Gianna Di Giovamberardino

Plasma Levels of Homocysteine and Cysteine Increased in Pediatric NAFLD and Strongly Correlated with Severity of Liver Damage

Non-alcoholic fatty liver disease (NAFLD) is a spectrum of metabolic abnormalities ranging from simple triglyceride accumulation in the hepatocytes to hepatic steatosis with inflammation, ballooning and fibrosis. It has been demonstrated that the pathogenesis of NAFLD involves increased oxidative stress, with consumption of the major cellular antioxidant, glutathione (GSH). Liver has a fundamental role in sulfur compound metabolism, although the data reported on plasma thiols status in NAFLD are conflicting. We recruited 63 NAFLD patients, and we analyzed all plasma thiols, such as homocysteine (Hcy), cysteine (Cys), cysteinylglycine (CysGly) and GSH, by high-performance liquid chromatography (HPLC) with fluorescence detection. Hcy, Cys and CysGly plasma levels increased in NAFLD patients (p < 0.0001); whereas GSH levels were decreased in NAFLD patients when compared to controls (p < 0.0001). On the contrary, patients with steatohepatitis exhibited lower levels of Hcy and Cys than subjects without. Furthermore, a positive correlation was found between Hcy and Cys and the presence of fibrosis in children with NAFLD. Taken together, these data demonstrated a defective hepatic sulfur metabolism in children with NAFLD, and that high levels of Hcy and Cys probably correlates with a pattern of more severe histological liver damage, due to mechanisms that require further studies.

Glutathione metabolism in cobalamin deficiency type C (cblC).

BackgroundMethylmalonic aciduria with homocystinuria, cblC defect, is the most frequent disorder of vitamin B12 metabolism. CblC patients are commonly treated with a multidrug therapy to reduce metabolite accumulation and to increase deficient substrates. However the long-term outcome is often unsatisfactory especially in patients with early onset, with frequent progression of neurological and ocular impairment. Recent studies, have shown perturbation of cellular redox status in cblC. To evaluate the potential contribution of oxidative stress into the patophysiology of cblC defect, we have analyzed the in vivo glutathione metabolism in a large series of cblC deficient individuals.MethodsLevels of different forms of glutathione were measured in lymphocytes obtained from 18 cblC patients and compared with age-matched controls. Furthermore, we also analyzed plasma cysteine and total homocysteine.ResultsWe found an imbalance of glutathione metabolism in cblC patients with a significant decrease of total and reduced glutathione, along with a significant increase of different oxidized glutathione forms.ConclusionsThese findings show a relevant in vivo disturbance of glutathione metabolism underlining the contribution of glutathione pool depletion to the redox imbalance in treated cblC patients. Our study may be helpful in addressing future research to better understanding the pathogenetic mechanism of the disease and in developing new therapeutic approaches, including the use of novel vitamin B12 derivatives.

High performance liquid chromatographic determination of plasma free and total tazobactam and piperacillin.

A high-pressure liquid chromatography (HPLC) method with ultraviolet detection was developed for the measurement of plasma free and total tazobactam and piperacillin. This method is simple and fast, requiring only 11 min for the HPLC run and a sample preparation of about 11 min for total drugs and 10 min for free drugs. The procedure for the assay involves the treatment of plasma with acetonitrile for total drugs determination, and the use of a centrifugal filter device to deproteinize plasma for free drugs determination. The HPLC column, a Hypersil-ODS, was equilibrated with an eluent mixture composed of acetonitrile-potassium phosphate (pH 2.6). CVs for repeatability of tazobactam and piperacillin measurements ranged from 4.30 to 6.60; CVs for reproducibility ranged from 5.60 to 9.40. Mean analytical recoveries ranged from 100.4 to 103%. A linear relationship was obtained between peak area and drugs concentration in the range studied (0-62.5mg/L for tazobactam and 0-500mg/L for piperacillin). The equation for regression line were y=19x-1.4 for tazobactam and y=1.7x-0.9 for piperacillin; correlation coefficients were >0.999. The lower limit of quantitation (LLQ) for standard samples was about 0.12 mg/L for tazobactam and 0.49 mg/L for piperacillin, respectively. The lower limit of detection (LLD) was 0.06 mg/L for tazobactam and 0.24 mg/L for piperacillin. This HPLC assay for tazobactam and piperacillin is sensitive and accurate, and provides a reliable determination of both free and total tazobactam and piperacillin in human plasma, thus allowing the determination of these analytes in patients receiving tazocillin therapy.

The proteome of cblC defect: in vivo elucidation of altered cellular pathways in humans

Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.

Pediatric reference intervals for muscle coenzyme Q10

Coenzyme Q10 (CoQ10) is present in humans in both the reduced (ubiquinol, CoQ10H2) and oxidized (ubiquinone, CoQ10) forms. CoQ10 is an essential cofactor in mitochondrial oxidative phosphorylation, and is necessary for ATP production. Total, reduced and oxidized CoQ10 levels in skeletal muscle of 148 children were determined by HPLC coupled with electrochemical detection, and we established three level thresholds for total CoQ10 in muscle. We defined as “severe deficiency”, CoQ10 levels falling in the range between 0.82 and 4.88 μmol/g tissue; as “intermediate deficiency”, those ranging between 5.40 and 9.80 μmol/g tissue, and as “mild deficiency”, the amount of CoQ10 included between 10.21 and 19.10 μmol/g tissue. Early identification of CoQ10 deficiency has important implications in children, not only for those with primary CoQ10 defect, but also for patients with neurodegenerative disorders, in order to encourage earlier supplementation with this agent also in mild and intermediate deficiency.

Pitfalls in the quantitative imaging of glutathione in living cells

Biothiols have crucial roles in maintaining redox homeostasis in biological systems. Glutathione (GSH) and its precursor, cysteine (Cys), are the two most abundant low-molecular weight thiols in living cells, and their intracellular abnormal levels are associated with diseases1. GSH is a ubiquitous thiol-containing tripeptide, which has a central role in cell biology. It is implicated in the cellular defense against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides2. The ratio of GSH to its oxidized form (glutathione disulfide, GSSG) is an indicator of the redox state of the cell, and the alteration of this ratio can lead to a number of human pathologies3. Consequently, the assessment of abnormal levels of thiol-containing substances in biological systems may provide valuable information for the early diagnosis of some diseases2,3. Recently, Jiang et al.4 reported the characterization of a fluorescent probe for quantitative real-time imaging of GSH in living cells. This probe is commercially available as RealThiol (RT, to be used for calibrations) and RealThiol AM Ester (RT-AM, to be used with cells) GSH Detection Probe (Kerafast, Boston, MA, USA). Authors performed an impressive number of experiments, aimed to demonstrate that RT preferentially reacts with GSH under physiological conditions. In contrast to that reported by Jiang et al.4, we show here that RT is able to react with both GSH and Cys (Fig. 1a, b). It should be noted that analytical methods for thiols assessment typically involve separation steps, and one of the most used method for the separation of biothiols is high performance liquid chromatography (HPLC) coupled either with fluorescence5 (HPLC-FD) , mass-spectrometry6 (HPLC-MS), or tandem massspectrometry detection7 (HPLC-MSMS). Although Jiang et al.4 used HPLC-MS for some of the GSH measurements, they do not show whether Cys is present or not in their HeLa cells before and after treatments with H2O2. In addition, they quantify thiols by HPLC-MS after reaction with N-methylmaleimide (NMM), using 100 μM NMM in some experiments, and 1 mM NMM in others. NMM is identical to the other chemical used for –SH groups alkylation, N-ethylmaleimide (NEM). Although NEM reacts with thiols by a very fast Michael addition reaction at neutral or slightly acidic pH, this reaction is reversible, and an excess of NEM should be used to avoid the possible migration of the maleimide group among different thiols3. This is not accomplished by Jiang et al.4, that use 100 μM or 1 mM NMM. These concentrations are insufficient to be sure that all thiols present are blocked, especially in cells where GSH levels are higher than 1 mM[3]. Over the years, great efforts have been devoted to develop reaction-based fluorescent sensors for the detection of thiols in living systems8. Nonetheless, Yin et al.8 just highlight that despite a large number of thiol-reactive reagents and probes have been commercially available for many years, filling an entire chapter of the Molecular Probes Handbook9, most of them are nonselective, forming covalent adducts with any sulfhydrylcontaining molecules. Notably, it is important to realize that such universal thiol-reactive probes are sometimes marketed or reported in the literature as GSH (or Cys) selective. This erroneous interpretation is often based on the assumption that GSH is believed to be the most abundant thiol in cells, and any probe signaling from an interaction with Cys or other thiols would be negligible. This may be true, but it has been clearly demonstrated that their concentrations varies not only depending on the cell type and treatments, but also during cell proliferation in comparison with quiescent or not dividing cells10. Indeed, in our more that ten-year experience on thiols measurements in various cells and tissues11–15, the concentration of GSH has been often well below 1 mM, and comparable to that of Cys (Fig. 1c). This is also true in H2O2-treated HeLa cells, where GSH and Cys levels become almost identical after treatment (Fig. 1d). On the basis of the literature reports and on our data, we believe that an unaware probe end-user could encounter artificially attributed high values of GSH that are arising instead from both GSH and Cys, including Cys-protein residues. Because of the possible presence of high Cys levels, the use of RT probe for GSH quantification must be applied cautiously. The reactivity of RT probe towards GSH is obviously not questioned, and the use of this probe could be useful after verifying, possibly by HPLC, that in an experimental system the amount of GSH is at least one order of magnitude greater than that of Cys, both before and after any treatment aimed to change thiols levels. DOI: 10.1038/s41467-018-04035-9 OPEN

Drastic reduction of piperacillin-tazobactam concentrations in an in-vitro model of continuous venovenous hemofiltration: proposal of an innovative modality of administration to maintain them at constant concentration.

BACKGROUND/AIMS Critically-ill patients often undergo continuous renal replacement therapy (CRRT) and need antimicrobial therapy. Piperacillin and tazobactam (Pip-Tzb) are cleared by CRRT. Our aim is to evaluate Pip-Tzb removal in an in-vitro-single-pool-model of continuous-veno-venous-hemofiltration (CVVH); we test a new method of Pip-Tzb administration during CRRT assuring constant levels of concentrations above the minimum inhibitory concentration (MIC). METHODS In an in-vitro-single-pool-model of CVVH, two solutions (Protein-Free-Solution, PFS and Fresh-Frozen- Plasma, FFP) added with Pip-Tzb were tested for Pip-Tzb removal and adsorption. Then, to keep concentrations constantly above the MIC during CVVH, we add Pip-Tzb in the reinfusion bags. RESULTS Pip-Tzb rapidly decreased than the MIC during CVVH. The adsorption was irrelevant in the test with FPS. Adding Pip-Tzb in the reinfusion bags of the CVVH system, we observed constant concentrations of Pip-Tzb over time. CONCLUSION The association of Pip-Tzb is rapidly cleared with a real risk of inadequate dosages in patients undergoing CRRT. Adding Pip-Tzb in the reinfusion bags above the MIC, we obtained stability of concentrations during CVVH.