915
AFLATOXIN B1 IN MOULD EXTRACTS USED FOR in both strains with activation. Because a level afaflaroxin-B) equal
DESENSITISATION
SIR,-In injection immunotherapy (hyposensitisation) mould
antigens are commonly used to treat patients with rhinitis, asthma,
or other conditions due to sensitisation to moulds.I’2 To investigate
the possibility that carcinogenic mycotoxins might be present in
Aspergillus mould extracts used for immunotherapy, we obtained
twelve samples from manufacturers or through allergy clinics in
Galveston and Houston and looked for aflatoxins. Mutagenicity
testing (Ames’ bioassay) was done directly on the commercial mould
extracts. Of the twelve samples analysed by a combination of thin
layer3 and high pressure liquid chromatography (HPLC)4’S four
contained 0054-1 -08 ppm aflatoxin Bj and the sample with 1 -08
ppm was highly mutagenic.
Chemicalanalysis was by thin-layer chromatography on silica gel G glass
plates (Analtech Inc, Newark, Delaware) in chloroform: acetone (90:10 v/v).3
Five plates were spotted with each allergenic sample (100 J11 plate). After
development of the plates, the region between Rf=O’ 01 and 0- 55 fluoresced
under ultraviolet light (signifying possible presence of aflatoxins and some
mycotoxins). It was scraped and the combined materials were extracted with
chloroform (2 x 100 ml). The chloroform extract was filtered and evaporated to
dryness under vacuum, and the residue was dissolved in 500 J11 methanol for
analysis by HPLC.
HPLC separations were done by using an Altex ’Ultrasphere 5 ODS’
reversed phase column with methanol: water (65:35 v/v) solvent at a flow rate of
1 ml/min, and the effluent was monitored by both ultraviolet (365 and 254 nm)
and fluorescence (excitation 360 nm, emission 455 nm) detectors in series.4,5
The lowest detectable amounts for aflatoxin-Bl by UV 365 nm and
fluorescence detection under these HPLC conditions were 5 and 0-01 ng,
respectively. After viewing the HPLC profile from each thin-layer
chromatographic extract in those samples in which aflatoxin Bl was found,
further HPLC quantitation was done by repeating the chromatography and
scraping and extracting the spot corresponding to the identified toxin. The
final amount of the toxin was then determined from a standard curve.
Ames assays of AspergIllus extracts were done by standard procedures.6
Usually, four concentrations of each extract were assayed with TA100 and
TA98 with and without the addition ofrat liver S-9 fraction. Negative controls
of spontaneous revertants for each strain with and without the addition of the
S-9 mm were included. In each experiment, positive mutagenesis controls (5pg
2-ammoanthracene per plate), were routinely included to confirm the
reversion properties of each strain. We considered a chemical to have a positive
response m the test ifthe number of induced revertants was equal to or greater
than twice the number of spontaneous revertants.
The table presents the Ames’ test results on the Aspergillus mixed
sample containing 1 ’08 ppm of aflatoxin-Bwithout purification.
This sample was highly mutagenic (>1000 net revertants per 100 111)
AMES’ TEST RESULTS ON ASPERGILLUS MIX* CONTAINING 1 - 08 ppm
OF AFLATOXIN B,
*Apphed directly without TLC, HPLC purification
t2-ammoanthracene, the posnve control.
net number after subtraction of mean for zero pl per plate in parentheses.
1 Baer H
Allergenic extracts. In: Middleton E, Jr, Reed CE, Ellis EF, eds Allergy:
Principles and practice: vol I. St Louis, CV Mosby, 1978: 217-30
2 Kaad PH, Ostergaard PA. The hazard of mould hyposensitization in children with
asthma. Clin Allergy 1982; 12: 317-20.
3 Beljaars PR, Verusdonk CAH, Paulsch WE, Liem DH Collaborative study of two-
dimensional thin layer chromatographic analysis of aflatoxin B1 in peanut butter
extracts, using the antidiagonal spot application technique J Assoc Off Anal Chem
1973, 56: 1444-451.
4. Davis ND, Diener UL. Confirmatory test for the high pressure liquid chromatographic
determination of aflatoxin B, J Assoc Off Anal Chem 1980; 63: 107-09
5 Gregory III JF, Manley DB. High performance liquid chromatographic quanititation of
aflatoxin metabolite in animal tissues. J Assoc Off Anal Chem 1982; 65: 869-75.
6 Ames BN, McCann J, Yamasaki E. Methods for detecting carcinogens and mutagens
with the Salmonella/mammalian microsome mutagenicity test. MutatRes 1975; 31:
347-64.
to 0 - 062 ppm (6 - 2 ng per 100 1 of the sample) is too low to reveal
any positive result in the Ames’ test, the other three samples with
low levels of aflatoxin B(0- 054, 0- 062, and 0- 096 ppm) gave only
± on the Ames’ test. The HPLC technique with fluorescence
detection was more sensitive for detecting these low levels of
aflatoxin Bl.
Our findings suggest that a careful screening of commercially
available allergenic mould extracts is warranted. There may be a risk
of iatrogenic aflatoxin-induced illness, such as Reye’s syndrome,7
from hyposensitisation immunotherapy.7
Based on a report presented at the 14th annual meeting of Environmental
Mutagen Society, in San Antonio, Texas, m March, 1983. Supported by the
McGregor Allergy Clinic, Houston, Texas.
MARVIN S. LEGATOR
GLENN KLINE
Division of Environmental Toxicology,
V. M. SADAGOPA RAMANUJAM
Department of Preventive Medicine BRUCE R. CUNNINGHAM
and Community Health
University of Texas Medical Branch, JONATHAN B. WARD, JR
Galveston, Texas 77550, USA MOHY M. GAD-EL KARIM
ROLE OF COXSACKIE B VIRUSES IN
INSULIN-DEPENDENT DIABETES MELLITUS
SIR,-We think Dr Orchard, Professor Eggers, and their co-
workers (Sept 10, p 631) for their interest in our studies on the role of
Coxsackie B virus in children with recently diagnosed insulin-
dependent diabetes mellitus (IDDM) (June 25, p 1397).
Orchard et al wonder whether we might have found a higher
frequency of Coxsackie B IgM responses in our control group if we
had tested them in the same months as the onset of IDDM in the
patient group. As stated in our paper, sera from our 290 controls
were collected during each month of 1982 (about 24 per month).
Nevertheless, we would have preferred our controls to have been
matched not .only in age and time but also geographically, a defect
we hope to remedy in future.
So far we have investigated only a small number of patients, but
the project continues; recent work on a few patients with IDDM
elsewhere, each matched with two controls admitted to hospital at
the same time, support our findings. We are now examining sera
from a much larger group of patients from different parts of the
world, and these will permit a more comprehensive assessment of
the role of Coxsackie B virus in the aetiology of IDDM.
Orchard and colleagues’ use of siblings as controls may be suitable
for investigating the genetics of IDDM. However, if the disease is
triggered by Coxsackie B viruses, spread to a high proportion of
family contacts would be likely because enteroviruses are readily
transmitted within such an environment. Indeed, the finding that a
high proportion of siblings of patients with recent-onset IDDM
have evidence of recent Coxsackie B virus infections, may
strengthen rather than weaken the association between Coxsackie B
virus and this disease.
Although our evidence suggested that Coxsackie-B-virus-specific
IgM responses do not cross-react with islet cell antibodies, we have
yet to exclude the possibility that they cross-react with other
IgM autoantibodies which occur in a high proportion of patients
with IDDM.
Orchard and colleagues’ failure to confirm the relationship
between homotypic responses and age may well be related to
technique; immunofluorescence methods for detecting virus-
specific IgM responses are likely to be less sensitive and less specific
than M-antibody capture enzyme-linked immunosorbent assays.
Eggers and colleagues drew attention to the broad antigenic cross-
reactions among enteroviruses in human sera and also estimated
that only about 10% of their patients with IDDM had specific
Coxsackie-B-virus-neutralising IgM antibodies. Although the dif-
ference in proportions may be a reflection of our ELISA test being
more sensitive, we acknowledge that the aetiology of IDDM is
complex; if viruses are involved, Coxsackie B may be only one of
7. PollackJD Models of chemical and virus interaction and their relation to a multiple
etiology of Reye’s syndrome. In: Crocker JFS, et al. eds. Reye’s syndrome II. New
York: Grune & Stratton, 1979: 341-60.
916
them. Perhaps the frequency of Coxsackie-B-virus-specific IgM
responses in patients with IDDM exhibits geographical and
temporal variation.
Our data and results from routine clinical practice confirm that
infants and younger children tend to exhibit homotypic responses.
Results on sera collected from older people may be more difficult to
interpret; one can only state that the patient has had a recent
enterovirus infection. However, clinicians appreciate such reports,
particularly in patients with pericarditis/myocarditis, since they
provide evidence of current or recent infection by a group of viruses
which conventional techniques have until now failed to provide.
MARY L. KING
Department of Virology,
St Thomas’ Hospital Medical School, D. BIDWELL
London SE1 7EH
A. VOLLER
and Institute of Zoology,
Zoological Society of London, JENNIFER BRYANT
London NW1 J. E. BANATVALA
ARE BRASSICA VEGETABLES AGGRAVATING
FACTORS IN TRIMETHYLAMINURIA
(FISH ODOUR SYNDROME)?
SiR,-Fish odour syndrome is caused by an inherent metabolic
defect involving the microsomal mixed-function oxidase which
converts trimethylamine (TMA) to its non-volatile N-oxide.
Excessive TMA in the sweat, breath, and urine produces the
offensive odour. There appear to be no other physical symptoms
though severe psychosocial problems may develop. The condition
seems to be more common than first supposed,23 and TMA
metabolism may also be impaired in patients with chronic liver
disease.4 TMA is released by the action of enteric bacteria on dietary
choline and TMA oxide, and the fishy odour is usually diminished
by a severe diet (no 3eggs, fish, or legumes).3 In some instances
neomycin has helped.3
Our (G. R. F., E. J. B.) interest in this condition arose from work
on fishy taint in eggs.5 This taint is also produced by TMA derived
from dietary choline and TMA oxide and is caused partly by an
inherited defect severely restricting the synthesis of TMA oxidase.
However, studies with Brassica napus and B campestris (rapeseed)
meals; which are fed as protein supplements to poultry, have
identified potent inhibitors of TMA oxidase in vitro and in
vivo-namely, a tannin (polyphenolic) fractionand a discrete 7
compound, 5-vinyloxazolidine-2-thione (OZT), a goitrogen
formed in the
gut by the enzymic breakdown of progoitrin (PG), a
thioglucoside. A daily PG dose of 10 mg/kg body weight can
depress TMA oxidation capacity in hens possessing the genetic
defect by well over 90%, and much lower doses may still increase the
egg’s TMA content above that necessary for taint (about 1 ppm).
OZT inhibits TMA oxidase by competing with substrate, and this
behaviour is attributable to the CSNH grouping9which is also a
feature of certain antithyroid drugs. Mammalian mixed-function
oxidase is also inhibited by thioamides in this way. Tannins appear
to inhibit TMA oxidase by a different mechanism.
The significance of our work in poultry for the fish odour
syndrome of man lies in the fact that PG (and hence OZT) and
1. Todd WA. Psychological problems as the major complication of an adolescent with
trimethylaminuria. J Pediatr 1979; 94: 936-37.
2 Brewster MA, Schedewie H. Trimethylaminuria. Ann Clin Lab Sci 1983; 13: 20-24.
3 Danks DM, Hammond J, Schlesinger P, Faull K, Burke D, Halpern B.
Trimethylaminuria: diet does not always control the fishy odor. N Engl J Med 1976;
295: 962.
4. Marks R, Dudley F, Wan A. Trimethylamine metabolism in liver disease. Lancet 1978;
i 1106-07.
5. Butler EJ, Fenwick GR. Trimethylamine and fishy taint in eggs. Wld Poultry Sci J (in
press).
6. Fenwick GR, Pearson AW, Greenwood NM, Butler EJ. Rapeseed meal tannins and egg
taint. Anim Fd Sci Technol 1981; 6: 421-31.
7. Pearson AW, Greenwood NM, Butler EJ, Fenwick GR. The inhibition of
trimethylamine oxidation in the domestic fowl (Gallus domesticus) by antithyroid
compounds. Comp Biochem Physiol 1981, 69C: 307-12
8. Fenwick GR, Heaney RK, Mullin WJ. Glucosinolates and their breakdown products
in food and food plants. CRC Crit Rev Fd Sci Nutr 1983; 18: 123-201.
9. Pearson AW, Greenwood NM, Butler EJ, Fenwick GR The effect ofthionamides and
related compounds on trimethylamine oxidase activity in hepatic microsomes
isolated from chickens (Gallus domesticus). Comp Biochem Physiol 1982; 73C:
389-93
tannins occur in many foods. Brussels sprouts, swede, and kohlrabi
are especially rich sources of PG, and significant amounts are also
found in cabbage and cauliflawer. Levels of PG in brussels sprouts
in excess of 80 mg per 100 g fresh weight (about 25mg OZT/100 g)
are not uncommon. A study funded by the Ministry of Agriculture,
Fisheries and Food has produced a figure of 7 mg for the average
daily intake of PG, although this varies with the seasonal production
of many brassicas, with variations in what people choose or can
afford to eat, and with the species itself and where it grows. The PG
content of typical helpings of cooked brussels sprouts and cabbage
range from 65 to 140 mg (20-40 mg OZT) and from 9 to 42 mg
(3-12 mg OZT), respectively.
We know of no report of the influence of green vegetables or
tannins on TMA metabolism in patients with fish odour syndrome,
but the similarities between this condition and egg taint merit
examination of the value of low-brassica (or low-tannin) diets in the
management of this socially undesirable disease.
ARC Food Research Institute,
Norwich, Norfolk NR4 7UA G. R. FENWICK
Houghton Poultry Research Station,
Huntingdon, Cambridgeshire E. J. BUTLER
Metabolic Laboratory, Arkansas Children’s Hospital,
Little Rock, Arkansas, USA M. A. BREWSTER
NEW TREATMENT FOR PROSTATIC CANCER
SIR,-Your Aug 20 editorial (p 438) discusses two analogues of
luteinising-hormone-releasing hormone (LHRH), ICI 118,630 and
buserelin (HOE 766), and states that both seem equally potent in the
treatment of prostatic cancer, but that buserelin given by intranasal
spray has the potential drawback of irregular dosing. We disagree.
Publicationsl-4 on buserelin treatment have reported long-term
(2-14 months) maintenance of castration levels of testosterone and
markedly decreased levels of LH and follicle-stimulating hormone
(FSH). So far at least 77 buserelin-treated cases of advanced
prostatic cancer have been recorded and 40 have been published.
Most of these patients were given subcutaneous injections for one
week, followed by intranasal spray. Peak levels of serum
testosterone were reached after 3-6 days of treatment. After 2
weeks’ treatment serum testosterone reductions of 60-90%
(comparable with results of parental therapy 5) were achieved, LH
being reduced by 60-7507o and FSH by 40-85%. In 37 patients
(92%) clinical response (relief of symptoms and/or regression of
bone lesions or tumour mass) has been observed. Though not
conclusive, these results do not support the view that intranasal
administration is likely to cause irregular dosing. Faure et al,6 on the
contrary, have demonstrated the effect of intranasal administration.
On practical grounds, after a short phase of parenteral treatment
maintenance therapy with pernasal buserelin treatment may be the
answer.7
Clinical Research Department,
DEV R. CHADHA
Hoechst Holland NV,
1100 AZ Amsterdam, Netherlands A. MATROOS
1. Borgmann V, et al. Sustained suppression oftestosterone production by the luteinising-
hormone releasing-hormone agonist buserelin in patients with advanced prostate
carcinoma. Lancet 1982; i: 1097-99.
2. Tolis G, al. Tumor growth inhibition in patients with prostatic carcinoma treated
et
with luteinising hormone-releasing hormone agonists. Proc Natl Acad Sci USA
1982, 79: 1658-62.
3. Wasman JH, et al. Treatment with gonadotrophin releasing hormone analogue in
advanced prostatic cancer. Br Med J 1983; 286: 1309-12.
4. Wenderoth UK, et al. Endocrine studies with a gonadotropin-releasing hormone
analogue to achieve withdrawal of testosterone in prostate carcioma patients Eur
Urol 1982; 8: 243-47.
5. Ahmed SR, et al. Treatment of advanced prostatic with LHRH analogue ICI
cancer
118630. Clinical response and hormonal mechanisms. Lancet 1983; ii: 415-19
6. Faure N, et al. Sensitivity of luteinising hormone and gonadal steroid responses to
single intranasal administration of an LHRH agonist (HOE 766) in young normal
adult men. J Endocrinol Invest 1982; 5: 355-60.
7. Jacobi GH, Wenderoth UK. Gonadotropin-releasing hormone analogues for prostate
cancer: Untoward side effects of high-dose regimens acquire a therapeutical
dimension Eur Urol 1982; 8: 129-34.