Bacterial type III effector-induced plant C8 volatiles elicit antibacterial immunity in heterospecific neighbouring plants via airborne signalling.

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.

Dual functionality of natural mixtures of bacterial volatile compounds on plant growth.

Bacteria emit volatile compounds that modulate plant growth. Previous studies reported the impacts of bacterial volatile compounds on plant growth; however, the results varied depending on bacterial nutrient availability. We investigated whether the effects of plant growth-inhibiting volatiles (PGIVs) and plant growth-promoting volatiles (PGPVs) depended on the perceived dose by evaluating the growth of Arabidopsis thaliana seedlings placed at 7, 14, and 21 cm away from Bacillus amyloliquefaciens GB03 colonies growing in rich medium. A large bacterial colony (500 µl inoculum) inhibited plant growth at 7 cm and promoted growth at 21 cm, whereas a small bacterial colony (100 µl inoculum) induced the opposite pattern of response. We identified pyrazine and 2,5-dimethylpyrazine as candidate PGIVs that significantly reduced plant growth at a distance of 7 cm. PGIV effects were validated by exposing plants to synthetic 2,5-dimethylpyrazine and bacteria emitting PGPVs, which showed that PGIVs overwhelm PGPVs to rapidly increase salicylic acid content and related gene expression. This is referred to as the defence-growth trade-off. Our results indicate that high PGIV concentrations suppress plant growth and promote immunity, whereas low PGPV concentrations promote growth. This study provides novel insights into the complex effects of bacterial volatile mixtures and fine-tuning of bacteria-plant interactions.

Phospholipase pPLAIIIα Increases Germination Rate and Resistance to Turnip Crinkle Virus when Overexpressed.

Patatin-related phospholipase As (pPLAs) are major hydrolases acting on acyl-lipids and play important roles in various plant developmental processes. pPLAIII group members, which lack a canonical catalytic Ser motif, have been less studied than other pPLAs. We report here the characterization of pPLAIIIα in Arabidopsis (Arabidopsis thaliana) based on the biochemical and physiological characterization of pPLAIIIα knockouts, complementants, and overexpressors, as well as heterologous expression of the protein. In vitro activity assays on the purified recombinant protein showed that despite lack of canonical phospholipase motifs, pPLAIIIα had a phospholipase A activity on a wide variety of phospholipids. Overexpression of pPLAIIIα in Arabidopsis resulted in a decrease in many lipid molecular species, but the composition in major lipid classes was not affected. Fluorescence tagging indicated that pPLAIIIα localizes to the plasma membrane. Although Arabidopsis pplaIIIα knockout mutants showed some phenotypes comparable to other pPLAIIIs, such as reduced trichome length and increased hypocotyl length, control of seed size and germination were identified as distinctive pPLAIIIα-mediated functions. Expression of some PLD genes was strongly reduced in the pplaIIIα mutants. Overexpression of pPLAIIIα caused increased resistance to turnip crinkle virus, which associated with a 2-fold higher salicylic acid/jasmonic acid ratio and an increased expression of the defense gene pathogenesis-related protein1. These results therefore show that pPLAIIIα has functions that overlap with those of other pPLAIIIs but also distinctive functions, such as the control of seed germination. This study also provides new insights into the pathways downstream of pPLAIIIα.

Integrated metabolomics and lipidomics reveals high accumulation of polyunsaturated lysoglycerophospholipids in human lung fibroblasts exposed to fine particulate matter.

Exposure to fine particulate matter (PM) comprising toxic compounds arising from air pollution is a major human health concern. It is linked to increased mortality and incidence of various lung diseases. However, the mechanisms underlying the toxic effects of PM on lung fibroblasts have not been fully explored. We used targeted quantitative metabolomics and lipidomics analysis along with cytotoxicity studies to comprehensively characterize the alterations in the metabolite profiles of human lung fibroblasts (HEL 299) upon exposure to PM2.5 and PM10. This exposure at 50 µg/mL for 72 h induced an abnormally high apoptotic response via triggering intracellular reactive oxygen species (ROS) production and mitochondrial dysfunction through an imbalance between pro- and anti-apoptotic signaling pathways. The cytotoxic effects of PM2.5 were more severe than those of PM10. Metabolomics and lipidomics analyses revealed that PM exposure triggered substantial changes in the cellular metabolite profile, which involved reduced mitochondria-related metabolites such as tricarboxylic acid (TCA) cycle intermediates, amino acids, and free fatty acids as well as increased lysoglycerophospholipids (LPLs) containing polyunsaturated fatty acids. The decrease in mitochondria-related metabolites suggested that PM exposure led to reduced TCA cycle capacity and energy production. Apoptotic and inflammatory responses as well as mitochondrial dysfunction were likely to be accelerated because of excessive accumulation of LPLs, contributing to the disruption of membrane rafts and Ca2+ homeostasis and causing increased mitochondrial ROS formation. These results provide valuable insights regarding the toxic effects of PM exposure. Our study also provides a new direction for research on PM exposure-related health disorders using different cell lines.

Shoot phytochrome B modulates reactive oxygen species homeostasis in roots via abscisic acid signaling in Arabidopsis.

Underground roots normally reside in darkness. However, they are often exposed to ambient light that penetrates through cracks in the soil layers which can occur due to wind, heavy rain or temperature extremes. In response to light exposure, roots produce reactive oxygen species (ROS) which promote root growth. It is known that ROS-induced growth promotion facilitates rapid escape of the roots from non-natural light. Meanwhile, long-term exposure of the roots to light elicits a ROS burst, which causes oxidative damage to cellular components, necessitating that cellular levels of ROS should be tightly regulated in the roots. Here we demonstrate that the red/far-red light photoreceptor phytochrome B (phyB) stimulates the biosynthesis of abscisic acid (ABA) in the shoots, and notably the shoot-derived ABA signals induce a peroxidase-mediated ROS detoxification reaction in the roots. Accordingly, while ROS accumulate in the roots of the phyb mutant that exhibits reduced primary root growth in the light, such an accumulation of ROS did not occur in the dark-grown phyb roots that exhibited normal growth. These observations indicate that mobile shoot-to-root ABA signaling links shoot phyB-mediated light perception with root ROS homeostasis to help roots adapt to unfavorable light exposure. We propose that ABA-mediated shoot-to-root phyB signaling contributes to the synchronization of shoot and root growth for optimal propagation and performance in plants.

Spatial Regulation of ABCG25, an ABA Exporter, Is an Important Component of the Mechanism Controlling Cellular ABA Levels.

The phytohormone abscisic acid (ABA) plays crucial roles in various physiological processes, including responses to abiotic stresses, in plants. Recently, multiple ABA transporters were identified. The loss-of-function and gain-of-function mutants of these transporters show altered ABA sensitivity and stomata regulation, highlighting the importance of ABA transporters in ABA-mediated processes. However, how the activity of these transporters is regulated remains elusive. Here, we show that spatial regulation of ATP BINDING CASETTE G25 (ABCG25), an ABA exporter, is an important mechanism controlling its activity. ABCG25, as a soluble green fluorescent protein (sGFP) fusion, was subject to posttranslational regulation via clathrin-dependent and adaptor protein complex-2-dependent endocytosis followed by trafficking to the vacuole. The levels of sGFP:ABCG25 at the plasma membrane (PM) were regulated by abiotic stresses and exogenously applied ABA; PM-localized sGFP:ABCG25 decreased under abiotic stress conditions via activation of endocytosis in an ABA-independent manner, but increased upon application of exogenous ABA via activation of recycling from early endosomes in an ABA-dependent manner. Based on these findings, we propose that the spatial regulation of ABCG25 is an important component of the mechanism by which plants fine-tune cellular ABA levels according to cellular and environmental conditions.

Systemic Immunity Requires SnRK2.8-Mediated Nuclear Import of NPR1 in Arabidopsis.

In plants, necrotic lesions occur at the site of pathogen infection through the hypersensitive response, which is followed by induction of systemic acquired resistance (SAR) in distal tissues. Salicylic acid (SA) induces SAR by activating NONEXPRESSER OF PATHOGENESIS-RELATED GENES1 (NPR1) through an oligomer-to-monomer reaction. However, SA biosynthesis is elevated only slightly in distal tissues during SAR, implying that SA-mediated induction of SAR requires additional factors. Here, we demonstrated that SA-independent systemic signals induce a gene encoding SNF1-RELATED PROTEIN KINASE 2.8 (SnRK2.8), which phosphorylates NPR1 during SAR. The SnRK2.8-mediated phosphorylation of NPR1 is necessary for its nuclear import. Notably, although SnRK2.8 transcription and SnRK2.8 activation are independent of SA signaling, the SnRK2.8-mediated induction of SAR requires SA. Together with the SA-mediated monomerization of NPR1, these observations indicate that SA signals and SnRK2.8-mediated phosphorylation coordinately function to activate NPR1 via a dual-step process in developing systemic immunity in Arabidopsis thaliana.

Functional characterization of the ribosome biogenesis factors PES, BOP1, and WDR12 (PeBoW), and mechanisms of defective cell growth and proliferation caused by PeBoW deficiency in Arabidopsis.

The nucleolar protein pescadillo (PES) controls biogenesis of the 60S ribosomal subunit through functional interactions with Block of Proliferation 1 (BOP1) and WD Repeat Domain 12 (WDR12) in plants. In this study, we determined protein characteristics and in planta functions of BOP1 and WDR12, and characterized defects in plant cell growth and proliferation caused by a deficiency of PeBoW (PES-BOP1-WDR12) proteins. Dexamethasone-inducible RNAi of BOP1 and WDR12 caused developmental arrest and premature senescence in Arabidopsis, similar to the phenotype of PES RNAi. Both the N-terminal domain and WD40 repeats of BOP1 and WDR12 were critical for specific associations with 60S/80S ribosomes. In response to nucleolar stress or DNA damage, PeBoW proteins moved from the nucleolus to the nucleoplasm. Kinematic analyses of leaf growth revealed that depletion of PeBoW proteins led to dramatically suppressed cell proliferation, cell expansion, and epidermal pavement cell differentiation. A deficiency in PeBoW proteins resulted in reduced cyclin-dependent kinase Type A activity, causing reduced phosphorylation of histone H1 and retinoblastoma-related (RBR) protein. PeBoW silencing caused rapid transcriptional modulation of cell-cycle genes, including reduction of E2Fa and Cyclin D family genes, and induction of several KRP genes, accompanied by down-regulation of auxin-related genes and up-regulation of jasmonic acid-related genes. Taken together, these results suggest that the PeBoW proteins involved in ribosome biogenesis play a critical role in plant cell growth and survival, and their depletion leads to inhibition of cell-cycle progression, possibly modulated by phytohormone signaling.

Root-mediated signal transmission of systemic acquired resistance against above-ground and below-ground pathogens.

Background and Aims Plants modulate defence signalling networks in response to various biotic stresses via inter-organ communications. The root-mediated transmission of systemic acquired resistance (SAR) against soil-borne and air-borne plant pathogens from SAR-induced plants to neighbouring plants subjected to local chemical and pathogen treatments was evaluated. Methods The first two plants out of ten Nicotiana benthamiana seedlings were pre-treated with the SAR-triggering chemical benzothiadiazole (BTH). All ten seedlings were then challenged with two pathogenic bacteria, i.e. the root (bacterial wilt) pathogen Ralstonia solanacearum and the leaf (wildfire) pathogen Pseudomonas syringae pv. tabaci, at 7 d after SAR induction. Key Results Disease severity was noticeably lower in BTH-pre-treated plants than in the control. Surprisingly, two plants located next to BTH-treated plants exhibited reduced disease symptoms indicating that SAR signal transmission occurred through the root system. Determinant(s) secreted from the root system were search for and it was found that salicylic acid (SA) is a major molecule involved in SAR transmission through the root. Analysis of the expression of the defence-related genes N. benthamiana pathogenesis-related gene 1a (NbPR1a) and NbPR2 confirmed that BTH treatment elicited SAR via root-root transmission between plants. Plants with knock-down of the multiple resistance component SGT1 and SA biosynthesis-related gene ICS1 by Tobacco rattle virus-mediated virus-induced gene silencing exhibited a lack of root-mediated SAR transmission. The biological relevance of this finding was validated by challenge with the SAR-inducing avirulent pathogen P. syringae pv. syringae instead of BTH, which produced similar results. Conclusions Our findings demonstrated that SAR is transmissible through the root system from SAR-triggered plants to neighbouring plants.

Identification of alkaloid constituents from Fangchi species using pH control liquid-liquid extraction and liquid chromatography coupled to quadrupole time-of-flight mass spectrometry.

RATIONALE: Fangchi (F.) species from four different origins have been widely used to treat or prevent diseases, and their main constitutes are several types of alkaloids. Identification of alkaloids in F. species is a necessary step to understand the therapeutic properties of each different origin, but this has not yet been fully performed. METHODS: Several types of alkaloids were extracted from F. species using ultrasonication with 70% CH(3)OH and the extract was partitioned at pH 2 and 12 to enrich alkaloid constituents and to remove interferences. The separation of alkaloids in the Fangchi extract was performed on a C18 column using gradient elution and their tandem mass spectra were obtained by quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) to perform accurate mass measurements of fragment ions for the alkaloid constituents. RESULTS: Several types of alkaloids were successfully separated and identified by LC/ESI-MS/MS. The structural assignment of individual alkaloids was performed based on convergence of MS/MS spectral data, pH partitioning behavior, LC retention behavior, and accurate mass measurements. The pH partition of the extract provided structural information about unknown alkaloids extracted from Fangchi species. A total of 28 compounds were identified and tentatively characterized, and of these 10 alkaloids were reported for the first time in the investigated F. species. CONCLUSIONS: The chemical profiling of alkaloids in F. species with different origins was performed for the first time and provided diagnostic ions for diverse alkaloids in F. species. Marker compounds were suggested based on the 28 characterized compounds for quality evaluation and the differentiation of Fangchi species with four different origins.

Simultaneous determination of structurally diverse compounds in different Fangchi species by UHPLC-DAD and UHPLC-ESI-MS/MS.

Two bisbenzylisoquinoline alkaloids, two morphine alkaloids, one aporphine alkaloid, syringaresinol and aristolochic acid І were selected as marker compounds and simultaneously analyzed using an ultra-high pressure liquid chromatography-diode array detection (UHPLC-DAD) method. These marker compounds were used for the quality control of Fangchi species of different origins, including Sinomenium acutum, Stephania tetrandra, Cocculus trilobus and Aristolochia fangchi. A reversed-phase UHPLC-DAD method was developed and validated for the simultaneous quantification of structurally diverse markers in different Fangchi species. In addition, an UHPLC-electrospray ionization tandem mass spectrometry (ESI-MS/MS) method was used for marker identification in Fangchi species, which provided diagnostic MS/MS spectral patterns that were dependent upon the marker structures. The UHPLC-MS/MS data were used to confirm and complement the UHPLC-DAD quality evaluation results. Additionally, magnoflorine and syringaresinol were observed for the first time in S. tetrandra and C. trilobus, respectively. Twenty different Fangchi species samples were analyzed for aristolochic acid I, syringaresinol and the alkaloids using the UHPLC-DAD and MS/MS method. Based on the levels of markers and principal component analysis (PCA), this method allowed for the clear classification of the samples into four different groups representing samples originating from the four species.

Proteomic analysis of zebrafish (Danio rerio) embryos exposed to benzyl benzoate.

Benzyl benzoate (BB) is widely used in the food, cosmetics, agriculture, and pharmaceutical industries and is discharged into the aquatic environment via various water sources, including wastewater. Research on the bioaccumulation and possible toxicity of BB has been conducted, but the biochemical responses to BB toxicity are not fully understood, and the specific molecular pathways by which BB causes toxicity remain unknown. In this study, label-free quantitative proteomics based on mass spectrometry was applied to investigate protein profiles in zebrafish (Danio rerio) embryos exposed to BB (1 µg/mL) for 7 days. A total of 83 differentially expressed proteins (DEPs) were identified, including 49 up-regulated and 34 down-regulated proteins. The biological functions of proteins regulated by BB were grouped into functional categories and subcategories, including the biosynthesis of organonitrogen compound biosynthetic process, translation, amide biosynthetic process, lipid transport, stress response, and cytoskeletal activity. The results provide novel insight into the molecular basis of the ecotoxicity of BB in aquatic ecosystems.

Analysis of saikosaponins in Bupleuri Radix and Caihu-shugan-san using reversed-phase HPLC with pulsed amperometric detection.

A simple and sensitive reversed-phase (RP) HPLC coupled with pulsed amperometric detection (PAD) method was developed to determine the saikosaponin content in Bupleuri Radix or Caihu-shugan-san. Four saikosaponins in Bupleuri Radix and Caihu-shugan-san were extracted with a 6:4 solution of 10 mM sodium phosphate buffer (pH 8)/100% ethanol. Pulsed amperometric detection of carbohydrates in four major saikosaponins was highly sensitive when used with a water-acetonitrile gradient on an alkaline RP column with a post-column delivery system. The limits of detection (S/N=3) and of quantification (S/N=10) of saikosaponins were 0.01-0.02 and 0.03-0.05 µg/mL, respectively. The intra- and inter-day precision (RSDs) were each <9.7% and the average recoveries were 95.0-97.6% in Bupleuri Radix. This method can be used to analyze saikosaponins in Bupleuri Radix and Caihu-shugan-san.

Achieving similar root microbiota composition in neighbouring plants through airborne signalling.

The ability to recognize and respond to environmental signals is essential for plants. In response to environmental changes, the status of a plant is transmitted to other plants in the form of signals such as volatiles. Root-associated bacteria trigger the release of plant volatile organic compounds (VOCs). However, the impact of VOCs on the rhizosphere microbial community of neighbouring plants is not well understood. Here, we investigated the effect of VOCs on the rhizosphere microbial community of tomato plants inoculated with a plant growth-promoting rhizobacterium Bacillus amyloliquefaciens strain GB03 and that of their neighbouring plants. Interestingly, high similarity (up to 69%) was detected in the rhizosphere microbial communities of the inoculated and neighbouring plants. Leaves of the tomato plant treated with strain GB03-released ß-caryophyllene as a signature VOC, which elicited the release of a large amount of salicylic acid (SA) in the root exudates of a neighbouring tomato seedling. The exposure of tomato leaves to ß-caryophyllene resulted in the secretion of SA from the root. Our results demonstrate for the first time that the composition of the rhizosphere microbiota in surrounding plants is synchronized through aerial signals from plants.

Development of an infant formula certified reference material for the analysis of organic nutrients.

Infant formula certified reference material (CRM, KRISS CRM 108-02-003) were developed for the analysis of organic nutrients. The CRM is a milk-based infant formula powder, packaged at 14 g per unit. Ten thousand units were prepared and stored at -70 °C. For the certification of each nutrient, ten units were analyzed for simultaneous value-assignment and homogeneity test. Analytical methods used were isotope dilution mass spectrometry (IDMS) based on liquid chromatography mass spectrometer (LC/MS) or gas chromatography mass spectrometer (GC/MS) as higher-order reference methods.13 vitamins, 3 fatty acids, and total cholesterol were certified. The between-unit relative standard deviation of measurement results for each nutrient ranged 0.2% to 2.5%, showing very good homogeneity. The expanded relative uncertainties of the certified values ranged from 1% to 8%, indicating that they have higher-order metrological quality. The values of proximates (proteins, lipids, carbohydrates, water, and ash) were assigned through inter-laboratory comparisons.

Physiological and Molecular Processes Associated with Long Duration of ABA Treatment.

Plants need to respond to various environmental stresses such as abiotic stress for proper development and growth. The responses to abiotic stress can be biochemically demanding, resulting in a trade-off that negatively affects plant growth and development. Thus, plant stress responses must be fine-tuned depending on the stress severity and duration. Abscisic acid, a phytohormone, plays a key role in responses to abiotic stress. Here, we investigated time-dependent physiological and molecular responses to long-term ABA treatment in Arabidopsis as an approach to gain insight into the plant responses to long-term abiotic stress. Upon ABA treatment, the amount of cellular ABA increased to higher levels, reaching to a peak at 24 h after treatment (HAT), and then gradually decreased with time whereas ABA-GE was maintained at lower levels until 24 HAT and then abruptly increased to higher levels at 48 HAT followed by a gradual decline at later time points. Many genes involved in dehydration stress responses, ABA metabolism, chloroplast biogenesis, and chlorophyll degradation were strongly expressed at early time points with a peak at 24 or 48 HAT followed by gradual decreases in induction fold or even suppression at later time points. At the physiological level, long-term ABA treatment caused leaf yellowing, reduced chlorophyll levels, and inhibited chloroplast division in addition to the growth suppression whereas short-term ABA treatment did not affect chlorophyll levels. Our results indicate that the duration of ABA treatment is a crucial factor in determining the mode of ABA-mediated signaling and plant responses: active mobilization of cellular resources at early time points and suppressive responses at later time points.

An Alcohol Dehydrogenase Gene from Synechocystis sp. Confers Salt Tolerance in Transgenic Tobacco.

Synechocystis salt-responsive gene 1 (sysr1) was engineered for expression in higher plants, and gene construction was stably incorporated into tobacco plants. We investigated the role of Sysr1 [a member of the alcohol dehydrogenase (ADH) superfamily] by examining the salt tolerance of sysr1-overexpressing (sysr1-OX) tobacco plants using quantitative real-time polymerase chain reactions, gas chromatography-mass spectrometry, and bioassays. The sysr1-OX plants exhibited considerably increased ADH activity and tolerance to salt stress conditions. Additionally, the expression levels of several stress-responsive genes were upregulated. Moreover, airborne signals from salt-stressed sysr1-OX plants triggered salinity tolerance in neighboring wild-type (WT) plants. Therefore, Sysr1 enhanced the interconversion of aldehydes to alcohols, and this occurrence might affect the quality of green leaf volatiles (GLVs) in sysr1-OX plants. Actually, the Z-3-hexenol level was approximately twofold higher in sysr1-OX plants than in WT plants within 1-2 h of wounding. Furthermore, analyses of WT plants treated with vaporized GLVs indicated that Z-3-hexenol was a stronger inducer of stress-related gene expression and salt tolerance than E-2-hexenal. The results of the study suggested that increased C6 alcohol (Z-3-hexenol) induced the expression of resistance genes, thereby enhancing salt tolerance of transgenic plants. Our results revealed a role for ADH in salinity stress responses, and the results provided a genetic engineering strategy that could improve the salt tolerance of crops.

A Systematic Approach for the Determination of B-Group Vitamins in Multivitamin Dietary Supplements by High-Performance Liquid Chromatography with Diode-Array Detection and Mass Spectrometry.

This paper describes a practical approach using an LC diode-array detector (DAD) and MS for the quantitation of B-group vitamins (thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxine, biotin, folic acid, and cyanocobalamin) in multivitamin supplements. The purpose of this study was to optimize the extraction and chromatographic conditions without application of internal standards to determine B-group vitamins in multivitamins. Chromatographic separation on a C18 column was optimized on the basis of chromatographic behavior depending on pH variation of the mobile phase. The optimized methods showed good r values, with RSD values lower than 2.44 and 3.09% for most of the B-group vitamins (except cyanocobalamin) using LC-DAD and LC-MS, respectively. The methods were also proved to be reproducible, with RSD values of 0.96 and 2.48% being obtained with LC-DAD and LC-MS, respectively. In addition, the developed analytical methods for B-group vitamins were validated using certified reference materials and were applied to commercially available multivitamin tablets.

Determination of proline-hydroxyproline dipeptide in rat urine by high-performance anion-exchange chromatography coupled with pulsed amperometric detection.

A method for determination of proline-hydroxyproline dipeptide (PHP) was developed using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). This method resulted in good separation of proline (Pro), hydroxyproline (Hyp), and PHP within 20min using a mobile phase of 1.2mM Ba(OH)2+1.5mM Ba(OAc)2. The linear dynamic ranges and their detection limits (S/N=3) were 1-100 (r(2)=0.9990-0.9999) and 0.05-0.3µM, respectively. Mean recoveries were 91.6-121.3% and 92.2-110.3% for intra- and inter-day assays, respectively. Our HPAEC-PAD method showed clear differences in the corrected PHP levels measured in urine samples from two groups of rats, sham-operated and ovariectomized, without the need for prior acid hydrolysis or sample derivatization.

Methionine/galactose ratio on newborn blood spots useful for reduction of false positives for homocystinuria and galactosemia by high-performance anion-exchange chromatography with pulsed amperometric detection.

BACKGROUND: Methionine (Met) in blood and urine is a useful diagnostic marker for homocystinuria (HCU). However, galactosemia could be misdiagnosed as HCU when Met is used as the sole marker, since elevated excretion of Met presents in both galactosemia and HCU. Use of a more specific diagnostic marker in addition to Met is therefore necessary for reduction of false positive results for HCU as well as confirmative diagnosis of HCU. METHODS: Chromatographic separation was performed using an anion-exchange column. The levels of Met and galactose (Gal) on blood were measured and Met/Gal ratios were calculated from blood spot samples from 300 normal volunteers, eight galactosemia patients, and three HCU patients. RESULTS: The Met/Gal ratio ranged 0-4.95 for normal blood spots (n=300), 0-0.22 for galactosemia samples (n=8), and >1250 for HCU patient samples. CONCLUSIONS: Separation, extraction, and deproteinization procedures were established for Met and Gal in blood spots. And Met/Gal ratio allowed HCU to clearly distinguish from galactosemia. As a way of second tier confirmative analysis, the ratio is the best way to reduce false positives. The assay is most appropriate to reduce false positives in labs that do not screen for galactosemia.