酒石酸对原代心肌细胞缺氧复氧损伤的保护作用

目的:考察酒石酸对新生大鼠原代心肌细胞缺氧复氧损伤的保护作用。方法:大鼠乳鼠进行心肌细胞原代培养,MTT法检测心肌细胞的活力。复制心肌细胞缺氧复氧损伤模型,考察酒石酸对心肌细胞缺氧复氧损伤所致的LDH漏出的影响,并进一步分别采用流式细胞术及Western blot考察酒石酸对心肌细胞凋亡,cleaved-caspase 3,Akt及P-Akt的影响。结果:(1)酒石酸31.25～500μg/ml范围对原代心肌细胞活力不产生影响。酒石酸400μg/ml均可显著降低心肌细胞缺氧复氧损伤后LDH漏出率,而200、100、50μg/ml浓度时作用均不明显;(2)酒石酸400、200μg/ml有抑制心肌细胞缺氧复氧损伤所致凋亡百分数增加的趋势;(3)酒石酸200μg/ml均可显著抑制心肌细胞缺氧复氧所致的cleaved-caspase 3蛋白表达增加,而酒石酸400μg/ml有降低cleaved-caspase 3蛋白表达的趋势,无统计学差异;(4)酒石酸400μg/ml可显著增加心肌细胞的p-Akt蛋白表达,而酒石酸200μg/ml对p-Akt蛋白表达未见明显影响。结论:酒石酸可以抑制心肌细胞缺氧复氧损伤所致的坏死和凋亡,可能是通过激活Akt的磷酸化而实现的。

Secreted Expression of S-adenosy-L-methionine Synthetase in Pichia pastoris

[Objective] The research aimed to study the secreted expression of S-edenosyl-L-methionine synthetase （SAMS） in Pichia pastoris. Method ] The gene coding SAMS, from the genomic DNA of Saccharomyces cerevisiae, was amplified by PCR and inserted into the secreted expression vector pPIC9K to get recombinant plasmid. The recombinant plasmid pPIC9K-sarr~ was integrated into Pichia pastoris GSl15 genome by electroporation and induced by methanol. The activity of the recombinant enzyme was measured using high-pedormance liquid chroma- tography （HPLC） by determining the production of S-adenosy-L-methionine （SAM） with the enzyme secreted. [ ResultJ The molecular weight of the expression protein identified by SDS-PAGE was about 50 kD, being larger than the theoretical molecular mass of SAMS, which might be due to the glycosytation in the process of secretion. Methanol-induction as well as preliminary purification could enhance the enzyme activity, espe- cially the latter, after which the specific activity of SAMS was improved to 61.48 U/rng. [Conclusion] SAMS with biological activity was secreted successfully in Pichia pastoris GSl15 for the first time. And it is the start for the genetic engineering strains to open up prospects for industrial production.

Mono-Feruloyl-R, R-(+)-Tartaric Acid from Salvia chinensis

报道了用半合成方法和光谱分析确证从华鼠尾草(Salvia chinensis Benth.)中分离的单阿魏酰-R,R-(+)-酒石酸的化学结构和绝对构型;合成了其 S,S-(-)-和 R,S-(meso)-酒石酸的衍生物,并分析了其间的光谱差异。

Breeding of Ferment Bacteria and Control of Methanol and Higher Alcohols in Jujube Wine

Jujube wine is a health drink with local characteristics and is worth pro- moting. Jujube wine fermentation can be divided into alcoholic fermentation and mal- olactic fermentation. Yeast is the key of alcoholic fermentation while lactic acid bac- teria is the key of malolactic fermentation, and therefore the breeding of yeast and lactic acid bacteria is crucial for the quality of jujube wine. Besides, the control of methanol is a major problem in production, and the control of higher alcohol is also difficult. Thus, we summarized the research related with the breeding of yeast and lactic acid bacteria, and the control of methanol and higher alcohols, and proposed that breeding specialized yeast and lactic acid bacteria was the future research di- rection. Moreover, the production mechanism of methanol and higher alcohols was investigated, and the content of methanol and higher alcohols was effectively con- trolled on the basis of quality guarantee, providing references for the production technology of jujube wine.

New method for preparation of D-valine

A method for preparing D-valine from L-valine by racemization and chemical resolution is presented. The resolving reagent, D-2,3-dibenzoyl tartaric acid was obtained by hydrolyzation of D-2,3-dibenzoyl tartaric anhydride prepared by reaction of benzoyl chloride with D-tartaric acid. DL-valine was prepared by racemization of L-valine in the presence of aldehyde in a medium of acetic acid at 100 to 110°C for 3 h. In the presence of mineral acid, reaction of D-2,3-dibenzoyl tartaric acid with DL-valine formed diastreroisomeric salts at 84 to 95°C. Salt composed of D-2,3-dibenzoyl tartaric acid and D-valine precipitated when the diastreroisomeric salts mixtures were cooled to 15°C. The salt was reacted with base giving D-valine with yield of 70% to 80% and optical purity of over 98%.

Double-blinded, Controlled, Randomized Study of Dihydrocodeine Tartrate vs Codeine Phosphate in Treating Cancer Pain

To compare the effects and adverse reactions of dihydrocodeine tartrate andcodeine phosphate in treating moderate cancer pain. Methods: Sixty-nine cases of cancer patientswith moderate pain were treated with dihydrocodeine tartrate or codeine phosphate respectively bydouble-blind, controlled randomized methods and the effects and adverse reactions were observed.Results: After administration of dihydrocodeine tartrate or codeine phosphate, in treatment group orcontrol group, the total effective rate was 86.6% and 93.6%, and common adverse symptoms includedconstipation (31.3%/12.9%), nausea (18.8%/19.7%), gastric trouble (18.8%/19.7%), skin pruritus(12.5%/10%), vomit (9.3% and 6.5%) with the difference being not significant. Conclusion: Theeffects of dihydrocodeine tartrate in treating moderate cancer pain are similar to codeinephosphate. Both them can be used to treat moderate cancer pain.

A Simplified Method for Purifying Osteoclasts from Human Giant Cell Tumor of Bone

Objective: To purify and identify the osteoclasts from the tissue of humangiant cell tumor of bone. Methods: We have developed a new method that allows the purification oflarge numbers of authentic osteoclasts (OCs). The OCs were isolated from tissue of human giant celltumor of bone by 0.25% trypsin and collagenase. We characterized OCs in terms of the expression ofdifferent phenotypic markers of OCs. The phenotypic markers of OC included Tartrate-resistant acidphosphatase staining (TRAP). The expression of calcitonin receptor (CTR), cathepsin K and receptoractivator of necrosis factor κB (RANK) mRNA were examined by RT-PCR. Results: The OC cell purifiedby above method functioned normally in vitro. The purity was about 79.7%. They showed the normalosteoclast phenotypes markers of OC. Conclusion: The method provides a system for performingbiochemical and molecular studies of OCs. The study indicates that the method of purifying theosteoclasts from human GCT cell can be used for research of bone metabolism.

Effect of Organic Ligands on Biological Availability of Inorganic Phosphorus in Soils

Citrate, oxalate, tartrate and malate were added into soils during the growthperiod of ryegrass to study the effect of different organic ligands on the release of variousinorganic P (Pi) fractions in a yellow-brown soil and a paddy soil. The results showed that oxalatewas most effective in promoting the release of total Pi in the yellow-brown soil and tartrate in thepaddy soil. The dominant Pi fractions released from the yellow-brown soil were calcium phosphate(Ca-P) and aluminum phosphate (Al-P) and those from the paddy soil were iron phosphate (Fe-P) andreductant soluble phosphate (O-P) mobilized by tartrate. Phosphorous-mobilizing capability oforganic acids in the yellow-brown soil revealed the following order: oxalate > citrate > malate >tartrate. In the paddy soil, the order was tartrate > citrate ≈ oxalate > malate. It wasdemonstrated that organic ligands were different in their capabilities of mobilizing Pi and the sameorganic ligand showed also a discrepancy in mobilizing P in different soils. Although the additionof organic ligands into soils could increase the amount of P taken up by ryegrass, the more uptakeof P, however, was not only due to the more release of Pi, but also partly from organic P. In manycases, organic ligands promoted the release of the total Pi, while different fractions showeddifferent trends: some increased and others decreased.

N-acetylcysteine attenuates alcohol-induced oxidative stess in rats

AIM:To investigate free-radical scavenger effect of n- acetylcysteine in rats intragastrically fed with ethanol. METHODS:Twenty-four rats divided into three groups were fed with ethanol (6 g/kg/day,Group 1),ethanol and n- acetylcysteine (1 g/kg,Group 2),or isocaloric dextrose (control group,Group 3) for 4 weeks.Then animals were sacrificed under ether anesthesia,and intracardiac blood and liver tissues were obtained.Measurements were made in both serum and homogenized liver tissues. Malondialdehyde (MDA) level was measured by TBARS method.Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) levels were studied by commercial kits. Kruskal-Wallis test was used for statistical analysis. RESULTS:ALT and AST in Group 1 (154 U/L and 302 U/L, respectively) were higher than those in Group 2 (94 U/L and 155 U/L) and Group 3 (99 U/L and 168 U/L) (P=0.001 for both).Serum and tissue levels of MDA in Group 1 (1.84 nmol/mL and 96 nmol/100 mg-protein) were higher than that in Group 2 (0.91 nmol/mL and 64 nmol/100 mg protein) and Group 3 (0.94 nmol/ml and 49 nmol/100 mg-protein) (P<0.001 for both).On the other hand,serum GSH-Px level in Group 1 (8.21 U/g Hb) was lower than that in Group 2 (16 U/g Hb) and Group 3 (16 U/g-Hb) (P<0.001).Serum and liver tissue levels of SOD in Group 1 (11 U/mL and 26 U/100 rag-protein) were lower than that in Group 2 (18 U/ mL and 60 U/100 mg protein) and Group 3 (20 U/mL and 60 U/100 rag-protein) (P<0.001 for both). CONCLUSION:Ethanol-induced liver damage was associated with oxidative stress,and co-administration of n-acetylolsteine attenuates this damage effectively in rat model.

Conductivity Prediction of Sodium and Potassium Hydrogen Tartrates in Aqueous Solution at Low Concentration

An ionic conductivity prediction equation at low concentration for two acid salts is proposed taking into account the dissociation and association equilibria among ions. The salts considered are sodium and potassium hydrogen tartrates. There is no additional parameter of high order terms except for the Onsager's coefficient of limited term in the new equation. Results show a complex conductance of acidic tartrates in aqueous solution. The molar conductivities of metal ions are nearly constant such that the contributions from hydrogen and tartrate ions decrease with concentration, while the molar conductivity of bitartrate ion increases with concentration.

Omega-3 fatty acids for the treatment of non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease （NAFLD） has been recognized as a major health burden. It is the most important cause of chronic liver disease and a major in- dependent cardiovascular risk factor. Lacking a definite treatment for NAFLD, a specific diet and an increase in physical activity represent the most commonly used therapeutic approaches. In this review, major literature data about the use of omega-3 polyunsaturated fatty ac- ids （n-3 PUFAs） as a potential treatment of NAFLD have been described, n-3 PUFAs, besides having a beneficial impact on most of the cardio-metabolic risk factors （hy- pertension, hyperlipidemia, endothelial dysfunction and atherosclerosis） by regulating gene transcription factors [i.e., peroxisome proliferator-activated receptor （PPAR） cz, PPARy, sterol regulatory element-binding protein-i, carbohydrate responsive element-binding protein], im- pacts both lipid metabolism and on insulin sensitivity. In addition to an enhancement of hepatic beta oxidation and a decrease of the endogenous lipid production, n-3 PUFAs are able to determine a significant reduction of the expression of pro-inflammatory molecules （tumor necrosis factor-~ and interleukin-6） and of oxygen reac- tive species. Further strengthening the results of the in vitro studies, both animal models and human interven- tion trials, showed a beneficial effect of n-3 PUFAs on the severity of NAFLD as expressed by laboratory pa- rameters and imaging measurements. Despite available results provided encouraging data about the efficacy of n-3 PUFAs as a treatment of NAFLD in humans, well- designed randomized controlled trials of adequate size and duration, with histological endpoints, are needed to assess the long-term safety and efficacy of PUFA, as well as other therapies, for the treatment of NAFLD and non-alcoholic steatohepatitis patients. It is worthwhile to consider that n-3 PUFAs cannot be synthesized by the human body and must be derived from exogenous sources （fish oil, flaxseeds, olive oil） which are typical foods of the Mediterranean diet, known for its beneficial effects in preventing obesity, diabetes and, in turn, cardiovascular events. According to these data, it is important to consider that most of the beneficial effects of n-3 PUFAs can also be obtained by an equilibrate nutrition program.

Effect of Propanoic Acid on Ethanol Fermentation by Saccharomyces cerevisiae in an Ethanol-Methane Coupled Fermentation Process

Propanoic acid accumulated in an ethanol-methane coupled fermentation process affects the ethanol fermentation by Saccharomyces cerevisiae. The effects of propanoic acid on ethanol production were examined in cassava mash under different pH conditions. Final ethanol concentrations increased when undissociated propanoic acid was <30.0 mmol·L-1 . Propanoic acid, however, stimulated ethanol production, as much as 7.6% under proper conditions, but ethanol fermentation was completely inhibited when undissociated acid was >53.2 mmol·L-1 . Therefore, the potential inhibitory effect of propanoic acid on ethanol fermentation may be avoided by controlling the undissociated acid concentrations through elevated medium pH. Biomass and glycerol production decreased with propanoic acid in the medium, partly contributing to increased ethanol concentration.

Coordination configurations of cupric tartrate in electronic industry wastewater

The coordination structure of cupric tartrate(Cu−TA)complex was investigated by ultraviolet−visible(UV-Vis)and liquid chromatography/mass spectrometer(LC-MS)firstly;furthermore,effective coordination configurations and electronic properties of Cu−TA in aqueous solution were systematically revealed by density functional theory(DFT)calculations.Consistently,Job plots show the possible existence of[Cu(TA)]and[Cu(TA)_(2)]^(2-)at 230 and 255 nm based on UV-Vis results.LC-MS results confirm the existence of the single and high coordination complexes[Cu_(2)(TA)_(2)]^(+),[Cu(TA)_(2)]^(+)and[Cu_(2)(TA)_(3)(H_(2)O)_(2)(OH)_(2)]^(2+).DFT calculation results show that carboxylic oxygen and hydroxyl oxygen of tartaric acid(TA)are preferred sites for Cu(Ⅱ)coordination.[Cu(TA)](1H,3H sites O of TA coordinated with Cu(Ⅱ)),[Cu(TA)_(2)]^(2-)(two 1^(C),2^(H) sites O of TA coordinated with Cu(Ⅱ)),and[Cu(TA)_(3)]^(4-)(three 2H,3H sites O of TA coordinated with Cu(Ⅱ))should be dominant coordination configurations of Cu−TA.The corresponding Gibbs reaction energies are-170.1,-136.2,and-90.2 kJ/mol,respectively.

Effectiveness of Saccharomyces boulardii in a rat model of colitis

AIM:To investigate the effects of Saccharomyces boulardii(S.boulardii) in an experimental rat model of trinitrobenzene sulfonic acid(TNBS)-induced colitis.METHODS:Thirty-two Wistar albino female rats were categorized into five groups.On the first day of the study,50 mg TNBS was administered via a rectal catheter in order to induce colitis in all rats,except those in the control group.For 14 d,the rats were fed a standard diet,without the administration of any additional supplements to either the control or TNBS groups,in addition to 1 mg/kg per day S.boulardii to the S.boulardii group,1 mg/kg per day methyl prednisolone(MP) to the MP group.The animals in the S.boulardii + MP group were coadministered these doses of S.boulardii and MP.During the study,weight loss,stool consistency,and the presence of obvious blood in the stool were evaluated,and the disease activity index(DAI) for colitis was recorded.The intestines were examined and colitis was macro-and microscopically scored.The serum and tissue levels of tumor necrosis factor-α(TNF-α) and nitric oxide(NO) were determined,and fungemia was evaluated in the blood samples.RESULTS:The mean DAI scores for the MP and S.boulardii + MP groups was significantly lower than the TNBS group(3.69 ± 0.61 vs 4.46 ± 0.34,P = 0.018 and 3.77 ± 0.73 vs 4.46 ± 0.34,P = 0.025,respectively).While no significant differences between the TNBS and the S.boulardii or MP groups could be determined in terms of serum NO levels,the level of serum NO in the S.boulardii + MP group was significantly higher than in the TNBS and S.boulardii groups(8.12 ± 4.25 μmol/L vs 3.18 ± 1.19 μmol/L,P = 0.013;8.12 ± 4.25 μmol/L vs 3.47 ± 1.66 μmol/L,P = 0.012,respectively).The tissue NO levels in the S.boulardii,MP and S.boulardii + MP groups were significantly lower than the TNBS group(16.62 ± 2.27 μmol/L vs 29.72 ± 6.10 μmol/L,P = 0.002;14.66 ± 5.18 μmol/Lvs 29.72 ± 6.10 μmol/L,P = 0.003;11.95 ± 2.34 μmol/Lvs 29.72 ± 6.10 μmol/L,P = 0.002,respectively).The tissue NO levels in the S.boulardii,MP and S.boulardii + MP groups were similar.The mean serum and tissue TNF-α levels were determined to be 12.97 ± 18.90 pg/mL and 21.75 ± 15.04 pg/mL in the control group,18.25 ± 15.44 pg/mL and 25.27 ± 11.95 pg/mL in the TNBS group,20.59 ± 16.15 pg/mL and 24.39 ± 13.06 pg/mL in the S.boulardii group,9.05 ± 5.13 pg/mL and 24.46 ± 10.85 pg/mL in the MP group,and 13.95 ± 10.17 pg/mL and 24.26 ± 10.37 pg/mL in the S.boulardii + MP group.Significant differences in terms of the levels of serum and tissue TNF-α and the macroscopic and microscopic scores were not found between the groups.S.boulardii fungemia was not observed in any of the rats.However,Candida fungemia was detected in one rat(14%) in the TNBS group,two rats(28%) in the S.boulardii group,three rats(50%) in the MP group,and three rats(42%) in S.boulardii + MP group.CONCLUSION:S.boulardii does not demonstrate considerable effects on the DAI,pathological scores,or cytokine levels but does decrease the tissue NO levels.

Adverse hepatic reactions associated with calcium carbimide and disulfiram therapy: Is there still a role for these drugs?

Disulfiram and calcium carbimide are two alcohol deterrants widely used in alcoholism treatment, however, there exist great concerns over their safety. Reports on hepatotoxicity, mainly related to disulfiram therapy, have been published. The hepatotoxic potential of calcium carbimide is less well characterized. Here, we describe four cases of liver damage related to this therapeutic group that were submitted to a Registry of hepatotoxicity and point out the limitations that we face when prescribing these compounds. A reassessment of the role of these compounds in the management of alcohol dependence is clearly needed.

Is the required therapeutic effect always achieved by racemic switch of proton-pump inhibitors?

Many of the drugs currently used in medical practice are racemates. The enantiomers of a racemic drug differ in pharmacodynamics and/or pharmacokinetics, thus in some cases it is preferable to develop pure enantiomers by racemic switch. In a recent study by Pai et al, dexrabeprazole [R(+)-rabeprazole] (10 mg) was found to be more effective than rabeprazole (20 mg) in the treatment of gastroesophageal reflux disease. We read with great interest in this study and discussed whether such racemic switch would be applicable to other proton-pump inhibitors (PPIs). A literature review indicates that stereoselective pharmacokinetics, rather than stereoselective pharmacological activity, is the main cause of differences in clinical efficacy between pure enantiomer and racemic PPI. Racemic switches of PPI provide the therapeutic advantages such as reducing metabolic load on the body, simplifying pharmacokinetics, providing benefit to the non-responders to standard dose of racemate, more homogenous response to treatment and better efficacy with equal safety. Further studies in quantitative structure-activity relationships (QSARs) are needed to address the fact that the preferred enantiomer of PPI is not always in the same absolute configuration, i.e., S-form is for omeprazole, pantoprazole and tenatoprazole whereas R-form is for lansoprazole and rabeprazole.

Oral administration of S-nitroso-N-acetylcysteine prevents the onset of non alcoholic fatty liver disease in rats

AIM： To evaluate the potential of S-nitroso-N-acetylcysteine （SNAC） in inhibition of lipid peroxidation and the effect of oral SNAC administration in the prevention of nonalcoholic fatty liver disease （NAFLD） in an animal model.METHODS： NAFLD was induced in Wistar male rats by choline-deficient diet for 4 wk. SNAC-treated animals （n=6） （1.4 mg/kg/day of SNAC, orally） were compared to 2 control groups： one （n=6） received PBS solution and the other （n=6） received NAC solution （7 mg/kg/d）. Histological variables were semiquantitated with respect to macro and microvacuolar fat changes, its zonal distribution, foci of necrosis, portal and perivenular fibrosis, and inflammatory infiltrate with zonal distribution. LOOHs from samples of liver homogenates were quantified by HPLC. Nitrate levels in plasma of portal vein were assessed by chemiluminescence. Aqueous low-density lipoprotein （LDL） suspensions （200 pg protein/mL） were incubated with CuCl2 （300 μmol/L） in the absence and presence of SNAC （300 μmol/L） for 15 h at 37 ℃ Extent of LDL oxidation was assessed by fluorimetry. Linoleic acid （LA） （18.8 μmol/L） oxidation was induced by soybean lipoxygenase （SLO） （0.056 μmol/L） at 37 ℃ in the presence and absence of N-acetylcysteine （NAC） and SNAC （56 and 560 pmol/L） and monitored at 234 nm. RESULTS： Animals in the control group developed moderate macro and microvesicular fatty changes in periportal area. SNAC-treated animals displayed only discrete histological alterations with absence of fatty changes and did not develop liver steatosis. The absence of NAFLD in the SNAC-treated group was positively correlated with a decrease in the concentration of LOOH in liver homogenate, compared to the control group （0.7±0.2 nmol/mg vs 3.2±0.4 nmol/mg protein, respectively, P〈0.05）, while serum levels of aminotransferases were unaltered. The ability of SNAC in preventing lipid peroxidation was confirmed in in vitro experiments using LA and LDL as model substrates. CONCLUSION： Oral administration of SNAC prevents the onset of NAFLD in Wistar rats fed with cholinedeficient diet. This effect is correlated with the ability of SNAC to block the propagation of lipid peroxidation in vitro and in vitro.

Electrochemical degradation of acidified reed pulp black liquor with three-dimensional electrode reactor

The electrochemical degradation of reed pulp black liquor containing lignin pretreated by acidification method was investigated using a three-dimensional electrode reactor. Using activated carbon as particle electrode, the effects of p H value, reaction temperature, electrolysis time and current on residual concentration of total organic carbon(TOC) were discussed in detail. The optimal conditions were obtained: pH 2.5, influent flow rate of 200 mL/min, 25 °C, 300 mA and 2h of electrolysis time, and the removal efficiency of TOC maintains at 35.57 %. The results of the electrochemical method indicate that ·OH radicals are produced in activated carbon anode in the electrolysis process and then adsorbed on the activated carbon surface. Microcell consists of ·OH radicals and the absorbed lignin. With the microcell reaction, the lignin is degraded, while the anodic polarized curve illustrates that the lignin is obviously oxidized in the anode. The contributions of direct and indirect electrolyses to the TOC removal ratio are about 50%, respectively.

Uncertainty Evaluation for the Determination of Dibutyl Phthalate(DBP) Concentration in Liqueur by GC-MS

The dibutyl phthalate （DBP） concentration in liqueur was measured by gas chromatography-mass spectrometry （GC-MS）, and the uncertainty during the mea-surement was evaluated in this study. The results showed that the combined stan-dard uncertainty was determined as 0.028 and the expanded uncertainty was 0.056 at confidence probability p=95%, coverage factor k=2, by fol owing the methods de-scribed in GB/T 21911-2008 ＂Determination of Phthalate Esters in Foods＂. The av-erage DBP concentration in the liqueur of eight repeated measurements was（0.985＆#177; 0.056） mg/kg finaly.

A Novel Membrane Prepared from Sodium Alginate Cross-linked with Sodium Tartrate for CO2 Capture

The membrane-based CO2 separation process has an advantage compared to traditional CO2 separation technologies. The membrane is the key of the membrane separation process. In this paper, preparation, characterization and laboratory testing of the membrane, which was prepared from sodium alginate, hydrogen bond cross-linked with sodium tartrate and used for CO2/N2 separation, were reported. The resistance to SO2 of the membrane was also investigated. The experimental results demonstrate that the membrane possesses a high resistance to SO2.Finally,based on experimental results, the economic feasibility of the membrane used for CO2/N2 separation was evaluated,indicating the two-stage membrane process can compete with the traditional chemical absorption method.