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Arachidonic acids and its metabolites modulate plenty of ligand-gated, voltage-dependent ion channels, and metabolically regulated potassium channels including ATP-sensitive potassium channels (KATP). KATP channels are hetero-multimeric complexes of sulfonylureas receptors (SUR1, SUR2A or SUR2B) and the pore-forming subunits (Kir6.1 and Kir6.2) likewise expressed in the pre-post synapsis of neurons and inflammatory cells, thereby affecting their proliferation and activity. KATP channels are involved in amyloid-β (Aβ)-induced pathology, therefore emerging as therapeutic targets against Alzheimer’s and related diseases. The modulation of these channels can represent an innovative strategy for the treatment of neurodegenerative disorders; nevertheless, the currently available drugs are not selective for brain KATP channels and show contrasting effects. This phenomenon can be a consequence of the multiple physiological roles of the different varieties of KATP channels. Openings of cardiac and muscular KATP channel subunits, are protective against caspase-dependent atrophy in these tissues and some neurodegenerative disorders, whereas in some neuroinflammatory diseases, benefits can be obtained through the inhibition of neuronal KATP channel subunits. For example, glibenclamide exerts an anti-inflammatory effect in respiratory, digestive, urological, and central nervous system (CNS) diseases, as well as in ischemia-reperfusion injury associated with abnormal SUR1-Trpm4/TNF-α or SUR1-Trpm4/ Nos2/ROS signaling. Despite this strategy being promising, glibenclamide may have limited clinical efficacy due to its unselective blocking action of SUR2A/B subunits also expressed in cardiovascular apparatus with pro-arrhythmic effects and SUR1 expressed in pancreatic beta cells with hypoglycemic risk. Alternatively, neuronal selective dual modulators showing agonist/antagonist actions on KATP channels can be an option.

Folic acid has been identified to be integral in rapid tissue growth and cell division during fetal development. Different studies indicate folic acid’s importance in improving childhood behavioral outcomes and underline its role as a modifiable risk factor for autism spectrum disorders. The aim of this systematic review is to both elucidate the potential role of folic acid in autism spectrum disorders and to investigate the mechanisms involved. Studies have pointed out a potential beneficial effect of prenatal folic acid maternal supplementation (600 µg) on the risk of autism spectrum disorder onset, but opposite results have been reported as well. Folic acid and/or folinic acid supplementation in autism spectrum disorder diagnosed children has led to improvements, both in some neurologic and behavioral symptoms and in the concentration of one-carbon metabolites. Several authors report an increased frequency of serum auto-antibodies against folate receptor alpha (FRAA) in autism spectrum disorder children. Furthermore, methylene tetrahydrofolate reductase (MTHFR) polymorphisms showed a significant influence on ASD risk. More clinical trials, with a clear study design, with larger sample sizes and longer observation periods are necessary to be carried out to better evaluate the potential protective role of folic acid in autism spectrum disorder risk.

Background & Objective: Multiple sclerosis (MS) is an inflammatory neurodegenerative disease characterized by destruction of oligodendrocytes, immune cell infiltration and demyelination. Inflammation plays a significant role in MS, and the inflammatory mediators such as eicosanoids, leukotrienes, superoxide radicals are involved in pro-inflammatory responses in MS. In this systematic review we tried to define and discuss all the findings of in vivo animal studies and human clinical trials on the potential association between arachidonic acid (AA) pathway and multiple sclerosis. Methods: A systematic literature search across Pubmed, Scopus, Embase and Cochrane database was conducted. This systematic review was performed according to PRISMA guidelines. Results: A total of 146 studies were included, of which 34 were conducted in animals, 58 in humans, and 60 studies reported the role of different compounds that target AA mediators or their corresponding enzymes/ receptors, and can have a therapeutic effect in MS. These results suggest that eicosanoids have significant roles in experimental autoimmune encephalomyelitis (EAE) and MS. The data from animal and human studies elucidated that PGI2, PGF2α, PGD2, isoprostanes, PGE2, PLA2, LTs are increased in MS. PLA2 inhibition modulates the progression of the disease. PGE1 analogues can be a useful option in the treatment of MS. Conclusions: All studies reported the beneficial effects of COX and LOX inhibitors in MS. The hybrid compounds, such as COX-2 inhibitors/TP antagonists and 5-LOX inhibitors can be an innovative approach for multiple sclerosis treatment. Future work in MS should shed light in synthesizing new compounds targeting arachidonic acid pathway.

Hyperhomocysteinemia (HHcy) has been considered a risk factor for different diseases, including cardiovascular disease (CVD), inflammation, neurological diseases, cancer, and many other pathological conditions. Likewise, arachidonic acid (AA) metabolism is implicated in both vascular homeostasis and inflammation, as shown by the development of CVD, following the imbalance of its metabolites. This review summarizes how homocysteine (Hcy) can influence the metabolism of AA. In silico literature searches were performed on PubMed and Scopus as main sources. Several studies have shown that altered levels of Hcy, through AA release and metabolism, can influence the synthesis and the activity of prostaglandins (PGs), prostacyclin (PGI2), thromboxane (TXA), epoxyeicosatrienoic acids (EETs), and hydroxyeicosatetraenoic acids (HETEs). It is believed that by targeting Hcy in the AA pathways, novel compounds with better pharmacological and pharmacodynamics benefits may be obtained and that this information is valuable for a dietician to manipulate diets to improve health.

Today the role of cytochrome P450 metabolites in inflammatory rheumatic disease, such as rheumatoid arthritis (RA) is still poorly understood. In this review we survey the current knowledge on cytochrome P450 metabolites in rheumatoid arthritis. The balance between CYP epoxygenase- and CYP ω- hydroxylase is correlated to the regulation of NF-κB. In RA patients synovial fluid there are higher levels of IL-6, which suppresses activities of CYP enzymes, such as CYP3A, CYP2C19, CYP2C9, and CYP1A2. EETs have anti-inflammatory effects, probably attributed to the PPARγ activation. EETs inhibit bone resorption and osteoclastogenesis, and can be considered as an innovative therapeutic strategy for rheumatoid arthritis. In reference to the CYP ɷ-hydroxylase pathway, 20-HETE is a pro-inflammatory mediator. While there is scarce information on the role of 20-HETE inhibitors and its antagonists in rheumatoid arthritis, the elevation of EETs levels by sEH inhibitors is a promising therapeutic strategy for rheumatoid arthritis patients. In addition, hybrid compounds, such as sEH inhibitors/FLAP inhibitors, or sEHI combined with NSAIDs/COXIBs are also important therapeutic target. However, studies investigating the effects of inflammation and rheumatic disease on CYP-mediated eicosanoid metabolism are necessary. Obtaining a better understanding of the complex role of CYP-derived eicosanoids in inflammatory rheumatic disease, such as rheumatoid arthritis will provide valuable insight for basic and clinical researchers investigation.

Introduction Edible algae, rich in biologically active components, may influence glycaemic control, lower blood lipids and increase antioxidant activities. The aim of this study was to evaluate the effect of prolonged supplementation with an extract of the brown edible seaweed Ascophyllum nodosum on anthropometric indexes and biomarkers of metabolic risk for cardiovascular disease in human volunteers.

Background: Low molecular weight thiols (cysteine, cysteinylglycine, glutathione [GSH] and homocysteine) are important intermediates in different metabolic pathways. Glutathione has a relevant role as an antioxidant in detoxification of toxic compounds and xenobiotics, and homocysteine represents a risk factor for cardiovascular, neurological, and congenital diseases. Homocysteine and GSH are metabolically related in a pathway including cysteine and cysteinylglycine as intermediates. For these reasons, determination of homocysteine and related thiols is of great importance in the diagnosis of several diseases. Methods: The measurement of these sulphur compounds can be performed by using different methods, such as liquid or gas chromatography/mass spectrometry, highperformance liquid chromatography (HPLC), or capillary electrophoresis. Results: This study describes an HPLC method coupled with fluorimetric detection for the simultaneous determination of cysteine, cysteinylglycine, GSH, and homocysteine in different biological fluids (blood, saliva, urine, and cerebrospinal fluid [CSF]). Conclusion: The comparison of the results obtained by other authors, as well as the method validation and the analytical costs, indicate that this HPLC method is particularly suitable for routine measurement of thiols in different body fluids.

Septic pulmonary embolism (SPE) is an uncommon, but life-threatening event that is usually associated with extrapulmonary infections. We report the first case of bilateral SPE secondary to a central venous catheter-related bloodstream infection involving pathogens commonly considered environmental contaminants: Tsukamurella tyrosinosolvens and Rhizobium radiobacter. Empirical levofloxacin treatment was confirmed by in vitro susceptibility data and produced prompt clinical improvement, but removal of the infected line proved indispensable for eradication of the infection. Laboratory personnel should be aware of the pathogenic potential of these environmental organisms, particularly in immunocompromised hosts with indwelling catheters.

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC-MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.

Background: 5,10-methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of homocysteine metabolism. Hyperhomocysteinemia, frequently associated with cardiovascular disease, congenital defects, pregnancy complications, and cancer, can derive from MTHFR impaired activity, depending on some common mutations such as C677T and A1298C. In this study we evaluated the genotype and the allele frequencies of the polymorphisms C677T and A1298C of MTHFR in 104 newborns from central-southern Italy. Materials and Methods: C677T and A1298C polymorphisms were analyzed by polymerase chain reaction (PCR-RFLP). Results: The 677TT and 1298CC genotypes showed a frequency of 25.0% and 12.5% respectively; the frequency of the mutated alleles was 50.5% for 677T and 32.7% for 1298C. The analysis of the genotype combination showed a significant negative linkage disequilibrium. Conclusions: The results showed interesting new data concerning the higher prevalence of the 1298CC genotype; furthermore, the frequency of the 677TT genotype could induce consideration of a nutrition policy based on folic acid supplementation.

BACKGROUND The authors evaluated serum levels of folate, homocysteine, and vitamin B12 in patients with head and neck squamous cell carcinoma (HNSCC) and in patients with laryngeal leukoplakia, a well known preneoplastic lesion. METHODS One hundred forty-four consecutive, untreated patients with HNSCC and 40 consecutive, untreated patients with laryngeal leukoplakia were enrolled in the Department of Otolaryngology at the authors' institution. Data from those patients were compared with data from one control group, which included 90 smokers, and from another control group, which included 120 nonsmokers. Serum levels of homocysteine, folate, and vitamin B12 were measured by an automated immunoassay method based on fluorescence polarization immunoassay technology. RESULTS Comparing groups by Student–Newman–Keuls test, serum folate levels were significantly lower in patients with HNSCC and in patients with laryngeal leukoplakia compared with serum folate levels in both the smoker control group and the nonsmoker control group. Serum homocysteine levels in patients with HNSCC were significantly higher compared with homocysteine levels both in the smoker and nonsmoker control groups and in patients with laryngeal leukoplakia. There were no statistically significant differences between groups in serum vitamin B12 levels. CONCLUSIONS A role for folate deficiency as a risk factor in head and neck carcinogenesis is plausible. A chemoprevention protocol with folate is both feasible and ethically correct and is in progress at the authors' institution. Homocysteine levels in patients with HNSCC probably are affected largely by the HNSCC phenotype. An accumulation of homocysteine may reveal a genetic defect, which, theoretically, may be a target for pharmacologic therapy, for example, with antifolic drugs. Cancer 2005. © 2004 American Cancer Society.

Background: Saliva is an easily available biological material that is not commonly analysed in clinical chemistry, while it could give useful information especially in several oral diseases. Methods: In this work, the sulphur containing compounds cysteine, cysteinylglycine and glutathione were analysed in saliva of control subjects and periodontopathic subjects by a HPLC method. The detection limit of the method is 0.5, 0.1 and 0.1 μmol/l for cysteine, cysteinylglycine and glutathione, respectively, and it is linear up to 10 mmol/l. Results: the median values for the control group are 1.2 μmol/l for cysteine and glutathione and 0.4 μmol/l for cysteinylglycine while those of periodontopathic patients are significantly increased (4.4, 2.1 and 11.0 μmol/l for cysteine, cysteinylglycine and glutathione, respectively).

BACKGROUND Local and systemic metabolic alterations are always present in cancer. Carcinogenesis is associated with biochemical disorders, often nonspecific, that might promote or derive from tumoral progression. Thus, analysis of metabolic alterations may be a valuable approach to understanding the biochemistry of tumors and may provide a means of identifying new targets for therapy. The methionine cycle in particular has been extensively studied in human cancer. METHODS The authors analyzed serum concentrations of two metabolites of such pathways, folate and homocysteine, in 42 patients affected by head and neck squamous cell carcinoma (HNSCC) in comparison with two control groups, composed of smokers and non smokers. RESULTS Mean folate level was 5.8 ± 2.1 ng/mL in carcinoma patients, 9.1 ± 2.7 ng/mL in smoking controls, and 9.7 ± 2.2 ng/mL in non smoking controls, with a statistically significant difference between carcinoma patients and smokers (mean difference: −3.3ng/mL; 95% confidence interval [CI]: −4.234 to −2.366; P < 0.0001) and between carcinoma patients and non smokers (mean difference: −3.9ng/mL; 95% CI: −4.67 to −3.13; P < 0.0001). Mean total homocysteine level was 10.4 ± 5.3 μM in carcinoma patients, 7.8 ± 2.5 μM in the non-smokers' group, and 8.3 ± 2.8 μM in the smokers' group, with statistically significant differences between carcinoma patients and smoking controls (mean difference: 2.1 μM; 95% CI: 0.7056 to 3.494; P = 0.0034) and between carcinoma patients and non smoking controls (mean difference: 2.6 μM; 95% CI: 1.381 to 3.819; P < 0.0001). CONCLUSIONS Differences in serum levels of folate and homocysteine might arise from tumor development and consequent metabolic alterations or might precede and promote tumor progression. If hypofolatemia is a risk factor for head and neck carcinogenesis, it might suggest a role for folate as a novel chemopreventive agent both in patients with precancerous lesions and in patients with treated HNSCC at risk for loco-regional recurrence and second primary tumors. Cancer 2002;94:1006–11. © 2002 American Cancer Society. DOI 10.1002/cncr.10343

Background: Breath condensate can give useful information on volatile compounds produced at alveolar level. Actual concentration of H2O2 in breath condensate is dependent on its production at alveolar level and on the efficacy of the detoxifying systems, catalase, glutathione peroxidase, etc. Methods: In the present paper, a simple chemiluminescent method for the determination of the H2O2 collected in exhaled breath is shown and data of both smokers and nonsmokers volunteers are presented. Results: The chemiluminescent response is linear up to 100 μmol/l H2O2. The analytical sensitivity is about 0.01 μmol/l. Most of the nonsmokers have a H2O2 content lower than 0.05 μmol/l, while smokers have a content ranging from 0.1 to 0.6 μmol/l.

Water-soluble extracts of cigarette smoke are easily formed in some body compartments, such as saliva or fluid lining alveolar spaces, and can act on both cellular and extracellular compartments. In this paper we have analysed the effect of aqueous smoke extract on some metabolic and functional aspects of polymorphonuclear leukocytes. In particular, the following cellular aspects were studied: chemiluminescence, glycolysis, membrane fluidity and microscopic interaction with zymosan particles. While chemiluminescence and glycolytic activity are highly inhibited, no effect of smoke extract on membrane fluidity was observed. Moreover, the response of luminol-dependent chemiluminescence was significantly delayed, while that of lucigenin-dependent chemiluminescence was anticipated. Furthermore, the phagocytic ability of neutrophils pretreated with aqueous smoke extract was also significantly hindered. All these results might indicate that the finely tuned activity of polymorphonuclear leukocytes is somehow hampered by the aqueous extract of cigarette smoke in a way which makes these cells less effective against bacteria and more noxious towards surrounding tissues.

Haemoglobin (Hb) J-Sardegna [α50(CE8)His → Asp] is a haemoglobin variant characteristic of subjects from the island of Sardinia. Here we report a study of the functional properties of both fetal and adult Hb J-Sardegna. The results indicate that adult Hb J-Sardegna displays an oxygen affinity that is higher than that of adult Hb only in the presence of 2,3-diphosphoglycerate (2,3-DPG). On the contrary, at 20 °C, the oxygen affinity of fetal Hb J-Sardegna is identical to that of normal fetal haemoglobin, both in the presence and in the absence of 2,3-DPG. A significant difference between these two systems (i.e. a higher oxygen affinity of fetal Hb J-Sardegna) shows up very clearly only when temperature is increased to 37 °C. Hence in fetal Hb, the main effect of the amino acid substitution is a decrease in the overall enthalpy change of oxygenation. The results outline the role of the α1-β1 interface in assessing the thermodynamics of oxygen binding. The functional properties of both adult and fetal Hb J-Sardegna have been interpreted at the structural level in light of the results obtained by a computational modelling approach performed in comparison with HbA and Hb Aichi, a variant characterized by a different mutation [α50(CE8)His → Arg] at the same position.

Cigarette smoke induces a vast cohort of deleterious effects on biological structures. In the present paper, the effect of aqueous extract of cigarette smoke on the activity of polymorphonuclear leukocytes was studied. Although the aqueous extract of cigarette smoke inhibits the luminol oxidation catalysed by horseradish peroxidase, it strongly interacts with polymorphonuclear leukocytes and inhibits their phorbol-induced chemiluminescence in the presence of either luminol or lucigenin. The results indicate that at least some of the components of the aqueous extract of cigarette smoke may strongly interfere with polymorphonuclear cells, contributing to the deleterious effects of smoke products. © 2000 John Wiley & Sons, Ltd.

The concentration of some of the purine nucleotides and their metabolites together with that of malondialdehyde (MDA) have been measured in resting and stimulated platelets of type 1 and type 2 diabetic patients. While control platelets show a net decrease of guanosine triphosphate (GTP) (3.1 vs. 2.3 nmol per 10(9) platelets) and guanosine diphosphate (GDP) (3.0 vs. 2.0 nmol per 10(9) platelets) and a significant increase of adenosine (0.04 vs. 0.55 nmol per 10(9) platelets) with platelet stimulation, platelets of type 1 and type 2 diabetic patients have a lesser change of these metabolites (GTP, 2.6 vs. 2.4; GDP, 2.3 vs. 2.4; adenosine, 0.04 vs. 0.30 (P < 0.05 vs. control) nmol per 10(9) platelets in type 1 diabetics; GTP, 2.4 vs. 2.7; GDP, 2.4 vs. 2.1; adenosine, 0.08 vs. 0.32 (P < 0.05 vs. control) nmol per 10(9) platelets in type 2 diabetics). These results indicate that the change (stimulated minus resting) of GTP, GDP and adenosine in diabetic platelets is significantly different from that of controls (P < 0.001). Moreover, the amount of MDA produced during platelet activation seems to be lower than controls only in type 2 diabetes (1.81 vs. 2.86 nmol per 10(9) platelets, P < 0.05). These results seem to indicate that a difference in the pattern of platelet nucleotides could be an important feature even in well-controlled diabetes, while MDA is probably modified only in association with the late vascular complications of diabetes.

Coenzyme Q and trace element contents of two antarctic fishes (Pagothenia bernacchii and Chionodraco hamatus) were investigated and compared with that of one temperate marine fish (Mugil cephalus). In particular, muscle and erythrocyte concentrations of coenzyme Q, vitamin E, selenium, copper and zinc were determined. Antarctic fishes showed, at the level of their muscles, higher levels of selenium, zinc and copper compared with M. cepalus. In antarctic fishes, in the place of Q10, the coenzyme Q form generally observed in temperature fishes, only the homolog coenzyme Q9 was found. This particular finding has been related to the difference in the crystallization temperature existing between CoQ10 and CoQ9. Moreover, the high levels of zinc and selenium, observed in C. hamatus, correlate very well with a recent report on the presence, in the liver of this fish, of a zinc-binding protein that, in addition, is characterized by an amino acid sequence closely similar to that of a selenium-containing protein isolated from ovine heart. All together, the results indicate, for the antarctic species, a high level of antioxidant defenses with respect to the mediterranean fish and may be considered as a stimulating basis for further studies on the oxidative metabolism of Antarctic organisms.

Two genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene (C677T and A1298C) can influence the plasma homocysteine (Hcy) levels, especially in the presence of an inadequate folate status. The aim of this study was to evaluate the frequencies of C677T and of A1298C MTHFR polymorphisms and their correlation with Hcy and serum folate concentrations in a population of blood donors living in a region of middle-southern Italy (the Molise Region). One hundred ninety seven blood donors were studied for total plasma Hcy, serum folate and C677T and A1298C MTHFR genotypes. The frequency of C677T genotypes was 20.8% (CC), 49.8% (CT) and 29.4% (TT); for the A1298C genotypes: 48.7% (AA), 43.7% (AC) and 7.6% (CC). Hcy and serum folate concentrations were significantly different among genotypes of the C677T polymorphism (CC versus CT versus TT

Background/Objectives: To compare the efficacy of a diet rich in natural folate and of two different folic acid supplementation protocols in subjects with “moderate” hyperhomocysteinemia, also taking into account C677T polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. Subjects/Methods: We performed a 13 week open, randomized, double blind clinical trial on 149 free living persons with mild hyperhomocyteinemia, with daily 200 μg from a natural folate-rich diet, 200 μg [6S]5-methyltetrahydrofolate (5-MTHF), 200 μg folic acid or placebo. Participants were stratified according to their MTHFR genotype. Results: Homocysteine (Hcy) levels were reduced after folate enriched diet, 5-MTHF or folic acid supplementation respectively by 20.1% (p < 0.002), 19.4% (p < 0.001) and 21.9% (p < 0.001), as compared to baseline levels and significantly as compared to placebo (p < 0.001, p < 0.002 and p < 0.001, respectively for enriched diet, 5-MTHF and folic acid). After this enriched diet and the folic acid supplementation, Hcy in both genotype groups decreased approximately to the same level, with higher percentage decreases observed for the TT group because of their higher pre-treatment value. Similar results were not seen by genotype for 5-MTHF. A significant increase in RBC folate concentration was observed after folic acid and natural folate-rich food supplementations, as compared to placebo. Conclusions: Supplementation with natural folate-rich foods, folic acid and 5-MTHF reached a similar reduction in Hcy concentrations.

Background and aims The relevance of folate, other B-vitamins and homocysteine (Hcy) for the occurrence or prevention of several diseases has induced growing interest. Unfortunately, little evidence is available regarding B-vitamin concentrations in Italy. This study evaluated in a region of middle-southern Italy, folate, vitamin B12 and Hcy concentrations and the prevalence of their ideal blood levels. The main determinants of B-vitamins and Hcy were also considered. Methods and results Male and female blood donors ( n =240), aged 18–66 years and living in Molise region (Italy), were enrolled in the study. They completed a brief questionnaire concerning fruit and vegetables intake, physical activity and smoking; serum and red blood cell (RBC) folate and serum vitamin B12 were measured by an immunoassay on an automated analyzer. Total Hcy was measured by high performance liquid chromatography (HPLC). Geometric means of serum folate, RBC folate and serum vitamin B12 were 10.8nmoll −1 , 426.0nmoll −1 and 245.0pmoll −1 , respectively. Only 22.5%, 24.2% and 16.3% of blood donors showed an adequate level of serum folate, RBC folate or serum vitamin B12 respectively. When a cut-off of RBC folate ≥906nmoll −1 was used no women of childbearing age had adequate levels. A geometric mean of 14.0μmoll −1 was found for total Hcy, with an ideal concentration in 12.1% of subjects. Folate concentration was higher in women and non-smokers and in subjects with higher consumption of fruit and vegetable. Conclusion This study shows a low-moderate B-vitamins status in middle-southern Italy, associated with an inadequate fruit and vegetable consumption. A public health strategy should be undertaken to encourage a B-vitamin-rich diet with the addition of vitamin supplements or vitamin fortified foods in population subgroups with special needs.

Asymmetric dimethylarginine (ADMA), an endogenous competitive inhibitor of nitric oxide synthase plays an important role in endothelial dysfunction processes. Recent studies have linked high ADMA levels with several pathological conditions. The interest as a marker of endothelial dysfunction has increased in the last few years. In this paper, a method for serum ADMA quantification by liquid chromatography tandem mass spectrometry has been described. To test the utility in a pathological condition ADMA levels in hypertensive subjects have been measured.HPLC separation was performed by hydrophilic interaction chromatography using acetonitrile/water containing 0.1% formic acid and 20 mmol/L ammonium formate. Selected reaction monitoring was performed following the transitions m/z 203.1→46.4 for ADMA and 210.1→46.3 for the internal standard [The method was linear up to 10 μmol/L, limit of detection and limit of quantification were 0.005 μmol/L and 0.01 μmol/L, respectively. Recovery was higher than 96%. Intra- and inter-assay imprecision were lower than 6%. The accuracy, expressed as bias %, was The method developed is selective and sensitive, thus suitable not only for research purposes, but also for routinely work.

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke-related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles. Copyright © 2010 John Wiley & Sons, Ltd.

We evaluated the prevalence of human papillomavirus (HPV) infection and correlated the molecular test results with the cytological examination data (PAP test) in 364 women living in central-southern Italy (Molise region), by means of polymerase chain reaction HPV DNA genotyping and of cervical cytology. One hundred and twenty-eight women resulted HPV positive (35.2%), HPV16 being the most frequent genotype. HPV positive women were significantly younger than negative patients (35.9 +/- 8.4 years and 38.2 +/- 9.1, respectively; p = 0.018); women with multiple infections were also significantly younger than those with single infection (31.7 +/- 6.9 and 37.6 +/- 8.3, respectively; p = 0.0002). Moreover, high risk HPV positive patients were significantly younger than low risk HPV positive women (35.1 +/- 7.7 and 40.5 +/- 10.5, respectively; p = 0.008). In the HPV positive group, 14 patients (10.9%) did not show any significant cytological alteration. Conversely, 7 out of 236 HPV negative women (3.0%) showed high grade squamous intraepithelial lesions (HSIL). Furthermore, HPV 16 or 18 were present in more than 70% of women positive for HSIL at cytology. Our data suggest the potential effectiveness of combined cytology and molecular test for further study of clinical cases with apparently laboratory conflicting results.

BACKGROUND Hyperhomocysteinemia is a risk factor for cardiovascular, cerebrovascular diseases and is also associated with congenital birth defects, pregnancy complications and cancer. Many homocysteine assays have been developed since the 1960s and most companies have commercially available kits for homocysteine determination. In this paper, we report the performance characteristics of the new Architect homocysteine assay and the correlation with HPLC and other routine analytical methods. METHODS Recovery, limit of detection (LOD), total imprecision and interferences were evaluated; furthermore, the method was compared with HPLC and with two routine methods. RESULTS Recovery was higher than 95%, LOD was lower than 1.0 micromol/L, the range for total imprecision was 1.7%-3.8%. No significant degree of interference was observed up to 5 g/L hemoglobin, 20 mg/dL (342.5 micromol/L) bilirubin and 60 g/L lipids. For the comparison study, this new method shows a lightly positive bias with HPLC (mean+/-SD: 0.03+/-1.7 micromol/L) with R(2)=0.96, a positive bias with the immunonephelometric method (mean+/-SD: 1.7+/-1.2 micromol/L) with R(2)=0.98, and a negative bias with the enzymatic method (mean+/-SD: -1.8+/-2.2 micromol/L) with R(2)=0.94. CONCLUSIONS This new method from Abbott can be included among the methods suitable for homocysteine routine analysis.

Background. We evaluated the concentrations of glutathione and uric acid, low molecular weight antioxidants, in saliva of patients with head and neck squamous cell carcinoma (HNSCC), in order to identify differences with normal subjects and to obtain information about biochemical alterations of human saliva during carcinogenesis. Methods. We compared 50 HNSCC patients, divided in 2 subsets on the basis of tumor site, with a control group of 77 subjects, without a previous diagnosis of HNSCC, matched for age, sex, alcohol consumption, and smoking status. Results. At tests for equality of means by Welch and Brown- Forsythe, differences between groups resulted probable for sali- vary levels of glutathione (p ¼ .004 and p < .001 respectively) but not for salivary levels of uric acid (p ¼ .228 and p ¼ .122 respectively). Comparing groups by Tamhane test, the patients with oral or pharyngeal cancer had significantly higher salivary levels of glutathione than both controls and patients with laryn- geal cancer. Conclusions. Salivary glutathione levels may be an index of oxidative stress at the level of the upper airways and in particular of oral cavity and pharynx. Therefore, high salivary glutathione may be an epidemiological marker to identify subjects with an increased risk of developing HNSCC, to submit to strict follow- up and chemoprevention. Metabolic alterations of saliva could be both an epidemiological marker and a target for chemopre- vention of oral and oropharyngeal carcinogenesis. V C 2007 Wiley Periodicals, Inc. Head Neck 29: 648-654, 2007

Recent studies describe the potential use of biochemical markers in the evaluation of the severity of periodontitis; moreover, patients suffering from periodontitis frequently complain of halitosis (breath malodour), mainly depending on volatile compounds (e.g. hydrogen sulphide, methyl mercaptan, etc.) produced by anaerobic metabolism of oral bacteria and involving sulphur-containing amino acids. In this study, salivary sulphur compounds, such as cysteine, cysteinylglycine and glutathione and some markers of cellular damage (lactate dehydrogenase and aspartate amino transferase), were measured in periodontitis patients and correlated with the periodontal probing pocket's depth.Twenty-two periodontitis patients and forty control subjects were studied for the salivary activities of lactate dehydrogenase and aspartate aminotransferase and cysteine, cysteinylglycine and glutathione concentrations. The periodontitis patients were divided into two subgroups based on the severity of periodontal disease, expressed as median periodontal probing pocket depth (or5 mm). Enzyme activities were measured by using an automated clinical analyzer; cysteine, cysteinylglycine and glutathione concentrations were measured by HPLC equipped with fluorescence detector.A statistically significant increase of the salivary parameters level (cysteine, cysteinylglycine, glutathione, aspartate aminotransferase and lactate dehydrogenase) was found in the patient subgroup with periodontal probing pocket depth5 mm, the salivary cysteine concentrations showing the most significant correlation.Salivary cysteine, a direct precursor of hydrogen sulphide, could be considered reliable markers for the oral tissue damage severity in periodontitis patients.

Background and objectives Increased plasma homocysteine, a sulphur amino acid closely related to methionine metabolism, is considered an independent risk factor for cardiovascular diseases. Over the last years, the request to clinical laboratories for homocysteine measurement is constantly increased and, for this reason, several new methods have been developed, mainly with the aim of using them on completely automated instruments for routine analyses. In this paper, we evaluated a new immunonephelometric method for homocysteine determination on the Dade Behring BNII nephelometer. Methods Linearity, recovery, limit of detection (LOD) and total imprecision and were assessed; moreover, the method was compared with a HPLC reference method and with an automated immunoassay method (AxSYM Abbott). Results Recovery range was 96.4–104.2%, LOD was 0.5 μmol/L and total imprecision ranged from 5.0% to 7.6%. For the comparison study, the immunonephelometric method showed a good correlation both with HPLC ( Y = 1.02 X − 0.79, R 2 = 0.99) and with the AxSYM method ( Y = 1.003 X + 0.06, R 2 = 0.98). The Bland–Altman plot analysis shows that the immunonephelometric method has a slight positive bias with both HPLC (mean: 1.03 μmol/L, 95% confidence interval: 0.28–1.79 μmol/L) and AxSYM methods (mean: 0.45 μmol/L, 95% confidence interval: − 0.03–0.94 μmol/L). Conclusion The new nephelometric method from Dade Behring, for its analytical performance, can be easily considered a suitable method for homocysteine routine measurement; moreover, it cannot be ruled out that the widespread availability of nephelometers in clinical laboratories play a leading role in the choice of this method.

Background and objectives: The increased need in clinical chemistry laboratories for methods of homocysteine determination, in correlation with cardiovascular diseases and nutritional deficient status, has led to the development of different analytical methods; fluorescent immunoenzymatic assays and, recently, new fully automated spectrophotometric methods are commercially available. In this paper, we compared data obtained from a new enzymatic method for homocysteine assay (Carolina Liquid Chemistries), with data obtained from a HPLC reference method and an immunoenzymatic method (Abbott AxSYM immunoassay). Results: The enzymatic method shows a good correlation with both the HPLC ( Y = −1.3 + 1.02 X ; R 2 = 0.93) and the immunoenzymatic method ( Y = 0.7 + 1.02 X ; R 2 = 0.92), although a bias enhancement was present in some samples. However, the enzymatic method shows a superior analytical feasibility because it needs only common laboratory instruments (UV–visible spectrophotometer) and can be easily adapted to large automatic clinical chemistry analyzers. Moreover, it lowers the laboratory cost of the analysis in comparison to both HPLC and immunoenzymatic methods. Conclusions: The enzymatic Carolina Liquid Chemistries method for homocysteine assay shows acceptable analytical performance and undoubtedly possesses technical and cost advantages.

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

Background : Plasmodium falciparum utilises the polyamine pathway, essential in proliferation and differentiation, and imposes an oxidative stress on host cell, enhancing the loss of glutathione. Methods : Standard hematological parameters were determined in 40 black African subjects with acute P. falciparum malaria, 30 aged 5–24 months, 5 aged 4–10 years and 5 aged 19–35 years. Plasma homocysteine, cysteine, glutathione and cysteinylglycine levels were measured by HPLC method. Twenty-eight healthy black children (15 aged 6–24 months and 13 aged 3–10 years) and 20 healthy black adults (aged 20–40 years) were also included as controls. Results : Plasma homocysteine levels were higher in all subjects with P. falciparum malaria and correlated positively with the disease severity and number of parasites, but negatively with Hb levels and patient ages. Cysteine level was found higher in all patients and markedly higher in 4–10 year old patients. Cysteinylglycine level was found lower particularly in 19–35 year old patients. Glutathione level was significantly lower in all patients. Conclusions : The elevated level of homocysteine during acute P. falciparum infection suggests an imbalance in the folate cycle, which could be a consequence of the reduced availability of NADPH and Vit B 12 , caused by increased oxidative stress. This may suggest a selection for the C677T MTHFR allele, driven by P. falciparum in sub-Saharan regions. Hence Hcy level could be useful as a predictive parameter of severity, as well as of treatment efficacy.

Tobacco smoke is involved in the pathogenesis of several diseases regarding different body systems, mainly cardiovascular and respiratory in addition to its local toxic effect in the oral cavity. The noxious effects of smoke compounds justify the high incidence of periodontal diseases, caries, and neoplastic diseases of oral tissues in smokers. Some toxic components of tobacco smoke, unsaturated and saturated aldehydes, could interact with thiol rich compounds, leading to structural and functional modification of these molecules. Previous papers have demonstrated an in vitro significant decrease of some enzymatic activities, both in plasma and in saliva, following external addition of aldehydes or exposure to cigarette smoke (CS). Furthermore, the same studies underlined the protective effect exerted by the addition of glutathione (GSH) against the damaging role of smoke aldehydes. In this study some salivary enzymes (lactic dehydrogenase [LDH], aspartate aminotransferase [AST] and amylase), and total GSH were measured in 20 volunteers smokers, before and just after smoking a single cigarette. All enzymatic activities showed a significant inhibition following a single cigarette, probably due to the interaction between smoke aldehydes and–SH groups of the enzyme molecules. Moreover, the percentage of the enzymatic inhibition showed a negative correlation with the basal level of salivary GSH. Our results emphasize that not only one cigarette is sufficient to impair the salivary enzymatic activities but also strengthen the proposed protective role of GSH against the noxious biochemical effects of CS.

Slightly elevated values of homocysteine are commonly associated with thromboembolic diseases, while high values can be found in patients with congenital metabolic defects or nutritional problems. The clinical use of homocysteine as an independent marker of cardiovascular disease was limited in the past by technical problems with its measurement, the instrumentation (HPLC, radioenzymatic assays, gas chromatography-mass spectrometry, etc.) and the necessary skills required. Commercially available immunoassays now permit a simpler and more rapid measurement of homocysteine, that is more suitable for routine clinical laboratories; in this paper we analyze the results obtained by using three fully automated methods for homocysteine determination (Abbott IMx immunoassay, Abbott AxSYM immunoassay and Immulite 2000 homocysteine immunoassay) and their correlation with the widely used HPLC method. The results clearly indicate that all three automated immunochemical methods correlate well with the HPLC method (slope 0.97-1.03; intercept 0.95-1.91 with a recovery above 95% for all three methods).

OBJECTIVES: Patients with inflammatory bowel disease (IBD) have an increased risk of thrombotic complications. Moreover, a hypercoagulable state has been hypothesized as a contributing factor in the pathogenesis of IBD. Recently, a growing amount of interest has focused on mild-to-moderate hyperhomocysteinemia as a risk factor for thromboembolic disease. We aimed to evaluate the prevalence of hyperhomocysteinemia in patients with IBD and to investigate the contribution of genetic defects in the enzymes involved in homocysteine (Hcy) metabolism and vitamin status in determining increased levels of plasma total Hcy (tHcy). METHODS: The concentrations of tHcy, folate, and vitamin B-12 as well as the prevalence of methylenetetrahydrofolate reductase (MTHFR) 677C to T mutation and the 68-bp insertion at exon 8 of cystathionine beta -synthase (CBS) were measured in patients with IBD and healthy controls. RESULTS: In all, 17 out of 64 IBD patients (26.5%) and four out of 121 (3.3%) controls had hyperhomocysteinemia with a statistically significant difference (p < 0.0001). No significant difference was found between IBD patients and controls with regard to the prevalence of homozygotes for the C677T variant (TT) of MTHFR or the prevalence of heterozygotes for the CBS-gene mutation (IN). Among the IBD patients the only independent factor significantly associated with hyperhomocysteinemia was folate deficiency (p = 0.0002), regardless of the MTHFR or the CBS genotype. CONCLUSIONS: MD patients have a higher prevalence of hyperhomocysteinemia than do healthy controls. Folate deficiency is the only independent risk factor in developing hyperhomocysteinemia. (Am J Gastroenterol 2001;96: 2677-2682. (C) 2001 by Am. Coll. of Gastroenterology).

Homocysteine is a non-protein-forming sulphur amino acid that plays an important role in remethylation and trans-sulphuration processes. In recent years, a high plasma homocysteine concentration has been implied as a possible pathophysiological factor in atherosclerosis and artery and deep vein thrombosis, probably through generation of H2O2, enhanced platelet activity and increased production of macrophage-derived tissue factor. Furthermore, an increase of polymorphonuclear leukocyte (PMN) activity mediated by homocysteine-generated H2O2 has also been reported. Because some preliminary experimental results in our laboratory did not confirm this effect of homocysteine on PMNs, we investigated the effect of homocysteine on the activity of PMNs, measured by their luminol-dependent chemiluminescence. Moreover, we also studied the effect of homocysteine in a luminol–hypochlorite chemiluminescent system. Our results clearly indicate that homocysteine at µmol/L concentrations (10–100 µmol/L) slightly inhibits neutrophil chemiluminescence, while it strongly inhibits the luminescence of the luminol–hypochlorite system. Therefore, the hypothesis that homocysteine induces an increase of H2O2-mediated neutrophil activity is not supported and, probably, the common opinion that views the H2O2 generated by homocysteine as a possible mechanism for cardiovascular damage should be reconsidered. Copyright © 2000 John Wiley & Sons, Ltd.

BACKGROUND: Human RBC metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of storage on this metabolic modulation and its relationship with the alteration of membrane protein composition. STUDY DESIGN AND METHODS: RBCs stored in CPD–saline-adenine-glucose-mannitol were assayed for glucose uptake and partition between glycolysis and the pentose phosphate pathway at high and low oxygen saturation by nuclear magnetic resonance spectroscopy after 1, 14, 21, 35, and 42 days of storage. Membrane protein composition was determined by SDS-PAGE on Days 1, 14, 35, and 42. Metabolic values and 2,3 DPG concentration were also measured after rejuvenation for 1 hour at 37°C with pyruvate-inosine-phosphate-adenine solution on Day 21. RESULTS: Metabolic differences between RBCs incubated at high and low oxygen saturation decreased during storage, and, on Day 35, the two groups did not have significant differences (p = 0.111). SDS-PAGE showed that membrane protein composition was concurrently modified. The percentage of unmodified band 3 decreased during storage, principally between Days 14 and 35. In rejuvenated RBCs, oxygen-dependent modulation was not restored. CONCLUSIONS: RBCs stored in CPD–saline-adenine-glucose-mannitol do show a progressive loss of oxygen-dependent metabolic modulation, which is not restored after rejuvenation and which seems partly related to modifications in membrane proteins, mainly band 3.

The effect of O2 exposure, expressed by mean daily fractional inspired oxygen concentration (FiO2), was evaluated during the first 6 d of life in the tracheobronchial aspirate fluid of 16 mechanically ventilated preterm infants in terms of both antioxidant response and oxidative damage, by measuring total antioxidant activity, uric acid concentrations and protein carbonyl content. Each day linear regression analysis was performed and a positive correlation was found between total antioxidant activity and FiO2 during the study period, especially on day 2 of life (r = 0.91, p < 0.0001), but uric acid correlated only in the first 3 d, especially on the 2nd day (r = 0.83, p < 0.0001). No correlation was found between carbonyl content and FiO2. The highest values of total antioxidant activity (416 and 790 micromol l(-1)) were found in 2 babies ventilated with highest FiO2: 1 and 0.80, respectively. Total antioxidant activity was not detectable or was very low in the babies not requiring O2 therapy. The highest value of uric acid (270 micromol l(-1)) was found in the baby ventilated with 100% oxygen. Uric acid concentrations obtained in these babies were much higher then those reported in the bronchoalveolar lavage fluid of adults. Preterm babies seem to have an antioxidant response in the tracheobronchial aspirate fluid following an oxidative stress and uric acid may be physiologically important as an antioxidant of the respiratory tract, especially during the first days of life.

Mild hyperhomocysteinaemia is an established risk factor for deep vein thrombosis (DVT); few data concerning its potential interaction with thrombophilic genotypes are available at the present time. We investigated 121 thrombosis-free individuals and 111 patients with at least one objectively confirmed episode of DVT. A thrombophilic condition (deficiency in antithrombin, protein C and S, factor V Leiden, prothrombin G20210A) was detected in 25.2% of the patients; mutant factor V or prothrombin genotypes were present in 6.6% of the controls. Hyperhomocysteinaemia was found in 14.4% of patients and 3. 3% of the controls, with a 3.7-fold increase in risk for DVT (95% CI 1.1-12.3). Adoption of different cut-off levels for definition of hyperhomocysteinaemia did not substantially change the magnitude of the risk. Carriership of both hyperhomocysteinaemia and factor V Leiden or prothrombin G20210A was detected in 2.7% of patients for each combination and in none of the controls. An approximate estimate of 30-fold increased risk in carriers of both hyperhomocysteinaemia and factor V Leiden and 50-fold increased risk in carriers of both hyperhomocysteinaemia and prothrombin G20210A was calculated, suggesting a synergistic interaction between hyperhomocysteinaemia and such thrombophilic genotypes. Yet statistical analysis is highly unstable due to the small number of individuals with combined defects. Further investigations on large series of patients are needed.

Concentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically significant difference between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were significantly (p < 0.05) higher in smokers. Smoking of a single cigarette induced a significant reduction in glutathione concentration (p < 0.05). Salivary antioxidant power may affect individual sensitivity toward tobacco stress.