A Revised Classification of Primary Iron Overload Syndromes.

Background and Aims: The clinical introduction of hepcidin25 (Hep25) has led to a more detailed understanding of its relationship with ferroportin (FP) and divalent metal transporter1 in primary iron overload syndromes (PIOSs). In 2012, we proposed a classification of PIOSs based on the Hep25/FP system, which consists of prehepatic aceruloplasminemia, hepatic hemochromatosis (HC), and posthepatic FP disease (FP-D). However, in consideration of accumulated evidence on PIOSs, we aimed to renew the classification. Methods: We reviewed the 2012 classification and retrospectively renewed it according to new information on PIOSs. Results: Iron-loading anemia was included in PIOSs as a prehepatic form because of the newly discovered erythroferrone-induced suppression of Hep25, and the state of traditional FP-D was remodeled as the BIOIRON proposal. The key molecules responsible for prehepatic PIOSs are low transferrin saturation in aceruloplasminemia and increased erythroferrone production by erythroblasts in iron-loading anemia. Hepatic PIOSs comprise four genotypes of HC, in each of which the synthesis of Hep25 is inappropriately reduced in the liver. Hepatic Hep25 synthesis is adequate in posthepatic PIOSs; however, two mutant FP molecules may resist Hep25 differently, resulting in SLC40A1-HC and FP-D, respectively. PIOS phenotypes are diagnosed using laboratory tests, including circulating Hep25, followed by suitable treatments. Direct sequencing of the candidate genes may be outsourced to gene centers when needed. Laboratory kits for the prevalent mutations, such as C282Y, may be the first choice for a genetic analysis of HC in Caucasians. Conclusions: The revised classification may be useful worldwide.

Distribution Analysis of Iron and Copper by STEM-EDX Spectroscopy of Hemosiderin Particles in the Liver of Rats Overloaded With Iron.

BACKGROUND/AIM: Our recent studies have indicated that trace copper co-existed with iron in hemosiderin particles of human genetic iron overload. To understand this phenomenon, we analyzed hemosiderin particles in iron-overloaded rat liver by using scanning transmission electron microscopy - energy-dispersive X-ray (STEM-EDX) spectroscopy. MATERIALS AND METHODS: Samples for STEM-EDX spectroscopy were prepared from the liver of rats administered an intraperitoneal injection of dextran iron. RESULTS: The micro-domain analysis with STEM-EDX spectroscopy showed that dense bodies contained high levels of iron and trace copper. Quantitative analysis of copper levels in the liver specimen using atomic spectrophotometry showed that copper concentration in the liver was not increased by iron overload. These findings suggest that the overload of iron induced distribution of trace copper to hemosiderin particles without changing cellular copper levels. CONCLUSION: Co-existence of copper with iron was observed in hemosiderin particles of the liver of an experimental model of iron overload, suggesting that iron overload induced distribution of trace copper into hemosiderin particles.

Effects of Ursodeoxycholic Acid and Insulin on Palmitate-Induced ROS Production and Down-Regulation of PI3K/Akt Signaling Activity.

In obese and diabetic patients, plasma free fatty acid (FFA) levels are often elevated and may play a causal role in insulin resistance and reactive oxygen species (ROS) production. We have previously shown that ursodeoxycholic acid (UDCA) has antioxidative activity through the phosphatidylinositol 3-kinase (PI3K)/Akt signaling-mediated glutathione production. In this study, we investigated the effects of UDCA on insulin response by analyzing intracellular ROS and the activation of the PI3K/Akt signaling pathway in HepG2 cells treated with palmitate. The level of ROS was quantified using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), and the activation of the PI3K/Akt signaling pathway was determined by Western blotting assay using appropriate antibodies. The intracellular ROS levels were increased by palmitate but were reduced by treatment with UDCA and insulin. Furthermore, insulin significantly stimulated the phosphorylation of Akt. When the cells were pre-treated with palmitate, insulin-induced Akt-phosphorylation was markedly inhibited. However, when the cells were treated with palmitate and UDCA, the effects of insulin were partially restored. UDCA may have protective effects against palmitate-induced decreases in responsiveness to insulin.

(-)-Epigallocatechin-3-gallate Down-regulates Doxorubicin-induced Overexpression of P-glycoprotein Through the Coordinate Inhibition of PI3K/Akt and MEK/ERK Signaling Pathways.

BACKGROUND/AIM: (-)-Epigallocatechin-3-gallate (EGCG) has been indicated to regulate the function of P-glycoprotein (P-gp), which is a drug transporter encoded by the MDR1 (ABCB1) gene. P-gp expression is induced by doxorubicin (DOX). We aimed to clarify the mechanisms and inhibitory effects of EGCG on DOX-induced P-gp expression in HepG2 cells. MATERIALS AND METHODS: Rhodamine 123 (Rho123) was used for P-gp substrate. Western blotting and polymerase chain reactions (PCRs) were conducted using specific antibodies and primer sets. RESULTS: The DOX-pretreated cells accumulated a significantly decreased amount of Rho123), than control cells; however, the cells pretreated with EGCG and DOX, in combination, accumulated Rho123 more than DOX-pretreated cells. DOX induced the overexpression of MDR1 mRNA and increased the phosphorylation of Akt, ERK1/2, p38 MAPK and JNK. EGCG significantly inhibited the phosphorylation of Akt and ERK. The DOX-induced P-gp overexpression was partially suppressed by an inhibitor of MEK1/2 (U0126), but not by a PI3K inhibitor (LY294002). Interestingly, the expression of P-gp was synergistically inhibited by combined treatment of U0126 with LY294002 and also inhibited by an mTORC1 inhibitor, rapamycin. CONCLUSION: EGCG inhibited DOX-induced overexpression of P-gp through the coordinate inhibitory action on MEK/ERK and PI3K/Akt signaling pathways.

Inclusion bodies of aggregated hemosiderins in liver macrophages.

Hemosiderin formation is a structural indication of iron overload. We investigated further adaptations of the liver to excess iron. Five patients with livers showing iron-rich inclusions larger than 2 µm were selected from our database. The clinical features of patients and structures of the inclusions were compared with those of 2 controls with mild iron overload. All patients had severe iron overload with more than 5000 ng/mL of serum ferritin. Etiologies were variable, from hemochromatosis to iatrogenic iron overload. Their histological stages were either portal fibrosis or cirrhosis. Inclusion bodies were ultra-structurally visualized as aggregated hemosiderins in the periportal macrophages. X-ray analysis always identified, in addition to a large amount of iron complexes including oxygen and phosphorus, a small amount of copper and sulfur in the mosaic matrixes of inclusions. There were no inclusions in the control livers. Inclusion bodies, when the liver is loaded with excess iron, may appear in the macrophages as isolated organella of aggregated hemosiderins. Trace amounts of copper-sulfur complexes were always identified in the mosaic matrices of the inclusions, suggesting cuproprotein induction against excess iron. In conclusion, inclusion formation in macrophages may be an adaptation of the liver loaded with excess iron.

Phenotypes and Chronic Organ Damage May Be Different among Siblings with Wilson's Disease.

Background and Aims: Cloning of ATP7B provided evidence that Wilson's disease is a hepatic copper toxicosis with a variety of extrahepatic complications. Affected siblings with the same genetic background and exposure to similar environmental factors may be a good model for the study of genotype-phenotype correlation. Methods: Twenty-three affected siblings in 11 families were selected from a database. The first phenotypes were determined according to the international proposal. The final types of chronic organ damage were re-evaluated for life-long management. Results: Phenotypes were identical in 5 of the families and different in 6 of the families. The acute hepatic phenotype H1 was found in 3 younger siblings and 1 older sibling. All survived an acute episode of hemolysis with underlying chronic liver disease. One also presented complication with neurological disease. The neurological phenotype N1 with neuropsychiatric symptoms and hepatic disease was found in 2 aged siblings of 1 family, in an older sibling in 3 families and in the oldest sibling in 1 family. Phenotypes in siblings were mainly split by either H1 occurring in random order or age-dependent N1. Types of chronic organ damage were identical in 8 of the families and different in 3 of the families. The same combination of chronic liver disease was found in 6 families and chronic liver disease complicated with neurological disease in 2 families. Split organ damage in siblings was found when an older sibling was complicated by neurological disease. There was no reverse combination of a younger sibling being complicated by neurological disease in any of the families. Conclusion: Phenotype combinations of siblings were mainly modified by externally-induced hemolytic episodes, while chronic organ damage in siblings was split by age-dependent neurological complications.

Coexistence of Copper in the Iron-Rich Particles of Aceruloplasminemia Brain.

The interaction between iron and copper has been discussed in association with human health and diseases for many years. Ceruloplasmin, a multi-copper oxidase, is mainly involved in iron metabolism and its genetic defect, aceruloplasminemia (ACP), shows neurological disorders and diabetes associated with excessive iron accumulation, but little is known about the state of copper in the brain. Here, we investigated localization of these metals in the brains of three patients with ACP using electron microscopes equipped with an energy-dispersive x-ray analyzer. Histochemically, iron deposition was observed mainly in the basal ganglia and dentate nucleus, and to lesser degree in the cerebral cortex of the patients, whereas copper grains were not detected. X-ray microanalysis identified two types of iron-rich particles in their brains: dense bodies, namely hemosiderins, and their aggregated inclusions. A small number of hemosiderins and most inclusions contained a significant amount of copper which was enough for distinct Cu x-ray images. These copper-containing particles were observed more frequently in the putamen and dentate nucleus than the cerebral cortex. Coexistence of iron and copper was supported by good correlations in the molecular ratios between these two metals in iron-rich particles with Cu x-ray image. Iron-dependent copper accumulation in iron-rich particles may suggest that copper recycling is enhanced to meet the increased requirement of cuproproteins in iron overload brain. In conclusion, the iron-rich particles with Cu x-ray image were found in the ACP brain.

Exposure characterization of three major insecticide lines in urine of young children in Japan-neonicotinoids, organophosphates, and pyrethroids.

The use of neonicotinoid (NEO) insecticides has increased over the past decade not only in Japan but also worldwide, while organophosphate (OP) and pyrethroid (PYR) insecticides are still conventionally used in agriculture and domestic pest control. However, limited data are currently available on the NEO exposure levels, especially in children, who are particularly vulnerable to environmental toxicants. Thus, the purpose of this study was to characterize the exposure to NEOs, as well as OPs and PYRs, in three-year-old Japanese children by assessing the range, distribution, and seasonal differences of the urinary concentrations of seven NEOs (acetamiprid, clothianidin, dinotefuran, thiacloprid, thiamethoxam, imidacloprid, and nitenpyram); four OP metabolites (dialkylphosphates [DAPs]), including dimethylphosphate, dimethylthiophosphate, diethylphosphate, and diethylthiophosphate; and three PYR metabolites (3-phenoxybenzoic acid, trans-chrysanthemumdicarboxylic acid, and 3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropane carboxylic acid). Urine samples were collected from 223 children (108 males and 115 females) in the summer and winter months. The detection rates of NEOs were 58% for dinotefuran, 25% for thiamethoxam, 21% for nitenpyram, and <16% for all other NEOs. The median and maximum concentrations of the sum of the seven NEOs (ΣNEO) were 4.7 and 370.2nmol/g creatinine, respectively. Urinary ΣNEO, dimethylphosphate, and all PYR metabolite concentrations were significantly higher in the summer than in the winter (p<0.05). The creatinine-adjusted concentration of ΣNEO significantly correlated with those of all DAPs (p<0.05) but not with those of the PYR metabolites. Moreover, the NEO-detected group showed higher urinary ΣDAP (sum of four OP metabolites) concentrations than the group without NEO detection. These findings suggest that children in Japan are environmentally exposed to the three major insecticide lines, and that the daily exposure sources of NEOs are common to those of OPs.

Copper Accumulates in Hemosiderins in Livers of Patients with Iron Overload Syndromes.

In biology, redox reactions are essential and sometimes harmful, and therefore, iron metabolism is tightly regulated by cuproproteins. Since the state of copper in iron overload syndromes remains unclear, we investigated whether copper metabolism is altered in these syndromes. Eleven patients with iron overload syndromes participated in this study. The clinical diagnoses were aceruloplasminemia (n=2), hemochromatosis (n=5), ferroportin disease (n=2), and receiving excess intravenous iron supplementation (n=2). Liver specimens were analyzed using a light microscope and transmission electron microscope equipped with an X-ray analyzer. In addition to a large amount of iron associated with oxygen and phosphorus, the iron-rich hemosiderins of hepatocytes and Kupffer cells contained small amounts of copper and sulfur, regardless of disease etiology. Two-dimensional imaging clearly showed that cuproproteins were distributed homogenously with iron complexes within hemosiderins. Copper stasis was unlikely in noncirrhotic patients. The enhanced induction of cuproproteins by excess iron may contribute to copper accumulation in hemosiderins. In conclusion, we have demonstrated that copper accumulates in hemosiderins in iron overload conditions, perhaps due to alterations in copper metabolism.

Acute Hepatic Phenotype of Wilson Disease: Clinical Features of Acute Episodes and Chronic Lesions Remaining in Survivors.

BACKGROUND AND AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism, and an international group for the study of WD (IGSW) has proposed three phenotypes for its initial presentation: acute hepatic, chronic hepatic, and neurologic phenotypes. Characterization of the acute hepatic phenotype may improve our understanding of the disease. METHODS: Clinical features of 10 WD patients with the acute hepatic phenotype and characteristics of chronic lesions remaining in survivors were assessed by the European Association for the Study of the Liver (EASL) guidelines. RESULTS: All six patients younger than 30 years had survived an acute episode of hemolytic anemia with residual liver disease of cirrhosis or chronic hepatitis. The acute episode was self-limiting in two of the four patients over the age of 30 years and progressed to acute liver failure in the other two patients. One of the two survivors had residual liver disease of chronic hepatitis, while the other had chronic hepatitis and neurologic disease. Neurologic disease remained in a patient who successfully received a liver transplantation. During acute episodes, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) changed rapidly along with anemia. Liver-specific ALT levels were age-dependently correlated with hemoglobin (Hb) concentrations. Enzyme reduction was milder for AST than ALT, which resulted in a high AST/ALT ratio in the anemic stage. The anemic stage in two patients transformed to acute liver failure. CONCLUSIONS: All survivors of an acute episode of the acute hepatic phenotype had residual liver disease or both liver and neurologic diseases. The rapid changes in liver enzymes during the acute episode and the liver and neurologic diseases remaining in survivors may provide a better understanding of WD.

A revised method for determination of dialkylphosphate levels in human urine by solid-phase extraction and liquid chromatography with tandem mass spectrometry: application to human urine samples from Japanese children.

OBJECTIVES: Biological monitoring of organophosphorus insecticide (OP) metabolites, specifically dialkylphosphates (DAP) in urine, plays a key role in low-level exposure assessment of OP in individuals. The aims of this study are to develop a simple and sensitive method for determining four urinary DAPs using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS), and to assess the concentration range of urinary DAP in Japanese children. METHODS: Deuterium-labeled DAPs were used as internal standards. Urinary dimethylphosphate (DMP) and diethylphosphate (DEP), which passed through the solid-phase extraction (SPE) column, and dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP), which were extracted from a SPE column using 2.5 % NH3 water including 50 % acetonitrile, were prepared for separation analysis. The samples were then injected into LC-MS/MS. The optimized method was applied to spot urine samples from 3-year-old children (109 males and 116 females) living in Aichi Prefecture in Japan. RESULTS: Results from the validation study demonstrated good within- and between-run precisions (<10.7 %) with low detection limits (0.4 for DMP and DMTP, 0.2 for DEP and 0.1 µg/L for DETP). The geometric mean values and detection rates of the urinary DAPs in Japanese children were 14.4 µg/L and 100 % for DMP, 5.3 µg/L and 98 % for DMTP, 5.5 µg/L and 99 % for DEP, and 0.6 µg/L and 80 % for DETP, respectively. CONCLUSIONS: The present high-throughput method is simple and reliable, and can thereby further contribute to development of an exposure assessment of OP. The present study is the first to reveal the DAP concentrations in young Japanese children.

Biochemical staging of the chronic hepatic lesions of Wilson disease.

BACKGROUND: Copper toxicity steadily affects the livers of patients with Wilson disease. However, the toxic effect of copper on serum aspartate and alanine aminotransferase levels remains to be clarified as a prerequisite for diagnostic tests. The clinical records of 33 cases were analyzed to clarify the natural history of Wilson disease. Phenotypes were simplified into hepatic, acute, and neurologic. The bio-low stage of both enzymes was less than 40 IU/L, the bio-moderate stage was intermediate between 40 and 200 IU/L, and the bio-high stage was more than 200 IU/L of either or both enzymes. Rebounded enzyme levels at the recovery period from anemia were presumed to be the chronic baselines when pre-anemic enzyme levels were not available in the acute phenotype. We investigated whether these enzyme levels may provide information useful for screening patients. The natural history of chronic Wilson disease consisted of the first increasing and second decreasing phases. The clinical courses of a 4-year-old boy and 12-year-old girl were representative of the 2 phases, respectively. All but one patient were in the decreasing phase. Negative correlations were obtained between age and enzyme level in the decreasing phase. The hepatic phenotype may be a prototype found throughout the 2 phases, and acute and neurologic phenotypes may be major complications in the bio-moderate and bio-low stages of the decreasing phase, respectively. Biochemical staging may provide a better understanding of Wilson disease when combined with phenotypes. Bio-high stage patients should be referred to a medical center for diagnosis.

Ursodeoxycholic acid inhibits overexpression of P-glycoprotein induced by doxorubicin in HepG2 cells.

The hepatoprotective action of ursodeoxycholic acid (UDCA) was previously suggested to be partially dependent on its antioxidative effect. Doxorubicin (DOX) and reactive oxygen species have also been implicated in the overexpression of P-glycoprotein (P-gp), which is encoded by the MDR1 gene and causes antitumor multidrug resistance. In the present study, we assessed the effects of UDCA on the expression of MDR1 mRNA, P-gp, and intracellular reactive oxygen species levels in DOX-treated HepG2 cells and compared them to those of other bile acids. DOX-induced increases in reactive oxygen species levels and the expression of MDR1 mRNA were inhibited by N-acetylcysteine, an antioxidant, and the DOX-induced increase in reactive oxygen species levels and DOX-induced overexpression of MDR1 mRNA and P-gp were inhibited by UDCA. Cells treated with UDCA showed improved rhodamine 123 uptake, which was decreased in cells treated with DOX alone. Moreover, cells exposed to DOX for 24h combined with UDCA accumulated more DOX than that of cells treated with DOX alone. Thus, UDCA may have inhibited the overexpression of P-gp by suppressing DOX-induced reactive oxygen species production. Chenodeoxycholic acid (CDCA) also exhibited these effects, whereas deoxycholic acid and litocholic acid were ineffective. In conclusion, UDCA and CDCA had an inhibitory effect on the induction of P-gp expression and reactive oxygen species by DOX in HepG2 cells. The administration of UDCA may be beneficial due to its ability to prevent the overexpression of reactive oxygen species and acquisition of multidrug resistance in hepatocellular carcinoma cells.

A non-invasive biomonitoring method for assessing levels of urinary pyrethroid metabolites in diapered children by gas chromatography-mass spectrometry.

The aim of this study was to develop a method for quantitative measurement of urinary metabolites of pyrethroid (PYR) insecticides, trans-chrysanthemumdicarboxylic acid (CDCA) and 3-phenoxybenzoic acid (3-PBA), extracted from disposable diapers. This study was approved by the university ethics committees, and informed consent was obtained from all the parents for their children and from adult volunteers. After extraction of PYR metabolites in the absorber of diapers with 5 ml acetone, the metabolites in the eluents were extracted with tert-butyl methyl ether, derivatized with 1,1,1,3,3,3-hexafluoroisopropanol and analyzed by gas chromatography-mass spectrometry. The limits of quantitation (LOQs) were 0.55 µg/l for CDCA and 0.09 µg/l for 3-PBA in 2 ml urine extracted from diapers. Within-series and between-day precisions were <14% (CV%) over the concentration range of metabolites from 0.4 to 20.4 µg/l urine. When concentrations of each metabolite were measured with the developed method after pouring 2 ml urine, which was obtained from adults both in a general population and pest control operators, on diapers, good correlations were shown between the measured results and the concentrations measured directly for the respective urine with the conventional method (Spearman's rank correlation coefficient 0.889 for CDCA and 0.989 for 3-PBA; n=27-28). The developed method would be applicable to epidemiological studies.

Quantitation of neonicotinoid metabolites in human urine using GC-MS.

A rapid and sensitive analytical method using gas chromatography-mass spectrometry (GC-MS) was developed for the measurement of neonicotinoid (NEO) metabolites 6-chloronicotinic acid (6CN), 2-chloro-1,3-thiazole-5-carboxylic acid (2CTCA) and 3-furoic acid (3FA) from human urine. After acid hydrolysis, the metabolites were extracted using solid phase extraction (SPE) column (Bond Elute Plexa PCX) and eluted with methanol. N,O-bis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane (BSTFA-TMCS, 99:1) was used for the derivatization of metabolites and analyzed by GC-MS with the electron ionization mode. The elution solvent, derivatization reagent and its conditions were mainly optimized for improved detection and quantitation of the metabolites based on signal-to-noise ratio, recoveries and reproducibility. Our present method offered a sufficiently low limit of detection (0.1µg/L for each metabolite) with satisfactory within-run and between-day accuracy and precision (variability less than 12.3%, R.S.D). This method is simple, sensitive and precise, and has been successfully applied to quantify low concentrations of urinary 6CN, 2CTCA and 3FA for the occupational NEO exposures survey.

Various copper and iron overload patterns in the livers of patients with Wilson disease and idiopathic copper toxicosis.

Wilson disease (WD) is a major type of primary copper toxicosis associated with hypoceruloplasminemia, while idiopathic copper toxicosis (ICT) is a minor type characterized by normoceruloplasminemia. Because ceruloplasmin is the major circulating ferroxidase, iron metabolism may be affected in patients with WD. Biopsied liver specimens obtained from patients with primary copper toxicosis were fixed with glutaraldehyde solution and embedded in epoxy resin. Ultrathin sections that had or had not been stained with uranyl acetate solution were examined under an electron microscope equipped with an energy dispersive X-ray analyzer. A 7-year-old boy with WD was free from any metal overloading at the pre-treatment stage. Pre-treatment liver specimens of another 16 patients showed a variety of copper and iron overload patterns, from isolated copper to evenly distributed combined overloading. A 19-year-old female patient was free from any metal overloading after 7 years of treatment. Post-treatment overloading in another 6 patients ranged between evenly distributed combined patterns and isolated iron patterns. All patients had hypoceruloplasminemia throughout treatment periods. A patient with normoceruloplasminemic ICT continued to display isolated copper overloading after 2.5 years of treatment. In conclusion, these observations support the hypothesis that iron accumulates in patients with hypoceruloplasminemia.

Clinicopathological study of Japanese patients with genetic iron overload syndromes.

In addition to hemochromatosis, aceruloplasminemia and ferroportin disease may be complicated by iron-induced multiple organ damage. Therefore, clinicopathological features should be evaluated in a wider range of genetic iron disorders. This study included 16 Japanese patients with genetic iron overload syndromes. The responsible genes were CP in four, HAMP in one, HJV in three, TFR2 in five, and SLC40A1 in three patients. No phenotype dissociation was observed in patients with the CP, TFR2, or HAMP genotypes. Two of the three patients with the HJV genotype displayed classic hemochromatosis instead of the juvenile type. Patients with the SLC40A1 genotype were affected by mild iron overload (ferroportin A) or severe iron overload (ferroportin B). Transferrin saturation was unusually low in aceruloplasminemia patients. All patients, except those with ferroportin disease, displayed low serum hepcidin-25 levels. Liver pathology showed phenotype-specific changes; isolated parenchymal iron loading in aceruloplasminemia, periportal fibrosis associated with heavy iron overload in both parenchymal and Kupffer cells of ferroportin B, and parenchyma-dominant iron-loading cirrhosis in hemochromatosis. In contrast, diabetes occurred in all phenotypes of aceruloplasminemia, hemochromatosis, and ferroportin disease B. In conclusion, clinicopathological features were partially characterized in Japanese patients with genetic iron overload syndromes.

Urinary concentrations of organophosphorus insecticide metabolites in Japanese workers.

A recent development in analytical chemistry has enabled us to monitor systemic organophosphorus insecticide (OP) exposure at individual levels. At present, however, limited data are currently available on urinary OP metabolite levels worldwide. The purpose of this study was to assess urinary dialkylphosphate (DAP) concentrations in Japanese workers. Urine samples were collected in both summer and winter from 339 Japanese adults who worked as food distributors (FDs, n=164), apple farmers (AFs, n=147) and pest control operators (PCOs, n=28). DAPs were measured by gas chromatography-mass spectrometry after derivatization with pentafluorobenzylbromide. Dimethylphosphate (DMP), diethylphosphate (DEP), dimethylthiophosphate (DMTP) and diethylthiophosphate (DETP) were detected in the urine of over 87% of the studied populations in both seasons. The geometric mean values of total DAPs (nmol g(-1) creatinine), DMP, DMTP, DEP and DETP (µg g(-1) creatinine) in summer and winter were 106.7 and 98.3, 7.0 and 3.8, 3.4 and 4.5, 0.8 and 1.5, and 0.3 and 0.2 for the FDs, 440.8 and 197.7, 33.1 and 10.8, 10.1 and 5.8, 4.2 and 4.7 and 1.6 and 0.8 for the AFs, and 473.4 and 284.6, 28.9 and 22.2, 17.6 and 4.6, 3.5 and 4.4, and 0.5 and 0.6 for the PCOs, respectively, thereby revealing significantly higher concentrations in AFs and PCOs groups than in the FDs in both seasons except for winter DMTP. These DAP concentrations were approximately the same or at lower levels compared with those reported in the previous literature. This is one of the first studies to demonstrate urinary DAP concentrations in Japanese adults.

Identification of a novel mutation in the HAMP gene that causes non-detectable hepcidin molecules in a Japanese male patient with juvenile hemochromatosis.

Hepcidin is an iron-regulatory hepatic peptide hormone encoded by the HAMP gene that downregulates iron export from enterocytes and macrophages into the blood plasma. In this study, we identified a novel mutation in the HAMP gene of a 58-year-old Japanese male patient with hemochromatosis. By direct sequencing of the five hereditary hemochromatosis-related genes, HFE, HAMP, HJV, TFR2, and SLC40A1, the previously unreported p.R75X mutation was identified, and the patient was found to be homozygous for the mutation. No other potentially pathogenic mutations were detected. In an LC-MS/MS analysis, hepcidin molecules were not detected in the patient's serum or urine. These results indicate that the p.R75X mutation causes iron overload by impairing the hepcidin system.

Potential of the international scoring system for the diagnosis of Wilson disease to differentiate Japanese patients who need anti-copper treatment.

AIM: Patients with Wilson disease show complex clinical features. Accurate diagnosis at the initial clinical manifestation is important for patients to receive effective treatment with anti-copper agents. In this study, we assessed whether the international scoring system for the diagnosis of Wilson disease is a reliable tool for screening Japanese patients with primary copper toxicosis requiring anti-copper treatment. METHODS: Twenty-three Japanese patients suspected of Wilson disease were enrolled in this study. We performed long-range polymerase chain reaction to detect ATP7B mutations in this series. Finally, we retrospectively assessed the reliability of using a diagnostic score of 4 or more points as the cut-off for this scoring system. RESULTS: Ten patients were homozygous or compound heterozygous for ATP7B mutations including a novel mutation of 3837 bp deletion including 3 exons. The mutation would have been missed by the traditional analysis. Six patients were heterozygous for ATP7B mutations. Three of these six patients had additional diagnostic points. The other three patients were diagnosed as carriers of a mutant gene based on their low scores. One of the seven patients free from ATP7B mutation was affected by copper toxicosis. Though the score was 3 points based on increased urinary copper and copper-positive cirrhosis, anti-copper treatment promptly improved liver failure, which was likely due to idiopathic copper toxicosis. CONCLUSION: The international scoring system for diagnosis of Wilson disease is a fairly reliable tool for screening Japanese patients who need anti-copper treatment. Caution is needed for patients with possible idiopathic copper toxicosis because the maximal score is 4 points.