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Plasma protein index of aflatoxin-fed ducklings
Life Saieacea Pol . 4, pp " 191-195, 1965 . Printed is Great Britain.
Pergaaoa Preen Ltd.
PIA8!!A PROTEIN INDER OF AFLATOXIN-FED DIICRLINGS P, R, Datta and R, J, Gajaa Food and Drug Administration Washington, D, C,
20204
(Received 14 June 1965)
An important but poorly documented area in the toxicology of fnagal metabolites of natural food products has come to atteatioa since the ontbreake of "turkey R" disease in England in 1960,
This disease has baen associated
with feeds which contained peanut meal from Brasil infected with Aspergillus flavus Link ex Frie~ (1),
English and Dutch iuveatigators (2) extracted and
crystallized two potent hepatotoxins
(Bl sad G1) from peanut meal contaminated
with the above strain of Aspergillus flavus , metabolites,
The structure of these two major
showing rnlationahip to synthetic coumarin, has been elucidated
(3), Ia general,
the livers of aflatoxia-fed ducklings show fatty changes,
pareachymal damage and proliferation of bile ducts,
These histopathological
changes are also observed in livers of aflatoxia-fed guinea pigs, chicks, pigs sad lambs,
In rate, however, hepatomae have been repeatedly observed after
chronic feeding of contaminated peanut meal for six months without antecedent morphological damage (4), The purpose of this cammunicatioa is to describe the application of a polarographic method based on Brdicka'e protein-reaction (5) to the plasma of ducklings fed crystalline aflatoxia B1 sad extracts of the Aspergillus flavus
Pza~~ pxomxia ixn~
1792
culture for a short time,
vol .
4, xo . 18
As a basis for comparison, the correlation of the
plasma protein index values with those of the hiatopathological indices of liver of a representative group of ducklings is also presented, Ia our experiments, one-day-old ducklings were fed different levels of crystalline aflatoxin B1 (purity based on physical data from T,L,C infrared, mesa spectra, N,M,R, and elementary analysis) or the extract from Aspergillus flayus culture (mold No, 4-95, T,L,C, analysis : for 7 days,
15% Bl, 9% Gl, < 1% B2 and G2)
At the end of the dosing period, the ducklings were decapitated
and oxylated plasma was obtained,
The plasma specimens were prepared for the
digest and filtrate teats and polarographed immediately as described by Muller (6), The polarograph used was a Davia Differential Cathode Ray Polarotrace, Model 1660,
This model can trace a complete polarographic wave on an
oscilloscopic screen every seven seconds, is slightly less than 30 seconds, permanent records,
The time required for each analysis
The polarotrace was photographed to obtain
In both tests, the wave heights of the second catalytic
protein wave, which has a peak potential at -1,4 ± 0,05 volts vs a mercury pool reference electrode, were measured at 25 ° ± 1 °C and expressed in units of wave height,
These units of wave height include the actual height of the wave
on the oscilloscope multiplied by the various sensitivity factors of the instrument,
The protein index was obtained from the ratio of the polsrographic
wave heights of the filtrate test to the digest teat, multiplied by an arbitrary constant of 15,
The data of the protein indices of the plasma
obtained from a representative group of ducklings show that the protein indices of plasma from ducklings fed 7 ug of crystalline aflatoxin B l are twice the value obtained from the plasma of control ducklings,
It is also noted that the
protein indices of ducklings fed Aflatoxin mold culture (determined by T,L,C, to be 20 uR R1) are 4 to 5 times higher than those of the control birds,
1793
PLABYII PBpTEIH IBDEä
Vol . 4, Bo . 18
TABLE 1 Average Protein Indices of Control and Aflatoxin-Fed Ducklings
Types of Ducklings
No, of Birds
Average Protein Index
Standard Deviation of the Mean
Range
20
28,8
± 4,0
21,3-36,3
Aflatoxin-fed 7 Rg crystalline Bl
4
60,0
± 1,0
59,0-61,2
Aflatoxin-fed mold culture extract concentrate *
12
122,3
± 18,2
102,0-198,0
Control
* T,L,C, analysis showed 20 Kg Bl The protein index values are relatively quantitative and show a relationship between dosage fed and abnormality of proteins present in experimental ducklings,
The described method is probably no more specific than
corresponding non-polarographic clinical tests, but it has advantages of simplicity of operation, sensitivity,
reproducibility,
speed, and wide scope of
application, Aistopathological eaaminatione of liner from individual ducklings were also carried out,
Both the protein index and the pathological index of a
iepreaentative group of control sad aflatoxin-fed ducklings were compared,
194
Vol . 4, Ao . 18
PIJ81(l PEO~ IIIDEâ TABLE 2
Correlation of Pathological Index of Liver with Protein Indea of Blood Plasma From a Representative Group of Control and Aflatoxin-Fed Ducklings Aflatozia-Fed u8 B1 calculated *
Protein 'Index
Pathological Index
Control
23,0
0
Control
21,3
0
Control
29,3
0
6,3 (cryst, Bl)
60,4
1-2
6,6 (cryet, Bl)
62,1
3
7
60,0
2
7
60,0
1-2
63
198,0
2-3
63
102,1
3
63
177,0
2-3
63
168,0
2-3
* Mold culture No, 4-95, T,L,C, analysis :
15X Sl, 9X Gl, < 1X B2 and
The pathological index values 0,1,2, and 3 represent none, alight, moderate, sad severe bile duct proliferation is liver, respectively,
The protein index
value correlates with the pathological index value for the individual ducklings and, in fact, reflects the pathological findings,
This agreement further
supports a quantitative relationship between the dosage fed and the protein index value obtained,
In a surveillance study on the hazard of peanuts, as
suggested by Lstham (7), the application of the protein index method as described here may therefore be of value is safeguarding public health,
Yol . 4, No .
18
PL~ß1tA PHOTEIH IADF.%
1~95
References 1,
R, Ai.T1R~FT and R, B, A, CARNAGHIi, Chem . Iad, ~0, Londoa (1963)=
Ibid ,
(1963), 2,
H, R, NESBITT, J, O'RELLY, R, SARGENT and A, SSERIDAN, Nature 195, 1062 (1962) ; A, 3, M, VAN DER ZLJDEN, W, A, A, BLANCHE ROELBNSMID, J, HOLDINGS, C, B, BARRETT, W, 0, ORD and J, PHILP, Nature 1=, 1060 (1962),
3,
T, ASAO, G, BDCSI, M, M, ABDSL-RODAR, S, B, CHANG, E, L, WICR and G, P, WOGAN, J . Am . Chum, Soc , 85, 1706
4,
(1963),
P, M, NEWHSRNE, G, N, WOGAN, W, W, CARLTON and M, M, A, RADER, Toxic, sad Appli . Phaxmco , !, 542 (1964),
5,
R, BRDICRA, Collectioa Czechoslovakia Chew, Comno4m , ~,
6,
0, H, MÜLLER, Method of Biochem, Analysis Vol, R1, 329 (1963),
7,
M, C, LATHAM, Brit . Med, J, 818 (Sept, 26, 1964),
112 (1933),
Pergaaoa Preen Ltd.
PIA8!!A PROTEIN INDER OF AFLATOXIN-FED DIICRLINGS P, R, Datta and R, J, Gajaa Food and Drug Administration Washington, D, C,
20204
(Received 14 June 1965)
An important but poorly documented area in the toxicology of fnagal metabolites of natural food products has come to atteatioa since the ontbreake of "turkey R" disease in England in 1960,
This disease has baen associated
with feeds which contained peanut meal from Brasil infected with Aspergillus flavus Link ex Frie~ (1),
English and Dutch iuveatigators (2) extracted and
crystallized two potent hepatotoxins
(Bl sad G1) from peanut meal contaminated
with the above strain of Aspergillus flavus , metabolites,
The structure of these two major
showing rnlationahip to synthetic coumarin, has been elucidated
(3), Ia general,
the livers of aflatoxia-fed ducklings show fatty changes,
pareachymal damage and proliferation of bile ducts,
These histopathological
changes are also observed in livers of aflatoxia-fed guinea pigs, chicks, pigs sad lambs,
In rate, however, hepatomae have been repeatedly observed after
chronic feeding of contaminated peanut meal for six months without antecedent morphological damage (4), The purpose of this cammunicatioa is to describe the application of a polarographic method based on Brdicka'e protein-reaction (5) to the plasma of ducklings fed crystalline aflatoxia B1 sad extracts of the Aspergillus flavus
Pza~~ pxomxia ixn~
1792
culture for a short time,
vol .
4, xo . 18
As a basis for comparison, the correlation of the
plasma protein index values with those of the hiatopathological indices of liver of a representative group of ducklings is also presented, Ia our experiments, one-day-old ducklings were fed different levels of crystalline aflatoxin B1 (purity based on physical data from T,L,C infrared, mesa spectra, N,M,R, and elementary analysis) or the extract from Aspergillus flayus culture (mold No, 4-95, T,L,C, analysis : for 7 days,
15% Bl, 9% Gl, < 1% B2 and G2)
At the end of the dosing period, the ducklings were decapitated
and oxylated plasma was obtained,
The plasma specimens were prepared for the
digest and filtrate teats and polarographed immediately as described by Muller (6), The polarograph used was a Davia Differential Cathode Ray Polarotrace, Model 1660,
This model can trace a complete polarographic wave on an
oscilloscopic screen every seven seconds, is slightly less than 30 seconds, permanent records,
The time required for each analysis
The polarotrace was photographed to obtain
In both tests, the wave heights of the second catalytic
protein wave, which has a peak potential at -1,4 ± 0,05 volts vs a mercury pool reference electrode, were measured at 25 ° ± 1 °C and expressed in units of wave height,
These units of wave height include the actual height of the wave
on the oscilloscope multiplied by the various sensitivity factors of the instrument,
The protein index was obtained from the ratio of the polsrographic
wave heights of the filtrate test to the digest teat, multiplied by an arbitrary constant of 15,
The data of the protein indices of the plasma
obtained from a representative group of ducklings show that the protein indices of plasma from ducklings fed 7 ug of crystalline aflatoxin B l are twice the value obtained from the plasma of control ducklings,
It is also noted that the
protein indices of ducklings fed Aflatoxin mold culture (determined by T,L,C, to be 20 uR R1) are 4 to 5 times higher than those of the control birds,
1793
PLABYII PBpTEIH IBDEä
Vol . 4, Bo . 18
TABLE 1 Average Protein Indices of Control and Aflatoxin-Fed Ducklings
Types of Ducklings
No, of Birds
Average Protein Index
Standard Deviation of the Mean
Range
20
28,8
± 4,0
21,3-36,3
Aflatoxin-fed 7 Rg crystalline Bl
4
60,0
± 1,0
59,0-61,2
Aflatoxin-fed mold culture extract concentrate *
12
122,3
± 18,2
102,0-198,0
Control
* T,L,C, analysis showed 20 Kg Bl The protein index values are relatively quantitative and show a relationship between dosage fed and abnormality of proteins present in experimental ducklings,
The described method is probably no more specific than
corresponding non-polarographic clinical tests, but it has advantages of simplicity of operation, sensitivity,
reproducibility,
speed, and wide scope of
application, Aistopathological eaaminatione of liner from individual ducklings were also carried out,
Both the protein index and the pathological index of a
iepreaentative group of control sad aflatoxin-fed ducklings were compared,
194
Vol . 4, Ao . 18
PIJ81(l PEO~ IIIDEâ TABLE 2
Correlation of Pathological Index of Liver with Protein Indea of Blood Plasma From a Representative Group of Control and Aflatoxin-Fed Ducklings Aflatozia-Fed u8 B1 calculated *
Protein 'Index
Pathological Index
Control
23,0
0
Control
21,3
0
Control
29,3
0
6,3 (cryst, Bl)
60,4
1-2
6,6 (cryet, Bl)
62,1
3
7
60,0
2
7
60,0
1-2
63
198,0
2-3
63
102,1
3
63
177,0
2-3
63
168,0
2-3
* Mold culture No, 4-95, T,L,C, analysis :
15X Sl, 9X Gl, < 1X B2 and
The pathological index values 0,1,2, and 3 represent none, alight, moderate, sad severe bile duct proliferation is liver, respectively,
The protein index
value correlates with the pathological index value for the individual ducklings and, in fact, reflects the pathological findings,
This agreement further
supports a quantitative relationship between the dosage fed and the protein index value obtained,
In a surveillance study on the hazard of peanuts, as
suggested by Lstham (7), the application of the protein index method as described here may therefore be of value is safeguarding public health,
Yol . 4, No .
18
PL~ß1tA PHOTEIH IADF.%
1~95
References 1,
R, Ai.T1R~FT and R, B, A, CARNAGHIi, Chem . Iad, ~0, Londoa (1963)=
Ibid ,
(1963), 2,
H, R, NESBITT, J, O'RELLY, R, SARGENT and A, SSERIDAN, Nature 195, 1062 (1962) ; A, 3, M, VAN DER ZLJDEN, W, A, A, BLANCHE ROELBNSMID, J, HOLDINGS, C, B, BARRETT, W, 0, ORD and J, PHILP, Nature 1=, 1060 (1962),
3,
T, ASAO, G, BDCSI, M, M, ABDSL-RODAR, S, B, CHANG, E, L, WICR and G, P, WOGAN, J . Am . Chum, Soc , 85, 1706
4,
(1963),
P, M, NEWHSRNE, G, N, WOGAN, W, W, CARLTON and M, M, A, RADER, Toxic, sad Appli . Phaxmco , !, 542 (1964),
5,
R, BRDICRA, Collectioa Czechoslovakia Chew, Comno4m , ~,
6,
0, H, MÜLLER, Method of Biochem, Analysis Vol, R1, 329 (1963),
7,
M, C, LATHAM, Brit . Med, J, 818 (Sept, 26, 1964),
112 (1933),