American Journal of Chemistry

2012; 2(4): 186-190 doi: 10.5923/j.chemistry.20120204.02 Cationic Surfactants from Arginine: Synthesis and Physicochemical Properties Pravin U. Singare 1, Jyoti D. Mhatre 2
1

Department of Chemistry, Bhavans College, Munshi Nagar, Andheri (West), Mumbai 4000058

Department of Chemistry, Shri. Jagdishprasad Jhabarmal Tibrewala University, Jhunjhunu, Rajasthan 333001 Correspondence to: Jyoti D. Mhatre , Department of Chemistry, Shri. Jagdishprasad Jhabarmal Tibrewala University, Jhunjhunu, Rajasthan 333001. Email: Copyright 2012 Scientific & Academic Publishing. All Rights Reserved. Abstract The present invention concerns the preparation of cationic surfactants derived from the condensation of an acid chloride, preferably a fatty acid with a number of carbon atoms 8, 9 and 14 with esterified amino acids, preferably basic-type amino acids, like (L)-arginine. The method comprises a first step in which the esterification of the amino acid with an alcohol is performed and a second step for the condensation with a chloride of fatty acid, using Schotten Baumann conditions. These surfactants constitute a novel class of chemicals of low toxicity with excellent surface properties and considerable antimicrobial activity. As in a conventional series of surfactants with different chain lengths, changes in the chain result in changes in the physicochemical properties. Excellent antimicrobial activity is observed for the homologue of 14 carbon atoms. Keywords: Cationic Surfactants, N-Acyl, Arginine, Schotten Baumann Article Outline 1. Introduction 2. Experimental 2.1. General reagents and Synthetic Method2.2. Physicochemical Behavior 2.3. Antimicrobial Activity 2.4. Results and Discussions 2.5. Conclusions

ACKNOWLEDGEMENTS 1. Introduction Arginine based cationic surfactants are amphiphilic compounds that possess excellent selfassembling properties, a low toxicity profile, high biodegradability and a broad antimicrobial activity, which make them candidates of choice as preservative and antiseptics in pharmaceutical, food and dermatological formulations[1-5]. The value of amino acids as raw materials for the preparation of surfactants was recognized as soon as they were discovered some 50 years ago. Initially they were used as preservatives for medical and cosmetic applications and were subsequently found to be active against various disease-causing bacteria, tumors and viruses. There is a large variety of amino acid/peptide structures and the fatty acid chains can vary in their structures, length and number, which explains their wide structural diversity and different physicochemical and biological properties[6]. In the last two decades, a group of scientists has published a number of papers addressing the synthesis and properties of biocompatible cationic amino acid based surfactants of different structures[7-10]. These surfactants show a low toxicity profile and an antimicrobial activity similar to those of conventional cationic surfactants. Lipoaminoacids derived from LArginine are a recently described family of nontoxic and biodegradable cationic surfactants with antimicrobial properties[1,3]. Arginine based surfactants constitute a promising alternative to other antimicrobial surfactants with high intrinsic toxicity and questioned biodegradability such as quaternary ammonium halides[11-12]. The antimicrobial activity of the argnine-based cationic surfactants is directly associated with the presence of the cationic charge of the protonated guanidine group of this amino acid[13]. Amino acid based surfactants have some distinctive structural features as shown by general chemical formula of N- acyl arginine derivatives, the surfactants object of this study (Fig. 1). (A) The special properties exhibited by these type of compounds are due to the strong hydrogen bonding of the amide bond located between the hydrophilic (amino acid residue) and hydrophobic part of the molecule. (B) Presence of asymmetric carbon atom in the molecule making formation of the chiral aggregates[14]. In this paper, the main part of the systematic study whose aim deals with the influence of terminal fatty acid chain on the properties of N- acyl arginine esters is reported. N- acyl arginine derivatives that contain basic amino acid (Arginine) as terminal amino acid have been prepared by peptide synthesis methods. These compounds have been synthesized as ethyl esters and their fundamental surfactant properties and antimicrobial activities have been evaluated. The properties of these compounds have been compared to the properties of the cationic monomer derivative methyl ester of N- lauroyl arginine and the amphoteric monomer derivative Nlauroyl arginine reported earlier. In this work, three arginine-derivative surfactants, ethyl esters of N- Octanoyl arginine, NNonanoyl arginine and N- Myristoyl arginine are studied. We report the chemical synthesis and the study of some physical properties such as critical micellar concentration. Biological property such as antimicrobial activity is also investigated. Figure 1. Molecular structure of the N-acyl arginine ethyl ester surfactants; n=6 CAE, n=7 NAE, n=14 MAE

If we consider the chemical structure of an amino acid, the fatty chain can be introduced via the amine or carboxylic function. However, the reactivity of the amine function in aqueous medium is widely higher than the one of carboxylic acid. Many pathways use organic solvents (1,15). Another pathway consists of synthesis by acylation using an acid chloride in water, following the Schotten-Baumann reaction (16,17). The preparation of the surfactants of our interest was carried out following the steps shown in following Fig.2 . It consisted of two steps using L-Arginine HCl as starting material. (I) Synthesis of L-Arginine ethyl ester dihydrochloride by esterification process. (II) Synthesis of N-acyl arginine ethyl ester by acylation of -Amino group of L-Arginine ethyl ester dihydrochloride with the corresponding long chain acid chloride. 2. Experimental MATERIALS The following N-acyl arginine monopeptides have been studied CAE: N--Octanoyl-L-Arginine ethyl ester NAE: N-Nonanoyl L-Arginine ethyl ester MAE: N-Myristoyl-L-Arginine ethyl ester 2.1. General reagents and Synthetic MethodL-Arginine was purchased from Ajinomoto Co., Octanoic acid, Nonanoic acid, Myristic acid and Sodium Dodecyl sulfate (SDS) were received from Sigma-Aldrich. LAE.HCl (N-Lauroyl arginine ethyl ester Hydrochloride) was supplied by local supplier. General reagents were of an analytical grade and higher purity. Solvents used were of analytical grade or higher purity and supplied by Sigma-Aldrich. All fatty acid chlorides are prepared in our lab. L-Arginine Hydrochloride is prepared following the procedure of literature (18). The homogeneity of compounds was checked by thin-layer chromatography on aluminium plates (Kieselgel G, Merck}. The solvent systems were (A) chloroform/methanol/acetic acid (8.5:10:5); and (B) chloroform/methanol (7:3). Ninhydrin developer solution was used for qualitative analysis of free amino groups. Nuclear Magnetic Resonance (1H NMR) and all the NMR measurements were performed with Bruker, Avance 300 spectrometer model at 300MHz in a 5mm direct probe (BBO BB-1H) using CDCl3 as a solvent. Surface Tension was measured using Stalagmometer with a Wilhelmy plate. Mass Spectroscopy with fast atom bombardment (FAB) was carried out with VG-QUATTRO from Fisons Instrument. Method for synthesis Figure 2. Schematic method of synthesis

Preparation of L-Arginine ethyl ester dihydrochloride In a 500ml round bottom flask is charged 250ml Ethyl alcohol followed by the addition of 0.25 equivalent of L-Arginine HCl at room temperature. Thionyl chloride (1.25 equivalents) is then charged slowly controlling exotherm. Heat is applied and reaction mixture is refluxed for 4-5 hours. After completion of the reaction, Ethanol is continuously removed under vacuum, with

intermediate additions of dry Ethanol. The residual mass is cooled to get crude L-Arginine ethyl ester dihydrochloride. Preparation of N-Acyl L-Arginine ethyl ester compounds by Schotten Baumann reaction. The crude reaction product obtained in the first step is dissolved in water and the pH of the solution is brought to a specific pH value 5.5-7 by the addition of aqueous sodium hydroxide. The pH of the reaction is carefully kept constant at this value until completion of the reaction. To this solution, add 0.96 equivalent of corresponding acid chloride drop-wise, whereby the temperature of the mixture is kept at a temperature of 10-15 C. After completion of the reaction, the stirring is maintained for a further two hours, after which the pH of the solution is adjusted to a final value of 5.5-7 with hydrochloric acid or sodium hydroxide. Finally, the crude reaction product is obtained either by filtration or by distillation. Compound: N--Octanoyl-L-Arginine ethyl ester (CAE) Prepared by reaction between LArginine Et ester diHCl and Octanoyl chloride in the presence of aqueous NaOH (Yield 85%). Clear Yellowish oil. Rf: 0.68; MW 328, ESI-MS; m/z 329 (m+H); 1H NMR: H (CDCl3), 0.89[t, 3H, (CH3 alkyl chain)], 1.29[s, 11H, (4CH2, alkyl chain), (OCH2-CH3)], 1.5-1.7[m, 4H, (-CH2-CH2-CH2-NH)], 2.048[s, 1H, (-CH2NH-)], 2.21-2.27[t, 2H, (-CH2CO-)], 3.1-3.3[m, 1H, (-CH2NH-)], 3.53.7[2H, (-CH2-CO-NH-)], 4.2[m, 2H, (-OCH2-CH3)], 4.44[m, 1H, (-NH-CH-COO-), 4.815[ m, 1H, (-CH2NH-)], 7.24-7.27[m, 2H, (-NH-C(=NH)-NH2)], 8.756[1H, (-NH-CH-COO)] Compound: N--Nonanoyl-L-Arginine ethyl ester (NAE) Prepared by reaction between LArginine Et ester diHCl and Nonanoyl chloride in the presence of aqueous NaOH (Yield 80%). Light brown sticky mass. Rf: 0.45; MW 342, ESI-MS; m/z 343 (m+H); 1H NMR: H (CDCl3), 0.87[t, 3H, (CH3 alkyl chain)], 1.27[s, 10H, 5CH2, alkyl chain], 1.59-1.83[m, 4H, (-CH2-CH2-CH2-NH-)], 2.21-2.27[t, 2H, (-CH2-CO-NH-)], 3.087-3.292[m, 1H, (-CH2NH-)], 3.5-3.7[1H, (-CH2-CO-NH-)] 4.2[m, 1H, (-OCH2-CH3), 4.456[m, 1H, (-NH-CH-COO-), 7.26[3H, (-NH-C(=NH)-NH2)], 8.958[1H, (-NH-CH-COO)] Compound: N--Myristoyl-L-Arginine ethyl ester (MAE) Prepared by reaction between LArginine Et ester diHCl and Myristoyl chloride in the presence of aqueous NaOH (Yield 78%). White solid Rf: 0.55; MW 412.6, ESI-MS; m/z 413.2 (m+H); 1H NMR: H (CDCl3), 0.855-0.899[t, 3H, (CH3 alkyl chain)], 1.251-1.299[m, 28H, CH2, OCH2CH3], 1.6-1.9[m, (-CH2-CH2-CH2-NH-)] , 2.26-2.34[t, 2H, (-CH2CO-)], 3.21-3.37[m, 2H, (-CH2NH-)], 4.2[m, 2H, (-OCH2-CH3), 4.43[m, 1H, (-NH-CH-COO-), 7.039[m, 3H, C(=NH)-NH2)], 7.22-7.26[t, 1H, (-CH2NH-)], 7.824[m, 1H, (-NH-CH-COO)]. 2.2. Physicochemical Behavior To check the behavior of the synthesized monopeptides of arginine as surfactants in solution, the concentration at which the surfactant molecules start to form micelles, known as critical micellar concentration (cmc), was determined. Water/surfactant solutions of different concentrations were prepared and allowed to equilibrate at 25C between 4 and 10 hr. The conductivity of these aqueous solutions was measured. The conductivity of the aqueous solutions rose linearly with increasing concentrations up to break points that correspond to the cmc of these surfactants. For the sake of comparison, the cmc value of pure commercially available LAE (N-Lauroylarginine

ethyl ester) was also determined (Table 1). Graphical representation of CMC versus carbon atoms in the hydrophobic chain for N-Acyl arginine surfactants,.is shown in Fig. 3. Table 1. Critical micellar concentration of N-acylarginine ester and references CMC (mg/L) (mN/m) CAE >1500 27.00.5 NAE 82050 26.10.5 LAE 41010 25.50.5 MAE 35030 24.00.5 The water solubility of CAE, NAE and MAE was partially studied at different pH values at a constant concentration of 1% (w/v) and at room temperature. CAE is clear in the range of pH 211.5, whereas solution of NAE is clear at pH 6-11, but insoluble at pH 4. MAE is soluble only in the pH range 1.5-5. This solubility data was compared with LAE solubility, which is showing clear solubility in the pH range 1-7.3 and insoluble at pH > 8 and 0.5 (Table 2). These results appear to indicate that the insolubility increases with increase in chain length (Hydrophobic Character) of the compound. Table 2. Water solubility of 1% aqueous solution at different pH

Figure 3. CMC versus carbon atoms in the hydrophobic chain for the N-Acyl arginine surfactants

2.3. Antimicrobial Activity The microbicidal effects of medium and long-chain fatty acids and their corresponding 1monoglycerides, of which the most active are the compounds with 12 carbon atoms in the alkyl chain, are well known (10,19). Lauric acid is known to the pharmaceutical industry for its good antimicrobial properties, and the monoglyceride derivative of lauric acid, monolaurin, is known to have more potent antimicrobial properties against enveloped viruses and numerous pathogenic Gram positive bacteria (20). The Antimicrobial activity cannot be determined by any given individual structural moiety alone. It is the right combination of positive charges and hydrophobic groups that provide the adequate hydrophilic-lipophilic balance (21). In order to study the effects of the introduction of the arginine amino acid in these structures on the antimicrobial properties, these compounds were evaluated against Gram positive and Gram

negative bacteria. Minimum Inhibitory Concentration (MIC) of molecules is defined as the lowest concentration of antimicrobial agent that inhibits the development of visible microorganism growth after incubation at 32C for 48 hrs and fungal growth at 25C for 4 days by Broth Dilution method. Sample preparation was done by simply mixing 1ml of the 1% solution in DMSO with 9ml of broth Tryptic Soy Broth (1000ppm solution). This stock solution was a clear solution. From the above stock solution 1ml was added to each of 12 consecutive sterile 13mm tubes containing 1ml TSB. Each tube is vortexed and aseptic transfer to give the concentration range of 0.25 to 500ppm. Each culture is grown in TSB>24hrs<48hrs at 32C. The culture is diluted to 10,000 cfu/ml and 10 l of this is added to each tube. Negative controls (NC) TSB confirm sterility of the TSB, Positive controls (PC) for each culture confirm organism capable of growth in the TSB. The antimicrobial activity of all synthesized compounds has been established by estimating their corresponding MIC values (in ppm) against Gram-positive and Gram-negative bacteria. For the sake of comparison, the MIC of LAM (N-Lauroylarginine methyl ester) has been assessed (see Table 3). Table 3. Comparison of Minimum Inhibitory Concentration of LAM (Methyl Lauroylarginate), CAE, NAE and MAE in ppm

From Table 3, it has been observed that N--Myristoyl-L-Arginine ethyl ester (MAE) showing greater antifungal activity and antimicrobial activity for Staphylococcus Aureus than that of commercially available Lauroyl derivative LAM (N-Lauroylarginine methyl ester) (22). N-Octanoyl-L-Arginine ethyl ester (CAE) is not active against Gram-positive, Gram-negative and Candida albicans. N--Nonanoyl-L-Arginine ethyl ester (NAE) exhibits antimicrobial property at the concentration of 62-250ppm against Gram-positive and Gram-negative bacteria, but requires higher concentrations against C.albicans and A.niger. This antimicrobial activity is dependent on several physicochemical properties (surface activity, solubility) and structural features (the length of alkyl chain) of Arginine derivatives (11). 2.4. Results and Discussions The low surface-tension values of solutions of our acylmonopeptides (27-24 nM/m) and the appearance of a CMC, suggest their utility as surfactants. These values are comparable to the 25.5 nM/m obtained for micellar solutions of commercially available surfactants: LAE.HCl (cationic surfactant). As expected, the cmc decreases when the alkyl chain increases as a

consequence of the higher hydrophobic content of the molecule. The most hydrophobic compound (MAE) showed the greatest ability to lower the surface tension and to form micelles. In view of the results of the antimicrobial activity of these compounds, the MAE homologue has a broader spectrum of antimicrobial activity than CAE, NAE and even commercial LAM compounds. Table 3 shows that the effectiveness of inhibiting the growth of bacteria decreases in the order of MAE > LAM > NAE > CAE. This optimum effect MAE homologue can be attributed to the combination of several physicochemical parameters: hydrophobicity, adsorption, cmc and aqueous solubility. 2.5. Conclusions The following conclusions may be drawn from the present study: i) N-Acylarginine ethyl ester can be synthesized in good yields using Schotten Baumann reaction conditions. ii) The surface activity was increased and the cmc decreased by raising the alkyl chain length and the hydrophobicity of the amino acid residue. iii) Increase in the carbon chain length of acyl group of N-Acylarginine ethyl ester improves antimicrobial properties. From this study we could summarize that the introduction of an appropriate long chain Narginine residue (In this case 14 carbon atoms) to the amino function of a amino acid yields an interesting multifunctional compound to be applied as a soft preservative peptidic surfactant in cosmetic, foods and dermopharmaceutical formulations. ACKNOWLEDGEMENTS We are indebted to Dr. Vilas Chopdekar and Dr. Richard Stockel for technical support to this project. We are also thankful to V & V Pharma Industries for providing Laboratory to conduct experiments. References

[1]

[2] [3] [4]

[5] [6]

Perez L, Torres J L, Manresa A et al. Synthesis, aggregation and biological properties of a new class of Gemini cationic amphiphilic compounds from arginine, bis(args). Langmuir (1996), vol. 12, pp. 5296-5301. Claps P, Morn C, Infante M R, Enzymatic synthesis of argnine-based cationic surfactants. Biotechnol Bioeng (1999), vol. 63, pp. 332-343 Piera E, Infante M R, Claps P, Chemo-enzymatic synthesis of arginine based Gemini surfactants. Biotechnol Bioeng (2000), vol. 70, pp. 323-331 Rodriguez E, Seguer J, Rocabayera X et al, Cellular effects of monohydrochloride of Larginine, N-lauroyl ethyl ester (LAE) on exposure to Salmonella typhimurium and Staphylococcus aureus. J. Appl. Microbiol. (2004), vol.96, pp. 903-912 Morn C, Claps P, Comelles F et al, Chemical structure/property relationship in singlechain arginine surfactants. Langmuir (2001), vol. 17, pp. 5071-5075 Presenz P, Lipoamino acids and lipopeptides as amphiphilic compounds. Pharmazie (1996), vol. 51, pp. 755-758

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Pegiadou S, Prez L and Infante M R, Synthesis, Characterization and surface properties of 1-N-L-Tryptophan-Glycerol Ether surfactants, J. Surf. Detergents (2000), vol. 3(4), pp. 517-525 Allouch M, Infante M R, Seguer J, Stebe MJ and Selve C., Nonionic Amphiphilic compounds from Aspartic and Glutamic acids as structural mimics of Lecithins, J.Am.Oil Chem.Soc. (1996), vol. 73(1), pp. 87-96 Prez L, Pinazo A, Vinardell P, Claps P, Angelet M and Infante M R, Synthesis and Biological properties of Dicationic arginine-diglycerides New J. Chem. (2002), vol. 26, pp. 1221-1227 Prez L, Pinazo A, Garcia MT, Morn C and Infante M R, Monoglyceride surfactants from arginine: Synthesis and biological properties. New J. Chem. (2004), vol. 28, pp. 1326-1334 Claps P. and Infante M. R., Amino acid-based surfactants. Enzymatic Synthesis, properties and Potential Applications, Biocatalysis and Biotransformation, (2002), vol. 20 (4), pp. 215-233 Pinazo A, Wen X, Prez L and Infante M R, Aggregation Behavior in Water of Monomeric and Gemini Cationic Surfactants Derived from Arginine, Langmuir (1999), vol. 15, pp. 3134-3142. Infante M. R., Erra P., Juli R., Prats M., Surface active molecules: Preparation and properties of long chain N-acyl-L-, , guanidine alkyl acid derivatives, Int. J. Cosmet. Sci., (1984), vol. 6, pp. 275-282. C. Solans, Ps M. A., Azemar N. and Infante M. R., LIpoamino acid surfactants: Phase behavior of long chain N- acyl arginine methyl esters, Progress in colloid and Polymer Science, (1990), vol. 81, pp. 144-150. Infante M.R., Pinazo A, Seguer J, Non conventional surfactants from Amino Acids and Glycolipids: Structure, Preparation and properties. Colloids and surfaces, A: Physicochemical and Engineering Aspects (1997), vol. 49, pp.123-124. Agustin C. M., Fransisco R.M., Joan S.B., Process for the preparation of cationic surfactants, PCT Int. Appl. (2001), WO 2001094292 Ghare V. S., Process for preparation of N-lauroyl-L-arginine ethyl ester hydrochloride salt as a cationic surfactant, U.S. Pat. Appl. Publ. (2010), US 20100152480 Natarajan V., Sivanesan T. and Pandi S., Third order non-linear optical properties of LArginine hydrochloride monohydrate single crystals by Z-scan technique, Indian Journal of Science & Technology, (2010), vol. 3(8), pp.897-899 Conley A. J. and Kabara J., Antimicrob. Agents Chemother. 1973, vol. 4(5), 501506 Bergsson G., Steingrimsson O. and Thormar H., Bactericidal effects of fatty acids and monoglycerides on Helicobacter pylori, Int. J. Antimicrob. Agents, (2002), vol. 20, pp. 258-262 Appelt C, Wessolowski A, Soderhall J A, Dathe M, Schmeieder P, Structure of the Antimicrobial, Cationic Hexapeptide Cyclo(RRWWRF) and Its Analogues in Solution and Bound to Detergent Micelles, ChemBioChem (2005), vol. 6, pp. 1654-1662

Chemistry education Journal An approach to chemical education at medical technologist training institutions in Japan

Hidetsugu Kohzaki1,2, Yoichi Fujita2 and Yoichi Ishida2

1 2

Department of Cell Biology, Institute for Virus Research, Kyoto Department of Medical Technology, Kyoto College of Health and Hygiene

University,

Corresponding author: Hidetsugu KOHZAKI, PhD, Department of Cell Biology, Institute for Virus Research, Kyoto University, Shogoin-kawahara-machi 53, Sakyo-ku, Kyoto, Japan 6068507. E-mail: charaznable.k@gmail.com Keywords Chemical education, Medical technologist, Training institutions Total number of words: 22,900 Short running title: Chemical education for medical technologists Abstract We teach chemistry at a medical technology school. Chemistry is not necessarily the favorite subject of Japanese students receiving paramedical education. However, many biochemical, microbiological, and advanced genetic tests are performed in the medical setting, and solutions and media also have to be prepared. Nurses and paramedics including medical technologists rarely prepare solutions because of automation in the medical setting and a lack of time. However, they might feel uneasy about certain aspects of medical practice, such as the use of infusions and injections, if they do not know how to prepare and label solutions. In addition, the annual national examinations include questions on concentration calculations. In this article, we introduce our approach to providing chemical education for medical technologists. Introduction In Japan, chemistry acted as the main driver of growth during the high-growth period. Thus, at that time, chemical education supported Japan. However, the academic ability of Japanese students has declined markedly because of the trend towards "science phobia" and pressure-free education; e.g., teaching the value of "pi" as 3 and not requiring students to memorize the periodic table of elements. Since the introduction of IT, information science has

been required in various fields [1]. However, this is not true of chemistry. Recently, many Japanese researchers have won the Nobel Prize. Thus, science education, especially chemical education, has come to be reevaluated. We teach chemistry at a medical technology school. Although many of our students are good at IT, they lack chemical knowledge. Japanese junior high school students must choose between humanities or science courses. However, adequate chemical education is provided at junior high school. Thus, even students who enroll in a humanities course should be able to answer the chemical questions on the National Center for University Entrance Examinations. However, the students on the humanities courses stop studying chemistry because their main goal is to pass the college entrance examination. Thus, chemistry is only studied at junior high school by the students who take science courses. In addition, some students lose interest in chemistry because of their desire for pressure-free education. Thus, chemistry has become unfamiliar to many students entering paramedic schools, such as nurses and medical technologists. In fact, many students have not seen the periodic table of the elements (ref.[2] Chapter 1 I, Table.2 Science I "Basic of chemistry", Chemistry "Chemical bond ") or cannot write the molecular formulae of water and/or carbon dioxide (ref.[2] Chapter 1 I.5, Table.2 Science I "Basic of chemistry ", Chemistry "Chemical bond "), much less the structural formulae of covalent (ref.[2] Chapter 1 II.9, Table.2 Science I "Basic of chemistry ", Chemistry "Chemical bond ") or ionic bonds (ref.[2] Chapter 1 II.8, Table.2 Science I "Basic of chemistry ", Chemistry "Chemical bond ") or amino acids (ref.[2] Chapter 5 IV.2, Table.2 Science I "Amphoteric electrolyte ", Chemistry "Chemistry of proteins"). However, paramedics should have some knowledge about amino acids (ref.2 Chapter 5 IV.2, Table.3 Science I "Amphoteric electrolytes ", Chemistry "Chemistry of proteins "), sugars (ref.[2] Chapter 5 IV.1, Table.2 Chemistry "Chemistry of sugars "), lipids (ref.[2] Chapter 5 IV.3, Table.2 Chemistry "Chemistry of lipids "), ions (ref.[2] Chapter 1 II.8, Table.2 Chemistry "Chemistry bond "), and osmotic pressure (ref.[2] Chapter 2 V). Laboratory test values should be correctly understood by all medical practitioners because they will encounter a wide variety of test parameters during their jobs, such as measures of in vivo enzyme function (enzymatic activity in blood is affected by metabolite levels and diseases) (Table.2 Science I "Enzyme reaction ", Table.4 1.B. "Unit of enzyme "), and the shortage of physicians in Japan is increasing the burden placed on medical institutions. In addition, there is also an increasingly broad range of medicines in use because of the rapid development of novel agents, and numerous diagnostic kits have also been developed. Furthermore, since the completion of the human genome project, chromosomal gene tests have become more common, and pharmacogenomics and personalized medicine have advanced. As a result, chemical questions have arisen on the National Examination for Paramedics (Table.4). Methods and results Medical technology schools produce curricula that help their students to pass the National Examination for Medical Technologists. Hence, they provide technical knowledge and skills, but allow no time for chemistry (Table 1). As such, students do not learn the chemistry they will require during medical practice. In addition, students cannot learn all the contents of basic textbooks for medical technology schools [2]. Thus, an exam was conducted to evaluate the basic scientific knowledge of our students. This written exam included questions pertaining to the measurement of acids and alkalis with litmus paper (ref. [2] Chapter 3 IV), the three states of

water (ref. [2] Chapter 2, I, II, III), the chemical formula for water (ref. [2] Chapter 1 I. 5), and how to calculate the percentage concentration of NaCl (ref. [2] Chapter 2 IV. 1). It was used to assess whether the students possessed academic knowledge that they were supposed to have learnt during middle school. In fact, about 20% of the enrolled students answered less than 50% of the questions in the science and chemistry tests correctly. Most of the enrolled students did not possess chemical knowledge because they did not take chemistry as an elective subject in high school due to a desire for pressure-free education. Thus, we used Mendeleev's periodic table to make them familiar with the chemical world (Table 2). In Science I, we taught chemical bonds, molar concentrations, solution preparation, specific gravity, etc. Colorimetry is important because it is used for the measurement of protein and nucleic acid concentrations, immunostaining, etc., in biochemistry (Table 2A). Thus, chemical knowledge is necessary to understand biochemistry. we also taught enzyme reactions and alcohol metabolism in the living body as well as the in vivo behaviors of proteins and sugars (Table 2B).

Table 1. Credits and time devoted to chemistry education at medical technology schools *Chemistry includes eScience If and eChemistryf, which are described in Table 2.

A. Science I Implementation term: First semester of the first year Time: 18 hours Teaching method: Lectures Number of units: One unit Contents: Exponent notation and significant figures Molecular weight and concentrations

Syllabus

B. Chemistry Implementation term: First semester of the first year Time: 30 hours Teaching method: Lectures Number of units: One unit Contents: Atoms and molecules; bonds; structures of carbon compounds and various linking groups; and the chemistry of sugars, lipids, and proteins

Syllabus

Table 2 Syllabus for chemistry education at medical technology schools A: Science I; B: Chemistry. In Science I, we teach basic chemistry. In Chemistry, we teach biochemistry and chemistry required by medical technologists. The education program was structured as follows (Remedial classes were introduced in 2008. Students who received these classes took the 55th or later National Examination.) 1. Remedial classes were organized for students who performed poorly in the tests conducted at enrollment in order to review the science and chemistry lessons provided to them at elementary and junior high school. This class usually contained about 10 students each year. Table 3 shows the remedial class of 2010, which contained 16 out of 40

enrolled students. They reviewed their exam results and deepened their understanding of chemistry after school for approximately 1.5 hours a week to help them understand the Science I and Chemistry classes (Table 2). 2. A quiz was given on the topics covered in the abovementioned class to improve the students' understanding. Topics associated with a low percentage of correct answers were repeated. 3. Further remedial classes were organized after school for the students who performed poorly in the quiz to provide thorough individualized instruction; i.e., they repeatedly solved the questions that they had found difficult. Details of the remedial classes for the "biology class" [3] and "mathematics class" [4] are submitted respectively. The achievements of the enrolled students were assessed by the final examination. The mean score exceeded 70 every year. All of the students that scored 50% or less in the enrollment tests achieved scores of 60% or above. Some students achieved increases in their scores of 60 points or more. Thus, the main goal of my program seemed to have been achieved. The effects of the remedial chemistry classes on chemistry scores were confirmed. The effects of the remedial mathematics and biology classes were also confirmed. Some students who took the remedial chemistry classes actually achieved higher scores in the Science I examination. On the other hand, the results of the mean score and score distribution of introductory statistics showed that the remedial mathematics classes were ineffective (Table 3A). However, they also showed that the remedial chemistry and mathematics classes were more effective at improving the studentsf chemistry scores than the remedial biology classes (Table 3B). The increases in the Science I and Chemistry scores of the students who took the remedial mathematics classes might have been due to improvements in their ability to calculate concentrations, such as molar concentrations. A

Table 3. Effects of the remedial classes on the final examination scores A: Effects of the remedial classes on the examination scores for Science I B: Effects of the remedial classes on the examination scores for Chemistry * Total number of students who took the remedial mathematics classes differs between Tables A and B, because one student quit school. The range of possible questions for the National Examination for Medical Technologists (5) is shown in Table 4, and some chemical questions are included on the test. In addition, chemistry is essential for clinical chemistry. Thus, chemistry is a fundamental subject that can

have

broad

influence

on

studen

ts' education.

Table 4. Range of possible chemistry questions for the National Examination for Medical Technologists Discussion

Japanese medical institutions have undergone considerable automation, and most test parameters are now automatically examined in clinical laboratories. In addition, the ISO15189 [6] guidelines have been established for clinical laboratories, and the standardization of test data has also been demanded. The following international standards are relevant to clinical laboratories: ISO15193 (In vitro diagnostic medical devices -- Measurement of quantities in samples of biological origin -- Requirements for content and presentation of reference measurement procedures) [7], ISO15194 (In vitro diagnostic medical devices -- Measurement of quantities in samples of biological origin -- Requirements for certified reference materials and the content of supporting documentation) [8], ISO17511 (In vitro diagnostic medical devices -Measurement of quantities in biological samples -- Metrological traceability of values assigned to calibrators and control materials) [9], and ISO18153(In vitro diagnostic medical devices -Measurement of quantities in biological samples -- Metrological traceability of values for catalytic concentration of enzymes assigned calibrators and control materials) [10]. ISO9001 (quality management systems) [11] is also applicable. Thus, it is vital that medical technologists are able to produce accurate test data. Considering the trends in exams over the past 7 years, a mean of 32 chemical questions, including biochemical questions, are set annually as clinical chemical questions in the National Examination for Medical Technologists [5]. However, chemistry includes the fields of biochemistry and clinical chemistry (Table 4). To become a medical technologist, students must pass the national examination, which requires a high score. We educate medical technologists and have prepared curricula that promote the understanding of biochemistry and clinical chemistry (Table 2). Correctly calculating the effects of our curricula is difficult. However, the results of the National Examination for Medical Technologists show that the mean score for Clinical Chemistry was 10% higher than those for Genetic Testing and Information Science, which we were also responsible for [12] (Table 5), but 4% lower than that for Biology & Biochemistry. The authors have been offering a remedial chemistry course because it is difficult to encourage students to undergo the basic course for medical technologist training institutions due to the recent reduction in students' interest in science and their lack of scientific knowledge. In fact, some students withdrew from the school because they could not keep up with the class, and the pass rate for the National Examination had begun to decrease (Tables 5 and 6). Although it has been only a few years since the remedial classes were adopted (Table 3), the National Examination scores of our students have increased by 4.4% for clinical science and 2.1% for all subjects, and the pass rate was also 4.2% higher among the students who took the remedial classes than among those who did not. The "Science I " and "Chemistry " curricula that we introduced are not the only the reasons for the increased pass rate; however, it is considered that increases in the students' scores for clinical chemistry contributed to it (Table 6). Basic chemistry provides a solid foundation for further studies in related sciences including clinical chemistry and gene testing, particularly biology and biochemistry. Therefore, we've admitted that our program was not a complete success; however, it seems to have helped the students to pass the National Examination (Figure 1, Table. 6).

Table 5. Scores for clinical chemistry on the National Examination for Medical Technologists

Chemistry is included in clinical chemistry. KCHH: data for the Department of Medical Technology, Kyoto College of Health and Hygiene. Ministry of Health, Labour, and Welfare: National Examination for Medical Technologists scores for the whole of Japan. SC: successful candidates. Number: the number of questions.

Table.6 Contribution of remedial classes to National Examination scores. *NE: National Examination * Because the pass rates of the 53rd or prior national examinations exceeded 90%, students did not require remedial classes. * Since the contents of examination markedly changed after the 53rd national examination, the test results before the 53rd national examination were excluded from this study.

Figure 1. Comparison of the scores obtained on the National Examination for Medical Technologists between our school and others across Japan [5]. The Japanese Association of Medical Technology Education (JAMTE) is composed of medical technology schools [13]. KCHH: the mean score for Kyoto College of Health and Hygiene, JAMTE: the mean score for the medical technology schools belonging to the JAMTE. CC: Clinical Chemistry, GT: Gene Testing, BB: Biology & Biochemistry, IS: Information Science, NE: National Examination. Furthermore, qualifications, such as the Qualified Class 1 and 2 Laboratory Technologist qualifications [14] and the Japan Society of Clinical Chemists qualification [15], have been established to improve the skills of medical technologists. Knowledge about clinical chemistry and the skills necessary for quality control should be obtained through these qualifications. Quality control is required to keep medical treatment fees low and also contributes to hospital management. In Japan, the management of toxic, highly poisonous, and hazardous substances is strictly regulated. These substances should be similarly handled in hospitals and clinical laboratories. Thus, paramedics should have chemical knowledge about these substances to allow them to handle them appropriately. Acknowledgements We thank my colleagues at Kyoto University, Osaka University, Kyushu University, and Kyoto Institute of Technology for their help with this study. This study was partially supported by the Japan Leukemia Research Fund (H.K). References

1. Kohzaki, H. A Proposal for Information Science Education for Paramedics/Medical Technologist Training in Japan. Submitted 2. Chemistry for Medical Technologists, Ishiyaku Co. Ltd. Japan. HP (http://www.ishiyaku.co.jp/search/details.aspx?bookcode=228940) (retrieved December 21, 2011). 3. Kohzaki, H. Biology and Biochemistry education for medical technologist/paramedics in Japan. Submitted. 4. Kohzaki, H.A proposal of Mathematics education for medical technologist/paramedics in Japan. Submitted. 5. Ministry of Health, Labour and Welfare, Japan ( http://www.mhlw.go.jp/topics/2009/05/dl/tp0513-1i.pdf)(retrieved December 21, 2011). 6. ISO15189(http://www.iso.org/iso/catalogue_detail?csnumber=26301)(retrieved December 21, 2011). 7. ISO15193(http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnu mber=42021)(retrieved December 21, 2011). 8. ISO15194(http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnu mber=42022)(retrieved December 21, 2011). 9. ISO17511(http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnu mber=30716)(retrieved December 21, 2011). 10. ISO18153(http://www.iso.org/iso/iso_catalogue/catalogue_tc/catalogue_detail.htm?csnu mber=31718)(retrieved December 21, 2011). 11. ISO9001(http://www.iso.org/iso/iso_catalogue/management_and_leadership_standards/q uality_management.htm)(retrieved December 21, 2011). 12. Kohzaki, H. Proposal regarding clinical genetics (genetics in medicine) education for paramedics/medical technologists in Japan. Journal of Genetic Counseling. Minor revised. 13. Japanese Association of Medical Technology Education (http://www.nitirinkyo.jp/link/index.html)(retrieved December 21, 2011). 14. College of Laboratory Medicine of Japan (CLMJ)( http://clmj.umin.jp/competency/first_class/sop.html or http://clmj.umin.jp/competency/second_stat/sop.html)(retrieved December 21, 2011). 15. Japan Society of Clinical Chemistry (http://www.jsccjp.gr.jp/approval/approval.html)(retrieved December 21, 2011).

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