A Frizzled-Like Cysteine-Rich Domain in Glypican-3 Mediates Wnt Binding and Regulates Hepatocellular Carcinoma Tumor Growth in Mice.

Wnt signaling is one of the key regulators of hepatocellular carcinoma (HCC) tumor progression. In addition to the classical receptor frizzled (FZD), various coreceptors including heparan sulfate proteoglycans (HSPGs) are involved in Wnt activation. Glypican-3 (GPC3) is an HSPG that is overexpressed in HCC and functions as a Wnt coreceptor that modulates HCC cell proliferation. These features make GPC3 an attractive target for liver cancer therapy. However, the precise interaction of GPC3 and Wnt and how GPC3, Wnt, and FZD cooperate with each other are poorly understood. In this study, we established a structural model of GPC3 containing a putative FZD-like cysteine-rich domain at its N-terminal lobe. We found that F41 and its surrounding residues in GPC3 formed a Wnt-binding groove that interacted with the middle region located between the lipid thumb domain and the index finger domain of Wnt3a. Mutating residues in this groove significantly inhibited Wnt3a binding, ß-catenin activation, and the transcriptional activation of Wnt-dependent genes. In contrast with the heparan sulfate chains, the Wnt-binding groove that we identified in the protein core of GPC3 seemed to promote Wnt signaling in conditions when FZD was not abundant. Specifically, blocking this domain using an antibody inhibited Wnt activation. In HCC cells, mutating residue F41 on GPC3 inhibited activation of ß-catenin in vitro and reduced xenograft tumor growth in nude mice compared with cells expressing wild-type GPC3. Conclusion: Our investigation demonstrates a detailed interaction of GPC3 and Wnt3a, reveals the precise mechanism of GPC3 acting as a Wnt coreceptor, and provides a potential target site on GPC3 for Wnt blocking and HCC therapy.

Association of Blood Pressure with Blood Lead and Cadmium Levels in Korean Adolescents: Analysis of Data from the 2010-2016 Korean National Health and Nutrition Examination Survey.

BACKGROUND: We evaluated the association of blood pressure (BP) with blood levels of cadmium, lead, and cadmium and lead together (cadmium + lead) in a representative sample of adolescents from Korea. METHODS: We used 2010-2016 data from the Korean National Health and Nutrition Examination Survey. This cross-sectional study enrolled adolescents aged at 10-18 years-old who completed a health examination survey and had blood measurements of lead and cadmium. The association of adjusted mean differences in diastolic and systolic BP with doubling of blood lead and cadmium were estimated by regression of BP against log2-transformed blood metals and their quartiles after covariate adjustment. Adjusted odds ratio for prehypertension were calculated for log2-transformed blood levels of lead and cadmium and their quartiles. RESULTS: Our analysis of adolescents in Korea indicated that blood levels of lead and cadmium were not significantly associated with increased BP or risk of prehypertension. However, the cadmium + lead level was associated with prehypertension. Previous studies showed that blood levels of lead and cadmium were associated with increased BP and risk of hypertension in adult populations. We found no such association in Korean adolescents. CONCLUSION: We found that the cadmium + lead level was associated with prehypertension. The differences between adults and adolescents are because adolescents generally have lower levels of these blood metals or because adolescents only rarely have hypertension.

Environmental exposures to lead, mercury, and cadmium among South Korean teenagers (KNHANES 2010-2013): Body burden and risk factors.

INTRODUCTION: Limited information is available on the association of age and sex with blood concentrations of heavy metals in teenagers. In addition, factors such as a shared family environment may have an association. We analyzed data from the Korean National Health and Nutrition Examination Survey (KNHANES, 2010-2013) to determine whether blood levels of heavy metals differ by risk factors such as age, sex, and shared family environment in a representative sample of teenagers. METHODS: This study used data obtained in the KNHANES 2010-2013, which had a rolling sampling design that involved a complex, stratified, multistage, probability-cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. Our cross-sectional analysis was restricted to teenagers and their parents who completed the health examination survey, and for whom blood measurements of cadmium, lead, and mercury were available. The final analytical sample consisted of 1585 teenagers, and 376 fathers and 399 mothers who provided measurements of blood heavy metal concentrations. RESULTS: Male teenagers had greater blood levels of lead and mercury, but sex had no association with blood cadmium level. There were age-related increases in blood cadmium, but blood lead decreased with age, and age had little association with blood mercury. The concentrations of cadmium and mercury declined from 2010 to 2013. The blood concentrations of lead, cadmium, and mercury in teenagers were positively associated with the levels in their parents after adjustment for covariates. CONCLUSION: Our results show that blood heavy metal concentrations differ by risk factors such as age, sex, and shared family environment in teenagers.

Recombinant immunotoxin for cancer treatment with low immunogenicity by identification and silencing of human T-cell epitopes.

Nonhuman proteins have valuable therapeutic properties, but their efficacy is limited by neutralizing antibodies. Recombinant immunotoxins (RITs) are potent anticancer agents that have produced many complete remissions in leukemia, but immunogenicity limits the number of doses that can be given to patients with normal immune systems. Using human cells, we identified eight helper T-cell epitopes in PE38, a portion of the bacterial protein Pseudomonas exotoxin A which consists of the toxin moiety of the RIT, and used this information to make LMB-T18 in which three epitopes were deleted and five others diminished by point mutations in key residues. LMB-T18 has high cytotoxic and antitumor activity and is very resistant to thermal denaturation. The new immunotoxin has a 93% decrease in T-cell epitopes and should have improved efficacy in patients because more treatment cycles can be given. Furthermore, the deimmunized toxin can be used to make RITs targeting other antigens, and the approach we describe can be used to deimmunize other therapeutically useful nonhuman proteins.

High carbohydrate diets are positively associated with the risk of metabolic syndrome irrespective to fatty acid composition in women: the KNHANES 2007-2014.

We evaluated that carbohydrates and fatty acids intake modifies the prevalence of metabolic syndrome (MetS) and we also determined gender-nutrient interaction in 38,766 adults in KNHANES (2007-2014). Carbohydrate intake was positively associated, and fat intake inversely associated, with the incidence of MetS. The association exhibited a gender interaction with the macronutrient intake; this association was significant in females. Furthermore, saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) intakes were inversely associated with MetS risk and only females showed the positive association. Both n-3 and n-6 fatty acids intake showed inverse associations with MetS risk, similar to PUFA intake. Among the MetS components, serum triglyceride levels and blood pressure had significant inverse associations with fatty acid intake irrespective of fatty acid types and exhibited a gender interaction. In conclusions, high carbohydrate intakes (≥74.2 En%) may increase the MetS risk and moderate fat intakes (≥20.7 En%), irrespective of fat types, may decrease it. These associations were significant only in women.

SymD webserver: a platform for detecting internally symmetric protein structures.

Internal symmetry of a protein structure is the pseudo-symmetry that a single protein chain sometimes exhibits. This is in contrast to the symmetry with which monomers are arranged in many multimeric protein complexes. SymD is a program that detects proteins with internal symmetry. It proved to be useful for analyzing protein structure, function and modeling. This web-based interactive tool was developed by implementing the SymD algorithm. To the best of our knowledge, SymD webserver is the first tool of its kind with which users can easily study the symmetry of the protein they are interested in by uploading the structure or retrieving it from databases. It uses the Galaxy platform to take advantage of its extensibility and displays the symmetry properties, the symmetry axis and the sequence alignment of the structures before and after the symmetry transformation via an interactive graphical visualization environment in any modern web browser. An Example Run video displays the workflow to help users navigate. SymD webserver is publicly available at http://symd.nci.nih.gov.

Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B-cell epitopes.

Recombinant immunotoxins (RITs) are hybrid proteins used to treat cancer. These proteins are composed of an Fv that reacts with cancer cells joined to a portion of Pseudomonas exotoxin A, which kills the cell. Because the toxin is a foreign protein, it can induce neutralizing antibodies and thereby limit the number of doses a patient can receive. We previously identified seven major mouse B-cell epitopes in the toxin, and subsequently silenced them using point mutations that converted large hydrophilic amino acids to alanine, yet retained full antitumor activity. Here we present results in which we identify and silence human B-cell epitopes in the RIT HA22. We obtained B cells from patients with antibodies to RITs, isolated the corresponding variable fragments (Fvs), and constructed a phage-display library containing Fvs that bind to the RITs. We then used alanine scanning mutagenesis to locate the epitopes. We found that human and mouse epitopes frequently overlap but are not identical. Most mutations that remove mouse epitopes did not remove human epitopes. Using the epitope information, we constructed a variant immunotoxin, HA22-LR-LO10, which has low reactivity with human antisera, yet has high cytotoxic and antitumor activity and can be given to mice at high doses without excess toxicity. The toxin portion of this RIT (LR-LO10) can be used with Fvs targeting other cancer antigens and is suitable for clinical development.

Self-rated Subjective Health Status Is Strongly Associated with Sociodemographic Factors, Lifestyle, Nutrient Intakes, and Biochemical Indices, but Not Smoking Status: KNHANES 2007-2012.

Despite advertised health warnings regarding the deadly hazards of smoking, many people have not heeded recommendations to quit smoking. We examined factors that affect self-rated subjective health status (SRH) scores among lifestyle, nutrient intake and biochemical parameters, and the association of SRH scores and smoking status in a large Korean adult population. Adjusted odd ratios for SRH were calculated for smoking status, selected biochemical data, and food and nutrient intake obtained using the 24-hr recall method after covariate adjustment in the 2007-2012 Korean National Health and Nutrition Examination Survey (27,534 men and women aged ≥ 20 yr). Age, sex, income, education, drinking, exercise and stress levels were associated with SRH scores, regardless of smoking status (P < 0.001). Interestingly, people in any smoking status groups considered the well-known indicators for metabolic diseases (HDL cholesterol, glucose, aspartate aminotransferase, and alanine aminotransferase in the circulation), and the intake of fiber, total vitamins A, and vitamin C as indicators of SRH. Especially in current smokers, higher intake of nutritious food groups such as grains (OR = 1.227), vegetables (OR = 1.944), and milk (OR = 2.26) significantly increased the adjusted odds ratio of SRH. However, smoking status was not associated with SRH scores. In conclusion, SRH is affected by the indices related to health but not smoking status in Korean adults. The development of a new indicator of the direct adverse effects of smoking at regular health check-ups might be required to modulate the SRH in smokers and a nutritional education should not include the possible attenuation of adverse effects of smoking by good nutrition.

Assessment of template-free modeling in CASP10 and ROLL.

We present the assessment of predictions for Template-Free Modeling in CASP10 and a report on the first ROLL experiment wherein predictions are collected year round for review at the regular CASP season. Models were first clustered so that duplicated or very similar ones were grouped together and represented by one model in the cluster. The representatives were then compared with targets using GDT_TS, QCS, and three additional superposition-independent score functions newly developed for CASP10. For each target, the top 15 representatives by each score were pooled to form the Top15Union set. All models in this set were visually inspected by four of us independently using the new plugin, EvalScore, which we developed with the UCSF Chimera group. The best models were selected for each target after extensive debate among the four examiners. Groups were ranked by the number of targets (hits) for which a group's model was selected as one of the best models. The Keasar group had most hits in both categories, with four of 19 FM and eight of 36 ROLL targets. The most successful prediction servers were QUARK from Zhang's group for FM category with three hits and Zhang-server for the ROLL category with seven hits. As observed in CASP9, many successful groups were not true "template-free" modelers but used remote templates and/or server models to obtain their winning models. The results of the first ROLL experiment were broadly similar to those of the CASP10 FM exercise.

Assessment of CASP10 contact-assisted predictions.

In CASP10, for the first time, contact-assisted structure predictions have been assessed. Sets of pairs of contacting residues from target structures were provided to predictors for a second round of prediction after the initial round in which they were given only sequences. The objective of the experiment was to measure model quality improvement resulting from the added contact information and thereby assess and help develop so-called hybrid prediction methods--methods where some experimentally determined distance constraints are used to augment de novo computational prediction methods. The results of the experiment were, overall, quite promising.

Definition and classification of evaluation units for CASP10.

For the 10th experiment on Critical Assessment of the techniques of protein Structure Prediction (CASP), the prediction target proteins were broken into independent evaluation units (EUs), which were then classified into template-based modeling (TBM) or free modeling (FM) categories. We describe here how the EUs were defined and classified, what issues arose in the process, and how we resolved them. EUs are frequently not the whole target proteins but the constituting structural domains. However, the assessors from CASP7 on combined more than one domain into 1 EU for some targets, which implied that the assessment also included evaluation of the prediction of the relative position and orientation of these domains. In CASP10, we followed and expanded this notion by defining multidomain EUs for a number of targets. These included 3 EUs, each made of two domains of familiar fold but arranged in a novel manner and for which the focus of evaluation was the interdomain arrangement. An EU was classified to the TBM category if a template could be found by sequence similarity searches and to FM if a structural template could not be found by structural similarity searches. The EUs that did not fall cleanly in either of these cases were classified case-by-case, often including consideration of the overall quality and characteristics of the predictions.

Familial interactions and physical, lifestyle, and dietary factors to affect bone mineral density of children in the KNHANES 2009-2010.

We examined familial bone mineral density (BMD) interactions between parents and children and lifestyle factors affecting BMD in the Korean general population of children under 20 and parents under 50 years of age. This cross-sectional study included 2,453 participants (667 daughters, 705 sons, 719 mothers, and 362 fathers) in the 2009-2010 Korean National Health and Nutrition Examination Survey. We calculated prevalence ratios and 95 % confidence intervals for BMD values of whole femur, femur neck, lumbar spine, and whole body excluding the head being in the low tertile in adolescents according to parental BMD tertile after adjusting for physical, lifestyle, and dietary factors. For daughters and sons, there were significant differences in BMD at the four bone sites according to age group, body fat percentage, regular walking and exercise, and milk consumption compared to the reference value for each classification category. Surprisingly, there were no differences in BMD according to serum 25-OH-D levels. Birth order affected BMD of only whole body except head, but its impact was less than that of lifestyle factors. The mean differences in BMD between daughters and sons in the first and third parental BMD tertiles were statistically significant. Notably, the prevalence ratio of whole body without head BMD being in the low tertile increased eight and ten-folds in adolescent daughters and sons, respectively, when parents were in the low BMD tertile. In specific bone regions, parental BMD had a greater effect on total femur in daughters but in the lumbar spine in sons. In conclusion, parental BMD positively influences BMD in daughters and sons after adjustment for environmental parameters. This suggests that the children from parents with low BMD need to make an extra effort to increase BMD through dietary and lifestyle changes.

Associations between blood mercury levels and subclinical changes in liver enzymes among South Korean general adults: analysis of 2008-2012 Korean national health and nutrition examination survey data.

INTRODUCTION: We herein used data from the Korean National Health and Nutritional Examination Survey (KNHANES) 2008-2012 to examine the associations between blood mercury levels and subclinical changes of liver function in a representative sample of the adult Korean population. METHODS: This study was based on data obtained from KNHANES, in which a rolling sampling design was used to perform a complex, stratified, multistage probability cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. The associations between subclinical hepatic changes and blood mercury levels were assessed after adjustment for various demographic and lifestyle factors. RESULTS: Multivariate linear regression analyses revealed that each doubling of blood mercury increased serum aspartate transaminase (AST) by 0.676U/L and serum alanine transaminase (ALT) by 1.067U/L. The mean differences (95% CI) in serum AST and ALT between the lowest and highest quartiles were statistically significant at 1.249 (0.263-2.235)U/L and 2.248 (0.648-3.848), respectively. Logistic regression analysis showed that the odd ratios for having serum AST and ALT levels above the median were statistically significant in both the models according to the increase of blood mercury. The risks of having serum AST and ALT levels higher than the median among subjects in 4th quartile of blood mercury were 1.524 and 1.947, respectively. DISCUSSION: The present findings show that subclinical changes of liver function are associated with blood mercury levels. This is the first study to show an association between blood mercury levels and mild liver dysfunction, as a possible proxy measure of nonalcoholic fatty liver disease (NAFLD), in Asian population.

Iron deficiency increases blood lead levels in boys and pre-menarche girls surveyed in KNHANES 2010-2011.

INTRODUCTION: We present data from the Korean National Health and Nutritional Examination Survey (KNHANES) 2010-11 on the distribution of blood lead levels, and examine their association with iron deficiency in a representative sample of the adolescent Korean population. METHODS: This study was based on data obtained from KNHANES, in which a rolling sampling design was used to perform a complex, stratified, multistage probability cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. Serum ferritin was categorized into three levels: low (serum ferritin <15.0µg/L), low normal (serum ferritin 15.0-30.0µg/L for girls and 15.0-50.0 for boys), and normal (serum ferritin ≥30.0µg/L for girls and ≥50.0 for boys), and its association with blood lead levels was assessed after adjustment for various demographic and lifestyle factors. RESULTS: The geometric mean (GM) of blood lead in the low serum ferritin group was significantly higher than that in the normal group among boys but not girls. After controlling for covariates, multiple regression analysis showed that blood lead was inversely correlated with serum ferritin levels in boys and pre-menarche girls only. DISCUSSION: The present study shows that iron deficiency increases blood lead levels in a representative sample of the male and pre-menarche female adolescent population, as evaluated in KNHANES. The confounding effect of estrogen on blood lead levels should be considered.

Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes.

Many nonhuman proteins have useful pharmacological activities, but are infrequently effective in humans because of their high immunogenicity. A recombinant immunotoxin (HA22, CAT8015, moxetumomab pasudotox) composed of an anti-CD22 antibody variable fragment fused to PE38, a 38-kDa portion of Pseudomonas exotoxin A, has produced many complete remissions in drug-resistant hairy-cell leukemia when several cycles of the agent can be given, but has much less activity when antibodies develop. We have pursued a strategy to deimmunize recombinant immunotoxins by identifying and removing B-cell epitopes. We previously reported that we could eliminate most B-cell epitopes using a combination of point mutations and deletions. Here we show the location and amino acid composition of all of the B-cell epitopes in the remaining 25-kDa portion of Pseudomonas exotoxin. Using this information, we eliminated these epitopes to produce an immunotoxin (HA22-LR-8M) that is fully cytotoxic against malignant B-cell lines, has high cytotoxic activity against cells directly isolated from patients with chronic lymphocytic leukemia, and has excellent antitumor activity in mice. HA22-LR-8M does not induce antibody formation in mice when given repeatedly by intravenous injection and does not induce a secondary antibody response when given to mice previously exposed to HA22. HA22-LR-8M also has greatly reduced antigenicity when exposed to sera from patients who have produced antibodies to HA22. The properties of HA22-LR-8M make it an excellent candidate for further clinical development.

Blood cadmium concentration of residents living near abandoned metal mines in Korea.

The purpose of this study was to investigate demographic and lifestyle variables and blood cadmium concentrations in residents living near abandoned metal mines in Korea. Blood cadmium concentrations were measured in 15,161 subjects living around abandoned metal mines (exposed group, n = 14,464) and compared with those living in designated control areas (control group, n = 697). A questionnaire was provided to all subjects to determine age, gender, mine working history, times of residence, smoking habits and dietary water type. The geometric mean (95% confidence intervals) of blood cadmium concentration (1.25 [1.24-1.27] µg/L) in the exposed group was significantly higher than in the control group (1.17 [1.13-1.22] µg/L). Mean residence time and mine working history in the exposed group were significantly higher than in the control group. Blood cadmium concentrations increased with increasing age, and residence time in both groups, and blood cadmium concentrations were higher in current-smokers than in non-smokers in both groups. This study shows the geometric mean of blood cadmium concentration in abandoned mining areas are higher than in non-mining areas in the general adult Korean population.

Blood levels of cadmium and lead in residents near abandoned metal mine areas in Korea.

We analyzed national data on blood lead levels (BLL) and blood cadmium levels (BCL) in residents living near 38 abandoned metal mining areas (n = 5,682, 18-96 years old) in Korea that were collected by the first Health Effect Surveillance for Residents in Abandoned Metal mines (HESRAM) from 2008 to 2011. The geometric mean BCL and BLL were 1.60 µg/L (95 % CI = 1.57-1.62 µg/L) and 2.87 µg/dL (95 % CI = 2.84-2.90 µg/dL), respectively, notably higher than levels in the general population in Korea and other countries. We found significantly higher BLL and BCL levels in people living within 2 km of an abandoned metal mine (n = 3,165, BCL = 1.87 µg/L, BLL = 2.91 µg/dL) compared to people living more than 2 km away (n = 2,517, BCL = 1.31 µg/L, BLL = 2.82 µg/dL; P < 0.0001) and to the general population values reported in the literature.

Association between the red cell distribution width and mortality in elderly patients with non-traumatic coma: An observational cohort study.

Elevated red blood cell distribution width (RDW) can be associated with disease severity. However, studies on RDW for the prognosis of elderly patients with non-traumatic coma (NTC) are lacking. This study aims to examine the relationship between RDW and outcomes in elderly patients with NTC. This observational cohort study included elderly patients (aged ≥ 65 years) with NTC between January 2022 and December 2022. We measured RDW upon patient arrival at the emergency department (ED). We conducted a multivariable analysis using logistic regression of relevant covariates to predict in-hospital mortality. Survival curves based on 30-day mortality were designed using the Kaplan-Meier method. The primary outcome was in-hospital mortality, and the secondary outcome was 30-day mortality. A total of 689 patients were included in the study, and in-hospital mortality was 29.6% (n = 204). Our results found that the RDWs of non-survivors were significantly greater than those of survivors (14.6% vs 13.6%). Multivariable analysis showed that RDWs at ED arrival were independently associated with in-hospital mortality (odds ratio, 1.126; 95% confidence interval, 1.047-1.212; P < .001). The Kaplan-Meier curve indicated that the survival probability of patients with a low RDW was greater than those with a high RDW. Having a high RDW at ED arrival was associated with in-hospital mortality in elderly patients with NTC.

DOMIRE: a web server for identifying structural domains and their neighbors in proteins.

SUMMARY: The DOMIRE web server implements a novel, automatic, protein structural domain assignment procedure based on 3D substructures of the query protein which are also found within structures of a non-redundant protein database. These common 3D substructures are transformed into a co-occurrence matrix that offers a global view of the protein domain organization. Three different algorithms are employed to define structural domain boundaries from this co-occurrence matrix. For each query, a list of structural neighbors and their alignments are provided. DOMIRE, by displaying the protein structural domain organization, can be a useful tool for defining protein common cores and for unravelling the evolutionary relationship between different proteins. AVAILABILITY: http://genome.jouy.inra.fr/domire CONTACT: jean.garnier@jouy.inra.fr.

Increased erythrocyte lead levels correlate with decreased hemoglobin levels in the Korean general population: analysis of 2008-2010 Korean National Health and Nutrition Examination Survey data.

INTRODUCTION: We present data from the Korean National Health and Nutrition Examination Survey 2008-2010 on the association between blood lead and hemoglobin levels in a representative sample of the adult South Korean population. METHODS: The analysis was restricted to participants ≥20 years of age who completed the health examination survey, including blood lead measurements (n = 5,951). Multivariate linear regression analyses were performed to estimate adjusted mean differences in hemoglobin level associated with doubling of whole blood and erythrocyte lead or quartiles of the metal after covariate adjustment. Odds ratios (ORs) for having borderline anemia or clinical anemia were calculated for log-transformed whole blood and erythrocyte lead or quartiles of the metal after covariate adjustment. RESULTS: A twofold increase in whole blood lead or erythrocyte lead was associated with a 0.285 g/dL increase or 0.088 g/dL decrease in hemoglobin level, respectively. There was a 0.416 g/dL increase or 0.143 g/dL decrease in hemoglobin, respectively, in the highest, compared with the lowest tertile of whole blood lead and erythrocyte lead, respectively. Based on ORs, doubling of whole blood lead or erythrocyte lead resulted in a 36.3 % decrease or 36.2 % increase, respectively, in the risk of borderline anemia. CONCLUSION: The association of whole blood lead versus erythrocyte lead with hemoglobin level was opposite. In the case of anemia, which is frequently caused by iron deficiency, the effect on the whole blood lead concentration may be very significant, leading to a considerable underestimation of the person's lead status. Therefore, hematocrit-adjusted blood lead level (i.e., erythrocyte lead) should be applied to the general population. To our knowledge, this is the first study to show that erythrocyte lead levels showed a significant inverse relationship with hemoglobin level at lead levels <10 µg/dL in the general Korean population. In conclusion, increased erythrocyte lead levels may be associated with mildly decreased hemoglobin levels, after adjusting for covariates, in a representative sample of the adult Korean population.