Farnesol and Selected Nanoparticles (Silver, Gold, Copper, and Zinc Oxide) as Effective Agents Against Biofilms Formed by Pathogenic Microorganisms.

Purpose: Biofilms, which are created by most microorganisms, are known for their widely developed drug resistance, even more than planktonic forms of microorganisms. The aim of the study was to assess the effectiveness of agents composed of farnesol and nanoparticles (silver, gold, copper, and zinc oxide) in the degradation of biofilms produced by pathogenic microorganisms. Methods: Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans were used to create the biofilm structure. Colloidal suspensions of silver, gold, copper, and zinc oxide (Ag, Au, Cu, ZnO) with the addition of farnesol (F) were used as the treatment factor. The size distribution of those composites was analyzed, their zeta potential was measured, and their structure was visualized by transmission electron microscopy. The viability of the microorganism strains was assessed by an XTT assay, the ability to form biofilms was analyzed by confocal microscopy, and the changes in biofilm structure were evaluated by scanning electron microscopy. The general toxicity toward the HFFF2 cell line was determined by a neutral red assay and a human inflammation antibody array. Results: The link between the two components (farnesol and nanoparticles) caused mutual stability of both components. Planktonic forms of the microorganisms were the most sensitive when exposed to AgF and CuF; however, the biofilm structure of all microorganism strains was the most disrupted (both inhibition of formation and changes within the structure) after AgF treatment. Composites were not toxic toward the HFFF2 cell line, although the expression of several cytokines was higher than in the not-treated group. Conclusion: The in vitro studies demonstrated antibiofilm properties of composites based on farnesol and nanoparticles. The greatest changes in biofilm structure were triggered by AgF, causing an alteration in the biofilm formation process as well as in the biofilm structure.

Evaluation of the Antimicrobial, Cytotoxic, and Physical Properties of Selected Nano-Complexes in Bovine Udder Inflammatory Pathogen Control.

Purpose: Mastitis in dairy cows is a worldwide problem faced by dairy producers. Treatment mainly involves antibiotic therapy, however, due to widespread antibiotic resistance among bacteria, such treatments are no longer effective. For this reason, scientists are searching for new solutions to combat mastitis, which is caused by bacteria, fungi, and algae. One of the most promising solutions, nanotechnology, is attracting research due to its biocidal properties. The purpose of this research was to determine the biocidal properties of nanocomposites as a potential alternative to antibiotics in the control of mastitis, as well as to determine whether the use of nanoparticles and what concentration is safe for the breeder and the animal. Patients and Methods: In this study, the effects of Ag, Au, Cu, Fe, and Pt nanoparticles and their complexes were evaluated in relation to the survival of bacteria and fungi isolated from cattle diagnosed with mastitis, their physicochemical properties, and their toxicity to bovine and human mammary epithelial cells BME-UV1 and HMEC (human microvascular endothelial cells). Moreover, E. coli, S. aureus, C. albicans, and Prototheca sp. invasion was assessed using the alginate bead (bioprinted) model. The NPs were tested at concentrations of 25, 12.5, 6.25, 3.125, 1.56 mg/l for Au, Ag, Cu and Fe NPs, and 10, 5, 2.5, 1.25, 0.625 mg/l for Pt. Results: With the exception of Fe and Pt, all exhibited biocidal properties against isolates, while the AgCu complex had the best effect. In addition, nanoparticles showed synergistic effects, while the low concentrations had no toxic effect on BME-UV1 and HMEC cells. Conclusion: Synergistic effects of nanoparticles and no toxicity to bovine and human cells might, in the future, be an effective alternative in the fight against microorganisms responsible for mastitis, and the implementation of research results in practice would reduce the percentage of dairy cows suffering from mastitis. The problem of increasing antibiotic resistance is posing a global threat to human's and animal's health, and requires comprehensive research to evaluate the potential use of nanoparticles - especially their complexes - as well as to determine whether nanoparticles are safe for the breeders and the animals. The conducted series of studies allows further consideration of the use of the obtained results in practice, creating a potentially new alternative to antibiotics in the treatment and prevention of mastitis in dairy cattle.

Ciprofloxacin-, Cefazolin-, and Methicilin-Soaked Graphene Paper as an Antibacterial Medium Suppressing Cell Growth.

This paper presents the results of research on the impact of graphene paper on selected bacterial strains. Graphene oxide, from which graphene paper is made, has mainly bacteriostatic properties. Therefore, the main goal of this research was to determine the possibility of using graphene paper as a carrier of a medicinal substance. Studies of the degree of bacterial inhibition were performed on Staphylococcus aureus and Pseudomonas aeruginosa strains. Graphene paper was analyzed not only in the state of delivery but also after the incorporation of the antibiotics ciprofloxacin, cefazolin, and methicillin into its structures. In addition, Fourier-Transform Infrared Spectroscopy, contact angle, and microscopic analysis of bacteria on the surface of the examined graphene paper samples were also performed. Studies have shown that graphene paper with built-in ciprofloxacin had a bactericidal effect on the strains of Staphylococcus aureus and Pseudomonas aeruginosa. In contrast, methicillin, as well as cefazolin, deposited on graphene paper acted mainly locally. Studies have shown that graphene paper can be used as a carrier of selected medicinal substances.

Effects of the Addition of Crude Fibre Concentrate on Performance, Welfare and Selected Caecal Bacteria of Broilers.

The study evaluated the effects of crude fibre concentrate supplementation on final body weight, mortality, feed conversion ratio, European Production Efficiency Factor, European Broiler Index, welfare parameters, colony-forming units of selected caecal bacteria (Enterobacteriaceae and lactic acid bacteria) and pH of broiler faeces and litter. The study comprised 990 Ross 308 male chicks divided into three groups, a control and two experimental groups, which were given crude fibre concentrate as a feed supplement. On the thirty-fifth day of rearing, the birds' welfare scores were evaluated, and 2 g of cecum was collected post-mortem from six chickens in each group. Subsequently, a series of ten-fold dilutions of the material was prepared, followed by cultures and measurement of pH in the faeces and litter. The inclusion of crude fibre concentrate resulted in a beneficial impact on the ultimate body mass (p ≤ 0.001), welfare standard (p ≤ 0.001), and quantity of colony-forming units of lactic acid bacteria (p ≤ 0.05) within the cecum. Furthermore, it had a positive influence on lowering the pH levels of both faeces and litter (p ≤ 0.05).

Silver Nanoparticles and Graphene Oxide Complex as an Anti-Inflammatory Biocompatible Liquid Nano-Dressing for Skin Infected with Staphylococcus aureus.

Background: Bacterial skin infections, including Staphylococcus aureus, are a powerful and still not fully resolved problem. The aim of this research was to determine the possibility of using a complex of graphene oxide (GO) encrusted with silver nanoparticles as an effective antibacterial agent against S. aureus and to assess its pro-inflammatory properties. Methods: The tests were carried out in vitro on EpiDerm™ Skin, an artificial skin model (MatTek in vitro Life Science Laboratories, Slovak Republic), and the fibroblast cell line (HFF-2 from ATCC, USA). Both models were infected with S. aureus bacteria (ATCC 25923) and then treated with antibiotics or our experimental factors: silver nanoparticles (AgNPs, Nano-koloid, Poland), graphene oxide (GO, NanoPoz, Poland), and complex AgNP-GO (hydrocolloid created by self-assembly). Results: The antibacterial effectiveness of the AgNP-GO complex was equivalent to that of the antibiotic. In addition, an increase in the level of pro-inflammatory cytokines was observed under the influence of antibiotic administration, in contrast to the effect of AgNP-GO, which showed very limited pro-inflammatory activity. Conclusion: Hydrocolloid of the AgNP-GO complex, administered in the form of a liquid dressing, may act as an antibacterial agent and also reduce inflammation induced by S. aureus infection.

The cytocompatibility of graphene oxide as a platform to enhance the effectiveness and safety of silver nanoparticles through in vitro studies.

The increasing emergence of antibiotic-resistant bacteria and the need to reduce the use of antibiotics call for the development of safe alternatives, such as silver nanoparticles. However, their potential cytotoxic effect needs to be addressed. Graphene oxide provides a large platform that can increase the effectiveness and safety of silver nanoparticles. Graphene oxide and silver nanoparticles complex applied as a part of an innovative material might have direct contact with human tissues, such as skin, or might be inhaled from aerosol or exfoliated pieces of the complex. Thereby, the safety of the prepared complex has to be evaluated carefully, employing a range of methods. We demonstrated the high cytocompatibility of graphene oxide and the graphene oxide-silver nanoparticles complex toward human cell lines, fetal foreskin fibroblasts (HFFF2), and lung epithelial cells (A549). The supporting platform of graphene oxide also neutralized the slight toxicity of bare silver nanoparticles. Finally, in studies on Staphylococcus aureus and Pseudomonas aeruginosa, the number of bacteria reduction was observed after incubation with silver nanoparticles and the graphene oxide-silver nanoparticles complex. Our findings confirm the possibility of employing a graphene oxide-silver nanoparticles complex as a safe agent with reduced silver nanoparticles' cytotoxicity and antibacterial properties.

Influence of GO-Antisense miRNA-21 on the Expression of Selected Cytokines at Glioblastoma Cell Lines.

Introduction: Graphene oxide (GO) is a single layer of carbon atoms with unique properties, which are beneficial due to its surface functionalisation by miRNA. miRNAs are a non-coding small form of RNA that suppress the expression of protein-coding genes by translational repression or degradation of messenger RNA. Antisense miRNA-21 is very promising for future investigation in cancer therapy. This study aimed to detect cytokine expression levels after the administration of GO-antisense miRNA-21 into U87, U118, U251 and T98 glioma cell lines. Methods: U87, U118, U251 and T98 glioma cell line were investigated in term of viability, human cytokine expression level at protein and genes after treatment with GO, GO-antisense miRNA-21 and antisense miRNA-21. The delivery of antisense miRNA-21 into the glioma cell at in vitro investigation were conducted by GO based transfection and electroporation. Results: The results of the protein microarray and gene expression profile showed that complexes of GO-antisense miRNA-21 modified the metallopeptidase inhibitor 2 (TIMP-2), interleukin-6 (IL-6), interleukin 8 (IL-8), intercellular adhesion molecule 1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) expression level compared to transfection by electroporation of antisense miRNA-21 at investigated glioblastoma cell lines. The TIMP-2 protein and gene expression level was upregulated after antisense miRNA-21 delivery by GO complex into U87, U251 and T98 glioblastoma cell lines comparing to the non-treated control group. The downregulation at protein expression level of ICAM - 1 was observed at U87, U118, U251 and T98 glioma cell lines. Moreover, the IL-8 expression level at mRNA for genes and protein was decreased significantly after delivery the antisense-miRNA-21 by GO compared to electroporation as a transfection method. Discussion: This work demonstrated that the graphene oxide complexes with antisense miRNA-21 can effectively modulate the cytokine mRNA and protein expression level at U87, U118, U251 and T98 glioma cell lines.

Antimicrobial Properties and Assessment of the Content of Bioactive Compounds Lavandula angustifolia Mill. Cultivated in Southern Poland.

Lavender is a valued plant due to its cosmetic, perfumery, culinary, and health benefits. A wide range of applications is related to the composition of bioactive compounds, the quantity and quality of which is determined by various internal and external factors, i.e., variety, morphological part of the plant, and climatic and soil conditions during vegetation. In the presented work, the characterization of antimicrobial properties as well as the qualitative and quantitative assessment of bioactive compounds in the form of polyphenols in ethanol extracts from leaves and flowers of Lavandula angustifolia Mill. intended for border hedges, cultivated in the region of southern Poland, were determined. The composition of the fraction of volatile substances and antioxidant properties were also assessed. The conducted research shows that extracts from leaves and flowers significantly affected the viability of bacterial cells and the development of mold fungi. A clear decrease in the viability of bacteria and C. albicans cells was shown in the concentration of 0.32% of extracts. Leaf extracts were characterized by a much higher content of polyphenols and antioxidant properties than flower extracts. The composition of volatiles measured by GC-MS was significantly different between the extracts. Linalyl acetate and ocimene isomers mix dominated in flower extracts, whereas coumarin, γ-cadinene, and 7-methoxycoumarin were identified as dominant in leaf extracts.

Effect of Melittin Complexes with Graphene and Graphene Oxide on Triple-Negative Breast Cancer Tumors Grown on Chicken Embryo Chorioallantoic Membrane.

One of the components of bee venom is melittin (M), which has strong lysing properties on membranes. M has high toxicity to cancer cells, but it also affects healthy cells, making it necessary to use methods for targeted delivery to ensure treatment. This research is a continuation of previous studies using graphene nanomaterials as M carriers to breast cancer cells. The studies described below are conducted on a more organized biological structure than what is found in vitro cells, namely, cancerous tumors grown on a chicken embryo chorioallantoic membrane. Caspase 3 and 8 levels are analyzed, and the level of oxidative stress markers and changes in protein expression for cytokines are examined. The results show that M complexes with nanomaterials reduce the level of oxidative stress more than M alone does, but the use of graphene (GN) as a carrier increases the level of DNA damage to a greater extent than the increase caused by M alone. An analysis of cytokine levels shows that the use of the M and GN complex increases the level of proteins responsible for inhibiting tumor progression to a greater extent than the increase occasioned by a complex with graphene oxide (GO). The results suggest that the use of GN as an M carrier may increase the toxic effect of M on structures located inside a cell.

Delivery of Melittin as a Lytic Agent via Graphene Nanoparticles as Carriers to Breast Cancer Cells.

Melittin, as an agent to lyse biological membranes, may be a promising therapeutic agent in the treatment of cancer. However, because of its nonspecific actions, there is a need to use a delivery method. The conducted research determined whether carbon nanoparticles, such as graphene and graphene oxide, could be carriers for melittin to breast cancer cells. The studies included the analysis of intracellular pH, the potential of cell membranes, the type of cellular transport, and the expression of receptor proteins. By measuring the particle size, zeta potential, and FT-IT analysis, we found that the investigated nanoparticles are connected by electrostatic interactions. The level of melittin encapsulation with graphene was 86%, while with graphene oxide it was 78%. A decrease in pHi was observed for all cell lines after administration of melittin and its complex with graphene. The decrease in membrane polarization was demonstrated for all lines treated with melittin and its complex with graphene and after exposure to the complex of melittin with graphene oxide for the MDA-MB-231 and HFFF2 lines. The results showed that the investigated melittin complexes and the melittin itself act differently on different cell lines (MDA-MB-231 and MCF-7). It has been shown that in MDA-MD-231 cells, melittin in a complex with graphene is transported to cells via caveolin-dependent endocytosis. On the other hand, the melittin-graphene oxide complex can reach breast cancer cells through various types of transport. Other differences in protein expression changes were also observed for tumor lines after exposure to melittin and complexes.

Bacterial Surface Disturbances Affecting Cell Function during Exposure to Three-Compound Nanocomposites Based on Graphene Materials.

Combating pathogenic microorganisms in an era of ever-increasing drug resistance is crucial. The aim of the study was to evaluate the antibacterial mechanism of three-compound nanocomposites that were based on graphene materials. To determine the nanomaterials' physicochemical properties, an analysis of the mean hydrodynamic diameter and zeta potential, transmission electron microscope (TEM) visualization and an FT-IR analysis were performed. The nanocomposites' activity toward bacteria species was defined by viability, colony forming units, conductivity and surface charge, cell wall integrity, ATP concentration, and intracellular pH. To ensure the safe usage of nanocomposites, the presence of cytokines was also analyzed. Both the graphene and graphene oxide (GO) nanocomposites exhibited a high antibacterial effect toward all bacteria species (Enterobacter cloacae, Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus), as well as exceeded values obtained from exposure to single nanoparticles. Nanocomposites caused the biggest membrane damage, along with ATP depletion. Nanocomposites that were based on GO resulted in lower toxicity to the cell line. In view of the many aspects that must be considered when investigating such complex structures as are three-component nanocomposites, studies of their mechanism of action are crucial to their potential antibacterial use.

Nanocomposites of Graphene Oxide-Silver Nanoparticles for Enhanced Antibacterial Activity: Mechanism of Action and Medical Textiles Coating.

The resistance of microorganisms to antibiotics is a crucial problem for which the application of nanomaterials is among a growing number of solutions. The aim of the study was to create a nanocomposite (composed of graphene oxide and silver nanoparticles) with a precise mode of antibacterial action: what enables textiles to be coated in order to exhibit antibacterial properties. A characterization of nanomaterials (silver nanoparticles and graphene oxide) by size distribution, zeta potential measurements, TEM visualization and FT-IR was performed. The biological studies of the nanocomposite and its components included the toxicity effect toward two pathogenic bacteria species, namely Pseudomonas aeruginosa and Staphylococcus aureus, interaction of nanomaterials with the outer layer of microorganisms, and the generation of reactive oxygen species and lipid peroxidation. Afterwards, antibacterial studies of the nanocomposite's coated textiles (cotton, interlining fabric, polypropylene and silk) as well as studies of the general toxicity towards a chicken embryo chorioallantoic membrane model were conducted. The toxicity of the nanocomposite used was higher than its components applied separately (zones of growth inhibition for P. aeruginosa for the final selected concentrations were as follows: silver nanoparticles 21 ± 0.7 mm, graphene oxide 14 ± 1.9 mm and nanocomposite 23 ± 1.6 mm; and for S. aureus were: silver nanoparticles 27 ± 3.8 mm, graphene oxide 14 ± 2.1 mm, and nanocomposite 28 ± 0.4 mm. The viability of P. aeruginosa and S. aureus after treatment with selected GO-Ag decreased to 27% and 31%, respectively, compared to AgNPs, when the viability of both species was 31% and 34%, accordingly). The coated textiles showed encouraging antibacterial features without general toxicity towards the chicken embryo chorioallantoic membrane model. We demonstrated that graphene oxide might constitute a functional platform for silver nanoparticles, improving the antibacterial properties of bare silver. Due to the application of the nanocomposite, the textiles showed promising antibacterial features with a low general toxicity, thereby creating a wide possibility for them to be used in practice.

Comparison of the Toxicity of Pristine Graphene and Graphene Oxide, Using Four Biological Models.

There are numerous applications of graphene in biomedicine and they can be classified into several main areas: delivery systems, sensors, tissue engineering and biological agents. The growing biomedical field of applications of graphene and its derivates raises questions regarding their toxicity. We will demonstrate an analysis of the toxicity of two forms of graphene using four various biological models: zebrafish (Danio rerio) embryo, duckweed (Lemna minor), human HS-5 cells and bacteria (Staphylococcus aureus). The toxicity of pristine graphene (PG) and graphene oxide (GO) was tested at concentrations of 5, 10, 20, 50 and 100 µg/mL. Higher toxicity was noted after administration of high doses of PG and GO in all tested biological models. Hydrophilic GO shows greater toxicity to biological models living in the entire volume of the culture medium (zebrafish, duckweed, S. aureus). PG showed the highest toxicity to adherent cells growing on the bottom of the culture plates-human HS-5 cells. The differences in toxicity between the tested graphene materials result from their physicochemical properties and the model used. Dose-dependent toxicity has been demonstrated with both forms of graphene.

Silver and Copper Nanoparticles Inhibit Biofilm Formation by Mastitis Pathogens.

Bovine mastitis is a common bovine disease, frequently affecting whole herds of cattle. It is often caused by resistant microbes that can create a biofilm structure. The rapidly developing scientific discipline known as nanobiotechnology may help treat this illness, thanks to the extraordinary properties of nanoparticles. The aim of the study was to investigate the inhibition of biofilms created by mastitis pathogens after treatment with silver and copper nanoparticles, both individually and in combination. We defined the physicochemical properties and minimal inhibitory concentration of the nanoparticles and observed their interaction with the cell membrane, as well as the extent of biofilm reduction. The results show that the silver-copper complex was the most active of all nanomaterials tested (biofilm was reduced by nearly 100% at a concentration of 200 ppm for each microorganism species tested). However, silver nanoparticles were also effective individually (biofilm was also reduced by nearly 100% at a concentration of 200 ppm, but at concentrations of 50 and 100 ppm, the extent of reduction was lower than for the complex). Nanoparticles can be used in new alternative therapies to treat bovine mastitis.

Phenylketonuria patients' and their parents' knowledge and attitudes to the daily diet - multi-centre study.

BACKGROUND: The aim of the study was to assess both patients' and their parents' knowledge of phenylketonuria (PKU) treatment and compliance with PKU diet. METHODS: The study included 173 PKU patients aged 10-19 and 110 parents of PKU children who were enrolled in the study on the basis of questionnaire data. The study also included 45 patients aged ≥20. RESULTS: Our study demonstrated that only 45% (n = 74) of PKU patients knew daily Phe intake recommendations, 27% of patients (n = 41) knew the Phe content in a minimum of three out of four researched food products. Patients' knowledge concerning Phe intake (p = 0.0181) and the knowledge of selected food products (p = 0.041819) improved with age. We did not establish such a correlation in the group of PKU children's parents. Approximately 31% of patients and 22% of parents reported helplessness, which increased with the child's age, associated with the necessity to adhere to the diet; 30% of patients reported feeling ashamed of the fact that they could not eat all food products. Regardless of age, children were more likely than parents to report helplessness (p = 0.032005). Among patients, 41.40% declared that they would wish to select products unassisted but their parents did not permit them to do so. The question of whether parents teach children self-reliance in meal preparation was answered affirmatively by 98% of parents and only 81% of children (p = 0.0001). CONCLUSION: Our data demonstrated that parents' and children's knowledge concerning treatment recommendations and food products does not have a direct impact on attitude to the PKU diet. Limiting children's independence in meal selection, growing helplessness in the face of dietary adherence and shame resulting from the necessity to follow a different diet observed in PKU families are responsible for shaping and perpetuating a consistently negative attitude to the diet. The care of PKU paediatric patients requires consistent, long-term family and individual therapy which may counteract the effects of learned helplessness. In regard to the educational effort, a good parent-child relationship as well as the teaching of behaviours motivating patients to comply with the diet are of great importance.

Phenylketonuria patients' and their parents' acceptance of the disease: multi-centre study.

PURPOSE: Phenylketonuria (PKU) still poses a therapeutic challenge for patients and medical professionals. The aim of the study was to assess both patients' and their parents' acceptance of the disease. METHODS: The study included 218 PKU patients and 178 parents of PKU children who were enrolled in the study on the basis of questionnaire data. RESULTS: Regarding attitude towards the disease, our study demonstrated that 63 (28.9 %) PKU patients did not accept the disease. Patients who found accepting the disease difficult, more frequently perceived themselves as inferior/different in comparison with their peers. In total, 36 % of patients did not want their friends to be aware of their condition, while only 18 % of parents believed that their children's peers should not know about their disease. In total, 42 % of parents wanted to talk to other parents of PKU children and only 13 % to a doctor. Only 20 % of patients saw the need to discuss their condition with a doctor. In total, 8 % of children, regardless of age, and 14 % of parents preferred to talk to a psychologist. CONCLUSION: Our data demonstrated that disease acceptance played an essential role in patients' social integration. The study also indicated the need to overcome communication barriers between patients and their healthy peers and for patients to find the courage to be open about the disease. The importance of support groups for PKU families and the significance of strict cooperation between patients and their families with PKU treatment teams were also revealed.

Molecular genetics of PKU in Poland and potential impact of mutations on BH4 responsiveness.

Tetrahydrobiopterin (BH4) has been recently approved as a treatment of patients with phenylketonuria. However, as a confirmation of BH4-responsiveness, it might require a very expensive trial treatment with BH4 or prolonged BH4-loading procedures. The selection of patients eligible for BH4-therapy by means of genotyping of the PAH gene mutations may be recommended as a complementary approach. A population-wide genotyping study was carried out in 1286 Polish phenyloketonuria-patients. The aim was to estimate the BH4 demand and to cover prospectively the treatment by a National Health Fund. A total of 95 types of mutations were identified. Genetic variants corresponding with probable BH4-responsiveness were found in 28.2% of cases. However, patients with mild or classical phenylketonuria who require continuous treatment accounted for 11.4% of the studied population only. Analysis of the published data shows similar percentage of the "BH4-responsive" variants of a PAH gene in patients from other countries of Eastern Europe. Therefore, it can be concluded, that the proportion of phenylketonuria-patients who could benefit from the use of BH4 reaches approximately 10% in the entire region.

Impact of lipophilic antioxidants and level of antibodies against oxidized low-density lipoprotein in Polish children with phenylketonuria.

The treatment of phenylketonuria (PKU) patients constitutes a phenylalanine (Phe) intake restriction in their diet, which is achieved by adding a special Phe-free amino acid mixture to the diet. It has been reported that this diet could have some micronutrient deficiency. Several authors have also reported an increased oxidative stress or impaired antioxidant status in human and experimental PKU. Our project assessed the concentrations of retinol, alpha-tocopherol, coenzyme Q10, and anti-oxidized low-density lipoprotein (ox-LDL) antibodies in PKU children's plasma. It was found that retinol concentration in PKU children remains within the norm despite a low intake. The lower plasma alpha-tocopherol concentration in PKU children compared with normal children was associated with the lower level of antibodies against ox-LDL. This raises the question whether higher than observed circulatory alpha-tocopherol is indeed beneficial to lower plasma ox-LDL levels. Further studies are needed to explain the genetic factor in PKU patients (e.g., CD36/FAT polymorphism gene). The open clinical question is whether daily supplementation of alpha-tocopherol changes the PKU patients' level of antibodies against ox-LDL.