Chapter 6
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Quantification of Limonin and Limonoate A-Ring Monolactone During Growth and Development of Citrus Fruit and Vegetative Tissues by Radioimmunoassay C. A. McIntosh Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614-0703 RIAs were developed using a polyclonal antibody raised against limonin-7-(0-carboxymethyl)oxime conjugated to BSA (1, 2). Using an iodinated tracer, sensitivity was 150 fmol limonin (less than 1 ppb) and only deoxylimonin showed significant cross-reactivity. The assay developed using H-limonol as tracer had a detection limit of 0.22 ng (2.2 ppb). In both systems, there was no cross-reactivity with limonoate Α-ring monolactone, therefore this compound could also be quantified. Since the RIAs required no prior purification of samples, up to 400 samples could be analyzed per day. Therefore, they were used to analyze natural variation of limonin levels in fruit and vegetative tissues, changes during growth and development, and variation in processed juice. RIA techniques can provide several advantages as compared to many other quantitative or qualitative methods. They are sensitive down to the fmol range, allow processing of hundreds of samples per day, and can be highly specific, depending on characteristics of antibodies used. The specificity of RIAfrequentlypermits substances to be measured or detected in crude samples. 3
Limonoids are a group of triterpenoid compounds that are widely distributed in Citrus sp. and other members of the Rutaceae. The naturally occurring limonoids are diverse in their chemistry (Figure 1), yet characteristic structural features exist (5). These are: 1) a furan ring attached to the D-ring at C-17; 2) oxygen containing functional groups at C-3, C-4, C-7, C-l6, and C-17; 3) a 14,15-epoxide group (except deoxylimonin); and 4) a methyl or oxymethylene at C-l9. One of the major areas of interest in the limonoids is the bitter properties of some of the naturally occurring forms. Limonin has long been known as a major bitter principle © 2000 American Chemical Society
In Citrus Limonoids; Berhow, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.
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74 in processed citrus (3), especially Navel oranges, Shamouti oranges, and grapefruit, and its presence greatly affects the consumer acceptance of these products. Limonin, nomilin, and ichangin are all bitter, while deacetylnomilin, obacunone, limonoate A-ring monolactone, and deoxylimonin (Figure 1) are regarded as non-bitter (4-7). As little as 0.5 ppm of limonin in juice can be detected by some people, however, below 6 ppm is normally considered to be non-bitter, 6-11 ppm is slightly bitter, and 18 ppm or more is very bitter (3). These values reflect a very narrow range of organoleptic tolerance when compared to the bitterness range of naringin, a flavonoid bitter principle in grapefruit. The range of organoleptic response to naringin isfrom20-1500 ppm (8, 9). The focus of the work described in this chapter was to learn as much as possible about the distribution and natural variation in limonin production by citrus fruit and vegetative tissues throughout growth and development. Additionally, studies were conducted to ascertain the natural variation in individual lots offreshlyprocessed juice. Another goal was to determine the feasibility of rapid identification of low limonin producing plants that could be used in the development of new and improved strains of citrus. Prior to 1980, only HPLC methods or semi-quantitative TLC methods were used for limonin detection (7, 10, 11). Neither of these methods were ideal for the planned studies. HPLC isrelatively sensitive, but fewer than 20 samples can be processed per day and each must be pre-purified. Our experimental designs required greater sensitivity and/or processing capacity than that afforded by either TLC or HPLC. Therefore, a radioimmunoassay for the detection of limonin in citrus, which is sensitive to the ppb range and allows the processing of over 400 unpurified samples per day, was developed by Mansell and Weiler (1, 2) in order to study the natural variation of limonin in citrus.
Immunoassay Technology: an Overview Quantification of molecules by immunoassay exploits the specificity of the mammalian immune system towards perceived infection by foreign agents. Microbes and/or large macromolecules are recognized as "non-self and antibodies are produced to neutralize the potential danger. Small compounds (usually
