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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
  • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y402/00—Carbon-oxygen lyases (4.2)
  • C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
  • C12Y402/02001—Hyaluronate lyase (4.2.2.1)

Definitions

• the invention relates to the method of determination of hyaluronic acid in complex samples by cleavage with SpnHyl enzyme followed by the reaction with 3-methyl-2- benzothiazolinonhydrazone (MBTH) to form a coloured product analysed by
• glycosaminoglycans these can be separated by precipitation before the cleavage, e.g. with use of cetyltrimethylammoniumbromide (CTAB) in acetate buffer and salt medium.
• CTAB cetyltrimethylammoniumbromide
• high content of reducing sugars e.g. glucose
• their influence can be further eliminated, after removing the glycosaminoglycans and before the cleavage, by precipitating hyaluronic acid from the formed supernatant and then redissolving this sediment in acetate buffer and performing the cleavage step.
• Hyaluronic acid (hyaluronan, hyaluronate, HA) is a linear glycosaminoglycan consisting of alternating molecules of glucuronic acid and N-acetylglucosamine. It is one of the basic building materials of connective tissue thanks to its exceptional rheological and mechanical properties.
• hyaluronic acid is widely used in pharmaceutical and cosmetics industry (Shimmura et al. 1995; Kogan et al. 2007); its production has been constantly increasing.
• the concentration of hyaluronic acid in blood serves as a biological marker of physiological state of joints and the whole moving apparatus.
• many methods of determination of hyaluronic acid concentration have been developed, differing in both their sensitivity and basic principles.
• the methods of determination of HA can be divided into nanogram, microgram, and milligram scales. According to the principle, the methods of determination of HA can be divided into the following categories:
• Gravimetric methods belong to the oldest and the least accurate methods, where HA is precipitated with alcohol, the precipitate is then dried and weighed. The processes of precipitation and drying are slow, which takes up to two days. The sensitivity of the method is low, usually hundreds of milligrams per litre, moreover, other polysaccharides can precipitate concurrently during the precipitation. Gravimetry is conventionally used in biotechnological production of HA, where the composition of the culture medium is known and constant, other polysaccharides are absent, and the analysis time does not play an important role.
• Turbidimetric methods exploit the properties of the complexes of glycosaminoglycans and aliphatic ammonium salts comprising at least one long aliphatic chain (cetylpyridinium chloride - CPC, or cetyltrimetylammonium bromide - CTAB). Under certain conditions these complexes form very stable colloid solutions.
• the content of HA in a sample can be determined according to the level of turbidity of the colloid solution of "HA - aliphatic ammonium salt" (Oueslati et al., 2014). This method has a great advantage of a very short time of analysis.
• the first colorimetric methods for determination of HA were based on the methods for detection of specific monosaccharides, such as N-acetylglucosamin in Elson-Morgan method with the Reissig's modification (Elson&Morgan, 1933; Reissig et al., 1955), and D-glucuronic acid in Dische method
• HA being the polysaccharide, must be depolymerized into monosaccharides prior the colour reaction. Depolymerization is performed by 80% sulfuric acid at 60 °C (Dische, 1947), or 2N-hydrochloric acid at 100 °C (Reissig et al., 1955). In some
• HA is cleaved with a lyase from S. hyalurolyticus into 4, 5 -unsaturated oligosaccharides. These are subsequently oxidized with periodic acid to formylpyruvate that reacts with
• HA thiobarbiturate.
• the sensitivity of this method is comparable with the Elson-Morgan method, but it is more user friendly.
• the drawbacks of the method are "slow" hyaluronan lyase and the use of highly toxic arsenic compounds.
• Chromatographic methods use a direct detection of HA at 201 nm, or at 232 nm in case of unsaturated HA forms (Gassier et al., 1993; Alkrad et al., 2002; Ruckmani et al., 2013). In both cases HA can be determined in pure solutions only, whereas the sensitivity is slightly lower than in colorimetric methods. On the contrary, the chromatography has the advantage of determination of molecular weight of HA. That is the main reason why the
• Spectrophotometry is considered to be a very accurate and sensitive tool. At the same time, spectrophotometry is relatively cheap and simple and thus is widely used in laboratories. In order to keep the affordable price while maintaining the high sensitivity, the spectrophotometric determination of HA must be based on the reaction forming an intensively coloured product that absorbs at longer wave lengths. There are many agents reacting with saccharides to form coloured products. Some of them have been used for determination of other polysaccharides, such as metyl-2-benzothiazolinonhydrazon (MBTH) which is characterized by a low interaction with salts, proteins, and nucleic acids (Moretti&Thorson, 2002).
• MTH metyl-2-benzothiazolinonhydrazon
• MBTH has been used primarily for the determination of aldehydes (Hauser&Cummins, 1964).
• the analysed aldehyde condenses with MBTH, and then follows the conversion into formazan upon reaction with the diazonium salt which is generated in the reaction medium by oxidation of the excess reagent. (Scheme 1). That forms highly a coloured product with the highest absorbance at620 nm (blue colour).
• HA of various molecular weight 24, 73, 211, 400, 549, 875, 1690, and 2049 kDa
• MBTH Anthon-Barrett method protocol
• Controlled degradation of HA leading to the formation of only one low molecular product solves simultaneously three problems.
• HA completely degraded in less than 5 minutes.
• the product of the first condensation of ⁇ 2 and MBTH is a yellow intermediate having the maximum absorbance at 390 nm.
• this yellow product can be used for the detection without the necessity of a further reaction.
• different amounts of HA cleaved in 200 were mixed with 200 ⁇ ⁇ of 1M NaOH and 600 ⁇ of 0.1 % (w/v) MBTH. Then the reaction mixture was heated at different temperatures from 60 to 95 °C. The samples were then cooled to room temperature and measured at 390 nm. It was found that the dependence of the absorbance on the concentration is not linear, the intensity of colouring is not stable during the time, and the sensitivity of the reaction is very low (extinction coefficient of 1100 mol "1 ). This experiment proved the necessity of the second reaction.
• the measurement error was less than 1%.
• the first reaction is performed at 75°C, for not less than 30 and not more than 50 minutes. Such conditions offer allowance of ⁇ 2°C and the sufficient time for the manipulation with the samples, which significantly improves the analysis repeatability and robustness.
• sample containing HA is mixed with the same volume of lOOmM acetate buffer pH 6 with lOOmM of NaCl.
• the resulting 400 ⁇ is divided into two equal parts of 200 ⁇ ,. One is marked as the “sample” (S), the other as the
• the absorbance is measured at 654 nm. After subtracting the absorbance of the "background” from the absorbance of the "sample", the concentration of HA can be calculated based of the extinction coefficient.
• the hyaluronan-based medical remedies and nutritional supplements for joint care and support usually contain high amounts of other glycosaminoglycans along with HA, the most often it is chondroitin sulfate (CS) (Table 1). Glycosaminoglycans have similar physical and chemical properties and play a similar role within the organism, especially in connective tissues. Therefore, the next step of investigation was to determine conditions of the SpnHyl- MBTH method which would be suitable for HA detection in the presence of high amounts of CS.of SpnHyl-MBTH
• the HA-CTAB complex was soluble in 50 mM acetic buffer pH 6 at concentrations of Na 2 S0 higher than 76 mM, whereas CS- CTAB dissolves at concentrations higher than 700 mM of Na 2 S0 4 ..
• the analysed sample is premixed with the same volume of 160mM Na 2 S0 4 in lOOmM acetate buffer of pH 6 (referred to as the "reagent A") - in this case 100 and 100 ⁇ .
• the thus treated sample is then mixed with l%w/v CTAB in 80mM Na 2 S0 4 , 50mM acetate buffer of pH 6 (referred to as the "reagent B").
• the hindering low-molecular-weight impurities such as reducing sugars could be removed by fractioning the glycosaminoglycans with the CTAB precipitation.
• the CS is removed by precipitation as described above.
• the object of the invention is a method of determination of hyaluronic acid in a sample comprising the following steps:
• the solution of a sample is prepared by mixing with acetate buffer containing monovalent salt so that pH of the final solution is within the range of 5 to 7, the concentration of acetate buffer is within the range of lOmM to 1M, and the ionic strength of the mixture corresponds to the solutions of monovalent salts with the concentration between 10 mM and 1M,
• an acidic solution of a ferric salt comprising citric acid and sulfamic acid is added to the mixture, the mixture is then cooled for 5 to 15 minutes to the room temperature,
• the solution of MBTH in step c) also includes dithiotreitol (DTT) in the concentrations 2-20mM.
• the monovalent salt in the step a) is preferably in the concentration 50 mM and is selected from the group comprising NaCl, KC1, CH 3 COONa, NaN0 3 , Na 2 S0 4 , and K 2 S0 4 .
• 1M aqueous solution of NaOH, KOH, or LiOH is preferably added as an alkali, wherein its final concentration in the reaction mixture is 0.25M.
• step d 50mM aqueous solution of FeCl 3 , 0.2M aqueous solution of citric acid, and l%w/v aqueous solution of sulfamic acid are added in step d).
• the step a) comprises a set of the following sub-steps:
• the sample is mixed with acetate buffer and aqueous solution of Na 2 S0 4 or K 2 S0 4 so that the final concentration of acetate buffer after mixing is in range of lOmM to lOOmM and the concentration of the sulfate is between 75 and 300mM,
• a2) 0.5-2%w/v aqueous solution of cetyltrimethylammoniumbromide or cetylpyridiniumchloride, comprising the same concentrations of acetate and sulfate as the sample after mixing in step al), is added to the sample solution from step al) in order to precipitate anionic glycosaminoglycans with a negative charge higher than HA, which lead to the sediment formation,
• step a3) the sediment formed in step a2) is removed.
• Na 2 S0 4 is added in steps al) and a2) so that its final concentration in the sample solution after mixing is 80mM, and acetate buffer is added so that its concentration in the sample solution after mixing is 50mM and pH is 6, and then the sediment is removed by centrifugation in step a3).
• step a4) an alkali, in an amount sufficient for reaching pH >12, is added to the supernatant obtained in step a3) to precipitate hyaluronic acid in form of a sediment,
• step a5) the sediment formed in step a4) is isolated and washed with 5-20mM of an alkali solution,
• the sediment from step a5) is dissolved in acetate buffer with the concentration from 10 to 100 mM, pH between 5.5 and 6.5, and containing sodium sulfate or potassium sulfate in the concentration 100-300 mM.
• the alkali in step a4) is selected from the group comprising NaOH, KOH, and LiOH, the concentration of the alkali is 1M, and the added volume is 20 to 40 ⁇ , per 200 to 500 ]xL of the supernatant, preferably 25 ⁇ ⁇ of 1M NaOH per 400 ⁇ , of supernatant.
• the sediment is separated preferably by centrifugation in step a5) and is washed with 5-20mM alkali selected from the group comprising NaOH, KOH, and LiOH, more preferably it is washed at least two times with lOmM NaOH.
• the acetate buffer in step a6) preferably has pH 6, its concentrationis lOOmM, and the sulfate is preferably Na 2 S0 4 with the concentration 160 mM.
• the concentration C(HA) of hyaluronic acid in the sample in mg/mL is calculated in step f) preferably according to the formula: r j r & _ ⁇ A s -Ab)*V ⁇ colored sample ⁇ )*M( HA2)
• a s is the absorbance of the sample
• Ab is the absorbance of the background taking into account the dilution after the colour development
• V(colored sample) is the final volume of the reaction mixture
• ⁇ ( ⁇ 2) is the molecular weight of the unsaturated disaccharide of hyaluronic acid, 379 g/mol
• ⁇ is the extinction coefficient, 34735 mol "1
• V(HA sample) is the volume of the original sample in the final volume of the reaction mixture.
• the activity of SpHyl was assayed on pure 0.1% HA, pure 0.1-2% chondroitin sulfate (CS) or on their mixes with constant 0.1% HA and 0.1-2% CS in a variety of buffers, which, however, always contained 50 mM sodium acetate buffer pH 6.
• the activity of SpHyl was defined as an increase in samples absorbance at 232 nm referred to 1 mg of enzyme.
• the slope of the linear part of the kinetic curve corresponded to enzyme activity according to the following formula: ⁇ 23 2 ⁇ 1000 T T r , -.
• Activity — [U/mg ] where ⁇ 232 - absorbance difference between the sample and background at 232 nm; At - time, min; ⁇ - extinction coefficient, 5500 mol.l ⁇ .cm "1 ; C - protein concentration, mg/mL.
• the unit of the activity is defined as the amount of enzyme necessary for the formation of one ⁇ of unsaturated bonds in 0.1%w/v HA at pH 6 at 37°C in one minute.
• the complete protocol was prepared for the HA samples contaminated with high concentrations of sulfated glycosaminoglycans (up to 5%w/v of CS) and reducing sugars (up to 5 %w/v of glucose). It describes the most preferred conditions of the method according to the invention.
• the process volumes were optimized for the standard spectrophotometric cell with the light path of 10 mm and volume approx. 1 mL.
• a s and Ab are absorbances of the "sample” and “background", taking into account the dilution after the colour development;
• V(colored sample) is the final volume of the reaction mixture, it is 1.3 mL for this protocol;
• M(AHA2) is the molecular weight of unsaturated disaccharide, 379 g/mol;
• ⁇ is the extinction coefficient, 34735 mol "1 ;
• V(HA sample) is the volume of the sample in the final volume of the reaction mixture, 0.04 mL for this protocol.
• the original protocol can be simplified, if the sample comprises small amounts of reducing sugars.
• concentration of the reducing sugars can be considered low, if the absorbance of "background" at 654 nm is less than 0.7.
• the absorbance of the "background” is determined as follows: mix 100 ⁇ of the sample with 100 of the solution A; add 200 ⁇ of the solution C and 400 ⁇ L of the solution D; incubate for 5 minutes at 95°C; let it cool and measure at 654 nm.
• steps 4-7 can be skipped and 200 ⁇ ⁇ of the supernatant from step 3 are pipetted to both the "sample” and the "background” tubes, then the procedure continues from the step 8 to 12 of the complete protocol.
• the modifications do not affect the formula; HA concentration is calculated in the same way as in the complete protocol.
• the protocol can be simplified further.
• concentration of sulfated glycosaminoglycans can be considered low, if the addition of 300 ⁇ of the solution B into the mixture of 100 ⁇ L of the sample and 100 ⁇ , of the solution A does not lead to the formation of a precipitate or turbidity. In that case, steps 2-3 are also omitted, besides the already omitted steps 4-7.
• the first step changes: 200 iL of a sample is mixed with 200 of the solution A, and after vortexing the solution is divided into equal parts: the "sample" and the "background". Then the procedure continues from step 8. An increase of the volume in the first step leads to a change of the formula for HA calculation:
• Fig. 1 represents the result of the reaction of 0.5% HA (weight average molecular weights from 24 kDa to 2049 kDa) with MBTH according to the Anthon-Barrett method.
• the first condensation reaction was performed at 80°C for 30 min.
• Fig. 2 represents the absorbance of the HA sample with the concentration of 0.1% after incubation with MBTH at various temperatures.
• the optical density increases in time due to the degradation of HA in a peeling reaction.
• Fig. 3 represents the enzymatic activity of S. pneumoniae hyaluronan lyase SpHyl.
• Fig. 4 represents the repeatability of the presented method at various temperatures and times of the reaction.
• HA samples were mixed with the solution A, digested with SpnHyl, then solutions C and D were added into the reaction mixture according to the protocol (see the Methods). Then the samples were subjected to heating at 73, 75, and 77°C for 30, 40, and 50 minutes. Immediately after heating, the solution E was added into the heated reaction mixture. After cooling to the room temperature and colour development, the absorbance spectra were measured in the range of 500-700 nm.
• Fig. 5 represents the influence of the molecular weight of HA on the accuracy of the presented method.
• the process volumes were optimized for the standard photometric cell with the light path of 10 mm and volume approx. 1 mL.
• the process volumes were optimized for the standard photometric cell with 10 mm light path and volume of approx. 1 mL.
• 100 ⁇ ⁇ of the analysed sample are mixed with 100 ⁇ . of the solution A (lOOmM acetate buffer of pH6, 160mM Na 2 S0 4 ) and the mixture is vortexed for 5 seconds.
• 300 ⁇ , of the solution B (l%w/v CTAB, 50mM acetate buffer of pH6, 80mM Na 2 S0 ) are added and the mixture is vortexed again for 5 seconds.
• the sediment is removed by centrifugation at >12000 g for 5 minutes.
• the process volumes were optimized for the standard photometric cell with 10 mm light path and volume of approx. 1 mL.
• the reducing sugars are often used as a component of cultivation media and veterinary and pharmaceutical preparations.
• the use of SpnHyl-MBTH method enables determination of HA in such samples.
• the process volumes were optimized for the standard photometric cell with 10 mm light path and volume of approx. 1 mL.
• Sample of HA comprising sulfated glycosaminoglycans but not comprising reducing sugars
• the process volumes were optimized for the standard photometric cell with 10 mm light path and volume of approx. 1 mL.
• 100 ⁇ , of the analysed sample and 100 ⁇ , of the solution A (lOOmM acetate buffer of pH 6,5, 200mM Na 2 S0 4 ) are mixed and the mixture is vortexed for 5 seconds.
• 300 ⁇ . of the solution B (l%w/v CTAB, 50mM acetate buffer pH 6, lOOmM Na 2 S0 4 ) are added and the mixture is vortexed again for 5 seconds.
• the sediment is removed by centrifugation at >12000 g for 5 minutes.
• the sediment (complex) is dissolved in 400 ⁇ , of the solution A, the microtube is marked as the "sample” and 200 ⁇ of the solution is pipetted into the second tube marked as the "background”.
• 1 ih of SpnHyl enzyme 1000 U/mg, 2 mg/mL is added into the neglectsample” tube and the mixture is incubated for 15 minutes at 37°C.
• 200 ⁇ of the solution C (1M NaOH) and 400 ⁇ , of the solution D (0.3%w/v MBTH and 5mM DTT) are added into both tubes - the "sample” and the "background". The mixture is mixed thoroughly and incubated at 95°C for 5 minutes.
• Geloren (Contipro, a.s.) is a gelatine gel tablet for dogs and horses.
• the "dog” version comprises 0.6% HA, 5% CS, 1.24% glucosamine, 7% hydrolysed collagen, 28% gelatine (http-V/wwvv ⁇ educationnizvirexz/kloubni-vyziva-pro-psy).
• the "horse” version comprises 0.4% HA, 1.9% CS, 15.5%> gelatine, and also a high amount of glucose (http://www.2011nizvire.cz vyziva-kloubu-kone). All percents indicated in the Geloren composition are weight percents, i.e. w/w.
• a slice of gel of the size of approx. 0.1 g was weighed and then melted in 9 volumes of 20mM Tris-HCl buffer pH 8 at 80°C. Then the sample was digested with pancreatin (10 mg of pancreatin per 1 mL of the melted gel) at 37°C for 1 hour. The digestion with pancreatin is necessary as the gelatine creates complexes with CTAB and thus inhibits the analysis (Ji et al., 2004), Then the sample is processed according to the Complete Protocol SpnHyl-MBTH including the removal of reducing sugars and CS. The measured concentration of HA was 0.408%> and 0.59% of HA for the horse and the dog version of Geloren, respectively.
• the MBTH-SpHyl method was used for the monitoring of HA content during the whole manufacturing process beginning from Streptococcus zooepidermicus (SEZ) cultivation, through to the downstream processes and ending with the final product. More than 500 samples were analyzed in total.
• Haserodt S, Aytekin M, Dweik RA, (201 1) A comparison of the sensitivity, specificity, and molecular weight accuracy of three different commercially available Hyaluronan ELISA-like assays. Glycobiology 21(2):175-83. doi: 10.1093/glycob/cwql45.

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