Association of ADIPOR1 polymorphisms with bone mineral density in postmenopausal Korean women

Adiponectin may affect bone through interactions with two known receptors, adiponectin receptors (ADIPOR) 1 and 2. We examined the association between polymorphisms of ADIPOR1 and ADIPOR2 and bone mineral density (BMD) in postmenopausal Korean women. Six polymorphisms in ADIPOR1 and four polymorphisms in ADIPOR2 were selected and genotyped in all study participants (n = 1,329). BMD at the lumbar spine and femur neck were measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment and the occurrence of non-vertebral fractures examined using self-reported data. P values were adjusted for multiple testing using Bonferroni correction (P(corr)). ADIPOR1 rs16850799 and rs34010966 polymorphisms were significantly associated with femur neck BMD (P(corr) = 0.036 in the dominant model; P(corr) = 0.024 and P(corr) = 0.006 in the additive and dominant models, respectively). Subjects with the rare allele of each polymorphism had lower BMD, and association of rs34010966 with BMD showed a gene dosage effect. However, ADIPOR2 single nucleotide polymorphisms and haplotypes were not associated with BMD at any site. Our results suggest that ADIPOR1 polymorphisms present a useful genetic marker for BMD in postmenopausal Korean women.     

Metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase polymorphisms: A metabolomic analysis

The aim of this study was to demonstrate the altered metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase (MTHFR) polymorphisms at first trimester and during delivery. Eight singleton pregnant women with MTHFR polymorphisms were compared with 10 normal pregnant women. Maternal blood samples were obtained twice during their pregnancy period (between the 11th and 14th gestational weeks and during delivery). Metabolomic analysis was performed using GC–MS. The GC–MS based metabolomic profile helped identify 95 metabolites in the plasma samples. In the MTHFR group, the levels of 1-monohexadecanoylglycerol, pyrophosphate, benzoin, and linoleic acid significantly decreased (P ˂ 0.05 for all), whereas the levels of glyceric acid, l -tryptophan, l -alanine, l -proline, norvaline, l -threonine, and myo-inositol significantly increased (P ˂ 0.01 for the first two metabolites, P ˂ 0.05 for the others) at 11–14 gestational weeks. Conversely, the levels of benzoin, 1-monohexadecanoylglycerol, pyruvic acid, l -proline, phosphoric acid, epsilon-caprolactam, and pipecolic acid significantly decreased in the MTHFR group, whereas metabolites such as hexadecanoic acid and 2-hydroxybutyric acid increased significantly in the study group during delivery. An impaired energy metabolism pathway, vitamin B complex disorders, tendency for metabolic acidosis (oxidative stress), and the need for cell/tissue support seem prevalent in pregnancies with MTHFR polymorphisms.     

Association of Paraoxonase 1 (PON1) polymorphisms with osteoporotic fracture risk in postmenopausal Korean women

There is increasing evidence of a biochemical link between lipid oxidation and bone metabolism. Paraoxonase 1 (PON1) prevents the oxidation of low-density lipoprotein (LDL) and metabolizes biologically active phospholipids in oxidized LDLs. Here, we performed association analyses of genetic variation in PON1 to ascertain its contribution to osteoporotic fractures (OFs) and bone mineral density (BMD). We directly sequenced the PON1 gene in 24 Korean individuals and identified 26 sequence variants. A large population of Korean postmenopausal women (n=1,329) was then genotyped for eight selected PON1 polymorphisms. BMD at the lumbar spine and femoral neck was measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment, and the occurrence of non-vertebral fractures (i.e., wrist, hip, forearm, humerus, rib, and pelvis) was examined using self-reported data. Multivariate analyses showed that none of the polymorphisms was associated with BMD at either site. However, +5989A>G and +26080T>C polymorphisms were significantly associated with non-vertebral and vertebral fractures, respectively, after adjustment for covariates. Specifically, the minor allele of +5989A>G exerted a highly protective effect against non-vertebral fractures (OR=0.59, P=0.036), whereas the minor allele of +26080T>C was associated with increased susceptibility to vertebral fractures (OR=1.73, P=0.020). When the risk for any OFs (i.e., vertebral or non-vertebral) was considered, the statistical significance of both polymorphisms persisted (P=0.002-0.010). These results suggest that PON1 polymorphisms could be one of useful genetic markers for OF risk in postmenopausal women.     

Perturbations of aromatic amino acids are associated with iron cluster assembly in ribonucleotide reductase

The β2 subunit of class Ia ribonucleotide reductases (RNR) contains an antiferromagnetically coupled μ-oxo bridged diiron cluster and a tyrosyl radical (Y122?). In this study, an ultraviolet resonance Raman (UVRR) difference technique describes the structural changes induced by the assembly of the iron cluster and by the reduction of the tyrosyl radical. Spectral contributions from aromatic amino acids are observed through UV resonance enhancement at 229 nm. Vibrational bands are assigned by comparison to histidine, phenylalanine, tyrosine, tryptophan, and 3-methylindole model compound data and by isotopic labeling of histidine in the β2 subunit. Reduction of the tyrosyl radical reveals Y122? Raman bands at 1499 and 1556 cm(-1) and Y122 Raman bands at 1170, 1199, and 1608 cm(-1). There is little perturbation of other aromatic amino acids when Y122? is reduced. Assembly of the iron cluster is shown to be accompanied by deprotonation of histidine. A p(2)H titration study supports the assignment of an elevated pK for the histidine. In addition, structural perturbations of tyrosine and tryptophan are detected. For tryptophan, comparison to model compound data suggests an increase in hydrogen bonding and a change in conformation when the iron cluster is removed. pH and (2)H(2)O studies imply that the perturbed tryptophan is in a low dielectric environment that is close to the metal center and protected from solvent exchange. Tyrosine contributions are attributed to a conformational or hydrogen-bonding change. In summary, our work shows that electrostatic and conformational perturbations of aromatic amino acids are associated with metal cluster assembly in RNR. These conformational changes may contribute to the allosteric effects, which regulate metal binding.     

The enabled homolog gene polymorphisms are associated with susceptibility and progression of childhood IgA nephropathy

The enabled homolog gene (ENAH, hMena) is abundantly expressed in mesangial tissue, and might play an important role in inflammatory processes of IgA nephropathy (IgAN). The present study was conducted to investigate the association between single nucleotide polymorphisms (SNPs) of the ENAH and childhood IgAN. We analyzed 12 SNPs of ENAH in 176 patients with childhood IgAN and 397 healthy controls. In addition, IgAN patients were dichotomized and compared with respect to several clinical and pathological parameters. Genotyping data showed significant differences between IgAN patients and controls in the frequency of rs2039620, rs12034829, and rs3795443. On comparison of patients with proteinuria to those without proteinuria (≤ or > 4 mg/m²/h), rs12043633 was significantly different between the two groups. With regard to maximum proteinuria (≤ or > 4 mg/m²/h), rs3795443, rs4653643, rs6751, rs10799319, rs7555139, rs576861, and rs487591 showed significant allele frequency differences. For patients with and without gross hematuria, rs4653643, rs6751, rs10799319, rs7555139, rs576861, and rs487591 showed significant allele frequency differences. The rs3795443 was found to be associated with progression of pathological findings. Our results suggest that ENAH polymorphisms are associated with increased susceptibility, development of proteinuria and gross hematuria, and pathological progression of childhood IgAN.     

Candidate gene polymorphisms for diabetes mellitus, cardiovascular disease and cancer are associated with longevity in Koreans

Long-lived people may have a unique genetic makeup that makes them more resistant than the general population to prevalent age-related diseases; however, not much is known about genes involved in the longevity. To identify susceptibility variants controlling longevity, we performed a high-throughput candidate gene study using 137 Koreans over 90 yr old and 213 young healthy Koreans. We evaluated 463 informative markers located in 176 candidate genes mostly for diabetes mellitus, cardiovascular disease and cancer under five genetic models. We estimated the odds ratios for each allele, genotype, haplotype, and gene-gene interaction using logistic regression analysis. Associations between 13 genes and longevity were detected at a P-value less than 0.01. Particularly, the rs671 (A) allele of the aldehyde dehydrogenase 2 family (mitochondrial) (ALDH2) gene was associated with longevity only in men (OR 2.11, P = 0.008). Four genes, proprotein convertase subtilisin/kexin type 1 (PCSK1, P = 0.008), epidermal growth factor receptor (EGFR, P = 0.003), paired box 4 (PAX4, P = 0.008), and V-yes-1 Yamaguchi sarcoma viral related oncogene homolog (LYN, P = 0.002) consistently yielded statistical evidence for association with longevity. The findings of the current study may provide a starting point for future studies to unravel genetic factors controlling longevity in Koreans.     

Liquid chromatography/mass spectrometry of pre-ionized Girard P derivatives for quantifying estrone and its metabolites in serum from postmenopausal women

An ultrasensitive stable isotope dilution liquid chromatography/selected reaction monitoring/mass spectrometry (LC/SRM/MS) assay has been developed for serum estrone, 16α‐hydroxyestrone, 4‐methoxyestrone, and 2‐ methoxyestrone. The enhanced sensitivity was obtained by the use of Girard P (GP) pre‐ionized derivatives coupled with microflow LC. The limit of detection for each estrogen using 0.5 mL of serum was 0.156 pg/mL and linear standard curves were obtained up to 20 pg/mL. Serum samples from 20 postmenopausal women (10 lifetime non‐smokers and 10 current smokers) were analyzed using this new assay. Mean serum concentrations of estrone and 2‐methoxyestrone were 14.06 pg/mL (±1.56 pg/mL) and 3.30 pg/mL (±1.00 pg/mL), respectively, for the 20 subjects enrolled in the study. The mean estrone concentration determined by our ultrasensitive and highly specific assay was significantly lower than that reported for the control groups in most previous breast cancer studies of postmenopausal women. In addition (and contrary to many reports) serum 16α‐hydroxyestrone was not detected in any of the subjects, and 4‐methoxyestrone was detected in only one of the subjects. Furthermore, there were no significant differences in the mean serum concentrations of estrone and 2‐methoxyestrone or the ratio of serum 2‐ methoxyestrone to estrone between the non‐smoking and smoking groups. Interestingly, the one subject with measurable serum 4‐methoxyestrone (2.3 pg/mL) had the lowest estrone and 2‐methoxyestrone concentrations. Using this assay it will now be possible to obtain definitive information on the levels of serum estrone, 4‐methoxyestrone, and 2‐methoxyestrone in studies of cancer risk using small serum volumes available from previous epidemiology studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Clinical evaluation of serum neuron-specific enolase levels in patients with lung cancer

Serum neuron-specific enolase (NSE) levels were studied in 105 patients with malignant neoplasms (lung cancer 38, others 67), 13 patients with various benign diseases and 7 healthy adults. The mean serum NSE level in adult control subjects was 7.4 +/- 0.8 ng/ml, and cut off level was decided 10 ng/ml. Serum NSE levels were elevated in 14/38 (37%) of patients with lung cancer and in 14/67 (21%) of patients with the other malignant neoplasms. In patients with benign diseases, serum NSE level was elevated only in one patient with pituitary adenoma. In 7 patients with small cell lung cancer, the positive rate was higher (86%) than in those with non-small cell lung cancer (26%), and serum NSE levels were higher than 25 ng/ml except one case. There was no correlation between serum NSE and CEA (carcinoembryonic antigen) levels in patients with small cell lung cancer, also in patients with lung cancer. The measurement of serum NSE level seemed to be useful for diagnosis in patients with small cell lung cancer.     

Methylenetetrahydrofolate reductase polymorphism, diet, and breast cancer in Korean women

To evaluate the interactive effect of methylenetetrahydrofolate reductase (MTHFR) genotype and dietary factors on the development of breast cancer, a hospital based case-control study was conducted in South Korean study population consisting of 189 histologically confirmed incident breast cancer cases and their 189 age-matched controls without present or previous history of cancer. A PCR-RFLP method was used for the genotyping of MTHFR (C677T) and statistical evaluations were performed by unconditional logistic regression analysis. Consumption of some dietary factors, such as green vegetables (OR = 0.3, 95% CI: 0.2-0.6), white vegetables (OR = 0.3, 95% CI: 0.1-0.7) mushrooms (OR = 0.4, 95% CI: 0.3-0.7), and meats (OR = 1.7, 95% CI: 1.1-2.8) significantly decreased or increased the risk of breast cancer. Although the breast cancer risk was 1.7-fold (95% CI: 0.8-3.2) increased in women with MTHFR TT genotype, the association was not statistically significant. Women with MTHFR TT genotype and low green vegetable intake increased 5.6-fold (95% CI: 1.2-26.3) risk of breast cancer compared to high green vegetable intake group containing MTHFR CC/CT genotype. However, the interaction was not significant (p for interaction = 0.96). Our findings suggest that MTHFR polymorphism did not influence individual susceptibility to breast cancer. However MTHFR (C667T) genotype and green vegetable intakes appeared to have the interactive effect in breast cancer development.     

Titanium release in serum of patients with different bone fixation implants and its interaction with serum biomolecules at physiological levels

Increased concentrations of circulating metal-degradation products derived from the use of Ti orthopaedic implants may have deleterious biological effects over the long term. Therefore, there is an increasing need to establish the basal level of Ti in the serum of the population (exposed and non-exposed) with appropriate highly sensitive techniques and strategies. With this aim, we have developed a quantitative strategy for the determination of total Ti concentration in human serum samples by isotope dilution analysis using a double-focussing inductively coupled plasma mass spectrometer. Minimizing sample handling and therefore contamination issues, we obtained detection limits of about 0.05 μg L⁻¹ Ti working at medium resolution (m/Δm 4000). Such extremely good sensitivity permitted us to establish the range of Ti concentration in serum of 40 control individuals (mean 0.26 μg L⁻¹) and also to compare it with the level in exposed patients with different Ti metal implants. On the other hand, Ti transport “in vivo” studies have been enabled by online coupling of liquid chromatography (anion-exchange) separation and double-focussing inductively coupled plasma mass spectrometry for sensitive detection of Ti. The development of a postcolumn isotope dilution strategy permitted quantitative characterization of the Ti-transporting biomolecules in human serum. The results for unspiked serum revealed that 99.8% of the Ti present in this fluid is bound to the protein transferrin, with column recoveries greater than 95%.     

MmpL genes are associated with mycolic acid metabolism in mycobacteria and corynebacteria

Mycolic acids are vital components of the cell wall of the tubercle bacillus Mycobacterium tuberculosis and are required for viability and virulence. While mycolic acid biosynthesis is studied extensively, components involved in mycolate transport remain unidentified. We investigated the role of large membrane proteins encoded by mmpL genes in mycolic acid transport in mycobacteria and the related corynebacteria. MmpL3 was found to be essential in mycobacteria and conditional depletion of MmpL3 in Mycobacterium smegmatis resulted in loss of cell wall mycolylation, and of the cell wall-associated glycolipid, trehalose dimycolate. In parallel, an accumulation of trehalose monomycolate (TMM) was observed, suggesting that mycolic acids were transported as TMM. In contrast to mycobacteria, we found redundancy in the role of two mmpL genes, in Corynebacterium glutamicum; a complete loss of trehalose-associated and cell wall bound corynomycolates was observed in an NCgl0228-NCgl2769 double mutant, but not in individual single mutants. Our studies highlight the role of mmpL genes in mycolic acid metabolism and identify potential new targets for anti-TB drug development.     

Evidence for a pterin-derivative associated with the molybdenum cofactor of Neurospora crassa nitrate reductase

Abstract— Following the method of Johnson and Rajagopalan (1982) for obtaining Form B of molybdopterin cofactors, we observed a prominent fluorescence band at480–482 nm in purified NR of Neurospora crassa mutant albino-band after boiling the enzyme at acidic pH and readjusting the sample to alkaline pH. This fluorescence band is maximally excited at 410 nm and maximally emitting at pH 11 (“F-480_pH11”); at pH4–7 only a featureless fluorescence band of low intensity remains (“F-480_pH5”). The fluorescence ΔF-480 = F-480_pH11 - F-480_pHS is examined here. ΔF-480 is associated specifically with NADPH-dependent and MVH-dependent nitrate reduction activities and with cytochrome b-557 absorption. In a protease-digested preparation lacking NADPH-dependent NR activity, ΔF-480 is associated with MVH-dependent nitrate reduction. The ΔF-480 signal is followed during the course of purification of NR. Its size increases with increasing purity of the enzyme. In partially purified NR preparations and especially in aqueous extracts from mycelia of N. crassa, a second, strong fluorescence signal with a pH-dependent emission maximum at around 450 nm (maximally excited at350–370 nm) was found beside ΔF-480. This “unspecific” signal was lost during NR purification. A procedure is developed to demonstrate AF-480 also in presence of the unspecific (350-370/450 nm) signal as well as flavins. We deduce that the ΔF-480 component is part of the Mo cofactor of N. crassa NR and that the signal is caused by a pterin derivative. From calculations of total content of the AF-480 component in mycelia it is likely that in vivo it is shared also by other enzymes.     

Problems associated with interferences in the analysis of serum for polychlorinated biphenyls

During a recent survey to determine serum concentrations of polychlorinated biphenyls (PCBs) among people living around New Bedford, MA, U.S.A., an unidentified contaminant precluded the quantification of some early eluting Webb and McCall peaks. Loss of data is estimated to have reduced reported serum levels by 12%. Efforts to identify the contaminant by gas chromatography with an electron-capture detector, a Hall electrolytic condutivity detector, and mass spectrometer were not successful. Researchers ascertained, however, that the contaminant is not a PCB, it does not contain halogens, but it may contain phthalates. Vacutainer tubes and closures for serum storage bottles are suspected sources of contamination.