Porphyrin profiles in blood and urine as a biomarker for exposure to various arsenic species

A sensitive method using HPLC with fluorescence detection has been established for the measurement of porphyrins in biological materials. The assay recoveries were 88.0 +/- 1.8% for protoporphyrin IX in the blood, and ranged from 98.3 +/- 2.7% to 111.1 +/- 7.4% for various porphyrins in the urine. This method was employed to investigate the altered porphyrin profiles in rats after a single dose of various arsenicals including soluble sodium arsenate and sodium arsenite, and the relatively insoluble calcium arsenite, calcium arsenate and arsenic-contaminated soils at dose rates of 5 mg/kg or 0.5 mg/kg body weight. Porphyrin concentrations increased within 24-48hr after the arsenic treatment in blood and urine. Protoporphyrin IX is the predominant porphyrin in the blood. In rats administered 5 mg As(III)/kg body weight, protoporphyrin IX concentration elevated to 123% of them control values in rats, 24 hr after the treatment. Higher increases were recorded in the urinary protoporphyrin IX (253% at 24 hr; 397% on day 2), uroporphyrin (121% at 24 hr; 208% on day 2) and coproporphyrin 111 (391% at 24 hr; 304% on day 2), while there was no significant increase (109% on day 3) observed in the urinary coproporphyrin I excretion. In rats administered 5 mg As(V)/kg, urinary excretion of protoporphyrin IX, uroporphyrin, coproporphyrin Ill and coproporphyrin I elevated to the maximum levels by 48 hr with the corresponding percentage values compared to the control being 177%, 158%, 224% and 143%, respectively. In rats dosed with 5 mg As(III)/kg, the increases (expressed as % of the control values) of protoporphyrin IX in the blood were in the order: sodium arsenite (144%) > sodium arsenate (125%) greater than or equal to calcium arsenite (123%) > calcium arsenate. In contrast, there was no significant increase of protoporphyrin K when the six arsenic-contaminated cattlei dip soils and nine copper chrome arsenate (CCA-contaminated) soils were administered to the rats. Probable explanations are discussed.

Porphyrins as early biomarkers for arsenic exposure in animals and humans

We studied the effect of arsenic exposure on the haem biosynthetic pathway in the rat and humans. Significant increases in protoporphyrin IX, coproporphyrin III, coproporphyrin I were observed in the blood, liver and kidney, and in the urine of rats after a single dose of arsenic. The level of increase was dependent on the arsenic species present. Most of porphyrin concentrations in the tissues increased within 24 hr and urinary excretion elevated within 48 hr. In the human study, we collected urine samples from 113 people who live in Xing Ren of Guizhou Province, a coal-borne arsenicosis endemic area in southwest of PR China and from 30 people who live in Xing Yi (about 80 km southwest of Xing Ren) where arsenicosis is not prevalent. We analyzed the urinary porphyrins using HPLC. Results indicate that all urinary porphyrins were higher in the arsenic exposed group than those in the control group. Women, children and older age people spend much of their time indoors, they had greater increases of urinary arsenic and porphyrins. They were the higher risk groups among the study subjects. A positive correlation between the urinary arsenic levels and porphyrin concentrations demonstrated the effect of arsenic on haem biosynthesis. Significant alteration in the porphyrin excretion profiles of the younger age (

Dietary interactions influence the effects of bovine folate-binding protein on the bioavailability of tetrahydrofolates in rats

The newborns of mammals have a high folate demand, yet obtain adequate folate nutrition solely from their mothers' milk despite its low folate content. Milk folate is entirely bound by an excess of folate-binding protein (FBP), prompting speculation that FBP may affect the bioavailability of the limited folate supply. Previous research has shown that FBP-bound folic acid is more gradually absorbed, thereby reducing the peak plasma folate concentration and preventing loss into the urine. Natural folates are reduced derivatives of folic acid, with milk predominantly containing 5-methyltetrahydrofolate, yet little research has been carried out to determine the role of FBP in the bioavailability of reduced folates. We studied the effect of FBP on folate nutrition of rats in both single-dose and 4-wk feeding experiments. The effect of FBP was influenced by the presence of other milk components. FBP increased bioavailability of dietary folate when it was consumed with other whey proteins or with soluble casein. However, in the presence of acid-precipitated casein or a whey preparation enriched in lipids, bioavailability was decreased. These results highlight the difficulties of extrapolating from experimental results obtained using purified diets alone and of studying interactions among dietary components. They suggest that the addition of FBP-rich foods to folate-rich foods could enhance the bioavailability of natural folates, but that the outcome of such a combination would depend on interactions with other components of the diet.

Development of TNBS-induced colitis: animal model to test new pharmacological approaches

IBD is a gastro-intestinal disorder marked with chronic inflammation of intestinal epithelium, damaging mucosal tissue and manifests into several intestinal and extra-intestinal symptoms. Currently used medical therapy is able to induce and maintain the patient in remission, however no modifies or reverses the underlying pathogenic mechanism. The research of other medical approaches is crucial to the treatment of IBD and, for this, it´s important to use animal models to mimic the characteristics of disease in real life. The aim of the study is to develop an animal model of TNBS-induced colitis to test new pharmacological approaches. TNBS was instilled intracolonic single dose as described by Morris et al. It was administered 2,5% TNBS in 50% ethanol through a catheter carefully inserted into the colon. Mice were kept in a Tredelenburg position to avoid reflux. On day 4 and 7, the animals were sacrificed by cervical dislocation. The induction was confirmed based on clinical symptoms/signs, ALP determination and histopathological analysis. At day 4, TNBS group presented a decreased body weight and an alteration of intestinal motility characterized by diarrhea, severe edema of the anus and moderate morbidity, while in the two control groups weren’t identified any alteration on the clinical symptoms/signs with an increase of the body weight. TNBS group presented the highest concentrations of ALP comparing with control groups. The histopathology analysis revealed severe necrosis of the mucosa with widespread necrosis of the intestinal glands. Severe hemorrhagic and purulent exsudates were observed in the submucosa, muscular and serosa. TNBS group presented clinical symptoms/signs and histopathological features compatible with a correct induction of UC. The peak of manifestations became maximal at day 4 after induction. This study allows concluding that it’s possible to develop a TNBS induced colitis 4 days after instillation.

Polio inactivated vaccine costs into routine childhood immunization in Brazil

OBJECTIVE To analyze the costs of vaccination regimens for introducing inactivated polio vaccine in routine immunization in Brazil.METHODS A cost analysis was conducted for vaccines in five vaccination regimens, including inactivated polio vaccine, compared with the oral polio vaccine-only regimen. The costs of the vaccines were estimated for routine use and for the “National Immunization Days”, during when the oral polio vaccine is administered to children aged less than five years, independent of their vaccine status, and the strategic stock of inactivated polio vaccine. The presented estimated costs are of 2011.RESULTS The annual costs of the oral vaccine-only program (routine and two National Immunization Days) were estimated at US$19,873,170. The incremental costs of inclusion of the inactivated vaccine depended on the number of vaccine doses, presentation of the vaccine (bottles with single dose or ten doses), and number of “National Immunization Days” carried out. The cost of the regimen adopted with two doses of inactivated vaccine followed by three doses of oral vaccine and one “National Immunization Day” was estimated at US$29,653,539. The concomitant replacement of the DTPw/Hib and HepB vaccines with the pentavalent vaccine enabled the introduction of the inactivated polio without increasing the number of injections or number of visits needed to complete the vaccination.CONCLUSIONS The introduction of the inactivated vaccine increased the annual costs of the polio vaccines by 49.2% compared with the oral vaccine-only regimen. This increase represented 1.13% of the expenditure of the National Immunization Program on the purchase of vaccines in 2011.

Survey on the clinical trial results achieved in Brazil comparing praziquantel and oxamniquine in the treatment of mansoni schistosomiasis

A random, double-blind, parallel group clinical trial program was carried out to compare praziquantel, a recently developed anti-helmintic drug, and oxamniquine, an already established agent for treating mansoni schistosomiasis. Both drugs were administered orally as a single dose, on the average, praziquantel 55 mg/kg and oxamniquine 16 mg/kg BWT. The diagnosis and the parasitological follow-up lasting for a minimum of six months, were based on stool examinations according to Kato/Katz technique. A patient was considered cured if all results were negative and if he had performed at least three post-treatment controls, each one comprising three stool examinations. The finding of a single S. mansoni egg in any stool examination indicated, a therapeutical failure. A total of 267, cases were treated with praziquantel and 272 with oxamniquine. The two groups were homogeneous in regard to patients, age, clinical form of the disease, risk of reinfection and worm burden, relevant factors in the therapeutical response. The incidence and severity of untoward, effects were similar in both groups but abdominal distress and diarrhoea were more frequently reported under praziquantel and dizzines under oxamniquine (p < 0.05). In the former group a marked urticariform reaction was observed whereas in the latter one patient presented convulsion. The laboratory work-up. failed to disclose any significant alteration although the AST, ALT and y-GT mean values revealed a tendence to increase on the 7th day after oxamniquine intake. The overall parasitological cure rates were 75.5% (139/ 184) with praziquantel and 69.8% (134/192) with oxamniquine (p > 0.05). Amongst the noncured aptients a reduction of 88.6% and 74.6% in the mean number of eggs/g of feces Was seen following the treatment with praziquantel and oxamniquine, respectively (p < 0.05). In conclusion, in spite of their different chemical, pharmacological and toxicological profiles as well as mechanisms-of-action, inclusively praziquantel already had proved to be 100% active against S. mansoni strains resistant to oxamniquine, both drugs showed comparable tolerance and therapeutical efficacy.

Schistosoma mansoni: preclinical studies with 9-acridanone-hydrazones in Cebus monkeys experimentally infected

Derivatives of acridine (9-Acridanone-hydrazones) were tested in Cebus monkeys experimentally infected with Schistosoma mansoni, at the dosages of 50, 25, and 12.5 mg/kg (p.o., single dose). At least, four compounds seemed to be very promising, promoting alterations in the oogram and reducing the worm burden drastically, even at the lowest dose (12.5 mg/kg). No side effects could be detected after drug administration.

Modulation of parasitemia and antibody responce to Trypanosoma cruzy by cyclophosphamide in Calomys callosus (Rodentia, Cricetidae)

Calomys callosus a wild rodent, previously described as harboring Trypanosoma cruzi, has a low susceptibility to infection by this protozoan. Experiments were designed to evaluate the contribution of the immune response to the resistance to T. cruzi infection exhibited by C. calossus. Animals were submitted to injections of high (200 mg/kg body weight) and low (20 mg/kg body weight) doses of cyclophosphamide on days -1 or -1 and +5, and inoculated with 4 x 10³ T. cruzi on day O. Parasitemia, mortality and antibody response as measured by direct agglutination of trypomastigotes were observed. Two hundred mg doses of cyclophosphamide resulted in higher parasitemia and mortality as well as in suppression of the antibody response. A single dose of 20 mg enhanced antibody levels on the 20th day after infection, while an additional dose did not further increase antibody production. Parasitemia levels were not depressed, but rather increased in both these groups as compared to untreated controls. Passive transfer of hyperimmune C. callosus anti-T. cruzi serum to cyclophosphamide immunosuppressed animals resulted in lower parasitemia and mortality rates. These results indicate that the immune response plays an important role in the resistance of C. callossus to T. cruzi.

Persistence of specific antibody response in different experimental infections of mice with Toxocara canis larvae

Anti-Toxocara antibody production and persistence were studied in experimental infections of BALB/c mice, according to three different schedules: Group I (GI) - 25 mice infected with 200 T. canis eggs in a single dose; Group II (GII) 25 mice infected with 150 T. canis eggs given in three occasions, 50 in the 1st, 50 in the 5th and 50 in the 8th days; Group III (GIII) - 25 mice also infected with 150 T. canis eggs, in three 50 eggs portions given in the 1st, 14th and 28th days. A 15 mice control group (GIV) was maintained without infection. In the 30th, 50th, 60th, 75th, 105th and 180th post-infection days three mice of the GI, GII and GIII groups and two mice of the control group had been sacrificed and exsanguinated for sera obtention. In the 360th day the remainder mice of the four groups were, in the same way, killed and processed. The obtained sera were searched for the presence of anti-Toxocara antibodies by an ELISA technique, using T. canis larvae excretion-secretion antigen. In the GI and GII, but not in the GIII, anti-Toxocara antibodies had been found, at least, up to the 180th post-infection day. The GIII only showed anti-Toxocara antibodies, at significant level, in the 30th post-infection day.

Ascariasis in the subdistrict of Cavacos, municipality of Alterosa (MG), Brazil: effect of mass treatment with albendazole on the intensity of infection

The clinical and epidemiologic aspects of infection with Ascaris lumbricoides were studied in a random stratified sample of the population of the subdistrict of Cavacos, municipality of Alterosa (Minas Gerais, Brazil). The effect of mass treatment with a single dose of albendazole on the prevalence and intensity of infection was also studied six months later in the same population. During the first phase of the study, a questionnaire was applied to 248 individuals to obtain information about the socioeconomic, sanitary and clinical conditions of the population surveyed. A total of 230 fecal samples were also examined by the Kato-Katz technique in order to determine the intensity of A. lumbricoides infection. Two hundred and two individuals were simultaneously submitted to blood counts and 70 children aged 12 years or less were evaluated for nutritional status. The presence of A. lumbricoides and other helminth eggs was also determined in 22 soil samples collected in the urban zone of Cavacos. Infection with enteroparasitic helminths was detected in 29.1% of the sample, with a predominance of A. lumbricoides (23.9%). Parasitism and/or intensity of A. lumbricoides infection were significantly correlated with age range (15 years or less), social class, sanitary and living conditions (water, sewage and domiciliary area per person), and presence of abdominal pain. However, these parameters were not correlated with nutritional status or hematocrit levels. During the second phase of the study, a slight but not statistically significant decrease in the prevalence of A. lumbricoides infection was detected after treatment with albendazole. However, an important and significant reduction in the amount of A. lumbricoides eggs eliminated through the feces was detected, indicating that the intensity of A. lumbricoides infection was lower in all the age ranges of the Cavacos population, especially among younger individuals, even six months after administration of the anthelminthic agent.

Use of doxycycline for leptospirosis after high-risk exposure in São Paulo, Brazil

A clinical trial pilot study, double-blinded, randomized, and controlled with a placebo to assess the effectiveness of oral doxycycline (200 mg, single dose) in preventing leptospirosis after high exposure to potentially contamined water was performed in São Paulo, SP, Brazil. Confirmed cases were defined as those with leptospira IgM antibody and symptoms; asymptomatic cases were those presenting with IgM antibodies but no symptoms; and suspected cases were individuals with symptoms but no IgM antibody. Forty subjects were given doxycycline and 42 were given placebo. In the drug-treated group there were 2 confirmed cases, 11 asymptomatic cases, and 6 suspected cases. In the placebo group there were 5 confirmed, 6 symptomatic, and 5 suspected cases. Even though we found a protective association of doxycycline for confirmed leptospirosis cases (RR = 2.3) and seroconversion only (RR = 2.0), the association was not statistically significant because of the small number of individuals enrolled in this pilot study. We observed that the 22% of the volunteers already had IgM antibodies to leptospirosis at the first sampling. Finally, the attack rate to confirmed, asymptomatic, and suspected cases of Leptospirosis was 8.5%, 22%, and 13%, respectively, in this population.

Thiamphenicol in the treatment of chancroid. A study of 1,128 cases

Thiamphenicol, an aminic derivate of hydrocarbilsulfonil propandiol, was used for the treatment of 1,171 chancroid bearing patients. Each patient was medicated with 5.0 g of granulated thiamphenicol, orally, in a single dose, and was reevaluated 3, 7 and 10 days after the treatment. Ten patients (0.89%) did not respond to the proposed treatment. 133 patients presented healed ulcers after 3 days of treatment, 976 patients healed chancres on the seventh day after the treatment, and 39 patients took 10 days to present healed chancres. The results of this study indicate that the rate of patients that were cured, the low incidence of side effects, and the practicality of administration make of thiamphenicol an excellent choice for the treatment of chancroid.

Significance of isolated hepatitis B core antibody in blood donors from São Paulo

The clinical significance of isolated anti-HBc is still a challenge. To elucidate the real importance of this finding in our blood donors, an investigation algorithm was tested. One hundred and twelve isolated anti-HBc seropositive blood donors underwent clinical evaluation and retesting of HBV markers. Those who presented repeatedly reactive isolated anti-HBc, received a single dose of hepatitis B recombinant vaccine to verify anti-HBs early response. A HBV-DNA determination by PCR was done for those who did not test positive to anti-HBs after vaccine. The level of anti-HBc was recorded as a ratio of the sample-to-cut-off values (S:C ratio) in 57 candidates at donation. Comparing true and false-positive anti-HBc results, the different S:C ratios of them were statistically significant and when less than 2, implying in a false-positive result probability over 80%. A high percent of false-positive results (16.07%) was verified after anti-HBc retesting. HBV immunity was characterized in 49.11%, either by anti-HBs detection in retesting (15.18%), or after a single dose HBV vaccination (33.93%). HBV-DNA was negative in all tested donors. In conclusion, this algorithm was useful to clarify the meaning of isolated anti-HBc in most of our blood donors.

Cystoscopy in the diagnosis and follow-up of urinary schistosomiasis in Brazilian soldiers returning from Mozambique, Africa

The assessment of urinary schistosomiasis in individuals coming from endemic areas often requires diagnostic resources not used in areas of exposure in order to determine complications or to establish more precise criteria of cure. Cystoscopy and 24-hour urine examination were performed, after treatments with praziquantel 40 mg/kg body weight, single dose, on 25 Brazilian military men who were part of a United Nations peace mission to Mozambique in 1994. The median age of the individuals was 29 years and all presented a positive urine parasitological exam. The alterations detected by cystoscopy were hyperemia and granulomas in the vesical submucosa in 59.1% of the individuals and only granulomas in 40.9%. A vesical biopsy revealed granulomas in all patients and viable eggs in 77.3% even after a period during which the patients no longer excreted eggs in urine. Cystoscopy after treatment, followed by biopsy and histopathological evaluation, performed in areas where the evolution of the disease can be better monitored, was found to be a safe criterion of parasitological cure.