Elisa Giocaliere

Proteinuria Impairs Podocyte Regeneration by Sequestering Retinoic Acid

In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

Expanded newborn screening by mass spectrometry: New tests, future perspectives

Tandem mass spectrometry (MS/MS) has become a leading technology used in clinical chemistry and has shown to be particularly sensitive and specific when used in newborn screening (NBS) tests. The success of tandem mass spectrometry is due to important advances in hardware, software and clinical applications during the last 25 years. MS/MS permits a very rapid measurement of many metabolites in different biological specimens by using filter paper spots or directly on biological fluids. Its use in NBS give us the chance to identify possible treatable metabolic disorders even when asymptomatic and the benefits gained by this type of screening is now recognized worldwide. Today the use of MS/MS for second-tier tests and confirmatory testing is promising especially in the early detection of new disorders such as some lysosomal storage disorders, ADA and PNP SCIDs, X-adrenoleucodistrophy (X-ALD), Wilson disease, guanidinoacetate methyltransferase deficiency (GAMT), and Duchenne muscular dystrophy. The new challenge for the future will be reducing the false positive rate by using second-tier tests, avoiding false negative results by using new specific biomarkers and introducing new treatable disorders in NBS programs.

Development of an UPLC–MS/MS method for the determination of antibiotic ertapenem on dried blood spots

Ertapenem (Invanz) is a newly developed carbapenem β-lactam antimicrobial agent. The drug usage in pediatric age needs an accurate drug monitoring for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to measure ertapenem concentration during treatment. The analysis was performed by UPLC-MS/MS operating in multiple reaction monitoring (MRM) mode. The calibration curve in matrix was linear in the concentration range of 0.5-100 mg/L with correlation coefficient value higher than 0.997. Performance parameters of this method like lower limit of detection (LLOD, 0.2 mg/L), lower limit of quantification (LLOQ, 0.5 mg/L), matrix effect (20%), intra- and inter-day imprecision (CV within than 15%) and accuracy (between 94 and 155%) of drug concentrations have been evaluated. The drug stability at different temperatures was tested for one month, to evaluate the risks of sample delivery at different climatic conditions. The reported method allows now ertapenem analysis and offers many advantages for patients including the possibility of collecting samples at home. This new assay is both precise and accurate and is especially suitable for therapeutic drug monitoring and pharmacokinetic studies in neonates in whom obtaining larger blood samples is not convenient or possible.

A rapid liquid chromatography tandem mass spectrometry-based method for measuring propranolol on dried blood spots.

Propranolol, a non-selective beta blocker drug, is used in young infants and newborns for treating several heart diseases; its pharmacokinetics has been extensively evaluated in adult patients using extrapolation to treat pediatric population. The purpose of the present study was to develop and validate a method to measure propranolol levels in dried blood spots. The analysis was performed by using liquid chromatography/tandem mass spectrometry operating in multiple reaction monitoring mode. The calibration curve in matrix was linear in the concentration range of 2.5-200 μg/L with correlation coefficient r=0.9996. Intra-day and inter-day precisions and biases were less than 8.0% (n=10) and 11.5% (n=10) respectively. The recoveries ranged from 94 to 100% and the matrix effect did not result in a severe signal suppression. Propranolol on dried blood spot showed a good stability at three different temperatures for one month. This paper describes a micromethod for measuring propranolol levels on dried blood spot, which determines a great advantage in neonates or young infants during pharmacokinetic studies because of less invasive sampling and small blood volume required.

Propranolol concentrations after oral administration in term and preterm neonates

Abstract Objective: While propranolol pharmacokinetics has been extensively studied in adults, this study reports the first evaluation of propranolol pharmacokinetics in term and preterm neonates. Methods: Propranolol concentrations were measured in four term and 32 preterm newborns treated with oral propranolol at the dose of 0.5 or 0.25 mg/kg every 6 h by serial dried blood spots. Results: The levels of propranolol, although with high inter-individual variability, were proportional with the administered dose. Pharmacokinetic parameters evaluated at the steady state in newborns treated with 0.5 mg/kg/6 h showed values of maximal (71.7 ± 29.8 ng/mL), minimal (42.2 ± 20.8 ng/mL) and average concentration (60.8 ± 25.0 ng/mL), time of maximal concentration (2.6 ± 0.9 h) and area under the time-concentration curve (364.7 ± 150.2 ng/mL/h) similar to those observed in adults. In both dosing groups, elimination half-life was significantly longer (14.9 ± 4.3 and 15.9 ± 6.1 h), and apparent total body clearance (27.2 ± 13.9 and 31.3 ± 13.3 mL/kg/min) lower than those reported in adults, suggesting a slower metabolism in newborns. No differences were observed between newborns with different gestational age or different sex. Conclusions: Neonates treated with propranolol-exhibited drug concentrations proportional with the dose, with significant long half-life.

Combined HPLC-DAD–MS, HPLC–MSn and NMR spectroscopy for quality control of plant extracts: The case of a commercial blend sold as dietary supplement

The efficiency of 1D and 2D NMR spectroscopy along with HPLC-DAD-MS analyses in characterising the content of a dietary supplement is demonstrated. Experiments directly performed on a lyophilised sample of a commercial product gave details on the quality control of the product. The lack of the marker constituents of some of the declared plant species (Crataegus oxyacantha, Olea europea, Capsella bursa-pastoris and Fumaria officinalis) and the presence of banned adulterants, responsible for the strong antihypertensive effect of the supplement were established. The analyses proved the presence of indole alkaloids belonging to the group of Rauwolfia sp., such as ajmaline, reserpine and yohimbine. Quantitative HPLC analysis showed that the content of reserpine in the product was in the therapeutic range and therefore responsible for the collapses of the patients.

The inclusion of ADA-SCID in expanded newborn screening by tandem mass spectrometry.

Severe combined immunodeficiency due to adenosine-deaminase defect (ADA-SCID) is usually deadly in childhood because of severe recurrent infections. When clinical diagnosis is done, permanent damages due to infections or metabolite accumulation are often present. Gene therapy, bone marrow transplantation or enzyme replacement therapy may be effective if started early. The aim of this study was to set-up a robust method suitable for screening with a minimized preparation process and with inexpensive running costs, for diagnosing ADA-SCID by tandem mass spectrometry. ADA-SCID satisfies all the criteria for inclusion in a newborn screening program. We describe a protocol revised to incorporate adenosine and 2-deoxyadenosine testing into an expanded newborn screening program. We assessed the effectiveness of this approach testing dried blood spots from 4 genetically confirmed early-onset and 5 delayed-onset ADA-SCID patients. Reference values were established on 50,000 healthy newborns (deoxyadenosine <0.09μmol/L, adenosine <1.61μmol/L). We also developed a second tier test to distinguish true positives from false positives and improve the positive predictive value of an initial abnormal result. In the first 18 months, the pilot project has identified a newborn with a genetically confirmed defect in adenosine deaminase (ADA) gene. The results show that the method having great simplicity, low cost and low process preparations can be fully applicable to a mass screening program.

Dried blood spot assay for the quantification of phenytoin using Liquid Chromatography-Mass Spectrometry

Phenytoin (PHT) is one of the most commonly used anticonvulsant drugs for the treatment of epilepsy and bipolar disorders. The large amount of plasma required by conventional methods for drug quantification makes mass spectrometry combined with dried blood spot (DBS) sampling crucial for pediatric patients where therapeutic drug monitoring or pharmacokinetic studies may be difficult to realize. DBS represents a new convenient sampling support requiring minimally invasive blood drawing and providing long-term stability of samples and less expensive shipment and storage. The aim of this study was to develop a LC-MS/MS method for the quantification of PHT on DBS. This analytical method was validated and gave good linearity (r(2)=0.999) in the range of 0-100mg/l. LOQ and LOD were 1.0mg/l and 0.3mg/l, respectively. The drug extraction from paper was performed in a few minutes using a mixture composed of organic solvent for 80%. The recovery ranged from 85 to 90%; PHT in DBS showed to be stable at different storage temperatures for one month. A good correlation was also obtained between PHT plasma and DBS concentrations. This method is both precise and accurate and appears to be particularly suitable to monitor treatment with a simple and convenient sample collection procedure.

Vitamin D3 quantification in a cod liver oil-based supplement

A reliable, accurate and reproducible method to quantify vitamin D3 (Vit. D3) in oily dietary supplements was developed after three Vit. D3 intoxications were diagnosed as reasonably resulting from a dietary administration of a cod liver oil based supplement. Liquid chromatography coupled to mass spectrometry operating in atmospheric pressure chemical ionization conditions (LC-APCI) and by using a deuterium labelled internal standard resulted to be an effective technique to reach the analytical aim. Due to the complexity of the oily matrix, the new analytical approach required a solid phase extraction step prior to analysis. The amount of Vit. D3 declared on the label of the cod liver oil based supplement for each soft capsule is 1.5μg. Consequently, the method was developed to quantify Vit. D3 amounts in the range 1-5μg/mL. To improve reliability of obtained data, both MS and MS/MS acquisition methods were employed. The method was evaluated by measuring the characteristic parameters such as linearity, precision, accuracy and robustness and cross checked against a certified pharmaceutical preparation. The LC-APCI-MS and MS/MS methods were applied in order to assess the Vit. D3 content in the dietary supplements taken by the intoxicated patients, found about three order of magnitude higher than that declared. The Vit. D3 content of other batches of the same commercial product was found as declared.

Anything to declare? Possible risks for patients' health resulting from undeclared plants in herbal supplements

Problems due to poor quality of herbal products continue to be a major international problem. Of particular concern is the deliberate or accidental addition of other plant species or synthetic drugs leading to adverse reactions (AR). As a result, labelling of herbal products may sometimes not accurately reflect their contents, leading to adverse events or interactions attributed to specific herbs, when in fact they are actually due to misidentified plants, mislabelling and/or adulteration. Since producers may include or omit safety information on product labels at their own discretion, labels themselves may lack clinically pertinent information. In this frame, the interpretation of specific ARs to herbal products might also be difficult [1]. During the last 3 years, Florence University Pharmacovigilance system received several reports from patients referring to the unexpected effectiveness of ‘Olivis’, a dietary supplement marketed in Italy by the company Ser-Vis as an adjunct to hypertension therapy. The declared components of this liquid preparation were extracts of leaves and buds of olive (Olea europea L.), leaves, flowers, fruits of hawthorn (Crataegus oxyacantha L.), flowers of fumitory (Fumaria officinalis L.) and shepherds purse (Capsella bursa pastoris L.). Furthermore an AR report was present in the National Institute of Health phytovigilance database concerning a female patient affected by hypertension, admitted to the hospital after an episode of hypotension and bradycardia. The patient had spontaneously replaced her antihypertensive therapy with 25 Olivis drops day−1. After an episode of hypotension, the dose was reduced to 8 drops day−1, maintaining good blood pressure control. One month later, she suspended the treatment and soon after faced a hypertensive crisis. The patient re-assumed 15 Olivis drops day−1, resulting in good blood pressure control and eventually in the reported AR. The reported AR raised suspicion as the available data on the ingredients of Olivis did not indicate significant hypotensive effects for olive and hawthorn and several studies on shepherds purse have shown both lowering and elevation of blood pressure [2]. Therefore the product was analyzed at Florence University Mass Spectrometry Centre and Department of Pharmaceutical Sciences Laboratory, by means of HPLC-ESI-ITMS and NMR, to assess the possible presence of synthetic drugs (ACE inhibitors, β-adrenoceptor blockers, angiotensin II receptor antagonists, calcium channel antagonists, α-adrenoceptor antagonists), as well as undeclared natural compounds with hypotensive activity. No synthetic drugs were found in the preparation, but analyses showed the presence of indole alkaloids, principally ajmaline and reserpine. The concentration of reserpine in the sample was 1.57 µg ml−1, corresponding to a suggested dose of 3.5–12.0 µg day−1, while the concentration of ajmaline was 861.8 µg ml−1, corresponding to a suggested dose of 1.8–6.5 mg day−1. Since both compounds are absent in label-declared herbal ingredients, addition of an extract from an undeclared Rauwolfia species was suspected [3]. In this case the significant effect observed on blood pressure raised suspicions of possible adulteration of Olivis with synthetic antihypertensive drugs. However, further laboratory investigation revealed that a reserpine/ajmaline containing plant species had been used in the product. Neither of these alkaloids is permitted in food supplements according to Italian regulations. Whilst both of them are effective in lowering blood pressure, only reserpine was present at an active concentration. Ajmaline is a well-known cardioactive drug with an anti-arrhythmic profile and the potential of induction of bradycardia and hypotension, but its side effects have been reported only after rapid intravenous injection of doses significantly higher than in the present case [4]. Reserpine, while being an authorized drug, is rarely prescribed nowadays due to its poor tolerability and unfavourable safety profile. We concluded therefore that the reported side effects were mainly due to reserpine present in the undeclared plant species. Information was transmitted to the Italian Ministry of Health, and it arranged for the withdrawal of the product from the market. After some months a new product with a similar composition was marketed by the same producer. A subsequent analysis conducted by us showed no undeclared Rauwolfia species in the new product. This case highlights the potentially serious health risks posed by the lack of effective controls on the quality of herbal products [5]. Clinicians should be aware of the potential for such cases to arise and the need to take a detailed history of herbal products used by patients. Where ARs to herbal products report significant pharmacological effects which are not expected with the declared herbal ingredients, then undeclared active ingredients are possible and should be fully investigated.