Kyouichi Sekine

Formation of Advanced Glycosylation End Products and Oxidative Stress in Young Patients with Type 1 Diabetes

Increased production of advanced glycosylation end products (AGEs) and augmented oxidative stress may contribute to vascular complications in diabetes. Little is known about the formation and accumulation of AGEs in young patients with type 1 diabetes. The aim of the present study was to investigate whether AGE production and oxidative stress are augmented in young patients with type 1 diabetes at early clinical stages of the disease. Urine samples of 38 patients with type 1 diabetes [mean age (±SD), 12.8 ± 4.5 y; diabetes duration, 5.7 ± 4.3 y; HbA1c, 8.0 ± 1.6%; urinary albumin excretion, 12.6 ± 14.4 mg/g creatinine (Cr)] and those of 60 age-matched healthy control subjects were assayed for AGEs, pentosidine and pyrraline, and markers of oxidative stress, 8-hydroxy-2′-deoxyguanosine (8-OHdG) and acrolein-lysine. Of these four markers, urinary concentrations of pentosidine, 8-OHdG, and acrolein-lysine were significantly higher in the patients with diabetes than in the healthy control subjects. For the patient group, pentosidine correlated significantly with 8-OHdG and acrolein-lysine, and pyrraline correlated significantly with acrolein-lysine. Urinary pentosidine, 8-OHdG, and acrolein-lysine but not pyrraline correlated significantly with urinary albumin excretion. Patients with microalbuminuria (≥15 mg/g Cr) showed significantly higher levels of all four markers than did normoalbuminuric patients and control subjects. The present study indicates that accumulation of AGEs, whose formation is closely linked to oxidative stress, and resultant endothelial dysfunction may start early in the course of type 1 diabetes. This means that the risk of vascular complications may be present at an early age and that the best possible glycemic control should be emphasized from the diagnosis of diabetes.

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.

Determination of asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, in umbilical blood.

Endothelial cells produce nitric oxide (NO), a potent vasodilator. Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of NO synthase. Little is known about the potential physiological roles of ADMA in a perinatal setting. This study measures concentrations of ADMA in umbilical blood using enzyme-linked immunosorbent assay and those of NO as nitrite/nitrate (NOx(-)) using the Griess assay. Their relationship to the degree of prematurity and maternal clinical condition is examined. Results show that ADMA concentrations in umbilical blood from control newborns were about twice as high as those of lactating women, healthy children, and healthy adults. Umbilical blood NOx(-) concentrations from control newborns were about half of those of lactating women, healthy children, and healthy adults. Consequently, the levels of ADMA relative to NOx(-) were about 4-fold higher in umbilical blood from control newborns than in blood from lactating women, healthy children, and healthy adults. Furthermore, the umbilical blood ADMA concentrations and the ratios of ADMA to NOx(-) in newborns were higher according to their birth prematurity and lower birth weight. The umbilical ADMA concentrations were independent of the delivery mode and maternal preeclampsia. We infer that the high ADMA levels play physiological roles in maintaining vascular tone and blood redistribution to vital organs during birth, thereby favoring the circulatory transition from fetal to neonatal life.

Concentrations of pentosidine, an advanced glycation end-product, in umbilical cord blood.

Advanced glycation end-products (AGEs) are formed over several weeks to months by non-enzymatic glycation and oxidation (“glycoxidation”) reactions between carbohydrate-derived carbonyl groups and protein amino groups, known as the Maillard reaction. Pentosidine is one of the best-characterized AGEs and is accepted as a satisfactory marker for glycoxidation in vivo. The present study was intended to measure pentosidine concentrations in umbilical cord blood from newborns with various gestational ages using our recently established high-performance liquid chromatography method [Tsukahara, H. et al. (2003) Pediatr. Res. 54, 419–424]. Our study demonstrates, for the first time, that pentosidine is detected in most of the umbilical blood samples. This study also shows that the umbilical blood concentrations of pentosidine are considerably lower than normal adult values, but that they increase with gestation progression and fetal growth. Umbilical pentosidine concentrations were significantly elevated in newborns of mothers with preeclampsia compared to those of mothers without preeclampsia. We conclude that accumulation of AGEs and oxidative stress occurs in fetal tissues and organs in utero at the early stage of human life and that their accumulation is augmented in the maternal preeclampsic condition.

Concentrations of thioredoxin, a redox-regulating protein, in umbilical cord blood and breast milk

Growing evidence indicates that oxidative stress occurs during the fetal-to-neonatal transition. Such stress plays an important role in the pathogenesis of many neonatal diseases. Thioredoxin (TRX), a redox-regulating protein with antioxidant activity, is induced in various cells against oxidative stress and is secreted extracellularly. This study was undertaken to examine the clinical and biological importance of TRX in the perinatal setting. We measured concentrations of TRX in umbilical cord blood and breast milk using a sandwich ELISA. Our study demonstrated that concentrations of TRX in umbilical cord blood were six to seven times higher than those in blood of healthy adults. This study also showed that umbilical concentrations of TRX were correlated significantly with the extent of prematurity of the newborn, and that they were elevated significantly in newborns of mothers with preeclampsia compared to those of mothers without preeclampsia. In contrast, concentrations of coenzyme Q10 and vitamin E in umbilical blood were lower than adult blood levels. Breast milk concentrations of TRX during the early postpartum period were seven to eight times higher than those in blood of lactating women. Those of the coenzyme Q10 were lower than adult blood levels, while those of vitamin E were comparable to adult blood levels. Our findings suggest that the systemic release of TRX is enhanced at birth, and that early breast milk is a rich source of this protein. Consequent high levels of TRX in newborns may provide a unique protective mechanism that allows the maintenance of redox balance during the fetal-to-neonatal transition.

The Effects of Intrinsic Vasopressin on Urinary Aquaporin-2 Excretion and Urine Osmolality During Surgery Under General Anesthesia

UNLABELLED A radioimmunoassay has been established to measure urinary aquaporin-2 excretion (u-AQP2). To elucidate how u-AQP2 changes when endogenous vasopressin is increased independently of plasma osmolality, we estimated u-AQP2 during general anesthesia for surgery. We collected urine and blood samples from 50 patients before and 90 and 180 min after anesthetic induction. Plasma (29.1+/-12.6 pg/mL) and urinary (565.1+/-207.0 ng/gCr) vasopressin levels were markedly increased after anesthetic induction. Although no significant alteration of plasma osmolality or serum sodium concentration was observed during 180 min, u-AQP2 was significantly increased (preinduction 224.5+/-24.2 fmol/ mgCr; 90 min 243.3+/-31.8; 180 min 331.4+/-45.9), paralleling an increase of plasma and urinary vasopressin. The plasma vasopressin concentration after anesthetic induction was far in excess of that expected based on plasma osmolality. Individual plasma and urinary vasopressin concentrations correlated significantly with u-AQP2. At 180 min after anesthesia, plasma osmolality did not change, but urine osmolality decreased despite increased u-AQP2, and a preanesthetic positive correlation between urine osmolality and u-AQP2 disappeared. Thus, although u-AQP2 correlates with increased intrinsic vasopressin levels, the increase in u-AQP2 did not directly contribute to urine concentration. Apparently, an escape from the physiologic effects of high vasopressin level occurs during anesthesia via a mechanism independent of aquaporin-2. We conclude that the anesthetic would interfere with the urinary concentrating capacity at the level of AQP2-action. IMPLICATIONS The excessive increase of intrinsic vasopressin exactly augmented urinary aquaporin-2 excretion, resulting in urine concentration; however, anesthesia seemed to modify this process possibly by interfering with the aquaporin-2 action.

Ubiquinol-10 and ubiquinone-10 levels in umbilical cord blood of healthy foetuses and the venous blood of their mothers

Abstract Despite their being good markers of oxidative stress for clinical use, little is known about ubiquinol-10 (reduced coenzyme Q10) and ubiquinone-10 (oxidized coenzyme Q10) levels in foetuses and their mothers. This study investigates oxidative stress in 10 healthy maternal venous, umbilical arterial and venous bloods after vaginal delivery by measuring ubiquinol-10 and ubiquinone-10 levels. Serum ubiquinol-10 and ubiquinone-10 levels were measured by HPLC with a highly sensitive electrochemical detector. Maternal venous ubiquinol-10 and ubiquinone-10 levels were significantly higher than umbilical arterial and venous levels (all p < 0.001). However, the ubiquinone-10/total coenzyme Q10 (CoQ10) ratio, which reflects the redox status, was significantly higher in umbilical arterial and umbilical venous blood compared to maternal venous blood (all p < 0.001). The ubiquinone-10/total CoQ10 ratio was higher in umbilical arterial than in umbilical venous blood (p < 0.01). The present study demonstrated that foetuses were under higher oxidative stress than their mothers.

Changes in 13, 14-dihydro-15-keto-prostaglandin F2α levels in saliva during pregnancy, labor and the postpartum period

Aims:  To examine changes in the levels of 13, 14‐dihydro‐15‐keto‐prostaglandin F2α (PGFM) in saliva during pregnancy, labor and the postpartum period, and to evaluate the relationship between cervical dilatation or contraction interval and salivary PGFM levels during labor.

Changes of plasma coenzyme Q10 levels in early infancy.

Abstract Rapid perfusion of oxygen in infants at birth may increase oxidative stress which has been incriminated in serious diseases including neonatal respiratory distress syndrome, chronic lung disease, and retinopathy of prematurity. Elucidating the antioxidant defense systems of neonates in clinical practice is important. Coenzyme Q10 is a widely distributed, redox-active quinoid compound originally discovered as an essential part of the mitochondrial respiratory chain in mammals. Although coenzyme Q10 is a powerful lipid antioxidant in vivo, few data pertain to plasma CoQ10 levels in infants. This is the first paper to report plasma coenzyme Q10 levels in preterm infants.