Reference intervals of 24 trace elements in blood, plasma and erythrocytes for the Slovenian adult population.

OBJECTIVES: The aim of the present study was to establish the population- and laboratory-specific reference intervals (RIs) for the Slovenian adult population for 24 trace elements (TEs) in blood, plasma and erythrocytes and to evaluate the impact of gender, age, seafood consumption, smoking habits and amalgam fillings on TEs levels. METHODS: TEs (Mn, Co, Cu, Zn, Se and Mo, Li, Be, V, Cr, Ni, Ga, As, Rb, Sr, Ag, Cd, Sn, Cs, Au, Hg, Tl, Pb and U) were determined in 192 a priori selected blood donors (107 women and 85 men, aged 18-65 years), using inductively coupled plasma mass spectrometry (ICP-MS) with the Octopole Reaction System. Participants filled out a questionnaire, and RIs were established according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for TEs. RESULTS: Uniform RIs for non-essential and gender-specific for essential TEs in blood, plasma and erythrocytes were established. In our population, higher blood and plasma Cu, and erythrocyte Mn levels in women were found. In men, blood Zn, plasma Zn, Mn and Se, and erythrocyte Cu levels were higher. Zn levels were higher in 30-39 years age group. Pb and Sr increased with age. Smoking positively affected Cd, Pb, Cs and Rb; seafood consumption increased As, Hg and Zn; and amalgam increased Hg, Ag and Cu levels. CONCLUSIONS: Essential TEs were inside recommended levels, and the non-essential ones were far below critical levels. Established RIs will provide an important foundation for clinical diagnostics, safety erythrocyte transfusions assessment, toxicology and epidemiological studies.

Maternal APOE ε2 as a possible risk factor for elevated prenatal Pb levels.

Lead (Pb) is a global contaminant associated with multiple adverse health effects. Humans are especially vulnerable during critical developmental stages. During pregnancy, exposure to Pb can occur through diet and release from maternal bones. Apolipoprotein E gene (APOE) variants (ɛ2, ɛ3, ɛ4 alleles) may influence sex steroid hormones, bone metabolism, and Pb kinetics. We examined the interplay among maternal APOE (mAPOE) genotypes, fetal sex, parity, and Pb in maternal and cord blood (mB-Pb, CB-Pb) using linear regression models. Our study involved 817 pregnant women and 772 newborns with measured adequate levels of zinc and selenium. We compared carriers of the ε2 and ε4 alleles to those with the ε3/ε3 genotype. The geometric means (range) of mB-Pb and CB-Pb were 11.1 (3.58-87.6) and 9.31 (1.82-47.0) ng/g, respectively. In cases with female fetuses, the maternal mAPOE ε2 allele was associated with higher, while the mAPOE ε4 allele was associated with lower mB-Pb and CB-Pb levels. Nulliparity increased the strength of the observed associations. These findings highlight the significance of mAPOE genetics, fetal sex, and parity in prenatal Pb kinetics. Notably, the maternal ε2 allele may increase the risk of Pb exposure.

ALAD and APOE polymorphisms are associated with lead and mercury levels in Italian pregnant women and their newborns with adequate nutritional status of zinc and selenium.

The impacts of single-nucleotide polymorphisms (SNPs) in ALAD and VDR genes on Pb health effects and/or kinetics are inconclusive at low exposure levels, while studies including APOE SNPs are rare. In this study, we examined the associations of ALAD, VDR and APOE SNPs with exposure biomarkers of Pb and other trace elements (TEs) in Italian pregnant women (N = 873, aged 18-44 years) and their newborns (N = 619) with low-level mixed-element exposure through diet, the environment or endogenously. DNA from maternal peripheral venous blood (mB), sampled during the second and third trimesters, was genotyped for ALAD (rs1800435, rs1805313, rs1139488, rs818708), VDR (rs2228570, rs1544410, rs7975232, rs731236) and APOE (rs429358, rs7421) using TaqMan SNP assays. Personal and lifestyle data and TE levels (mB, maternal plasma, hair and mixed umbilical cord blood [CB]) from the PHIME project were used. Multiple linear regression models, controlling for confounding variables, were performed to test the associations between SNPs and TEs. The geometric means of mB-Pb, mB-Hg, mB-As and mB-Cd (11.0 ng/g, 2.16 ng/g, 1.38 ng/g and 0.31 ng/g, respectively) indicated low exposure levels, whereas maternal plasma Zn and Se (0.72 µg/mL and 78.6 ng/g, respectively) indicated adequate micronutritional status. Variant alleles of ALAD rs1800435 and rs1805313 were negatively associated with mB-Pb levels, whereas a positive association was observed for rs1139488. None of the VDR SNPs or their haplotypes had any association with Pb levels. Regarding APOE, the ϵ4 allele was associated with lower mB-Hg and CB-Hg, while a positive association was found with the ϵ2 allele and CB-Pb when the model included only newborn girls. The observed associations indicate possible modification effects of ALAD and APOE SNPs on Pb or Hg kinetics in women and their newborns with low exposure to non-essential TEs, as well as an adequate nutritional status of Zn and Se.

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.

Empagliflozin-Metformin Combination Has Antioxidative and Anti-Inflammatory Properties that Correlate with Vascular Protection in Adults with Type 1 Diabetes.

Methods: 40 individuals with type 1 diabetes (average age of 44.7 ± 2.5 years) were randomized into four groups: (1) control (placebo), (2) empagliflozin 25 mg daily, (3) metformin 2000 mg daily, and (4) empagliflozin-metformin combination (25 mg and 2000 mg daily, respectively). At inclusion and after 12 weeks of treatment, the blood samples were collected, and the oxidative stress (total antioxidative status (TAS), superoxide dismutase (SOD), glutathione peroxidase (GPx), uric acid, advanced oxidation protein products (AOPP), advanced glycosylation end products ((AGE) and isoprostane), and inflammation (C-reactive protein (CRP) and interleukin-6 (IL-6)) parameters were determined. Results: The empagliflozin-metformin combination increased levels of the antioxidants (TAS, SOD, and GPx up to 1.1-fold; P < 0.01), decreased the levels of prooxidants (AOPP and isoprostanes up to 1.2-fold, P < 0.01; AGE up to 1.5-fold, P < 0.01), and decreased inflammatory parameters (up to 1.5-fold, CRP P < 0.01; IL-6 P < 0.001). Antioxidative action was associated with the improvement in arterial function (obtained in the previous study) in the empagliflozin-metformin combination group. Conclusion: Empagliflozin-metformin combination has strong antioxidative and anti-inflammatory capacity, in adults with type 1 diabetes that is greater than that for the individual drugs. Its antioxidative activity at least partially explains the improvement in arterial function. Therefore, it appears that the combination provides the most powerful vascular protection.

Sub-therapeutic doses of fluvastatin and valsartan are more effective than therapeutic doses in providing beneficial cardiovascular pleiotropic effects in rats: A proof of concept study.

BACKGROUND: Statins and sartans can, in therapeutic doses, induce pleiotropic cardiovascular effects. Similar has recently been shown also for sub-therapeutic doses. We thus explored and compared the cardiovascular pleiotropic efficacy of sub-therapeutic vs. therapeutic doses. METHODS: Wistar rats were randomly divided into 7 groups receiving fluvastatin, valsartan and their combination in sub-therapeutic and therapeutic doses, or saline. After 6weeks, the animals were euthanised, their hearts and thoracic aortas isolated, and blood samples taken. Endothelium-dependent relaxation of the thoracic aortae and ischaemic-reperfusion injury of the isolated hearts were assessed along with the related serum parameters and genes expression. RESULTS: Fluvastatin and valsartan alone or in combination were significantly more effective in sub-therapeutic than therapeutic doses. The sub-therapeutic combination greatly increased thoracic aorta endothelium-dependent relaxation and maximally protected the isolated hearts against ischaemia-reperfusion injury and was thus most effective. Beneficial effects were accompanied by increased levels of nitric oxide (NO) and decreased levels of asymmetric dimethylarginine (ADMA) in the serum (again prominently induced by the sub-therapeutic combination). Furthermore, nitric oxide synthase 3 (NOS3) and endothelin receptor type A (EDNRA) genes expression increased, but only in both combination groups and without significant differences between them. In the therapeutic dose groups, fluvastatin and valsartan decreased cholesterol values and systolic blood pressure. CONCLUSION: Sub-therapeutic doses of fluvastatin and valsartan are more effective in expressing cardiovascular pleiotropic effects than therapeutic doses of fluvastatin and/or valsartan. These results could be of significant clinical relevance.

Levels of metals in the blood and specific porphyrins in the urine in children with autism spectrum disorders.

The aim of the present study was to determine the levels of metals in blood (zinc (Zn), copper (Cu), aluminium (Al), lead (Pb) and mercury (Hg)), as well as the specific porphyrin levels in the urine of patients with autism spectrum disorder (ASD) compared with patients with other neurological disorders. The study was performed in a group of children with ASD (N = 52, average age = 6.2 years) and a control group of children with other neurological disorders (N = 22, average age = 6.6 years), matched in terms of intellectual abilities (Mann-Whitney U = 565.0, p = 0.595). Measurement of metals in blood was performed by atomic absorption spectrometry, while the HPLC method via a fluorescence detector was used to test urinary porphyrin levels. Results were compared across groups using a multivariate analysis of covariance (MANCOVA). In addition, a generalized linear model was used to establish the impact of group membership on the blood Cu/Zn ratio. In terms of blood levels of metals, no significant difference between the groups was found. However, compared to the control group, ASD group had significantly elevated blood Cu/Zn ratio (Wald χ (2) = 6.6, df = 1, p = 0.010). Additionally, no significant difference between the groups was found in terms of uroporphyrin I, heptacarboxyporphyrin I, hexacarboxyporphyrin and pentacarboxyporphyrin I. However, the levels of coproporphyrin I and coproporphyrin III were lower in the ASD group compared to the controls. Due to observed higher Cu/Zn ratio, it is suggested to test blood levels of Zn and Cu in all autistic children and give them a Zn supplement if needed.