Biol. Pharm. Bull. 30(2) 237—241 (2007)

teins of the body and it is present in the skin, blood vessels, organs, bone and connective tissue. Collagen also accounts for approximately 70% of the dermal structural ingredient of skin and it is the primary ingredient of the extracellular matrix surrounding the outer membrane of the cell body (scaffold) which supports the cell population. Gelatin on the other hand is collagen with a triple helical structure that has lost its g and b chains following the application of heat, and is also called heat-denatured collagen. Gelatin is used not only in food but also in basic ingredients used in cosmetic products and as a base material of soft and hard capsules used in the preparation of medicinal products. Gelatin like collagen is widely used as biomaterial that forms the scaffold supporting cell populations and is also widely used to coat the surfaces of sterile containers facilitating cell attachment and improving the ability of cells to proliferate. In medical practice, collagen and gelatin are used as biomaterials that promote the regeneration of true skin-like tissue in injuries affecting the whole layer of skin. However, there are no set standards for the endotoxin levels of gelatin which is listed in the Japanese Pharmacopoeia and which is also widely used as a material for medical products. The primary ingredient of endotoxin is lipopolysaccharide (LPS) which is a conserved component of the outer membrane of gram-negative bacteria. LPS is constantly being released in bacterial surroundings similar to the constant shedding of the horny layer of human skin. When bacteria undergo auto-destruction, all the endotoxin is released from the bacteria. When LPS invades the body, even a minute amount can trigger fever, shock, a fall in blood pressure and in worst cases may cause death. There are also several reports that endotoxin damages various types of cells suggesting that this substance must be eliminated as much as possible from raw materials that are to be used as biomaterials in regenerative medicine. Endotoxin may be inactivated by heating it to a high temperature of 250 °C for 60 min or more ruling out autoclaving at 121 °C which is the setting used for sterilization. In general, distillation, reverse osmosis, absorption, molecular weight cut-off and ultrafiltration (UF) using a membrane of several kDa etc., are the methods most commonly used for removing endotoxin from solutions such as water. However, for various reasons, it is difficult to remove endotoxin from collagen and gelatin solutions. For example, with the distillation method, it is possible to completely remove the endotoxin in water; however, it is impossible to isolate the endotoxin in high-molecular weight protein solutions. When gelatin is subjected to continuous heat of a high temperature, it turns brown, and the quality is extremely decreased making its application impossible. With reverse osmosis, endotoxin is removed together with high molecular proteins such as gelatin and so its application is also impossible. In addition, there is a procedure which uses an absorbent, nylon 66 membrane, that is exposed to zeta potential and endotoxin is filtered by absorption. However, gelatin solutions have a higher ionic strength than that of water and this therefore affects the removal of endotoxin. Thus, an effective rate of removal as is the case with water is difficult. On the other hand, the UF procedure is used as the method of choice for removing endotoxin from water and solutions with low molecular-weight ingredients as in the case of injectable drugs that are highly controlled for endotoxin levels because they are administered directly into blood vessels. Use of an UF membrane makes it possible to remove harmful endotoxin from the filtrate while low molecular-weight substances such as antibiotics, salts, sugars and low molecular weight proteins remain. However, the molecular weight cut-off of the UF membrane that is being used is about several kDa, and so proteins with a molecular weight of approximately 10 kDa or higher are removed. As such, this procedure can only be used for the removal of substances in low molecularweight solutions. Due to that, there are no procedures that use UF to remove endotoxin from solutions of gelatin that are high molecular weight proteins of approximately 50— 100 kDa. In this paper, we report a method using UF membrane with an appropriate molecular weight cut-off for the removal of endotoxin from gelatin solutions to produce low endotoxin gelatin of a higher quality which meets the standards for “puFebruary 2007 237