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.     

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.     

Distribution of dimethylsulfide and dimethylsulfoniopropionate and its relation with phytoneuston in the surface microlayer of the western North Atlantic during summer

One of the key steps towards predicting dimethylsulfide (DMS) emission to the atmosphere is to understand the distribution and cycling of biogenic sulfur in the microlayer. In this study, we examined the distribution of DMS and dissolved and particulate fractions of dimethylsulfoniopropionate (DMSPd and DMSPp) in the surface microlayer and bulk water of the western North Atlantic during July 2003. DMS concentrations in the bulk water varied from 0.71 to 7.65 nM. In contrast, DMS concentrations in the surface microlayer were fairly low (0.17–1.33 nM). Average concentrations of DMSPd and DMSPp in the bulk water were 2.09 (1.87–6.25) and 44.1 (8.06–119.8) nM, respectively, and those in the surface microlayer were 15.4 (4.06–54.3) and 29.9 (7.32–97.0) nM. In general, DMS was depleted in the microlayer (mean concentration: 0.60 nM) relative to the bulk water (mean concentration: 2.38 nM) with enrichment factors (the ratio of the microlayer concentration to bulk water concentration) ranging from 0.13 to 0.54. There was no consistent enrichment of DMSPp and chlorophyll a in the microlayer. On the contrary, DMSPd appeared to be highly enriched in the microlayer with an average EF of 4.89. The concentration of phaeopigments was also generally greater in the microlayer than in the bulk water, presumably due to enhanced photo-oxidation of chlorophyll a under high surface light intensities in the microlayer. In the study area, the concentration of DMSPp was significantly correlated with the abundance of dinoflagellates in the microlayer. Moreover, a significant correlation between the distributions of DMS, DMSPp, chlorophyll a and phaeopigment concentrations in the microlayer and the bulk water demonstrated that the biogenic materials in the microlayer come primarily from the bulk water below.     

Metabolism of dimethylsulfoniopropionate and glycine betaine by a marine bacterium

Abstract The metabolism of the methylated osmolytes glycine betaine (GB) and dimethylsulfoniopropionate (DMSP) was studied in a bacterium (strain MD 14–50) isolated from a colony of the cyanobacterium Trichodesmium . MD 14–50 when grown on DMSP cleaved dimethylsulfide (DMS) from DMSP and oxidized acrylate. In contrast to DMSP, GB was metabolized by sequential N-demethylations. Low concentrations (100 μM) of DMSP or GB allowed the growth of MD 14–50 on glucose at higher salinities than in their absence. At elevated salinities, DMSP was accumulated intracellularly with less catabolism and DMS production. Thus, DMSP and GB were catabolized by different mechanisms but functioned interchangeably as osmolytes.     

Cleavage of dimethylsulfoniopropionate and reduction of acrylate by Desulfovibrio acrylicus sp. nov.

From anoxic intertidal sediment, a dimethylsulfoniopropionate (DMSP)-cleaving anaerobe (strain W218) was isolated that reduced the acrylate formed to propionate. The bacterium was vibrio- to rod-shaped and motile by means of multiple polar flagella. It reduced sulfate, thiosulfate, and acrylate, and used lactate, fumarate, succinate, malate, pyruvate, ethanol, propanol, glycerol, glycine, serine, alanine, cysteine, hydrogen, and formate as electron donors. Sulfate and acrylate were reduced simultaneously; growth with sulfate was faster than with acrylate. Extracts of cells grown in the presence of DMSP contained high DMSP lyase activities (9.8 U/mg protein). The DNA mol% G+C was 45.1. On the basis of its characteristics and the 16S rRNA gene sequence, strain W218 was assigned to a new Desulfovibrio species for which the name Desulfovibrio acrylicus is proposed. A variety of other sulfate-reducing bacteria (eight of them originating from a marine or saline environment and five from other environments) did not reduce acrylate. Received: 22 March 1996 / Accepted: 8 May 1996     

马里亚纳海沟沉积物可培养异养细菌的多样性及其DMSP降解能力

[目的]马里亚纳海沟是地球上最深的海沟,具有超高静水压力、低温、无光等生境特征,蕴含独特的微生物资源.二甲基巯基丙酸内盐(dimethylsulphoniopropionate,DMSP)是海洋环境中最丰富的有机硫分子之一,海洋异养微生物可裂解DMSP产生"冷室气体"二甲基硫(dimethyl sulfide,DMS)...     

中国东海二甲基巯基丙酸内盐(DMSP)合成与降解菌的水平和垂直分布

【目的】二甲基巯基丙酸内盐(dimethylsulfoniopropionate,DMSP)及其裂解产物二甲基硫(dimethyl sulfide,DMS)在海洋硫循环中发挥重要作用。目前关于DMSP降解细菌的分布已有部分报道,但其合成细菌的研究才刚刚起步。本文拟研究中国东海水体DMSP合成与降解菌及基因的水平和垂直分布(1000m水深)差异,分析其对环境梯度变化的响应。【方法】利用流式细胞仪计数海水样品中微微型浮游生物的数量,通过荧光定量PCR和高通量测序手段定量测定DMSP合成基因(dsy B和mmt N)及物种、DMSP降解基因(ddd P和dmd A)及物种的丰度,分析其在东海海域水平及垂直方向上的分布差异。【结果】在垂直方向上,聚球藻、原绿球藻、微微型真核生物和异养细菌丰度随着水深的增加而先增后减,最大值位于30–50m附近。表层(4m左右)水体的DMSP合成及降解基因丰度最高,DMSP合成菌(如Alteromonas、Phaeobacter和Pelagibaca等)丰度也最高;随着水深增加,表层以下水体中DMSP合成及降解基因和物种丰度先增加后降低,峰值均出现在100–150 m;100 m以下,DMSP降解基因丰度迅速下降,而合成基因丰度下降程度较低,而且接近底层(500 m)时出现随水深逐渐增加的趋势。水平方向二者变化规律不明显。浅层水体(≤100 m)和深层水体(100 m)细菌群落结构存在显著差异,前者拥有较高比例的黄杆菌纲、放线菌纲和蓝细菌纲细菌,后者α变形菌纲细菌丰度较高。【结论】100m及以浅和100m以深的浮游细菌群落结构存在显著差异。表层水体中DMSP合成和降解细菌的丰度最高,100–150 m水体次之,但100–1022 m介导的DMSP合成和降解细菌丰度的变化趋势有较大差别。     

Direct quantification of dimethylsulfoniopropionate (DMSP) in marine micro- and macroalgae using HPLC or UPLC/MS

A simple method for the direct quantification of dimethylsulfinopropionate (DMSP) using HPLC or UPLC coupled to UV and/or MS detection is introduced. The protocol is applied for the determination of DMSP from marine micro- and macroalgae. The method is based on the derivatisation of DMSP using 1-pyrenyldiazomethane followed by reversed phase HPLC or UPLC separation. The detection limit is 590 nM, corresponding to 1 ng DMSP per injection. Using a combination of UV and MS detection the calibration curves were linear in the range of 2.93 microM to 11.7 mM concentrations. We show that direct determination of DMSP is possible from macroalgal tissue and microalgal cultures if DMSP-lyase activity is suppressed during work-up.     

The distribution and activity of sulphate reducing bacteria in estuarine and coastal marine sediments

Sulphate-reducing bacteria (SRB) play a vital role both the carbon and sulphur cycles and thus are extremely important components of the global microbial community. However, it is clear that the ecology, the distribution and activity of different SRB groups is poorly understood. Probing of rRNA suggests that different sediments have distinctly different patterns of SRB with complex factors controlling the activity of these organisms. The linking of community structure and function using sediment slurry microcosms suggests that certain groups of SRB, e.g., Desulfobacter and Desulfobulbus, can be linked to the use of specific substrates in situ. However, it is still unclear what environmental substrates are utilised by the majority of known SRBs. The work to date has greatly enhanced our understanding of the ecology of these organisms and is beginning to suggest patterns in their distribution and activity that may be relevant to understanding microbial ecology in general. This revised version was published online in August 2006 with corrections to the Cover Date.     

Biodegradation of polyethylene terephthalate (PET) by diverse marine bacteria in deep-sea sediments

PET plastic waste entering the oceans is supposed to take hundreds of years to degrade and tends to accumulate in the deep sea. However, we know little about the bacteria capable of plastic degradation therein. To determine whether PET-degrading bacteria are present in deep-sea sediment, we collected the samples from the eastern central Pacific Ocean and initiated microbial incubation with PET as the carbon source. After enrichment with PET for 2 years, we gained all 15 deep-sea sediment communities at five oceanic sampling sites. Bacterial isolation for pure culture and further growth tests confirmed that diverse bacteria possess degradation ability including Alcanivorax xenomutans BC02_1_A5, Marinobacter sediminum BC31_3_A1, Marinobacter gudaonensis BC06_2_A6, Thalassospira xiamenensis BC02_2_A1 and Nocardioides marinus BC14_2_R3. Furthermore, four strains were chosen as representatives to reconfirm the PET degradation capability by SEM, weight loss and UPLC-MS. The results showed that after 30-day incubation, 1.3%–1.8% of PET was lost. De-polymerization of PET by the four strains was confirmed by the occurrence of the PET monomer of MHET and TPA as the key degradation products. Bacterial consortia possessing PET-degrading potential are prevalent and diverse and might play a key role in the removal of PET pollutants in deep oceans.     

Determination of selenium and its compounds in marine organisms

In this study, we investigate the type and quantity of selenium compounds in fish and marine organisms, using ion-pair reversed phase LC–ICP-MS, developed and applied for the analysis of Atlantic cod, Atlantic salmon, Greenland halibut, Atlantic herring, blue mussel, common crab, scallop, calanus, and Euphasia super. Of the samples examined, the lowest level of selenium was found in farmed Atlantic salmon (0.17 mg Se kg⁻¹ dm). The total selenium extraction efficiency by phosphate buffer was 2.5 times higher in sea plankton and shellfish samples than in fish samples. Analysis of Se species in each hydrolysate obtained by proteolysis showed the presence of selenomethionine, which constituted 41.5% of the selenium compounds detected in hydrolysates of Atlantic herring and 98.4% of those in extracts of Atlantic salmon. Inorganic compounds, such as selenates and selenites, were detected mainly in sea plankton and shellfish samples (<0.13 mg Se kg⁻¹ wm), although no correlation was found between the presence of inorganic compounds and total selenium concentration. The accuracy of the total selenium determination was validated using a certified reference material (oyster tissue (NIST 1566b)). A lyophilised powder of cod (Gadus morhua) was used to validate speciation analysis, enzymatic hydrolysis of lyophilised powder of cod recovered 54 ± 6% of total selenium, and SeMet constituted 83.5 ± 5.28% of selenium detected in hydrolysates. The chromatographic detection limits were, respectively, 0.30 ng mL⁻¹, 0.43 ng mL⁻¹, 0.54 ng mL⁻¹, 0.55 ng mL⁻¹, 0.57 ng mL⁻¹ and 0.72 ng mL⁻¹ for selenate, selenomethionine, selenite, Se-methyl-selenocysteine, selenocystine and selenomethionine selenoxide.The data on selenium concentrations and speciation presented here could be useful in estimating levels of selenium intake by seafood consumption.     

Microbial production and consumption of dimethyl sulfide (DMS) in a sea grass (Zostera noltii)-dominated marine intertidal sediment ecosystem (Bassin d'Arcachon, France)

The relation between net dimethyl sulfide (DMS) production and changes in near surface (0-5 mm) oxygen concentrations in a sea grass (Zostera noltii Hornem)-covered intertidal sediment ecosystem was examined during a diel cycle. Sediment covered with Zostera was found to be more oxygenated than uncovered sediment during the period of photosynthesis. This phenomenon was probably caused by radial oxygen loss of the Zostera root-rhizome system. The population sizes of the three functional groups of microbes mainly responsible for the concentration of DMS, the dimethylsulfoniopropionate (DMSP)-demethylating, DMSP-cleaving and DMS-oxidizing bacteria, were quantified by most probable number (MPN) methodologies. Sediments with Zostera supported substantially higher populations of both aerobic (149x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) and anaerobic (43x10(6) cm(-3) DMSP-utilizing and 0.4x10(6) cm(-3) DMS-oxidizing) microorganisms than sediments without Zostera (DMSP-utilizing aerobes and anaerobes both 2x10(6) cm(-3) and DMS-oxidizing aerobes and anaerobes both 0.2x10(6) cm(-3)). Experiments conducted with sediment cores and sediment slurries suggested that the net production of DMS in these sediments was significantly lower during oxic periods than during anoxic periods. Intact sediment cores with and without Zostera produced DMS when incubated under anoxic/dark conditions (97.0 and 53.6 nmol DMS m(-2) h(-1), respectively), while oxic/light-incubated cores did not produce detectable amounts of DMS. In addition, kinetic parameter values (V(max) and K(m)) for DMSP degradation in cell suspensions of isolated DMSP-demethylating and DMSP-cleaving bacteria were measured and compared to documented values for other strains. Both V(max) and K(m) values for DMSP-demethylating organisms were found to be relatively low (14.4-20.1 nmol DMSP mg protein(-1) min(-1) and 4.1-15.5 μM, respectively) while these parameter values varied widely in the group of the DMSP-cleaving organisms (6.7-1000 nmol DMSP mg protein(-1) min(-1) and 2-2000 μM, respectively). It was hypothesized that a diel rhythm in DMS emission occurred, with a relatively low net production during the day and a high net production during the night. Environmental changes which result in increased anoxic conditions in coastal sediments, such as an increase in eutrophication, may therefore result in increased atmospheric DMS emission rates.     

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.     

Quantification of total and particulate dimethylsulfoniopropionate (DMSP) in five Bermudian coral species across a depth gradient

The symbiotic dinoflagellate microalgae of corals (Symbiodinium spp.) contain high concentrations of dimethylsulfoniopropionate (DMSP), a multifunctional metabolite commonly found in many species of marine algae and dinoflagellates. A photoprotective antioxidant function for DMSP and its breakdown products has often been inferred in algae, but its role(s) in the coral–algal symbiosis remains elusive. To examine potential correlations between environmental and physiological parameters and DMSP, total DMSP (DMSP_t, from the host coral and zooxanthellae), particulate DMSP (DMSP_p, from the zooxanthellae only), coral surface area, and total protein, as well as zooxanthellae density, chlorophyll concentration, cell volume and genotype (i.e., clade) were measured in five coral species from the Diploria-Montastraea-Porites species complex in Bermuda along a depth gradient of 4, 12, 18, and 24 m. DMSP_t concentrations were consistently greater than DMSP_p concentrations in all species suggesting the possible translocation of DMSP from symbiont to host. D. labyrinthiformis was notably different from the other corals examined, showing DMSP_p and DMSP_t increases (per coral surface area or tissue biomass) with increasing water depth. However, overall, there were no consistent depth-related patterns in DMSP_p and DMSP_t concentrations. Further research, investigating dimethylsulfide (DMS), dimethylsulfoxide, and acrylate levels and DMSP-lyase activity in correlation with other biomarker endpoints that have been shown to be depth (i.e., temperature and light) responsive are needed to substantiate the significance of these findings.     

Effect of different selenium source (sodium selenite and selenium yeast) on broiler chickens

A feeding experiment was carried out to compare the effects of supplementing a poultry meal-based diet with selenium as sodium selenite or selenium yeast on broiler chickens. Three groups with three replicates of broiler chickens (mean weight 710 ± 5.3 g) were given a basal diet either unsupplemented (control) or supplemented with 0.2 mg Se kg⁻¹ as sodium selenite (trial 1) or selenium yeast (trial 2) respectively, for 21 days. There was significant difference (P<0.05) in Feed Conversion Ratio (FCR) of trials 1 and 2 compared with the control. However, there were no significant differences (P>0.05) in FCR between trials 1 and 2. Final weight, survival rate and Daily Gain (DG) were not affected by the dietary Se source. Chickens fed the basal diet showed lower (P<0.05) selenium content in muscle, kidney, liver and pancreas compared to that fed selenium supplements (trials 1 and 2). Furthermore, trial 2 showed the highest value (P<0.05) among these treatments. However, there was no significant difference (P>0.05) in muscle selenium content of chickens between trials 1 and 2. Glutathione peroxidase (GSH-Px) activities in broiler chickens plasma and liver of all selenium treatment groups (trials 1 and 2) were significantly different (P<0.05) from that of the control. The GSH-Px activity in plasma was higher (P<0.05) in trial 2 compared with trial 1 and the control. However, there was no difference (P>0.05) in hepatic glutathione peroxidase between trials 1 and 2 although the average value of GSH-Px activity in trial 2 presented the trend of increase.