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2
Laboratories of
Food Hygiene and
*
Large-Scale Catering, Nutrition Research Section, Musashigaoka College, 111 Yoshimi-cho, Hiki-gun, Saitama-ken, 355-0154, Japan, and
**
Nodai Institute, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, 156-8502 Japan
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • thiaminase • thiamin • entomophagy • ataxia • silk worm • Anaphe venata
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Wright and Morley 1958
). The syndrome is deduced to be caused by an acute thiamin
deficiency based on the fact that the administration of thiamin could
overcome the clinical severity in a randomized, double-blind test
(Adamolekun et al. 1994
). The traditional practice of
Anaphe venata entomophagy also has been implicated in the
etiopathogenesis of the seasonal ataxia (Adamolekun 1993a
, Adamolekun et al. 1994
). However, the
larvae of Anaphe are a protein source, with a nutritional
value comparable to that of chicken eggs, and mass rearing as an
alternative protein source has been advocated (Adamolekun 1993b
, Ashiru 1988
).
We investigated the possibility of detoxifying the Anaphe
larvae, since Australian Aborigines detoxified a heat-stable
thiaminase in a plant called Marsilea drummondii, which was
harmful enough to kill three expeditionists (Earl and McCleary 1994
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Silk nests containing pupae of Anaphe spp. (A. infracta or A. panda) were collected in a suburb of Kampara, Uganda, through the National Sericulture Development Center. Nearly 40% of the dried cocoons contained pupae while the remainder held moths. Dry pupae (3 g) were extracted with 20 mL of 0.2 mol/L sodium potassium phosphate buffer, pH 6.5, by grinding in a mortar with 2 g of purified sea sand. The ground mixture was centrifuged at 4°C and 15,000 x g for 10 min, and the supernatant was used as the crude extract. The total amount of protein extracted was ~100 mg.
Thiaminase assay.
The enzyme sample was incubated at 37°C in 0.1 mol/L TrisHCl buffer,
pH 8.0, with 10-5 mol/L thiamin and 4 x 10-3 mol/L pyridoxine for 30 min. The remaining thiamin
was oxidized with the addition of 1.5 mL BrCN and 1.0 mL of 200 g/L
NaOH and assayed fluorometrically (Nishimune et al. 1988
). Reagents used were obtained from Wako Pure Chemicals
(Osaka, Japan), and thiamin analogs were purchased from Sigma Chemical
Co. (St. Louis, MO).
Protein assay.
The amount of protein was assayed using bicinchoninic acid–Cu+1 complex reagent (Pierce, Rockford, IL).
Estimation of molecular size.
The crude extract of Anaphe pupae was concentrated ~10
times with a Millipore Ultrafree Biomax 10K (Millipore Co., Bedford,
MA) and loaded on a Hiprep Sephacryl S300 (16/60) column (Amersham
Pharmacia Biotech, Uppsala, Sweden) equilibrated with 0.05 mol/L
sodium, potassium phosphate buffer, pH 7.2 at 20°C and eluted at a
flow rate of 0.8 mL/min. The elution position of
thiamin-decomposing activity was detected after fractionation of
the eluate into 4-mL fractions. The size was estimated against a gel
filtration standard (Bio-Rad Laboratories, Hercules, CA) which
contained bovine thyroglobulin; 670 kDa, bovine 
-globulin; 158 kDa,
chick ovalbumin; 44 kDa, equine myoglobin; 17 kDa and vitamin B-12;
1350 Da. Absorbance at 280 nm was monitored continuously.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The thiamin-decomposing activity detected was shown to be
time-dependent, co-substrate-requiring, temperature-dependent
and heat-labile. The active co-substrates at a concentration of
4 x 10-3 mol/L were pyridoxine (Fig. 1
) and compounds shown in Table 1.
4-Amino-pyridine enhanced the activity the most. The enzyme was
practically inactive when thiamin phosphate esters were substrates
(Table 2
). Thiamin analogs competed with thiamin to various degrees (Table 2)
. The
thiaminase activity was also nondialyzable through a cellophane
dialysis bag that excluded molecular weights above ~10 kDa.
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Optimal temperature and heat stability.
Thiamin-decomposing activity was assayed at various temperatures for 15
min at pH 6.5 using a 1:20 dilution of crude extract, and the highest
activity was detected at ~70°C (Fig. 2
). Even at 100°C some activity remained. The crude enzyme was thus
relatively heat-resistant. Heat treatment at 100°C for 15 min
before the standard assay at 37°C inactivated the crude extract of
Anaphe pupae nearly completely (data not shown).
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The highest thiaminase activity, after subtracting the nonenzymatic alkaline destruction of the substrate, was observed at pH 8.0–8.5 in TrisHCl buffer (data not shown).
Estimation of molecular size.
When the concentrated crude extract was analyzed on a column of
Sephacryl S300, thiaminase activity was recovered as a single peak in a
position different from the major A280 nm elution position
(Fig. 3
), where it was expected that fragments of various cell structures and
membrane-bound enzymes would elute. As a result, ~10 times higher
activity per unit protein [90 nmol/(mg protein · min)] was
obtained. The size of the thiaminase was estimated to be 200 kDa.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), plants, fish and shellfish (Evans 1975
).
However, this is the first report of the enzyme in insects, and the
hypothesis presented by Adamolekun (1992)
concerning the
etiopathogenesis of the seasonal ataxia of Nigerians has been
confirmed. The characteristics of Anaphe thiaminase include
its high heat resistance, which could explain why the symptoms of
seasonal ataxia occur within hours of consuming a carbohydrate meal
with a stew containing roasted larvae of A. venata. The
Nardoo thiaminase (obtained from M. drummondii) has a high
optimal temperature of 65°C (McCleary and Chick 1977
).
Anaphe thiaminase in a crude extract preparation showed an
even higher optimal temperature of 70°C. Although our measurement was
accomplished using fairly diluted enzyme, possibly the optimum would
shift to one slightly lower when the measurement is made in a purified
sample. The heat stability of animal enzymes is, however, higher in
many cases when they are located in organs or tissue matrices compared
to when they are extracted into buffer solutions. Many bacterial food
poisonings have arisen because the temperature of the inner part of
foodstuffs is much lower throughout the cooking process than the
temperature of the cooking oil or cooking pan. Thus, it is entirely
possible that a roasted or stewed larvae of A. venata still
retains its thiaminase activity.
As a rough estimate, we calculated the amount of thiamin decomposed by
100 g of the pupae in 1 h to be 0.9 g, using the optimal
reaction rate in Figure 2
[14 nmol thiamin/(mg protein · min),
100 mg of the extracted protein from 3 g of the pupae]. At the
human body temperature of 37°C, the reaction rate was nearly
one-half of the optimum (Fig. 2)
. The assay method we used, on the
other hand, could not give the maximal rate when the percentage of
thiamin decomposed exceeded ~30%. Furthermore, the pH in the small
intestine is more alkaline than pH 6.5, and nearer to the optimal pH of
this enzyme. Although the amount of Anaphe larvae consumed
by seasonal ataxia patients in Nigeria has not been reported, the
above-estimated value suggests that the entomophagy is a possible
reason for thiamin deficiency. On the other hand, thiaminase activities
in fresh fish and fish products on the market have been reported
(Hilker and Peter 1966
). The amount of thiamin destroyed
varied from 0 to 1.0 mg by 100 g of the tissue in 1 h. Raw
carp entrails, with which a typical thiamin avitaminosis was produced
in foxes, destroyed ~10.0 mg of thiamin/100 g wet entrails in 1 h. (Spitzer et al. 1941
). Thiaminase activity in bracken
ferns has been reviewed (Fujita 1954
), and the amounts
of thiamin destroyed are 12.0–13.4 mg/h by 100 g of the tissue.
In the latter two cases, numerous victims of thiaminase have been
reported in foxes and ruminants such as sheep and cattle (Evans 1975
, Green et al. 1941
). A similar level of
thiaminase activity seems to be present in a Nardoo fern
(M. drummondii) (McCleary and Chick 1977
). We
are interested in the fluctuations of thiaminase activity in the larvae
of Anaphe during their growth stages. However, we have not
yet measured this due to the difficulty of obtaining samples. As
Adamolekun (1993b)
has indicated, a monotonous diet of
carbohydrates and the resulting marginal thiamin deficiency may play a
role in the etiology of seasonal ataxia.
From the heat-resistant nature of Anaphe thiaminase, we
expected a smaller molecular weight than was observed, and a similar
molecular size to those reported for thiaminases of other origins. The
reported molecular sizes of thiaminases are 42–44 kDa (bacterial
thiaminase I) (Abe et al. 1987
, Agee et al. 1973
), and 100–115 kDa (bacterial thiaminase II
(Ikehata 1960
) and thiaminase I of plant and shellfish
(McCleary and Chick 1977
)). The size of
Anaphe thiaminase was ca. double that of any previously
studied enzyme. The importance of the larger molecular weight of
Anaphe thiaminase remains to be determined.
In M. drummondii, which contains plant thiaminase I,
detoxification was accomplished by Australian Aborigines through
washing the Nardoo powder with a large amount of water.
Baking the Nardoo powder without washing it did not reduce
the thiaminase activity because of its stability to heat (Earl and McCleary 1994
). A similar procedure seems to have been
ineffective with Anaphe larvae, since this enzyme can
utilize some amino acids as the second substrate, like the
Nardoo thiaminase, and the larvae are expected to be a
source of protein.
Although thiaminases were discovered many decades ago, the
physiological importance of these enzymes has been an enigma. Since the
enzyme reaction can decrease cellular free thiamin concentration,
carbohydrate metabolism or energy production can be impaired. The
enzyme could play a role in the metamorphosis of insects. We are
interested in its gene expression in the various steps of silkworm
metamorphosis, which has many apoptosis-like steps. A thiaminase I
gene has been sequenced (Costello et al. 1996
) using a
bacterial gene that we have cloned (Abe et al. 1987
). We
have preliminarily measured thiaminase activity in Japanese silkworms
(Bombyx mori) and found thiaminase I activity to be less
than one-third that of Anaphe spp. The practice of
eating silk worm pupae is declining in Japan, and the eating of larvae
was last documented in 1919. In the event of a severe food shortage in
the future, we have to be careful to heat-cook the pupae to avoid
suffering from thiamin deficiency.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received October 14, 1999. Revision accepted February 21, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1. Abe M., Ito S., Kimoto M., Hayashi R., Nishimune T. Molecular studies on thiaminase I. Biochim. Biophys. Acta 1987;909:213-221[Medline]
2. Adamolekun B. A seasonal ataxic syndrome in southwestern Nigeria: An etiological hypothesis of acute thiamin deficiency. Ethnicity & Disease 1992;2:185-186
3. Adamolekun B. Epidemiological studies of the etiology of a seasonal ataxia in Nigerians. Neurology 1993a;43:1419
4. Adamolekun B. Anaphe venata entomophagy and seasonal ataxic syndrome in southwest Nigeria. Lancet 1993b;341:629
5.
Adamolekun B., Adamolekun W. E., Sonibare A. D., Sofowara G. A double-blind, placebo-controlled study of the efficacy of thiamin hydrochloride in a seasonal ataxia in Nigerians. Neurology 1994;44:549-551
6. Adamolekun B., Ibikunde F. R. Investigation of an epidemic of seasonal ataxia in Ikare, western Nigeria. Acta Neurol. Scand. 1994a;90:309-311[Medline]
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Agee C., Wilkins J. H., Airth R. C. Cell-bound thiaminase I of Bacillus thiaminolyticus. J. Bacteriol. 1973;115:949-956
8. Ashiru M. O. The food value of the larvae of Anaphe venata. Butler. Ecol. Food Nutr. 1988;22:313-320
9.
Costello C. A., Kelleher N. L., Abe M., McLafferty T. W., Beyloy T. P. Mechanistic studies on thiaminase I. J. Biol. Chem. 1996;271:3445-3452
10. Earl J. W., McCleary B. V. Mystery of the poisoned expedition. Nature Apr 1994;21;368:683-684
11. Evans W. C. Thiaminases and their effects on animals. Vitamins & Hormones 1975;33:467-504[Medline]
12. Fujita A. Studies on thiaminase. Vitamins (Japan) 1954;7:1-11
13. Green R. G., Carlson W. E., Evans C. A. A deficiency disease of foxes produced by feeding fish (B1 avitaminosis analogous to Wernicke’s disease of man). J. Nutr. 1941;21:243-256
14. Hilker D. M., Peter O. F. Anti-thiamine activity in Hawaii fish. J. Nutr. 1966;89:419-421
15. Ikehata H. The purification of thiaminase II. J. Gen. Appl. Microbiol. 1960;6:30-39
16. McCleary B. V., Chick B. F. The purification and properties of a thiaminase I enzyme from Nardoo (Marsilea drummondii). Phytochem 1977;16:207-213
17. Nishimune T., Ito S., Abe M., Kimoto M., Hayashi R. Conditions for thiamin assay by cyanogen bromide oxidation. J. Nutr. Sci. Vitaminol. 1988;34:543-552
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Shida O., Takagi H., Kadowaki K., Yano H., Abe M., Udaka S., Komagata K. Bacillus aneurinolyticus sp. nov., nom. rev. Int. J. Syst. Bacteriol. 1994;44:143-150
19. Spitzer E. H., Coombes A. I., Elvehjem C. A., Wisnicky W. Inactivation of vitamin B1 by raw fish. Proc. Soc. Exp. Biol. Med. 1941;48:376-379
20. Wright J., Morley D. C. Encephalitis tremens. Lancet 1958;1:871-873[Medline]
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