number of oxidative [e.g., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-guanine, 5-(hydroxymethyl)uracil], non-oxidative (cyclobutane thymine dimers) and oligomeric DNA products in urine. We analysed spot urine samples from 20 healthy subjects, and 20 age- and sex-matched cancer patients. Mononuclear cell DNA 8-oxodG levels were assessed by LC-EC. The data support our proposal that urinary DNA lesion products are predominantly derived from DNA repair. Furthermore, analysis of DNA and urinary 8-oxodG in cancer patients and controls suggested reduced repair activity towards this lesion marker in these patients.     

Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels using mass spectrometer and urine albumin creatinine ratio as a predictor of development of diabetic nephropathy

Abstract Background. Measurement of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) has recently become more popular as a means of assessing oxidative stress in the human body. The aim of this study is to compare the levels of urine 8-OHdG in patients with type 2 diabetes with and without nephropathy and to evaluate its role as a biochemical marker for distinguishing these patients from healthy and patients without complications. Methods. For this purpose, 52 patients with type 2 diabetes mellitus (32 with nephropathy (DMN), 20 without nephropathy (DM)) and 20 healthy control subjects (C) were included in this study. The urine concentrations of 8-OHdG were measured by modified LC-MS/MS method and compared with the first morning voiding urine albumin/creatinine ratio (UACR) and HbA1c values of the same patients. Results. The concentrations of urine 8-OHdG in DMN and DM patients were higher than those of the control subjects (3.47?±?0.94, 2.92?±?1.73, 2.1?±?0.93 nmol/mol creatinine, respectively). But there was no statistical difference between DMN and DM (p =?0.115). There is significant correlation between urinary 8-OHdG and UACR (r =?0.501, p 相似文献   

Increased DNA Oxidation and Decreased Levels of Repair Products in Alzheimer's Disease Ventricular CSF

Abstract : One of the leading etiologic hypotheses regarding Alzheimer's disease (AD) is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Although several recent studies show an increase in levels of brain DNA oxidation in both aging and AD, there have been no studies of levels of markers of DNA oxidation in ventricular CSF. This is a study of levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), the predominant marker of oxidative DNA damage, in intact DNA and as the "free" repair product that results from repair mechanisms. Free 8-OHdG was isolated from CSF from nine AD and five age-matched control subjects using solidphase extraction columns and measured using gas chromatography/mass spectrometry with selective ion monitoring. Intact DNA was isolated from the same samples and the levels of 8-OHdG determined in the intact structures. Quantification of results was carried out using stable isotope-labeled 8-OHdG. By using this sensitive methodology, statistically significant elevations ( p < 0.05) of 8-OHdG were observed in intact DNA in AD subjects compared with age-matched control subjects. In contrast, levels of free 8-OHdG, removed via repair mechanisms, were depleted significantly in AD samples ( p < 0.05). Our results demonstrate an increase in unrepaired oxygen radical-mediated damage in AD DNA as evidenced by the increased presence of 8-OHdG in intact DNA and decreased concentrations of the free repair product. These data suggest that the brain in AD may be subject to the double insult of increased oxidative stress, as well as deficiencies in repair mechanisms responsible for removal of oxidized bases.     

Urinary excretion of 8-hydroxy-2'-deoxyguanosine measured by high-performance liquid chromatography with electrochemical detection

There is good evidence that oxidative DNA damage permanently occurs in living cells. The oxidative DNA damage product 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of the predominant forms of radical-induced lesions to DNA, and has therefore been widely used as a biomarker for oxidative stress, either in cellular DNA or as DNA repair product in urine. In this paper we describe the use of a high-performance liquid chromatographic procedure with electrochemical detection for the measurement of urinary 8-OHdG. Our study has addressed the questions (i) of baseline urinary levels of 8-OHdG in spot urine and 24-h urine, (ii) of inter- and intra-individual variation of this biomarker, and (iii) of confounding factors for the excretion of 8-OHdG. No significant difference between the mean group levels of 8-OHdG/creatinine in spot urine (2.03+/-1.21 micromol/mol, n=148) and in 24-h urine (1.86+/-1.09 micromol/mol, n=67) was observed. However, when only 24-h urine was used for analysis, 8-OHdG was found to be statistically significantly higher in smokers. By multiple linear regression analysis, urinary creatinine was identified as the only predictor of 8-OHdG/24 h (r(p)=0.33, P=0.007). High intra-individual coefficients of variation of 8-OHdG/24 h were observed in two healthy subjects over a period of 10 consecutive days (37 and 57%, respectively), indicating that the intra-individual fluctuation of urinary 8-OHdG has so far been underestimated. Therefore, we suggest that single values of 8-OHdG should be considered with caution, in particular in small study groups and when spot urine is used.     

Effect of ionizing radiation on the pteridine metabolic pathway and evaluation of its cytotoxicity in exposed hospital staff

Investigations carried out to estimate the effect of long-term occupational exposure to low levels of external ionizing radiation indicated that exposed hospital staff showed an increase in chromosome aberrations. The purpose of this study was to evaluate whether genomic instability or an alteration in pteridine synthesis could be used as a marker of the potential hazard of ionizing radiation in hospital workers. Twenty gamma-radiation- and 33 X-ray-exposed technicians working in radiotherapy and radio-diagnostic units were included in this study, along with 22 healthy matched individuals. Plasma concentrations of nitrite plus nitrate (NO(x)) were measured to estimate reactive nitrogen species. Urinary neopterin, biopterin and creatinine concentrations were measured by high-performance liquid chromatography to determine metabolic activity along the pteridine pathway. Sister chromatid exchange was used as a measure of mutagenicity. Apoptosis was evaluated morphologically and also with a DNA-fragmentation test. The plasma NO(x) levels of both gamma-radiation- and X-ray-exposed technicians were significantly higher than those of the healthy controls (p<0.05). While the urinary biopterin concentrations were significantly higher in radiation-exposed groups compared with the healthy subjects (p<0.05), urinary neopterin concentrations remained unchanged. The apoptosis rates of gamma-radiation- and X-ray-exposed workers were significantly elevated in comparison with those in the control group (both p<0.05). Also, the increase in sister chromatid exchange frequency was significant in each of the radiation-exposed groups (exposed groups versus controls; p<0.05). These results indicate that long-term exposure to low-dose ionizing radiation, even below the permitted levels, could result in increased oxidative stress, which may lead to DNA damage and mutagenicity.     

Spontaneous oxidative DNA damage in bovine retained and nonretained placental membranes

Retention of fetal membranes (RFM) is believed to be associated with conditions of oxidative stress. In this study, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) was used for the determination of spontaneous oxidative DNA lesions in maternal and fetal parts of bovine retained and nonretained placentas. Placental specimens were collected directly after spontaneous delivery or during cesarean section from cows divided into 6 groups: (A) cesarean section before term without RFM, (B) with RFM, (C) cesarean section at term without RFM, (D) with RFM, (E) spontaneous delivery at term without RFM and (F) with RFM. Isolated DNA was hydrolyzed and analyzed by HPLC; native nucleosides were monitored at 254 nm and 8-OH-dG by electrochemical detection. No significant differences in 8-OH-dG levels between retained and nonretained placental tissues were found in all samples from preterm groups (mean concentrations between 13 and 42 micromol/mol deoxyguanosine (dG)). In the term cesarean section group with RFM a significant increase in 8-OH-dG concentration in DNA from maternal (8-fold) and fetal (18-fold) membranes were detected when compared to the respective nonretained tissues. Also, in the term spontaneous delivery groups maternal nonretained placental tissues showed increased levels of 8-OH-dG in comparison to the respective tissues of the retained placenta group. In placental tissues oxidative DNA lesions appear to be controlled by responsive mechanisms which, possibly following exhaustion, give rise to increased 8-OH-dG levels.