John R. Barr

Approved IFCC reference method for the measurement of HbA1c in human blood.

Abstract HbA1c is the stable glucose adduct to the N-terminal group of the β-chain of HbA0. The measurement of HbA1c in human blood is most important for the long-term control of the glycaemic state in diabetic patients. Because there was no internationally agreed reference method the IFCC Working Group on HbA1c Standardization developed a reference method which is here described. In a first step haemoglobin is cleaved into peptides by the enzyme endoproteinase Glu-C, and in a second step the glycated and non-glycated N-terminal hexapeptides of the β-chain obtained are separated and quantified by HPLC and electrospray ionisation mass spectrometry or in a two-dimensional approach using HPLC and capillary electrophoresis with UV-detection. Both principles give identical results. HbA1c is measured as ratio between the glycated and non-glycated hexapeptides. Calibrators consisting of mixtures of highly purified HbA1c and HbA0 are used. The analytical performance of the reference method has been evaluated by an international network of reference laboratories comprising laboratories from Europe, Japan and the USA. The intercomparison studies of the network showed excellent results with intra-laboratory CVs of 0.5 to 2% and inter-laboratory CVs of 1.4 to 2.3%. Possible interferences have been carefully investigated. Due to the higher specificity of the reference method the results are lower than those generated with most of the present commercial methods which currently are calibrated with unspecific designated comparison methods. The new reference method has been approved by the member societies of the International Federation of Clinical Chemistry and Laboratory Medicine and will be the basis for the future uniform standardization of HbA1c routine assays worldwide.

Intermittent Prophylaxis with Oral Truvada Protects Macaques from Rectal SHIV Infection

Treating monkeys with single doses of an antiretroviral drug before and after exposure to SHIV provides protection against infection, a schedule that may prove practical in humans. Rearranging Retroviral Regimens for HIV Antiretroviral drugs have transformed the lives of HIV-infected people by preventing progression to full-blown AIDS. These drugs also dramatically reduce HIV transmission from mothers to infants during pregnancy and breastfeeding, and work in monkeys suggests that daily doses can also reduce transmission from unprotected sex. But prophylactic treatment with antiretroviral drugs is costly and impractical—even if confined to a high-risk population. García-Lerma et al. now show that in monkeys a more realistic medication schedule may work just as well as daily doses. To simulate how people are likely to be infected with HIV, the authors exposed macaque monkeys rectally to 14 weekly doses of simian-human immunodeficiency virus (SHIV) engineered to resemble the human virus. Control macaques treated in this way became infected within the first five exposures to SHIV. Researchers then assessed whether oral, human-equivalent doses of antiretroviral agents could prevent infection in monkeys. The best protection—equivalent to that provided by daily antivirals—occurred when the drug Truvada was given 1, 3, or 7 days before virus exposure followed by a second dose 2 hours after exposure. Less effective, but still better than no treatment at all, was a schedule in which the drug was given 2 hours before or after exposure and then again 24 hours later. Drugs given only 24 or 48 hours after exposure did not safeguard against infection. The results of this study are preliminary, largely because each of the groups had only six macaques, but they are nevertheless promising. If ongoing clinical trials in healthy people show that daily antiretroviral therapy can diminish the chances of acquiring HIV after exposure, a reasonable next step would be to evaluate more practical, less costly drug schedules in humans. For example, a weekly dose followed by a second dose after a possible exposure could prove both effective and tractable. It will also be important to evaluate treatments based solely on exposure, as these would not require ongoing prophylactic drug treatment and would minimize any drug toxicity. If one or more of these therapeutic regimens is successful, antiretroviral drugs may expand the transformation they have already engendered by preventing many more new infections as well as controlling existing ones. HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care, preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid transmission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada (TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans.

Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry

A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described.

A multi-analyte method for the quantification of contemporary pesticides in human serum and plasma using high-resolution mass spectrometry

We have developed a sensitive and accurate analytical method for quantifying 29 contemporary pesticides in human serum or plasma. These pesticides include organophosphates, carbamates, chloroacetanilides, and synthetic pyrethroids among others and include pesticides used in agricultural and residential settings. Our method employs a simple solid-phase extraction followed by a highly selective analysis using isotope dilution gas chromatography-high-resolution mass spectrometry. Our method is very accurate, has limits of detection in the low pg/g range and coefficients of variation of typically less than 20% at the low pg/g end of the method linear range. We have used this method to measure plasma pesticide concentrations in females living in an urban area. We found detectable concentrations of carbaryl/naphthalene, propoxur, bendiocarb, chlorpyrifos, diazinon, dicloran, captan and folpet or their metabolites in more than 20% of the plasma samples tested.

Xanthurenic Acid Induces Gametogenesis in Plasmodium, the Malaria Parasite

A small, heat stable chromophore extracted from mosquitoes has recently been implicated as the signal that induces mating of Plasmodium, the malaria parasite. We have used high resolution electrospray mass spectrometry to determine that this gamete activation factor (GAF) has a m/z = 205.0450, suggesting a molecular species composition of C10H7NO4. Xanthurenic acid (XA), a product of tryptophan catabolism, was determined to have an elemental composition, ultraviolet absorbance maxima, and mass spectrum consistent with those characteristics of GAF. XA activated gametogenesis of Plasmodium gallinaceum and P. falciparum in vitro at concentrations lower than 0.5 μm in saline buffered to pH 7.4. A structural analog of XA, kynurenic acid (C10H6NO3), also activated gametogenesis but only at higher concentrations and with less effect. We propose that XA is GAF. This is the first evidence that XA has induction activity.

Personalized exposure assessment : Promising approaches for human environmental health research

New technologies and methods for assessing human exposure to chemicals, dietary and lifestyle factors, infectious agents, and other stressors provide an opportunity to extend the range of human health investigations and advance our understanding of the relationship between environmental exposure and disease. An ad hoc Committee on Environmental Exposure Technology Development was convened to identify new technologies and methods for deriving personalized exposure measurements for application to environmental health studies. The committee identified a “toolbox” of methods for measuring external (environmental) and internal (biologic) exposure and assessing human behaviors that influence the likelihood of exposure to environmental agents. The methods use environmental sensors, geographic information systems, biologic sensors, toxicogenomics, and body burden (biologic) measurements. We discuss each of the methods in relation to current use in human health research; specific gaps in the development, validation, and application of the methods are highlighted. We also present a conceptual framework for moving these technologies into use and acceptance by the scientific community. The framework focuses on understanding complex human diseases using an integrated approach to exposure assessment to define particular exposure–disease relationships and the interaction of genetic and environmental factors in disease occurrence. Improved methods for exposure assessment will result in better means of monitoring and targeting intervention and prevention programs.

Ambient generation of fatty acid methyl ester ions from bacterial whole cells by direct analysis in real time (DART) mass spectrometry

Direct analysis in real time (DART) is implemented on a time-of-flight (TOF) mass spectrometer, and used for the generation of fatty acid methyl esters (FAMEs) ions from whole bacterial cells.

A Case of Naturally Acquired Inhalation Anthrax: Clinical Care and Analyses of Anti-Protective Antigen Immunoglobulin G and Lethal Factor

This report describes the first case of naturally acquired inhalation anthrax in the United States since 1976. The patients clinical course included adjunctive treatment with human anthrax immunoglobulin. Clinical correlation of serologic assays for the lethal factor component of lethal toxin and anti-protective antigen immunoglobulin G are also presented.

Measurement of bisphenol A levels in human urine

We report a new approach for assessing human exposure to bisphenol A (BPA) by measuring BPA in urine after enzymatic deglucuronidation. This method involves addition of 13C 12-labeled BPA, enzymatic deconjugation, solid-phase extraction, and derivatization with pentafluorobenzyl bromide. The product of the derivatization is separated by gas chromatography followed by mass spectrometric detection using negative chemical ionization and selected ion monitoring. Using this analysis method, urine samples fortified with both a constant level of labeled BPA and a range of unlabeled BPA levels (0.27–10.6 ng/ml) demonstrated constant percentage recovery. In addition, a range of urine sample volumes (0.25–10.0 ml) with constant amounts of added internal standard produced a linear response (r 2=0.99). The method limit of detection was 0.12 ng/ml. This method was validated by duplicate analyses using gas chromatography coupled to a high-resolution mass spectrometer.

New high-resolution mass spectrometric approach for the measurement of polychlorinated biphenyls and organochlorine pesticides in human serum

To increase our analytical throughput for measuring polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides without sacrificing data quality, we have developed and validated a combined PCB/OC pesticide gas chromatography-high-resolution mass spectrometry (GC-HRMS) analysis. In a single GC-HRMS analysis, both selected PCBs and OC pesticides are detected and quantified. Previously, this has been difficult, if not impossible, because of the major difference in masses of the most abundant electron-impact ions. However, we have identified slightly less abundant ions to monitor that allow us to successfully combine these analytes into a single analysis without sacrificing any analytical sensitivity or instrument reliability. Consequently, we have been able to double our analytical throughput by modification of mass spectrometric parameters alone. Our new methodology has been validated against our current GC-HRMS method, which entails using two separate injections, one for PCB analysis and one for OC pesticide analysis. The two methods differ by less than 4% overall, with no systematic bias. We used this method to analyze approximately 350 serum samples over a period of several months. We found that our new method was as reliable in automated, overnight runs as our current method.