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 B-1. 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 (r(2) = 0.95) and 3.3 (r(2) = 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.

Isotope dilution gas chromatography/mass spectrometry method for the determination of methionine sulfoxide in protein

We have developed a new technique for quantifying methionine sulfoxide (MetSO) in protein to assess levels of oxidative stress in physiological systems. In this procedure, samples are hydrolyzed with methanesulfonic acid (MSA) in order to avoid the conversion of MetSO to methionine (Met) that occurs during hydrolysis of protein in HCl. The hydrolysate is fractionated on a cation exchange column to remove the nonvolatile MSA from amino acids, and the amino acids are then derivatized as their trimethylsilyl esters for analysis by selected ion monitoring-gas chromatography/mass spectrometry. The limit of detection of the assay is 200 pmol of MetSO per analysis, and the interassay coefficient of variation is 5.8%. Compared to current methods, the SIM-GC/MS assay avoids the potential for conversion of Met to MetSO during sample preparation, requires less sample preparation time, has lower variability, and uses mass spectrometry for sensitive and specific analyte detection.

The practical implications of using standardized estimation equations in calculating the prevalence of chronic kidney disease

Background. Kidney Disease Outcomes Quality Initiative (KDOQI) chronic kidney disease (CKD) guidelines have focused on the utility of using the modified four-variable MDRD equation (now traceable by isotope dilution mass spectrometry IDMS) in calculating estimated glomerular filtration rates (eGFRs). This study assesses the practical implications of eGFR correction equations on the range of creatinine assays currently used in the UK and further investigates the effect of these equations on the calculated prevalence of CKD in one UK region Methods. Using simulation, a range of creatinine data (30–300 µmol/l) was generated for male and female patients aged 20–100 years. The maximum differences between the IDMS and MDRD equations for all 14 UK laboratory techniques for serum creatinine measurement were explored with an average of individual eGFRs calculated according to MDRD and IDMS 30 ml/min/1.73 m2. Observed data for 93,870 patients yielded a first MDRD eGFR 3 months later of which 47 093 (71%) continued to have an eGFR

Reduced estimated glomerular filtration rate in Alzheimer's disease

OBJECTIVES:

Renal disease is increasingly regarded as an independent risk factor for vascular disease which in itself is believed to influence risk of AD. Alterations in amyloid homeostasis via reduced renal clearance of peripheral beta-amyloid (A|*beta*|) may represent another potential role for variation in renal function leading to increased risk of AD. We sought to examine estimates of glomerular filtration rate in AD and control groups.
METHODS:

AD patients were randomly recruited from the Memory Clinic of the Belfast City Hospital (n = 83). Genomic DNA was extracted from peripheral leucocytes and was genotyped for Apolipoprotein E using standard methods. Using creatinine values, age and gender, estimated Glomerular Filtration Rates (eGFR) were calculated using the isotope dilution mass spectrometry (IDMS)-traceable Modification of Diet in Renal Disease (MDRD) Study equation (using the United Kingdom National External Quality Assessment Scheme (UKNEQAS) correction factor). IDMS eGFR values were then compared between AD and control groups.

RESULTS:

Significant baseline differences in age, diastolic blood pressure, education level attained and APOE |*epsilon*|4 carriage were noted between cases and controls. The AD group had a significantly lower eGFR versus controls (69 vs 77 ml/min) which persisted after adjustment for possible confounders (p = 0.045).

CONCLUSIONS:

This case-control analysis suggests that using a relatively accurate estimate of renal function, patients with AD have greater renal impairment than cognitively normal controls. This may reflect impaired renal clearance of peripheral A|*beta*| or be a marker of shared vascular processes altering cerebral and renal functioning.

Nicotinamide Riboside Is a Major NAD+ Precursor Vitamin in Cow Milk

Background: Nicotinamide riboside (NR) is a recently discovered NAD+ precursor vitamin with a unique biosynthetic pathway. Although the presence of NR in cow milk has been known for more than a decade, the concentration of NR with respect to the other NAD+ precursors was unknown.

Objective: We aimed to determine NAD+ precursor vitamin concentration in raw samples of milk from individual cows and from commercially available cow milk.

Methods: LC tandem mass spectrometry and isotope dilution technologies were used to quantify NAD+ precursor vitamin concentration and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was used to test for NR binding to substances in milk.

Results: Cow milk typically contained ∼12 μmol NAD+ precursor vitamins/L, of which 60% was present as nicotinamide and 40% was present as NR. Nicotinic acid and other NAD+ metabolites were below the limits of detection. Milk from samples testing positive for Staphylococcus aureus contained lower concentrations of NR (Spearman ρ = −0.58, P = 0.014), and NR was degraded by S. aureus. Conventional milk contained more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an NMR spectrum consistent with association with a protein fraction in skim milk.

Conclusions: NR is a major NAD+ precursor vitamin in cow milk. Control of S. aureus may be important to preserve the NAD+ precursor vitamin concentration of milk.