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Urinary chromatographic profiles in schizophrenia
BIOL PSYCHIATRY 199o;27:1127-1132
1127
Urinary Chromatographic Profiles in Schizophrenia J.J. Gilroy, I.N. Ferrier, T.J. Crow, and F.J. Rowell
In a series of articles, Trygstad et al. (1980) suggested that specific patterns of peptide excretion and increased levels of peptide material are found by chromatographic analysis of the urine of patients with a variety of psychiatric illness including schizophrenia. As these results have not been independently replicated in another laboratory, we conducted an investigation of urine samples from 5 DSM-III classified schizophrenic patients and 4 normal subjects using the techniques described by the Norwegian group, and with a series of modifications. The chromatographic profiles obtained differ widely from those repotted by Trygstad et al. No significant differences were detected between patients with schizophrenia and controls. The methods used by Trygstad et al. are complex and we have defined several parts of the methods which are subject to variability. Our findings lend no support to the view that patients with psychiatric illness can be readily distinguished from normal subjects by tb~eamount or profile of peptide ;xcretion in urine.
Introduction A series of publications from a grc.ep of Norwegian workers have promulgated the hypothesis that excess of neuropeptides is central to the development of psychotic disorders. They suggest that "overproduction" of peptides may lead to "hyperfunctioning" of some neuronal systems and to an overflow of peptide into the urine. Trygstad st al. (1980) studied the benzoic acid-treated urines of schizophrenics by Sephadex G-25 gel chromatography and reported two reproducible pattet'ns which are both substantially different from controls. These findings were confirmed in subsequent studies from the same group (Reichelt et al. 1981, 1985) who reported increased levels of benzoic acid precipitable aromatic compounds together with glycoprotein- and uric ac!d,~s~iated Ix:pride material in urine of schizophrenics. When this material was furthe, fractionated, the hydrolyzed fractions were found to contain significantly greater amounts of amino acids in the schizophrenic group compared with the control group. However, no evidence has been presented to show that this putative peptide material originates in the brain and th-~re is no evidence to suggest a generalized abnormality of peptides in scldzophrenia. As these results have not been independentlyreplicated in any other laboratory [indeed one group (Isranglmn et al. 1983) found it impossible to reproduce the G-25 profiles from
Frcm the Department of Pharmacy, Royal Victoria Infummy, Newcastle Upon Tyne (LJ.G.); I~{e,~.,:c¢~'rin~logy Unit, Newcastle General Hospital, Newcastle upon Tyne (I.N.F.); Division of Psychiatry, MRC Ctinit.ai Research Centre, Harrow, Middlesex (T.LC.); and Depamnent of Pharmaceutical Chemistry, Sunderland Polytechnic0 Lungham Tower, Sunderland (FJ.R.), England. Address reprint requeststo John J. Gilmy, Departmentof Pharmacy, Royal VictoriaInfirmary,Queen Victoria Road, Newcastle upon Tyne NEI 4LP, England. Received June 8° 1987; revised April 6, 1989.
@ 1990 Society of Biological Psychiatry
0006-3223/~0/$03.50
1128
BIOLPSYCHIATRY
J.J. Gilroy et al.
1990;27:1127-1132
normal controls], we felt it appropriate to ~ttempt to replicate the findings of the Norwegian group. M a t e r i a l s and M e t h o d s Five patients recently admitted to Northwick Park Hospital research ward with episodes of acute psychosis that met DSM-III criteria for schizophrenia were included. There were 3 men (ages 27, 29, and 33 years) and 2 women (ages 29 and 38 years). Urine samples were taken in the ~irst week of admission while the patients were all suffering from psychotic symptoms. Two patients had been drug free for several months. One had received 300 mg of Chlorpromazine following admission, and the other 2 had received no neuroleptic medication following admission but their previous neuroleptic status was uncertain. Control samples were obtained from the laboratory staff: 2 men (ages 31 and 40 years) and 2 women (ages 34 and 36 years). The methods used for precipitation, washing, and G-25 and P-2 chromatography are as described by the Norwegian group (Reichelt et al. 1985). P-2 cta'omatography was performed on material extracted from region 2 (elution volume: 800-1400 ml) of the G25 chromatogram. Fractions corresponding to the maxima of peaks from the G-25 chromatogram were screened for protein using a modified Lowry method (Leggett-Bailey 1967). Results
Sephadex G-25 Chromatography Irrespective of psychiatric status, two chromatographic patterns were found: one for men and one for women. The distinction between them occurs at 2100 ml where there is a large peak in the male chromatogram (Figure 1). The chromatograr,~ may be divided into three elution volume regions: 400-600 ml, 800-1400 ml, and i400-5000 rnl (Figure 1). There is no significant difference between schizophrenic aed control areas under the curve in region 2. In region 1, there is an increase in ares in the schizophrenic group although the difference is not statistically significant. In ,egion 3, there are insufficient results to perform statistical analysis but no trend is aPl~ga'ent(Table 1). For all individuals in the study, a positive reaction for protein was obtairJed from the peaks in region 1 only.
Biogel P-2 Chromatography Irrespective of gender or psychiatric status, there are two chromatographic patterns---one for unhydrolyzed fractions and one for hydrolyz~d fractions (Figure 2). The distinction between them occurs at 154 ml (the bed volume) where, except for I male schizophrenic, there is a large peak in the unhydrolyzed chromatogram. The areas under the curve for the hydrolyzed and unhydrolyzed chromatograms show no significant differences between the two groups (Table 1).
G-25 Methodology The materialelutingin region 3 of the G-25 chromatograms is produced in largeramounts by men than by women. However, if "IMS 99" (ethanol ~> 98.5% with methanol, moisture, etc. ~ 1.5%) without methylisobutylketone(MIBK) was used as the washing
Urinary Chromatography in Schizophrenia
BIOL PsYCmATRY
1129
1990;27:1127-1132
,° I 7.0
6,0
E 5.0
Normal male
~
4.0
4.0
3.0
3.0
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2.0
No~'n'~lfen,ale
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Schizophrenic
1.5 1.2 O,9 0.6 0.3
o.-i?
~
0
• Eiulion vol
|0o NO 1200 BOO
)~ 600 ~mo119o ISO0
(ml)
Figure 1. (3-25 Chromatographic profiles of benzoic acid-treated urine. Top row: chromatograms from control men and control women (chromatograms for schizophrenic men and women were respectively the same); middle and bottom rows: region beyond 1500 ml elution volume deleted and absorbance axis scale expanded; middle row: normal group; bottom row: schizophrenic group.
alcohol, there was considerable variation in the size of the peaks in region 3 from both genders. In addition, we found that when the urinarj precipitate was washed using "IMS 99" without MIBK there was a loss of peaks h region 2 of the G 2 5 chromatogram. Therefore, the presence of MIBK in the washing alcohol is essential.
P-2 Methodology Fractions in the 140-160 ml region were boiled for 2 hr with distilled water and then tested with Ninhydrin. Results suggest that because the peak in this region of the unhydrolyzed chromatogram is lost following alkaline hydrolysis, it is produced by ammonia. The difference in absorbance at 570 nm of an aliquot of sample before and after
1130
BIOLPSYCHIATRY
J.J. Gilroy et al.
1990;27:, 127-1132
Table 1. Area Under Curve (cm2) for Peaks in the G-25 and P-2 Column Chromatograms in Schizophrenics and Controls
G-25 peak 1
Schizophrenics
Controls
Male schizophrenics
Male controls
(n = 5)
(n = 4)
( n = 3)
( n = 2)
4.8 ± 2.1
2.4 - 0.8 a
3.3 ± 1.8
2.5 ± 0.7
(400-600 rid) G-25 peak 2
m
(800-1400 ml) G-25 peak 3 (1400-5000 nil)
P-2 unhydrolyzed P-2 hydrolyzed
87/130/37
~
25.1 ± 18.8 12.5 ± 9.7
140/121
19.7 ± 8.9 8.8 - 1.3
ap = 0.063. Mann-Whitney U test (2 = tailed), no significant differences. Means - SD. Peak 3 on G-25 is only fom~d in males (see text).
alkaline hydrolysis suggests that the p e ~ is sample related. The Norwegian group have used either rotary evaporation (Saelid et al. 1985) or lyophilization (Reichelt~et al. 1985) to concentrate their G-25 fractions with no change in the results. We used rotary evaporation to concentrate our large volume samples (approximately 400 ml). However, because a significant amount of ammonium bicarbonate remained (as measu~d by change in pH), we found it necessary to follow evaporation with lyophilization. Discussion For normal individuals, the Norwegian group reports one chromatogram from Se,phadex G-25 chromatography whereas we demonstrate a large male-female difference in our chromatograms. For schizophrenic patients, they report two disease-related chromatograms. Our results also show two chcomatograms in schizophrenic patients which, like those from normal individuals, are gender-related. We find no significant differences between chromatograms from the schizophrenic: group and the controls. We were only able to produce the chromatograms reported here following a later publication (Reichelt et al. 1985) and personal communication with the Norwegian group which indicate that the following additional steps are used: (1) The pH of the precipitated urine is adjusted to between 4.0 and 4.3. (2) The precipitate Js washed such that only part of the added benzoic acid is removed. (3) The washing alcohol contains 2% by volume of MIBK. Without MIBK we have shown that the selective removal of benzoic acid could not be achieved. [This would explain why Israngkun et al. (1983) were unable to replicate the results of the Norwegian group.] Our work proceeded to a second separation stage---Biogel I'-2 chromatography-because the absorbance of the G-25 co!unto eluate is due to the presence of any molecule that absorbs at this wavelength and not just to the presence of proteins and peptides containing amino acid residues such as tryptophan, tyrosine, or phenylalanine (our screen for proteinaceous material found protein to be present in region 1 but absent from regions 2 and 3). The separation was carried out on material extracted from region 2, as the Norwegian group have reported a diverse range of bioactivities from this material (Reichelt et al. 1981). Using Biogel P-2 chromatography to separate this material, the Norwegian group report one chromatogram for normal individuals and chromatograms for their
Urinary Chromatography in Schizophrenia
BIOLPSYCHIATRY
113 l
1990;27: 1127-1132 UNHYDROLYSED 5.0
4.ol
[ E 3.0
HYDROLYSED
3.0
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30 |0 90120 IS0110H0 0 30 H gO 1|0150110|10
Figure 2. Po2: Chromatographic profiles of the extract of pooled fractions from the 800-1400 ml elution volume region of the G-25 chromatogmms~ Top row: cltromatograms from fractions pre- and posthydrolysis; middle and bottom rows: chromatograansfrom fractions posthydrolysis; middle row: normal group; bottom row: schizoFhrer.ic gToup. schizophrenic patients that are much more varied (Reichelt et ai. 1985), whereas we found that there is only one chromatogram for all individuals in our study. Following hydrolysis, the Norwegian group report a significant increase in Ninhydrinpositive material in both their schizophrenic subgroups when compared to that obtained from their normal population, whereas we found that there is no significant difference between our two groups. The Norwegian group have not reported chromatograms prior to hydrolysis. However, as Ninhydrin will react with any compound containing free amino groups to produce a blue color, it follows that an unhydrolyzed control must be performed. The marked differences between our results from G-25 and P-2 chromatography and those of the Norwegian group cannot be explained by differences in the met.beds used
1132
BIOLPSYCHIATRY • 1990;27:1127-1132
J.J. Gilroy et al.
as we have used their more recently reported method. This method is complex and extremely lengthy (up to 6 days for G-25 chromatography and 3 days for P-2 chromatography) and thus many sources of variability may occur. No evidence was found to support the basic observation of Trygstad et al. (1980) and Reichelt et al. (1981, 1985) that the urine of schizophrenic patients contains increased levels of benzoic acid precipitable aromatic compounds and putative peptide material. The complexity of and possible sources of variation in the methods are emphasized and we suggest that further research and methodological comparisons need to be performed.
References Israngk~mP, Patel ST, Newman HAI, Taylor WA (1983): Pattern of urinary putative neuropeptides in control and autistic subjects. Clin Chem 29:1287. Leggett-Bailey J (1967): Techniques in Protein Chemistry (2rid revised and expanded edition). Amsterdam: Elsevier, pp 340--341. Reichelt KL, Edminson PD, Toft KG (1985): Urinary pepfides in schizophrenia and depression. Stress Med 1:169-181. Reichelt KL, Hole K, Hamberger A, et al (1981): Biologically active peptide-containing fractions in schizophrenia and childhood autism. Adv Biochem Psychopharmacol 28:627-643. Saelid G, Haug JO, Heiberg T, Reichelt KL (1985): Peptide-containing fractions in depression. Biol Psychiatry 20:245-256. Trygstad OE, Reichelt KL, Foss I, et al (1980): Patterns of peptides and protein-associated peptide complexes in psychiatric disorders. Br J Psychiatry 136:59-72.
1127
Urinary Chromatographic Profiles in Schizophrenia J.J. Gilroy, I.N. Ferrier, T.J. Crow, and F.J. Rowell
In a series of articles, Trygstad et al. (1980) suggested that specific patterns of peptide excretion and increased levels of peptide material are found by chromatographic analysis of the urine of patients with a variety of psychiatric illness including schizophrenia. As these results have not been independently replicated in another laboratory, we conducted an investigation of urine samples from 5 DSM-III classified schizophrenic patients and 4 normal subjects using the techniques described by the Norwegian group, and with a series of modifications. The chromatographic profiles obtained differ widely from those repotted by Trygstad et al. No significant differences were detected between patients with schizophrenia and controls. The methods used by Trygstad et al. are complex and we have defined several parts of the methods which are subject to variability. Our findings lend no support to the view that patients with psychiatric illness can be readily distinguished from normal subjects by tb~eamount or profile of peptide ;xcretion in urine.
Introduction A series of publications from a grc.ep of Norwegian workers have promulgated the hypothesis that excess of neuropeptides is central to the development of psychotic disorders. They suggest that "overproduction" of peptides may lead to "hyperfunctioning" of some neuronal systems and to an overflow of peptide into the urine. Trygstad st al. (1980) studied the benzoic acid-treated urines of schizophrenics by Sephadex G-25 gel chromatography and reported two reproducible pattet'ns which are both substantially different from controls. These findings were confirmed in subsequent studies from the same group (Reichelt et al. 1981, 1985) who reported increased levels of benzoic acid precipitable aromatic compounds together with glycoprotein- and uric ac!d,~s~iated Ix:pride material in urine of schizophrenics. When this material was furthe, fractionated, the hydrolyzed fractions were found to contain significantly greater amounts of amino acids in the schizophrenic group compared with the control group. However, no evidence has been presented to show that this putative peptide material originates in the brain and th-~re is no evidence to suggest a generalized abnormality of peptides in scldzophrenia. As these results have not been independentlyreplicated in any other laboratory [indeed one group (Isranglmn et al. 1983) found it impossible to reproduce the G-25 profiles from
Frcm the Department of Pharmacy, Royal Victoria Infummy, Newcastle Upon Tyne (LJ.G.); I~{e,~.,:c¢~'rin~logy Unit, Newcastle General Hospital, Newcastle upon Tyne (I.N.F.); Division of Psychiatry, MRC Ctinit.ai Research Centre, Harrow, Middlesex (T.LC.); and Depamnent of Pharmaceutical Chemistry, Sunderland Polytechnic0 Lungham Tower, Sunderland (FJ.R.), England. Address reprint requeststo John J. Gilmy, Departmentof Pharmacy, Royal VictoriaInfirmary,Queen Victoria Road, Newcastle upon Tyne NEI 4LP, England. Received June 8° 1987; revised April 6, 1989.
@ 1990 Society of Biological Psychiatry
0006-3223/~0/$03.50
1128
BIOLPSYCHIATRY
J.J. Gilroy et al.
1990;27:1127-1132
normal controls], we felt it appropriate to ~ttempt to replicate the findings of the Norwegian group. M a t e r i a l s and M e t h o d s Five patients recently admitted to Northwick Park Hospital research ward with episodes of acute psychosis that met DSM-III criteria for schizophrenia were included. There were 3 men (ages 27, 29, and 33 years) and 2 women (ages 29 and 38 years). Urine samples were taken in the ~irst week of admission while the patients were all suffering from psychotic symptoms. Two patients had been drug free for several months. One had received 300 mg of Chlorpromazine following admission, and the other 2 had received no neuroleptic medication following admission but their previous neuroleptic status was uncertain. Control samples were obtained from the laboratory staff: 2 men (ages 31 and 40 years) and 2 women (ages 34 and 36 years). The methods used for precipitation, washing, and G-25 and P-2 chromatography are as described by the Norwegian group (Reichelt et al. 1985). P-2 cta'omatography was performed on material extracted from region 2 (elution volume: 800-1400 ml) of the G25 chromatogram. Fractions corresponding to the maxima of peaks from the G-25 chromatogram were screened for protein using a modified Lowry method (Leggett-Bailey 1967). Results
Sephadex G-25 Chromatography Irrespective of psychiatric status, two chromatographic patterns were found: one for men and one for women. The distinction between them occurs at 2100 ml where there is a large peak in the male chromatogram (Figure 1). The chromatograr,~ may be divided into three elution volume regions: 400-600 ml, 800-1400 ml, and i400-5000 rnl (Figure 1). There is no significant difference between schizophrenic aed control areas under the curve in region 2. In region 1, there is an increase in ares in the schizophrenic group although the difference is not statistically significant. In ,egion 3, there are insufficient results to perform statistical analysis but no trend is aPl~ga'ent(Table 1). For all individuals in the study, a positive reaction for protein was obtairJed from the peaks in region 1 only.
Biogel P-2 Chromatography Irrespective of gender or psychiatric status, there are two chromatographic patterns---one for unhydrolyzed fractions and one for hydrolyz~d fractions (Figure 2). The distinction between them occurs at 154 ml (the bed volume) where, except for I male schizophrenic, there is a large peak in the unhydrolyzed chromatogram. The areas under the curve for the hydrolyzed and unhydrolyzed chromatograms show no significant differences between the two groups (Table 1).
G-25 Methodology The materialelutingin region 3 of the G-25 chromatograms is produced in largeramounts by men than by women. However, if "IMS 99" (ethanol ~> 98.5% with methanol, moisture, etc. ~ 1.5%) without methylisobutylketone(MIBK) was used as the washing
Urinary Chromatography in Schizophrenia
BIOL PsYCmATRY
1129
1990;27:1127-1132
,° I 7.0
6,0
E 5.0
Normal male
~
4.0
4.0
3.0
3.0
2.0
2.0
No~'n'~lfen,ale
< i
1,0
1.0 |
0
4O0O
SO00
1600
Elullo~ ~ I (n~l
1.8,
2000 Elution
,
3000 v~d (ml)
400Q
f
S(X)O
]
Normal
1,5, 1.2.
osi 0.6 0.3
i ° ! < 1.8
Schizophrenic
1.5 1.2 O,9 0.6 0.3
o.-i?
~
0
• Eiulion vol
|0o NO 1200 BOO
)~ 600 ~mo119o ISO0
(ml)
Figure 1. (3-25 Chromatographic profiles of benzoic acid-treated urine. Top row: chromatograms from control men and control women (chromatograms for schizophrenic men and women were respectively the same); middle and bottom rows: region beyond 1500 ml elution volume deleted and absorbance axis scale expanded; middle row: normal group; bottom row: schizophrenic group.
alcohol, there was considerable variation in the size of the peaks in region 3 from both genders. In addition, we found that when the urinarj precipitate was washed using "IMS 99" without MIBK there was a loss of peaks h region 2 of the G 2 5 chromatogram. Therefore, the presence of MIBK in the washing alcohol is essential.
P-2 Methodology Fractions in the 140-160 ml region were boiled for 2 hr with distilled water and then tested with Ninhydrin. Results suggest that because the peak in this region of the unhydrolyzed chromatogram is lost following alkaline hydrolysis, it is produced by ammonia. The difference in absorbance at 570 nm of an aliquot of sample before and after
1130
BIOLPSYCHIATRY
J.J. Gilroy et al.
1990;27:, 127-1132
Table 1. Area Under Curve (cm2) for Peaks in the G-25 and P-2 Column Chromatograms in Schizophrenics and Controls
G-25 peak 1
Schizophrenics
Controls
Male schizophrenics
Male controls
(n = 5)
(n = 4)
( n = 3)
( n = 2)
4.8 ± 2.1
2.4 - 0.8 a
3.3 ± 1.8
2.5 ± 0.7
(400-600 rid) G-25 peak 2
m
(800-1400 ml) G-25 peak 3 (1400-5000 nil)
P-2 unhydrolyzed P-2 hydrolyzed
87/130/37
~
25.1 ± 18.8 12.5 ± 9.7
140/121
19.7 ± 8.9 8.8 - 1.3
ap = 0.063. Mann-Whitney U test (2 = tailed), no significant differences. Means - SD. Peak 3 on G-25 is only fom~d in males (see text).
alkaline hydrolysis suggests that the p e ~ is sample related. The Norwegian group have used either rotary evaporation (Saelid et al. 1985) or lyophilization (Reichelt~et al. 1985) to concentrate their G-25 fractions with no change in the results. We used rotary evaporation to concentrate our large volume samples (approximately 400 ml). However, because a significant amount of ammonium bicarbonate remained (as measu~d by change in pH), we found it necessary to follow evaporation with lyophilization. Discussion For normal individuals, the Norwegian group reports one chromatogram from Se,phadex G-25 chromatography whereas we demonstrate a large male-female difference in our chromatograms. For schizophrenic patients, they report two disease-related chromatograms. Our results also show two chcomatograms in schizophrenic patients which, like those from normal individuals, are gender-related. We find no significant differences between chromatograms from the schizophrenic: group and the controls. We were only able to produce the chromatograms reported here following a later publication (Reichelt et al. 1985) and personal communication with the Norwegian group which indicate that the following additional steps are used: (1) The pH of the precipitated urine is adjusted to between 4.0 and 4.3. (2) The precipitate Js washed such that only part of the added benzoic acid is removed. (3) The washing alcohol contains 2% by volume of MIBK. Without MIBK we have shown that the selective removal of benzoic acid could not be achieved. [This would explain why Israngkun et al. (1983) were unable to replicate the results of the Norwegian group.] Our work proceeded to a second separation stage---Biogel I'-2 chromatography-because the absorbance of the G-25 co!unto eluate is due to the presence of any molecule that absorbs at this wavelength and not just to the presence of proteins and peptides containing amino acid residues such as tryptophan, tyrosine, or phenylalanine (our screen for proteinaceous material found protein to be present in region 1 but absent from regions 2 and 3). The separation was carried out on material extracted from region 2, as the Norwegian group have reported a diverse range of bioactivities from this material (Reichelt et al. 1981). Using Biogel P-2 chromatography to separate this material, the Norwegian group report one chromatogram for normal individuals and chromatograms for their
Urinary Chromatography in Schizophrenia
BIOLPSYCHIATRY
113 l
1990;27: 1127-1132 UNHYDROLYSED 5.0
4.ol
[ E 3.0
HYDROLYSED
3.0
! "~ 2.0
2.0
J
,<
1.0
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o
,
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60
..
9 0 / f 120 150 160 Elution vol. (ml)
210 240
30
60
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120 150 180 Elulion voL (ml)
210 240
Normal 1.S 12
0.9 06 0.3 O-
~-
----
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0 ]0 60 90 1Z0150110]~10240~10
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30 i0 S0 lZ01|0110210
n 10 SO 90 t,101S0Ul0Zl0|40~r|0 [:lulson ~1 |ml|
30 |0 90120 IS0110H0 0 30 H gO 1|0150110|10
Figure 2. Po2: Chromatographic profiles of the extract of pooled fractions from the 800-1400 ml elution volume region of the G-25 chromatogmms~ Top row: cltromatograms from fractions pre- and posthydrolysis; middle and bottom rows: chromatograansfrom fractions posthydrolysis; middle row: normal group; bottom row: schizoFhrer.ic gToup. schizophrenic patients that are much more varied (Reichelt et ai. 1985), whereas we found that there is only one chromatogram for all individuals in our study. Following hydrolysis, the Norwegian group report a significant increase in Ninhydrinpositive material in both their schizophrenic subgroups when compared to that obtained from their normal population, whereas we found that there is no significant difference between our two groups. The Norwegian group have not reported chromatograms prior to hydrolysis. However, as Ninhydrin will react with any compound containing free amino groups to produce a blue color, it follows that an unhydrolyzed control must be performed. The marked differences between our results from G-25 and P-2 chromatography and those of the Norwegian group cannot be explained by differences in the met.beds used
1132
BIOLPSYCHIATRY • 1990;27:1127-1132
J.J. Gilroy et al.
as we have used their more recently reported method. This method is complex and extremely lengthy (up to 6 days for G-25 chromatography and 3 days for P-2 chromatography) and thus many sources of variability may occur. No evidence was found to support the basic observation of Trygstad et al. (1980) and Reichelt et al. (1981, 1985) that the urine of schizophrenic patients contains increased levels of benzoic acid precipitable aromatic compounds and putative peptide material. The complexity of and possible sources of variation in the methods are emphasized and we suggest that further research and methodological comparisons need to be performed.
References Israngk~mP, Patel ST, Newman HAI, Taylor WA (1983): Pattern of urinary putative neuropeptides in control and autistic subjects. Clin Chem 29:1287. Leggett-Bailey J (1967): Techniques in Protein Chemistry (2rid revised and expanded edition). Amsterdam: Elsevier, pp 340--341. Reichelt KL, Edminson PD, Toft KG (1985): Urinary pepfides in schizophrenia and depression. Stress Med 1:169-181. Reichelt KL, Hole K, Hamberger A, et al (1981): Biologically active peptide-containing fractions in schizophrenia and childhood autism. Adv Biochem Psychopharmacol 28:627-643. Saelid G, Haug JO, Heiberg T, Reichelt KL (1985): Peptide-containing fractions in depression. Biol Psychiatry 20:245-256. Trygstad OE, Reichelt KL, Foss I, et al (1980): Patterns of peptides and protein-associated peptide complexes in psychiatric disorders. Br J Psychiatry 136:59-72.