Research Spotlight

Posted February 19th 2016

Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

Erland Arning Ph.D.

Erland Arning, Ph.D.

Arning, E. and T. Bottiglieri (2016). “Quantitation of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 255-262.

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We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a diagnostic test. SAM and SAH are key metabolic intermediates of methionine metabolism and the methylation cycle. Determination of SAM and SAH in plasma was performed by high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards ((2)H3-SAM and (2)H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 muL sample with 180 muL of internal standard solution consisting of heavy isotope labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 muL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP((R)) (AB Sciex). Chromatographic separation was achieved on a 250 mm x 2.0 mm EA:faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100 % methanol with retention times of 6.0 and 5.7 min, respectively. The observed m/z values of the fragment ions were m/z 399 –> 250 for SAM, m/z 385 –> 136 for SAH, m/z 402 –> 250 for (2)H3-SAM, m/z 203 –> 46. The calibration curve was linear over the ranges of 12.5-5000 nmol/L for SAM and SAH.


Posted February 19th 2016

Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

Erland Arning Ph.D.

Erland Arning, Ph.D.

Arning, E. and T. Bottiglieri (2016). “Quantitation of 5-Methyltetrahydrofolate in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 175-182.

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We describe a simple stable isotope dilution method for accurate and precise measurement of cerebrospinal fluid (CSF) 5-methyltetrahydrofolate (5-MTHF) as a clinical diagnostic test. 5-MTHF is the main biologically active form of folic acid and is involved in regulation of homocysteine and DNA synthesis. Measurement of 5-MTHF in CSF provides diagnostic information regarding diseases affecting folate metabolism within the central nervous system, in particular inborn errors of folate metabolism. Determination of 5-MTHF in CSF (50 muL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). 5-MTHF in CSF is determined by a 1:2 dilution with internal standard (5-MTHF-(13)C5) and injected directly onto the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve (25-400 nM) and has an analytical measurement range of 3-1000 nM.


Posted February 19th 2016

Quantification of gamma-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.

Erland Arning Ph.D.

Erland Arning, Ph.D.

Arning, E. and T. Bottiglieri (2016). “Quantification of gamma-Aminobutyric Acid in Cerebrospinal Fluid Using Liquid Chromatography-Electrospray Tandem Mass Spectrometry.” Methods Mol Biol 1378: 109-118.

Full text of this article.

We describe a simple stable isotope dilution method for accurate and precise measurement of gamma-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of GABA in CSF (50 muL) was performed utilizing high performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Determination of total GABA in CSF requires additional sample preparation in order to hydrolyze all the bound GABA in the sample to the free form. This requires hydrolyzing the sample by boiling in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90 % acetonitrile/0.1 % formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 nM to 1000 nM and 0.63 muM to 80 muM for free and total GABA, respectively.


Posted February 19th 2016

Penicillin skin testing is a safe method to guide beta-lactam administration in the intensive care unit.

Mercedes Arroliga 

M.D.

Mercedes Arroliga, M.D.

Arroliga, M. E., A. Vazquez-Sandoval, J. Dvoracek and A. C. Arroliga (2016). “Penicillin skin testing is a safe method to guide beta-lactam administration in the intensive care unit.” Ann Allergy Asthma Immunol 116(1): 86-87.

Full text of this article.

Geng et al. analyzed factors associated with negative histamine control reactions in patients evaluated with a penicillin skin test (PST) for allergy to β-lactams. In a case–control study of 52 patients with a negative response and 125 controls with a normal response, they used histamine dihydrochloride at a concentration of 6 mg/mL for prick or puncture skin testing but did not perform intradermal testing.1 Admission to an intensive care unit (ICU), older age, and treatment with systemic steroids and H2 blockers were associated with negative histamine responses. Geng et al1 cited our work and suggested that it was difficult to assess the precise risk of a negative histamine response during skin testing in the ICU owing to the heterogeneity of conditions, comorbidities, and medications. The response to histamine is influenced by many factors, including but not limited to age, sex, reactivity to allergens, and photoaging of the skin. Histamine response decreases with aging (>65 years), particularly in women. Patients with multiple allergen sensitizations as indicated by multiple positive skin test responses tend to have larger histamine wheals. Medications such as H2 blockers and photoaging are associated with a weaker response to histamine. These factors might be of importance in the study by Geng et al. because there were differences in age between patients with negative histamine responses and controls. Patients with negative histamine responses were older than the patients in our cohorts. There are many differences besides age between our reports and the report of Geng et al. Geng et al. did not use intradermal testing owing to concerns of subjecting patients to a higher risk of exposure to intradermal antibiotics that could lead to anaphylaxis. All our patients underwent intradermal testing as part of our PST protocol. The PST is safe provided that strict protocols are followed. Intradermal testing must be preceded by a negative prick or puncture test reaction to increase safety. Intradermal tests have higher sensitivity than prick or puncture tests when testing for penicillin allergy. Positive responses are those that have a wheal of at least 5 mm in diameter and a flare larger than the wheal with a negative response to the control saline solution and a positive response to histamine. Our studies dealt with the questions of whether patients with a history of penicillin allergy admitted to an ICU could take penicillins safely. In a cohort of 596 patients of whom 300 were admitted to the ward and 145 to the ICU, the PST was safe and helped to guide the administration of β-lactams. The PST response was negative in 88%, positive in 8%, and indeterminate (negative histamine response) in 3.4%. Most of our positive results were with PrePen intradermal injection (36 of 49) and only 0.17% of patients developed an urticarial reaction after an intradermal PST. From 39%4 to 57%9 were challenged safely with a β-lactam (the percentage of patients challenged in the ICU was 70%) with a negative predictive value for an IgE-mediated event of 99.3%. Two of 290 patients had a reaction that included flushing and urticarial and a pruritic rash within 12 hours of administration of a β-lactam. Although we did not do it in our studies, current recommendations include the administration of a single oral dose of amoxicillin to confirm adequate tolerance after a negative PST response. The PST adequately performed in patients labeled allergic to penicillins could have an important impact from the public health point of view. Patients labeled as allergic to penicillins or first-generation cephalosporins without formal evaluation have more hospital use and have increased rates of infections with Clostridium difficile, vancomycin-resistant Enterococcus species, and methicillin-resistant Staphylococcus aureus. In patients with life-threatening infections, such as methicillin-sensitive S aureus, allergy evaluation with a history-appropriate PST is preferred. In summary, the PST is safe and a very useful tool in the management of the ambulatory and hospitalized patient with a history of penicillin allergy, including the critically ill. Allergists and immunologists working together with infectious diseases specialists and critical care physicians are in a unique position to help make the correct diagnosis. Making the correct diagnosis will have financial and epidemiologic consequences for the patient and for the entire health care system.


Posted February 19th 2016

A phase II trial of trabectedin in triple-negative and HER2-overexpressing metastatic breast cancer.

Joanne L. Blum M.D.

Joanne L. Blum. M.D.

Blum, J. L., A. Goncalves, N. Efrat, M. Debled, P. Conte, P. D. Richards, D. Richards, P. Lardelli, A. Nieto, M. Cullell-Young and S. Delaloge (2016). “A phase II trial of trabectedin in triple-negative and HER2-overexpressing metastatic breast cancer.” Breast Cancer Res Treat 155(2): 295-302.

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Trabectedin is an alkylating agent that binds to the minor groove of DNA. Early studies with trabectedin suggested efficacy in triple-negative and HER2-overexpressing metastatic breast cancer (MBC). The efficacy and safety of trabectedin in pretreated patients with these tumors were evaluated in this parallel-cohort phase II trial. Patients received a 3-h infusion of trabectedin 1.3 mg/m(2) intravenously every 3 weeks until progression or unmanageable/unacceptable toxicity. The primary objective was to evaluate the efficacy using the objective response rate (ORR) as per Response Evaluation Criteria In Solid Tumors (RECIST). Secondary objectives comprised time-to-event endpoints and safety assessed with the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v.3.0. Patients with heavily pretreated triple-negative (n = 50) or HER2-overexpressing (n = 37) MBC were enrolled. No confirmed responses were found in triple-negative MBC patients, with median progression-free survival (PFS) of 2.2 months (95 % CI 1.3-2.7 months). Confirmed partial responses occurred in 4 of 34 evaluable HER2-overexpressing MBC patients (ORR = 12 %; 95 % CI 3-27 %) and lasted a median of 12.5 months (95 % CI, 6.2-14.7 months); median PFS was 3.8 months (95 % CI, 1.8-5.5 months). Most trabectedin-related adverse events were mild or moderate, and the most frequent were fatigue, nausea, vomiting, constipation, and anorexia. Severe neutropenia and transaminase increases were non-cumulative and transient and were mostly managed by infusion delays or dose reductions. Single-agent trabectedin is well tolerated in aggressive MBC and has moderate activity in HER2-overexpressing tumors. Further studies are warranted to evaluate trabectedin combined with HER2-targeted treatments in this subtype.