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Towards a possible aetiology for depressions?

Towards a possible aetiology for depressions? Background: Since a genetic disposition for depression is probable, there ought to be biochemical changes. Increased peptide levels with relevant bioactivities have been found in urine in a previous investigation, which may be such changes. Methods: Urine from patients with severe depression according to ICD 10 have been run on reversed phase High Performance Liquid Chromatography, and off line mass spectrometry was performed on some of these peptides. Results: We find overlapping patterns of peptide peaks in severe depression, but with considerable individuality. Mass spectrometry shows that some of these peptides are probably of dietary origin, because their sequences are found only in certain dietary proteins. Opioids from casein and gliadin are typical examples. Conclusion: Our data show that the disposition must be polygenetic because some peptide peaks with the same bioactivity are of different length in different patients, but with the same diagnosis. However, some of the peaks are common Peptide increase in urine is found when break down is deficient, and the data presented agree with reports on peptidase deficiencies in depression. Antidepressant drugs decrease the peptide level after about 3 weeks. Background profiles from subjects with depression, we wanted to Considerable evidence indicates a genetic disposition for study a severely ill group to try to tease out what is typical. severe depressions [1-4], which of necessity entails chem- Some of the peptides have been purified guided by serot- ical changes. The disease takes time to develop, which onin uptake stimulation in platelets [7], opioid receptor probably points to unknown substances increasing and, binding and/or antibody binding assay [8]. Other pep- or decreasing until they reach a critical level. We have pre- tides have been purified using their mass-spectrometric viously found increased low molecular weight peptides molecular weight as a guide. (fragments of proteins) in urine from patients with depression [5,6] diagnosed according to The Diagnostic Patients and methods rd Manual of Mental Disorder, 3 edition (DSMIII). A pep- Patients were diagnosed mainly by our psychiatrist (TH) tide fraction was found that stimulated the uptake of sero- according to ICD 10. However, single cases were obtained tonin (5-HT) into platelets [7] Compounds with opioid from various psychiatrists over many years. All 36 patients activity were also found. On account of the confusing and were severely depressed, needing hospital care and were varied patterns and levels of compounds found in urinary without medication for at least five weeks. Twentyfour Page 1 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 females and 12 males were included in the untreated 02139, USA) with cellulose acetate filters of pore radius group with an age range of 26–58 years. Eight were 0.22 µm and centrifuged at 4000 × g for 30 minutes at depressed bipolar (F31.5) and we could see no systematic 20°C. Filtrate equivalent to 250 nano-moles of creatinine difference in pattern and level of peptides comparing was applied to the column. The column was a C-18 depressed with ICD-10 diagnosis F 32.3, (n = 13) and F reverse phase column(Vydac C-18 column 0.5 × 25 cm, 33.3 (n = 15) compared to F 31.5 (n = 8). Therefore all Hesperia, Ca, USA) detailed elsewhere [9]. Standards depressions were treated as one group. The treated group obtained from Calbiochem-Novabiochem, AG, Läufelin- used tricyclic anti-depressant or selective serotonin gen, CH-4448, Switzerland and Bachem (Bubendorf, reuptake inhibitors. No difference was found between the Switzerland) were analyzed after every 11 HPLC runs and two medicated groups and they were therefore treated as spiked urine runs were used when needed. one group made up of 18 patients. Twelve females and 6 males with an age range of 23–60. Three males and 8 Gel filtration females were part of the original untreated group, and Was performed on Sephadex G-25 columns to separate reanalyzed after 5 weeks of anti-depressive treatment high and low molecular weight compounds (Dimensions (Table 1). Normal controls were obtained from the hospi- 1.6 × 90 cm run in 0.5 M acetic acid at 0.4 ml/min, appli- tal staff, nurses, teachers and the Kings Guard regiment. cation volume 10 ml). After rota-vapor concentration 10 Of these controls none had seen a psychiatrist or psychol- ml of filtered low MW fractions were run on P2 gels (1.6 ogist or had suicidal ideas. Their age ranged from 16 to 65 × 90 cm) again in 0.5 M acetic acid. Off line alkaline and 118 were female and 99 males. We here report on the hydrolysis and neutralization of 5% aliquots and ninhy- urinary state in severe depression only. We have previ- drin colouring in an acetate/cyanide buffer was read at ously found a lack of peptides during mania/hypomania 570 nm [8]. This has the advantage that tryptophan is not [6]. destroyed by the hydrolysis and the amino acids have equimolar absorption at this wavelength with this Urine collection method. For pattern analysis the first morning urine and for purifi- cation purposes a complete 24 diuresis were collected Mass spectrometry Material obtained from the chromatography peaks was under supervision (The pattern and levels of compounds were not statistically different comparing morning urine analyzed on the PeSciex API 2000 LC/MS/MS system. The to a 24 h diuresis) and frozen. After thawing, the pH was peptides were dissolved in 50% by volume methanol/ measured and creatinine determined by the Clinical water/0.01 M formic acid, filtered through Millex GS 0.22 Chemical Laboratory at Rikshospitalet using standard µm filter unit from Millipore, and run in the positive ion technique. 0.5 ml urine was pipetted into Costar Spin-x mode, and if possible also in the negative ion mode. A centrifuge filter units (205 Broadway, Cambridge Ma blank was run prior to each test sample and because of the high ability for peptides to bind to surfaces, prolonged washing was required. Fragmentation patterns (MS/MS) Table 1: Severe Depression and the level of peptides. were compared to that of commercially available standard Disorder Depr. Depr. Treated Control Control peptides. The theoretical mass was calculated from tables Female Male Female Male provided by Micromass UK Ltd, Manchester, UK and com- pared to the found mass. We chose the average mass dif- Average 812 766 335 252 221 ferent from the mono-isotopic mass. This is the isotopic SD 263 257 106 101 69 mix usually found in nature today. To calculate the MW N 241318 118 99 95% CI the MW for each amino acid with one Hydrogen removed Lower 701 611 254 234 208 and an OH group removed is added with addition of one value H for the N terminal amino acid and either water (17 dal- Higher 923 921 417 271 235 tons) or 16 daltons for C terminal amide and +1 for the value positive charge N terminally of NH3 +. The reason for this t: 20.8 17.7 df: 340 140 is that one molecule of water is lost for each peptide bond p < 0.001 0.001 formed. Area is measured in µmeter under the 215 nanometer curve Platelet preparation integrated by computer. 215 nano-meters was taken to represent Platelets were obtained from healthy post-puberty males the peptide bonds mostly. To control for drugs the UV 280 nm and were prepared as described [7]. Because the Fura-2 absorption is measured simultaneously. It was difficult to get untreated patients hence the small numbers. P values were obtained calcium marker leaks out of platelets that were cooled to using students t test is for females against controls and males against 4°C during the preparation, the platelets were prepared controls. All treated patients (male and female) were on tricyclic with glucose and pyruvate to ensure viability at 20°C [7]. anti-depressants or cipramil. Page 2 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 For buffers and details see Pedersen et al 1999 [7]. Platelet NORMAL count was adjusted to 1.5 × 10 platelets per ml with 0.200 0.200 buffer. Serotonin uptake into platelets The re-suspended platelets were divided in aliquots of 450 µl stored at 4°C [7] and when used pre-incubated for 10 min at 37°C. For some unknown reason far more stable uptake data were obtained if stored at 4 degrees [10]. The aliquots were incubated with various concentrations of the peptides in 25 µl buffer and for 4 min, and after 2 min 0.00 0.00 ( C)-5-HT (Amersham Life Science, UK) in 25 µl was Peptides added to a final concentration of 1 µM and 41530 cpm and uptake run for 2 min, the time when the uptake is still 20 40 60 80 Ml eluate linear. For details see Pedersen et al 1999 [7]. This factor and other peptides were purified as described DEPRESSION [7,8]. Briefly: a complete 24 h diuresis was reduced by evaporation under reduced pressure (rota-vapor) to 10 ml 0.200 0.200 and applied to gel filtration on G-25 to separate protein from low MW compounds. Peptides were separated from amino acids and salts as described [11] and subsequently the low MW fractions were filtered on P2 gels in 0.5 M ace- 0.100 0.100 tic acid. Composition of purified peptides was found by hydrolysis in 6 M HCl (Merck) with traces of phenol to protect tyrosine and phenylalanine, in closed glass ampoules at 110°C. for 14 hours. HCL was removed over 0.00 0.00 KOH and P O in vacuum. Amino acid analysis was per- 2 5 Main peptide area formed on the Alpha plus amino acid analyzer (Pharma- cia, Uppsala, Sweden) using the ninhydrin technique as 20 40 60 80 ordained for the apparatus. The average from three analy- ses is given under each peptide. Peptide separation form The top from a con Figure 1 trace is a trol on c-18 typica reverse phase column l 215 nm normal elution pattern amino acids is as described [11]. The top trace is a typical 215 nm normal elution pattern from a control on c-18 reverse phase column. (Vydac C-18 Results protein and peptide column, Vydac, Hesperia, Ca 250 × 4.5 Figure 1 shows a typical HPLC peptidogram from the mm) at 30 degrees C [11]. First morning urine equivalent to urine of an asymptomatic control and 1b below is from a 250 nano-moles of creatinine was applied. The peaks after psychotic, depressive female age 32(F 33.3). The urinary hippuric acid were integrated by the HPLC attached compu- peptide pattern of two depressed persons (Figures 2a and ter. The bottom trace is from a severely depressed female 2b) both males with ICD 10 diagnosis F 33.3 or serious patient age 33 yr (Diagnosis: F: 33.3). recurring depressive episode with psychotic symptoms. They both had pronounced symptoms like depressed mood, feeling of guilt, loss of interest and work ability, psychic anxiety and depersonalization. Considerable dif- and female controls and treated patients had a significant ferences were found in patients with the same sex, age, dis- lower level of peptide excretion (p < 0.05) order and degree of depression. For statistical analysis of the peaks eluting after hippuric acid and prior to thymol, After gel filtration of urines the most commonly seen pat- the peaks were expressed as area under the 215 nano- tern (n = 15) of three patients with diagnosis F 33.3 is pre- meter curve (Table 1). The peptide nature of the com- sented in Figure 3 The gel-filtration constants and the pounds has been shown by hydrolysis and re-chromatog- frequency of their occurrence in depression are shown in raphy of the released amino acids on an amino acid Table 2. The table again illustrates the chemical heteroge- analyzer [8] In Table 1 the quantitative results of neity of these disorders. We could not classify depressions untreated, treated and controls is presented. The differ- according to profiles. ence is statistically very significant (Table 1), where male Page 3 of 7 (page number not for citation purposes) Absorbance 215 nm Absorbance 215 nm Hippuric acid Absorbance 280 nm Absorbance 280 nm Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 Table 2: Gel filtration characteristics of low MW peaks separated by size. 0.200 0.200 Kav Frequency 0.20 3/27 0.46 11/27 0.100 0.100 Thymol 0.50 25/27 0.61 26/27 0.66 20/27 0.74 8/27 0.83 1/27 Main peptide area The frequency of the low MW peak maxima typical for depressions 20 40 60 ml analyzed by gel Filtration expressed as Kav values (A gel-filtration constant) is shown. Kav = Elution volume – Void volume/Total volume – Void volume. Depression shown here is of psychomotorically retarded psychotic depression. All were drug free and collected over 18 years. 0.200 0.200 The great variation in peptide chain lengths with the same bio-activity from one patient to the next, makes it important to measure total hydrolysis releasable amino acids. This also increases the sensitivity of the assay because splitting peptide bonds increase the amino groups Thymol that can be coloured with ninhydrin [8]. 0.100 0.100 compounds found in different gel filtration peaks in some, but not all patients are seen in Table 3. Spiking the samples with standards and co-chromatography on HPLC as well as correct amino acid composition was a prerequi- Main peptide area site for inclusion in the table. Examples of mass spectrum of the compounds found in the Kav = 0.61 and 0.66 peaks 20 40 60 ml after gel filtration, is given below. The MW is average of three and the following peptides can all be seen in Figure 2a and 2 ho a Figure 2 nd age (35 yr) spitalized males b HPLC chromatogram from with the same diagnos two depressed is (ICD 10: F33.3) and , 5: 2a and 2b HPLC chromatogram from two depressed and hospitalized males with the same diagnosis (ICD 10: F33.3), P2 gel filtration and age (35 yr). Note the difference in pattern in spite of 1,6 similar diagnosis, sex and age. The reproducibility of the points of elution running controls after each buffer renewal: 1,4 For beta-casomorphine (bovine) 1–7: X ± SD = 62.18 ± 0.24 1,2 ml (n = 38); 95% Confidence interval = 62.10–62.26. For the beta-casomorphine like peak 1–5: 50.14 ± 0.19 (n = 38) and Patient 1 Patient 2 0,8 95% Confidence interval = 50.08–50.20. Patient 3 0,6 0,4 Serotonin uptake into platelets 0,2 In fig 4 the uptake of serotonin into platelets is shown for the final purification step of a tri-peptide with MW = 63 75 87 99 111 123 135 147 159 195 207 171 183 372.4(expected weight = 372.36) with the M+1 species ml 373.4 (Figure 5). The probable amino acid E = 1 and G = Gel filtration on 24 h complete u si Figure 3 on P 2 rine of gels 1.6 × three p90 cm in 0.5 M acetic atients suffering from depres- acid of 1.3 and W (tryptophan) was calculated form UV absorp- Gel filtration on P 2 gels 1.6 × 90 cm in 0.5 M acetic acid of tion at 280 nm comparing to synthetic Pyroglu- 24 h complete urine of three patients suffering from depres- TrpGlyNH2(pE-W-GNH2)[7]. The uptake curve is typi- sion. (ICD F33.3). The patients were on usual food supply cally bell shaped and is found for many peptides [12], and but hospitalized. Whatman 3 MM paper filters was used to the optimal dose is different from the control with p = filter urine. Elution was monitored by measuring 5% aliquots 0.001 (n = 9), two tailed. We therefore propose that this by off line alkaline hydrolysis and ninhydrin colour developed peptide may be responsible for serotonin removal into as described [9], and with an equimolar absorption coeffi- cient for peptides measured at 570 nm for each amino acid. platelets and synapses as found for the amidated tri-pep- tide [7]. Peptides seen in fig 5 are discussed below, while Page 4 of 7 (page number not for citation purposes) Absorbance 215 nm Absorbance 215 nm Absorbance 280 nm Absorbance 280 nm OD570nm Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 Table 3: Probable structure of some mass spectrometric peaks not shown in fig 5. 5. 4e6 102.0 357.2 5. 0e6 185.2 Kav of Mass Mass Structure HPLC Co- 4. 5e6 peak Found Expected chromatography with 313.2 4. 0e6 401.3 3. 5e6 0.61 579.6 579.6 Y-P-F-P- beta-casomorphineb 3. 0e6 445.4 G-NH2 1–5 amide 269.1 2. 5e6 489.3 0.66 522.3 522.3 Y-P-F-P beta-casomorphineb 461.3 505.4 1–4 2. 0e6 373.4 417.6 533.5 0.50 888.05 888.05 Y-P-F-P- beta-casomorphineb 158.2 352.2 577.4 1. 5e6 402.4 G-P-I-P 1–8* 335.2 528.4 1. 0e6 291.2 637.4 621.5 201.2 0.50 790.9 790.9 Y-P-F-P- beta-casomorphineb 467.3 681.5 186.1 253.2 423.3 385.2 5. 0e5 G-P-I 1–7 605.6 769.5 0. 0 100 200 300 400 500 600 700 800 b stands for bovine. * Imuno assay for casomorphine 1–8 with m/z, amu antibodies provided by Professor Dr Teschemacher, Giessen, Germany in 4 patients with this HPLC peak came to 120 fentomoles Mass spectrometry of the peak 0.6 with Kav = 0.66 Figure 5 1 characteristically partially ov from ge erlappl f ing with the peak iltration with Kav = ± 30 (n = 4) while control values from the same peak area in 4 normal controls came to 15 ± 6 (n = 4). Mass spectrometry of the peak from gel filtration with Kav = 0.61 characteristically partially overlapping with the peak with Kav = 0.66. The serotonin uptake stimulator is seen 1 From the composite peak with Kav = 0.61 a compound with mass +1 = 373.4 and a peptide with unknown function with MW+1 = 505.4 (fig 5) yielded on amino acids Y = and mass +1 of 505.4(Y-P-E-P) which fits deamidated gliadi- 0.8; P = 1.9; traces of G and E = 1. Found mass+1 = 505.4 nomoprhine tetrapeptide. Also casomorphine 3–6 with mass (Expected mass +1 = 505.54). This fits gliadin-morphine +1 = 417.6 is seen (F-P-G-P). A peptide with MW+1 = 489.3 1–4 deamidated or Y-P-E-P. (Expected = 489.5) that may be derived from kappa-casein has a probable structure of L-P-Y-P (casein κ 56–59). Several 2 Also in the Kav = 0.66 and 0.61 peak beta-casomorphine other peptides will be published separately when elucidated. The figure illustrates the heterogeneity of the gel filtration 3–6 with amino acid composition F = 0.9; P = 2; G = 1.4. peaks and the amount of work needed to characterize the (Glycine always appear higher than expected even in many eluted compounds. standard synthetic compounds). Found MW +1 = 417.5(Expected = 417.4), which thus agrees with F-P-G-P. (Figure 5, the MW came out as 417.6) or casomorphine 3– 6. 3 MW+1 = 357.2 from the Kav = 0.66 peak likewise fits Lactoferrin 310–313 or SP-P-G or theoretical MW+1 = 357.37 and hydrolysis gave P(2), S(0,9) and G (1.4). 4 With a MW+1 of MW+1 = 489.3 (Expected = 489.5) probably from kappa-casein has a reasonable structure of L-P-Y-P (casein kappa 56–59). Amino acid composition was quite varying but V = 1, Y = 0.6, P = 2. (Glycine was sometimes found but varied from 0 to 1.) We also obtained a peak that eluted with substance P (co- chromatography after spiking). However, it clogged (for unknown reason) the capillary electrode in the mass spec- trometer, so we were unable to obtain its mass and frag- mentation pattern. control - 12 - 10 - 8 - 6 - 14 Discussion We have found substantial increase in peptide excretion in CONCENTRATION OF TRIPEPTIDE severe depression. To our surprise the level was found to be decreased in treated patients. Increase in peptides espe- Ser Figure 4 otonin uptake stimulation of the pure peptide cially in urine is usually due to peptidase deficiency or Serotonin uptake stimulation of the pure peptide. Concen- inhibition [13,14]. The presented data indicate that tration of peptide along the abcissa. Platelets were prepared -14 severely depressed patients show hyper-peptiduria as as outlined in methods. -14 is 10 to the power of -14 M(10 M). The typical hormetic dose response curve is shown. would be expected if peptidase defects were present. Pepti- dase deficiencies have been found in depression and mel- Page 5 of 7 (page number not for citation purposes) 5-HT UPTAKE AS C. P. M. Intensity, cps Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 ancholic states [15-19]. Because the level and pattern of human caso-morphines [42] and shows depressive traits. peptides differ, and differing chain lengths of peptides Since peptides in general are excellent peptidase inhibi- with the same activities are found, this points to a hetero- tors [43] and peptides also have strong tendencies to form geneity of genetics. Different families may have a different complexes [44,45] and bind to other molecules and mem- sets of dysfunctional enzymes probably peptidases or branes [46,47], a rather complex situation with varied peptidase binding proteins. This may explain the diffi- results can be envisioned. Bell shaped dose responses are culty of pinpointing a specific gene as the genetic disposi- common to many peptides and add to this complicated tion in depression. We suggest that we have depression picture [12]. The nature of some of these peptides is causing genes which may cause formation of different but shared with schizophrenia and may constitute the com- overlapping peptidases or peptidase regulating proteins, mon features of these disorders [48]. Possibly relevant to and that the mediators of depression may be peptides reg- a gut-brain axis in depression is epidemiological data ulating the uptake and release of different transmitters. showing very high frequency of depression in irritable Since antidepressant medication reduced the level of pep- bowel syndrome [49-51]. Future work is needed to eluci- tides (table 1) this could be due to peptidase induction as date if there is a correlation of peptide increase and degree has been shown for neuroleptic medication. It remains to of depression and to look for any correlations between be seen if peptide level correlates with the degree of individual peptide peaks and specific symptoms of this depression. The presence of opioids may explain the psy- disorder. chotic features of our patients because opioids have been shown to cause increased dopamine in the synaptic cleft Conclusion by inhibiting reuptake [20] An exogenous supply of pep- In severe cases of hospitalized patients with depression, tides may also explain the often seen and peculiar fluctu- we find peptide increase in urine. After treatment the level ating course of the disorder with morning worsening and is decreased. Some of these peptides affect serotonin afternoon a relative high or agitation. Increased reuptake uptake and others show exorphine like characteristics. of serotonin into platelets may have relevance to the Our study is based on a small number of patients due to reported changes in serotonin transport into platelets in problems of getting untreated ones. We do not know how depression [21,22]. Both the tri-cyclic antidepressants and general or specific these findings are to the depression selective serotonin re-uptake inhibitors have the opposite spectrum. However, our data are compatible with effect on serotonin uptake. We have previously found the reported decreased peptidase levels found by others in tri-peptide but amidated, in autistic patients' urine and depression. If correct the profound heterogeneity casts this tri-peptide stimulated the serotonin transporter medi- doubt on most double blind treatment trials in depres- ated uptake of serotonin in hamster ovarial cells trans- sion. fected with the human serotonin transporter gene [23]. That tri-peptide doubled the serotonin content of platelets Authors' contributions when injected into pups subcutaneously [24]. 5 HT 2a LY carried out the serotonin uptake studies, TH the diag- receptors are increased in brain tissue and platelets in nosis of patients and urine collection, KLR did the urine depression [25] which would agree with decreased levels analysis, mass spectrometry and administration of the in the synaptic cleft due to stimulated uptake. Also the project. decreased 5HIAA (5-hydroxy indole-acetic acid) in sui- cidal patients fit an increased uptake [26]. The tri-peptide Acknowledgements We wish to thank Major Ecbo's Foundation and Haldis and Josef Andresens sequence has only been found in reelin, which is a matrix foundation for support. proteinase [27]. That food derived peptides are taken up has been demonstrated [28,29] and is increased by pepti- References dase defects [30]. Depression has been found in cases of 1. Allan MG: Twin studies of affective illness. Arch Gen Psychiatry coeliac disease [31,32] indicating that such a mechanism 1976, 33:1476-1489. is not unreasonable. Considerable differences in rates in 2. Bertelsen A, Harvald B, Hauge M: A Danish twin study of manic- depressive disorders. Br J Psychiatry 1977, 130:330-351. different cultures may also thus be explainable. Other 3. Cavoret RJ, O'Gorman TW, Heywood EC, Troughton E: Genetic groups have found increases in some peptides in depres- and environmental factors in major depression. J Affect Disord 1985, 9:155-164. sion. Thus TRH (pE-H-P-NH2) has been found increased 4. Wender PH, Kety SS, Rosenthal D, Schulsinger F, Ortmann J, Lunde in CSF [33,34]; beta-endorphin [35] and an unspecified I: Psychiatric Disorders in the Biological and adoptive fami- opioid fraction 1 measured by receptor binding [36,37]. lies of adopted individuals with affective disorders. Arch Gen Psychiatry 1986, 43:923-929. Substance P was also found increased in CSF [38], and 5. Sælid G, Haug JO, Heiberg T, Reichelt KL: Peptide containing frac- delta sleep factor increased in plasma [39,40]. Also tions in depression. Biol Psychiatry 1985, 20:245-256. 6. Reichelt KL, Edminson PD, Toft KG: Urinary peptides in schizo- plasma arginine vasopressin increase in depression was phrenia and depression. Stress Med 1984, 1:169-181. inversely related to daytime motor activity [41]. Further- more post partum psychosis may be mediated by different Page 6 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 7. Pedersen OS, Liu Y, Reichelt KL: Serotonin uptake stimulating 30. Mahe S, Tome D, Dumontier AM, Desjeux JF: Absorption of intact peptide found in plasma of normal individuals and in some morphiceptin by diisopropylfluorophosphate-treated rabbit autistic urines. J Pept Res 1999, 53:641-646. ileum. Peptides 1989, 10:45-52. 8. Reichelt WH, Reichelt KL: The possible role of peptides derived 31. Hallert C, Åstrøm J, Sedvall G: Psychic disturbances in adult from food proteins in diseases of the nervous system. In Epi- celiac disease III. Reduced central momoamine metabolism lepsy and other Neurological Disorders in Coeliac Disease Edited by: and signs of depression. Scand J Gastroenterol 1982, 17:25-28. Gobbi G. London: John Libbey & Comp Ltd; 1997:225-235. 32. Corvaglia L, Catamo R, Pepe G, Lazzari R, Corvaglia E: Depression 9. Reichelt WH, Ek J, Stensrud MB, Reichelt KL: Peptide excretion in in adult untreated celiac subjects: diagnosed by the pediatri- celiac disease. J Pediatr Gastroenterol Nutr 1998, 26:305-309. cian. Am J Gastroenterol 1999, 94:839-843. 10. Lingjaerde O: Uptake of serotonin in blood platelets: depend- 33. Kirkegaard C, Faber J, Hummer L, Rogowski P: Increased levels of ence on sodium and chloride, and inhibition by choline. FEBS TRH in cerebrospinal fluid from patients with endogenous Lett 1969, 3:103-106. depression. Psychoneuroendocrinology 1979, 4:227-235. 11. Böhlen P, Castillo F, Ling R, Guillemin R: An efficient procedure 34. Banki CM, Bissette G, Arato M, Nemeroff CB: Elevation of Immu- for the separation of peptides from amino acids and salts. Int noreactive CSF TRH in depressed patients. Am J Psychiatry J Pept Protein Res 1980, 16:306-310. 1988, 145:1526-1531. 12. Calabrese EJ, Baldwin LA: Hormesis: U-shaped dose responses 35. Darko DF, Risch SC, Gillin JC, Golshan S: Association of beta- and their centrality in toxicology. Trends Pharmacol Sci 2001, endorphin with specific clinical symptoms of depression. Am 22:285-291. J Psychiatry 1992, 149:1162-1167. 13. Abassi Z, Golomb E, Keiser HR: Neutral endopeptidase inhibi- 36. Agren H, Terenius L: Depression and CSF endorphin fraction 1: tion increases urinary excretion and plasma level of seasonal variation and higher levels in unipolar than bipolar Endothelin. Metabolism 1992, 41:683-685. patients. Psychiatry Res 1983, 10:303-311. 14. Watanabe Y, Kojima-Kumatsu T, Iwaki-Egewa S, Fujimoto Y: 37. Terenius L, Wahlstrom A, Agren T: Naloxone (narcan)treatment Increased excretion of proline-containing peptides in dipep- in depression: Clinical observations and effects on CSF tidyl-peptidase IV deficient rats. Res Commun Chem Pathol Phar- endorphins and monoamine metabolites. Psychopharmacology macol 1993, 81:323-350. (Berl) 1997, 54:31-33. 15. Maes M, Demeester I, Vanhoof G, Scharpe S, Bosmans E, Vandervorst 38. Rimon R, Le Greves P, Nyberg F, Heikkila L, Salmela L, Terenius L: C, Verkerk R, Minner B, Suy E, Raus J: Decreased serum dipepti- Elevation of substance P-like peptides in the CSF of psychiat- dyl-peptidase IV activity in major depression. Biol Psychiatry ric patients. Biol Psychiatry 1984, 19:509-516. 1991, 30:577-586. 39. Westrin A, Ekman R, Träskman-Bendz L: High delta sleep-induc- 16. Maes M, Scharpe S, Meltzer HY, Suy E, Cosyns P, Calabrese J: Lower ing peptide-like immuno-reactivity in plasma in suicidal angiotensin 1 converting enzyme activity in melancholic sub- patients with major depressive disorder. Biol Psychiatry 1998, jects: a pilot study. Biol Psychiatry 1992, 32:621-624. 43:734-739. 17. Maes M, Goosens F, Scharpe S, Meltzer HY, D'Hondt P, Cosyns P: 40. Westrin Å, Engström G, Ekman R, Träskman-Bendz L: Correlations Lower serum prolyl-endopeptidase enzyme activity in major between plasma-neuropeptides and temperament dimen- depression: further evidence that peptidases play a role in sions differ between suicidal patients and healthy controls. J patho-physiology of depression. Biol Psychiatry 1994, 35:545-552. Affect Disord 1998, 49:45-54. 18. Maes M, DeMeester I, Verkerk R, DeMedts P, Wauters A, Vanhoof 41. Van Londen L, Kerkhof GA, Van den Berg F, Goekoop JG, Zwinder- G, Vandoolaeghe E, Neels H, Scharpe S: Lower serum dipeptidyl man KH, Frankhuijzen-Siervogel AC, et al.: Plasma arginine vaso- peptidase IV activity in treatment resistant major depres- pressin and motor activity in major depression. Biol Psychiatry sion: relationships with immune-inflammatory markers. Psy- 1998, 43:196-204. choneuroendocrinology 1997, 22:65-78. 42. Lindstrøm LH, Nyberg F, Terenius , Bauer K, Besev G, Gunne LM, 19. Elgun S, Keskinege A, Kumbassar H: Dipeptidyl peptidase IV and Lyrenaas S, Wildeck-Lund G, Lundberg B: CSF and plasma beta- adenosine deaminase activity. Decrease in depression. Psy- casomorphin like opioid peptides in post-partum psychosis. choneuroendocrinology 1999, 24:823-832. Am J Psychiatry 1984, 141:1059-1066. 20. Hole K, Bergslien AA, Jørgensen H, Berge O-G, Reichelt KL, Trygstad 43. LaBella FL, Geiger JD, Glavin G: Administration of peptides OE: A peptide containing fraction from schizophrenia which inhibits the degradation of endogenous peptides. The stimulates opiate receptors and inhibits dopamine uptake. dilemma of distinguishing direct from indirect effects. Pep- Neuroscience 1979, 4:1139-1147. tides 1985, 6:645-660. 21. Paul SM, Rehavi M, Skolnic P, Ballenger JC, Goodwin FK: Depressed 44. Burhol K, Jensen TG, Florholmen TG, Jorde H, Vonen B, Olsen R: patients have decreased binding of tritiated imipramine to Protein-binding and aggregation of somatostatin in human platelet "tansporter". Arch Gen Psychiatry 1981, 38:1315-1317. plasma. Ital J Gastroenterol 1966, 18:1-6. 22. Oxenkrug GF: The content and uptake of 5-HT by blood plate- 45. Kastin AJ, Casillanos PF, Fischman PJ, Profitt JK, Graf MV: Evidence lets in depressive patients. J Neural Transm 1979, 45:285-289. for peptide aggregates. Pharmacol Biochem Behav 1984, 23. Keller F: 2nd annual report to EU Commission project BMH4-CT 96-0730 21:969-974. 1998:1-10. 46. Rocetti G, Venturella F, Roda IG: Enkephalin binding system in 24. Persico AM, Baldi A, Reichelt KL, Gonzales A, Keller F: Serotonin human plasma III Comparative protection of different pep- uptake-stimulating peptides extracted for urines of autistic tides. Neurochem Res 1988, 13:221-224. patients: potential significance for the pathogenesis of autis- 47. Meneszo NY, Khatchaturian C: Peptides bound to albumin. Life tic disorders. Amer Neurosci. Meet Los Angeles 1998. Abs no 2 Sci 1986, 39:. 25. Pandey GN: Altered serotonin function in suicide. Ann N Y Acad 48. Boteva K, Lieberman J: Reconsidering the classification of schiz- Sci 1997, 836:182-200. ophrenia and manic-depressive illness-a critical analysis and 26. Åsberg ML, Träskman L, Thoren P: 5-HIAA in the cerebrospinal new conceptual model. World J Biol Psychiatry 2003, 4:81-92. fluid: A biochemical suicide predictor. Arch Gen Psychiatry 1976, 49. Addolorato G, Capristo E, Stefanini GG, Gasbarrini G: Inflamma- 38:1193-1197. tory bowel disease: a study of the association between anxi- 27. Quattrocchi CC, Wannenes F, Persico AM, Ciafre SA, d'Arcangelo G, ety and depression, physical morbidity, and nutritional Farace MG, Keller F: Reelin is a Serine Protease of the Extra- status. Scand J Gastroenterol 1997, 32:1013-1021. cellular Matrix. J Biol Chem 2002, 277:303-309. 50. Haug TT, Mykletun A, Dahl AA: Are anxiety and depression 28. Chabance B, Marteau P, Rambaud JC, Migliore-Samour D, Boynard M, related to gastrointestinal symptoms in the general popula- Perrotin P, Guillet R, Jolles P, Fiat AM: Casein peptide release and tion? Scand J Gastroenterol 2005, 37:294-298. passage to the blood in humans during digestion of milk and 51. Alander T, Svärdsudd K, Johansson SE, Agreus L: Psychological ill- yogurt. Biochimie 1998, 80:155-165. ness is commonly associated with functional gastrointestinal 29. Gardner MLG: Absorption of intact proteins and peptides. In disorders and is important to consider during patient consul- Physiology of the Gastrointestinal Tract 3rd edition. Edited by: Johnson tation: a population-based study. BMC Med 2005, 3:8-20. LR. New York: Raven Press; 1994:1795-1782. Page 7 of 7 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral and Brain Functions Springer Journals

Towards a possible aetiology for depressions?

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Springer Journals
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Copyright © 2007 by Liu et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
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1744-9081
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10.1186/1744-9081-3-47
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17868435
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Abstract

Background: Since a genetic disposition for depression is probable, there ought to be biochemical changes. Increased peptide levels with relevant bioactivities have been found in urine in a previous investigation, which may be such changes. Methods: Urine from patients with severe depression according to ICD 10 have been run on reversed phase High Performance Liquid Chromatography, and off line mass spectrometry was performed on some of these peptides. Results: We find overlapping patterns of peptide peaks in severe depression, but with considerable individuality. Mass spectrometry shows that some of these peptides are probably of dietary origin, because their sequences are found only in certain dietary proteins. Opioids from casein and gliadin are typical examples. Conclusion: Our data show that the disposition must be polygenetic because some peptide peaks with the same bioactivity are of different length in different patients, but with the same diagnosis. However, some of the peaks are common Peptide increase in urine is found when break down is deficient, and the data presented agree with reports on peptidase deficiencies in depression. Antidepressant drugs decrease the peptide level after about 3 weeks. Background profiles from subjects with depression, we wanted to Considerable evidence indicates a genetic disposition for study a severely ill group to try to tease out what is typical. severe depressions [1-4], which of necessity entails chem- Some of the peptides have been purified guided by serot- ical changes. The disease takes time to develop, which onin uptake stimulation in platelets [7], opioid receptor probably points to unknown substances increasing and, binding and/or antibody binding assay [8]. Other pep- or decreasing until they reach a critical level. We have pre- tides have been purified using their mass-spectrometric viously found increased low molecular weight peptides molecular weight as a guide. (fragments of proteins) in urine from patients with depression [5,6] diagnosed according to The Diagnostic Patients and methods rd Manual of Mental Disorder, 3 edition (DSMIII). A pep- Patients were diagnosed mainly by our psychiatrist (TH) tide fraction was found that stimulated the uptake of sero- according to ICD 10. However, single cases were obtained tonin (5-HT) into platelets [7] Compounds with opioid from various psychiatrists over many years. All 36 patients activity were also found. On account of the confusing and were severely depressed, needing hospital care and were varied patterns and levels of compounds found in urinary without medication for at least five weeks. Twentyfour Page 1 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 females and 12 males were included in the untreated 02139, USA) with cellulose acetate filters of pore radius group with an age range of 26–58 years. Eight were 0.22 µm and centrifuged at 4000 × g for 30 minutes at depressed bipolar (F31.5) and we could see no systematic 20°C. Filtrate equivalent to 250 nano-moles of creatinine difference in pattern and level of peptides comparing was applied to the column. The column was a C-18 depressed with ICD-10 diagnosis F 32.3, (n = 13) and F reverse phase column(Vydac C-18 column 0.5 × 25 cm, 33.3 (n = 15) compared to F 31.5 (n = 8). Therefore all Hesperia, Ca, USA) detailed elsewhere [9]. Standards depressions were treated as one group. The treated group obtained from Calbiochem-Novabiochem, AG, Läufelin- used tricyclic anti-depressant or selective serotonin gen, CH-4448, Switzerland and Bachem (Bubendorf, reuptake inhibitors. No difference was found between the Switzerland) were analyzed after every 11 HPLC runs and two medicated groups and they were therefore treated as spiked urine runs were used when needed. one group made up of 18 patients. Twelve females and 6 males with an age range of 23–60. Three males and 8 Gel filtration females were part of the original untreated group, and Was performed on Sephadex G-25 columns to separate reanalyzed after 5 weeks of anti-depressive treatment high and low molecular weight compounds (Dimensions (Table 1). Normal controls were obtained from the hospi- 1.6 × 90 cm run in 0.5 M acetic acid at 0.4 ml/min, appli- tal staff, nurses, teachers and the Kings Guard regiment. cation volume 10 ml). After rota-vapor concentration 10 Of these controls none had seen a psychiatrist or psychol- ml of filtered low MW fractions were run on P2 gels (1.6 ogist or had suicidal ideas. Their age ranged from 16 to 65 × 90 cm) again in 0.5 M acetic acid. Off line alkaline and 118 were female and 99 males. We here report on the hydrolysis and neutralization of 5% aliquots and ninhy- urinary state in severe depression only. We have previ- drin colouring in an acetate/cyanide buffer was read at ously found a lack of peptides during mania/hypomania 570 nm [8]. This has the advantage that tryptophan is not [6]. destroyed by the hydrolysis and the amino acids have equimolar absorption at this wavelength with this Urine collection method. For pattern analysis the first morning urine and for purifi- cation purposes a complete 24 diuresis were collected Mass spectrometry Material obtained from the chromatography peaks was under supervision (The pattern and levels of compounds were not statistically different comparing morning urine analyzed on the PeSciex API 2000 LC/MS/MS system. The to a 24 h diuresis) and frozen. After thawing, the pH was peptides were dissolved in 50% by volume methanol/ measured and creatinine determined by the Clinical water/0.01 M formic acid, filtered through Millex GS 0.22 Chemical Laboratory at Rikshospitalet using standard µm filter unit from Millipore, and run in the positive ion technique. 0.5 ml urine was pipetted into Costar Spin-x mode, and if possible also in the negative ion mode. A centrifuge filter units (205 Broadway, Cambridge Ma blank was run prior to each test sample and because of the high ability for peptides to bind to surfaces, prolonged washing was required. Fragmentation patterns (MS/MS) Table 1: Severe Depression and the level of peptides. were compared to that of commercially available standard Disorder Depr. Depr. Treated Control Control peptides. The theoretical mass was calculated from tables Female Male Female Male provided by Micromass UK Ltd, Manchester, UK and com- pared to the found mass. We chose the average mass dif- Average 812 766 335 252 221 ferent from the mono-isotopic mass. This is the isotopic SD 263 257 106 101 69 mix usually found in nature today. To calculate the MW N 241318 118 99 95% CI the MW for each amino acid with one Hydrogen removed Lower 701 611 254 234 208 and an OH group removed is added with addition of one value H for the N terminal amino acid and either water (17 dal- Higher 923 921 417 271 235 tons) or 16 daltons for C terminal amide and +1 for the value positive charge N terminally of NH3 +. The reason for this t: 20.8 17.7 df: 340 140 is that one molecule of water is lost for each peptide bond p < 0.001 0.001 formed. Area is measured in µmeter under the 215 nanometer curve Platelet preparation integrated by computer. 215 nano-meters was taken to represent Platelets were obtained from healthy post-puberty males the peptide bonds mostly. To control for drugs the UV 280 nm and were prepared as described [7]. Because the Fura-2 absorption is measured simultaneously. It was difficult to get untreated patients hence the small numbers. P values were obtained calcium marker leaks out of platelets that were cooled to using students t test is for females against controls and males against 4°C during the preparation, the platelets were prepared controls. All treated patients (male and female) were on tricyclic with glucose and pyruvate to ensure viability at 20°C [7]. anti-depressants or cipramil. Page 2 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 For buffers and details see Pedersen et al 1999 [7]. Platelet NORMAL count was adjusted to 1.5 × 10 platelets per ml with 0.200 0.200 buffer. Serotonin uptake into platelets The re-suspended platelets were divided in aliquots of 450 µl stored at 4°C [7] and when used pre-incubated for 10 min at 37°C. For some unknown reason far more stable uptake data were obtained if stored at 4 degrees [10]. The aliquots were incubated with various concentrations of the peptides in 25 µl buffer and for 4 min, and after 2 min 0.00 0.00 ( C)-5-HT (Amersham Life Science, UK) in 25 µl was Peptides added to a final concentration of 1 µM and 41530 cpm and uptake run for 2 min, the time when the uptake is still 20 40 60 80 Ml eluate linear. For details see Pedersen et al 1999 [7]. This factor and other peptides were purified as described DEPRESSION [7,8]. Briefly: a complete 24 h diuresis was reduced by evaporation under reduced pressure (rota-vapor) to 10 ml 0.200 0.200 and applied to gel filtration on G-25 to separate protein from low MW compounds. Peptides were separated from amino acids and salts as described [11] and subsequently the low MW fractions were filtered on P2 gels in 0.5 M ace- 0.100 0.100 tic acid. Composition of purified peptides was found by hydrolysis in 6 M HCl (Merck) with traces of phenol to protect tyrosine and phenylalanine, in closed glass ampoules at 110°C. for 14 hours. HCL was removed over 0.00 0.00 KOH and P O in vacuum. Amino acid analysis was per- 2 5 Main peptide area formed on the Alpha plus amino acid analyzer (Pharma- cia, Uppsala, Sweden) using the ninhydrin technique as 20 40 60 80 ordained for the apparatus. The average from three analy- ses is given under each peptide. Peptide separation form The top from a con Figure 1 trace is a trol on c-18 typica reverse phase column l 215 nm normal elution pattern amino acids is as described [11]. The top trace is a typical 215 nm normal elution pattern from a control on c-18 reverse phase column. (Vydac C-18 Results protein and peptide column, Vydac, Hesperia, Ca 250 × 4.5 Figure 1 shows a typical HPLC peptidogram from the mm) at 30 degrees C [11]. First morning urine equivalent to urine of an asymptomatic control and 1b below is from a 250 nano-moles of creatinine was applied. The peaks after psychotic, depressive female age 32(F 33.3). The urinary hippuric acid were integrated by the HPLC attached compu- peptide pattern of two depressed persons (Figures 2a and ter. The bottom trace is from a severely depressed female 2b) both males with ICD 10 diagnosis F 33.3 or serious patient age 33 yr (Diagnosis: F: 33.3). recurring depressive episode with psychotic symptoms. They both had pronounced symptoms like depressed mood, feeling of guilt, loss of interest and work ability, psychic anxiety and depersonalization. Considerable dif- and female controls and treated patients had a significant ferences were found in patients with the same sex, age, dis- lower level of peptide excretion (p < 0.05) order and degree of depression. For statistical analysis of the peaks eluting after hippuric acid and prior to thymol, After gel filtration of urines the most commonly seen pat- the peaks were expressed as area under the 215 nano- tern (n = 15) of three patients with diagnosis F 33.3 is pre- meter curve (Table 1). The peptide nature of the com- sented in Figure 3 The gel-filtration constants and the pounds has been shown by hydrolysis and re-chromatog- frequency of their occurrence in depression are shown in raphy of the released amino acids on an amino acid Table 2. The table again illustrates the chemical heteroge- analyzer [8] In Table 1 the quantitative results of neity of these disorders. We could not classify depressions untreated, treated and controls is presented. The differ- according to profiles. ence is statistically very significant (Table 1), where male Page 3 of 7 (page number not for citation purposes) Absorbance 215 nm Absorbance 215 nm Hippuric acid Absorbance 280 nm Absorbance 280 nm Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 Table 2: Gel filtration characteristics of low MW peaks separated by size. 0.200 0.200 Kav Frequency 0.20 3/27 0.46 11/27 0.100 0.100 Thymol 0.50 25/27 0.61 26/27 0.66 20/27 0.74 8/27 0.83 1/27 Main peptide area The frequency of the low MW peak maxima typical for depressions 20 40 60 ml analyzed by gel Filtration expressed as Kav values (A gel-filtration constant) is shown. Kav = Elution volume – Void volume/Total volume – Void volume. Depression shown here is of psychomotorically retarded psychotic depression. All were drug free and collected over 18 years. 0.200 0.200 The great variation in peptide chain lengths with the same bio-activity from one patient to the next, makes it important to measure total hydrolysis releasable amino acids. This also increases the sensitivity of the assay because splitting peptide bonds increase the amino groups Thymol that can be coloured with ninhydrin [8]. 0.100 0.100 compounds found in different gel filtration peaks in some, but not all patients are seen in Table 3. Spiking the samples with standards and co-chromatography on HPLC as well as correct amino acid composition was a prerequi- Main peptide area site for inclusion in the table. Examples of mass spectrum of the compounds found in the Kav = 0.61 and 0.66 peaks 20 40 60 ml after gel filtration, is given below. The MW is average of three and the following peptides can all be seen in Figure 2a and 2 ho a Figure 2 nd age (35 yr) spitalized males b HPLC chromatogram from with the same diagnos two depressed is (ICD 10: F33.3) and , 5: 2a and 2b HPLC chromatogram from two depressed and hospitalized males with the same diagnosis (ICD 10: F33.3), P2 gel filtration and age (35 yr). Note the difference in pattern in spite of 1,6 similar diagnosis, sex and age. The reproducibility of the points of elution running controls after each buffer renewal: 1,4 For beta-casomorphine (bovine) 1–7: X ± SD = 62.18 ± 0.24 1,2 ml (n = 38); 95% Confidence interval = 62.10–62.26. For the beta-casomorphine like peak 1–5: 50.14 ± 0.19 (n = 38) and Patient 1 Patient 2 0,8 95% Confidence interval = 50.08–50.20. Patient 3 0,6 0,4 Serotonin uptake into platelets 0,2 In fig 4 the uptake of serotonin into platelets is shown for the final purification step of a tri-peptide with MW = 63 75 87 99 111 123 135 147 159 195 207 171 183 372.4(expected weight = 372.36) with the M+1 species ml 373.4 (Figure 5). The probable amino acid E = 1 and G = Gel filtration on 24 h complete u si Figure 3 on P 2 rine of gels 1.6 × three p90 cm in 0.5 M acetic atients suffering from depres- acid of 1.3 and W (tryptophan) was calculated form UV absorp- Gel filtration on P 2 gels 1.6 × 90 cm in 0.5 M acetic acid of tion at 280 nm comparing to synthetic Pyroglu- 24 h complete urine of three patients suffering from depres- TrpGlyNH2(pE-W-GNH2)[7]. The uptake curve is typi- sion. (ICD F33.3). The patients were on usual food supply cally bell shaped and is found for many peptides [12], and but hospitalized. Whatman 3 MM paper filters was used to the optimal dose is different from the control with p = filter urine. Elution was monitored by measuring 5% aliquots 0.001 (n = 9), two tailed. We therefore propose that this by off line alkaline hydrolysis and ninhydrin colour developed peptide may be responsible for serotonin removal into as described [9], and with an equimolar absorption coeffi- cient for peptides measured at 570 nm for each amino acid. platelets and synapses as found for the amidated tri-pep- tide [7]. Peptides seen in fig 5 are discussed below, while Page 4 of 7 (page number not for citation purposes) Absorbance 215 nm Absorbance 215 nm Absorbance 280 nm Absorbance 280 nm OD570nm Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 Table 3: Probable structure of some mass spectrometric peaks not shown in fig 5. 5. 4e6 102.0 357.2 5. 0e6 185.2 Kav of Mass Mass Structure HPLC Co- 4. 5e6 peak Found Expected chromatography with 313.2 4. 0e6 401.3 3. 5e6 0.61 579.6 579.6 Y-P-F-P- beta-casomorphineb 3. 0e6 445.4 G-NH2 1–5 amide 269.1 2. 5e6 489.3 0.66 522.3 522.3 Y-P-F-P beta-casomorphineb 461.3 505.4 1–4 2. 0e6 373.4 417.6 533.5 0.50 888.05 888.05 Y-P-F-P- beta-casomorphineb 158.2 352.2 577.4 1. 5e6 402.4 G-P-I-P 1–8* 335.2 528.4 1. 0e6 291.2 637.4 621.5 201.2 0.50 790.9 790.9 Y-P-F-P- beta-casomorphineb 467.3 681.5 186.1 253.2 423.3 385.2 5. 0e5 G-P-I 1–7 605.6 769.5 0. 0 100 200 300 400 500 600 700 800 b stands for bovine. * Imuno assay for casomorphine 1–8 with m/z, amu antibodies provided by Professor Dr Teschemacher, Giessen, Germany in 4 patients with this HPLC peak came to 120 fentomoles Mass spectrometry of the peak 0.6 with Kav = 0.66 Figure 5 1 characteristically partially ov from ge erlappl f ing with the peak iltration with Kav = ± 30 (n = 4) while control values from the same peak area in 4 normal controls came to 15 ± 6 (n = 4). Mass spectrometry of the peak from gel filtration with Kav = 0.61 characteristically partially overlapping with the peak with Kav = 0.66. The serotonin uptake stimulator is seen 1 From the composite peak with Kav = 0.61 a compound with mass +1 = 373.4 and a peptide with unknown function with MW+1 = 505.4 (fig 5) yielded on amino acids Y = and mass +1 of 505.4(Y-P-E-P) which fits deamidated gliadi- 0.8; P = 1.9; traces of G and E = 1. Found mass+1 = 505.4 nomoprhine tetrapeptide. Also casomorphine 3–6 with mass (Expected mass +1 = 505.54). This fits gliadin-morphine +1 = 417.6 is seen (F-P-G-P). A peptide with MW+1 = 489.3 1–4 deamidated or Y-P-E-P. (Expected = 489.5) that may be derived from kappa-casein has a probable structure of L-P-Y-P (casein κ 56–59). Several 2 Also in the Kav = 0.66 and 0.61 peak beta-casomorphine other peptides will be published separately when elucidated. The figure illustrates the heterogeneity of the gel filtration 3–6 with amino acid composition F = 0.9; P = 2; G = 1.4. peaks and the amount of work needed to characterize the (Glycine always appear higher than expected even in many eluted compounds. standard synthetic compounds). Found MW +1 = 417.5(Expected = 417.4), which thus agrees with F-P-G-P. (Figure 5, the MW came out as 417.6) or casomorphine 3– 6. 3 MW+1 = 357.2 from the Kav = 0.66 peak likewise fits Lactoferrin 310–313 or SP-P-G or theoretical MW+1 = 357.37 and hydrolysis gave P(2), S(0,9) and G (1.4). 4 With a MW+1 of MW+1 = 489.3 (Expected = 489.5) probably from kappa-casein has a reasonable structure of L-P-Y-P (casein kappa 56–59). Amino acid composition was quite varying but V = 1, Y = 0.6, P = 2. (Glycine was sometimes found but varied from 0 to 1.) We also obtained a peak that eluted with substance P (co- chromatography after spiking). However, it clogged (for unknown reason) the capillary electrode in the mass spec- trometer, so we were unable to obtain its mass and frag- mentation pattern. control - 12 - 10 - 8 - 6 - 14 Discussion We have found substantial increase in peptide excretion in CONCENTRATION OF TRIPEPTIDE severe depression. To our surprise the level was found to be decreased in treated patients. Increase in peptides espe- Ser Figure 4 otonin uptake stimulation of the pure peptide cially in urine is usually due to peptidase deficiency or Serotonin uptake stimulation of the pure peptide. Concen- inhibition [13,14]. The presented data indicate that tration of peptide along the abcissa. Platelets were prepared -14 severely depressed patients show hyper-peptiduria as as outlined in methods. -14 is 10 to the power of -14 M(10 M). The typical hormetic dose response curve is shown. would be expected if peptidase defects were present. Pepti- dase deficiencies have been found in depression and mel- Page 5 of 7 (page number not for citation purposes) 5-HT UPTAKE AS C. P. M. Intensity, cps Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 ancholic states [15-19]. Because the level and pattern of human caso-morphines [42] and shows depressive traits. peptides differ, and differing chain lengths of peptides Since peptides in general are excellent peptidase inhibi- with the same activities are found, this points to a hetero- tors [43] and peptides also have strong tendencies to form geneity of genetics. Different families may have a different complexes [44,45] and bind to other molecules and mem- sets of dysfunctional enzymes probably peptidases or branes [46,47], a rather complex situation with varied peptidase binding proteins. This may explain the diffi- results can be envisioned. Bell shaped dose responses are culty of pinpointing a specific gene as the genetic disposi- common to many peptides and add to this complicated tion in depression. We suggest that we have depression picture [12]. The nature of some of these peptides is causing genes which may cause formation of different but shared with schizophrenia and may constitute the com- overlapping peptidases or peptidase regulating proteins, mon features of these disorders [48]. Possibly relevant to and that the mediators of depression may be peptides reg- a gut-brain axis in depression is epidemiological data ulating the uptake and release of different transmitters. showing very high frequency of depression in irritable Since antidepressant medication reduced the level of pep- bowel syndrome [49-51]. Future work is needed to eluci- tides (table 1) this could be due to peptidase induction as date if there is a correlation of peptide increase and degree has been shown for neuroleptic medication. It remains to of depression and to look for any correlations between be seen if peptide level correlates with the degree of individual peptide peaks and specific symptoms of this depression. The presence of opioids may explain the psy- disorder. chotic features of our patients because opioids have been shown to cause increased dopamine in the synaptic cleft Conclusion by inhibiting reuptake [20] An exogenous supply of pep- In severe cases of hospitalized patients with depression, tides may also explain the often seen and peculiar fluctu- we find peptide increase in urine. After treatment the level ating course of the disorder with morning worsening and is decreased. Some of these peptides affect serotonin afternoon a relative high or agitation. Increased reuptake uptake and others show exorphine like characteristics. of serotonin into platelets may have relevance to the Our study is based on a small number of patients due to reported changes in serotonin transport into platelets in problems of getting untreated ones. We do not know how depression [21,22]. Both the tri-cyclic antidepressants and general or specific these findings are to the depression selective serotonin re-uptake inhibitors have the opposite spectrum. However, our data are compatible with effect on serotonin uptake. We have previously found the reported decreased peptidase levels found by others in tri-peptide but amidated, in autistic patients' urine and depression. If correct the profound heterogeneity casts this tri-peptide stimulated the serotonin transporter medi- doubt on most double blind treatment trials in depres- ated uptake of serotonin in hamster ovarial cells trans- sion. fected with the human serotonin transporter gene [23]. That tri-peptide doubled the serotonin content of platelets Authors' contributions when injected into pups subcutaneously [24]. 5 HT 2a LY carried out the serotonin uptake studies, TH the diag- receptors are increased in brain tissue and platelets in nosis of patients and urine collection, KLR did the urine depression [25] which would agree with decreased levels analysis, mass spectrometry and administration of the in the synaptic cleft due to stimulated uptake. Also the project. decreased 5HIAA (5-hydroxy indole-acetic acid) in sui- cidal patients fit an increased uptake [26]. The tri-peptide Acknowledgements We wish to thank Major Ecbo's Foundation and Haldis and Josef Andresens sequence has only been found in reelin, which is a matrix foundation for support. proteinase [27]. That food derived peptides are taken up has been demonstrated [28,29] and is increased by pepti- References dase defects [30]. Depression has been found in cases of 1. Allan MG: Twin studies of affective illness. Arch Gen Psychiatry coeliac disease [31,32] indicating that such a mechanism 1976, 33:1476-1489. is not unreasonable. Considerable differences in rates in 2. Bertelsen A, Harvald B, Hauge M: A Danish twin study of manic- depressive disorders. Br J Psychiatry 1977, 130:330-351. different cultures may also thus be explainable. Other 3. Cavoret RJ, O'Gorman TW, Heywood EC, Troughton E: Genetic groups have found increases in some peptides in depres- and environmental factors in major depression. J Affect Disord 1985, 9:155-164. sion. Thus TRH (pE-H-P-NH2) has been found increased 4. Wender PH, Kety SS, Rosenthal D, Schulsinger F, Ortmann J, Lunde in CSF [33,34]; beta-endorphin [35] and an unspecified I: Psychiatric Disorders in the Biological and adoptive fami- opioid fraction 1 measured by receptor binding [36,37]. lies of adopted individuals with affective disorders. Arch Gen Psychiatry 1986, 43:923-929. Substance P was also found increased in CSF [38], and 5. Sælid G, Haug JO, Heiberg T, Reichelt KL: Peptide containing frac- delta sleep factor increased in plasma [39,40]. Also tions in depression. Biol Psychiatry 1985, 20:245-256. 6. Reichelt KL, Edminson PD, Toft KG: Urinary peptides in schizo- plasma arginine vasopressin increase in depression was phrenia and depression. Stress Med 1984, 1:169-181. inversely related to daytime motor activity [41]. Further- more post partum psychosis may be mediated by different Page 6 of 7 (page number not for citation purposes) Behavioral and Brain Functions 2007, 3:47 http://www.behavioralandbrainfunctions.com/content/3/1/47 7. Pedersen OS, Liu Y, Reichelt KL: Serotonin uptake stimulating 30. Mahe S, Tome D, Dumontier AM, Desjeux JF: Absorption of intact peptide found in plasma of normal individuals and in some morphiceptin by diisopropylfluorophosphate-treated rabbit autistic urines. J Pept Res 1999, 53:641-646. ileum. Peptides 1989, 10:45-52. 8. Reichelt WH, Reichelt KL: The possible role of peptides derived 31. Hallert C, Åstrøm J, Sedvall G: Psychic disturbances in adult from food proteins in diseases of the nervous system. In Epi- celiac disease III. Reduced central momoamine metabolism lepsy and other Neurological Disorders in Coeliac Disease Edited by: and signs of depression. Scand J Gastroenterol 1982, 17:25-28. Gobbi G. London: John Libbey & Comp Ltd; 1997:225-235. 32. Corvaglia L, Catamo R, Pepe G, Lazzari R, Corvaglia E: Depression 9. Reichelt WH, Ek J, Stensrud MB, Reichelt KL: Peptide excretion in in adult untreated celiac subjects: diagnosed by the pediatri- celiac disease. J Pediatr Gastroenterol Nutr 1998, 26:305-309. cian. Am J Gastroenterol 1999, 94:839-843. 10. Lingjaerde O: Uptake of serotonin in blood platelets: depend- 33. Kirkegaard C, Faber J, Hummer L, Rogowski P: Increased levels of ence on sodium and chloride, and inhibition by choline. FEBS TRH in cerebrospinal fluid from patients with endogenous Lett 1969, 3:103-106. depression. Psychoneuroendocrinology 1979, 4:227-235. 11. Böhlen P, Castillo F, Ling R, Guillemin R: An efficient procedure 34. Banki CM, Bissette G, Arato M, Nemeroff CB: Elevation of Immu- for the separation of peptides from amino acids and salts. Int noreactive CSF TRH in depressed patients. Am J Psychiatry J Pept Protein Res 1980, 16:306-310. 1988, 145:1526-1531. 12. Calabrese EJ, Baldwin LA: Hormesis: U-shaped dose responses 35. Darko DF, Risch SC, Gillin JC, Golshan S: Association of beta- and their centrality in toxicology. Trends Pharmacol Sci 2001, endorphin with specific clinical symptoms of depression. Am 22:285-291. J Psychiatry 1992, 149:1162-1167. 13. Abassi Z, Golomb E, Keiser HR: Neutral endopeptidase inhibi- 36. Agren H, Terenius L: Depression and CSF endorphin fraction 1: tion increases urinary excretion and plasma level of seasonal variation and higher levels in unipolar than bipolar Endothelin. Metabolism 1992, 41:683-685. patients. Psychiatry Res 1983, 10:303-311. 14. Watanabe Y, Kojima-Kumatsu T, Iwaki-Egewa S, Fujimoto Y: 37. Terenius L, Wahlstrom A, Agren T: Naloxone (narcan)treatment Increased excretion of proline-containing peptides in dipep- in depression: Clinical observations and effects on CSF tidyl-peptidase IV deficient rats. Res Commun Chem Pathol Phar- endorphins and monoamine metabolites. Psychopharmacology macol 1993, 81:323-350. (Berl) 1997, 54:31-33. 15. Maes M, Demeester I, Vanhoof G, Scharpe S, Bosmans E, Vandervorst 38. Rimon R, Le Greves P, Nyberg F, Heikkila L, Salmela L, Terenius L: C, Verkerk R, Minner B, Suy E, Raus J: Decreased serum dipepti- Elevation of substance P-like peptides in the CSF of psychiat- dyl-peptidase IV activity in major depression. Biol Psychiatry ric patients. Biol Psychiatry 1984, 19:509-516. 1991, 30:577-586. 39. Westrin A, Ekman R, Träskman-Bendz L: High delta sleep-induc- 16. Maes M, Scharpe S, Meltzer HY, Suy E, Cosyns P, Calabrese J: Lower ing peptide-like immuno-reactivity in plasma in suicidal angiotensin 1 converting enzyme activity in melancholic sub- patients with major depressive disorder. Biol Psychiatry 1998, jects: a pilot study. Biol Psychiatry 1992, 32:621-624. 43:734-739. 17. Maes M, Goosens F, Scharpe S, Meltzer HY, D'Hondt P, Cosyns P: 40. Westrin Å, Engström G, Ekman R, Träskman-Bendz L: Correlations Lower serum prolyl-endopeptidase enzyme activity in major between plasma-neuropeptides and temperament dimen- depression: further evidence that peptidases play a role in sions differ between suicidal patients and healthy controls. J patho-physiology of depression. Biol Psychiatry 1994, 35:545-552. Affect Disord 1998, 49:45-54. 18. Maes M, DeMeester I, Verkerk R, DeMedts P, Wauters A, Vanhoof 41. Van Londen L, Kerkhof GA, Van den Berg F, Goekoop JG, Zwinder- G, Vandoolaeghe E, Neels H, Scharpe S: Lower serum dipeptidyl man KH, Frankhuijzen-Siervogel AC, et al.: Plasma arginine vaso- peptidase IV activity in treatment resistant major depres- pressin and motor activity in major depression. Biol Psychiatry sion: relationships with immune-inflammatory markers. Psy- 1998, 43:196-204. choneuroendocrinology 1997, 22:65-78. 42. Lindstrøm LH, Nyberg F, Terenius , Bauer K, Besev G, Gunne LM, 19. Elgun S, Keskinege A, Kumbassar H: Dipeptidyl peptidase IV and Lyrenaas S, Wildeck-Lund G, Lundberg B: CSF and plasma beta- adenosine deaminase activity. Decrease in depression. Psy- casomorphin like opioid peptides in post-partum psychosis. choneuroendocrinology 1999, 24:823-832. Am J Psychiatry 1984, 141:1059-1066. 20. Hole K, Bergslien AA, Jørgensen H, Berge O-G, Reichelt KL, Trygstad 43. LaBella FL, Geiger JD, Glavin G: Administration of peptides OE: A peptide containing fraction from schizophrenia which inhibits the degradation of endogenous peptides. The stimulates opiate receptors and inhibits dopamine uptake. dilemma of distinguishing direct from indirect effects. Pep- Neuroscience 1979, 4:1139-1147. tides 1985, 6:645-660. 21. Paul SM, Rehavi M, Skolnic P, Ballenger JC, Goodwin FK: Depressed 44. Burhol K, Jensen TG, Florholmen TG, Jorde H, Vonen B, Olsen R: patients have decreased binding of tritiated imipramine to Protein-binding and aggregation of somatostatin in human platelet "tansporter". Arch Gen Psychiatry 1981, 38:1315-1317. plasma. Ital J Gastroenterol 1966, 18:1-6. 22. Oxenkrug GF: The content and uptake of 5-HT by blood plate- 45. Kastin AJ, Casillanos PF, Fischman PJ, Profitt JK, Graf MV: Evidence lets in depressive patients. J Neural Transm 1979, 45:285-289. for peptide aggregates. Pharmacol Biochem Behav 1984, 23. Keller F: 2nd annual report to EU Commission project BMH4-CT 96-0730 21:969-974. 1998:1-10. 46. Rocetti G, Venturella F, Roda IG: Enkephalin binding system in 24. Persico AM, Baldi A, Reichelt KL, Gonzales A, Keller F: Serotonin human plasma III Comparative protection of different pep- uptake-stimulating peptides extracted for urines of autistic tides. Neurochem Res 1988, 13:221-224. patients: potential significance for the pathogenesis of autis- 47. Meneszo NY, Khatchaturian C: Peptides bound to albumin. Life tic disorders. Amer Neurosci. Meet Los Angeles 1998. Abs no 2 Sci 1986, 39:. 25. Pandey GN: Altered serotonin function in suicide. Ann N Y Acad 48. Boteva K, Lieberman J: Reconsidering the classification of schiz- Sci 1997, 836:182-200. ophrenia and manic-depressive illness-a critical analysis and 26. Åsberg ML, Träskman L, Thoren P: 5-HIAA in the cerebrospinal new conceptual model. World J Biol Psychiatry 2003, 4:81-92. fluid: A biochemical suicide predictor. Arch Gen Psychiatry 1976, 49. Addolorato G, Capristo E, Stefanini GG, Gasbarrini G: Inflamma- 38:1193-1197. tory bowel disease: a study of the association between anxi- 27. Quattrocchi CC, Wannenes F, Persico AM, Ciafre SA, d'Arcangelo G, ety and depression, physical morbidity, and nutritional Farace MG, Keller F: Reelin is a Serine Protease of the Extra- status. Scand J Gastroenterol 1997, 32:1013-1021. cellular Matrix. J Biol Chem 2002, 277:303-309. 50. Haug TT, Mykletun A, Dahl AA: Are anxiety and depression 28. Chabance B, Marteau P, Rambaud JC, Migliore-Samour D, Boynard M, related to gastrointestinal symptoms in the general popula- Perrotin P, Guillet R, Jolles P, Fiat AM: Casein peptide release and tion? Scand J Gastroenterol 2005, 37:294-298. passage to the blood in humans during digestion of milk and 51. Alander T, Svärdsudd K, Johansson SE, Agreus L: Psychological ill- yogurt. Biochimie 1998, 80:155-165. ness is commonly associated with functional gastrointestinal 29. Gardner MLG: Absorption of intact proteins and peptides. In disorders and is important to consider during patient consul- Physiology of the Gastrointestinal Tract 3rd edition. Edited by: Johnson tation: a population-based study. BMC Med 2005, 3:8-20. LR. New York: Raven Press; 1994:1795-1782. Page 7 of 7 (page number not for citation purposes)

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