Biochemical markers in autism spectrum disorder

Avtizem je razvojna, pervazivna nevrološko-biološka motnja, za katero so značilne motnje v socialnih stikih, teţave z verbalno in neverbalno komunikacijo ter nenavadne, ponavljajoče in zelo omejene spretnosti in zanimanja. Ker se fenotipsko izraţa na zelo različne načine in zaradi zelo širokega spektra kliničnih značilnosti avtizma, so strokovnjaki uvedli terminologijo »spekter avtističnih motenj« ali SAM. Prevalenca SAM se giblje od 9 do 11,6 na 1000 otrok. Razmerje med dečki in deklicami se giblje med 2:1 in 6,5:1. Podatki študij, ki so bile objavljene v zadnjih petdesetih letih, prepričljivo kaţejo, da so za razvoj SAM odločilni genetski dejavniki, vendar predvidevajo, da so v razvoj SAM vpleteni tudi dejavniki okolja. Zelo pogosto kot dejavnike okolja raziskujejo infekcije, cepiva, zdravila in številne teţke kovine, najpogosteje med njimi ţivo srebro, svinec, aluminij, cink in baker, vendar vzroki nastanka bolezni ostajajo večinoma neznani. Čeprav je avtizem nevrobiološka motnja, diagnostika sloni predvsem na zdravniških pregledih z zbiranjem natančnih anamnestičnih podatkov o razvoju otroka, opazovanjem otroka v različnih razmerah ter psihološkem testiranju. Trenutno še nimamo biološkega testa ali specifičnega označevalca, s katerim bi jo lahko diagnosticirali. V naši raziskavi smo v skupini s spektrom avtističnih motenj vključili 86 otrok, v kontrolni skupini, ki so jo sestavljali otroci z drugimi nevrološkimi motnjami pa 21 otrok. Ţeleli smo ugotoviti ali obstajajo razlike v koncentracijah ţivega srebra, svinca, aluminija, cinka in bakra v krvi ter posameznih porfirinskih frakcijah v urinu med skupino s spektrom avtističnih motenj in kontrolno skupino ter populacijskimi vrednostmi otrok, starih od 1 do 15 let. Ţivo srebro v krvi smo določali z atomsko absorpcijsko spektroskopijo hladnih par, svinec v krvi in aluminij v serumu smo določali z metodo elektrotermične atomske absorpcijske spektrometrije. Cink in baker v serumu smo določali z metodo plamenske absorpcijske spektrometrije. Posamezne frakcije porfirinov v urinu smo določali z metodo tekočinske kromatografije visoke ločljivosti. Naši rezultati kaţejo, da so povprečne vrednosti ţivega srebra v krvi, aluminija v serumu in svinca v krvi pri skupini SAM nekoliko višje kot pri kontrolni skupini, vendar statistično značilnih razlik nismo našli. Povprečne vrednosti bakra, cinka in razmerja med bakrom in cinkom v serumu so pri skupini SAM niţje kot pri kontrolni skupini, vendar razlike niso bile statistično značilne. Primerjava vrednosti skupine SAM z vrednostmi populacije otrok starih med 1 in 15 let pa je pokazala, da so vrednosti v skupini SAM za ţivo srebro v krvi, aluminij in baker statistično značilno višje, za cink pa značilno niţje glede na populacijske vrednosti otrok, starih med 1 in 15 leti. Kot biološke označevalce za toksične učinke teţkih kovin smo v urinu določili tudi celokupne porfirine in porfirinske frakcije. Primerjave porfirinskih frakcij med skupino SAM in kontrolno skupino so pokazale, da med skupinama ni statistično pomembnih razlik, z izjemo pentakarboksiporfirina, ki je v skupini SAM značilno višji. V primerjavi s povprečnimi vrednostmi za tipično nevrološko razvite otroke so vrednosti naše skupine SAM za uroporfirin, heptakarboksiporfirin, heksakarboksiporfirin, pentakarboksiporfirin in koproporfirin višje. Ugotovili smo negativno statistično pomembno povezavo med serumskim cinkom in koproporfirnom I ter celokupnimi porfirini. Statistično pomembno pozitivno povezavo smo ugotovili med ţivim srebrom v urinu, izraţenim na kreatinin, in razmerjem koproporfirin III / koproporfirin I. Študija predstavlja prvo raziskavo biokemičnih dejavnikov pri osebah z spektrom avtističnih motenj v Sloveniji in je pomembna obogatitev na tem področju tudi v mednarodnem prostoru, kjer so bile izvedene ţe številne podobne raziskave, vendar večinoma brez primerjave z ustrezno kontrolno skupino, ki je bila v naši raziskavi sestavljena iz otrok z različnimi drugimi nevrološkimi motnjami.Autism spectrum disorders (ASD) are pervasive neural developmental disorders characterized by varying degrees of impairment in communication skills, social interactions, and restricted, repetitive and stereotyped patterns of behavior. Autism spectrum disorders prevalence is approximately 9 to 11,6 per 1000 children and it affects more boys than girls (from 2:1 to 6,5:1). Autism spectrum disorders have a strong genetic basis, but there are possible environmental factors that also contribute to ASD such as heavy metals, drugs and other toxins, infections and vaccines. Most frequently mentioned heavy metals are mercury, lead, aluminum, copper and zinc. Although ASD have neuro-biological base there are no biological tests or specific biochemical markers for ASD. The main scope of our study was to discover differences in concentrations of toxic metals such as mercury, lead, aluminum, copper and zinc in blood between group of children with ASD, control group, which consisted of children with neurological disturbances other than ASD and population group, which consisted of children aged between 1 and 15 years. Comparison of urine porphyrins in group with ASD with those of control group was performed too. The study included 86 ASD children and 21 children in control group. Determinations of mercury, aluminum, lead, copper and zinc were performed by atomic absorption spectrometry. Analysis of urinary porphyrins was performed by high performance liquid chromatography. There were no statistical differences in concentrations of toxic metals between ASD group and control group, but there were significant differences when comparing the concentrations between ASD group and population group. Mercury, lead, aluminum and copper were significantly higher and zinc were significantly lower in the ASD group. Porphyrins in urine were determined as biological markers of intoxication with heavy metals. There were no statistical differences between the ASD group and control group except for pentacoproporphyrin which was significantly higher in the ASD group. Comparing porphyrins in the ASD group to group of children with normal neurological development we found significantly higher concentrations for uroporphyrin, heptacarboxyporphyrin, hexacarboxyporphyrin, pentacarboxyporphyrin and coproporphyrin in the ASD group. Significant connection between mercury in urine and ratio between coproporphyrin III and coproporphyrin I was discovered. This study is first investigation of possible biochemical markers in ASD in Slovenia and it is also the first one to include children with neurological disorder other than ASD as the control group

Arsenic metabolites ; selenium ; and AS3MT, MTHFR, AQP4, AQP9, SELENOP, INMT and MT2A polymorphisms in Croatian-Slovenian population from PHIME-CROME study

The relationships between inorganic arsenic (iAs) metabolism, selenium (Se) status, and genetic polymorphisms of various genes, commonly studied in populations exposed to high levels of iAs from drinking water, were studied in a Croatian- Slovenian population from the wider PHIME-CROME project. Population consisted of 136 pregnant women in the 3rd trimester and 176 non-pregnant women with their children (n = 176, 8–9 years old). Their exposure to iAs, defined by As (speciation) analyses of biological samples, was low. The sums of biologically active metabolites (arsenite + arsenate + methylated As forms) for pregnant women, non-pregnant women, and children, respectively were: 3.23 (2.84–3.68), 1.83 (1.54– 2.16) and 2.18 (1.86–2.54) ng/mLSG ; GM (95 CI). Corresponding plasma Se levels were: 54.8 (52.8– 56.9), 82.3 (80.4–84.0) and 65.8 (64.3–67.3) ng/mL ; GM (95 CI). As methylation efficiency indexes confirmed the relationship between pregnancy/childhood and better methylation efficiency. Archived blood and/or saliva samples were used for single nucleotide polymorphism (SNP) genotyping of arsenic(3+) methyltransferase - AS3MT (rs7085104, rs3740400, rs3740393, rs3740390, rs11191439, rs10748835, rs1046778 and the corresponding AS3MT haplotype) ; methylene tetrahydrofolate reductase - MTHFR (rs1801131, rs1801133) ; aquaporin - AQP 4 and 9 (rs9951307 and rs2414539) ; selenoprotein P1 - SELENOP (rs7579, rs3877899) ; indolethylamine N- methyltransferase - INMT (rs6970396) ; and metallothionein 2A - MT2A (rs28366003). Associations of SNPs with As parameters and urine Se were determined through multiple regression analyses adjusted using appropriate confounders (blood As, plasma Se, ever smoking, etc.). SNPs’ influence on As methylation, defined particularly by the secondary methylation index (SMI), confirmed the ‘protective’ role of minor alleles of six AS3MT SNPs and their haplotype only among non-pregnant women. Among the other investigated genes, the carriers of AQP9 (rs2414539) were associated with more efficient As methylation and higher urine concentration of As and Se among non-pregnant women ; poorer methylation was observed for carriers of AQP4 (rs9951307) among pregnant women and SELENOP (rs7579) among non- pregnant women ; MT2A (rs28366003) was associated with higher urine concentration of AsIII regardless of the pregnancy status ; and INMT (rs6970396) was associated with higher As and Se concentration in non-pregnant women. Among confounders, the strongest influence was observed for plasma Se ; it reduced urine AsIII concentration during pregnancy and increased secondary methylation index among non-pregnant women. In the present study of populations with low As exposure, we observed a few new As–gene associations (particularly with AQPs). More reliable interpretations will be possible after their confirmation in larger populations with higher As exposure levels