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A method for determining polysaccharide content in biological samples
Accepted Manuscript Title: A method for determining polysaccharide content in biological samples Authors: Duoduo Xu, Wei Zheng, Yanqiu Zhang, Qipin Gao, Mingxing Wang, Yang Gao PII: DOI: Reference:
S0141-8130(17)33314-7 http://dx.doi.org/10.1016/j.ijbiomac.2017.09.045 BIOMAC 8222
To appear in:
International Journal of Biological Macromolecules
Received date: Accepted date:
30-8-2017 14-9-2017
Please cite this article as: Duoduo Xu, Wei Zheng, Yanqiu Zhang, Qipin Gao, Mingxing Wang, Yang Gao, A method for determining polysaccharide content in biological samples, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.09.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A method for determining polysaccharide content in biological samples Duoduo Xuab, Wei Zhengc, Yanqiu Zhangd，Qipin Gaoab, Mingxing Wangd, Yang Gaoab, * a Center of Research & Development, Changchun University of Chinese Medicine, Changchun, Jilin 130117, China b Macromolecule of Chinese Medicine Key Lab of Jilin Province, Changchun 130117, Jilin, China c Departments of Ophthalmology, The Affiliated Hospital, Changchun University of Chinese Medicine, Changchun, Jilin 130021, China d Preparation center, The Affiliated Hospital, Changchun University of Chinese Medicine, Changchun, Jilin 130021, China
Abstract: Difficulties in the determination of polysaccharide content in biological samples has been one of the bottlenecks limiting the development of polysaccharides pharmacokinetics, greatly due to the complicated chemical structure of these compounds. In this study, we established a simple, reproducible and reliable method for the determination of polysaccharide content in biological samples. Polysaccharides were replaced by fluorescein isothiocyanate (FITC) and iodine to generate a complex. The iodine content in the complex was measured by inductively coupled plasma-mass spectrometry (ICP-MS) to indirectly reflect polysaccharide content. We investigated the stability of the complex, carried on methodological validation and proceeded to determine the polysaccharide content in rat blood and organs after oral administration of iodine substituted polysaccharides using ICP-MS. The results showed that the iodine complex was stable in vivo and in vitro. In the bioavailability of polysaccharides, after 1h, the absorption rate of polysaccharides in blood was the highest, reaching 5.84%. After 3h, the bioaccessibility was differently distributed in various organs, reaching 1% on average across organs.
Keywords: Polysaccharide, Iodine substitution, FITC, ICP-MS; Biological sample 1. Introduction In recent years, polysaccharides have received increased attention due to their broad application potential, including the development of new drugs and health products. Having relatively low toxicity and minor side effects [1], their biological activities include hepatoprotection [2], antioxidant effects [3], antitumour properties [4], lipid-accumulation inhibition [5], and anti-inflammatory [6]. However, in natural medicine, the study of polysaccharides has been difficult due to their very complex chemical structure, large molecular size, strong polarity and intricate spatial structure. Polysaccharide separation, purification, characterization and determination procedures are challenging, which hinders the development and use of polysaccharide drugs compared to other compounds. Determination of polysaccharide content in biological samples remains a major bottleneck in the development of polysaccharide pharmacokinetics, including the study of the mechanisms of action of novel polysaccharide-based drugs, and the design and optimization of dosage regimens. The methods used for detection of polysaccharides in biological samples include chromatography [7.8], radioactive isotope labelling [9-11], fluorescence assay [12-14] and the ELISA assay [15,16]. Currently, these methods present important limitations, such as low sensitivity and specificity. In addition, radioactive isotope labelling requires a high-tech operating environment and has low operability. Here, we established a simple, reliable and reproducible method for the determination of polysaccharide content in biological samples. Previously, our group developed a
method for glycoprotein content determination in biological samples [17]. In this method, glycoprotein was substituted by iodine to form an iodide-glycoprotein complex. Then, the iodine content in the iodide-glycoprotein complex was further assayed by ICP-MS, which is a technique widely used in qualitative and quantitative analysis of inorganic elements. In the present study, we prepared stable polysaccharide-iodine complexes as in with slight modifications and carried out a method validation procedure. We then used this method to determine polysaccharide content of in rat blood and organs. 2. Materials and methods 2.1. Materials and chemicals The polysaccharide (EPA-1) was isolated by ourselves from Pleurotus eryngii supplied by Mushroom and Vegetable base of Jilin Agricultural University (Jilin, China). Sephadex G-50 was purchased from the GE Healthcare Ltd. (Chalfront St. Guiles, UK). Cell culture (RPM 450) was purchased from Thermo, Rockford, IL USA. The no iodine feeds of SPF rat were purchased from Beijing Aokexieli Feed Co., Ltd. (Beijing, China). The rats were purchased from College of Pharmacy, Jilin University (Jilin, China), certification of conformity for the SCXK (2012-0003). Several reagents were marked in articles, and other reagents were of analytical grade (China). 2.2. Sample preparation A sample of polysaccharide EPA-1 was obtained from the mushroom Pleurotus eryngii and used in this study because its physical and chemical properties, structure and biological activity had already been characterized. EPA-1 is a homogeneous
polysaccharide with 98.6% sugar content, with no acidic sugar or protein components, and a molecular weight of 1.0 × 105 Da. It consists of mannose, glucose and galactose, and the main linkage mode was the (1→6)-linked galactose residue. Activity tests indicated that EPA-1 possessed good immunoregulatory activity [18]. 2.3. The Reaction of FITC labelling of Polysaccharides The method described in Belder [19] was used. First, 1g of EPA-1 was dissolved in a 10mL methyl sulphoxide solution plus five drops of pyridine. The sample was then mixed ultrasonically. Secondly, 0.1g FITC and 20mg dibutyltin dilaurate were added to the mixture, which was vigorously shaken and heated for 2h at 95 ℃. Finally, after cooling, a volume of 95 % (v/v) ethanol was slowly added to the mixture with constant stirring until the ethanol content of the mixture was 85%. This mixture was centrifuged and the precipitate repeatedly cleaned by ethanol washes. The precipitate containing the complex (EPA-1F) formed by EPA-1 and FITC was dried and stored. 2.4. Iodine Substitution Reaction The reaction for iodine substitution was performed according to Keck [20] with small modifications. The dried EPA-1F extract (1g) was dissolved in 5 mL of water by ultrasonic mixing. Next, 2 mL of NaI (0.5 mol/L) and 5 mL of chloramine T buffer solution (0.1 g/mL) were added to mixture, followed by mixing for 1 min on the turbine mixer. After this, 5 mL of sodium metabisulfite buffer solution (0.1 g/mL) and 5 mL of potassium iodide (0.1 g/mL) were added to the mixture and dissolved by shaking and mixing. The mixture was centrifuged (5000 rpm, 5 min), the supernatant was collected to get the iodine-substituted Pleurotus eryngii polysaccharides. We
prepared three replicates. The buffer solution was prepared by adding 81mL of disodium hydrogen phosphate (71.6mg/mL) and 19mL of sodium dihydrogen phosphate (31.2mg/mL) to a 1000 mL volumetric flask and diluted to the mark with water. 2.5 Supernatant Purification 30 mL of supernatant containing iodine-substituted polysaccharides was separated by medium-pressure preparative liquid chromatography (CHEETAHTM MP, Agela, Tianjun, China) on a Sephadex G-10 column (5×50 cm) and eluted using distilled water. The main fraction was pooled by carbohydrate colourimetric detection method and electrical conductivity testing. 20 mL of the main fraction (EPA-1FI) were collected, concentrated and lyophilized. 2.6 Stability Study of EPA-1FI in vivo and in vitro For the in vitro assay, 100 mg of EPA-1FI were dissolved in 50 mL of cell culture (RPM1640) in a shaker-incubator at the 37 °C. At 1, 2 and 3 h, 10 mL of this cell culture were taken out of the shaker-incubator at the 37 °C and stored. For the in vivo assay, the blood, collected from rats that had been orally administered EPA-1FI, was dissolved in pure water to investigate the stability of iodine-substituted polysaccharides in vivo. The samples obtained for the in vivo and in vitro studies were separated on a Sephadex G-50 column (1.2×30 cm) and eluted using distilled water. All fractions, excluding extracellular water, were collected to analyze the content of iodine by ICP-MS. The volume of intracellular and extracellular water was marked with blue dextran and glucose.
2.7 Samples Digestion Individual samples, probably EPA-1FI or biological samples, were placed in 10 mL headspace sampling bottles. Subsequently, 4 mL of H2O, 3 mL of 25% tetramethylammonium hydroxide (TMAH) and 0.1 mL of H2O2 were added to the sampling bottles. The bottles were closed tightly using a capping device. The samples were digested by ultrasonic treatment at 80 °C for 30 min. After digestion and cooling, the reactant in headspace sampling bottle was transferred to a 25 mL flask, using 1% TMAH solution as a control. 2.8 ICP-MS Analysis Method ICP-MS was performed using Agilent 7700 (Agilent, Palo Alto, CA, USA). Tuning solution contained 7Li, 59Co, 89Y, 140Ce, 205Ti. The internal standard was 1mg/mL indium solution. The conditions of detection were same as described in published literature [12]. The ICP-MS system was washed for 90 seconds between injections to reduce memory effects. 2.9 Determination of the Rate of Iodine Substitution of EPA-1 10 mg EPA-1FI was digested as described in section 2.7. The samples were diluted 1000 times and filtered through a 0.45 µm filter membrane before ICP-MS determination. The formula of the iodine substitution rate of polysaccharides was as follows. RI =
×
× 100%
Note: RI: iodine substitution rate M: iodine content of sample measured from the ICP-MS D: dilution times G: weight of EPA-1FI
2.10 Determination of EPA-1 content in blood and organ samples All the SD rats were fed for three days and fasted for 12h, with water ad libitum, before experiment. Twenty rats were randomly divided into two groups including the control and drug groups. The rats were orally administered EPA-1FI (dosage, 100 mg/100g) in the drug group, while the control group received the same volume of pure water. About 1 mL of blood were collected from the eye vein of diethyl ether-anesthetized rats, at 1 and 2 hours after administration. At 3 hours, the rats were anesthetized with diethyl ether and then sacrificed. The samples of blood from abdominal aorta and organs, such as liver, spleen, kidney, lung and heart were collected. After repeatedly rinsing the organs using saline solution, these were freeze-dried and powdered. 1 mL of blood or 100 mg of powdered organs were placed in a headspace sampling bottle for digestion and prior determination were diluted to a suitable multiple to make them in linear range respectively. At last, samples were analyzed using ICP-MS. The formula for the content of polysaccharides in blood and organ respectively was as follows. Cb = Co = R=
(
×
×L×10-6
×
× )
×10-6
× 100%
Note: Cb: the content of EPA-1 in blood of rat Co: the content of EPA-1 in organ. R: the absorptivity of blood or organ of EPA-1 L: rat blood volume. In general, the blood volume of rats is 7.4% of the body weight. Wo: the weighe of rat organ Y: the dose of oral administration to rat
3. Results 3.1 The reaction of FITC labelling and iodine substitution of polysaccharides In general, iodine can react with amino acids in proteins. Therefore, while glycoproteins can be directly substituted by iodine, that is not the case for polysaccharides. Therefore, in this study, the polysaccharide was initially attached to FITC, and subsequently, EPA-1 was replaced by iodine. In this way, the polysaccharides are indirectly determined by iodine measurements. The reaction mixture was purified using gel-filtration in order to eliminate free iodine and excess small molecule reagents in case these interfered with the sample content. The elution pattern (Figure 1) showed the 10-21 fractions that could be detected by the phenol-sulfuric colourimetric method. Also, it showed that salts, including reagents salts, were eluted from the column after fraction 34 as detected by electric conductivity. Thus, the 18-32 fractions (corresponding to EPA-1FI) were collected, concentrated and dried. 3.2 The Stability of Iodinated Polysaccharides The stability of the iodine label is very important when detecting iodinated sugar complexes in biological samples. In our study, the stability of complexes was tested in vitro and in vivo and the portion of small molecules was collected using a Sephadex G-50 column according to intracellular and extracellular water volume marked by glucose and blue dextran. The results showed no free iodine was detected in the collected fractions using ICP-MS regardless of the samples being of in vivo or in vitro origin, suggesting that the EPA-1FI was stable in biologic samples.
3.3 Optimization of Digestion Method Biological samples should be digested in order to release iodine before content determination. To improve sample digestion, three methods including microwave, acid, and alkali digestion were compared. We found that the method based on alkali digestion was the most stable and the easiest to perform and therefore, it was chosen for this study. 3.4 Method Validation and Determination of the Rate of Iodine substitution of EPA-1 For the assessment of linearity and linear range, a standard iodide gradient was made by NaI using a series of concentrations from a stock solution of 1 % TMAH, including 1 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL and 300 ng/mL of iodine solution. The iodine content was detected by ICP-MS as described in section 2.8. The standard curve equation was y=1.0058X+0.1001 with a correlation coefficient of 0.9999, and it showed satisfactory linearity in the range of 0-300 ng/mL. In the precision experiment, the relative standard deviation (RSD) of iodine measurements was 0.7% which indicates high instrument precision. For the repeatability tests, samples of EPA-1FI were digested and analyzed on ICP-MS in replicates of three, and the RSD was 0.89 % (n=6), which indicates good repeatability for this method. For the stability tests, a digested sample was placed at room temperature and iodine determinations were made at 2, 4, 8, 12, 24 and 48h. The results show that this method was stable within 48 hours (RSD=1.25%). In addition, this method had good accuracy with recoveries in the range of 96.4–104% (RSD, 2.2%). The iodine substitution rate of the three replicate polysaccharide samples were
6.20%, 7.15% and 6.89%. The results show that this newly developed method for the quantitative determination of the iodinated polysaccharides is accurate and therefore, it was used for the analysis of biological samples in this study. 3.5 Detection of EPA-1 in Biological Samples 3.5.1 EPA-1 Content in Rat Blood Using the developed standard curve, the concentration of iodine was read from the instrument directly, and the content of EPA-1 and EPA-1 absorption rate in rat blood is shown in Table 1. These results showed that 1h after treatment, the absorption rate in blood was at its highest, reaching 5.84%. From then, the absorption rate in blood decreased. 3.5.2 EPA-1 Content in Rat Organs 3 hours after of administration, the content and distribution of EPA-1 in the organs of rats was shown in Table 2. EPA-1 had a differential distribution in primary organs at 3h. Specifically, the spatial distribution of EPA-1 in the spleen was the highest, while the EPA-1 content per unit of weight was the highest in the lung. In ascending order, depending on the amount of EPA-1FI of organ, the main organs were listed as spleen> liver > kidney > lung >heart. In ascending order, depending on the amount per unit of weight, the main organs were listed as lung > spleen > kidney > heart > liver. The total absorption rate in the viscera was very low, only 0.9%. In total, approximately 5 % of EPA-1 could be found in the rat body, suggesting that the absorption and utilization rate of oral polysaccharides in vivo is very low, which is
one of the obstacles to further development of new polysaccharide drugs.
4. Conclusion The detection of polysaccharides in vivo has been one of the bottlenecks in the study of pharmacokinetics of polysaccharides. In this study, we successfully developed a new method to determine the content of polysaccharides in the biological samples. At first, the polysaccharide was indirectly linked with iodine by FITC to form the complex of iodine-FITC-polysaccharide. Then, the stability of the purified complex was investigated in vivo and in vitro. Next, the content of iodine was determined using ICP-MS, in this case, to indirectly assess the amount of polysaccharide. The iodine substitution rate of three replicate batches of EPA-1FI was measured and was 6.20%, 7.15% and 6.89% using validated method. In order to further validate the method and investigate the absorption rate of polysaccharides in vivo, EPA-1FI were orally administered to rats. The contents of polysaccharides in the blood of rats at 1h, 2h and 3h after administration and the distribution of polysaccharides in the main organs at 3h were measured. The results showed that after 1h, the absorption rate in blood was at its highest, reaching 5.84%. After 3h, EPA-1FI have a differential distribution in the different organs. The total absorbance of EPA-1FI in vivo was very low, which emphasized polysaccharides low bioavailability after oral administration. In summary, this method for polysaccharides content determination in biological samples is accurate, reliable and reproducible. Acknowledgements This research work was supported by the National Foundation of Major Drug
Discovery (grant 20142x09301306-10) and the Foundation of New Drug Research and Development (grant: 20140203003YY), Jilin Province, China.
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Figure captions Fig. 1. Purification of EPA-1FA on Sephadex G-50 column
Note: Vo and Ve was internal and external water volume determined by glucose and glucose, tespectively.
S0141-8130(17)33314-7 http://dx.doi.org/10.1016/j.ijbiomac.2017.09.045 BIOMAC 8222
To appear in:
International Journal of Biological Macromolecules
Received date: Accepted date:
30-8-2017 14-9-2017
Please cite this article as: Duoduo Xu, Wei Zheng, Yanqiu Zhang, Qipin Gao, Mingxing Wang, Yang Gao, A method for determining polysaccharide content in biological samples, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.09.045 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A method for determining polysaccharide content in biological samples Duoduo Xuab, Wei Zhengc, Yanqiu Zhangd，Qipin Gaoab, Mingxing Wangd, Yang Gaoab, * a Center of Research & Development, Changchun University of Chinese Medicine, Changchun, Jilin 130117, China b Macromolecule of Chinese Medicine Key Lab of Jilin Province, Changchun 130117, Jilin, China c Departments of Ophthalmology, The Affiliated Hospital, Changchun University of Chinese Medicine, Changchun, Jilin 130021, China d Preparation center, The Affiliated Hospital, Changchun University of Chinese Medicine, Changchun, Jilin 130021, China
Abstract: Difficulties in the determination of polysaccharide content in biological samples has been one of the bottlenecks limiting the development of polysaccharides pharmacokinetics, greatly due to the complicated chemical structure of these compounds. In this study, we established a simple, reproducible and reliable method for the determination of polysaccharide content in biological samples. Polysaccharides were replaced by fluorescein isothiocyanate (FITC) and iodine to generate a complex. The iodine content in the complex was measured by inductively coupled plasma-mass spectrometry (ICP-MS) to indirectly reflect polysaccharide content. We investigated the stability of the complex, carried on methodological validation and proceeded to determine the polysaccharide content in rat blood and organs after oral administration of iodine substituted polysaccharides using ICP-MS. The results showed that the iodine complex was stable in vivo and in vitro. In the bioavailability of polysaccharides, after 1h, the absorption rate of polysaccharides in blood was the highest, reaching 5.84%. After 3h, the bioaccessibility was differently distributed in various organs, reaching 1% on average across organs.
Keywords: Polysaccharide, Iodine substitution, FITC, ICP-MS; Biological sample 1. Introduction In recent years, polysaccharides have received increased attention due to their broad application potential, including the development of new drugs and health products. Having relatively low toxicity and minor side effects [1], their biological activities include hepatoprotection [2], antioxidant effects [3], antitumour properties [4], lipid-accumulation inhibition [5], and anti-inflammatory [6]. However, in natural medicine, the study of polysaccharides has been difficult due to their very complex chemical structure, large molecular size, strong polarity and intricate spatial structure. Polysaccharide separation, purification, characterization and determination procedures are challenging, which hinders the development and use of polysaccharide drugs compared to other compounds. Determination of polysaccharide content in biological samples remains a major bottleneck in the development of polysaccharide pharmacokinetics, including the study of the mechanisms of action of novel polysaccharide-based drugs, and the design and optimization of dosage regimens. The methods used for detection of polysaccharides in biological samples include chromatography [7.8], radioactive isotope labelling [9-11], fluorescence assay [12-14] and the ELISA assay [15,16]. Currently, these methods present important limitations, such as low sensitivity and specificity. In addition, radioactive isotope labelling requires a high-tech operating environment and has low operability. Here, we established a simple, reliable and reproducible method for the determination of polysaccharide content in biological samples. Previously, our group developed a
method for glycoprotein content determination in biological samples [17]. In this method, glycoprotein was substituted by iodine to form an iodide-glycoprotein complex. Then, the iodine content in the iodide-glycoprotein complex was further assayed by ICP-MS, which is a technique widely used in qualitative and quantitative analysis of inorganic elements. In the present study, we prepared stable polysaccharide-iodine complexes as in with slight modifications and carried out a method validation procedure. We then used this method to determine polysaccharide content of in rat blood and organs. 2. Materials and methods 2.1. Materials and chemicals The polysaccharide (EPA-1) was isolated by ourselves from Pleurotus eryngii supplied by Mushroom and Vegetable base of Jilin Agricultural University (Jilin, China). Sephadex G-50 was purchased from the GE Healthcare Ltd. (Chalfront St. Guiles, UK). Cell culture (RPM 450) was purchased from Thermo, Rockford, IL USA. The no iodine feeds of SPF rat were purchased from Beijing Aokexieli Feed Co., Ltd. (Beijing, China). The rats were purchased from College of Pharmacy, Jilin University (Jilin, China), certification of conformity for the SCXK (2012-0003). Several reagents were marked in articles, and other reagents were of analytical grade (China). 2.2. Sample preparation A sample of polysaccharide EPA-1 was obtained from the mushroom Pleurotus eryngii and used in this study because its physical and chemical properties, structure and biological activity had already been characterized. EPA-1 is a homogeneous
polysaccharide with 98.6% sugar content, with no acidic sugar or protein components, and a molecular weight of 1.0 × 105 Da. It consists of mannose, glucose and galactose, and the main linkage mode was the (1→6)-linked galactose residue. Activity tests indicated that EPA-1 possessed good immunoregulatory activity [18]. 2.3. The Reaction of FITC labelling of Polysaccharides The method described in Belder [19] was used. First, 1g of EPA-1 was dissolved in a 10mL methyl sulphoxide solution plus five drops of pyridine. The sample was then mixed ultrasonically. Secondly, 0.1g FITC and 20mg dibutyltin dilaurate were added to the mixture, which was vigorously shaken and heated for 2h at 95 ℃. Finally, after cooling, a volume of 95 % (v/v) ethanol was slowly added to the mixture with constant stirring until the ethanol content of the mixture was 85%. This mixture was centrifuged and the precipitate repeatedly cleaned by ethanol washes. The precipitate containing the complex (EPA-1F) formed by EPA-1 and FITC was dried and stored. 2.4. Iodine Substitution Reaction The reaction for iodine substitution was performed according to Keck [20] with small modifications. The dried EPA-1F extract (1g) was dissolved in 5 mL of water by ultrasonic mixing. Next, 2 mL of NaI (0.5 mol/L) and 5 mL of chloramine T buffer solution (0.1 g/mL) were added to mixture, followed by mixing for 1 min on the turbine mixer. After this, 5 mL of sodium metabisulfite buffer solution (0.1 g/mL) and 5 mL of potassium iodide (0.1 g/mL) were added to the mixture and dissolved by shaking and mixing. The mixture was centrifuged (5000 rpm, 5 min), the supernatant was collected to get the iodine-substituted Pleurotus eryngii polysaccharides. We
prepared three replicates. The buffer solution was prepared by adding 81mL of disodium hydrogen phosphate (71.6mg/mL) and 19mL of sodium dihydrogen phosphate (31.2mg/mL) to a 1000 mL volumetric flask and diluted to the mark with water. 2.5 Supernatant Purification 30 mL of supernatant containing iodine-substituted polysaccharides was separated by medium-pressure preparative liquid chromatography (CHEETAHTM MP, Agela, Tianjun, China) on a Sephadex G-10 column (5×50 cm) and eluted using distilled water. The main fraction was pooled by carbohydrate colourimetric detection method and electrical conductivity testing. 20 mL of the main fraction (EPA-1FI) were collected, concentrated and lyophilized. 2.6 Stability Study of EPA-1FI in vivo and in vitro For the in vitro assay, 100 mg of EPA-1FI were dissolved in 50 mL of cell culture (RPM1640) in a shaker-incubator at the 37 °C. At 1, 2 and 3 h, 10 mL of this cell culture were taken out of the shaker-incubator at the 37 °C and stored. For the in vivo assay, the blood, collected from rats that had been orally administered EPA-1FI, was dissolved in pure water to investigate the stability of iodine-substituted polysaccharides in vivo. The samples obtained for the in vivo and in vitro studies were separated on a Sephadex G-50 column (1.2×30 cm) and eluted using distilled water. All fractions, excluding extracellular water, were collected to analyze the content of iodine by ICP-MS. The volume of intracellular and extracellular water was marked with blue dextran and glucose.
2.7 Samples Digestion Individual samples, probably EPA-1FI or biological samples, were placed in 10 mL headspace sampling bottles. Subsequently, 4 mL of H2O, 3 mL of 25% tetramethylammonium hydroxide (TMAH) and 0.1 mL of H2O2 were added to the sampling bottles. The bottles were closed tightly using a capping device. The samples were digested by ultrasonic treatment at 80 °C for 30 min. After digestion and cooling, the reactant in headspace sampling bottle was transferred to a 25 mL flask, using 1% TMAH solution as a control. 2.8 ICP-MS Analysis Method ICP-MS was performed using Agilent 7700 (Agilent, Palo Alto, CA, USA). Tuning solution contained 7Li, 59Co, 89Y, 140Ce, 205Ti. The internal standard was 1mg/mL indium solution. The conditions of detection were same as described in published literature [12]. The ICP-MS system was washed for 90 seconds between injections to reduce memory effects. 2.9 Determination of the Rate of Iodine Substitution of EPA-1 10 mg EPA-1FI was digested as described in section 2.7. The samples were diluted 1000 times and filtered through a 0.45 µm filter membrane before ICP-MS determination. The formula of the iodine substitution rate of polysaccharides was as follows. RI =
×
× 100%
Note: RI: iodine substitution rate M: iodine content of sample measured from the ICP-MS D: dilution times G: weight of EPA-1FI
2.10 Determination of EPA-1 content in blood and organ samples All the SD rats were fed for three days and fasted for 12h, with water ad libitum, before experiment. Twenty rats were randomly divided into two groups including the control and drug groups. The rats were orally administered EPA-1FI (dosage, 100 mg/100g) in the drug group, while the control group received the same volume of pure water. About 1 mL of blood were collected from the eye vein of diethyl ether-anesthetized rats, at 1 and 2 hours after administration. At 3 hours, the rats were anesthetized with diethyl ether and then sacrificed. The samples of blood from abdominal aorta and organs, such as liver, spleen, kidney, lung and heart were collected. After repeatedly rinsing the organs using saline solution, these were freeze-dried and powdered. 1 mL of blood or 100 mg of powdered organs were placed in a headspace sampling bottle for digestion and prior determination were diluted to a suitable multiple to make them in linear range respectively. At last, samples were analyzed using ICP-MS. The formula for the content of polysaccharides in blood and organ respectively was as follows. Cb = Co = R=
(
×
×L×10-6
×
× )
×10-6
× 100%
Note: Cb: the content of EPA-1 in blood of rat Co: the content of EPA-1 in organ. R: the absorptivity of blood or organ of EPA-1 L: rat blood volume. In general, the blood volume of rats is 7.4% of the body weight. Wo: the weighe of rat organ Y: the dose of oral administration to rat
3. Results 3.1 The reaction of FITC labelling and iodine substitution of polysaccharides In general, iodine can react with amino acids in proteins. Therefore, while glycoproteins can be directly substituted by iodine, that is not the case for polysaccharides. Therefore, in this study, the polysaccharide was initially attached to FITC, and subsequently, EPA-1 was replaced by iodine. In this way, the polysaccharides are indirectly determined by iodine measurements. The reaction mixture was purified using gel-filtration in order to eliminate free iodine and excess small molecule reagents in case these interfered with the sample content. The elution pattern (Figure 1) showed the 10-21 fractions that could be detected by the phenol-sulfuric colourimetric method. Also, it showed that salts, including reagents salts, were eluted from the column after fraction 34 as detected by electric conductivity. Thus, the 18-32 fractions (corresponding to EPA-1FI) were collected, concentrated and dried. 3.2 The Stability of Iodinated Polysaccharides The stability of the iodine label is very important when detecting iodinated sugar complexes in biological samples. In our study, the stability of complexes was tested in vitro and in vivo and the portion of small molecules was collected using a Sephadex G-50 column according to intracellular and extracellular water volume marked by glucose and blue dextran. The results showed no free iodine was detected in the collected fractions using ICP-MS regardless of the samples being of in vivo or in vitro origin, suggesting that the EPA-1FI was stable in biologic samples.
3.3 Optimization of Digestion Method Biological samples should be digested in order to release iodine before content determination. To improve sample digestion, three methods including microwave, acid, and alkali digestion were compared. We found that the method based on alkali digestion was the most stable and the easiest to perform and therefore, it was chosen for this study. 3.4 Method Validation and Determination of the Rate of Iodine substitution of EPA-1 For the assessment of linearity and linear range, a standard iodide gradient was made by NaI using a series of concentrations from a stock solution of 1 % TMAH, including 1 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL and 300 ng/mL of iodine solution. The iodine content was detected by ICP-MS as described in section 2.8. The standard curve equation was y=1.0058X+0.1001 with a correlation coefficient of 0.9999, and it showed satisfactory linearity in the range of 0-300 ng/mL. In the precision experiment, the relative standard deviation (RSD) of iodine measurements was 0.7% which indicates high instrument precision. For the repeatability tests, samples of EPA-1FI were digested and analyzed on ICP-MS in replicates of three, and the RSD was 0.89 % (n=6), which indicates good repeatability for this method. For the stability tests, a digested sample was placed at room temperature and iodine determinations were made at 2, 4, 8, 12, 24 and 48h. The results show that this method was stable within 48 hours (RSD=1.25%). In addition, this method had good accuracy with recoveries in the range of 96.4–104% (RSD, 2.2%). The iodine substitution rate of the three replicate polysaccharide samples were
6.20%, 7.15% and 6.89%. The results show that this newly developed method for the quantitative determination of the iodinated polysaccharides is accurate and therefore, it was used for the analysis of biological samples in this study. 3.5 Detection of EPA-1 in Biological Samples 3.5.1 EPA-1 Content in Rat Blood Using the developed standard curve, the concentration of iodine was read from the instrument directly, and the content of EPA-1 and EPA-1 absorption rate in rat blood is shown in Table 1. These results showed that 1h after treatment, the absorption rate in blood was at its highest, reaching 5.84%. From then, the absorption rate in blood decreased. 3.5.2 EPA-1 Content in Rat Organs 3 hours after of administration, the content and distribution of EPA-1 in the organs of rats was shown in Table 2. EPA-1 had a differential distribution in primary organs at 3h. Specifically, the spatial distribution of EPA-1 in the spleen was the highest, while the EPA-1 content per unit of weight was the highest in the lung. In ascending order, depending on the amount of EPA-1FI of organ, the main organs were listed as spleen> liver > kidney > lung >heart. In ascending order, depending on the amount per unit of weight, the main organs were listed as lung > spleen > kidney > heart > liver. The total absorption rate in the viscera was very low, only 0.9%. In total, approximately 5 % of EPA-1 could be found in the rat body, suggesting that the absorption and utilization rate of oral polysaccharides in vivo is very low, which is
one of the obstacles to further development of new polysaccharide drugs.
4. Conclusion The detection of polysaccharides in vivo has been one of the bottlenecks in the study of pharmacokinetics of polysaccharides. In this study, we successfully developed a new method to determine the content of polysaccharides in the biological samples. At first, the polysaccharide was indirectly linked with iodine by FITC to form the complex of iodine-FITC-polysaccharide. Then, the stability of the purified complex was investigated in vivo and in vitro. Next, the content of iodine was determined using ICP-MS, in this case, to indirectly assess the amount of polysaccharide. The iodine substitution rate of three replicate batches of EPA-1FI was measured and was 6.20%, 7.15% and 6.89% using validated method. In order to further validate the method and investigate the absorption rate of polysaccharides in vivo, EPA-1FI were orally administered to rats. The contents of polysaccharides in the blood of rats at 1h, 2h and 3h after administration and the distribution of polysaccharides in the main organs at 3h were measured. The results showed that after 1h, the absorption rate in blood was at its highest, reaching 5.84%. After 3h, EPA-1FI have a differential distribution in the different organs. The total absorbance of EPA-1FI in vivo was very low, which emphasized polysaccharides low bioavailability after oral administration. In summary, this method for polysaccharides content determination in biological samples is accurate, reliable and reproducible. Acknowledgements This research work was supported by the National Foundation of Major Drug
Discovery (grant 20142x09301306-10) and the Foundation of New Drug Research and Development (grant: 20140203003YY), Jilin Province, China.
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Figure captions Fig. 1. Purification of EPA-1FA on Sephadex G-50 column
Note: Vo and Ve was internal and external water volume determined by glucose and glucose, tespectively.