S. L. Vimokesant,3
D. M.
Hilker,4
ABSTRACT consumption
Thiamin of
food
Thailand
indicated
Thailand,
correlation
prevalence Abstention
both
exists
of thiamin from
increased
significantly
destroyed
thiaminase,
could
thiamin
Betel
and and
subjects
resume
in a significant
supplementation
effect. This amount of thiamin was not sufficient to neutralize
nuts
chewing
(10
and S. Dhanamitta6
inadequate surveys
of subjects of food
raw
fermented
fish
fermented
fish
effect.
The
their
chewing
could
further
the effect nut chewing.
of raw
were
activity.
resulted
in
effect
again
of fermented
pyrophosphate thiamin
fermented Am.J. C/in.
In
and the
antithiamin
Cooking
or
sufficient.
pyrophosphate
their
thiamin
factors
consumption
habits. reduce
studies
possess
of
in northeastern
antithiamin
thiamin
of thiamin
intake
conducted
under
containing
raw
decrease
mg/day)
could counteract the effect ofbetel
from
Dietary
intake
pyrophosphate
the
resulted
either
factors.
the consumption
nut
thiamin
when
result
and caloric
between
betel of
K. Rungruangsak,5
antithiamin
deficiency.
both
reduction
Thiamin
deficiency
containing
that
significant
subjects.
S. Nakornchai,5
fish on Thais”2
effect
a
fish of
the
pyrophosphate
fish consumption Nutr. 28: 1458-
but 1463,
l975.
Although decreasing in occurrence, beribeni is still a major health problem in countries where polished rice is the main dietary constituent (I -4). In western countries, this disease essentially has been eliminated except when associated with alcoholism (5). In Thailand, thiamin deficiency has been reported to be high in northern and northeastem provinces (6). Thiamin pyrophosphate (TPP) effect of 21.6% of I I I northern Thai subjects were greater than 20% stimulation and 25% of 198 subjects in northeastern provinces were found to be thiamin deficient by the same criteria. In the northern provinces approximately 80% of the adults chew fermented tea leaves as a stimulant while betel nut chewing is common in other areas of Thailand. Both tea leaves (7) and betel nuts (Vimokesant, unpublished observation) contam high antithiamin factors (ATF). In northeastern provinces. fermented fish, which contains thiaminase, is eaten daily (8). A preliminary study, made at Fang, a district in Chiengmai Province, showed that both tea drinking and chewing fermented tea leaves could have a significant deleterious effect on the thiamin status of the subjects under study as determined by the erythrocyte transketolase thiamin pyrophosphate effect (9). The present study was conducted in order 1458
The
A merican
Journal
of Clinical
Nutrition
to determine the effects of chewing betel nuts and consumption of raw fermented fish on the thiamin status of subjects in a village near Ubol in northeastern Thailand. A 3-day dietany survey was also made in order to relate the adequacy of thiamin intake to the transketolase levels.
Experimental Subjects The study was conducted at Nongjarn, a village of Ubol Province in northeastern Thailand where preliminary health surveys by Dhanamitta have indicated a high incidence of subclinical beriberi. This was determined by I Department of Biochemistry. Faculty of Science, Department of Pediatrics, Ramathibodi Hospital. Mahidol University, Bangkok, Thailand and Department of Food and Nutrition Sciences, University’ of Hawaii, Honolulu, Hawaii 96822. #{176}Thiswork is supported in part by the University of Hawaii Office of Research Administration, The Rockefeller Foundation, The Fund for Overseas Research
Grants and Education, Inc., and the United States Operating Mission. 3 Chairman, Department of Biochemistry. Faculty of Science. Mahidol University. Bangkok. Thailand. 4 Associate Professor, Department of Food and Nutrition Sciences, College of Tropical Agriculture. University of Hawaii. 5Lecturer, Department of Biochemistry, Faculty of Science. Mahidol University. ‘Assistant Professor, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital. Bangkok. Thailand.
28:
DECEM
BER
l975,
pp.
1458-
1463.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
Effects of betel nut and fermented the thiamin status of northeastern
BETEL
Group
Betel PRow
Betel
I
nut fermented
L
AND
nut
FERMENTED
FISH
DayO
14 period
4
Group
I
II
.
THIAMIN
Betel nut +Cooked fermented
period
.-
fish
2
Cooked
DayO
period
-s
period
I
fermented
period
FIG.
fish
2
42 i-period
‘
fermented
antithiamin
activity
(ATA)
in the
betel
nut before chewing and in the expectorated material were made as described by Hilker et al. (7). In this method, 2.5 jag of standard thiamin solution was incubated with betel nut extract (appropriately diluted to destroy 40 60% of added thiamin) in phosphate buffer pH 7.5 at 60 C for 3 hours. The thiamin remaining after incubation was determined by thiochrome method. Somogyi and Bonicke ( lO) showed that the antithiamin activity of various phenolic compounds assayed by thiochrome method agreed well with the microbiological method. The blood transketolase activity was estimated by the method of Dreyfus ( I I ). The values are expressed in terms ofTPP effect (% stimulation by thiamin pyrophosphate of blood transketolase per g/l00 ml Hb). Hemo-
fish
B1 /day
42
period
3
4
. ..-period
-a.
plan.
TABLE I Composition and betel nut chew
antithiamin
activity
Betel nut Composition in grams Betel nut Betel pepper leaves Lime Tobacco leaves Bark Average weight per serving A ntithiamin avg
activity
of4
of betel
of
chew 4.0 1.0 0.1 0.7 1.2 7.0 nut
chews
exp
Tg B destroyed/serving per 3 hours
Betel nut chew
60C
37C Beforechewing
A nalyses
fermented
‘
32
I. Experimental
consumed raw fermented fish daily. while the 21 villagers in group II normally consumed raw fermented fish daily but did not chew betel nuts. The experimental plans are shown in Fig. I. To test only the effects of betel nut chewing, subjects in group I consumed cooked fermented fish throughout the experimental period (cooking destroys the thiaminase activity). Fingertip blood was taken from subjects in group I on days 0. l4, 25. 35 and 42 and from subjects in group II on days 0. 14, 25, 32 and 42. The composition of the betel nut chewed by the subjects is shown in Table I. Ten milligram ofthiamin in capsules was given daily to group I subjects during period 4 and to group II subjects during periods 3 and 4.
of
4
Raw
fish
+ 10 mq B
0
deep tendon reflexes (knee and biceps). complaints of extremity numbness and anorexia, weakness and aching of calf muscles. The subjects were volunteer adults. male and female ranging from 18 to 50 years of age. Two groups of subjects were used, group I consisted of 30 villagers who habitually chewed betel nuts and normally
Analysis
3
25
-‘
fish
consumers
4 4-
fermented B1 /doy
35
Cooked fish
+Cooked + 10 mq
fish
25
Fermented
fermented
1459
STATUS
chewers
Withdraw betel nut +Cooked fermented fish
fish
ON
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
I
NUT
16.l
After chewing (expectorated) Difference in ATA (swallowed) Betel nut chew preincubated I hour with saliva
13.2 x lO
4.6
38.4
I 1.5 18.7
l3.l 12.5
x
l0 l0
globin was determined by the cyanomethemoglobin method (l2). Blood samples were placed in dry ice then kept in a deep freezer ( - 20 C) until analyzed. Dietary
survet
Eighteen 21 subjects dietary
of the 30 subjects in group II were
survey.
All
foods
eaten
in group I and selected randomly’ by the
subjects
16 of the for the during
a
1460
VIMOKESANT
ET
sumption. was given
Table I shows antithiamin activity (ATA) of the extract of betel nut chew and the expectorated material mixed with saliva collected after chewing. The assays were done both at the physiological temperature of 37 C and 60 C, temperatures generally used for the assay of antithiamin activity. The antithiamin activity at 60 C is much higher as compared to 37 C. Even if the antithiamin activity at 37 C is low, it still has some biological activity. The difference in ATA before and after chewing is estimated to be the amount of the ATA swallowed by the subjects. To see if saliva has any effect on the antithiamin activity of the betel nut chew, extract from betel nut chew was incubated with saliva (using the same dilution as found in the expectorate) at 37 C for 1 hour and assayed for ATA both at 37 C and at 60 C. The antithiamin activity of betel nut chews was not altered after incubation with saliva. The effects of chewing betel nuts on thiamm status of the subjects are shown in Table 2. There was no significant change in the TPP effect between the beginning and the end of the control period. However, when the betel nuts were withdrawn and the fermented fish was cooked during period 2, the TPP effect dropped significantly. The TPP effect again increased significantly during period 3 when the subjects began chewing betel nuts and fermented fish was still cooked before con-
Period
I 2 3 4
2 ofchewing
betel
nuts
on thiamin
status
Day blood sample taken 0 l4 25 35 42
(group
40
35
30
25
#{149}
+ +
S S
!
o
0 &
is :
ii
S
I
S
:
I
I
.
I 5
I
l
8
#{149}
5
8 S S
a
It
.15I Piflod
esisi
(sit
Skiiues a the
and
FIG. 2. Individual after withdrawing
(group
2
P.,i.d
I
and
ROWF.F i5ouiol3O)i5% IOautof3O)20%
ith&ow betel COOkSdF.F 4cutof3O>i5%
nut
P,flod and
repmenl
iatnbe
values of the betel nuts and
nut
5 and
COOk.dFF iiauiof3O)i5./.
i autof3O>20% bad;sis
Se$ei
lOutO(3O)2O./. of
adrilcis
TPP effect before raw fermented fish
I).
I)
Treatment
Normal (betel nut Normal (betel nut Withdraw betel nut Betel nut + cooked Betel nut + cooked
supplementation effect decreased,
albeit nonsignificantly. Figure 2 shows individual values of the transketolase TPP effect before and after withdrawing betel nuts and fermented fish. Ten of the 30 subjects chewing betel nuts and consuming raw fermented fish had TPP slimulation values greater than 20% and could be considered deficient by Sauberlich’s criteria (14), and 5 subjects were in the 16-20% range which is considered low and marginal. After withdrawal of the betel nuts and the consumption of cooked fermented fish was con-
Results
TABLE Effects
When thiamin in period 4, TPP
raw fermented fish) raw fermented fish) + cooked fermented fish fermented fish fermented fish + 10 mg B,/day
%TPP mean
19.4 16.4 6.6 12.7 9.4
effect ±
± ± ± ± ±
P Value
si
1.7 l.5 1.1 2.0 1.4
0.001
-i
20%
of calories
2
Betel nut chewers (N= 18) Fermented fish consumers (N - 16)
and Calorie intake/day
Subject
decreased nuts and
Cooked
Individual values of the raw and cooked fermented
TABLE 4 Daily intakes
.
2
P,r.od
fish
2,476.6 ±135.5 2,767.3 ±136.0
thiamin
fermented
fish
Out
of
21>15%
out
of
2 I >2O’Y
TPP effect fish (group
after II).
of the subjects
‘
mg B per day 1. I 3 ±0.10 1.24 ±0.10
intake per keal 0.44 ±0.02 0.45 ±0.04
TPP effect on withdrawal of betel fermented fish and the increased TPP effect after reconsumption was consistent with the previous findings on tea (9). In the present study the 10 mg thiamin supplementation alleviated the effects of the fermented fish and generally the effects of the chewing of betel nuts although the decrease in
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
I 2 3 4
fermented
NUT
1462
VIMOKESANT
(
AL.
source of protein, the practice of eating them raw may contribute to thiamin deficiency. The diet of the people in Nongjarn village consists mainly of glutinous rice, fermented fish, cooked fish and green vegetables. Papaya salad, prepared from green papaya, is consumed frequently. Chicken, beef, dried pork or sausages and eggs were occasionally eaten (23 times/week). Coconut was also used as coconut meat. Fruits consumed included tamarind, lemon and lime cooked in food, banana, pomelo, guava. jujube, ripe papaya and pineapple. Measurement of erythrocyte transketolase and, particularly, the stimulation of this enzyme by TPP has been used as a sensitive index ofthiamin status (14, 20, 21), although limitations have been reported. Fenelly et al. (22) reported that blood transketolase was frequently low in hospitalized alcoholics with thiamin deficiency and cirrhosis, but their transketolase activity was not stimulated by the addition ofTPP. Ifconsideration is based only on the TPP effect, these patients would be considered normal. More recently, it has been reported that transketolase is altered in uremia (23). The subjects in our study showed no overt clinical signs. Those having TPP effects above 20% may be considered to have subclinical thiamin deficiency which could proceed to clinical beriberi in the presence of stress, decreased thiamin intake or increased work and caloric intake. These data lend weight to the contention of Thongmitr et al. (24) that malnutrition is not the important cause of beriberi in Thailand but rather thiaminase and other ATA agents are responsible. The authors Aree Valyasevi, ibodi Hospital.
wish to express their sincere thanks to Dr. Dean, Faculty of Medicine, Ramathfor his support in the field studies.
References I.
Nutrition Survey in the Kingdom of Thailand. A Report by ICNND, l962, p. 32 36. 2. Nutrition Survey in the Republic of Vietnam. A Report by ICNND. 1960, p. 3. 3. Nutrition Survey in the Federation of Malaya. A Report by ICNND, 1964, p. 61, 129. 4. Interdepartmental Committee on Nutrition for National Defense. Nutrition Survey Union of Burma. Washington. D. C.: U. S. Govt. Printing Office, 1963. 5. ToMAstlo, P. A., R. H. M. KATER ANI) F. L. IBER.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
TPP effect when thiamin was given to the betel nut chewers was not significant. It should be noted that the subjects chewing the betel nut also consumed raw fermented fish during their normal dietary period. However, the fish were cooked during later periods in order to obtain the effects of the betel nut only. This may explain why the TPP effect of the resumption of betel nut chewing in period 3 I 2.7%) did not increase to the #{231}riod 1 level of 16.4%. Also, the 10-day period of chewing may not have been long enough to get the complete metabolic effects. The biological mechanism by which betel nut and/or the other ingredients produce thiamin deficiency, as evidenced by high TPP stimulation of blood transketolase activity, is not known nor have the active compounds been identified. The antithiamin activity of tea has been related to its tannin content (7). Betel nuts possess high tannic acid content and are widely used in tanning industry. Preliminary data from our laboratory indicated that the reaction between tannic acid and thiamin was biphasic. The first step occurred very fast and was independent of reactant concentrations and oxygen while the second step was both oxygen and concentration dependent (ms in preparation). Many compounds with orthodihydroxyphenol groups have been reported to contain high ATA (10, 16). Such compounds are common in plant materials. The enzyme thiaminase I (EC 2.5.1.2) has been studied in fermented fish collected in the Ubol area (8). Thiaminase I catalyses the nucleophilic displacement of the methylene group of thiamin by basic substances (17) such as aniline and pyridine and also by some amino acids, e.g., lysine, histidine etc. The work of Melnick et al. (18) indicated that the ingestion of raw clams which contain thiaminase. can lead to considerable destruction of thiamin in the gastrointestinal tract of men. The experiments of Nimitmongkol (19) have shown that the addition of thiaminase enzyme extracted from clams to the drinking water of rats resulted in an increased TPP effect after 10 days although there was no difference in growth rate. The present work indicates that the consumption of raw fish containing thiaminase can have nutritionally significant nesuits in human. While these fish are a good
ET
BETEL Impairment Am.
6.
8.
9.
10.
1 1.
12.
13. 14.
15.
of
thiamin
Nutr. A., S.
21:
absorption
AND
FERMENTED in
FISH
THIAMIN
STATUS
1463
Allowances Acad. Sci.
alcoholism.
1968. VALYASEVI, LOCHAYA. R. A. OLSON AND C. KAMPANART-SANYAKORN . Epidemiological. clinical and biochemical studies of beriberi in infants and adults in Thailand. Ann. Progr. Rept. SEATO Med. Res. Lab. Clin. Res. Ctr., Bangkok, Thailand, 1966. HILKER, D. M., K. C. CHAN, R. CHE,.. ANI) R. I.. SMITH. Anti-thiamin effects of tea. Temperature and pH dependence. Nutr. Rept. Intern. 4: 223, 1971. LOCHAYA, S. L., B. W. LANGER ANI) C. KHUANKONG. Am. Progr. Rept. SEATO Clin. Res. Lab. 1966. Possible role of thiaminase in the etiology of beriberi in Thailand. VIMOKESANT, S. L., S. NAKORNCIIAI, S. DHANAMITTA AND D. M. HII,KER. Effect of tea consumption on thiamin status in man. Nutr. Rept. Intern. 9: 37l, 1974. SOMOGYI, J. C., ANI) R. BONICKE. Connection between chemical structure and antithiamin activity of various phenol derivatives. Intern. Z. Vitaminforsch. 39: 65. l969. DREYFUS, P. M. Clinical application of blood transketolase determinations. New EngI. J. Med. 267: 596, 1962. Laboratory Procedures in Clinical Hematology. Dctermination of hemoglobin. Department of the Army Technical Manual T. M. 8-227-4. Washington, D. C., 1963. Tables of Food Composition in Thailand. By Nutrition Division, Health Promotion Dept. 1970. SAUBERLICH, H. E. Biochemical alterations in thiamm deficiency and their interpretation. Am. J. Clin. Nutr. 20: 529, 1967. Food and Nutrition Board. Recommended Dietary
ON
1341,
16.
17.
18.
19.
20. 21.
22.
23.
24.
(7th rev. ed). Washington. D. C.: NatI. Publ. no. 1964, 1968. HASEGAWA, E., S. SAKAMOTO, K. NAGA’AMA ANI) A. FUJITA. Studies on thiamin decomposing thermostable factors. J. Vitaminol., Kyoto 2: 31, 1956. FUJITA, A., Y. NOSE. S. KOZUKA, T. TASIJIRO, K. UETA AND S. SAKAsioTo. Studies on thiaminase. I. Activation of thiamine breakdown by organic bases. J. Biol. Chem. 196: 289, 1952. MELNICK, D., M. HoCKRERc ANt) B. L. OSER. Physiological availability of vitamins. II. The effect of dietary thiaminase in fish products. J. Nutr. 30: 81, 1945. NIMITM0NGKoI,, N. Role of thiaminase in the etiology of beriberi in Thailand. A thesis submitted on partial fulfillment for the degree of Master of Science, Mahidol University. Bangkok, I 970. BRIN, M. Erythrocyte transketolase in early thiamin deficiency. Ann. N. Y. Acad. Sci. 98: 528, 1962. TANPHA1CHITR, V., S. L. VIMoKESANT, S. DHANAMITTA ANI) A. VAI.YASE\I. Clinical and biochemical studies ofadult beriberi. Am. J. Clin. Nutr. 23: 1017, l970. FENEI,I,Y, J., 0. FRANK. H. BAKER ANI) C. M. LEEV . Red blood cell transketolase activity in malnourished alcoholics with cirrhosis. Am. J. Clin. Nutr. 20: 946, 1967. KOPPIE, D., 0. V. DIRIiE, M. JACoB, M. WANG AND M. SENENISEII. Transketolase activity in red cells in chronic uremia. Trans. Am. Soc. Artificial Internal Organs 18: 250. 1972. THONGMITR, V., Y. SL’kLSIAI(’IIANTRA, V. TANPHAICHITR ANt) B. JL’SIBALA. Hemodynamic studies in adult beriberi. J. Med. Assoc. Thailand 54: 103, 1973.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
7.
J. Clin.
NUT
D. M.
Hilker,4
ABSTRACT consumption
Thiamin of
food
Thailand
indicated
Thailand,
correlation
prevalence Abstention
both
exists
of thiamin from
increased
significantly
destroyed
thiaminase,
could
thiamin
Betel
and and
subjects
resume
in a significant
supplementation
effect. This amount of thiamin was not sufficient to neutralize
nuts
chewing
(10
and S. Dhanamitta6
inadequate surveys
of subjects of food
raw
fermented
fish
fermented
fish
effect.
The
their
chewing
could
further
the effect nut chewing.
of raw
were
activity.
resulted
in
effect
again
of fermented
pyrophosphate thiamin
fermented Am.J. C/in.
In
and the
antithiamin
Cooking
or
sufficient.
pyrophosphate
their
thiamin
factors
consumption
habits. reduce
studies
possess
of
in northeastern
antithiamin
thiamin
of thiamin
intake
conducted
under
containing
raw
decrease
mg/day)
could counteract the effect ofbetel
from
Dietary
intake
pyrophosphate
the
resulted
either
factors.
the consumption
nut
thiamin
when
result
and caloric
between
betel of
K. Rungruangsak,5
antithiamin
deficiency.
both
reduction
Thiamin
deficiency
containing
that
significant
subjects.
S. Nakornchai,5
fish on Thais”2
effect
a
fish of
the
pyrophosphate
fish consumption Nutr. 28: 1458-
but 1463,
l975.
Although decreasing in occurrence, beribeni is still a major health problem in countries where polished rice is the main dietary constituent (I -4). In western countries, this disease essentially has been eliminated except when associated with alcoholism (5). In Thailand, thiamin deficiency has been reported to be high in northern and northeastem provinces (6). Thiamin pyrophosphate (TPP) effect of 21.6% of I I I northern Thai subjects were greater than 20% stimulation and 25% of 198 subjects in northeastern provinces were found to be thiamin deficient by the same criteria. In the northern provinces approximately 80% of the adults chew fermented tea leaves as a stimulant while betel nut chewing is common in other areas of Thailand. Both tea leaves (7) and betel nuts (Vimokesant, unpublished observation) contam high antithiamin factors (ATF). In northeastern provinces. fermented fish, which contains thiaminase, is eaten daily (8). A preliminary study, made at Fang, a district in Chiengmai Province, showed that both tea drinking and chewing fermented tea leaves could have a significant deleterious effect on the thiamin status of the subjects under study as determined by the erythrocyte transketolase thiamin pyrophosphate effect (9). The present study was conducted in order 1458
The
A merican
Journal
of Clinical
Nutrition
to determine the effects of chewing betel nuts and consumption of raw fermented fish on the thiamin status of subjects in a village near Ubol in northeastern Thailand. A 3-day dietany survey was also made in order to relate the adequacy of thiamin intake to the transketolase levels.
Experimental Subjects The study was conducted at Nongjarn, a village of Ubol Province in northeastern Thailand where preliminary health surveys by Dhanamitta have indicated a high incidence of subclinical beriberi. This was determined by I Department of Biochemistry. Faculty of Science, Department of Pediatrics, Ramathibodi Hospital. Mahidol University, Bangkok, Thailand and Department of Food and Nutrition Sciences, University’ of Hawaii, Honolulu, Hawaii 96822. #{176}Thiswork is supported in part by the University of Hawaii Office of Research Administration, The Rockefeller Foundation, The Fund for Overseas Research
Grants and Education, Inc., and the United States Operating Mission. 3 Chairman, Department of Biochemistry. Faculty of Science. Mahidol University. Bangkok. Thailand. 4 Associate Professor, Department of Food and Nutrition Sciences, College of Tropical Agriculture. University of Hawaii. 5Lecturer, Department of Biochemistry, Faculty of Science. Mahidol University. ‘Assistant Professor, Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital. Bangkok. Thailand.
28:
DECEM
BER
l975,
pp.
1458-
1463.
Printed
in U.S.A.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
Effects of betel nut and fermented the thiamin status of northeastern
BETEL
Group
Betel PRow
Betel
I
nut fermented
L
AND
nut
FERMENTED
FISH
DayO
14 period
4
Group
I
II
.
THIAMIN
Betel nut +Cooked fermented
period
.-
fish
2
Cooked
DayO
period
-s
period
I
fermented
period
FIG.
fish
2
42 i-period
‘
fermented
antithiamin
activity
(ATA)
in the
betel
nut before chewing and in the expectorated material were made as described by Hilker et al. (7). In this method, 2.5 jag of standard thiamin solution was incubated with betel nut extract (appropriately diluted to destroy 40 60% of added thiamin) in phosphate buffer pH 7.5 at 60 C for 3 hours. The thiamin remaining after incubation was determined by thiochrome method. Somogyi and Bonicke ( lO) showed that the antithiamin activity of various phenolic compounds assayed by thiochrome method agreed well with the microbiological method. The blood transketolase activity was estimated by the method of Dreyfus ( I I ). The values are expressed in terms ofTPP effect (% stimulation by thiamin pyrophosphate of blood transketolase per g/l00 ml Hb). Hemo-
fish
B1 /day
42
period
3
4
. ..-period
-a.
plan.
TABLE I Composition and betel nut chew
antithiamin
activity
Betel nut Composition in grams Betel nut Betel pepper leaves Lime Tobacco leaves Bark Average weight per serving A ntithiamin avg
activity
of4
of betel
of
chew 4.0 1.0 0.1 0.7 1.2 7.0 nut
chews
exp
Tg B destroyed/serving per 3 hours
Betel nut chew
60C
37C Beforechewing
A nalyses
fermented
‘
32
I. Experimental
consumed raw fermented fish daily. while the 21 villagers in group II normally consumed raw fermented fish daily but did not chew betel nuts. The experimental plans are shown in Fig. I. To test only the effects of betel nut chewing, subjects in group I consumed cooked fermented fish throughout the experimental period (cooking destroys the thiaminase activity). Fingertip blood was taken from subjects in group I on days 0. l4, 25. 35 and 42 and from subjects in group II on days 0. 14, 25, 32 and 42. The composition of the betel nut chewed by the subjects is shown in Table I. Ten milligram ofthiamin in capsules was given daily to group I subjects during period 4 and to group II subjects during periods 3 and 4.
of
4
Raw
fish
+ 10 mq B
0
deep tendon reflexes (knee and biceps). complaints of extremity numbness and anorexia, weakness and aching of calf muscles. The subjects were volunteer adults. male and female ranging from 18 to 50 years of age. Two groups of subjects were used, group I consisted of 30 villagers who habitually chewed betel nuts and normally
Analysis
3
25
-‘
fish
consumers
4 4-
fermented B1 /doy
35
Cooked fish
+Cooked + 10 mq
fish
25
Fermented
fermented
1459
STATUS
chewers
Withdraw betel nut +Cooked fermented fish
fish
ON
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
I
NUT
16.l
After chewing (expectorated) Difference in ATA (swallowed) Betel nut chew preincubated I hour with saliva
13.2 x lO
4.6
38.4
I 1.5 18.7
l3.l 12.5
x
l0 l0
globin was determined by the cyanomethemoglobin method (l2). Blood samples were placed in dry ice then kept in a deep freezer ( - 20 C) until analyzed. Dietary
survet
Eighteen 21 subjects dietary
of the 30 subjects in group II were
survey.
All
foods
eaten
in group I and selected randomly’ by the
subjects
16 of the for the during
a
1460
VIMOKESANT
ET
sumption. was given
Table I shows antithiamin activity (ATA) of the extract of betel nut chew and the expectorated material mixed with saliva collected after chewing. The assays were done both at the physiological temperature of 37 C and 60 C, temperatures generally used for the assay of antithiamin activity. The antithiamin activity at 60 C is much higher as compared to 37 C. Even if the antithiamin activity at 37 C is low, it still has some biological activity. The difference in ATA before and after chewing is estimated to be the amount of the ATA swallowed by the subjects. To see if saliva has any effect on the antithiamin activity of the betel nut chew, extract from betel nut chew was incubated with saliva (using the same dilution as found in the expectorate) at 37 C for 1 hour and assayed for ATA both at 37 C and at 60 C. The antithiamin activity of betel nut chews was not altered after incubation with saliva. The effects of chewing betel nuts on thiamm status of the subjects are shown in Table 2. There was no significant change in the TPP effect between the beginning and the end of the control period. However, when the betel nuts were withdrawn and the fermented fish was cooked during period 2, the TPP effect dropped significantly. The TPP effect again increased significantly during period 3 when the subjects began chewing betel nuts and fermented fish was still cooked before con-
Period
I 2 3 4
2 ofchewing
betel
nuts
on thiamin
status
Day blood sample taken 0 l4 25 35 42
(group
40
35
30
25
#{149}
+ +
S S
!
o
0 &
is :
ii
S
I
S
:
I
I
.
I 5
I
l
8
#{149}
5
8 S S
a
It
.15I Piflod
esisi
(sit
Skiiues a the
and
FIG. 2. Individual after withdrawing
(group
2
P.,i.d
I
and
ROWF.F i5ouiol3O)i5% IOautof3O)20%
ith&ow betel COOkSdF.F 4cutof3O>i5%
nut
P,flod and
repmenl
iatnbe
values of the betel nuts and
nut
5 and
COOk.dFF iiauiof3O)i5./.
i autof3O>20% bad;sis
Se$ei
lOutO(3O)2O./. of
adrilcis
TPP effect before raw fermented fish
I).
I)
Treatment
Normal (betel nut Normal (betel nut Withdraw betel nut Betel nut + cooked Betel nut + cooked
supplementation effect decreased,
albeit nonsignificantly. Figure 2 shows individual values of the transketolase TPP effect before and after withdrawing betel nuts and fermented fish. Ten of the 30 subjects chewing betel nuts and consuming raw fermented fish had TPP slimulation values greater than 20% and could be considered deficient by Sauberlich’s criteria (14), and 5 subjects were in the 16-20% range which is considered low and marginal. After withdrawal of the betel nuts and the consumption of cooked fermented fish was con-
Results
TABLE Effects
When thiamin in period 4, TPP
raw fermented fish) raw fermented fish) + cooked fermented fish fermented fish fermented fish + 10 mg B,/day
%TPP mean
19.4 16.4 6.6 12.7 9.4
effect ±
± ± ± ± ±
P Value
si
1.7 l.5 1.1 2.0 1.4
0.001
-i
20%
of calories
2
Betel nut chewers (N= 18) Fermented fish consumers (N - 16)
and Calorie intake/day
Subject
decreased nuts and
Cooked
Individual values of the raw and cooked fermented
TABLE 4 Daily intakes
.
2
P,r.od
fish
2,476.6 ±135.5 2,767.3 ±136.0
thiamin
fermented
fish
Out
of
21>15%
out
of
2 I >2O’Y
TPP effect fish (group
after II).
of the subjects
‘
mg B per day 1. I 3 ±0.10 1.24 ±0.10
intake per keal 0.44 ±0.02 0.45 ±0.04
TPP effect on withdrawal of betel fermented fish and the increased TPP effect after reconsumption was consistent with the previous findings on tea (9). In the present study the 10 mg thiamin supplementation alleviated the effects of the fermented fish and generally the effects of the chewing of betel nuts although the decrease in
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I 2 3 4
fermented
NUT
1462
VIMOKESANT
(
AL.
source of protein, the practice of eating them raw may contribute to thiamin deficiency. The diet of the people in Nongjarn village consists mainly of glutinous rice, fermented fish, cooked fish and green vegetables. Papaya salad, prepared from green papaya, is consumed frequently. Chicken, beef, dried pork or sausages and eggs were occasionally eaten (23 times/week). Coconut was also used as coconut meat. Fruits consumed included tamarind, lemon and lime cooked in food, banana, pomelo, guava. jujube, ripe papaya and pineapple. Measurement of erythrocyte transketolase and, particularly, the stimulation of this enzyme by TPP has been used as a sensitive index ofthiamin status (14, 20, 21), although limitations have been reported. Fenelly et al. (22) reported that blood transketolase was frequently low in hospitalized alcoholics with thiamin deficiency and cirrhosis, but their transketolase activity was not stimulated by the addition ofTPP. Ifconsideration is based only on the TPP effect, these patients would be considered normal. More recently, it has been reported that transketolase is altered in uremia (23). The subjects in our study showed no overt clinical signs. Those having TPP effects above 20% may be considered to have subclinical thiamin deficiency which could proceed to clinical beriberi in the presence of stress, decreased thiamin intake or increased work and caloric intake. These data lend weight to the contention of Thongmitr et al. (24) that malnutrition is not the important cause of beriberi in Thailand but rather thiaminase and other ATA agents are responsible. The authors Aree Valyasevi, ibodi Hospital.
wish to express their sincere thanks to Dr. Dean, Faculty of Medicine, Ramathfor his support in the field studies.
References I.
Nutrition Survey in the Kingdom of Thailand. A Report by ICNND, l962, p. 32 36. 2. Nutrition Survey in the Republic of Vietnam. A Report by ICNND. 1960, p. 3. 3. Nutrition Survey in the Federation of Malaya. A Report by ICNND, 1964, p. 61, 129. 4. Interdepartmental Committee on Nutrition for National Defense. Nutrition Survey Union of Burma. Washington. D. C.: U. S. Govt. Printing Office, 1963. 5. ToMAstlo, P. A., R. H. M. KATER ANI) F. L. IBER.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
TPP effect when thiamin was given to the betel nut chewers was not significant. It should be noted that the subjects chewing the betel nut also consumed raw fermented fish during their normal dietary period. However, the fish were cooked during later periods in order to obtain the effects of the betel nut only. This may explain why the TPP effect of the resumption of betel nut chewing in period 3 I 2.7%) did not increase to the #{231}riod 1 level of 16.4%. Also, the 10-day period of chewing may not have been long enough to get the complete metabolic effects. The biological mechanism by which betel nut and/or the other ingredients produce thiamin deficiency, as evidenced by high TPP stimulation of blood transketolase activity, is not known nor have the active compounds been identified. The antithiamin activity of tea has been related to its tannin content (7). Betel nuts possess high tannic acid content and are widely used in tanning industry. Preliminary data from our laboratory indicated that the reaction between tannic acid and thiamin was biphasic. The first step occurred very fast and was independent of reactant concentrations and oxygen while the second step was both oxygen and concentration dependent (ms in preparation). Many compounds with orthodihydroxyphenol groups have been reported to contain high ATA (10, 16). Such compounds are common in plant materials. The enzyme thiaminase I (EC 2.5.1.2) has been studied in fermented fish collected in the Ubol area (8). Thiaminase I catalyses the nucleophilic displacement of the methylene group of thiamin by basic substances (17) such as aniline and pyridine and also by some amino acids, e.g., lysine, histidine etc. The work of Melnick et al. (18) indicated that the ingestion of raw clams which contain thiaminase. can lead to considerable destruction of thiamin in the gastrointestinal tract of men. The experiments of Nimitmongkol (19) have shown that the addition of thiaminase enzyme extracted from clams to the drinking water of rats resulted in an increased TPP effect after 10 days although there was no difference in growth rate. The present work indicates that the consumption of raw fish containing thiaminase can have nutritionally significant nesuits in human. While these fish are a good
ET
BETEL Impairment Am.
6.
8.
9.
10.
1 1.
12.
13. 14.
15.
of
thiamin
Nutr. A., S.
21:
absorption
AND
FERMENTED in
FISH
THIAMIN
STATUS
1463
Allowances Acad. Sci.
alcoholism.
1968. VALYASEVI, LOCHAYA. R. A. OLSON AND C. KAMPANART-SANYAKORN . Epidemiological. clinical and biochemical studies of beriberi in infants and adults in Thailand. Ann. Progr. Rept. SEATO Med. Res. Lab. Clin. Res. Ctr., Bangkok, Thailand, 1966. HILKER, D. M., K. C. CHAN, R. CHE,.. ANI) R. I.. SMITH. Anti-thiamin effects of tea. Temperature and pH dependence. Nutr. Rept. Intern. 4: 223, 1971. LOCHAYA, S. L., B. W. LANGER ANI) C. KHUANKONG. Am. Progr. Rept. SEATO Clin. Res. Lab. 1966. Possible role of thiaminase in the etiology of beriberi in Thailand. VIMOKESANT, S. L., S. NAKORNCIIAI, S. DHANAMITTA AND D. M. HII,KER. Effect of tea consumption on thiamin status in man. Nutr. Rept. Intern. 9: 37l, 1974. SOMOGYI, J. C., ANI) R. BONICKE. Connection between chemical structure and antithiamin activity of various phenol derivatives. Intern. Z. Vitaminforsch. 39: 65. l969. DREYFUS, P. M. Clinical application of blood transketolase determinations. New EngI. J. Med. 267: 596, 1962. Laboratory Procedures in Clinical Hematology. Dctermination of hemoglobin. Department of the Army Technical Manual T. M. 8-227-4. Washington, D. C., 1963. Tables of Food Composition in Thailand. By Nutrition Division, Health Promotion Dept. 1970. SAUBERLICH, H. E. Biochemical alterations in thiamm deficiency and their interpretation. Am. J. Clin. Nutr. 20: 529, 1967. Food and Nutrition Board. Recommended Dietary
ON
1341,
16.
17.
18.
19.
20. 21.
22.
23.
24.
(7th rev. ed). Washington. D. C.: NatI. Publ. no. 1964, 1968. HASEGAWA, E., S. SAKAMOTO, K. NAGA’AMA ANI) A. FUJITA. Studies on thiamin decomposing thermostable factors. J. Vitaminol., Kyoto 2: 31, 1956. FUJITA, A., Y. NOSE. S. KOZUKA, T. TASIJIRO, K. UETA AND S. SAKAsioTo. Studies on thiaminase. I. Activation of thiamine breakdown by organic bases. J. Biol. Chem. 196: 289, 1952. MELNICK, D., M. HoCKRERc ANt) B. L. OSER. Physiological availability of vitamins. II. The effect of dietary thiaminase in fish products. J. Nutr. 30: 81, 1945. NIMITM0NGKoI,, N. Role of thiaminase in the etiology of beriberi in Thailand. A thesis submitted on partial fulfillment for the degree of Master of Science, Mahidol University. Bangkok, I 970. BRIN, M. Erythrocyte transketolase in early thiamin deficiency. Ann. N. Y. Acad. Sci. 98: 528, 1962. TANPHA1CHITR, V., S. L. VIMoKESANT, S. DHANAMITTA ANI) A. VAI.YASE\I. Clinical and biochemical studies ofadult beriberi. Am. J. Clin. Nutr. 23: 1017, l970. FENEI,I,Y, J., 0. FRANK. H. BAKER ANI) C. M. LEEV . Red blood cell transketolase activity in malnourished alcoholics with cirrhosis. Am. J. Clin. Nutr. 20: 946, 1967. KOPPIE, D., 0. V. DIRIiE, M. JACoB, M. WANG AND M. SENENISEII. Transketolase activity in red cells in chronic uremia. Trans. Am. Soc. Artificial Internal Organs 18: 250. 1972. THONGMITR, V., Y. SL’kLSIAI(’IIANTRA, V. TANPHAICHITR ANt) B. JL’SIBALA. Hemodynamic studies in adult beriberi. J. Med. Assoc. Thailand 54: 103, 1973.
Downloaded from https://academic.oup.com/ajcn/article-abstract/28/12/1458/4716614 by East Carolina University user on 15 January 2019
7.
J. Clin.
NUT
