Angelo Minucci

Glucose-6-phosphate Dehydrogenase Laboratory Assay: How, When, and Why?

Glucose 6‐phosphate dehydrogenase (G6PD) deficiency is the most common defect of red blood cells. Although some different laboratory techniques or methods are employed for the biochemical screening, a strict relationship between biochemists, clinicians, and molecular biologists is necessary for a definitive diagnosis. This article represents an overview on the current laboratory tests finalized to the screening or to the definitive diagnosis of G6PD‐deficiency, underlying the problems regarding the biochemical and molecular identification of heterozygote females other than those regarding the standardization of the clinical and laboratory diagnostic procedures. Finally, this review is aimed to give a flow‐chart for the complete diagnostic approach of G6PD‐deficiency.

Inhibition of salivary enzymes by cigarette smoke and the protective role of glutathione

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.

Serum levels of folate, homocysteine, and vitamin B12 in head and neck squamous cell carcinoma and in laryngeal leukoplakia.

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.

Is homocysteine a pro-oxidant?

High plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association. In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine. Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin. All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage.

Gilbert and Crigler Najjar syndromes: An update of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene mutation database

UGT1A1 enzyme defects are responsible of both Gilbert syndrome (GS) and Crigler-Najjar syndrome (CNS). GS depends on a variant TATAA element (which contains two extra TA nucleotides as compared to the wild type genotype) in the UGT1A1 gene promoter resulting in a reduced gene expression. On the contrary, CNS forms are classified in two types depending on serum total bilirubin concentrations (STBC): the more severe (CNS-I) is characterized by high levels of STBC (342-684μmol/L), due to total deficiency of the UGT1A1 enzyme, while the milder one, namely CNS-II, is characterized by partial UGT1A1 deficiency with STBC ranging from 103 to 342μmol/L. GS and CNS are caused by genetic lesions involving a complex locus encoding the UGT1A1 gene. The present report provides an update of all reported UGT1A1 gene mutations associated to GS and CNS.

Salivary glutathione and uric acid levels in patients with head and neck squamous cell carcinoma

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.

A fast chemiluminescent method for H2O2 measurement in exhaled breath condensate.

BACKGROUND Breath condensate can give useful information on volatile compounds produced at alveolar level. Actual concentration of H(2)O(2) 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 H(2)O(2) 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 micromol/l H(2)O(2). The analytical sensitivity is about 0.01 micromol/l. Most of the nonsmokers have a H(2)O(2) content lower than 0.05 micromol/l, while smokers have a content ranging from 0.1 to 0.6 micromol/l.

Homocysteine Lowering by Folate-Rich Diet or Pharmacological Supplementations in Subjects with Moderate Hyperhomocysteinemia

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.

Rapid UGT1A1 (TA)(n) genotyping by high resolution melting curve analysis for Gilbert's syndrome diagnosis

BACKGROUND The basis of Gilberts syndrome is a 70% reduction in bilirubin glucuronidation which, in the Caucasian population, is the result of a homozygous TA insertion into the promoter region of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene (UGT1A128 allele). In addition, homozygous subjects for UGT1A128 genotype may suffer from severe irinotecan toxicity or jaundice during treatment with the protease inhibitor atazanavir. For these reasons it is very important to perform a correct molecular diagnosis. In this study, we describe for the first time a new high resolution melting (HRM) analysis for a rapid UGT1A1 (TA)(n) genotyping. METHODS We screened the TA number repetitions of the TATA-box promoter region of the UGT1A1 gene in 30 patients attending the Gemelli Hospital. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and sequencing. RESULTS Since the TA insertion modifies the derivative melting curve shape and the melting temperature (T(m)), all possible genotypes for the 6 and 7 repeat alleles were successfully identified. CONCLUSIONS HRM analysis for the UGT1A1 (TA)(n) genotyping is a simple, rapid, sensitive and low cost method, very useful in diagnostics.

Secretory phospholipase A2 pathway during pediatric acute respiratory distress syndrome: A preliminary study

Objective: To verify if secretory phospholipase A2 (sPLA2) is increased in pediatric acute respiratory distress syndrome (ARDS) triggered or not by respiratory syncytial virus infection and to clarify how the enzyme may influence the disease severity and the degree of ventilatory support. Design: Prospective pilot study. Setting: Two academic pediatric intensive care units. Patients: All infants <6 months old hospitalized for severe respiratory syncytial virus bronchiolitis, who developed ARDS (respiratory syncytial virus-ARDS group); all infants <6 months old diagnosed with ARDS secondary to other causes (ARDS group); and infants <6 months old who needed ventilation for reasons other than any lung disease (control group). Interventions: None. Measurements and Main Results: We enrolled six respiratory syncytial virus -ARDS babies, five ARDS babies, and six control infants. The sPLA2 activity and tumor necrosis factor (TNF)-&agr; were significantly higher in the bronchoalveolar lavage of ARDS infants. Worst oxygenation, ventilation, and longer pediatric intensive care unit stay and ventilation time were present in ARDS babies. No differences were found in Clara cell secretory protein and in serum cytokines levels. Because there is no correlation between bronchoalveolar lavage protein content (a marker of permeability) and sPLA2, the enzyme seems mainly produced in the alveoli. TNF-&agr;, the main inductor of sPLA2 expression, significantly correlates with the enzyme level in the bronchoalveolar lavage. Significant positive correlations exist between sPLA2, TNF-&agr; and oxygen need, mean airway pressure, ventilatory index, and the Murrays lung injury score. Negative correlations were also found between sPLA2, TNF-&agr;, and Pao2/Fio2 ratio. Conclusions: The sPLA2 and TNF-&agr; are increased in ARDS and seem correlated with clinical severity, higher oxygen requirement, and more aggressive ventilation. This correlation confirms findings from adult experience and should guide further investigations on pediatric ARDS pathophysiology.