‘Opioid peptides and dipeptidyl peptidase in autism’

‘Opioid peptides and dipeptidyl peptidase in autism’ SIR–We read with interest the paper by Hunter and colleagues1 which proposes that previously published studies suggesting a role for opioid peptide in autism are ‘inaccurate’. They also suggest that their study ‘questions the validity of the opioid excess theory’. It is encouraging that such theories are taken seriously and it is important that appropriate investigations are performed. It was particularly pleasing to see that detailed assessments of the study participants had been performed, as this is all too frequently ignored. It was also refreshing to see such a detailed description of the chemical analyses. Perhaps these details provide us with some clues as to why these results differ from those obtained by others. We would like to draw particular attention to the following points. The table of results (Table II) on page 125 appears to be unrelated to the mass spectrograms it claims to record. The retention data differ markedly from those shown in the profiles – even the order of elution is different. It is difficult to see how conclusions can be drawn from such contradictory findings. Most assays involving complex substrates, such as urine, consist of two stages. The preliminary preparation, or cleanup stage is designed to remove unwanted elements and to concentrate the material being identified or quantified in the following estimation stage. If the estimation stage is fairly nonspecific (as with ultraviolet [UV] detection) then it is vital that the first clean-up stage is efficient. Where the final stage is very specific, as with the mass spectrometric (MS) methods, the requirements of the initial stage are less stringent. Hunter et al. used a clean-up process specific to an MS method2 that falls far short of that necessary for UV detection. For example, the crude extract is washed in a 0.15% acetonitrile solution, as against the standard 10% solution; and furthermore, they prepare their final extract in 100% acetonitrile solution, as against the standard 40% solution. The MS clean-up process leaves the urine largely unchanged and unsuitable for UV analysis. The result would be the uninterpretable chromatograms described and illustrated in this paper. Even in heavily spiked samples it would be impossible, as Hunter et al. admit, to identify individual peptides from such a mixture with UV analysis. The concentration of bovine casomorphin used to spike the urine is not given, and its appearance in the MS spectrograms may reflect the technique’s greater sensitivity but might also be explained by the use of greater sample quantities: 560 microlitres for the (very sensitive) MS analysis compared to 40 microlitres for the (comparatively non-sensitive) UV method. To identify bovine casomorphins under these circumstances would be very difficult; to determine the very low levels of deltorphins or dermorphin which could be present would be very much more difficult still. The authors failed to detect bovine casomorphin 1-7 in any of the 10 samples from children with autism (or from the sibling samples they used as controls). Like Dr Karl Reichelt (University of Oslo, personal communication), we detect this particular peptide in measurable quantities in only some 25–30% of our case series with an almost total bias towards those children whose autism has been present from a very early age. Of the 10 children, three were on diets devoid of casein (as well as gluten) and seven had undergone regression in their second year. The introduction misattributes the advocacy of caseinfree diets to Whiteley3 and in support of its own failure to identify peptides quotes two earlier papers: Zhang’s paper4 is a brief (200 word) abstract of a non-peer reviewed conference poster; the paper by Pavone and Fiumara5 (actually published in 1997 and not 1996) discussed the relationship of autism with coeliac disease and reported on the absence of antibodies to gluten, but made no mention of peptides. Given these significant methodological shortcomings, we question the validity of the findings and, therefore, the basis of the comments on the opioid excess theory of autism.