Probiotics and Autism
Letter for Doctors & Patients April 2001
The theory of casein and gluten derived
exorphins and their influence on autism via gut disbiosis is
well known. This theory
has given rise to a demand for foods and supplements, which are
both casein- and gluten-free. However, as more test results have
been reported on different products, there has been an appearance
of often-spurious results. This has been especially true of probiotic
supplements. Until now, the issues surrounding such wide-ranging
results have not been addressed. This report discusses the technical
issues involved in testing and presents a concluding commentary
as to the relevance of such tests to the autistic community.
Currently, there is a demand in the autism community for foods
and supplements, which are both casein and gluten free (CGF).
There is also an increasing demand for probiotic organisms.
Question has arisen regarding the ability to produce probiotic
organisms, which can qualify as CGF. The CGF issue presents
several technical issues, which have remained un-addressed.
At present, the overwhelming majority of probiotics are produced
in growth media, which at some point contain at least one dairy
product. However, it is generally agreed that through proper
attention to growth conditions as well as processing methods,
the state of the art is such that for all intents-and-purposes,
the final products can be considered milk or casein free. This
is because during normal growth and processing, the bacteria
consume the dairy constituents of the growth media and residuals
are separated during the concentration/purification of the probiotics.
However, there are often problems demonstrating this property
due to the inherent problems of current assay methods.
There are several types of assay
methods currently employed for the detection of casein. The
first is precipitation and quantification
by "total protein" methods such as the Kjeldahl procedure.
The second is by an enzyme-linked immunosorbent assay (ELISA).
The ELISA method utilizes antibodies to detect the casein as
a target antigen with subsequent reporter systems (e.g., colorometric).
The former method evolved out of the food processing industry
as a way to test milk products for casein. The method relies
on the fact that the vast majority of protein present in milk
is casein and for that industry, the method proved useful. Additionally,
the probiotics are living organisms producing a wide variety
of proteins. These too can contribute to a false positive signal
from the reporter system due to similarity to casein in sequence.
The chance of this happening when using a mAb is much less than
when a pAb is employed, however, the standard "kit" used
to detect casein contain pAbs not mAbs. It is worth emphasizing
that different bacteria produce different levels of various proteins
so there can be what appear as spurious results from species
to species and even strain to strain. Because the probiotic organisms
contain thousands of different proteins at any given time, the
method is not appropriate for the determination of casein in
any given culture. Similarly, the later method has several drawbacks,
which are discussed below.
First, in the ELSA, it is most
desirable to use a monoclonal antibody (mAb) vs. a polyclonal
antibody (pAb) for reasons of
specificity. With a mAb, the chances of a false positive are
much less because the mAb is much more specific for the desired
target (in this case casein) than the pAb is. The pAb by its
very definition is specific for several if not many different
targets. The reason is that a pAb is not one single antibody,
but consists of many different Abs and hence the "poly." For
many purposes, a pAb is sufficient. The reasons for the use of
a pAb over a mAb range from time to cost. A pAb can be produced
much faster than a mAb and at much less cost.
Those are just the first problems
associated with the ELISA assay. While ELISA is a very good
assay technique, it is less
than desirable for assaying casein in probiotics. The reason
for this, in addition to those above, has to do with how the "reporter" portion
of the ELISA functions. While there are different ways to perform
the ELISA assay, they all can be generalized as follows. During
the ELISA, when a target molecule binds to the Ab, a subsequent
enzymatic reaction (typically the enzyme is "linked" to
the antibody) is used to "report" that the binding
occurred. This enzymatic reaction, more often than not, involves
a peroxidase or phosphatase. Herein lies the problem, because
probiotics produce proxidases and phosphatases. The problem is
further confounded because different bacteria produce different
levels of these enzymes. For instance, Fitzsimmon and Berry (1994,
pp 125-33) showed that lactobacillus acidophilus (LA) produce
peroxidase. Not surprisingly, LA shows up as a positive using
the ELISA even when produced using the very same procedure for
other strains which show up as negatives. Further, there are
probably other enzymes produced by the bacteria, which can similarly
trigger the reporter system resulting in a false positive.
There is a third, though less-common method for detecting casein,
which utilizes gel (typically, SDS-polyacrylamide) chromatography.
Here, cellular extracts are placed in an electric field at one
end of a gel matrix. The matrix allows the smaller proteins to
move through first and the larger proteins to migrate more slowly
and hence lag relative to the smaller proteins. This differential
mobility in the gel affords a separation of proteins and protein
fragments. The main problem with this method is that it is not
only possible, but probable, that two very different proteins
(based on sequence) can have the same mobility in the gel. This
limitation can be overcome, but only to a degree, using isoelectric
focusing gels. This process also has similar problems associated
As a final note, it should
be kept in mind that the reason for the concern over the casein
in the first place is the fear of
the production of exorphins from the casein. This is important
because the probiotic organisms that are being subjected to the
casein analysis, themselves contain enzymes capable of breaking-down
such exorphins. Varmanen et al (2000, pp. 146-54) recently showed
that probiotic organisms, currently utilized as health supplements,
contain analogues of the Dipeptidyl peptidase IV enzyme (e.g.,
PepX2) which is known to be able to digest exorphins. Noteworthy
is the fact that the higher the concentration of probiotics a
supplement might contain, the higher the chances it will test
as a false positive for casein while concurrently producing extraordinarily
large amounts of the DPPIV analogues.
Taken as a whole, this information serves to clarify several
standing issues regarding both probiotic supplementation in autism,
as well as reported test discrepancies. In light of recent advances
in understanding the underlying enzymology of probiotic organisms,
it is prudent to view casein testing in light of the actual biological
significance that any detected presence might have. Further,
the current state of the art of casein testing should be given
Fitzsimmon N, Berry DR. Inhibition of Candida albicans by Lactobacillus
acidophilus:evidence for the involvement of a peroxidase system.
Varmanen P, Savijoki K, Avall S, Palva A, Tynkkynen S54X-prolyl
dipeptidyl aminopeptidase gene (pepX) is part of the glnRA operon
inLactobacillus rhamnosus. J Bacteriol 2000 Jan;182(1):146-54.
Please feel free to contact
Mark A. Brudnak PhD, ND
957 Lake Shore Road
Grafton, WI 53024
(Wisconsin is in the Central