Full text of DOI:10.1093/chromsci/40.8.441

Journal of Chromatographic Science, Vol. 40, September 2002
High-Performance Liquid Chromatographic
Analysis of Amino Acids in Ackee Fruit with
Emphasis on the Toxic Amino Acid Hypoglycin A
K.D. Golden*, O.J. Williams, and Y. Bailey-Shaw
Department of Basic Medical Sciences (Biochemistry Section), University of the West Indies, Mona Campus, Kingston 7, Jamaica W.I.
and Pogson (3) have shown that in the mode of action, the
hypoglycin metabolite methylene cyclopropylpyruvate inhibits
gluconeogenesis by inhibiting a key enzyme (pyruvate car-
boxylase) in gluconeogenesis. Hypoglycin has also been impli-
cated in the inhibition of fatty acid metabolism (β-oxidation),
in which there is inhibition of the acyl–CoA dehydrogenases
that are active on medium and short-chain fatty acids (5). The
excretion of unusual dicarboxylic acids in urine is directly
related to the inhibition of the acyl–CoA dehydrogenases (6).
Although the mechanism of action of hyp-A poisoning has
been elucidated, some individuals in rural Jamaica still have
difficulty with ackee preparation because the water used to
cook the fruit is often used as stock for soup or rice. This has
been further complicated by the fact that the dose of hyp-A
needed to cause fatality in humans per kilogram body weight
(the LD50) is unknown. Other factors of importance are the
maturity of the ackee fruit at harvest and the amount of water
used for cooking ackee fruit per unit fresh weight arilli.
Ackee forms a part of the Jamaican national dish (ackee and
saltfish) and is consumed extensively locally. The fruit is also of
commercial importance because it is widely exported to various
countries worldwide. The purpose of this study was to quanti-
tate the levels of hyp-A and other amino acids in ackee fruit and
evaluate the effect of boiling on residual levels of hyp-A in
cooked and uncooked ackee fruit.
Abstract
High-performance liquid chromatography is used to determine the
amino acid content of ripe and unripe ackee fruit. Specific
emphasis is placed on the level of the toxic amino acid
hypoglycin A (hyp-A) in the unripe and ripe ackee fruit and seed.
Unripe samples are found to contain significantly higher quantities
(P < 0.05) of hyp-A when compared with ripe samples. Uncooked
unripe fruit is found to contain 124.4 6.7 mg/100 g fresh weight
and uncooked ripe fruit 6.4 1.1 mg/100 g fresh weight. The seed
of the uncooked unripe fruit is found to contain 142.8 8.8
mg/100 g fresh weight, and the seed of uncooked ripe fruit has
106.0 5.4 mg/100 g fresh weight. Boiling fruit in water for
approximately 30 min is efficient in removing hyp-A from the
edible arilli; however, low levels of 0.54 0.15 mg/200 mL are
detected in the water that was used to cook the ripe fruit. The
average %recovery of the amino acids was 80.34%.
Introduction
The ackee plant (Blighia sapida) is widespread in the island
of Jamaica and its fleshy fruit (the arillus) is a staple in the
Jamaican diet. The ackee plant contains two amino acids of
interest, hypoglycin A (L-α-amino-β-methylene cyclopropane
propionic acid) (hyp-A) and hypoglycin B (γ-glutamyl hypo-
glycin) (hyp-B). Hyp-A has assumed greater importance
because it is a toxic amino acid that is present in the seed and
flesh of the ackee fruit, and hyp-B (which is far less toxic) is
found only in the seed (1).
Experimental
Hyp-A is the causative agent of ackee poisoning, commonly
known as Jamaican vomiting sickness (2). Jamaican vomiting
sickness results from the ingestion of immature or improperly
prepared ackee fruit. Patients suffering from hyp-A poisoning
experience vomiting approximately 4 h after ingestion. The
condition is accompanied by hypoglycemia (4), during which
the patient becomes comatose and death usually occurs. Kean
Chemicals
All chemicals were of analytical grade unless otherwise noted.
Standard amino acids, phenylisothiocianate (PITC), ninhydrin,
and triethanolamine (TEA) were obtained from Sigma (St.
Louis, MO). Amberlite (IR-45) resin was obtained from BDH
Chemicals (Poole, U.K.), and a Waters-Spherisorb reversed-
phase column was obtained from Supelco (Bellefonte, PA).
* Author to whom correspondence should be written.
Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
441

Journal of Chromatographic Science, Vol. 40, September 2002
Plant material
To determine the efficiency of the extraction procedure,
samples were spiked with a known amount of standard amino
acids (10 mg each) and carried through the extraction and
derivatization processes as previously described.
Ackee fruit was obtained locally from fruit trees on the Mona
campus of the University of the West Indies.
Sample preparation
Ackee fruit (100 g) was homogenized with 195 mL of 80%
ethanol and strained through 2 layers of cheesecloth. The
extract was then centrifuged at 2000 × g for 10 min in a bench
centrifuge. The supernatant (205 mL) was extracted twice with
200-mL portions of toluene to remove the residual fat. The
ethanol layer (200 mL) was removed and filtered through
Whatman No. 4 filter paper (Whatman International Ltd., Kent,
U.K.). Samples from ackee seeds were prepared similarly to
samples from the arilli with the exception that the seed coat
was removed before homogenization. Cooked fruit was pre-
pared by boiling 100 g of tissue in 230 mL of water for 30
min. The water, which also contained 0.4 g of NaCl, was
decanted and retained for analysis. The cooked fruit was
homogenized and extracted as previously described for other
samples.
Filtered extracts (200 mL) were reduced to 10 mL using a
rotary evaporator Model No. 506 (Buchler Instruments, Fort
Lee, NJ) and made acidic by adding 1 mL of 0.5N HCl. The mix-
ture (10 mL) was applied to Amberlite resin IR-45 (9 × 5 cm
column dimensions) and washed with water (100 mL) followed
by 0.4M ammonium acetate (100 mL) at pH 9.4. Thirty-five
fractions (5 mL each) were collected using an ISCO Model 328
fraction collector (ISCO, Lincoln, Nebraska). Each fraction
was assayed for amino acids using 0.4% ninhydrin solution. All
positive fractions were pooled (105 mL) and retained. The
column was then washed with 0.4M ammonium acetate (100
mL) at pH 12.7. Positive fractions were again pooled (60 mL)
and the combined-pooled fractions (165 mL) reduced to 20 mL
by rotary evaporation. Hyp-A was isolated from the seeds of the
ripe fruit as outlined in reference 7.
High-performance liquid chromatographic apparatus
Chromatographic analysis was performed using a Beckman
(System Gold-Nouveau) chromatographic system (Beckman,
Fullerton, CA) consisting of a solvent module (Model 126), a UV
detector (Model 168), and an autosampler (Model 508) fitted
with a 20-µL injection loop. The solvent system consisted of
two buffers. Buffer A was 0.05M ammonium acetate (pH 6.8)
and buffer B was 0.1M ammonium acetate in acetonitrile–
methanol–water (44:10:46, v/v/v) at pH 6.8. The flow rate
through the column was 1 mL/min, and 20 µL of the samples
were injected onto the column (Waters-Spherisorb ODS2,
5-µm film thickness, 250 × 4.6 mm). Amino acids were identi-
fied by UV detection at 254 nm. A gradient elution program was
employed for analysis. Stepwise elution was used beginning
with 100% A for 0.2 min and changing from 100–50% A in 35
min. Fifty percent A was changed to 25% A in 5 min, the
system was held at 25% A for another 5 min and finally
changed from 25% to 100% A in 10 min.
Quantitation and identification
The internal standard method was used to quantitate the
amino acids in the samples according to the formula below (9):
K = (Ai) (Wx) / (Ax) (Wi)
Eq. 1
where Wx is the weight of component x, K the relative response
factor for the particular amino acid, Ai the area under the peak
of the internal standard, Ax the area under the peak of compo-
nent x, and Wi the weight of the internal standard.
The relative retention time (the ratio of the retention time
of the standard amino acids to that of the internal standard)
(RRT) was used to identify the amino acids that were present in
the sample.
Derivatization of amino acids using PITC
A stock solution containing 2.5 µmol/mL of each standard
amino acid in 0.1N HCl was prepared. A partially purified hyp-A
sample (1 mL) of unknown concentration was also prepared to
be used for the quantitative identification of hyp-A. From each
preparation, 20 µL was pipetted into separate eppendorf tubes
and 10 µL of norleucine (10 µmol/mL) was added.
Results and Discussion
The amino acid derivatization process of the standard amino
acids and hypoglycin is a modification of the method of
González-Castro et al. (8). After ion-exchange chromatography
100 µL of the extract of the ripe and unripe fruit was pipetted
into separate eppendorf tubes and 10 µL of norleucine (10
µmol/mL) was added to each. The solutions were dried under
vacuum in a Savant (Farmingdale, NY) speed vacuum at 65°C.
To the residue, 60 µL of methanol–water–TEA (2:2:1, v/v) was
added, and the resulting solution was evaporated under
nitrogen. Sixty microliters of the derivatizing reagent, which
consisted of PITC in methanol–water–TEA (7:1:1, v/v/v), was
added and the tubes were left at room temperature (26°C) for
20 min. The solvents were evaporated under a stream of
nitrogen and 300 µL of 0.05M ammonium acetate was added to
the residue prior to analysis.
Figure 1 shows the chromatogram of the standard amino
acids and the internal standard (norleucine = peak 16).
Although there was some variation in the retention time of the
amino acids, the RRT of the amino acids showed little variation
(Tables I–III). The percent recovery of some standard amino
acids is shown in Table IV, and these data were typical of all the
samples analyzed (ripe fruit, unripe fruit, and cooked ripe
fruit). With an average %recovery of 80.34% and with the K for
each of the amino acids being unity, the efficiency of the extrac-
tion and derivatization procedures may be described as good.
Based on the assessment of the percent recovery, phenylala-
nine, valine, and isoleucine (which like hypoglycin contain
hydrophobic R-groups) gave %recoveries of 98.5%, 75%, and
63.1%, respectively. These amino acids and their representative
recoveries were used to estimate the %recovery of hyp-A
442

Journal of Chromatographic Science, Vol. 40, September 2002
because pure hyp-A is not commercially available and because
a particular family of biological compounds are expected to
show similar trends and properties.
Figure 2 shows the chromatogram of partially purified hypo-
glycin (86.4%), in which the amino acids most prominent as
impurities were isoleucine (3.04%) and leucine (4.46%), which
are the amino acids that usually coelute or elute close to hypo-
glycin (7). Alanine (2.14%), tyrosine (0.523%), valine (0.594%),
phenylalanine (0.704%), and lysine
unripe ackee fruit, respectively, and the amino acid content of
the ackee fruit and seed is shown in Tables I and II, respectively.
The essential amino acids arginine, histidine, isoleucine,
leucine, valine, lysine, phenylalanine, threonine, and trypto-
phan are all present in the unripe fruit and the ripe fruit (Table
I). Similar results were obtained for the seed, with the excep-
tion that leucine and tryptophan were not detected in the ripe
seed (Table II). Isoleucine was the only essential amino acid
(2.139%) were also detected in the hypo-
glycin sample. McGowan et al. (10) have
also reported on the separation of
isoleucine, leucine, and hypoglycin from
partially purified hypoglycin samples with
similar results. Chase et al. (11) used ion-
exchange chromatography to determine
the level of hypoglycin in canned ackee
fruit; however, they were unable to sepa-
rate isoleucine from methionine. The
problem of isolating pure hypoglycin still
remains because isoleucine, leucine, and
hypoglycin have very similar chromato-
graphic properties. To date, high-perfor-
mance liquid chromatography (HPLC) is
one of the few chromatographic methods
that have been able to separate these three
amino acids with success.
Figures 3 and 4 show typical chro-
matograms of the amino acids that are
present in the ripe ackee fruit and the
Figure 1. HPLC of the PITC derivative of the standard amino acids: aspartic acid, 1; glutamic acid, 2;
hydroxy-proline, 3; serine, 4; glycine, 5; threonine, 6; alanine, 7; histidine, 8; proline, 9; arginine, 10;
tyrosine, 11; valine, 12; methionine, 13; isoleucine, 14; leucine, 15; norleucine (internal standard), 16;
phenylalanine, 17; tryptophan, 18; and lysine, 19.
Table I. Amino Acid Content of Ackee Fruit (Arillus)*
Unripe fruit
Ripe fruit
(Mg/100 g wwf)
†
‡
Amino acid
K
RRTAA
RRTsam
(Mg/100 g wwf§)
Aspartic acid
Glutamic acid
Hydroxy-proline
Serine
Glycine
Threonine
Alanine
Histidine
Proline
Arginine
Tyrosine
Valine
1.00
0.99
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.99
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.99
1.00
0.16 0.01
0.20 0.01
0.31 0.01
0.35 0.01
0.38 0.01
0.45 0.00
0.47 0.00
0.50 0.00
0.52 0.00
0.59 0.01
0.77 0.00
0.79 0.00
0.83 0.01
0.95 0.00
0.96 0.01
0.98 0.01
1.08 0.01
1.13 0.00
1.16 0.01
0.17 0.00
0.21 0.00
0.30 0.01
0.35 0.01
0.38 0.01
0.45 0.00
0.47 0.00
0.50 0.00
0.51 0.01
0.59 0.00
0.77 0.00
0.79 0.00
0.83 0.00
0.95 0.00
0.96 0.00
0.98 0.00
1.08 0.00
1.12 0.01
1.17 0.01
4.37 0.24
80.80 7.20
1.64 0.15
9.58 1.50
11.34 0.48
26.86 4.09
34.46 2.84
6.38 1.10
1.45 0.16
53.54 5.99
2.02 0.40
2.52 0.15
2.32 0.21
2.47 0.55
5.92 1.04
124.4 6.70
6.03 1.56
1.46 0.49
5.68 0.75
ND**
26.51 0.35
1.40 0.12
3.06 0.28
7.13 0.64
5.73 0.44
14.42 2.90
2.84 0.29
0.45 0.01
62.88 5.87
0.58 0.08
0.80 0.11
5.67 0.45
0.18 0.00
3.77 0.38
6.40 1.10
2.65 0.27
0.24 0.00
10.56 1.67
Methionine
Isoleucine
Leucine
Hyp-A
Pheylalanine
Tryptophan
Lysine
*
Values are mean standard deviation of duplicates.
RRT of the standard amino acids.
RRT of the amino acids found in the sample.
wwf, wet weight fruit.
†
‡
§
** ND, not detected.
443

Journal of Chromatographic Science, Vol. 40, September 2002
Table II. Amino Acid Content of Ackee Seed*
Unripe seed
Ripe seed
(Mg/100 g wwf)
†
‡
Amino acid
RRTAA
RRTsam
(Mg/100 g wwf§)
Aspartic acid
Glutamic acid
Hydroxy-proline
Serine
Glycine
Threonine
Alanine
Histidine
Proline
Arginine
Tyrosine
Valine
0.16 0.01
0.20 0.01
0.31 0.01
0.35 0.01
0.38 0.01
0.45 0.01
0.47 0.00
0.50 0.00
0.52 0.00
0.59 0.01
0.77 0.00
0.79 0.00
0.83 0.01
0.95 0.00
0.96 0.01
0.98 0.01
1.08 0.01
1.13 0.00
1.16 0.01
ND**
ND
0.21 0.00
0.31 0.01
0.36 0.00
0.39 0.01
0.46 0.01
0.47 0.00
0.50 0.01
0.52 0.00
0.59 0.00
0.76 0.00
0.78 0.00
0.82 0.00
0.95 0.01
0.97 0.00
0.98 0.01
1.07 0.01
1.12 0.01
1.15 0.00
25.71 3.41
2.50 0.29
7.39 0.95
6.49 1.89
24.55 3.78
41.04 4.86
17.45 2.35
30.38 1.95
23.28 1.79
2.98 0.13
9.86 0.87
2.81 0.33
7.63 2.04
4.97 0.58
142.8 8.80
4.93 0.20
5.95 0.56
15.50 3.99
29.30 4.99
4.98 0.67
13.56 2.26
15.0 2.51
23.15 3.87
19.53 3.27
44.46 4.43
ND
40.43 3.52
1.11 0.37
5.92 0.98
1.28 0.43
4.18 0.40
ND
Methionine
Isoleucine
Leucine
Hyp-B
106.0 5.40
4.36 0.46
ND
Phenylalanine
Tryptophan
Lysine
2.10 0.70
*
Values are the mean standard deviation of duplicates.
RRT of the standard amino acids.
RRT of the amino acids found in the sample.
wwf, wet weight fruit.
†
‡
§
** ND, not detected.
Table III. Amino Acids Present in Cooked Ripe Fruit and the Water Used to Cook the Ripe Fruit*
Cooked ripe fruit
Water used to cook ripe fruit
(Mg/200 mL)
†
‡
Amino acids
RRTAA
RRTsam
(Mg/100 g wwf§)
Aspartic acid
Glutamic acid
Hydroxy-proline
Serine
Glycine
Threonine
Alanine
Histidine
Proline
Arginine
Tyrosine
Valine
Methionine
Isoleucine
Leucine
Hyp-A
Hyp-A
Phenylalanine
Tryptophan
Lysine
0.16 0.01
0.20 0.01
0.31 0.01
0.35 0.01
0.38 0.01
0.45 0.00
0.47 0.00
0.50 0.00
0.52 0.00
0.59 0.01
0.77 0.00
0.79 0.00
0.83 0.01
0.95 0.00
0.96 0.01
0.98 0.01
0.99 0.01
1.08 0.01
1.13 0.00
1.16 0.01
0.17 0.01
0.18 0.00
0.31 0.01
0.35 0.00
0.39 0.00
0.46 0.01
0.47 0.00
0.49 0.00
0.51 0.00
0.59 0.00
0.76 0.00
0.78 0.00
0.82 0.00
0.94 0.01
0.97 0.01
0.98 0.01
0.99 0.01
1.08 0.00
1.12 0.00
1.16 0.00
2.23 0.12
45.15 4.41
2.26 0.76
11.09 3.66
1.63 0.35
9.62 1.35
9.21 0.99
7.38 0.69
8.58 0.95
43.57 4.99
1.58 0.35
3.00 0.75
11.00 1.31
ND**
2.25 0.24
ND
(1.17 0.35)
5.12 0.79
4.79 3.47
2.09 0.46
0.28 0.06
4.17 1.03
0.22 0.04
2.65 0.46
1.64 0.29
0.64 0.12
5.33 0.79
2.60 0.24
9.25 0.76
11.23 2.46
2.04 0.39
2.58 0.46
1.29 0.11
1.62 0.18
0.20 0.02
0.54 0.15
(4.96 0.50)
1.29 0.10
2.20 0.28
1.98 0.24
*
Values are the mean standard deviation of duplicates. Values in parentheses were determined from a separate sample of the cooked ripe fruit.
†
RRT of the standard amino acids.
RRT of the amino acids found in the sample.
wwf, wet weight fruit.
‡
§
** ND, not detected.
444

Journal of Chromatographic Science, Vol. 40, September 2002
that was not detected in cooked ripe fruit (Table III).
The presence of hypoglycin in the ackee fruit has been a
major concern over the years. Although the level in the ripe
fruit (6.38 1.06 mg/100 g fresh weight) is much less than that
in the unripe fruit (124.43 6.73 mg/100 g fresh weight) there
is still concern, because the LD50 level of hypoglycin for
humans is unknown. The LD50 for rats is approximately 90–100
mg/kg body weight (12). The level of hypoglycin that was
detected in the ripe fruit compared well with the value (8
mg/100 g) reported by Ellington (13). Chase et al. (11) deter-
mined that canned ackee fruit contains on an average 8.47
mg/100 g. This value compares well with the value obtained
from the ripe fruit (6.38 1.06 mg/100 g).
In this study, the level of hypoglycin in the cooked ripe fruit
and the water that was used to cook the ripe fruit was deter-
mined. Data in Table III shows that in one set of samples, hyp-
A was not detected in the cooked ripe fruit; however, it was
detected in the water that was used to cook the ripe fruit, the
amount being 0.54 0.15 mg/200 mL water. This implies that
most if not all of the hypoglycin leached from the ripe fruit into
the water. It is very unlikely that boiling at 100°C could have
destroyed the hypoglycin, in light of the fact that hypoglycin has
a melting point of 280°C up to 284°C (14). In some instances we
detected hypoglycin in the cooked ripe fruit (1.17 0.35 mg/100
g fresh weight fruit), and hypoglycin at 4.96 0.50 mg/200 mL
was detected in the water used to cook the fruit (Table III). We
can only conclude that the amount of hypoglycin present in the
ripe fruit is significantly reduced when the fruit is cooked.
It is well-known that people (Jamaicans) who prepare the
ripe ackee fruit for a meal discard the water in which the ripe
ackee fruit is cooked, thus the likelihood of being poisoned by
the ackee is significantly minimized. In recent times, with
people being more aware of the danger of (a) cooking and
eating unripe ackee and (b) using the pot water in which the
ackee was cooked to cook rice, incidences of ackee poisoning
has declined significantly over the past
presence of essential amino acids in the ackee fruit and the
findings of Odutuga et al. (16) that the ackee fruit contains
fatty acids (with linoleic, palmitic, and stearic acid being the
major fatty acids in the lipids and with linoleic acid (an essen-
tial fatty acid) accounting for over 55% of the total fatty
acids), then it is reasonable to conclude that the nutritional
value of the ackee fruit is indeed good.
Conclusion
Ackee fruit, when adequately cooked loses significant
amounts of its toxic hyp-A to the surrounding water in which
it is cooked. The leaching of this toxic amino acid in this
manner may be the single most important factor in the pre-
vention of the Jamaican vomiting sickness. Although the actual
Table IV. Percent Recovery of Standard Amino Acids*
Observed value
(mg)
Expected value
(mg)
Amino acid
%Recovery†
Serine
Hydroxy-proline
Alanine
Threonine
Valine
Arginine
Isoleucine
Phenylalanine
Methionine
Lysine
2.30
2.74
2.78
2.35
2.50
2.77
2.10
3.28
2.39
3.54
3.33
3.33
3.33
3.33
3.33
3.33
3.33
3.33
3.33
3.33
69.1
82.3
83.5
70.6
75.0
83.2
63.1
98.5
71.8
106.3
* Data are typical for the analyses of the ripe fruit, unripe fruit, and the cooked ripe
fruit. Analyses were done in duplicate.
†
The average %recovery was 80.34%.
twenty years (1).
Based on the results presented in this
study, ripe ackee fruit that is properly pre-
pared should not be toxic, because the
level of hypoglycin is much lower in the
cooked ripe fruit (1.17 0.35 mg/100 g
wet weight fruit) (Table III) than in the
uncooked ripe fruit (6.38 1.06 mg/100 g
wet weight fruit) (Table I).
Although the amount of each essen-
tial amino acid present in the ripe fruit is
relatively small (being that the daily
requirement for humans is on an average
1.46 g/day (15)), nevertheless the pres-
ence of the essential amino acids does
add to the daily intake whenever the ripe
fruit is consumed. Even more interesting
is the fact that arginine is present in the
largest quantity (62.88 5.87 mg/100 g)
Figure 2. HPLC of the PITC derivative of amino acids in partially purified hypoglycin: alanine, 1; tyro-
sine, 2; valine, 3; isoleucine, 4; leucine, 5; hypoglycin, 6; norleucine (internal standard), 7; phenylala-
nine, 8; and lysine, 9.
(Table I), the irony being that although
arginine is essential the precise require-
ment is not yet established (15). The
445

Journal of Chromatographic Science, Vol. 40, September 2002
amount of hyp-A that causes food poisoning is unknown, it is
clear that boiling effectively reduces the residual amount of
hyp-A in ripe ackee fruit, rendering it nontoxic.
References
1. E.A. Kean. Toxicants of Plant Origin. P.R. Cheeke, Ed. Boca Raton,
FL, 1998, Chapter 10, p 229.
2. K.R. Hill. The vomiting sickness of Jamaica: a review. West.
Indian Med. J. 1: 243–64 (1952).
3. E.A. Kean and C.I. Pogson. Inhibition of gluconeogenesis in iso-
lated rat liver cells by methylenecyclopropylpyruvate (ketohypo-
glycin). Biochem. J. 182: 789–96 (1979).
4. B.D. Jelliffe and K.L. Stuart. Acute toxic hypoglycaemia occurring
in the vomiting sickness of Jamaica. Br. Med. J. 1: 175–77 (1954).
5. E.A. Kean. Selective inhibition of acyl-CoA dehydrogenase by a
metabolite of hypoglycin. Biochim. Bio-
phys. Acta 422: 8–14 (1976).
Acknowledgments
This work was supported by a grant from Research and Pub-
lications (University of the West Indies, Mona). The technical
assistance of Mrs. Janet Golden is also appreciated.
6. K.D. Golden and E.A. Kean. The biogen-
esis of dicarboxylic acids in rats given
hypoglycin. Biochim. Biophys. Acta 794:
83–88 (1983).
7. E.A. Kean. Improved method for isolation
of hypoglycin A and B from fruit of
Blighia sapida. J. Pharm. Pharmacol. 26:
639–40 (1974).
8. M.J. González-Castro, J. López-Hernán-
dez, J. Simal-Lozano, and M.J. Oruña-
Concha. Determination of amino acids in
green beans by derivatization with
phenylisothiocianate and high-perfor-
mance liquid chromatography with ultra-
violet detection. J. Chromatogr. Sci.
35(4): 181–85 (1997).
9. K.D. Golden and O.J. Williams. Amino
acid, fatty acid, and carbohydrate con-
tent of Artocarpus altilis (breadfruit).
J. Chromatogr. Sci. 39(6): 243–50 (2001)
10. C. McGowan, V.A. Wiley, and P. Bates.
Application of methodology for RP-HPLC
amino acid analysis to the measurement
of hypoglycin A. Biochromatography 4:
161–64 (1989).
Figure 3. HPLC of the PITC derivative of amino acids in ripe ackee fruit: glutamic acid, 1; hydroxy-pro-
line, 2; serine, 3; glycine, 4; threonine, 5; alanine, 6; histidine, 7; proline, 8; arginine, 9; tyrosine, 10;
valine, 11; methionine, 12; isoleucine, 13; hypoglycin, 14; norleucine (internal standard), 15; phenyl-
alanine, 16; tryptophan, 17; and lysine, 18. Unlabelled peaks could not be identified.
11 G.W. Chase, W.O. Landen, Jr., L.T. Gel-
baum, and A.M. Soliman. Ion-exchange
chromatographic determination of hypo-
glycin A in canned ackee fruit. J. Assoc.
Off. Anal. Chem. 72: 374–77 (1989).
12. P.C. Feng and S.J. Patrick. Studies of the
action of hypoglycin A, an hypoglycemic
substance. Br. J. Pharmacol. 13: 125–30
(1958).
13. E.V. Ellington. Hypoglycin. E.A. Kean, Ed.
Academic Press, New York, NY, 1975,
p 1.
14. C.H. Hassall and K. Reyle. Hypoglycin A
and B: two biologically active ‘polypep-
tides’ from Blighia sapida. Biochem.
J. 60: 334–39 (1955).
15. A.L. Lehninger. Principles of Biochem-
istry. Worth Publishers, New York, NY,
1982, pp. 762–63.
16. A.A. Odutuga, H.N. Asemota, I. Musac,
K.D. Golden, and E.A. Kean. Fatty acid
composition of arilli from ackee fruit
(Blighia sapida). Jamaican J. Sci. Tech. 3:
30–32 (1992).
Figure 4. HPLC of the PITC derivative of amino acids in unripe ackee fruit: aspartic acid, 1; glutamic acid,
2; hydroxy-proline, 3; serine, 4; glycine, 5; threonine, 6; alanine, 7; proline, 8; arginine, 9; tyrosine, 10;
valine, 11; methionine, 12; isoleucine, 13; hypoglycin, 14; norleucine (internal standard), 15; pheny-
lalanine, 16; tryptophan, 17; and lysine, 18. Unlabelled peaks could not be identified.
Manuscript accepted June 14, 2002.
446