Measurement method for hydroxylated polychlorinated biphenyls in the blood of Yusho patients by liquid chromatography-electrospray tandem mass spectrometry.

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 developed a measurement method for OH-PCBs in blood samples by LC/MS/MS. This method is effective at determining the concentrations of PCDDs, PCDFs, Co-PCBs and OH-PCBs from the same sample without special treatment of the sample. The concentration of OH-PCBs in the blood of Yusho patients was examined using this method. The major OH-PCB metabolites were 4-OH-CB187 (54-906 pg/g-wet), 4-OH-CB146 + 3-OH-CB153 (32-527 pg/g-wet), 4-OH-CB109 (ND-229 pg/g-wet) and 4'-OH-CB172 (ND-143 pg/g-wet). The total OH-PCBs ranged from 95 to 1740 pg/g-wet.

[Relationship between half-lives of blood dioxins and possible metabolic mechanism].

Blood levels of dioxins in Yusho patients have been measured for 10 years. The purposes of this study were to determine the half-lives of dioxins on the basis of the data obtained from Yusho patients and to compare the half-lives of the compounds. Linear regression analysis was performed using the binary logarithmic value of each dioxin level as the dependent variable and the year of measurement as the independent variable. The linear coefficient obtained from this linear regression analysis was the reciprocal number of the half-life. The relationship between the blood dioxin levels estimated in 2004 and the reciprocal number of half-life was evaluated. Of the studied dioxins, the concentrations of some compounds were strongly correlated with that of 2,3,4,7,8-PeCDF. For 2 such compounds, the correlation coefficient of the reciprocal numbers of half-life were greater than the correlation coefficient of the logarithmic values of the estimated concentrations. Of these 2 compounds, the concentration of 3,3,4,4,5,5-HxCB was at least 50 times less than that of the 2,3,4,7, 8-PeCDF in rice oil: however, their current concentrations are equivalent. Patients with high levels of 2,3,4,7,8-PeCDF also showed high levels of 3,3,4,4,5,5-HxCB. Yusho patients may have a disease-specific mechanism to supply 3,3,4,4,5,5-HxCB.

[Time serial changes in the concentrations of the etiological agents of fetal Yusho--PCDDs and PCDFs].

We determined polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) in 6 preserved umbilical cords of fetal Yusho patients and in 11 preserved umbilical cords of Yusho suspected persons who were born to mothers with Yusho from 1970 to 2002, which were Yusho group. As a control, we also analyzed PCDFs and PCDDs in 15 preserved umbilical cords of babies who were born to healthy mothers, which was healthy group, in the same period of time. As a result, 2,3,4,7,8-pentachlorodibenzofuran and 1,2,3,4,7,8-hexachlorodibenzofuran, true causal agents of fetal Yusho, were only determined in the umbilical cords of fetal Yusho patients, except for one umbilical cords of Yusho suspected persons. Decreasing rate in concentrations of PCDFs and PCDDs seemed to greater in Yusho group than in healthy group during this period of time. Therefore, we considered due to high exposure to PCDFs some drug metabolizing enzymes such as aryl hydrocarbon hydroxylase were induced and the excretion of PCDFs and PCDDs were enhanced from the bodies of Yusho group. In order to clarify this hypothesis, further more detail researches are required.

[Time serial changes in the concentrations of the related agents to fetal Yusho--dioxin-like PCBs and PCBs].

We determined dioxin-like polychlorinated biphenyls (PCBs) and PCBs in 6 preserved umbilical cords of fetal Yusho patients and in 11 preserved umbilical cords of Yusho suspected persons who were born to mothers with Yusho from 1970 to 2002, which were Yusho group. As a control, we also analyzed dioxin-like PCBs and PCBs in 15 preserved umbilical cords of babies who were born to healthy mothers, which was healthy group, in the same period of time. As a result, concentrations of three dioxin-like PCBs, that is, #156, #157 and #189 which were 6 to 20 times higher in fetal Yusho patients than in healthy babies were still 4 to 6 times greater in Yusho group than in healthy group about 20 years after the outbreak of Yusho, but could not recognize this characteristic anymore about 30 years after the outbreak. Decreasing rate in concentrations of dioxin-like PCBs and PCBs seemed to higher in Yusho group than in healthy group during this period of time. Therefore, we considered due to heavy exposure to PCDFs some drug metabolizing enzymes such as aryl hydrocarbon hydroxylase were induced and excretion of the related agents to fetal Yusho were enhanced from the bodies of Yusho group. In order to clarify this hypothesis, further more detail studies are needed.

[Promotive excretion of causative agents of Yusho by fermented brown rice with Aspergillus oryze in Yusho patients].

Forty two years have passed since the outbreak of Kanemi rice oil poisoning, namely, Yusho in the western Japan. However, even now the Yusho patients have been still suffering from several objective and subjective symptoms. In order to improve or, if possible, to cure such symptoms, the most important therapeutic treatment is considered to actively excrete the causative agents, that is, polychlorinated dibenzofurans (PCDFs) from the bodies of the patients and to reduce their body burdens. In rats, chlorophyll and dietary fiber have been shown to promote the fecal excretion of PCDFs and polychlorinated dibenzo-p-dioxins (PCDDs) and to reduce their levels in rats. In this study, we have examined whether such kinds of effect are also observed by fermented brown rice with Aspergillus oryzae (FBRA) containing 5% spirulina, which is so-called spirulina HI * GENKI, the health food and relatively rich with chlorophyll and dietary fiber, in eighteen Yusho patients. They were divided into two groups, namely group A, ten patients (3 males and 7 females) with the mean age of 67.7 years old and group B, eight patients (4 males and 4 females) with the mean age of 64.1 years old. Among the patients of group A, three patients were especially highly contaminated with PCDFs and we classified them into group A (High). Respective mean concentrations of PCDFs in the blood just before initiating this study were as follows; group A : 145 pg/g lipid, group A (High) : 371 pg/g lipid and group B : 52.1 pg/g lipid. Contamination levels of PCDFs, PCDDs and dioxin-like polychlorinated biphenyls (PCBs) in group A (High) were 1.7 to 2.6 times higher than those in group A and 2.4 to 7.1 times higher than those in group B. Accordingly, concentrations of dioxins (PCDFs + PCDDs + dioxin-like PCBs) in the blood of groups A, A (High) and B were, respectively, 194, 458 and 85 pg-TEQ/g lipid. Concentrations of PCBs were also the highest in group A (High) : 1399 ng/g lipid, in group A : 748 ng/g lipid and the lowest in group B : 456 ng/g lipid. Groups A and A (High) took around 7.0 g of spirulina HI * GENKI after each meal and three times a day for the first one year and for the second one year, they did not take spirulina HI * GENKI anymore. Group B took spirulina HI * GENKI with the same manner as the group A only for the second one year. The concentrations of PCDFs, PCDDs, dioxin-like PCBs and PCBs in the blood were also measured at the end of the first and second year, respectively. Assuming the body fat is also contaminated with these compounds at their concentrations on lipid weight basis in the blood and the content of body fat is 20% of 60 kg body weight, we computed the average amounts in their net excretion from the body of the patients due to spirulina HI * GENKI in the three groups. As a result, in group A (High), 341 ng-TEQ/patient of dioxins was excreted from the body, which was 3.4 times greater than that in group A and 12 times higher than that in group B. Therefore, promotive excretions of causative agents of Yusho were the most effective in group A (High) and we consider spirulina HI * GENKI is more effective from the therapeutic point of view in more highly contaminated Yusho patients.

[The difference between male and female 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 polychlorinated dibenzofuran (PCDFs) in Kanemi rice oil. Extensive studies have been performed since 1995 by the Yusho study group involving follow-up surveys of human blood concentrations of the casual compounds in Yusho patients as well as clinical trials of the acceleration of the excretion of these compounds in Yusho patients. We have previously measured dioxin concentrations in the blood of 649 Yusho patients in annual medical examinations from 2001 to 2009. We determined that 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 twice as high levels to as those of normal controls. In this study, we compared 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 male Yusho patients with those of female Yusho patients. As a result, it was found that the mean 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 female Yusho patients were 2.9, 3.7, 2.4, and 1.3 times higher, respectively, than those of male Yusho patients.

[Promotive excretion of causative agents of Yusho by intake of fermented brown rice with Aspergillus oryze in patients with Yusho --with regard to PCDFs and PCDDs].

Forty years have passed since the outbreak of Kanemi rice oil poisoning, namely, Yusho in the western Japan. However, even now the patients with Yusho have been still suffering from several objective and subjective symptoms. In order to improve or, if possible, to cure such symptoms, the most important therapeutic treatment is considered to actively excrete the causative agents, that is, polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) from the bodies of the patients and to reduce their body burdens. In rats, chlorophyll and dietary fiber have been shown to promote the fecal excretion of PCDFs and PCDDs and to reduce their levels in rats. In this study, we have examined whether such kinds of effect are also observed by fermented brown rice with Aspergillus oryzae (FBRA) containing 5% spirulina, which is so-called spirulina HI*GENKI, the health food and relatively rich with chlorophyll and dietary fiber, in eighteen patients with Yusho. They were divided into two groups, namely group A, ten patients (3 males and 7 females) with the mean age of 67.7 years old and group B, eight patients (4 males and 4 females) with the mean age of 64.1 years old. Respective mean concentrations of the three PCDF congeners, that is, 2,3,4,7,8-PenCDF, 1, 2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF in the blood on lipid weight basis just before initiating this study were as follows; group A: 413, 152 and 45.7 pg/g lipid, and group B: 151, 42.7 and 17.3 pg/g lipid. Contamination levels of these PCDF congeners in group A were 2.6 to 3.6 times higher than those in group B. In respective mean concentrations of PCDFs, PCDDs and PCDFs/DDs in both groups were as follows; group A: 228, 30.9 and 258 pg-TEQ/g lipid, and group B: 82.4, 19.7 and 102 pg-TEQ/g lipid. Contamination levels of PCDFs and PCDDs were around 2 times higher in group A than in group B. Group A took around 7.0 g of spirulina HI*GENKI after each meal and tree times a day for the first one year and for the second one year, they did not take spirulina HI*GENKI any more. Group B took spirulina HI*GENKI with the same manner as the group A only for the second one year. The concentrations of PCDFs and PCDDs in the blood of groups A and B were also measured at the end of the first and second year, respectively. Assuming the body fat is also contaminated with PCDFs and PCDDs at their concentrations on lipid weight basis in the blood and the content of body fat is 20% of 60 kg body weight, we computed the average amounts in the net excretion of PCDFs and PCDFs/DDs from the body of the patients due to the intake of spirulina HI*GENKI in groups A and B. As a result, in group A, 85.0 and 99.6 ng-TEQ/patient, respectively were excreted from the body of the patients. In group B, only 38.1 nd 40.0 ng-TEQ/patient were excreted. Accordingly, promotive excretion of PCDFs and PCDDs from the patients with Yusho seemed much effective in group A, of which their concentrations in the blood were much higher than those of group B.

[Trend of 2,3,4,7,8-PeCDF concentrations in the blood of Yusho patients over four years].

In the Yusho medical checkup, measurement of dioxin has been performed since 2001. Although the time trend of dioxin concentrations in blood from 2001 to 2005 has been reported for the whole measurement candidate, details in the trend are not known. Therefore, to verify the trend, we divided the Yusho patients into four quatertile groups based on the dioxin concentrations. About 39 years have passed since Yusho occurred, and it is said that a big change in the concentrations of 2,3,4,7,8-PeCDF in blood is now no longer seen. However, when the Yusho patients were divided into the four groups, a downward tendency was even now found in the higher dioxin groups, but not in the lower dioxin groups.

[Blood PCB concentrations and their characteristics examined in Fukuoka 2006 annual inspection for Yusho].

We analyzed sixty-eight PCB isomers and determined their concentrations and characteristics in fifty-eight individual blood samples collected during the annual Yusho inspection in 2006. The method used in this study consists of a rapid GPC clean-up and sensitive PCB isomer-specific identification with HRGC/HRMS. As a result, the highest total PCB concentration in inspected subjects was 6.6 ppb on a blood weight basis, which was 8.1 times higher than that of a control blood sample. When blood PCB patterns were compared among twenty-seven persons in undergoing examinations both 2004 and 2006, the longitudinal classifications in the respective individuals hardly changed between these years. Mean concentrations of each PCB isomer in the blood of pattern A subjects (n=4) were compared with those in the blood collected from Fukuoka prefecture residents (n=127). Consequently, the respective blood concentrations of PCB #157, #156, #189, #137, #195, #170, #99 were obviously higher than those of Fukuoka residents, while those of PCB #118, # 05 was evidently lower than those of Fukuoka residents.

Excretion of causative PCDFs congeners of Yusho by one year intake of FBRA in patients with Yusho.

Thirty-eight years have passed since the outbreak of Kanemi rice oil poisoning, namely, Yusho in the western Japan. However, even now the patients with Yusho have been still suffering from several objective and subjective symptoms. In order to improve or, if possible, to cure the such symptoms, the most important therapeutic treatment is considered to actively excrete the causative agents, that is, polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) from the bodies of the patients and to reduce their body burdens. In rats, dietary fiber and chlorophyll have been shown to promote the fecal excretion of dioxins and to reduce their levels in rat liver. In this study, we examined whether such kinds of effect were also observed by FBRA, which was the health food and relatively rich with dietary fiber and chlorophyll, in eighteen patients with Yusho, which were divided into two groups, namely group A, ten patients (Male: 3 and Female: 7) with the mean age of 67.7 years old and group B, eight patients (Male: 4 and Female: 4) with the mean age of 64.1 years. Respective mean concentrations of the three PCDF congeners, that is, 2,3,4,7,8-PenCDF, 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF in the blood on whole weight basis just before initiating this study were as follows; group A: 1.36, 0.491 and 0.150 pg/g, and group B: 0.571, 0.159 and 0. 064 pg/g. Contamination levels of these PCDF congeners in group A were 2 to 3 times higher than those in group B. Group A took 7.0 to 10.5g of FBRA after each meal and tree times a day for the first one year and for second one year, they did not take FBRA any more. Group B took FBRA with the same manner as the group A only for the second one year. The concentrations of these PCDFs congeners in the blood of groups A and B were also measured at the end of first and second year, respectively. Assuming that the lipid content of the blood is 0.3% in order to convert their concentrations on whole weight basis to those on lipid weight basis and also that the body fat is contaminated with these PCDF congeners at their concentrations on lipid weight basis and the content of body fat is 20% of the body weight (60 kg), we computed the average amounts in the net excretion of these PCDF congeners from the body of the patients due to the intake of FBRA in groups A and B. As a result, in group A, 120, 372 and 96 ng/patient of 2,3,4,7,8-PenCDF, 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF, respectively, were excreted from the body of the patients. In group B, however, 36 ng/patient of 2,3,4,7,8-PenCDF only was excreted, but other two PCDF congeners were not. Accordingly, promotive excretion of theses PCDF congeners from the patients with Yusho seemed much effective in group A, of which their concentrations in the blood were much higher than those of group B.

Dioxin concentration in the blood of patients collected during medical check-up for Yusho in 2004-2005.

We measured the concentrations of polychlorinated dizenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and non-ortho coplanar polychlorinated biphenyls (non-ortho PCBs) in blood collected from 242 Yusho patients and 74 Yusho-suspected persons in 2004 and 237 Yusho patients and 114 Yusho-suspected persons in 2005. The sums of toxic equivalents (TEQ) concentrations of PCDDs, PCDFs, and non-ortho PCBs in the blood of Yusho patients in 2004 and 2005 were 126.1 and 124.2 pg TEQ/g lipid, respectively, and the concentrations were 3.4 and 3.3 times higher than those of normal controls that had been previously reported, respectively. Those of the Yusho-suspected persons were about 0.8 and 1.0 times higher than those of normal controls, respectively. Although the TEQ concentrations of PCDDs and non-ortho PCBs among Yusho patients, Yusho-suspected persons, and normal controls were nearly the same, the PCDFs levels of Yusho patients were about 9.8 and 9.5 times higher than those of normal controls in 2004 and 2005, respectively. The concentration of 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF), which was the highest among PCDFs congeners for Yusho patients, was about 10. 6 and 10.2 times higher than that of the normal controls in 2004 and 2005, respectively. In the case of Yusho-suspected persons, the concentrations were 0.9 and 1.4 times higher than those of normal controls, respectively. However, some of the Yusho-suspected persons showed a high concentration of 2,3,4,7,8-PeCDF that was approximately 15 times greater than concentrations in normal controls. Of 74 Yusho-suspected persons measured in 2004, 7 persons were officially registered as Yusho patients based on the "New Diagnostic Criteria" that officially became the diagnostic criteria for Yusho exposure on September 29, 2004, which included a concentration of 2,3,4,7,8-PeCDF in the blood, and in 2005, 14 persons of 114 Yusho-suspected persons were officially registered as Yusho patients.

[Congener specific determination of PCBs in human blood using gel permeation chromatography (GPC) and high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS)].

We studied the progressive analytical method for polychlorinated biphenyls (PCBs) in blood samples collected from Yusho patients. This study established a systematic procedure for determining both "total concentration of PCBs and their profiles" and "concentrations of mono-ortho PCBs as dioxin-like compounds", providing a significant index for diagnosis. The method we developed consists of a rapid clean-up using gel permeation chromatography (GPC) and sensitive identification with high resolution gas chromatography/high resolution mass spectrometry (HRGC/HRMS). This method provides identification of all of PCB congeners typically detected in human blood samples, especially it is worth noting that nine significant congeners on Yusho diagnosis (IUPAC #105, #114, #118, #123, #153, #156, #157, #167, #189) were detected from a five-gram blood sample. Using the present method, we analyzed sixty-six individual blood samples collected during the annual Yusho inspection in the 2004, and could identified sixty-eight PCB isomers in all samples. As a result, ten patients were classified into pattern A, the typical Yusho pattern. The mean total PCB concentration of pattern A subjects was 2.95 ppb, while that of control subjects was 0.77 ppb, at whole blood basis respectively.

Follow-up survey of dioxin concentrations in the blood of Yusho patients in 2002-2003.

We measured the concentrations of polychlorinated dizenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and non-ortho-coplanar polychlorinated biphenyls (Co-PCBs) in blood collected from 279 Yusho patients and 92 Yusho-suspected persons living in Japan in 2002, 269 Yusho patients and 74 Yusho-suspected persons living in Japan in 2003 and 128 normal controls living in Fukuoka Prefecture, and compared with the concentrations of these compounds among the groups. The total TEQ concentrations of PCDDs, PCDFs, and Co-PCBs in the blood of Yusho patients were 3.6 times and 3.3 times higher than those in the normal controls in 2002 and 2003, respectively. In the case of Yusho-suspected persons, the concentrations were 1.1 times and 1.0 times higher in 2002 and 2003, respectively. Among the TEQ concentrations of PCDDs, PCDFs, and Co-PCBs, the concentrations of PCDFs in the blood of Yusho patients were 10.3 times and 9.4 times higher than those of the normal controls in 2002 and 2003, respectively, whereas the concentrations of PCDDs and Co-PCBs in blood were identical in Yusho patients, Yusho-suspected persons and normal controls. Yusho patients indicated the highest concentrations ratio of PCDFs to the total TEQ concentrations. Moreover, of the PCDFs congeners for Yusho patients, the concentrations of 2,3,4,7,8-PeCDF were about 11.0 times and 10.1 times higher in 2002 and 2003 than those in normal controls. These results indicated that Yusho patients have a much higher concentration of 2,3,4,7,8-PeCDF in their blood than do unaffected persons even though over 34 years have passed since the outbreak of Yusho.

[Follow-up survey of dioxins in the blood of Yusho patients (in 2001)].

Follow-up survey of the blood concentration of polychlorinated dibezo-p-dioxin (PCDDs), polychlorinated dibenzofuran (PCDFs), Non-ortho-coplaner PCBs (Non-Co-PCB), and Mono-ortho-coplaner PCBs (Mo-Co-PCB) in Yusho patients is very important for their health control. We determined the blood concentration of these dioxin-like isomers in 78 blood samples collected in 2001 using by a high-resolution gas chromatograph/high-resolution mass spectrometer (HRGC/HRMS) equipped with a solvent-cut large volume injection system (SCLV). The accelerated solvent extraction (ASE) method was employed for the treatment of blood samples. The method employed here can allow a reduced amount of blood collecting from Yusho patients compared with the conventional method. In typical Yusho patients (Group A of PCB pattern), the mean TEQ concentrations of PCDDs, PCDFs, Non-Co-PCBs, and Mono-Co-PCBs in blood collected in 2001 were 36, 260, 9.3, and 64 pg-TEQ/g lipid, respectively. As well, the toxic contribution of PCDDs, PCDFs, Non-Co-PCBs, and Mono-Co-PCBs in typical Yusho patients (Group A of PCB pattern) were 10, 70, 3, and 17% of the total TEQ value, respectively. The toxic contribution rate of PCDFs TEQ was still at high levels in relation to total TEQ in typical Yusho patients as mention above. The Yusho patients showing B pattern also had the same A pattern contribution rate. In the patients of group C, however, the mean blood levels of dioxin TEQ in 2001 was two times those of the normal controls determined in 2000 in Fukuoka prefecture. As well, the contribution rates were similar to those of normal subjects, although some patients in this group showed a high toxic contribution rate of PCDFs TEQ in relation to total TEQ. Thirty-three years had passed since the Yusho accident, and the total blood concentrations of PCDFs in the blood of Yusho patients were still higher than those of normal subjects. Among the different groups of PCB patterns, the concentration of 2,3,4,7,8-PeCDF, 1,2,3,4,7,8-HxCDF and 1,2,3,6,7,8-HxCDF showed significant differences. These findings indicate that these isomers are not metabolized in the human body since traces of them continued to be detected in these patients since they had been contaminated by the rice oil in the original incident.