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Lubomir Skladany,1 Jana Vnencakova,1 Lukas Laffers,2 Beata Skvarkova,1 Eva Hrubá,1 Pavol Molcan,1 Tomas Koller3 1Hepatology, Gastroenterology and Transplantation (HEGITO) Department of the 2nd Department of Medicine, Slovak Medical University, FD Roosevelt Hospital, Banska Bystrica, Slovakia; 2Department of Mathematics, Faculty of Natural Sciences, Matej Bel University, Banska Bystrica, Slovakia; 3Gastroenterology and Hepatology Subdiv. of the 5th Department of Medicine, Comenius University Faculty of Medicine, University Hospital Bratislava, Bratislava, SlovakiaCorrespondence: Tomas KollerGastroenterology and Hepatology Subdiv. of the 5th Department of Medicine, Comenius University Faculty of Medicine, University Hospital Bratislava, Ruzinovska 6, Bratislava 82606, SlovakiaTel +42190518692Email koller.tomas@gmail.comPurpose: Patients with advanced chronic liver disease (ACLD) often have a poor nutritional status. In the management, current guidelines recommend dietary counseling and oral nutritional supplements (ONS). Nutritional goals and adherence to ONS are difficult to achieve while studies addressing adherence are scarce. We aimed to evaluate adherence to ONS, the associated factors, and its impact on outcome among ALCD patients who are discharged from the hospital.Patients and Methods: We identified consecutive hospitalized patients with ACLD from the cirrhosis registry and ONS prescription at discharge. Baseline demographics, anthropometrics, hand-grip strength (HGS), nutritional, and laboratory parameters were recorded. Adherence was assessed at 30, 90, and 180 days, but not in patients who did not survive or in those who underwent liver transplantation (LT) before the time-point.Results: From the registry containing 1004 patients, we included 450 cases, the median age was 56.3 (IQR 47– 62), 60% were males, 63.8% had alcoholic etiology, and the median model for end-stage liver disease score (MELD) was 16 (11– 21). During follow-up, 13.6%, 23.6%, and 31.1% of patients have died within 30, 90, and 180 days, respectively, and 21 underwent LT. Adherence to ONS in surviving patients was observed in 46%, 26.1%, and 16.9% within 30, 90, and 180 days, respectively. Baseline refractory ascites (HR=0.43, 0.24– 0.76), HGS (HR=1.03, 1.01– 1.06), and mid-arm circumference (HR=0.93, 0.88– 0.99) were independently associated with 30-day adherence. Among patients who survived beyond 30 days, adherents for > 30 days had improved synthetic liver function, HGS, a higher probability of LT (HR=1.7, 1.03– 2.8) and lower risk of death (HR=0.65, 0.45– 0.89), particularly those with MELD> 16 (OR=0.55, 0.36– 0.85) and low HGS (OR=0.61, 0.39– 0.93).Conclusion: In ACLD patients after discharge, adherence to ONS steeply declined and was associated with baseline refractory ascites and low muscle strength. Adherence to ONS also improved liver function, muscle strength, and survival.Keywords: adherence, nutritional supplements, malnutrition, cirrhosis, outcome, prognosis

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Background: Ameloblastic carcinoma and metastasising ameloblastoma are rare epithelial odontogenic tumours with aggressive features. Distinguishing between these two lesions is often clinically difficult but necessary to predict tumour behaviour or to plan future therapy. Here, we provide a brief review of the literature available on these two types of lesions and present a new case report of a young man with an ameloblastoma displaying metastatic features. We also use this case to illustrate the similarities and differences between these two types of tumours and the difficulties of their differential diagnosis., Case Presentation: Our histopathological analyses uncovered a metastasising tumour with features of ameloblastic carcinoma, which developed from the ameloblastoma. We profiled the gene expression of Wnt pathway members in ameloblastoma sample of this patient, because multiple molecules of this pathway are involved in the establishing of cell polarity, cell migration or for epithelial-mesenchymal transition during tumour metastasis to evaluate features of tumor behaviour. Indeed, we found upregulation of several cell migration-related genes in our patient. Moreover, we uncovered somatic mutation BRAF p.V600E with known pathological role in cancerogenesis and germline heterozygous FANCA p.S858R mutation, whose interpretation in this context has not been discussed yet., Conclusions: In conclusion, we have uncovered a unique case of ameloblastic carcinoma associated with an alteration of Wnt signalling and the presence of BRAF mutation. Development of harmful state of our patient might be also supported by the germline mutation in one FANCA allele, however this has to be confirmed by further analyses., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Carcinomas of the oral cavity and oropharynx belong among the ten most common malignancies in the human population. The prognosis of head and neck squamous cell carcinoma (HNSCC) is determined by the degree of invasiveness of the primary tumor and by the extent of metastatic spread into regional and distant lymph nodes. Moreover, the level of the perineural invasion itself associates with tumor localization, invasion's extent, and the presence of nodal metastases. Here, we summarize the current knowledge about different aspects of epigenetic changes, which can be associated with HNSCC while focusing on perineural invasion (PNI). We review epigenetic modifications of the genes involved in the PNI process in HNSCC from the omics perspective and specific epigenetic modifications in OSCC or other neurotropic cancers associated with perineural invasion. Moreover, we summarize DNA methylation status of tumor-suppressor genes, methylation and demethylation enzymes and histone post-translational modifications associated with PNI. The influence of other epigenetic factors on the HNSCC incidence and perineural invasion such as tobacco, alcohol and oral microbiome is overviewed and HPV infection is discussed as an epigenetic factor associated with OSCC and related perineural invasion. Understanding epigenetic regulations of axon growth that lead to tumorous spread or uncovering the molecular control of axon interaction with cancer tissue can help to discover new therapeutic targets for these tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hurník, Chyra, Ševčíková, Štembírek, Trtková, Gaykalova, Buchtová and Hrubá.)

Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD/MTPD) and medium chain acyl-CoA dehydrogenase deficiency (MCADD) were included in the expanded neonatal screening program (ENBS) in Czechia in 2009, allowing for the presymptomatic diagnosis and nutritional management of these patients. The aim of our study was to assess the nationwide impact of ENBS on clinical outcome. This retrospective study analysed acute events and chronic complications and their severity in pre-ENBS and post-ENBS cohorts. In total, 28 children (12 before, 16 after ENBS) were diagnosed with LCHADD/MTPD (incidence 0.8/100,000 before and 1.2/100,000 after ENBS). In the subgroup detected by ENBS, a significantly longer interval from birth to first acute encephalopathy was observed. In addition, improvement in neuropathy and cardiomyopathy (although statistically non-significant) was demonstrated in the post-ENBS subgroup. In the MCADD cohort, we included 69 patients (15 before, 54 after ENBS). The estimated incidence rose from 0.7/100,000 before to 4.3/100,000 after ENBS. We confirmed a significant decrease in the number of episodes of acute encephalopathy and lower proportion of intellectual disability after ENBS ( p < 0.0001). The genotype-phenotype correlations suggest a new association between homozygosity for the c.1528C > G variant and more severe heart involvement in LCHADD patients.

Deciphering the role of the aryl hydrocarbon receptor (AhR) in lung cancer cells may help us to better understand the role of toxic AhR ligands in lung carcinogenesis, including cancer progression. We employed human lung carcinoma A549 cells to investigate their fate after continuous two-week exposure to model AhR agonists, genotoxic benzo[a]pyrene (BaP; 1 μM) and non-genotoxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 10 nM). While TCDD increased proliferative rate of A549 cells, exposure to BaP decreased cell proliferation and induced epithelial-to-mesenchymal transition (EMT)-like phenotype, which was associated with enhanced cell migration, invasion, and altered cell morphology. Although TCDD also suppressed expression of E-cadherin and activated some genes linked to EMT, it did not induce the EMT-like phenotype. The results of transcriptomic analysis, and the opposite effects of BaP and TCDD on cell proliferation, indicated that a delay in cell cycle progression, together with a slight increase of senescence (when coupled with AhR activation), favors the induction of EMT-like phenotype. The shift towards EMT-like phenotype observed after simultaneous treatment with TCDD and mitomycin C (an inhibitor of cell proliferation) confirmed the hypothesis. Since BaP decreased cell proliferative rate via induction of p21 expression, we generated the A549 cell model with reduced p21 expression and exposed it to BaP for two weeks. The p21 knockdown suppressed the BaP-mediated EMT-like phenotype in A549 cells, thus confirming that a delayed cell cycle progression, together with p21-dependent induction of senescence-related chemokine CCL2, may contribute to induction of EMT-like cell phenotype in lung cells exposed to genotoxic AhR ligands., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Exposure to persistent ligands of aryl hydrocarbon receptor (AhR) has been found to cause lung cancer in experimental animals, and lung adenocarcinomas are often associated with enhanced AhR expression and aberrant AhR activation. In order to better understand the action of toxic AhR ligands in lung epithelial cells, we performed global gene expression profiling and analyze TCDD-induced changes in A549 transcriptome, both sensitive and non-sensitive to CH223191 co-treatment. Comparison of our data with results from previously reported microarray and ChIP-seq experiments enabled us to identify candidate genes, which expression status reflects exposure of lung cancer cells to TCDD, and to predict processes, pathways (e.g. ER stress, Wnt/β-cat, IFNɣ, EGFR/Erbb1), putative TFs (e.g. STAT, AP1, E2F1, TCF4), which may be implicated in adaptive response of lung cells to TCDD-induced AhR activation. Importantly, TCDD-like expression fingerprint of selected genes was observed also in A549 cells exposed acutely to both toxic (benzo[a]pyrene, benzo[k]fluoranthene) and endogenous AhR ligands (2-(1H-Indol-3-ylcarbonyl)-4-thiazolecarboxylic acid methyl ester and 6-formylindolo[3,2-b]carbazole). Overall, our results suggest novel cellular candidates, which could help to improve monitoring of AhR-dependent transcriptional activity during acute exposure of lung cells to distinct types of environmental pollutants., (Copyright © 2018 Elsevier B.V. All rights reserved.)

PCB 136 is an environmentally relevant chiral PCB congener, which has been found in vivo to be present in form of rotational isomers (atropisomers). Its atropselective biotransformation or neurotoxic effects linked with sensitization of ryanodine receptor suggest that it might interact also with other intracellular receptors in a stereospecific manner. However, possible atropselective effects of PCB 136 on nuclear receptor transactivation remain unknown. Therefore, in this study, atropselective effects of PCB 136 on nuclear receptors controlling endocrine signaling and/or expression of xenobiotic and steroid hormone catabolism were investigated. PCB136 atropisomers were found to exert differential effects on estrogen receptor (ER) activation; (+)-PCB 136 was estrogenic, while (-)-PCB 136 was antiestrogenic. In contrast, inhibition of androgen receptor (AR) activity was not stereospecific. Both PCB136 stereoisomers induced the constitutive androgen receptor (CAR)-dependent gene expression; however, no significant stereospecificity of PCB 136 atropisomers was observed. PCB136 was a partial inducer of the pregnane X receptor (PXR)-dependent gene expression. Here, (-)-PCB 136 was a significantly more potent inducer of PXR activity than (+)-PCB 136. Taken together, the present results indicate that at least two nuclear receptors participating in endocrine regulation or metabolism, ER and PXR, could be regulated in an atropselective manner by chiral PCB 136. The enantioselective enrichment of PCB atropisomers in animal and human tissues may thus have significant consequences for endocrine-disrupting effects of chiral ortho-substituted PCB congeners.

Phthalate esters are ubiquitous environmental pollutants widely used as plasticizers, which have been shown to interfere with both endocrine regulation and development of reproductive organs. In the present study, we examined the impact of diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) on the proliferation of androgen-sensitive human prostate carcinoma LNCaP cells and related events. The results showed that both compounds were able to inhibit cell cycle progression in a dose-dependent manner. However, only DEHP was found to weakly reduce androgen receptor (AR) protein levels after long-term exposure, while only DBP partially inhibited expression of the prostate-specific antigen (KLK3) gene, a model AR transcriptional target. This indicated that inhibition of cell proliferation was likely independent of any AR modulations. Both phthalates induced suppression of cell proliferation, but none of them affected the levels of markers associated with neuroendocrine transdifferentiation (NED) in LNCaP cells. Taken together, the presented data indicate that phthalates may exert long-term negative effects on the proliferation of prostate epithelial cells derived from the carcinoma model, which are, nevertheless, largely independent of the modulation of AR expression/activity, and which do not alter further processes associated with NED.

Plakoglobin is an important component of intercellular junctions, including both desmosomes and adherens junctions, which is known as a tumor suppressor. Although mutations in the plakoglobin gene (Jup) and/or changes in its protein levels have been observed in various disease states, including cancer progression or cardiovascular defects, the information about endogenous or exogenous stimuli orchestrating Jup expression is limited. Here we show that the aryl hydrocarbon receptor (AhR) may regulate Jup expression in a cell-specific manner. We observed a significant suppressive effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a model toxic exogenous activator of the AhR signaling, on Jup expression in a variety of experimental models derived from rodent tissues, including contact-inhibited rat liver progenitor cells (where TCDD induces cell proliferation), rat and mouse hepatoma cell models (where TCDD inhibits cell cycle progression), cardiac cells derived from the mouse embryonic stem cells, or cardiomyocytes isolated from neonatal rat hearts. The small interfering RNA (siRNA)-mediated knockdown of AhR confirmed its role in both basal and TCDD-deregulated Jup expression. The analysis of genomic DNA located ~2.5kb upstream of rat Jup gene revealed a presence of evolutionarily conserved AhR binding motifs, which were confirmed upon their cloning into luciferase reporter construct. The siRNA-mediated knockdown of Jup expression affected both proliferation and attachment of liver progenitor cells. The present data indicate that the AhR may contribute to negative regulation of Jup gene expression in rodent cellular models, which may affect cell adherence and proliferation.

Although the tumor-promoting effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), coplanar polychlorinated biphenyls (PCBs), and related compounds in liver tissue are primarily attributed to the activation of the aryl hydrocarbon receptor (AhR), the underlying molecular mechanisms are still unclear. Liver progenitor (oval) cells have been suggested to constitute a potential target for hepatocarcinogenic chemicals. To better understand AhR-driven pathways, we analyzed the transcriptional program in response to coplanar PCB 126 in contact-inhibited rat liver progenitor WB-F344 cells using high-density microarrays. After 6-h treatment, we identified 145 significantly deregulated genes considered to be direct AhR-dependent target genes. The number of differentially regulated genes increased to 658 and 968 genes after 24 and 72 h, respectively. Gene ontology analysis revealed that these genes were primarily involved in drug and lipid metabolism, cell cycle and growth control, cancer developmental processes, cell-cell communication, and adhesion. Interestingly, the Wnt and TGF-β signaling pathways, both being involved in developmental and tumorigenic processes, belonged to the most affected pathways. AhR- and ARNT-dependent regulation of selected target genes of interest was then confirmed using TCDD as a model AhR agonist, together with pharmacological inhibition of the AhR and by RNA-interference techniques. We demonstrated AhR-dependent regulation of emerging and novel AhR target genes, such as Fst, Areg, Hbegf, Ctgf, Btg2, and Foxq1. Among them, the transcription factor Foxq1, recently suggested to contribute to tumor promotion and/or progression, was found to be regulated at both mRNA and protein levels by AhR/ARNT activation.

Crosstalk between the aryl hydrocarbon receptor (AhR) and transforming growth factor-β1 (TGF-β1) signaling has been observed in various experimental models. However, both molecular mechanism underlying this crosstalk and tissue-specific context of this interaction are still only partially understood. In a model of human non-tumorigenic prostate epithelial cells BPH-1, derived from the benign prostatic hyperplasia, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) persistently activates the AhR signaling pathway and induces expression of xenobiotic metabolizing enzymes, such as CYP1A1 or CYP1B1. Here we demonstrate that TGF-β1 suppresses the AhR-mediated gene expression through multiple mechanisms, involving inhibition of AhR expression and down-regulation of nuclear AhR, via a SMAD4-dependent pathway. In contrast, TCDD-induced AhR signaling does not affect either TGF-β1-regulated gene expression or epithelial-to-mesenchymal transition. These observations suggest that, in the context of prostate epithelium, TGF-β1 signaling plays a dominant role in the crosstalk with AhR signaling pathway. Given the importance of TGF-β1 signaling in regulation of prostate epithelial tissue homeostasis, as well as the recently revealed role of AhR in prostate development and tumorigenesis, the above findings contribute to our understanding of the mechanisms underlying the crosstalk between the two signaling pathways in the prostate-specific context., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Differences between tissues in the expression of drug-metabolizing enzymes may substantially contribute to tissue-specificity of chemical carcinogens. To verify this hypothesis, the spontaneously immortalized human keratinocytes HaCaT were used, in order to evaluate the genotoxic potential of 7H-dibenzo[c,g]carbazole (DBC), a known hepatocarcinogen and sarcomagen, and its synthetic tissue-specific derivatives, 5,9-dimethyl-DBC (DiMeDBC) and N-methyl-DBC (N-MeDBC), which manifest specific tropism to the liver and skin, respectively. HaCaT cells mainly express cytochrome P4501A1 (CYP1A1), which is involved in metabolism of DBC and N-MeDBC, but not DiMeDBC [10]. Both DBC and the sarcomagen N-MeDBC induced significant levels of DNA strand-breaks, micronuclei, and DNA adducts followed by the phosphorylation of the p53 protein and histone H2AX in HaCaT cells. In contrast, the specific hepatocarcinogen DiMeDBC was devoid of any significant genotoxic activity in this cell line. Our study demonstrates that the absence of drug-metabolizing enzyme(s) involved in DiMeDBC metabolism may contribute substantially to the tissue-specific genotoxicity of this hepatocarcinogen., (© 2012 Elsevier B.V. All rights reserved.)

Alzheimer's disease is a progressive neurodegenerative disorder mainly manifested by memory loss, personality changes, and cognitive dysfunction. Despite the fact that tireless research is being conducted, up-to-date pharmacotherapy of AD is presented only by two groups diverging in the mechanism of action. The larger one uses acetylcholinesterase inhibitors, and the second group is represented by the N-methyl-D-aspartate antagonist memantine. Even though the etiology of Alzheimer's disease is unknown, several different therapeutic approaches are being investigated. The aim of this paper is to provide an overview of the present state of intended therapeutics for AD, describing their mechanism of action if known, displaying chemical structures, and the state of clinical trials if any.

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) are known as efficient mutagens and ligands of the aryl hydrocarbon receptor (AhR), which has been suggested to play an important role in prostate carcinogenesis. In order to evaluate the complex relationship between the genotoxicity and the AhR-mediated activity of PAHs in prostate cells, we selected benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as model genotoxic and nongenotoxic AhR ligands, respectively, to explore global changes in gene expression in LNCaP cells by microarray analysis. We identified 112 genes that were differentially expressed in cells treated for 24h with BaP, TCDD or both compounds. Our data indicated that the impacts of BaP and TCDD on transcriptome of LNCaP cells significantly overlap, since over 64% of significantly up-regulated genes and 47% of down-regulated genes were similarly affected by both AhR ligands. This suggested that the activation of AhR played a prominent role in the nongenotoxic effects of BaP in the prostate carcinoma cell model LNCaP. Both AhR ligands suppressed expression of genes associated with cell cycle progression, DNA replication, spindle assembly checkpoint or DNA repair, which probably occurred secondary to inhibition of cell cycle progression. In contrast, we identified Wnt5a, an important regulator of prostate cancer progression, to be induced as early as 6h after exposure to both AhR ligands. The AhR ligand-induced Wnt5a upregulation, together with other observed alterations of gene expression, may further contribute to enhanced cell plasticity of prostate carcinoma cells., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The goal of this study was to investigate the genotoxicity of 7H-dibenzo[c,g]carbazole (DBC), a ubiquitous environmental pollutant, and its methyl derivatives, 5,9-dimethylDBC (DiMeDBC), a strict hepatocarcinogen, and N-methylDBC (N-MeDBC), a specific sarcomagen in human hepatoma HepG2 cells, and to infer potential mechanisms underlying the biological activity of particular carcinogen. All dibenzocarbazoles, regardless the tissue specificity, induced significant DNA strand break levels and micronuclei in HepG2 cells; though a mitotic spindle dysfunction rather than a chromosome breakage was implicated in N-MeDBC-mediated micronucleus formation. While DBC and N-MeDBC produced stable DNA adducts followed with p53 protein phosphorylation at Ser-15, DiMeDBC failed. A significant increase in DNA strand breaks following incubation of exposed cells with a repair-specific endonuclease (Fpg protein) suggested that either oxidative DNA damage or unstable DNA-adducts might underlie DiMeDBC genotoxicity in human hepatoma cells. DiMeDBC and N-MeDBC increased substantially also the amount of CYP1A1/2 expression in HepG2 cells. Pretreatment of cells with substances affecting AhR-mediated CYP1A family of enzymes expression; however, diminished DiMeDBC and N-MeDBC genotoxicity. Our data clearly demonstrated differences in the mechanisms involved in the biological activity of DiMeDBC and N-MeDBC in human hepatoma cells; the genotoxicity of these DBC derivatives is closely related to CYP1A1/2 expression., (Copyright © 2011 Wiley-Liss, Inc.)

Exposure to polycyclic aromatic hydrocarbons (PAHs) has been positively associated with prostate cancer, but knowledge of the formation of PAH-DNA adducts and related genotoxic events in prostatic cells is limited. In the present study, benzo[a]pyrene (BaP), a potent mutagenic PAH, formed significant levels of DNA adducts in cell lines derived from human prostate carcinoma. When analyzing the effect of BaP on the induction of CYP1 enzymes participating in the metabolic activation of PAHs in LNCaP cells, we found that BaP induced expression of CYP1A1 and CYP1A2, but not CYP1B1 enzyme. Despite a significant amount of DNA adducts being formed by BaP and, to a lesser extent also by another strong genotoxin, dibenzo[a,l]pyrene, neither apoptosis nor cell-cycle arrest were induced in LNCaP cells. LNCaP cells were not sensitized to the induction of apoptosis by PAHs even through inhibition of the phosphoinositide-3-kinase/Akt pro-survival pathway. The lack of apoptosis was not due a disruption of expression of pro-apoptotic and pro-survival members of the Bcl-2 family of apoptosis regulators. In contrast to other genotoxic stimuli, genotoxic PAHs failed to induce DNA double-strand breaks, as illustrated by the lack of phosphorylation of histone H2AX or checkpoint kinase-2. BaP did not activate p53, as evidenced by the lack of p53 accumulation, phosphorylation at Ser15, or induction of p53 transcriptional targets. Taken together, although genotoxic PAHs produced significant levels of DNA adducts in a model of human prostate carcinoma cells, they did not activate the mechanisms leading to elimination of cells with significant damage to DNA, presumably due to their failure to activate the p53-dependent DNA damage response., ((c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: Serious hematological, metabolic and neurological complications owing to the nutritional deficiency of vitamin B12 may occur in infants of mothers on a strict vegetarian diet., Methods and Results: The mother of the first child was a strict vegetarian. She had an elevated urinary methylmalonic acid level and a low concentration of serum vitamin B12. Her 13-month-old daughter was exclusively breast-fed until the age of 9 month and then she was fed only vegetables. Physical examination revealed psychomotoric retardation, apathy, muscular hypotonia, abnormal movements and failure to thrive. Laboratory analysis showed a megaloblastic anaemia, a low level of vitamin B12 and methylmalonic aciduria. MRI of the brain revealed diffuse frontotemporoparietal atrophy and retardation of myelination. After treatment with vitamin B12 supplements, abnormal movements disappeared and development improved, but a mild generalised hypotonia continued. A cranial MRI 9 months after treatment still showed signs of retardation of myelination. The second patient, an 8 month-old male, son of a strict vegetarian mother too, was referred for investigation of psychomotoric retardation, hypotonia, dyskinesia, failure to thrive and microcephaly. He was breast-fed and from 6 month of age he had also received fruit juices. Laboratory analysis revealed megaloblastic anaemia, high methylmalonic aciduria and homocystinuria. The patient's and his mother's serum level of vitamin B12 were low. After treatment with vitamin B12 supplements, biochemical and metabolic markers of disease were normal but there continued a generalised hypotonia, microcephaly and language delay., Conclusion: Our observations emphasize the health complications of nutritional cobalamine deficiency and a requirement of clinical, biochemical and metabolic monitoring in infants within strict vegetarian families.

Background: The most frequent manifestation of mitochondrial DNA (mtDNA) mutation 8344 A-->G is MERRF syndrome (Myoclonic Epilepsy and Myopathy with Ragged Red Fibres). Less frequent symptoms include ataxia, perceptive type of deafness, cardiomyopathy or external ophthalmoplegia and mental and motor retardation in children. We describe heterogeneity of clinical symptoms and results of biochemical and molecular investigations in four families with the heteroplasmic mutation 8344 A-->G in mtDNA., Methods and Results: In co-operation with paediatric, neurological and genetic specialists from the Czech and Slovak Republic we found in 1993-1998 at the enzymatic or molecular level more than 90 children and adults with impaired mitochondrial energy metabolism. Heteroplasmic mutation 8344 A-->G in mtDNA was found in four families. Ataxia and progressive muscle weakness appeared in the first proband with 50% of mutated copies of mtDNA in muscle at the age of 30 years. The second proband with 95% of mutated mtDNA had his first clinical symptoms--muscle hypotonia, cardiomyopathy and mental and motor retardation--in infancy while his four relatives with 25-50% mutated mtDNA lack so far clinical symptoms. In a female from the third family with 50% mutated mtDNA in muscle the disease manifested at the age of 42 years with progressive external ophthalmoplegia (PEO) and muscle weakness. In the fourth proband with 50% of mutated mtDNA in blood the disease started in infancy with spastic quadruparesis and arrested mental and motor development. Enzymatic and histochemical investigation in muscle biopsy in two probands revealed lower cytochrom c oxidase activity. Ragged-red fibres were found only in one adult patient., Conclusions: MtDNA mutation 8344 A-->G can manifest by heterogeneous symptoms. A higher percentage of mutated mtDNA is usually associated with more serious forms of the disease, but there is not always a correlation between the degree of heteroplasmy and severity of the disease or the age of the first clinical symptoms.

Background: Secondary lactate acidosis is found in children with hypoxaemia, with impaired tissue perfusion, in hepatic and renal failure or in intoxications. Primary lactate acidosis is usually caused by hereditary metabolic disorders. The objective of the trial was to analyze the causes which lead in childhood to the development of primary hyperlactacidaemia., Methods and Results: The authors examined during 1995-1996 the lactate and pyruvate concentration in 479 children referred by paediatric and neurological departments with a suspect hereditary metabolic disturbances. A raised lactate in blood or cerebrospinal fluid > 2.3 mmol/l was found in 230 children incl. 49 where a metabolic disorder was detected. Ten children had impaired cytochrome c oxidase, two children had a combined deficience of NADH dehydrogenase and cytochrome c oxidase, three children had a deficience of the pyruvate dehydrogenase (PDH) complex, one child had a deficience of ATP synthase and seven children suffered from impaired beta-oxidation. Glycogenosis type I, III or IX was found in 13 children. In three children organic aciduria was found, two children had an impaired urea cycle and three children impaired fructose metabolism. In five children a low level of free and total carnitene was found as a result of valproate treatment. A significant increase of the lactate level by more than 1 mmol/l during an oral glucose load was found in 11 of 16 children with impairment of the respiratory chain or PDH complex. In 58 children concurrently lactate in blood and cerebrospinal fluid assessed but no correlation of lactate levels was found., Conclusions: In patients with suspect hereditary metabolic disorders examination of lactate, pyruvate and alanine levels can be considered a screening test for detection of mitochondrial disorders. It remains difficult to reveal the cause of hyperlactacidaemia in a sick child even if a wide range of laboratory methods are used which contribute to the diagnosis of hereditary metabolic disorders.