Direct quantification of dimethylsulfoniopropionate (DMSP) with hydrophilic interaction liquid chromatography/mass spectrometry

A simple, derivatization free method for the direct determination of dimethylsulfoniopropionate (DMSP) using hydrophilic interaction liquid chromatography (HILIC)/mass spectrometry is introduced. DMSP is a zwitterionic osmolyte which is produced from marine plankton, macro algae and higher plants. Due to its central role in climate relevant geochemical processes as well as in plant physiology and chemical ecology there is a great interest in methods for its quantification. Since DMSP is labile and difficult to extract currently most protocols for quantification are based on indirect methods. Here we show that ultra performance liquid chromatography/mass spectrometry using a HILIC stationary phase is suitable for the direct quantification of DMSP from aqueous samples and microalgal extracts. The protocol requires minimal sample preparation and phytoplankton samples can be investigated after filtration of small volumes. The limit of detection is 20nM and the calibration curve is linear in the range of 60nM to 50μM. The use of [(2)H(6)]-DMSP as internal standard allows prolonged sample storage since it is transformed with the same kinetics as natural DMSP. This makes the method suitable for both laboratory and field studies.     

Prey-dependent retention of dimethylsulfoniopropionate (DMSP) by mixotrophic dinoflagellates

We investigated the retention of dimethylsulfoniopropionate (DMSP) in phototrophic dinoflagellates arising from mixotrophy by estimating the cellular content of DMSP in Karlodinium veneficum (mixotrophic growth) fed for 7-10 days on either DMSP-rich Amphidinium carterae (phototrophic growth only) or DMSP-poor Teleaulax sp. (phototrophic growth only). In K. veneficum fed on DMSP-poor prey, the cellular content of DMSP remained almost unchanged regardless of the rate of feeding, whereas the cellular content of DMSP in cells of K. veneficum fed on DMSP-rich prey increased by as much as 21 times the cellular concentration derived exclusively from phototrophic growth. In both cases, significant fractions (10-32% in the former case and 55-65% in the latter) of the total DMSP ingested by K. veneficum were transformed into dimethylsulfide and other biochemical compounds. The results may indicate that the DMSP content of prey species affects temporal variations in the cellular DMSP content of mixotrophic dinoflagellates, and that mixotrophic dinoflagellates produce DMS through grazing on DMSP-rich preys. Additional studies should be performed to examine the universality of our finding in other mixotrophic dinoflagellates feeding on diverse prey species.     

A novel inhibitory interaction between dimethylsulfoniopropionate (DMSP) and the denitrification pathway

Dimethylsulfoniopropionate (DMSP) is an abundant organic sulfur compound in marine algae and denitrification influences nitrogen availability to primary producers, the key regulators of coastal eutrophication. In this study, we tested the effect of DMSP on the nitrous oxide (N₂O) reduction step of denitrification in sediments and biofilms from the Douro and Ave estuaries (NW Portugal) and in pure cultures of a denitrifying bacterium, Ruegeria pomeroyi. N₂O accumulation rates were monitored in sediment slurries and bacterial cell suspensions amended with DMSP concentrations ranging from 0 to 5 mM. In these treatments N₂O accumulation rates increased linearly with DMSP concentration (R ² from 0.89 to 0.99, p < 0.001), suggesting an inhibitory effect of DMSP on the nitrous oxide reductase activity. The addition of DMSP to sediments and bacterial culture resulted in accumulation of dimethylsulfide (DMS) as well as N₂O. However, no direct inhibition on N₂O reductase activity by DMS was observed. Natural concentrations of DMSP in the different estuarine sites were found to be linearly correlated to natural N₂O effluxes (R ² = 0.64, p < 0.001), suggesting that DMSP may negatively affect N₂O reductase in situ. This newly identified interaction between DMSP and N₂O emissions may have a significant ecological role as the inhibition of the nitrous oxide reduction enhances nitrogen loss via N₂O. Since N₂O is a powerful greenhouse gas, the results from our study may be important for evaluating climate change scenarios.     

Glycerophosphocholine molecular species profiling in the biological tissue using UPLC/MS/MS

A strategy consisting of a two-phase analytical procedure was used to obtain detailed molecular species composition for glycerophosphocholines (GPCs) profiling in biological tissue using ultra performance liquid chromatography coupled with a triple quadrupole mass spectrometer operating under electrospray mode. In phase one of the analytical procedure, the precursor ion scan was first conducted to obtain the preliminary lipid profile that revealed the composition of the molecular species possessing phosphocholine structure in the biological tissue. In phase two of the analytical procedure, each product ion spectrum obtained for the GPC components in the profile was sequentially acquired for the determination of the molecular structure. A simple guide with high differentiability was proposed for the diacyl-, alkyl-acyl- and alk-1-enyl-acyl-GPC, and related lyso-GPCs molecular structure decision. Total 93 GPCs molecular species were identified in the fetal mouse lung with the relative amounts from 14.39% to less than 0.01% (normalizing by the total GPCs signal). The optimized chromatographic conditions were also proposed in the analytical procedure based on the compromise between the separation efficiency and electrospray signal response. The plate number of the probing GPCs was obviously improved to above 30,000 and the detection limits of the probing GPCs were between 0.002 and 0.016 ng/μL. The practical usability of the analytical procedure has been validated using a study of chemically induced early lung maturation. The metabolic difference between chemically treated and untreated fetal mouse lung was clearly distinguished by the composition of GPCs with several characteristics of molecular structure. The overall results showed that this two-phase analytical procedure was reliable for comprehensive GPC profiling.     

Macroscale patterns of the biological cycling of dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS) in the Northwest Atlantic

The influence of the seasonal development of microplankton communities on the cycling of dimethylsulfide (DMS) and its precursor dimethylsulfoniopropionate (DMSP) was investigated along a South–North gradient (36–59^°N) in the Northwest (NW) Atlantic Ocean. Three surveys allowed the sampling of surface mixed layer (SML) waters at stations extending from the subtropical gyre to the Greenland Current during May, July and October 2003. Pools and transformation rates of DMSP and DMS were quantified and related to prevailing physical and biochemical conditions, phytoplankton abundance and taxonomic composition, as well as bacterioplankton abundance and leucine uptake. The South–North progression of the diatom bloom, a prominent feature in the NW Atlantic, did not influence the production of DMS whereas conditions in the N Atlantic Drift lead to a persistent bloom of DMSP-rich flagellate-dominated phytoplankton community and high net DMS production rates. Macroscale patterns of the observed variables were further explored using principal component analysis (PCA). The first axis of the PCA showed a strong association between the spatio-temporal distribution of DMSP and the abundance of several phytoplankton groups including dinoflagellates and prymnesiophytes, as well as with microbial-mediated DMSP_d consumption and yields and rates of the conversion of DMSP into DMS. The second axis revealed a strong association between concentrations of DMS and SML depth and photosynthetically active radiation, a result supporting the prominent role of solar radiation as a driver of DMS dynamics.     

Effects of increased temperature on dimethylsulfoniopropionate (DMSP) concentration and methionine synthase activity in Symbiodinium microadriaticum

Dinoflagellate algae of the genus Symbiodinium occur as endosymbionts in a variety of hosts including coral. The response of Symbiodinium spp. to environmental changes could dictate survival of their hosts and the ecological success of coral reef ecosystems. Oxidative stress has been linked to a breakdown in this symbiotic relationship, known as bleaching. Increased temperature is one of the primary environmental changes linked to this phenomenon. Preliminary studies have established high concentrations of the sulfur compound dimethylsulfoniopropionate (DMSP) in Symbiodinium spp., with increased temperature. To examine the potential use of DMSP as an antioxidant, a 5?day incubation experiment was conducted at two temperatures with the algae S. microadriaticum (CCMP1633) isolated from the cnidarian host Aiptasia pulchella. An HPLC assay for the activity of the enzyme B₁₂-dependent methionine synthase was modified and used to determine the link between de novo production of methionine, a precursor to DMSP, and temperature induced oxidative stress. DMSP concentrations per cell increased approximately 38?% in the 33?°C treatment cultures over 120?h. However, these cells also increased more than 2-fold in biovolume (127?±?43?%), and SYTO-BC stain indicated increased DNA content (approximately 4-fold), suggesting arrested cell division. Normalization of DMSP to biovolume revealed that the concentrations actually decreased approximately 49?% after 2?days in cultures exposed to elevated temperature (33?°C), but were not significantly different from the control treatment at 120?h (27?°C). Concomitant changes in the 33?°C treatment relative to the control (after 120?h) resulted in an approximately 8-fold increase in reactive oxygen species, a 37?% (±7?%) decrease in photosynthetic efficiency of photosystem II, and a 5-fold increase in xanthophyll cycling. Methionine synthase activity (MSA) correlated to the decrease in DMSP concentration (R ²?=?0.778), with decreasing activity at the high temperature. Given this decrease in MSA, the increase in DMSP per cell may be due to DMSP production utilizing methionine from protein turnover, and not de novo synthesis via MSA. The findings of this study provide insight into the responses of algal symbionts to environmental changes, shed light on the potential use of DMSP and other known photo-protective mechanisms such as xanthophyll cycling under temperature induced oxidative stress, and support the suspected cessation of cell division under these conditions. This information could be crucial to understanding cellular responses to environmental changes and the ability of these organisms to survive under elevated sea surface temperatures projected for the near future.     

Nuclear magnetic resonance analysis of [1-13C]dimethylsulfoniopropionate (DMSP) and [1-13C]acrylate metabolism by a DMSP lyase-producing marine isolate of the alpha-subclass of Proteobacteria.

The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-(13)C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-(13)C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-(13)C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-(13)C]DMSP and [1-(13)C]beta-hydroxypropionate ([1-(13)C]beta-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation. When [1-(13)C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-(13)C]beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and beta-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR.     

HPLC MS/MS method for quantification of meprobamate in human plasma: application to 24/7 clinical toxicology

We described the development and full validation of rapid and accurate liquid chromatography method, coupled with tandem mass spectrometry detection, for quantification of meprobamate in human plasma with [(13)C-(2)H(3)]-meprobamate as internal standard. Plasma pretreatment involved a one-step protein precipitation with acetonitrile. Separation was performed by reversed-phase chromatography on a Luna MercuryMS C18 (20 mm×4 mm×3 μm) column using a gradient elution mode. The mobile phase was a mix of distilled water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The selected reaction monitoring transitions, in electrospray positive ionization, used for quantification were 219.2→158.2 m/z and 223.1→161.1m/z for meprobamate and internal standard, respectively. Qualification transitions were 219.2→97.0 and 223.1→101.1 m/z for meprobamate and internal standard, respectively. The method was linear over the concentration range of 1-300 mg/L. The intra- and inter-day precision values were below 6.4% and accuracy was within 95.3% and 103.6% for all QC levels (5, 75 and 200 mg/L). The lower limit of quantification was 1 mg/L. Total analysis time was reduced to 6 min including sample preparation. The present method is successfully applied to 24/7 clinical toxicology and demonstrated its usefulness to detect meprobamate poisoning.     

The effect of light intensity and daylength on the β-dimethylsulphoniopropionate (DMSP) content of marine green macroalgae from Antarctica*

Abstract. The β-dimethylsulphoniopropionate (DMSP) concentrations of 5 Antarctic green algae grown at 2, 30 and 55 μmol photons m^?2 s^?1 were determined during a period of one year, cultivated under fluctuating daylengths mimicking the conditions of the natural habitat at King George Island. Antarctica. The intracellular DMSP content of all species decreased simultaneously with decreasing daylengths and vice versa. Additionally, the DMSP level was affected by the light intensity: the higher the photon fluence rate the greater the algal DMSP concentration. Under conditions of darkness, there was a degradation of the DMSP pool in members of the Acrosiphoniales, while the DMSP content in members of the Ulvales did not change. The results indicate a light-dependent DMSP accumulation in algae. Therefore, they may help to explain the seasonal variability of DMSP and its cleavage product dimethylsulphide (DMS) in coastal waters.     

Metabolic profiling of urine in young obese men using ultra performance liquid chromatography and Q-TOF mass spectrometry (UPLC/Q-TOF MS)

Obesity is currently epidemic in many countries worldwide. In the young adult, obesity often accompanies hyperlipemia, which is strongly related to the occurrence and development of obesity-related chronic diseases such as diabetes mellitus, hypertension and cardiovascular disease. This study investigated the differences in metabolomic profiling between obese (with hyperlipemia, n=30) and normal-weight (n=30) young men. Anthropometric parameters and conventional metabolites were measured. There were no significant differences between obese and normal-weight young men in age, height and fasting plasma glucose level. Obese young men showed increased weight, body mass index, fat mass, systolic blood pressure, and triglyeride, total cholesterol and insulin levels, and lower levels of testosterone. The endogenous metabolite profile of urine was investigated by UPLC/Q-TOF MS (ultra performance liquid chromatography and Q-TOF mass spectrometry) with electrospray ionization (ESI). Partial least squares (PLS) enabled clusters to be visualized. Eight urine principal metabolites contributing to the clusters were identified; these included increased L-prolyl-L-proline, leucyl-phenylalanine, and decanoylcarnitine in positive ESI mode (m/z 213.1267, 279.1715 and 316.2459, respectively) and N-acetylornithine, 17-hydroxypregnenolone sulfate, 11β-hydroxyprogesterone, 5a-dihydrotestosterone sulfate and glucosylgalactosyl hydroxylysine in negative ESI mode (m/z 173.0931, 411.1883, 331.185, 369.1751 and 485.1875, respectively). These metabolite changes in obese men suggested early changes of metabolism in young-male obesity with hyperlipemia. The study may further aid the clinical prevention and treatment of obesity and related chronic disease.     

High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography/tandem mass spectrometry (LC-MS/MS)

Phytoestrogens are currently the subject of intense study owing to their potential protective effects against a number of complex diseases. However, in order to investigate the interactions between phytoestrogens and disease state effectively, it is necessary to have analytical methods which are sensitive, reproducible, and require low sample volumes. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (equol and O-desmethylangolensin), three lignans (secoisolariciresinol, enterodiol, and enterolactone), and one flavanone (naringenin) in human urine and serum. A high throughput of samples has been achieved via the use of 96-well plate sample extraction and liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis incorporating column switching, thus making the assay suitable for use on large sample numbers, such as those found in epidemiological studies. The robustness of the assay was proven via the comparison of data generated on two different LC-MS/MS systems, with and without column switching.     

Demethylation and cleavage of dimethylsulfoniopropionate in marine intertidal sediments

Abstract Demethylation and cleavage of dimethylsulfoniopropionate (DMSP) was measured in three different types of intertidal marine sediments: a cyanobacterial mat, a diatom-covered tidal flat and a carbonate sediment. Consumption rates of added DMSP were highest in cyanobacterial mat slurries (59 μmol DMSP 1⁻¹) and lower in slurries from a diatom mat and a carbonate tidal sediment (24 and 9 μmol DMSP 1⁻¹ h⁻¹, respectively). Dimethyl sulfide (DMS) and 3-mercaptopropionate (MPA) were produced simultaneously during DMSP consumption, indicating that cleavage and demethylation occurred at the same time. Viable counts of DMSP-utilizing bacteria revealed a population of 2 × 10⁷ cells cm⁻³ sediment (90% of these cleaved DMSP to DMS, 10% demethylated DMSP to MPA) in the cyanobacterial mat, 7 × 10⁵ cells cm⁻³ in the diatom mat (23% cleavers, 77% demethylators), and 9 × 10⁴ cells cm⁻³ (20% cleavers and 80% demethylators) in the carbonate sediment. In slurries of the diatom mat, the rate of MPA production from added 3-methiolpropionate (MMPA) was 50% of the rate of MPA formation from DMSP. The presence of a large population of demethylating bacteria and the production of MPA from DMSP suggest that the demethylation pathway, in addition to cleavage, contributes significantly to DMSP consumption in coastal sediments.     

A rapid and accurate UPLC/MS/MS method for the determination of benzodiazepines in human urine

A rapid, sensitive, and specific ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) assay method for simultaneous determination of 13 benzodiazepine compounds in human urine was developed and validated. Aliquots of 0.5 mL of urine specimens were used for the analysis and the benzodiazepines were extracted by single step methanol (containing 0.2% formic acid) precipitation and then separated on a BEH C18 (50 mm × 2.1 mm, 1.7 μm) analytical column with the temperature maintained at 45°C. The mobile phases consisted of methanol and water (both containing 0.2% formic acid) and the flow rate was 0.4 mL/min. The TQ detector, equipped with an electrospray ionization ion source, was set up with a positive mode. The acquisitions were performed in multiple-reaction monitoring (MRM) and the limit of quantification was 20 ng/mL for all of the 13 compounds. The low limits of detections (LODs) of the benzodiazepines in this method were between 0.5 and 2 ng/mL. The chromatographic separation time was 4 min and calibration curves in human urine were generated over the range of 20-2000 ng/mL. The method validation parameters such as accuracy, precision, carryover, recovery, stability, and specificity for all of the 13 compounds were within the acceptable range. This method is suitable for the high throughput screening of benzodiazepines in clinical laboratories.     

Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS

Malonyl-CoA is a key intermediate involved in lipid synthesis and lipid oxidation. Here, we report on a novel method for the quantification of malonyl-CoA and seven other short-chain acyl-CoAs in various rat and mouse tissues using ion-pairing reversed-phase HPLC/MS. This method is capable of measuring malonyl-CoA, free coenzyme A (CoASH), acetyl-CoA, beta-hydroxyl-butyryl-CoA (HB-CoA), 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), propionyl-CoA, succinyl-CoA, and isobutyryl-CoA simultaneously with a dynamic linear range over two orders of magnitude in a 7.0 min HPLC gradient run. The lower limit of quantification (LLOQ) was 0.225 pmol for all acyl-CoAs studied, except for HMG-CoA which had a higher LLOQ of 0.90 pmol. The interference of HB-CoA on the quantification of malonyl-CoA in animal tissues was also explored for the first time.     

Absolute quantification of endogenous angiotensin II levels in human plasma using ESI-LC-MS/MS

Background

Angiotensin II acts as a peptide hormone and component of renin-angiotensin- system (RAS) regulating the blood pressure, and seems to be involved in renal and vascular disorders. There is no reliable quantification method for angiotensin II available until now and the angiotensin II plasma levels described in the literature are correspondingly strongly divergent. Therefore, we developed and validated a sensitive, selective and reliable LC-ESI-MS/MS method for absolute quantification of angiotensin II concentration in human plasma based on the AQUA strategy.

Methods

Plasma samples were extracted using MAX Oasis cartridges and were subjected to a further immunoaffinity-purification using immobilized anti-angiotensin II antibodies in order to isolate endogenous angiotensin II. Stable isotope (¹³C- and ¹⁵ N-) labeled angiotensin II was used as an internal standard. The fractionated samples were analysed using LC-ESI-MS/MS.

Results

The calibration curve was established in plasma in the concentration range 6–240 pM (r² > 0.999). The developed and validated method was successfully applied for quantification of endogenous angiotensin II in human plasma of healthy volunteers and chronic kidney disease (CKD-5D) patients. The mean plasma angiotensin II levels were found to be 18.4 ± 3.3 pM in healthy subjects and 64.5 ± 32.4 pM in CKD-5D patients (each n =9).

Conclusion

The LC-ESI-MS/MS-based method for quantification of angiotensin II levels in human plasma was successfully evaluated within the study. This method is applicable for clinical applications aiming at the validation of the impact of highly physiologically and pathophysiologically active angiotensin II.