Armando Soldarini

Simultaneous determination of human plasma levels of four selective serotonin reuptake inhibitors by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (HPLC) method with fluorimetric detection, which allows the simultaneous determination of plasma concentrations of four selective serotonin reuptake inhibitors (SSRIs) is presented. Fluvoxamine, paroxetine, sertraline, and fluoxetine were extracted from plasma with ethyl acetate and then derivatized with dansyl chloride. The analytes were separated using Hypersyl ODS C18 (5 microm) 250 x 4.6 mm column (ThermoQuest, Runcorn, UK). For continuous gradient separation, the mobile phase consists of two eluents, acetonitrile and potassium phosphate buffer (10 mmol/L, pH 7.2) at total flow rate of 1.5 mL/min. Detection was carried out at lambda exc = 366 nm and lambda em = 490 nm. The authors found recoveries of 90% to 95% for fluvoxamine, 94% to 100% for paroxetine, 88% to 95% for sertraline, 93% to 100% for fluoxetine, and 97% to 100% for internal standard (nortriptyline). Imprecision of the method ranged from 2.5% to 8.9%. The assay was linear from 10 to 1500 ng/mL for sertraline, and from 5 to 1500 ng/mL for the other drugs. The authors conclude that this method is suitable for monitoring antidepressant therapy. In addition, the authors report the effects of adding paroxetine to fluvoxamine on plasma levels in a group of patients in combined drug therapy.

Immunological Basis for IgE Hyper-Production in Enfuvirtide-Treated HIV- Positive Patients

We previously reported that enfuvirtide (ENF) treatment is accompanied by a selective increase of serum IgE. We asked whether ENF had intrinsic capability to direct B-lymphocytes to switch to IgE and/or if it could drive CD4 T cells to a Th2 phenotype. ENF was added in vitro: (a) to B-lymphocytes stimulated with IgE-switch inducing stimuli; (b) to peripheral blood mononuclear cells. Total IgE production by B cells and IL4 and IFN-γ production by CD4 T lymphocytes were evaluated, respectively. ENF had no measurable effect on the IgE production by B-lymphocytes. In contrast, it sharply increased the IL4 to IFN-γ (a correlate of the Th2 phenotype) when added in vitro to T cells from healthy donors or from single ENF-treated patients. The hyper-IgE production in ENF-treated patients is associated with the in vitro induction of a type-2 phenotype in CD4 T cells.

Copper metabolism and green serum during pregnancy

Karaca and colleagues describe a 24-year-old woman who was found to have green serum identified during routine blood tests during her 26th week of pregnancy [1]. During the diagnostic evaluation, a disorder of copper (Cu) metabolism was suspected but was not confirmed with laboratory testing [1]. In fact, the authors state that “the level of serum copper detected was 0.172 μmol/L (reference interval 0.141–0.173 μmol/L, SI unit) and of ceruloplasmin was 600 mg/L (reference interval: 260–630mg/L, SI unit),within the normal reference interval” concluding that there was no apparent issue with Cu metabolism [1]. However, Karaca and colleagues do not cite any study to support their reference intervals for serum copper and ceruloplasmin [1]. In this regard, we would like to point out the possible incorrect transcription of the reference interval for serumcopper,which is reported to be 11–22 μmol/L [2], not 0.141–0.173 μmol/L, as was published in the case report [1]. Furthermore, other important related tests involving Cumetabolism (i.e., zinc, iron, selenium) and urine copper concentrations [3], were not reported and are often performed in cases of suspected copper metabolism disorders in humans [3,4]. In fact, the case study by Karaca and co-workers is more difficult to interpret than their conclusions might suggest. When total serum copper and ceruloplasmin measurements are used for the diagnosis of Cu-related disorders, (i.e., Wilsons disease, Menkes disease, and Cu poisoning), both total serum Cu and serum ceruloplasmin should be measured with assays and methods traceable to the international standards for total serum/ plasma Cu and serum ceruloplasmin [2,5–7]. The concentration of serum Cu reported in the case report might well be above physiological limits if measured with a traceable method. This also applies to the reported serum ceruloplasmin result in the case report, as elevated ceruloplasmin concentrations have been often noted to occur in parallel with the level of the total serum Cu during the second part of pregnancy [4,8,9].

Phleum pratense manganese superoxide dismutase identified by proteomic: A new candidate grass allergen

The identification of allergen-specific sensitization is a clue to Briefly, the western blot reaction of sera from patients 101 and establish the correct diagnosis and treatment of allergic diseases. Currently available forms of diagnosis still rely prevalently on allergen extracts prepared from various allergen-containing biological sources (pollen, fruits, animal hair, etc). These extracts contain different allergenic and nonallergenic components and are often awkward to standardize. Diagnosis based on single allergens obtained by recombinant cloning technology or as purified components has progressively increased in recent years. There are advantages and limits to the 2 approaches, whose discussion is beyond the purpose of the present work. However, a possible limit of an allergen-componentebased diagnosis is related to the actual availability of each component, which is relevant to identify the entire population of patients sensitized to a given allergen source. This phenomenon is difficult to quantify, because for most common allergen sources, at least 1 allergen component has been shown to perform as a highly efficient marker of sensitization for the entire allergen source.1,2 Nevertheless, theremight be uncommonpatients who have a diagnosis based on a positive reaction to an allergen extract and a coherent clinical history but a negative score when tested with the set of allergen components available to the clinical laboratory. These patients are experiments of nature demonstrating the clinical relevance of a sensitizing component missing from the diagnostic toolbox based on a component-resolved diagnosis and showing shortcuts to the discovery of new allergens. The authors previously used a proteomic approach to detect grass allergens from a natural protein extract. They identified 6 of 8 expected clinically relevant allergens in the natural grass extract, including different molecular isoforms of single allergens.3 In the present work, the authors investigated the IgE reactivity of 1 patient (number 101) with seasonal allergic rhinoconjunctivitis and a positive skin prick test reaction to grass extract who had a negative score for IgE to the 8 allergen components of Phleum pratense (Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 7, Phl p 11, and Phl p 12) included in the most recent version of a commercial allergen test (ImmunoCAP ISAC, Thermo Fisher Diagnostics, Waltham, Massachusetts). A skin test for house dust mite extract also showed a positive reaction in this patient, whereas other allergens relevant in the area where she lived had a negative score (eTable 1). In addition, the authors studied the serum from another adult patient (number 202) with allergywho had been sensitized to grass according to the skin prick test reaction and had coherent seasonal symptoms. The diagnoses and results from the total in vitro specific IgE determination from these 2 patients are listed in eTable 1. To identify the putative allergen recognized by the IgE in patient 101, the authors used a previously described approach.3