Liver disease and erythropoietic protoporphyria:A concise review

The porphyries are a group of metabolic disorders characterized by deficiencies in the activity of enzymes involved in the biosynthesis of heme.In erythropoietic protoporphyria(EPP),in the majority of cases an autosomal dominant disease,there is a mutation of the gene that encodes ferrochelatase(FECH).FECH deficiency is associated with increased concentrations of protoporphyrin in erythrocytes,plasma,skin and liver.The prevalence of this inherited disorder oscillates between 1:75 000 and 1:200 000.Clinical manifestations of EPP appear in early infancy upon first exposure to the sun.Nevertheless,approximately 5%-20% of patients with EPP develop liver manifestations.Retention of protoporphyrin in the liver is associated with cholestatic phenomena and oxidative stress that predisposes to hepatobiliary disease of varying degrees of severity,such as cholelithiasis,mild parenchymal liver disease,progressive hepatocellular disease with end-stage liver disease and acute liver failure.Liver damage is the major risk in EPP patients,so surveillance and frequent clinical and biochemical liver follow-up is mandatory.The diagnostic approach consists in detecting increased levels of protoporphyrin,decreased activity of FECH and genetic analysis of the FECH gene.A variety of nonsurgical therapeutic approaches have been adopted for the management of EPP associated with liver disease,but none of these has been shown to be unequivocally efficacious.Nevertheless,some may have a place in preparing patients for liver transplantation.Liver transplantation does not correct the constitutional deficiency of FECH.Consequently,there is a risk of recurrence of liver disease after liver transplantation as a result of continuing overproduction of protoporphyrin.Some authors recommend that bone marrow transplantation should be considered in liver allograft recipients to prevent recurrence of hepatic disease.

Effect of heme oxygenase-1 on renal function in rats with liver cirrhosis

AIM: To investigate the role of heme oxygenase-1 (HO-1) in pathogenesis of experimental hepatorenal syndrome (HRS). METHODS: Rats were divided into liver cirrhotic group, zinc protoporphyrin IX (ZnPP) treatment group, cobalt protoporphyrin (CoPP) treatment group and sham group. Biliary cirrhosis was established by bile duct ligation in the first three groups. Rats in the ZnPP and CoPP treatment groups received intraperitoneal injection of ZnPP and CoPP, respectively, 24 h before sample collection. Expression of HO-1 mRNA in kidney was detected by reverse-transcription polymerase chain reaction, while protein expression was determined by immunohis-tochemical analysis. Hematoxylin and eosin staining was performed to observe liver cirrhosis and renal structure. Renal artery blood flow, mean arterial pressure and portal vein pressure, 24 h total urinary volume, serum and urine sodium concentrations, and creatinine clearance rate (Ccr) were also measured.RESULTS: The HO-1 mRNA and protein expression levels in kidney, 24 h total urinary volume, renal artery blood flow, serum and urine sodium concentration and Ccr were lower in cirrhotic group than in sham group (P < 0.05). However, they were significantly lower in ZnPP treatment group than in cirrhotic group and significantly higher in CoPP treatment group than in cirrhotic group (P < 0.05). CONCLUSION: Low HO-1 expression level in kidney is an important factor for experimental HRS.

Influence of heme oxygenase-1 expression on immune liver fibrosis induced by cobalt protoporphyrin in rats

AIM:To investigate the effect of heme oxygenase-1 (HO-1)expression on immune liver fibrosis induced by cobalt protoporphyrin(CoPP)in rats. METHODS:An immune liver fibrosis model of rat was established by administering human serum albumin (HSA).The rats were divided into CoPP,liver fibrosis and normal control groups.Rats in the CoPP group received intraperitoneal CoPP concurrently with HSA. Expression of HO-1 protein was observed by Western blotting and immunohistochemistry.Hematoxylin and eosin(HE)staining was performed to assess fibrosis proliferation and distribution,proliferation extent of fibroblasts,and alterations in hepatocytes and inflammatory cells.TypeⅠandⅢcollagens were detected with Van Gieson’s(VG)staining and Foot’s reticular fiber staining,respectively.In addition, spindle-shaped cells existing at perisinusoidal locations beyond portal and septa areas were investigated with HE staining. RESULTS:Western blotting and immunohistochemistry showed that the expression of HO-1 protein was higher in the CoPP group than in the liver fibrosis group(P<0.05).Compared with the liver fibrosis group,the serological index of hepatic fibrosis in the CoPP group decreased significantly(P<0.05).HE,VG and Foot’s staining revealed that administration of CoPP reduced the extent of hepatic fibrosis.The levels of serological indicators and the number of spindle-shaped cells at perisinuous locations beyond the portal and septa areas were reduced in the CoPP group.Only a few inflammatory cells were seen around the portal areas and central veins in the CoPP group. CONCLUSION:Increased endogenous HO-1 may suppress liver fibrosis by protecting liver cells, inhibiting inflammatory cell infiltration and hepatic stellate cell transformation.

Recent advances in photodynamic diagnosis of gastriccancer using 5-aminolevulinic acid

Photodynamic diagnosis based on 5-aminolevulinic acidinduced protoporphyrin IX has been clinically applied in many fields based upon its evidenced efficacy and adequate safety. In order to establish a personalized medicine approach for treating gastric cancer patients, rapid intraoperative detection of malignant lesions has become important. Feasibility of photodynamic diagnosis using 5-aminolevulinic acid for gastric cancer patients has been investigated, especially for the detection of peritoneal dissemination and lymph node metastasis. This method enables intraoperative realtime fluorescence detection of peritoneal dissemination, exhibiting higher sensitivity than white light observation without histopathological examination. The method also enables detection of metastatic foci within excised lymph nodes, exhibiting a diagnostic accuracy comparable to that of a current molecular diagnostics technique. Although several complicating issues still need to be resolved, such as the effect of tissue autofluorescence and the insufficient depth penetration of excitation light, this simple and rapid method has the potential to become a useful diagnostic tool for gastric cancer, as well as urinary bladder cancer and glioma.

Heme oxygenase-1 inhibits neuropathic pain in rats with diabetic mellitus

A diabetes mellitus model was established through single intraperitoneal injection of streptozotocin into rats. Seven days later, model rats were intraperitoneally administered zinc protoporphyrin, a heme oxygenase-1 inducer, and cobalt protoporphyrin, a heme oxygenase-1 inhibitor, once every two days, for 5 successive weeks. After administration, the paw withdrawal mechanical threshold of diabetic mellitus rats significantly decreased, the myelin sheath of the sciatic nerve thickened or showed vacuole defects, the number of spinal dorsal horn neurons reduced, some neurons degenerated and were necrotic, and heme oxygenase-1 was visible in the cytoplasm of spinal dorsal hom neurons. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling demonstrated that the number of apoptotic neurons increased, which could be inhibited by cobalt protoporphyrin, however, zinc protoporphyrin led to an opposite effect. Our experimental findings indicate that heme oxygenase-1 attenuates neuropathic pain in diabetic mellitus rats through amelioration of peripheral neuropathy and inhibition of spinal dorsal horn neuron apoptosis.

A Comparative Study on the Enhancement Efficacy of Specific and Non-specific Iron Chelators for Protoporphyrin Ⅸ Production and Photosensitization in HaCat Cells

The iron chelators can be utilized in target cells to improve 5-aminolaevulinic acid （ALA）-based photodynamic therapy （PDT）. The purpose of this study is to compare the effect of two kinds of iron chelators, desferrioxamine （DFO） and ethylenediaminetetraacetic acid （EDTA） on the enhancement of ALA-PDT. HaCat cells were cultured in medium containing 2.0 mmol/L of ALA and 0.5 mmol/L of DFO or EDTA. After 3-h incubation in the dark, the concentration of cellular pro-toporphyrin Ⅸ （PpⅨ） was detected by high performance liquid chromatography （HPLC）, and the fluorescence of PpⅨ was observed at 630 nm emission under confocal laser scanning microscope. For PDT, HaCat cells were irradiated using 632.8 nm laser, and the fractions of apoptotic and necrotic cells were flow cytometrically assayed. Related differences in morphology and ultrastructure of Ha-Cat cells were observed using optical microscope or transmission electron microscope. Compared to incubation with ALA alone, the addition of DFO or EDTA increased the concentration of cellular PpⅨ and the fluorescent density of PpⅨ, and also increased cell death ratio after PDT. PDT using ALA plus DFO produced the highest cellular PpⅨ level, greatest cell death ratio and most severe structural damage to the cells. It was concluded that both DFO and EDTA could enhance ALA-based PpⅨ production and PDT. Compared to the non-specific iron chelator of EDTA, the specific chelator, DFO, showed more potential for the enhancement.

Letter to the editor: Diagnosis of erythropoietic protoporphyria with severe liver injury-a case report

Erythropoietic protoporphyria(EPP)is an extremely rare disease which is often unrecognized as diagnosis.In the recent article Lui et al describe a patient with a new diagnosis of EPP with severe liver injury.Approximately 5%-20%of patients with EPP develop liver manifestations.The most severe complication of EPP is an hepatic crisis,which is a medical emergency requiring urgent treatment.Intensive treatment should consist of(exchange)transfusions and preferably in a center that performs liver transplantations.

Glucose and δ-Aminolevulinic Acid Stimulate the Dark Chlorophyll Synthesis of Rice Seedlings

This research was to examine if rice (Oryza sativa L.), a monocotyledon of angiosperm, was able to synthesize chlorophyll (Chl) in complete darkness. Five-cm-tall etiolated seedlings of rice were used as starting materials and treated with or without various concentrations of glucose and/or δ-aminolevulinic acid (ALA) in the dark. Leaves harvested at the indicated time were determined for their contents of Chl, protoporphyrin Ⅸ (Proto), Mg-protoporphyrin Ⅸ (Mg-Proto) and protochlorophyllide (Pchlide). The mole percentage of porphyrin was calculated. The Chl content in the etiolated rice seedlings slightly increased from about 2.5 μg/g to 7.5 μg/g within 12 d in the dark, but the total Chl of dark-grown rice increased from 0.36 μg/g to 3.6 μg/g. While the mole percentages of Proto, Mg-Proto and Pchlide in the dark-grown seedlings without any treatment were about 65%, 27.5% and 7.5% at the beginning, respectively, those in the light-grown seedlings were about 42.5%, 35% and 22.5%, respectively. The mole percentage of porphyrin of etiolated seedlings resumed its normal ratio within 2 d after treatment with glucose. While the Chl content of etiolated seedlings grown in culture solution with 3% and 6% glucose increased 2.5 and 4.0 folds, respectively, those with 3% and 6% glucose and 1 mmol/L ALA increased 22 and 24 folds, respectively. It is concluded that angiosperm might be able to synthesize a small amount of Chl in complete darkness, that either glucose or ALA could stimulate dark Chl synthesis in angiosperm, and that a combination of glucose and ALA exhibited an additional effect. It is still unknown and remains to be further explored what is the mechanism of the effect of glucose and ALA on the Chl synthesis of rice in the dark.

Effect of zinc protoporphyrin on carbon monoxide/heme oxygenase-1 system in rats subjected to recurrent febrile convulsion

BACKGROUND： Studies on febrile convulsion （FC）-caused brain injury are disputed in many aspects. How FC cause nervous system injury in the developmental period and what are the characteristics of these pathological injury are unknown. The current studies have demonstrated that berne oxygenase-1 （HO-1） exerts effects on brain injury mainly by catalyzing hemoglobin to produce degradation products, and HO-1 not only has neuroprotective effects, but also has neurotoxic effects during the FC-caused brain injury. Study on the effect of zinc protoporphyrin （ZnPP） on brain injury is still in the stage of animal experiment. OBJECTIVE： To observe the effects of ZnPP on carbon monoxide （CO）/HO-1 system of rats subjected to FC, and to analyze the action pathway of ZnPP in brain protective effect. DESIGN： A randomized controlled animal experiment. SETTING： Department of Pediatrics, First Hospital Affiliated to Jiamusi University. MATERIALS： Sixty-five Wistar rats, of either gender, were involved in this study. They were randomized into normal control group（ n =14, 37℃ water bath） and febrile treatment group （n =51, 44.5℃ hot water bath）. Febrile treatment group was sub-divided into febrile non-convulsion group （FNC group, n =16） and FC group （n =35）. FC group was further sub-divided into simple convulsion group （n =20） and ZnPP treatment group （n =15）. HO-1 mRNA in situ hybridization kit was provided by Boster Bioengineering Co.,Ltd. ZnPP（dark brown powder） was the product of Jingmei Bioengineering Company. METHODS： This study was carried out in the postgraduate laboratory of Jiamusi University between January 2004 and January 2007. Rats in the febrile treatment group were placed in the 44.5℃ hot water bath box. If rats did not convulse in the water within 5 minutes, they were taken out, namely FNC group （n = 16）, and those, which were convulsed within 5 minutes, were taken out immediately when they presented such a phenomenon, namely FC group （n =35）. Convulsion induction was conducted once every other day, totally 10 times. Rats were euthanized for analysis at 24 hours after the last induction. Rats in the control group were placed in the 37℃ water. Rats in the ZnPP treatment group were intraperitoneally injected with ZnPP at 45 μ mol/kg before FC attack. Rats in the simple convulsion group were only induced to be convulsed but not administrated. MAIN OUTCOME MEASURES： CO level in the brain tissue homogenate and plasma of rats in each group was detected with a spectrophotometer. HO-1 mRNA expression in the hippocampal CAI region, CA3 region and dentate gyrus of rats was observed by in situ hybridization technique. RESULTS： Sixty-five Wistar rats were involved in the study. Two rats died respectively due to drowning and convulsion in the FC group. One rat died due to convulsion drowning in the ZnPP treatment group. ①Plasma CO concentration of control group and ZnPP treatment group was significantly lower than that of the FC group （P 〈 0.01）, and was significantly higher in the ZnPP treatment group than in the FNC group （P 〈 0.05）. ②CO level in the brain tissue homogenate was significantly lower in the control group and ZnPP treatment group than in the FC group （P 〈 0.01）, and was very significantly higher in the ZnPP treatment group than in the control group （P 〈 0.01）. ③HO-1 mRNA expressions in the neuron of hippocampal CAl region, CA3 region and dentate gyrus of the control group were the lowerest, and those in the FC group were the highest. HO-1 mRNA expression in the neuron of dentate gyrus in the FC group was significantly higher than that in the ZnPP treatment group （P 〈 0.01）, and those in the FNC group and control group was significantly lower than that in the ZnPP treatment group （P 〈 0.01）. CONCLUSION： FC can cause brain injury. Over-expression of HO-I mRNA and the increase of CO are involved in the patho-physiological process of FC. ZnPP can inhibit HO-lmRNA activity and decrease CO level, which is one of pathways for protecting brain.

基于ALA-PDT中PpIX亚细胞分布位点光漂白实验研究

随着光动力学疗法(photodynamic therapy,PDT)基础研究的不断深入和临床应用的广泛开展,光敏剂的光漂白现象日渐成为PDT研究的热点问题。实验在ALA的终浓度为10 mM/ml,辐射光波长为410 nm,辐照光功率为5 mW/cm2等条件下采用荧光探针标记技术在自行设计的光动力疗法反应室进行了PDT实验研究。Cell Counting Kit-8 (CCK-8)法用来检测细胞的活性,通过获得的荧光光谱研究了亚细胞的光漂白情况。结果表明:以每种细胞器光照前的平均荧光强度为基准,则光照60 min后线粒体、溶酶体、内质网的荧光强度下分别降低了95%、91.5%和51.2%。线粒体区域光漂白速率较快,其荧光强度的变化基本表征了HL60细胞活性的变化,推断线粒体是光动力效应的主要作用靶点之一。

Preliminary study of photodynamic diagnosis using 5-aminolevulinic acid in gastric and colorectal tumors

AIM:To investigate the utility of photodynamic diagnosis(PDD) using 5-aminolevulinic acid(5-ALA) to detect gastric/colorectal tumors.METHODS:This prospective single-center study investigated inter-subject variability in patients with early-stage gastric/colorectal tumor indicated for endoscopic resection.Subjects were patients with gastric or colorectal tumors who had undergone endoscopic resection between November 2012 and November 2013.Selection criteria included age 20-80 years,either sex,and provision of informed consent.Patients were orally administered 20 mg/kg of 5-ALA enteric-coated capsules(SBI ALApromo Co.,Tokyo,Japan).Administration of5-ALA was followed by endoscopic resection of gastric or colorectal tumors,and the resected specimens were examined using a video autofluorescence processor and a fluorescence endoscope(SAFE-3000 and EB-1970 AK,respectively;Pentax,Tokyo,Japan).The primary endpoint was the presence of fluorescence in tumors.Endoscopic,macroscopic,and histopathologic findings of tumors were assessed.We also evaluated adverse events of the present procedure as a secondary endpoint and examined each patient for the presence of known adverse effects of 5-ALA,namely,hematocytopenia,liver dysfunction,hypotension,nausea,and photosensitivity.RESULTS:We enrolled 10 patients(7 men,3 women)(n = 13 lesions:10 gastric/3 colorectal tumors).Fluorescence was detected in 7/13(53.8%) lesions.No significant differences in sex(male:55.6%vs female:50.5%,P = 1.00),age(67.1 ± 1.9 years vs 65.0 ±2.0 years,P= 0.45),tumor color(reddish:60.0%vs discolored:33.3%,P = 0.56),tumor diameter(15.0± 2.1 mm vs 14.2 ± 2.3 mm,P= 0.80),macroscopic type(protruded:70.0%vs depressed 0%,P = 0.07),histologic type(differentiated type:58.3%vs 0%,P = 0.46),invasion depth(mucosal layer:55.6%vs submucosal layer:33.3%,P = 1.00),lymphatic invasion(present:33.3%vs absent:50.0%,P= 1.00),venous invasion(present:0%vs absent:54.5%,P= 1.00) or procedure time of endoscopic resection(36.3 ± 8.3 min vs 36.7 ± 9.0 min,P = 0.98) were observed between the patients with and without fluorescence.Fluorescence detection rate tended to be high for elevated lesions.Liver dysfunction developed in 4/10(40.0%) patients.The extent of the liver dysfunction was a slight increase in transaminases and total bilirubin levels,which spontaneously improved in the patients.None of the patients developed photosensitivity.CONCLUSION:Results of this preliminary study suggest the utility of PDD using 5-ALA for screening of gastric and colorectal cancers.

Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin

AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved.

Parallel fluorescence and phosphorescence monitoring of singlet oxygen photosensitization in rats

The time-and spectral-resolved set-up for measurements of weak infrared luminescence of photosensitizers(PSs)and singlet oxygen using optical lightguides was used on skin of laboratory animals in vivo.Wistar rats with abdominal incisions treated by methylis aminolevulinitis(MAL)were used as a model.A control group of animals with abdominal incisions was also tested.Spectrally resolved fluorescence of the PS was acquired during the treatment from the same spot.The intensity and spectral profile of the fiuorescence signal from the skin can be used to guide the detection setup to the investigated spots in the lesion.The rate of bleaching of Protoporphyrin IX band and appearance of a band of its photoproducts during the treatment can be characterized by the exposition ED,under which the latter becomes dominant feature infiuorescence spectrum.ED value differs statistically significantly between the normal skin and the lesion treated by MAL.No direct proportionality was found between the fluorescence signal and singlet oxygen production.Nevertheless,the strong fluorescence signal is necessary but not a su±cient condition for higher singlet oxygen production in vivo.ED value correlates rather well with production of singlet oxygen,but differently in lesion and normal skin.Lifetimes of singlet oxygen differ between spots outside and in the lesion.PS triplet state lifetimes exhibit weak di®erence between spots treated and untreated by MAL.

Influence of Montmorillonite Nanocomposite on Tissue Levels of Lead and Blood Biochemical Parameters in Pigs Exposed to Lead

ABSTRACT： Eighteen （Duroc x Landrace x Yorkshire） castrated boars, 55 days of age and weig- hing approximately 19 kg, were used to investigate the effects of montmorillonite nanocomposite on tissue levels of lead as well as changes in blood biochemical parameters in pigs fed lead contaminated diets. The barrows were randomly assigned to one of three treat- ments （n = 6） including a control which were fed a basal diet based on corn and soybean meal. The re- maining two groups were fed the basal diet supple- mented with 10 mg/kg lead either with or without 0.5% montmorillonite nanocomposite for 50 days. Pigs treated with lead had significantly increased lev- els of lead in their tissues. Lead accumulated in all tis- sues of the body with the highest accumulation found in the hair. When montmorillonite nanocomposite was given along with lead, tissue concentrations of lead did not differ from the control. Fecal excretion of leadincreased significantly in lead-exposed pigs treated with montmorillonite nanocomposite. A progressive and appreciable accumulation of lead was seen in blood with a concomitant increase in zinc protopor- phyrin levels during the course of treatment. Howev- er, zinc protoporphyrin levels did not differ from the control when montmorillonite nanocomposite was ad- ministered along with lead. The activity of ^-amino le- vulinic acid dehydratase in blood was significantly in- creased in lead treated pigs compared with the con- trol. However, in pigs treated with lead and montmo- riltonite nanocomposite in combination, the activity of δ-amino levulinic acid dehydratase was similar to con- trol pigs. Our results indicate that montmorillonite nanocomposite treatment of pigs exposed to lead re- suited in reduced lead concentrations in body tissues. It appears that montmorillonite nanocomposite func- tions by increasing fecal excretion of lead.

<i>In Vitro</i>Culture of Shell Gland Epithelial Cells in Japanese Quail (<i>Coturnix coturnix japonica</i>)

To investigate the secretion of protoporphyrin IX (PpIX), superficial eggshell pigment, from shell gland cells of Japanese quail, the epithelial cells of the gland were collected and isolated for cultivation in vitro. An analysis of a peak for PpIX in the cells was performed using a fluorescence microplate reader. The measurement showed that PpIX has a peak of excitation wavelength at 410 nm and emission wavelength at 606 nm in the culture medium (HamF12 + 4% HCl). Volumes of PpIX in the medium after 4 hour culture of the cells were measured with a microplate reader using filter set of excitation wavelength 400/30nm and emission wavelength 620/40nm. However the cells did not secrete significantly PpIX during 4 hour incubation in this culture system, addition of quail plasma to the medium resulted in significantly higher secretion. A cultivation system in this study is able to use for the study on the mechanism of the secretion of eggshell pigment, PpIX from Japanese quail shell gland epithelial cells.

Calcium Participates in Secretion of Porphyrin from Shell Gland Epithelial Cells of Japanese Quail (<i>Coturnix coturnix japonica</i>)

To investigate factors involved in the secretion of protoporphyrin IX (PpIX), a superficial eggshell pigment, from shell gland epithelial cells of Japanese quail, we cultured cells in Ham’s F12 medium with calcium chloride and quail plasma. The addition of hormones (prostaglandin F2α, progesterone, estradiol-17β) to the medium did not change the PpIX concentration in the culture supernatant, but changing the calcium chloride (CaCl2) concentration did: a lower concentration of CaCl2 led to a higher PpIX concentration;0 mM CaCl2 enhanced the secretion of PpIX from epithelial cells prepared at 5 or 7 mM CaCl2. The result suggests that a drop in concentration of CaCl2 mimics the end of shell calcification and stimulates rapid secretion of PpIX in vivo. Bovine serum albumin was almost as effective as quail plasma for PpIX secretion in culture, and would facilitate further study of the mechanism of PpIX secretion.

Action of protoporphyrin-IX (PP-IX) in the lifespan of <i>Drosophila melanogaster</i>deficient in endogenous antioxidants, Sod and Cat

Protoporphyirin-IX (PP-IX) is a precursor of the biosynthesis of the hemo group, most of the cytochromes and the chlorophylls. The PP-IX is used for medical purposes, and recently a report indicated that it exhibits a dual action since it can decrease or increase the genetic damage caused by N-nitroso-N-ethylurea (ENU) in somatic cells of Drosophila. PP-IX is known to be able to act as an anti-or pro-oxidant agent. The aim of the present research was to study the role of PP-IX on the lifespan of Drosophila melanogaster, taking into account the fact that increasing levels of ROS can accelerate the aging process. The Canton-S strain (CS) was used as well as Sod and Cat which are deficient in the endogenous enzymes, superoxide dismutase and catalase, respectively. Groups of females and males were treated separately with 5 mg/ml of PP-IX solution. The comparison of survival curves indicates that this pigment extended the lifespan of CS. In contrast, Sod strain showed that the opposite effect and had no effect in Cat strain. The fact that PP-IX reduces the mean lifespan in Sod deficient strain might suggest a pro-oxidant action of PP-IX, and consequently the cumulating of ROS as a superoxide could have a mutagenic effect as was shown recently. The results presented evidence of the dual effect of PP-IX.

Synthesis of Porphyrin-Appended Pyridine as a Fluorescent Chemosensor for Cd（Ⅱ） and Hg（Ⅱ）

In this research, specific molecular sensors are classified according to the type of receptor-cation interaction, that is ligand-metal interactions. Receptors are based on a multidentate protoporphyrin-appended pyridine platform, which leaves at least a vacant coordination site for the incoming metal ions. A protoporphyrin-appended pyridine, 2,5-pyridine dicarboxyamidyl-8,13-bis（vinyl）-3,7,18,17-tetramethyl-21 H, 23 H-porphyrin（P-PTP）, was designed and synthesized. Its application as potential fluoroionophore for recognition of cadmium and mercury ions is reported. P-PTP shows chelation-enhanced fluorescence effect with Cd（Ⅱ） and Hg（Ⅱ） via the interruption of photoinduced electron transfer （PET） process, which has been utilized as the basis of the fabrication of the metal ions-sensitive fluorescent chemosensor. The analytical performance characteristics of the proposed Cd（Ⅱ）- and Hg（Ⅱ）- sensitive chemosensors were investigated. It shows a linear response toward Cd（Ⅱ） and Hg（Ⅱ） in the concentration range of 1.0×10-3 to 1.0×10-7 M with a limit of detection of 1.0×10-7 M and 0.5×10-7M for Cd（Ⅱ） and Hg（Ⅱ）, respectively. The chemosensor shows good selectivity for Cd（Ⅱ） over a large number of other transition metal ions, i.e., Cu（Ⅱ）, Zn（Ⅱ） and mixed metal ions.

Low Frequency Ultrasonication Induced Antitumor Effect in 5-Aminolevulinic Acid Treated Malignant Glioma

We investigated the feasibility of sonodynamic therapy for glioma by low frequency ultrasoundwith5-aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX) in heme synthetic process. In vivo tumor model was made by inoculating human glioma cell line U87-MG subcutaneously in nude mice. The tumor was sonicated by 25-kHz ultrasound 4 hours following administration of 5-ALA. The tumor size decreased in 5-ALA administered (ALA(+)US(+)) mice, while increased in non-5-ALA administrated (ALA(-)US(+)) mice and non-sonicated mice (ALA(+)US(-)). The immunohistochemical analysis revealed an apoptotic change in tumor tissue of ALA(+)US(+) mice. The results showed the therapeutic effect of 25 kHz ultrasound for the glioma in 5-ALA administered tumor-bearing mice by inducing apoptotic change of tumor cells. This is a first report to elucidate the feasibility of therapeutic use of 25 kHz, relatively low frequency, ultrasound in sonodynamic therapy using 5-ALA as a sonosensitizer precursor. The utilization of this frequency will contribute to the development of sonodynamic therapy for gliomas and the spread of this technique in many hospitals that possess ultrasonic aspirators.

Preoperative Prediction of Whether Intraoperative Fluorescence of Protoporphyrin IX Can Be Achieved by 5-Aminolevulinic Acid Administration

Protoporphyrin IX (PPIX) fluorescence-guided brain tumor resection, using 5-aminolevulinic acid (5-ALA), is among the most valuable tools for determining tumor removal area. However, PPIX fluorescence is not necessarily achieved during an operation visually even when 5-ALA is used, and we do not know until tumor exposure to the excitation light of the ultraviolet region whether PPIX fluorescence has been achieved. When a particular biopsy and frozen section diagnosis is made, the reason for lack of PPIX fluorescence in the tissue cannot be judged. We do not know whether the tumor fails to fluoresce or no fluorescence is seen because it is not the main body of the tumor. We investigated whether the presence or absence of tumor fluorescence could be predicted by examining urinary porphyrin before surgery, at the time of intraoperative fluorescence diagnosis using 5-ALA. The urine of brain tumor patients 2 hours after 5-ALA administration was irradiated with a 405 ± 1 nm laser light. The patients were divided into a fluorescent urine group and negative fluorescent urine group. Red fluorescence was observed in response to the 405 ± 1 nm laser beam for all tumors in the fluorescent urine group. Clear red fluorescence was not observed even with 405 ± 1 nm laser beam irradiation in any tumors in the negative fluorescent urine group. Preoperative prediction of the intraoperative fluorescence of PPIX can be achieved by observation of urine 2 hours after 5-ALA administration with exposure to a 405 ± 1 nm laser light.