Monica Gelzo

A powerful couple in the future of clinical biochemistry: in situ analysis of dried blood spots by ambient mass spectrometry

Since the early 1960s, dried blood spots (DBS) on filter paper have been used in clinical applications. The first key milestone in the use of DBS was the screening of phenylketonuria and other inborn errors of metabolism using microbiological and enzymatic analytical methods. 20 years after its introduction, advanced mass spectrometers and new soft ionization techniques have permitted the coupling of liquid chromatography with MS and tandem MS (MS/MS) and since the 1990s, DBS analysis by LC-MS/MS expanded screening to many inborn errors of metabolism simultaneously. Recently, DBS-LC-MS/MS analysis has been used in other fields such as pharmacology, toxicology and forensic sciences. Today, new ambient ionization techniques, coupled to MS, directly desorb/ionize molecules from solid samples. This presents new opportunities for the in situ analysis of DBS. Most likely, ambient MS methods will be used to analyze DBS, increasing the clinical applications of MS within the next 10 years.

Direct analysis of sterols from dried plasma/blood spots by an atmospheric pressure thermal desorption chemical ionization mass spectrometry (APTDCI-MS) method for a rapid screening of Smith-Lemli-Opitz syndrome

Here is proposed a rapid and sensitive method involving atmospheric pressure thermal desorption chemical ionization mass spectrometry (APTDCI-MS) for specific laboratory screening of the Smith-Lemli-Opitz syndrome (SLOS), an inherited defect of cholesterol biosynthesis. Biochemical findings in the blood of SLOS patients are low cholesterol (Chol), high 7- and 8-dehydrocholesterol (DHCs) levels and high DHCs/Chol ratios. The APTDCI proposed method is able to ionize sterols for qualitative and quantitative analysis directly from dried plasma/blood spots. Critical APTDCI parameters--desolvation gas flow and temperature--were optimized analyzing Chol, 7-DHC and cholesteryl stearate standards spotted onto a glass slide acquiring the full scan spectra in positive ion mode. Chol levels in dried plasma spots of unaffected controls (n = 23) obtained by the proposed method were compared with those of the enzymatic method (y = 0.9166x + 0.3811; r = 0.8831) while Chol and DHCs of SLOS patients (n = 9) were compared with the gas chromatography flame ionization detection (GC-FID) method (y = 0.8214x + 0.7388; r = 0.8288). The APTDCI-MS method is also able to differentiate normal from SLOS samples directly analyzing whole blood and washed red cells spotted on paper. In conclusion, the intrinsic analytical high-throughput of APTDCI-MS method for sterol analysis could be useful to screen SLO syndrome.

Sterol profiles in plasma and erythrocyte membranes in patients with Smith-Lemli-Opitz syndrome: a six-year experience

Abstract Background: This study reports our experience over the last six years in the diagnosis of Smith-Lemli-Opitz syndrome and other inborn errors of cholesterol biosynthesis. Methods: Gas chromatography/mass spectrometry was used to obtain sterol profiles in plasma and erythrocyte membranes of suspected patients. Results: Plasma sterol reference values calculated in unaffected subjects (n=276) were in agreement with those previously reported. Among patients investigated from 2005 to 2010, we report 16 patients affected by Smith-Lemli-Opitz syndrome, three of whom represent new cases and 13 of whom were follow-up patients. In this period we also identified a new case of chondrodysplasia punctata 2 X-linked. The estimated incidence obtained for Smith-Lemli-Opitz syndrome was 1:93 suspected patients (1.08%). We also studied the effect of storage on the dehydrocholesterols/cholesterol ratio in plasma and erythrocyte membranes of patients affected by Smith-Lemli-Opitz syndrome stored at –20°C for up to 22 and 20 months, respectively. A significant negative linear correlation between storage time and the dehydrocholesterols/cholesterol ratio was identified in both plasma and erythrocyte membranes. The decrease in the dehydrocholesterols/cholesterol ratio in erythrocyte membranes was at least two-fold higher than in plasma. Conclusions: The results of this study may be helpful for diagnosis and interpretation of data in patients with findings suggestive of a cholesterol biosynthesis defect.

Polar and non-polar organic binder characterization in Pompeian wall paintings: comparison to a simulated painting mimicking an "a secco" technique.

The use of Fourier transform infrared spectromicroscopy and mass spectrometry (MS) allowed us to characterize the composition of polar and non-polar binders present in sporadic wall paint fragments taken from Pompeii’s archaeological excavation. The analyses of the polar and non-polar binder components extracted from paint powder layer showed the presence of amino acids, sugars, and fatty acids but the absence of proteinaceous material. These results are consistent with a water tempera painting mixture composed of pigments, flours, gums, and oils and are in agreement with those obtained from a simulated wall paint sample made for mimicking an ancient “a secco” technique. Notably, for the first time, we report the capability to discriminate by tandem MS the presence of free amino acids in the paint layer.

Evaluation of cytotoxic effects of 7-dehydrocholesterol on melanoma cells

Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. There is no therapy for advanced-stage melanoma and its metastasis because of their high resistance to various anticancer therapies. Human skin is an important metabolic organ in which occurs photoinduced synthesis of vitamin D3 from 7-dehydrocholesterol (7-DHC). 7-DHC, the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHC-derived compounds. The intracellular levels of 7-DHC or its derivatives can have deleterious effects on cellular functionality and viability. In this study we evaluated the effects on melanoma cell lines of 7-DHC as such and for this aim we used much care to minimize 7-DHC modifications. We found that from 12 to 72 h of treatment 82-86% of 7-DHC entered the cells, and the levels of 7-DHC-derived compounds were not significant. Simultaneously, reactive oxygen species production was significantly increased already after 2h. After 24 h and up to 72 h, 7-DHC-treated melanoma cells showed a reduction in cell growth and viability. The cytotoxic effect of 7-DHC was associated with an increase in Bax levels, decrease in Bcl-2/Bax ratio, reduction of mitochondrial membrane potential, increase in apoptosis-inducing factor levels, unchanged caspase-3 activity, and absence of cleavage of PARP-1. These findings could explain the mechanism through which 7-DHC exerts its cytotoxic effects. This is the first report in which the biological effects found in melanoma cells are mainly attributable to 7-DHC as such.

Comparison of binder compositions in Pompeian wall painting styles from Insula Occidentalis

AbstractBackgroundAlthough the pigment composition of Pompeian wall paintings has been the object of several studies, a comprehensive characterization of paint binder components is still lacking. This work aimed investigated at a molecular level the binder composition differences among wall paintings belonging to different periods of Pompeiis history. Analytical investigations were performed on representative samples of the first, second, third, and fourth painting styles excavated from the house of Marcus Fabius Rufus (Insula Occidentalis). The application of sensitive experimental methodologies was complemented by historical knowledge to gain insight in painting techniques and materials used by Pompeian artists.ResultsFourier transform infrared spectroscopy and Raman spectroscopy were used to investigate the organic components and pigments present in powders obtained from samples of the four painting styles. No proteinaceous components were detected in the samples with liquid chromatography-electrospray ionization-hybrid quadrupole/time-of-flight mass spectrometry. Liquid chromatography, gas chromatography with flame-ionization detection, and gas chromatography–mass spectrometry of polar and non-polar components extracted from powders were used to evaluate and compare the free amino acids, sugars, and fatty acids profiles.ConclusionsPigments and natural products (lipids, gums and wheat flours) were the main components of all samples. This supports the hypothesis that artists likely used water tempera for Pompeian wall paintings. Graphical AbstractScheme of the multi-analytical approach followed to compare Pompeian paint binders composition.Scheme of the multi-analytical approach followed to compare Pompeian paint binders composition.

A routine method for cholesterol and 7-dehydrocholesterol analysis in dried blood spot by GC-FID to diagnose the Smith-Lemli-Opitz syndrome.

This work was aimed to implement a fast and simple method to quantify cholesterol (CHOL) and 7-dehydrocholesterol (7-DHC) in dried blood spot (DBS) to diagnose the Smith-Lemli-Opitz syndrome (SLOS), an inborn error of CHOL biosynthesis. We developed and validated a GC-FID method for separation and quantification of underivatized CHOL and 7-DHC using a DBS disc of 6mm with a run time of 9 min. Correlation coefficients (r) of calibration curves ranged from 0.998 to 0.999 for CHOL and from 0.997 to 0.998 for 7-DHC. Within-day and between-day imprecision (CV%), accuracy (%), carry-over, and extraction efficacy (%) were also evaluated for validation. CHOL and 7-DHC were analyzed in DBS and plasma samples from 8 SLOS patients and 30 unaffected subjects. In SLOS patients, 7-DHC/CHOL ratios in DBS and plasma samples ranged from 0.035 to 1.448 and from 0.012 to 0.926, respectively. Results from calibration curves, quality controls and patient samples reveal that the method is suitable to analyze DBS to screen patients affected by SLOS.

Characterization of pigments and ligands in a wall painting fragment from Liternum archaeological park (Italy)

Spectroscopic and MS techniques were used to characterize the pigments and the composition of polar and nonpolar binders of a stray wall painting fragment from Liternum (Italy) archaeological excavation. X-ray fluorescence and diffraction analysis of the decorations indicated mainly the presence of calcite, quartz, hematite, cinnabar, and cuprorivaite. Infrared spectroscopy, GC coupled to flame-ionization detector, and MS analysis of the polar and nonpolar components extracted from paint layers from three different color regions revealed the presence of free amino acids, sugars, and fatty acids. Interestingly, LC-MS shotgun analysis of the red painting region showed the presence of αS1-casein of buffalo origin. Compared to our previous results from Pompeiis wall paintings, even though the Liternum painting mixture contained also binders of animal origin, the data strongly suggest that in both cases a tempera painting technique was utilized.

Reduced absorption and enhanced synthesis of cholesterol in patients with cystic fibrosis: a preliminary study of plasma sterols.

Abstract Background: Low cholesterol is typically observed in the plasma of patients with cystic fibrosis (CF) contrasting with the subcellular accumulation of cholesterol demonstrated in CF cells and in mice models. However, the homeostasis of cholesterol has not been well investigated in patients with CF. Methods: We studied the plasma of 26 patients with CF and 33 unaffected controls campesterol and β-sitosterol as markers of intestinal absorption and lathosterol as a marker of de novo cholesterol biosynthesis by gas chromatography (GC-FID and GC-MS). Results: Plasma campesterol and β-sitosterol results were significantly (p=0.01) lower while plasma lathosterol was significantly higher (p=0.001) in patients with CF as compared to control subjects. Plasma cholesterol results were significantly lower (p=0.01) in CF patients. Conclusions: Our data suggest that the impaired intestinal absorption of exogenous sterols in patients with CF stimulates the endogenous synthesis of cholesterol, but the levels of total cholesterol in plasma remain lower. This may be due to the CFTR dysfunction that reduces cholesterol blood excretion causing the accumulation of cholesterol in liver cells and in other tissues contributing to trigger CF chronic inflammation.

Sterol and steroid catabolites from cholesterol produced by the psychrophile Pseudoalteromonas haloplanktis

Pseudoalteromonas haloplanktis, a psychrotrophilic marine bacterium of biotechnological interest, shows anti-biofilm properties and is particularly relevant for cold storage of vacuum packed seafood. We focused our interest on the activation of cholesterol metabolism in this bacterium as the presence in its genome of a putative 3-ketosteroid-Δ(1) -dehydrogenase. This study reports GC-MS and LC-MS/MS profiles of sterols/steroids and their derivatives found in cell extracts of P. haloplanktis grown in a medium with a low content of cholesterol. Here, for the first time, we suggest that P. haloplanktis produces some intermediates of cholesterol catabolism, putatively identified as 24-hydroxycholest-1,4-dien-3-one-26-oic acid, chol-1,4-dien-3-one-24-oic acid, 26-hydroxycholest-4-en-3-one, and pregn-4-en-3-one-20-carboxylic acid, a finding already reported in other microorganisms. The presence of these compounds, also considered steroid precursors, produced by P. haloplanktis in vacuum packed seafood could be of interest for healthy of consumers, as well as, for biotechnological applications in pharmaceutical industry.