ABSTRACT:The unbound "free" bilirubin concentration (B f ), not the total bilirubin concentration, is the critical determinant of cellular uptake and toxicity of bilirubin. We compared B f measured by a modified peroxidase method with published data obtained with ultrafiltration and examined conditions that affect the affinity (K F ) of human (HSA) and bovine (BSA) serum albumin for bilirubin. The peroxidase and ultrafiltration methods yielded similar K F values that decreased with increasing HSA concentration and the presence of 50 mM chloride. When related to ionic strength, inhibition of BSAbilirubin binding by chloride, bromide, and sulfate were similar, whereas phosphate buffer had a smaller effect. K F was lower at 37°C than at 25°C for HSA but not for BSA. K F for BSA was similar at pH 7.4 and 8. T he unbound ("free") concentration of unconjugated bilirubin (B f ) in plasma, although less than 0.1% of total bilirubin concentration, is the principal determinant of tissue uptake and toxicity of bilirubin, and plays a critical role in the pathogenesis of bilirubin encephalopathy in jaundiced newborns (1,2) and in patients with Crigler Najjar disease (3). Notwithstanding its biologic significance, B f has rarely been measured in either clinical evaluation of jaundiced newborns or in vitro studies of bilirubin effects and toxicity. This avoidance is due in part to the perceived complexity of B f assays and scepticism regarding their clinical value or accuracy (4 -6).Bilirubin-albumin binding has been studied using a variety of techniques, including fluorescent quenching, bilirubin fluorescence, circular dichroism, Sephadex gel filtration, optical rotary dispersion, dialysis, ultrafiltration, spectrophotometry, and enzymatic oxidation of bilirubin (peroxidase method). Reported association constants for HSA range from 6.7 ϫ 10 6 M Ϫ1 to Ͼ10 8 M Ϫ1 (7-10). The binding constant of HSA has been estimated to be 2-13 times greater than that of BSA (11-13). Considerable variation in serum binding of bilirubin has been reported in newborn infants (2). These differences may be due to direct binding competitors (e.g. sulfonamides), allosteric effects, electrolyte environment, or simply the assay technique, e.g. serum sample dilution (14).The dependency of bilirubin binding affinity on HSA concentration as well as chloride concentration was demonstrated by Weisiger et al. (15) using a complicated procedure in which binding affinity of 14 C-bilirubin was calculated after the sequential removal of labeled impurities by serial ultrafiltration. A more practical technique for measuring B f in clinical or laboratory settings was developed by Jacobsen and Wennberg (16) based on the observation that albumin-binding protects bilirubin from oxidation by HRP. Ahlfors (14,17) has modified the peroxidase method, emphasizing the need to measure B f with two or more HRP concentrations and under the same conditions and albumin concentrations existing in plasma or incubation medium.In this investigation, we replicated and extended the exp...

A mtDNA A1555G base substitution in a highly conserved region of the 12S rRNA gene has been reported to be the main cause of aminoglycoside induced deafness. This mutation is found in approximately 3% of Japanese and 0.5-2.4% of European sensorineural deafness patients. We report a high prevalence (5.3%) of the A1555G mutation in sensorineural deafness patients in Sulawesi (Indonesia). Our result confirms the importance of determining the prevalence of the mtDNA A1555G mutation in different populations, and the need for mutation detection before the administration of aminoglycoside antibiotics.

BackgroundHigh concentrations of plasma leptin and the release of pro-inflammatory cytokines in leptin-resistance in obesity have been reported to trigger endothelial dysfunction. The objective of this study was to elucidate the role of quercetin in modulating leptin-induced inflammation as assessed by the levels of Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion in umbilical vein endothelial cells (HUVECs) in vitro.FindingsHUVECs were exposed to either control levels (0 ng/ml) or 500 ng/mL leptin (L) for 48 hours, followed by control or 125 uM quercetin (Q) for another 6 h. The experimental groups were as follows: L0Q0, L0Q125, L500Q0, L500Q125. The presence of the short chain leptin receptor isoform Ob-Ra in HUVECs was determined by Western blot and immunocytochemistry analyses. Ob-Ra expression, ERK1/2 phosphorylation, NF-kappa B activation and TNF-alpha secretion were quantified by ELISA, and NF-kappa B activationby immunofluorescence staining. Our results showed that Ob-Ra expression, ERK1/2 phosphorylation and NF-kappa B activation increased significantly after 500 ng/mL leptin exposure (1.8x, 1.5x, 6.2x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), but were reduced by addition of 125 uM quercetin (0.7x, 0.3x and 0.4x for Ob-Ra, ERK1/2 and NF-kappa B, respectively), and that quercetin could also partially suppress leptin-induced TNF-alpha secretion (3.8x) by 0.8x.ConclusionExposure of HUVECs to leptin up-regulated Ob-Ra expression and elevated ERK1/2 phosphorylation and NFkB activation, and increased TNF-alpha secretion. These effects strongly suppressed by quercetin, with the exception of TNF-alpha which was partially suppressed. The findings might be of clinical significance, as endothelial dysfunction that could lead to cardiovascular disease is preventable, and quercetin is a natural compound found in various plants and fruits.

BackgroundUncoupling protein 2 (UCP2) gene polymorphisms have been reported as genetic risk factors for obesity and type 2 diabetes mellitus (T2DM). We examined the association of commonly observed UCP2 G(−866)A (rs659366) and Ala55Val (C > T) (rs660339) single nucleotide polymorphisms (SNPs) with obesity, high fasting plasma glucose, and serum lipids in a Balinese population.MethodsA total of 603 participants (278 urban and 325 rural subjects) were recruited from Bali Island, Indonesia. Fasting plasma glucose (FPG), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC) were measured. Obesity was determined based on WHO classifications for adult Asians. Participants were genotyped for G(−866)A and Ala55Val polymorphisms of the UCP2 gene.ResultsObesity prevalence was higher in urban subjects (51%) as compared to rural subjects (23%). The genotype, minor allele (MAF), and heterozygosity frequencies were similar between urban and rural subjects for both SNPs. All genotype frequencies were in Hardy-Weinberg equilibrium. A combined analysis of genotypes and environment revealed that the urban subjects carrying the A/A genotype of the G(−866)A SNP have higher BMI than the rural subjects with the same genotype. Since the two SNPs showed strong linkage disequilibrium (D’ = 0.946, r2 = 0.657), a haplotype analysis was performed. We found that the AT haplotype was associated with high BMI only when the urban environment was taken into account.ConclusionsWe have demonstrated the importance of environmental settings in studying the influence of the common UCP2 gene polymorphisms in the development of obesity in a Balinese population.

Background Vitamin D deficiency (VDD) is a common problem in reproductive-aged women and has become a major public health problem worldwide. The effect of VDD in pregnancy has been associated with several adverse pregnancy outcomes. This study aims to assess the serum levels of 25-hydroxyvitamin D (25(OH)D) in the first trimester and its associated factors (socio-demographics, pregnancy profiles, dietary intake, and maternal anthropometry measurements) for the determination of vitamin D deficiency status in early pregnancy. Methods A cross-sectional study of 239 pregnant mothers in West Sumatra, Indonesia was conducted. We measured lifestyle, socio-demographics and pregnancy profile through a structured questionnaire and interview process. A semi quantitative-food frequency questionnaire (SQ-FFQ) was used to analyse the dietary intake of the pregnant women. Serum 25(OH)D concentrations were measured at < 13 weeks gestation using ELISA and logistic regression models were employed to identify the predictors of low vitamin D status. Results The prevalence of first-trimester maternal VDD and sufficiency were 82.8 and 17.2% respectively. The median 25(OH)D was 13.15 ng/mL (3.00–49.29 ng/mL). The significant independent predictors were no working status (OR: 0.029;0.001–0.708) ( p = 0.030); nulliparous parity status (OR: 7.634;1.550–37.608) ( p = 0.012); length of outdoor activity status of less than an hour (OR: 9.659;1.883–49.550) ( p = 0.007); and no consumption of supplements before pregnancy (OR: 4.49;1.081–18.563) ( p = 0.039). Conclusions The prevalence of VDD is common in early pregnancy among Minangkabau women. Recommendations and policies to detect and prevent such insufficiency during pregnancy should be developed by considering the associated factors.

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Background The Supplementation with Multiple Micronutrients Intervention Trial (SUMMIT) in Lombok, Indonesia showed that maternal multiple micronutrients (MMN), as compared with iron and folic acid (IFA), reduced fetal loss, early infant mortality, and low birth weight. Mitochondria play a key role during pregnancy by providing maternal metabolic energy for fetal development, but the effects of maternal supplementation during pregnancy on mitochondria are not fully understood. Objective The aim of this study was to assess the impact of MMN supplementation on maternal mitochondrial DNA copy number (mtDNA-CN). Methods We used archived venous blood specimens from pregnant women enrolled in the SUMMIT study. SUMMIT was a cluster-randomized double-blind controlled trial in which midwives were randomly assigned to distribute MMN or IFA to pregnant women. In this study, we selected 108 sets of paired baseline and postsupplementation samples (MMN = 54 and IFA = 54). Maternal mtDNA-CN was determined by real-time quantitative polymerase chain reaction in baseline and postsupplementation specimens. The association between supplementation type and change in mtDNA-CN was performed using rank-based estimation for linear models. Results In both groups, maternal mtDNA-CN at postsupplementation was significantly elevated compared with baseline (P < 0.001). The regression revealed that the MMN group had lower postsupplementation mtDNA-CN than the IFA group (β = −4.63, P = 0.003), especially for women with mtDNA-CN levels above the median at baseline (β = −7.49, P = 0.007). This effect was rapid, and observed within 33 d of initiation of supplementation (β = −7.39, P = 0.017). Conclusion Maternal MMN supplementation rapidly stabilized mtDNA-CN in pregnant women who participated in SUMMIT, indicating improved mitochondrial efficiency. The data provide a mechanistic basis for the beneficial effects of MMN on fetal growth and survival, and support the transition from routine IFA to MMN supplementation. This trial was registered at www.isrctn.com as ISRCTN34151616.

Purpose Adverse effects of maternal vitamin D deficiency have been linked to adverse pregnancy outcomes. We investigated the relationship between maternal vitamin D status and newborn anthropometry measurements using a genetic approach and examined the interaction between genetic variations in involved in vitamin D synthesis and metabolism and maternal vitamin D concentrations on newborn anthropometry. Methods The study was conducted in 183 pregnant Indonesian Minangkabau women. Genetic risk scores (GRSs) were created using six vitamin D–related single nucleotide polymorphisms and their association with 25-hydroxyvitamin D [25(OH)D] levels and newborn anthropometry (183 infants) were investigated. Results There was no significant association between maternal 25(OH)D concentrations and newborn anthropometry measurements (P > 0.05, for all comparisons). After correction for multiple testing using Bonferroni correction, GRS was significantly associated with 25(OH)D in the third trimester (P = 0.004). There was no association between GRS and newborn anthropometric measurements; however, there was an interaction between GRS and 25(OH)D on head circumference (P = 0.030), where mothers of neonates with head circumference < 35 cm had significantly lower 25(OH)D if they carried ≥4 risk alleles compared to those who carried ≤3 risk alleles. Conclusion Our findings demonstrate the impact of vitamin D-related GRS on 25(OH)D and provides evidence for the effect of vitamin D-related GRS on newborn anthropometry through the influence of serum 25(OH)D levels among Indonesian pregnant women. Even though our study is a prospective cohort, before the implementation of vitamin D supplementation programs in Indonesia to prevent adverse pregnancy outcomes, further large studies are required to confirm our findings.