Effect of colestimide on the concentrations of polychlorinated dibenzo-p-dioxins, polychlorinated dizenzofurans, and polychlorinated biphenyls in blood of Yusho patients.

BACKGROUND: Oral colestimide was reported to lower the concentration of PCDDs, PCDFs, and PCB in the blood of humans. A pilot study showed that the arithmetic mean total TEQ concentrations of PCDDs, PCDFs, and PCBs in the blood of subjects after the trial decreased approximately 20 % compared to pre-trial levels, suggesting that colestimide could decrease human dioxin levels. We designed the current clinical trial study based on this information. In this study, we examined whether colestimide could reduce the individual congener concentrations of PCDDs, PCDFs, and PCBs in the blood of Yusho patients. METHODS: Out of the 36 Yusho patients who participated in the clinical trial, 26 patients self-administered colestimide 3 g/day orally for 6 months. The concentrations of PCDDs, PCDFs and PCBs in the blood of 26 Yusho patients before the trial were compared with those after the trial. RESULTS: The arithmetic mean total TEQ concentrations of PCDDs, PCDFs, non-ortho PCBs, and mono-ortho PCBs in the blood of the 26 Yusho patients before and after the clinical trial were 42-303 (mean: 130, median: 120) and 43-283 (mean: 132, median: 118) pg TEQ/g lipid, respectively. The sums of the concentrations of 58 PCB congeners measured in the blood of Yusho patients before and after the trial were 321-2643 (mean: 957, median: 872) and 286-2007 (mean: 975, median: 806) ng/g lipid, respectively, indicating that the concentrations of PCDDs, PCDFs, and PCBs after the trial were almost the same as those before the trial. Among congeners of PCDDs, PCDFs, dioxin-like PCBs, and non-dioxin-like PCBs, most congeners of these compounds did not show a statistically significant decrease after the trial. CONCLUSION: Colestimide may not be beneficial in reducing the high blood levels of dioxin-like compounds in Yusho patients.

Pitfalls in Erythrocyte Protoporphyrin Measurement for Diagnosis and Monitoring of Protoporphyrias.

BACKGROUND: Laboratory diagnosis of erythropoietic protoporphyria (EPP) requires a marked increase in total erythrocyte protoporphyrin (300-5000 µg/dL erythrocytes, reference interval <80 µg/dL) and a predominance (85%-100%) of metal-free protoporphyrin [normal, mostly zinc protoporphyrin (reference intervals for the zinc protoporphyrin proportion have not been established)]; plasma porphyrins are not always increased. X-linked protoporphyria (XLP) causes a similar increase in total erythrocyte protoporphyrin with a lower fraction of metal-free protoporphyrin (50%-85% of the total). CONTENT: In studying more than 180 patients with EPP and XLP, the Porphyrias Consortium found that erythrocyte protoporphyrin concentrations for some patients were much higher (4.3- to 46.7-fold) than indicated by previous reports provided by these patients. The discrepant earlier reports, which sometimes caused the diagnosis to be missed initially, were from laboratories that measure protoporphyrin only by hematofluorometry, which is intended primarily to screen for lead poisoning. However, the instrument can calculate results on the basis of assumed hematocrits and reports results as "free" and "zinc" protoporphyrin (with different reference intervals), implying separate measurements of metal-free and zinc protoporphyrin. Such misleading reports impair diagnosis and monitoring of patients with protoporphyria. SUMMARY: We suggest that laboratories should prioritize testing for EPP and XLP, because accurate measurement of erythrocyte total and metal-free protoporphyrin is essential for diagnosis and monitoring of these conditions, but less important for other disorders. Terms and abbreviations used in reporting erythrocyte protoporphyrin results should be accurately defined.

A Study on Polychlorinated Biphenyls Specifically--Accumulated in Blood of Yusho Patients Collected from Medical Check-Ups in 2012.

In this study, we analyzed polychlorinated biphenyls (PCBs) in the blood of Yusho patients collected from medical check-ups, which were conducted in 2012. The results show that 65 PCB isomers, not including non-ortho PCBs, were detected in the blood samples, and the total concentration was 620 ng g(-1) lipid. This value was comparable to the concentration in blood samples collected in 2005, and indicated that PCB concentrations in Yusho patients remained unchanged from 2005 to 2012. Here, we focused on major and specific PCB isomers in the blood samples of Yusho patients and normal controls. Examples of the former include hexaCB-153, hexaCB-138, and heptaCB-180, which are detected in human blood, while the latter include hexaCB-156, hexaCB-157, and heptaCB-189, and are highly detected in the blood of Yusho patients. Additionally, we tried to determine why the specific isomers were highly accumulated in the blood of Yusho patients as compared to the normal controls. We therefore analyzed these isomers in the contaminated rice oil, and found that the concentrations of hexaCB-156, hexaCB-157, and heptaCB-189 were 1800, 450, and 190 ng g(-1), respectively. Notably, previous studies indicated that these isomers might not be easily metabolized in humans. Therefore, these findings demonstrated that these isomers were highly accumulated in the blood of Yusho patients.

[Serum Levels of Regulatory T Cell in the Yusho Patients].

Dioxin and dioxin-like compounds receptor (Ahr) mainly expressed on the surface of regulatory T (Treg) cell and Th17 cell could regulate immunological functions in the Yusho patients. We prospectively analyzed data obtained in a total of 56 cases of Yusho, which include patients identified ('Nintei' ) or non-identified ( 'Minintei') or identified as a family member, at the annual health check in 2014. The number of Treg cell showed lower among identified patients compared with non-identified group or family identified group (p = 0.4184 and p = 0.291, respectively). There was also a strong correlation between serum levels of neutral fat and the number of Treg cells (p = 0.0313). These results suggest that Treg cell plays a principal role in the immune response among Yusho patients.

[The Distribution of Dioxin Concentrations in the Blood of Yusho Patients].

In 1968, the contamination of cooking oil by heat-degraded polychlorinated biphenyls (PCBs) caused a case of mass poisoning, the so-called Yusho incident. The cause of Yusho disease is thought to be ingested toxic substances, including not only PCBs but also polychlorinated dibenzofuran (PCDFs) in Kanemi rice oil. We previously measured dioxins concentrations in the blood of 854 Yusho patients by annual medical examination from 2001 to 2013. We determined the concentrations of 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF, 1,2,3,6,7,8-HxCDF and 3,3',4,4',5,5'-HxCB (#169) in the blood of Yusho patients were more than double those of normal controls. In 2012, a new Yusho criteria was established. Family members living with a Yusho patient were also certified as Yusho patients and called Yusho family cohabitant patients. In this study, we compared the distribution of 2,3,4,7,8-PeCDF concentration in the blood of Yusho patients and Yusho family cohabitant patients. It was suspected that the distribution of 2,3,4,7,8-PeCDF concentration in Yusho family cohabitant patients would be lower than that of Yusho patients.

[Blood PCB Concentrations and their Tendencies Examined in Fukuoka 2011-2014 Annual Inspections for Yusho].

We measured PCBs of blood of 154 people in the Yusho medical check-up from January 2011 through December 2014 and analyzed their PCB blood patterns. Eleven examinees were newly certified as Yusho patients during these 4 years. Of these 11, we identified three Yusho patients with the A pattern, five with the B pattern, and three with the BC pattern. We found no Yusho patients with the C pattern. The B pattern was found in 27 of the 154 examinees, such that the probability that an examinee with the B pattern would be certified as a Yusho patient was about 20%. Since 2012, any family members living with a Yusho patient have also been certified as Yusho patients. The number of such family cohabitants was 16, among whom the PCB pattern was uniformly the C pattern. There were 20 examinees born after 1989, and all those examinees were Yusho-suspected persons. The total PCB concentrations of this younger-age group was less than 0.5 ng/g on average and was low compared with the normal controls.

[ALLELES C282Y AND H63D HFE GENE, INSULIN RESISTANCE AND SUSCEPTIBILITY TO DISTURBANCE OF PORPHYRIN METABOLISM IN NON-ALCOHOLIC FATTY LIVER DISEASE].

THE PURPOSE OF THE STUDY: The aim of the present work was to study the frequency of genotypes and alleles of C282Y and H63D HFE gene that may be associated with impaired porphyrin metabolism, as well as possible reasons for the formation of dysmetabolism porphyrins with NAFLD. MATERIALS AND METHODS: The study involved 65 patients (52 men and 13 women) aged 21 to 69 years (mean age 48.5±1.5 years). Excretion uroporphyrin, coproporphyrin, 6-aminolevulinic acid of porphobilinogen in urine was determined by chromatography and spectrophotometry calculated total excretion of porphyrins. Allele frequencies C282Y and H63D were determined during the molecular genetic analysis of DNA using the polymerase chain reaction followed by analysis of length polymorphism restraktsionnyh fragments. Condition of carbohydrate metabolism was evaluated by the level of fasting blood glucose and standard glucose tolerance test. Diagnosis of insulin resistance was performed according to the criteria proposed by the European Group for the Study of insulin resistance (EGIR). RESULTS: Skill test for the C282Y mutation carriage and H63D in the HFE gene in 65 patients with non-alcoholic fatty liver disease. Disturbances in the metabolism of porphyrins were recorded in 43 (66.2%) patients. H63D and C282Y mutations were found in 18 (27.7%) patients, of whom 13 (72.2%) people with different options dismetabolism porphyrins and signs of insulin resistance. In 47 (72.3%) patients without mutations studied porphyrin metabolism disorders were detected in 30 (63.8 %), of which insulin resistance is registered only in 16 (34.0 %). CONCLUSION: Detection of mutations C282Y and H63D in the HFE gene in combination with disorders of porphyrin metabolism on the background of insulin resistance is likely to allow such patients considered as candidates for inclusion in the higher risk of formation of diabetes.

Purpuric eruption in a transfused neonate receiving phototherapy.

We describe the clinical and biochemical findings in a neonate requiring multiple blood transfusions and phototherapy for alloimmune hemolytic anemia and unconjugated hyperbilirubinemia, respectively. In this newborn, a severe photosensitivity reaction developed and laboratory testing revealed elevated serum and urine porphyrins at the time of the eruption. The cause of the transient porphyrinemia was likely multifactorial. Possible mechanisms include poor hepatic metabolism and reticulocyte hemolysis. However, the exact pathogenesis remains unclear at this time.

Concentration of hydroxylated polychlorinated biphenyls (OH-PCBs) in the blood of Yusho patients in 2010.

Hydroxylated polychlorinated biphenyls (OH-PCBs) are formed as major metabolites of PCBs by cytochrome P450 enzyme-mediated oxidation. It has been reported that their total concentration in serum samples of Yusho patients ranged from 390 to 1300 pg/g. We measured the concentration of OH-PCBs in blood collected from 183 Yusho patients living in Japan in 2010. The major OH-PCB metabolites were 4-OH-CB187 (ND-1300 pg/g-wet), 4-OH-CB146 + 3-OH-CB153 (8.4-1200 pg/g-wet), 4-OH-CB109 (ND-530 pg/g-wet) and 4'-OH-CB172 (ND-380 pg/g-wet). The total OH-PCBs ranged from 36 to 3800 pg/g-wet. A positive relationship between the concentrations of OH-PCBs and PCBs was observed, but no significant relationship between the concentrations of OH-PCBs and PCDD/DFs was observed.

[Serum levels of IL-21 and TGF-beta in the Yusho patients].

Aryl hydrocarbon receptor (AhR), recognized as a dioxin receptor, is expressed on the surface of helper T (Th) 17 cells. As PCBs and PCDFs are still detected in the sera of the Yusho patients, we hypothesized dysregulation of Th17 cells in the Yusho patients. In the present study, we measured IL-21 and TGF-beta in the Yusho patients which induce differentiation from Th0 to Th17 cells. Serum levels of IL-21 were lower than those of controls (p < 0.05). Meanwhile, serum levels of TGF-beta were decreased relative to controls, but not significant. These results may imply differentiation from Th0 cells to Th17 cells is not induced in the Yusho patients.

[Increased rate of serum uric acid levels in Yusho sufferers].

We measured serum uric acid levels in Yusho sufferers annually from 2007 to 2012 in Nagasaki prefecture. We observed an increased rate of serum uric acid levels in 38.2% of the male and 5.5% of the female sufferers. There was no relation among serum uric acid value, Body Mass Index, liver function, blood polychlorinated biphenyls and hypersensitive C reactive protein. We conclude that it is unclear if blood polychlorinated biphenyls may play a role in the increase of serum uric acid levels in Yusho sufferers.

[Adipokine profile of Yusho patients].

Serum levels of adipokines among Yusho patients and normal controls were measured. Compared with normal controls, serum levels of leptin were significantly lower, while those of RBP4 were significantly higher in Yusho patients. Dioxins may impair production of adipokines from adipose tissue, which would increase the risk to develop lifestyle-related diseases, such as diabetes mellitus and hypertension.

[Serum levels of IL-10 and IL-35 in Yusho patients].

We thought that dioxin and dioxin-like compound receptor AhR expressed on the surface of regulatory T (Treg) cells and Th17 cells could regulate immunological functions in the Yusho patients. In the present study, we measured Treg cell related cytokines IL-10 and IL-35 in the Yusho patients. Serum levels of IL-10 were higher, but not significant (p = 0.06), and serum levels of IL-35 were increased (p = 0.006) in comparison with healthy controls. These results imply Treg cell activation in the Yusho patients.

The changes in dioxin concentrations in the blood of Yusho patients from 2004 to 2010.

We measured the concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) in blood collected from Yusho patients during medical health examinations performed from 2004 to 2010. Out of the 242 and 200 Yusho patients who received medical health examinations in 2004 and 2010, respectively, the concentrations of PCDDs, PCDFs, and PCBs in the blood of 136 patients were measured in both of those years. The concentrations of individual congeners of PCDDs, PCDFs, and PCBs in the blood of these 136 Yusho patients measured in 2004 were compared with those measured in 2010. Among individual congeners of PCDDs, PCDFs, and PCBs, most congeners of these compounds did not significantly decrease from 2004 to 2010. However, the concentrations of 1, 2, 3, 4, 6, 7, 8-heptaCDD, octaCDD, 1, 2, 3, 4, 7, 8-hexaCDF, tetraCB 52/69, pentaCB 101, octaCB 198/201, and octaCB 196/203 in the blood of Yusho patients were significantly decreased from 2004 to 2010. In addition, the concentrations of 1, 2, 3, 6, 7, 8-hexaCDD, 2, 3, 4, 7, 8-pentaCDF, and 1, 2, 3, 6, 7, 8-hexaCDF tended to decrease slightly from 2004 to 2010. These findings suggest that the PCDDs, PCDFs, and PCBs have remained in the blood of Yusho patients for a very long time, with over 40 years having passed since the outbreak of Yusho.

Development of a newly large-volume injection system for dioxin determinations in blood of Yusho patients.

We developed a more effective method to measure the concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and non-ortho-coplanar polychlorinated biphenyls (non-ortho PCBs) in the blood of Yusho patients using high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS) equipped with a newly large-volume injection system. The new injection system linked a LaviStoma system with a unique stomach-shaped inlet liner (SSIL) and a solvent-cut large-volume (SCLV) injection system. This approach made it possible to introduce volumes up to 200 microl into the HRGC/HRMS in comparison with the 20microl volume of the previously reported conventional SCLV method. Based on experiments conducted using the same blood sample, the concentrations of PCDDs, PCDFs, and non-ortho PCBs obtained by the developed method showed a close correlation to that by the conventional SCLV method. By improving the injection method, the operation time and labor for the purification procedure from blood could be reduced. Furthermore, the developed method was more effective than the conventional SCLV method for recovery of PCDDs, PCDFs, and non-ortho PCBs.

[The laboratory diagnostics of disorders of porphyrin metabolism: a lecture].

The lecture presents data concerning biosynthesis of haem and mechanisms of its regulation in bone marrow and liver. The basic pathogenic mechanisms of porphyrias development and their classification are exposed. The optimal list of laboratory tests to diagnose porphyrias is presented. The role and significance of various laboratory analysis techniques to diagnose porphyrias are demonstrated. The technology of laboratory analysis in case of porphyria suspicion is described.

Afterglow Amplification for Fast and Sensitive Detection of Porphyria in Whole Blood.

Porphyria is a group of genetic photodermatoses that cause too much porphyrin to accumulate in the blood, skin, and liver, resulting in skin photosensitivity and damage, liver disease, or potential liver failure. Conventional detection methods include high-performance liquid chromatography and fluorescence spectrometry. However, these methods usually require complicated pretreatment and time-consuming processes. Therefore, efficient and fast detection of porphyria is urgently needed. Herein, we develop a molecular afterglow reporter-based sensing scheme for the detection of porphyrins in whole blood. The afterglow reporter can respond to the production of singlet oxygen (1O2) of porphyrins after light excitation, and the detection signals can be amplified through adjusting the amount of singlet oxygen and afterglow reporter molecules. Moreover, without the use of a real-time excitation source, afterglow signals can avoid the scattering and autofluorescence interference in biological samples, thereby reducing background noise. More importantly, we prove the applicability of the afterglow reporter in the quantitative detection of porphyrins in whole blood and demonstrate its great clinical potential.

[Clinical and biochemical syndromes of cadmium-induced acute porphyrinopathy].

AIM: To study the specific features of porphyrin metabolic disturbances in cadmium poisoning. MATERIAL AND METHODS: The paper describes a patient who has developed clinical and biochemical syndromes of acute porphyrinopathy after exposure to cadmium-containing paint the vapors. The levels of delta-aminolevulinic acid, porphobilinogen, coproporphyrin, and uroporphyrin in urine and those of coproporphyrin and protoporphyrin in feces were measured. The concentrations of lead, cadmium, and copper were determined in whole blood and urine; selective screening of amino acids for hereditary metabolic diseases was made. RESULTS: The clinical signs of acute porphyrinopathy developed in the patient mimicked those of acute porphyries known by the current classification. The biochemical syndrome more corresponded to lead poisoning. However, the blood and urinary lead levels were not greater than the normal values, but the blood showed a 4-fold increase in cadmium, which seemed to induce porphyrin dysmetabolism.

Cholesterol hydroperoxide formation in red cell membranes and photohemolysis in erythropoietic protoporphyria.

3beta-Hydroxy-5alpha-hydroperoxy-Delta(6)-cholestene is produced in protoporphyrin-containing red blood cell ghosts irradiated with approximately 400-nanometer light in the presence of oxygen. Incorporation of this cholesterol photooxidation product into normal red blood cells leads to increased osmotic fragility and eventual hemolysis. These results may be relevant to photohemolysis associated with erythropoietic protoporphyria.

Erythropoietic protoporphyria and lead intoxication: the molecular basis for difference in cutaneous photosensitivity. I. Different rates of disappearance of protoporphyrin from the erythrocytes, both in vivo and in vitro.

In lead intoxication photosensitivity is usually absent, despite concentrations of protoporphyrin in the erythrocytes equal to or greater than in erythropoietic protoporphyria. Profound differences in the distribution of protoporphyrin in aging erythrocytes were demonstrated by age-dependent fractionation of cells on discontinuous density gradients. In erythropoietic protoporphyria the concentration of protoporphyrin declined extremely rapidly with erythrocyte age; the bulk of the protoporphyrin was lost in less than 3 days and the concentration of fluorescent erythrocytes in the gradient paralleled the decline of protoporphyrin. In lead intoxication the protoporphyrin concentration declined only slightly with cell aging and erythrocytes of all ages fluoresced. In the bone marrow from a patient with erythropoietic protoporphyria all reticulocytes, but only occasional late normoblasts, fluoresced, suggesting a single population. Sterile incubation in plasma (pH 7.5) demonstrated rapid diffusion of protoporphyrin from the erythrocytes in erythropoietic protoporphyria, but not in lead intoxication. Plasma protoporphyrin was elevated in erythropoietic protoporphyria, but not in lead intoxication. Estimates of the daily loss of protoporphyrin from erythropoietic tissue in erythropoietic proporphyria suggested an order of magnitude similar to the total blood protoporphyrin. Therefore, it is not necessary to postulate a preponderant extraerythropoietic source to explain the amount of fecal excretion. A significant amount of the diffused protoporphyrin probably reaches the skin with resulting photosensitivity. In contrast, in lead intoxication protoporphyrin remains within the erythrocyte throughout its life span ; there is no diffusion into the plasma and hence no photosensitivity.