Peter F. Scholl

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.

Mass spectrometry for genotyping: an emerging tool for molecular medicine

Recent technological innovations have made proteins and nucleic acids accessible to mass spectrometric analysis. As a result of their inherently high specificity, accuracy and throughput, there is considerable interest in developing mass spectrometric methods for genotype analysis in clinical diagnostic and research applications. This review outlines some of the most promising genotyping methods developed using electrospray and matrix-assisted laser-desorption-ionization mass spectrometry.

Molecular biomarkers for aflatoxins: from adducts to gene mutations to human liver cancer

Over the past 30 years there have been extensive efforts to investigate the association between aflatoxin exposure and human liver cancer. These studies have been hindered by the lack of adequate dosimetry data on aflatoxin intake, excretion, and metabolism in people, as well as by the general poor quality of worldwide cancer morbidity and mortality statistics. These realities have spurred the efforts to develop new technologies to assess exposure status and risk for aflatoxins, and these agents are among the few environmental carcinogens for which quantitative risk assessments have been attempted. One of the goals of these risk assessments has been the development of primary and secondary preventive intervention methods to lower the human health impact from aflatoxin exposures. The long-term goal of the research described herein is the application of biomarkers to the development of preventive interventions for use in human populations at high risk for cancer. Several of the aflatoxin-specific biomarkers have been validated in epidemiologic studies and are now available for use as intermediate biomarkers in prevention trials. The development of these aflatoxin biomarkers has been based upon the knowledge of the biochemistry and toxicology of aflatoxins gleaned from both experimental and human studies. These biomarkers have been utilized subsequently in experimental models to provide data on the modulation of the markers under different situations of disease risk. This systematic approach provides encouragement for preventive interventions and should serve as a template for the development for the development and validation of other chemical-specific biomarkers and their application to cancer or other chronic diseases.

Human Aflatoxin Albumin Adducts Quantitatively Compared by ELISA, HPLC with Fluorescence Detection, and HPLC with Isotope Dilution Mass Spectrometry

Essential to the conduct of epidemiologic studies examining aflatoxin exposure and the risk of heptocellular carcinoma, impaired growth, and acute toxicity has been the development of quantitative biomarkers of exposure to aflatoxins, particularly aflatoxin B1. In this study, identical serum sample sets were analyzed for aflatoxin-albumin adducts by ELISA, high-performance liquid chromatography (HPLC) with fluorescence detection (HPLC-f), and HPLC with isotope dilution mass spectrometry (IDMS). The human samples analyzed were from an acute aflatoxicosis outbreak in Kenya in 2004 (n = 102) and the measured values ranged from 0.018 to 67.0, nondetectable to 13.6, and 0.002 to 17.7 ng/mg albumin for the respective methods. The Deming regression slopes for the HPLC-f and ELISA concentrations as a function of the IDMS concentrations were 0.71 (r2 = 0.95) and 3.3 (r2 = 0.96), respectively. When the samples were classified as cases or controls, based on clinical diagnosis, all methods were predictive of outcome (P < 0.01). Further, to evaluate assay precision, duplicate samples were prepared at three levels by dilution of an exposed human sample and were analyzed on three separate days. Excluding one assay value by ELISA and one assay by HPLC-f, the overall relative SD were 8.7%, 10.5%, and 9.4% for IDMS, HPLC-f, and ELISA, respectively. IDMS was the most sensitive technique and HPLC-f was the least sensitive method. Overall, this study shows an excellent correlation between three independent methodologies conducted in different laboratories and supports the validation of these technologies for assessment of human exposure to this environmental toxin and carcinogen. (Cancer Epidemiol Biomarkers Prev 2008;17(7):1653–7)

Quantitative Comparison of Aflatoxin B1 Serum Albumin Adducts in Humans by Isotope Dilution Mass Spectrometry and ELISA

Metabolic activation of the hepatocarcinogenic mycotoxin aflatoxin B1 (AFB1) results in the covalent attachment of AFB1 to serum albumin. Digestion of adducted albumin releases AFB1-lysine, a biomarker of exposure status. AF-albumin adducts have been most frequently measured in precipitated serum albumin using an immunoassay (ELISA); however, a sensitive and specific isotope dilution mass spectrometric (IDMS) assay for measurement of AFB1-lysine in serum has recently been developed. The ELISA and IDMS methods were compared using 20 human sera collected in Guinea, West Africa, where AF exposure is endemic. Measurement of AFB1-lysine adduct concentrations by IDMS in serum and albumin precipitated from the same sample revealed that precipitation has no effect on the measured adduct levels. The concentration of AF-albumin adducts measured by ELISA and AFB1-lysine measured by IDMS in 2 mg of albumin were well correlated (R = 0.88, P < 0.0001); however, AF-albumin adduct concentrations measured by ELISA were on average 2.6-fold greater than those of the AFB1-lysine adduct. Although these data suggest that the ELISA is measuring other AF adducts in addition to AFB1-lysine, these biomarkers are comparable in their ability to assess AF exposure at AF-albumin concentrations ≥3 pg AFB1-lysine equivalents/mg albumin. Identification of other adducts may clarify the mechanistic basis for using AF-protein biomarkers to assess exposure status in future epidemiologic studies of liver cancer. (Cancer Epidemiol Biomarkers Prev 2006;15(4):823–6)

Long-term Stability of Human Aflatoxin B1 Albumin Adducts Assessed by Isotope Dilution Mass Spectrometry and High-Performance Liquid Chromatography–Fluorescence

The measurement of the aflatoxin B1-lysine serum albumin adduct in human blood samples is the most facile biomarker for the assessment of chronic exposure to aflatoxin B1. Many technologies have been developed for the measurement of this protein adduct including immunoassays, high-performance liquid chromatography (HPLC) with fluorescence detection, and a newly developed isotope-dilution mass spectrometry method. Irrespective of the technology used to determine this adduct level, an important question remains about the long-term stability of this damage product in stored samples. To address this issue, 19 human serum samples that had been previously analyzed for the aflatoxin B1-lysine adduct by high-performance liquid chromatography–fluorescence in 1989 were re-analyzed by isotope dilution mass spectrometry after storage at −80°C. The adduct concentrations measured by these two techniques were identical within 4% over the range 5 to 100 pg of aflatoxin B1-lysine/mg albumin. In addition, the specific chemical structure of the aflatoxin B1-lysine adduct in human samples was confirmed for the first time by collision-induced dissociation full scan mass spectrometry analysis of the protonated adduct molecular ion. These results illustrate that the aflatoxin B1-lysine serum albumin adduct can be stable in human serum stored at −80°C since 1989, and this provides confidence for the measurement of this biomarker in repository samples from epidemiologic investigations. (Cancer Epidemiol Biomarkers Prev 2008;17(6):1436–9)

Immunoaffinity-based phosphorescent sensor platform for the detection of bacterial spores

Consideration of emergency response plans to an attack with biological weapons such as anthrax spores has spawned renewed interest in the development of inexpensive, rapid, and sensitive field portable sensors for use by non- specialists. The conceptual feasibility of such a device is demonstrated via the immunoaffinity capture of spores of the anthrax simulant B. globigii on a column followed by their washing, elution and phosphorescent detection. Spores are generically detected via the rapid extraction of dipicolinic acid (DPA) followed by its chelation with terbium to yield a phosphorescent complex. Chemical, thermal and mechanical methods of DPA extraction were evaluated. Simple extraction in HNO3 released up to 5 percent of the spore weight as DPA within 60 seconds. Extraction in H2O liberated 7 percent of the spore weight as DPA. Sonication with glass beads in H2O for 45 seconds released up to 4 percent of the spore weight as DPA. It is estimated that implementation of these techniques will permit development of a device requiring 3-5 minutes per analysis with a limit of detection on the order of 500 ng spore/mL. This approach is not intended to replace more specific methods of analysis. However, it is proposed for consideration as an inexpensive, simple and rapid means of spore detection by non-specialists in emergency situations.

Nosocomial Malaria and Saline Flush

An investigation of malaria in a US patient without recent travel established Plasmodium falciparum molecular genotype identity in 2 patients who shared a hospital room. P. falciparum can be transmitted in a hospital environment from patient to patient by blood inoculum if standard precautions are breached.

Quantitative characterization of hemozoin in Plasmodium berghei and vivax

The incidence and global distribution of chloroquine resistant (CR) Plasmodium vivax infection has increased since emerging in 1989. The mechanism of resistance in CR P. vivax has not been defined. The resistance likely relates to the formation and disposition of hemozoin as chloroquines primary mechanism of action involves disruption of hemozoin formation. CR P. berghei strains, like CR P. vivax strains, are confined to reticulocyte host cells and reportedly they do not accumulate appreciable intraerythrocytic hemozoin. Reports comparing hemozoin production between P. vivax strains and CR to chloroquine sensitive (CS) P. berghei are absent. Here we compare in vivo patterns of hemozoin formation and distribution in blood, spleen and liver tissue of male Swiss mice infected with CS or CR P. berghei not treated with chloroquine and CR P. berghei also treated with chloroquine. Light microscopy, laser desorption mass spectrometry and a colorimetric hemozoin assay detect trace hemozoin in the blood of CR P. berghei infected mice but significant hemozoin accumulation in liver and spleen tissue. Field emission in lens scanning electron microscopy reveals CR P. berghei hemozoin crystals are morphologically smaller but similar to those formed by CS parasites. CR P. berghei produces approximately five-fold less total hemozoin than CS strain. Lipid analysis of CS and CR P. berghei sucrose gradient purified bloodstage hemozoin indicates a similar lipid environment around the isolated hemozoin, predominately monopalmitic glycerol and monostearic glycerol. In contrast to CR and CS P. berghei, colorimetric hemozoin analysis of P. vivax strains indicates similar amounts of hemozoin are produced despite differing chloroquine sensitivities. These results suggest CR P. berghei forms significant hemozoin which accumulates in liver and spleen tissues and that the P. vivax chloroquine resistance mechanism differs from P. berghei.

Immunoaffinity-based biosensor for polycyclic aromatic hydrocarbons

An automated biosensor for 1-hydroxypyrene-glucuronide (OHPG) has been developed using a sensor platform initially designed for aflatoxin. The platform is based on the properties of immunoaffinity for sample purification and concentration, and fluorescence for detection. Experiments have demonstrated capture, wash, elution, and quantitation with very good results. The device is handheld and battery- driven, and can be miniaturized further. The bed volume of the column was 0.1 ml with a capacity of about 400 ng of the OHPG. Analysis time was 10 - 11 minutes. The sensitivity was about 0.5 ppb. The antibodies for the system were developed previously and recognize OHPG. The biosensor relies upon microprocessor-controlled minifluidics and fluorometry.