A significant relationship between mercury exposure from dental amalgams and urinary porphyrins: a further assessment of the Casa Pia children’s dental amalgam trial

Previous studies noted specific changes in urinary porphyrin excretion patterns associated with exposure to mercury (Hg) in animals and humans. In our study, urinary porphyrin concentrations were examined in normal children 8–18 years-old from a reanalysis of data provided from a randomized, prospective clinical trial that was designed to evaluate the potential health consequences of prolonged exposure to Hg from dental amalgam fillings (the parent study). Our analysis examined dose-dependent correlations between increasing Hg exposure from dental amalgams and urinary porphyrins utilizing statistical models with adjustments for the baseline level (i.e. study year 1) of the following variables: urinary Hg, each urinary porphyrin measure, gender, race, and the level of lead (Pb) in each subject’s blood. Significant dose-dependent correlations between cumulative exposure to Hg from dental amalgams and urinary porphyrins associated with Hg body-burden (pentacarboxyporphyrin, precoproporphyrin, and coproporphyrin) were observed. Overall, 5–10% increases in Hg-associated porphyrins for subjects receiving an average number of dental amalgam fillings in comparison to subjects receiving only composite fillings were observed over the 8-year course of the study. In contrast, no significant correlations were observed between cumulative exposure to Hg from dental amalgams and urinary porphyrins not associated with Hg body-burden (uroporphyrin, heptacarboxyporphyrin, and hexacarboxyporphyrin). In conclusion, our study, in contrast to the no-effect results published from the parent study, further establishes the sensitivity and specificity of specific urinary porphyrins as a biomarker for low-level Hg body-burden, and also reveals that dental amalgams are a significant chronic contributor to Hg body-burden.     

The Relative Efficacy of Connectivity Guided and Symptom Based EEG Biofeedback for Autistic Disorders

Autism is a neurodevelopmental disorder characterized by deficits in communication, social interaction, and a limited range of interests with repetitive stereotypical behavior. Various abnormalities have been documented in the brains of individuals with autism, both anatomically and functionally. The connectivity theory of autism is a recently developed theory of the neurobiological cause of autisic symptoms. Different patterns of hyper- and hypo-connectivity have been identified with the use of quantitative electroencephalogray (QEEG), which may be amenable to neurofeedback. In this study, we compared the results of two published controlled studies examining the efficacy of neurofeedback in the treatment of autism. Specifically, we examined whether a symptom based approach or an assessment/connectivity guided based approach was more effective. Although both methods demonstrated significant improvement in symptoms of autism, connectivity guided neurofeedback demonstrated greater reduction on various subscales of the Autism Treatment Evaluation Checklist (ATEC). Furthermore, when individuals were matched for severity of symptoms, the amount of change per session was significantly higher in the Coben and Padolsky (J Neurother 11:5–23, 2007) study for all five measures of the ATEC. Our findings suggest that an approach guided by QEEG based connectivity assessment may be more efficacious in the treatment of autism. This permits the targeting and amelioration of abnormal connectivity patterns in the brains of people who are autistic.     

Effect of l-Carnosine as adjunctive therapy in the management of children with autism spectrum disorder: a randomized controlled study

l-Carnosine is an amino acid that acts as an anti-oxidant, anti-toxic and neuroprotective agent. There is a paucity of data about the effectiveness of l-Carnosine in the management of autism spectrum disorder (ASD) in children. This study aimed at investigating the effectiveness of l-Carnosine as adjunctive therapy in the management of ASD. This was a randomized controlled trial. Children aged 3–6 years with a diagnosis of mild to moderate ASD were assigned to standard care arm (occupational and speech therapy) and intervention care arm (l-Carnosine, 10–15 mg/kg in 2 divided doses) plus standard care treatment. The children were assessed at the baseline and the end of 2 months for the scores of Childhood Autism Rating Scale, Second Edition—Standard Version (CARS2-ST), Autism Treatment Evaluation Checklist (ATEC), BEARS sleep screening tool and 6-item Gastrointestinal Severity Index (6-GSI). Of the sixty-seven children enrolled, sixty-three children had completed the study. No statistically significant difference (p > 0.05) was observed for any of the outcome measures assessed. Supplementation of l-Carnosine did not improve the total score of CARS2-ST, ATEC, BEARS sleep screening tool and 6-GSI scores of children with ASD. Further investigations are needed with more objective assessments to critically validate the effectiveness of l-Carnosine on ASD children for more decisive results.

     

In vitro toxicity of palladium(II) and gold(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth

The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.     

Effects of a dolphin interaction program on children with autism spectrum disorders - an exploratory research

ABSTRACT: BACKGROUND: Interaction programs involving dolphins and patients with various pathologies or developmental disorders (e.g., cerebral palsy, intellectual impairment, autism, atopic dermatitis, post-traumatic stress disorder, depression) have stimulated interest in their beneficial effects and therapeutic potential. However, the true effects observed in different clinical and psycho-educational setups are still controversial. RESULTS: An evaluation protocol consisting of the Childhood Autism Rating Scale (CARS), Psychoeducational Profile-Revised (PEP-R), Autism Treatment Evaluation Checklist (ATEC), Theory of Mind Tasks (ToM Tasks) and a custom-made Interaction Evaluation Grid (IEG) to evaluate behavioural complexity during in-pool interactions was applied to 10 children diagnosed with Autism Spectrum Disorders. The ATEC, ToM Tasks and CARS results show no benefits of the dolphin interaction program. Interestingly, the PEP-R suggests some statistically significant effects on 'Overall development score', as well as on their 'Fine motor development', 'Cognitive performance' and 'Cognitive verbal development'. Also, a significant evolution in behavioural complexity was shown by the IEG. CONCLUSIONS: This study does not support significant developmental progress resulting from the dolphin interaction program.     

Prospective,Blinded Exploratory Evaluation of the PlayWisely Program in Children with Autism Spectrum Disorder

The purpose of the study was to explore a low-cost intervention that targets an increasingly common developmental disorder. The study was a blinded, exploratory evaluation of the PlayWisely program on autism symptoms and essential learning foundation skills (attention, recognition, and memory skills) in children with a diagnosis of autism, autism spectrum disorder (ASD), pervasive developmental disorder – not otherwise specified (PDD-NOS), and Asperger syndrome (AS). Eighteen children, 1 to 10 years of age, were evaluated using the Childhood Autism Rating Scale, Second Edition (CARS2); the PlayWisely Interactive Test of Attention, Recognition, and Memory Skills; Autism Treatment Evaluation Checklist (ATEC), and the Modified Checklist for Autism in Toddlers (M-CHAT). There were significant treatment effects for the PlayWisely measure on the Yellow Sets that examine recognition; Purple Sets that examine brain region agility and early memory skills; Blue Sets that examine phonemic awareness and recognition; and for the Total Sets, with a similar trend toward improvement in the Green Sets that examine perception and Red Sets that examine attention. No other measures reached statistical significance. The results suggest that PlayWisely can improve recognition, brain region agility, phonemic awareness, letter recognition, and early memory skills in ASD. It was observed by the parents, coaches, and study investigators that the children who were less than 3 years of age showed improvements in autism symptoms; however, the group was too small to reach statistical significance. Future studies are needed to see if this intervention can mitigate autism symptoms in very young children with ASD.     

The Relationship between Temperament and Autistic Traits in a Non-Clinical Students Sample

Since temperament affects the development of social behaviours and interpersonal relations, the possible links between autistic traits and temperament are of particular interest. The purpose of the study was to explore the relationships between autistic traits and temperamental characteristics in the framework of the Regulative Temperament Theory by Strelau, and the Emotionality, Activity and Sociability theory by Buss and Plomin, with particular emphasis on gender differences. The Autism Spectrum Quotient (AQ), Formal Characteristics of Behaviour – Temperament Inventory and Temperament Survey for Adults were administered. The participants were 593 university students, including 364 females and 229 males. Results showed positive correlations between autistic traits and Emotional Reactivity, Perseveration, Distress, Fear and Anger, and negative correlations with Activity, Briskness, Endurance and Sociability. The results of multiple regression analyses involving the Autism Spectrum Quotient score as a dependent measure were different for females and males. Results of exploratory PCA analysis showed that AQ score, Sociability and Activity loaded one factor (with AQ loading being opposite to two others). High AQ scorers demonstrated higher Emotional Reactivity, Perseveration, Distress and Anger, and lower Briskness, Endurance, Activity and Sociability as compared to norms for the general population. In this study we showed that temperament measures were able to identify items that correlated in parts with autistic traits, while other items were obverse. The relationships between temperament and autistic traits differ slightly between genders. We assume that with regard to the broader autism phenotype, temperaments might be helpful in characterizing healthy control samples.     

A comparison of urinary mercury between children with autism spectrum disorders and control children

Background

Urinary mercury concentrations are used in research exploring mercury exposure. Some theorists have proposed that autism is caused by mercury toxicity. We set out to test whether mercury concentrations in the urine of children with autism were significantly increased or decreased compared to controls or siblings.

Methods

Blinded cohort analyses were carried out on the urine of 56 children with autism spectrum disorders (ASD) compared to their siblings (n = 42) and a control sample of children without ASD in mainstream (n = 121) and special schools (n = 34).

Results

There were no statistically significant differences in creatinine levels, in uncorrected urinary mercury levels or in levels of mercury corrected for creatinine, whether or not the analysis is controlled for age, gender and amalgam fillings.

Conclusions

This study lends no support for the hypothesis of differences in urinary mercury excretion in children with autism compared to other groups. Some of the results, however, do suggest further research in the area may be warranted to replicate this in a larger group and with clear measurement of potential confounding factors.     

A review of candidate urinary biomarkers for autism spectrum disorder

Context: Autism is a complex, heterogeneous neurodevelopmental condition with a strong genetic component potentially impacted by various environmental factors influencing susceptibility. There are no reliable laboratory tests available to confirm an autism diagnosis.

Objective: To examine the published literature and identify putative urinary biomarkers of autism.

Methods: A comprehensive literature search was conducted using electronic bibliographic databases.

Results: Putative autism biomarkers were identified that could be categorized according to the key theories that exist regarding the etiology of autism: gastrointestinal factors, immune dysregulation, heavy metal toxicity, neurotransmitter abnormalities, and oxidative stress.

Conclusion: There is scope for specific urinary biomarkers to be useful for identification of autistic metabolic phenotypes.     

Toxic effects of mercury(II), cadmium(II) and lead(II) porphyrins on Trypanosoma brucei brucei growth

The effects of free mercury(II), cadmium(II) and lead(II) ions and their metalloporphyrin-derivatives on Trypanosoma brucei brucei growth in culture were studied. All experiments were conducted in the dark. IC(50) values on growth obtained in 24-h time-course experiments were 1.5 x 10(-7), 2.4 x 10(-6), 4.4 x 10(-6) and 2.6 x 10(-5) M for mercury(II) porphyrin, cadmium(II) porphyrin, lead(II) porphyrin and free base porphyrin, respectively. While the IC50 values for Hg2+, Cd2+ and Pb2+ were 3.6 x 10(-6), 1.5 x 10(-5) and 1.6 x 10(-5) M, respectively. These results clearly indicate that the toxicity of the metalloporphyrin complexes of mercury(II), cadmium(II) and lead(II) to T. b. brucei parasites was much higher compared to their free metal ions and free base porphyrin at low concentrations. It was also observed after 8 h incubation that the metalloporphyrins were effective in inhibiting the division of the parasites at concentrations >1.25 x 10(-7) M for mercury(II) porphyrin, concentrations >1.2 x 10(-6) M for cadmium(II) and lead(II) porphyrins and at concentrations >3.6 x 10(-6) M for Hg2+ ion. These observations were not detected in samples treated with the free metal ions and the free base porphyrin at the same concentrations. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. The chemotherapeutic potential of the metalloporphyrins of H2TMPyP for treatment of African trypanosomiasis is 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 (<20 y) arsenic exposed group suggested that porphyrins could be used as early warning biomarkers for chronic exposure to arsenic.     

Determination of porphyrins and biliverdin in bile and excreta of birds by a single liquid chromatography-ultraviolet detection analysis

Methods developed for porphyrin analysis have low recoveries and/or poor precision for the less polar protoporphyrin IX. We describe a simple method of analysis of porphyrins and biliverdin in bile and excreta of birds based on extraction with HCl 3N: acetonitrile and HPLC/UV analyses. Recoveries were good for protoporphyrin IX and other porphyrins (>79%). Applications of this method showed that porphyrins and biliverdin in birds excreta are mainly of biliary-fecal origin rather than urinary origin. Biliverdin and protoporphyrin IX increased proportionately more than the rest of the porphyrins and coproporphyrin III increased more than coproporphyrin I in the bile of Pb-poisoned mallards.     

Magnetic circular dichroism spectroscopy: A definitive method for the determination of urinary porphyrins

The application of MCD for the determination of urinary porphyrins is assayed by specific application to the screening of workers exposed to lead arsenate. The definitive nature of this new spectroscopic method arises from: (1) its sensitivity to as little as 0.02 μg/ml of porphyrin dication; (2) its freedom from interference caused by other substances; (3) the highly characteristic shape of the MCD bands exhibited by porphyrin dications; and (4) the high information content of the MCD spectrum.     

A new rapid method for isolation of naturally occurring porphyrins and their quantitation after high performance liquid chromatography

High performance liquid chromatography (HPLC) has been used to separate porphyrin methyl esters isolated from porphyric patients. Fecal porphyrins were esterified directly with boron trifluoride: urinary porphyrins were absorbed on talcum and then treated with boron trifluoride. HPLC permits rapid, efficient, and quantitative detection and identification of porphyrins in complex natural mixtures.     

Spontaneous porphyria of the Long-evans cinnamon rat: an animal model of Wilson's disease

To confirm and extend our previous microspectrophotometric observations of 30-week-old male Long-Evans Cinnamon (LEC) rats, an animal model of human Wilson's disease, we analyzed the porphyrin patterns of the organs, urine, and plasma of LEC rats. Abnormal accumulation of porphyrins, especially highly carboxylated porphyrins (uro- and heptaporphyrin), in the kidneys and liver was seen in male and female LEC rats aged 30 weeks and also in 10-week-old rats, before the onset of spontaneous hepatic dysfunction. Accumulation of copper and iron in the kidneys was not observed in the 10-week-old rats. Massive accumulation of porphyrins was observed only in the kidneys of the 30-week-old male LEC rat, indicating that this symptom is related to sex and age. Renal accumulation of porphyrins was reflected in the rate of urinary porphyrin excretion. Hepatic accumulation of porphyrins appeared to be independent of sex and age. These results indicate that neither renal nor hepatic porphyrin accumulation is the result of renal deposition of metals or of spontaneous hepatic dysfunction and that porphyrinuria in the LEC rat is closely related to the renal accumulation of porphyrins. In contrast to these organs, a reduction in the porphyrin levels was observed in the brain of the LEC rat. This was independent of sex and age. The present work stresses the existence of an abnormal heme metabolism in the LEC rat, and thus, the necessity to study the heme metabolism in human Wilson's disease is strongly suggested.     

Kinetics of porphyrin accumulation in cultured epithelial cells exposed to ALA

• 1.1. The kinetics of porphyrin accumulation in cultured mammalian epithelial cells (CNCM-I-221) during exposure to ALA was investigated.
• 2.2. The total porphyrin synthesized is a function of ALA concentration and the incubation time. The cellular porphyrin content exhibited a saturation pattern, reaching a plateau at about 0.04 fmol porphyrins/cell. A biphasic time-dependent increase in the total porphyrin synthesized was observed.
• 3.3. After 3 hr of exposure to ALA the rate of synthesis increased to ahnost twice the initial rate, reaching between 0.02 and 0.05 fmol porphyrins/cell/hr depending on serum concentration in the medium.
• 4.4. Two effects of FBS on ALA-stimulated porphyrin accumulation were observed. Greater total porphyrin synthesis was found when incubations were made in 10% FBS compared to those in 1% FBS.
• 5.5. The higher serum concentration also caused a greater release into the medium of the porphyrins generated in the cells with a calculated half-life of 24 min in 10% serum-supplemented medium compared with 62 min in 1% serum.
• 6.6. The results obtained from cell synchronization experiments suggest that there is little obvious cell cycle-dependent variation in the synthesis of porphyrins from ALA.
• 7.7. The small differences in the intracellular porphyrin content that were observed may be attributed to a slight reduction in the rate of loss of porphyrins in G2/M cells.

     

Total syntheses of three copper (II) tetracarboranylphenylporphyrins containing 40 or 80 boron atoms and their biological properties in EMT-6 tumor-bearing mice

Three carboranyltetraphenylporphyrins containing 40 or 80 boron atoms were synthesized and evaluated for their biodistribution and toxicity in EMT-6 tumor-bearing mice. Copper (II) meso-5,10,15,20-tetrakis[3-methoxy-4-(o-carboranylmethoxy)phenyl]porphyrin, 6, and copper (II) meso-5,10,15,20-tetrakis[3-hydroxy-4-(o-carboranylmethoxy)phenyl]porphyrin, 8, are B40 congeners with different lipophilicities, each less than their B80 congener, copper (II) meso-5,10,15,20-tetrakis[m-(3,5-di-o-carboranylmethoxybenzyloxy)phenyl]porphyrin, 18. Two days after the last of a series of i.p. injections in BALB/c mice bearing EMT-6 mammary tumors, a dose of 185 mg/kg 6 (54 mg/kg B) delivered over 3.5 times the concentration of boron to tumor (169 microg/g B) than did 118 mg/kg 8 (36 mg/kg B), which delivered 35 microg/g B, or 87 mg/kg 18 (30 mg/kg B), which delivered 46 microg/g B. The tumor-to-blood and tumor-to-brain boron concentration ratios at that time for all three porphyrins exceeded 80:1. Two days after the last injection, there resulted moderate thrombocytopenia that essentially disappeared two days later from 6 and 18, and mild leukocytosis from 6, 8, and 18, all of which were clinically inconsequential. Thus, 6 may rank among the most clinically promising carboranyl porphyrins ever made to deliver 10B to tumors for boron neutron-capture therapy (BNCT) that has also been tested for its toxicity in vivo.     

Detection of antinuclear and antilaminin antibodies in autistic children who received thimerosal-containing vaccines

Autism, a neurodevelopmental disorder, may involve autoimmune pathogenesis. Since mercury is potentially a risk factor for autoimmunity, we conducted a study of mercury-induced antinuclear and antilaminin antibodies in autistic and normal children who had been pre-administered with thimerosal-containing vaccines. Laboratory analysis by different immunoassays showed that the serum level of these two autoimmune markers did not significantly differ between autistic and normal children. This finding suggests that the mercury as in thimerosal-containing vaccines is likely not related to autoimmune phenomenon in autism.     

Attenuation of Typical Sex Differences in 800 Adults with Autism vs. 3,900 Controls

Sex differences have been reported in autistic traits and systemizing (male advantage), and empathizing (female advantage) among typically developing individuals. In individuals with autism, these cognitive-behavioural profiles correspond to predictions from the “extreme male brain” (EMB) theory of autism (extreme scores on autistic traits and systemizing, below average on empathizing). Sex differences within autism, however, have been under-investigated. Here we show in 811 adults (454 females) with autism and 3,906 age-matched typical control adults (2,562 females) who completed the Empathy Quotient (EQ), the Systemizing Quotient-Revised (SQ-R), and the Autism Spectrum Quotient (AQ), that typical females on average scored higher on the EQ, typical males scored higher on the SQ-R and AQ, and both males and females with autism showed a shift toward the extreme of the “male profile” on these measures and in the distribution of “brain types” (the discrepancy between standardized EQ and SQ-R scores). Further, normative sex differences are attenuated but not abolished in adults with autism. The findings provide strong support for the EMB theory of autism, and highlight differences between males and females with autism.     

Evidence for the interference of aluminum with bacterial porphyrin biosynthesis

Aluminum (0.74 mm) was found to retard bacterial growth, and enhance porphyrin formation and excretion in Arthrobacter aurescens RS-2. Coproporphyrin III was shown to be the main porphyrin excreted by aluminum-exposed A. aurescens RS-2 cultures and by RS-2 cultures grown under anoxic conditions. Synthesis and excretion of porphyrins in A. aurescens RS-2 increased in a dose-dependent manner when the bacteria were exposed to increasing aluminum concentrations. Incubation of A. aurescens RS-2 with -aminolevulinic acid (-ALA, 1.2 mm) brought about the intense formation and excretion of porphyrins by the cells, in the presence or absence of aluminum. -ALA slightly enhanced the toxicity of aluminum towards RS-2 bacteria. Furthermore, the intracellular concentration of heme was reduced by 63.9 ± 8.67% in aluminum-exposed RS-2 bacteria when compared with control cultures. The results are discussed in light of the recent finding concerning aluminum toxicity and porphyrin biosynthesis in microorganisms.