Takao Morinobu

Raised concentrations of aldehyde lipid peroxidation products in premature infants with chronic lung disease

AIM To indicate the extent of lipid peroxidation induced by oxidative stress, by measuring aldehyde end products in biological samples. METHODS A highly specific gas chromatography and mass spectrometry (GC/MS) method was used to measure plasma concentrations of aliphatic aldehydes within the first week of life in 13 premature infants who subsequently developed chronic lung disease (CLD) and 11 infants without CLD (non-CLD). The oxime-tert-butyldimethylsilyl derivatives of aldehydes were analysed using 2,2,6,6-d4-cyclohexanone as the internal standard. RESULTS All of the aldehydes measured were raised in those infants with CLD compared with non-CLD infants. Plasma concentrations of heptanal, 2-nonenal, and 4-hydroxynonenal (HNE) were significantly increased in CLD infants on the day of birth, while the differences in all aldehydes between the two groups were not significant at 4–6 days of age. Logistic regression analysis showed that the increase in these three aldehydes within the first 24 hours of life independently showed significant associations with the development of CLD. In particular, an HNE concentration of ⩾ 200 nM on day 0 was the best predictor for the early detection of CLD (odds ratio = 32.0), followed by a 2-nonenal concentration of ⩾ 150 nM (odds ratio = 16.0). CONCLUSIONS These findings suggest that lipid peroxidation may have a role in the pathogenesis of neonatal CLD.

New Evidence for the Involvement of Oxygen Radicals in Triggering Neonatal Chronic Lung Disease

We measured the plasma concentration of allantoin, an oxidation product of uric acid and an “in vivo” marker of free radical generation, within 24-48 h after birth in 10 premature infants who subsequently developed chronic lung disease (CLD) and 9 infants without CLD(non-CLD). The plasma allantoin level (mean ± SD, 25.9 ± 9.8μM for CLD versus 11.0 ± 5.7 μM for non-CLD,p < 0.01) and the allantoin/urate ratio (5.8 ± 2.0% for CLD infants versus 2.4 ± 0.9% for non-CLD infants,p < 0.01) were significantly higher in the CLD group than those in the non-CLD group. These observations suggest the possible involvement of oxygen radicals in triggering CLD. In addition, the plasma allantoin concentration and the allantoin/urate ratio may be useful early predictors of the development of CLD.

Antioxidant Vitamin Levels in Plasma and Low Density Lipoprotein of Obese Girls

To investigate the antioxidant status of obese children, we analyzed beta-carotene and alpha-tocopherol levels in plasma and low density lipoprotein (LDL). We also analyzed the fatty acid composition of LDL as a substrate for oxidative stress. The plasma beta-carotene and alpha-tocopherol levels were relatively lower in obese girls than in normal controls. However, the plasma alpha-tocopherol/lipids ratio was significantly lower in obese girls than in normal controls. Both LDL beta-carotene and LDL alpha-tocopherol levels were significantly lower in obese girls than in normal controls, although no obvious differences were observed in plasma levels. In obese girls LDL contained more polyunsaturated fatty acid (PUFA) compared with normal controls. When the peroxidizability index (PI) was calculated to estimate the susceptibility of lipids to oxidative stress, obese girls had significantly higher PI values than normal controls. Both the LDL beta-carotene/PI ratio and the LDL alpha-tocopherol/PI ratio were significantly lower in obese girls than in normal controls. These results indicate the increased susceptibility of LDL to oxidative stress in obese girls which may promote atherosclerosis later in life.

Non-Protein-Bound Transition Metals and Hydroxyl Radical Generation in Cerebrospinal Fluid of Newborn Infants with Hypoxic Ischemic Encephalopathy

Among various hypothetical mechanisms for the in vivo production of reactive oxygen species, transition metal-catalyzed reactions in cooperation with a biologic reducing agent like ascorbic acid or superoxide may be some of the most important. In the present study, we retrospectively examined the existence of non-protein-bound metal ions, an essentially hazardous pro-oxidant form of various transition metals, and the occurrence of metal-catalyzed reactive oxygen species production in cerebrospinal fluid (CSF) of 10 infants with hypoxic ischemic encephalopathy (HIE) subsequent to perinatal asphyxia and 12 control infants within 72 h of birth. Non-protein-bound iron was detected in eight out of 10 CSF samples from the HIE infants and its level was significantly correlated with Sarnats clinical stage, whereas none of the control infants had detectable non-protein-bound iron levels. Non-protein-bound copper was below the detection limit in all CSF samples from both groups. Ascorbic acid was significantly increased in the CSF of HIE infants when compared with that of controls (means, 664.9 versus 449.4 μM, p = 0.008). ortho-Tyrosine and meta-tyrosine, which are highly specific and sensitive markers of protein oxidation induced by hydroxyl radicals, were significantly higher in HIE infants than in controls when evaluated by the ratio relative to their source amino acid, phenylalanine [means, 110.5 versus 75.4, p = 0.018 for ortho-tyrosine/phenylalanine; 104.6 versus 67.7 (nM/μM × 102), p = 0.048 for meta-tyrosine/phenylalanine]. Both ratios were significantly correlated with non-protein-bound iron, but not with ascorbic acid. Our preliminary observations provide direct evidence that hydroxyl radicals are generated in the CNS during asphyxiation. Iron chelation therapy could be worth developing as a neuroprotective strategy for perinatal asphyxia.

α-Tocopherol Transfer Protein Expression in Rat Liver Exposed to Hyperoxia

f -Tochopherol transfer protein ( f TTP), a 32 kDa protein exclusively expressed in liver cytosol, has a high binding affinity for f -tochopherol. The factors that regulate the expression of hepatic f TTP are not clearly understood. In this study, we investigated whether or not exposure to hyperoxia (>95% O 2 for 48 h) could alter the expression of hepatic f TTP. We also examined the association between the expression of antioxidant enzymes (hepatic glutathione peroxidase (GPX) and Mn-superoxide dismutase (Mn-SOD)) and the expression of hepatic f TTP. The levels of thiobarbituric acid-reactive substances (TBARS) in both plasma and liver were significantly higher after rats were exposed to hyperoxia for 48 h when compared with the levels in control rats. Northern blotting showed a decrease in the expression of f TTP messenger RNA (mRNA) after hyperoxia, although the f TTP protein level remained constant. Expression of Mn-SOD mRNA and protein, as well as the expression of GPX mRNA, were stable after hyperoxia. These findings indicate that mRNA for hepatic f TTP, rather than Mn-SOD or GPX, may be highly responsive to oxidative stress.

Determination of β-carotene in plasma, blood cells and buccal mucosa by electrochemical detection

The β-carotene concentrations in plasma, blood cells and buccal mucosal cells were determined by high-performance liquid chromatography with electrochemical detection. This method was 1,000 times more sensitive than the conventional spectrophotometric method. Polymorphonuclear cells and red blood cells had lower β-carotene levels than the other cells. After oral administration of 580 mg/day of all-trans β-carotene to human male volunteers for a week, the β-carotene concentrations in all cell types increased at least several times above the original levels.

9-cis β-carotene in human plasma and blood cells after ingestion of β-carotene

For 44 wk, thirty male volunteers were given daily either 60 mg of synthesized all-trans β-carotene, a naturally-occurring β-carotene derived fromDunaliella bardawil, or a placebo. Basal levels of 9-cis β-carotene in plasma, platelets, and mononuclear cells were 10, 20 and 25% of those of the all-trans form, respectively. The plasma levels reached a maximum after two weeks of administration and plateaued thereafter in the subjects who took the β-carotene preparations. The all-trans β-carotene level in the subjects given the synthesized all-trans form was almost twice that for theDunaliella preparation. The plasma 9-cis level was found to be higher in the all-trans β-carotene group than in theDunaliella group, despite no intake of the 9-cis form in the all-trans group and the higher intake of the 9-cis form in theDunaliella group. This finding suggests that isomerization of the all-trans form to the 9-cis form may occur in the body either during or after absorption.

Plasma KL-6 Predicts the Development and Outcome of Bronchopulmonary Dysplasia

Circulating KL-6 is a specific indicator of pulmonary injury affecting the alveolar epithelium and interstitium. Our preliminary study suggested the usefulness of plasma KL-6 as a marker of bronchopulmonary dysplasia (BPD). To confirm the diagnostic value of KL-6 for BPD as well as to determine the reference range, we conducted a larger prospective study in 135 preterm infants <32 wk GA. Among the infants without oxygen dependence at a postconceptional age of 36 wk, the plasma KL-6 level showed no significant association with GA at any time. Among 42 infants <28 wk GA, plasma KL-6 levels were significantly higher in those with moderate/severe BPD compared with those with no/mild BPD. A plasma level of 199 U/mL at 1 wk or 232 U/mL at 2 wk was an excellent predictor of moderate/severe BPD <28 wk GA (positive predictive value of 83% and 80%, respectively). Unlike nonspecific markers of inflammation or fibrosis, KL-6 objectively reflects the severity of pulmonary injury irrespective of the treatment or the radiographic changes. Therefore, not only as a good marker, measurement of KL-6 may also help to provide new insights into the pathogenesis of BPD.

Pharmacokinetics of All-trans Retinoic Acid in Pediatric Patients with Leukemia

Since all‐trans retinoic acid (ATRA) induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL), and its effectiveness appears to be related to the plasma or serum level, a pharmacokinetic study of ATRA was undertaken in nine patients with various leukemias. After oral administration at a dose of 30 mg/m2, the time required to reach the peak plasma level of ATRA (20–1198 ng/ml) was between 120 and 240 min and the apparent plasma elimination half life was 21–51 min. In addition, 13‐cis retinoic acid was detected in the plasma of seven patients, indicating the occurrence of ATRA isomerization in vivo. ATRA therapy did not induce complete remission in all patients, even when high plasma levels were achieved. Among the six APL patients given ATRA therapy, one who failed to respond had a very low plasma ATRA level. These findings suggest that it may be useful to monitor plasma levels during oral ATRA therapy in order to achieve an appropriate treatment regimen.

Antioxidant enzymes and vitamins in Down's syndrome

Abstract Twenty-two children with Donws syndrome and 25 age-matched control children were examined to determine their levels of antioxidant enzymes and vitamins. Cu, Zn-superoxide dismutase (SOD), Mn-SOD, glutathione peroxidase (GSH-Px) and catalase were studied as antioxidant enzymes, while α-tocopherol, β-carotene, and retinol were studied as antioxidant vitamins. The Cu, Zn-SOD activity and immoreactive protein levels in plasma and red blood cells were higher in Downs syndrome children than in control children, while plasma Mn-SOD was lower in Downs syndrome children than in controls. Red cell GSH-Px and catalase activities were lower in Downs syndrome than in the controls, but the difference was not significant. There were also no significant differences in the α- and γ-tocopherol levels in plasma, platelets, mononuclear cells, polymorphonuclear cells, and buccal mucosal cells between the Downs syndrome children and the control children. Finally, no significant differences were observed in plasma β-carotene and retinol levels.