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Determination of solanidine in human plasma by radioimmunoassay
Fd Chem. Toxk'. Vol. 21, No. 5, pp. 637-640, 1983
0278-6915/83 $3.00 + 0.00 Copyright :t'~ 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
D E T E R M I N A T I O N OF S O L A N I D I N E IN H U M A N BY R A D I O I M M U N O A S S A Y
PLASMA
J. A. MATTHEW, M. R. A. MORGAN, R. MCNERNEY, H. W.-S. CHAN and D. T. COXON ARC Food Research Institute, Colney Lane, Norwich NR4 7UA, England (Received 5 October 1982; revision received 28 November 1982)
radioimmunoassay for solanidine, the major hydrolysis product of potato glycoalkaloids, has been established and validated for application to human plasma. All 34 samples of human plasma tested contained solanidine, with a mean of 1.27 + 0.97 ng/ml (3.18 + 2.43 nmol/litre). Abstract--A
INTRODUCTION
~-Solanine and ~-chaconine constitute 95% of the total glycoalkaloid fraction found in commercially available potatoes, Solanum tuberosum (Paseshnichenko & Guseva, 1956). Normal levels of this fraction, which are determined by genetic and environmental factors, are between 5.9 and 15.1 mg/100 g fresh tuber (Coxon, Price & Jones, 1979). Hydrolysis of the glycosidic side chains of ct-solanine and ~-chaconine yields a common aglycone, the steroidal alkaloid solanidine (Fig. 1). The presence of glycoalkaloids in potato foliage is thought to be desirable for resistance to disease and insect infestation (Allen & Kuc, 1968; Sinden, Sanford & Osman, 1980), but a high foliar concentration is generally correlated with an undesirably high tuber content of glycoalkaloid. In breeding for disease and pest resistance one must therefore ensure that an excessively high glycoalkaloid content is not introduced into the tubers. Such tubers would have a bitter taste (Sinden & Deahl, 1976) and would be potentially toxic. There have been several reports of human H CH 3 CH s
RO"
Fig.
1.
Structure of ~-solanine
(R = ~alact°se-gluc°se / rhamnose
poisoning being associated with consumption of potatoes with a high glycoalkaloid concentration (McMillan & Thompson, 1979), and the poisoning of farm animals by greened or sprouting potatoes is a well-recognized hazard. A correlation between human neural tube defects, such as spina bifida, and potato glycoalkaloids has been suggested (Renwick, 1972; Renwick, Possami & Manday, 1974) but not proven (Nevin & Merrett, 1975). Experiments with pure glycoalkaloids have shown the teratogenic effects of, for example, ~-chaconine in mice (Pierro, Haines & Osman, 1977) and hamsters (Renwick, 1982) but have also shown that toxicity is acutely dependent upon the method of glycoalkaloid administration. Thus in mice the LD~ for ~-solanine given intraperitoneally is 42 mg/kg, but mice are unharmed by 1000mg/kg given orally (Nishie, Gumbmann & Keyl, 1971). This could be explained by absorption properties, since ~-chaconine has been shown to be poorly adsorbed from the rat gastrointestinal tract (Norred, Nishie & Osman, 1976), but it does not explain the observations of human poisoning caused by potatoes, since in these cases the glycoalkaloid levels in the tubers, whilst high, are not dramatically elevated (Abbot, Field & Johnson, 1960; McMillan & Thompson, 1979). McMillan & Thompson (1979), noting this anomaly, have suggested the presence in the toxic potatoes of a further factor which acts to modify absorption of glycoalkaloid. Studies to define any possible hazard to man through either the toxic or the teratogenic effects of glycoalkaloids have been hindered by the lack of suitable analytical methods that can be applied routinely to body fluids. We have set up a radioimmunoassay for solanidine and applied it to the detection and determination of this compound in human plasma.
/' EXPERIMENTAL
c~-chaconine
Chemicals. ~-Solanine, ~-chaconine and solanidine were obtained from etiolated potato sprouts and purified (Coxon et al. 1979). Demissidine was prepared by hydrogenation of solanidine. Rubijervine was obtained from ICN Pharmaceuticals (Houston, TX, USA), solasodine and fl-sitosterol from Koch
(R = ~luc°se-rhamn°se / rhamnose
/'
and solanidine (R = H). 637
638
J. A. MATTHEWet al.
Light Laboratories (Colnbrook, Bucks.), tomatidine from Fluka AG (Buchs, Switzerland) and keyhole limpet haemocyanin (KHLH) from C. P. Laboratories (Bishops Stortford, Herts.). Bovine serum albumin (BSA), activated charcoal, dextran (mol wt 40,000), cholesterol, tomatine and stigmasterol were from Sigma Chemical Co. (London). [16,22-3H]Solanidine, specific activity approximately 10mCi/ mg, was obtained from Professor J. H. Renwick, London School of Hygiene and Tropical Medicine. All other chemicals and solvents were obtained from BDH Chemicals Ltd (Poole, Dorset). Chloroform was re-distilled prior to use. Animals and plasma samples. Male New Zealand white rabbits (2-3 kg) were obtained from Bantin and Kingman Ltd (Hull). Plasma samples were collected by Dr M. McMillan, Consultant Chemical Pathologist, Lewisham Hospital, London, and were taken in the morning, before lunch. Procedure
Rabbits were immunized as described previously (Whittaker, Fuller & Morgan, 1981) against a solanine-BSA conjugate synthesized by a periodatecleavage method (Butler &Chen, 1967). The immunogen had a molar glycoalkaloid:protein ratio of 3:1, as determined by acid hydrolysis and gas liquid chromatographic quantitation of the liberated solanidine (Coxon et al. 1979) and of 9:1 as determined by utilizing the immunoassay described. The immune serum was stored at - 4 0 ° C and diluted in phosphate-buffered saline, pH 7.4, containing BSA (0.1~o, w/v) as required prior to use. Throughout the present study, one particular bleed was used at a final dilution of 1/3000. The radioimmunoassay methodology was described previously (Morgan, Whittaker, Fuller & Dean, 1980). The sample or standard to be assayed (0.1 ml) and [3H]solanidine (2400 dpm, 0.1 ml), both in phosphate-buffered saline, together with antisera (0.2ml) were incubated for 15hr at 2°C. Free and antibody-bound fractions were separated using dextran-coated charcoal. After determination of the radioactivity present in the supernatant (antibodybound fraction), sample solanidine concentrations were calculated by reference to standard curves. Plasma samples were extracted with 4 vols chloroform. The organic layer was either transferred to assay tubes and evaporated under a stream of nitrogen prior to assay or chromatographed overnight on Whatman No. 1 paper in butanol-ethanol (80:20, v/v). The putative solanidine was eluted into assay tubes, taken to dryness and assayed. Bulked human plasma was extracted with dextran-charcoal overnight at +4°C, filtered and assayed as above.
Table 1. Cross reaction of various compounds for the solanidineantiserum Cross reaction Compound % -Chaconine Cholesterol Demissidine Rubijervine fl-Sitosterol Solanidine ~-Solanine Salasodine Stigmasterol Tomatidine Tomatine
100 < 0.1 100 4.0 < 0.1 100 100 < 0.1 < 0.1 < 0.1 < 0.1
chloroform before assay. Chloroform extracts of pre-immune rabbit plasma and charcoal-extracted human plasma assayed as for zero solanidine. All chloroform extracts of human plasma assayed as positive for solanidine. Quantification of solanidine in analyte-free media was checked as follows: standard solutions of solanidine in buffer were (i) used directly, (ii) added to rabbit plasma or (iii) added to charcoal-extracted human plasma, and were subsequently extracted with chloroform and assayed. Fig. 2 shows the results of these assays. Chromatography of chloroform extracts of human plasma followed by elution and assay of the solanidine fraction gave results that were the same (when corrected for recovery) as those obtained by assay of the initial extracts. These extracts also showed linearity of response over a range appropriate to the standard curve. For example, 0.5, 0.75 and 1.0 ml of one sample gave solanidine concentrations of 1.6, 1.6 and 1.7 ng/ml, respectively. Table 2 shows the solanidine concentrations of 34 human plasma samples obtained at random from a hospital clinic. Repeated assay of a plasma pool gave an inter-assay coefficient of variation of 18.8~o ( n - - 1 0 ) and an intra-assay coefficient of 17.8% (n = 10). The lower limit of detection of the standard curve was 200 pg (0.5 pmol). Equivalents of 1 ml of plasma were assayed, giving an assay sensitivity of 0.2 ng/ml (0.5 nmol/litre). 60
50
i~ 40 o o
30
~ 2o o m
IO RESULTS
Table 1 shows the cross-reactions of various compounds tested against solanidine. Cross-reaction was defined according to Abraham (1969) but was corrected for molecular weight. Superimposable standard curves were obtained when solanidine in buffer was assayed directly and when the solanidine in buffer was extracted with
I
I
OI
I
Solanidine
I IO
[ngl
Fig. 2. Standard curves obtained from assay of standard solutions of solanidine in buffer (0), in rabbit plasma (©) and in charcoal-extracted human plasma (A). Radioactivity present in the antibody-bound fraction (as a percentage) is plotted against the amount of added solanidine.
639
Solanidine immunoassay Table 2. Plasma-solanidine concentrations of 34 human adults Solanidine concn in plasma Subject no. ng/ml nmol/litre Males
1 2 3 4 5 6 7
0.85 1.45 4.15 1.12 1.37 0.80 1.17 Mean + SD* 1.56_+ 1.17
2.13 3.63 10.38 2.80 3.43 2.00 2.93 3.88+2.92
Females
1 2 3 4 5 6 7 8 9 10 11 12
0.33 0.80 0.36 1.50 0.85 1.42 1.75 5.00 0.89 2.95 0.67 0.57
0.83 2.00 0.90 3.75 2.13 3.55 4.38 12.50 2.23 7.38 1.68 1.43
13
1.40
3.50
14 15 16 17 18 19 20 21 22 23 24 25 26 27
1.07 2.68 0.58 1.45 1.50 3.75 0.75 1.88 1.15 2.88 0.96 2.40 1.50 3.75 1.07 2.68 0.80 2.00 1.30 3.25 0.98 2.45 0.80 2.00 0.88 2.20 0.52 1.30 Mean _+SD* 1.20 + 0.93 3.00 __+2.33 *Overall mean + SD for males and females was 1.27__-0.97ng/ml (3.18 _ 2.43 nmol/litre).
DISCUSSION O u r evidence suggests t h a t the a n t i s e r u m raised is extremely specific. Closely related c o m p o u n d s such as tomatine, t o m a t i d i n e a n d solasodine h a d no affinity for the antiserum. Neither did the sterols tested interfere with the assay. Rubijervine (12~-hydroxysolanidine) showed only 4% cross-reaction. The cross-reactions o f ~-chaconine, ~-solanine a n d demissidine (all 100%) would be anticipated when solanine is coupled via the 3-OH position of the sterol to BSA in order to synthetize the i m m u n o g e n . Demissidine (5-dihydrosolanidine) a n d its glycoside demissine, if they occur, are only very m i n o r c o m p o n e n t s o f the total glycoalkaloid fraction o f commercial p o t a t o varieties. These two, together with ~-solanine a n d ~ - c h a c o n i n e (the m a j o r c o m p o n e n t s ) , would not n o r m a l l y form a significant p a r t o f the c h l o r o f o r m extract o f h u m a n plasma. The validity o f the assay is further indicated by the superimposability of the s t a n d a r d curves o b t a i n e d from extracts o f solanidine in buffer, in r a b b i t p l a s m a a n d in charcoal-extracted h u m a n plasma (Fig. 2). Solanidine would n o t be expected to occur either in the plasma of rabbits fed on n o r m a l l a b o r a t o r y a n i m a l diets or in the charcoal-extracted h u m a n
plasma, a n d indeed such plasma samples assayed as for zero solanidine in o u r system. N o r m a l h u m a n p l a s m a always gave positive results which could not be eliminated by c h r o m a t o g r a p h y a n d subsequent elution o f relevant b a n d s for assay. W e believe that this r a d i o i m m u n o a s s a y is specific for solanidine in the c h l o r o f o r m extracts o f h u m a n plasma, a n d therefore that solanidine is present in the h u m a n plasma samples examined. Solanidine has previously been detected a n d quantified in h u m a n urine using a gas c h r o m a t o g r a p h y - m a s s spect r o m e t r y procedure (D. T. Coxon, personal c o m m u nication, 1982), but this is the first report o f direct analysis of plasma. Tritiated solanidine has been identified by thin-layer c h r o m a t o g r a p h y of extracts of rat urine after oral a d m i n i s t r a t i o n of labelled solanine (Nishie et al. 1971). These results o f the analysis of 34 h u m a n plasma samples show a solanidine range of 0.33 5.00 ng/ml (0.83-12.50nmol/litre). The apparently widespread occurrence o f this c o m p o u n d m a y be due to several factors. Potatoes occur extensively in n o r m a l h u m a n diets, a n d they are not easy to avoid. In 1980/81, 21.2% of the U K p o t a t o crop was used for processing (Potato M a r k e t i n g Board, 1981) a n d glycoalkaloids have been found to be very stable during cooking (Bushway & P o n n a m p a l a m , 1981). Whilst there m a y be a significance in the ubiquitous occurrence o f solanidine in h u m a n plasma, assessment o f such significance must await the results o f further studies. Acknowledgements--We would like to thank Dr M. McMillan (Lewisham Hospital) and Professor V. Marks and Mr B. A. Morris (Department of Biochemistry, University of Surrey) for their assistance and advice.
REFERENCES
Abbot D. C., Field K. & Johnson E. I. (1960). Observations on the correlation of anti-cholinesterase effect with solanine content of potatoes. Analyst 85, 375. Abraham (3. E. (1969). Solid-phase radioimmunoassay of estradiol-17 beta. J. clin. Endocr. Metab. 29, 866. Allen E. H. & Kuc J. (1968). ~-Solanine and c~-chaconine as fungitoxic compounds in extracts of Irish potato tubers. Phytopathology 58, 776. Bushway R. J. & Ponnampalam R. (1981). ~-Chaconine and ~-solanine content of potato products and their stability during several modes of cooking. J. Agric. Fd Chem. 29, 814. Butler V. P., Jr & Chen J. P. (1967). Digoxin-specific antibodies. Proc. natn. Acad. Sci. U.S.A. 57, 71. Coxon D. T., Price K. R. & Jones P. G. (1979). A simplified method for the determination of total glycoalkaloids in potato tubers. J. Sci. Fd Agric. 30, 1043. McMillan M. & Thompson J. C. (1979). An outbreak of suspected solanine poisoning in schoolboys: examination of criteria of solanine poisoning. Q. Jl Med. 48 (190), 227. Morgan M. R. A., Whittaker P. G., Fuller B, P. & Dean P. D. G. (1980). A radioimmunoassay for equilin in postmenopausal plasma: plasma levels of equilin determined after oral administration of conjugated equine oestrogens (Premarin). J. steroid Biochem. 13, 551. Nevin N. C. & Merrett J. D. (1975). Potato avoidance during pregnancy in women with a previous infant with either anencephaly and/or spina bifida. Br. J. prey. soc. Med. 29, 111. Nishie K., Gumbmann M. R. & Keyl A. C. (1971). Pharmacology of solanine. Toxic. appl. Pharmac. 19, 81.
640
J . A . MATTHEW et al.
Norred W. P., Nishie K. & Osman S. F. (1976). Excretion, distribution and metabolic fate of 3H-~-chaconine. Res. Commun. chem. Path. Pharmac. 13, 161. Paseshnichenko V. & Guseva A. R. (1956). Quantitative determination of potato glycoalkaloids and their preparative separation. Biochemistry, U S S R 21, 606. Pierro C. J., Haines J. S. & Osman S. F. (1977). Teratogenicity and toxicity of purified ~-chaconine and ~-solanine. Teratology 15, 31A. Potato Marketing Board (1981). Potato Processing in Great Britain. PMB, London. Renwick J. H. (1972). Hypothesis: anencephaly and spina bifida are usually preventable by avoidance of a specific but unidentified substance present in certain potato tubers. Br. J. prey. soc. Med. 26, 67.
Renwick J. H. (1982). Vitamin supplementation and neural tube defects. Lancet I, 748. Renwick J. H., Possami A. M. & Manday M. R. (1974). Potatoes and spina bifida. Proc. R. Soc. Med. 67, 10. Sinden S. L. & Deahl K. L. (1976). Effect of glycoalkaloids and phenolics on potato flavour. J. Fd Sci. 41, 520. Sinden S. L., Sanford L. L. & Osman S. F. (1980). Glycoalkaloids and resistance to the Colorado potato beetle in Solanum chacoense bitter. Am. Pot. J. 57, 331. Whittaker P. G., Fuller B. P. & Morgan M. R. A. (1981). Rabbit anti-steroid antisera: a study of titers and specificities over a 22-week period. Experientia 37, 1203. specificities over a 22-week period. Experientia 37, 1203.
0278-6915/83 $3.00 + 0.00 Copyright :t'~ 1983 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
D E T E R M I N A T I O N OF S O L A N I D I N E IN H U M A N BY R A D I O I M M U N O A S S A Y
PLASMA
J. A. MATTHEW, M. R. A. MORGAN, R. MCNERNEY, H. W.-S. CHAN and D. T. COXON ARC Food Research Institute, Colney Lane, Norwich NR4 7UA, England (Received 5 October 1982; revision received 28 November 1982)
radioimmunoassay for solanidine, the major hydrolysis product of potato glycoalkaloids, has been established and validated for application to human plasma. All 34 samples of human plasma tested contained solanidine, with a mean of 1.27 + 0.97 ng/ml (3.18 + 2.43 nmol/litre). Abstract--A
INTRODUCTION
~-Solanine and ~-chaconine constitute 95% of the total glycoalkaloid fraction found in commercially available potatoes, Solanum tuberosum (Paseshnichenko & Guseva, 1956). Normal levels of this fraction, which are determined by genetic and environmental factors, are between 5.9 and 15.1 mg/100 g fresh tuber (Coxon, Price & Jones, 1979). Hydrolysis of the glycosidic side chains of ct-solanine and ~-chaconine yields a common aglycone, the steroidal alkaloid solanidine (Fig. 1). The presence of glycoalkaloids in potato foliage is thought to be desirable for resistance to disease and insect infestation (Allen & Kuc, 1968; Sinden, Sanford & Osman, 1980), but a high foliar concentration is generally correlated with an undesirably high tuber content of glycoalkaloid. In breeding for disease and pest resistance one must therefore ensure that an excessively high glycoalkaloid content is not introduced into the tubers. Such tubers would have a bitter taste (Sinden & Deahl, 1976) and would be potentially toxic. There have been several reports of human H CH 3 CH s
RO"
Fig.
1.
Structure of ~-solanine
(R = ~alact°se-gluc°se / rhamnose
poisoning being associated with consumption of potatoes with a high glycoalkaloid concentration (McMillan & Thompson, 1979), and the poisoning of farm animals by greened or sprouting potatoes is a well-recognized hazard. A correlation between human neural tube defects, such as spina bifida, and potato glycoalkaloids has been suggested (Renwick, 1972; Renwick, Possami & Manday, 1974) but not proven (Nevin & Merrett, 1975). Experiments with pure glycoalkaloids have shown the teratogenic effects of, for example, ~-chaconine in mice (Pierro, Haines & Osman, 1977) and hamsters (Renwick, 1982) but have also shown that toxicity is acutely dependent upon the method of glycoalkaloid administration. Thus in mice the LD~ for ~-solanine given intraperitoneally is 42 mg/kg, but mice are unharmed by 1000mg/kg given orally (Nishie, Gumbmann & Keyl, 1971). This could be explained by absorption properties, since ~-chaconine has been shown to be poorly adsorbed from the rat gastrointestinal tract (Norred, Nishie & Osman, 1976), but it does not explain the observations of human poisoning caused by potatoes, since in these cases the glycoalkaloid levels in the tubers, whilst high, are not dramatically elevated (Abbot, Field & Johnson, 1960; McMillan & Thompson, 1979). McMillan & Thompson (1979), noting this anomaly, have suggested the presence in the toxic potatoes of a further factor which acts to modify absorption of glycoalkaloid. Studies to define any possible hazard to man through either the toxic or the teratogenic effects of glycoalkaloids have been hindered by the lack of suitable analytical methods that can be applied routinely to body fluids. We have set up a radioimmunoassay for solanidine and applied it to the detection and determination of this compound in human plasma.
/' EXPERIMENTAL
c~-chaconine
Chemicals. ~-Solanine, ~-chaconine and solanidine were obtained from etiolated potato sprouts and purified (Coxon et al. 1979). Demissidine was prepared by hydrogenation of solanidine. Rubijervine was obtained from ICN Pharmaceuticals (Houston, TX, USA), solasodine and fl-sitosterol from Koch
(R = ~luc°se-rhamn°se / rhamnose
/'
and solanidine (R = H). 637
638
J. A. MATTHEWet al.
Light Laboratories (Colnbrook, Bucks.), tomatidine from Fluka AG (Buchs, Switzerland) and keyhole limpet haemocyanin (KHLH) from C. P. Laboratories (Bishops Stortford, Herts.). Bovine serum albumin (BSA), activated charcoal, dextran (mol wt 40,000), cholesterol, tomatine and stigmasterol were from Sigma Chemical Co. (London). [16,22-3H]Solanidine, specific activity approximately 10mCi/ mg, was obtained from Professor J. H. Renwick, London School of Hygiene and Tropical Medicine. All other chemicals and solvents were obtained from BDH Chemicals Ltd (Poole, Dorset). Chloroform was re-distilled prior to use. Animals and plasma samples. Male New Zealand white rabbits (2-3 kg) were obtained from Bantin and Kingman Ltd (Hull). Plasma samples were collected by Dr M. McMillan, Consultant Chemical Pathologist, Lewisham Hospital, London, and were taken in the morning, before lunch. Procedure
Rabbits were immunized as described previously (Whittaker, Fuller & Morgan, 1981) against a solanine-BSA conjugate synthesized by a periodatecleavage method (Butler &Chen, 1967). The immunogen had a molar glycoalkaloid:protein ratio of 3:1, as determined by acid hydrolysis and gas liquid chromatographic quantitation of the liberated solanidine (Coxon et al. 1979) and of 9:1 as determined by utilizing the immunoassay described. The immune serum was stored at - 4 0 ° C and diluted in phosphate-buffered saline, pH 7.4, containing BSA (0.1~o, w/v) as required prior to use. Throughout the present study, one particular bleed was used at a final dilution of 1/3000. The radioimmunoassay methodology was described previously (Morgan, Whittaker, Fuller & Dean, 1980). The sample or standard to be assayed (0.1 ml) and [3H]solanidine (2400 dpm, 0.1 ml), both in phosphate-buffered saline, together with antisera (0.2ml) were incubated for 15hr at 2°C. Free and antibody-bound fractions were separated using dextran-coated charcoal. After determination of the radioactivity present in the supernatant (antibodybound fraction), sample solanidine concentrations were calculated by reference to standard curves. Plasma samples were extracted with 4 vols chloroform. The organic layer was either transferred to assay tubes and evaporated under a stream of nitrogen prior to assay or chromatographed overnight on Whatman No. 1 paper in butanol-ethanol (80:20, v/v). The putative solanidine was eluted into assay tubes, taken to dryness and assayed. Bulked human plasma was extracted with dextran-charcoal overnight at +4°C, filtered and assayed as above.
Table 1. Cross reaction of various compounds for the solanidineantiserum Cross reaction Compound % -Chaconine Cholesterol Demissidine Rubijervine fl-Sitosterol Solanidine ~-Solanine Salasodine Stigmasterol Tomatidine Tomatine
100 < 0.1 100 4.0 < 0.1 100 100 < 0.1 < 0.1 < 0.1 < 0.1
chloroform before assay. Chloroform extracts of pre-immune rabbit plasma and charcoal-extracted human plasma assayed as for zero solanidine. All chloroform extracts of human plasma assayed as positive for solanidine. Quantification of solanidine in analyte-free media was checked as follows: standard solutions of solanidine in buffer were (i) used directly, (ii) added to rabbit plasma or (iii) added to charcoal-extracted human plasma, and were subsequently extracted with chloroform and assayed. Fig. 2 shows the results of these assays. Chromatography of chloroform extracts of human plasma followed by elution and assay of the solanidine fraction gave results that were the same (when corrected for recovery) as those obtained by assay of the initial extracts. These extracts also showed linearity of response over a range appropriate to the standard curve. For example, 0.5, 0.75 and 1.0 ml of one sample gave solanidine concentrations of 1.6, 1.6 and 1.7 ng/ml, respectively. Table 2 shows the solanidine concentrations of 34 human plasma samples obtained at random from a hospital clinic. Repeated assay of a plasma pool gave an inter-assay coefficient of variation of 18.8~o ( n - - 1 0 ) and an intra-assay coefficient of 17.8% (n = 10). The lower limit of detection of the standard curve was 200 pg (0.5 pmol). Equivalents of 1 ml of plasma were assayed, giving an assay sensitivity of 0.2 ng/ml (0.5 nmol/litre). 60
50
i~ 40 o o
30
~ 2o o m
IO RESULTS
Table 1 shows the cross-reactions of various compounds tested against solanidine. Cross-reaction was defined according to Abraham (1969) but was corrected for molecular weight. Superimposable standard curves were obtained when solanidine in buffer was assayed directly and when the solanidine in buffer was extracted with
I
I
OI
I
Solanidine
I IO
[ngl
Fig. 2. Standard curves obtained from assay of standard solutions of solanidine in buffer (0), in rabbit plasma (©) and in charcoal-extracted human plasma (A). Radioactivity present in the antibody-bound fraction (as a percentage) is plotted against the amount of added solanidine.
639
Solanidine immunoassay Table 2. Plasma-solanidine concentrations of 34 human adults Solanidine concn in plasma Subject no. ng/ml nmol/litre Males
1 2 3 4 5 6 7
0.85 1.45 4.15 1.12 1.37 0.80 1.17 Mean + SD* 1.56_+ 1.17
2.13 3.63 10.38 2.80 3.43 2.00 2.93 3.88+2.92
Females
1 2 3 4 5 6 7 8 9 10 11 12
0.33 0.80 0.36 1.50 0.85 1.42 1.75 5.00 0.89 2.95 0.67 0.57
0.83 2.00 0.90 3.75 2.13 3.55 4.38 12.50 2.23 7.38 1.68 1.43
13
1.40
3.50
14 15 16 17 18 19 20 21 22 23 24 25 26 27
1.07 2.68 0.58 1.45 1.50 3.75 0.75 1.88 1.15 2.88 0.96 2.40 1.50 3.75 1.07 2.68 0.80 2.00 1.30 3.25 0.98 2.45 0.80 2.00 0.88 2.20 0.52 1.30 Mean _+SD* 1.20 + 0.93 3.00 __+2.33 *Overall mean + SD for males and females was 1.27__-0.97ng/ml (3.18 _ 2.43 nmol/litre).
DISCUSSION O u r evidence suggests t h a t the a n t i s e r u m raised is extremely specific. Closely related c o m p o u n d s such as tomatine, t o m a t i d i n e a n d solasodine h a d no affinity for the antiserum. Neither did the sterols tested interfere with the assay. Rubijervine (12~-hydroxysolanidine) showed only 4% cross-reaction. The cross-reactions o f ~-chaconine, ~-solanine a n d demissidine (all 100%) would be anticipated when solanine is coupled via the 3-OH position of the sterol to BSA in order to synthetize the i m m u n o g e n . Demissidine (5-dihydrosolanidine) a n d its glycoside demissine, if they occur, are only very m i n o r c o m p o n e n t s o f the total glycoalkaloid fraction o f commercial p o t a t o varieties. These two, together with ~-solanine a n d ~ - c h a c o n i n e (the m a j o r c o m p o n e n t s ) , would not n o r m a l l y form a significant p a r t o f the c h l o r o f o r m extract o f h u m a n plasma. The validity o f the assay is further indicated by the superimposability of the s t a n d a r d curves o b t a i n e d from extracts o f solanidine in buffer, in r a b b i t p l a s m a a n d in charcoal-extracted h u m a n plasma (Fig. 2). Solanidine would n o t be expected to occur either in the plasma of rabbits fed on n o r m a l l a b o r a t o r y a n i m a l diets or in the charcoal-extracted h u m a n
plasma, a n d indeed such plasma samples assayed as for zero solanidine in o u r system. N o r m a l h u m a n p l a s m a always gave positive results which could not be eliminated by c h r o m a t o g r a p h y a n d subsequent elution o f relevant b a n d s for assay. W e believe that this r a d i o i m m u n o a s s a y is specific for solanidine in the c h l o r o f o r m extracts o f h u m a n plasma, a n d therefore that solanidine is present in the h u m a n plasma samples examined. Solanidine has previously been detected a n d quantified in h u m a n urine using a gas c h r o m a t o g r a p h y - m a s s spect r o m e t r y procedure (D. T. Coxon, personal c o m m u nication, 1982), but this is the first report o f direct analysis of plasma. Tritiated solanidine has been identified by thin-layer c h r o m a t o g r a p h y of extracts of rat urine after oral a d m i n i s t r a t i o n of labelled solanine (Nishie et al. 1971). These results o f the analysis of 34 h u m a n plasma samples show a solanidine range of 0.33 5.00 ng/ml (0.83-12.50nmol/litre). The apparently widespread occurrence o f this c o m p o u n d m a y be due to several factors. Potatoes occur extensively in n o r m a l h u m a n diets, a n d they are not easy to avoid. In 1980/81, 21.2% of the U K p o t a t o crop was used for processing (Potato M a r k e t i n g Board, 1981) a n d glycoalkaloids have been found to be very stable during cooking (Bushway & P o n n a m p a l a m , 1981). Whilst there m a y be a significance in the ubiquitous occurrence o f solanidine in h u m a n plasma, assessment o f such significance must await the results o f further studies. Acknowledgements--We would like to thank Dr M. McMillan (Lewisham Hospital) and Professor V. Marks and Mr B. A. Morris (Department of Biochemistry, University of Surrey) for their assistance and advice.
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