High cysteine diet reduces insulin resistance in SHR-CRP rats.

Increased plasma total cysteine (tCys) has been associated with obesity and metabolic syndrome in human and some animal studies but the underlying mechanisms remain unclear. In this study, we aimed at evaluating the effects of high cysteine diet administered to SHR-CRP transgenic rats, a model of metabolic syndrome and inflammation. SHR-CRP rats were fed either standard (3.2 g cystine/kg diet) or high cysteine diet (HCD, enriched with additional 4 g L-cysteine/kg diet). After 4 weeks, urine, plasma and tissue samples were collected and parameters of metabolic syndrome, sulfur metabolites and hepatic gene expression were evaluated. Rats on HCD exhibited similar body weights and weights of fat depots, reduced levels of serum insulin, and reduced oxidative stress in the liver. The HCD did not change concentrations of tCys in tissues and body fluids while taurine in tissues and body fluids, and urinary sulfate were significantly increased. In contrast, betaine levels were significantly reduced possibly compensating for taurine elevation. In summary, increased Cys intake did not induce obesity while it ameliorated insulin resistance in the SHR-CRP rats, possibly due to beneficial effects of accumulating taurine.

HFE and ALK3 act in the same signaling pathway.

Hepcidin deficiency leads to iron overload by increased dietary iron uptake and iron release from storage cells. The most frequent mutation in Hfe leads to reduced hepcidin expression and thereby causes iron overload. Recent findings suggested that HFE activates hepcidin expression predominantly via the BMP type I receptor ALK3. Here, we investigated whether HFE exclusively utilizes ALK3 or other signaling mechanisms also. We generated mice with double deficiency of Hfe and hepatocyte-specific Alk3 and compared the iron overload phenotypes of these double knockout mice to single hepatocyte-specific Alk3 deficient or Hfe knockout mice. Double Hfe-/-/hepatic Alk3fl/fl;Alb-Cre knockouts develop a similar iron overload phenotype compared to single hepatocyte-specific Alk3 deficient mice hallmarked by serum iron levels, tissue iron content and hepcidin levels of similar grades. HFE protein levels were increased in Alk3fl/fl;Alb-Cre mice compared to Alk3fl/fl mice, which was caused by iron overload - and not by Alk3 deficiency. The data provide evidence by genetic means that 1. HFE exclusively uses the BMP type I receptor ALK3 to induce hepcidin expression and 2. HFE protein expression is induced by iron overload, which further emphasizes the iron sensing function of HFE.

Dissecting the role of Folr1 and Folh1 genes in the pathogenesis of metabolic syndrome in spontaneously hypertensive rats.

Increased levels of plasma cysteine predispose to obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed mutated Folr1 (folate receptor 1) on chromosome 1 as a quantitative trait gene associated with reduced folate levels, hypercysteinemia and metabolic disturbances. The Folr1 gene is closely linked to the Folh1 (folate hydrolase 1) gene which codes for an enzyme involved in the hydrolysis of dietary polyglutamyl folates in the intestine. In the current study, we obtained evidence that Folh1 mRNA of the BN (Brown Norway) origin is weakly but significantly expressed in the small intestine. Next we analyzed the effects of the Folh1 alleles on folate and sulfur amino acid levels and consecutively on glucose and lipid metabolism using SHR-1 congenic sublines harboring either Folr1 BN and Folh1 SHR alleles or Folr1 SHR and Folh1 BN alleles. Both congenic sublines when compared to SHR controls, exhibited significantly reduced folate clearance and lower plasma cysteine and homocysteine levels which was associated with significantly decreased serum glucose and insulin concentrations and reduced adiposity. These results strongly suggest that, in addition to Folr1, the Folh1 gene also plays an important role in folate and sulfur amino acid levels and affects glucose and lipid metabolism in the rat.

Hereditary xanthinuria is not so rare disorder of purine metabolism.

Hereditary xanthinuria (type I) is caused by an inherited deficiency of the xanthine oxidorectase (XDH/XO), and is characterized by very low concentration of uric acid in blood and urine and high concentration of urinary xanthine, leading to urolithiasis. Type II results from a combined deficiency of XDH/XO and aldehyde oxidase. Patients present with hematuria, renal colic, urolithiasis or even acute renal failure. Clinical symptoms are the same for both types. In a third type, clinically distinct, sulfite oxidase activity is missing as well as XDH/XO and aldehyde oxidase. The prevalence is not known, but about 150 cases have been described so far. Hypouricemia is sometimes overlooked, that´s why we have set up the diagnostic flowchart. This consists of a) evaluation of uric acid concentrations in serum and urine with exclusion of primary renal hypouricemia, b) estimation of urinary xanthine, c) allopurinol loading test, which enables to distinguish type I and II; and finally assay of xanthine oxidoreductase activity in plasma with molecular genetic analysis. Following this diagnostic procedure we were able to find first patients with hereditary xanthinuria in our Czech population. We have detected nine cases, which is one of the largest group worldwide. Four patients were asymptomatic. All had profound hypouricemia, which was the first sign and led to referral to our department. Urinary concentrations of xanthine were in the range of 170-598 mmol/mol creatinine (normal < 30 mmol/mol creatinine). Hereditary xanthinuria is still unrecognized disorder and subjects with unexplained hypouricemia need detailed purine metabolic investigation.

Iron Overload Causes Alterations of E-Cadherin in the Liver.

Iron overload causes tissue damage in the liver, but its initial effects at the molecular and cellular level are not well understood. Epithelial cadherin (E-cad) is a major adhesion protein in adherens junctions and is associated with several signal transduction pathways. Dysfunction of E-cad causes instability of adherens junctions, which leads to cell invasion, cell migration, and carcinogenesis. We found in liver samples from iron-overloaded mice that the apparent molecular mass of E-cad was reduced from 125 to 115 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions and immunoblotting, and that the cellular expression of E-cad was decreased in immunohistochemistry. The mRNA level of E-cad, however, did not change significantly, suggesting that the alterations are posttranslational. Interestingly, incubation of control liver extracts with Fe2+ alone also produced the same mobility shift. Neither an oxidant nor an antioxidant influenced this shift in vitro, suggesting that reactive oxygen species, which are generated by iron and known to cause damage to macromolecules, are not involved. Treatment of the 115 kDa E-cad with deferoxamine, an iron chelator, thus removing Fe2+, shifted the molecular mass back to 125 kDa, demonstrating that the shift is reversible. The observation also implies that the alteration that causes the mobility shift is not due to transcriptional control, deglycosylation, and proteolysis. This reversible mobility shift of E-cad has not been previously known. The alteration of E-cad that causes the mobility shift might be an initial step to liver diseases by iron overload.

Enzymatic diagnosis of homocystinuria by determination of cystathionine-ß-synthase activity in plasma using LC-MS/MS.

BACKGROUND: Cystathionine ß-synthase (CBS) is released into plasma from organs expressing this enzyme. Decreased plasma CBS activity has been demonstrated in CBS-deficient patients with 16 different genotypes. The aim of this study was to determine plasma CBS activity in patients carrying 11 additional genotypes using two LC-MS/MS methods. Patients and methods CBS activity was measured in EDTA or heparin plasma using either a previously described or a newly developed LC-MS/MS method optimized for analysis of the reaction product, 3,3-(2)H2-cystathionine, as its butyl ester derivative. We analyzed plasma samples from 26 CBS-deficient patients with known genotypes and 57 controls. RESULTS: We developed a new LC-MS/MS method for simple and sensitive determination of CBS activity. Plasma CBS activity was low (i.e., 0.001-0.036 of the multiples of median control values, MoM) in patients homozygous for the prevalent Hispanic mutation c.572C>T (p.T191M) but was highly elevated (2.95 MoM) in a single patient homozygous for the c.1330G>A (p.D444N) mutation. Patients with the remaining nine genotypes exhibited decreased activities (0.00-0.22 MoM), which did not overlap with the controls (0.29-2.10 MoM). CONCLUSIONS: The determination of CBS activity in plasma is a rapid and non-invasive procedure for detecting a subgroup of CBS-deficient patients with distinct genotypes.

Decreased hemojuvelin protein levels in mask mice lacking matriptase-2-dependent proteolytic activity.

Matriptase-2, a membrane protein encoded by the Tmprss6 gene, is a negative regulator of hepcidin expression. Although matriptase-2 has been proposed to cleave membrane hemojuvelin, we have recently found decreased hemojuvelin protein levels in Tmprss6 -/- mice. The purpose of this study was to confirm this observation by determining hemojuvelin protein levels in another strain of mice with disrupted Tmprss6 gene, and to determine the effect of matriptase-2 deficiency on the expression of other membrane proteins participating in the bone morphogenetic protein signal transduction. Mask mice, which lack the proteolytic domain of matriptase-2, displayed decreased liver hemojuvelin protein content, while Id1 mRNA level, an indicator of hemojuvelin-dependent signal transduction, was increased. Protein levels of bone morphogenetic protein receptors Alk3 and Acvr2a were unchanged, and transferrin receptor 2 and neogenin protein levels were slightly decreased. The results confirm that the loss of matriptase-2 increases bone morphogenetic protein-dependent signaling, while paradoxically decreasing liver hemojuvelin protein content. The regulation of transferrin receptor 2 protein levels by transferrin saturation was not affected in mask mice. How the loss of matriptase-2 proteolytic activity leads to decreased hemojuvelin protein levels is at present unclear.

Novel mutations in the tyrosine hydroxylase gene in the first Czech patient with tyrosine hydroxylase deficiency.

Tyrosine hydroxylase deficiency manifests mainly in early childhood and includes two clinical phenotypes: an infantile progressive hypokinetic-rigid syndrome with dystonia (type A) and a neonatal complex encephalopathy (type B). The biochemical diagnostics is exclusively based on the quantitative determination of the neurotransmitters or their metabolites in cerebrospinal fluid (CSF). The implementation of neurotransmitter analysis in clinical praxis is necessary for early diagnosis and adequate treatment. Neurotransmitter metabolites in CSF were analyzed in 82 children (at the age 1 month to 17 years) with clinical suspicion for neurometabolic disorders using high performance liquid chromatography (HPLC) with electrochemical detection. The CSF level of homovanillic acid (HVA) was markedly decreased in three children (64, 79 and 94 nmol/l) in comparison to age related controls (lower limit 218-450 nmol/l). Neurological findings including severe psychomotor retardation, quadruspasticity and microcephaly accompanied with marked dystonia, excessive sweating in the first patient was compatible with the diagnosis of tyrosine hydroxylase (TH) deficiency (type B) and subsequent molecular analysis revealed two novel heterozygous mutations c.636A>C and c.1124G>C in the TH gene. The treatment with L-DOPA/carbidopa resulted in the improvement of dystonia. Magnetic resonance imaging studies in two other patients with microcephaly revealed postischaemic brain damage, therefore secondary HVA deficit was considered in these children. Diagnostic work-up in patients with neurometabolic disorders should include analysis of neurotransmitter metabolites in CSF.

Hepcidin expression in the liver of mice with implanted tumour reacts to iron deficiency, inflammation and erythropoietin administration.

Cancer is known to be an important cause of anaemia due to several factors including iron deficiency and inflammation. Hepcidin, a key regulator of iron metabolism, is up-regulated by iron and inflammatory stimuli such as interleukin 6, and decreased by iron deficiency, enhanced erythropoiesis and hypoxia. It is supposed to play a crucial role in changes of iron metabolism in anaemia of chronic disease, which is characterized by sequestering iron in macrophages and decreasing its availability for red blood cell production. To study the effect of tumour growth on hepcidin expression, we implanted human melanoma cells into mice and studied the changes of the amount of liver hepcidin mRNA by real-time PCR. We observed development of anaemia, which correlated with the size of the tumour. Hepcidin expression significantly decreased with the anaemia development, but in late stages we observed an increase of its expression together with an increase of mRNA for interleukin 6. However, the increase of hepcidin expression could be inhibited by exogenous erythropoietin administration. In our model of tumour growth, hepcidin expression reflected anaemia development and iron deficiency, erythropoietin administration and inflammation, and we suppose that it could therefore serve as a useful marker of these clinical situations common in cancer patients and play a role in the pathogenesis of cancer-associated anaemia.

Oligodendroglia from ADSL-deficient patient produce SAICAribotide and SAMP.

Succinylpurines accumulate in the body fluids of patients with adenylosuccinate lyase (ADSL) deficiency but their source in the cerebrospinal fluid remains obscure. Study based on the incorporation of 13C-stable isotope-labeled glycine into cultured oligodendroglia from ADSL-deficient patient and the measurement of labeled products by LC/MS/MS showed total intracellular concentrations of succinylpurines from 45 to 99µmol/l and so these results suggest that these cells can be the source of the compounds in vivo.

Hepcidin downregulation by repeated bleeding is not mediated by soluble hemojuvelin.

Hepcidin is a key regulator of iron homeostasis, while hemojuvelin is an important component of the hepcidin regulation pathway. It has been recently proposed that soluble hemojuvelin, produced from hemojuvelin by the protease furin, decreases hepcidin expression. The aim of the presented study was to examine the downregulation of hepcidin by chronic bleeding in hemojuvelin-mutant mice. Male mice with targeted disruption of the hemojuvelin gene (Hjv-/- mice) and wild-type littermates were maintained on an iron-deficient diet and subjected to weekly phlebotomies for 7 weeks. Gene expression was examined by real-time PCR. In wild type mice, repeated bleeding decreased hepcidin mRNA by two orders of magnitude. In Hjv-/- mice, basal hepcidin expression was low; however, repeated bleeding also decreased hepcidin mRNA content by an order of magnitude. Phlebotomies reduced hepatic iron overload in Hjv-/- mice by 80 %. Liver and muscle furin mRNA content was not significantly changed. No effect on hepatic Tmprss6 mRNA content was observed. Results from the study indicate that soluble hemojuvelin is not the sole factor responsible for hepcidin downregulation. In addition, the presented data suggest that, under in vivo conditions, tissue hypoxia does not transcriptionally regulate the activity of furin or TMPRSS6 proteases.

D-ribose therapy in four Polish patients with adenylosuccinate lyase deficiency: absence of positive effect.

Deficiency of adenylosuccinate lyase (ADSL) (OMIM 103050) is an autosomal recessive disorder of the purine de novo synthesis pathway and purine nucleotide cycle, diagnosed so far in approximately 50 patients. The clinical presentation is characterized by severe neurological involvement including hypotonia, seizures, developmental delay and autistic features. Epilepsy in ADSL deficiency is frequent and occurs in approximately two-thirds of patients, beginning either early in the neonatal period or after the first year of life. At present there is no treatment of proven clinical efficacy. Despite of the increasing number of ADSL-deficient patients reported, there are only a few communications of therapeutic considerations or efforts. Among them only two showed some beneficial effects in ADSL-deficient patients. D-ribose, a simple and relatively cheap therapy, has been associated with improvement of behaviour and progressive reduction of the seizure frequency in one 13-year-old patient with ADSL deficiency. In this study we have re-examined D-ribose treatment in four ADSL-deficient patients. Assessments consisted of biochemical markers and neurological outcome. The 12-month trial of D-ribose failed to show any clinical benefit in ADSL patients with both milder and severe phenotype. D-ribose administration was accompanied by neither reduction in seizure frequency nor growth enhancement. Additionally, patients with milder type II presented the first seizure after 4 and 8 months of the D-ribose treatment. Therefore, we could not confirm a positive effect of D-ribose as previously reported.

Plasma cysteine concentrations in uncomplicated pregnancies.

OBJECTIVE: To measure levels of total plasma cysteine, homocysteine, cysteinylglycine and glutathione of normotensive primiparous pregnant women in the second and the third trimester. METHODS: Two consecutive blood samples were taken from 65 healthy primiparous women in the 19th to 21st weeks of pregnancy and then in the 30th to 32nd weeks. Plasma total cysteine, homocysteine, cysteinylglycine and glutathione were determined by HPLC method. Women were followed until delivery. Sixty-two pregnant women were normotensive throughout the pregnancy and 3 developed pre-eclampsia. Median levels of thiols in the second and the third trimesters were compared using paired t test. RESULTS: Levels (median [range], micromol/l) of plasma total cysteine in normotensive pregnant women were significantly lower in the third than in the mid-trimester (176.1 [163.0, 189.4] vs. 187.4 [178.7, 205.2], p < 0.001). Concentrations of total homocysteine, cysteinylglycine and glutathione were not different. CONCLUSION: Plasma total cysteine (t-Cys) is significantly lower in the third compared to the second trimester. Urinary excretion of t-Cys does not differ in the second compared to the third trimester. The decrease of t-Cys might indicate that cysteine is essential for the fetus.

[Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)]. / Mitochondriální neurogastrointestinální encefalomyopatie (syndrom MNGIE).

BACKGROUND: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a disorder with autosomal recessive inheritance caused by mutations in the gene encoding thymidine phosphorylase (TP). TP deficiency results in imbalance of mitochondrial pool of nucleotides leading secondary to multiple deletions and depletion of mitochondrial DNA (mtDNA) and impairment of oxidative phosphorylation system. The disease is clinically characterized by gastrointestinal dysmotility with symptoms of pseudo-obstruction, severe failure to thrive, ptosis, leukoencephalopathy, peripheral neuropathy and myopathy. We present results of the clinical, histochemical, biochemical and molecular analyses of the first Czech patient with MNGIE syndrome. METHODS AND RESULTS: Man, 33-years old with twenty-year history of failure to thrive (height 168 cm, weight 34 kg) and progressive gastrointestinal dysmotility, external ophthalmoplegia, leucoencephalopathy and peripheral neuropathy was recommended to metabolic center. Histochemical analyses in muscle biopsy showed the presence of "ragged red fibers" with focal decrease of cytochrome c oxidase activity, but spectrophotometric analyses in isolated muscle mitochondria revealed normal activities of all respiratory chain complexes. Metabolic investigation revealed markedly increased plasma level of thymidine (6.6 micromol/l, controls <0.05 micromol/l) and deoxyuridine (15 micromol/l, controls <0.05 micromol/l). The activity of TP in isolated lymphocytes was low (0.02 micromol/hour/mg protein, reference range 0.78 +/- 0.18). Molecular analyses in muscle biopsy revealed multiple mtDNA deletions and homozygous mutation 1419G>A (Gly145Arg) was found in gene for TP. Both parents are heterozygotes. CONCLUSIONS: MNGIE has to be considered in patients presenting with a combination of gastrointestinal and neurological symptoms. Plasma level of thymidine may serve as the best method for laboratory screening of MNGIE, but molecular analyses are necessary for genetic counselling and prenatal diagnosis in affected families.

Effect of lipopolysaccharide and bleeding on the expression of intestinal proteins involved in iron and haem transport.

Haem carrier protein 1 (Hcpl) is a component of the haem-iron uptake pathway in the small intestine. Using quantitative real-time PCR, we examined the expression of Hcp1 and other intestinal iron-transporting proteins in male C57BL/6 mice with experimentally altered iron homeostasis. Intestinal Hcp1 mRNA content was not significantly changed by iron overload (600 mg/kg); however, it was increased to 170 % of controls 72 h after withdrawal of 0.7 ml of blood; the same treatment increased intestinal Cybrd1 mRNA to 900 % of controls. LPS treatment (1 mg/kg, 6 h) decreased intestinal Hcp1 mRNA content to 66 % of controls and Flvcr mRNA content to 65 % of controls, while Cybrd1 mRNA, Dmt1 mRNA and Fpn1 mRNA decreased to 6 %, 43 % and 32 %, respectively. In 129SvJ mice with targeted disruption of the hemojuvelin (Hfe2) gene, which display very low expression of liver hepcidin, Cybrd1 mRNA content increased to 1040 %, Dmt1 mRNA content to 200 % and Fpn1 mRNA to 150 % when compared to wild-type mice; changes in Hcp1, Abcg2 and Flver mRNA content were only minor. Overall, these results suggest that, during inflammation, the intestinal haem-iron uptake pathway is not as strongly transcriptionally downregulated as the non-haem iron uptake pathway. A decrease in circulating hepcidin increases the expression of proteins participating in non-haem iron uptake, but has no significant effect on Hcp1 mRNA content.

Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis.

Hepcidin, a key regulator of iron metabolism, decreases intestinal absorption of iron and its release from macrophages. Iron, anemia, hypoxia, and inflammation were reported to influence hepcidin expression. To investigate regulation of the expression of hepcidin and other iron-related genes, we manipulated erythropoietic activity in mice. Erythropoiesis was inhibited by irradiation or posttransfusion polycythemia and stimulated by phenylhydrazine administration and erythropoietin. Gene expression of hepcidin and other iron-related genes (hemojuvelin, DMT1, ferroportin, transferrin receptors, ferritin) in the liver was measured by the real-time polymerase chain reaction. Hepcidin expression increased despite severe anemia when hematopoiesis was inhibited by irradiation. Suppression of erythropoiesis by posttransfusion polycythemia or irradiation also increased hepcidin mRNA levels. Compensated hemolysis induced by repeated phenylhydrazine administration did not change hepcidin expression. The decrease caused by exogenous erythropoeitin was blocked by postirradiation bone marrow suppression. The hemolysis and anemia decrease hepcidin expression only when erythropoiesis is functional; on the other hand, if erythropoiesis is blocked, even severe anemia does not lead to a decrease of hepcidin expression, which is indeed increased. We propose that hepcidin is exclusively sensitive to iron utilization for erythropoiesis and hepatocyte iron balance, and these changes are not sensed by other genes involved in the control of iron metabolism in the liver.

Preparation of 5-amino-4-imidazole-N-succinocarboxamide ribotide, 5-amino-4-imidazole-N-succinocarboxamide riboside and succinyladenosine, compounds usable in diagnosis and research of adenylosuccinate lyase deficiency.

The enzyme adenylosuccinate lyase (ADSL) intervenes twice in the biosynthesis of adenine nucleotides. ADSL deficiency is an inherited metabolic disease characterized by various degrees of psychomotor retardation and accumulation of dephosphorylated enzyme substrates 5-amino-4-imidazole-N-succinocarboxamide riboside (SAICAr) and succinyladenosine (SAdo) in body fluids. Severity of symptoms seems to correlate with residual activity of mutant enzyme and with SAdo/SAICAr concentration ratio in cerebrospinal fluid. To better understand the pathogenetic mechanisms of the disease symptoms, studies of catalytic properties of mutant enzymes together with in vitro and in vivo experiments utilizing SAICAr and SAdo must be performed. Such studies require availability of both ADSL substrates, 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAICAR) and succinyladenosine 5'-monophosphate (SAMP) and their dephosphorylated products in sufficient amounts and purity. Except for SAMP, none of these compounds is commercially available and they must therefore be synthesized. SAICAR was prepared by recombinant human ADSL-catalysed reaction of AICAR (5-aminoimidazole-4-carboxamide) with fumarate and isolated by thin-layer chromatography. SAICAr and SAdo were prepared by calf intestine alkaline phosphatase-catalysed dephosphorylation of SAICAR and SAMP and isolated on cation- and anion-exchange resin columns. The procedures described are easily scalable and provide high yields of sufficiently pure products for use in experiments related to studies of pathogenetic mechanisms in ADSL deficiency.

Adenosine and methotrexate polyglutamate concentrations in patients with juvenile arthritis.

OBJECTIVE: In contrast to the anti-proliferative properties of high-dose methotrexate (MTX) its anti-inflammatory mechanism of action in rheumatic diseases has been attributed to increased adenosine accumulation, most likely caused by long-lived intracellular MTX polyglutamates. The aim of this study was to assess adenosine concentrations in MTX-treated and untreated children and to relate it to MTX polyglutamate concentration measured in erythrocytes and to the therapeutic efficacy. METHODS: Adenosine and MTX-polyglutamate concentrations in erythrocytes (EMTX) were assessed in venous blood samples taken before the next MTX dose in 30 patients treated long-term for juvenile idiopathic arthritis (JIA) and in 16 untreated matched controls. The blood concentration of adenosine was measured by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) method and EMTX by an enzymatic assay. Therapeutic efficacy was assessed using the preliminary definition of improvement in JIA patients. RESULTS: Mean blood adenosine concentration in MTX-treated patients was 48.05 nmol/l (s.d. 10.1) vs 49.6 nmol/l (s.d. 12.5) in untreated controls (P=0.55). Mean EMTX was 215.56 nmol/l (s.d. 212.9). No significant correlation was found between adenosine concentrations and MTX dose or EMTX (P=0.8 and 0.6, respectively). Adenosine concentration did not differ in clinical responders when compared with non-responders (P=0.9). CONCLUSIONS: We have shown that there is no impact of effective MTX dose represented by EMTX on blood adenosine concentration in JIA patients. If MTX anti-inflammatory action is mediated by adenosine it is likely that local release of adenosine at inflamed tissues is responsible for its action which may not be reflected by sustained increase of its blood concentration.

Maternal hyperphenylalaninemias in healthy Czech population of pregnant women: 30 years experience with screening, prevention and treatment.

INTRODUCTION: The increased level of phenylalanine (Phe) in maternal blood--hyperphenylalaninemia (mHPA) has a detrimental effect on the early development of healthy foetus (1965). The toxic effect causes spontaneous abortion or retards intrauterine growth, skeletal malformation, cardiac anomalies can appear. However the most frequent are microcephaly, mental retardation and hypotrophy. PATIENTS AND METHODS: Simultaneously with the introduction of obligatory "Newborn Screening Program" in CR also the facultative screening for mHPA was introduced ("Maternal Hyperphenylalaninemia Preventive Screening Program"). Since 1975 till now 222,990 healthy pregnant women (16-47 yrs) from city Prague and its area (cca 2 mil. inh.) have been screened for increased Phe in blood by Efron's chromatographic screening test (1964); Phe cut off value: 240 micromol/l. Nonfasting venous blood has been taken in 2nd-3rd month of pregnancy during the first antenatal visit. All positive cases have been verified with quantitative Phe estimation on amino acid analyzer incl. pterines analysis in urine. For differentiation of detected mHPAs the Güttler's scheme (1980) has been used. Mutations for Phe-hydroxylase gene analyzed by restriction enzyme digestion after Guldberg (1994). RESULTS: The average incidence of mHPA detected at the beginning of pregnancy was found 1:8675. The major part (65.3%) of all detected mHPA belongs to mild or moderate form of phenylketonuria (PKU) with most frequent PAH gene mutations R408W, Y414C, IVS11 nt8g-a, R158Q, IVS12ntlg-a and R261Q. 19.2% corresponds to atypical or classical PKU with prevailing mutation R408W. Only in 15.3% were detected non-PKU (persistent HPA) with mutations R408W, Y414C, IVS12ntlg-a, IV11nt8g-a and A403V. 28 offsprings born from pregnancies on low-phenylalanine diet (LPD) introduced at least 2 months before the conception and during the whole pregnancy show normal psychomotoric development. In 7 offsprings without LPD or after delayed introducing or on PLD or badly monitored showed malformations (microcephaly, hypotrophy, skeletal malformations) or died. DISCUSSION: Relatively high incidence of mHPA detected in healthy population of pregnant women of Prague area differs from findings of Buist (1989) or Levy (1994) from American pregnant women screened for mHPA from umbilical blood. We consider that screening performed at the beginning of pregnancy from nonfasting venous blood is more effective compared to umbilical blood from two reasons: the Phe level in maternal blood is increased during first trimester of pregnancy due to succing effect of placenta in comparison to decreased Phe level at the end of labour. Umbilical blood for screening of mHPA is not quite suitable to detect the atypical or mild forms of Phe disturbances which prevailed in our Slavonic population of pregnant women. (Tab. 5, Fig. 7, Ref. 16.)

Inhibitory effects of Bcl-2 on mitochondrial respiration.

In contrast to the well-established anti-apoptotic effect of Bcl-2 protein, we have recently demonstrated that Bcl-2 overexpression by vaccinia virus causes apoptosis in BSC-40 cells, while it prevents apoptosis in HeLa G cells. Given the key role of mitochondria in the process of apoptosis, we focused on effects of Bcl-2 expression on mitochondrial energetics of these two cell lines. In this study we present data indicating that BSC-40 cells derive their ATP mainly from oxidative phosphorylation whereas HeLa G cells from glycolysis. More importantly, we show that in both cell lines, Bcl-2 inhibits mitochondrial respiration and causes a decrease of the ATP/ADP ratio. However, it appears that BSC-40 cells cannot sustain this decrease and die, while HeLa G cells survive, being adapted to the low ratio of ATP/ADP maintained by glycolysis. Based on this observation, we propose that the outcome of Bcl-2 expression is determined by the type of cellular ATP synthesis, namely that Bcl-2 causes apoptosis in cells relying on oxidative phosphorylation.