Natural rubber has been available commercially for more than a century. Its latex-dipped products, particularly gloves, have been used preferentially as effective barriers to transmitted diseases such as AIDS. However, the occurrence of Type I allergy has recently been reported by some hypersensitive users, especially in the West. This awareness has promptly initiated a great deal of research addressing the problem, not only in the consumer countries in Europe and the USA, but also in the latex product manufacturing countries, particularly Malaysia. While clinical research in the West places emphasis on the development of the best diagnosis method for the affected users and their welfare management, work in Malaysia focusses on the manufacturing of better and safer latex products.

As the reported Type I allergy has been shown (1) to be due to the residual soluble proteins containing the allergens found in some latex products, intensive study on these proteins is thus carried out in Malaysia, with special reference to their removal or reduction in latex products. This paper discusses some of our findings particularly pertaining to the residual proteins and the allergic response elicited in latex hypersensitized persons. Since this allergy problem is thought to have also affected the NR dry rubber products, their extractable protein contents and allergenicity are also examined.

The starting material for all latex-dipped products, the latex concentrate, is derived from Hevea brasiliensis latex obtained from the tree on tapping. When fresh, the Hevea latex consists of about 35% rubber particles and 15% of non-rubber particles, all suspended in an ambient serum. Of the non-rubber substances present, proteins constitute approximately 1% - 1.5% (of total latex weight), of which about ¼ is associated with surfaces of the dispersed latex particles, and the remaining present as soluble proteins in the serum phase (2). When converted into latex concentrate with a rubber content of 60%, some of the soluble proteins in the serum phase are removed. The proportion of the surface bound proteins is thus increased from ¼ to about ½, while that of the soluble proteins is decreased accordingly. Although there is still a certain quantity of proteins present, the amount extractable when latex concentrate is processed into latex gloves or products is, however, only very small; it often does not exceed a few percent of the total. It is noteworthy that it is this small fraction that constitutes the residual extractable protein fraction implicated in the Type I allergic reaction. Improvements of latex products therefore invariably involve the reduction or removal of this undesirable protein fraction, particularly the allergens. Removal of the latter alone is unfortunately not yet possible due to incomplete information regarding their identities - inspite of the very active research currently in progress in many laboratories.

As has been well demonstrated, the residual extractable protein (EP) content of latex products prepared from the same latex can vary, depending on the processing conditions they are subjected to. For example, it increases (3) after compounding and when the products are vulcanized or drying at elevated temperature of 100° - 120°C. It decreases, on the other hand, when the products are leached in water, either during the wet-gel stage or the dry-film stage after vulcanization/drying, as well as after chlorination of the products (4). Hence, the EP values can range from as high as more than 1mg/g rubber for the unleached and untreated gloves to as low as less than 0.03mg/g or even lower for the well leached or chlorinated samples, as shown by both the RRIM modified Lowry microassay (5) and the size-exclusion high performance liquid chromatography (SE-HPLC) method (6) for EP quantitations.

Consequently, the utilisation of appropriate processing procedures provides one means of reducing these proteins. With the application of suitable wet-gel leaching and dry-film washing conditions during processing, our findings (7) reveal that EP of latex gloves prepared from normal latex concentrate, can be reduced to less than 0.1mg/g of glove (as measured by the RRIM modified Lowry microassay). EP reduction can also be effected during the latex stage by the use of low protein latices such as LOPROL (8), a partially deproteinized latex, or the recentrifuged prevulcanized latex (7). Unleached products from both have 70% - 80% less EP than their controls. The most effective approach by far is the use of a low protein latex combined with appropriate wet-gel and dry-film leaching conditions during processing. Values of EP of as low as 0.03mg/g in gloves have been achieved, reaching levels shown by chlorinated gloves known to have extremely low EP content. It may however be mentioned that the chlorination process, if not properly controlled, could often give rise to inferior physical properties of the gloves. On the other hand, low EP gloves produced by processes mentioned, frequently exhibit good physical properties when tested under both unaged and aged conditions.

Reduction of residual proteins would be more meaningful if such reduction could be related to allergenicity of the resulting product. However, much information on such relationship is still lacking, particularly of one that relates allergenicity to EP values obtained by the method employed during production. In view of the absence of a universally standardardized procedure for EP determination, the methods of choice in in Malaysia are those of the RRIM modified Lowry and the SE-HPLC. Athough both are equally sensitive and very reproducible, the former is a much faster test. Hence it is often used for quick routine product monitoring. This procedure involves initially the precipitation of soluble proteins from the extract, using trichloroacetic and phosphotungstic acids. The precipitated proteins are then resolubilised and concentrated in sodium hydroxide before subjecting to the colorimetric measurement of the Lowry reactions. Results are then calibrated against bovine serum albumin standard. The entire test takes less than 3 hours to complete.

A study is therefore carried out whereby extracts from 39 lots of commercial medical gloves and gloves produced under different processing conditions, are analysed and clinically tested. The EP contents are determined and the allergic responses elicited are assessed by the skin-prick test (9) performed on a total of 59 latex hypersensitive subjects (in 5 groups) in Finland.

The skin-prick test is known to be a rapid test of high sensitivity for IgE-mediated allergy. Besides being used identify sensitized patients, it can also be used to detect the presence of protein allergens in latex products (10,11). Allergic responses are evaluated with reference to a positive control of histamine. Resulting wheal size of the same size as or larger than that of the histamine is a clear or strong positive reaction, and a wheal size of at least half of that of the histamine control denotes a weak reaction. Very small wheals are not considered to be positive. Expressing the allergenicity measured as percentage of negative allergic responses shown by the subjects tested, results are shown in Figure 1.

It is apparent that gloves with higher EP contents are associated with higher degrees of positive allergic reaction, or rather low negative responses. Gloves with low EP levels, on the other hand, tend to show comparatively low or negligible responses as clearly reflected in the much higher percentages of negative response recorded. Regression analysis reveals that the EP content is very significantly correlated with the allergic response (r = 0.83 at P<0.0001). This result is consistent with that recently reported by Yunginger et al (12), who studied 15 lots of powdered gloves and showed a correlation of r = 0.60 at P<0.02, between the extractable proteins (as measured by a modified ninhydrin method) and the allergen content determined by an inhibition immunoassay.

Although threshold levels of extractable proteins below which relatively few sensitized people are affected have not been established, the study has nevertheless indicated that gloves with EP content of about 0.4 mg/g and lower, generally produce more than 60% negative allergic responses. At even lower levels of 0.1mg/g and less, higher degree of negative allergic reaction, or very low/negligible allergenicity can be achieved. Furthermore, while inadequate leaching always result in high content of the undesirable extractable proteins in the final product, application of proper processing conditions can produce low EP gloves of hypoallergenic quality. These findings are therefore of considerable relevance not only to the glove manufacturers, but also to the users assisting them in their choice of gloves.

The processing of Hevea latex into dry rubbers and their products is different from that of latex products. Instead of dipping, latex is first converted into dry rubber usually by acid coagulation. This is followed by crumbling/creping with extensive washing in water, and then dried at 100E-130EC. Fabrication of dry rubber products often involves compounding and curing of the dry rubber at elevated temperature, sometimes reaching as high as 160EC. In view of such processing steps, particularly the extensive washing, extractable protein contents of the final products are expected to be relatively low. This is in fact found to be so, as can be seen in Table 1., which shows EP content of 14 dry rubber samples from various commercial grades and 5 different dry rubber products. The values are so low that most of them reach the limits of the Lowry method used.

To study the allergenicity of dry rubbers and their products, extracts from the samples mentioned above are skin-prick tested on a total of 39 latex hypersensitive subjects (in 3 groups). It is apparent from Table 1. that the extremely low EP levels of the products show very little or no allergic response in latex hypersensitive patients tested in all cases . Such negative responses are strongly substantiated by the positive reactions elicited in the same patients by extracts from certain latex gloves known for their allergenicity.

Hence, the protein allergy problem of some latex articles does not necessarily affect the NR dry rubber products. Consequently, the reported campaign against NR dry rubber products by some non-NR producers is therefore unwarranted. This is particularly so in the case of cut threads, since these are generally covered by fabric thereby minimising any contact with the human skin. Furthermore, there are relatively fewer dry rubber products used in the healthcare sector where prevalence of latex hypersensitivity is often reported.

The authors wish to thank the Director of the Rubber Research Institute OF permission to present this paper.

1 Proceedings of "International Latex Conference: Sensitivity to latex medical devices" (1992) held on November 5 - 7, in Baltimore, Maryland, USA

2 Tata, S. J. (1980) Distribution of proteins between the fractions of Hevea latex separated by ultracentrifugation. J. Rubb. res. Inst. Malaysia, 28(2), 77-85

3 Amir Hashim Mohd. Yatim (1993) Effect of leaching on extractable protien content. Latex Proteins and Glove Industry, Rubb. Res. Inst. Malaysia, 51-58.

4 Nor Aisah Abd.Aziz (1993) Chlorination of gloves. Latex Proteins and Glove Industry, Rubb. Res. Inst. Malaysia 59-67.

5 Faridah Yusof and Yeang, Y. H. (1992) Quantitation of proteins from natural rubber latex gloves. J. nat. Rubb. Res., 7(3), 206-218.

6 Esah Yip (1993) Determination of extractable proteins in natural rubber products by high performance liquid chromatography (HPLC). Latex Proteins and Glove Industry, Rubb. Res. Inst. Malaysia, 41-50.

7 Ng, K. P., Esah Yip and Mok, K. L. (1994) Production of natural rubber latex gloves with low extractable protein content - some practical recommendations. J. nat. nat. Rubb. Res., 9(2), in press.

8 Hafsah Mohd. Ghazaly (1993) Properties of natural rubber low protein latex. Latex Proteins and Glove Industry, Rubb. Res. Inst. Malaysia, 81-91.

9 Dreborg, S. (1989) Skin test used in Type I allergy testing. Position paper. Allergy, 44, Suppl.10.

10 Turjanmaa, K., Reunala, T. and Rasanen, L. (1988) Comparison of diagnosis in latex surgical glove contact urticaria. Contact Derm., 19, 241-247.

11 Turjanmaa, K., Lauren, K., Mäkinen-Kiljunen, S. and Reunala, T. (1988) Rubber contact urticaria: Allergenic properties of 19 brands of latex gloves. Contact Derm., 19, 362-367.

12 Yunginger, J. W., Jones, R. T., Fransway, A. F., Kelso, J. M., Warner, M. A. and Hunt, L. W. (1994) J. Allergy Clinical Immunology, 93(5), 836-842.

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