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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.

http://dx.doi.org/10.1016/j.eurpsy.2016.01.839

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)

( Table 1 o

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

Table 1 ,

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.

http://dx.doi.org/10.1016/j.eurpsy.2016.01.840

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