A Metabolomics Assay to Diagnose Citrus Huanglongbing Disease and to Aid in Assessment of Treatments to Prevent or Cure Infection.

Citrus greening disease, or Huanglongbing (HLB), has devastated citrus crops globally in recent years. The causal bacterium, 'Candidatus Liberibacter asiaticus', presents a sampling issue for qPCR diagnostics and results in a high false negative rate. In this work, we compared six metabolomics assays to identify HLB-infected citrus trees from leaf tissue extracted from 30 control and 30 HLB-infected trees. A liquid chromatography-mass spectrometry-based assay was most accurate. A final partial least squares-discriminant analysis (PLS-DA) model was trained and validated on 690 leaf samples with corresponding qPCR measures from three citrus varieties (Rio Red grapefruit, Hamlin sweet orange, and Valencia sweet orange) from orchards in Florida and Texas. Trees were naturally infected with HLB transmitted by the insect vector Diaphorina citri. In a randomized validation set, the assay was 99.9% accurate to classify diseased from nondiseased samples. This model was applied to samples from trees receiving plant defense-inducer compounds or biological treatments to prevent or cure HLB infection. From two trials, HLB-related metabolite abundances and PLS-DA scores were tracked longitudinally and compared with those of control trees. We demonstrate how our assay can assess tree health and the efficacy of HLB treatments and conclude that no trialed treatment was efficacious.

Validation and modification of dried blood spot-based glycosylated hemoglobin assay for the longitudinal aging study in India.

OBJECTIVES: This study aims to validate a modified dried blood spot (DBS)-based glycosylated hemoglobin (HbA1c) assay protocol, after a pretest in India showed poor correlation between the original DBS-based protocol and venous results. METHODS: The original protocol was tested on different chemistry analyzers and then simplified at the University of Washington (UW). A second pretest was conducted in India to validate the modified assay protocol, using 44 quality control specimens. RESULTS: Data from UW indicated that, using the original protocol, the correlation coefficients between DBS and venous results were above 0.98 on both Bio-Rad and Olympus chemistry analyzers. The protocol worked equally well on filter paper, with or without pre-treatment, and when the recommended amount of blood spot material, or less, was used. A second pretest of the modified protocol confirmed that DBS-based levels from both Olympus and Roche chemistry analyzers were well correlated with DBS results from UW (correlation coefficients were above 0.96), as well as with venous values (correlation coefficients were above 0.94). CONCLUSIONS: The DBS-based HbA1c values are highly correlated with venous results. The pre-treatment of filter paper does not appear to be necessary. The poor results from the first pretest are probably due to factors unrelated to the protocol, such as problems with the chemistry analyzer or assay reagents.

The functional polymorphism of the hemoglobin-binding protein haptoglobin influences susceptibility to idiopathic Parkinson's disease.

Oxidative stress and iron have been widely implicated in the etiology of Parkinson's disease (PD). Hemoglobin is the richest source of iron in the body. The human Haptoglobin (Hp) protein is a plasma alpha-2 glycoprotein that removes free Hemoglobin from the circulation and tissues and is important in protection from oxidative stress, in immune system regulation, and angiogenesis. A common genetic polymorphism of Hp exists in the population, where the Hp 1-1, Hp 2-1, and Hp 2-2 forms exhibit profound functional differences. In this study, the Hp genotype corresponding to phenotypes Hp 1-1, 2-1 and 2-2 was determined in 312 idiopathic PD patients and 420 normal control subjects. A significant increase in the number of subjects carrying the Hp 2-1 genotype was present among PD patients. The distribution of Hp genotypes among PD patients (16.0% Hp 1-1, 56.4% Hp 2-1, 27.6% Hp 2-2) was significantly different from the distribution in controls (15.2% Hp 1-1, 48.1% Hp 2-1, 36.7% Hp 2-2) (chi(2) = 6.99, P = 0.030). The odds ratios for PD risk for Hp 2-1 and Hp 1-1 versus Hp 2-2 genotype were 1.51 (1.07-2.12) and 1.36 (0.86-2.15), respectively. Overall, the association of Hp-1 allele with PD resulted stronger among subjects who were never-smokers as compared to ever-smokers. Also, among ever-smokers, Hp genotypes were significantly associated with PD only among women, but not men, indicating the presence of a gene x gender x smoking interaction. To our knowledge, this is the first study that investigates the association of Hp genotypes with the risk of PD.

Quantification of urinary 8-iso-PGF2alpha using liquid chromatography-tandem mass spectrometry and association with elevated troponin levels.

OBJECTIVES: Increased lipid peroxidation (i.e. "oxidative stress") has been identified as a central mechanism in the development of atherosclerosis and inflammatory vascular damage. Measurement of 8-iso-PGF(2alpha) has demonstrated to be a reliable indicator of in vivo oxidative stress levels. The purpose of this study was to develop a rapid, sensitive, and specific LC-MS/MS method for detection of urinary 8-iso-PGF(2alpha), establish reference intervals, and correlate isoprostane levels with cardiac troponin I. DESIGN AND METHODS: Urinary 8-iso-PGF(2alpha) was detected after direct injection onto a C18 silica column and monitored in the MRM mode using m/z transitions of 353.2>193.25 (8-iso-PGF(2alpha)) and 357.2>197.25 (8-iso-PGF(2alpha)-d(4)). The LC-MS/MS method was also compared to an ELISA kit. Reference interval studies were evaluated against a separate population of patients presenting with chest pain that had positive cTnI values. RESULTS: Elution of 8-iso-PGF(2alpha) was achieved after 7 min, with a total run time of 10 min. Inter-assay CVs were 13.8-20.0% and intra-assay CVs were 10.9-17.0%. Linearity ranged from 100 pg/mL to 100 ng/mL. Deming regression of ELISA and LC-MS/MS methods for 8-iso-PGF(2alpha) levels yielded poor correlation, with a slope of 0.0265, y-intercept of 0.255 ng/mL, and R(2) value of 0.0434. Urine 8-iso-PGF(2alpha) concentrations in samples obtained from healthy individuals (n=34) ranged from 57 to 390 ng/g creatinine with a mean of 221 ng/g creatinine. 8-iso-PGF(2alpha) levels were statistically significant in troponin-positive (n=35) versus troponin-negative (n=36) patients (p<0.0049). CONCLUSIONS: This LC-MS/MS method provides a rapid, accurate, sensitive, and cost-effective alternative to other methods for detection of 8-iso-PGF(2alpha) in urine. 8-iso-PGF(2alpha) has potential to be a great prognostic risk indicator in individuals with a high probability for future coronary events.