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Assessment of dietary exposure to the natural toxin hypoglycin in ackee (Blighia sapida) by Jamaican consumers
Food Research International 37 (2004) 833–838 www.elsevier.com/locate/foodres
Assessment of dietary exposure to the natural toxin hypoglycin in ackee (Blighia sapida) by Jamaican consumers Orane A. Blake b
a,1
, Jose C. Jackson
a,*
, Maria A. Jackson b, C.L. Andre Gordon
c
a Department of Chemistry, University of the West Indies, Mona Campus, Kingston 7, Jamaica Department of Community Health and Psychiatry, University of the West Indies, Mona Campus, Kingston 7, Jamaica c Technological Solutions Limited, 71/2 Retirement Road, Kingston 5, Jamaica
Received 29 March 2004; accepted 2 May 2004
Abstract Dietary exposure to hypoglycin (HG), the natural toxin found in the ackee fruit of Jamaica, was determined for children and adults using ackee consumption data and quantifying HG levels in typical ackee diets. Ackee consumption was highest in the lower socio-economic group, particularly in children. HG occurrence levels in typical ackee diets ranged from 1.21 to 89.28 lg HG/g ackee. Dietary exposure to HG was highest in young children, who lived in lower socio-economic rural areas of Jamaica. This study is the first to quantify dietary exposure to HG by Jamaican consumers, providing some basis to guide risk assessment. The findings also concur with observations that young children in lower socio-economic groups are the most vulnerable to HG toxicity, when it occurs. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Ackee; Natural toxin; Hypoglycin; Ackee consumption; Dietary exposure; Jamaica
1. Introduction Ackee is the national fruit of Jamaica. It is widely consumed by Jamaicans around the world, by Haitians and in parts of Western Africa. Millions of visitors to Jamaica over the years from various countries around the world have also eaten ackee during their visits to Jamaica. Dr. Thomas Clarke, the first island botanist, brought the fruit to Jamaica in 1778, after obtaining the seeds from a West African slave ship (Bressler, Corridor, & Brendel, 1969; Hill, 1952). The young fruit produced is a green pod which becomes red or yellow upon maturation and then splits open to reveal two to four shiny
*
Corresponding author. Present address: Department of Research and Development, National Food Technology Research Centre, P.O. Box 70498, Gaborone, Botswana. Tel.: +267-71464330/267-393-6243; fax: +267-393-6243. E-mail address: [email protected] (J.C. Jackson). 1 Present Address: Department of Food Science, Purdue University, West Lafayette, IN 47907. 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.05.003
black seeds, each having a cream-colored fleshy body, the arillus, at its base (Fig. 1). For many years, it was recognized that the unripe ackee fruit may be poisonous (Hassall & Hill, 1955; Hill, 1952), containing a natural toxin, which exists as the cyclic amino acid, hypoglycin A (HG-A), and its c-glutamyl derivative, hypoglycin B (HG-B). The level of the toxin is elevated in the unripe fruit at concentrations of 100–111 mg/100 g, while present at an innocuous concentration of less than 10 mg/100 g when the fruit is ripe (Brown, Bates, Mcgowan, & Cornell, 1992). Several studies have also been conducted that establishes the levels of HG in canned ackees exported to Canada, the US and England. These studies have shown that the HG levels are generally well within the limit of 100 ppm set by the Food and Drug Administration (FDA) and Health Canada (unpublished data). In 1973, because of concerns of this natural toxin HG, the FDA imposed an import alert on canned ackee from Jamaica; this was upgraded to ‘‘ackees in all forms’’ in 1993. Although the ban did not extend to
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O.A. Blake et al. / Food Research International 37 (2004) 833–838
Fig. 1. Mature ripe ackee fruit showing ackee arillus, seeds and pod (Picture courtesy of Dr. Andre Gordon).
Canada, it was a major loss to the Jamaican ackee processing industry since it effectively removed over one million consumers from the accessible market (FDA, 2000; Personal Communication, 2000). The ban was lifted in 2000 after the adoption of HACCP based programs in a number of commercial enterprises and a public and private sector partnership that provided training and testing facilities to these enterprises. Consumption of the unripe fruit produces an acute toxic effect, usually within 6–48 h, with symptoms including nausea, vomiting, drowsiness and hypoglycemia (Bressler et al., 1969; Brown et al., 1992; Hassall & Hill, 1955; Hill, 1952). Coma and death occur within 12 h in severe cases (Brown et al., 1992; Hassall & Hill, 1955; Hill, 1952). This illness is commonly known as Jamaican vomiting sickness (JVS) or more accurately toxic hypoglycaemic syndrome (THS) (Baldwin & Parker, 1987). It has been reported to affect young children more adversely than adults in a number of reported cases (Bressler et al., 1969; Brown et al., 1992; Hassall & Hill, 1955; Hill, 1952). When ingested the toxin follows a similar metabolic pathway as a branched chained amino acid, thus producing the active metabolite methylenecyclopropylacetyl-CoA (MCPA-CoA). MCPA-CoA then irreversibly binds to flavin adenine dinucleotide (FAD) and thereby inhibits the activity of medium chain acyldehydrogenases. Medium chain and short chain acyldehydrogenases are critical to the complete b-oxidation of fats. Hence, their inactivation will have adverse effects on blood serum short chain fatty acid concentrations, urinary hydroxy and dicarboxylic acid concentrations, as well as plasma and urinary amino acid concentrations (Tanaka, Kean, & Johnson, 1976; Von Holt & Bohm, 1966). Although significant research has been conducted on the biochemistry of HG (Baldwin, Adlington, Bebbington, & Russell, 1994; Baldwin & Parker, 1987; Kean, 1974; Kean, 1989; Manchester, 1974; Ming-tain Lai, Eugene, & Hung-wen, 1991, 1992, 1993; Tanaka, Is-
selbacher, & Shih, 1972; Tanaka et al., 1976), there has been no previous research that studied the HG exposure of Jamaican consumers. The goal of this study is to quantify the dietary exposure to ackee HG by Jamaican consumers.
2. Materials and methods 2.1. Survey research design An assessment of the quantity of ackee consumed and hence, exposure to the natural toxin HG was conducted in urban (the Parish of Kingston and St. Andrew) and rural communities (the Parish of St. Thomas) in Jamaica. The study was undertaken over a three-month period from January to March 2000. Selection of the study area was based on a process of multistage, stratified, random sampling that was determined by the Statistical Institute of Jamaica (STATIN) as representative of the Jamaican population. Every fourth household was selected after a random starting point. 2.2. Survey instrument A twenty-six-item survey instrument, based on a quantitative food frequency questionnaire developed in Jamaica (Jackson et al., 2001) was used. The instrument was pilot tested on a comparable sample of consumers for clarity and validity; adjustments were made where necessary. The revised questionnaire was divided into three sections: (i) socio-economic and demographic characteristics of the respondents, (ii) consumers’ ackee consumption practices and frequency, and (iii) consumers’ body weight. It was then administered to 300 households from both parishes. Informed consent was obtained by reading a statement to prospective respondents seeking permission for the interview and affirming that the data would be treated confidentially. Data were collected on weekends, and weekday afternoons when a
O.A. Blake et al. / Food Research International 37 (2004) 833–838
member of the selected target population would most likely be at home. Each instrument took an average of 30 min to administer. 2.3. Hypoglycin isolation and characterization An analytically pure standard of HG was isolated from ackee seeds obtained from trees around the University of the West Indies (UWI), Mona Campus, Jamaica, using a modification of the methods of Kean (1974), Fowden (1978), Billington, Osmundsen, and Sherratt (1978), (Fig. 2). The standard was used to quantify the presence of HG in a typical ackee dish consumed in Jamaica. The purity of the standard obtained was determined at the South–East Regional Laboratory, Food and Drug Administration, USA and the Department of Chemistry, UWI, Mona Campus, Jamaica. The purity was assessed using carbon-13 NMR, utilizing D2 0 as a solvent and HPLC coupled to a light scattering detector (LSD) Ground ackee seeds 80% ethanol
Ackee seed extract Extract fat, with hexane Fat free extract
835
with phenylisothiocyanate as the derivatizing agent. About 10.70 mg of the HG-A was dissolved in 25 ml of water and 10 ll of this was then injected onto a C-18 column (5 lm particle size, 16% carbon load). Gradient elution commenced at 1 ml/min with 100% 3 mM trichloroacetic acid (solvent A) to 50% solvent A and 50% acetonitrile (solvent B); the nitrogen flow rate and column temperature were set at 1 ml/min and 27 °C, respectively. A Perkin–Elmer 1600 FT-IR was used to record infrared spectra of the HG-A standard. Samples were analysed as thin films between sodium chloride discs. 2.4. Hypoglycin intake in Jamaican consumers Samples of ackee and salted fish (cod fish), which is the typical way of consuming ackee in Jamaica, were collected in the areas in which the ackee consumption assessment was done. A total of 11 samples were collected from households and restaurants in the geographically diverse areas of Liguanea, New Kingston, Downtown Kingston, Papine, Constant Spring, Red Hills, and Yallahs. The salted fish and other vegetable seasonings were removed from the ackee mixture and the quantity of hypoglycin present in each sample was then determined using the Pico-tag method reported by Sarwar and Botting (1994). The daily hypoglycin intake relative to the body weight of children, adolescents, adults, and the elderly was calculated using the following formula: Intake ðlg=dy=kgBWÞ ¼ ackee consumed ðg=day=kg BWÞ HG occurrence ðlg=gÞ:
Apply to anion exchange resin
2.5. Statistical analysis
Absorbed (acidic amino acids, hypoglycin B)
Unabsorbed (neutral and basic amino acids)
The data were analyzed using SPSS Version 11 (SPSS, 2001), and mean responses and percentages generated. The effect of gender, socio-economic status (SES), and geographical location on the ackee consumed and hypoglycin intake was determined using multivariate analysis of variance.
Gradient elution with 0 - 3M acetic acid HG-B and impurities Hydrolyze HG-B with 2M formic acid
3. Results and discussion 3.1. Profile of the respondents
HG-A and impurities Apply to anion exchange resin Crude HG-A Recrystallize twice using 70% ethanol HG-A > 95% pure Fig. 2. HG-A isolation and purification from ackee seeds.
Table 1 shows the profile of the respondents that participated in the study. A total of 1278 persons participated, of which 55% were females and 45% males. The majority of them (76%) came from the lower socioeconomic status, while only 14% and 10% were from the middle and upper SES in Jamaica, respectively. Eighty nine percent (89%) of them had at least primary and secondary education, while only about 11% had tertiary
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Table 1 Socio-demographic profile of respondents in ackee consumption survey
Table 2 Effect of SES, geographical location, gender and age on ackee consumption
Frequency
Percent
Parameter
Gender Male Female
569 709
44.5 55.5
Age group (yrs) 0–12 13–19 20–59 >60
329 217 636 96
25.7 17 49.8 7.5
Socio-economic status (SES) Upper SES Middle SES Lower SES
0.19 0.13a 0.19 0.13a 0.34 0.28b
Geographical location Kingston/St. Andrew (urban) St. Thomas (rural)
0.22 0.18a 0.34 0.27b
Socio-economic status Lower Middle Upper
961 182 127
75.7 14.3 10.0
Gender Male Female
0.36 0.27a 0.29 0.24b
Location Kingston/St. Andrew St. Thomas
790 488
61.8 38.2
Education Primary Secondary Tertiary
Age Children Adolescents Adults Elderly
0.35 0.26a 0.32 0.15a 0.28 0.14b 0.28 0.13b
413 693 135
33.3 55.8 10.9
education. Most respondents (62%) lived in Kingston/ St. Andrew (urban) and 38% lived in St. Thomas (rural). The majority of the participants (50%) were adults, 26% children, 17% adolescents and about 7% were elderly (>60 yrs old). These findings were consistent with the overall population profile of Jamaica (STATIN, 2001). 3.2. Ackee consumption Table 2 indicated that there were significant differences (p < 0:05) in ackee consumption between gender, individuals from rural and urban locations, different SES and age. Males consumed a greater quantity of ackee (0.36 0.27 g/day/kg BW) than females (0.29 0.24 g/ day/kg BW). Individuals from Kingston and St. Andrew, that is urban areas of Jamaica, consumed significantly less (p < 0:05) ackee (0.22 0.17 g/day/kg BW) than individuals from St. Thomas (0.34 0.27 g/day/kg BW), considered a rural location in Jamaica. In the lower SES, which includes almost all of St. Thomas and a significant number of the respondents from Kingston and St. Andrew, individuals consumed more ackee (0.34 0.28 g/ day/kg BW), in comparison to their counterparts from the middle and upper SES (0.19 0.13 g/day/kg BW). Children also consumed a greater quantity of ackee (0.35 0.26 g/day/kg BW), than adolescents, adults and the elderly (0.32 0.15, 0.28 0.14, and 0.28 0.13 g/day/kg BW, respectively). 3.3. Hypoglycin purity and characterization Approximately 81 mg of white HG-A crystals was extracted from 3890 g of ackee seeds, giving an extrac-
Ackee consumed (g/day/kg BW)
Different letters in each row for a specific parameter indicate significant differences.
tion rate of 0.02%. Previous studies indicated that the extraction rate of HG-A from ackee seeds via the hydrolysis of HG-B is between 0.03% and 0.04% (Billington et al., 1978; Fowden, 1978; Kean, 1974). Therefore the modified method used did not produce significantly different yields from established methods. The purity of the isolated HG-A was established using 13 C NMR and HPLC techniques, which indicated that the HG isolated was greater than 95% pure. The 13 C NMR spectrum of HG-A confirmed that the molecule consisted of seven carbons which were attributed to d 174.67 (C7 O2 H), 134.92 (C1 @CH2 ), 104.15, 104.12 (C@4 CH2 ), 55.32 (d, 6 CHCH2 ), 55.19, 34.03 (5 CH2 ), 11.18 (d, cyclopropyl 2 CH), 10.96, 9.30 (cyclopropyl 3 CH2 ), 9.12. HMQC and COSY spectra confirmed the proton chemical shifts of the HG molecule, i.e. d 5.45 (1H, m, C@CH4a H4b ), 5.42 (m), 5.37 (1H, m, C@CH4a H4b ), 5.36 (m), 3.74 (1H, t, aH), 3.75 (t), 1.77 to 1.91 (2H, m, CH2ð5a;bÞ ), 1.42 (1H, m, cyclopropyl CH2a ), 1.29 (1H, m, cyclopropyl CH3a H3b ), 0.82 (1H, m, cyclopropyl CH3a H3b ). An IR spectrum of isolated HG-A showed absorption bands at 3444 cm1 (NH2 ); 2938– 3000 cm1 (CH2 , CH3 ) and 1583, 1408 cm1 (CO2 ). 3.4. Hypoglycin dietary occurrence The HG concentration found in the prepared ackee dishes from restaurants and homes in the study location ranged from 1.21–89.28 lg HG/mg ackee (Table 3). This wide range may be due to the varying preparation and cooking methods used by individuals from these locations. Traditionally, ackee is firstly cleaned to remove the seeds, pod and raphe (commonly called the placenta or membrane), all of which is thought to contain ap-
O.A. Blake et al. / Food Research International 37 (2004) 833–838 Table 3 HG occurrence in ackee collected from 11 different locationsa Location
HG-A (ppm)
1 2 3 4 5 6 7 8 9 10 11
7.48 0.14 17.73 0.74 27.19 0.83 12.85 2.29 8.44 0.09 89.28 30.73 64.15 6.42 1.40 0.25 21.76 10.06 3.26 1.09 1.21 0.03
a
Mean HG ¼ 23.16 28.56 ppm.
preciable levels of hypoglycin. It is then boiled and sometimes sauteed. The level of hypoglycin in the fruit could therefore be affected by the efficiency of cleaning, particularly in removing the raphe. The boiling time used in preparing ackee dishes is often dependent on the variety and maturity of the ackee and the individual preparing the meal. Ackee varieties may differ in texture, which will affect the boiling time required. Since HG-A is water soluble, varying the boiling times will affect its concentration in the final product. Using ackee with a range of maturities will also naturally affect the hypoglycin content of the final product. 3.5. Hypoglycin intake assessment The mean and maximum HG intake with respect to age, SES, rural and urban locations and gender is illustrated in Table 4. The mean HG intake was calculated by multiplying the average ackee consumption and the average HG concentration (23.16 mg/g) in the ackee dishes. Similarly, the maximum HG intake (lg/day/kg BW) was calculated by multiplying the maximum ackee consumption (average + 3 SD), by the maximum HG concentration found in the ackee dishes (89.28 mg/g). The maximum HG intake was useful for assessing potential HG intake of high consumers of ackee in Jamaica. HG intake differences by age, SES, rural and urban locations and gender followed similar trends as observed for ackee consumption. The data in Table 4 show that children consumed a greater quantity of HG (8.18 6.04 lg HG/day/kg BW) than adolescents, the elderly and adults (7.41 3.52, 6.58 2.90, and 6.55 3.12 lg HG/day/kg BW, respectively). Individuals from the lower SES consumed more HG (7.87 6.48 lg/ day/kg BW) than individuals from the upper and middle SES (4.40 3.01 lg/day/kg BW). Similarly, individuals from St. Thomas consumed 7.87 6.25 lg/day/kg BW, a much larger quantity than individuals from Kingston and St. Andrew (5.10 4.17 lg /day/kg BW). Males also consumed a greater quantity of HG than females (8.34 6.25 and 6.72 5.56 lg/day/kg BW, respec-
837
Table 4 Dietary intake of HG by Jamaican consumers of different ages, SES, geographical location and gender Parameter
Avg. HG intake (lg/day/kg BW)
Max. HG intake (lg/day/kg BW)
Age Children Adolescent Adult Elderly
8.18 6.04a 7.41 3.52b 6.55 3.12c 6.58 4.06c
115.2 79.30 69.90 66.95
SES Upper SES Middle SES Lower SES
4.40 3.01a 4.40 3.01a 7.87 6.48b
51.79 51.79 105.36
5.10 4.17a
67.86
Geographical location Kingston/St. Andrew (urban) St. Thomas (rural)
7.87 6.25b
102.68
Gender Male Female
8.34 6.25a 6.72 5.56b
104.46 90.18
Different letters in each row for a specific parameter indicate significant differences.
tively). This suggests that males would be more likely to have higher incidences of ackee toxicity than females. These findings could account for the higher incidences of ackee toxicity occurring in children, particularly from the lower SES and rural areas in Jamaica (Hill, 1952; MOH, 1999).
4. Conclusion This study indicates that consumers in Jamaica are exposed to varying levels of the natural toxin HG found in the ackee fruit depending on their age, SES, location and gender. Children appear to be the most at risk in experiencing ackee toxicity than other members of a family, since they consume far greater quantities relative to body weight. Since their young bodies are still developing, there is thus greater potential for HG to affect their systems, than in other persons. Individuals from the lower SES are also at greater risk of experiencing ackee toxicity since they also consume a far greater quantity of ackee and HG than individuals in the middle or upper SES. Similarly persons from rural locations and males are also at greater risk. These findings explain the observation in the literature that most of the reported cases of ackee toxicity in Jamaica have involved young male children from the lower SES in rural areas.
Acknowledgements This work represents partial fulfillment for the M.Phil degree in Chemistry and was supported by a grant from
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O.A. Blake et al. / Food Research International 37 (2004) 833–838
the Organization of American States, Inter American Agency for Cooperation and Development (IACD); the International Foundation for Science (IFS); and the University of the West Indies, Research and Publications Committee. The authors thank Dr. George Ware of the US FDA, South Eastern Regional Office for technical assistance with the analysis and Dr. Donna Minott of the Department of Chemistry, University of the West Indies for reviewing of the thesis.
References Baldwin, J. E., Adlington, R. M., Bebbington, D., & Russell, A. T. (1994). Asymmetric total synthesis of the Individual diastereomers of hypoglycin A. Tetrahedron, 50(41), 12015. Baldwin, J. E., & Parker, D. W. (1987). Stereospecific (methylenecyclopropyl) acetyl CoA inactivation of general acy CoA dehydrogenase from pig kidney. Journal of Organic Chemistry, 52(8), 1475–1477. Billington, D., Osmundsen, H., & Sherratt, H. S. (1978). Mechanisms of the metabolic disturbances caused by hypoglycin and by pent 4-enoic acid in vivo studies. Biochemical Pharmacology, 27, 2891–2900. Bressler, R., Corridor, C., & Brendel, K. (1969). Hypoglycin and hypoglycin like compounds. Pharmacological Reviews, 21(2), 105. Brown, M., Bates, R. P., Mcgowan, C., & Cornell, J. A. (1992). Influence of fruit maturity on the hypoglycin A level in ackee (Blighia sapida). Journal of Food Safety, 12(2), 167. Food and Drug Administration (FDA). Center for Food Safety and Applied Nutrition (CFSAN). (2000). Import Alert # 21-11, Detention without physical examination of Ackees (All Types) due to contamination by natural toxins. Fowden, L. (1978). Hypoglycin and related compounds: Occurrence, isolation and biosynthesis. In E. A. Kean (Ed.), Hypoglycin. New York: Academic Press. Hassall, C. H., & Hill, K. R. (1955). The toxicity of the ackee and its relationship to the vomiting sickness of Jamaica. West Indian Medical Journal, 4, 83. Hill, K. R. (1952). The vomiting sickness of Jamaica. West Indian Medical Journal, 1, 243. Jackson, M., Walker, S., Cade, J., Forrester, T., Cruickshank, J. K., & Wilks, R. (2001). Reproducibility and validity of a quantitative
food frequency questionnaire among Jamaicans of African origin. Public Health Nutrition, 4(5), 971–980. Kean, E. A. (1974). Improved method for isolation of hypoglycins A and B from fruit of Blighia sapida. Journal of Pharmacology and Pharmacognosy, 26, 639. Kean, E. A. (1989). Hypoglycin. In P. R. Cheeke (Ed.), Toxicants of plant origin: Proteins and amino acids (p. 229). Boca Raton, FL: CRC Press Inc. Manchester, K. L. (1974). Biochemistry of hypoglycin. FEBS Letters, 40, S133–S139. Ming-tain Lai, D. L., Eugene, Oh, & Hung-wen, Liu (1991). Mechanistic study on the inactivation of general Acyl-CoA dehydrogenase by a metabolite of hypoglycin A. Journal of the American Chemical Society, 113, 7388–7397. Ming-tain Lai, D. L., Eugene, Oh, & Hung-wen, Liu (1992). Studies of the inactivation of general Acyl-CoA dehydrogenase by (1R) and (1S)-(methylenecyclopropyl) acetyl-CoA. Bioorganic and Medical Chemistry Letters, 2, 1423. Ming-tain Lai, D. L., Eugene, Oh, & Hung-wen, Liu (1993). Inactivation of medium-chain acyl-CoA dehydrogenase by a metabolite of hypoglycin: Characterization of the major turnover product and evidence suggesting an alternative flavin modification pathway. Journal of the American Chemical Society, 115, 5. Ministry of Health (MOH), Jamaica. (1999). Poisonous substances surveillance report. Epidemiology Unit, Ministry of Health, Jamaica. Personal Communication (2000). The Ackee Industry of Jamaica. Sarwar, G., & Botting, H. G. (1994). Reversed-phase liquid chromatographic determination of hypoglycin A (HG-A) in canned ackee fruit samples. Journal of AOAC International., 77, 5. Statistical Institute of Jamaica (STATIN) (2001). Demographic statistics 2001. The statistical institute of Jamaica, Kingston 10, Jamaica. Tanaka, K., Isselbacher, K. J., & Shih, V. (1972). Isovaleric and amethylbutyric acidemias induced by hypogycin A: Mechanism of Jamaican vomiting sickness. Science, 175, 69. Tanaka, K., Kean, E. A., & Johnson, B. (1976). Jamaican vomiting sickness, biochemical investigation of two cases. New England Journal of Medicine, 295(9), 461–467. Von Holt, V. H. M., & Bohm, H. (1966). Metaboblic effects of methylenecyclo-propaneacetic acid, a metabolite of hypoglycin. Biochimica et Biophysica Acta, 125(1), 1.
Assessment of dietary exposure to the natural toxin hypoglycin in ackee (Blighia sapida) by Jamaican consumers Orane A. Blake b
a,1
, Jose C. Jackson
a,*
, Maria A. Jackson b, C.L. Andre Gordon
c
a Department of Chemistry, University of the West Indies, Mona Campus, Kingston 7, Jamaica Department of Community Health and Psychiatry, University of the West Indies, Mona Campus, Kingston 7, Jamaica c Technological Solutions Limited, 71/2 Retirement Road, Kingston 5, Jamaica
Received 29 March 2004; accepted 2 May 2004
Abstract Dietary exposure to hypoglycin (HG), the natural toxin found in the ackee fruit of Jamaica, was determined for children and adults using ackee consumption data and quantifying HG levels in typical ackee diets. Ackee consumption was highest in the lower socio-economic group, particularly in children. HG occurrence levels in typical ackee diets ranged from 1.21 to 89.28 lg HG/g ackee. Dietary exposure to HG was highest in young children, who lived in lower socio-economic rural areas of Jamaica. This study is the first to quantify dietary exposure to HG by Jamaican consumers, providing some basis to guide risk assessment. The findings also concur with observations that young children in lower socio-economic groups are the most vulnerable to HG toxicity, when it occurs. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Ackee; Natural toxin; Hypoglycin; Ackee consumption; Dietary exposure; Jamaica
1. Introduction Ackee is the national fruit of Jamaica. It is widely consumed by Jamaicans around the world, by Haitians and in parts of Western Africa. Millions of visitors to Jamaica over the years from various countries around the world have also eaten ackee during their visits to Jamaica. Dr. Thomas Clarke, the first island botanist, brought the fruit to Jamaica in 1778, after obtaining the seeds from a West African slave ship (Bressler, Corridor, & Brendel, 1969; Hill, 1952). The young fruit produced is a green pod which becomes red or yellow upon maturation and then splits open to reveal two to four shiny
*
Corresponding author. Present address: Department of Research and Development, National Food Technology Research Centre, P.O. Box 70498, Gaborone, Botswana. Tel.: +267-71464330/267-393-6243; fax: +267-393-6243. E-mail address: [email protected] (J.C. Jackson). 1 Present Address: Department of Food Science, Purdue University, West Lafayette, IN 47907. 0963-9969/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2004.05.003
black seeds, each having a cream-colored fleshy body, the arillus, at its base (Fig. 1). For many years, it was recognized that the unripe ackee fruit may be poisonous (Hassall & Hill, 1955; Hill, 1952), containing a natural toxin, which exists as the cyclic amino acid, hypoglycin A (HG-A), and its c-glutamyl derivative, hypoglycin B (HG-B). The level of the toxin is elevated in the unripe fruit at concentrations of 100–111 mg/100 g, while present at an innocuous concentration of less than 10 mg/100 g when the fruit is ripe (Brown, Bates, Mcgowan, & Cornell, 1992). Several studies have also been conducted that establishes the levels of HG in canned ackees exported to Canada, the US and England. These studies have shown that the HG levels are generally well within the limit of 100 ppm set by the Food and Drug Administration (FDA) and Health Canada (unpublished data). In 1973, because of concerns of this natural toxin HG, the FDA imposed an import alert on canned ackee from Jamaica; this was upgraded to ‘‘ackees in all forms’’ in 1993. Although the ban did not extend to
834
O.A. Blake et al. / Food Research International 37 (2004) 833–838
Fig. 1. Mature ripe ackee fruit showing ackee arillus, seeds and pod (Picture courtesy of Dr. Andre Gordon).
Canada, it was a major loss to the Jamaican ackee processing industry since it effectively removed over one million consumers from the accessible market (FDA, 2000; Personal Communication, 2000). The ban was lifted in 2000 after the adoption of HACCP based programs in a number of commercial enterprises and a public and private sector partnership that provided training and testing facilities to these enterprises. Consumption of the unripe fruit produces an acute toxic effect, usually within 6–48 h, with symptoms including nausea, vomiting, drowsiness and hypoglycemia (Bressler et al., 1969; Brown et al., 1992; Hassall & Hill, 1955; Hill, 1952). Coma and death occur within 12 h in severe cases (Brown et al., 1992; Hassall & Hill, 1955; Hill, 1952). This illness is commonly known as Jamaican vomiting sickness (JVS) or more accurately toxic hypoglycaemic syndrome (THS) (Baldwin & Parker, 1987). It has been reported to affect young children more adversely than adults in a number of reported cases (Bressler et al., 1969; Brown et al., 1992; Hassall & Hill, 1955; Hill, 1952). When ingested the toxin follows a similar metabolic pathway as a branched chained amino acid, thus producing the active metabolite methylenecyclopropylacetyl-CoA (MCPA-CoA). MCPA-CoA then irreversibly binds to flavin adenine dinucleotide (FAD) and thereby inhibits the activity of medium chain acyldehydrogenases. Medium chain and short chain acyldehydrogenases are critical to the complete b-oxidation of fats. Hence, their inactivation will have adverse effects on blood serum short chain fatty acid concentrations, urinary hydroxy and dicarboxylic acid concentrations, as well as plasma and urinary amino acid concentrations (Tanaka, Kean, & Johnson, 1976; Von Holt & Bohm, 1966). Although significant research has been conducted on the biochemistry of HG (Baldwin, Adlington, Bebbington, & Russell, 1994; Baldwin & Parker, 1987; Kean, 1974; Kean, 1989; Manchester, 1974; Ming-tain Lai, Eugene, & Hung-wen, 1991, 1992, 1993; Tanaka, Is-
selbacher, & Shih, 1972; Tanaka et al., 1976), there has been no previous research that studied the HG exposure of Jamaican consumers. The goal of this study is to quantify the dietary exposure to ackee HG by Jamaican consumers.
2. Materials and methods 2.1. Survey research design An assessment of the quantity of ackee consumed and hence, exposure to the natural toxin HG was conducted in urban (the Parish of Kingston and St. Andrew) and rural communities (the Parish of St. Thomas) in Jamaica. The study was undertaken over a three-month period from January to March 2000. Selection of the study area was based on a process of multistage, stratified, random sampling that was determined by the Statistical Institute of Jamaica (STATIN) as representative of the Jamaican population. Every fourth household was selected after a random starting point. 2.2. Survey instrument A twenty-six-item survey instrument, based on a quantitative food frequency questionnaire developed in Jamaica (Jackson et al., 2001) was used. The instrument was pilot tested on a comparable sample of consumers for clarity and validity; adjustments were made where necessary. The revised questionnaire was divided into three sections: (i) socio-economic and demographic characteristics of the respondents, (ii) consumers’ ackee consumption practices and frequency, and (iii) consumers’ body weight. It was then administered to 300 households from both parishes. Informed consent was obtained by reading a statement to prospective respondents seeking permission for the interview and affirming that the data would be treated confidentially. Data were collected on weekends, and weekday afternoons when a
O.A. Blake et al. / Food Research International 37 (2004) 833–838
member of the selected target population would most likely be at home. Each instrument took an average of 30 min to administer. 2.3. Hypoglycin isolation and characterization An analytically pure standard of HG was isolated from ackee seeds obtained from trees around the University of the West Indies (UWI), Mona Campus, Jamaica, using a modification of the methods of Kean (1974), Fowden (1978), Billington, Osmundsen, and Sherratt (1978), (Fig. 2). The standard was used to quantify the presence of HG in a typical ackee dish consumed in Jamaica. The purity of the standard obtained was determined at the South–East Regional Laboratory, Food and Drug Administration, USA and the Department of Chemistry, UWI, Mona Campus, Jamaica. The purity was assessed using carbon-13 NMR, utilizing D2 0 as a solvent and HPLC coupled to a light scattering detector (LSD) Ground ackee seeds 80% ethanol
Ackee seed extract Extract fat, with hexane Fat free extract
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with phenylisothiocyanate as the derivatizing agent. About 10.70 mg of the HG-A was dissolved in 25 ml of water and 10 ll of this was then injected onto a C-18 column (5 lm particle size, 16% carbon load). Gradient elution commenced at 1 ml/min with 100% 3 mM trichloroacetic acid (solvent A) to 50% solvent A and 50% acetonitrile (solvent B); the nitrogen flow rate and column temperature were set at 1 ml/min and 27 °C, respectively. A Perkin–Elmer 1600 FT-IR was used to record infrared spectra of the HG-A standard. Samples were analysed as thin films between sodium chloride discs. 2.4. Hypoglycin intake in Jamaican consumers Samples of ackee and salted fish (cod fish), which is the typical way of consuming ackee in Jamaica, were collected in the areas in which the ackee consumption assessment was done. A total of 11 samples were collected from households and restaurants in the geographically diverse areas of Liguanea, New Kingston, Downtown Kingston, Papine, Constant Spring, Red Hills, and Yallahs. The salted fish and other vegetable seasonings were removed from the ackee mixture and the quantity of hypoglycin present in each sample was then determined using the Pico-tag method reported by Sarwar and Botting (1994). The daily hypoglycin intake relative to the body weight of children, adolescents, adults, and the elderly was calculated using the following formula: Intake ðlg=dy=kgBWÞ ¼ ackee consumed ðg=day=kg BWÞ HG occurrence ðlg=gÞ:
Apply to anion exchange resin
2.5. Statistical analysis
Absorbed (acidic amino acids, hypoglycin B)
Unabsorbed (neutral and basic amino acids)
The data were analyzed using SPSS Version 11 (SPSS, 2001), and mean responses and percentages generated. The effect of gender, socio-economic status (SES), and geographical location on the ackee consumed and hypoglycin intake was determined using multivariate analysis of variance.
Gradient elution with 0 - 3M acetic acid HG-B and impurities Hydrolyze HG-B with 2M formic acid
3. Results and discussion 3.1. Profile of the respondents
HG-A and impurities Apply to anion exchange resin Crude HG-A Recrystallize twice using 70% ethanol HG-A > 95% pure Fig. 2. HG-A isolation and purification from ackee seeds.
Table 1 shows the profile of the respondents that participated in the study. A total of 1278 persons participated, of which 55% were females and 45% males. The majority of them (76%) came from the lower socioeconomic status, while only 14% and 10% were from the middle and upper SES in Jamaica, respectively. Eighty nine percent (89%) of them had at least primary and secondary education, while only about 11% had tertiary
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Table 1 Socio-demographic profile of respondents in ackee consumption survey
Table 2 Effect of SES, geographical location, gender and age on ackee consumption
Frequency
Percent
Parameter
Gender Male Female
569 709
44.5 55.5
Age group (yrs) 0–12 13–19 20–59 >60
329 217 636 96
25.7 17 49.8 7.5
Socio-economic status (SES) Upper SES Middle SES Lower SES
0.19 0.13a 0.19 0.13a 0.34 0.28b
Geographical location Kingston/St. Andrew (urban) St. Thomas (rural)
0.22 0.18a 0.34 0.27b
Socio-economic status Lower Middle Upper
961 182 127
75.7 14.3 10.0
Gender Male Female
0.36 0.27a 0.29 0.24b
Location Kingston/St. Andrew St. Thomas
790 488
61.8 38.2
Education Primary Secondary Tertiary
Age Children Adolescents Adults Elderly
0.35 0.26a 0.32 0.15a 0.28 0.14b 0.28 0.13b
413 693 135
33.3 55.8 10.9
education. Most respondents (62%) lived in Kingston/ St. Andrew (urban) and 38% lived in St. Thomas (rural). The majority of the participants (50%) were adults, 26% children, 17% adolescents and about 7% were elderly (>60 yrs old). These findings were consistent with the overall population profile of Jamaica (STATIN, 2001). 3.2. Ackee consumption Table 2 indicated that there were significant differences (p < 0:05) in ackee consumption between gender, individuals from rural and urban locations, different SES and age. Males consumed a greater quantity of ackee (0.36 0.27 g/day/kg BW) than females (0.29 0.24 g/ day/kg BW). Individuals from Kingston and St. Andrew, that is urban areas of Jamaica, consumed significantly less (p < 0:05) ackee (0.22 0.17 g/day/kg BW) than individuals from St. Thomas (0.34 0.27 g/day/kg BW), considered a rural location in Jamaica. In the lower SES, which includes almost all of St. Thomas and a significant number of the respondents from Kingston and St. Andrew, individuals consumed more ackee (0.34 0.28 g/ day/kg BW), in comparison to their counterparts from the middle and upper SES (0.19 0.13 g/day/kg BW). Children also consumed a greater quantity of ackee (0.35 0.26 g/day/kg BW), than adolescents, adults and the elderly (0.32 0.15, 0.28 0.14, and 0.28 0.13 g/day/kg BW, respectively). 3.3. Hypoglycin purity and characterization Approximately 81 mg of white HG-A crystals was extracted from 3890 g of ackee seeds, giving an extrac-
Ackee consumed (g/day/kg BW)
Different letters in each row for a specific parameter indicate significant differences.
tion rate of 0.02%. Previous studies indicated that the extraction rate of HG-A from ackee seeds via the hydrolysis of HG-B is between 0.03% and 0.04% (Billington et al., 1978; Fowden, 1978; Kean, 1974). Therefore the modified method used did not produce significantly different yields from established methods. The purity of the isolated HG-A was established using 13 C NMR and HPLC techniques, which indicated that the HG isolated was greater than 95% pure. The 13 C NMR spectrum of HG-A confirmed that the molecule consisted of seven carbons which were attributed to d 174.67 (C7 O2 H), 134.92 (C1 @CH2 ), 104.15, 104.12 (C@4 CH2 ), 55.32 (d, 6 CHCH2 ), 55.19, 34.03 (5 CH2 ), 11.18 (d, cyclopropyl 2 CH), 10.96, 9.30 (cyclopropyl 3 CH2 ), 9.12. HMQC and COSY spectra confirmed the proton chemical shifts of the HG molecule, i.e. d 5.45 (1H, m, C@CH4a H4b ), 5.42 (m), 5.37 (1H, m, C@CH4a H4b ), 5.36 (m), 3.74 (1H, t, aH), 3.75 (t), 1.77 to 1.91 (2H, m, CH2ð5a;bÞ ), 1.42 (1H, m, cyclopropyl CH2a ), 1.29 (1H, m, cyclopropyl CH3a H3b ), 0.82 (1H, m, cyclopropyl CH3a H3b ). An IR spectrum of isolated HG-A showed absorption bands at 3444 cm1 (NH2 ); 2938– 3000 cm1 (CH2 , CH3 ) and 1583, 1408 cm1 (CO2 ). 3.4. Hypoglycin dietary occurrence The HG concentration found in the prepared ackee dishes from restaurants and homes in the study location ranged from 1.21–89.28 lg HG/mg ackee (Table 3). This wide range may be due to the varying preparation and cooking methods used by individuals from these locations. Traditionally, ackee is firstly cleaned to remove the seeds, pod and raphe (commonly called the placenta or membrane), all of which is thought to contain ap-
O.A. Blake et al. / Food Research International 37 (2004) 833–838 Table 3 HG occurrence in ackee collected from 11 different locationsa Location
HG-A (ppm)
1 2 3 4 5 6 7 8 9 10 11
7.48 0.14 17.73 0.74 27.19 0.83 12.85 2.29 8.44 0.09 89.28 30.73 64.15 6.42 1.40 0.25 21.76 10.06 3.26 1.09 1.21 0.03
a
Mean HG ¼ 23.16 28.56 ppm.
preciable levels of hypoglycin. It is then boiled and sometimes sauteed. The level of hypoglycin in the fruit could therefore be affected by the efficiency of cleaning, particularly in removing the raphe. The boiling time used in preparing ackee dishes is often dependent on the variety and maturity of the ackee and the individual preparing the meal. Ackee varieties may differ in texture, which will affect the boiling time required. Since HG-A is water soluble, varying the boiling times will affect its concentration in the final product. Using ackee with a range of maturities will also naturally affect the hypoglycin content of the final product. 3.5. Hypoglycin intake assessment The mean and maximum HG intake with respect to age, SES, rural and urban locations and gender is illustrated in Table 4. The mean HG intake was calculated by multiplying the average ackee consumption and the average HG concentration (23.16 mg/g) in the ackee dishes. Similarly, the maximum HG intake (lg/day/kg BW) was calculated by multiplying the maximum ackee consumption (average + 3 SD), by the maximum HG concentration found in the ackee dishes (89.28 mg/g). The maximum HG intake was useful for assessing potential HG intake of high consumers of ackee in Jamaica. HG intake differences by age, SES, rural and urban locations and gender followed similar trends as observed for ackee consumption. The data in Table 4 show that children consumed a greater quantity of HG (8.18 6.04 lg HG/day/kg BW) than adolescents, the elderly and adults (7.41 3.52, 6.58 2.90, and 6.55 3.12 lg HG/day/kg BW, respectively). Individuals from the lower SES consumed more HG (7.87 6.48 lg/ day/kg BW) than individuals from the upper and middle SES (4.40 3.01 lg/day/kg BW). Similarly, individuals from St. Thomas consumed 7.87 6.25 lg/day/kg BW, a much larger quantity than individuals from Kingston and St. Andrew (5.10 4.17 lg /day/kg BW). Males also consumed a greater quantity of HG than females (8.34 6.25 and 6.72 5.56 lg/day/kg BW, respec-
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Table 4 Dietary intake of HG by Jamaican consumers of different ages, SES, geographical location and gender Parameter
Avg. HG intake (lg/day/kg BW)
Max. HG intake (lg/day/kg BW)
Age Children Adolescent Adult Elderly
8.18 6.04a 7.41 3.52b 6.55 3.12c 6.58 4.06c
115.2 79.30 69.90 66.95
SES Upper SES Middle SES Lower SES
4.40 3.01a 4.40 3.01a 7.87 6.48b
51.79 51.79 105.36
5.10 4.17a
67.86
Geographical location Kingston/St. Andrew (urban) St. Thomas (rural)
7.87 6.25b
102.68
Gender Male Female
8.34 6.25a 6.72 5.56b
104.46 90.18
Different letters in each row for a specific parameter indicate significant differences.
tively). This suggests that males would be more likely to have higher incidences of ackee toxicity than females. These findings could account for the higher incidences of ackee toxicity occurring in children, particularly from the lower SES and rural areas in Jamaica (Hill, 1952; MOH, 1999).
4. Conclusion This study indicates that consumers in Jamaica are exposed to varying levels of the natural toxin HG found in the ackee fruit depending on their age, SES, location and gender. Children appear to be the most at risk in experiencing ackee toxicity than other members of a family, since they consume far greater quantities relative to body weight. Since their young bodies are still developing, there is thus greater potential for HG to affect their systems, than in other persons. Individuals from the lower SES are also at greater risk of experiencing ackee toxicity since they also consume a far greater quantity of ackee and HG than individuals in the middle or upper SES. Similarly persons from rural locations and males are also at greater risk. These findings explain the observation in the literature that most of the reported cases of ackee toxicity in Jamaica have involved young male children from the lower SES in rural areas.
Acknowledgements This work represents partial fulfillment for the M.Phil degree in Chemistry and was supported by a grant from
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the Organization of American States, Inter American Agency for Cooperation and Development (IACD); the International Foundation for Science (IFS); and the University of the West Indies, Research and Publications Committee. The authors thank Dr. George Ware of the US FDA, South Eastern Regional Office for technical assistance with the analysis and Dr. Donna Minott of the Department of Chemistry, University of the West Indies for reviewing of the thesis.
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