Mycotoxins and Other Fungal Related Food Problems Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 09/22/18. For personal use only.
12 The Ochratoxins and Other Related Compounds STANLEY NESHEIM Division of Chemistry and Physics, Food and Drug Administration, 200 "C" Street, S. W., Washington, D.C. 20204
The ochratoxins are toxic metabolites elaborated by several species of Aspergillus ochraceus as well as by Penicillium viridicatum. The discovery, chemistry, history, sources, oc currence, toxicology, and fate in foods and feeds of these metabolites are reviewed. Emphasized are the analytical methods for the detection and estimation of the ochratoxins with reference to their applicability in commodities such as grains, nuts, beer, and animal tissues. Similarities and dif ferences in sampling procedures, extraction methods and solvents, extract purification, toxin detection and estimation, and chemical and biological techniques of confirmation of identity are described. Other metabolites which interfere with current methods are listed. Suggestions to direct fur ther efforts in developing analytical methods of ochratoxins are proposed.
In a screening program to determine possible causes of certain animal diseases in South Africa, Scott showed that three of five strains of Aspergillus ochraceus Wilh isolated from local domestic legumes and cereal products were toxigenic (1). The toxic metabolites were later isolated and chemically characterized (2, 3, 4). The major toxic com pound was named ochratoxin A; several related compounds were also isolated and identified (Figure 1). More recently one or more of the ochratoxins have been isolated from various mold species of the Asper gillus and Penicillium genera (5-14). These molds are commonly found in soils and on food and feed commodities such as nuts, dried fish, grains, beans, peas, coffee, spices, alfalfa, and meats (9, 10, 12, 15-24). Ochra toxin A, the 7-carboxy-5-chloro-8-hydroxy-3,4-dihydro-3-R-methyl isocou marin amide of L-β-phenylalanine (Figure 1), is the most toxic of the 276
12.
277
Ochratoxins
NESHEIM
ochratoxins ( 2 5 )
a n d is p r o d u c e d i n h i g h e s t y i e l d ( 2 6 ) .
s t r u c t u r e has b e e n c o n f i r m e d b y synthesis ( 2 7 , 2 8 ) . t o x i n B , its d e c h l o r o a n a l o g , a r e a c i d i c c o m p o u n d s a n d under ultraviolet light.
Its c h e m i c a l
Both it and ochra fluoresce
intensely
T h e y are r e l a t i v e l y h e a t stable, r e s i s t i n g a u t o -
claving i n oatmeal a n d cereal (29).
H o w e v e r , t h e y are l a r g e l y d e s t r o y e d
i n coffee b y h e a t i n g to 2 0 0 ° C f o r 5 m i n o r m o r e u n d e r s i m u l a t e d coffee roasting conditions (21).
B o t h ochratoxins A a n d Β h a v e b e e n f o u n d as
n a t u r a l c o n t a m i n a n t s of f o o d a n d feeds s u c h as w h e a t , b a r l e y , oats, coffee beans, c o r n , r y e , a n d d r i e d w h i t e beans ( 9 , 1 0 , 1 2 , 1 4 , 29-39). d e t e c t e d r a n g e d f r o m < 1 0 / * g / k g to 28 m g / k g .
T h e levels
T h e h i g h e r levels w e r e
f o u n d i n l o w e r g r a d e or m o l d y m a t e r i a l s . I n D e n m a r k , w h e r e
samples
w e r e t a k e n f r o m districts e x p e r i e n c i n g a h i g h i n c i d e n c e of s w i n e n e p h r o p a t h y , 5 8 % of 33 samples w e r e c o n t a m i n a t e d , a n d a v e r a g e d 3 m g / k g .
In
o t h e r surveys there a n d elsewhere i n v o l v i n g n o n s e l e c t e d samples t h e i n c i d e n c e a n d degree of c o n t a m i n a t i o n w a s l o w . T o c o n t r o l , r e d u c e o r e l i m i nate o c h r a t o x i n c o n t a m i n a t i o n , factors w h i c h c a n m o d i f y m o l d ment (including moisture, temperature, aeration, time, a n d h a v e b e e n i n v e s t i g a t e d (21, 40, 41).
develop substrate)
T h e f a t e of o c h r a t o x i n i n the n o r m a l
use of c o n t a m i n a t e d p r o d u c t s has also b e e n s t u d i e d . A s m e n t i o n e d a b o v e , u n d e r s i m u l a t e d coffee r o a s t i n g c o n d i t i o n s t h e ochratoxins are l a r g e l y d e s t r o y e d (21),
b u t 3 0 % r e m a i n e d i n t a c t i n o a t m e a l o r r i c e cereals after
3 h r of a u t o c l a v i n g ( 2 9 ) .
O n m a l t i n g a n d b r e w i n g beer f r o m barley con
t a m i n a t e d w i t h 1-5000 / x g / k g o c h r a t o x i n A the b e e r c o n t a i n e d f r o m /Ag/1.; this corresponds to a 2 - 7 % Several
investigators
transmission
studied
the
effects p r o d u c e d b y t h e ochratoxins.
6-20
(42).
toxicological
and
pathological
T h e L D o for ochratoxin A a d m i n 5
i s t e r e d per os varies f r o m 2.1-4.67 m g / k g for the c h i c k , s w i n e , a n d t r o u t to 22 m g / k g for the f e m a l e r a t (12, 25, 30, 33, 43-57).
O c h r a t o x i n A has
b e e n r e p o r t e d as teratogenic to m i c e a n d rats ( 5 1 , 59, 5 9 )
a n d to t h e
c h i c k e n e m b r y o (60);
teratogenic
other investigators h o w e v e r r e p o r t n o
effects i n c h i c k e n e m b r y o s
(61,
62).
A dose of 200 /xg o c h r a t o x i n A / k g
b o d y w e i g h t has b e e n r e p o r t e d to p r o d u c e nephrosis i n p i g s (31,
Figure 1. Ochratoxin A: R =H,R = Cl; ochratoxin B: Ri = R2 = H; ochratoxin C: R = -CH -CH , R = Cl 1
2
1
2
S
2
52).
278
MYCOTOXINS
T h e d e p o s i t i o n of o c h r a t o x i n A i n the l i v e r , k i d n e y , fat, a n d m u s c l e tissues of s w i n e a n d the e x c r e t i o n of o c h r a t o x i n A i n c o w s ' m i l k h a v e b e e n r e ported
(30,63,64).
T o h e l p detect, c o n t r o l , a n d e l i m i n a t e o c h r a t o x i n f r o m h u m a n a n d a n i m a l foods a n d feeds, sensitive a n a l y t i c a l m e t h o d s screening methods have been developed.
i n c l u d i n g some
S o m e of these h a v e b e e n u s e d
f o r t h e s u r v e i l l a n c e of v a r i o u s c o m m o d i t i e s a n d to detect the n a t u r a l o c c u r r e n c e of ochratoxins.
T w o of the o c h r a t o x i n m e t h o d s h a v e b e e n
tested i n i n t e r l a b o r a t o r y c o l l a b o r a t i v e studies (65, 66)
and were adopted
"official first a c t i o n ' for i n c l u s i o n i n O f f i c i a l M e t h o d s of A n a l y s i s " of t h e A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s (67).
S u c h a d o p t i o n means
t h a t the m e t h o d has b e e n v a l i d a t e d b y t e s t i n g i n s e v e r a l laboratories o n i d e n t i c a l sets of samples c o n t a i n i n g ochratoxins at u n k n o w n levels o c c u r r i n g b o t h as a n a t u r a l c o n t a m i n a n t a n d as a n a d d e d c o n t a m i n a n t . A l t h o u g h s e v e r a l g o o d g e n e r a l r e v i e w s c o v e r i n g b r o a d areas of t h e subject ( 1,12,14,33,47, 72, 73)
68-71
) a n d r e v i e w s of some of the m e t h o d s
(25,
h a v e b e e n p r e p a r e d , n o i n - d e p t h r e v i e w of t h e m e t h o d o l o g y has
been made.
T h i s c h a p t e r attempts to s u m m a r i z e a l l the w o r k o n m e t h
o d o l o g y i n c l u d i n g m u c h t h a t has b e e n scattered as parts of reports i n o t h e r areas, fields, o r d i s c i p l i n e s . M o s t of the p u b l i s h e d o c h r a t o x i n m e t h o d s d e s c r i b e d b e l o w
apply
o n l y to t h e analysis of o c h r a t o x i n s A a n d B . A f e w m e t h o d s h a v e b e e n a p p l i e d to t h e analysis of the m e t h y l a n d e t h y l esters (2-4,15, 67, 74-78).
26, 43,
65,
A l t h o u g h the esters of o c h r a t o x i n A are as t o x i c as the p a r e n t
c o m p o u n d s (47),
t h e y h a v e b e e n f o u n d o n l y i n substrates m o l d e d u n d e r
l a b o r a t o r y c o n d i t i o n s a n d at m u c h l o w e r concentrations t h a n those ochratoxins A a n d Β
of
(4).
A U o c h r a t o x i n m e t h o d s consist of t w o or m o r e of the f o l l o w i n g b a s i c steps: lot s a m p l i n g , a n a l y t i c a l s a m p l e p r e p a r a t i o n , e x t r a c t i o n of the toxins, p u r i f i c a t i o n o r c o n c e n t r a t i o n , d e t e c t i o n , q u a n t i t a t i o n , a n d c o n f i r m a t i o n of i d e n t i t y of the t o x i n . T h e m e t h o d s differ i n c o m m o d i t y a p p l i c a b i l i t y , sen s i t i v i t y ( o r m i n i m u m d e t e c t i o n l e v e l ) , a c c u r a c y , p r e c i s i o n , a m o u n t of v a l i d a t i o n d a t a s u p p o r t i n g t h e m e t h o d , cost of m a t e r i a l s , e q u i p m e n t , t i m e , a n d labor. T h e c h o i c e of a m e t h o d is d i c t a t e d b y factors s u c h as t y p e a n d n u m b e r of samples t o b e a n a l y z e d a n d i n f o r m a t i o n r e q u i r e d for the p a r t i c u l a r s t u d y . T h e a n a l y s t s h o u l d n o t b e l i m i t e d to a n y specific m e t h o d .
For a
s u r v e y , f o r e x a m p l e , h e c a n a n d s h o u l d use t h e s i m p l e s t r e l i a b l e m e t h o d a v a i l a b l e (i.e.,
a screening m e t h o d )
to e l i m i n a t e the m a n y
negative
samples a n d t h e n p e r h a p s a p p l y a n o t h e r , m o r e r i g o r o u s m e t h o d to the p o s i t i v e samples. O f course t h e s c r e e n i n g m e t h o d s h o u l d h a v e a s e n s i t i v i t y a d e q u a t e to the needs of t h e s u r v e y . M e t h o d s are c o n t i n u a l l y i m p r o v e d as n e w t e c h n i q u e s a p p e a r a n d
Ochratoxins
279
are a p p l i e d to o l d p r o b l e m s .
T h e a n n u a l r e p o r t of t h e G e n e r a l R e f e r e e
12.
NESHEiM
o n M y c o t o x i n s o f t h e A s s o c i a t i o n o f O f f i c i a l A n a l y t i c a l C h e m i s t s is p u b l i s h e d e v e r y y e a r i n t h e M a r c h issue o f t h e Journal Official
Analytical
Chemists
ments i n m e t h o d o l o g y . s u c h as Ό c h r a t o x i n s ,
of the Association
of
a n d provides a continuing review of develop
A s s o c i a t e Referees a r e assigned t o separate topics ,,
" A f l a t o x i n s i n Coffee," " M y c o t o x i n s i n G r a i n s , "
to n a m e a f e w , u n d e r t h e r e v i e w of t h e G e n e r a l Referee.
T h e Associate
Referees also r e p o r t progress i n t h e i r assignments b u t o n a less r e g u l a r basis.
Lot Sampling F o r m y c o t o x i n s i n g e n e r a l o r f o r ochratoxins specifically, t h e c o n t a m i n a t i o n i n t h e c o m m o d i t y m a y b e i n s m a l l pockets o r i n a f e w o f m a n y u n i t s of t h e c o m m o d i t y .
A . ochraceus
is k n o w n to p r o d u c e as h i g h as
1.3-1.5 m g o c h r a t o x i n A / k g o n c h o p p e d c o r n , p o l i s h e d r i c e , o r w h e a t b r a n ( 2 9 ) a n d 3.9 g / k g o n s h r e d d e d w h e a t ( 7 9 ) . T h e s e h i g h levels c a n b e associated w i t h v e r y s m a l l portions o f a t o t a l lot. T h e r e f o r e , l a r g e samples m u s t b e t a k e n to increase t h e p r o b a b i l i t y of i n c l u d i n g c o n t a m i n a t e d p o r t i o n s o f heterogeneous lots.
C o m m o n p r a c t i c e f o r aflatoxin
analysis is to t a k e a 25 k g s a m p l e f o r larger k e r n e l m a t e r i a l s s u c h as nuts a n d a five k g s a m p l e f o r c o m m o d i t i e s s u c h as s m a l l grains. T h e c o n s i d e r a tions i n v o l v e d i n s a m p l i n g peanuts h a v e b e e n p u b l i s h e d
(80-82).
Analytical Sample Preparation T h e effectiveness of s o m e a p p r o a c h e s to r e d u c t i o n of t h e l o t s a m p l e to a n a n a l y t i c a l s a m p l e , 1 0 - 3 5 0 g, has b e e n s t u d i e d ( 8 3 , 84).
Generally
10 g is c o n s i d e r e d too s m a l l f o r a d e q u a t e a n a l y t i c a l s a m p l i n g ; c u r r e n t l y a 100-g s a m p l e is r e c o m m e n d e d .
F o r s m a l l samples (
100 m g f r o m
10-g samples of c o m m o d i t i e s s u c h as grains or nuts. T h i s m i x t u r e is t h e r e f o r e f u r t h e r f r a c t i o n a t e d ( u s u a l l y b y T L C ) , a n d the toxins are d e t e r m i n e d b y v i s u a l o r i n s t r u m e n t a l c o m p a r i s o n o n t h i n l a y e r plates of the
fluores
c e n c e intensities of the u n k n o w n spots w i t h spots of the T L C standards. T h e m a n y solvents a n d absorbents w h i c h h a v e b e e n u s e d for the T L C d e t e c t i o n a n d e s t i m a t i o n of ochratoxins a n d some t y p i c a l R
f
values are
l i s t e d i n T a b l e V . I t s h o u l d b e e m p h a s i z e d t h a t n e a r l y a l l the
methods
d e p e n d o n g o o d T L C r e s o l u t i o n of t h e toxins f r o m e a c h other a n d f r o m i n t e r f e r i n g m a t e r i a l s . T h e T L C R values are not v e r y r e p r o d u c i b l e , a n d t
u n k n o w n spots are best i d e n t i f i e d b y c h r o m a t o g r a p h y w i t h k n o w n o c h r a t o x i n standards a d d e d to the extract either i n s o l u t i o n or o n the o r i g i n s p o t o n the T L C p l a t e . I d e n t i c a l R values of u n k n o w n a n d s t a n d a r d , e v e n t
i n s e v e r a l T L C systems, d o not p o s i t i v e l y i d e n t i f y t h e u n k n o w n .
Usually
f u r t h e r c o n f i r m a t i o n of i d e n t i t y m u s t b e m a d e . T h i s is discussed
below.
12.
285
Ochratoxins
NESHEIM
R
Table V .
V a l u e s ' of Ochratoxins for T h e i r Separation, Detection and Quantitation
t
Ochratoxins: Esters" of Solvent"
Α Thin Layer
Β
Chromatography
A (Silica
C e H - H O A c , 25:1 C H -HOAc,9:l
45
24
C H6-HOAc,8:2 C H -H0Ac,3:l C H -HOAc,4:l
50 69
35 45
65
C H - H O A c - H 0 90:10:1 C H - M e O H - H O A c , 18:1:1
32 65
50
100
C e H e - M e O H - H O A c , 12:2:1 C H e - M e O H - H O A c , 24:2:1
60 60
52 35
14
0
0
e
55 67
Β Gel) 30 46
e
e
e
e
6
6
e
6
c
2
e
C H -CCl -nBuOH-EtOAcformic acid, 35:25:20:19:1 C H - M e O H - E t O A c , 15:3:1 C H - E t O A c - f o r m i c acid, 70:30:1 C H - a c e t o n e - f o r m i c acid, 80:20:1 C H -BuOH-EtOAc-formic acid, 69:15:12:4 C He-BuOH-EtOAc-formic acid, 50:15:33:2 Toluene-EtOAc-formic acid, 5:4:1 e
6
4
100
4,27 1, 5 - 9 , 1 3 , 26, 3 7 , 7 5 , 79,90 111,112 78,86,110 27, 6 2 , 1 0 4 , 108,111 113 35-38,96 8,21,35,36, 6 7 , 7 4 , 76 77,96,117 1-3,86,111 9,10,85,87 100,115-117 15 14, 9 7 , 1 0 5 28
e
6
31 50
6
e
46
e
e
74
65
81
15
71
63
80
15
e
6
e
Toluene-EtOAc-formic acid, 6:3:1 EtOAc-t-PrOH-H 0,5:2:1 Et20-MeOH-H 0-formic acid, 95:4:1:1 CHCI -acetone,93:7 CHCls-EtOAc-formic acid, 60:40:1 CHCls-acetone-i-PrOH, 85:12.5:2.5 2
2
3
74
References
28
70
55-70 42 27
25
95
13,18,21,28, 3 5 , 3 6 , 75, 87, 9 0 , 9 1 , 94-96,99102, 111, 118 6,7,9,10,98, 116 97 10 91 119 15
286
MYCOTOXINS
Table V .
Continued Ochratoxins: Esters' of
Solvent*
Α
CHCl -acetone-formic acid, 80:20:1 C H C l - M i B K , 4:1 (Oxalic acid impregnated silica) D i - i s o p r o p y l ether ( O x a l i c acid impregnated silica) Hexane-acetone-HOAc, 18:2:1
A
Β
References
65
3
48
3
Thin Layer
Β
28,102 20
1,120
35
28 50
Chromatography
67,74,76
(Alumina)
CHCl -acetone,4:l
9
3
Paper i-PrOH-3iV (ΝΗ ) 00 ,3:1 Hexane (formamide i m p r e g n a t e d paper) 4
Silica
2
3
Gel Mini-Column
Chromatography 65 50 Chromatography
18
2,3,86 4
Detection
Toluene-EtOAc-formic acid, 5:4:1
4
89
° RfxlOO.
See a, Table I. • Methyl or ethyl esters of ochratoxins A and B . Fluorescent band detected on 4 mm X 7 cm silica gel column.
b
d
T h e m e t h o d of v i s u a l c o m p a r i s o n of fluorescence intensities suffers f r o m p o o r p r e c i s i o n ( > db 2 0 % ) ( 6 5 ) a n d d e p e n d s g r e a t l y o n t h e a n a lyst's a b i l i t y a n d experience. T h e T L C d e n s i t o m e t r y is m o r e objective b u t suffers m o r e f r o m i n s t r u m e n t a l p r o b l e m s , interferences, a n d p o o r r e s o l u t i o n i n T L C . T h e types of T L C s c a n n i n g i n s t r u m e n t s , l i n e a r ranges a n d sensitivities o b t a i n e d w i t h t h e m are s h o w n i n T a b l e V I . A l s o l i s t e d i n T a b l e V I are o t h e r a n a l y t i c a l m e t h o d s . T h e s e are less sensitive o r m o r e c o m p l i c a t e d to use b u t h a v e b e e n u s e d i n s p e c i a l c i r c u m s t a n c e s as i n d i c a t e d i n references l i s t e d i n t h e t a b l e .
Chemical Confirmation of Identity T h e presence of ochratoxins i n a s a m p l e is i n i t i a l l y b a s e d o n t h e i d e n t i t y of t h e R values of t h e u n k n o w n s w i t h those o f s t a n d a r d o c h r a toxins. A d d i t i o n a l p r o o f is m o s t f r e q u e n t l y o b t a i n e d b y d e v e l o p i n g c h r o m a t o g r a m s i n several solvent systems. T h i s is c o n c l u s i v e e v i d e n c e o n l y i n a negative sense—i.e., t h a t t h e i n i t i a l suspect spots are n o t o c h r a toxins. F o r p o s i t i v e p r o o f t h e toxins are separated a n d p u r i f i e d b y p r e t
12.
Table V I .
287
Ochratoxins
NESHEIM
Instrumental Methods for Quantitation of Ochratoxins (ng/spot) Linear Range
Instrument
Sensitivity
CV
h
References
TLC Fluorodensitometry P h o t o v o l t , m o d e l 530 10-100 7.1 15,26,91,101 Ozumor,SD-91,Asuka 0-5000 170 14,105 Kogyo Co., Tokyo H i t a c h i F l u o r o spec6 5 , 74 trometer, M P F - 2 A , T L C accessory 018-0057 Schoeffel, S D 3000 3-10 65,77 A m i n c o - B o w m a n spectro 0-10 0.5-1.0 3.6 5 , 4 3 , 8 5 , 1 1 4 , 121 photo fluorometer A m i n c o - B o w m a n spectro 0.25-5 0.25 52,117 p h o t o fluorometer (after exposing T L C spots t o N H ) F o u r different i n s t r u 6.4 32,42, 52,65,67, ments ( P h o t o v o l t , 74 Schoeffel, A m i n c o Bowman and Hitachi) Miscellaneous Techniques U V spectrophotometry sensitivity 15,18,26, 28,43-45, 0.5-5 /xg/ml 67,74, 85,98,103, 109,110,112,118, 121-123 U V spectrophotometric dialysis 121 and titration Phosphorescence a n d fluorescence 118 H y d r o l y s i s followed b y G L C 43,121 analysis for phenylalanine R a d i o c h e m i c a l methods 62,99,108,110 a
3
In acid medium : excitation maximum 310^340 nm, emission maximum 440-475 n m ; i n basic medium: excitation maximum 360-390 nm, emission maximum 427445 nm. br* m - χ £ · χ· standard deviation Χ 100 ° Coefficient of variation = average paratory T L C a n d identified b y one or m o r e of the p h y s i c a l c h e m i c a l or β
bioassay techniques ( T a b l e V I I ) . A l m o s t every instrument for measuring p h y s i c a l parameters has been a p p l i e d t o t h e i d e n t i f i c a t i o n o f o c h r a t o x i n s ( i r , u v , N M R , a n d mass spec trometry).
P h y s i c a l a n d c h e m i c a l p r o p e r t i e s s u c h as m e l t i n g p o i n t s ,
e l e m e n t a l a n a l y s i s , a n d c h e m i c a l d e r i v a t i v e f o r m a t i o n h a v e also u s e d . T h e b a s i c T L C sprays l i s t e d i n T a b l e V c h a n g e t h e
been
fluorescence
of
o c h r a t o x i n s A a n d Β f r o m g r e e n t o b l u e b y f o r m i n g a salt w i t h t h e phenolic functional group.
288
MYCOTOXINS
Table VII.
Chemical Confirmation of Identity of Ochratoxins
TLC Sprays
References
N E U O H or N H fumes 3
Triethylamine NaHC0 FeCl K O H (20%) A1C1 3
3
3
Repeated TLC with up to 10 different solvent systems Solubility in NaHCO solution Chemical derivatives M e t h y l esters a n d ethers s
5 - 7 , 1 0 , 1 3 , 3 5 , 36, 87, 9 0 , 9 1 , 93, 94,99-101,117,119 87 67,74 7,104,120 75 6,7 4 , 9 , 27, 2 8 , 3 5 , 3 6 , 8 6 , 8 7 , 90, 95 9, 9 0 , 9 4 , 3 5 - 3 8 2-4, 7,10, 26,31, 3 5 , 3 7 , 3 8 , 9 4 , 9 9 , 104 4, 26, 6 5 , 6 7 , 52, 74 15,100 15 15
E t h y l esters Acetates Trifluoroacetates T r i m e t h y l s i l y l ether Spectral methods V i s u a l c o m p a r i s o n u n d e r short a n d long u v light 76,94 Fluorescence spectrophotometry, 5 acid-base Ultraviolet spectrophotometry 2-4, 9,15,18,26,28,44, 98,100, 102,122 Infrared spectrophotometry 2-4,13,15,26-28,102 Optical rotatory dispersion and 2 - 4 , 1 3 , 2 7 , 28,104 polarimetry N u c l e a r m a g n e t i c resonance 2-4,13,26, 27,31, 69,102,104,124 M a s s spectrometry 2-4, 26-28, 69,104,125 M e l t i n g points, C , H , N analysis 2-4,13, 26,27,98,102,124 A T L C i d e n t i f i c a t i o n m a y b e m a d e b y p r e p a r i n g t h e esters or ethers of b o t h the u n k n o w n a n d s t a n d a r d o c h r a t o x i n s a n d c o m p a r i n g t h e T L C characteristics ( R / s ) of these d e r i v a t i v e s .
Biological Tests T a b l e V I I I lists s o m e of t h e m o r e sensitive tests w h i c h h a v e b e e n d e v i s e d to test o c h r a t o x i n f o r b i o l o g i c a l a c t i v i t y . T h e s e are f o r t h e m o s t p a r t q u a l i t a t i v e i n n a t u r e . I n c o n j u n c t i o n w i t h other p h y s i c a l a n d c h e m i c a l d a t a some of t h e b i o l o g i c a l tests are o c c a s i o n a l l y u s e d for c o n f i r m i n g t h e i d e n t i t y of o c h r a t o x i n s .
Ochratoxin-Kelated Metabolites (Table IX) T h e f e w c h e m i c a l l y r e l a t e d d e r i v a t i v e s of t h e ochratoxins r e p o r t e d so f a r i n c l u d e , besides t h e e t h y l a n d m e t h y l esters of ochratoxins A a n d
12.
289
Ochratoxins
NESHEIM
Table VIII.
B i o l o g i c a l Tests f o r O c h r a t o x i n
Type Response
System
Sensitivity
C h i c k e n embryo
death
LD
Z e b r a fish l a r v a Brine shrimp
death death
20% mortality
Brine shrimp larvae
death
LC
0 . 0 1 - 17 μg/egg
5 0
5
1.7
0
/xg/ml
A Reference 25,60-62,70, 78, 126 129 70,130
1 /Ag/ml
megaterium g r o w t h inhibition growth Bacillus cereus mycoides inhibition growth Tetrahymena pyriinhibition formis H S M H e L a cell cytotoxicity T r a c h e a l organ death culture D a y old duckling death D a y old chick death Bacillus
131 LC l O r t / m l ; 17% m o r t a l i t y 0.2 /xg/disc 35, 3 6 , 1 2 7 , 1 fig/disc 128 127 1.5 j t t g / d i s c 5 0
1%200
ftg/ml
10/xg/ml LC
5
0
150
LD
1.7
ftg/ml
132 22, 6 8 , 9 7 133
1-4 1 3 5 - 1 6 6 / A g / c h i c k 107, 1 2 2 , 1 3 4
/Ag/duckling 5 0
B , 4-hydroxy-ochratoxin A a n d ochratoxin a and β ( F i g u r e 1).
Methods
f o r one o r m o r e of t h e m e t h y l a n d e t h y l esters h a v e b e e n p u b l i s h e d 65, 74-77).
T h e y are as t o x i c as the p a r e n t c o m p o u n d s ( 2 5 )
p r o d u c e d i n r a t h e r l o w y i e l d b y the m o l d ( 4 ) .
(4,
b u t are
Ochratoxin D reported b y
Scott is s y n o n y m o u s w i t h 4 - h y d r o x y o c h r a t o x i n A . O c h r a t o x i n a is d e r i v e d f r o m ochratoxin A b y r e m o v i n g the phenylalanine moiety.
S e v e r a l of t h e
other c o m p o u n d s s h o w n i n T a b l e I X are r e l a t e d to the o c h r a t o x i n s o n l y i n t h a t t h e y are p r o d u c e d b y the same m o l d s a n d h e n c e c a n i n t e r f e r e b o t h i n c h e m i c a l analyses, tests, a n d i n b i o l o g i c a l assays or t o x i c i t y studies. T h e d e r i v a t i v e s f o r the most p a r t are r e l a t i v e l y n o n t o x i c ( 2 5 ) .
Methods
of analysis are a v a i l a b l e for some of these substances b u t h a v e n o t b e e n r e f e r e n c e d b e c a u s e t h e y are b e y o n d the scope of this r e v i e w . Conclusion T h e m u l t i p l i c i t y of m e t h o d s i n use as o u t l i n e d here i n d i c a t e s the l a c k of a single m e t h o d a d e q u a t e f o r a l l tasks. M e t h o d o l o g y w i l l p r o b a b l y b e g r e a t l y i m p r o v e d i n the near f u t u r e w h e n t h e p r o m i s i n g t e c h n i q u e h i g h efficiency l i q u i d c h r o m a t o g r a p h y
of
(already successfully a p p l i e d to
t h e analysis of t h e m y c o t o x i n s , aflatoxins, p a t u l i n , a n d s t e r i g m a t o c y s t i n ) is a p p l i e d to o c h r a t o x i n s . I t is h o p e d t h a t t h e f u t u r e w i l l b r i n g r e l i a b l e , r a p i d , i n e x p e n s i v e , a n d e v e n a u t o m a t e d m e t h o d s to effectively a n d e c o n o m i c a l l y protect the food a n d feed supply.
290
MYCOTOXINS
Table I X . Ochratoxins, Related Metabolites and Derivatives Produced by T w o Major Ochratoxin-Producing Fungi Compound Ochratoxin A and Β M e t h y l a n d e t h y l esters of ochratoxins A and Β 4 - H y d r o x y ochratoxin A Ochratoxin T and T O c h r a t o x i n a a n d β* A
a
c
a
L - A l a n i n e and L-leucine de r i v a t i v e s of o c h r a t o x i n a 4-Hydroxyaspergillic acid Aspocracin Brevianamide A and Β Citrinin Emodin 3- (1,2-Epoxy propyl) -5,6dihydro-5-hydroxy-6methyl-2-one Ergosterol Erythritol Griseofulvin M e l l e i n (ochracin) 4-Hydroxymellein Oxalic acid Penicillic acid
L-Prolyl-L-leucine anhydride L-Prolyl-L-valine anhydride Secalonic acid Viridicatin Viridicatol Viridicatum toxin Unidentified toxins
A. ochraceus
P. viridi catum
+ + + +
+
1,12,13,93,98,119 4
+ +
1 2 , 9 8 , 104 78 2 9 , 4 4 , 88, 9 9 , 1 0 4 , 107, 111, 1 1 2 , 1 1 3 , 117 136
+ + + + + + + + +
+ +
+ + +
+ + + +
+ + +
Reference
4,97 137,138 139 9,10,12,31,50,119 14 140
13,14 14 141 1, 22, 125 96,135 9,50 4, 5, 1 4 , 2 2 , 9 1 , 9 3 , 9 7 , 1 0 3 , 1 1 9 , 142, 143 1 1 14,145 146 147 141 11,16, 2 3 , 9 3 , 9 7 , 119,144,148-152
Ochratoxins A and C in which the phenylalanine moiety has been replaced with tyrosine. * Ochratoxins in which the phenylalanine moiety has been removed. β
Literature Cited
1. Steyn, P. S., "Microbial Toxins," Vol. VI, A. Ciegler, S. Kadis, S. J. Ajd, Eds., pp. 179-205, Academic Press, New York, 1971. 2. Van der Merwe, K. J., Steyn, P. S., Fourie, L., Scott, de Β., Theron, J. J., Nature (1965) 205, 1112. 3. Van der Merwe, K. J., Steyn, P. S., Fourie, L., J. Chem. Soc. (1965) 7083.
12.
NESHEIM
Ochratoxins
291
4. Steyn, P. S., Holzapfel, C. W., J. S. Afr. Chem. Inst. (1967) 20, 186. 5. Ciegler, Α., Can. J. Microbiol. (1972) 18, 631. 6. Ciegler, H., Fennel, D. J., Mintzlaff, H . J., Leistner, L., Naturwissenschaften (1972) 59, 365. 7. Ciegler, Α., Mintzlaff, H . J., Machnik, W., Leistner, L., Fleischwirtschaft (1972) 52, 1311. 8. Hesseltine, C. W., Vandegraft, Ε. E., Fennell, D. I., Smith, M. L., Shotwell, O. J., Mycologia (1972) 64, 539. 9. Scott, P. M., van Walbeek, J. W., Harwig, J., Fennell, D. I., Can. J. Plant Sci. (1970) 50, 583. 10. Scott, P. M., van Walbeek, W., Kennedy, B., Anyeti, D., J. Agric. Food Chem. (1972) 20, 1103. 11. Semeniuk, G., Harshfield, G. S., Carlson, C. W., Hesseltine, C. W., Kwolek, W. F., "Proceedings First U.S.-Japan Conference on Toxic Micro-Organisms," U.S. Department of Interior, Washington, D.C. (1970) 185-190. 12. van Walbeek, W., Can. Inst. Food Technol. J. (1973) 6, 96. 13. van Walbeek, W., Scott, P. M., Harwig, J., Lawrence, J. W., Can. J. Microbiol. (1969) 15, 1281. 14. Yamazaki, M., "Mycotoxins in Human Health," I. F. H . Purchase, Ed., p. 107, Macmillan Press Ltd., London, 1971. 15. Broce, D., Ph.D. Thesis, Louisiana State University, Baton Rouge, 1970. 16. Doupnik, B., Jr., Bell, D. K., Appl. Microbiol. (1971) 21, 1104. 17. Leistner, L., Mintzlaff, H . J., Maing, I. Y., Abstracts of the 2nd Interna tional Union of Pure and Applied Chemistry Sponsored International Symposium Mycotoxins in Food, Pulaway, Poland (1974) July 23-25. 18. Moore, J. H., Ph.D. Thesis, Auburn University, Auburn, Ala., 1971. 19. Nesheim, S., J. Ass. Offic. Anal. Chem. (1967) 50, 370. 20. Tsunoda, H., "Proceedings First U.S.-Japan Conference on Toxic Micro organisms," U.S. Department of Interior, Washington, D.C. (1970) 143. 21. Levi, C. P., Trenk, H. L., Mohr, H . K., J. Ass. Offic. Anal. Chem. (1974) 866. 22. Umeda, M., Yamashita, T., Saito, M., Sekita, S., Takahashi, C., Yoshihira, K., Natori, S., Kurata, H., Udagawa, S., Jap. J. Exp. Med. (1974) 44, 83. 23. Saito, M., Ohtsuba, K., Umeda, M., Enomoto, M., Kurata, H., Udagawa, S., Sakabe, F., Ichinoe, M., Jap. J. Exp. Med. (1971) 40, 1. 24. Saito, M., Ishiko, T., Enomoto, M., Ohtsubo, K., Umeda, M., Kurata, H., Udagawa, S., Taniguchi, S., Sekita, S., Jap. J. Exp. Med. (1974) 44, 63. 25. Chu, F. S., Crit. Rev. Toxicol. (1974) 2, 499. 26. Nesheim, S., J. Ass. Offic. Anal. Chem. (1969) 52, 975. 27. Steyn, P. S., Holzapfel, C. W., Tetrahedron (1967) 23, 4449. 28. Roberts, J. C., Woollven, P., J. Chem. Soc, C (1970) 278. 29. Trenk, H. L., Butz, M. E., Chu, F. S., Appl. Microbiol. (1971) 21, 1032. 30. Krogh, P., Abstracts of the 2nd International Union of Pure and Applied Chemistry Sponsored International Symposium Mycotoxins in Food, Pulaway, Poland (1974) July 23-25. 31. Krogh, P., Hald, B., Pedersen, E. J., Acta Pathol. Microbiol. Scand., Sect. Β (1973) 81, 689. 32. Krogh, P., Hald, B., Englund, P., Rutqvist, L., Swahn, O., Acta Pathol. Microbiol. Scand. Sect. Β (1974) 82, 301. 33. Munro, I. C., Scott, P. M., Moodie, C. Α., Willes, R. F., J. Amer. Vet. Med. Ass. (1973) 163, 1269. 34. Patterson, D. S. P., Roberts, Β. Α., Abstracts of the 2nd International Union of Pure and Applied Chemistry Sponsored International Sympo sium Mycotoxins in Food, Pulaway, Poland (1974) July 23-25.
292
MYCOTOXINS
35. Shotwell, Ο. L., Hesseltine, C. W., Goulden, M . L., Appl. Microbiol. (1969) 17, 765. 36. Shotwell, O. L., Hesseltine, C. W., Goulden, M. L., J. Ass. Offic. Anal Chem. (1969) 52, 81. 37. Shotwell, O. L., Hesseltine, C. W., Goulden, M. L., Vandegraft, Ε. E., Cereal Chem. (1970) 47, 700. 38. Shotwell, O. L., Hesseltine, C. W., Vandegraft, Ε. E., Goulden, M . L., Cereal Sci. Today (1971) 16, 266. 39. Shotwell, O. L., Goulden, M. L., Fennell, D. L., Hesseltine, C. W., 88th Ann. Meetg., Association of Analytical Chemists, Washington, D.C., Oct. 14-17, 1974. 40. Lillehoj, Ε. Β., J. Amer. Vet. Med. Ass. (1973) 163, 1281. 41. Harwig, J., Chen, Y.-K., Can. J. Plant Sci. (1974) 54, 17. 42. Krogh, P., Hald, B., Gjertsen, P., Myken, F., Appl. Microbiol. (1974) 28, 31. 43. Chu, F. S., Biochem. Pharmacol. (1974) 23, 1105. 44. Doster, R. C., Sinnhuber, R. O., Food Cosmet. Toxicol. (1972) 10, 389. 45. Doster, R. C., Arscott, G. H., Sinnhuber, R. O., Poultry Sci. (1973) 52, 2351. 46. Elling, F., Moeller, T., Bull World Health Org. (1973) 49, 411. 47. Food Cosmet. Toxicol. (1973) 11, 903. 48. Galtier, P., More, J., Bodin, G., Ann. Rech. Veter. (1974) 5, 233. 49. Huff, W. E., Wyatt, R. D., Tucker, T. L., Hamilton, P. B., Poultry Sci. (1974) 53, 1585. 50. Krogh, P., Hasselager, E., Friis, P., Acta Pathol.Microbiol.Scand. Sect. Β (1970) 78, 401. 51. More, J., Galtier, P., Ann. Rech. Veter. (1974) 5, 167. 52. Krogh, P., Axelsen, Ν. H., Elling, F., Gyrd-Hansen, N., Hald, B., Hyldgaard-Jensen, J., Larsen, A. E., Madsen, Α., Mortensen, H. P., Moeller, T., Petersen, O. K., Ravnskov, U., Rostgaard, M., Aalund, O., Acta Pathol Microbiol. Scand. Sect. A (1974) Supplement 246, 1. 53. Meisner, H., Chan, S., Biochemistry (1974) 13, 2795. 54. Munro, I. C., Moodie, C. Α., Kuiper-Goodman, T., Scott, P. M., Grice, H. C., Toxicol.Appl.Pharmacol. (1974) 28, 180. 55. Szczech, G. M., Carlton, W. W., Tuite, J., Toxicol. Appl. Pharmacol. (1973) 25, 456. 56. Szczech, G. M., Carlton, W. W., Tuite, J., Vet. Pathol. (1973) 10, 219. 57. Szczech, G. M., Carlton, W. W., and Tuite, J. Vet.Pathol.(1973) 10, 135. 58. Hayes, A. W., Hood, R. D., Toxicol. Appl. Pharmacol. (1973) 25, 457. 59. Hayes, A. W., Hood, R. D., Lee, H. L., Teratology (1974) 9, 93. 60. Verrett, M. J., unpublished data (1974). 61. Mintzlaff, H. J., Christ, W., Fleischwirtschaft (1972) 52, 1174. 62. Yamazaki, M., Suzuki, S., Sakakibara, Y., Miyaki, K., Jap. J. Med. Sci. Biol. (1971) 24, 245. 63. Madsen, Α., Mortensen, H . P., Larsen, A. E., Flengmark, P., Hald, B., Krogh, P., Elling, F., Ugeskr. Agron. Hort. (1973) 6, 100. 64. Ribelin, W. E., Smalley, Ε. B., Strong, F. M., Abstract of the 2nd Int. Cong. Plant Pathol., Minneapolis, Minn. (1973) Sept. 5-12. 65. Nesheim, S., J. Ass. Offic.Anal.Chem. (1973) 56, 822. 66. Levi, C. P., Presented at the 88th Annual Meeting of the Association of Official Analytical Chemists, Washington, D.C. (1974) Oct. 14-17. 67. "Changes in Methods," J. Ass. Offic. Anal. Chem. (1973) 56, 486. 68. Enomoto, M., Saito, M., Annu. Rev. Microbiol. (1972) 26, 279. 69. Ferreira, N. P., "Biochemistry of Some Foodborne Microbiological Toxins," Mateles, R. I., Wogan, G. N., Eds., p. 157, MIT Press, Cambridge, Mass. 1967.
12.
NESHEIM
Ochratoxins
293
70. Brown, R. F., "Proceedings First U.S.-Japan Conference on Toxic Micro organisms," U.S. Department of Interior, Washington, D.C., (1970) pp. 12-18. 71. Mirocha, C. J. Christensen, C. M., Ann. Rev. Phytopathol. (1974) 12, 303. 72. Fishbein, L., Falk, H . L., Chromatogr. Rev. (1970) 12, 42. 73. Stoloff, L., Clin. Toxicol. (1972) 5, 465. 74. Nesheim, S., Hardin, N . F., Francis, O. J., Jr., Langham, W. S., J. Ass. Offic. Anal Chem. (1973) 56, 817. 75. Vorster, L. J., Analyst (1969) 94, 136. 76. Stoloff, L., Nesheim, S., Yin, L., Rodricks, J. V., Stack, M., J. Ass. Offic. Anal. Chem. (1971) 54, 91. 77. Stoloff, L., "Quantitative Thin-Layer Chromatography," J. Touchstone, Ed., p. 95, John Wiley & Sons, New York, 1973. 78. Wei, R , Chu, F. S., Experientia (1974) 30, 174. 79. Schindler, A. F., Nesheim, S., J. Ass. Offic. Anal. Chem. (1970) 53, 89. 80. Tiemstra, P. J., J. Amer. Oil Chem. Soc. (1971) 46, 1307. 81. Whitaker, T. B., Dickens, J. W., Wiser, E. H., J. Amer. Oil Chem. Soc. (1970) 47, 501; Whitaker, T. B., Dickens, J. W., Monroe, R. J., ibid. (1974) 51, 214. 82. Whitaker, T. B., Wiser, Ε. H., J. Amer. Oil Chem. Soc. (1969) 46, 377. 83. Stoloff, L., Dantzman, J., J. Amer. Oil Chem. Soc. (1972) 49, 264. 84. Stoloff, L., Campbell, A. D., Beckwith, A. C., Nesheim, S., Winbush, J. S., Jr., Fordham, Ο. M., Jr., J. Amer. Oil Chem. Soc. (1969) 46, 678. 85. Chu, F. S., Butz, Μ. Ε., J. Ass. Offic. Anal. Chem. (1970) 53, 1253. 86. Steyn, P. S., van der Merwe, K. J., Nature (1966) 211, 418. 87. Scott, P. M., Hand, T. B., J. Ass. Offic. Anal. Chem. (1967) 50, 366. 88. van Walbeek, W., Moodie, C. Α., Scott, P. M., Harwig, J., Grice, H . C., Toxicol. Appl. Pharmacol. (1971) 20, 439. 89. Hald, B., Krogh, P., J. Ass. Offic. Anal Chem. (1975) 58, 156. 90. Eppley, R. M., J. Ass. Offic. Anal Chem. (1968) 51, 74. 91. Escher, F. E., Koehler, P. E., Ayres, J. C., Appl. Microbiol. (1973) 26, 27. 92. Yin, L., Ass. Offic. Anal Chem. (1969) 52, 880. 93. Bullerman, L. B., J. Milk Food Technol. (1974) 37, 1. 94. Davis, N . D., Searcy, J. W., Diener, U. L., Appl. Microbiol. (1969) 17, 742. 95. Lai, M., Semeniuk, C., Hesseltine, C. W., Appl. Microbiol. (1970) 19, 542. 96. Moore, J. H., Davis, N . D., Diener, U. L., Appl. Microbiol. (1972) 23, 1067. 97. Natori, S., Sakaki, S., Kurata, H., Udagawa, S., Ichine, M., Saito, M., Umeda, M., Chem. Pharm, Bull. (1970) 18, 2259. 98. Scott, P. M., Kennedy, B., van Walbeek, W., J. Ass. Offic. Anal. Chem. (1971) 54, 1445. 99. Searcy, J. W., Davis, N. D., Diener, U . L., Appl. Microbiol. (1969) 18, 622. 100. van Walbeek, W., Scott, P. M., Thatcher, F. S., Can. J. Microbiol. (1968) 14, 131. 101. Wu, M. T., Ayres, J. C., J. Agric. Food Chem. (1974 ) 22, 537. 102. Galtier, P., Ann. Rech. Veter. (1974) 5, 155. 103. Bacon, C. W., Sweeney, J. G., Robbins, J. D., Burkick, D., Appl. Micro biol (1973) 26, 155. 104. Hutchison, R. D., Steyn, P. S., Thompson, D. C., Tetrahedron Lett. (1971) 43, 4033. 105. Yamazaki, M., Maebayashi, Y., Miyaki, K., Appl. Microbiol. (1970) 20, 452. 106. Davis, N . D., Sansing, G. Α., Ellenburg, T. V., Diener, U . L., Appl. Microbiol. (1972) 23, 433. 107. Chu, F. S., Chang, C. C., J. Ass. Offic. Anal. Chem. (1971) 54, 1032.
294
MYCOTOXINS
108. Maebayashi, Y., Miyaki, Κ., Yamazaki, M., Chem. Pharm. Bull. (1972) 20, 2172. 109. Pitout, M. J., Toxicol. Appl. Pharmacol. (1968) 13, 299. 110. Wei, R., Strong, F. M., Smalley, Ε. B., Appl. Microbiol. (1971) 22, 276. 111. Nel, W., Purchase, I. F. H., J. S. Afr. Chem. Inst. (1968) 21, 87. 112. Pitout, M. J., Biochem. Pharmacol. (1969) 18, 485. 113. Steyn, P. S., Holzapfel, C. W., Ferreira, N . P., Phytochemistry (1970) 9, 1977. 114. Lemieszek, K., Abstracts of the 2nd International Union of Pure and Applied Chemistry Sponsored International Symposium Mycotoxins in Food. Pulaway, Poland (1974) July 23-25. 115. Chu, F. S., J. Ass. Offic. Anal. Chem. (1970) 53, 696. 116. Scott, P. M., Lawrence, J. W., van Walbeek, W., Appl. Microbiol. (1970) 20 839 117. Trenk, H.L., Chu, F. S., J. Ass. Offic. Anal. Chem. (1971) 54, 1307. 118. Neely, W. C., West, A. D., J. Ass. Offic. Anal. Chem. (1972) 55, 1305. 119. Ciegler, Α., Fennell, D. I., Sansing, G. Α., Detroy, R. W., Bennett, G. Α., Appl. Microbiol. (1973) 26, 271. 120. Steyn, P. S., J. Chromatogr. (1969) 45, 473. 121. Shu, F. S., Arch. Biochem. Biophys. (1971) 147, 359. 122. Chu, F. S., Noh, I., Chang, C. C., Life Sci. (1972) 11, Part I, 503. 123. Pitout, M . J., "Mycotoxins in Human Health," I. F. H . Purchase, Ed., p. 53, Macmillan, London, 1971. 124. Yamazaki, M., Maebayashi, Y., Miyaki, K., Tetrahedron Lett. (1971) 25, 2301. 125. Doster, R. C., Sinnhuber, R. O., Wales, J. H., Food Cosmet. Toxicol. (1972) 10, 85. 126. Choudhury, H., Carlson, C. W., Poultry Sci. (1973) 52, 1202. 127. Broce, D., Grodner, R. M., Killebrew, R. L., Bonner, F. L., J. Ass. Offic. Anal. Chem. (1970) 53, 616. 128. Clements, N. L., Abstracts of the American Association of Cereal Chemists —American Oil Chemists Society Joint Meeting, Washington, D.C. (1968). 129. Abedi, Ζ. H., Scott, P. M., J. Ass. Offic. Anal. Chem. (1969) 52, 963. 130. Brown, R. F., J. Amer. Oil Chem. Soc. (1969) 46, 119. 131. Harwig, J., Scott, P. M., Appl. Microbiol. (1971) 21, 1011. 132. Hayes, A. W., Melton, R., Smith, S. J., Bull. Environ. Contam. Toxicol. (1974) 11, 321. 133. Cardeilhac, P. T., Nair, K. P. C., Colwell, W. M., J. Ass. Offic. Anal. Chem. (1972) 55, 1120. 134. Peckham, J. C., Doupnik, B., Jr., Jones, Ο. H., Jr., Appl. Microbiol. (1971) 21, 492. 135. Cole, R. J., Moore, J. H., Davis, N . D., Kirksey, J. W., Diener, U . L., J. Agric. Food Chem. (1971) 19, 909. 136. Pawlowski, Ν. E., Doster, R. C., Lee, D. J., Nixon, J. E., Sinnhuber, R. O., "Abstracts of Papers," 167th National Meeting, ACS, March 31-April 5, 1974, Los Angeles, Calif. 137. Myokei, R., Sakurai, Α., Chang, C., Kodaira, Y., Takahashi, N., Tamura, S., Tetrahedron Lett. (1969). 138. Myokei, R., Sakurai, Α., Chang, C., Kodaira, Y., Takahashi, N., Tamura, S., Agric. Biol. Chem. (1969) 33, 1491. 139. Wilson, B. J., Yang, D. T. C., Harris, T. M., Appl. Microbiol. (1973) 26, 633. 140. Moore, J. H., Murray, T. P., Marks, M. E., J. Agric. Food Chem. (1974) 22, 697. 141. Hutchison, R. D., Steyn, P. S., van Rensburg, S. J., Toxicol. Appl. Pharmacol. (1973 ) 24, 507.
12.
NESHEIM
Ochratoxins
295
142. Udagawa, S., Ichinoe, M., Kurata, H., "Proceedings First U.S.-Japan Conference on Toxic Micro-Organisms," U.S. Department of Interior, Washington, D.C. (1970) pp. 174-187. 143. Wu, M. T., Ayres, J. C., Kohler, P. E., Appl. Microbiol. (1974) 27, 427. 144. Budiarso, I. T., Carlton, W. W., Tuite, J. F., Vet. Pathol. (1970) 7, 531. 145. Yamazaki, M., Maebayashi, Y., Miyaki, K., Chem. Pharm. Bull. (1971) 19, 199 146. Cunningham, K. G., Freeman, G. G., Biochem. J. (1953) 53, 328. 147. Luckner, M., Mohammed, Y. S., Tetrahedron Lett. (1964) 29, 1987. 148. Carlton, W. W., Tuite, J., Caldwell, R., J. Amer. Vet. Med. Ass. (1973) 163, 1295. 149. McCracken, M . D., Carlton, W. W., Tuite, J., Food Cosmet. Toxicol. (1974) 12, 79. 150. Ibid., 89. 151. Ibid., 99. 152. Zwicker, G. M., Carlton, W. W., Tuite, J., Food Cosmet. Toxicol. (1973) 11, 989. RECEIVED December 16, 1974.
