European Psychiatry - Volume 33 - March 2016

29 / 812

24th European Congress of Psychiatry / European Psychiatry 33S (2016) S18–S55 S25
permeability and stress-related psychiatric disorders. Front Cell
Neurosci 2015;9.
[3] Mass M, Kubera M, Leunis JC. The gut-brain barrier in major
depression: intestinal mucosal dysfunction with an increased
translocation of LPS from gram negative enterobacteria (leaky
gut) plays a role in the inflammatory pathophysiology of depres-
sion. Neuroendocrinol Lett 2008;29(1):117–24.
S24
Can the pathophysiology of autism be
explained by the nature of the
discovered urine peptides and dietary
antigens?
K.L. Reichelt
Oslo University, Blindern, Oslo and lab 1, Sandvika, Norway
Purpose A: 1. To develop the urine analysis for exorphins for rou-
tine use in blood and cerebrospinal fluid (CSF).
2. Disorders where patient related validation must be carried out:
schizophrenia, depression (uni- and bipolar) and autism.
Method A: HPLC-MS/MS (fragmentation mass spectrometry)
technology.
With both a specific HPLC retention time and MS/MS (fragmen-
tation) this method is close to an absolute technique for peptide
recognition.
B: ELISA against specific proteins (gliadin, gluten and casein and
transglutaminase 6) g 2).
Background A: schizophrenia: increased opioid peptide levels
have been found in Schizophrenia using HPLC, immune assay and
behavioral tests. [1–6] as part of a general peptide increase in urine.
Since peptides are signaling compounds inhibition of peptidases
during transport and work up of samples is critical to prevent break
down, which is as expected fast at room temperature.
Strongly supporting is view is the data on postpartum psychoses
(a very symptom rich psychosis) where also amino acid sequence
of human casomorphin found increased, has been done [7–8].
The opioids can explain most of the symptoms of the psychotic
schizophrenic state [6]. It is of paramount importance then to mea-
sure these peptides in carefully diagnosed patients on and without
medication, in urine, blood and spinal fluid.
As can be seen in it is important to measure IgA and IgG
antibodies against the precursor proteins for the exorphins, which
are found increased by several groups, and also have direct effects
on the nervous system [9].
B. In depression increase levels of peptides has been found
[18,28,29] and also opioid levels measured as opium receptor
binding peptides [28]. In schizoaffective psychosis MS/MS exact
detection of exorphins have been published [6]. Also in this syn-
drome it is critical to be able to measure the exorphins in blood
and CSF, especially since the peptidases involved in break down
of exorphins are decreased in depressions [30,31]. Inflammatory
interleukins are also increased in depressions both uni- and bipo-
lar [32] indicative of inflammatory processes probably in the gut.
Inflammatory interleukins increase the permeability of epithelial
membranes [33].
C. Autism. Considerable work has been done using HPLC with
UV detection and co-chromatography [12,34–40]. However, with
HPLC–MS/MS we can ensure that we are measuring only the exor-
phins and not chromatographically similar peaks that hide inside
the main peak [41–43]. We therefore need to validate the new
method in autism for both urine, blood and CSF (CSF collected only
when spinal tap has to be done in any case).
Inhibition of break down in urine, blood and cerebrospinal fluid (CSF)
After extensive testing we have been left with three inhibitors.
Citric acid 0.2M; acetic acid 0.2M and aprotenine [44,45].
These body fluids will be provided by Prof Dr E. Severance and
Prof Dr R. Yolken (Johns Hopkins Univ.) and Prof Dr. Cunningham
(Uppsala Univ. Sweden). Lab 1 provides monovettes with citric acid
as peptidase inhibitor for urine collection. Blood will be collected
in EDTA – aprotenin vacuum test tubes (Vacutainer) as will be CSF.
HPLC and MS/MS detection.
The amount of urine analyzed on the HPLC after work up = 250
nanomles creatinine. To pick out generally active peptides in any
one disorder, five andfive autistic children or schizophrenic derived
and depressive derived urines are mixed, creatinine re-determined
and rerun. Peaks that are common to all patients increase or remain
the same, while individual peaks of material on the HPLC runs are
diluted out.
The complete procedure is published in detail [48]. If we use
reporter ions we do not have to match all the peaks as shown in
attached figures. On Fig. 1, synthetic bovine -casomorphine 1-4
(Y-P-F-P) is compared to biologically isolated compound from a
batch of five autistic children. On Fig. 2, the faster routine analy-
sis using reporter ions is shown for bovine -casomorhne 1-4. Top
trace is synthetic casomorphin 1-4 and bottom trace is biologically
isolated compound. The complete analysis for a series of opioids is
published [48].
Program is then in sequence:
– A: further validation of method for urine in the different disor-
ders;
– B: validation of method for blood in the same disorders;
– C: validation of method for CSF (spinal fluid) in schizophrenics
and depressive patients.
NB.
To avoid overlooking new compounds a complete HPLC run with
UV 215 nm (peptide bonds); 280 nm (aromatic groups) and 325 nm
(Indolyl-acryloid) shall be run for urines. If sufficient serum is avail-
able and spinal fluid these will also be run on HPLC in addition to
MS/MS detection.
Antibody assays will be done at Johns Hopkins using ELISA, Trans-
glutaminase 6 antibodies at Lab 1 also using ELISA assay.
Figures and references not available in the abstract.
Table 1 Antibodies of type IgA and IgG increased in relevant
disorders.
Disorder References
Autism spectrum Reichelt et al. [10]; Lucarelli et al. [11];
Cade et al. [12]; Vojdani et al. [13];
Kawashti et al. [14]; Trajkowski et al. [15];
Lau et al. [16]; de Magistris et al. [17]
Depression Sælid et al. [18]; Maes [19]
Bipolar Severance et al. [20]
Schizophrenia Dohan et al. [21]; Reichelt and Landmark
[22]; Samaro et al. [23]; Dickerson et al.
[24]; Severance et al. [25]; Jin et al. [26];
Niebuhret al. [27]
Reference no in parenthesis is found in the reference list. The anti-
bodies are of the IgA and IgG type and not IgE often found in allergic
pathology.
Disclosure of interest The authors have not supplied their decla-
ration of competing interest.
S25
Gastroenterology issues in
schizophrenia: Why the gut matters
E. Severance
Johns Hopkins University School of Medicine, Department of
Pediatrics, Baltimore, MD, USA
Numerous risk factors for schizophrenia can be reconciled through
a common enteric source. These risk factors include systemic